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Development of a microbicide that prevents rectal transmission of human immunodeficiency virus (HIV) is a vital component in reducing HIV spread. We recently demonstrated that a formulation of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan reduced vaginal infection of macaques with simian immunodeficiency virus SIVmac239 with HIV-1HxB2 reverse transcriptase (SHIV-RT). Herein, we performed the first testing of MIV-150–carrageenan against rectal infection. Rhesus macaques were treated rectally with MIV-150–carrageenan or methyl cellulose (MC) placebo gel up to 4 h prior to rectal challenge with 103 or 104 50% tissue culture infective doses (TCID50) of SHIV-RT. Infection was assessed by measuring plasma virus RNA as well as T and B cell responses. MIV-150–carrageenan protected all animals challenged with 103 TCID50 when gel was applied either 30 min or 4 h prior to challenge, while 100% of the MC-treated animals became infected (n = 4 each; P < 0.03). Partial protection (2 of 4 animals) by MIV-150–carrageenan was observed for rectal challenge with 10-fold more virus applied 4 h after the gel. Sequencing of the RT gene from plasma virus RNA isolated at peak viremia confirmed that both of these animals (like infected MC controls) were infected with wild-type virus. Infection correlated with the development of SIV-specific T and B cell responses. MIV-150 was detected in the rectal fluids and tissues 4 h after gel application but was not detected in the blood at any time (0.5 to 24 h). These data are promising for the development of NNRTI-containing gels to prevent rectal HIV transmission.

Abstract

We previously showed that a carrageenan (CG) gel containing 50 μM MIV-150 (MIV-150/CG) reduced vaginal simian/human immunodeficiency virus (SHIV)-RT infection of macaques (56%, p>0.05) when administered daily for 2 weeks with the last dose given 8 h before challenge. Additionally, when 100 mg of MIV-150 was loaded into an intravaginal ring (IVR) inserted 24 h before challenge and removed 2 weeks after challenge, >80% protection was observed (p<0.03). MIV-160 is a related NNRTI with a similar IC50, greater aqueous solubility, and a shorter synthesis. To objectively compare MIV-160 with MIV-150, herein we evaluated the antiviral effects of unformulated MIV-160 in vitro as well as the in vivo protection afforded by MIV-160 delivered in CG (MIV-160/CG gel) and in an IVR under regimens used with MIV-150 in earlier studies. Like MIV-150, MIV-160 exhibited potent antiviral activity against SHIV-RT in macaque vaginal explants. However, formulated MIV-160 exhibited divergent effects in vivo. The MIV-160/CG gel offered no protection compared to CG alone, whereas the MIV-160 IVRs protected significantly. Importantly, the results of in vitro release studies of the MIV-160/CG gel and the MIV-160 IVR suggested that in vivo efficacy paralleled the amount of MIV-160 released in vitro. Hundreds of micrograms of MIV-160 were released daily from IVRs while undetectable amounts of MIV-160 were released from the CG gel. Our findings highlight the importance of testing different modalities of microbicide delivery to identify the optimal formulation for efficacy in vivo.

Background

Repeated use, coitus-independent microbicide gels that do not contain antiretroviral agents also used as first line HIV therapy are urgently needed to curb HIV spread. Current formulations require high doses (millimolar range) of antiretroviral drugs and typically only provide short-term protection in macaques. We used the macaque model to test the efficacy of a novel combination microbicide gel containing zinc acetate and micromolar doses of the novel non-nucleoside reverse transcriptase inhibitor MIV-150 for up to 24 h after repeated gel application.

Methods and Findings

Rhesus macaques were vaginally challenged with SHIV-RT up to 24 h after repeated administration of microbicide versus placebo gels. Infection status was determined by measuring virologic and immunologic parameters. Combination microbicide gels containing 14 mM zinc acetate dihydrate and 50 µM MIV-150 afforded full protection (21 of 21 animals) for up to 24 h after 2 weeks of daily application. Partial protection was achieved with the MIV-150 gel (56% of control at 8 h after last application, 11% at 24 h), while the zinc acetate gel afforded more pronounced protection (67% at 8–24 h). Marked protection persisted when the zinc acetate or MIV-150/zinc acetate gels were applied every other day for 4 weeks prior to challenge 24 h after the last gel was administered (11 of 14 protected). More MIV-150 was associated with cervical tissue 8 h after daily dosing of MIV-150/zinc acetate versus MIV-150, while comparable MIV-150 levels were associated with vaginal tissues and at 24 h.

