Despite the decline in HIV-1–related disease and death in the United States over the past 8 years, because of the availability of antiretroviral therapies, there are major gaps in our current HIV-1 prevention strategies. Although an HIV-1 vaccine will be important to the control of the global epidemic, no effective vaccine against HIV-1 is available thus far. In the absence of an effective vaccine against HIV-1, there is an increasing recognition that topical microbicides for vaginal use that can prevent HIV-1 transmission during sexual intercourse may be the most viable current option in controlling the epidemic.1–3 Most of the antiviral properties of microbicides have been evaluated by examining their effect on the replication of virus in human primary T lymphocytes–transformed T-cell lines and epithelial cells.4–9 However, there are concerns that these assays may have less relevance when applied to sexual transmission of HIV-1 across epithelial cells of vaginal and cervical origin.
To address these concerns, a number of investigators have used primary cervical/vaginal tissue–based organ culture and organotypic cervicovaginal tissues to evaluate toxicity and antiviral activity of microbicides.10–18 We have previously established a quantitative high-throughput tissue transmission assay using the organ culture model to evaluate potential topical microbicides for their ability to block HIV-1 transmission across the epithelial mucosal barrier.11,19,20 Compounds evaluated in this system include reverse transcriptase (RT) inhibitors (PMPA and UC781), various HIV entry–inhibiting antimicrobial peptides (WLBU-2, LL37, and UC781), and membrane cholesterol–dissolving drugs, such as beta-cyclodextrin.19,20 In addition, the organ culture assay has allowed us to directly test the potential cellular toxicity of microbicide candidates by measuring immune and nonimmune cellular markers and proinflammatory cytokine responses. Our organ culture assay thus provides an ideal system for evaluating potential topical microbicides in their ability to block HIV-1 transmission across the epithelial mucosa and directly test the potential toxicity of microbicide candidates in cervical tissue.
A limited number of anti-HIV-1 microbicides that can be applied topically in the vagina have been developed. First generation of microbicides, consisting of surfactants and polyanionic polymers that are supposed to inactivate HIV-1 upon contact, were found to be either toxic or ineffective in blocking HIV-1 transmission in clinical trials.21–23 A second generation of HIV-1 RT-inhibiting compounds with potent antiviral activity in vitro has been recently described. Gel formulations of both nucleoside analog PMPA (Tenofovir) and nonnucleoside analogs UC781 and TMC120 are currently in clinical trials.24–27 Vaginal application of 1% Tenofovir gel has recently been shown to reduce HIV-1 transmission by 40%.28 Another class of microbicides in development blocks viral attachment to CD4, co-receptor (CCR5 and CXCR4) interactions, or gp41-mediated fusion. Such compounds may provide a highly effective strategy for preventing localized mucosal infection because they prevent the initial stages of the infectious life cycle.29,30 Unlike surfactant-based microbicides, such an approach is highly unlikely to perturb the protective effects of resident microflora or have contraceptive potential. Cellular co-receptor antagonists, such as CMPD167 and AOP-RANTES (CCR5 inhibitors) and AMD3465 (X4 inhibitor), have been tested in monkeys 30,31 and are being considered potential microbicides in humans.
The cyclic antimicrobial peptide retrocyclin RC-101 is a cationic β-sheet 18-residue peptide that interacts with gp41 and prevents fusion. It is considered a potential microbicide candidate because of its anti-HIV activity against a wide variety of HIV, with no toxicity in cell culture.32–34 In this report, we have evaluated the antiviral and cytotoxic profile of RC-101 in our cervical tissue–based organ culture model. Our data indicate that RC-101 blocks HIV-1 transmission in the cervical tissue–based organ culture. Furthermore, RC-101 showed no cytotoxicity as measured by a variety of immune and nonimmune functions.
RC-101 Formulation as Intravaginal Films
The 18-amino acid RC-101 peptide was prepared on a 0.25 mmol scale with an ABI 431A peptide synthesizer using FastMoc chemistry. The purified RC-101 was subsequently oxidized and cyclized as described elsewhere.32 The peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm homogeneity and that the measured mass agrees well with its expected mass. RC-101 was formulated by solvent casting technique as a quick dissolving polymeric 27.5 × 33.5-mm2 vaginal film, composed of 2.0 mg of RC-101, 6% polyvinyl alcohol (Kuraray America, Inc, New York, NY), 0.12% hydroxypropyl methylcellulose 6 (Sigma, St Louis, MO), and 3% glycerin (Dow Chemical Co., Midland, MI) as described by Sassi et al.35 All films were stored in PET/aluminum foil pouches (Amcor Flexibles Healthcare, Inc, Mundelein, IL) until use.
