SIV Infection Is Associated with Transient Acute-Phase Steatosis in Hepatocytes In Vivo.

Metabolic-dysfunction-associated fatty liver disease (MAFLD) is a major cause of morbidity and mortality in HIV-infected individuals, even those receiving optimal antiretroviral therapy. Here, we utilized the SIV rhesus macaque model and advanced laparoscopic techniques for longitudinal collection of liver tissue to elucidate the timing of pathologic changes. The livers of both SIV-infected (N = 9) and SIV-naïve uninfected (N = 8) macaques were biopsied and evaluated at four time points (weeks -4, 2, 6, and 16-20 post-infection) and at necropsy (week 32). SIV DNA within the macaques' livers varied by over 4 logs at necropsy, and liver SIV DNA significantly correlated with SIV RNA in the plasma throughout the study. Acute phase liver pathology (2 weeks post-infection) was characterized by evidence for fat accumulation (microvesicular steatosis), a transient elevation in both AST and cholesterol levels within the serum, and increased hepatic expression of the PPARA gene associated with cholesterol metabolism and beta oxidation. By contrast, the chronic phase of the SIV infection (32 weeks post-infection) was associated with sinusoidal dilatation, while steatosis resolved and concentrations of AST and cholesterol remained similar to those in uninfected macaques. These findings suggest differential liver pathologies associated with the acute and chronic phases of infection and the possibility that therapeutic interventions targeting metabolic function may benefit liver health in people newly diagnosed with HIV.

Herpes Simplex Virus Type 2 Prevalence and Association with Inflammatory Cytokines Among Sexual and Gender Minorities Living With and Without HIV-1 from Lagos, Nigeria.

Herpes simplex virus type 2 (HSV-2) is common globally and contributes significantly to the risk of acquiring HIV-1, yet these two sexually transmitted infections have not been sufficiently characterized for sexual and gender minorities (SGM) across Sub-Saharan Africa. To help fill this gap, we performed a retrospective study using plasma and serum samples from 183 SGM enrolled at the Lagos site of the TRUST/RV368 cohort in Nigeria, assayed them for HSV-2 antibodies with the Kalon ELISA and plasma cytokines and chemokines with Luminex, and correlated the findings with HIV-1 viral loads (VLs) and CD4 counts. We found an overall HSV-2 prevalence of 36.6% (49.5% and 23.9% among SGM with and without HIV-1, respectively, p < .001). Moreover, HSV-2-positive status was associated with high circulating concentrations of CCL11 among antiretroviral therapy-treated (p = .031) and untreated (p = .015) participants, and with high concentrations of CCL2 in the untreated group (p = .004), independent of VL. Principal component analysis revealed a strong association of cytokines with HIV-1 VL independent of HSV-2 status. In conclusion, our study finds that HSV-2 prevalence among SGM with HIV-1 is twice as high than HSV-2 prevalence among SGM without HIV-1 in Lagos and suggests that this is associated with higher levels of certain systemic cytokines. Additional work is needed to further characterize the relationship between HSV-2 and HIV-1 in SGM and help develop targeted therapies for coinfected individuals.

Optimization and use of near infrared imaging to guide lymph node collection in rhesus macaques (Macaca mulatta).

BACKGROUND: Identification of lymph nodes (LNs) draining a specific site or in obese macaques can be challenging. METHODS: Indocyanine Green (ICG) was administered intradermal (ID), intramuscular, in the oral mucosa, or subserosal in the colon followed by Near Infrared (NIR) imaging. RESULTS: After optimization to maximize LN identification, intradermal ICG was successful in identifying 50-100% of the axillary/inguinal LN at a site. Using NIR, collection of peripheral and mesenteric LNs in obese macaques was 100% successful after traditional methods failed. Additionally, guided collection of LNs draining the site of intraepithelial or intramuscular immunization demonstrated significantly increased numbers of T follicular helper (Tfh) cells in germinal centers of draining compared to nondraining LNs. CONCLUSION: These imaging techniques optimize our ability to evaluate immune changes within LNs over time, even in obese macaques. This approach allows for targeted serial biopsies that permit confidence that draining LNs are being harvested throughout the study.

