As women account for close to 50% of newly acquired HIV-1 infections worldwide , the development of an effective and well tolerated vaccine or topical compounds to prevent HIV transmission remains a major goal in the control of the AIDS pandemic. Although there has been some encouraging news on the development of a vaccine  and a topical microbicide  to prevent HIV-1 vaginal transmission, the effect seen is only partial. Thus, increasing emphasis is now placed on combining intervention strategies to enhance their efficacy [4–6]. Although vaccines that elicit protective anti-HIV envelope antibodies are desirable, the challenges involved in eliciting broadly neutralizing antibodies remain formidable . Thus, HIV vaccines have been developed primarily to elicit T-cell immunity and, as such, are unlikely to block establishment of the founder population. Rather, they are anticipated to restrict local expansion and/or dissemination of the transmitted virus, leading to lower set-point levels of HIV replication, slower disease progression, and reduction in secondary virus transmission . Interventions that can lower the inoculum dose and/or slow amplification of the virus through virus inactivation, therefore, are likely to be good candidates for evaluation in combination with T-cell-based vaccines, as they would provide a wider window of opportunity within which a vaccine-induced immune response at the mucosal surface can be effective.
The HIV-1 nucleocapsid zinc fingers have long been considered prime targets for therapeutic and microbicide intervention because they are mutationally intolerant and are required for multiple steps of the viral replication cycle [9–15]. We have demonstrated that S-acyl-2-mercaptobenzamide thioester (SAMT) zinc finger inhibitors (ZFIs) penetrate cell-free HIV-1 virions and inactivate the zinc fingers via covalent modification and metal ejection [16–18]. SAMTs reduce virus infectivity and dissemination in cell-based assays and ex-vivo cervical tissue explants [19–21]. In both an HIV-1 transgenic mouse model and a simian immunodeficiency virus (SIV) nonhuman primate (NHP) model, they reduced the level of infectious virus without a significant or consistent reduction in plasma virus RNA copy [22,23] and a 1% gel formulation of SAMT-247 protected five of six rhesus macaques from vaginal challenge with a CCR5 (R5)-tropic SHIV . Furthermore, this class of ZFI has recently been shown to have a unique mechanism of action in which the active drug is regenerated intracellularly  that may account for the prolonged reduction of the infectivity of the virus produced by infected cells .
Because a vaccine is considered the best strategy for controlling HIV infection, we initiated studies in a NHP model of vaginal HIV-1 transmission to investigate whether the modest-protective effect of a prime-boost T-cell-based vaccine could be enhanced when used in combination with a ZFI topical microbicide. We hypothesized that by reducing the infectious dose, the length of the eclipse phase of infection prior to systemic viremia would be extended, allowing the vaccine a greater interval of efficacy. Indeed, Barouch and coworkers recently showed that by reducing the inoculum dose used in a mucosal challenge infection model, the time to the first positive peripheral plasma SIV RNA levels was lengthened . To rigorously test our hypothesis, a vaccine containing only two transgenes (SIV Gag and Pol) was used to limit immunogenicity and effectiveness and a suboptimal concentration (0.1%) of SAMT-247 was selected for the microbicide. This dose was shown not to have a significant effect on HIV infection in cervical explant assays but was effective at reducing the initial viral infectivity and infectivity of progeny virus . A rhesus macaque model of vaginal infection consisting of weekly R5 SHIVSF162P3 exposures was employed. Our primary endpoint was prevention of SHIV acquisition, with postinfection viremia measurements serving as secondary endpoints. The results show that a poorly effective T-cell-based HIV-1 vaccine may actually help to protect against the spread of the AIDS virus if combined with another prevention strategy that is able to reduce the challenge inoculum.
Materials and methods
Forty-six female rhesus macaques (Macaca mulatta) of Indian origin housed individually at the Tulane National Primate Research Center (TNPRC) in compliance with the Guide for the Care and Use of Laboratory Animals were used. Studies were reviewed and approved by the Institutional Animal Care and Use Committee at TNPRC. Animals were confirmed negative for SIV, simian T-cell lymphotropic virus, and retrovirus type D. Additionally, animals were screened for the presence of the Mamu-A*01, Mamu-B*17, and Mamu-B*08 class I alleles expression using standard PCR with allele-specific primers . The macaques were not treated with Depo-provera to thin the vaginal epithelium and were sedated with ketamine-HCl (10 mg/kg) for all procedures. At the end of the study period, the animals were euthanized by intravenous administration of ketamine-HCl followed by an overdose of sodium pentobarbital.