Virus and Cell Culture
Cell-free HIV-1 BAL was grown in phytohemagglutinin (PHA)-stimulated CD8-depleted PBMCs from seronegative persons and titered in the same cells. CD8-depleted peripheral blood mononuclear cells were prepared from blood bank donors by the use of anti-CD8 monoclonal antibody–coated immunomagnetic beads (Dynal, Oslo, Norway) as described previously.36
Testing Antiviral Activity of RC-101 in Organ Culture
Cervical tissues were obtained from seronegative premenopausal women aged 50 years or younger undergoing hysterectomy or anterior/posterior repair procedures at the Magee Women's Hospital under an institutional review board–approved protocol of the University of Pittsburgh. The organ culture was set up with CD8-depleted PBMCs (500,000) from a seronegative normal donor as indicator cells in the bottom chamber of the Transwell system as previously described.11,20 A transwell with agarose only in the top chamber served as a negative control, whereas transwells with the membrane only served as a positive control. To measure antiviral activity, cell-free HIV-1 BAL or IIIB (1 × 105 TCID50) were preincubated with RC-101 (10–40 μg/mL) for 1 hour. RC-101 and virus mixture were added to the top chambers of the tissue. As a control, HIV-1 incubated with media for 1 hour was added to another tissue well, and agarose control and membrane control wells, and incubated at 37°C for 3–4 days. After incubation, the top chamber of the well was removed, and culture of CD8-depleted cells in the bottom chamber was continued for an additional 10 days. Viral growth was monitored by measuring HIV-1 p24 antigen levels in the culture supernatant in the bottom chamber of the tissue well. A 3-fold or greater increase in HIV-1 p24 during the 2-week period was taken as positive virus infection of the CD8-depleted cells. On the last day of culture, leakiness of the organ culture system was routinely monitored by examining transmission of blue dextran, a 2 × 106 Da polysaccharide, through the tissue-containing Transwell and the agarose control into the bottom chamber as described previously.19 The amount of blue dextran transmitted was less than 1% in agarose control well, and tissue sample wells routinely showed less blue dextran transmission than did the agarose control wells. Most of the experiments were done with tissues from 2 to 3 subjects in duplicate or triplicates depending on the availability of tissues.
Measurement on Intracellular Ki67 and Cytokeratin Protein
The level of intracellular Ki67 and cytokeratin (AE1/AE3) was determined by quantitative immunostaining as described previously.11
Measurement of Proinflammatory Cytokines Response to RC-101
The level of proinflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) messages was measured in the tissues by real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Total RNA from tissues was isolated with RNA-Bee (Tel-Test, Inc, Friendswood, TX) and followed by reverse transcription with TaqMan Reverse Transcription Reagents (Applied Biosystems, Carlsbad, CA) according to the manufacturer's protocols. Thirty microliters of PCR mixture consist of 3 μL of cDNA (12 ng of total RNA equivalent), 2× TaqMan Universal PCR Master Mix, and 20× TaqMan Pre-Developed Assay Reagents (Applied Biosystems). ABI Prism 7000 Sequence Detection System was used to carry out real-time PCR under the following cycling condition: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. We selected human β2M primers and probe labeled with VIC/TAMRA for endogenous control. Human TNF-α, IL-1β, IL-6, and IL-8 primers and probes labeled with FAM/MGB were used for proinflammatory cytokine measurements. Assays were performed under conditions suggested by the manufacturer, which were designed to exclude the detection of genomic DNA. Each sample was run in duplicate. No template control was applied in each assay to ensure no cross contamination. Relative gene expression data were analyzed with relative quantification (ΔΔCt) method with 7000 System SDS software (Applied Biosystems). Our preliminary data showed that the amplification efficiency of β2M and other cytokines was approximately equal; therefore, using the ΔΔCt calculation was valid in our assay system (User bulletin #2; Applied Biosystems). The cytokine gene expression results were reported as relative fold change. Secreted cytokines in culture supernatant were measured by Luminex technology according to the manufacturer's (Bio-Rad Laboratory, Hercules, CA) instruction.