Conclusions

A combination MIV-150/zinc acetate gel and a zinc acetate gel provide significant protection against SHIV-RT infection for up to 24 h. This represents a novel advancement, identifying microbicides that do not contain anti-viral agents used to treat HIV infection and which can be used repeatedly and independently of coitus, and underscores the need for future clinical testing of their safety and ability to prevent HIV transmission in humans.

Topical microbicides that block the sexual transmission of HIV and herpes simplex virus 2 (HSV-2) are desperately needed to reduce the incidence of HIV infections worldwide. Previously we completed phase 3 testing of the carrageenan-based gel Carraguard. Although the trial did not show that Carraguard is effective in preventing HIV transmission during vaginal sex, it did show that Carraguard is safe when used weekly for up to 2 years. Moreover, Carraguard has in vitro activity against human papillomavirus (HPV) and HSV-2 and favorable physical and rheological properties, which makes it a useful vehicle to deliver antiviral agents such as zinc acetate. To that end, we previously reported that a prototype zinc acetate carrageenan gel protects macaques against vaginal challenge with combined simian-human immunodeficiency virus reverse transcriptase (SHIV-RT). Herein, we report the safety and efficacy of a series of zinc acetate and/or carrageenan gels. The gels protected mice (75 to 85% survival; P < 0.001) against high-dose (106-PFU) HSV-2 vaginal or rectal challenge. In contrast, zinc acetate formulated in HEC (hydroxyethylcellulose; or the Universal Placebo) failed to protect mice against the high-dose vaginal HSV-2 challenge (similar to aqueous zinc acetate solution and the placebo controls). The gels were found to be effective spreading gels, exhibited limited toxicity in vitro, caused minimal damage to the architecture of the cervicovaginal and rectal mucosae in vivo, and induced no increased susceptibility to HSV-2 infection in a mouse model. Our results provide a strong rationale to further optimize and evaluate the zinc acetate/carrageenan gels for their ability to block the sexual transmission of HIV and HSV-2.

Anti-HIV microbicides are being investigated in clinical trials and understanding how promising strategies work, coincident with demonstrating efficacy in vivo, is central to advancing new generation microbicides. We evaluated Carraguard® and a new generation Carraguard-based formulation containing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (PC-817). Since dendritic cells (DCs) are believed to be important in HIV transmission, the formulations were tested for the ability to limit DC-driven infection in vitro versus vaginal infection of macaques with RT-SHIV (SIVmac239 bearing HIV reverse transcriptase). Carraguard showed limited activity against cell-free and mature DC-driven RT-SHIV infections and, surprisingly, low doses of Carraguard enhanced infection. However, nanomolar amounts of MIV-150 overcame enhancement and blocked DC-transmitted infection. In contrast, Carraguard impeded infection of immature DCs coincident with DC maturation. Despite this variable activity in vitro, Carraguard and PC-817 prevented vaginal transmission of RT-SHIV when applied 30 min prior to challenge. PC-817 appeared no more effective than Carraguard in vivo, due to the limited activity of a single dose of MIV-150 and the dominant barrier effect of Carraguard. However, 3 doses of MIV-150 in placebo gel at and around challenge limited vaginal infection, demonstrating the potential activity of a topically applied NNRTI. These data demonstrate discordant observations when comparing in vitro and in vivo efficacy of Carraguard-based microbicides, highlighting the difficulties in testing putative anti-viral strategies in vitro to predict in vivo activity. This work also underscores the potential of Carraguard-based formulations for the delivery of anti-viral drugs to prevent vaginal HIV infection.

