Molecular Insights Into HIV Biology

Introduction
Bringing the global HIV epidemic under control will require more effective approaches to prevent the
spread of the retrovirus, as well as broader use of existing and future antiretroviral drugs. These
interventions must be applicable in the developing world, where HIV has the most severe impact.
Understanding the dynamic interplay of HIV with its cellular host provides the biological basis for
controlling the epidemic. This chapter reviews current understanding of the HIV life cycle, with particular
attention to the interactions between viral proteins and cellular machinery, and highlights promising future
points of attack.

Binding and Entry

The genetic material of HIV, an RNA molecule 9 kilobases in length, contains 9 different genes encoding
15 proteins. Considerable insights have been gained into the function of these different gene products.
(Figure 1) To productively infect a target cell, HIV must introduce its genetic material into the cytoplasm of
this cell. The process of viral entry involves fusion of the viral envelope with the host cell membrane and
requires the specific interaction of the envelope with specific cell surface receptors. The two viral
envelope proteins, gp120 and gp41, are conformationally associated to form a trimeric functional unit
consisting of three molecules of gp120 exposed on the virion surface and associated with three molecules
of gp41 inserted into the viral lipid membrane. Trimeric gp120 on the surface of the virion binds CD4 on
the surface of the target cell, inducing a conformational change in the envelope proteins that in turn allows
binding of the virion to a specific subset of chemokine receptors on the cell surface.(1)(Figure 2) These
receptors normally play a role in chemoattraction, in which hematopoietic cells move along chemokine
gradients to specific sites. Although these receptors, which contain seven membrane-spanning domains,
normally transduce signals through G proteins,(2) signaling is not required for HIV infection.

Twelve chemokine receptors can function as HIV coreceptors in cultured cells, but only two are known to
play a role in vivo.(2) One of these, CCR5, binds macrophage-tropic, non-syncytium-inducing (R5)
viruses, which are associated with mucosal and intravenous transmission of HIV infection. The other,
CXCR4, binds T-cell-tropic, syncytium-inducing (X4) viruses, which are frequently found during the later
stages of disease.(3) In up to 13% of individuals of northern European descent, a naturally occurring
deletion of 32 base pairs in the CCR5 gene results in a mutant CCR5 receptor that never reaches the cell
surface.(4,5) Individuals homozygous for this mutation (1-2% of the Caucasian population) are almost
completely resistant to HIV infection.(4,5) These observations emphasize the pivotal role of CCR5 in the
spread of HIV and suggest that small molecules that prevent HIV interaction with CCR5 might form a
promising new class of antiretroviral drugs.

Both CD4 and chemokine coreceptors for HIV are found disproportionately in lipid rafts in the cell
membrane.(6) These cholesterol- and sphingolipid-enriched microdomains likely provide a better
environment for membrane fusion, perhaps by mirroring the optimal lipid bilayer of the virus.(7) Removing
cholesterol from virions, producer cells, or target cells greatly decreases the infectivity of HIV.(8) Studies
currently under way are exploring whether cholesterol-depleting compounds might be efficacious as
topically applied microbicides to inhibit HIV transmission at mucosal surfaces. The development of
effective microbicides represents an important component of future HIV prevention strategies.

The binding of surface gp120, CD4, and the chemokine coreceptors produces an additional radical
conformational change in gp41.(9) Assembled as a trimer on the virion membrane, this coiled-coil protein
springs open, projecting three peptide fusion domains that "harpoon" the lipid bilayer of the target cell.
The fusion domains then form hairpin-like structures that draw the virion and cell membranes together to
promote fusion, leading to the release of the viral core into the cell interior.(9) The fusion inhibitors T-20
and T-1249 act to prevent fusion by blocking the formation of these hairpin structures.

HIV virions can also enter cells by endocytosis. Usually, productive infection does not result, presumably
reflecting inactivation of these virions within endosomes. However, a special form of endocytosis has
been demonstrated in submucosal dendritic cells. These cells, which normally process and present
antigens to immune cells, express a specialized attachment structure termed DC-SIGN.(10) This C-type
lectin binds HIV gp120 with high affinity but does not trigger the conformational changes required for
fusion. Instead, virions bound to DC-SIGN are internalized into an acidic compartment and subsequently
displayed on the cell surface after the dendritic cell has matured and migrated to regional lymph nodes,
where it engages T cells.(11) Thus, dendritic cells expressing DC-SIGN appear to act as "Trojan horses"
facilitating the spread of HIV from mucosal surfaces to T cells in lymphatic organs.

Cytoplasmic Events
Once inside the cell, the virion undergoes uncoating, likely while still associated with the plasma
membrane.(Figure 2) This poorly understood process may involve phosphorylation of viral matrix proteins
by a mitogen-activated protein (MAP) kinase(12) and additional actions of cyclophilin A(13) and the viral
proteins Nef(14) and Vif.(15) Nef associates with a universal proton pump, V-ATPase,(16) which could
promote uncoating by inducing local changes in pH in a manner similar to that of the M2 protein of
influenza.(17) After the virion is uncoated, the viral reverse transcription complex is released from the
plasma membrane.(18) This complex includes the diploid viral RNA genome, lysine transfer RNA
(tRNALys) which acts as a primer for reverse transcription, viral reverse transcriptase, integrase, matrix
and nucleocapsid proteins, viral protein R (Vpr), and various host proteins. The reverse transcription
complex docks with actin microfilaments.(19) This interaction, mediated by the phosphorylated matrix, is
required for efficient viral DNA synthesis. By overcoming destabilizing effects of a recently identified
protein termed CEM15/APOBEC3G, Vif stabilizes the reverse transcription complex in most human cells.
(15-20)

Reverse transcription yields the HIV preintegration complex (PIC), composed of double-stranded viral
cDNA, integrase, matrix, Vpr, reverse transcriptase, and the high mobility group DNA-binding cellular
protein HMGI(Y).(21) The PIC may move toward the nucleus by using microtubules as a conduit.(22)
Adenovirus and herpes simplex virus 1 also dock with microtubules and use the microtubule-associated
dynein molecular motor for cytoplasmic transport. This finding suggests that many viruses use these
cytoskeletal structures for directional movement. How the switch from actin microfilaments to
microtubules is orchestrated remains unknown.

Recent studies have revealed a mechanism by which the target cell defends against the HIV intruder.
(23,24) Within 30 minutes of infection, select host proteins including the integrase interactor 1 (also
known as INI-1, SNF5, or BAF47), a component of the SWI/SNF chromatin remodeling complex, and
PML, a protein present in promyelocytic oncogenic domains, translocate from the nucleus into the
cytoplasm.(24)(Figure 2) Addition of arsenic trioxide sharply blocks PML movement and enhances the
susceptibility of cells to HIV infection raising the possibility that the normal function of PML is to oppose
viral infection.(24) The binding of integrase to integrase interactor 1 may be a viral adaptation that recruits
additional chromatin remodeling factors. Whether these complexes influence the site of viral integration or
improve subsequent proviral gene expression is not known.

Crossing the Nuclear Pore

Unlike most animal retroviruses, HIV can infect nondividing cells, such as terminally differentiated
macrophages.(25) This requires an ability to cross the intact nuclear membrane. With a Stokes radius of
approximately 28 nm or roughly the size of a ribosome, the PIC is roughly twice as large as the maximal
diameter of the central aqueous channel in the nuclear pore.(26) The 3 µm contour length of viral DNA
must undergo significant compaction, and the import process must involve considerable molecular
gymnastics.

One of the most contentious areas of HIV research involves the identification of key viral proteins that
mediate the nuclear import of the PIC. Integrase,(27) matrix,(28) and Vpr(29) have been implicated.
(Figure 2) Because plus-strand synthesis is discontinuous in reverse transcription, a triple helical DNA
domain or "DNA flap" results that may bind a host protein containing a nuclear targeting signal.(30) Matrix
contains a canonical nuclear localization signal that is recognized by the importins alpha and beta, which
are components of the classical nuclear import pathway. However, a recent publication calls into question
the contributions both of the nuclear import signal in integrase and of the DNA flap to the nuclear uptake
of the PIC.(31) The HIV Vpr gene product contains at least three noncanonical nuclear targeting signals.
(32) Vpr may bypass the importin system altogether, perhaps mediating the direct docking of the PIC with
one or more components of the nuclear pore complex. The multiple nuclear targeting signals within the
PIC may function in a cooperative manner or play larger roles individually in different target cells. For
example, while Vpr is not needed for infection of nondividing, resting T cells,(33) it enhances viral
infection in nondividing macrophages.(34) The finding that both matrix(35) and Vpr(32) shuttle between
the nucleus and cytoplasm explains their availability for incorporation into new virions.

Integration
Once inside the nucleus, the viral PIC can establish a functional provirus.(Figure 2) Integration of double-
stranded viral DNA into the host chromosome is mediated by integrase, which binds the ends of the viral
DNA.(21) The host proteins HMGI(Y) and barrier to autointegration (BAF) are required for efficient
integration, although their precise functions remain unknown.(36) Integrase removes terminal nucleotides
from the viral DNA, producing a two-base recess and thereby correcting the ragged ends generated by
the terminal transferase activity of reverse transcriptase.(21) Integrase also catalyzes the subsequent
joining reaction that establishes the HIV provirus within the chromosome.

Not all PICs that enter the nucleus result in a functional provirus. The ends of the viral DNA may be joined
to form a 2-LTR circle containing long terminal repeat sequences from both ends of the viral genome, or
the viral genome may undergo homologous recombination yielding a single-LTR circle. Finally, the viral
DNA may auto-integrate into itself, producing a rearranged circular structure. Although some circular
forms may direct the synthesis of the transcriptional transactivator Tat or the accessory protein Nef, none
produces infectious virus.(37) In a normal cellular response to DNA fragments, the nonhomologous end-
joining (NHEJ) system may form 2-LTR circles to protect the cell.(38) This system is responsible for rapid
repair of double-strand breaks, thereby preventing an apoptotic response. A single double-strand break
within the cell can induce G1 cell-cycle arrest. The ability of the free ends of the viral DNA to mimic such
double-strand chromosomal breaks may contribute to the direct cytopathic effects observed with HIV.

Transcriptional Controls
Integration can lead to latent or transcriptionally active forms of infection.(39) HIV's transcriptional latency
explains the inability of potent antiviral therapies to eradicate the virus from the body. Moreover, despite a
vigorous immune response early in infection, these silent proviruses are a reservoir that allows
reemergence of HIV when the body's defenses grow weaker. Understanding latency and developing
approaches to target latent virus are essential goals if eradication of HIV infection is ever to be achieved.

The chromosomal environment likely shapes the transcriptional activity of the provirus.(40) For example,
proviral integration into repressed heterochromatin might result in latency.(Figure 3) Other causes of
latency may include cell type differences in the availability of activators that bind to the transcriptional
enhancer in the HIV LTR or the lack of Tat. However, of the multiple copies of provirus that are usually
integrated in a given infected cell, at least one is likely to be transcriptionally active. This fact may explain
why the number of latently infected cells (105-106) in infected patients is small.

In the host genome, the 5´ LTR functions like other eukaryotic transcriptional units. It contains
downstream and upstream promoter elements, which include the initiator (Inr), TATA-box (T), and three
Sp1 sites.(41) These regions help position the RNA polymerase II (RNAPII) at the site of initiation of
transcription and to assemble the preinitiation complex. Slightly upstream of the promoter is the
transcriptional enhancer, which in HIV-1 binds nuclear factor [kappa]B (NF-[kappa]B), nuclear factor of
activated T cells (NFAT), and Ets family members.(42) NF-[kappa]B and NFAT relocalize to the nucleus
after cellular activation. NF-[kappa]B is liberated from its cytoplasmic inhibitor, I[kappa]B, by stimulus-
coupled phosphorylation, ubiquitination, and proteosomal degradation of the inhibitor.(43) NFAT is
dephosphorylated by calcineurin (a reaction inhibited by cyclosporin A) and, after its nuclear import,
assembles with AP1 to form the fully active transcriptional complex.(44) NF-[kappa]B, which is composed
of p50 and p65 (RelA) subunits, increases the rates of initiation and elongation of viral transcription.(45)
Since NF-[kappa]B is activated after several antigen-specific and cytokine-mediated events, it may play a
key role in rousing transcriptionally silent proviruses

When these factors engage the LTR, transcription begins, but in the absence of Tat described below the
polymerase fails to elongate efficiently along the viral genome.(Figure 3) In the process, short
nonpolyadenylated transcripts are synthesized, which are stable and persist in cells due to the formation
of an RNA stem loop called the transactivation response (TAR) element.(46)

Tat significantly increases the rate of viral gene expression. With cyclin T1 (CycT1), Tat binds to the TAR
RNA stem-loop structure and recruits the cellular cyclin-dependent kinase 9 (Cdk9) to the HIV LTR.(47)
(Figure 3) Within the positive transcription elongation factor b (P-TEFb) complex, Cdk9 phosphorylates
the C-terminal domain of RNAPII, marking the transition from initiation to elongation of eukaryotic
transcription.(48) Other targets of P-TEFb include negative transcription elongation factors (N-TEF), such
as the DRB-sensitivity inducing (DSIF) and negative elongation (NELF) factors.(48) The high efficiency
with which the HIV LTR attracts these negative transcription factors in vivo may explain why the LTR is a
poor promoter in the absence of Tat. The arginine-rich motif (ARM) within Tat binds the 5´ bulge region in
TAR. A shorter ARM in cyclin T1, which is also called the Tat-TAR recognition motif (TRM), binds the
central loop of TAR.(47)

Binding of the Tat cyclin T1 complex to both the bulge and loop regions of TAR strengthens the affinity of
this interaction. All of these components are required for Tat transactivation. In the presence of the
complex between Tat and P-TEFb, the RNAPII elongates efficiently. Because murine CycT1 contains a
cysteine at position 261, the complex between Tat and murine P-TEFb binds TAR weakly.(49) Thus, Tat
transactivation is severely compromised in murine cells. Cdk9 also must undergo autophosphorylation of
several serine and threonine residues near its C-terminus to allow productive interactions between Tat, P-
TEFb, and TAR.(50) Additionally, basal levels of P-TEFb may be low in resting cells or only weakly active
due to the interaction between P-TEFb and 7SK RNA.(51) All of these events may contribute to
postintegration latency.

Viral Transcripts
Transcription of the viral genome results in more than a dozen different HIV-specific transcripts.(52) Some
are processed cotranscriptionally and, in the absence of inhibitory RNA sequences (IRS), transported
rapidly into the cytoplasm.(53) These multiply spliced transcripts encode Nef, Tat, and Rev. Other singly
spliced or unspliced viral transcripts remain in the nucleus and are relatively stable. These viral transcripts
encode the structural, enzymatic, and accessory proteins and represent viral genomic RNAs that are
needed for the assembly of fully infectious virions.

Incomplete splicing likely results from suboptimal splice donor and acceptor sites in viral transcripts. In
addition, the regulator of virion gene expression, Rev, may inhibit splicing by its interaction with alternate
splicing factor/splicing factor 2 (ASF/SF2)(54) and its associated p32 protein.(55)

Transport of the incompletely spliced viral transcripts to the cytoplasm depends on an adequate supply of
Rev.(53) Rev is a small shuttling protein that binds a complex RNA stem-loop termed the Rev response
element (RRE), which is located in the env gene. Rev binds first with high affinity to a small region of the
RRE termed the stem-loop IIB.(56)(Figure 4) This binding leads to the multimerization of Rev on the
remainder of the RRE. In addition to a nuclear localization signal, Rev contains a leucine-rich nuclear
export sequence (NES).(53) Of note, the study of Rev was the catalyst for the discovery of such NES in
many cellular proteins and led to identification of the complex formed between CRM1/exportin-1 and this
sequence.(53)

The nuclear export of this assembly (viral RNA transcript, Rev, and CRM1/exportin 1) depends critically
on yet another host factor, RanGTP. Ran is a small guanine nucleotide-binding protein that switches
between GTP- and GDP-bound states. RanGDP is found predominantly in the cytoplasm because the
GTPase activating protein specific for Ran (RanGAP) is expressed in this cellular compartment.
Conversely, the Ran nucleotide exchange factor, RCC1, which charges Ran with GTP, is expressed
predominantly in the nucleus. The inverse nucleocytoplasmic gradients of RanGTP and RanGDP
produced by the subcellular localization of these enzymes likely plays a major role in determining the
directional transport of proteins into and out of the nucleus. Outbound cargo is only effectively loaded onto
CRM1/exportin-1 in the presence of RanGTP. However, when the complex reaches the cytoplasm, GTP
is hydrolyzed to GDP, resulting in release of the bound cargo. The opposite relationship regulates the
nuclear import by importins alpha and beta, where nuclear RanGTP stimulates cargo release.(53)

For HIV infection to spread, a balance between splicing and transport of viral mRNA species must be
achieved. If splicing is too efficient, then only the multiply spliced transcripts appear in the cytoplasm.
Although required, the regulatory proteins encoded by multiply spliced transcripts are insufficient to
support full viral replication. However, if splicing is impaired, adequate synthesis of Tat, Rev, and Nef will
not occur. In many non-primate cells, HIV transcripts may be overly spliced, effectively preventing viral
replication in these hosts.(57)

HIV Replication
In contrast to Tat and Rev, which act directly on viral RNA structures, Nef modifies the environment of the
infected cell to optimize viral replication.(2)(Figure 4) The absence of Nef in infected monkeys and
humans is associated with much slower clinical progression to AIDS.(58,59) This virulence caused by Nef
appears to be associated with its ability to affect signaling cascades, including the activation of T-cell
antigen receptor,(60) and to decrease the expression of CD4 on the cell surface.(61,62) Nef also
promotes the production and release of virions that are more infectious.(63,64) Effects of Nef on the PI3-
K signaling cascade--which involves the guanine nucleotide exchange factor Vav, the small GTPases
Cdc42 and Rac1, and p21-activated kinase PAK--cause marked changes in the intracellular actin
network, promoting lipid raft movement and the formation of larger raft structures that have been
implicated in T-cell receptor signaling.(65) Indeed, Nef and viral structural proteins colocalize in lipid rafts.
(64,66) Two other HIV proteins assist Nef in downregulating expression of CD4.(67) The envelope protein
gp120 binds CD4 in the endoplasmic reticulum, slowing its export to the plasma membrane,(68) and Vpu
binds the cytoplasmic tail of CD4, promoting recruitment of TrCP and Skp1p.(Figure 5) These events
target CD4 for ubiquitination and proteasomal degradation before it reaches the cell surface.(69)

Nef acts by several mechanisms to impair immunological responses to HIV. In T cells, Nef activates the
expression of FasL, which induces apoptosis in bystander cells that express Fas,(70) thereby killing
cytotoxic T cells that might otherwise eliminate HIV-1 infected cells. Nef also reduces the expression of
MHC I determinants on the surface of the infected cell(71)(Figure 4) and so decreases the recognition
and killing of infected cells by CD8 cytotoxic T cells. However, Nef does not decrease the expression of
HLA-C,(72) which prevents recognition and killing of these infected cells by natural killer cells.

Nef also inhibits apoptosis. It binds and inhibits the intermediate apoptosis signal regulating kinase-1
(ASK-1)(73) that functions in the Fas and TNFR death signaling pathways and stimulates the
phosphorylation of Bad leading to its sequestration by 14-3-3 proteins.(74)(Figure 4) Nef also binds the
tumor suppressor protein p53, inhibiting another potiential initator of apoptosis.(75) Via these different
mechanisms, Nef prolongs the life of the infected host cell, thereby optimizing viral replication.

Other viral proteins also participate in the modification of the environment in infected cells. Rev-
dependent expression of Vpr induces the arrest of proliferating infected cells at the G2/M phase of the cell
cycle.(76) Since the viral LTR is more active during G2, this arrest likely enhances viral gene expression.
(77) These cell-cycle arresting properties involve localized defects in the structure of the nuclear lamina
that lead to dynamic, DNA-filled herniations that project from the nuclear envelope into the cytoplasm.(78)
(Figure 4) Intermittently, these herniations rupture, causing the mixing of soluble nuclear and cytoplasmic
proteins. Either alterations in the lamina structure or the inappropriate mixing of cell cycle regulators that
are normally sequestered in specific cellular compartments could explain the G2 arresting properties of
Vpr.

Viral Assembly
New viral particles are assembled at the plasma membrane.(Figure 5) Each virion consists of roughly
1500 molecules of Gag and 100 Gag-Pol polyproteins,(79) two copies of the viral RNA genome, and Vpr.
(80) Several proteins participate in the assembly process, including Gag polyproteins and Gag-Pol, as
well as Nef and Env. A human ATP-binding protein, HP68 (previously identified as an RNase L inhibitor),
likely acts as a molecular chaperone, facilitating conformational changes in Gag needed for the assembly
of viral capsids.(81) In primary CD4 T lymphocytes, Vif plays a key but poorly understood role in the
assembly of infectious virions. In the absence of Vif, normal levels of virus are produced, but these virions
are noninfectious, displaying arrest at the level of reverse transcription in the subsequent target cell.
Heterokaryon analyses of cells formed by the fusion of nonpermissive (requiring Vif for viral growth) and
permissive (supporting growth of Vif-deficient viruses) cells have revealed that Vif overcomes the effects
of a natural inhibitor of HIV replication.(20,82) Recently this factor, initially termed CEM15/APOBEC3G,
was identified(83) and shown to share homology with APOBEC1, an enzyme involved in RNA editing.
Whether the intrinsic antiviral activity of CEM15 involves such an RNA editing function remains unknown.
CEM15 is expressed in non-permissive but not in permissive cells and when introduced alone is sufficient
to render permissive cells nonpermissive.

Virion Budding
The Gag polyproteins are subject to myristylation,(84) and thus associate preferentially with cholesterol-
and glycolipid-enriched membrane microdomains.(85) Virion budding occurs through these specialized
regions in the lipid bilayer, yielding virions with cholesterol-rich membranes. This lipid composition likely
favors release, stability, and fusion of virions with the subsequent target cell.(7)

The budding reaction involves the action of several proteins, including the "late domain"(86) sequence
(PTAP) present in the p6 portion of Gag.(87)(Figure 5) The p6 protein also appears to be modified by
ubiquitination. The product of the tumor suppressor gene 101 (TSG101) binds the PTAP motif of p6 Gag
and also recognizes ubiquitin through its ubiquitin enzyme 2 (UEV) domain.(88,89) The TSG101 protein
normally associates with other cellular proteins in the vacuolar protein sorting pathway to form the
ESCRT-1 complex that selects cargo for incorporation into the multivesicular body (MVB).(90) The MVB
is produced when surface patches on late endosomes bud away from the cytoplasm and fuse with
lysosomes, releasing their contents for degradation within this organelle. In the case of HIV, TSG101
appears to be "hijacked" to participate in the budding of virions into the extracellular space away from the
cytoplasm.

Summary and Conclusions

As the AIDS pandemic continues, advances in antiretroviral therapies have slowed its advance in the
industrialized world, but have had little effect in developing countries. Because of its high rate of mutation,
HIV is able to refine and optimize its interactions with various host proteins and pathways, thereby
promoting its growth and spread. The virus ensures that the host cell survives until the viral replicative
cycle is completed. Possibly even more damaging, HIV establishes stable latent forms that support the
chronic nature of infection. Eradication of the virus appears unlikely until effective methods are developed
to purge these latent viral reservoirs.

Basic science will clearly play a leading role in future attempts to solve the mysteries of viral latency and
replication. A small-animal model that recapitulates the pathogenic mechanisms of HIV is sorely needed
to study the mechanisms underlying viral cytopathogenesis. Virally induced cell death is not limited to
infected targets but also involves uninfected bystander cells.(91) Murine cells support neither efficient
virion assembly nor release of virions from the cell surface.(92) Currently, this defect represents a major
impediment to the successful development of a rodent model of AIDS.

Proposed mechanisms for HIV killing of T cells include the formation of giant cell syncytia through the
interactions of gp120 with CD4 and chemokine receptors,(93) the accumulation of unintegrated linear
forms of viral DNA, the proapoptotic effects of the Tat,(94) Nef,(95) and Vpr(96) proteins, and the adverse
effects conferred by the metabolic burden that HIV replication places on the infected cell.(97) Of note,
expression of Nef alone as a transgene in mice recapitulates many of the clinical features of AIDS,
including immunodeficiency and loss of CD4-positive cells.(98) All of these mechanisms suggest potential
points of therapeutic intervention. Finally, future therapies will likely target viral proteins other than the
reverse transcriptase, protease, and integrase enzymes. Clinical trials are already underway to study
small molecules or short peptides that block the binding of HIV to cell-surface chemokine receptors or
interfere with the machinery of viral-host cell fusion. Although not as advanced in development, small
molecules have been found that block Tat transactivation(99) and Rev-dependent export of viral
transcripts from the nucleus to the cytoplasm.(100) As a proof of principle, dominant-negative mutants of
Tat, Rev, and Gag proteins have been shown to block viral replication. By increasing the number of
antiviral compounds available to target different steps in the viral replicative cycle, in particular drugs that
can be deployed in developing countries, research at the cellular level can serve to extend survival and to
improve the quality of life for infected individuals, and to inhibit the spread of AIDS.

Executive Summary

The development and application of an effective vaccine against HIV is our best hope for
stemming the devastating consequences of the AIDS pandemic. This is particularly true because
HIV infection has caused enormous social and economic losses in the developing world and
because of the high costs and other barriers to behavioral or biomedical interventions against
HIV transmission or infection. Unfortunately, mounting difficulties seriously threaten the
creation of an effective vaccine.
One significant concern is the present lack of basic knowledge needed by private enterprise to
meaningfully enter AIDS vaccine development. Another concern, despite proof of principle in
some nonhuman primate models, is a widespread perception that an effective vaccine against
HIV is highly unlikely, will be extremely difficult to develop, and is far in the future.
Surprisingly, HIV vaccine research and development programs of the NIH currently receive the
least funding of any of the major AIDS research disciplines as defined by the Office of AIDS
Research (OAR). Thus, the combined response from industry, Government, and the public is
disproportionately low compared with the immediate and long-term public health benefits that an
effective AIDS vaccine would offer worldwide.

There is a growing recognition that the NIH must now bear the major responsibility for driving
research toward the development of a vaccine against HIV. The role of the NIH is particularly
important since new concepts and strategies may be required to design a vaccine against this
unique human pathogen. HIV-1 is a retrovirus that attacks the immune system and is distinctive
in a number of ways from other viruses against which vaccines have already been developed.
Making a vaccine to counter this unusual virus may require an increased understanding of the
human immune system and its specific antiviral response. The role of the NIH in funding
research for the acquisition of medical knowledge has become ever more critical for HIV vaccine
development. Yet the NIH must be prepared to go beyond its traditional role, for the discovery
and development of a vaccine demands more than just the acquisition of fundamental
knowledge; it requires that the information be applied and resultant vaccine strategies
appropriately evaluated. Thus, NIH-funded research must become the primary "discovery
engine" to power vaccine development by the commercial sector or, if needed, by the Federal
Government. Without a strong stimulus from NIH that includes much needed basic information,
the waning private sector interest in an HIV vaccine may vanish altogether.

A discovery engine for an AIDS vaccine entails striking an appropriate balance between
fundamental and applied research, the preclinical testing of vaccine concepts in primate models,
and the conduct of human clinical trials of appropriate vaccine candidates. Having recognized
the necessity for a multicomponent AIDS vaccine research and development program, the
National Institute of Allergy and Infectious Diseases (NIAID) has set in place the framework for
such an effort. The NIAID program represents the major scientific thrust of the vaccine effort
supported by NIH. Its principal components are Basic Research, Targeted Research, and Clinical
Trial networks constituting a small but well-integrated vaccine development activity. The
Vaccine Research and Development Area Review Panel evaluated each of these areas separately
and together.

Basic Research

The Basic Research effort, as defined by the portfolio of R01 grants encoded as AIDS/ "vaccine-
related," was considered to be vastly insufficient. AIDS grants that were appropriately coded
cover only a fraction of the research activities necessary for vaccine development. It became
apparent during this Panel's review that some of the research that should be regarded as vaccine-
related actually had been coded as Etiology and Pathogenesis and was under review of the
Etiology and Pathogenesis Panel; nonetheless, many scientific aspects of vaccine research
demand additional attention. Chief among these is the need for a better understanding of the
immune system and its response to HIV infection, both in humans and in the nonhuman primate
vaccine models. Also lacking is a basic understanding of correlates of protection and of the HIV
immunogens that are required to induce vaccine responses of appropriate breadth and duration.
Of particular importance are studies concerning the basic immunology of the female and male
genital tracts and exploration of effective immunization routes. Attempts to stimulate interest in
vaccine-related research in general (not only in AIDS) through Requests for Applications (RFAs)
or Program Announcements (PAs) have been largely unsuccessful because of limited funds, the
one-time nature of funding for RFAs, and the failure of many applications responsive to PAs to
obtain fundable scores. In addition, the "one-time" aspect of RFAs is not, by definition, the
appropriate method to maintain a sustained effort to develop a vaccine against HIV-1.

During this review it became apparent that even applications at the cutting edge of vaccine
research tended to fare poorly in Initial Review Groups (IRGs) or study sections, perhaps
because their empirical nature was not always appreciated by reviewers. The Panel has
considered these issues at length and recommends several strategies to enhance basic research
that is relevant and appropriate to HIV vaccine research and development. Although it is
essential to increase the general level of support for basic research, the Panel believes that an
effective solution requires more than this. The Panel is convinced that it is necessary to create a
"culture" in which vaccine research is both supportable and an attractive area of research for
investigators. This must be a culture that will entice leading immunologists and virologists from
within and outside the AIDS field to contribute their expertise to overcome the critical and
challenging problems associated with developing an HIV vaccine. Crucial to this culture is the
development of a peer-review mechanism that will have the broad expertise and continuity
required to evaluate vaccine-related proposals. At present, no single study section has the
essential combination or depth of talents in microbiology, host defense mechanisms,
immunology, and chemistry that is pertinent to vaccine biology, especially when considering an
agent as complex as HIV-1. The need for vaccines in human and veterinary medicine is now so
obvious that the Panel recommends the creation of a vaccine-dedicated study section. If
necessary, it may be established at the expense of an existing one. Such a study section need not
be restricted to research on an AIDS vaccine since multidisciplinary interactions might best be
fostered by a broader approach.

Recommendations

• Create an IRG that would be dedicated to broad aspects of vaccine research,

including both HIV and other pathogens.

• Continue NIH/NIAID efforts to encourage and solicit the research community to

submit applications in vaccine biology and immunology.

• Selectively target vaccine-related research areas for special consideration during

review and funding.

• Increase the access of basic research scientists, who are interested in HIV
interactions with the human immune system, to clinical materials emerging from
studies within existing networks such as the Multicenter AIDS Cohort Study
 (MACS), the AIDS Vaccine Evaluation Group (AVEG), and the HIV Network for
Efficacy Trials (HIVNET) and from certain animal model studies.

Targeted Research

Targeted Research, as defined by the Panel, encompasses several elements within broader areas
such as vaccine design and preclinical vaccine evaluation. Targeted Research can include both
investigator-initiated and "directed" research activities that range from R01 grants on basic AIDS
vaccine strategies to highly directed contract research mechanisms. The major programs include
the National Cooperative Vaccine Development Groups (NCVDGs), the AIDS Cooperative
Adjuvant Group (now largely terminated), the Collaborative Mucosal Immunology Groups
(CMIGs), the Correlates of HIV Immune Protection Laboratory Contract (coded under
Pathogenesis and Etiology, to be terminated June 1997), and both the Simian Vaccine Evaluation
and Chimpanzee Vaccine Resource Units. Support services for these and other vaccine-related
activities include a Resources Support Contract and a Preclinical Master Agreement contract
program for reagents, services, and animal studies.

The Panel recognized the value of this network of resources and the continuing need for these
programs. However, the links to related efforts with substantial budgets in other NIH Institutes,
Centers, and Divisions (ICDs) such as the National Cancer Institute (NCI) and the National
Center for Research Resources (NCRR) are not well-established, and better ways to bridge the
NIAID programs to related activities in other ICDs must be found. The Panel examined the way
in which animal models for HIV vaccine research have been utilized and concluded that such
studies often have not been conducted in such a manner as to provide the essential information
needed for vaccine development. The Panel concluded that this area requires considerably more
oversight and coordination than has been applied in the past.

Recommendations

• The Panel specifically supports and commends the recent establishment of a

Vaccine Design Focus Group by NIAID. This group is composed of intramural and
extramural investigators from both academia and industry who have direct
experience in vaccine design and immunogenicity and are empowered to make
appropriate priority choices.

• The charge of this group should be expanded to an NIH-wide effort to: (1)
understand the early steps in viral infection and pathogenesis in the host, (2)
evaluate candidate vaccines in a systematic manner that will allow promising
approaches to be identified, (3) determine correlates of immunity, and (4) establish
primary (prevention of infection) and secondary (attenuation of infection such that
disease is prevented and transmission is curtailed) vaccine goals. Another critical
role of the expanded Vaccine Design Focus Group should be to seek out and
evaluate new vaccine candidates, so that it can recommend the most promising
concepts for clinical testing by the AIDS Vaccine Evaluation Group (AVEG) (see
below).

