Given that there is currently no vaccine, there is an immediate need for effective agents to decrease the significant number of new HIV transmissions, recently estimated to be at ~7000 new infections/day globally [12010.WHO-UNAIDS Report on the Global AIDS Epidemic http://www.unaids.org/ globalreport/global_report.htm]. Non-vaccine Biomedical Prevention (NBP) methods in the form of topical or systemic PrEP are attractive strategies to control AIDS dissemination [2Cohen MS, Gay C, Kashuba AD, Blower S, Paxton L. Narrative review: Antiretroviral therapy to prevent the sexual transmission of HIV-1. Ann Intern Med 2007; 146: 591-601.
[http://dx.doi.org/10.7326/0003-4819-146-8-200704170-00010] -15Turpin JA. Topical microbicides to prevent the transmission of HIV: Formulation gaps and challenges. Drug Deliv Transl Res 2011; 1: 194-200.
[http://dx.doi.org/10.1007/s13346-011-0034-2] ]. Several clinical trials, based on regimens of tenofovir (TFV), frequently in combination with emtricitabine (FTC), provided evidence that PrEP could significantly reduce HIV infection in individuals [16Abdool Karim Q, Abdool Karim SS, Frohlich JA, et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 2010; 329: 1168-74.
[http://dx.doi.org/10.1126/science.1193748] -24McCormack S, Dunn DT, Desai M, et al. Pre-exposure prophylaxis to prevent the acquisition of HIV-1 infection (PROUD): Effectiveness results from the pilot phase of a pragmatic open-label randomised Trial. Lancet 2016; 387: 53-60.
[http://dx.doi.org/10.1016/S0140-6736(15)00056-2] ].The CAPRISA 004 trial provided the first demonstration that a topical microbicide could prevent HIV transmission in humans. Specifically, a 1% tenofovir (TVF) gel used pericoitally decreased the incidence of HIV transmission in South African women by 39% [16Abdool Karim Q, Abdool Karim SS, Frohlich JA, et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 2010; 329: 1168-74.
[http://dx.doi.org/10.1126/science.1193748] ]. However, two additional trials with 1% tenofovir gel with pericoital (VOICE) [25Delany-Moretlwe S, Smith E, Myer L, et al. FACTS 001: Characteristics of participants enrolled in a phase III randomised controlled trial of tenofovir gel for prevention of HIV-1 and HSV-2. AIDS Res Hum Retroviruses 2014; 30: A281-2.
[http://dx.doi.org/10.1089/aid.2014.5637.abstract] ] and daily (FACTS) [26Marrazzo JM, Ramjee G, Richardson BA, Gomez K, Mgodi N, Nair G, et al. Tenofovir-based preexposure prophylaxis for HIV infection among african women. Obstet Gynecol Surv 2015; 70: 444-6.
[http://dx.doi.org/10.1097/01.ogx.0000466878.37011.6f] ] dosing regimens failed to provide efficacy against new sexual HIV infections. In the ASPIRE trial of a monthly intravaginal ring delivering dapivirine, incidence of HIV infection was significantly reduced for women who wore the ring consistently, but low overall efficacy (27% risk reduction) resulted from poor adherence in certain sub-groups, particularly among younger women [27Baeten JM, Palanee-Phillips T, Brown ER, Schwartz K, Soto-Torres LE, Govender V, et al. Use of a vaginal ring containing dapivirine for HIV-1 prevention in women. N Engl J Med 2016; 375: 2121-32.
[http://dx.doi.org/10.1056/NEJMoa1506110] ]. Indeed, poor adherence to the prophylaxis regimens may be a primary factor in this lack of efficacy; although additional factors cannot be ruled out to account for the disparate range of efficacy in the results, as indicated in a randomized pharmacokinetic crossover study [28Hendrix CW. Exploring concentration response in HIV pre-exposure prophylaxis to optimize clinical care and trial design. Cell 2013; 155: 515-8.
[http://dx.doi.org/10.1016/j.cell.2013.09.030] ], where the tissue concentration advantage (>100x) seen topically as compared to oral dosing was not reflected in the seroconversion data of the CAPRISA and VOICE trials. This may indicate that factors beyond the purely antiviral effect may reduce topical PrEP efficacy. Potential factors include concentration-dependent tissue toxicity from TFV, tenofovir-diphosphate (TFV-DP) effects, or dose frequency-dependent effects from the gel itself. Issues such as these may be difficult to adequately assess, and may be outside the scope of standard safety evaluation. In any case, the conflicting trial results certainly point out the multiple factors at work in the interactions between HIV and the host at the mucosal surfaces during initiation of infection, and highlight an urgent need for suitable in vivo model systems that can illuminate the various possible contributions to the contradictory clinical results.