Vaccine construction and immunization
A DNA vaccine expressing SIVmac239 Gag and Pol as a fusion protein was constructed (see Figure, Supplemental Digital Content 1A, http://links.lww.com/QAD/A155). Using a published algorithm , the SIVmac239 gag and pol genes were optimized to reflect the codon characteristics of eukaryotic expression systems and synthesized by overlapping oligonucleotides. To reduce insertion fragment size for plasmid stabilization, the pol gene was truncated at both the N- (60aa) and C- (150aa) termini. The deleted regions were shown not to contain known T-cell epitopes (Los Alamos Data Base). The gag/pol fragment was cloned into the pVAX1 (Invitrogen, Carlsbad, California, USA) DNA vaccine vector. Recombinant E1/E3-deleted Ad5 construct was generated by cloning gag/pol fragments into a shuttle vector and subsequently transferring the entire expression cassette into the adenovirus genome vector (Ad-HQ system; Vector Laboratories, Eagleville, Pennsylvania, USA). Endotoxin-free DNA vaccine and rAd5 particles were prepared by Aldevron (Fargo, North Dakota, USA) and Vector Laboratories, respectively. Rhesus macaques were primed intramuscularly with 5 mg vaccine at weeks 0 and 4. All animals then received a single intramuscular immunization of 1011 viral particles of rAd5 vectors at week 12, with challenges beginning at week 24 (see Figure, Supplemental Digital Content 1B, http://links.lww.com/QAD/A155).
Simian human immunodeficiency virus challenge
The challenge virus, SHIVSF162P3, was generated through successive rapid transfer in rhesus macaques of the CCR5 molecular clone SHIVSF162. The recovered virus was propagated and titered in RhPBMC. Weekly vaginal challenges were conducted for a total of 20 exposures or until the animals were confirmed to be infected. The initial 10 challenges were performed with 300 TCID50 (10.8 × 106 copies of SHIVSF162P3), followed by five inoculations with 1000 TCID50 virus and five with 3000 TCID50 (see Figure, Supplemental Digital Content 1C, http://links.lww.com/QAD/A155). Whole blood samples were collected weekly for 30 weeks following the first challenge and at biweekly intervals thereafter for an additional 10 weeks. For ZFI-treated rhesus macaques, 2 ml of 0.1% SAMT-247 formulated in 2% hydroxyl ethyl cellulose  was applied to the vaginal cavity 20 min prior to each virus inoculation. Plasma virus was quantified by a branch DNA signal amplification assay  (Siemens Medical Solutions Diagnostic Clinical Lab, Emeryville, California, USA).
SHIV gp120-specific IgG antibodies were measured by ELISA using a 1 : 10 dilution of plasma, as previously described . Briefly, 250 ng of purified SF162 gp120 protein was coated onto 96-well Costar plates in phosphate-buffered saline (PBS) overnight at 4°C. After blocking the plates in PBS containing 5% nonfat dry milk and 0.05% bovine serum albumin (BSA), plasma samples were added and incubated at 37°C for 2 h. Gp120-specific antibodies were detected by adding secondary HRP-conjugated antimonkey IgG (Sigma-Aldrich, St Louis, Missouri, USA) and the signal was developed by addition of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Invitrogen, Carlsbad, California, USA). The reaction was terminated by adding 1N H2SO4 and the absorbance at 450 nm was measured. Between each incubation step, the plates were washed five times with PBS, 0.005% Tween-20. SHIV T-cell immunity was determined by intracellular staining. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation from heparinized blood and resuspended in complete medium [RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin]. Cells (2 × 106) were incubated for 2 h at 37°C with medium as a negative control, overlapping 15-mer peptides spanning SIV Gag peptide (2 μg/ml), or staphylococcus enterotoxin B (SEB, 5 μg/ml; Sigma-Aldrich) as the positive control. Brefeldin A (10 μg/ml) was then added and incubation continued for an additional 5 h. Following activation, the cultured cells were stained with CD3, CD4, and CD8-specific antibodies. After being fixed and permeabilized using the GOLGI PLUG kit (BD Biosciences; Franklin Lakes, New Jersey, USA), cells were then stained with antibodies against gamma interferon (IFN-γ, clone 4S-B3) and interleukin-2 (IL-2, clone MQ1–17H12). Approximately, 105 lymphocytes, gated through forward and side scatter, chosen to be large enough to include lymphocyte blasts were acquired on a LSR II instrument (BD Biosciences) and analyzed using FloJo software (TreeStar, Ashland, Oregon, USA). Isotype controls were processed in parallel for each sample and used to set the gate for intracellular staining. Specific cytokine responses were determined by subtracting background staining seen in negative control (typically below 0.02% of gated CD4+ or CD8+ lymphocytes).
