J Me d Al l i e d S c i 201 4 ; 4 ( 1 ) : 4 5 - 53

www. j ma s . i n
Pr i nt I SSN: 22311696 Onl i ne I SSN: 2231170X
Journal of
Medi cal
&Allied
Sciences



Review
Vaccinology: Reflections and the way forward
Anubrolu Naveen, Uday Shanker Araga, Mangamoori Lakshmi Narasu
Center for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University
Hyderabad, Kukatpally, Hyderabad-500085, Andhra Pradesh, India.
Article history: Abstract

Received 14 February 2014
Revised 20 February 2014
Accepted 22 February 2014
Early online 25 February 2014
Print 28 February 2014



In recent times vaccine development exploration accelerates the
new strategies based on genomics, proteomics, functional ge-
nomics and glycochemistry (synthetic chemistry). The evolving Ad-
vanced technologies have the potential to identify potential targets
(antigens-proteins, cell wall components, polysaccharides), produc-
tion of genetically engineered proteins (antigenic or carriers) and
synthesizing the complex peptidoglycans / lipopolysacchrides. The
review primarily focuses on vaccines for bacteria (Mycobacterium
tuberculosis); the notorious success of Mycobacterium tuberculosis
a highly adapted human pathogen rests intracellular (Latent phase),
interferes membrane trafficking in infected macrophages, blocks
the process of phagolysosome and thereby protect the patho-
gen/organism against lysis. The world is witnessing an escalation
of multidrug and extreme drug resistant tubercle bacilli challenging
the normal therapeutic practices and increased mortality rate. The
sequencing of the M. tuberculosis genome has thrown light on
newer target proteins that could be used for vaccine development.
Corresponding author

Mangamoori Lakshmi Narasu
Center for Biotechnology,
Institute of Science and Technology,
Jawaharlal Nehru Technological University
Hyderabad, Kukatpally,
Hyderabad-500085,
Andhra Pradesh, India.
Phone: +91 40 23156129
Email: Mangamoori@gmail.com

Key words: conjugate vaccines, DNA vaccines, humoral and cell
mediated immunity, immunization

2014 Deccan College of Medical Sciences. All rights reserved.

f all living organisms, microbes are the most
diverse living forms, and life itself stems from
them. The majority of these are bacteria
which have adapted themselves to survive within di-
verse environments such as hot springs, ice and
snow, bare rock, airborne particles, and also upon
and within other living creatures
1,2
. Many bacterial
species are intimately involved in the health and dis-
ease of humans and animals
3,4
.
Infectious diseases were the leading cause of death
worldwide
5
during the last century. However, the
scenario changed dramatically due to the concept
of vaccination introduced by Edward Jenner in 1798
and the advent of antibiotic- based therapies that
followed. Vaccines are one our greatest triumphs,
and have been responsible for eradicating smallpox
globally
6-8
(announced by the WHO 1980)
9
. A global
drive to eliminate polio and tuberculosis is now
underway
10
. A few decades ago, the battle
against bacterial infections was generally be-
lieved to be won- until the development of drug
resistance in these bacteria due to the increased
selection pressure as a consequence of the over-
use of antibiotics.
Since the introduction of that first vaccine more
than 200 years ago, vaccination has controlled
nine major diseases: smallpox, diphtheria, pertus-
sis, tetanus, yellow fever, poliomyelitis, measles,
mumps and rubella; and achieved eradication of
smallpox (WHO 2005)
11,12
. Vaccines can be di-
vided into two broad categories i.e. active and
passive. An active vaccine stimulates the bodys
immune system to produce specific antibodies
(humoral response), cellular immune responses
O
45
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

(e.g. cytotoxic T-lymphocytes), or both. A passive
vaccine is a preparation of antibodies that is admin-
istered before, at, or around the time of exposure.
Active vaccines can in turn be divided into four main
categories viz. live attenuated vaccines, killed
vaccines, subunit vaccines and DNA vaccines
11
.
(Table 1)
Table 1: A comparison of important properties of different vaccine types
11
Vaccine type Advantages Drawbacks
Live vaccines
(attenuated)
One or few doses normally re-
quired
Long lasting protection
Both humoral and cellular re-
sponses
Controlled attenuation normally re-
quired
Risk of reversion to pathogenicity
Certain risk of transmission to non-
vaccinated contacts
Poorly defined composition
Killed vaccines No risk of reversion to patho-
genicity
No risk of transmission to non-
vaccinated contacts
Multiple doses typically required
Poorly defined composition
Antigen must be produced by culti-
vation of a pathogen
Mainly humoral response
Adjuvants normally needed
Subunit vaccine
(Non-recombinant)
Defined composition
Various delivery systems availa-
ble
Antigen must be produced and pu-
rified by cultivation of a pathogen
Multiple doses typically required
Adjuvants needed
Subunit vaccine
(Recombinant)