A vaginal gel containing 1% tenofovir (TFV) was found to be safe and effective in reducing HIV infection in women when used pericoitally. Because of the long intracellular half-life of TFV and high drug exposure in vaginal tissues, we hypothesized that a vaginal gel containing TFV may provide long-lasting protection. Here, we performed delayed-challenge experiments and showed that vaginal 1% TFV gel protected 4/6 macaques against vaginal simian-human immunodeficiency virus (SHIV) exposures occurring 3 days after gel application, demonstrating long-lasting protection. Despite continued gel dosing postinfection, neither breakthrough infection had evidence of drug resistance by ultrasensitive testing of SHIV in plasma and vaginal lavage. Analysis of the active intracellular tenofovir diphosphate (TFV-DP) in vaginal lymphocytes collected 4 h to 3 days after gel dosing persistently showed high TFV-DP levels (median, 1,810 fmol/106 cells) between 4 and 24 h that exceed the 95% inhibitory concentration (IC95), reflecting rapid accumulation and long persistence. In contrast to those in peripheral blood mononuclear cells (PBMCs) following oral dosing, TFV-DP levels in vaginal lymphocytes decreased approximately 7-fold by 3 days, exhibiting a much higher rate of decay. We observed a strong correlation between intracellular TFV-DP in vaginal lymphocytes, in vitro antiviral activity, and in vivo protection, suggesting that TFV-DP above the in vitro IC95 in vaginal lymphocytes is a good predictor of high efficacy. Data from this model reveal an extended window of protection by TFV gel that supports coitus-independent use. The identification of protective TFV-DP concentrations in vaginal lymphocytes may facilitate the evaluation of improved delivery methods of topical TFV and inform clinical studies.

Tenofovir gel (1%) is being developed as a microbicide for the prevention of human immunodeficiency virus (HIV) infection and has been shown to reduce transmission to women by 39%. The gel also prevents infection in macaques when applied intravaginally or intrarectally prior to challenge with simian-human immunodeficiency virus (SHIV), but very little pharmacokinetic information for macaques is available to help extrapolate the data to humans and thus inform future development activities. We have determined the pharmacokinetics of tenofovir in macaques following intravaginal and intrarectal administration of 0.2, 1, and 5% gels. Plasma and vaginal and rectal fluid samples were collected up to 24 h after dosing, and at 24 h postdosing biopsy specimens were taken from the vaginal wall, cervix, and rectum. Following vaginal and rectal administration, tenofovir rapidly distributed to the matrices distal to the site of administration. In all matrices, exposure increased with increasing dose, and with the 1% and 5% formulations, concentrations remained detectable in most animals 24 h after dosing. At all doses, concentrations at the dosing site were typically 1 to 2 log units higher than those in the opposite compartment and 4 to 5 log units higher than those in plasma. Exposure in vaginal fluid after vaginal dosing was 58 to 82% lower than that in rectal fluid after rectal dosing, but plasma exposure was 1- to 2-fold greater after vaginal dosing than after rectal dosing. These data suggest that a tenofovir-based microbicide may have the potential to protect when exposure is via vaginal or anal intercourse, regardless of whether the microbicide is applied vaginally or rectally.

Three gel formulations (1%, 3%, and 5% [wt/wt]) of SPL7013, a dendrimer known to have antiviral (anti-human immunodeficiency virus and anti-herpes simplex virus) activities, completed a range of preclinical tests in the pigtailed macaque models for vaginally and rectally applied topical microbicide safety assessments. The vaginal safety profile of the 3% SPL7013 gel formulation was equal to that of the 1% formulation but was superior to that of the 5% formulation. The 3% SPL7013 gel was further evaluated for rectal safety and for antichlamydial efficacy with cervical challenge with Chlamydia trachomatis. This first-generation dendrimer-based product was shown to be safe to the vaginal and rectal microenvironments with repeated daily use. However, a single intravaginal application of the 3% (wt/wt) SPL7013 gel did not provide protection from the acquisition of cervical chlamydial infection.