Infection-free survival curves for the naive untreated, vaccinated, ZFI-treated and combination-treated monkeys were estimated using the Kaplan–Meier method with the number of challenges treated as the time scale. Differences in time to infection between groups were assessed using log-rank tests. Differences in peak and viral loads (log transformed) between naive untreated, vaccinated, ZFI-treated and combination-treated animals were examined using Mann–Whitney U-tests. A P value less than 0.05 was considered statistically significant.
A prime-boost strategy with DNA prime and rAd5 virus boost was employed. This vaccine platform had previously been shown to suppress SHIV and SIV replication in the monkey model with vaccines that express multiple immunogens [31,32]. To ensure that protection would be suboptimal, the constructs we used contained only SIV gag, pol without env, and the animals received two instead of three DNA immunizations. Vaccinees were exposed in the absence (n = 8) and presence (n = 15) of 0.1% SAMT-247, along with eight naive controls and 15 ZFI-treated monkeys, to repeated vaginal R5 SHIVSF162P3 challenges. The dose of the ZFI used was also designed to be largely ineffective , and 20 weekly exposures using escalating doses of the virus were performed to more closely mimic high-risk exposure of women to HIV as well as to increase the sensitivity of efficacy assessment . Our criterion for infection was detectable plasma viremia (assay threshold of 165 RNA copies/ml) at 2 consecutive weeks and challenges were terminated upon systemic infection. Levels of SHIV replication in the infected macaques were monitored for over 20 weeks.
Combination treatment delays simian human immunodeficiency virus acquisition
The eight naive control macaques were infected after a median of 6.5 exposures (range 3–18; Table 1). Among the vaccinated monkeys, six were infected after a median of 10 exposures (range 1–17), with two (25%) remaining free of systemic infection after 20 challenges. Fourteen of 15 macaques challenged in the presence of the ZFI-microbicide alone were also infected, requiring a median of eight exposures (range 1–20), with one (6%) showing no evidence of systemic infection. In comparison, 12 of 15 vaccinees challenged in the presence of the ZFI-microbicide (combination) were infected after a median of 13.5 exposures (range 3–19), but three (20%) remained protected from systemic infection after receiving the maximum numbers of challenges in the study protocol. Kaplan–Meier infection-free survival curves for each group are given in Fig. 1. As anticipated from the study design, neither vaccine nor ZFI-microbicide alone reduced the rate of SHIVSF162P3 infection after repeated vaginal challenges. However, vaccine in combination with the ZFI-microbicide significantly delayed SHIV acquisition (log-rank test, P = 0.0416).
Simian human immunodeficiency virus replication is significantly reduced in combination-treated macaques
In comparison with infected naive control animals, combination-treated animals had significantly lower peak plasma virus replication during the first 3 weeks of infection (Mann–Whitney U-test, P = 0.0387) as well as reduced SHIV cumulative RNA levels during the first 20 weeks of infection (P = 0.0252); this was not observed in monkeys that were vaccinated or treated with ZFI alone (Fig. 2). Peak plasma virus replication was lower in naive control monkeys infected with low-dose virus (300 TCID50) than in those infected with 10-fold higher doses, suggestive of a dose effect (Fig. 3). Moreover, the postinfection virus replication pattern was more variable and atypical in naive macaques infected with low in comparison to high inoculum doses, with multiple spikes in viremia seen in the former. No obvious difference in replication pattern was seen among the ZFI-treated macaques infected with different inoculum doses, but the postinfection replication pattern in the infected vaccinees appeared to be the converse of that seen in the infected naive controls. Plasma viremia was higher and the replication pattern was less variable in vaccinees infected with low dose in comparison to those infected with high inoculum sizes. In contrast, an effect of the vaccine in combination with the ZFI-microbicide in controlling SHIV viremia was readily apparent. Virus replication was attenuated in the combination-treated animals, regardless of inoculum size. None of the aviremic macaques seroconverted. Moreover, two infected monkeys in the combination-treated group which had transient and low viremia also failed to seroconvert (FN85, FP24; see Figure, Supplemental Digital Content 2, http://links.lww.com/QAD/A155 showing anti-SHIV gp120 antibody responses in infected macaques). Thus, if infection is determined by seroconversion, as is usually the case in HIV-1 infection in humans, then combination treatment reduced the risk of contracting SHIV by as much as a third, in comparison to none in the treatment-naive group, 25% in the vaccination alone group and 6% in the ZFI alone group. Collectively, the data show that vaccination in combination with the ZFI-microbicide improved protection against infection and significantly reduced the levels of SHIV replication in macaques that were infected after repeated vaginal exposure.