Natural Killer Cell–Mediated Target Cell Lysis Assay
PBMCs (5 × 106/mL) from normal donor were stimulated with PHA (1 μg/mL) and IL-2 (200 U/mL) and exposed to the indicated doses of RC-101. Three days posttreatment, cells were washed and degranulation of natural killer (NK) cells within PBMCs was measured after coincubation of total PBMCs with K562 (PBMC/K562 ratio = 1:1 in a total volume of 1 mL) for 2 hours, including a last 1 hour with Golgy stop. Surface staining was performed using CD3-ECD (electron coupled dye/PE-Texas Red), CD56-phycoerythrin (PE), and CD107a-Fluorescein isothiocyanate (FITC) conjugated antibodies. Expression of CD107a in CD3−/C56+ gated NK cells was determined by flow cytometry. As a vehicle control cells were treated with 20 μL of 0.1% acetic acid per ml culture volume. Number in quadrant represents percentage of CD3− CD56+ cells expressing CD107a.37
Cell Proliferation Assay
Cell proliferation assay was done as described previously.38 Briefly, 1 × 105 PBMCs were incubated with carboxyfluorescein succinimidyl ester dye (10 μg of CFSE in 1 mL of 0.2% bovine serum albumin–phosphate-buffered saline; Molecular Probes, Eugene, OR) at 37°C for 10 minutes. After incubation, 5 mL of cold complete medium was added and incubated on ice for 5 minutes. Cells were washed with cold RPMI 1640 medium containing 10% heat-inactivated human AB+ serum (Sigma), 1% L-glutamine, 1% HEPES buffer, and 1% penicillin–streptomycin and cultured for 6 days in various concentration of RC-101 (10–40 μg/mL) at 37°C. After 6 days of incubation, the cells were harvested, washed, and stained for surface markers by using anti-CD8-peridinin chlorophyll protein and anti-CD4-phycoerythrin monoclonal antibody (BD Biosciences, San Jose, CA). After staining, the cells were washed, fixed, and analyzed in a FACS Canto II flow cytometer (BD Immunocytometry Systems, San Diego, CA). Negative controls (medium only) and positive controls (phytohemagglutinin, 5 μg/mL; Sigma) were included in each assay. The results are expressed as net percentage of CFSE-positive T cells (percentage of positive peptide-stimulated T cells − percentage of medium control).
Chemotaxis assays were performed as described previously.39 Briefly, PBMCs were loaded onto top well of a 96-well ChemoTx Chemotaxis System (5-m pore; NeuroProbe, Gaithersburg, MD). RC-101 or the control chemokines were added in the bottom chamber. Cells were incubated for 5 hours at 37°C in 5% CO2; the cells on top of the membrane were removed with a scraper, and the migrated cells in the bottom wells were counted using a hemocytometer.
Antiviral Activity of Unformulated and Formulated RC-101 in Organ Culture
RC-101 was solubilized in water and tested in organ culture for its ability to block HIV-1 transmission across cervical mucosa. As shown in Figure 1, RC-101 blocked transmission of HIV-1 in a dose-dependent manner. Antiviral activity was demonstrated against both R5 HIV-1 BAL and X4 HIV-1 IIIB. More than 90% inhibition was obtained at 40 μg/mL of RC-101. Antiviral activity was similar to that observed with an RT-inhibiting microbicide UC781.19,20 We also examined the antiviral activity of RC-101 against 2 other clades of HIV-1 of African origin in organ culture. They were ZA/97/003, clade A, R5 HIV-1 and UG/92/037, clade A, and X4 HIV-1. As shown in Figure 1, RC-101 effectively blocked transmission of both African strains across cervical mucosa, although it showed higher antiviral activity against African isolate ZA/97/003.
Next, we measured antiviral activity of film-formulated RC-101. For this purpose, one film containing 2 mg of RC-101 was dissolved quickly in 1 mL of culture medium and tested at various dilutions in duplicate for antiviral activity in cervical organ culture. As shown in Figure 2, film-formulated RC-101 blocked HIV-1 transmission in a dose dependent manner, with more than 80% inhibition at 200 μg/mL. Interestingly, the placebo film also exhibited some antiviral activity.