New-generation gels that deliver potent antiretroviral drugs against human immunodeficiency virus type 1 have renewed hopes for topical prophylaxis as a prevention strategy. Previous preclinical research with monkey models suggested that high concentrations and drug combinations are needed for high efficacy. We evaluated two long-acting reverse transcriptase inhibitors, tenofovir (TFV) and emtricitabine (FTC), by using a twice-weekly repeat challenge macaque model and showed that a preexposure vaginal application of gel with 1% TFV alone or in combination with 5% FTC fully protected macaques from a total of 20 exposures to simian-human immunodeficiency virus SF162p3. FTC and TFV were detected in plasma 30 min after vaginal application, suggesting rapid absorption. FTC was detected more frequently than TFV and showed higher levels, reflecting the fivefold-higher concentration of this drug than of TFV. Two of 12 repeatedly exposed but protected macaques showed limited T-cell priming, which did not induce resistance to infection when macaques were rechallenged. Thus, single drugs with durable antiviral activity can provide highly effective topical prophylaxis and overcome the need for noncoital use or for drug combinations which are more complex and costly to formulate and approve.

The nonnucleoside reverse transcriptase inhibitor UC781 is under development as a potential microbicide to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1). Two gel formulations of UC781 (0.1% and 1.0%) were evaluated in a range of preclinical safety assessments, including systemic absorption analysis following topical application in the pig-tailed macaque models for vaginally and rectally applied topical microbicides. High-sensitivity high-performance liquid chromatography analysis of serum samples showed that no systemic absorption of UC781 was detected after repeated vaginal or rectal application of either product. However, high levels of UC781 were detectable in the cervicovaginal lavage samples up to 6 h after product exposure. Both formulations were safe to the vaginal microenvironment, even with repeated daily use, as evidenced by colposcopy, cytokine analysis, and lack of impact on vaginal microflora. By contrast, rectal application of the 1.0% UC781 formulation caused an increased expression of numerous cytokines not observed after rectal application of the 0.1% UC781 formulation. These results provide additional support for the continued development of UC781 formulations as anti-HIV microbicides.

Maraviroc (MVC) gels are effective at protecting rhesus macaques from vaginal SHIV transmission, but breakthrough infections can occur. To determine the effects of a vaginal MVC gel on infecting SHIV populations in a macaque model, we analyzed plasma samples from three rhesus macaques that received a MVC vaginal gel (day 0) but became infected after high-dose SHIV-162P3 vaginal challenge. Two infected macaques that received a placebo gel served as controls. The infecting SHIV-162P3 stock had an overall mean genetic distance of 0.294±0.027%; limited entropy changes were noted across the envelope (gp160). No envelope mutations were observed consistently in viruses isolated from infected macaques at days 14–21, the time of first detectable viremia, nor selected at later time points, days 42–70. No statistically significant differences in MVC susceptibilities were observed between the SHIV inoculum (50% inhibitory concentration [IC50] 1.87 nM) and virus isolated from the three MVC-treated macaques (MVC IC50 1.18 nM, 1.69 nM, and 1.53 nM, respectively). Highlighter plot analyses suggested that infection was established in each MVC-treated animal by one founder virus genotype. The expected Poisson distribution of pairwise Hamming Distance frequency counts was observed and a phylogenetic analysis did not identify infections with distinct lineages from the challenge stock. These data suggest that breakthrough infections most likely result from incomplete viral inhibition and not the selection of MVC-resistant variants.

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV162P4. Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.

Background

In a previous study, progesterone treatment of female monkeys immunized with live, attenuated SHIV89.6 abrogated the generally consistent protection from vaginal simian immunodeficiency virus (SIV) challenge. The mechanisms responsible for the loss of protection remain to be defined. The objective of the present study was to determine whether Depo-Provera® administration alters protection from intravenous SIV challenge in SHIV-immunized female macaques.