HIV-1 Establishes a Sanctuary Site in the Testis by Permeating the BTB Through Changes in Cytoskeletal Organization.

Studies suggest that HIV-1 invades the testis through initial permeation of the blood-testis barrier (BTB). The selectivity of the BTB to antiretroviral drugs makes this site a sanctuary for the virus. Little is known about how HIV-1 crosses the BTB and invades the testis. Herein, we used 2 approaches to examine the underlying mechanism(s) by which HIV-1 permeates the BTB and gains entry into the seminiferous epithelium. First, we examined if recombinant Tat protein was capable of perturbing the BTB and making the barrier leaky, using the primary rat Sertoli cell in vitro model that mimics the BTB in vivo. Second, we used HIV-1-infected Sup-T1 cells to investigate the activity of HIV-1 infection on cocultured Sertoli cells. Using both approaches, we found that the Sertoli cell tight junction permeability barrier was considerably perturbed and that HIV-1 effectively permeates the BTB by inducing actin-, microtubule-, vimentin-, and septin-based cytoskeletal changes in Sertoli cells. These studies suggest that HIV-1 directly perturbs BTB function, potentially through the activity of the Tat protein.

Liver Bacterial Dysbiosis With Non-Tuberculosis Mycobacteria Occurs in SIV-Infected Macaques and Persists During Antiretroviral Therapy.

Liver disease is a significant contributor to morbidity and mortality in HIV-infected individuals, even during successful viral suppression with combination antiretroviral therapy (cART). Similar to HIV infection, SIV infection of rhesus macaques is associated with gut microbiome dysbiosis and microbial translocation that can be detected systemically in the blood. As microbes leaving the intestines must first pass through the liver via the portal vein, we evaluated the livers of both SIV-infected (SIV+) and SIV-infected cART treated (SIV+cART) rhesus macaques for evidence of microbial changes compared to uninfected macaques. Dysbiosis was observed in both the SIV+ and SIV+cART macaques, encompassing changes in the relative abundance of several genera, including a reduction in the levels of Lactobacillus and Staphylococcus. Most strikingly, we found an increase in the relative abundance and absolute quantity of bacteria within the Mycobacterium genus in both SIV+ and SIV+cART macaques. Multi-gene sequencing identified a species of atypical mycobacteria similar to the opportunistic pathogen M. smegmatis. Phosphatidyl inositol lipoarabinomannan (PILAM) (a glycolipid cell wall component found in atypical mycobacteria) stimulation in primary human hepatocytes resulted in an upregulation of inflammatory transcriptional responses, including an increase in the chemokines associated with neutrophil recruitment (CXCL1, CXCL5, and CXCL6). These studies provide key insights into SIV associated changes in hepatic microbial composition and indicate a link between microbial components and immune cell recruitment in SIV+ and SIV+cART treated macaques.

A Tat/Rev Induced Limiting Dilution Assay to Measure Viral Reservoirs in Non-Human Primate Models of HIV Infection.

The establishment of latent infection and poorly characterized viral reservoirs in tissues represent major obstacles to a definitive cure for HIV. Non-human primate (NHP) models of HIV infection are critical to elucidate pathogenic processes and an essential tool to test novel therapeutic strategies. Thus, the availability of novel assays to measure residual viral replication and reservoirs in NHP models may increase their utility in the search for an HIV cure. We developed a tat/rev induced limiting dilution assay to measure the frequency of CD4+ T cells that express multiply-spliced(ms)_SIV RNA in presence and absence of stimulation. We validated the assay using cell lines and cells from blood and lymph nodes of SIV infected macaques. In vitro, SIV/SHIV TILDA detects only cells expressing viral proteins. In SIV/SHIV-infected macaques, CD4+ T cells that express msSIV/SHIV RNA (TILDA data) were detected also in the setting of very low/undetectable viremia. TILDA data were significantly higher after stimulation and correlated with plasma viral load (pVL). Interestingly, TILDA data from early cART initiation correlated with peak and AUC pVL post-cART interruption. In summary, we developed an assay that may be useful in characterizing viral reservoirs and determining the effect of HIV interventions in NHP models.

Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo.

Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) strategies with proven in vivo efficacy rely on antiretroviral drugs, creating the potential for drug resistance and complicated treatment options in individuals who become infected. Moreover, on-demand products are currently missing from the PrEP development portfolio. Griffithsin (GRFT) is a non-antiretroviral HIV entry inhibitor derived from red algae with an excellent safety profile and potent activity in vitro. When combined with carrageenan (CG), GRFT has strong activity against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) in vitro and in vivo. Here, we report that GRFT/CG in a freeze-dried fast dissolving insert (FDI) formulation for on-demand use protects rhesus macaques from a high dose vaginal SHIV SF162P3 challenge 4 h after FDI insertion. Furthermore, the GRFT/CG FDI also protects mice vaginally against HSV-2 and HPV pseudovirus. As a safe, potent, broad-spectrum, on-demand non-antiretroviral product, the GRFT/CG FDI warrants clinical development.

Experimental Oral Herpes Simplex Virus-1 (HSV-1) Co-infection in Simian Immunodeficiency Virus (SIV)-Infected Rhesus Macaques.

Herpes simplex virus 1 and 2 (HSV-1/2) similarly initiate infection in mucosal epithelia and establish lifelong neuronal latency. Anogenital HSV-2 infection augments the risk for sexual human immunodeficiency virus (HIV) transmission and is associated with higher HIV viral loads. However, whether oral HSV-1 infection contributes to oral HIV susceptibility, viremia, or oral complications of HIV infection is unknown. Appropriate non-human primate (NHP) models would facilitate this investigation, yet there are no published studies of HSV-1/SIV co-infection in NHPs. Thus, we performed a pilot study for an oral HSV-1 infection model in SIV-infected rhesus macaques to describe the feasibility of the modeling and resultant immunological changes. Three SIV-infected, clinically healthy macaques became HSV-1-infected by inoculation with 4 × 108 pfu HSV-1 McKrae on buccal, tongue, gingiva, and tonsils after gentle abrasion. HSV-1 DNA was shed in oral swabs for up to 21 days, and shedding recurred in association with intra-oral lesions after periods of no shedding during 56 days of follow up. HSV-1 DNA was detected in explant cultures of trigeminal ganglia collected at euthanasia on day 56. In the macaque with lowest baseline SIV viremia, SIV plasma RNA increased following HSV-1 infection. One macaque exhibited an acute pro-inflammatory response, and all three animals experienced T cell activation and mobilization in blood. However, T cell and antibody responses to HSV-1 were low and atypical. Through rigorous assessesments, this study finds that the virulent HSV-1 strain McKrae resulted in a low level HSV-1 infection that elicited modest immune responses and transiently modulated SIV infection.

A model of genital herpes simplex virus Type 1 infection in Rhesus Macaques.

BACKGROUND: Although HSV-2 is the major cause of genital lesions, HSV-1 accounts for half of new cases in developed countries. METHODS: Three healthy SHIV-SF162P3-infected Indian rhesus macaques were inoculated with 4×108 pfu of HSV-1 twice, with the second inoculation performed after the vaginal mucosa was gently abraded with a cytobrush. RESULTS: HSV-1 DNA was detected in vaginal swabs 5 days after the second but not the first inoculation in all three macaques. An increase in inflammatory cytokines was detected in the vaginal fluids of the animals with no or intermittent shedding. Higher frequency of blood α4 ß7high CD4+ T cells was measured in the animals with consistent and intermitted shedding, while a decrease in the frequency of CD69+ CD4+ T cells was present in all animals. CONCLUSIONS: This macaque model of genital HSV-1 could be useful to study the impact of the growing epidemic of genital HSV-1 on HIV infection.

An intravaginal ring that releases three antiviral agents and a contraceptive blocks SHIV-RT infection, reduces HSV-2 shedding, and suppresses hormonal cycling in rhesus macaques.