Evaluation of Cytotoxicity of RC-101 in Organ Culture
Subtle microbicide-induced cytotoxicity may not be fully ascertained by physical examination or histological examination of the vaginal/cervical tissues. However, measurements of the level of inflammatory cytokines and cellular markers cytokeratin and Ki67 in response to microbicides in tissues in an organ culture model are excellent approaches to monitor cellular injury and cytotoxicity caused by microbicides. Therefore, we determined the effect of RC-101 on the level of intracellular Ki67 protein, a cell division marker, and cytokeratin, an epithelial cell differentiation marker. Tissues in the organ culture format with no indicator cells in the bottom well were exposed to unformulated RC-101 (40 μg/mL) for 24–72 hours at 37°C. After incubation, tissues were harvested from top well, fixed, and analyzed for Ki67 and cytokeratin (AE1/AE3) by quantitative immunostaining, as described previously.11 As shown in Table 1, the relative percentage of Ki67 and cytokeratin in RC-101-exposed cervical tissues ranged between 20.4 and 26.1 for Ki67 and between 36.9 and 44.5 for cytokeratin. This was similar to levels recovered from controls (17.5%–24.7% for cytokeratin and 38%–41% for cytokeratin). In contrast, treatment with N-9, a known cytotoxic microbicide, resulted in approximately 3-fold reduction of Ki67 (7.3%) and 2-fold reduction in cytokeratin (19.7%) markers.
Next, we measured, in our organ culture, the inflammatory cytokine response to unformulated RC-101. Our standard organ culture was set up as described above, with no indicator cells in the bottom. Cervical tissues were exposed to RC-101 (40 μg/mL) for various lengths of time. Tissues were then harvested and examined for IL-1β, IL-6, IL-8, and TNF-α cytokine messages by real-time RT-PCR and secreted cytokines in the supernatant by the Bio-Rad Bio-Plex System using the Luminex technology according to the manufacturer's (Bio-Rad Laboratory) instruction. The RT-PCR assay conditions were designed to exclude the detection of genomic DNA. Each sample was run in duplicate, and average CT values were used for gene expression calculation. Results are expressed as the fold increase in cytokine expression as compared with tissue incubated in media alone. Tissues treated with RC-101 for 24–48 hours exhibited low level of cytokine messages (see Figure, Supplemental Digital Content 1, http://links.lww.com/QAI/A322) and secreted cytokine proteins (Fig. 3) similar to controls. Longer incubation resulted in slightly higher levels of TNF-α protein (Fig. 3). In contrast, N9, a microbicide known to cause epithelial irritation in women,40–42 elicited more than 5 folds of TNF-α messages within 24 hours of exposure of tissue with 4% N9, reaching a maximum level of more than 30 folds with 1% and 4% N9 after 48 hours. A 3-fold increase in IL-1β messages was also noted after 48-hour culture, after exposure to 1% N9 for 1 hour (data not shown). There was no change in the level of expression of IL-6 and IL-8. Loss of inflammatory response at 72 hours in 4% N9 was due to loss of cell viability at 72 hours at that concentration. Because there were no indicator cells in the bottom well, we conclude that cytokine response was elicited by the tissues.
Effect of RC-101 on Immune Functions
Cervical tissues exposed to HIV-1 induce a number of immune activators to control viral infection. We evaluated the effect of unformulated RC-101 on the following 3 common immune parameters: NK cell activity, chemotactic activity, and cell proliferation. Because these experiments are difficult to perform in tissues, we evaluated effects of RC-101 in lymphocyte cell cultures. It is noteworthy that RC-101 also exhibits profound antiviral activity in PBMC (P. Gupta, manuscript in preparation). As shown in Supplemental Digital Content 2 (see Figure, http://links.lww.com/QAI/A323), RC-101 up to 20 μg/mL had no significant effect on NK cell activity, although it showed some inhibition at 40 μg/mL. Similarly, RC-101 up to a concentration of 40 μg/mL had no significant chemotactic activity of lymphocytes compared with positive control chemokines CXCL11 and CCL21 (see Figure, Supplemental Digital Content 3, http://links.lww.com/QAI/A324). RC-101 up to a concentration of 40 μg/mL also showed little effect on proliferation of CD4 and CD8 lymphocytes. As a positive control, PHA showed high-degree proliferation (see Figure, Supplemental Digital Content 4, http://links.lww.com/QAI/A325).