The use of microbicides in studies, by the fact of their tissue-specific nature, requires the use of animal models [3Derdelinckx I, Wainberg MA, Lange JM, Hill A, Halima Y, Boucher CA. Criteria for drugs used in pre-exposure prophylaxis trials against HIV infection. PLoS Med 2006; 3: e454.
[http://dx.doi.org/10.1371/journal.pmed.0030454] , 23Marcus JL, Hurley LB, Hare CB, et al. Preexposure prophylaxis for HIV prevention in a large integrated health care system: Adherence, renal safety, and discontinuation. J Acquir Immune Defic Syndr 2016; 73: 540-6.
[http://dx.doi.org/10.1097/QAI.0000000000001129] -25Delany-Moretlwe S, Smith E, Myer L, et al. FACTS 001: Characteristics of participants enrolled in a phase III randomised controlled trial of tenofovir gel for prevention of HIV-1 and HSV-2. AIDS Res Hum Retroviruses 2014; 30: A281-2.
[http://dx.doi.org/10.1089/aid.2014.5637.abstract] ]. One animal model often used in the study of vaginal HIV transmission is the Rhesus macaque; in this case, the animals are infected with either Simian Immunodeficiency Virus (SIV) or SIV/HIV (SHIV) chimeric viruses [29Veazey RS, Springer MS, Marx PA, Dufour J, Klasse PJ, Moore JP. Protection of macaques from vaginal SHIV challenge by an orally delivered CCR5 inhibitor. Nat Med 2005; 11: 1293-4.
[http://dx.doi.org/10.1038/nm1321] -34Williams R, Bokhari S, Silverstein P, Pinson D, Kumar A, Buch S. Nonhuman primate models of NeuroAIDS. J Neurovirol 2008; 14: 292-300.
[http://dx.doi.org/10.1080/13550280802074539] ]. However, this model has limitations, as it does not support HIV replication, as well as requiring sometimes prohibitive cost for primates whose accessibility is limited, especially with regard to the females. In addition, it faces disparities in host SIV and SHIV vulnerability since the primate colonies are outbred. Another animal model currently in use for testing of microbicidal candidates is the BLT (human Bone marrow, Liver, Thymus) mouse [35Wege AK, Melkus MW, Denton PW, Estes JD, Garcia JV. Functional and phenotypic characterization of the humanized BLT mouse model. Curr Top Microbiol Immunol 2008; 324: 149-65.
[http://dx.doi.org/10.1007/978-3-540-75647-7_10] -47Chateau ML, Denton PW, Swanson MD, McGowan I, Garcia JV. Rectal transmission of transmitted/founder HIV-1 is efficiently prevented by topical 1% tenofovir in BLT humanized mice. PLoS One 2013; 8: e60024.
[http://dx.doi.org/10.1371/journal.pone.0060024] ]. These humanized BLT mice are created by the implantation of human fetal liver and thymus tissues under the kidney capsule of an immunodeficient NOD scid gamma (NSG = NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) mouse, followed three weeks later by the injection of autologous human fetal liver CD34+ cells (human hematopoietic stem cells, HSC). In the BLT mouse, a complete systemic reconstitution is achieved for all of the major human hematopoietic lineages, including T, B, monocyte/macrophage, dendritic, and natural killer cells. In addition, the developing T cell education occurs in the human thymic tissue. Fortuitously, for their use in the testing of microbicides, this widespread systemic and genital mucosal reconstitution with human lymphoid cells make female humanized BLT mice a useful tool to test both vaginal and rectal HIV transmission.