Clinical Trials
The Clinical Trials program encompasses the NIAID-supported AVEG which conducts Phase
I/II clinical evaluations of AIDS vaccine candidates for safety and immunogenicity, and the
HIVNET, which is designed to provide baseline seroincidence information in high-risk cohorts
and support for efficacy studies. The latter effort has formal and informal links to training and
infrastructure efforts of the Fogarty International Center (FIC), to cohorts for epidemiological
and intervention studies supported by the National Institute on Drug Abuse (NIDA), and to
studies on AIDS and sexually transmitted diseases (STDs) at the Centers for Disease Control and
Prevention (CDC). Together, these sites and cohorts are well-suited to conducting the evaluation
of vaccine candidates in humans.

The dilemma that now confronts both AVEG and HIVNET is related to the paucity of promising
new AIDS vaccine candidates. In addition, the relative size and use of the infrastructure of
HIVNET should be reexamined when vaccine candidates are not available.

Recommendations

• AVEG should work closely with the Vaccine Design Focus Group to encourage the
preclinical development of promising vaccine candidates that are suitable for
clinical evaluation in Phase I and II studies as well as those that might prove worthy
of evaluation for efficacy.

• Guidelines should be established for the advancement of a vaccine product to

efficacy trials sponsored by the NIH. Although the precise criteria might vary with
the nature of the concept under evaluation, a product ideally should be shown to
induce humoral and cellular immunity that is broad, durable, and likely to provide
a significant barrier to natural HIV infection. If and when appropriate animal
models become available, demonstration of protection in the preclinical evaluation
with such models should support the entry of a vaccine into efficacy trials.

• AVEG also should invest more effort on in-depth comprehensive assessments of

human immune responses to HIV antigens. Greater emphasis should be placed on
the laboratory analysis of immune responses in vaccinees, even if this necessitates
the study of many fewer individuals with any one vaccine candidate. Among
immune system parameters that should be evaluated in more detail are the
generation, function, and specificity of cytotoxic T lymphocytes (CTLs); the
relevance of neutralizing and nonneutralizing antibodies; the importance of Th1
and Th2 subsets of helper T cells; the targeting of immune cells to mucosal sites; the
sensitivity to infection and function of antigen-presenting cells (particularly
dendritic cells); and the roles of Type 1 versus Type 2 cytokines in specific and
nonspecific immunity to viral infection. The participation of non-AVEG
investigators in such studies is essential and should be encouraged.

• NIAID should rapidly reassess the status of the HIVNET program. It is likely that
few expanded (Phase II or efficacy) vaccine trials will be conducted within the next 5
years; thus, a careful reevaluation of the size and nature of HIVNET programs is
now needed. The seronegative cohorts that have been established for determination
of seroincidence can and should be used to evaluate biomedical and/or behavioral
 strategies designed for reduction in HIV transmission, as has been proposed by
HIVNET. If appropriately sampled, these and future cohorts also would be of value
for studies of primary HIV infection and pathogenesis, and studies of early
treatment of acute infection. However, because the principal mission of HIVNET
has been vaccine preparedness, it is not obvious that HIVNET has the intrinsic
expertise or infrastructure to move effectively beyond its original mission. This
raises the question of where and how such expanded studies are best undertaken.
NIAID should promptly compile a comprehensive research plan for the HIVNET
effort that addresses these issues. This plan should be reviewed by a panel of experts
in behavioral, epidemiologic, prevention, and pathogenesis research. The Panel also
urges NIAID to prepare an overall funding strategy for HIVNET that is congruent
with plans for vaccine development, and that should be reviewed by the OAR.
Finally, NIAID should strengthen the ties between AVEG and HIVNET so that each
group can benefit from the expertise of the other.

In general, the Panel felt that NIAID, with additional advice and leadership, is well-positioned to
assume even more responsibility for the direction of an AIDS vaccine discovery and
development program. This will be aided by forming stronger partnerships with other ICDs,
academia, and industry. NIAID programs currently are constrained by having insufficient funds
to adequately support all areas of extramural research and development. These programs would
benefit from increased flexibility in funding, the rapid deployment of resources, and the creation
of new mechanisms for enhancing extramural research in areas that are vital to vaccine research
and development. NIAID's efforts in vaccine research and development would be enhanced by
seeking much more extensive involvement of the extramural vaccine research community in the
critical decision-making process of the program.

The Panel also reviewed substantial portfolios of HIV vaccine-related activities at the NCI and
NCRR and a much smaller program at the National Institute of Dental Research (NIDR). The
Panel expressed serious reservations about the cost- effectiveness of these programs. The NCI
HIV/AIDS vaccine effort is almost exclusively an intramural program, dependent on dispersed
investigator-initiated research that is only periodically assessed by external program review.
NCRR support intersects with AIDS vaccine research and development primarily in two areas:
the Regional Primate Research Centers (RPRCs) and the General Clinical Research Centers
(GCRCs). These Centers are training and infrastructure programs that have highly diverse AIDS
components and even further variability in the amount of effort actually applied to AIDS vaccine
development. The true contribution to vaccine development of the substantial NCRR expenditure
coded in this area was difficult to assess, and a more thorough evaluation of the NCRR activities
related to HIV vaccine research is necessary.

Recommendation

• The Panel found NIAID's extramural vaccine efforts to be well-integrated; however,

the vaccine-related activities at the NCI, NIDR, and the NCRR appear to be
critically lacking in oversight and coordination. The entire HIV vaccine effort of
NIH would best be coordinated by the OAR with centralization in a single ICD,
such as NIAID, and with appropriate linkages to other ICDs. Establishment of such
 a program requires that a strong, effective, and visible leadership structure be
created that includes non-Government experts who have had extensive experience in
vaccine research and development. Such an organizational structure will also enable
OAR to address the problems related to a balanced allocation of annual resources
(and discretionary resources) where they are most needed for AIDS Vaccine
Research and Development.

Summary

The primary public health goal of eradicating AIDS nationally and globally can best be met by
an effective vaccine that is safe and affordable. However, a combination of economic and
scientific obstacles seriously threatens the ability to sustain an HIV vaccine research and
development program. This has raised questions of whether the combined response of
Government and industry has been sufficient and whether new approaches are required to
promote an invigorated effort in HIV vaccine research and development. To this end, the Panel
strongly urges NIH to undertake the primary responsibility for ensuring that a more vigorous and
effective research program in HIV vaccines is established and pursued. This will maximize the
potential to exploit the emerging knowledge needed for the future design and development of an
effective vaccine. Such a program should include expanded efforts to fund basic vaccine
research, a stronger and more coordinated effort in preclinical research, and an appropriately
balanced clinical trial infrastructure. The OAR, in concert with the participating ICDs, must
ensure that this new initiative with expanded scope is optimally structured, balanced, and
coordinated. The program should be visible and guided by effective leadership that involves
active participation of non-Government vaccine experts. Its primary mission should be to forge
effective partnerships with the biomedical community and private enterprise that would result in
development and production of an HIV vaccine for the public good.

Introduction

The Vaccine Research and Development Area Review Panel was organized on a thematic basis,
covering three broad areas: Basic Research, Targeted Research, and Clinical Trials Research.
Each of these areas was reviewed in depth by a subpanel which provided an evaluation and
specific recommendations. From these reports, an Executive Summary was crafted reflecting the
major recommendations of each subpanel along with several overarching issues that emerged
from discussions among Panel members. Several problems also were identified that were not
specific to vaccine research; these are grouped in the section entitled Special Issues. Some of
these points resonate with the observations of other Area Review Panels (ARPs).

The major theme of this report is that HIV vaccine research and development is in crisis. This
crisis currently extends from the difficulty that new vaccine concepts encounter in peer review;
through the lack of coordination and a shortage of funds for targeted, developmental testing of
these concepts in animal models and Phase I clinical trials; to an insufficient technical base to
engage industry or private sector initiatives in meaningful vaccine development. In addition,
there are administrative concerns about the distribution of funds among different competing
ICDs and the relative balance between intramural and extramural funding, particularly in the
NCI. The crisis in AIDS vaccine research and development requires immediate attention if we
are to achieve the public health goal of halting the AIDS pandemic. Both the overall evaluation
embodied in the Executive Summary and the more specific subpanel reports reflect this deep
concern and have prompted recommendations that are intended to generate an invigorated and
effective national effort to develop an HIV vaccine.

A. Panel Goals

The Panel was asked to review the status of current research in the field to determine what was
needed for effective AIDS vaccine development, and to determine whether current funding or
planned funding would meet those needs.

The Panel's initial goal was to attempt to answer a number of key questions related to HIV
vaccine research in each of the following areas:

Basic Research

1. Is basic research providing the fundamental information necessary to design and evaluate
AIDS vaccines? Are there gaps in scientific areas that should be addressed?

2. How can NIH encourage additional high-caliber investigator-initiated research pertinent

to HIV vaccines?

Targeted Research

1. How successful are targeted research programs for HIV vaccine design? How successful
are they for vaccine development?

2. Is effective use being made of animal resources for preclinical vaccine evaluation?

Clinical Trials Research

1. To what level should established infrastructures for evaluating vaccines in people be

maintained considering the limited number of new vaccine candidates available for
testing?

2. What other valuable research might be accomplished within these units until promising
vaccine candidates are available for testing?

Additional questions spanning these areas were readily apparent:

1. What is the appropriate balance in resources allocated for investigator-initiated research,

targeted research programs, and clinical trials? How should resources be allocated
between intramural and extramural programs?

2. Are current funding mechanisms adequate, or are there novel funding mechanisms that
would be particularly well-suited to HIV vaccine research and development?
 3. How can NIH best encourage the private sector to participate more actively in HIV
vaccine development?

4. Is there duplication or overlap between different programs? Is there sufficient

cooperation and collaboration between ICDs and between NIH and other Federal
agencies? What role should OAR play in this area?

5. What should the NIH and other agencies of the Federal Government do to collect the
technical information essential to ensuring the engagement of private industry in the
vaccine enterprise? What should NIH do if industry's role is even further diminished?
What role(s) should NIH and the Federal Government assume in AIDS vaccine research
relative to that of the private sector (industry, non-Governmental organizations,
independent research institutions, international efforts, etc.)?

6. What priority should vaccine research receive relative to other AIDS research priorities?

B. Panel and Subpanel Structure and Process

Recognizing that focus on any one type of vaccine effort would be too narrow, the Panel initially
resolved to establish subpanels to review three areas that roughly reflect the development of an
AIDS vaccine: the basic research on AIDS vaccines, including vaccine design and discovery;
targeted AIDS vaccine development focused largely on preclinical testing in animal models for
safety and efficacy; and AIDS vaccine clinical trials efforts, primarily in uninfected subjects. The
Panel met for the first time May 3, 1995, when tasks were assigned to individual Panel members.

A balance of expertise and research interests was considered in the selection of members for
each subpanel, recognizing the potential for conflict of interest in the different scientific areas, so
that competing interests might provide both a multidisciplinary and balanced review of the
information. Thus, basic scientists were included in the review of the Clinical Trials Program and
clinical investigators were involved in the review of Basic Research. All members had the
opportunity to review all subpanel reports at various stages of development, and a consensus
document was developed.

C. Methodology

The Panel reviewed extensive scientific and budgetary documents provided primarily by NIAID,
NCI, NCRR, and FIC. The Panel also invited presentations from officials and staff scientists
representing the above ICDs. Recent NIH program reviews by external ad hoc groups or
program summaries, when available, were provided to the Panel by the ICDs as a starting
framework. Additional information was provided by NICHD, NHLBI, NIDR, and NIDDK
where projects were expected to have impact on AIDS vaccine research areas. All relevant recent
RFAs, PAs, and contract solicitations also were provided to the Panel members by the ICDs,
through the OAR. Representatives from NIAID and NCI presented update and planning
information to the Panel at the July 10-11, 1995, meeting.

Budgetary information was independently confirmed by review of the ARIS database, which
identified only small AIDS vaccine research efforts in ICDs other than those named above.
Information about IRGs and study sections managed by the Division of Research Grants (DRG)
was prepared for a subgroup of the panel.

To obtain a more complete picture of how the NIH AIDS vaccine basic science research
programs relate to work supported by other Governmental and non-Government agencies, the
Panel invited presentations at the July meeting from representatives from the Walter Reed Army
Institute of Research, Department of Defense (WRAIR, DoD), and The Rockefeller Foundation.

Initial reports, prepared by individual Panel members, were distributed, read, and discussed at a
meeting on August 30, 1995, attended by nearly all of the members. These topic reports were
then fused into subpanel reports. Subpanel conference calls and electronic mail discussions of
selected topics were used to consolidate concerns. Revisions and refinements of the reports
continued until all major issues were resolved.

The Panel held an open session on October 16, 1995, to hear public testimony from individuals
interested in expressing their views on NIH-funded vaccines research and how it interfaces with
companies and communities involved. Testimony also was solicited from a group of basic
research scientists who have expressed interest, but have encountered difficulty, in acquiring
funding for AIDS or AIDS-related research.

Detailed reports and specific recommendations from the subpanels are a direct result of these
activities. The views of each subpanel within their specific area as well as in areas covered by
other subpanels are included in this document. Thus, some overlap, as well as some minor
differences of opinion, can be found in the detailed summaries. The subpanels employed
different formats to best express their respective evaluations. On the whole, however, there was a
remarkable degree of congruity in the overall assessment of the NIH vaccine program. This is
reflected in the Executive Summary, which synthesizes the evaluations and most important
recommendations of the three subpanels.

I. Basic Research Subpanel Report

Introduction

The creation of an effective AIDS vaccine requires interlocking contributions from many sectors
of the biomedical research community. The role of academic scientists working in the intramural
and extramural AIDS programs supported by the NIH who define themselves as "basic vaccine
researchers" is to obtain the fundamental scientific information that is necessary for the rational
design of AIDS vaccines. Their contributions are critical for the interpretation of information
accruing from vaccine evaluation in humans and relevant animal models.

The inadequate proportion of the total AIDS research funds that have been allocated to vaccine
research and development was the greatest concern identified by the Basic Science Subpanel,
and this finding was strongly endorsed by the other Vaccine Subpanels. The cost of all of the
NIH's AIDS vaccine programs in FY 1994 was limited to only approximately $112.9 million
under the present coding system. However, this Subpanel review revealed that the true
expenditure on the core topics of vaccine development was substantially less, because of the
tendency to code under the category of vaccine research and development some programs that
bore only a marginal relevance to the subject. It was noted that the problem of underfunding was
particularly acute for unsolicited extramural "investigator-initiated" basic research projects aimed
at providing the necessary intellectual framework for rational vaccine design, development, and
testing. Unlike some of the programs covered by the other two Vaccine Subpanels where a more
targeted or directed approach is rightly favored, basic research is most effective if it is not
directed. The Basic Science Subpanel strongly endorsed the R01 mechanism for supporting
investigator-initiated basic vaccine research, while recognizing that timely guidance might be
provided by NIH through PAs to identify areas of research considered to be of the highest
priority. Further concerns about funding basic vaccine research through existing AIDS-related
study sections are discussed below.

Substantially increased support should be focused on acquiring more knowledge of the

fundamental workings of the human and primate immune systems, on encouraging innovative
approaches to vaccine development, and on creating novel or improved mechanisms or end
points for the conduct of definitive animal trials. At this point in AIDS vaccine development, it is
essential to provide incentives for the engagement of a larger group of creative immunologists
with vaccine biology interests to work on AIDS vaccine issues. A "culture" needs to be created
that will permit full investigation of the immunological consequences of HIV infection as well as
vaccine immunization. The information gained from such studies could be vital for the continued
improvement of HIV vaccine concepts.

A. Scientific Priorities: Opportunities, Needs, and Gaps

Scientific Issue from the FY 1998 NIH Plan for HIV-Related Research:

4.A More basic knowledge is needed to define protective immune responses to HIV in order to
facilitate the development of vaccines and other biomedical intervention strategies to prevent
and control HIV infection.

1. Host Defense Mechanisms

Status

For many years, most AIDS vaccine researchers were focused on approaches designed to induce
antibodies able to neutralize cell-free HIV in vitro. The essential goal was that of inducing
"sterilizing" immunity in vivo. The importance of cell-mediated immunity (CMI), particularly the
role of CTLs in suppressing HIV replication, was underappreciated by many in the AIDS
vaccine research community. The difficulties posed by the available assays contributed to the
limited analysis of CMI responses to HIV antigens. This was a problem exacerbated by the
reluctance of basic immunologists with expertise in viral immunity to join the AIDS vaccine
effort. Now it is obvious that HIV has much in common with other intracellular pathogens, and
thus targeting the cells in which HIV initially replicates is an important vaccine-design strategy.
This does not necessarily dismiss humoral immunity as a contributor to a successful
immunization stratagem; both antibody- and cell-mediated responses may be required. Recent
findings indicate that the current AIDS vaccine candidates are unlikely to induce sterilizing
immunity. While the induction of sterilizing immunity should remain a goal for HIV vaccine
research, a vaccine that suppresses viral replication to nonpathogenic levels is arguably more
achievable. This concept is already a major research focus in animal models and clinical trials.
One consequence of such a strategic switch in emphasis is an absolutely compelling need to
better understand T-cell responses in humans and nonhuman primates.

As most cases of HIV infection involve the sexual transmission of virus across mucosal surfaces,
it is desirable for the immune system to intervene at this earliest stage of the infection cycle. This
requires the induction by a vaccine of mucosal immunity, preferably involving both humoral and
cellular components. Although there have been some successes in detecting mucosal antibodies
to HIV after intramuscular inoculation, these responses have been generally weak and primarily
limited to immunoglobin G (IgG). Furthermore, the nature and location of the cellular immune
responses functioning in the genital and rectal mucosa and adjacent tissues are relatively obscure.
It is essential to determine the mechanism of HIV transmission across mucosal surfaces and its
initial dissemination to understand the nature of the immune responses that will be required to
combat it. The contribution of the Collaborative Mucosal Immunity Group (CMIG) awards
notwithstanding, the knowledge of the mucosal immune responses to different types of vaccines,
and to sexually transmitted diseases in general, is limited at best. In the case of AIDS vaccines,
the dearth of knowledge on mucosal immunity to HIV is particularly troubling.

Among the critical questions that remain to be answered regarding viral immunogenicity are:

• What are the true correlates of immunity against HIV infection?

• Why are neutralizing antibody titers found at only very low levels in patient sera, and
why do they develop so slowly?
• Can the induction of neutralizing antibodies be improved and targeted to mucosal
surfaces?
• What is required to induce CTLs and target them to mucosae?
• What is required to induce long-term memory in B, Th, and CTL compartments?
• Can improved methods be found to monitor bulk CTL activity?
• Can better reagents be generated for analyzing and manipulating the macaque immune
system?
• What is the composition and role of factors (interleukins or chemokines) secreted from
CD8+ cells in suppressing HIV replication? Can cells that release protective cytokines be
induced by a vaccine?

Scientific Issue from the FY 1998 Plan for HIV-Related Research:

4.B The identification of viral antigens and vaccine delivery methods that elicit long-lasting
protective immune responses against a broad range of HIV isolates will facilitate development
of effective vaccines.

2. Vaccine Design and Animal Testing

Status
Initial AIDS vaccine candidates were based on inducing immunity to defined neutralization
epitopes on the virus surface. Among these immunogens were the gp120 and gp160 envelope
subunit vaccines and complex peptides designed to induce antibody responses to the V3 loop of
gp120. With the appreciation that it was necessary to stimulate multiple components of the
immune system to respond to multiple components of the virus, additional vaccine designs
involving combination approaches were developed. These included the recombinant poxvirus-
based immunogens that incorporated a subunit protein boost (the prime-boost strategy). These
second-generation vaccines are worthy of continued support, but they may not induce a
sufficiently powerful immune response to be the definitive AIDS vaccine. Other concepts under
evaluation in animals include attenuated strains of SIV and HIV-1, following on the outstanding
success of this approach in the macaque model. However, safety--but not efficacy--concerns
continue to cloud the development of attenuated virus vaccines for human use. Killed virus
vaccines were evaluated extensively in simian models in the early part of this decade, but
protection in these models was almost exclusively associated with an immune response to
"contaminating" heterologous (xeno) cell proteins in these vaccines. It is not obvious how this
observation can be practically exploited for a human vaccine against natural, human-derived
HIV.

A very limited number of novel vaccine designs are developed and tested each year, usually
through the NIAID-supported National Cooperative Vaccine Development Group (NCVDG)
cooperative agreements (U01 awards), and the Simian Vaccine Evaluation Units (SVEUs), in the
NCRR-supported RPRCs or in the intramural research programs of NCI and NIAID. Often these
approaches have little or no private sector support to enable the development of clinical-grade
vaccine products for human testing. Because the science and knowledge base at present is
insufficient to engage meaningful participation of the private sector in HIV/AIDS vaccine
development, it is essential that NIH assert leadership for the collection of information essential
to bridge the gap between basic research and industrial product development, an area that is
definable as targeted research. If the private sector fails to respond to evolving opportunities in
vaccine discovery and development, the NIH should take responsibility for development of
immunogen design and product production. Facilities administered by the NIH or the DoD could
be used to prepare clinical-grade candidate vaccines, at least for pilot lots. Alternatively, vaccine
production could be allocated to the private sector under Government contracts.

Basic research on vaccine adjuvants has been focused primarily on the empirical testing of
products, rather than on defining and understanding their mechanisms of action. How most
vaccines containing adjuvants engage and stimulate the immune system to produce greater and
more appropriate immune responses than aqueous preparations is an important area in which
knowledge and understanding is lacking. Neither is it clear what effect adjuvants have on the
conformation of complex glycosylated proteins such as HIV-1 gp120 or gp160, either as subunit
proteins or as part of virion or pseudovirion structures.

In the absence of any "perfect" animal model for human AIDS, it was initially appropriate to
evaluate rigorously the characteristics of as many plausible models as possible. The inevitable
consequence of this policy is that, in the simian model alone, there are now too many different
test monkey species and too many different SIV and HIV-2 challenge stocks for all the models to
be sustained at adequate levels of funding. Distributing what are inevitably limited funds among
too many models has not permitted any of them to be characterized in as much detail as
desirable. Furthermore, vaccine experiments in primates tend to use only a few monkeys per
experiment, which usually leads to nondefinitive end-points and a limited opportunity for
followup analyses. While this is understandable for experiments on chimpanzees because of their
restricted availability and cost, it would certainly be feasible to conduct critical basic research
experiments on a larger scale with macaques if funds were available. (See further discussion in
the section on targeted vaccine development.)

The issue of HIV variation has always been a critical one in the development of an effective
AIDS vaccine. HIV varies within an individual, within populations in a geographically restricted
area such as the United States, and among different areas of the world. Hence there is substantial
concern over the impact of HIV variation on the development of an effective AIDS vaccine.
Nine major envelope sequence subtypes or clades within what is now known as the Main (M)
group of HIV-1, designated A through I, have been identified, along with a separate, highly
divergent group of strains referred to as the Outlier (O) group. Almost all documented HIV-1
infections within the United States have been B-subtype strains, but rare cases of infections with
strains from the A-, D- and E-subtypes have been identified in North America. Early predictions
that variation in the HIV-1 env gene would continue to increase at about 1 percent per year have
been borne out, and there is every reason to expect that the recognized subtypes will continue to
be disseminated, both globally and within the United States. While a limited degree of cross-
subtype virus neutralization by some HIV-1+ sera has been observed, its significance for vaccine
development is not yet clear. There is an imprecise concordance between neutralization
serotypes and the genetic subtypes, and much remains to be learned about neutralization and
whether a vaccine can be developed to induce such cross-neutralizing antibodies.

An extensive and expensive framework for performing vaccine studies in animals and humans
has been put in place without adequate funds or mechanisms to generate vaccine concepts and
products necessary to utilize this framework efficiently. In contrast, it was estimated by the Panel
that the annual cost of the unsolicited R01s ($8.6 million) and other grants ($1.6 million) in the
portfolio on basic and preclinical vaccine research, including some animal models research, was
about $11 million in FY 1994. This was less than 10 percent of the total AIDS vaccine research
and development budget for that year. This is in stark contrast to the greater funds allocated to
contract-supported preclinical testing in animal models, clinical trials, and vaccine trial training
and infrastructure. (See accompanying reports from the other Vaccine ARP subpanels.) The
extensive commitment of vaccine-related funds to mechanisms other than R01s is evident, and
the Panel felt strongly that this imbalance should be redressed. It was recognized that other
solicited, investigator-initiated programs also support basic as well as preclinical vaccine
research: the NCVDGs, funded through the U01 mechanism, and the CMIGs, interactive R01s
funded through an RFA, as well as some intramural projects. Further, the Panel noted that
several areas of basic research related to vaccine development that is likely to be funded by
R01s, such as the mechanisms of antigen presentation, mucosal immunity, and human
immunology in general, have probably not been coded by the ICDs as vaccine-related, and
perhaps not even as AIDS-research related. Problems in coding research accurately were a
recurrent theme that hindered the Panel's ability to gain a precise picture of the scale of basic
research relevant to AIDS vaccine development. Nevertheless, the shortage of unsolicited
investigator-initiated research in HIV vaccine research is critically low and this category of
research has been identified as a high priority for increased support.

B. Review of Vaccine Research and Development at the NIH by Scientific Priorities

1. Host Immune Defense Mechanisms

Considering those grants identified by the Division of AIDS (DAIDS), NIAID, to be part of the
AIDS vaccine research and development portfolio, the Panel felt that there was an overemphasis
on humoral immunity at the expense of studies on cellular immunity. At least seven independent
groups are currently funded to develop and characterize human or rodent monoclonal antibodies
to HIV proteins, while there is significantly less emphasis on approaches to induce and
characterize effective cellular immune responses to HIV or its antigens.

The contribution of the CMIG R01 awards to the overall portfolio ensures that mucosal
immunity is evaluated to some extent. However, it is not clear how these grants will fare in
recompetition. It was not determined whether NIAID or other ICDs plan to continue specific set-
aside funding to this area. Because the induction of mucosal immunity by an HIV-1 vaccine may
be essential to its success in preventing or limiting sexual transmission of HIV, research in this
area must remain a high scientific priority.

2. Vaccine Design and Animal Testing

A reasonable number of grantees are slowly developing new immunogens for vaccine
development. However, there still appears to be too much emphasis on subunit vaccines or
chimeric proteins and too little on more imaginative approaches. Nonreplicating vaccines based
on the expression of a small fragment of the HIV-1 core or envelope proteins, especially from
the variable regions of gp120, are unlikely to induce an immune response of sufficient breadth or
potency to mediate significant protection from HIV. Almost all of the "live" vector approaches
currently being pursued involve poxvirus variants; other vectors should be pursued and
supported.

Research on adjuvants was relatively well-supported by NIAID in 1994, mostly under U01
Cooperative Agreements or a Simian Vaccine Evaluation Unit contract rather than through the
R01 mechanism. However, these grants have expired and most have not competed successfully
in the IRGs. This may create a shortfall of research in this important area.

Recent studies supported by NIAID and other organizations such as the World Health
Organization (WHO) have indicated that the envelope subtypes defined by genetic sequencing
do not correlate with neutralization serotype, despite both qualities being based on properties of
the viral envelope. Although neutralization serotypes clearly exist, their number and
compositions remain uncertain; there is an imprecise concordance between neutralization
serotypes and genetic subtypes. This is not to say, however, that the genetic subtypes have no
relevance to vaccine development: First, they can influence neutralization serotypes, and second,
the CTL response is directed at linear epitopes. Variation in the presentation of these subtype
epitopes to the individual's immune system is likely to correspond very closely to the subtype-
defining, primary genetic sequence variation.

Notwithstanding the major problems imposed by HIV-1 sequence variation, the Panel considered
that this issue could almost be considered secondary to that of creating a vaccine able to prevent
or limit infection even of an HIV-1 strain of a genotype closely related to the immunogen.
However, the subtypes of HIV-1 strains, defined by genetic sequence, that are circulating at the
likely site(s) of vaccine efficacy trials should be taken into account at the time the immunogen is
prepared. There is a compelling case that efficacy trials are best conducted in areas with the
highest rates of incident infections. This is not likely to be within the United States or other
countries within the highly industrialized world, where HIV-1 strains with envelope sequence
subtype B predominate. Thus, future immunogens might be based most usefully on the (almost
exclusively non-B) envelope sequence subtypes that are endemic to potential international test
sites. That the subtype of a test immunogen is not necessarily matched to virus strains circulating
in the United States should not preclude its initial evaluation for safety and immunogenicity in
Phase I trials in this country. Information gleaned from these initial trials could readily be used to
determine whether further trials of the concept would be warranted in the geographic area for
which the immunogen was initially designed. Finally, a multivalent vaccine is likely to be
needed for citizens of any country engaged in global travel or business in the international
community.

The Panel felt that a better return could have been received from the enormous investment that
has been made by NIAID, NCI, and NCRR in nonhuman primate models for HIV vaccines. A
serious problem imposed by the present structure of funding nonhuman primate model research
is that the traditional level of funding available through the R01 and U01 mechanisms is too low
to permit examination of more than approximately two or three variables, and appropriate
controls, during the 2- to 3-year life span of projects. NCVDG funding is presently at a level that
permits, on average, the use of 12 monkeys per year. The effect of these restrictions is that
experiments often contain a number of experimental and control animals inadequate to obtain
definitive end points. Historically, this situation has caused significant problems of data
interpretation, and the Panel felt that it would be wrong to repeat the errors of the past. It has
been argued by NIH program administrators, and supported by study sections, that general
increases in the funds available for nonhuman primate studies through the R01 or NCVDG
mechanisms would be wasteful in the long run, given that most of the experimental approaches
will be failures. However, a definitive end point can often be valuable, even if the approach
under evaluation is an intrinsic failure. Too often, nondefinitive end points are reached with
small animal studies that are ultimately a wasted effort or that turn out to mislead the field,
causing even greater losses.

In an attempt to confirm or refute initial findings in animal models that are made by NCVDGs
and other investigator-initiated programs, the Master Contract mechanism was developed. In the
past, this system has involved DAIDS Program Staff deciding internally whether or not to
provide additional support for concepts they deem to be sufficiently promising. The Panel has
learned that funding has been provided for 410 monkeys via Master Contracts, a significant
multiple of the number of animals supported by investigator-initiated funding mechanisms. The
Panel viewed the Master Contract procedure as laudable in principle but having potential
shortcomings in practice. The Panel believes that external review of administrative decisions is
essential, given the scale of the awards being made. The recently established NIAID Vaccine
Design Group may resolve concerns about this issue.

C. Review of Vaccine Research and Development at the NIH by Institute, Center, or

Division (ICD)

1. NCI

In FY 1994, NCI spent in excess of $16 million on projects that were defined as relevant to
AIDS vaccine research and development. These projects are conducted both on the central NIH
campus in Bethesda and at the Frederick Cancer Research and Development Facility (FCRDC).
Basic, preclinical, clinical, and animal model research activities contribute to the overall
program. Experimental approaches include vaccination with cellular antigens, peptides, DNA,
pseudovirions, recombinant adenoviruses, or poxviruses.

Until 1990, the vaccine-related programs at NCI were organized as an AIDS Task Force. This
was disbanded and currently there is no central organization of the programs and no attempt to
coordinate them towards a common goal. Indeed, even within the geographically restricted
FCRDC site, cooperation between the various groups appears to be minimal or nonexistent.
There have been only limited attempts to coordinate studies between NCI and NIAID or NCRR.
The justification provided by NCI for its current structure is that the individual groups are
considered to be conducting investigator-initiated research programs that neither need nor would
benefit from any central direction. This was seriously questioned by the Panel.

The Panel had many concerns about the way in which the NCI AIDS vaccine research programs
were organized and conducted. In principle, investigator-initiated research intramural programs
can play a key role in the development of an AIDS vaccine. However, the analogy drawn by NCI
between the NCI intramural programs and extramural investigator-initiated programs was
considered by the Panel to be inexact and inappropriate. Extramural programs are subject to
prospective, expert peer review from other AIDS investigators. The Panel was concerned that,
while the NCI vaccine programs were peer-reviewed, they have not been reviewed as vaccine
programs per se. This was an inevitable consequence of the lack of central organization of the
NCI AIDS vaccine research programs; the individual programs often were reviewed as minor
components of a larger entity, with a consequent lack of specialist input. One consequence of
this system is that NCI has no means to assess the relative merits of the individual vaccine
research programs it administers. This lacuna would clearly hinder any internal attempts to
prioritize those programs to preserve under conditions of greater financial stringency than apply
today.

The Panel recommends an expert peer review of the entire vaccine research program
administered by NCI as soon as possible. In the absence of such a comparative review, the Panel
was able to provide only general opinions on the value of the individual vaccine research
programs within this Institute. Within these limitations, the overall quality of the NCI vaccine
research programs was considered mixed. There are clearly projects that are highly rated and
internationally competitive, and there are others that are not contributing significantly to either
AIDS vaccine research or even to AIDS-related research. Although many of the programs raised
concerns, better quality programs included those on virion-associated cellular antigens and their
roles in protection against SIV, the DNA vaccine program, collaboration on the Macaca
nemestrina model with NIAID-sponsored groups, collaborative studies on poxvirus vectors that
help underpin the prime-boost vaccine strategy, and a peptide vaccine program.