No risk of pathogenicity since the
pathogenic organism is not pre-
sent
Defined composition
Various delivery systems availa-
ble
Simplified large scale production
Further engineering possible
Multiple doses typically required
Adjuvants needed
DNA vaccine Simple production
Induces both humoral and cellu-
lar responses
Highly stable
Multiple epitopes can be en-
coded
Does not require cultivation of in-
fectious agents Innate adjuvatic-
ity
Does not require expression and
purification of antigen
Inefficient transfection
Risk of integration into host chro-
mosomes
Inefficient delivery
Variable immune responses
Reproduced with modification from Hansson M et al. Design and production of recombinant subunit vaccines. Biotechnol. Appl. Bio-
chem. (2000) 32, 95107. Copyright 2000 Portland Press Ltd, Great Britain.
Different approaches to vaccine design
Conventional vaccines consist either of live-attenu-
ated microbes, killed microbes, purified microbial
components, polysaccharide-carrier protein conju-
gates or recombinant proteins. The two basic types
of vaccines, based on the microorganism in an at-
tenuated but live form, or on the killed inactivated
microorganism, were among the first vaccines gen-
erated. Traditionally, live attenuated vaccines were
prepared by repeated passage of the infectious
agent in tissue culture or within animal hosts, until
its virulence was greatly reduced but its immuno-
genicity was retained. Live attenuated vaccines can
potentially replicate in the host, eliciting both hu-
moral and cellular immunity, and only a single or a
few doses may give lifelong protection. But a major
drawback of attenuated live vaccines is the risk of
reversion into their original pathogenic forms, espe-
cially in immune-compromised individuals and in-
fants. Moreover, there are chances of some live
vaccine strains being transmitted from the vac-
cinated to an unvaccinated individual
11,13
.
46
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

Killed vaccines do not replicate or multiply in the
host and therefore are not pathogenic and cannot
be transmitted to another individual. The killed vac-
cine is usually administered along with an adjuvant
to enhance its immunogenicity and multiple doses
are necessary for obtaining long-term protective im-
munity. Killed vaccines usually function by stimulat-
ing the humoral immune response, as well as by
priming for immunological memory. The production
of such vaccines requires large-scale in vitro cultur-
ing of organism, which can be associated with both
safety risks and increased costs of production chal-
lenge in the preparation of killed vaccines, is to kill
pathogenic organisms without destroying their pro-
tective antigens
7,11,14
.
A third type of vaccine i.e. the subunit vaccine is pre-
pared using only part or the secretory product (toxin)
of the infectious micro-organism to raise a protec-
tive immune response, and hence there is no risk of
pathogenicity. The composition of a sub-unit vac-
cine can be clearly defined, which is a significant ad-
vantage in terms of safety considerations and mini-
mization of side effects
11.
In order to elicit a vigorous
immune response, subunit vaccines often require
multiple doses, as well as the use of adjuvants. Sub-
unit vaccines can be based on peptides, proteins or
polysaccharides that have been shown to contain
protective epitopes. The first subunit based vac-
cines were developed in the 1920s for diphtheria
and tetanus by chemical detoxification of toxins to
yield the non-toxic toxoid. Subunit conjugate vac-
cines based on polysaccharides are commonly de-
livered coupled to a protein carrier such as the teta-
nus toxoid e.g. Hemophilus influenza type b (Hib)
vaccines
15
. Toward the end of the 20
th
century, re-
combinant DNA techniques brought a revolution in
microbiology providing better tools for vaccine re-
search
16
. In this approach, specific antigens, se-
lected on the basis of immunological data from pa-
tients, are purified from heterologous systems in
which the corresponding genes have been cloned
and tested for safety and efficacy. This approach
generated the first recombinant vaccine for human
use i.e. the hepatitis B vaccine in 1984
17
.
DNA vaccines
DNA vaccines are considered the third generation
vaccines
18
. DNA-mediated immunization involves
the direct introduction of a plasmid DNA which en-
codes an antigenic protein that is expressed within
cells of the organism. The in vivo expression of that
protein using the patients own cellular machinery
leads to the induction of antigen-specific immune
responses
18-20
. DNA-based immunization most
closely resembles a virus infection. DNA vaccines
usually consist of plasmid vectors (derived from
bacteria) that contain heterologous genes
(transgenes) inserted under the control of a eukar-
yotic promoter, allowing protein expression in mam-
malian cells. The basic requirements for the back-
bone of a plasmid DNA vector are a eukaryotic pro-
moter, a cloning site, a polyadenylation sequence,
a selectable marker and a bacterial origin of repli-
cation
21
. A DNA vaccine was first demonstrated to
be effective using influenza as a model, where it
was shown that DNA-encoding nucleoprotein (NP)
induced cytotoxic T lymphocytes (CTLs) and cross-
strain protection in mice. DNA vaccines have been
effective for viral diseases such as Hepatitis B and
C, Influenza, Coxsackievirus B3, Rabies, Herpes,
HIV; bacterial diseases such as M. tuberculosis, B.
anthracis, S. aureus; and parasitic diseases such
as malaria. The first clinical trials using injections of
DNA to stimulate an immune response against a
foreign protein began for HIV in 1995. DNA vac-
cination can over-come most disadvantages of con-
ventional vaccine strategies and has the potential
to become the vaccination of the future. However,
today after 20 years of research, no such commer-
cial product has reached the market. One explana-
tion is the technique's failure to induce an efficient-
enough immune response in humans, but safety is
also a fundamental issue
21-23
.
Reverse vaccinology
The field of bioinformatics has revolutionized the
field of vaccinology, leading to the identification of
potential novel vaccine candidates without the need
for cultivating the pathogens. This approach has
been termed as reverse vaccinology, since the
process of vaccine discovery starts in silico using
genetic information rather than the pathogen itself:
computer-software is utilized, to visualize all possi-
ble molecular interactions based on the pathogens
genome, and any promising molecules/processes
then selected for lab-testing. It reduces the time and
cost required for the identification of vaccine candi-
dates, and provides new solutions for those dis-
eases for which vaccines are not available. The re-
verse vaccinology approach involves the in silico
analysis of the microbial genome sequence and
was first used to identify novel and effective vaccine
candidates against sero-group B Meningococ-
cus
24,25
. The same approach has been successfully
applied to other important human pathogens,
demonstrating the feasibility of developing vaccines
against any infectious disease
25,26
.
The reverse approach to vaccine development
takes advantage of the pathogens genome se-
47
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