The short amphipathic helical peptide SWLRDIWDWICEVLSDFK called C5A, was originally identified by the Chisari lab as an active anti-hepatitis C virus (HCV) agent [48Cheng G, Montero A, Gastaminza P, et al. A virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2008; 105: 3088-93.
[http://dx.doi.org/10.1073/pnas.0712380105] ]. The C5A amino acid sequence encompasses the region that permits the anchoring of the HCV nonstructural protein 5A (NS5A) into the ER membrane by an in-plane amphipathic alpha-helix [49Brass V, Bieck E, Montserret R, et al. An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 2002; 277: 8130-9.
[http://dx.doi.org/10.1074/jbc.M111289200] , 50Penin F, Brass V, Appel N, et al. Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J Biol Chem 2004; 279: 40835-43.
[http://dx.doi.org/10.1074/jbc.M404761200] ] (Fig. 1). In collaboration with the Chisari lab, we demonstrated that C5A also exerts antiviral activities against HIV [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. We found that C5A inhibits a broad range of primary HIV isolates at an nM-µM peptide concentration range and blocks infection of the three major in vivo targets of HIV; CD4⁺ T-lymphocytes, macrophages and dendritic cells [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. Using an in vitro transwell chamber assay, which mimics HIV transmigration through primary human genital epithelial cells, we also demonstrated that C5A prevents the passage of HIV through this in vitro genital epithelial barrier [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. Moreover, we showed that C5A prevents HIV transfer from Langerhans and dendritic cells to CD4⁺ T-lymphocytes [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. Importantly, we obtained evidence for the antiviral mechanism of action of C5A. We demonstrated that C5A disrupts the membrane integrity of the HIV virion as well as the integrity of the conical capsid core that surrounds the viral genome, leading to non-infectious particles [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. Most importantly, although C5A destabilizes the viral membrane, we showed that it preserves the integrity of cellular membranes and is not toxic to cervical epithelial cells even when used at a concentration of 10- to 100-fold greater than that which blocks HIV infection [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ].

In this study, we tested the in vivo microbicidal efficacy of C5A against vaginal and rectal HIV challenges using the BLT mouse model. Additionally, we tested a panel of C5A variants with various degrees of anti-infectivity efficacy for their capacities to prevent vaginal and rectal HIV transmission. The main goal of this study was to investigate the microbicidal potency of C5A in vivo and to examine the molecular requirements of the structure and composition of C5A that determine its microbicidal activity. These data will be used to determine the most effective combination of C5A derivatives and well-characterized, small molecule ARVs with anti-HIV mechanisms of action distinct from that of C5A (i.e., elvitegravir, FTC or TDF), for formulation as an intravaginal ring nBP product.