The Panel reviewed the titles and abstracts of those intramural projects deemed by NCI to be
AIDS vaccine-related at both the Bethesda and Frederick campuses. It was clear to the Panel that
many of the projects are, at best, of only indirect relevance to AIDS vaccine research and
development. At least two projects at Bethesda focused on vaccines against cancer; other
projects are designed to characterize the basic molecular virology of HTLV-1 and to develop an
HTLV-1 vaccine. There also are projects to study the functions of HIV-1 regulatory genes and
sequences and to develop antisense oligonucleotide and gene therapy antiviral strategies. While
plausibly promising projects, these studies are considered to fall outside the definition of AIDS
vaccine research and should not be considered as part of the NIH AIDS vaccine research budget.
Indeed, some of the studies should not be considered part of the AIDS research budget at all. A
preliminary estimate is that up to half of the total budget that was classified by NCI as AIDS
vaccine research and development (in particular, $6.3 million indicated only as Operations and
Technical Support) may have been misclassified. The Panel also expressed concerns about the
scale and nature of several of the support contracts awarded to Bethesda-based biotechnology
companies ($1.9 million) and considered that the value and need for these contracts should be
closely scrutinized before they are recompeted.

It is clear that the FCRDC has virus, antigen, and antibody production facilities that are unique
among NIH-supported programs. These facilities could be a critical national resource for any
centrally directed national program to develop an AIDS vaccine. Some NCI-supported scientists,
especially several of those groups working at the FCRDC, could make vital contributions
towards such a program. However, the Panel felt that, at present, the NIH was not receiving
sufficient return on an annual investment of over $14.8 million in the NCI AIDS vaccine
programs to warrant continued support at this level. The Panel recommends cessation of the use
of AIDS monies for projects not pertinent to AIDS vaccine development and critical review of
the remaining projects within the NCI to allocate resources where they might be most productive
for the HIV/AIDS vaccine efforts.

2. NIAID

The NIAID intramural program on HIV vaccine research development is smaller than the NCI
program: Yet, the intramural AIDS vaccine research projects (Z01 awards) at NIAID received
$7.2 million in FY 94. The Panel did not examine these programs in any rigorous sense, because
a detailed project review was beyond the scope of this review. However, it noted that the
intramural budget for HIV/AIDS vaccines was nearly equivalent to the entire portfolio of
extramural R01 grants in basic and preclinical vaccine research ($8.6 million). The Panel
recommends that a future review of intramural research at the NIAID be conducted under
guidelines similar to those recommended above for the NCI intramural programs. Again, the
Panel noted that several of the NIAID intramural programs classified as AIDS vaccines research,
including some intramural contracts, bore only a tenuous connection to the subject, at least in so
far as could be judged from the titles and abstracts of the awards. For example, research on the
mechanisms of pathogenesis of murine leukemia virus-induced "AIDS" in mice might be more
appropriately coded under AIDS etiology and pathogenesis. Similarly, a major program on the
molecular genetics of eukaryotic cells and their viruses has yielded outstanding results but is
mostly directed at topics more closely related to HIV pathogenesis. The Panel was concerned
that such misclassification within the intramural programs leads to the perception that more
AIDS resources are allocated to vaccine development than is truly the case.

In FY 1994, a total of $4.34 million was allocated to Research Management Support for AIDS
vaccine research, the administrative costs of the many vaccine programs supported by NIAID. In
addition to support for grants and contract management, this budget covers workshops, meetings
such as the NCVDG, and travel for extramural investigators advising NIAID and is proportional
to its role in extramural funding.

3. NCRR

The Regional Primate Research Centers (RPRCs) funded by NCRR constitute a critical national
resource for AIDS vaccine development. When the AIDS problem was recognized, limited
expertise in viral diseases was in place at many of these facilities to take immediate advantage of
these resources. As the priorities for use of these Centers began to shift to AIDS research,
supplemental funds were initially added to support these new priorities. Some additional funds
were subsequently included in the Base Grants to the RPRC, but these supplemental funds did
not keep pace with the growth in the rest of the AIDS research field. Individual centers focused
on gathering basic data in many different SIV models, which led to a proliferation of SIV,
mutant SIV, HIV-2, and SHIV model systems. This has been extremely valuable for studies of
viral pathogenesis as well as vaccine research. The differential pathogenicity observed in these
models now needs to be exploited with in-depth immunological studies and comparative studies
of vaccines tested under different "stringency" of pathogenic challenge now possible with the
range of viral strains available.

There is a need for the RPRCs to be more proactive in (1) the development of refined and
comprehensive studies, particularly involving immunologic analysis, (2) incorporation of outside
complementary expertise to enhance the scientific breadth of studies, and (3) an effective policy
that makes these animal resources and facilities, as well as viral resources, even more accessible
through competitive peer-reviewed research from the broader scientific community. The Panel
noted that the directed programs of the Medical Research Council of the United Kingdom in
association with the primate centers of the European Economic Community (EEC) have yielded
a high return from a relatively small investment of targeted funds. The European consortium has
developed a supplemental reimbursement system for funding cross-center collaborative vaccine
studies, which are conducted in addition to center-specific studies.

D. Special Issues

1. Administrative Aspects
The Panel was, in general, satisfied with the way in which the limited resources designated as
basic AIDS vaccine research were distributed by NIAID, and by the proactive approach of the
Program Staff of the Preclinical Branch of the DAIDS, NIAID, in supporting investigators with
novel ideas and approaches. However, it was demonstrated to the Panel that investigator-initiated
R01 applications that propose testing or comparative studies of the immunogenicity of different
immunogens or adjuvants in whole animals (even mice) tend to compete poorly in Initial Review
Groups or study sections, compared with projects with a more molecular or mechanistic focus in
immunology (e.g., transduction factors, signal transduction, activation of membrane receptors,
etc.). Even more worrisome is that projects to define basic principles of immunogenicity and
tolerance induction in humans and nonhuman primates are often viewed by IRGs as "lateral"
research, i.e., merely extending or translating principles established in mice to other species. This
has occurred despite repeated indications that human and murine systems are divergent in many
aspects that dramatically affect lymphocyte development or induction and maintenance of
immunity to specific antigens or pathogens. As a result, basic immunogenicity issues that are
fundamental to vaccine development, not only for HIV but also for other pathogens, remain
poorly supported and fail to attract many of the best established immunology and virology
laboratories as well as many of the best young investigators in these fields.

Assessment of the current portfolio of grants administered by the Vaccine and Prevention
Research Program of the DAIDS is complicated by the grant-coding system currently in use.
Under the current system, many awards to study human immunology (particularly CTL research)
and the mechanisms of HIV transmission and pathogenesis are designated to categories other
than those dedicated to vaccine research. It is probable that many of these grants have been
classified as "etiology and pathogenesis" or, in the case of awards for understanding human
immunology, classified as non-AIDS and not reviewed by any of the Area Review Panels. While
the Panel was not overly concerned about these administrative minutiae, they did affect its ability
to gain a good understanding of the scope of current basic and preclinical, vaccine-related
research.

Increasing the pool of resources available for investigator-initiated basic and preclinical vaccine
research through the R01 mechanism--the principal need identified by the Panel--will be difficult
without a substantial infusion of additional funds, either de novo or from other research
activities. The Panel considered that the true scale of the deficiency in grant funding is even
worse than it might seem, because of techniques used to artificially inflate the number of grants
funded and give the false impression that more work is being conducted than is actually the case.
For example, although an extremely low proportion of highly rated R01 applications is presently
funded, this proportion is also inflated by the present policy of cutting funding for successful
applicants by 17 percent or more and by delaying their start dates to release funds for additional
awards.

The Panel also felt that the interactive research project grants (IRPGs) using the R01 mechanism
to conduct collaborative multidisciplinary studies of potential AIDS vaccines is not working well
and that this should be rectified by increased use of U01 or U19 cooperative agreement awards
or by reconfiguring the IRPG/R01 mechanism and its review. The principal value of the IRPGs
is to realize the political aim of funding a greater number of R01 grants. But, review committees
do not always evaluate or appreciate the requirements for the collaborating entity before deciding
the merit of the individual awards. For example, it is much harder to obtain adequate funding
through the R01 mechanism of the IRPG for the vital but scientifically less intriguing grants that
largely provide critical cohort maintenance and sample acquisition.

The Panel received information from DAIDS as to how the issue of R01 underfunding was being
addressed. This included the use of Select Pay with oversight by the NIAID Advisory Council,
which the Panel endorsed under certain circumstances. However, the Panel urged a widespread
dissemination of the criteria used by staff to determine the suitability of an award for Select Pay,
because these procedures are poorly understood.

The RFA mechanism for targeting vaccine research was supported in principle by the Panel,
provided that the standards of solicited awards remained reasonably high and comparable to
successful unsolicited awards. For example, the Panel considered it advisable to retain the
current policy whereby a fixed sum is not preallocated to an RFA; only those proposals
considered sufficiently meritorious should be funded. The former policy of large set-aside funds
allowed many less-deserving awards to be supported.

The Master Contract mechanism provides a third means by which DAIDS staff have responded
to the shortfall for specific needs not met by R01 funding; the operation of this mechanism is
also discussed below and in the section on the Animal Models Programs. The Panel considered
NIAID's establishment of the Vaccine Design Group as a positive step towards the external
oversight of development and analysis of new immunogens. However, without adequate
financial resources at its disposal, the potential of this group may not be fully realized.

E. Conclusions and Recommendations

Recommendations by Scientific Areas

• NIH should increase total funds allocated to basic research in support of vaccine
design and development. In particular, basic research on immune responses in
humans and macaques should be targeted as an area of the highest priority.

• The Division of Research Grants (DRG) should develop a newly configured separate
study section for vaccine-related research.

The obvious solution to address a deficiency in a specific area of research is a separate

study section for that area. Separate, ad hoc study sections, such as those that have been
constituted for review of RFAs, do not have continuity built into their structure and thus
fail to address the long-term practical issues associated with flexibility for consideration
of newly emerging concepts and reevaluation of revised applications. A continuing study
section on vaccines would require a very broad expertise in many related areas.

• NIH should allocate specific funding for vaccine-related research to stimulate the
fundamental research needed in immunology and vaccine design.
 Even if a new permanent Vaccine Study Section could be established in the near future,
the problem requires an immediate intervention. The ICDs, in collaboration with OAR,
should be encouraged to allocate a defined amount of funding in each fiscal year for
targeted areas of basic research that are specified as high priority for vaccine
development. All grant applications that are received would be examined for their
relevance to these areas and would be designated for special consideration by DRG staff
and the chair of the study section. The study section members reviewing the application
might be requested to give the grants a "relevance score" from 1 to 5 based on their
perception of the application's relevance to the targeted areas. The study section would be
informed that these designated applications would be funded from the separate allocation
of funds, because this research was a national priority. The payline for these applications
might be much higher than the payline for all other applications reviewed by that study
section. Care should be taken by DRG, ICD, and OAR staff to recognize artificial
maneuvers to utilize "buzz words" in the abstracts or Specific Aims that trigger
designation for targeted review. Placing applications in this separate category should be
done thoughtfully and fairly, and should also involve input from the chair of the study
section at the time of the review.

The Immunobiology Study Section or the AIDS-related Research-A (ARRA) study

section might be an appropriate study sections for these grants; if necessary, a few new
members could be added with specific expertise and interest in vaccines.

• All future research devoted to the development and analysis of vaccination

strategies should involve forging more extensive collaborative links with cellular
immunologists.

This applies to both nonhuman primate and clinical human AIDS vaccine trials, and
could enable a better understanding of the consequences of immunization. It is
recognized that many of the leading immunologists in the United States have not been
involved in AIDS vaccine research to any significant extent. The Panel felt that it is vital
to develop ways to change this situation. A culture must be created that will lead to
improving our understanding of immunogenicity to viral antigens. Thus the Panel
strongly recommends development of funding mechanisms designed to attract creative
young investigators and experienced immunologists to this complex and challenging area
of research.

• Clinical samples, from vaccine trials and relevant NIH-supported natural history
cohorts, as well as viral stocks for experimental animal models, should be made
more readily available to researchers, with appropriate research plans, outside the
groups involved with the initial sample and data acquisition.

Approval of these plans should fall within the purview of an independent committee, and
not, as at present, solely under the control of the group which has collected the samples.
The supply of materials available, and competing demands upon them, should be taken
into account. The Panel noted the success of the NIAID AIDS Reference and Reagent
 Program in providing virus stocks and other reagents obtained from clinical sites and
basic researchers for distribution to the research community.

• NIH should reevaluate the relative distribution of resources between clinical

evaluation of current vaccine candidates and the development of new ones with
potentially greater promise. Redundant funding for clinical trials of similar vaccine
candidates should be eliminated in favor of new vaccine concepts. NIH should
establish mechanisms for identifying novel approaches to vaccine development and
rapidly testing their feasibility.

• NIAID and NCI research programs on HIV-1 genetic variation should be better
coordinated, both within the NIH and with other agencies pursuing this problem,
including the DoD, CDC, EEC, and WHO.

Although NIAID has been proactive in this regard, HIV genetic variation research
programs of the newly created HIVNET are likely to duplicate other activities already
supported by NIAID, including NIAID-sponsored work by the WHO (WHO AIDS
research activities will be subsumed within a new United Nations structure, the UNAIDS
Program, during 1996).

• A rationalization of the simian animal model field and a redistribution of funding

for a significant fraction of the current animal models is imperative. The Subpanel
recommends performance of fewer, but more detailed, studies each using an
increased number of animals is advised.

A top-level, independent expert group (see the Targeted Vaccine Subpanel Report)
should develop criteria for the continuation or expanded utilization of an animal model in
vaccine research and determine which of the current models meet those criteria. Among
the current simian models, there is a spectrum of viral virulence and consequent
pathogenicity. Some virus strains in some animals replicate only to low levels, cause only
limited disease, and tend to be relatively easy to protect against infection by
immunization with SIV antigens. Conversely, other models replicate to high levels in
vivo, cause rapid disease, and are hard to protect against. The Panel felt that, in principle,
it was important to retain one simian model at each end of the spectrum and perhaps one
in the middle for larger comparative vaccine studies. When developing a new vaccine
concept, it is advantageous to show that it confers protection under favorable conditions,
yet a rigorous test of the concept is also important. The considered development and use
of assays to measure virus load in plasma and tissues is essential for assessing the
comparative worth of simian models and for determining the outcome of vaccine trials.

• NIH-supported basic vaccine research on the HIV-1/Chimpanzee model should

focus almost exclusively on the development of a challenge strain that would
replicate to high titers in these animals and cause disease.

The current stocks of virus that replicate poorly in chimpanzees have limited value,
except for initial "proof of concept" studies.
 • NIH should continue to encourage the use of small animal models (such as cats and
mice) for the development and assessment of vaccine concepts.

These animals provide the opportunity to examine large numbers of variables and group
sizes in a relatively short time frame and at relatively modest cost. However, the value of
large-animal studies not involving primates or an SIV or HIV challenge should be judged
critically for their potential contribution against the substantial cost of these models.

Recommendations on Administrative Issues

General

• NCI and NIAID should reevaluate their code assignment procedures to ensure that
a more accurate representation of what basic and preclinical research is devoted to
the NIH vaccine program.

The Panel believes that the approximately $113 million sum defined as the cost of the
vaccine research and development by NIH for FY 1994 significantly overestimated the
true expenditure on these programs. Miscoding also might have underestimated the
expenditure on basic research in some areas of immunology.

• NIH should place a very high priority on the creation of a new study section
dedicated to fundamental vaccine research topics, either de novo or from existing
AIDS-related study sections.

OAR should work with DRG to ensure that this is done as rapidly and as efficiently as
possible. The academic standards of successfully competing awards should be maintained
in vaccine research at a common, high level, as in all areas of AIDS research. Because
some vaccine research may not be considered cutting edge science by the existing study
sections, the new study section should include individuals who recognize the imperative
need for translational research in vaccines. Such individuals should be sought to ensure
adequate unsolicited R01 funding.

• Both NIAID and NCI should minimize the amount of funds allocated by Program
Staff without outside peer review from contractual or other resources available
within these Institutes.

A detailed breakdown of such supplementary funds should be provided to OAR on an

annual basis. Distribution of funds through the Master Contract (in NIAID) and other
contracts in NCI should be determined by a review group comprised of both intramural
and extramural scientists, in addition to NIH Program Staff and administrators.

NIAID

• A relatively small sum of seed money (perhaps derived from the OAR Director's
Discretionary Funds) could be placed under the control of an expert peer-review
 group, specifically to allow vaccine concepts to be evaluated in primates before
submission of a more formal research proposal.

The Panel fully appreciated that the R01 mechanism was not necessarily the most
suitable way to fund some key areas of AIDS vaccine research and development. Vaccine
research can have an empirical nature that is not well-recognized by study sections, and
can be unusually expensive compared with certain other areas of AIDS research. A less
abstract problem is that study sections require, not unreasonably, evidence of preliminary
data that support the feasibility of a concept under review. Without such data, an
application may well be denied funding, irrespective of its intrinsic merit. Yet without
such funds, it may be impossible to obtain appropriately convincing data, especially if
experiments involving primates are necessary. This vicious cycle needs resolution.

• Basic research activities should not be supported under the existing contract
mechanisms.

Contracts are time-consuming and expensive to compete for and to administer, both in
terms of financial costs and of demands on scientists' time. The necessarily rigid legal
constraints imposed by the nature of a contract are not always compatible with the
flexibility that is an integral component of cutting-edge research. NIAID basic vaccine
research funds currently allocated to contracts should be shifted to grant mechanisms.
Targeted R01 awards or cooperative agreements (U01s) were perceived to be superior
mechanisms for enabling key areas to be studies, while allowing investigators the
independence to achieve scientific goals in the way they consider most appropriate.

• The Panel urges Review Committees and Program Staff to recognize the clear
requirement for more support for investigator-initiated research in both basic and
applied studies in human and nonhuman primates and encourage approval of the
necessary support in appropriate circumstances.

In many cases, small-to-medium-sized R01 grants are suitable, and the scientific return
on such investments can be considerable. However, funding limits often preclude the
support of such studies through the conventional R01 mechanism. Three mechanisms by
which adequate resources might be obtained are: increasing the funding ceiling on R01
awards under certain circumstances; reinstituting Program Project (P01) awards similar to
those provided by other ICDs; and increasing funds for NCVDGs or unsolicited
cooperative agreements (U01s).

• The U01 and U19 mechanism used to support the NCVDGs for conducting multi-
disciplinary studies of potential AIDS vaccines merits increased support, whereas
the interactive R01 mechanism for multidisciplinary studies should be abolished.

The U01 and U19 cooperative agreements retain administrative flexibility and encourage
cross-talk between basic research investigators and preclinical study investigators.
However, the Subpanel was particularly concerned that the current cap on funds for these
awards precludes or substantially limits basic concept testing in primate models.
 • To enhance the functioning of both the vaccine development oversight group
proposed above (discussed further in the Targeted Vaccine Research Subpanel
report) and the efforts of extramural investigators, NIAID should establish and
monitor a comprehensive computer database for recording and analyzing
information derived from vaccine trials in animals and humans.

This information should be routinely available to all grantees involved in AIDS vaccine
research to permit coordinated efforts to critically evaluate different experimental
approaches and develop valid cross-comparisons of data.

• The need for an RFA should be reviewed by an expert group comprised of both
extramural non-Government scientists and DAIDS Program Staff.

The charge to that group would include a careful review of study section-approved
proposals to address whether work supported by the proposed solicitation would overlap
with other supported or planned programs. NIAID should retain its current policy of
funding only those proposals considered sufficiently meritorious and not pre-allocating a
fixed sum to an RFA.

NCI

• NCI should immediately conduct a rigorous review of all components of its

intramural program presently designated as contributing to AIDS vaccine research
and development and use this review to determine which of its current vaccine-
related programs should be preserved, and at what level of support.

The Panel was concerned that many projects coded in this area bore little or no relevance
to the reality of vaccine research and development. Thus their inclusion within the
vaccine budget substantially distorts the perception of the actual NIH effort expended on
vaccine development. Indeed, a substantial proportion of dollars assigned to NCI
intramural vaccine research probably should not be considered as AIDS-related in any
sense.

• The Subpanel proposed that AIDS vaccine research activities of the NIH might best
be served if the most relevant and competitive intramural programs at the NCI
were placed within a single Division, with administrative control by the OAR, with
the view of assimilation into the NIAID intramural program.

In nearly all cases, it should be possible to achieve this goal without the necessity for
physical relocation of any research groups. The facilities at the FCRDC could be used for
a targeted, coordinated national effort towards developing an AIDS vaccine. At the very
least, the coordination of AIDS programs at NCI needs to be under the administrative
review of the OAR until the creation of a unified, cohesive AIDS vaccine program within
the NIH.
II. Targeted Research Subpanel Report

A. Scientific Priorities: Opportunities, Needs, and Gaps

Scientific Issues from the FY 1998 NIH Plan for HIV-Related Research:

4.A More basic knowledge is needed to define protective immune responses to HIV in order to
facilitate the development of vaccines and other biomedical intervention strategies to prevent
and control HIV infection.

4.B The identification of viral antigens and vaccine delivery methods that elicit long-lasting
protective immune responses against a broad range of HIV isolates will facilitate development
of effective vaccines.

1. Status

Targeted Research: The Link Between Basic Research and Industrial Product
Development

Targeted research involves the design of vaccines, preparation of suitably characterized viral
challenge stocks, and preclinical evaluation of vaccines in animal models (See Figure 1, second
box from left). In reviewing the status of NIH-sponsored vaccine research, the Panel found a
critical need for a strengthened program on targeted vaccine development. The Panel therefore
recommended the creation of a strong, targeted vaccine initiative to provide an essential,
currently missing bridge between NIH-sponsored basic research and commercial product
development.

Figure 1. Targeted Research

A Key Link Between Basic Research and Industrial Product Development
Early stage vaccine development and basic vaccine research, which has historically been the
strength of the NIH, is distinct from commercial product development. The latter relies on and is
made possible by the successful accumulation of research knowledge of sufficient depth to
support a commercial undertaking. Industry-sponsored vaccine development represents the
preparation and testing of candidate vaccines in large-scale Phase III clinical tests to prove safety
and efficacy. This is accompanied by engineered scale-up to commercial production levels,
submission and obtaining of product licenses, and development of distribution and sales. Such a
degree of costly activities cannot be justified for products that do not have the clear scientific
basis for safety and efficacy. In short, for industry to undertake product development, a scientific
foundation for this undertaking must be provided by targeted development of basic research
findings from investigator-initiated research.

Overview of the Current NIH Efforts on Targeted Vaccine Research

During the past decade, NIH funds for targeted vaccine research have supported the development
of different approaches to AIDS vaccines and the development of animal models for testing
candidate vaccines. (For the major ICDs participating in preclinical/targeted vaccine research
and budgets, see Figure 2.) A number of different approaches to AIDS vaccines have been
supported. These include peptide subunits of viral proteins, purified viral proteins, inactivated
viral particles, recombinant viral and bacterial vectors expressing immunodeficiency virus
proteins, plasmid DNAs expressing immunodeficiency virus proteins, and live attenuated
immunodeficiency viruses. A number of different animal models, with particular emphasis on
nonhuman primates, have been developed to test the safety, immunogenicity, and efficacy of
candidate vaccines: These use HIV-1 infections in chimpanzees; HIV-2 infections in baboons;
and HIV-2, SIV, and SHIV (chimeras of HIV and SIV) infections in macaques. The NIH also
has supported solicited research on particular problems in vaccinology, such as the use of
adjuvants and induction of immune responses at mucosal surfaces. The primary and most
important deficiencies in the program have been its failure to define broad spectrum
antigens/epitopes, to achieve immune responses of appropriate quality (both humoral and cell-
mediated) to provide protective immunity, and to achieve immunity with long-term memory at
the mucosal site of infection. These are the principal hurdles for future research.

Figure 2. Financial Support and Coordination of NIH-Sponsored Targeted Vaccine

Development

Lack of Critical Coordination of NIH-Supported Targeted Research

To date, no overall coordination has been provided for NIH-supported targeted research. Current
mechanisms for coordination of programs are Institute-specific. Even within an Institute, the
level of coordination and oversight has been different for extramural and intramural initiatives.
Overall, extramural programs (where researchers compete for support) have received the most
oversight. For example, the Vaccine and Prevention Research Program (VPRP) of the DAIDS
has provided coordination for extramural programs supported by the NIAID, and these programs
and their management have had intermittent oversight in the past by the NIAID Advisory
Council as well as an external review group (Summary of the report to the NIAID Advisory
Council provided to the Panel).
To a large extent, the lack of central coordination for the NIH-sponsored vaccine development
effort reflects the historic role of the NIH in supporting investigator-initiated basic science
research. Given the dwindling involvement of private industry, the lead responsibility in targeted
vaccine initiatives will fall to the NIH. The Panel recommends that careful consideration be
given as to how the funding structures of the NIH can be adapted for the effective
accomplishment of targeted research.

2. Problems Confounding the Development of an AIDS Vaccine

Several major problems confound the development of an AIDS vaccine (Table 1). These include
(1) the failure to have identified candidate vaccines that have a chance to be both safe and highly
efficacious, (2) the confusion of preclinical trial results by the use of animal models with virus
challenges that differ in virulence, (3) the lack of adequate sample size to definitively answer
questions in trials in nonhuman primates, and (4) the failure to develop and implement tests for
immunity that distinguish responses that are of substance for protection (correlates for
protection).

a. Failure to Identify Candidate Vaccines With Perceived Safety and Efficacy

A number of different approaches to an AIDS vaccine have been tried. Discouragingly, none
have resulted in the development of a clear candidate for an HIV vaccine. In monkey
experiments, the most protective vaccines appear to be live-attenuated, genetically defective
immunodeficiency viruses. Such live vaccines, however, are fraught with concerns for safety,
such as the potential to cause cancer by insertional mutagenesis, the risk of disease in
immunocompromised or immunologically immature (newborn) hosts, and the potential for
regaining virulence by recombination in the recipient host. In contrast to the live-attenuated
vaccines, the perceived safe vaccines (such as protein subunits) have shown little promise for
protective efficacy. Furthermore, in most instances, evaluations of the perceived safe vaccines in
animal models have been performed where the challenge virus was administered at the peak of
the immune response after booster vaccinations, making it difficult to project how approaches
that have protected or modified disease would translate into vaccines with long-term efficacy for
heterologous infection. Finally, multicomponent vaccine strategies now being tested for safety
and immunogenicity in humans have had limited opportunity for evaluation in animal models,
which afford the opportunity to use viral challenges to assess protective efficacy.

Table 1. Problems Faced by NIH-Sponsored Targeted Research for AIDS Vaccines

1. No clear choice as to which vaccine approaches or combination of vaccine approaches offer

the most promise for both safety and efficacy.
2. No one animal model in which vaccines are being tested. No clear certainty as to which of the
nonhuman primate models best mimic human infections with HIV.
3. Failure to identify the kinds of immune responses that provide correlates of protection.
Confusion over role of correlates in different test models.
4. Failure to define antigens/epitopes and adjuvants or optimal vectors to be used in a vaccine.
5. Failure to achieve durable induction of protective immune responses (with memory) at
mucosal sites.
6. Failure to adequately develop models of maternal/infant transmission for testing vaccines in
the newborn or pregnant animal.

b. Need for Comparative Testing of Candidate Vaccines in the Same Animal Models

Through NIH-sponsored investigator-initiated research, competing vaccine approaches have

been evaluated in different animal models. Relatively little work has been done (or has been
possible to do) to directly compare the efficacy of different vaccine approaches. Given that the
independent trials have offered some hope for a vaccine, yet failed to provide a vaccine
preparation that is clearly worthy of further pursuit, direct comparisons of different vaccine
approaches must be undertaken to identify those that indeed offer the most promise. This will
require centralized coordination of a targeted vaccine development effort and a transition from
independent investigator-initiated trials to a venue of centralized coordination, evaluation, and
problem solving.

c. Need for the Identification of Correlates of Protection

The development of an HIV/AIDS vaccine also has been hampered by the failure to identify
immune responses that may provide a correlate for protective efficacy. The identification of
correlates of protection is central to vaccine development. Such correlates provide a clear focus
for the types of immune responses that must be raised by a candidate vaccine. The identification
of correlates of protection would provide a strong justification for trials in humans. Existence of
meaningful correlates also would provide a preliminary means for evaluating the success of
vaccination in human trials.

The failure of investigator-initiated research to identify correlates of protection is due in part to

the evaluation of candidate vaccines in different animal models. These animal model infections
are inadequate because of the nonstandardized virulence of the challenge viruses (how long the
infection takes to cause AIDS and/or death in test animals) and in the variable susceptibility of
the challenge viruses to neutralizing antibody. Thus, immune responses, such as production of
neutralizing antibody, that have correlated with protection in some challenge models (e.g., T cell
tropic HIV in chimpanzees) have not correlated with protection in other models (e.g., SIV in
macaques). This has led to immense confusion, since a relevant correlate for one vaccine model
often has not proved a meaningful correlate in a model using a more vigorous challenge. Vaccine
challenge studies also should include experiments to address the role of mucosal immune
responses in the prevention of infection with SIV, HIV, or chimeric viruses and the potentiation
of virus infectivity (enhancement).

d. Need for the Evaluation of Vaccine Approaches in More Than One Model
At present it is not clear which of the various nonhuman primate infections is the best surrogate
of the HIV-1 and HIV-2 infections that occur in humans. As mentioned above, animal models
differ in the virulence of the challenge infection and in the host response to that infection. These
differences affect the outcomes of vaccine trials. In general, protection has been more easily
achieved against relatively avirulent infections (in which disease occurs after several years) than
for highly virulent infections in which disease occurs within the first year. Given the uncertainty
about which animal model most closely represents the minimum predictor for success in human
trials, it is important that vaccine approaches be evaluated in animal models of different
virulence. Such systematic evaluations would provide a rational base for the decision as to which
approaches (and eventually as to which models) are appropriate to making meaningful
predictions for human vaccines.

B. Recommendations

1. NIH should institute a mechanism for accomplishing targeted vaccine research.

Given that the NIH is the Federal Government's lead agency with the responsibility for
developing an AIDS vaccine, it is essential that the NIH institute a mechanism for a targeted
research program that will provide the missing link between basic research and the Phase I, II,
and III clinical trials that lead to industrial development (See Figure 1). This will require
centralized coordination of initiatives that are currently supported by different ICDs and by both
extramural and intramural mechanisms. Centralized direction, review, analysis, and decision-
making will be essential to sort out what is valid from that which is not valid in order to achieve
progress from the multitude of approaches and models that have emerged from independent
investigator-initiated projects.

The Panel considered various organizational structures that might support the achievement of
targeted objectives by the NIH (see list at the end of this Subpanel report). The Panel decided
that a mechanism that provides centralized leadership for independent projects by different
investigators/organizations could combine the strengths and traditions of NIH-supported research
with an effective program for targeted research (See below, Figure 3). The leadership of this
group must be scientifically strong and constituted with authority for distribution and
redistribution of funds between the ICDs within the NIH. The charge of this team might best be
achieved if the targeted vaccine program were centered in a single ICD such as the NIAID.

Figure 3. Proposed Mechanism for the NIH to Accomplish Preclinical Targeted Research
for AIDS Vaccines.
2. NIH should establish a team of extramural-intramural experts for centralized direction
of HIV/AIDS targeted vaccine research.

The Panel recommends that an extramural-intramural team or task force be formed to review,
define, and guide all NIH-sponsored efforts on targeted vaccine research (Figure 3). The team
should be comprised of experts in vaccinology, in the immunology and pathogenesis of HIV-1,
and in the commercial developmental process. This team would assume responsibility to set
priorities and allocate resources in the area of targeted vaccine development. The primary goal of
such an effort would be to evaluate, compare, and improve the vaccine approaches that have
emerged from NIH-sponsored, investigator-initiated research to identify candidates for clinical
trials.

Responsibilities of the team or task force:

• Provide coordination for preclinical vaccine trials by choosing vaccine approaches,

antigens, desired immune responses, animal models, challenge strains, and assays for
initial evaluation of candidate vaccines.

• Evaluate the results of comparative trials to identify those approaches and vaccine
compositions or strategies that warrant further development.

• Identify correlates of protection for the purpose of evaluating each of the more promising
approaches.

• Recommend contract research to solve problems that will overcome deficiencies in

promising approaches.
 • Ensure the rapid evaluation of promising new vaccine candidates or animal models to test
vaccine concepts.

3. The NIH should provide additional funds for targeted vaccine development.

Given the importance of the development of an effective AIDS vaccine, it is recommended that
additional funds be identified to provide a budget for the implementation of expanded vaccine
efforts. These funds should be used to implement competitive contracts and solicited peer-
reviewed research. It is estimated that a budget of $25 to $50 million per year would be
necessary for the extramural-intramural team to achieve an effective multi-investigator targeted
vaccine research effort. This allocation of funds should be exclusive of current support for basic
science, clinical evaluation, and all other AIDS research and should be provided without
diminution of current support for basic research programs.

4. Importance of establishing new funds for a new area of NIH activity.

The assumption of a major role in the targeted development of an HIV/AIDS vaccine represents
an expanded, but critically important, activity for the NIH. This role is being undertaken because
developing an HIV/AIDS vaccine has proved to be more complex than anticipated and still lacks
the substantive scientific knowledge required for a realistic industrial effort. Because of the
importance of protecting the Nation and potentially other areas of the world against the
expansion of the AIDS epidemic, the budget for these activities needs to be provided from new
funds, not from the redirection of funds from the traditional basic science program of the NIH,
nor from the extramural funds currently categorized as "Basic AIDS Vaccine Research."