quence, which provides a catalog of virtually all pro-
tein antigens that the pathogen can express at any
time. As shown in figure 1, this approach starts from
the genomic sequence, wherein computer analysis
predicts those antigens that are most likely to be
vaccine candidates, independent of their abun-
dance and immunogenicity during infection and
without the need to grow the pathogen in vitro
27,28
.
This involves the in silico analysis of microbial ge-
nome sequences followed by the high-throughput
expression of the genes of interest. The recombi-
nant proteins are then used to immunize mice and
the post-immunization sera are analyzed to assess
the ability of the polypeptide to elicit a quantitative
and qualitative immune response.

Fig 1. Flow chart of the genome-based approach to vaccine de-
velopment [Reproduced with modification from Mora M et al. Re-
verse vaccinology. Drug Discovery Today 2003; 8(10):459 464.
Copyright 2003 Elsevier Science Ltd.]
28
This approach allows not only the identification of all
the antigens seen by the conventional methods, but
also the discovery of novel antigens that work on a
totally different paradigm. Therefore, this method al-
lows the discovery of novel mechanisms of immune
intervention. The feasibility of this approach relies
heavily on the availability of a high-throughput sys-
tem to screen protective immunity using which all
the potential genes of a pathogen can be tested,
without any bias of any type.
Unfortunately, owing to limited knowledge of vac-
cine immunology, good correlates of protection are
rare and, therefore, screening for protective immun-
ity is the rate-limiting step of reverse vaccinology.
The other limitations of this approach are: the inabil-
ity to identify non-protein antigens such as polysac-
charides which are also important components of
many successful vaccines; and the difficulty in iden-
tification of CD1 restricted antigens such as glycoli-
pids, which represent new promising vaccine candi-
dates
28-30
. The steps involved in vaccine develop-
ment by conventional and reverse vaccinology are
depicted in figure 2 and a comparison of conven-
tional and genomic approaches to vaccine develop-
ment is given in table 2.
In silico analysis of genomes
Secreted/extracellular proteins are more easily ac-
cessible immunogens than intracellular proteins,
and therefore ideal vaccine candidates. Various al-
gorithms currently exist to identify proteins with
these features from data bank sequences but com-
puter algorithms are not always able to correctly
predict the cellular localization of proteins. Several
approaches can be used to mine genomic se-
quences, and the appropriate combination of the
various algorithms and the critical evaluation of the
information generated are essential for the proper
selection of the antigens
28
.
A primary screen for coding capacity is carried out
on DNA segments or contigs [contiguous se-
quences] using database and computer programs
included in the Wisconsin package version 10.0
(Genetics Computer Group (GCG), http://www.ac-
celrys.com). As a second step, all the predicted
open reading frames (ORFs) are used for homology
searches against a database with BLASTX,
BLASTN and TBLASTX programs to identify DNA
segments with potential coding regions. The ORFs
coding for known cytoplasmic functions or known
antigens are excluded, whereas the other coding re-
gions are selected for further analysis. A third
screening step, designed to identify putative pro-
teins with a cellular localization spanning the inner
membrane to outside the bacterium, is applied.
48
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

