ABSTRACT
A safe and effective vaginal microbicide could decrease human immunodeficiency virus (HIV) transmission in women. Here, we evaluated the safety and microbicidal efficacy of a short amphipathic peptide, C5A, in a rhesus macaque model. We found that a vaginal application of C5A protects 89% of the macaques from a simian-human immunodeficiency virus (SHIV-162P3) challenge. We observed no signs of lesions or inflammation in animals vaginally treated with repeated C5A applications. With its noncellular cytotoxic activity and rare mechanism of action, C5A represents an attractive microbicidal candidate.
This article is contribution 29185 from the Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.
TEXT
Approximately 35 million people are living with human immunodeficiency virus (HIV), 5,000 people are newly infected per day, and 1.5 million people die of AIDS per year. Half of individuals living with HIV are women, who acquire the virus mainly by heterosexual exposure (1–4). Due to restrained economic choices and gender inequity, often women are unable to negotiate sexual encounters, leaving them defenseless to unwanted pregnancy and sexually transmitted diseases, including HIV-1 infection. Due to a lack of a vaccine, vaginally or rectally administered microbicides represent an option to interrupt HIV transmission.
The Chisari lab previously identified a short amphipathic helical peptide (SWLRDIWDWICEVLSDFK), called C5A, that exhibits high antiviral activity against hepatitis C virus (HCV) (5, 6) (Fig. 1A). In collaboration with the Chisari laboratory, we demonstrated that C5A also neutralizes HIV-1 in vitro at a nanomolar-to-micromolar range, depending on the size of the viral inoculum, the type of virus (laboratory-adapted or primary viruses), or target cells (CD4+ T cells, macrophages, and CD4+ HeLa cells) (6). The C5A sequence matches amino acids 3 to 20 of the N-terminal α-helical region of the HCV nonstructural protein 5A (NS5A). This α-helical region targets NS5A into the endoplasmic reticulum (ER) membrane (7, 8). We demonstrated that C5A disrupts the HIV-1 membrane without affecting the cellular membrane and that its amphipathic structure is absolutely required for its antiviral activity (5, 7). We also showed that C5A blocks herpes simplex virus 1 (HSV-1) and HSV-2 infection of epithelial cells both in vitro and ex vivo by disrupting the integrity of the viral membrane (9). Thus, C5A is an attractive microbicidal candidate because it neutralizes both HIV-1 and HSV via a rare mechanism of antiviral action. Supporting this notion, the Garcia lab demonstrated that vaginal administration of C5A fully protects humanized bone marrow-liver-thymus (BLT) mice against a vaginal HIV-1 challenge (10).
(A) Amino acid sequence and three-dimensional structure of the short helical peptide C5A. Shown are the l-amino acid sequence and three-dimensional structure of the short helical peptide C5A (amino acids 1 to 18) deduced from the experimental nuclear magnetic resonance (NMR) structure of NS5A N-terminal membrane anchor in dodecylphosphocholine detergent used as a membrane mimetic as described previously (6) The image was generated from structure coordinates using VMD (http://www.ks.uiuc.edu/Research/vmd/). (B) Protection of rhesus macaques by C5A. Placebo (PBS; 9 animals) or 200 μM C5A (in PBS; 9 animals) was applied in a 4-ml volume to the vagina of rhesus macaques 30 min before vaginal challenge with RT-SHIV-162P3. The outcome of the challenge was determined by measuring plasma viremia at weekly intervals. Data are expressed as viral load (RNA copies per milliliter). The limit of detection was 85 viral RNA copies/ml plasma, as indicated by the dashed line.
Progression of C5A development led to a safety and efficacy evaluation of the peptide in the nonhuman primate model. This animal model involved a 30-day pretreatment of rhesus macaques with progesterone (Depo-Provera) that synchronizes the menstrual cycle, thins the vaginal epithelium, and most importantly, enhances vaginal viral transmission, at least in macaques challenged with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) (11, 12). On the day of viral challenge, 4 ml containing 200 μM C5A synthesized with d-stereoisomers (GenScript) in phosphate-buffered saline (PBS) was applied atraumatically to the vagina 30 min prior to SHIV challenge. Nine animals were vaginally dosed with 4 ml of placebo (PBS only) and nine with C5A. The CCR5-using RT-SHIV-162P3 virus (13) was atraumatically vaginally applied at a concentration of 500 50% tissue culture infective doses (TCID50) per ml of RPMI containing 5% fetal calf serum (FCS) (RPMI-5) in a 1-ml volume. We have used this virus specifically in vaginal challenge studies and have shown that it is readily transmissible by the vaginal route and useful for microbicide testing (14). Controls were vaginally challenged with 1 ml RPMI-5 alone. A detailed description of the experimental methodology for the SHIV-162P3 vaginal transmission macaque model was described previously (11, 15, 16). Successful infection was monitored by quantifying plasma viral loads at day 0, 7, 14, 21, 28, 42, and 56 post-SHIV-162P3 challenge using a quantitative reverse transcription-PCR (RT-PCR) for viral gag RNA as previously described (17). Specifically, an optimized SIV plasma viral load quantitative PCR was used to quantify the number of copies of SIV genomic RNA per milliliter of plasma of each macaque. These studies were reviewed and approved by the IACUC of the Tulane National Primate Research Center. Importantly, 7 of 9 placebo-treated animals were infected, whereas 8 of 9 C5A-treated animals remained uninfected. Thus, the short amphipathic helical peptide C5A provides protection from challenge with a CCR5-using SHIV. The differences were highly significant by a one-tailed Fisher's exact probability test (P value of 0.0076).