List of Supporting Materials

Hilleman, M.R., Whether and when an AIDS vaccine? Nature Medicine 1:1126-1129, 1995.

Hilleman, M.R., Overview: Practical insights from comparative immunology and pathogenesis
of AIDS, hepatitis B, and measles for developing an HIV vaccine. Vaccine 13:1733-1740, 1995.

Report of the "Division of AIDS Ad Hoc Review of the AIDS Preclinical Vaccine Research,"
May 22-23, 1995. Scott Koenig, Chair. (Provided by NIAID)

III. Clinical Trials Research Subpanel

Introduction

Clinical trials research in the AIDS vaccine area encompasses several widely divergent scientific
issues. To be successful, this endeavor must channel the efforts of a diverse group of experts:
basic research scientists, clinicians, community representatives, company sponsors, statisticians,
and epidemiologists in active functional networks. Because even small trials can require up to 2
years to complete, resources and energy have to be committed for extended periods of time by
clinical staff and trial participants at evaluation units, with little likelihood of individual gain on
the part of the volunteers. Therefore, tightly linked, preclinical research should be used to guide
and support these efforts wherever this is rational and feasible. As safety and immunogenicity
studies are expanded and populations at risk of HIV-1 exposure are included, issues of
counselling, informed consent, accurate assessment of risk-taking behavior, and social harms
become increasingly important.

A. Scientific Priorities: Opportunities, Needs, and Gaps

1. Scientific Issue from the FY 1998 NIH Plan for HIV-Related Research:

4.B The identification of viral antigens and vaccine delivery methods that elicit long-lasting
protective immune responses against a broad range of HIV isolates will facilitate development
of effective vaccines.

Status

The optimization of vaccine design and delivery requires increased collaboration among
preclinical investigators, vaccine developers, and clinical trials groups. Such collaborations have
begun to occur and are critical to continued progress.

Opportunities/Needs/Gaps

Tighter links should be forged between preclinical research in vaccine design, including the area
of primate model testing, and clinical trials groups, so that findings in all areas rapidly stimulate
improvements in vaccine candidates. Stronger links are specifically needed between contract
programs, between Institutes, and between those programs and groups conducting HIV/AIDS
vaccine trials outside of the NIH.

In addition to the need for continued testing of existing vaccine concepts, the Panel sees an
opportunity for human clinical vaccine trial networks to contribute to an understanding of
protective immunity by conducting comprehensive studies of the human immune response to
vaccination and infection.

2. Scientific Issue from the FY 1998 NIH Plan for HIV-Related Research:

4.D Suitable candidate vaccines need to be selected and evaluated in Phase I and Phase II trials
to determine safety and immunogenicity.

Status

Without better animal models that are predictive both of the immune responses observed in
humans and of the impact of HIV candidate vaccines on disease development or clear correlates
of immune protection, an empirical approach is necessary to evaluate candidate HIV vaccines in
human Phase I and II trials.
To date, the NIAID AVEG clinical trials network has conducted Phase I trials for most of the
currently available HIV vaccine products as well as a Phase II study of two recombinant
envelope glycoprotein subunits (see Appendix A). Neither the AVEG nor other research groups
have identified vaccine approaches that have progressed to efficacy trials.

Selection of vaccine candidates for Phase I and II testing operates smoothly through the Vaccine
Selection Committee and the AVEG. Infrastructure and investigators are in place to continue to
conduct clinical trials at current or higher levels of enrollment. Funding to six sites is committed
for the next several years. (A 5-year cycle of funding was initiated in FY 1994 to the AVEG sites
and for supporting and laboratory and statistical centers.)

Opportunities/Needs/Gaps

The primary gap identified by the Panel is the shortage of promising new candidate vaccines for
human testing and the low level of commitment from private industry. This situation is not likely
to change in the next several years.

There is, at present, no acknowledged process for developing HIV vaccine candidates that do not
have the backing of private companies (although the capability theoretically exists either at
FCRDC or through contract mechanisms for the NIH to produce small lots of vaccine for
testing). This was identified as a need, particularly for small research groups.

Strategies not actively pursued by private industry have not been moved into human trials as
rapidly as industry-sponsored products.

3. Scientific Issue from the FY 1998 NIH Plan for HIV-Related Research:

4.C Distinct study designs and vaccine or intervention strategies are needed to achieve
protection of newborns and infants because of the unique nature of their immune responses and
modes of exposure to HIV.

Status

Encouraged by the results of ACTG 076, which demonstrated the ability of AZT to significantly
reduce HIV transmission from mother to infant, researchers continue to place a priority on
developing methods to prevent transmission, especially for settings where long-term antiviral
treatment may not be available.

Opportunities/Needs/Gaps

The probability that a proportion of maternal/infant HIV transmission occurs perinatally rather
than in utero provides an opportunity for immunoprophylaxis of the infant or immunotherapy of
the mother (see Appendix C).
Recent reports suggest that, in rare instances, infants may clear an initial infection. If additional
such cases are identified, they might provide a unique opportunity to define protective immune
responses to HIV, since clearance of infection would be one end point for a successful vaccine.

4. Scientific Issue from the FY 1998 NIH Plan for HIV-Related Research:

4.E Preparation for HIV vaccine efficacy trials requires characterization of the biological and
behavioral factors in affected populations, including seroincidence, development of an
infrastructure and a series of studies and planning activities to ensure trial feasibility.

Status

NIAID has established the HIV Network for efficacy trials (HIVNET), a network of domestic
and international sites for trials of various strategies to prevent HIV infection, including but not
limited to HIV vaccine candidates (see Appendix B).

Domestic and international HIVNET sites have succeeded in rapidly enrolling large numbers of
high-risk individuals into cohorts for baseline studies. These sites subsequently have begun to
gather data on risk and incidence of HIV infection, to assess willingness to participate in future
vaccine trials, and to evaluate consent procedures.

Because of the current status of Phase I/II trials, randomized efficacy trials of alternative vaccine
concepts could begin in the United States no earlier than 1998.

Trials of other prevention interventions have already begun at some of the international HIVNET
sites. Several domestic HIVNET sites also have received funding and are currently developing
protocols for non-vaccine prevention interventions, i.e., microbicide/barrier/STD and behavioral
studies that would last 1 to 2 years.

Opportunities/Needs/Gaps

The HIVNET domestic and international sites, laboratory, and statistical center represent a
substantial financial investment in HIV prevention and vaccine evaluation (see Appendix B).

The total AIDS research funding for the HIVNET in FY 1994 was $21.2 million, of which $16.6
million was coded as vaccine research (Infrastructure for Vaccine Efficacy Trials). This includes
funding allocated early in FY 1994 to start up the Domestic Master Contract (DMC) and the
International Master Contract (IMC) as well as the second portion allocated in late FY 1994 to
fund a statistical center, the network's central laboratory, and the individual sites funded through
the DMC and IMC, whose subcontracts were actually initiated in early FY 1995. The FY 1995
renewal funding was $16.5 million, of which only $3.66 million was identified as vaccine-related
research, and a much larger portion of funding was coded as epidemiology and natural history.*

*
This represents a shift of nearly $13 million or 10 percent of the NIH vaccine-related research category.
Some current HIVNET work, particularly in the United States, is now more accurately defined as
epidemiology and natural history; its major focus has been preparation for evaluation of vaccine
candidates to prevent HIV infection.

At present, the dilemma for NIAID is the potential for a gap in time between the completion of
HIVNET baseline seroincidence studies and the availability of adequate data from Phase I/II
studies of the next most likely vaccine candidates (or other prevention interventions) to be able
to decide on their advancement to Phase III efficacy trials. If current vaccine candidates are not
advanced, this gap may be substantial. If other non-vaccine interventions are advanced, it is not
clear when or how vaccine candidates available at a somewhat later date can be advanced.

5. Scientific Issue from the FY 1998 NIH Plan for HIV-Related Research:

4.F Conduct HIV vaccine efficacy trials of the most promising candidate vaccines in well-
characterized high-risk populations to identify effective vaccines for the control of HIV infection.

Status

The most promising vaccine concepts should be advanced to efficacy trials, if preliminary data
justify doing so. It has not been determined how decisions will be made to advance candidate
vaccines into Phase III, although NIAID has begun to define criteria for advancement of vaccine
candidates on a product-by-product basis.

Opportunities/Needs/Gaps

Criteria for advancement of candidate HIV vaccines should be determined in advance. The
decision-making process needs to be open, i.e., including participation of both the larger
scientific and affected community, more clearly defined, with a specified person or group that
has final decision-making authority.

Industry would prefer that criteria for selection of candidate vaccines for advancement to
efficacy trials be determined beforehand.

6. Scientific Issue:

Determine whether vaccines can play a role in post-infection treatment of HIV disease.

Status

In the past, the same HIV vaccine candidates designed for prevention have been placed into
treatment trials. The rationale for such trials is that a new anti-HIV, vaccine-induced immune
response would be cross-reactive with host virus and control the virus. A different type of effort
is required to evaluate these and other immunogens in HIV-infected individuals.

After reviewing information available from current therapeutic vaccine trials, the Panel believes
that therapeutic HIV vaccine trials should be considered strictly as part of AIDS therapeutics
research, where they should compete against other immune-based, antiviral or combination
therapies for funding.

Opportunities/Needs/Gaps

The Panel members seriously question the value of continued pursuit of a therapeutic approach
with existing envelope vaccine candidates. The completion within the next year of several large,
industry-sponsored, therapeutic vaccine trials in the United States, Canada, and Europe
(including three NIH co-sponsored trials) and publication of the results should provide a more
definitive picture of any potential benefit.

B. Review of Clinical Trials Vaccine Research at the NIH by Scientific Priorities

The Panel's primary concern is ensuring that a pipeline of promising vaccine approaches and
products exists, while maintaining Phase I/II capability and readiness for eventual efficacy trial
opportunities.

1. Identify Vaccine Components and Delivery Methods

Although vaccine candidates are largely identified through basic research and animal model
studies, human Phase I and II trials play an important role in HIV vaccine design and
development. In vitro assays and immune response data from animals are often more robust and
may not reflect effective human immune responses to HIV vaccines. Standardization,
improvement, and development of new assays for measuring immunogenicity should be
priorities of the clinical trials networks.

When possible, efforts should be focused toward using clinical trial networks and cohorts to
advance the overall knowledge about human immune response to different HIV vaccine
strategies. In turn, this knowledge should be utilized to improve and refine vaccine design.

2. Select and Evaluate Candidate Vaccines in Phase I and Phase II Trials

Without better animal models or clear correlates of immune protection, an empirical approach is
necessary to evaluate and advance the most promising vaccine concepts. By their very nature,
human trials of candidate HIV vaccines will be more definitive, but also riskier and more
expensive than other avenues of vaccine research. Extreme care must be taken to ensure safety,
ethics, and scientifically sound clinical study designs. Both preclinical and clinical milestones
should be developed that determine to what extent a candidate vaccine is advanced.

3. Explore Ways To Achieve Protection in Newborns and Infants

Domestic and international trials are in progress to evaluate envelope vaccines and the ability of
human polyvalent anti-HIV immunoglobulin (HIVIG) products to prevent HIV-1 transmission
from mother to infant. These studies may provide answers to important issues of immunity to
genetic variants and advance the knowledge base for this line of research. (Note: In FY 1994,
these studies were not coded as vaccines but as therapeutics.)
Trials of HIV vaccines in the infants born to HIV-infected mothers should be conducted
independently of trials in other high-risk populations, because the risk of infection, even with
AZT treatment, still exceeds that in most at-risk adult populations. This effort should be
coordinated between NIAID and NICHD.

4. Prepare for HIV Vaccine Efficacy Trials

The amount of resources allocated for vaccine efficacy trials (through HIVNET, a few related
R01s, and other contracts) seems high, as it is largely for vaccine preparedness, other prevention
research, and infrastructure. However, ultimately it will be very valuable to establish such
programs and maintain readiness for conducting successful efficacy trials. Funding levels should
reflect prospects for use of the cohorts for multiple, investigator-initiated, high-value, high-
quality investigations in line with the HIVNET mission to test efficacy of products and
approaches to prevent HIV transmission. Recommendations for this relatively new program are
outlined below.

5. Conduct HIV Vaccine Efficacy Trials

The relative importance of vaccine efficacy trials in relation to other vaccine objectives
(understanding host defense mechanisms, developing new vaccine strategies, and protecting
newborns) will change unpredictably with time, but is likely to move toward more emphasis on
human trials as progress is made in other areas. New vaccines or encouraging results in early
trials would require increases in funding or at least a substantial shift of resources from activities
now being pursued in HIVNET in order to initiate and conduct vaccine efficacy trials.

6. Determine the Role of Vaccines in Treatment of HIV Disease

Ongoing trials are likely to determine whether current approaches in HIV vaccines have any
promise for therapeutic treatment of HIV disease without further resources. Furthermore, if it is
desirable to modify the immune response to shift the balance or the specificity of the immune
response during HIV infection, distinct approaches, different from those taken for preventive
HIV envelope candidate vaccines, probably will be required. Such approaches should be
prioritized and pursued as part of a therapeutic agenda.

C. Review of Clinical Trials Vaccine Research at the NIH by ICD

National Institute of Allergy and Infectious Diseases (NIAID)

Of the $46.7 million expenditures designated in FY 1994 as AIDS vaccine clinical trials (OAR
category 4C, $9.4 million) and development of cohorts and infrastructure for vaccine efficacy
trials (4B, $27.3 million), 78 percent ($36.5 million) was managed through the DAIDS, NIAID.
The bulk of this NIAID-sponsored effort is conducted through various extramural contracting
mechanisms, with Research Management Support (RMS) accounting for 4.5 percent of the total.
This concentration of these efforts in one Institute has led to the development of a more coherent
and unified vaccine research program than found in other NIH AIDS research efforts.
Nonetheless, several improvements can be made.
The caveat for this directed approach is that particular attention must be paid to accommodating
alternative research approaches and incorporating peer review: both periodic program reviews
and prospective review for studies begun after contracts are in place.

The Panel believes that NIAID should continue to direct all NIH HIV vaccine clinical testing, as
long as their program is subject to regular input and objective thorough evaluation by non-
Governmental extramural experts. Because of the relatively high cost of clinical trials and the
need for comparative data, the structure of contract groups with common laboratories and data
centers seems desirable. NIAID did not conduct intramural clinical trials for preventive vaccines
in FY 1994 but has done so in the past. As with NCI intramural research (see below), the Panel
recommends using existing extramural contract groups designated for this purpose whenever
possible to avoid duplication of resources and to ensure comparability of analyses.

Scientific Advances and Gaps

Virtually all of the scientific advances and gaps described above in Section A, Status and
Opportunities/Needs/Gaps, for vaccine clinical trials can be ascribed to NIAID.

Mechanisms

The bulk of NIAID's funding of vaccine clinical trials research including laboratory and
statistical centers is through various extramural contracting mechanisms, primarily the AVEG
and HIVNET, which are reviewed in greater detail in Appendices A and B. These two different
networks differ in organizational aspects: The AVEG is a group of vaccine investigators on
individual contracts administered directly by NIAID Program Staff. The HIVNET operates under
two master contractors, in which the prime contract interaction is between Program Staff in
NIAID and the principal investigators of the two Master Contracts. Distinct problems have arisen
with lines of communication between investigators at subcontract sites, the linked statistical
center contract, and Program Staff.

Funding

The primary program for conducting Phase I/II trials is the AVEG network and laboratories,
which received 31 percent of the total vaccine clinical trials funds (OAR codes 4B and 4C, in FY
1994). Approximately 36 percent of the FY 1994 budget coded for vaccine clinical trials was
allocated to HIVNET, and an additional 4.8 percent was encumbered by R01s, interagency
agreements, and other contracts associated with vaccine field-site preparedness. The coding of
the many and varied activities of vaccine trial preparedness and prevention interventions as
"vaccine trials and infrastructure" overstates the amount supporting actual clinical trials. The FY
1996 NIH budget for AIDS research defines these scientific issues more clearly into prevention,
epidemiology, and behavioral risk assessment, making the relative expenditures easier to
compare and evaluate. Unfortunately these data were not complete and available for the Panel to
review.

Vaccine trials to interrupt mother-to-infant transmission of HIV-1 have been conducted through
the AVEG (vaccines) and the ACTG (therapeutics) networks. Together, with expenditures at the
GCRCs (funded by NCRR) and the Centers for AIDS Research (CFARs) in FY 1994, these trials
accounted for roughly $8 million of the total AIDS research budget.

Future Directions/New Initiatives Needed

The size and components of the AVEG and HIVNET groups should be as flexible as possible, so
that the best resources are included, all available expertise inside and outside these programs is
utilized, and unnecessary duplications of effort are eliminated. Continuity and long-term
commitment are essential.

The Subpanel commends the Program Staff at the DAIDS for their management of these
programs. However, the AVEG represents the vaccine clinical trials research activity of only a
few researchers who focus on these projects. In the absence of vaccine candidates with proven
efficacy, the wider research community must become involved in investigating human immune
responses which would lead to more promising vaccine approaches and candidates. The time has
come to engage other scientists with strong backgrounds in human immunology, HIV
pathogenesis, and non-HIV vaccines in the HIV vaccine effort.

National Center for Research Resources (NCRR)

Non-NIAID expenditures for vaccine clinical trials (OAR category 4C) are predominantly
through the NCRR General Clinical Research Centers (GCRCs) ($2.56 million).

Scientific Advances and Gaps

The Panel recognizes the flexibility and diversity of the national GCRCs and their ability to
support local clinical research. AIDS vaccine research, including some Phase I studies of novel
vectors, is being carried out effectively at some centers. In addition, training and access to GCRC
facilities are being provided to some NIAID-sponsored programs, contracts, or grants. In FY
1994, several ACTG sites also were conducting therapeutic or pediatric vaccine trials.

Mechanisms

GCRC projects and utilization of an individual Center's funds are approved locally by Center
review boards. During the review, the Panel found that NCRR staff are responsible for coding
GCRC projects to specific AIDS research categories, and this coding is based on annual reports
of patient visits which may not accurately reflect the level of a GCRC's support or resource
allocation to ongoing programs or individual projects. Furthermore, this coding of projects to
OAR scientific categories is done after the fact, delaying reporting up to an additional year.

Funding

Unfortunately, using the current information reporting system, it is virtually impossible to

determine whether GCRC expenditures for clinical vaccine research areas are being allocated
wisely or accurately at the sites to AIDS vaccine-related research.
Future Directions/New Initiatives Needed

OAR should require more accountability over the amount of AIDS funds going to GCRCs for
project-related research, so that the level of expenditure accurately reflects contribution to the
vaccine development effort.

NCRR should develop a more direct budget-tracking mechanism to justify the utilization of
AIDS funds for clinical trials of HIV vaccine candidates or other training and infrastructure
support. Furthermore, NCRR must audit reports of patient visits provided by the Centers to
ensure the accuracy of the reports filed by the Center principal investigators.

National Cancer Institute (NCI)

At present, most NCI vaccine research is conducted by intramural scientists and is largely
preclinical (see the previous two sections on Basic Vaccine Research and Development).
However, one trial in HIV-infected individuals now is being conducted with a peptide vaccine.

Scientific Advances and Gaps

Basic researchers within NCI have developed several potential vaccine candidates but have
chosen not to approach the Vaccine Selection Committee of NIAID and are proceeding to
produce the product and evaluate their first vaccine candidates in clinical trials.

Mechanisms

In FY 1994, no NCI funds were ascribed to clinical vaccine trials, but NCI now is testing an
HIV-1 peptide vaccine for safety in HIV-infected individuals, utilizing NCI intramural resources.

Future Directions/New Initiatives Needed

As NCI vaccine concepts reach the stage for testing in clinical trials for safety and efficacy,
every effort should be made to advance them into existing NIAID clinical trial programs to avoid
duplications of effort and to utilize mechanisms of external review and oversight already in place
for this aspect of HIV/AIDS vaccine clinical trials. NCI is undergoing major organizational
changes that may refocus its AIDS vaccine research effort significantly and address this problem.

For a better test of immunogenicity, the Panel recommends testing the vaccine candidates
through the AVEG in low-risk HIV-uninfected volunteers rather than in HIV-infected subjects.

Fogarty International Center (FIC)

In FY 1994, the FIC supported a substantial portfolio ($8.85 million) of international

infrastructure and epidemiological research training awards with AIDS research dollars. Of this
sum, $6.82 million was defined as Infrastructure/Vaccine Clinical Trials (OAR research code
4B); this represents nearly 25 percent of all of the monies in this category and 6 percent of the
total NIH budget for AIDS vaccine research. The remaining $2 million was assigned to OAR
code 6A (Training); this represents over half of the total NIH budget for AIDS research training.
This level of international training is appropriate, considering where the epidemic is having its
greatest impact, but points up the unfortunately low priority placed on training in AIDS research
in general.

Scientific Advances and Gaps

The FIC has the flexibility, through investigator-initiated projects in its AIDS International
Training and Research Program (AITRP), to pursue areas of scientific research in AIDS
important to vaccine research. Individual training fellowships allow scientists from international
sites to acquire training, primarily in epidemiology but also in virology, immunology, or
molecular biology in basic research or clinical laboratories in the United States.

Mechanisms

In FY 1994, the FIC funded 10 Individual Training Awards (F05, F06); 25 International
Fellowships through its 11 AITRP (D43) awards for training and infrastructure in AIDS
research; 9 International Postdoctoral Fellowships (T22) awarded to 4 centers; and 2 Fogarty
Center Fellowships. The FIC also awarded 8 small grants (R03) designed to provide supplements
to encourage established investigators to participate in international AIDS vaccine research
studies.

Future Directions/New Initiatives Needed

The Panel raised serious concerns about the relative size of the budget for the FIC program
compared with the overall NIH effort in AIDS vaccines and with the extraordinary amount of
funds allocated to training relative to the entire AIDS training budget.

In many cases, the FIC AITRP awards appeared to be linked to NIAID-sponsored HIVNET sites
and should be considered a formal part of these networks and reviewed accordingly.

The FIC should assess its training programs and focus them to truly fulfill the defined goals of
assisting the AIDS vaccine research programs in developing countries. There is concern that, in
some cases, the training programs are merely providing inexpensive help for highly technical
HIV research activities of a few host institutions in the United States, research that cannot be
readily utilized in the international site.

D. Special Issues

Funding mechanisms

NIAID

AVEG is funded through individual contracts with each participating unit, laboratory, and data
center. The program is managed by DAIDS Program Staff and is largely self-directed by an
executive committee of extramural investigators. This structure has worked well for a group this
size.

HIVNET is funded through a Master Contract mechanism in which a private, non-Government

organization is engaged to conduct contract functions that might ordinarily be undertaken by
NIAID Program Staff. Two Master Contracts serve HIVNET: (1) the International Master
Contract (IMC) for international sites and (2) the Domestic Master Contract (DMC) for domestic
sites. One readily apparent consequence of the use of this mechanism is the absence of clear
leadership of HIVNET. No single individual seems to have overall responsibility for the
scientific direction of this program, especially for the domestic component, which, unlike
international sites, has adopted a uniform study design applied to all sites.

Whether leadership eventually will emerge is not clear as yet. Thus, the Panel has serious
questions about the Master Contract mechanism. Does this help or hinder the project? Does it
dissipate leadership? Is it really needed? Is duplication of international and domestic contracts
appropriate? The Master Contract mechanism for HIVNET appears to create layers and distance
between NIAID Program Staff and the researchers at the sites that have not been conducive to
rapid development of working arrangements.

Funding Issues in Other ICDs

As explained elsewhere in this report, the amount actually spent for vaccine clinical trials in FY
1994 is overstated as a result of inaccurate assignment of AIDS vaccine trials research codes to
what appears to be epidemiology and natural history research. This was noted not only in the
NIAID-sponsored HIVNET but also in the FIC AIDS epidemiology and training programs. In
one case, the NCRR-supported GCRCs appear to have reported duplicate funding for research
efforts supported by the NIAID-sponsored AVEG. In FY 1994 NICHD did not report any
funding in the AIDS vaccine research categories, although it appears that vaccine trials research
was conducted at some of the NICHD-funded pediatric clinical trial sites. In addition, some of
the basic research in NICHD's grant portfolio should be reviewed for its relevance to HIV
vaccines.

Peer Review

Because so much of the vaccine clinical trials program is funded through contracts, there is a
constant need to be aware of and promote periodic peer review, both before and after significant
projects are undertaken. The Panel sees the need to establish a broader base of investigators on
peer-review panels to ensure adequate review and to identify opportunities to work creatively
with collaborators outside the clinical trials networks to accomplish basic science and other
research goals.

Cross-Institute and Interagency Collaborations

Through interagency agreements, NIAID has incorporated expertise from NIDA, FIC, the CDC,
the U.S. Department of Energy, and the U.S. Department of Veterans Affairs into its vaccine
development program. This effort seems altogether appropriate if NIAID is to spearhead the
development of vaccines and related prevention research, which necessarily involves areas
outside its primary expertise.

Cross-Disciplinary Research: Overlaps With Other Panels

The HIVNET program bridges several disciplines, especially vaccines, epidemiology, and
prevention. Thus it transcends the individual purview of several Area Review Panels. It is
desirable for large programs to make scientific contributions across several areas; however,
difficulties arise when appropriate expertise is not available or efforts duplicate other programs.
Therefore, it is important for DAIDS to collaborate with and to incorporate expertise from other
programs in planning its prevention agenda in general and in any nonvaccine efforts it pursues.

Links Among Research, Services, and Communities

Two specific points related to communities potentially involved in HIV/AIDS vaccine research
require attention: (1) transfer and exchange of research findings and provider and community
concerns and (2) participation of community representatives in AIDS research activities. NIAID
has been a pioneer in involving communities in its research programs through open scientific
meetings, local and national Community Advisory Boards, and the inclusion of representatives
from the affected community on peer-review panels at all levels. This effort has been of benefit
to all parties and to AIDS research programs as a whole. The Panel commends NIAID for these
actions.

However, a great deal more must be done to involve the public in the difficult processes and
decisions that face development of an AIDS vaccine. Significantly more attention and resources
should be devoted to educating communities that will participate in trials.

Links Between the Public and Private Sectors

NIAID has brought companies together as collaborators through the NCVDG and for particular
AVEG trials, e.g., a trial of avipox HIV vaccine vectors (Pasteur-Merieux Connaught and
Virogenetics) plus envelope subunit boost (Chiron). This model should be strongly encouraged
as a particular role for Government in a public health crisis.

The Panel was provided with an overview of the Department of Defense (DoD) AIDS vaccine
effort by staff of the Walter Reed Army Institute of Research (WRAIR). The Panel supports the
DoD's highly focused and coherent research effort on preventive vaccines and encourages
WRAIR and DAIDS staff to continue to work together on strategies for investigating newer
vaccine approaches to evoke antibodies to conformational cross-strain determinants that may be
needed, especially in the international vaccine effort.

Dr. Seth Berkley of the Rockefeller Foundation provided the Panel with an overview of the
newly established International AIDS Vaccine Initiative (IAVI). The stated goal of this new
initiative is to quicken the pace of vaccine development so that a vaccine suitable for use
throughout the world becomes a reality. The intent is to support research and development
activities on vaccine concepts not being pursued by industry by working with Government,
private industry, funding groups, and regulatory authorities to create a more favorable
environment that will encourage increased investment in HIV vaccine research and development.
If adequately supported by new independent funds, this effort should enlarge the intellectual and
financial investment in the development of vaccine candidates.

Poor economic incentives for vaccine development appear to be an obstacle for industry
involvement at many levels. As a high priority, OAR should invest resources to analyze options
and propose solutions for this problem.

Access and Ownership of the Products of NIH-Supported AIDS Research

It may be time to consider cooperative or public ownership arrangements for AIDS vaccine
products, especially for riskier approaches, since private industry has largely been reluctant to
enter this area at levels required if there is to be an active program.

E. Conclusions and Recommendations

The many challenges for developing HIV vaccines have created a tension over what resources
should be devoted to efforts to understand more about the basic biology of the virus (i.e.,
immune responses to HIV through in vitro studies, animal models, and studies of both
chronically infected individuals who have controlled viral load as well as multiply-exposed,
uninfected individuals) and what resources should be allocated to clinical studies to evaluate
candidate vaccines in human subjects.

Some scientists have voiced the opinion that no human clinical efficacy trials are justified until
more is understood about the virus. Others have argued that some questions, such as
identification of the correlates of immune protection, may be answered only in human clinical
trials in populations at high-risk for exposure to HIV-1 once protection can be demonstrated.
Most scientists have taken an intermediate position.

The NIAID position has been to undertake both basic and empirical research, since success with
either approach alone cannot be predicted. The Panel concurs with this view, because it has
become evident that the predictive value of animal studies has its limitations. Further, HIV/AIDS
vaccine candidates can be tested effectively and safely in low- and high-risk populations. Finally,
the information gathered on comparative immunogenicity and safety of individual candidate
vaccines evaluated in Phase I/II clinical trials will be essential to determine future directions for
vaccine development.

The capability to do clinical trials of AIDS vaccines in humans must remain a high priority.
Given uncertainties about mechanisms of immune protection, studies must be designed to gather
samples that could be used in ancillary studies to answer fundamental scientific questions and
help direct future vaccine development. However, NIAID needs to promptly develop concrete
plans and timelines for making decisions about whether and when to launch efficacy trials. Such
plans will help ensure that clinical trials networks are effectively utilized or downsized or
redirected to other prevention or risk intervention research if appropriate vaccine strategies do
not become available.
Key Recommendations for HIV/AIDS Vaccine Clinical Trials Research

The poor economic prospects for vaccine development appear to be a restraining force on the
entire vaccine development effort.

• OAR, as a high priority, should invest resources in analyzing and proposing

solutions to this problem. It may need to attract more industrial-sector participation
or consider cooperative or public ownership arrangements for AIDS vaccine
products, especially for riskier approaches.

Tighter links must be forged among basic research efforts, animal studies, vaccine developers,
and clinical trials groups. Because clinical trials are conducted largely through contracted
programs, the Panel sees the potential danger of NIH/NIAID clinical programs missing
opportunities to work creatively with new or outside collaborators.

• It is essential that AVEG and DAIDS Program Staff establish a mechanism to allow
scientists to request collaboration with contracted programs for studies with clinical
samples, with some funds set aside to support such collaborations.

Criteria for selection of candidate vaccines for advancement to efficacy trials have appeared to
some to be arbitrary and influenced by small "inside" groups of investigators.

• Criteria for advancement of candidate HIV vaccines should be determined in

advance. The decision-making process must be open (i.e., including participation of
both the larger scientific and affected community), more clearly defined, and with a
specified person or group that has final decision-making authority.

Efficacy cannot be predicted for preventive HIV vaccines, and it seems unlikely that the first
ones tested will be highly effective.

• Therefore, new Phase I and Phase II trials should continue to be a high priority
even if one or more vaccines advance into efficacy trials.

Given the OAR mandate to allocate funds for specific scientific priorities, the Panel finds it
difficult to accept that the amounts coded by ICDs for vaccine clinical trials are actually
supporting such trials, directly or indirectly. Both coding systems, by Functional Category
(Mason Code) and by NIH Scientific Objective, are subject to miscoding through mistakes and
inconsistencies in interpretation by ICDs. Accuracy of coding is essential for OAR to influence
research direction.

• OAR should develop a process to assist with and review the allocation of resources
and coding of projects in the AIDS budget.

The major impact of HIV/AIDS is not in the developed world, but on developing countries.
Important vaccine research is now being carried out worldwide, and a vaccine must be available
to the developing world.
 • NIH should retain the commitment to international trials and scientific
collaborations, given the global nature of this disease.

Recommendations for Specific ICDs (For more details and recommendations, also see
Appendices A and B.)

NIAID

The Panel sees great opportunities for the existing human trials networks to conduct laboratory-
based human immunology studies that could contribute to an understanding of protective
immune response, response to vaccination, and infection.

• Significant resources should be directed toward using clinical trial networks (AVEG
and HIVNET cohorts) to advance our overall knowledge of the immune responses
to vaccine strategies.

• Laboratories linked to the clinical trials effort should focus on automation of

routine assays, development and validation of new and better assays, and more
comprehensive studies of the human immune responses to vaccination. It is
particularly important that methods to quantify cellular responses, especially CTL,
be encouraged.

• NIAID should streamline mechanisms for ensuring access to specimens for

investigators outside of AVEG/HIVNET and ensuring available funding for
specialized studies.

• NIAID should be more active in seeking out and assisting in the production of pilot
lots of vaccine candidates, if needed, to test new vaccine strategies.

• NIAID should dedicate additional personnel or resources to facilitate the trials

"start up" process (protocol development, satisfying FDA requirements, filing
investigational new drug [IND] applications, making arrangements with
manufacturers) to enter candidate vaccines into Phase I/II trials and ensure
effective use of the AVEG trials network

The Panel's extensive discussions of the HIVNET program and the many uncertainties in the
timing of vaccine development led to the following series of recommendations:

• NIAID should establish a rigorous plan and schedule for deciding the future
direction of HIVNET as soon as possible. HIVNET, or selected components, should
be decreased in scope or eliminated after baseline studies are completed, unless
clearly appropriate vaccine or nonvaccine intervention trials are undertaken or
anticipated in the near future.

It will remain important to maintain a presence in and commitment to the participating

communities for future vaccine trials.
 • More of HIVNET's resources should be devoted to educating communities that will
eventually participate in trials.