Table 2: Comparison of conventional and genomic approaches to vaccine development
27
Conventional vaccinology Reverse vaccinology
Essential features
Most abundant antigens during disease
All antigens immunogenic during disease
Cultivable microorganism
Animal models essential
Correlates of protection useful
All antigens immunogenic during disease
Antigens even if not immunogenic during dis-
ease
Antigens even in non-cultivable microorganisms
Animal models essential
Correlates of protection very important
Correct folding in recombinant expression im-
portant
High-throughput expression/analysis important
Advantages
Polysaccharides may be used as antigens
Lipopolysaccharide-based vaccines are pos-
sible
Glycolipids and other CD-1 restricted anti-
gens can be used
Fast access to virtually every single antigen
Non-cultivable microorganisms can be ap-
proached
Non abundant antigens can be identified
Antigens that are not immunogenic during infec-
tion can be identified
Antigens that are transiently expressed during
infection can be identified
Antigens not expressed in vitro can be identified
Non-structural proteins can be used
Disadvantages
Long time required for antigens identification
Antigenic variability of many of the identified
antigens
Antigens not expressed in vitro cannot be
identified
Only structural proteins are considered
Non protein antigens cannot be used (polysac-
charide, lipopolysaccharides, glycolipids and
other CD-1 restricted antigens)
BLAST, FASTA, MOTIFS, FIND-PATTERNS
PSORT, Epitope Prediction (MHC-Peptide binding)
and Analysis Tools (IEDB Analysis Resource), in
addition to ProDom, Pfam and Blocks databases,
are used to predict features typical of surface-asso-
ciated proteins
31-33
and for modeling the interaction
between potentially antigenic peptides and Major
Histocompatibility Complex (MHC) molecules for
identifying potential T-cell and B-cell epitopes
34
.
As the number of complete genome sequences
available is increasing, it is now possible to com-
pare the sequences of related bacteria and patho-
gens against commensals of the same or related
species, and even bacteria with different or similar
pathogenic profiles, leading to rapid identification of
putative disease-related genes or the complete set
of genes potentially responsible for acquisition of
virulence (comparative genomics). Functional ge-
nomics approaches include the large-scale analysis
of gene transcription by DNA microarrays and iden-
tification of the whole set of proteins en-coded by an
organism (proteomics) by two-dimensional gel elec-
trophoresis and mass spectrometry
35
.
Following the success of Meningococcus B vac-
cine, many scientists have used this approach to
identify vaccine candidates against human patho-
gens. Pathogens that have been studied using re-
verse vaccinology include Bacillus anthracis, Strep-
tococcus pneumoniae, Staphylococcus aureus,
Chlamydia pneumoniae, Porphyromonas gingivalis,
Edwardsiella tarda and Mycobacterium tuberculo-
sis
26
.
Mycobacterium tuberculosis
Tuberculosis (TB) has afflicted mankind throughout
history. Approximately one third of the worlds pop-
ulation is currently infected with Mycobacterium tu-
berculosis and nearly two million people die of TB
anually
36,37
. Within the past decade, there has been
a remarkable emergence of multi-drug resistant var-
iants of M. tuberculosis (MDR-TB)
38-40
that poses a
challenge to normal therapeutic practices and in-
creases the mortality rates worldwide. Although the
49
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

emergence of any drug resistance is of concern, the
appearance of multi-drug resistant strains of M. tu-
berculosis is particularly disturbing, since few drugs
are effective against tuberculosis. Resistance to
each of these agents has been reported.
The notorious success of Mycobacterium tuberculo-
sis as a highly adapted human pathogen rests upon
the ability of this microorganism to interfere with in-
tracellular membrane trafficking in infected macro-
phages. M. tuberculosis blocks the biogenesis of
the phagolysosome, the very organelle responsible
for routine elimination of microorganisms by phago-
cytic cells. The propensity of M. tuberculosis to en-
ter the host macrophages, where it is safely stored
in a pathogen-friendly phagosome, which does not
mature into the phagolysosome, is central to tuber-
culosis infection, latency, disease activation,
spread, and suppression of immunological detec-
tion by the host
41-47
.