In the absence of a safe and effective vaccine, there is an imperative demand for effective agents to reduce the 7,000 new HIV infections occurring every day. An attractive possibility is to identify the best drugs and drug combinations, delivered by optimal nBP methods such Intravaginal Rings (IVRs) for vaginal delivery and gels or enemas for rectal protection. We previously reported that C5A, a short, amphipathic, 18 amino acid (SWLRDIWDWICEVLSDFK) helical peptide, inhibits in vitro infectivity of a broad range of primary HIV isolates in various primary target cells [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. In this study, we evaluated the microbicidal efficacy of C5A in vivo in a humanized mouse model, and investigated the structural requirements of C5A to block HIV transmission in vivo. These studies show that parental C5A efficiently inhibits both vaginal and rectal HIV transmission in humanized mice. This is in accordance with a previous study demonstrating that topical vaginal administration of 200 µM C5A in PBS confers 100% protection against a vaginal challenge of HIV in humanized mice [38Denton PW, Othieno F, Martinez-Torres F, et al. One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates. J Virol 2011; 85: 7582-93.
[http://dx.doi.org/10.1128/JVI.00537-11] ]. This is also in accordance with our previous study that showed that C5A maintains its antiviral activities in genital fluids including seminal plasma, cervical and vaginal fluids [57de Witte L, Bobardt MD, Chatterji U, et al. HSV neutralization by the microbicidal candidate C5A. PLoS One 2011; 6: e18917.
[http://dx.doi.org/10.1371/journal.pone.0018917] ]. Moreover, this is in agreement with our recent study that showed that a vaginal application of C5A protects 89% of macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge [58Veazey RS, Chatterji U, Bobardt M, et al. C5A protects macaques from vaginal simian-human immunodeficiency virus challenge. Antimicrob Agents Chemother 2015; 60: 693-8.
[http://dx.doi.org/10.1128/AAC.01925-15] ]. Although the vaginal pH varies between species – 3.5 to 4.5 for humans and 6 to 7 for mice and macaques is 6 to 7 [59Zeitlin L, Hoen TE, Achilles SL, et al. Tests of Buffergel for contraception and prevention of sexually transmitted diseases in animal models. Sex Transm Dis 2001; 28: 417-23.
[http://dx.doi.org/10.1097/00007435-200107000-00010] , 60Daggett GJ Jr, Zhao C, Connor-Stroud F, et al. Comparison of the vaginal environment in rhesus and cynomolgus macaques pre- and post-lactobacillus colonization. J Med Primatol 2017; 46: 232-8.
[http://dx.doi.org/10.1111/jmp.12264] ] - we previously reported that C5A keeps is antiviral activity between pH 5 and 7, but loses its activity at pH 8 [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. For the humanized mouse studies reported here, 50% effective concentrations (EC₅₀) of C5A calculated using the traditional sigmodal dose-response model were 32.5 and 25.7 µM, respectively, for vaginal and rectal HIV challenge (Fig. 2). A major advantage of the humanized mouse model is the ability to create large groups of animals from tissues from a single donor. We routinely generate 60-80 humanized mice per liver/thymus tissue. Another advantage of this model is the capacity of using high numbers of animals per group/treatment (n = 10) to attain a satisfactory statistical power. This further confirms that the BLT mouse model is an ideal tool to screen microbicidal candidates.

Applying the median-effect model to the humanized mouse dose-ranging efficacy datasets allowed EC values in the 50-97% protection range to be calculated (Fig. 3). The slope, m, of the linear fit to the log-log median-effects plots Figs. (3A and 3B) is analogous to the Hill coefficient and provides a quantitative estimation of the sigmoidicity of the dose-effect curve. Slope parameters (m) for C5A of 7.6 ± 1.2 and 7.7 ± 1.2 were calculated for the vaginal and rectal compartments, respectively. Fig. (3) summarizes the results from this expanded analsyis, where F_a represents the fraction affected and F_u the fraction unaffected. The high regression coefficients (r > 0.95) support the applicability of the model. Shen et al. used an ex vivo HIV model to show that ARV agents has a characteristic slope, ranging from 1 for nucleoside reverse transcriptase inhibitors (NRTIs) to 1.8-4.5 protease inhibitors (PIs) [61Shen L, Peterson S, Sedaghat AR, et al. Dose-response curve slope sets class-specific limits on inhibitory potential of anti-HIV drugs. Nat Med 2008; 14: 762-6.
[http://dx.doi.org/10.1038/nm1777] ]. The much higher slopes (m) of the C5A dose-response curves (7.6 and 7.7 for vaginal and rectal dosing, respectively) observed in vivo suggest that this microbicide has significant potential as an efficatious PrEP agent, as illustrated in Fig. (3C). To our knowledge, this is the first demonstration that the humanized BLT mouse model allows the determination of ECs of a drug against vaginal or rectal HIV transmission.

Fig. (3) Median-effect plots describing the impact of (A) vaginal (regression coefficient, r = 0.956) and (B) rectal (regression coefficient, r = 0.955) C5A dosing on HIV preventions in humanized BLT mice. The model allows a range (50-97%) of C5A effective concentrations to be calculated for both compartments (C).

We found that the genital administration of 25 or 50 µM of C5A provides only a partial protection. We believe that these concentrations of the virucidal peptide in the genital compartment do not neutralize (rupture) 100% of incoming viruses, allowing some infectious particles to cross the genital epithelium and to reach the peripheral blood where CD4+ T lymphocytes will be infected and will support robust HIV replication.

