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Background. Herpes simplex virus type 2 (HSV-2) infection is common among human immunodeficiency virus
(HIV)infected persons, and HSV reactivation increases plasma and genital HIV-1 levels. We studied HIV-1 levels
during HSV suppression in coinfected persons in a placebo-controlled crossover trial.
Methods. Twenty antiretroviral therapy (ART)naive HIV-1/HSV-2seropositive men who have sex with men in
Lima, Peru, with CD4 cell counts 200 cells/L were randomized to receive either valacyclovir at 500 mg twice daily
or placebo for 8 weeks, after which they underwent a 2-week washout period and then received the alternative regimen
for 8 weeks. Specimens included daily anogenital swabs (for HSV DNA polymerase chain reaction [PCR]), thrice
weekly rectal mucosal secretions (for HIV-1 RNA and HSV DNA PCR) obtained by anoscopy, and weekly plasma (for
HIV-1 RNA PCR). Outcomes were rectal and plasma HIV-1 RNA levels by treatment arm.
Results. HIV-1 was detected in 73% of 844 rectal and 99% of 288 plasma specimens. HSV was detected in 29%
and 4% of mucocutaneous specimens obtained during placebo and valacyclovir administration, respectively
(P .001). Valacyclovir resulted in a 0.16 (95% confidence interval [CI], 0.07 0.25; P .0008; 33% decrease) log10
copies/mL lower mean within-subject rectal HIV-1 level and a 0.33 (95% CI, 0.23 0.42; P .0001; 53% decrease)
log10 copies/mL lower plasma HIV-1 level, compared with values for placebo.
Conclusions. Valacyclovir significantly reduces rectal and plasma HIV-1 levels in HIV-1/HSV-2 coinfected men.
HSV suppression may provide clinical benefits to persons not receiving highly active ART as well as public health benefits.
Trial registration. ClinicalTrials.gov identifier: NCT00378976.
Most HIV-infected persons are also infected with herpes transmission is higher in HIV-1 serodiscordant couples
simplex virus type 2 (HSV-2) [1]. The risk of HIV-1 when the source partner has reported recent genital ul-
cers [2]. Plasma and genital HIV-1 levels are increased
during both symptomatic and asymptomatic HSV reac-
Received 2 April 2007; accepted 27 April 2007; electronically published 31
October 2007. tivations [3 6]. In vitro studies have demonstrated that
Potential conflicts of interest: C.C. has received research grant support from the HSV proteins increase HIV-1 expression [79], HSV
National Institutes of Health (NIH), the Bill and Melinda Gates Foundation, and
GlaxoSmithKline (GSK) and has served on an advisory board for GSK. J.S. has coinfection of HIV-infected cells [10], and levels of pro-
received grant support from the NIH and GSK. A.W. has received grant support inflammatory cytokines during HSV reactivation, which
from the NIH, GSK, Antigenics, 3M, Roche, and Vical; she is a consultant for
Novartis, PowderMed, and MediGene and is a speaker for Merck Vaccines. The
University of Washington Virology Division Laboratories have received grant Presented in part: 44th Annual Meeting of the Infectious Diseases Society of
funding from GSK and Novartis to perform herpes simplex virus serologic assays America, Toronto, 1115 October 2006 (abstract LB-25).
and polymerase chain reaction assays for studies funded by these companies. L.C. Financial support: GlaxoSmithKline (research grant R103); National Institutes of
directs these laboratories. He receives no salary support from these grants. Health (Centers for AIDS Research Clinical Research and Laboratory Core Grants
The Journal of Infectious Diseases 2007; 196:1500 8 AI-27757 and AI-38858, R37 AI-42528, and HSV Program Project Grant AI-30731).
2007 by the Infectious Diseases Society of America. All rights reserved. Reprints or correspondence: Dr. Connie Celum, University of Washington,
0022-1899/2007/19610-0013$15.00 Harborview Medical Center, Box 359927, 325 9th Ave., Seattle, WA 98104
DOI: 10.1086/522523 (ccelum@u.washington.edu).