Fig 2. Approaches to vaccine development
27
[Reproduced with modification from Rappuoli R. Reverse Vaccinology. Curr Opin Mi-
crobiol 2000; 3(5):445-450. Copyright 2000 Elsevier Science Ltd.]
The world is witnessing an escalation of the tuber-
culosis (TB) epidemic, particularly in sub-Saharan
Africa and South-East Asia. The problem has been
compounded by the evolution of the human immu-
nodeficiency virus pandemic and this emergence of
multidrug-resistant tubercle bacilli
48-50
.
50
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

BCG is the most frequently used vaccine in the
world, but there is no scientifically-based evidence
that it prevents primary pulmonary tuberculosis
51,52
.
Recent years have seen a renewed effort to develop
new vaccines against tuberculosis. As a result, sev-
eral promising avenues of research have been-
pursued
53-56
including the production of recombinant
BCG vaccines (like BCG30, a secretory protein)
Ag85B, ESAT-6, BCG:RD1, BCG and rBCG:ureC-
Hly
57,58
; auxotrophic vaccines(PhoP mutant of M.
tb)
59-61
; DNA vaccines (using a recombinant attenu-
ated Vaccinia virus MVA strain construct carrying
the M. tb secretory Ag85A and non-replicative ade-
novirus vector expressing Ag85A)
62
; and subunit
vaccines Mtb32/Mtb39, Mtb72F fusion protein and
ESAT-6/Ag85B fusion protein
63
. All these vaccines
have been shown to be safe in animals but their
evaluation in humans is still met with the problem of
safety and stability. One of the double mutants ap-
pears to provide protection at least equivalent to
standard BCG in a relevant guinea pig challenge
model, and this vaccine is expected to enter phase-
I clinical trial later this year.
Glycobiology and glycochemistry
Bacterial engineered protein conjugates show
promising applications for development on an array
of diagnostics and immunoprotective opportunities.
Capsular polysaccharides are T-cell independent
antigens that induce transient antibody responses
(mainly of IgM and IgG2 isotypes). Furthermore, pol-
ysaccharides do not induce any immunological
memory and repeated immunizations are unable to
increase antibody titers and in some cases, provoke
tolerance in adults
64
. These drawbacks were solved
by covalently linking the sugar to a carrier protein
(conjugation). This procedure converts T-cell inde-
pendent antigens into T-cell dependent antigens by
providing a source of appropriate T-cell epitopes
(present in the carrier protein). This technology has
now been successfully applied to develop very effi-
cacious vaccines against several encapsulated bac-
teria.
Current research into a tuberculosis vaccine is
based on the similarity of primary infection caused
by Mycobacterium tuberculosis and the other cap-
sulated bacterial respiratory pathogens like Menin-
gococci, Pneumococci and Hemophilus influenza
type B. A notable feature in all these infections is
that they show a similarity of age distribution to that
of M. tuberculosis and other mycobacteria. The M.
tuberculosis has a polysaccharide capsule in vitro
and in vivo similar to the other above mentioned
pathogens, and their capsular protein components
prolong the survival inside phagosomes, which ex-
plains the overlap in age distribution. BCG whole
cell protein components prolong the survival but do
not protect animals against challenge with wild-type
M. tuberculosis
65
.
The observation that mutants of encapsulated bac-
teria without the capsule are non-pathogenic (being
highly sensitive to serum complement), led to the
use of the capsular vaccines. Glycolipid conjugate
vaccines provide effective prophylaxis against bac-
terial infections. To date, however, no commercial
vaccine has been made available in which the key
glycolipid antigens are produced synthetically and
proteins by cloning. A conjugate vaccine based on
synthetic glycolipid trehalose dimycolic acid analogs
(TDM) and MCE operon-encoded surface proteins,
could be an effective approach as it arrests the ba-
cillary invasion in the macrophages.
The currently used BCG vaccine, and most vaccine
candidates against Tuberculosis, augments cell me-
diated immune response more than humoral re-
sponse. Using monoclonal antibodies, researchers
demonstrated modification of various aspects of
mycobacterial infection to the benefit of the host; by
eliciting protective responses that are thought to
eliminate the infectious inoculum.
With such increasing understanding of the disease,
and concurrent sequencing of the genome of M. tu-
berculosis H37Rv
66,67
, newer aspects of vaccine de-
velopment and tuberculosis prevention are being
both intensely and widely pursued.
The historically elusive dream of control of con-
sumption, is now perhaps more realizable than
ever before.
Acknowledgments: None
Conflict of interest: None
References
1. Martiny JB, Bohannan BJ, Brown JH, Colwell RK, Fuhrman
JA, Green JL, et al. Microbial biogeography: putting micro-
organisms on the map. Nat. Rev. Microbiol. 2006; 4:102-
112.
2. Head IM, Saunders JR, Pickup RW. Microbial evolution, di-
versity and ecology: A decade of ribosomal RNA analy-sis
of uncultivated microorganisms. Microb. Ecol 1998; 35:1-
21.
3. Relman DA. New technologies, human-microbe interactions
and the search for previously unrecognized patho-gens. J.
Infect. Dis. 2002; 186 Suppl 2:S254-258.
4. Sherratt D. Bacteria rule! The start of a new era in bacterial
microbiology? EMBO Rep. 2001; 2:175-176.
5. Anderson RM. The Croonian lecture. Populations, infec-
tious disease and immunity: A very nonlinear world. Philos
Trans R Soc Lond B Biol Sci 1994; 346(1318):457-505.
6. Plotkin SA and Fantini B. Vaccinia, vaccination, vaccinol-
ogy. Jenner, Pasteur and their successors. Proceedings of
51
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