Women globally need access to multipurpose prevention technologies (MPTs) that prevent human immunodeficiency virus (HIV), sexually transmitted infections that increase HIV acquisition/transmission risk, and unintended pregnancy. Seeking an MPT with activity against HIV, herpes simplex virus-2 (HSV-2), and human papillomavirus (HPV), we developed a prototype intravaginal ring (IVR), the MZCL IVR, which released the antiviral agents MIV-150, zinc acetate, and carrageenan (MZC for short) and the contraceptive levonorgestrel (LNG). Previously, we showed that an MZC gel has potent activity against immunodeficiency viruses, HSV-2, and HPV and that the MZCL (MZC with LNG) IVR releases all four components in macaques in vivo at levels associated with efficacy. Vaginal fluid from treated macaques has in vitro activity against HIV, HSV-2, and HPV. Herein, we assessed the ability of the MZCL IVR to protect macaques against repeated co-challenge with HSV-2 and SHIV-RT (simian immunodeficiency virus [SIV] containing the reverse transcriptase gene from HIV) and prevent hormonal cycling. We evaluated in vivo drug release in co-challenged macaques by measuring drug levels in blood and vaginal fluid and residual drug levels in used IVRs. The MZCL IVR significantly prevented SHIV-RT infection, reduced HSV-2 vaginal shedding, and prevented cycling. No non-nucleoside HIV reverse transcriptase inhibitor (NNRTI)-resistant SHIV was detected in macaques that became infected after continuous exposure to MZC from the IVR. Macaques wearing the MZCL IVR also had carrageenan levels in vaginal fluid expected to protect from HPV (extrapolated from mice) and LNG levels in blood associated with contraceptive efficacy. The MZCL IVR is a promising MPT candidate that warrants further development.

PolyICLC Exerts Pro- and Anti-HIV Effects on the DC-T Cell Milieu In Vitro and In Vivo.

Myeloid dendritic cells (mDCs) contribute to both HIV pathogenesis and elicitation of antiviral immunity. Understanding how mDC responses to stimuli shape HIV infection outcomes will inform HIV prevention and treatment strategies. The long double-stranded RNA (dsRNA) viral mimic, polyinosinic polycytidylic acid (polyIC, PIC) potently stimulates DCs to focus Th1 responses, triggers direct antiviral activity in vitro, and boosts anti-HIV responses in vivo. Stabilized polyICLC (PICLC) is being developed for vaccine adjuvant applications in humans, making it critical to understand how mDC sensing of PICLC influences HIV infection. Using the monocyte-derived DC (moDC) model, we sought to describe how PICLC (vs. other dsRNAs) impacts HIV infection within DCs and DC-T cell mixtures. We extended this work to in vivo macaque rectal transmission studies by administering PICLC with or before rectal SIVmac239 (SIVwt) or SIVmac239ΔNef (SIVΔNef) challenge. Like PIC, PICLC activated DCs and T cells, increased expression of α4ß7 and CD169, and induced type I IFN responses in vitro. The type of dsRNA and timing of dsRNA exposure differentially impacted in vitro DC-driven HIV infection. Rectal PICLC treatment similarly induced DC and T cell activation and pro- and anti-HIV factors locally and systemically. Importantly, this did not enhance SIV transmission in vivo. Instead, SIV acquisition was marginally reduced after a single high dose challenge. Interestingly, in the PICLC-treated, SIVΔNef-infected animals, SIVΔNef viremia was higher, in line with the importance of DC and T cell activation in SIVΔNef replication. In the right combination anti-HIV strategy, PICLC has the potential to limit HIV infection and boost HIV immunity.

Rectal HSV-2 Infection May Increase Rectal SIV Acquisition Even in the Context of SIVΔnef Vaccination.

Prevalent HSV-2 infection increases the risk of HIV acquisition both in men and women even in asymptomatic subjects. Understanding the impact of HSV-2 on the mucosal microenvironment may help to identify determinants of susceptibility to HIV. Vaginal HSV-2 infection increases the frequency of cells highly susceptible to HIV in the vaginal tissue of women and macaques and this correlates with increased susceptibility to vaginal SHIV infection in macaques. However, the effect of rectal HSV-2 infection on HIV acquisition remains understudied. We developed a model of rectal HSV-2 infection in macaques in combination with rectal SIVmac239Δnef (SIVΔnef) vaccination and our results suggest that rectal HSV-2 infection may increase the susceptibility of macaques to rectal SIVmac239 wild-type (wt) infection even in SIVΔnef-infected animals. Rectal SIVΔnef infection/vaccination protected 7 out of 7 SIVΔnef-infected macaques from SIVmac239wt rectal infection (vs 12 out of 16 SIVΔnef-negative macaques), while 1 out of 3 animals co-infected with SIVΔnef and HSV-2 acquired SIVmac239wt infection. HSV-2/SIVmac239wt co-infected animals had increased concentrations of inflammatory factors in their plasma and rectal fluids and a tendency toward higher acute SIVmac239wt plasma viral load. However, they had higher blood CD4 counts and reduced depletion of CCR5+ CD4+ T cells compared to SIVmac239wt-only infected animals. Thus, rectal HSV-2 infection generates a pro-inflammatory environment that may increase susceptibility to rectal SIV infection and may impact immunological and virological parameters during acute SIV infection. Studies with larger number of animals are needed to confirm these findings.