HIVNET investigators now have assessed the willingness of participants at risk for infection in
their cohorts to volunteer in vaccine trials. Valuable trials experience would be gained by the
sites if smaller trials were run in preparation for larger trials.

• HIVNET should participate in expanded Phase II studies of HIV vaccines,

especially when such trials involve people at some risk of HIV infection.

HIV-infected individuals identified through HIVNET provide an important human resource for
understanding the dynamics of the earliest stages of HIV infection.

• Options should be developed to allow individuals to enroll in studies of the clinical

benefit of early treatment, especially if the scope of such studies are expanded to
include people with documented recent infections who are already antibody positive.

The Master Contract mechanism may be complicating the ability of NIAID to manage its
HIVNET program.

• NIAID should exert closer oversight of HIVNET and the Master Contract
mechanisms and initiate regular reevaluation of the funding mechanisms and
funding level as changes in vaccine development plans occur.

• A reasonable percentage of the cost of HIVNET should be coded for clinical vaccine
trial preparedness. The rest of the cost must be justified by the strength of the other
research and coded appropriately under objectives in the annual NIH Plan for HIV-
Related Research. (This issue has been addressed in FY 1995. See Appendix 2 on
Coding of HIV Vaccine Resources.)

• Trials of HIV vaccines in the infants born to HIV-infected mothers should be

conducted independently of trials in other high-risk populations, because the risk of
infection, even with AZT treatment, still exceeds that in most at-risk adult
populations.

NIAID should establish a strong link between AVEG and HIVNET. Consideration should be
given to possibly merging the two efforts.

• NIAID should explore ways that bring AVEG and HIVNET together that build on
the strengths of each group and facilitate sharing of knowledge and skills.

• NIAID should consider if and how its intramural program can contribute to the
clinical vaccine development effort. This effort should be coordinated with the
extramural AVEG efforts.

NCI
 • As NCI vaccine concepts reach the stage for testing in clinical trials for safety and
immunogenicity, every effort should be made to advance them into existing NIAID
Phase I/II trial programs to avoid duplication of resources and effort.

NCRR

With its current monitoring system, it is virtually impossible for NCRR to accurately determine
whether GCRC expenditures are being allocated wisely or accurately to AIDS vaccine-related
research.

• NCRR and OAR should work together to develop a mechanism for direct budgeting
and tracking of AIDS research funds allocated to vaccine clinical trials.

FIC

• A substantial proportion of the budget assigned to AIDS vaccine clinical trials

infrastructure and AIDS training requires reexamination. FIC and OAR should
work together to ensure that assignment of codes for vaccine clinical trials is
appropriately applied.

F. Reports for the Clinical Trials Subpanel

Appendix A: NIAID AIDS Vaccine Evaluation Group (AVEG)

[Note: The Subpanel did not review the AVEG directly, but relied on a recent review by an ad
hoc panel of extramural scientists, presentations from NIAID, summary information, as well as
trial synopses provided by DAIDS Program Staff and the experience of Panel members, some of
whom receive funding from this program.]

In the AVEG, NIAID has put in place a well-coordinated and funded group of clinically oriented
AIDS vaccine evaluation units (AVEUs). The AVEG consists of six AVEUs, each with some
laboratory capability on site. There is one central immunology laboratory (CIL) at Duke
University that performs routine assessments for all trials. In addition, there is one specialized
laboratory for mucosal immunity at the University of Alabama and a centralized data center, the
EMMES Corp., Potomac, MD. This combination of dispersed clinical sites and centralized
analysis components allows for efficiency and flexibility.

The mission of AVEG is to evaluate promising candidate HIV-1 vaccines in Phase I/II trials. To
this end, it is charged with:

• Testing strategies that represent the range of options available (i.e., subunits, peptides,
live vectors, etc.).

• Carrying out a comprehensive evaluation of the safety of each approach.

• Assessing the humoral and cellular immune responses induced by each candidate vaccine
using state-of-the-art assays.
 • Facilitating further studies of each candidate vaccine by making specimens, etc.,
available to qualified investigators outside of this program who have interesting and
scientifically sound proposals.

• Providing the information, obtained in Phase I/II trials, that is necessary for making
decisions regarding the advancement of strategies to Phase III efficacy trials.

The Panel concurs with the conclusions of the recent external review conducted by NIAID and
believes that the AVEG is functioning well and has been able to test most of the currently
available HIV vaccine strategies. Approaches that have been tested by AVEG include the
following:

• Recombinant envelope glycoprotein subunits produced in various expression systems

(yeast, baculovirus, CHO cells, vaccinia) ranging from nonglycosylated denatured
polypeptides to fully glycosylated native proteins.

• Envelope glycoprotein subunits combined with novel adjuvants.

• Genetically engineered pox virus vectors (vaccinia, avipox) expressing HIV envelope as
well as other HIV-1 gene products.

• Various peptides and complex peptide constructs and formulations.

• Combination strategies consisting of priming with genetically engineered pox viruses and
boosting with subunits (Vaccinia env + rgp120 subunit; avipox env + rgp120 subunit; and
others).

• Virus-like particles.

AVEG has been successful in the recruitment and retention of both low-risk and high-risk
volunteers. It has active Community Advisory Boards (CABs) at each site and a national CAB
that includes a representative from the HIV-affected community on its Executive Committee.
Particular efforts have been made to improve minority recruitment at every site and to create
opportunities for younger clinical and basic research investigators.

To date, the full potential of the AVEG has been hampered by a shortage of candidate vaccines.
The AVEG has the capacity to evaluate a larger number of candidate vaccines with little or no
additional manpower, but it probably does not have capacity to do more than one Phase II study
at a time without additional resources.

A considerable effort has been devoted to laboratory assessment of immune responses of

vaccinees. Approximately 10 percent of the AVEG budget supports the CIL activities at Duke.
Additional resources are allocated at each of the AVEUs for laboratory assessments; this is
estimated to represent 20 percent of the AVEG site budget. However, the Panel felt that an even
greater proportion of AVEG resources should be devoted to analyzing the consequences of
immunization, even if this necessitates smaller members of individuals studied within Phase I/II
trials.
The CIL performs a wide variety of routine immunologic assessments, including various ELISAs
to assess antibody responses to the candidate vaccine; neutralization assays; and, on a more
limited basis, lymphoproliferation assessments and CTL assays. The laboratory evaluation
historically has focused heavily on assessments of humoral immunity because of difficulties in
measuring CTL responses, and the nature of the candidate vaccines evaluated (envelope and
subunit). More CTL assays are being done presently, especially with pox vector vaccines.
Studies quantifying CTLs and assessment of nonenvelope CTLs are ongoing.

Consensus protocols exist for cell-mediated immunity assays performed in the central laboratory
and at individual AVEU sites. Additional exploratory and ancillary studies are performed at the
sites or at other laboratories; these are of value, but all data should be reported to the central
database to avoid nonuniformity and for quality control. The capability of doing routine
immunologic assessment does not appear to be limited, as the group is discovering that more
selective sampling may give an accurate picture of a particular vaccine candidate once the basic
pattern of the immune response is established.

Because there is no clear-cut correlate of immunity to measure, the Panel believes that there
should be a more concerted effort by the CIL to broaden studies of immune responses.
Therefore, the relative emphasis on immunological analysis of vaccine candidates might best be
shifted from a large-scale effort using traditional assays that catalogue the magnitude of humoral
and cellular immune response toward more focused, comprehensive studies of human immune
response to vaccination. Examples of such immunity studies would be studies of the balance
between TH1 and TH2 responses, the role of CD8 cells in suppressing HIV replication, and the
roles of antibodies and cells involved in viral clearance or viral dissemination.

Research Opportunities

Clinical trials of HIV/AIDS candidate vaccines provide a unique opportunity to study the
response of the human immune system to HIV gene products. Since correlates of immunity to
HIV infection remain undefined, it is not possible to select one form of immunity (e.g., humoral,
cellular, mucosal) over another or to focus on traditional functional immune responses (e.g.,
neutralizing antibodies, CTLs) in order to characterize the immunogenicity or likelihood of
efficacy of a particular vaccine candidate.

The CIL spends much of its effort manually performing routine assays. Automation of these
assays, if possible, would free up human resources. More emphasis and resources should be
placed on the development and validation of assays to assess cell-mediated immunity. It is
particularly important that methods to quantify cellular responses, especially CTLs, be
encouraged.

The Panel felt that the development of assays to assess mucosal immunity was adequately
covered by the central Mucosal Immunity Laboratory (MIL) and the CMIGs. However, when
these assays move into large-scale use, shifting of resources may be required.
The AVEG has sometimes taken unexpectedly long to move concepts/products into trials. More
effort should be dedicated to streamlining the trial protocol development process so the trials
network can be utilized more effectively.

Specimens

The specimens generated by the AVEG during the course of vaccine trials represent an
extremely valuable resource. When appropriate, research projects beyond the scope of the initial
trial utilizing these materials should be encouraged and funded. AVEG has been cooperative
with outside investigators interested in obtaining specimens for their research. They have also
been sensitive to the needs of the industry sponsors. However, the procedure for obtaining
specimens also should be streamlined. The bureaucracy is still cumbersome, and it often takes
considerable time to obtain approval and receive the materials.

General Conclusions

The clinical evaluation of candidate vaccines is the end of a pipeline. AVEG is currently
suffering from a thin base of new products and strategies to feed this pipeline.

If new vaccine concepts and approaches do not generate candidate HIV vaccines, it will be
difficult to justify sustaining AVEG at its current funding level. Industry involvement in AIDS
vaccine development has been rapidly declining. However several concepts from small groups of
investigators using DNA vaccines and other vectors have been moving toward clinical trials.
Strategies for keeping industry involved need to be developed. In addition, NIAID should be
even more active in seeking out and assisting both industry or academic investigators in the
production of pilot lots of new candidate vaccines for testing in "proof of concept" trials in the
AVEG.

Appendix B: NIAID HIV Vaccine Efficacy Trials Network (HIVNET)

[Note: The Panel did not review the effectiveness of HIVNET directly, since it is a new program.
It relied on presentations from NIAID Program Staff; documents provided by NIAID, including
the RFP and summary information provided to the Panel; and the experience of panel members,
some of whom receive funding from this program.]

In 1993 and 1994, NIAID established HIVNET, a network of domestic and international sites for
trials of various strategies to prevent HIV infection, including HIV vaccines, through two Master
Contracts and as a solicitation for proposals from individual study sites. The overall objective of
HIVNET was to create the infrastructure and gather baseline data to assess the feasibility and
design of future efficacy trials in high-risk uninfected individuals and, eventually, to conduct
trials of preventive HIV vaccines and other appropriate interventions to prevent HIV
transmission. (Because of the perceived urgency in early FY 1994, this effort was "jump started"
with supplements to several unsolicited R01 grants already funded by NIDA or NIAID and two
interagency agreements with the CDC and the VA. These sites were required to compete with
other sites for funding through the Master Contractor.)
Master Contractors were funded in late 1993 (FY 1994), and the associated Statistical Center,
Laboratory Contract, and Specimen Repository were funded early in 1994. Proposals for
domestic and international sites were received in early 1994, and domestic and international sites
were funded in August-October 1994 (with a second allocation of funds to the master contractors
in September 1994, i.e., also FY 1994). Altogether, eight domestic contracts (one each in San
Francisco, Seattle, Denver, Chicago, Boston, and Philadelphia, and two in New York City) were
funded for studies of homosexual men, injection drug users (IDUs), and women at high risk
through heterosexual contact. Internationally, nine sites were funded (in Thailand, India, Uganda,
Malawi, Kenya, Zimbabwe, Haiti, Senegal, and Brazil) for studies in high-risk adults and in
infants born to HIV-infected women.

Since that time, domestic and international HIVNET studies have succeeded in rapidly enrolling
large numbers of high-risk individuals into cohorts for baseline studies and have begun to gather
data on risk and incidence of HIV infection and to assess the willingness of enrolled subjects to
participate in future vaccine efficacy trials and to evaluate informed consent procedures.

NIAID's AIDS Research Advisory Committee (ARAC) recommended in June 1994 that large-
scale efficacy trials of recombinant gp120 vaccines (those most advanced in testing at the time)
be put "on hold" in the United States. This presented HIVNET, especially the domestic
component, with the dilemma of potentially preparing for a vaccine efficacy trial when no
vaccine candidate was likely to be available for several years.

In contrast to the NIAID ARAC recommendation, a World Health Organization (WHO)

committee recommended that efficacy trials of gp120 vaccines could be conducted in countries
where the incidence of HIV infection has been found to be extraordinarily high, where other
interventions either have been ineffective or could not be applied, and where the local
government endorsed the trial. Investigators at one of the HIVNET international sites (Thailand)
are participating with the DoD in its Phase I/II studies of gp120 vaccine matching the local HIV
clades.

The HIVNET Laboratory Contract functions differently from some of the major laboratories
under other NIAID contracts. Its task is to do assays that can be performed on a large scale on
specimens from large numbers of participants, rather than the labor-intensive assays required of
other laboratories focused more on detailed immunological assessments or pathogenesis. Thus,
the emphasis is on use of standard assays to detect HIV and immune response to the virus,
quantitation of virus in infected participants, and broad identification of HIV variants seen in
HIVNET populations that would help target appropriate vaccine candidates.

Clinical Trials

Trials of nonvaccine interventions to prevent HIV infection have already begun at some of the
international sites. Although proposals for the domestic HIVNET sites included plans for
nonvaccine trials after the baseline studies, NIAID has not committed to funding such trials until
recently. The current commitment to domestic HIVNET sites for the completion of baseline
studies in preparation for vaccine efficacy trials ends in 1997. The international HIVNET sites
that already are conducting other nonvaccine interventions have commitments for funding to
complete the currently approved trials.

From the outset, several international HIVNET sites proposed to undertake trials assessing the
efficacy of nonvaccine interventions while gathering baseline data for eventual vaccine trial
design:

1. A clinical efficacy trial of HIV immune globulin (HIVIG) for newborns is being planned
in Haiti.

2. A trial of vaginal cleansing to prevent maternal/infant transmission was conducted in

Malawi and is being modified to assess the efficacy of vitamin A treatment of the
mothers.

3. Sex workers in Kenya are participating in Phase II/III trials of low-dose nonoxynol-9 as a
topical microbicide to prevent HIV transmission.

4. A randomized trial of peer counseling to reduce risk behavior is being carried out in
Zimbabwe.

Recently, additional funds have been allocated ($7 million) for the international and domestic
HIVNET to carry out pilot studies of trials of nonvaccine interventions in 1996 and 1997.
Proposals include evaluation of topical microbicides to prevent sexual transmission, an antiviral
trial to prevent perinatal transmission, a trial of the efficacy of prophylaxis of other STDs on the
incidence of HIV infection, behavioral interventions, and computer reporting of risk behavior.
These trials of specific nonvaccine interventions are being designed and selected after the
contracts are in place, so they would benefit from high-quality, critical peer review of their value
and design. Most of these nonvaccine studies have been reviewed by the Natural History,
Epidemiology, and Biomedical Prevention Research ARP or the Behavioral, Social Science, and
Prevention Research ARP (see Cross-Disciplinary Research: Overlaps with Other Panels).

Intermediate-Sized Efficacy Trials

A Workshop on Alternative Trial Design, sponsored by AVEG, HIVNET, and NIAID, held in
April 1995, concluded that trials in populations with an incidence of HIV infection as low as 2
percent per year (the incidence currently seen in the highest risk U.S. populations) could
determine whether a vaccine (or some other intervention) was "reasonably" effective (i.e., had 60
percent efficacy or greater) or not (i.e., less than 30 percent efficacy), with prevention of HIV
infection as the primary end point using a sample size as low as 1,500 per arm of the study. Prior
considerations of study design had estimated a need for 3,000 participants per arm to estimate
efficacy with a precision of +/- 10 percent.

Additionally, based on data on the spectrum of immune responses to candidate vaccines, a study
of this size also would be sufficient to determine whether a difference as small as two-fold in the
level of vaccine-induced immune responses (which occurred in greater than 80 percent of the
vaccinees) can predict protection against HIV in vaccinated individuals. Only if CTLs are
detected in a larger fraction of vaccinees with the newer HIV candidate vaccines will it be
possible for intermediate-sized efficacy trials to establish CTL levels as a correlate of protection.

Trials in populations with an incidence substantially higher than the 2 percent (discussed in the
Workshop) would require an even smaller sample size to determine efficacy with the same
precision. Thus, trials with vaccines having a limited expectation of efficacy might be most
practically carried out among high-risk populations in certain international settings.

An advantage of conducting trials in domestic populations, however, is the relative ease of

collecting, processing, and testing the kinds of specimens necessary to evaluate potential
correlates of protection (especially cellular immune responses), information critical to future
vaccine development if the first vaccines tested do not work or are only partially effective.

At the time of this review, at least one live recombinant vaccine (based on insertion of one or
more HIV genes into an avipox virus capable of infecting human cells but not replicating)
together with an envelope subunit vaccine was under consideration for human Phase II testing by
the AVEG. One potential advantage of the avipox vaccine (possibly used as a priming
vaccination with a gp120 boost) is induction of cellular immune responses in some vaccine
recipients.

If study results indicate that it would be appropriate to test the efficacy of these vaccines against
HIV infection, the earliest that randomized trials of such alternative vaccine concepts could
begin in the United States is 1998. To meet this schedule, innovative ways of planning and
conducting Phase II trials must be developed.

Recommendations

With the above considerations, the Subpanel recommends the following:

NIAID should establish a rigorous plan and schedule for deciding the future direction of
HIVNET as soon as possible, including consideration of which candidate vaccines may
be adequately tested to be considered for Phase III efficacy trials through the current
HIVNET mechanism and the clear criteria for making that decision.

HIVNET proposals for trials of the efficacy of nonvaccine interventions that have not
undergone peer review or have been reviewed only by contract-associated review groups
should be reviewed by an outside panel of experts to determine their quality and
relevance to the field of HIV prevention.

Collaboration with investigators in other fields of HIV prevention should be sought when
studies beyond the current HIVNET expertise are undertaken. If, for example, efficacy
trials of behavioral interventions are proposed, collaboration with behavioral scientists
would be essential for such efforts. Dual support between NIAID and other relevant
Institutes also might be appropriate in such cases.
 The potentially competing objectives (vaccine and nonvaccine trials for prevention of
HIV transmission) must be carefully coordinated so that the ability to carry out HIV
vaccine efficacy trials is not jeopardized once the opportunity and need for them arises.

To ensure smooth coordination of the entire clinical vaccine trials effort, NIAID should
establish a strong link between AVEG and HIVNET, possibly even merging the two
efforts to minimize duplication and competition. Mutual representation on Steering
Committees might be considered as well as collaboration between Central Laboratories.
HIVNET also might be included in expanded Phase II studies of HIV candidate vaccines,
especially when such trials involve individuals at risk of HIV infection. HIVNET has
ready access to high-risk populations and involvement in such studies would help prepare
the investigators and their communities for future efficacy trials.

HIVNET, or selected components, should be eliminated or decreased in scope after

baseline studies are completed, if no clearly appropriate vaccine or nonvaccine
intervention trials are contemplated in the near future. However, it would be important to
maintain a presence in and commitment to the participating communities for future
vaccine trials. NIAID should consider mechanisms and activities for achieving this goal.

NIAID should establish means to link HIVNET with non-HIVNET investigators so that
resources generated by HIVNET efforts can be quickly and effectively utilized toward
advancing the vaccine effort. For example, HIVNET studies will detect a relatively large
number of early HIV infections during its baseline studies. Specimens from such cases
could be used for more detailed investigation of HIV variants and pathogenesis to
supplement HIVNET objectives, without unnecessary duplication. Additionally, HIV
infections identified through HIVNET could provide an important human resource for
trials evaluating the impact of early treatment of HIV infection, especially if the scope of
such trials were expanded to include people with documented recent infections who are
already antibody positive.

NIAID should clearly identify what proportion of the HIVNET cost is related to HIV
vaccine development and evaluation and what would be better assigned to other areas of
AIDS research, such as epidemiology, pathogenesis, and biomedical or behavioral
nonvaccine prevention. FY 1994 coding of HIVNET as nearly all vaccine-related
misleads both by exaggerating the vaccine effort and by underrepresenting HIVNET's
contribution to other areas of epidemiology and prevention research.

Appendix C: HIV Vaccine Trials in Newborns

Infants born to HIV-infected women must almost certainly be exposed to substantial amounts of
virus in utero across the placenta and during delivery with exposure to maternal blood and body
fluids. Yet only about one of four of these infants become infected, and this number can be
reduced to less than 10 percent by the use of AZT. Results of the clinical trial ACTG 076
demonstrated that treatment of the mother and infant with AZT reduced HIV mother-to-infant
transmission from 24 percent to 8 percent. Despite this success, reducing maternal-infant
transmission remains a high priority, because even at the reduced level, HIV transmission
exceeds that observed in most other high-risk populations. The probability that a high proportion
of maternal-infant HIV transmission occurs perinatally rather than in utero provides a strong
rationale for using immunoprophylaxis with antibodies or vaccines, even if begun postexposure
at the time of birth. Recent preliminary data with passive antibody in infant or juvenile macaques
support the continued interest in research in this area. Finally, in settings where extended
antiviral treatment may not be applicable or possible due to costs, preventive vaccine or passive
antibody interventions may be cost-effective.

Passive immunity studies with HIV-intravenous immunoglobulin (HIVIG) in mother-infant pairs

in the United States (ACTG 185) and in Uganda are under way. Studies in Haiti also are planned
for immunoprophylaxis in infants who are breastfed by their HIV-positive mothers. Studies with
monoclonal antibodies or related products are under consideration. With the success of AZT
therapy, passive immunity approaches should be considered only as adjuncts to antiretroviral
therapy in developed countries.

Additionally, the parallels between perinatal HIV and hepatitis B virus (HBV) transmission
encourages investigations of active vaccine plus passive immunity approaches. Active
immunization with hepatitis B vaccine begun at birth, with or without passive immunization with
hepatitis B immune globulin (HBIG), is highly effective in preventing perinatal HBV infection
and the development of chronic HBV infection. A logical extension of current studies of HIVIG
to protect infants born to HIV-infected women would be vaccine trials in the infants, also with or
without HIV immune globulin. Failure of HIVIG to protect infants should not deter undertaking
vaccine approaches. Passive immunoprophylaxis had little effect in preventing HBV infection
(although it clearly delayed the onset of infection and reduced the chronic carrier rate) whereas
active induction of immunity by vaccine alone or vaccine with HBIG was highly effective. Thus,
trials of HIV vaccines in high-risk newborns should be considered if populations and appropriate
vaccines are available.

Several Phase I studies on HIV-1 envelope candidate vaccines in HIV-infected pregnant women
or in infants born to HIV-infected mothers are either nearing completion or continuing followup.
These pediatric or maternal transmission clinical trials with HIV vaccines have been conducted
through collaborations between the AVEG and the Pediatric ACTG. A Pediatric Vaccine
Working Group with representatives from AVEG and ACTG have collaborated on these
activities. These trials were not coded in the vaccine area by either NIAID or by NICHD.

Infants born to women in HIV vaccine clinical trials provide a unique opportunity to study
mechanisms of infection and protection, and if adequate samples are available, may be the
optimal way to answer critical questions about transmission and acquisition of disease. Recent
anecdotal observations that, in rare cases, infants may clear HIV infection could provide a
remarkable tool to examine the protective immune responses to HIV, if additional such infants
are identified. Because data on reduction of viral load do not appear to explain the reduction in
transmission observed, surveillance of AZT-treated women and their infants should be increased
to identify possible rare transient virus infections for detailed immunology studies.

As with vaccines for adult populations, the basic research underpinning the clinical trials effort
for either active or passive immunity in children is meager. As part of long-term vaccine goals,
studies in infant primates with vaccine vectors or candidate vaccines specifically designed for
childhood vaccines should be initiated as soon as constructs are available. This is essential
because the strategy that would be most effective in preventing transmission of HIV as well as
other STDs in young adults (now recommended for Hepatitis B) would be vaccination in
childhood with subsequent boosting in preteen, school-age children. This would ensure that
strong recall priming of memory T cells would be possible and might permit the incorporation of
an AIDS vaccine in a standard childhood vaccine regimen.

IV. Appendices on Special Issues

Appendix 1. Links to Other Panel Reports

A number of programmatic components and general issues that concerned the Panel are also
discussed in reviews performed by the other Area Review Panels. These cross-cutting issues are
identified here (please see the reports of the other Area Review Panels for their independent
assessments).

Identification of HIV Vaccine and AIDS-Related Activities

As noted in the body of the report, the Panel had considerable difficulties in accurately
determining how much actual vaccine-related research was being performed in different vaccine
research programs and distinguishing between vaccine-related and other categories of HIV/AIDS
research. These difficulties were complicated by the current system of coding, which is not easily
aligned with programmatic areas. In some instances, projects or budgetary items appeared to be
assigned arbitrarily to the different AIDS coding categories; in others, misclassification of
resources was systematic. Two areas of particular concern to this Panel were (1) the systematic
failure to code many of the HIV/AIDS or AIDS-related immunology projects on correlates of
immune protection to the Basic Vaccine Research category and (2) the coding, by both NIAID
and FIC, of a substantial block of epidemiology and prevention projects as
Infrastructure/Vaccine Clinical Trials. Other Area Review Panels identified similar problems,
indicating that it is necessary to institute a major overhaul of the coding system used to track the
allocation of resources allocated within the HIV/AIDS research budget.

As the coding system and information provided by the ICDs to the Panel was sometimes
confusing and imprecise, OAR is urged to undertake an in-depth assessment of this problem to
accurately identify the use of AIDS-related funds.

Reprogramming of Resources

The Panel uncovered a small but significant pool of resources that were allocated to projects
coded as HIV vaccine-related research but whose title and abstract bore little or no apparent
connection to HIV/AIDS research. This was largely a problem in the NCI intramural program at
FCRDC, in the FIC Fellowship and small grants projects, and in the high levels of AIDS funds
allocated to nonhuman primate research by NCRR. Such monies should be reprogrammed to
bona fide HIV vaccine research activities.
Peer Review

The Panel concluded that one of its major concerns about the current peer review system was the
lack of synchrony with the mission or goals for the HIV/AIDS vaccine programs defined in the
NIH Plan for HIV-Related Research. Otherwise stated, there is a major disconnect between
programmatic goals and the current review process. Consequently, many worthwhile research
activities that could provide essential knowledge for vaccine development are not appreciated by
review groups that are either unaware of programmatic needs or are not experts in the vaccine
area and do not recognize the critical but somewhat pragmatic nature of translational research.
The Panel calls for the establishment of a special review group for the vaccine area as one of its
major recommendations and appreciates that this is a problem shared by other thematic areas.

NCRR

No formal review of NCRR-sponsored HIV/AIDS vaccine-related activities was undertaken.

However, Panel members identified several concerns about how AIDS resources were allocated
and used within Regional Primate Research Centers (RPRCs). The problem of developing
vaccines in the SIV macaque model has been hampered by the use of experimental groups with
only a few animals in each and the inability to do comparative studies with different virus
challenges. Both of these problems stem from the lack of sufficient nonhuman primate resources,
either at the RPRCs at other sites performing AIDS vaccine studies. Furthermore, there are
serious concerns that there may not be adequate resources in the Centers to provide sufficient
housing for the numbers of animals infected with AIDS viruses. The need for long-term housing
of experimental nonhuman primates has greatly increased because vaccinated animals, protected
from disease and not simply from virus infection, may need to be studied over long periods of
time for viral load assessments and other measures of vaccine efficacy. Related problems were
identified by other Panels.

Recommendations

• OAR and NCRR should make AIDS resources available through open, competitive
review to investigators who seek to perform "pilot" vaccine studies. NCRR should devise
a system to ensure access to animals for AIDS vaccine studies by non-RPRC
investigators.

• NCRR should work with OAR and research teams studying AIDS vaccine approaches to
provide adequate animal resources for testing novel vaccines, both at the RPRCs as well
as at other sites.

• NCRR should better coordinate and integrate its HIV/AIDS vaccine research activities
with other ICDs to allow better planning for nonhuman primate research resources.

These recommendations should be considered in tandem with parallel reviews of NCRR by the
Drug Discovery and Etiology and Pathogenesis Review Panels.

NCI
The Panel's review of NCI's HIV vaccine-related activities revealed an atypical distribution of
resources relative to other ICDs. There appeared to be an excessive emphasis on intramural
research in the HIV/AIDS vaccine area. In addition, there was also a large proportion of the
funds associated with contracts to further support these and other intramural activities. Other
ARP reviews reached similar conclusions. The NCI vaccine effort is poorly coordinated within
the Institute and with other NIH vaccine-related activities. The Panel urges that the NCI
activities in HIV vaccine research be carefully evaluated, streamlined, centrally coordinated and
closely linked, wherever possible, to other ICD efforts.

The HIVNET Program of NIAID

The HIVNET program was reviewed by this Panel as well as by the Natural History,
Epidemiology, and Prevention Panel and the Behavioral, Social Science, and Prevention Panel.
Although the vaccine preparedness activities are amply covered by the Vaccine Panel, other
activities of HIVNET that include behavioral and biomedical interventions as well as the
potential epidemiological value of its initial study cohorts are best addressed in the reports of
these other Panels. Collectively, these Panels have called for a reassessment of HIVNET by an
expert panel to address issues of how to best maintain this valuable network for future vaccine
and prevention research studies.*

*
NIAID has responded to the NIH AIDS Research Program Evaluation Working Group Report and has constituted,
with OAR input and approval, an ad hoc review panel to review the several complex and interrelated areas of
research and the agenda proposed by the HIVNET investigators.

Appendix 2. Coding of HIV Vaccine Resources

Expenditures on HIV/AIDS vaccines in FY 1994 was reported at about $113 million according
to the present ARIS database and the current coding system for projects as applied by various
ICDs. The Vaccine Research and Development Area Review Panel examined the extramural and
intramural components of NIH vaccine-related activities and concluded the following:

1. The present coding system presents significant difficulties in accurately assessing the
level of resources within the areas defined by the NIH Plan for HIV-Related Research
(e.g., vaccines versus pathogenesis, behavioral, or epidemiologic research). It appears
that a substantial proportion (up to 20 percent) of the research coded as HIV/AIDS
vaccines actually should be coded in other areas of AIDS or non-AIDS research.

2. Misclassification of non-AIDS research expenditures in AIDS or AIDS-related categories

has occurred in some ICDs.

3. It was difficult to assess the level of funds expended for specific areas of the NIH Plan
because some contract research, reagent repositories, and administrative support activities
cannot be divided easily by either scientific project or NIH Plan areas. If the support
activities cover several different areas, the amounts specified can change from year to
year.
Thus, often it was not possible to accurately determine whether the allocation and expenditure of
resources for HIV/AIDS vaccine research and development was appropriate. The assessment that
follows can therefore only be considered a "best effort" estimate that outlines the current
situation and provides a starting point for a more precise definition of the scope of the NIH
vaccine programs.

Of the nearly $113 million coded as HIV/AIDS vaccine research, approximately $89 million
appears to have been appropriately designated. The remaining projects represent (1) research that
may have been designated appropriately as AIDS or AIDS-related research that is only
tangentially related, at best, to vaccine efforts (about $18 million) and (2) research that does not
appear to be AIDS-related (about $6 million). Overall, this analysis suggests that considerably
fewer dollars were actually spent on bona fide HIV/AIDS vaccine research than has been
reported for FY 1994.*

*
During the process of review and writing this report, several of the ICDs, namely NIAID, NCI, and FIC, addressed
some issues of coding that were identified by this Panel. NIAID coded the HIVNET project in FY 1995 to more
adequately reflect the epidemiological and natural history AIDS research being conducted in those cohorts. The NCI
administration has begun an extensive revision of its intramural budgeting and coding process.

Some examples of the projects totalling $18 million that were appropriately identified as AIDS-
related but which were probably inappropriately coded as vaccine-related research are:

1. Intramural research at NIAID that would be more appropriately coded as AIDS

pathogenesis research ($2.1 million).

2. All costs of chimpanzee and specific pathogen free (SPF) macaque breeding funded
through NCRR ($4.1 million and $2.1 million respectively). These funds are independent
of any research funds spent on experimental vaccine studies.

3. Many aspects of the FIC training programs in epidemiology (in particular the AITRP;
$6.8 million coded to the vaccine clinical trials infrastructure category, 4B) are more
clearly epidemiology than vaccine-related. (This is fully discussed in the Clinical Vaccine
Trials Subpanel section of this review.)

4. Support mechanisms for reagents, shared resources, and services that extend beyond
vaccines. This was a particular problem for a series of four large-budget allocations
(totalling $6.3 million) identified by NCI only as "Operations and Technical Support" for
FCRDC. This allocation was in addition to a budget item ($2.98 million) identified as
"Evaluating Lentivirus Compounds as Vaccines in Animals," which was described as
monies transferred to DoD for "overhead" at the FCRDC facility. These allocations were
not clearly associated with any projects, but it was estimated that at least $3 million could
not be justified in the basic vaccine research category.
It also was determined that some bona fide vaccine-related research had been coded to topics in
Etiology and Pathogenesis and, to a lesser extent, as Epidemiology or Prevention research. Thus,
some fraction of each of those categories also appropriately might be reclassified.