International Meeting on the History of Vaccinology. Paris:
Elsevier, 1996.
7. Janeway CA, Travers P, Shlomchik MJ. Immunobiology: the
immune system in health and disease, 5
th
ed, Garland pub-
lishing, 2001.
8. Silvers MJ and Steptoe MM. Historical overview of vac-
cines. Prim. Care 2001; 28:685-695.
9. WHO. The global eradication of smallpox: in History of In-
ternational Public Health No.4. WHO, Geneva, 1980.
10. Dowdle WR, DeGourville E, Kew OM, Pallansch MA, Wood
DJ. Polio Eradication: The OPV Paradox. Rev Med Virol
2003; 13(5):277-291.
11. Hansson M, Nygren PA, Stahl S. Design and production of
recombinant subunit vaccines. Biotechnol. Appl. Biochem.
2000 32:95-107.
12. WHO. Vaccine preventable disease: Monitoring system
2005 global summary. Available at www.who.int/vaccines-
documents/
13. Rappuoli R and Del Giudice G. Identification of vaccine tar-
gets. In Vaccines: From Concept to Clinic. Paoletti LC,
McInnes PM (eds). Boca Raton: CRC Press, pp.1-17, 1999.
14. Ada G. The immunology of vaccination. In: Plotkin SA and
Orenstein WA (eds), Vaccines, 4
th
ed, Philadelphia, USA:
Saunders, pp. 3145, 2003.
15. Mkel PH. Vaccines against Haemophilus influenza type
B. In: Ala Aldeen DAA and Hormaeche CE (eds). Molecular
and clinical aspects of bacterial vaccine development, John
Wiley and Sons, pp. 4191, 1995.
16. Eldred BE, Dean AJ, McGuire TM, Nash AL. Vaccine com-
ponents and constituents: Responding to consumer con-
cerns. Med J Aust 2006; 184(4):170-175.
17. McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller
WJ, Hilleman MR. Human Hepatitis B vaccine from recom-
binant yeast. Nature 1984; 307:178-180.
18. Whalen RG. DNA vaccines for emerging infectious dis-
eases: what if? Emerg. Infect. Dis. 1996; 2:168-175.
19. Huygen K, Content J, Denis O, Montgomery DL, Yawman
AM, Deck RR, et al. Immunogenicity and protective efficacy
of a tuberculosis DNA vaccine. Nat. Med. 1996; 2:893-898.
20. Tuteja R. DNA vaccines: A ray of hope. Crit Rev. Bio-chem.
Mol. Biol. 1999; 34:1-24.
21. Sheikh NA and Morrow WJ. Guns, genes and spleen: A
coming of age for rational vaccine design. Methods 2003;
31(3):183-192.
22. Delcayre A, Peake JS, White DJ, Yuan S, McDonald MK,
Liang A, Tan PL, Watson JD. A genome-based functional
screening approach to vaccine development that combines
in vitro assays and DNA immunization. Vaccine 2003;
21(23):3259-3264.
23. Wack A and Rappuoli R. Vaccinology at the beginning of
the 21
st
century. Curr. Opin. Immunol. 2005; 17:411-418.
24. Pizza M, Scarlato V, Masignani V, Giuliani MM, Arico B, Co-
manducci M, et al. Identification of vaccine candidates
against serogroup B Meningococcus by whole-genome se-
quencing. Science 2000; 287:1816-1820.
25. Serruto D, du-Bobie J, Capecchi B, Rappuoli R, Pizza M,
Masignani V. Biotechnology and vaccines: application of
functional genomics to Neisseria meningitidis and other
bacterial pathogens. J. Biotechnol. 2004; 113:15-32.
26. Scarselli M, Giuliani MM, du-Bobie J, Pizza M, Rappuoli R.
The impact of genomics on vaccine design. Trends Bio-
technol. 2005; 23:84-91.
27. Rappuoli R. Reverse vaccinology. Curr Opin Microbiol
2000; 3(5):445-450.
28. Mora M, Veggi D, Santini L, Pizza M, Rappuoli R. Re-verse
vaccinology. Drug Discov. Today 2003; 8:459-464.
29. Du-Bobie J, Capecchi B, Serruto D, Rappuoli R, Pizza M.
Two years into reverse vaccinology. Vaccine 2003; 21:605-
610.
30. Nguyen TK, Koets AP, Santema WJ, van Eden W, Rutten
VP, van Rhijn I. The Mycobacterial glycolipid glucose mono-