HSV Suppression to Reduce HIV Levels JID 2007:196 (15 November) 1501
HIV-1 RNA quantitation assays. HIV-1 RNA was quanti- of AE buffer. A fluorescent probe based rt-PCR (TaqMan; Ap-
fied using the TaqMan real-time RNA PCR (rt-PCR) amplifica- plied Biosystems) assay was used to quantitate HSV, using 10 L
tion assay [21] or the Amplicor HIV Monitor assay (Roche Mo- of the extracted DNA for each PCR, with primers and probe
lecular Systems). The rt-PCR assay was modified to include a sequences and PCR conditions as described eslewhere [22, 23].
second gag oligonucleotide probe with a 5'-carboxyfluorescein To ensure that negative results were not due to nonspecific in-
(FAM) reporter dye and a 3'-minor groove binder/nonfluorescent hibition, each PCR also contained 50,000 copies of EXO DNA,
quencher (AR8: 6FAM-CTA TCC CAT TCT GC-3MGBNFQ) to 30 mmol/L of primers EXO186F and EXO315R, and 50 nmol/L
enhance sensitivity. HIV-1 RNA external standards and positive of probe EXO-P, labeled at the 5' end with VIC (Perkin-Elmer
and negative controls were included on each 96-well plate. To Cetus) and at the 3' end with TAMRA [24]. All negative HSV
ensure that negative results were not due to loss during nucleic PCR results required detection of EXO DNA. One positive con-
acid extraction or nonspecific inhibition of the PCR assay, 300 trol with 5000 copies of HSV was coprocessed with specimens.
copies of an external synthetic sequence control (Gene Am- Specimens were processed in parallel with aliquots of 1 PBS.
plimer pAW 109 RNA [ABI catalogue N808-0037]) were added Wells without DNA also were included in each PCR run.
to and copurified with participant and control samples. Each
PCR also contained 800 nmol/L each of the forward primer Statistical Methods
(GCC TGG GTT CCC TGT TCC) and reverse primer (CGA Sample size. The primary study end point was the effect of
CGT ACC CCT GAC ATG G) and 100 nmol/L of the labeled valacyclovir on rectal mucosal HIV-1 levels. On the basis of on
pAW probe (VIC-CCA GGC CAA TGT CTC ACC AAG CTC our previous studies among MSM [25] and assuming a 10%
TG-MGBNFQ). The laboratory was certified by the National
dropout rate, we estimated that a sample size of 20 men would
Table 2. Rates of herpes simplex virus (HSV) and HIV-1 detection and mean log10 HIV-1 levels for 20 HIV-1/HSV-2 coinfected men who
have sex with men.
Arm
NOTE. Data are no. with detectable HIV-1 RNA or HSV DNA by polymerase chain reaction/no. of samples obtained, unless otherwise indicated. Observations
begin on the second day for each study arm. Undetectable HIV-1 levels have been imputed to the midpoint between zero and the lower limit of detection.
a
P .001, compared with placebo.
b
P .02, compared with placebo.
HSV Suppression to Reduce HIV Levels JID 2007:196 (15 November) 1503
Table 3. Potential predictors of HIV-1 level in rectal mucosa and plasma, in univariate and multivariate models.
NOTE. Estimates represent the effect of the predictors on HIV-1 plasma or rectum levels (log10 copies/mL) in either univariate or multivariate (backward-
elimination) models. HSV, herpes simplex virus; NA, not applicable (interaction terms were not tested univariately); ND, not done (indicated for terms that were
not included in multivariate analysis; only 1 predictor of HSV detection was used in multivariate analysisrectal HSV detection was used as a potential predictor
in rectal HIV detection, and any HSV detection [home or clinic] was used to predict HIV detection in plasma); NS, not statistically significant when included in the
multivariate model (therefore, the term was removed).
a
Each 100-cell/L increase in CD4 cell count.
b
For HSV detection, any indicates detectable HSV-2 from any swab acquired in the study (home or clinic), and rectal indicates detectable HSV-2 DNA in
a rectal swab obtained by anoscopy.
c
Interaction term included to determine whether the treatment effect differed by CD4 cell count (each 100-cell/L increase).