For example, essentially all of the grants on cellular immunology and CTLs, even those
investigating T-cell epitopes. have been assigned to an Etiology and Pathogenesis budget
code, 2A, whereas many of the grants on humoral immunity have been assigned to basic
vaccine research category, 4A. Both of these areas fit logically into basic research for the
design of vaccines to induce protective immune responses. Another example is the
Correlates of HIV Immune Protection Contract, which was coded as category 2A rather
than 4A, although one of the stated goals of that contract was to evaluate the immune
responses of vaccinees who failed to be protected by candidate vaccines. Furthermore,
research on vaccinees as well as basic research on correlates of immunity relevant to
vaccine design has been conducted under that contract. At the same time, an interagency
agreement for the statistical analysis of studies performed on the contract is appropriately
coded 4C. Both projects are administered through the DAIDS.*

*
The Correlates of HIV Immune Protection Contract is now scheduled for termination in June 1997. NIAID intends
to shift the $3 million currently allocated for this contract to investigator-initiated, peer-reviewed grants. The need
for other research and development contracts in the vaccine areas will be reviewed by NIAID with the OAR if they
are proposed for renewed funding.

The Panel identified a number of projects that were clearly non-AIDS activities misclassified as
HIV vaccine-related research including the following:

• About $1.5 million was coded for one component of NCRR's GCRC awards (M01). This
appeared to duplicate funding for AIDS projects that were funded by an NIAID-
sponsored AVEG contract and represented about half of the total budget allocated to
AIDS vaccine clinical trials research by NCRR.

• Intramural research at the NCI (Z01) relating to peptide vaccines against cancer viruses
and oncogenes ($257,000) was coded as AIDS vaccine research. NCI research on HIV
peptides is funded appropriately under this same project.

• Several projects supported through the FIC small grants programs had titles and abstracts
that were related to infectious diseases in developing countries but had little direct
relevance to AIDS research.

• An excessive amount specified for "Operations and Technical Support" for intramural
research at NCI, FCRDC was coded as HIV/AIDS vaccine research. A precise estimate
of the miscoding is difficult, but the sum is likely to be in excess of $3 million (about half
of the budget for this item) in this category.

These issues notwithstanding, Table 2 presents a rough approximation of the resources according
to the vaccine categories evaluated by this Panel--Basic Research, Targeted Research, and
Clinical Trial Research. Clearly evident from this breakdown is the paucity of resources for
support of extramural research in both the Basic ($12,428,823) and Targeted ($17,122,643)
research categories, compared with funds for Clinical Trials networks ($32,943,191). With the
exception of Clinical Trials Research, intramural research received roughly one-third of the total
funding in these areas, despite a great disparity in the number of investigators being supported:
many more extramural investigators (45 research project grants) than intramural investigators
(14 Z01 projects).

In view of these and other considerations expressed in this report, the first priority for
additional or reprogrammed funds should be to increase the level of extramural
investigator-initiated research.

Table 2. NIH HIV-Related Vaccine Research FY 1994

Basic Targeted Clinical Trials

Research Research Research Other Total
$12,428,823 $17,122,643
Extramural $32,943,191 (c) $62,494,657
(a) (b)
Intramural 6,626,524 (d) 8,768,728 (d) [000] (e) 15,395,252
Primate Resources 14,519,676 (f) 14,519,676
General
8,334,446 (g) 8,334,446
Infrastructure (g)
Administration (h) 1,649,548 1,900,300 3,659,895 4,955,988 12,165,731
TOTAL $20,704,895 $42,311,347 $44,937,532 $4,955,988 $112,909,762

a. Extramural Basic Research includes all unsolicited, investigator-initiated grants (R01, R29, R03, R37, R43, R44;
Physician-scientist and fellowship awards K08, K11, F05, F06 and components of a P01 coded HIV vaccine
research).

b. Targeted Extramural Research includes grants and contracts solicited through RFAs and RFPs; CMIG R01s,
NCVDG and Adjuvant cooperative agreements (U01s), R & D contracts on genetic variation, and related smaller
efforts.

c. Extramural Clinical Trials Research consists of the AVEG and HIVNET efforts, including all of the associated
laboratory and statistical center contracts. Amounts for FY 1994 are artificially high for 2 reasons: First, the timing
of expenditures for the HIVNET and AVEG resulted in 15 to 18 months of funds being allocated in a single fiscal
year. Second, both NIAID and FIC coded substantial amounts of their AIDS budget to the development of
Infrastructure for Vaccine Efficacy Trials, when some of these projects or activities might have been more
appropriately coded as natural history and epidemiology research.

d. Intramural HIV/AIDS vaccine research, conducted at NCI and NIAID (coded 4A) was divided by project content.
Several projects similar to NCVDG awards with accompanying primate resources were included in the Targeted
Research.
e. Intramural HIV/AIDS vaccine clinical trials have been conducted by NIAID and NCI in the past, and are ongoing
in FY 1996; no funds were identified for this activity in FY 1994.

f. Primate Resources included are: RPRCs, $2.5 million; SVEUs $3.1 million; Contracts for primate resources for
intramural investigators, $2.3 million; SPF macaque breeding, $2.1 million; and chimpanzee breeding, $4.1 million.
RPRCs funds also support some Basic Research on vaccines as do resources for some of the cooperative agreements
and other investigator-initiated, solicited projects included in the targeted research.

g. General Infrastructure represents the GCRC support, $2.56 million; and FIC (training programs: D43 awards $5
million, and T22 awards, $0.77 million). The coding of GCRC activities at one Center by NCRR resulted in an
inflated amount of funds attributed to vaccine research at clinical centers.

h. Administration included research management support (RMS), which covers staff salaries, services, program
reviews, conferences, meetings, etc., intramural support (FCRDC) contracts that did not appear to fit a single
category.

Appendix 3. NCRR-Supported Nonhuman Primate Resources

Two factors initiated the early disease model development and AIDS vaccine experimentation in
nonhuman primates. The first was the identification of several SIV isolates that induced AIDS in
rhesus or pigtailed macaques at four of the Regional Primate Research Centers (RPRCs). The
second was the discovery that some early isolates of HIV could infect chimpanzees, a species
that had proven extremely valuable for the development of vaccines against hepatitis virus.

A major component of nonhuman primate resources dedicated to AIDS research are the RPRCs.
These Centers, administered by NCRR, were established 30 to 35 years ago so that the
biomedical research community could benefit from regional facilities with expertise in research
with nonhuman primates. There are seven RPRCs in the country: California, Georgia (Yerkes),
Louisiana, Massachusetts, Oregon, Washington, and Wisconsin. While each is a distinct
organization affiliated with a major academic institution, each receives operating support from
NIH.

RPRC Location Host Institution

New England Southborough, MA Harvard Medical School
California Davis, CA University of California, Davis
Washington Seattle, WA University of Washington
Oregon Portland, OR Oregon Health Sciences Center
Wisconsin Madison, WI University of Wisconsin
Yerkes Atlanta, GA Emory University
Tulane University (Delta) Covington, LA Tulane University

Recent program guidelines issued by NCRR for the RPRCs state that the overall objective of the
program is to provide specialized resources (physical facilities, technology, professional and
technical staffing, and a variety of primate species) for research studies applicable to the solution
of human health problems. Of importance for this review on AIDS vaccine-related research, it is
noted that RPRC funds should be utilized to:

1. Conduct meritorious basic and applied biomedical research in areas requiring the use of
primates.

2. Develop improved practices of primate breeding, husbandry, and genetic definition to

help meet research needs for pedigreed, disease-free animals of defined quality, and to
assure the continued availability of species of biomedical research importance whose
wild populations are considered threatened or endangered.

3. Provide opportunities for research development and experience in primatology to trainees

at various levels of experience.

4. Provide regional and national resources to support primate-related research, including

data collection and management, consultative expertise, biologic and genetic material,
and specialized facilities and equipment.

Each RPRC receives operating base grant support from NCRR. Base grant funds awarded by
NCRR provide support for infrastructure and resource-related expenses as well as a portion of
each Center's programs in AIDS and non-AIDS research. Other disease-specific research
activities at RPRCs, including AIDS research, are also supported by NIH grants from other ICDs
and from other sources including pharmaceutical firms and non-Government research funding
groups. Base grant support from NCRR accounts for approximately 40 to 60 percent of the
research and operating costs of each of the RPRCs. AIDS research supplements were initially
awarded to the individual Centers based on their capability of pursuing specific AIDS-related
research activities. These AIDS "supplements" were subsequently incorporated into RPRC Base
Grant renewals and this development has resulted in some of the concerns that have been raised
by the Panel in its review of this portfolio of grants.

The RPRC program has contributed substantially to the overall AIDS effort since AIDS-targeted
support was initiated in 1984. The RPRC Base Grants funded the following: the original
identification and isolation of SIV and its association with an AIDS-like disease; reproduction of
AIDS like-disease in readily available laboratory primates with both cloned and uncloned SIV;
development and testing of vaccine concepts; evaluation of the relative effectiveness of different
vaccine concepts; and investigation of fundamental mechanisms of viral pathogenesis. Reliance
on primate models to better understand pathogenesis and immune-mediated control of AIDS
viruses has been made possible principally through this previous long-term commitment to high-
quality primate resources. While far from ideal, the RPRC program has clearly made critical
contributions to the nation's AIDS research effort and to the HIV/AIDS vaccine efforts in
particular.

In addition, funds coded solely to the AIDS vaccine effort have been allocated by NCRR for
specific pathogen-free (SPF) macaques. The scientific need and the extent to which these
resources are used for AIDS research, and specifically the HIV/AIDS vaccine effort, should be
reviewed carefully. It was noted that SPF macaques were recently utilized in experiments to
address critical co-infection issues with attenuated virus vaccine concepts. AIDS pathogenesis
questions may require further use of these animals, and some budget allocation might be more
appropriately coded as AIDS pathogenesis.

In response to concerns previously raised about the research conducted at RPRCs and about
access to nonhuman primate resources, the RPRCs have recently increased efforts to include
scientists not affiliated with the RPRC and to institute local systems for external evaluation of
meritorious proposals. Some RPRCs have been responsive to investigators seeking access to
experimental resources for small "test of concept" types of vaccine experiments. Part of the
constraint is the limited availability of resources, particularly holding space for lentivirus-
infected animals, beyond those previously committed to funded studies by primate center
investigators. Finite resources at the RPRCs do not allow for expansion. It is not always
appreciated that independent support for research activities at the RPRCs is usually required,
even for investigators at the RPRCs. Furthermore, in some cases investigators at the RPRCs are
forced by limitations at the Centers to conduct a large part of their investigations on contract with
private firms outside of the RPRCs. Nevertheless, if the pool of resources to support animal
research at RPRCs is increased and opened to competition (see below), this configuration could
change to permit the rapid inclusion of additional novel meritorious research. To achieve this
goal, the Panel recommends that a system for evaluating AIDS-related proposals on a national
basis through NIH (DRG) study sections rather than by the individual RPRC would be more
equitable. It also should be possible to reserve a portion of each Center's AIDS-related resources
strictly for access by investigators not affiliated with the Center. To ensure the appropriate use of
these valuable nonhuman primate models, new non-RPRC investigators proposing studies could
work with RPRC investigators on study design issues. To maximize the quality of research, the
Panel recommends that NCRR (or DRG) institute ongoing review mechanisms, including experts
in other areas of AIDS research, to ensure that AIDS research programs at RPRCs are stringently
evaluated and that funds are awarded in a competitive fashion. Simultaneous peer review of
AIDS-targeted Base Grant support to all of the seven RPRCs should also be considered, in
contrast to the nonconcurrent, cyclical review of this research which is now linked to general
review of the individual Center.

The Panel commends RPRCs that have successfully recruited high-caliber staff in immunology
and virology and built interactive teams to perform research on AIDS vaccines. The Panel wants
to ensure that such programs are not dismantled because of a failure to recognize the importance
of translational research, particularly in immunology. Guidelines should be formulated for the
award of AIDS research funds from NCRR such that stronger interdisciplinary AIDS research
programs are formed and appropriately funded.

In the Panel deliberations, questions were raised about the extent of AIDS-related primate
research that is conducted at nonhuman primate facilities not associated with the RPRCs, and
about how this is funded and linked with research that is conducted at RPRCs. It is readily
acknowledged that the RPRCs do not have the capacity or the flexibility to accommodate all of
the AIDS-related research that would be optimal for AIDS vaccine research and development.
However, funding of nonhuman primate research at diverse sites should be incorporated as part
of the complete NIH Plan for HIV-Related Research to ensure that small primate facilities with
valuable resources and only one or two investigators are kept informed of rapidly moving
research goals.
As a result of strong NIH support for investigator-initiated studies in the United States, a broad
array of pathogenic SIV, HIV-2, and SHIV isolates for analysis of pathogenesis and vaccine-
related issues have been developed. This diversity can be viewed as a healthy exploration of
isolates with different pathogenic potential, comparable to the range of isolates that exist in
human HIV-1 disease. However, one inherent problem of investigator-initiated research with
primate models in the vaccine research area is the difficulty in comparing results from different
laboratories using different animal models to evaluate vaccine approaches. For example, it is
difficult to compare the results of one laboratory using virus A, neutralization assay B, and virus
quantitation assay C with the results of a second laboratory using virus X, neutralization assay Y,
and virus quantitation assay Z. This has been a problem in the United States for at least 6 years.
The comparison of different models might be resolved by cross-comparison of end points with
new forms of measurement such as viral load. A plan for systematic, comparative evaluation of
vaccine approaches and associated variables is still desirable. Large comparative studies in
monkeys can be achieved either through a centralized testing facility (the approach taken by the
Medical Research Council in England) or through team-organized simian vaccine evaluation
studies (the approach of the AIDS vaccine study group supported by the European Economic
Community). A third approach has been instituted by NIAID for experimental design approval
by a group of extramural, non-Government research scientists and Program Staff for utilization
of contract-supported nonhuman primate resources for studies, some of which are conducted at
RPRC sites. Coordinated support through NCRR and NIAID, and other relevant ICDs, should be
developed for well-designed comparative AIDS vaccine activities.

Finally, OAR should facilitate the reevaluation of the issue of AIDS-related chimpanzee
resources with two objectives in mind. One objective is to determine whether the cost being
borne by the NIH AIDS research budget (about $4.1 million) for chimpanzees is appropriate.
These costs are independent of costs for use of the animals in vaccine experiments because most
of the chimpanzees are now at breeding facilities where AIDS research cannot be conducted
because biohazard containment facilities are not available. The second objective is to determine
how to support testing and availability of HIV stocks for chimpanzees and the type of viral
stocks that are needed for critical vaccine evaluation. Because the National Research Council is
evaluating the broader issue of the use of chimpanzees in biomedical research and their long-
term care, the OAR, through the NIH AIDS Vaccine Coordinating Committee, should consider
findings from that review in its evaluation.

Appendix 4. Target Areas for Application of Additional Resources in HIV/AIDS Vaccine

Research

A principal recommendation of the Vaccine Research and Development Area Review Panel is
that NIH should expand the scope and dimensions of the HIV/AIDS vaccine program. One of the
highest priorities is to increase funding for basic research as defined by the Basic Research
Subpanel. These funds should encourage and support research on human and nonhuman primate
systemic and mucosal immunology, and on novel concepts in design of immunogens and
delivery systems. Investigation is needed to understand the early events in natural HIV infection
that limit the HIV replication level in some individuals. As the Panel noted previously, such
funds must be applied in concert with establishment of a supportive "culture" for both basic and
pragmatic aspects of all vaccine research to yield success for this endeavor.
Appreciable funds also will be necessary to support the preclinical evaluation of vaccines in
animal models, with derivation of immune correlates as described in the Targeted Vaccine
Research Subpanel report. Although such funds are not of higher priority than those for Basic
Research, they can be applied more rapidly, as determined by the NIAID Vaccine Design Focus
Group guiding this area. Nevertheless, a careful assessment must be undertaken promptly to
determine the needs for comparative vaccine testing relative to the needs for evaluation of novel
vaccine concepts worthy of testing.

The area described as Clinical Trials Research, which includes AVEG and HIVNET, does not
appear to have an immediate need for additional funds for vaccine research. The Panel
recommends that NIAID review the HIVNET program and use its review to make the best use of
the cohorts and funds currently available. This may require that some funds be redirected to other
areas of AIDS vaccine research. At the present time, it is unlikely that an efficacy trial could
begin before 1998. If the overall HIV/AIDS vaccine program is expanded and becomes
invigorated as recommended by this review, the vaccine pipeline should be filled with promising
HIV candidate vaccines, making it likely that additional resources will be required in the near
future for both the AVEG and HIVNET.

Appendices
Supporting Materials

A. Schedule of Meetings/Conference Calls and Deadlines for the Panel

January 29, Evaluation Working Group and Area Review Panels (ARP), Chairs meeting,
1995 Washington, D.C.
Vaccine Research and Development Area Review Panel (Vaccine ARP) meeting;
May 3
Gaithersburg Hilton, Gaithersburg, MD.
Vaccine ARP Conference call with members not able to attend May 3 meeting,
May 5
2:00 pm EDT.
May 18 Vaccine ARP Clinical Evaluation Trials Subpanel teleconference, 4:00 pm EDT.
May 31 Vaccine ARP Clinical Evaluation Trials Subpanel teleconference, 4:00 pm EDT.
June 7 Vaccine ARP teleconference, 1:00 pm EDT.
June 16 Executive Secretaries meeting with Dr. Marvin Kalt, NCI.
June 20 Targeted Vaccine Subpanel, teleconference at 1:00 pm EDT.
July 10 Evaluation Working Group and ARP Chairs meeting, Bethesda, MD.
Vaccine ARP meeting, Bethesda, MD. Presentations by ICD Program Staff and
July 10-11
representatives of selected non-NIH vaccine programs.
July 20 Teleconference with Drs. Levine and Paul with ARP Chairs, 11:00 am EDT.
August 10-11 Evaluation Working Group and ARP Chairs meeting, Bethesda, MD
August 24 Vaccine ARP Clinical Trials Subpanel conference call, 11:00 am, EDT.
Vaccine ARP meeting, NIH, Bethesda, MD. Discussion of rough drafts of vaccine
August 30
subpanel and topic reports; identification of issues for further investigation.
Evaluation Working Group and ARP Chairs meeting, Bethesda, MD. (Public
September 13
Session)
October 2 Joint ARP conference call with NIAID staff about HIVNET, 3:00 pm EDT.
October 12- Evaluation Working Group and ARP Chairs meeting, Bethesda, MD. (First draft of
13 Executive Summary reviewed by Vaccine Panel Members.)
Vaccine Research and Development ARP meeting, Crystal City, VA (Public
October 16
session).
December 14- Evaluation Working Group and ARP chairs meeting, Hyatt, Bethesda, MD. (First
15 complete draft of Subpanel reports. Sent December 23 to all Panel members.)
February 6,
Revised draft of Vaccine ARP Subpanel reports distributed to Panel for comment.
1996
February 15-
Evaluation Working Group and ARP Chairs meeting, Bethesda, MD.
16
February 26-
Evaluation Working Group and ARP Chairs meeting, Bethesda, MD.
27
Completed draft report distributed to Evaluation Working Group, Office of AIDS
March 6
Research Advisory Council (OARAC), and ICD Directors.
June 13-18 Final draft report, with revised appendices, sent to Panel members.
Final report sent to Evaluation Working Group, and OARAC for approval. Copies
June 25
provided to ICD Directors.
Note: This list does not contain several Subpanel teleconferences at which fewer than 4 members
addressed subtopic issues.

Introduction
Measurement of immune responses directed specifically against HIV is critical for understanding the
interplay between the virus and the host immune system. By characterizing the immunological correlates
of protection against HIV infection, such measurements will aid in the development of efficacious
prophylactic vaccines. Immune responses to HIV can be measured by a variety of methods. Humoral
immunity is detected by neutralizing antibody assays, while cellular immunity is investigated using a
variety of techniques, including cytotoxic T-lymphocyte (CTL) assays for detection of CD8+ T cells with
cytolytic ability; ELISpot and intracellular cytokine staining assays for detection of cytokine-secreting T
cells; HLA tetramer binding assays for the enumeration of antigen-specific T cells; and lymphoproliferative
assays (LPAs) for detection of HIV-specific CD4+ T cells.
Humoral Immune Responses

Neutralizing Antibody Assays

Sterilizing immunity against HIV infection is expected to require the presence of HIV-specific neutralizing
antibodies. Such antibodies prevent infection by binding to regions of the HIV envelope that are required
for viral attachment and entry into host cells. Antibody-mediated neutralization of different T-cell line
adapted HIV strains is measured by MT-2 cell killing assays.(1) MT-2 is a CD4+ human lymphoblastoid
cell line that is highly permissive to cytopathic infection with CXCR4-utilizing strains of HIV.(2) In these
assays, cell-free virus is incubated with multiple dilutions of serum samples at 37°C for 1 hour before MT-
2 cells are added. Any neutralizing antibodies present in the serum will bind to the virus and limit its ability
to infect the MT-2 cells. Neutralization is measured by staining viable cells with Finter's neutral red when
cytopathic effects in control wells (cells plus virus but no serum sample) are >70% but <100%.
Neutralizing titers are expressed as the reciprocal of the plasma dilution required to protect 50% of cells
from virus-induced killing. This 50% cutoff corresponds to a 90% reduction in viral antigen synthesis.(3)
For standardization purposes, positive control serum samples with known average titers are tested in
parallel. Deviation of more than 2-fold in experimental titer of the positive control serum from its known
average titer requires that the assay be repeated. Serum samples are heat treated prior to use in these
assays in order to inactivate the complement system, because components of activated complement can
deposit on virions and target them to complement receptors on the MT-2 cells, enhancing infection and
interfering with assessment of antibody neutralization. Activation of complement is not an issue for PBMC
assays (see below) because normal CD4+ T cells do not express complement receptors.

Although MT-2 killing assays are useful for determining neutralization against T-cell line adapted
(CXCR4-tropic) strains of virus, they are not useful for determining neutralization against most primary
isolates, which are CCR5-tropic. Neutralization of primary isolates of HIV is assayed in
phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) by monitoring for a
reduction in p24 Gag core antigen synthesis.(4) As with the MT-2 assays, known concentrations of virus
are incubated with various dilutions of serum samples for 1 hour at 37°C before addition of PHA-
stimulated cells. After an overnight incubation the cells are washed and then cultured in fresh interleukin-2
(IL-2) containing growth medium. Culture supernatants are collected on a daily basis and assayed for p24
using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Concentrations of p24 in virus
control wells (virus plus cells but with no serum) are calculated for each harvest day and are used to
construct a viral replication curve. Neutralization is measured at a time before peak p24 production occurs
as determined from this curve. Neutralization titers are the reciprocal of the dilution at which p24
synthesis is reduced by 80% relative to a negative control serum.
Cellular Immune Responses
The immunological correlates of protection against HIV are not known. While neutralizing antibodies may
be necessary to achieve sterilizing immunity, they have proven difficult to induce by vaccination.(5) It is
likely that cellular immunity, particularly CD8+ cytotoxic T lymphocyte, will be critical in limiting viral
replication if initial HIV infection cannot be prevented.(6,7) Indeed, depletion of CD8+ lymphocytes in
nonhuman primate models results in an increase in plasma viral load.(8-10) In addition, increasing
evidence suggests that induction of HIV-specific CD4+ T cells may also be critical for sustained control of
viral replication.(11-14) For these reasons, current trials of potential HIV vaccines are designed to induce
HIV-specific T-cell responses in addition to neutralizing antibodies.

CD8+ T cells exert their antiviral effects by direct lysis of infected cells and by secretion of antiviral
cytokines (eg, interferon gamma [IFN-gamma]) or chemokines (eg, MIP1-alpha, RANTES). Cellular
immunoassays have been developed to measure both of these types of effector function.

Cytotoxic T-Lymphocyte (CTL) Assays

See Figure 1.

Chromium release assays determine the ability of CD8+ T cells to lyse target cells expressing specific
HIV antigens. Briefly, freshly isolated PBMCs from an infected or vaccinated individual are stimulated in
vitro with HIV antigens for 2 weeks in order to expand HIV-specific T cells. Such expansion is required
because HIV-specific CD8+ T cells are normally present at frequencies that are too low to detect by this
method if cells are used directly ex vivo. These expanded T-cell lines are then incubated with autologous
target cells (normally B-lymphoblastoid cell lines derived by transformation of autologous B cells with
51
Epstein-Barr virus) that have been labeled with the radioisotope Cr and infected with vaccinia
expressing the HIV antigens of interest. HIV-specific T cells will recognize and lyse the target cells,
resulting in liberation of the radioactive label into the culture supernatant. Spontaneous lysis is measured
in replicate wells containing radiolabeled targets in the absence of effector cells, and maximum lysis is
measured in replicate wells containing detergent. Radioactivity of the supernatants is measured using
scintillation counting. Specimens with HIV-1-specific lysis exceeding 15% [calculated as: (lysis minus
spontaneous lysis) /(maximum lysis minus spontaneous lysis) X 100] after correcting for lysis against
mock-infected target cells are considered positive.

CTL assays are extremely laborious due to the in vitro stimulation period and preparation of autologous
target cells required for these assays. In addition, they can only be reliably performed on freshly isolated,
not cryopreserved, PBMCs. For these reasons, a number of assays designed to measure other effector
functions of CD8+ T cells, notably cytokine secretion, have been developed.

Interferon gamma (IFN-gamma) ELISpot Assays

These assays measure the ability of T cells to secrete IFN-gamma in response to a specific antigen. They
are based on ELISA methodology. PBMCs from the individual being tested are added along with specific
HIV peptides to a 96-well nitrocellulose plate that has been coated with antibodies to IFN-gamma. Control
wells containing PBMCs and medium alone, without the HIV peptides, are assayed in parallel. During an
overnight incubation at 37°C HIV-specific T cells present among the PBMCs will be stimulated by their
cognate HIV peptide to secrete IFN-gamma. The plate-bound anti-IFN-gamma antibody captures this
IFN-gamma. The following day, the PBMCs are removed and the wells washed before addition of a
second anti-IFN-gamma antibody, which is labeled with biotin. This second antibody recognizes a
different epitope on IFN-gamma than the first coating antibody. A "sandwich" of IFN-gamma between two
anti-IFN-gamma antibodies is thus produced. Excess unbound antibody is removed by washing before
addition of a streptavidin-enzyme conjugate. Streptavidin binds with extraordinarily high avidity to the
biotin linked to the second anti-IFN-gamma antibody, creating a very stable biotin/streptavidin complex
bound to the enzyme (typically horseradish peroxidase [HRP]). The enzyme substrate is added, which
results in the formation of a colored product, or spot, where the streptavidin/biotin complex resides. Each
spot corresponds to an antigen-specific cell that has secreted IFN-g in response to its cognate HIV
peptide. The number of spots corresponds to the number of antigen-specific T cells present among the
PBMCs. HIV-specific CD8 T-cell responses can be distinguished from HIV-specific CD4 T-cell responses
in these assays by depletion of CD8+ T cells prior to the assay using magnetic beads labeled with anti-
CD8 antibodies. These CD8-depleted cells are tested in parallel with the undepleted PBMC. If responses
are seen in the PBMC wells but not the corresponding CD8-depleted wells, the responses are due to HIV-
specific CD8+ T cells. If responses persist in the CD8-depleted wells, these are due to HIV-specific CD4+
T cells.

Because antigen-specific effector T cells secrete a number of cytokines and chemokines in addition to
IFN-gamma that may play an antiviral role, measurement of IFN-gamma alone is not ideal, since it is
likely that discrete populations of cells secreting other cytokines and chemokines will be missed. ELISpot
assays have been developed to enumerate cells secreting a number of other cytokines, such as IL-2 or
tumor necrosis factor alpha (TNF-alpha).

ELISpot Movie
See the ELISpot movie, courtesy of Martin Horton. (Requires Macromedia Flash Player to view.)
Intracellular Cytokine Staining (ICS)
See Figure 2.

Intracellular cytokine staining (ICS) is a flow cytometry-based method for enumeration of antigen-specific,
cytokine-secreting T cells. PBMCs from the subject being tested (typically an infected or vaccinated
individual) are stimulated with HIV peptides in the presence of costimulatory antibodies against CD28 and
CD49d. Negative control stimulations with PBMCs and costimulatory antibodies but no HIV peptide, and
positive control stimulations with superantigens such as staphylococcal enterotoxin B (SEB) are set up in
parallel. Stimulations are allowed to proceed for 6 hours at 37°C in the presence of brefeldin A or
monesin, which inhibit protein transport through the golgi. Thus any cytokines induced by the HIV
peptides in HIV-specific T cells will be prevented from being secreted and will accumulate within the cells.
Responding cells can be visualized using fluorescently labeled antibodies that are specific for the
cytokines of interest. Responding cells can be further characterized by additional, lineage-specific
antibody markers (eg, CD3 and CD8 antibodies for CD8+ T cells). Once fluorescently labeled, the cells
are analyzed using a flow cytometer, which possesses lasers and filters to excite specific fluorescent
antibodies and measure the released light, permitting enumeration of the responding T cells.

ICS and ELISpot both measure the same effector function but have distinct advantages. In general,
ELISpot requires fewer cells and is less technically demanding than ICS. ICS, however, allows precise
immunophenotyping using lineage-specific markers to identify the responding cell population or
populations. A major advantage of both ICS and ELISpot assays is that, unlike chromium release assays,
they can be performed on cryopreserved cells.

MHC Tetramer Binding Assays

Whereas the cellular assays mentioned thus far measure effector function, tetramers measure the
absolute number of cells that recognize a particular epitope, without providing any information regarding
the functionality of these cells.(15,16) HIV-infected cells digest HIV proteins into short peptidic fragments
(epitopes), which are bound to MHC class I molecules and displayed at the cell surface. T cells recognize
their targets through the interaction of their T-cell receptors (TCR) with these MHC/HIV epitope
complexes. MHC tetramers are reagents consisting of four MHC class I molecules bound to the HIV
peptide of interest, linked together by a fluorescently labeled streptavidin molecule. These reagents will
bind specifically to the TCR of all T cells that recognize that particular MHC/peptide complex. This is a
very powerful tool for precise, easy, and rapid enumeration of HIV-specific T cells.

Tetramer technology is limited by several factors. The exact HIV peptide and its restricting MHC class I
molecule must be known in order to make the tetramers. Since there are hundreds of different HIV
epitopes whose restricting MHC molecules are not defined, this constitutes a major limitation.
Furthermore, although MHC class II tetramers are now available for the detection of specific CD4+ T
cells, these are even more limited than the class I tetramers by the lack of defined epitopes and known
restricting MHC class II molecules. In addition, tetramer staining gives no information regarding the
functionality of the stained T cells.

Lymphoproliferative Assays
See Figure 3.

LPAs measure the ability of HIV-specific CD4+ T cells to proliferate in response to their cognate antigen.
LPAs are generally performed by incubation of PBMCs with HIV proteins for 6 days at 37°C. The proteins
are endocytosed, processed, and presented to HIV-specific CD4+ T cells by macrophages present
among the PBMCs. These HIV-specific T cells will start to proliferate in response to activation. After 5
days, thymidine radiolabeled with 3H is added to the culture wells. The proliferating cells take up the
radioactive thymidine and incorporate it into newly synthesized DNA. The following day the cells are
harvested onto fiber filter mats and the amount of radioactivity is measured. The amount of radioactivity is
directly proportional to the amount of proliferation, and therefore, to the number of HIV-specific CD4+ T
cells present. LPAs generally do not show proliferation of CD8+ T cells because protein antigens, after
endocytosis by macrophages, are processed through the class II antigen-processing pathway. The
resulting peptides are presented on MHC class II molecules for recognition by CD4+ T cells.
Measurements are normally recorded as stimulation indices (SI). The stimulation index is the counts per
minute (cpm) of radioactivity measured in the wells containing HIV protein divided by the cpm measured
in control wells containing PBMC but no HIV protein. An SI of 5.0 (ie, a 5-fold increase over background)
is considered positive.
Introduction and History
Widespread awareness of HIV disease began with a brief report in 1981, published in the Morbidity and
Mortality Weekly Report, of a rare pneumonia caused by Pneumocystis carinii (now known as P jiroveci)
as well as other unusual infections in 5 young homosexual men in Los Angeles.(1) Awareness that a
significant epidemic was developing grew as case reports mounted and similar immune deficiency
syndromes were described in New York, California,(2,3) and elsewhere among homosexual men,
intravenous drug users, Haitians,(4) hemophiliacs,(5) recipients of blood transfusions,(6) infants,(7)
female sexual partners of infected men,(8,9) prisoners,(10) and Africans.(11) As researchers began to
describe the epidemiology and risk factors in a systematic way, many theories emerged regarding the
cause of the mysterious disease. An infectious agent was postulated, and, in 1983, a novel human
retrovirus was isolated as the putative etiologic agent.(12-14) That virus was eventually named human
immunodeficiency virus, or HIV.(15) Despite dramatic advances in basic virology and clinical
management, HIV infection has developed into a worldwide pandemic, with tens of millions of individuals
infected by the virus and many millions more affected by it. Clinicians treating HIV are challenged by a
clinically complex illness with relatively limited resources for treatment in most settings.