mycolate induces a memory T cell response comparable to
a model protein antigen and Non B cell response upon ex-
perimental vaccination of cattle. Vaccine 2009;
27(35):4818-4825.
31. de Groot AS, Bosma A, Chinai N, Frost J, Jesdale BM, Gon-
zalez MA, Martin W, Saint-Aubin C. From genome to vac-
cine: In silico predictions, ex vivo verification. Vaccine 2001;
19(31): 4385-4395.
32. Masignani V, Balducci E, Serruto D, Veggi D, Arico B, Co-
manducci M, Pizza M, Rappuoli R. In silico identification of
novel bacterial ADP-ribosyl transferases. Int. J. Med. Micro-
biol. 2004; 293:471-478.
33. Meinke A, Henics T, Hanner M, Minh DB, Nagy E. Anti-
genome technology: A novel approach for the selection of
bacterial vaccine candidate antigens. Vaccine 2005;
23:2035-2041.
34. Salomon J and Flower DR. Predicting class II MHC-peptide
binding: A kernel based approach using similarity scores.
BMC Bioinformatics 2006; 7:501.
35. Pucci MJ. Use of genomics to select antibacterial targets.
Biochem. Pharmacol. 2006; 71:1066-1072.
36. Glickman MS and Jacobs WR Jr. Microbial pathogenesis of
Mycobacterium tuberculosis: Dawn of a discipline. Cell
2001; 104(4):477-485.
37. Young DB, Gideon HP and Wilkinson RJ. Eliminating la-tent
tuberculosis. Trends Microbiol 2009; 17(5):183-188.
38. Shah NS, Wright A, Bai GH, Barrera L, Boulahbal F, Martin-
Casabona N, et al. Worldwide emergence of extensively
drug-resistant tuberculosis. Emerg Infect Dis 2007;
13(3):380-387.
39. Migliori GB, Besozzi G, Girardi E, Kliiman K, Lange C,
Toungoussova OS, et al. Clinical and operational value of
the extensively drug-resistant tuberculosis definition. Eur
Respir J 2007; 30(4):623-626
40. Shah NS, Richardson J, Moodley P, Moodley S, Babaria P,
Ramtahal M, et al. Increasing drug resistance in extensively
drug-resistant tuberculosis, South Africa. Emerg Infect Dis
2011; 17(3):510-513.
41. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC.
Consensus statement. Global burden of tuberculosis: Esti-
mated incidence, prevalence, and mortality by country.
WHO Global Surveillance and Monitoring Project. JAMA
1999; 282(7):677-686.
42. Hanekom WA, Mendillo M, Manca C, Haslett PA, Siddiqui
MR, Barry C III, Kaplan G. Mycobacterium tuberculosis in-
hibits maturation of human monocyte-derived dendritic cells
in vitro. J Infect Dis 2003; 188(2):257-266.
43. Hmama Z, Gabathuler R, Jefferies WA, de Jong G, Reiner
NE. Attenuation of HLA-DR expression by mononuclear
phagocytes infected with Mycobacterium tuberculosis is re-
lated to intracellular sequestration of immature class II het-
erodimers. J Immunol 1998; 161(9):4882-4893.
44. Mustafa T, Phyu S, Nilsen R, Bjune G, Jonsson R. In-
creased expression of Fas ligand on Mycobacterium tuber-
culosis infected macrophages: A potential novel mechanism
of immune evasion by Mycobacterium tuberculosis? Inflam-
mation 1999; 23(6): 507-521.
45. Pancholi P, Mirza A, Schauf V, Steinman RM, Bhardwaj N.
Presentation of mycobacterial antigens by human dendritic
cells: Lack of transfer from infected macrophages. Infect Im-
mun 1993; 61(12):5326-5332.
46. Stenger S, Niazi KR, Modlin RL. Down-regulation of CD1 on
antigen-presenting cells by infection with Mycobacterium tu-
berculosis. J Immunol 1998; 161(7):3582-3588.
47. Ting LM, Kim AC, Cattamanchi A, Ernst JD. Mycobacterium
tuberculosis inhibits IFN-gamma transcriptional responses
without inhibiting activation of STAT1. J Immunol 1999;
163(7):3898-3906.
52
Naveen A et al. Vaccinology: Reflections and the way forward