A novel intravaginal ring to prevent HIV-1, HSV-2, HPV, and unintended pregnancy.

Women urgently need a self-initiated, multipurpose prevention technology (MPT) that simultaneously reduces their risk of acquiring HIV-1, HSV-2, and HPV (latter two associated with increased risk of HIV-1 acquisition) and prevents unintended pregnancy. Here, we describe a novel core-matrix intravaginal ring (IVR), the MZCL IVR, which effectively delivered the MZC combination microbicide and a contraceptive. The MZCL IVR contains four active pharmaceutical ingredients (APIs): MIV-150 (targets HIV-1), zinc acetate (ZA; targets HIV-1 and HSV-2), carrageenan (CG; targets HPV and HSV-2), and levonorgestrel (LNG; targets unintended pregnancy). The elastomeric IVR body (matrix) was produced by hot melt extrusion of the non-water swellable elastomer, ethylene vinyl acetate (EVA-28), containing the hydrophobic small molecules, MIV-150 and LNG. The solid hydrophilic core, embedded within the IVR by compression, contained the small molecule ZA and the macromolecule CG. Hydrated ZA/CG from the core was released by diffusion via a pore on the IVR while the MIV-150/LNG diffused from the matrix continuously for 94 days (d) in vitro and up to 28 d (study period) in macaques. The APIs released in vitro and in vivo were active against HIV-1ADA-M, HSV-2, and HPV16 PsV in cell-based assays. Serum LNG was at levels associated with local contraceptive effects. The results demonstrate proof-of-concept of a novel core-matrix IVR for sustained and simultaneous delivery of diverse molecules for the prevention of HIV, HSV-2 and HPV acquisition, as well as unintended pregnancy.

Short communication: a repeated simian human immunodeficiency virus reverse transcriptase/herpes simplex virus type 2 cochallenge macaque model for the evaluation of microbicides.

Epidemiological studies suggest that prevalent herpes simplex virus type 2 (HSV-2) infection increases the risk of HIV acquisition, underscoring the need to develop coinfection models to evaluate promising prevention strategies. We previously established a single high-dose vaginal coinfection model of simian human immunodeficiency virus (SHIV)/HSV-2 in Depo-Provera (DP)-treated macaques. However, this model does not appropriately mimic women's exposure. Repeated limiting dose SHIV challenge models are now used routinely to test prevention strategies, yet, at present, there are no reports of a repeated limiting dose cochallenge model in which to evaluate products targeting HIV and HSV-2. Herein, we show that 20 weekly cochallenges with 2-50 TCID50 simian human immunodeficiency virus reverse transcriptase (SHIV-RT) and 10(7) pfu HSV-2 results in infection with both viruses (4/6 SHIV-RT, 6/6 HSV-2). The frequency and level of vaginal HSV-2 shedding were significantly greater in the repeated exposure model compared to the single high-dose model (p<0.0001). We used this new model to test the Council's on-demand microbicide gel, MZC, which is active against SHIV-RT in DP-treated macaques and HSV-2 and human papillomavirus (HPV) in mice. While MZC reduced SHIV and HSV-2 infections in our repeated limiting dose model when cochallenging 8 h after each gel application, a barrier effect of carrageenan (CG) that was not seen in DP-treated animals precluded evaluation of the significance of the antiviral activity of MZC. Both MZC and CG significantly (p<0.0001) reduced the frequency and level of vaginal HSV-2 shedding compared to no gel treatment. This validates the use of this repeated limiting dose cochallenge model for testing products targeting HIV and HSV-2.