By 1985, serologic assays had been developed to test for HIV infection in asymptomatic persons, to
identify new infections by seroconversion, and to screen blood donations.(16) Early trials of antiviral
treatments for HIV and immune modulators were fraught with disappointment.(17-22) In 1987, zidovudine
(AZT, or azidothymidine) became the first drug approved by the U.S. Food and Drug Administration (FDA)
for the treatment of AIDS.(23) Early excitement over the life-extending effects of the drug soon waned, as
patients treated with this single-drug therapy began to experience disease progression leading in most
cases, to death. However, understanding of the epidemiology, treatment, and prophylaxis of opportunistic
infections (OIs) associated with HIV-induced immune deficiency led to significant life-saving advances,
particularly in the areas of infection with Pneumocystis jiroveci and Mycobacterium avium complex (MAC).
(24,25)

The introduction of protease inhibitors (PIs) in the mid-1990s revolutionized the treatment of HIV.(26)
Effective combination antiretroviral therapy (ART) became the standard of care in the United States and
Western Europe. Very soon thereafter, countries in which effective ART was available began to note
sharply declining morbidity and mortality associated with HIV infection.(27)

Studies of patients receiving the new therapies shed light on HIV pathogenesis. Patients treated with
potent ART showed precipitous decreases in the amount of HIV RNA circulating in their serum, indicating
interference with HIV replication (which, unimpeded, can produce more than 10 billion viral particles per
day). Additionally, after successful inhibition of viral replication, CD4 T-cell counts began to increase in
treated individuals, demonstrating the regenerative capacity of the damaged immune system.(28)
Corroborating this understanding of the dynamic interaction between viral replication and the host
immune system, studies began to show the value of HIV RNA measurement (viral load) as both a
predictor of disease progression and a measure of treatment success.(29-33)

Potent therapy was not without complications, however; and the dogma of the late 1990s, "hit early, hit
hard," (34) became balanced by realization that long-term medication toxicity was likely among individuals
who were now living longer, healthier lives with HIV infection. Once again, the paradigm of HIV treatment
underwent revision, and treatment was now recommended primarily for individuals with more advanced
disease.(35)

This chapter provides a general overview of issues relevant to clinical practitioners. More extensive
discussions on the topics covered here are available in other chapters of the HIV InSite Knowledge Base.

Basics of HIV Virology and Immunology

(See chapters "Molecular Insights Into HIV Biology" and "Structure, Expression, and Regulation of the
HIV Genome")
Virology
HIV-1 and the less common HIV-2 belong to the family of retroviruses. HIV-1 contains a single-stranded
RNA genome that is 9 kilobases in length and contains 9 genes that encode 15 different proteins.(36,37)
The major viral proteins (some of which contain >1 protein subunit) are classified as structural proteins
(Gag, Pol, and Env), regulatory proteins (Tat and Rev), and accessory proteins (Vpu, Vpr, Vif, and Nef).

Three major classes of HIV-1 have emerged: M (main), N (new), and O (outlier). Among M group viruses,
which account for >90% of HIV infections worldwide, there are 9 subtypes, called clades, designated by
the letters A-D, F-H, J, and K, as well as many recombinant forms.(38,39) Variation between one clade
and another in the amino acid sequences of the envelope protein may exceed 30%. Clade B, the most
common subtype in the Americas and Western Europe, differs considerably from those clades found in
Asia and Africa, where the majority of HIV-infected individuals reside (see Figure 1). Viral diversity is
greatest in sub-Saharan Africa. To date, most HIV drug development has targeted clade B. As HIV
treatment is extended into regions where non-B clades predominate, issues of differential drug response,
drug mutation patterns, and reliability of viral testing (ie, viral loads and resistance testing) may emerge.
(40,41) Sequence diversity among various clades also needs to be considered in vaccine development,
as most HIV-specific neutralizing antibodies (42) and some cytotoxic T-lymphocyte (CTL) responses (43)
are type specific.

HIV infection of a host cell begins with the binding of the virus particle (virion) to the host cell. This
process is initiated when the surface envelope protein (Env, which consists of 3 copies each of the 2
subunit proteins gp120 and gp41) engages its primary receptor, the CD4 molecule on the surface of the
target cell.(44) Initial binding to CD4 exposes another portion of the Env trimer, which then binds to a
coreceptor, usually the chemokine receptor CXCR4 (in the case of T-cell-tropic, or syncytium-inducing
strains of HIV) or the chemokine receptor CCR5 (in the case of macrophage-tropic, or nonsyncytium-
inducing strains).(45) This coreceptor binding causes the gp41 trimer portion of the envelope molecule to
spring open and "harpoon" the lipid bilayer of the target cell membrane. The "hairpin" domains of gp41
then fold together to pull the virus and host cell membranes together, allowing fusion to occur.(46) The
viral contents, including copies of the viral genetic material and the Pol protein (reverse transcriptase, or
RT) thus enter the cytoplasm of the host cell. Reverse transcription, that is, the copying of the viral
genetic material from RNA into DNA can then occur.

The preintegration complex (PIC), composed of the copied DNA (cDNA) and a number of viral and host
proteins, then enters the cell nucleus, where the viral enzyme integrase mediates the insertion of the viral
cDNA into the host chromosomal DNA.(47) The resulting integrated DNA virus (also called a provirus, to
distinguish it from the virion form) may remain latent for hours to years before becoming active through
transcription (copying of DNA into RNA).(48) Transcription of the viral genome is under complex control of
a number of proteins, including Tat and cellular DNA transcription factors.(49) Transport of the transcribed
viral RNA out of the nucleus also depends on a number of host and viral factors, including Rev.(50) The
transcribed viral RNA may be transported out of the nucleus in its full-length form to serve as genetic
material for new virions, or it may be partially or fully spliced. The unspliced, partially spliced, and fully
spliced versions of viral RNA direct the synthesis of different viral proteins by the cell ribosomes. New
viral particles are assembled at the plasma membrane and incorporate Gag subunits, Pol, Nef, Env, Vpr,
and viral genomic RNA.(51) The HIV viral protease enzyme acts following virion assembly to cleave viral
proteins into functional structural and enzymatic components. Gag then functions in the budding of
mature virions from the plasma membrane.(52) The Nef protein acts on the cellular environment to
promote replication by inhibiting the host immunologic response to HIV (53-55) and inhibiting death of
infected cells by apoptosis.(56)

Current HIV therapies inhibit the viral replication process at the binding and entry stage (fusion inhibitors),
the reverse transcription stage (nucleoside and nonnucleoside reverse transcriptase inhibitors [NRTIs and
NNRTIs, respectively]), or the protein cleavage stage (PIs). Inhibitors of coreceptor binding, integration,
and maturation are in clinical trials.

Immunology
Individuals infected with HIV show both cellular and humoral (antibody) immune responses to the virus,
but these responses are unable to prevent the ultimate progression of disease in the great majority of
infected individuals. Cellular responses are mediated by CTLs (CD8 cells) and helper T lymphocytes
(CD4 cells). CTLs inhibit HIV replication both directly, by recognizing and killing infected cells, and
indirectly, by producing soluble chemokine antiviral factors.(57,58)

CTL-mediated killing of virally infected host cells occurs through direct contact, whereby the T-cell
receptor on the surface of the CTL recognizes a fragment (epitope) of an HIV protein bound to a major
histocompatibility complex (MHC) class I molecule on the surface of the infected host cell. After this
interaction, the CTL releases enzymes that kill the infected cell. CTL responses directed against certain
epitopes of the Gag protein have been associated with slower HIV disease progression than CTL
responses against other epitopes.(59) CTLs also exert effects through soluble factors such as RANTES,
macrophage inflammatory protein (MIP)-1-alpha, and MIP-1-beta, which inhibit HIV from infecting new
cells by blocking HIV coreceptors.(60)

CD4 responses to HIV are important in viral control, and strong HIV-specific CD4 responses are
associated with lower HIV viral loads.(61) CD4 cells respond to HIV antigens presented in conjunction
with MHC class II molecules on the surface of infected cells. The fact that HIV infects CD4 cells
themselves is an evolutionary strategy with a number of consequences. Because productive HIV infection
occurs in activated CD4 cells, infection and killing of CD4 cells that are responding to HIV infection itself
may cause a selective decrease in the number of HIV-specific CD4 cells. (HIV can also exist in
nonactivated CD4 cells in a preintegrated form, which can become integrated if activation occurs within a
few days.[62]) Additionally, as some of the activated, infected CD4 cells differentiate into resting memory
CD4 cells, they may carry copies of the HIV genome in a postintegrated form that can persist for decades.
(63) Current antiretroviral medications cannot efficiently eliminate the virus from cells in the resting state,
leading to persistence of infection even in the presence of suppressive therapy.(63) Moreover, HIV
continues to evolve under the selection pressure of the immune response that occurs in each infected
individual, and mutations in the viral epitopes recognized by the immune system may enable the virus to
escape the control of even broad and robust CD4 and CD8 HIV-specific responses.(64)

Depletion of CD4 lymphocytes is the hallmark of HIV infection, and predicts an individual's risk for
infection with opportunistic pathogens as well as other complications of HIV disease. Evidence has shown
that both increased peripheral destruction and decreased production of CD4 cells likely play a role in this
decline.(65-69)

Humoral immunity appears to be less effective in controlling viremia than cellular responses, as HIV is
remarkably effective at evading host antibody responses, and broadly neutralizing antibodies are rare.
(70,71) The difficulty in eliciting broadly neutralizing antibody responses against HIV has posed a
particularly difficult challenge to the development of a protective HIV vaccine.

Epidemiology
There are currently approximately 900,000 persons living with HIV/AIDS in the United States, 180,000
(20%) of whom are women and 10,000 (1.1%) of whom are children.(72) Young men who have sex with
men (MSM) remain heavily affected. In a sample of 15- to 22-year-old MSM in 7 urban areas, 7% were
already infected with HIV. Within this group, there were higher percentages of HIV infection among
African Americans (14%) and Hispanics (7%) than whites (3%). In 2000 in the United States, over half of
reported HIV infections among males aged 13-24 years were attributed to male-to-male sexual contact.
Ethnic and racial minorities make up a disproportionate number of new AIDS cases in the United States.
In 2000, the incidence rate among African Americans was 58.1 per 100,000 population, among Hispanics,
22.5, and among whites, 6.6.(73)

Worldwide, UNAIDS estimates that 40,000,000 persons are living with HIV/AIDS, 18,500,000 (44%) of
whom are women, and 3,000,000 (7.1%) of whom are children (72,74). The most heavily affected area of
the world is sub-Saharan Africa, with almost 30,000,000 people infected with HIV.(75) In countries with
the highest prevalence such as Botswana and Zimbabwe, rates of HIV infection may exceed 30% in the
general population and may be >50% among selected groups, including pregnant women, male patients
at sexually transmitted infection clinics, and female sex workers.(75) Southeast Asia is estimated to have
6,000,000 infections, and China and the former Soviet Union are estimated to have more than 1,000,000
infections each. Infection rates are increasing in China, India, and Eastern Europe, fueled by high rates of
intravenous drug use, increasing prevalence of other sexually transmitted diseases, and public health
systems that are poorly prepared to prevent the spread of HIV.(76,77)

There were 2,400,000 deaths from AIDS in 2001, and some 14,000,000 children orphaned by the disease
are living in the world.(78)

Transmission and Risk Factors

The primary method of spread of HIV infection worldwide is through sexual exposure. In the United States
and Europe, acquisition of the virus through homosexual contact remains important, and there is some
evidence of increasing incidence of infection among young gay men and ethnic minorities.(79) MSM,
however, now account for <50% of new infections in the United States.(79) In the areas of highest HIV
prevalence globally, heterosexual intercourse is the primary mode of transmission, accounting for
approximately 70% of the overall sexual transmission.(80)

HIV has been isolated from blood, seminal fluid, pre-ejaculate, vaginal secretions, cerebrospinal fluid,
saliva, tears, and breast milk of infected individuals.(81-84) HIV-1 DNA sequences have also been
detected in pre-ejaculatory fluid.(85) In genital fluids, HIV may be found in both cell-free and cell-
associated compartments, but it is unknown which is responsible for productive infection.(86) Viral
concentrations in tears and saliva are comparatively low, and there are substances in saliva that appear
to inhibit infectivity. No cases of HIV infection have been documented to arise from contact with
nonbloody saliva or tears.

Transmission of HIV occurs more frequently through penile-anal intercourse and penile-vaginal
intercourse than through fellatio, although clear cases of transmission through oral sex exist.(87) Female-
to-female HIV transmission has been reported, but is rare.(88) In a meta-analysis, the overall efficacy of
condoms in reducing HIV transmission was 69%.(89)

Sexual activity that is associated with exposure to infected blood increases the risk of transmission, as
does the presence of genital ulcers.(90-92) Serum HIV viral load is strongly associated with heterosexual
transmission between HIV-serodiscordant African sexual partners, where transmission was noted to be
rare at viral loads <1,500 copies/mL.(93) The effect of viral load reduction with ART on HIV transmission
is being investigated. Intervention with antiretroviral medications soon after high-risk sexual exposures
has been proven to be safe and may be effective in preventing transmission of HIV (as discussed in the
chapter "Prophylaxis Following Nonoccupational Exposure to HIV").(94)

Nonsexual HIV transmission can occur through transfusion with contaminated blood products, injection
drug use, occupational exposure, or accidental needlesticks. The risk from occupational needlesticks to
health care workers from known HIV-positive source patients in case series performed prior to the
availability of potent ART was found to be 0.33-0.5%.(95,96) Factors increasing the risk of HIV acquisition
from an occupational needlestick include deep injury, injury with a visibly bloody device, or injury with a
device that had been previously used in the source patient's vein or artery.(96) Postexposure prophylaxis
(PEP) has been associated with a reduction of HIV transmission after occupational needlestick events of
approximately 80%.(96,97)

HIV transmission through transfusion of contaminated blood products was recognized early in the
epidemic.(6) With current testing methods, the risk of acquiring HIV from a unit of transfused blood in the
United States is 1 in 676,000,(98) but is significantly higher in many developing countries.

In the absence of interventions, mother-to-child transmission occurs in approximately 25% of live births to
HIV-infected mothers.(99) Various regimens of antiretrovirals can reduce the rate of perinatal
transmission by 50% or more.(99-102,103). Breast-feeding is also a risk factor for HIV transmission.
Approximately one-third of cases of mother-to-child transmission result from breast-feeding, and the risk
increases with the duration of breast-feeding.(104) Thus, interventions to prevent mother-to-child
transmission at delivery may be largely negated if mothers are not provided with safe alternatives to
breast-feeding.

Classification of HIV Disease

HIV damages the immune system, leaving the infected person vulnerable to a variety of infections (called
"opportunistic" infections to indicate that they arise in the setting of immune impairment). The effect of HIV
on the immune system is monitored by measuring the CD4 (helper) lymphocyte count in the blood. A
normal CD4 count (between approximately 600 and 1,200 cells/µL) indicates that the immune system has
not undergone sufficient damage to put the individual at risk for opportunistic illness. However, recent
studies have demonstrated that even those with CD4 counts above 350-500 cells/µL are at elevated risk
for a number of conditions that were not previously recognized as related to HIV infection. These include
cardiovascular disease, kidney and liver disease, malignancies, and neurocognitive decline. As a result,
experts have increasingly recommended initiation of treatment at higher CD4 counts. CD4 counts <500
cells/µL indicate that impairment of immune function is present, and are an indication for ART. CD4
counts <200 cells/µL indicate imminent risk of serious OIs or other complications of HIV disease, and
prompt treatment is recommended.(35) [Editor's note: This paragraph was updated on December 8,
2009, to reflect new guidelines]

Untreated HIV disease is chronic and progressive. Primary HIV infection, often marked by a
mononucleosis-like acute viral syndrome, is followed by a period of clinical latency typically lasting several
years, during which high levels of viral replication and CD4 cell turnover lead to progressive immune
dysfunction, eventually resulting in clinical disease progression. The distinction between "HIV infection"
and "AIDS" is important, as it has clinical and prognostic implications, as well as utility in research.

The U.S. Centers for Disease Control and Prevention (CDC) definition of AIDS, initially published in 1986
and revised in 1993, is based on certain clinical conditions, infections, and malignancies associated with
HIV infection (see Table 1). Additionally, AIDS may be defined by a CD4 count of <200 cells/µL or <14%
of all lymphocytes, even in the absence of the listed conditions (see Table 2).(105)

The World Health Organization has developed a clinical staging system for HIV infection (see Table 3).
(106) This system relies more heavily on clinical rather than laboratory evaluation, and has been used
widely in resource-constrained areas where laboratory testing is not widely available.

Natural History of Untreated HIV Infection

Primary HIV Infection

Primary HIV infection is defined as the time period from initial infection with HIV to the development of an
antibody response detectable by standard tests. Data from careful prospective evaluations of populations
at risk for HIV infection demonstrate that up to 87% of individuals who acquire HIV may experience some
symptoms of primary HIV infection.(107) The acute viral syndrome of primary HIV infection (sometimes
referred to as "seroconversion illness") was first defined in 1985, with symptoms resembling those of
mononucleosis appearing within days to weeks following exposure to HIV.(108,109). Symptoms may be
mild or severe and may last from a few days to several weeks, with the average duration being 14 days.
The most common presenting symptom is fever, seen in over 75% of patients.(110) Other commonly
reported symptoms include fatigue, lymphadenopathy, headache, and rash. The rash, which is present in
40-80% of cases, may be evanescent, is typically maculopapular in character, and typically involves the
trunk.(110) Evaluation of cohorts from Kenya (111) and India (112) found more frequent reports of joint
pains, night sweats, and mucosal candidiasis and less frequent rash and pharyngitis in these study
populations. A more severe clinical syndrome in primary HIV infection has been associated with a more
rapid subsequent clinical course of HIV disease.(113)

The nonspecific symptoms of primary HIV infection may make diagnosis a challenge. In a study of high-
risk individuals presenting with symptoms consistent with primary HIV infection, only 25% were diagnosed
during their initial presentation.(107)

Diagnosis of HIV during the acute seroconversion phase requires not only high clinical suspicion but also
an understanding of appropriate testing strategies. Routine HIV antibody testing may be negative for
several weeks or even months after exposure in the so-called "window period."(114) During primary
infection with HIV, plasma viral load often reaches very high levels in the range of millions of RNA
copies/mL.(115,116) Thus, for individuals in whom primary HIV infection is clinically suspected, HIV RNA
assays, which have a sensitivity approaching 100% and specificity of 97.4% in this setting,(117) should
be included in the diagnostic evaluation. HIV RNA tests are not licensed for the diagnosis of HIV infection,
and positive RNA tests during acute infection should be confirmed by documentation of subsequent HIV
antibody conversion. The high levels of viremia seen in primary infection do not persist, however,(116)
providing evidence of a host immune response capable of bringing the infection under some degree of
control, at least in the short term.

During primary HIV infection, HIV-specific CD8 cells undergo a marked clonal expansion and express
high levels of activation markers such as CD38 and human leukocyte antigen (HLA)-DR.(118) The
breadth and strength of this CTL response correlate positively with the degree of viral control and
inversely with the rapidity of clinical progression.(119-122)

CD4 counts and CD4 function may decline during primary HIV infection, occasionally to levels that allow
OIs to develop.(123-125) Absolute CD4 count often rebounds after the primary infection, but may not
return to a normal baseline. In patients with clinical progression of HIV disease, CD4 responses against
HIV itself appear to remain particularly impaired following primary infection.(126)

After the initial reduction of viremia, a viral "set-point" is established in each infected individual. The
magnitude of this set-point correlates with the rate of progression of HIV disease (see Table 5).
(31,127,128) Studies of individuals during primary HIV infection have raised the question of whether the
set-point might be reduced by early treatment.(129) Although early antiretroviral therapy may preserve
immune function,(130) rapid control of viremia may also inhibit the full development of a mature
immunologic response.(131) Carefully supervised interruptions of antiretroviral treatment after initial
control during acute infection may permit the development of an effective immune response in the short
term,(129) but long-term follow-up suggests that increases in viral load and emergence of drug resistance
may occur in such patients.(132) The best strategy for treatment of acute HIV infection remains a matter
of investigation.

Chronic HIV Infection

After the period of acute HIV infection--during which CD4 counts and viral load change dramatically--a
relative equilibrium between viral replication and the host immune response is reached, and individuals
may have little or no clinical manifestations of HIV infection. This time between initial infection and the
development of AIDS may be long, averaging 10 years, even in the absence of treatment.(133)

Despite the relative clinical latency of this stage of HIV infection, viral replication and CD4 cell turnover
remain active, with millions of CD4 cells and billions of virions produced and destroyed each day.(28)
During this period, most infected individuals will have progressive loss of CD4 lymphocytes and
perturbation of immune function.(134-137) On average, CD4 counts will drop by 50-90 cells/µL per year in
asymptomatic individuals, usually with an acceleration of this rate over time.(138)

The rate of progression of infection may vary considerably. In adults, progression from infection to clinical
AIDS is rare in the first 2 years of infection; however, reports describe rapid disease progression in infants
infected by blood transfusion.(139) In a well-characterized cohort of HIV seroconverters who were
identified in a retrospective analysis of stored serum samples from hepatitis B vaccine trials in the 1970s,
87% of infected individuals had developed AIDS by 17 years postseroconversion. Twelve percent
maintained a CD4 count >500 cells/µL at 10 years, but only 3% maintained a CD4 count >500 cells/µL at
16 years after seroconversion.(140)

During chronic HIV infection, HIV RNA levels in plasma correlate with the rate of CD4 decline, with higher
plasma viral loads predicting more rapid progression to AIDS and death.(141,142) An undetectable HIV
RNA level in peripheral blood is associated with stable CD4 lymphocyte counts, and increases in HIV
RNA correlate with more rapid rates of CD4 cell decline.(143,144)

The analogy of a train on a track (attributed to John Coffin of Tufts University, circa 1996) has been
helpful in illustrating the independent contributions of CD4 count and HIV viral load in an individual
person. If the infected individual is imagined as being on that train traveling toward a clinical event--such
as acquiring an OI or dying from AIDS--the CD4 count provides information on the distance of the train
from that destination, whereas the viral load provides information on the speed of the train in reaching the
destination (see Figure 2).

Clinical AIDS
According to CDC criteria (see Table 1 and Table 2), AIDS is defined by either diagnosis of one of the
AIDS-defining conditions, or by measurement of CD4 levels <200 cells/µL. Progression to AIDS from time
of infection occurs, on average, 2 years earlier when defined by laboratory criteria (CD4 levels <200
cells/µL) compared to clinical criteria (development of an opportunistic illness).(145,146) Survival time
from the development of AIDS varies according to the AIDS-defining event. In the Multicenter Hemophilia
Cohort Study, median survival after a single AIDS-defining condition ranged from 3 to 51 months for the
10 most common conditions.(147) The mean survival time after diagnosis of AIDS in the United States
prior to the availability of antiretroviral treatment was 10-12 months.(147)

Special Considerations in Disease Progression

Host Factors
A number of host factors influence HIV disease progression. Individuals who acquire HIV at an older age
tend to have more rapid disease progression (134) and shorter survival times.(148) Variation in HIV
coreceptor molecules, notably CCR5, influences both HIV susceptibility and disease progression. A
mutant allele of CCR5 with a 32-base-pair deletion, CCR5-delta-32, is frequent in populations of
European origin (10-15% of Caucasians are heterozygous, and 1% are homozygous), and encodes a
nonfunctional truncated protein that is not transported to the cell surface. Homozygotes for the delta-32
allele exhibit a strong, although not complete, resistance to HIV infection, whereas heterozygotes display
nearly normal rates of infection, but delayed progression to AIDS.(149)

Genetic differences in HLA alleles have also been shown to influence HIV disease susceptibility (150,151)
and disease progression.(152-157) The class I alleles B35 and Cw4 have been associated with
accelerated progression of disease,(158-160) as has general HLA homozygosity.(161) Because HLA
class I alleles determine which viral epitopes can be presented to CD8 cells, greater diversity of HLA
(heterozygosity) in an individual may reflect greater options for effective cell-mediated immunity to HIV.
Conversely, HLA B27 and B57 have been associated with long-term nonprogression of HIV disease.(153)
In particular, HLA B*5701 has been found to be highly overrepresented in long-term nonprogressors.(157)

Behavioral or psychological host factors may also influence HIV disease progression. More rapid HIV
disease progression has been reported with unprotected anal intercourse,(162) smoking,(163) poor
nutrition,(164) and depression (165); however, not all studies confirm these findings. Drug use might be
expected to influence HIV disease progression, but studies of that question have produced mixed results.
(162,166) Additionally, differences in disease course based on the route of HIV transmission have been
difficult to prove.(167,168)

Viral Factors
HIV virions infect human cells by first binding to the CD4 receptor on the cell surface. This alone is not
sufficient for the virus to enter the host cell; binding to an additional coreceptor is also required.
Macrophage- or M-tropic viruses preferentially infect monocytes and macrophages, using the cell surface
protein CCR5 (R5) as the preferred coreceptor to enter cells, and produce a nonsyncytium-inducing (NSI)
phenotype in cell culture. Conversely, thymocyte- or T-tropic viruses preferentially infect T cells, use
CXCR4 (X4) as the preferred coreceptor to enter cells, and produce a syncytium-inducing (SI) phenotype
in cell culture.(169) Dual-tropic viruses, which may use either CCR5 or CXCR4 coreceptors, also exist. M-
tropic viruses are frequently found in early HIV infection, and a switch to T-tropic strains in the course of
disease is associated with rapid CD4 cell depletion.(170-172)

The concept of viral "fitness" refers to the pathogenicity of certain strains of HIV. HIV replicative capacity
(RC) has been studied as a component of viral fitness. RC is a measure of the ability of a given virus to
replicate successfully in a given environment.(173-177) During the course of drug treatment, mutations
arise in the HIV reverse transcriptase and protease enzymes that make the virus resistant to particular
drugs, thus conferring a selective advantage to that subpopulation that arises from a resistant variant.
(178-180) Several of these mutations have been shown to cause a reduction in RC in the absence of drug
when compared to wild-type virus.(173,174,176) Further accumulation of mutations over time under drug
selection pressure may increase the "fitness" of the drug-resistant variant by further increasing phenotypic
resistance,(178,181,182) or by increasing RC of the resistant virus.(174,183) The role of viral fitness on
individual disease progression is just beginning to be understood.

Other viral factors may be important as well. For example, faster rates of disease progression have been
observed in Ugandan individuals infected with subtype D compared with subtype A isolates.(184)
Additionally, rare individuals who are infected with variant HIV strains, particularly those with a defective
nef gene product, may experience slower disease progression.(185)

Coinfections
The development of OIs during HIV disease not only indicates the degree of immunosuppression, but
may also influence disease progression itself. When stratified by CD4 counts, patients with prior histories
of OIs have higher mortality rates than those without prior histories of OIs.(186)

Hepatitis C coinfection is common in HIV-infected patients, present in up to 40-50% of all patients in

urban setting and in 90% of intravenous drug users.(187) HIV clearly leads to more rapid HCV disease
progression; however, the effect of HCV infection on HIV progression is less clear. In a study of the Swiss
HIV Cohort, HCV coinfection was associated with poorer CD4 responses to ART, development of new
AIDS-defining events, and increased mortality (188); however, other authors have not found these
associations.(189)

Long-Term Nonprogressors
A small subset of individuals infected with HIV--probably <5%--remain free of symptoms, achieve good
control of HIV viral replication, and maintain high CD4 counts in the absence of antiretroviral medications
over many years of infection, although some individuals initially identified as long-term nonprogressors
(LTNPs) have experienced disease progression over time.(140) In general, LTNPs appear to have strong
cellular immune responses to a variety of HIV antigens.(126,190,191)
Laboratory Testing

HIV Antibody Testing

(See chapter "HIV Antibody Assays")

HIV infection is usually diagnosed by testing serum for antibodies to HIV using a commercially available
enzyme-linked immunosorbent assay (ELISA or EIA). Because the ELISA test is not entirely specific,
positive results are confirmed with a Western blot assay, which identifies antibodies to specific
components of HIV.(192,193). The 2-step process may mean that a patient must wait for a week or more
to receive test results.

ELISA is quite sensitive in chronic HIV infection (although decline in antibody responses have been
reported in advanced AIDS), but because antibody production does not occur immediately upon infection,
an infected individual may test ELISA negative during a "window period" that varies in length from a few
weeks to a few months after infection, depending on the individual case and assay used. Despite
negative antibody testing during this window period, an individual may have high viral load and be at high
risk of transmitting infection.

Newer methodologies allow antibody testing on saliva (194,195) and urine (195,196) specimens, although
positive results should be confirmed with serologic testing. Home testing methods are also available.(197)
Rapid HIV serum testing, with results available in 3-30 minutes, has shown 99-100% sensitivity and
specificity compared to ELISA when tested in clinical settings,(198) including in resource-poor settings
(199-201) and in pooled specimens.(202) In recent years, with the availability of rapid tests such as
OraQuick (Abbott Laboratories, Abbott Park, IL) and Reveal (MedMira, Halifax, Nova Scotia, Canada),
rapid testing protocols are being implemented in many countries, and will likely become commonplace.

"Detuned" Antibody Testing

By decreasing the sensitivity of ELISA assays, relatively recent infection (in which antibodies are present
in lower concentrations and bind to HIV less effectively) can be distinguished from established infection
(in which antibodies reach stable levels and have been selected for more avid binding to HIV). Soon after
infection (but after the window period) an individual will test positive on the standard ELISA, but negative
on the less-sensitive ("detuned") test. After maturation of the antibody response, both tests will give a
positive result. Such "sensitive/less sensitive" or detuned ELISA testing strategies can be used to identify
individuals who are in the early months of HIV infection and can help to identify incident infections in
epidemiologic studies.(203,204)

CD4 Testing
The CD4 cell count in blood correlates with the risk of OIs in HIV disease,(205,206) and is therefore a
useful marker for HIV disease staging. CD4 count is the main criterion for clinical decision making in
guidelines developed in the United States for the prophylaxis of OIs (24,207) and for HIV treatment.(208)

The CDC recommends CD4 testing every 3-6 months in all HIV-infected persons,(209) but different
intervals may be appropriate to the individual case. More than 1.6 million CD4 cell measurements are
performed annually by approximately 600 testing laboratories in the United States.(210)

Because CD4 cells are a subset of all T lymphocytes, which are in turn a subset of all white blood cells,
variations in CD4 count can occur in response to a variety of variables including concurrent infection,
medications, stress, malnutrition, vitamin deficiencies, and normal diurnal variation. Often, these variables
affect many subsets of lymphocytes and not exclusively CD4 cells; thus, the percentage of T lymphocytes
that are CD4 positive will remain relatively stable. On the contrary, the depletion of T lymphocytes in HIV
disease primarily affects CD4 cells, causing a relative CD4 cytopenia and a drop in the CD4 cell
percentage. Additionally, an inversion of the normal CD4/CD8 cell ratio, which is usually >1 in non-HIV-
infected individuals, may be seen with progressive CD4 cell depletion due to HIV. Thus, the CD4 percent
and the CD4/CD8 ratio may help the clinician determine if a change in absolute CD4 count is due to the
effects of HIV disease or to some other factor.

Until recently, most absolute CD4 cell counts were determined using 2 instruments, a hematology
analyzer and a flow cytometer (dual-platform technology [DPT]). The CD4 count produced from DPT is
the product of 3 laboratory measurements: the white blood cell count, the percentage of white blood cells
that are lymphocytes (differential), and the percentage of lymphocytes that are CD positive (determined
by flow cytometry). Single-platform technology (SPT) is designed to enable determinations of both
absolute and percentage lymphocyte subset values using a single tube.(211) SPT, introduced for clinical
application in 1996 is becoming the preferred method of CD4 count determination in a number of
laboratories.(212)

Both SPT and DPT flow cytometry technology for CD4 count determination require specialized equipment
and technician training. In resource-limited settings where CD4 count may be unavailable, the total
lymphocyte count (TLC), which can be determined simply and cheaply, may be used as a surrogate for
CD4 in determining stage of HIV infection.(213-215) For example, in a cohort of HIV-positive people in
south India, a TLC of <1,400 cells/µL has been shown to be a good predictor of a CD4 count <200
cells/µL and thus an appropriate surrogate marker for initiating cotrimoxazole prophylaxis.(215) TLC may
also have applications in monitoring response to antiretroviral therapy in place of or in conjunction with
CD4 count. In an analysis of patients initiating a triple antiretroviral drug regimen, an increase in TLC was
associated with an increase in CD4 count and a decrease in plasma viral load.(216)

HIV Viral Load Testing

(See chapter "HIV Viral Load Assays")

Three technologies exist to measure HIV viral load in serum: reverse transcription polymerase chain
reaction (RT-PCR), branched DNA (bDNA), and nucleic acid sequence-based amplification assay
(NASBA). The basic principles underlying these assays are similar--HIV is detected using DNA
sequences that bind specifically to those in the virus--but results may vary between tests. Whereas early
versions of the bDNA and RT-PCR techniques showed 2- to 2.5-fold differences in results, the version 3.0
bDNA assay and version 1.5 RT-PCR test yield values that are highly correlated (r = 0.96) and in good
agreement (92.7%).(217,218) Testing of non-clade B HIV-1 viruses using these methods may not yield
such highly correlated results.(219) Thus, it is advised that clinicians consistently use the same test when
possible to compare results over time.