Methods and Findings

Two groups of female macaques were immunized with attenuated SHIV89.6 and then challenged intravenously with SIVmac239. Four weeks before challenge, one animal group was treated with Depo-Provera®, a commonly used injectable contraceptive progestin. As expected, SHIV-immunized monkeys had significantly lower peak and set-point plasma viral RNA levels compared to naïve controls, but in contrast to previously published findings with vaginal SIV challenge, the Depo-Provera® SHIV-immunized animals controlled SIV replication to a similar, or even slightly greater, degree than did the untreated SHIV-immunized animals. Control of viral replication from week 4 to week 20 after challenge was more consistent in the progesterone-treated, SHIV-immunized animals than in untreated, SHIV-immunized animals. Although levels of interferon-γ production were similar, the SIV-specific CD8+ T cells of progesterone-treated animals expressed more functions than the anti-viral CD8+ T cells from untreated animals.

Conclusions

Depo-Provera® did not diminish the control of viral replication after intravenous SIV challenge in female macaques immunized with a live-attenuated lentivirus. This result contrasts with the previously reported effect of Depo-Provera® on protection from vaginal SIV challenge and strongly implies that the decreased protection from vaginal challenge is due to effects of progesterone on the genital tract rather than to systemic effects. Further, these results demonstrate that the effects of hormonal contraceptives on vaccine efficacy need to be considered in the context of testing and use of an AIDS vaccine.

Dapivirine is a nonnucleoside reverse transcriptase inhibitor being developed as a topical microbicide for the prevention of human immunodeficiency virus infection. The distribution of radioactivity and drug in plasma and in vaginal, cervical, and draining lymph node tissues was investigated after daily application of a vaginal gel formulation of [14C]dapivirine to rhesus macaques. This was preceded by a preliminary study with rabbits. Following the intravaginal administration of [14C]dapivirine (∼0.1 mg/ml [15 μCi/ml]) to rabbits (0.5 ml/day) and macaques (1 ml/day) for 7 days, the dapivirine levels associated with vaginal and cervical tissue samples 1 h after the final dose were high (quantities of μg/g of tissue) and remained detectable at 24 h (mean, ≥2.5 ng/g in rabbits) and 48 h (mean, >80 ng/g in macaques). Radioactivity levels were low in the plasma and very low or unquantifiable in the draining lymph nodes of the macaques. Microautoradiography identified drug-related material (DRM) on the surfaces of the vaginal and cervical tissues of the rabbits and macaques. Although DRM was primarily associated with the outermost layer of shedding cells in rabbits, two animals showed some evidence of small quantities in the mucosal epithelium of the cervix. In macaques, DRM was seen within the keratinized layer of the vaginal epithelium and and was found to extend into the superficial cellular layers, and in at least one animal it appeared to be present in the deepest (germinal) layer of the epithelium and in submucosal tissues. The persistence of biologically significant concentrations of dapivirine in vaginal and cervical tissues for >24 h supports the development of dapivirine as a microbicide for once daily application.

An effective vaginal microbicide could reduce human immunodeficiency virus type 1 (HIV-1) transmission to women. Among microbicide candidates in clinical development is Maraviroc (MVC), a small molecule drug that binds the CCR5 co-receptor and impedes HIV-1 entry into cells. Delivered systemically, MVC reduces viral load in HIV-1-infected people, but its ability to prevent transmission is untested. We have now evaluated MVC as a vaginal microbicide, using a stringent model involving challenge of rhesus macaques with a high-dose of a CCR5-using virus, SHIV-162P3. Gel-formulated, prescription-grade MVC provided dose-dependent protection, half-maximally at 0.5 mM (0.25 mg/ml). The duration of protection was transient; the longer the delay between MVC application and virus challenge, the less protection (T1/2 ~ 4 h). As expected, MVC neither protected against challenge with a CXCR4-using virus, SHIV-KU1, nor exacerbated post-infection viremia. These findings validate MVC development as a vaginal microbicide for women, and should guide clinical programs.

We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIVmac251 at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.

Background

Worldwide, ~90% of all HIV transmissions occur mucosally; almost all involve R5 strains. Risks of sexual HIV acquisition are highest for rectal, followed by vaginal and then oral exposures.

Methods

Mucosal lacerations may affect the rank-order of susceptibility to HIV but cannot be assessed in humans. We measured relative virus transmissibility across intact mucosae in macaques using a single stock of SHIV-1157ipd3N4, a simian-human immunodeficiency virus encoding a primary R5 HIV clade C env (SHIV-C).