Sex hormones selectively impact the endocervical mucosal microenvironment: implications for HIV transmission.

Several studies suggest that progesterone and estrogens may affect HIV transmission in different, possibly opposing ways. Nonetheless, a direct comparison of their effects on the mucosal immune system has never been done. We hypothesize that sex hormones might impact the availability of cells and immune factors important in early stages of mucosal transmission, and, in doing so influence the risk of HIV acquisition. To test this hypothesis, we employed 15 ovarectomized rhesus macaques: 5 were treated with Depot Medroxy Progesterone Acetate (DMPA), 6 with 17-ß estradiol (E2) and 4 were left untreated. All animals were euthanized 5 weeks after the initiation of hormone treatment, a time post-DMPA injection associated with high susceptibility to SIV infection. We found that DMPA-treated macaques exhibited higher expression of integrin α4ß7 (α4ß7) on CD4+ T cells, the gut homing receptor and a marker of cells highly susceptible to HIV, in the endocervix than did the E2-treated animals. In contrast, the frequency of CCR5+ CD4+ T cells in DMPA-treated macaques was higher than in the E2-treated group in vaginal tissue, but lower in endocervix. α4ß7 expression on dendritic cells (DCs) was higher in the DMPA-treated group in the endocervical tissue, but lower in vaginal tissue and on blood DCs compared with the E2-treated animals. Soluble MAdCAM-1, the α4ß7 ligand, was present in the vaginal fluids of the control and E2-treated groups, but absent in the fluids from DMPA-treated animals. Both hormones modulated the expression and release of inflammatory factors and modified the distribution of sialomucins in the endocervix. In summary, we found that sex hormones profoundly impact mucosal immune factors that are directly implicated in HIV transmission. The effect is particularly significant in the endocervix. This may increase our understanding of the potential hormone-driven modulation of HIV susceptibility and potentially guide contraceptive policies in high-risk settings.

A potent combination microbicide that targets SHIV-RT, HSV-2 and HPV.

Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p = 0.0187) infection of mice when 10(6) pfu HSV-2 were applied immediately after vaginal challenge and also when 5×10(3) pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×10(6) HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use.

Exposure to MIV-150 from a high-dose intravaginal ring results in limited emergence of drug resistance mutations in SHIV-RT infected rhesus macaques.

When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides.

A combination microbicide gel protects macaques against vaginal simian human immunodeficiency virus-reverse transcriptase infection, but only partially reduces herpes simplex virus-2 infection after a single high-dose cochallenge.

Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.

A modified zinc acetate gel, a potential nonantiretroviral microbicide, is safe and effective against simian-human immunodeficiency virus and herpes simplex virus 2 infection in vivo.

We previously showed that a prototype gel comprising zinc acetate (ZA) in carrageenan (CG) protected mice against vaginal and rectal herpes simplex virus 2 (HSV-2) challenge as well as macaques against vaginal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) challenge. In this work, we modified buffers and cosolvents to obtain a stable, nearly iso-osmolal formulation and evaluated its safety and efficacy against SHIV-RT and HSV-2. In vitro toxicity to lactobacilli and Candida albicans was determined. Macaques were given daily doses of ZA and CG (ZA/CG) or CG alone vaginally for 14 days and challenged with SHIV-RT 24 h later. Mice were challenged vaginally or rectally with HSV-2 immediately after a single gel treatment to measure efficacy or vaginally 12 h after daily gel treatment for 7 days to evaluate the gel's impact on susceptibility to HSV-2 infection. The modified ZA/CG neither affected the viability of lactobacilli or C. albicans nor enhanced vaginal HSV-2 infection after daily ZA/CG treatment. Vaginal SHIV-RT infection of macaques was reduced by 66% (P = 0.006) when macaques were challenged 24 h after the last dose of gel. We observed 60% to 80% uninfected mice after vaginal (P < 0.0001) and rectal (P = 0.008) high-dose HSV-2 challenge. The modified ZA/CG gel is safe and effective in animal models and represents a potential candidate to limit the transmission of HIV and HSV-2.