The studies with modified C5A-C and C5A-N demonstrate that the removal of four amino acids at either end of C5A does not alter the microbicidal efficacy of the peptide. In sharp contrast, the simultaneous removal of four amino acids at each end (C5A-CN) abolishes the microbicidal capabilities of C5A, suggesting that the peptide requires a sufficient length to effectively block both vaginal and rectal HIV transmissions.

The lack of protection observed for C5A-NA demonstrates that the amphipathic structural nature of C5A is critical for its microbicidal properties. Similarly, C5A-HS offered no protection vaginally or rectally, indicating that the hydrophobic face of C5A (or NS5A), thought to mediate anchoring into the ER membrane, is also critical for the ability of the peptide to inhibit HIV infection.

Following these studies with unformulated C5A solutions applied topically, the next goal is to formulate C5A, alone and in combination with other antiretroviral agents, into murine intravaginal rings to evaluate sustained topical delivery approaches for protection against vaginal and rectal HIV challenges. We plan on using a combination of drugs with distinct mechanisms of action. Remarkably, C5A possesses a unique anti-HIV mechanism of action. We demonstrated that C5A disrupts the stability of the membrane of HIV virions as well as that of the capsid core, surrounding the viral genome, resulting into non-infectious particles [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. Our working hypothesis is that anchoring C5A molecules via their hydrophobic surface into the HIV membrane, destabilizes and ruptures the integrity of the viral membrane. This means that C5A theoretically would act in the lumen (vaginal or rectal), much like the traditional microbicides. This mode of action would be highly complementary to small-molecule ARV PrEP candidates (e.g., TDF and FTC) as these inhibit viral replication in immune cells, presumably in tissues (vaginal or rectal), a different anatomic compartment. Importantly, C5A does not alter the cellular membranes even when used at a concentration of 10- to 100-fold greater than that which blocks HIV infection [51Bobardt MD, Cheng G, de Witte L, et al. Hepatitis C virus NS5A anchor peptide disrupts human immunodeficiency virus. Proc Natl Acad Sci USA 2008; 105: 5525-30.
[http://dx.doi.org/10.1073/pnas.0801388105] ]. This is in accordance with our recent study that demonstrated that a vaginal application of C5A protects 89% of macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge and that no signs of lesions or inflammation were observed in animals vaginally treated with repeated C5A applications [57de Witte L, Bobardt MD, Chatterji U, et al. HSV neutralization by the microbicidal candidate C5A. PLoS One 2011; 6: e18917.
[http://dx.doi.org/10.1371/journal.pone.0018917] ]. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate to be combined with other drugs, including ARVs, with different antiviral mechanisms of action.

An additional attractive property that C5A offers is that it also efficiently inhibits HSV infection [58Veazey RS, Chatterji U, Bobardt M, et al. C5A protects macaques from vaginal simian-human immunodeficiency virus challenge. Antimicrob Agents Chemother 2015; 60: 693-8.
[http://dx.doi.org/10.1128/AAC.01925-15] ], potentially acting as a single-agent multipurpose protection technology (MPT). Previous studies have shown that C5A inhibits HSV infection of Vero cells, human dendritic cells and primary genital epithelial cells in vitro as well as human epidermis (epidermal sheets) ex vivo, and that it inhibits acyclovir- and ganciclovir-resistant HSV isolates [58Veazey RS, Chatterji U, Bobardt M, et al. C5A protects macaques from vaginal simian-human immunodeficiency virus challenge. Antimicrob Agents Chemother 2015; 60: 693-8.
[http://dx.doi.org/10.1128/AAC.01925-15] ]. Remarkably, in an in vitro transwell assay, C5A prevented HIV transmigration by preserving the integrity of the genital epithelium that is normally severely compromised by HSV pre-infection [58Veazey RS, Chatterji U, Bobardt M, et al. C5A protects macaques from vaginal simian-human immunodeficiency virus challenge. Antimicrob Agents Chemother 2015; 60: 693-8.
[http://dx.doi.org/10.1128/AAC.01925-15] ]. Because genital herpes is a major risk factor in acquiring HIV infection, C5A represents a multipurpose microbicide candidate, which may interfere with HIV transmission via a dual mechanism of action – neutralizing both HSV and HIV.