Results

The penetrability of rhesus macaque mucosae differed significantly, with rectal challenge requiring the least virus, followed by the vaginal and then oral routes. These findings imply that intrinsic mucosal properties are responsible for the differential mucosal permeability. The latter paralleled the rank-order reported for humans, with relative risk estimates within the range of epidemiologic human studies. To test whether inflammation facilitates virus transmission – as predicted from human studies – we established a macaque model of localized buccal inflammation. Systemic infection occurred across inflamed, but not normal buccal mucosa.

Conclusion

Our primate data recapitulate virus transmission risks observed in humans, thus establishing R5 SHIV-1157ipd3N4 in macaques as a robust model system to study cofactors involved in human mucosal HIV transmission and its prevention.

The presence, at the time of challenge, of antiviral effector T cells in the vaginal mucosa of female rhesus macaques immunized with live-attenuated simian-human immunodeficiency virus 89.6 (SHIV89.6) is associated with consistent and reproducible protection from pathogenic simian immunodeficiency virus (SIV) vaginal challenge (18). Here, we definitively demonstrate the protective role of the SIV-specific CD8+ T-cell response in SHIV-immunized monkeys by CD8+ lymphocyte depletion, an intervention that abrogated SHIV-mediated control of challenge virus replication and largely eliminated the SIV-specific T-cell responses in blood, lymph nodes, and genital mucosa. While in the T-cell-intact SHIV-immunized animals, polyfunctional and degranulating SIV-specific CD8+ T cells were present in the genital tract and lymphoid tissues from the day of challenge until day 14 postchallenge, strikingly, expansion of SIV-specific CD8+ T cells in the immunized monkeys was minimal and limited to the vagina. Thus, protection from uncontrolled SIV replication in animals immunized with attenuated SHIV89.6 is primarily mediated by CD8+ T cells that do not undergo dramatic systemic expansion after SIV challenge. These findings demonstrate that despite, and perhaps because of, minimal systemic expansion of T cells at the time of challenge, a stable population of effector-cytotoxic CD8+ T cells can provide significant protection from vaginal SIV challenge.

Single-genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection. Use of this strategy as a means for identifying transmitted viruses suggested that intrarectal simian immunodeficiency virus (SIV) inoculation of macaques recapitulates key features of human rectal infection. However, no studies have used the SGA strategy to identify vaginally transmitted virus(es) in macaques or to determine how early SIV diversification in vaginally infected animals compares with HIV-1 in humans. We used SGA to amplify 227 partial env sequences from a SIVmac251 challenge stock and from seven rhesus macaques at the earliest plasma viral RNA-positive time point after low- and high-dose intravaginal inoculation. Sequences were analyzed phylogenetically to determine the relationship of transmitted/founder viruses within and between each animal and the challenge stock. In each animal, discrete low-diversity env sequence lineages were evident, and these coalesced phylogenetically to identical or near-identical env sequences in the challenge stock, thus confirming the validity of the SGA sequencing and modeling strategy for identifying vaginally transmitted SIV. Between 1 and 10 viruses were responsible for systemic infection, similar to humans infected by sexual contact, and the set of viruses transmitted to the seven animals studied represented the full genetic constellation of the challenge stock. These findings recapitulate many of the features of sexual HIV-1 transmission in women. Furthermore, the SIV rhesus macaque model can be used to understand the factors that influence the transmission of single versus multiple SIV variants.

Background

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an important category of drugs for both chemotherapy and prevention of human immunodeficiency virus type 1 (HIV-1) infection. However, current non-human primate (NHP) models utilizing simian immunodeficiency virus (SIV) or commonly used chimeric SHIV (SIV expressing HIV-1 envelope) are inadequate due to the insensitivity to NNRTIs. To develop a NHP model for evaluation of NNRTI compounds, we characterized a RT-SHIV virus that was assembled by replacing the SIVmac239 reverse transcriptase (RT) with that of HIV-1HXB2. Since RT-SHIV exhibited in vitro characteristics of high infectivity, CCR5-usage, and sensitivity to HIV-1 specific NNRTIs, this virus was thought to be suitable for mucosal transmission and then was used to carry out a vaginal transmission study in pigtail macaques (Macaca nemestrina).