To be considered a microbicidal candidate, C5A should also be evaluated for safety with repeated use. We thus conducted safety studies in 4 of the macaques that had previously resisted infection in weekly repeated vaginal C5A dosing experiments. Five weekly dosings of 200 μM C5A in 4 ml PBS were vaginally administered to uninfected 4 animals at weeks 0, 1, 2, 3, and 4. Two completely naive animals were vaginally dosed with 4 ml PBS alone as controls on weeks 1 to 4. Vaginal fluids were atraumatically collected with Merocel sponges prior to dosing (predose) and weekly after each dose from the C5A-treated animals and weekly for 4 weeks from the PBS sham controls to assess changes in soluble inflammatory mediators throughout the study. Pure vaginal fluid was extracted from sponges and final volumes calculated as previously described (18, 19). In experimental animals, vaginal pinch biopsy specimens were collected with a 3-mm alligator biopsy forceps from the lateral walls (alternating sites) 3 weeks prior (predose) and on week 2 (1 week after first dosing), and at weeks 3 and 5 (1 week after last dosing) to look for evidence of inflammation after repeated dosing. Vaginal biopsy specimens were collected from PBS controls at weeks 2, 3, and 5 of the study. Based on histologic examination of vaginal biopsy specimens, we observed no evident lesions or inflammation in animals treated with 5 successive applications of 200 μM C5A (Fig. 2A). Moreover, using a Luminex 29-plex cytokine/chemokine bead array performed on vaginal fluids, we found no evidence of increased inflammatory responses between placebo, baseline, and multiple C5A vaginal applications (Fig. 2B). Specifically, levels of the inflammation markers epidermal growth factor (EGF), eotaxin, fibroblast growth factor (FGF-basic), granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), gamma interferon (IFN-γ), interleukin-1β (IL-1β), interleukin-1 receptor alpha (IL-1RA), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, I-TAC, monocyte chemoattractant protein 1 (MCP-1), macrophage-derived chemokine (MDC), migration inhibitory factor (MIF), monokine induced by IFN-γ (MIG), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and IFN-γ-induced protein 10 (IP-10) in vaginal fluids were unchanged or very similar between controls, baseline samples, and after repeated dosing with C5A (Fig. 2B). Luminex array values (in picograms per milliliter) are presented in Table 1.
Safety of multiple vaginal applications of C5A. (A) Representative histology of vaginal biopsy specimens of four macaques exposed to multiple C5A dosing. Note that there are no significant signs of inflammation. (B) Representative levels of key inflammatory cytokines/chemokines in vaginal fluids collected from macaques before and after repeated C5A dosing (n = 4) or from sham-dosed controls (n = 2). Vaginal fluids were analyzed for 29 inflammatory mediators by Luminex bead arrays before dosing and weekly after each dosing, including 2 weeks after the last dose. Note that samples from the two sham controls were first examined at week 1, and missing values after that point reflect analytes that were below the limit of detection. Note that there are no significant changes in any of the animals even after 5 weekly dosings of C5A. Although only representative cytokines are shown here, no significant changes were detected in any of the 23 analytes measured before and after repeated dosing.
Results from the Luminex cytokine arrays
In conclusion, we showed that C5A offers protection of rhesus macaques challenged with a CCR5-using virus. This macaque vaginal transmission model entails the thinning of the vaginal epithelium using the progesterone Depo-Provera to facilitate the penetration of the virus through the epithelium in order to reach the underlying cells susceptible to infection. In this model, 80 to 90% of macaques become infected after a single vaginal challenge. Accordingly, we found in this study that 7/9 of placebo-treated animals were infected. Remarkably, only 1/9 of C5A-treated animals were infected, indicating that a single topical application of the short amphipathic helical peptide may reduce the rate of vaginal transmission of the CCR5-using virus.
A strength of C5A is the rare mechanism of antiviral action of C5A that is the disruption of the envelope of CCR5-, CXCR4- and CCR5/CXCR4-using viruses (6). Another is that C5A neutralizes viruses that are resistant to drugs currently used worldwide, such as reverse transcriptase inhibitors, protease inhibitors, and T20 (6). Another is that C5A prevents the in vitro transfer of HIV-1 from dendritic or Langerhans cells to T cells and blocks HIV-1 infection of Langerhans cells ex vivo in human epidermal sheets (6). On the other hand, the peptidic nature of C5A may present a couple of weaknesses. Despite being short (18 amino acids), its synthesis on a large scale may not be cost-effective. Another possible weakness of C5A could be a short half-life in the vaginal environment, although we showed above that its endurance in the genital compartment (30 min) was sufficient to prevent virus transmission. To improve its endurance, we synthesized C5A with d-stereoisomers instead of l-stereoisomers. Antiviral activities of C5A peptides synthesized with either d-stereoisomers or l-stereoisomers were comparable in vitro. Moreover, we demonstrated that the mechanical incorporation of C5A into matrices comprising subliming solids yielded a controlled release and preserved anti-HIV-1 activity of the peptide for up to 2 months in ectocervical explant models of HIV-1 infection (20, 21). Furthermore, in collaboration with the Baum and Moss labs, we are developing new intravaginal rings that can be loaded with antivirals with distinct mechanisms of action and which represent a promising coitally independent, long-term sustained-release microbicidal strategy (22, 23). We are currently testing these drug-loaded rings for efficacy against vaginal and rectal HIV challenges in humanized mice and macaques.
ACKNOWLEDGMENTS
We thank Meagan Watkins and Megan Gardner for technical support.
This work was supported by U.S. Public Health Service grants AI087470 and AI079782 from the National Institute of Allergy and Infectious Diseases (NIAID).
FOOTNOTES
- Received 7 August 2015.
- Returned for modification 9 September 2015.
- Accepted 5 November 2015.
- Accepted manuscript posted online 9 November 2015.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.