levels was mediated through HSV suppression. Our detection of The mean decrease in plasma HIV-1 level was 0.33 log10 cop-
HSV in samples from the distal rectum, where mucosal secre- ies/mL over the 2 months of HSV suppression, with a more pro-
tions were sampled to quantify rectal HIV-1 shedding, further nounced effect among men with higher CD4 cell counts. Our
suggests a direct association between anogenital HSV reactiva- findings are consistent with those of a recent trial in Burkina
tion and HIV-1 replication. Thus, these findings support a sub- Faso in women, which demonstrated a mean reduction of 0.53
stantial body of research in which HSV-2 enhanced HIV-1 rep- log10 copies/mL in plasma HIV level with valacyclovir suppres-
lication in vitro and HSV-2 reactivation increased plasma and sion [14]. Natural history studies indicate that such a reduction
anogenital HIV-1 load in vivo, indicating that HSV-2 may en- in plasma HIV-1 levels may result in clinical benefits if associated
hance HIV-1 infectiousness in coinfected persons [2, 26 29]. with increased CD4 cell counts and delayed HIV disease progres-
HSV Suppression to Reduce HIV Levels JID 2007:196 (15 November) 1505
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Figure 2. Difference in mean plasma HIV level between the valacyclovir and placebo arms in 20 men who have sex with men enrolled in a
randomized crossover trial, by CD4 cell count at screening. The figure shows a greater reduction in HIV levels at higher CD4 cell counts (P .018)
sion [30, 31]. Comparable reductions in plasma HIV-1 levels biological factors and greater variability in rectal HIV-1 mea-
(0.5 log10 copies/mL) with zidovudine monotherapy have surement, given the small volumes collected by Snostrips and the
translated into clinical benefit [32], although the benefit was at- dilution for sufficient volume for the HIV-1 RNA assays. Com-
tenuated by HIV rebound due to resistance to zidovudine. How- plex factors likely influence HIV replication in the gut-
ever, this would not be expected with antiherpes therapy, be- associated lymphoid tissue, which contains more CD4 T cells
cause it acts via suppression of a cofactor in HIV-1 up-regulation than lymph nodes or the peripheral blood [34]. Further studies,
rather than directly on HIV-1 transcription. Interestingly, the including immunological and virological analyses of rectal bi-
Burkina Faso study, during which the study drug was adminis- opsy samples, will be required to understand the pathogenesis of
tered for 3 months, noted an increased effect on HIV-1 over anorectal HSV reactivation and HIV-1 replication.
time, suggesting that longer duration of HSV-2 suppression may Most HSV reactivations in this cohort were asymptomatic, so
achieve greater reductions in HIV-1 levels. In comparison, the HSV interventions directed at decreasing HIV-1 levels will re-
present crossover trial involved 2 months of administration for quire the use of suppressive rather than episodic treatment.
each arm, and no temporal effect in HIV-1 levels was observed Given the small proportion who shed HSV-2 during valacyclovir
(data not shown). Both trials showed an increased effect with a administration in this trial, additional studies are needed to as-
higher CD4 cell count. sess the effect of greater HSV-2 suppression on HIV replication.
HSV replication occurs in the anogenital mucosa, and the Valacyclovir, the hydrochloride salt of the L-valyl ester of acy-
mechanisms by which HSV reactivation increases HIV-1 levels clovir, is rapidly and completely metabolized to acyclovir,
in plasma are not well understood. Increased levels of proinflam- achieves higher plasma levels than do similar doses of acyclovir,
matory genital cytokines and chemokines were observed in a and is very safe. Studies with comparable doses of acyclovir,
cross-sectional study of HIV-1/HSV-2 coinfected African which is available generically at lower cost, have demonstrated
women who were shedding HSV-2, compared with those in comparable efficacy to valacyclovir in suppressing HSV recur-
women who were not shedding HSV-2 [33]. Among HIV/HSV- rences and shedding [35, 36]. Although acyclovir-resistant
2 coinfected MSM, anorectal HSV reactivation could directly strains of HSV are observed among HIV-1 and HSV-2infected
increase HIV replication in the gut lymphoid tissue, which con- persons [3739], the prevalence remains low (5%). Genital
tains large numbers of CD4 lymphocytes, dendrocytes, and mac- herpes lesions that are clinically refractory to acyclovir therapy
rophages. The greater reduction in plasma HIV-1 than rectal because of acyclovir resistance are much less common [39, 40],
HIV-1 levels during HSV suppression is likely a reflection of and transmission of acyclovir-resistant strains is rare [41].
HSV Suppression to Reduce HIV Levels JID 2007:196 (15 November) 1507
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