Results

RT-SHIV exhibited in vitro characteristics of an infectious CCR5-tropic chimeric virus. This virus was not only highly sensitive to HIV-1 RT specific NNRTIs; its replication was also inhibited by a variety of NRTIs and protease inhibitors. For in vivo vaginal transmission studies, macaques were either pretreated with a single dose of DMPA (depot medroxyprogesterone acetate) or left untreated before intravaginal inoculation with 500 or 1,000 TCID50 of RT-SHIV. All macaques became systemically infected by 2 or 3 weeks post-inoculation exhibiting persistent high viremia, marked CD4+T cell depletion, and antiviral antibody response. DMPA-pretreated macaques showed a higher mean plasma viral load after the acute infection stage, highly variable antiviral antibody response, and a higher incidence of AIDS-like disease as compared with macaques without DMPA pretreatment.

Conclusion

This chimeric RT-SHIV has exhibited productive replication in both macaque and human PBMCs, predominantly CCR5-coreceptor usage for viral entry, and sensitivity to NNRTIs as well as other anti-HIV compounds. This study demonstrates rapid systemic infection in macaques following intravaginal exposure to RT-SHIV. This RT-SHIV/macaque model could be useful for evaluation of NNRTI-based therapies, microbicides, or other preventive strategies.

Although priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, followed by HIV/SIV envelope boosting, has proven highly immunogenic, resulting in protection from SIV/simian-human immunodeficiency virus (SHIV) challenges, Ad5hr recombinant distribution, replication, and persistence have not been examined comprehensively in nonhuman primates. We utilized Ad5hr-green fluorescent protein and Ad5hr-SIV recombinants to track biodistribution and immunogenicity following mucosal priming of rhesus macaques by the intranasal/intratracheal, sublingual, vaginal, or rectal route. Ad recombinants administered by all routes initially targeted macrophages in bronchoalveolar lavage (BAL) fluid and rectal tissue, later extending to myeloid dendritic cells in BAL fluid with persistent expression in rectal mucosa 25 weeks after the last Ad immunization. Comparable SIV-specific immunity, including cellular responses, serum binding antibody, and mucosal secretory IgA, was elicited among all groups. The ability of the vector to replicate in multiple mucosal sites irrespective of delivery route, together with the targeting of macrophages and professional antigen-presenting cells, which provide potent immunogenicity at localized sites of virus entry, warrants continued use of replicating Ad vectors.

Immunization with attenuated lentiviruses is the only reliable method of protecting rhesus macaques (RM) from vaginal challenge with pathogenic simian immunodeficiency virus (SIV). CD8+ lymphocyte depletion prior to SIVmac239 vaginal challenge demonstrated that a modest, Gag-specific CD8+ T cell response induced by immunization with simian-human immunodeficiency virus 89.6 (SHIV89.6) protects RM. Although CD8+ T cells are required for protection, there is no anamnestic expansion of SIV-specific CD8+ T cells in any tissues except the vagina after challenge. Further, SHIV immunization increased the number of viral target cells in the vagina and cervix, suggesting that the ratio of target cells to antiviral CD8+ T cells was not a determinant of protection. We hypothesized that persistent replication of the attenuated vaccine virus modulates inflammatory responses and limits T cell activation and expansion by inducing immunoregulatory T cell populations. We found that attenuated SHIV infection decreased the number of circulating plasmacytoid dendritic cells, suppressed T cell activation, decreased mRNA levels of proinflammatory mediators, and increased mRNA levels of immunoregulatory molecules. Three days after SIV vaginal challenge, SHIV-immunized RM had significantly more T regulatory cells in the vagina than the unimmunized RM. By day 14 postchallenge, immune activation and inflammation were characteristic of unimmunized RM but were minimal in SHIV-immunized RM. Thus, a modest vaccine-induced CD8+ T cell response in the context of immunoregulatory suppression of T cell activation may protect against vaginal HIV transmission.