Simian human immunodeficiency virus challenge elicits anamnestic cellular responses
Analysis of IFN-γ and IL-2 secretion from PBMCs freshly isolated from the group of combination-treated monkeys in response to SIV Gag antigen stimulation 2 weeks after rAd5 virus boost showed SHIV-specific T-lymphocyte responses in a majority of the DNA-primed monkeys, demonstrating that the vaccine is immunogenic. Response was largely restricted to IFN-γ secretion, with comparable percentages of CD4+ and CD8+ cells responding (Fig. 4a). A SHIV-specific T-cell response could also be detected in peripheral blood 1–2 weeks after the first virus exposure (Fig. 4b), with a particularly robust response in four of 15 animals: FP24, which was infected after three virus exposures, EE78 which required 19 exposures to be infected, and two of the three resistant monkeys (FM42, FP96; Table 1). Notably, SIV gag-specific CD4+ T cells were more readily demonstrable than CD8+ T cells in the macaque that was infected early (FP24) than in those that were infected late (EE78) or resisted SHIV challenges (FM42, FP96). Because virus-specific CD4+ T cells are the preferred targets for virus infection , the biased CD4 cell response in FP24 may have contributed to the ready susceptibility of this monkey to infection, but which was rapidly controlled. Conversely, the robust and balanced CD4 and CD8 anamnestic responses seen in EE78, FM42, and FP96 suggested that they may have played a role in delaying (EE78) or containing (FM42, FP96) systemic infection.
Control of simian human immunodeficiency virus replication in combination-treated macaques cannot be explained by differences in the major histocompatibility complex class I loci
None of the animals used in this study expressed the restrictive MamuB*08 and B*17 genotypes, but three and five of the ZFI-treated and combination-treated SHIV-infected monkeys, respectively, expressed the MamuA*01 major histocompatibility complex. However, we did not find lower peak or chronic viremia in the SHIV-infected MamuA*01-positive animals in comparison to the MamuA*01-negative monkeys (see Figure, Supporting Digital Content 3 showing the effect of MamuA*01 allelic expression on SHIV replication), consistent with a previous report that this haplotype has a weak effect on viral control . There was also no apparent correlation between virus replication levels and the allelic polymorphisms of TRIM5α (data not shown).
This proof-of-concept study demonstrates that a partially effective T-cell-based vaccine, in combination with a suboptimal dose of a topically applied microbicide, delays vaginal SHIV acquisition in macaques that were repeatedly challenged and weakens pathogenicity of the infection in those that became infected. Peak viremia and total RNA copies were significantly lower in the combination-treated infected macaques than in the naive control monkeys. As the levels of HIV viremia correlate with the likelihood of virus transmission and disease [36–39], our findings suggest that the combined use of a T-cell-based vaccination and a topical microbicide that reduces the viral challenge and infectivity could slow the spread of HIV-1.