J Med Allied Sci 2014; 4(1)

48. Aaron L, Saadoun D, Calatroni I, Launay O, Memain N, Vin-
cent V, et al. Tuberculosis in HIV-infected patients: A com-
prehensive review. Clin Microbiol Infect 2004; 10(5):388-
398.
49. Corbett EL, Marston B, Churchyard GJ, de Cock KM. Tu-
berculosis in Sub-Saharan Africa: Opportunities, challenges
and change in the era of antiretroviral treatment. Lancet
2006; 367(9514):926-937.
50. Lawn SD and Zumla AI. Tuberculosis. Lancet 2011;
378(9785):57-72.
51. Fine PE. Variation in protection by BCG: implications of and
for heterologous immunity. Lancet 1995; 346(8986):1339-
1345.
52. Andersen P and Doherty TM. The success and failure of
BCG - Implications for a novel Tuberculosis vaccine. Nat
Rev Microbiol 2005; 3(8):656-662.
53. Skeiky YA and Sadoff JC. Advances in Tuberculosis vac-
cine strategies. Nat Rev Microbiol 2006; 4(6):469-476.
54. Brennan MJ, Clagett B, Fitzgerald H, Chen V, Williams A,
Izzo AA, Barker LF. Preclinical evidence for implementing a
prime-boost vaccine strategy for Tuberculosis. Vaccine
2012; 30(18):2811-2823.
55. Kaufmann SH. Future vaccination strategies against tuber-
culosis: Thinking outside the box. Immunity 2010;
33(4):567-577.
56. Kaufmann SH. Learning from natural infection for rational
tuberculosis vaccine design: from basic science to transla-
tional research. Hum Vaccine 2010; 6(8):614-618.
57. Bastos RG, Borsuk S, Seixas FK, Dellagostin OA. Recom-
binant Mycobacterium Bovis BCG. Vaccine 2009;
27(47):6495-6503.
58. Horowitz MA, Harth G, Dillon BJ, Maslesa-Galic S. Recom-
binant Bacillus Calmette-Guerin (BCG) vaccines expressing
the Mycobacterium tuberculosis 30-kDa major secretory
protein induce greater protective immunity against tubercu-
losis than conventional BCG vaccines in a highly suscepti-
ble animal model. Proc Natl Acad Sci 2000; 97(25):13853-
13858.
59. Orme IM. New vaccines against Tuberculosis. The status
of current research. Infect Dis Clin North Am 1999;
13(1):169-185.
60. Jackson M, Phalen SW, Lagranderie M, Ensergueix D,
Chavarot P, Marchal G, McMurray DN, Gicquel B, Guilhot
C. Persistence and protective efficacy of a Mycobacterium
tuberculosis auxotroph vaccine. Infect Immun 1999;
67(6):2867-2873.
61. Sampson SL, Mansfield KG, Carville A, Magee DM, Quitu-
gua T, Howerth EW, Bloom BR, Hondalus MK. Extended
safety and efficacy studies of a live attenuated double leu-
cine and pantothenate auxotroph of Mycobacterium tuber-
culosis as a vaccine candidate. Vaccine 2011; 29(30):4839-
4847.
62. Okada M, Kita Y, Nakajima T, Hashimoto S, Nakatani H,
Nishimatsu S, et al. The study of novel DNA vaccines
against tuberculosis: Induction of pathogen-specific CTL in
the mouse and monkey models of tuberculosis. Hum Vaccin
Immunother 2012; 9(3).
63. Dietrich J, Aagaard C, Leah R, Olsen AW, Stryhn A, Do-
herty TM, Andersen P. Exchanging Esat6 with Tb10.4 in an
Ag85b fusion molecule-based Tuberculosis subunit vac-
cine: Efficient protection and Esat6-based sensitive moni-
toring of vaccine efficacy. J Immunol 2005; 174(10):6332-
6339.
64. Granoff DM, Gupta RK, Belshe RB, Anderson EL. Induction
of immunologic refractoriness in adults by Meningococcal C
polysaccharide vaccination. J Infect Dis 1998; 178(3):870-
874.
65. Brandt L, Skeiky YA, Alderson MR, Lobet Y, Dalemans W,
Turner OC, et al. The protective effect of the Mycobacterium
bovis BCG vaccine is increased by co-administration with
the Mycobacterium tuberculosis 72-Kilodalton fusion poly-
protein Mtb72f in M. tuberculosis-infected guinea pigs. In-
fect Immun 2004; 72(11):6622-6632.
66. Camus JC, Pryor MJ, Medigue C, Cole ST. Re-annotation
of the genome sequence of Mycobacterium tuberculosis
H37rv. Microbiology 2002; 148(10):2967-2973.
67. Cole ST and Barrell BG. Analysis of the genome of Myco-
bacterium tuberculosis H37rv. Novartis Found Symp 1998;
217:160-172; discussion 172-177.
53