Objective

Sexual transmission of HIV is the most common route of HIV transmission throughout the world. To prevent sexually transmitted HIV infection, a vaccine is urgently needed. A previous report demonstrated the targeted immunization of the iliac lymph nodes with simian immunodeficiency virus (SIV) subunits protects rhesus macaques from rectal challenge with SIV. We sought to determine whether this immunization strategy could protect rhesus macaques from vaginal challenge with SIV.

Design

Macaques were immunized with either whole-killed SIV or envelope and core subunit antigen vaccines. Using three independent groups, with three macaques in each group, macaques were immunized by the targeted iliac lymph-node (TILN) route, injecting the vaccine close to the iliac lymph nodes that drain the genital tract.

Results

The TILN immunization procedure induced high-titer SIV-specific immunoglobulin (Ig) G antibodies in serum in all animals and anti-SIV IgG and IgA antibodies in the cervicovaginal secretions of most animals. After a series of three or four TILN immunizations, the animals were intravaginally challenged with SIVmac251. All animals became virus isolation-positive, except one animal immunized with SIV p27 and gp120. This animal was virus isolation-negative but SIV DNA proviral sequences were detected in peripheral blood mononuclear cells.

Conclusions

In this series of studies, reliable protection from vaginal transmission of SIV was not achieved by the TILN immunization procedure.

Antiretroviral entry inhibitors are now being considered as vaginally administered microbicide candidates for the prevention of the sexual transmission of human immunodeficiency virus. Previous studies testing the entry inhibitors maraviroc and CMPD167 in aqueous gel formulations showed efficacy in the macaque challenge model, although protection was highly dependent on the time period between initial gel application and subsequent challenge. In this paper, we describe the sustained release of maraviroc and CMPD167 from matrix-type silicone elastomer vaginal rings both in vitro and in vivo. Both inhibitors were released continuously during 28 days from rings in vitro at rates of 100 to 2,500 μg/day. In 28-day pharmacokinetic studies in rhesus macaques, the compounds were measured in the vaginal fluid and vaginal tissue; steady-state fluid concentrations were ∼106-fold greater than the 50% inhibitory concentrations (IC50s) for simian human immunodeficiency virus 162P3 inhibition in macaque lymphocytes in vitro. Plasma concentrations for both compounds were very low. The pretreatment of macaques with Depo-Provera (DP), which is commonly used in macaque challenge studies, was shown to significantly modify the biodistribution of the inhibitors but not the overall amount released. Vaginal fluid and tissue concentrations were significantly decreased while plasma levels increased with DP pretreatment. These observations have implications for designing macaque challenge experiments and also for ring performance during the human female menstrual cycle.

Abstract

Understanding factors that affect heterosexual transmission of HIV in women is of great importance. Lactobacilli in the lower genital tract of women utilize glycogen in vaginal epithelial cells as an energy source and produce lactic acid. The resultant vaginal acidity is believed to provide protection against HIV infection. Conversely, bacterial vaginosis (BV) is characterized by less lactic acid and a higher pH, and is associated with increased susceptibility to HIV infection. Because vaginal infection of macaques with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) is used as a model to study HIV sexual transmission, and because previous studies have shown a paucity of lactobacilli in rhesus macaques' lower genital tract, we compared lactic acid and glycogen levels in the genital fluid of rhesus and pigtail macaques with levels found in humans. The levels of lactic acid were lower in both rhesus (median=1.2 mol lactate/mg protein) and pigtail macaques (median=0.7 mol/mg) compared to women with healthy genital microbiota (median=4.2 mol/mg). Glycogen levels were significantly lower in both rhesus (median=0.004 μg glycogen/μg protein) and pigtail macaques (median=0 μg/μg) than in women (median=0.2 μg/μg). No significant differences in glycogen or lactate levels were observed comparing longitudinally collected samples from cycling pigtail macaques. These data show that the previously reported scarcity of lactobacilli in macaques correlates with low glycogen and lactic acid levels. These findings have important implications for studies of vaginal infection of macaques with SIV or SHIV and further our understanding of how the bacterial microbiota influences HIV infection.