We find that the SIVgag/pol DNA prime/rAd5 boost vaccine alone may afford some immune control. Although the differences did not reach statistical significance, only six of eight vaccinees were infected relative to all eight in the naive control group, with increased number of exposures needed to establish systemic infection. As expected from the experimental design, 0.1% SAMT-247 alone was ineffective in preventing or controlling SHIV infection in comparison to the naive control macaques. Fourteen of the 15 ZFI-treated macaques were infected, with no reduction in viremia as compared to the naive control animals. In this regard, NCp7 inhibitor action has been associated with the inactivation of newly produced virus in the absence of decreases in viral RNA copy, p24, or supernatant reverse transcriptase activity in cellular models , transgenic mouse model of HIV-1 infection , and a therapeutic model in cynomolgus macaques . Moreover, the dose of SAMT-247 used (0.1%) was chosen based on previous data showing that it did not significantly affect p24 production from cervical explants, but reduced the initial viral infectivity as well as infectivity of the progeny virus . As recent studies show that the time to the first positive peripheral plasma SIV RNA levels was lengthened by reducing the infectious dose used in a mucosal challenge infection model , our finding of reduction of viremia in the combination group supports the hypothesis that the DNA prime-rAd virus boost vaccine established sufficient immunity for control of SHIV infection and pathogenesis if assisted by a microbicide that expands the potential window of vaccine effectiveness by reducing the initial virus seeding to slow virus spread and replication.
This proof-of-concept study does have several limitations. First, the SIV gag and pol in the immunogens and challenge virus were genetically matched, potentially overestimating efficacy. Thus, future studies will be required to evaluate the utility of vaccine/microbicide combination against heterologous virus challenge. Second, we made the assumption that the susceptibilities to infection were equal for all animals within each group. We monitored for major histocompatibility complex (MHC) I and TRIM5α expression in the monkeys used in this study and did not observe inherent genetic differences at these loci that could explain differences in SHIV susceptibility. Nevertheless, an examination of the extent of animal-to-animal variation in susceptibility will require a much larger number of animals than the estimation of prevention efficacy. Third, the number of animals used, in particular the vaccine arm alone group, was too few to allow a robust direct comparison of time to SHIV infection between the experimental groups. Finally, SHIV T-cell immunity was analyzed using peripheral blood cells, which may not accurately reflect mucosal events. However, a repeat-challenge model prohibits sampling of mucosal and lymphoid tissue sites due to concerns that such procedures may alter SHIV susceptibility.
In summary, the combined use of a topical microbicide to lower the initial viral seeding/spread and a modestly effective T-cell-based vaccine to immunologically contain the early virological events of mucosal transmission holds promise as a preventive approach to control the spread of the AIDS epidemic. Conceivably, the use of a more potent topical gel (20–50% efficacy) in combination with a more effective vaccine that includes an Env component could increase protection against infection. Our findings represent an important first step toward improving HIV-1 prevention measures and have implications for the design of combination packages against HIV-1. A 31% protection against HIV-1 acquisition was reported in the recently completed RV144 study in Thailand using a recombinant poxvirus vector combined with a recombinant envelope protein, with subgroup analyses suggesting that the vaccine may be more effective at blocking low-risk than high-risk HIV-1 transmission . Thus, it is tempting to speculate that the modest protective effect of the RV144 vaccine might be enhanced if the individuals were using a microbicide at the same time. Furthermore, a 1% vaginal gel formulation of tenofovir reduced HIV acquisition by an estimated 39% overall and by 54% in women with high gel adherence , suggesting that a major obstacle to microbicide effectiveness will be adherence. A vaccine could compensate for compliance issues in the use of topical microbicides. Further studies will be required to determine whether the additive or possibly synergistic protection provided by the combination of a modestly effective vaccine and a topical microbicide depends on the mechanism of action of the microbicide or the vaccine components.
The authors thank L. Stamatatos for sgp120, and T.M. Rasmussem and R. Hayashi for technical assistance. SIV gag peptides were obtained through the NIH AIDS Research and Reagent Program, Division of AIDS, NIAID, NIH. C.C.M., Y.H., M.L.S., and E.A. conceived experiments. A.G., L.T., W.R., M.S., S.T.S., N.T., and J.B. performed experiments. Y.L. performed statistical analysis. C.C.M., Y.H., L.M.M.J., M.L.S., and E.A. prepared the manuscript.
Conflicts of interest
The present work was supported in part by the NIH Intramural AIDS Targeted Antiretroviral Program (IATAP) (E.A.), the Tulane National Primate Research Center Base grant RR00164, and NIH grant AI069991 (C.C.M.). The authors declare no competing financial interests or conflicts of interest.
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HIV; nucleocapsid inhibitor; prevention; topical microbicide; vaccine
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