Because viral load may vary by orders of magnitude, results of viral load testing are often expressed in
log units, where each increase of 1 log corresponds to a factor of 10. Thus, a viral load of 1,000 would be
3 log units, and the difference between a viral load of 1,000 and 10,000 would be 1 log unit. A change in
viral load of >0.5 log copies/mL (approximately 3-fold) exceeds assay and diurnal variations, and may be
considered to represent a true biological event, whereas a change of <0.5 log copies/mL cannot be
distinguished from random variability. Diurnal variation in stable HIV viral loads is approximately 0.4 log
copies/mL.(220) Acute intercurrent infection (221) or immunization (222,223) may also transiently
increase viral load.

HIV Antigen Testing

Assays for HIV antigens, notably the p24 antigen encoded by the gag gene, can be used to screen
donated blood products.(224) Measurement of p24 antigen may also be a less expensive yet effective
alternative to HIV RNA testing in monitoring response to treatment.(225) Testing for p24 may also be
used to diagnose early HIV infection, because this viral antigen can be detected in the blood of infected
individuals prior to the development of antibodies (seroconversion) detectable by ELISA or Western blot
tests. In identifying primary HIV infection, p24 is more specific (99% vs. 95-97%) but less sensitive (79%
vs. 100%) than HIV RNA determinations (either PCR or bDNA).(117)

HIV Resistance Testing

(See chapters "Assays for Antiretroviral Resistance" and "Genotypic Testing for HIV")

Resistance to antiretroviral drugs is unfortunately common in treated populations. Resistance testing can
be useful in determining which drugs not to use in a treatment-experienced patient whose viral load is
increasing despite therapy, or in a previously untreated individual who may have been infected with HIV
resistant to one or more drugs.
Two types of HIV resistance testing are available. Genotypic assays detect genetic mutations in the
coding regions of the protease and reverse transcriptase enzymes in HIV isolated from the patient. Using
the results, standardized algorithms are applied to predict resistance to various antiretrovirals. Phenotypic
assays are more similar to standard bacteriologic sensitivity assays in that they are performed by
culturing a fixed inoculum of HIV genetic material isolated from the patient with serial dilutions of
individual antiretroviral drugs.

Prospective trials of genotypic (226-228) and phenotypic (229) assays have shown benefit with each of
these assays in achieving virologic control in patients failing antiretroviral regimens. Additionally, with
increasing incidence of drug resistance in individuals recently infected with HIV,(230) resistance testing
during acute or early HIV infection may have important long-term clinical relevance. Resistance testing in
chronically HIV-infected individuals provides information only on resistance to the medications being
taken at the time of the test. In individuals who have changed or interrupted antiretroviral treatment, HIV
harboring resistance mutations selected by prior treatment may be "archived" as proviral DNA in long-
lived resting lymphocytes or macrophages, and may not be detected by resistance tests. Archived strains,
however, can be expected to return to dominance if selected by the drugs to which they possess
resistance. Results of resistance testing are therefore not a substitute for the patient's clinical
antiretroviral history, which must also be taken into account. Further, because many resistance mutations
tend to become outgrown by wild-type virus when the drug in question is no longer present to select for
the resistance mutation, resistance testing in chronically infected individuals who are not on ART at the
time of testing is unlikely to be of use and may provide misleading information.

In general, genotypic assays are more easily available, cheaper, and more rapidly performed than
phenotypic assays. A genotype result is more likely than a phenotype to detect resistance from a minor
variant or population mixture. Genotype testing identifies only the dominant strains representing >10-20%
of virus circulating in blood at the time of testing.(231,232). Genotypic assays require an HIV viral load of
>1,000 copies/mL to be reliable. Different laboratories use different algorithms for determining drug
resistance from a given genotype result, and those different algorithms often produce discordant results,
particularly for NRTIs.(233)

A phenotypic resistance test has the advantage that results are generated in a minimal inhibitory
concentration (MIC) format that is more familiar to many clinicians and also emphasizes the gradation of
resistance that often exists; however, the phenotypic threshold value that correlates with clinical
resistance is still debated for certain antiretrovirals. A phenotype gives results for one drug at a time and
is unable to predict the effects of combinations of drugs. Phenotypic resistance assays may be
particularly useful when combined with monitoring of drug levels in treating individuals with highly
resistant virus.

The "virtual phenotype" approach combines databases of matched genotypes and phenotypes from the
same viral isolates to predict the phenotypic susceptibility of viruses with known genotypic sequences.

Current guidelines in the United States recommend resistance testing in cases of acute or recent HIV
infection, for certain patients who have been infected as long as 2 years or more prior to initiating therapy,
in cases of antiretroviral failure, and during pregnancy.(234)

It is worth reemphasizing that, given the limitations of all currently available resistance assays to detect
archived mutations and minor variants, results of resistance testing must always be interpreted in the
context of prior antiretroviral history and previous resistance testing.

Therapeutic Drug Monitoring

The measurement of antiretroviral drug concentrations in patient serum may be used to predict toxicity,
(235) maximize efficacy,(236) assess effects of drug-drug interactions,(237,238) and provide evidence
regarding medication adherence.(239)

Therapeutic drug monitoring (TDM) requires proper timing of sampling relative to dosing and meals, and
sampling the appropriate body compartment. For PIs and NNRTIs, drug concentrations are measured in
the plasma compartment, whereas for nucleoside and nucleotide reverse transcriptase inhibitors,
measurement of intracellular metabolites is necessary.

In general, data on the efficacy of TDM in clinical practice are mixed. In certain circumstances, such as in
pregnant or pediatric patients, TDM may provide data on drug concentrations that have not otherwise
been well characterized, but data from large studies are lacking to support its routine use in clinical care.
(240)

Other HIV Testing Techniques

ELISA or HIV viral load testing of fluids other than blood (seminal and vaginal fluid, cerebrospinal fluid,
urine, and saliva) are currently available or under investigation. The clinical applications of some of these
methods are well proven (ie, diagnosis of HIV infection with saliva or urine ELISA testing [241]), whereas
for others it is less so (ie, serum viral load as a predictor of infectivity [242] or testing of semen for use in
in vitro fertilization [243]).

Various culture techniques are available to isolate HIV from patient specimens.(244) HIV can be
quantitated by determination of proviral DNA in peripheral blood mononuclear cells.(245) Proviral DNA
has been used to test babies for HIV infection who were born to HIV-positive mothers.(246)

Assays of HIV antigens, notably p24 antigen, can be used to screen donated blood products.(224) It may
also be an effective, inexpensive alternative to HIV RNA testing in monitoring response to treatment.(225)
Additionally, because it is a direct viral antigen, p24 can be detected in the blood of infected individuals
prior to the seroconversion detected by ELISA or Western blot tests. When comparing p24 antigenemia to
HIV RNA determinations (either PCR or bDNA) in identifying primary HIV infection, p24 is equally specific
(99% vs. 95-97%) but less sensitive (79% vs. 100%).(117)

Treatment of HIV Infection

Overview of Antiretroviral Medications

(See "Overview of Antiretroviral Drugs" )

Currently, all FDA-approved antiretroviral medications work by inhibiting 1 of 3 steps in the life cycle of
HIV:

1. Blocking the reverse transcriptase (RT) enzyme. The RT enzyme is used by the virus to convert
its RNA into DNA after the virus enters the cell but before it enters the nucleus. All nucleoside and
nucleotide analogues as well as NNRTIs function by interfering with the activity of this enzyme.
2. Blocking the protease enzyme. Protease inhibitors, as their name indicates, inhibit the action of
the HIV protease, namely, cleaving protein products of the viral structural genes into the
functional subunits needed to create new infectious virions.
3. Inhibiting fusion of the viral and host membranes. By attaching itself to the HIV envelope
glycoprotein gp41, fusion inhibitors prevent formation of the "hairpin" structure (see "Virology",
above) required for fusion of the HIV and host cell membranes, and thus prevent viral entry into
the host cell.

New antiretroviral medications that are in development include improved formulations of currently
approved drugs (to enhance bioavailability, increase half-life, or reduce adverse effects); new drugs in the
same classes as currently approved drugs (such as PIs or NNRTIs with fewer adverse effects or unique
resistance patterns); and drugs with novel mechanisms of action (eg, integrase inhibitors, entry inhibitors,
and HIV coreceptor blockers).

Initiating Treatment
Not all HIV-infected individuals require ART at a given point in time. For those whose CD4 count and
clinical assessment indicate a low risk of imminent disease progression, the potential adverse effects of
immediate treatment would be expected to outweigh any benefit. Others may have psychosocial barriers
to adherence that preclude effective ART, at least in the short term, until conditions related to these
issues can be improved (eg, through stable housing, substance abuse counseling, or treatment of
medical or psychiatric conditions). Because incomplete adherence can rapidly lead to lasting resistance
against available antiretrovirals, it is usually worthwhile to address issues that are likely to impair
adherence before starting ART. Still other individuals may opt for complimentary/alternative medicine
(CAM) for management of their disease. In one large population-based study, 3% of individuals with HIV
chose to use CAM as a substitute for standard therapy.(247) Unfortunately, there is no convincing
evidence that CAM is effective in improving clinical status or survival in HIV infection.

Individuals for whom initiation of ART is not indicated must be monitored closely for changes in immune
status (eg, CD4 counts) that might signal increased risk of OIs and thus trigger initiation of ART, OI
prophylaxis, or other intervention.(24,207) In general, determination of CD4 counts and viral loads should
be made every 3-6 months in individuals with stable infection. Measurements of CD4 count may be
transiently decreased and measurements of HIV viral load transiently increased during acute infection
with another pathogen (248-251) or immediately after vaccination,(252-255) and providers should avoid
measurement of these parameters in such situations.

The decision to initiate (or reinitiate) ART should be made with clear goals that address the concerns of
the patient as well as the provider. Active viral replication in the presence of antiretroviral drugs can be
expected to result in selection of drug-resistant virus leading ultimately to treatment failure. Therefore, any
antiretroviral regimen should ideally be prescribed with the intention of complete viral suppression. When
complete suppression is not possible, secondary goals of treatment may include partial virologic
suppression (which might be the best attainable goal if the virus is highly resistant to antiretrovirals),
immunologic reconstitution, or alleviation of symptoms (such as fever, night sweats, or weight loss).
Sufficient patient education on the antiretroviral regimen chosen--dosing schedule, meal restrictions,
anticipated adverse effects and adverse effect management--is also crucial to the success of any
treatment regimen. Individual adherence is one of the most challenging and most important aspects of
treatment success.(256,257)

In initiating or changing an antiretroviral regimen, patients and clinicians must be aware of the
development of potential drug adverse effects or toxicities--in both the short and long term (see Table 4).

Monitoring ART
The efficacy of an antiretroviral regimen should be monitored by regular determinations of HIV viral load
and CD4 count. The first indication of successful treatment is a decline in viral load. The decline in viral
load is biphasic.(258) The first (fast) decay phase occurs during the first 2 weeks of treatment for most
patients; a second (slow) phase of decay is evident thereafter. The first phase may result primarily from
antiviral effects of medications, whereas immune factors such as CTL antiviral activity may contribute to
the elimination of virally infected cells in the second phase.(259) The kinetics of response are variable
among individuals, and do not appear to be entirely determined by adherence or drug levels. Moreover,
although viral decay rates are not entirely predictive of subsequent virologic failure,(259) virologic
response at 4 weeks of treatment has been shown to correlate with response at 48 weeks.(260) As a rule
of thumb, in a successful regimen the viral load should decline by at least 1 log by 4 weeks after
treatment initiation; slower rates should prompt closer assessment of patient adherence, viral resistance,
and possible drug-drug interactions. Even a successful regimen, however, may take 4-6 months or even
longer to attain undetectable viral loads.

As viral load declines, the number of circulating CD4 cells begins to increase. Initial increases in CD4
count during the first 1-3 months of therapy are believed to be caused primarily by a redistribution of cells
trapped from lymphoid tissue.(261) The subsequent rate of improvement in CD4 levels is variable among
individuals, and gradual CD4 count increases may continue for many years with therapy that effectively
suppresses HIV replication.(262-264) Individuals with lower nadir CD4 counts may have a slower and
less complete CD4 recovery, whereas those who start therapy with higher CD4 counts and maintain
continued viral suppression with antiretroviral medications may reach CD4 counts similar to those of HIV-
uninfected individuals.(264)

As the immune system begins to reconstitute itself in the early phases of ART, it may mount vigorous
inflammatory responses against certain pathogens. The restoration of immune function may therefore
cause paradoxical worsening of certain coexisting OIs, a phenomenon known as "immune reconstitution
syndrome." Infections for which immune reconstitution syndrome has been reported include
pneumocystosis,(265) cryptococcosis,(266) cytomegalovirus (CMV) retinitis,(267) Mycobacterium avium
complex infection,(268) and tuberculosis.(269) Immune reconstitution syndrome generally presents as
inflammation of tissues involved in the infection (eg, marked lymphadenopathy in tuberculosis, potentially
sight-threatening inflammation of the vitreous in CMV retinitis), presumably reflecting localized antigen
load. The syndrome appears to occur most frequently in individuals with low CD4 counts and develops in
the initial weeks of ART. It may present a diagnostic challenge to the clinician, and should be evaluated
thoroughly to exclude other etiologies such as failure of therapy or other infections. Exact diagnostic
criteria and guidelines for management of immune reconstitution syndrome are being developed. No clear
guidelines for the management of immune reconstitution syndrome exist; however some approaches
include using systemic corticosteroid treatment, or stopping antiretrovirals in the most severe cases. For
more information on immune reconstitution inflammatory syndromes, see "Clinical Implications of Immune
Reconstitution in AIDS".
Changing ART
Antiretroviral regimens may fail to suppress viral replication for a number of reasons, including incomplete
adherence, poor absorption, toxicity leading to missed or lowered doses, pharmacokinetic interaction,
suboptimal antiviral potency, and preexisting drug resistance. Virologic failure has been reported in as
many as 63% of patients in population-based studies.(270,271) However, virologic outcomes may be
improving over time; in a recent large cohort study, 72% of subjects on therapy attained HIV RNA <500
copies/mL at 6 months.(272)

At the time of changing antiretroviral regimens, careful assessment of the reasons for changing should be
undertaken. Understanding the factors of adherence, toxicity, and resistance that prompted the switch will
aid in designing subsequent regimens and optimizing patient outcomes. Changes to regimens for reasons
of toxicity may be as simple as exchanging a new drug for the one that was causing the toxicity, provided
that virus resistant to the other drugs in the regimen has not emerged. Patients who have failed multiple
regimens or who have highly drug-resistant virus may require a more complicated "salvage" regimen.
Such regimens may contain more drugs, may involve more complicated dosing schedules and may
include experimental agents. Complete viral suppression may not be an achievable goal in some patients,
decisions to change or continue treatment should take into account immunologic and clinical stability,
(217) as well as the observation that selective pressure by antiretrovirals can maintain drug-resistant
mutations that impair viral RC.(273,274)

Treatment Interruption
Situations arise when interruption of therapy is necessary in a given patient--toxicity, severe illness, and
other circumstances making adherence impossible. In these situations, clinicians and patients should be
aware of the recommendations to stop all antiretrovirals at once (or possibly stagger according to each
drug's half-life) to minimize the possible emergence of resistance.(35)

Intentional supervised (or structured) treatment interruption (STI) presents several theoretical benefits:
minimizing drug exposure, possibly decreasing short- and long-term adverse effects; "autovaccinating"
individuals with their own virus in hopes of boosting the host immune control of HIV; allowing reversion of
resistant virus to wild type, potentially creating a more drug-sensitive target for salvage therapy; and
reducing the cost of therapy.

Studies conducted thus far demonstrate that STI does not increase HIV-specific immune response by
acting as "autoimmunizations," and does not lead to persistent control of viremia.(275-281) A possible
exception is in the unique situation of treatment during acute infection.(129,282) Studies of STI in
conjunction with immune modulators such as interleukin (IL)-2 or therapeutic vaccination are underway.

A trial of intermittent therapy (2 months on, 1 month off) demonstrated no significant difference in virologic
or immunologic markers compared to continuous therapy, but did show increased development of drug
resistance in the intermittent therapy arm (3 of 8 patients on efavirenz-based regimens developed NNRTI
resistance).(283) Using STIs composed of shorter cycles (eg, 1 week on, 1 week off) or guided by CD4
count or viral load parameters, other investigators have shown that these types of intermittent therapy
may be able to reduce drug toxicity, reduce time on drug, reduce cost, and improve quality of life without
sacrificing clinical control of HIV.(284,285)

Two large trials have examined STI as a part of salvage therapy. In 1 study, highly treatment-experienced
patients with advanced disease and drug-resistant virus were randomized to immediate salvage therapy
versus salvage therapy following a 4-month treatment interruption.(286) That trial showed no benefit to
the STI group, which in fact demonstrated a higher rate of AIDS-associated events, causing accrual to be
closed early. In a second study, highly treatment-experienced patients were randomized to a 2-month STI
versus no STI prior to initiating a 7- to 8-antiretroviral drug + hydroxyurea salvage regimen.(287) In this
study, STI patients showed a benefit in viral load and CD4 cell response through 48 weeks. The disparate
results in these trials are not easily reconciled. Differences in the patient population and duration of STI
exist, but still fail to completely explain the differences in outcomes.

In general, studies of the safety and efficacy of STIs do not support their use in routine care, and STI
cannot be recommended outside of a research setting.(35)

In a modification of this concept, the idea of partial treatment interruptions has emerged as a possibility
for treating drug-resistant HIV.(288) In a small, selected cohort of patients with persistent viremia on a
stable PI- and NRTI-containing antiretroviral regimen, discontinuation of the PI component of the regimen
resulted in stable viremia, reduced toxicity, and halted accumulation of new PI mutations. Discontinuation
of the NRTI component of the regimen, however, was associated with rapid rises in viral load.(274) This
concept, too, must still be considered experimental and cannot be recommended for routine practice.

Immune Modulators for HIV Treatment

Control of HIV viremia depends on host immune response as well as exogenous intervention with
antiretroviral medications. In an effort to enhance host immune response to HIV, several techniques of
immune modulation have been studied. Early attempts at immune modulation through bone marrow
transplantation, donor lymphocyte infusions, and cytokine infusions demonstrated no consistent, durable
clinical benefit. Immunomodulatory approaches may prove more effective in combination with effective
ART.

In the context of established infection, therapeutic immunization has been studied as a means to enhance
HIV-specific immune responses. No study to date has demonstrated sustained clinical benefit to such
"therapeutic vaccination." Some studies have demonstrated improved HIV-specific immune responses
after therapeutic vaccination,(289) but the clinical significance of these responses remains unclear.

IL-2, a cytokine released by activated CD4 cells that regulates T-cell proliferation and maturation, has
been studied as an immune modulator in HIV-infected patients. Administration of IL-2 to individuals with
controlled HIV viremia leads to increased CD4 counts,(290-292) expansion of both memory and naive
CD4 pools,(292) and decreased T-cell activation.(292) IL-2 administration is accompanied by numerous
adverse effects and toxicities. No clinical benefits of IL-2 have yet been demonstrated. Large clinical trials
of IL-2 are underway. Other cytokines, such as IL-12 (293) and IL-4 (294) are being studied, as is IL-2 in
combination with therapeutic vaccination.

Techniques of immune manipulation through infusion of antigen-presenting cells that have been activated
in vitro,(295) infusion of expanded (296) or activated (297) CD4 cells, and genetic manipulation of cells to
induce anti-HIV CTL activity (298) are also under investigation, but even if such resource-intensive,
individualized approaches are shown to be effective, it is doubtful that they will become accessible to
most people infected with HIV.

Administration of human growth hormone (hGH) as a method of enhancing CD4 recovery in HIV is
another technique being studied, spurred by the observations of increased circulating naive CD4 cells and
increased thymic mass, suggesting increased thymopoeisis, during hGH treatment.(299)

Prophylaxis Against OIs

Many commonly encountered OIs may be prevented by administering prophylactic antibiotics to those at
risk, based on pathogen-specific CD4 cell count thresholds.(207) In geographic areas where local
epidemiology of opportunistic pathogens varies from that of the United States, different recommendations
for primary prophylaxis (treatment to prevent an initial episode of an OI) may be appropriate. In general,
secondary prophylaxis (treatment to prevent recurrence of an OI) should be provided as long as immune
impairment persists.

Among individuals who experience reconstitution of immunologic function with ART, as assessed by
sustained increases in CD4 count to levels above those associated with OI, discontinuation of primary
prophylaxis or secondary prophylaxis has been shown to be safe.(300)

Routine Health Care Maintenance in HIV Infection

All individuals, whether on ART or not, will have other health care needs, some related to HIV infection
and some not. Individuals with HIV infection must be considered to be at risk for other blood-borne
pathogens and sexually transmitted infections. All HIV-infected individuals should be screened for viral
hepatitis A, B, and C, and immunized (against A and/or B) or treated as appropriate. Routine screening
for syphilis, chlamydia, gonorrhea, and other sexually transmitted diseases should be done according to
the individual's risk behavior. Age- and gender-appropriate cancer screening should also be done in HIV-
infected individuals, with special recommendations for increased screening for cervical and anal dysplasia
associated with human papillomavirus (HPV) disease.(301-303) Annual screening for tuberculosis with
purified protein derivative (PPD) testing is indicated, particularly in high incidence areas.

Hyperlipidemia, glucose intolerance, and insulin resistance are common consequences of antiretroviral
therapy.(304-306) With indications of potential increases in cardiac events among HIV-infected
individuals, careful attention should be paid to modifiable cardiac risk factors.(307) Routine monitoring of
fasting lipid panels, particularly among patients on antiretrovirals, and lipid management that follows the
guidelines of the National Cholesterol Education Program (NCEP) is recommended.(308)

Routine immunizations for HIV-infected individuals should follow standard guidelines with a few
exceptions. Oral polio vaccine and smallpox vaccine are contraindicated in HIV-infected individuals and in
those with whom they have significant contact. The risks of other live vaccines such as measles, mumps,
and rubella must be weighed against the potential benefits of vaccination. Data on varicella vaccination in
HIV-infected adults are lacking. Pneumococcal vaccination is recommended every 5 years for those with
HIV infection, as is annual influenza vaccine. Vaccination may be more effective in individuals with
relatively intact immune systems (CD4 count >200 cells/µL), and for individuals beginning ART, may be
delayed until such a goal is reached.

Discussions of sexual health should involve education to reduce risk of transmitting HIV to uninfected
partners, as well as preventing other sexually transmitted diseases. Contraceptive options and issues of
family planning should be addressed regularly with individuals of reproductive potential.

Other routine health measures such as blood pressure determination, depression and domestic violence
screening, smoking cessation interventions, drug and alcohol counseling, and dental and ophthalmologic
evaluation should be used with HIV-infected individuals, just as with HIV-uninfected individuals.

Summary and Future Directions

Clinical care of patients with HIV requires familiarity with a wide range of medical, social, economic, and
scientific issues. Despite the limitations of current forms of ART, it is clear that HIV replication can be
effectively suppressed. Current challenges include improving upon existing antiretroviral therapies,
developing new therapies to control and eliminate virus and enhance the immune system, extending
current and future therapies to all individuals in need of treatment worldwide, and developing effective
strategies to prevent HIV infection.

Nucleoside/Nucleotide Analogues
Abacavir (Ziagen, ABC)

Didanosine (Videx, ddI)

Emtricitabine (Emtriva, FTC)

Lamivudine (Epivir, 3TC)

Stavudine (Zerit, d4T)

Tenofovir (Viread, TDF)

Zalcitabine (Hivid, ddC) Discontinued by manufacturer 12/31/06

Zidovudine (Retrovir, AZT, ZDV)

Nonnucleoside Reverse Transcriptase Inhibitors
Delavirdine (Rescriptor, DLV)

Efavirenz (Sustiva, Stocrin, EFV)

Etravirine (Intelence, TMC 125)

Nevirapine (Viramune, NVP)

Protease Inhibitors
Amprenavir (Agenerase, APV) Discontinued by manufacturer 10/07

Atazanavir (Reyataz, ATV)

Darunavir (Prezista, DRV, TMC 114)

Fosamprenavir (Lexiva, Telzir, FPV)

Indinavir (Crixivan, IDV)

Lopinavir/Ritonavir (Kaletra)

Nelfinavir (Viracept, NFV)

Ritonavir (Norvir, RTV)

Saquinavir (Invirase, SQV)

Tipranavir (Aptivus, TPV)

Fusion Inhibitors
Enfuvirtide (Fuzeon, ENF, T-20)

Chemokine Coreceptor Antagonists

Maraviroc (Selzentry, Celsentri, MVC)

Integrase Inhibitors
Raltegravir (Isentress, RAL)
Lymphoma

Kaposi Sarcoma and Human Herpesvirus 8

Human Papilloma Virus-Associated Neoplasms

Parasitic
Cryptosporidiosis, Cyclosporiasis, and Isosporiasis

Microsporidiosis

Toxoplasmosis

Viral
Viral Hepatitis

Cytomegalovirus

Herpes Simplex Virus

Varicella-Zoster Virus

Fungal
Blastomycosis

Penicilliosis

Paracoccidioidomycosis

Coccidioidomycosis

Aspergillosis

Histoplasmosis

Cryptococcosis

Candidiasis

Pneumocystosis

Bacterial
Tuberculosis

Mycobacterium avium Complex and Atypical Mycobacterial Infections

Syphilis

Bartonella: Bacillary Angiomatosis and Peliosis

Pneumococcal Infection

Simian immunodeficiency viruses (SIV) also known as the African Green Monkey Virus, are retroviruses that are
found in over 40 species of African primates.

Viruses from two of these primate species, SIVsmm in sooty mangabeys and SIVcpz in chimpanzees, are believed
to have crossed the species barrier into humans, resulting in HIV-2 and HIV-1, respectively. The most likely route of
transmission of HIV-1 to humans involves contact with the blood of chimps that are often hunted for bushmeat in
Africa.

Unlike HIV-1 and HIV-2 infections in humans, SIV infections in their natural hosts are widely believed to be non-
pathogenic. Extensive studies in sooty mangabeys have established that SIVsmm infection does not cause any
disease in these animals, despite high levels of circulating virus. However, if this virus infects an Asian rhesus
Macaque, the animal will develop simian AIDS. A recent study of SIVcpz in wild living chimpanzees suggests that
infected chimpanzees experience an AIDS-like illness similar to HIV-1 infected humans. The later stages of SIV
turnes into SAIDS much like human AIDS

Immunodeficiency resembling human AIDS was reported in captive monkeys in the United States beginning in
1983.[1][2][3] SIV was isolated in 1985 from some of these animals, captive rhesus macaques suffering from simian
AIDS (SAIDS).[2] The discovery of SIV was made shortly after HIV-1 had been isolated as the cause of AIDS and
led to the discovery of HIV-2 strains in West Africa. HIV-2 was more similar to the then-known SIV strains than to
HIV-1, suggesting for the first time the simian origin of HIV. Further studies indicated that HIV-2 is derived from
the SIVsmm strain found in sooty mangabeys, whereas HIV-1, the predominant virus found in humans, is derived
from SIV strains infecting chimpanzees (SIVcpz).
Interim WHO clinical staging of HIV and AIDS, and HIV and AIDS case definitions for
surveillance
(2005) are made up of the following:

 Primary HIV infection - may be asymptomatic or experienced as Acute retroviral syndrome

 Clinical stage 1 - asymptomatic or generalized swelling of the lymph nodes
 Clinical stage 2 - includes minor weight loss, minor mucocutaneous manifestations, and
recurrent upper respiratory tract infections
 Clinical stage 3 - includes unexplained chronic diarrhoea, unexplained persistent fever, oral
candidiasis or leukoplakia, severe bacterial infections, pulmonary tuberculosis, and acute
necrotizing inflammation in the mouth. Some persons with clinical stage 3 have AIDS.
 Clinical stage 4 - includes 22 opportunistic infections or cancers related to HIV. All persons
with clinical stage 4 have AIDS.

Most of these conditions are opportunistic infections that can be treated easily in healthy people.
SOURCE: UNAIDS

Primary HIV infection is the first stage of HIV disease, typically lasting only a week or two, when
the virus first establishes itself in the body. Some researchers use the term acute HIV infection to
describe the period of time between when a person is first infected with HIV and when antibodies
(proteins made by the immune system in response to infection) against the virus are produced by the
body (usually 6 to 12 weeks) and can be detected by an HIV test.

Up to 70% of people newly infected with HIV will experience some "flu-like" symptoms during this
stage. These symptoms, which usually last no more than several days, might include fevers, chills,
night sweats, and rashes. Afterward, the infected person returns to feeling and looking completely
well. The remaining percentage of people either do not experience symptoms of acute infection or
have symptoms so mild that they may not notice them.

Seroconversion

This term refers to the time when an HIV positive person's immune system responds to the infection
by producing antibodies to the virus. Most people develop antibodies within three months after
infection, and some can take up to six months.

If an antibody test is done before seroconversion is complete, it may give a "false negative" result
because sufficient antibodies have not yet been developed by the body. A three-month window period
between infection and production of antibodies is normal for most of the population. Very, very rarely
(i.e., in only a few cases ever), a person may take six months to produce antibodies. To be certain of
your HIV status, take an HIV antibody test three months or longer after you were exposed to the
virus. For even greater certainty, get tested again six months after the exposure occurred.

The Asymptomatic Stage

After the acute stage of HIV infection, people infected with HIV continue to look and feel completely
well for long periods, usually for many years. During this time, the only indication that you are
infected with HIV is that you will test positive on standard (antibody) HIV tests and you may have
swollen lymph glands.

This means that you look and feel healthy but can infect other people through unprotected sex or
through needle sharing -- especially if you have not been tested and do not know that you are
infected.

Even though an infected person may appear perfectly healthy, HIV is still very active and is continuing
to weaken the immune system during this stage. In some individuals, the virus appears to slowly
damage the immune system over a number of years. In most people, however, a faster decline of the
immune system occurs at some point, and the virus rapidly replicates. This damage can be seen in
blood tests before any actual symptoms are experienced.

HIV positive people should seek medical care and begin monitoring their immune systems as soon as
possible after receiving a positive test result. Periodic immune monitoring tests, such as CD4 count
and viral load tests, can give you and your doctor a better picture of your immune health and disease
progression, and can help you make smart choices about treatment.

Seeking early care for HIV disease can give people better chances of survival and improved quality of
life. People with HIV are encouraged to see a doctor regularly, even if they feel fine at the moment,
because the virus could be already damaging the immune system. Early and regular care enables HIV
positive individuals and their medical care providers to take control of their treatment before
symptoms appear.

Early- and Medium-Stage HIV Symptomatic Disease

When the immune system is compromised by HIV infection, many people begin to experience some
mild HIV disease symptoms, such as skin rashes, fatigue, night sweats, slight weight loss, mouth
ulcers, and fungal skin and nail infections. Most, though not all, will experience mild symptoms such
as these before developing more serious illnesses. Although one's prognosis varies greatly depending
on a number of factors, it is generally believed that it takes five to seven years for the first mild
symptoms to appear. These symptoms mark the early and medium stages of HIV symptomatic
disease.

As the disease progresses, some individuals may become quite ill even if they have not yet been
diagnosed with AIDS, the late stage of HIV disease. Typical problems include chronic oral or vaginal
thrush (a fungal rash or spots), recurrent herpes blisters on the mouth (cold sores) or genitals,
ongoing fevers, persistent diarrhea, and significant weight loss.
These symptoms are not necessarily specific to HIV or the development of AIDS. However, they
should be of concern to people who have tested positive for HIV. Usually, symptoms occur when the
virus has already caused considerable damage to the immune system. For that reason, people with
HIV should not wait until symptoms appear to get medical treatment. Also, people with high risk for
HIV infection should not wait to for symptoms to appear before getting tested.

Late-Stage HIV Disease (AIDS)

When immune system damage is more severe, HIV positive individuals may experience opportunistic
infections (called "opportunistic" because they are caused by organisms which do not ordinarily induce
illness in people with normal immune systems, but take the opportunity to flourish in people with
compromised immune systems). Some of the most common opportunistic infections include
Pneumocystis carinii pneumonia (PCP), Mycobacterium avium complex (MAC) disease,
cytomegalovirus (CMV), toxoplasmosis, and candidiasis.

According to the Centers for Disease Control and Prevention (CDC), an AIDS diagnosis can be given
to an HIV positive person who has a CD4 count of less 200/mm3 or a history of an "AIDS-defining
illness" (such as one of the opportunistic infections mentioned above). For more information on what
defines AIDS, including a complete list of AIDS-defining illnesses, see "Mortality Trends" from the
Winter 2005 BETA.

It is important to note that this definition of AIDS may apply to HIV positive individuals who have
never experienced symptoms of HIV disease.

Receiving an AIDS diagnosis does not necessarily mean that the diagnosed person will die soon; some
people have lived for many years after their diagnosis. This is even more the case today with the
availability of highly active antiretroviral therapy (HAART), which has helped extend the lives of
thousands of people living with HIV and AIDS. In addition, many opportunistic infections can be
prevented or treated successfully. This has substantially increased the longevity and quality of life of
people living with HIV/AIDS.

Does everyone who has HIV eventually develop AIDS? We don't know for certain. Studies show that
the majority of untreated people do eventually become ill from HIV. However, with regular medical
care and other positive lifestyle factors, such as emotional support, many long-term survivors have
been living with HIV/AIDS for upwards of two decades. As existing treatments are used earlier in the
course of HIV disease and new treatments are developed, it has become possible to further postpone,
and perhaps even prevent, illness.