José A. Fernández-Romero

An antiretroviral/zinc combination gel provides 24 hours of complete protection against vaginal SHIV infection in macaques.

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.

An Intravaginal Ring That Releases the NNRTI MIV-150 Reduces SHIV Transmission in Macaques

An intravaginal ring loaded with the NNRTI MIV-150 prevents transmission of the HIV/SIV chimera SHIV-RT in macaques. HIV Protection That Has a Ring to It An ounce of prevention is better than a pound of cure. This is especially true for HIV, where no cure exists. Pre-exposure prophylaxis is showing promising results in preventing HIV transmission in early clinical trials, but the means of delivery of active pharmaceutical ingredients remains a major challenge. Singer et al. now show that the non-nucleoside reverse transcriptase inhibitor MIV-150 delivered by intravaginal rings can protect macaques from simian/HIV (SHIV) infection. For pre-exposure prophylaxis to be successful, individuals must remember and be willing to treat themselves regularly. Intravaginal rings are well tolerated among women and—through sustained release—can help overcome this adherence bottleneck. The authors test the efficacy of MIV-150 when delivered by intravaginal rings made of either ethylene vinyl acetate or silicone. MIV-150 was successfully delivered to vaginal fluids and tissues and protected macaques from SHIV infection. If these studies hold true in people, intravaginal rings containing MIV-150 may help prevent HIV infection. Microbicides may prevent HIV and sexually transmitted infections (STIs) in women; however, determining the optimal means of delivery of active pharmaceutical ingredients remains a major challenge. We previously demonstrated that a vaginal gel containing the non-nucleoside reverse transcriptase inhibitor MIV-150 partially protected macaques from SHIV-RT (simian/HIV reverse transcriptase) infection, and the addition of zinc acetate rendered the gel significantly protective. We test the activity of MIV-150 without the addition of zinc acetate when delivered from either ethylene vinyl acetate (EVA) or silicone intravaginal rings (IVRs). MIV-150 was successfully delivered, because it was detected in vaginal fluids and tissues by radioimmunoassay in pharmacokinetic studies. Moreover, EVA IVRs significantly protected macaques from SHIV-RT infection. Our results demonstrate that MIV-150–containing IVRs have the potential to prevent HIV infection and highlight the possible use of IVRs for delivering drugs that block HIV and other STIs.

Zinc Acetate/Carrageenan Gels Exhibit Potent Activity In Vivo against High-Dose Herpes Simplex Virus 2 Vaginal and Rectal Challenge

ABSTRACT 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.

The Nonnucleoside Reverse Transcriptase Inhibitor MIV-150 in Carrageenan Gel Prevents Rectal Transmission of Simian/Human Immunodeficiency Virus Infection in Macaques

ABSTRACT 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.

Multipurpose prevention technologies: the future of HIV and STI protection

Every day, more than 1 million people are newly infected with sexually transmitted infections (STIs) that can lead to morbidity, mortality, and an increased risk of human immunodeficiency virus (HIV) acquisition. Existing prevention and management strategies, including behavior change, condom promotion, and therapy have not reduced the global incidence and prevalence, pointing to the need for novel innovative strategies. This review summarizes important issues raised during a satellite session at the first HIV Research for Prevention (R4P) conference, held in Cape Town, on October 31, 2014. We explore key STIs that are challenging public health today, new biomedical prevention approaches including multipurpose prevention technologies (MPTs), and the scientific and regulatory hurdles that must be overcome to make combination prevention tools a reality.

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 106 pfu HSV-2 were applied immediately after vaginal challenge and also when 5×103 pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×106 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.

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.

Griffithsin and Carrageenan Combination To Target Herpes Simplex Virus 2 and Human Papillomavirus

ABSTRACT Extensive preclinical evaluation of griffithsin (GRFT) has identified this lectin to be a promising broad-spectrum microbicide. We set out to explore the antiviral properties of a GRFT and carrageenan (CG) combination product against herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) as well as determine the mechanism of action (MOA) of GRFT against both viruses. We performed the experiments in different cell lines, using time-of-addition and temperature dependence experiments to differentiate inhibition of viral attachment from entry and viral receptor internalization. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins, and immunoprecipitation and immunohistochemistry were used to identify the specific glycoprotein involved. We determined the antiviral activity of GRFT against HSV-2 to be a 50% effective concentration (EC50) of 230 nM and provide the first evidence that GRFT has moderate anti-HPV activity (EC50 = 0.429 to 1.39 μM). GRFT blocks the entry of HSV-2 and HPV into target cells but not the adsorption of HSV-2 and HPV onto target cells. The results of the SPR, immunoprecipitation, and immunohistochemistry analyses of HSV-2 combined suggest that GRFT may block viral entry by binding to HSV-2 glycoprotein D. Cell-based assays suggest anti-HPV activity through α6 integrin internalization. The GRFT-CG combination product but not GRFT or CG alone reduced HSV-2 vaginal infection in mice when given an hour before challenge (P = 0.0352). While GRFT significantly protected mice against vaginal HPV infection when dosed during and after HPV16 pseudovirus challenge (P < 0.026), greater CG-mediated protection was afforded by the GRFT-CG combination for up to 8 h (P < 0.0022). These findings support the development of the GRFT-CG combination as a broad-spectrum microbicide.

Validation of a dye stain assay for vaginally inserted hydroxyethylcellulose-filled microbicide applicators.

Background: The reliability and validity of self-reports of vaginal microbicide use are questionable given the explicit understanding that participants are expected to comply with study protocols. Our objective was to optimize the use of Population Councils previously validated dye stain assay (DSA) and related procedures, and to establish predictive values for the DSAs ability to identify vaginally inserted single-use, low-density polyethylene microbicide applicators filled with hydroxyethylcellulose gel. Methods: Applicators, inserted by 252 female sex workers enrolled in a microbicide feasibility study in Southern India, served as positive controls for optimization and validation experiments. Before validation, optimal dye concentration and staining time were ascertained. Three validation experiments were conducted to determine sensitivity, specificity, and negative and positive predictive values. Results: The dye concentration of 0.05% (wt/vol) FD&C Blue No. 1 Granular Food Dye (Prime Ingredients, Inc, Saddlebrook, NJ) and staining time of 5 seconds were determined to be optimal and were used for the 3 validation experiments. There were a total of 1848 possible applicator readings across validation experiments; 1703 (92.2%) applicator readings were correct. On average, the DSA performed with 90.6% sensitivity, 93.9% specificity, and had a negative predictive value of 93.8% and a positive predictive value of 91.0%. No statistically significant differences between experiments were noted. Conclusions: The DSA was optimized and successfully validated for use with single use, low-density polyethylene applicators filled with hydroxyethylcellulose gel. We recommend including the DSA in future microbicide trials involving vaginal gels so as to identify participants who have low adherence to dosing regimens. In doing so, we can develop strategies to improve adherence as well as investigate the association between product use and efficacy.

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.