Effect of Seminal and Vaginal Fluids on the Antiviral Activity of RC-101 in Cervical Organ Culture
The antiviral activity of RC-101 was evaluated in the presence of 10% human seminal and vaginal fluids. For this purpose, unformulated RC-101 was incubated for 1 hour at 37°C with HIV-1 BAL, in the presence and the absence of 10% seminal fluid or vaginal fluid obtained from seronegative control men and women, respectively. Because of the reported toxicity of seminal fluid on cells/tissues, we have recovered HIV-1 after incubation from seminal fluid or by high-speed centrifugation of the semen/microbicide mixture. Pelleted virus was resuspended in small volume of medium and then added onto the tissue and cultured for 3–4 days. Transmitted virus was measured by its growth in the indicator cells in the bottom well. Because vaginal fluid is not toxic for cells, after incubation, we added vaginal fluid/HIV-1/RC-101 mixture directly into cervical tissue in the organ culture. As shown in Figure 4, seminal fluid or vaginal fluid did not significantly affect antiviral activity of RC-101 in cervical tissues.
Retrocyclins are members of a class of antimicrobial peptides called θ-defensins. Although monkeys produce θ-defensin peptides, humans cannot produce these peptides because the gene contains a premature stop codon in the peptide's signal sequence.43 Previous studies have demonstrated that retrocyclins, such as RC-101, can protect primary T cells from in vitro infection by both X4 and R5 strains of HIV-1 and are much more active in vitro than other closely related defensin molecules.32–34 However, the relevance of these assays is uncertain when considered in the context of sexual transmission of HIV across epithelial cells of vaginal and cervical origin. Therefore, in this report, we evaluated RC-101 in a cervical tissue matrix in an organ culture that closely mimics in vivo conditions. We have shown that RC-101 blocks transmission of both R5 and X4 HIV-1 across cervical mucosa in this organ culture model. Furthermore, film-formulated RC-101 retained antiviral activity in organ culture. However, it needed higher concentration of RC-101 when film formulated to achieve same level of suppression by the unformulated RC-101. This is not unexpected because similar situation was reported for other microbicides, for example, 1% RC-101 produces same amount of antiviral activity as 0.01% RC-101 in aqueous form.35 Both vaginal and seminal fluids had no deleterious effect on the antiviral activity of RC-101 in organ culture, although seminal plasma itself has some antiviral activity, which is not due to toxic effect. It is possible that semen has innate immunity against HIV-1. Regardless, these data further illustrate the importance of RC-101 as microbicide by demonstration of its antiviral activity in the presence of semen and vaginal fluids.
Because microbicides will be topically applied to the vagina, it is important to determine their cytotoxicity in vaginal/cervical tissues. The failure of N9 and cellulose phosphate in phase 3 clinical trial21,22,44 warned us that safety evaluation of a microbicide candidate should be performed as early as possible. Although monitoring for lesions in the cervix/vagina has been used to test for cytotoxicity in phase 1 clinical trials for microbicides,21,22,45 it cannot detect subtle changes in the mucosal barrier, such as induction of clinically inapparent inflammation. Although proinflammatory responses are beneficial to control vaginal bacterial infection, inflammatory cytokines may enhance HIV-1 transcription in infected cells and increase HIV-1 transmission.46,47 The rabbit vaginal irritation model has also been used to study the inflammation and toxicity of the drug formulation to the genital mucosa. Its usage is limited because of anatomical differences of the vaginal and cervical mucosa between human and rabbit. The cervical tissues in our organ culture conditions have been shown to maintain histological and cellular (immunological and nonimmunological) markers during the 6 days of incubation period 11 and therefore provide a relevant and convenient model to evaluate microbicides for potential cytotoxicity in the tissue matrix. Using this organ culture, we have demonstrated that RC-101 had no cytotoxic effect as evidenced by lack of any inhibition on 2 key cellular proteins Ki67, a cell proliferation marker, and cytokeratin, an epithelial cell marker. Furthermore, RC-101 did not induce proinflammatory response in tissues up to 72 hours after exposure. RC-101 also did not alter key immune functions such as NK cell activity and cell proliferation of CD4 and CD8 cells and did not show chemotactic activity of lymphocytes. These results together with previously reported in vitro data showing its nonhemagglutinating properties32 strongly suggest that RC-101 would be a safe microbicide for application in human. This suggestion gained support from our recent collaborative study in the nonhuman primate model showing no vaginal cytotoxicity.48
Currently, microbicides based on nucleoside RT inhibitors (UC781 and TMC120) and one with a nucleoside RT inhibitor (Tenofovir) are in Phase 2 and 3 clinical trials. However, there is a definite need to identify new antiviral compounds as backup microbicidal agents because many drugs with promising preclinical properties fail during advanced clinical evaluation, as they may be ineffective in preventing sexual transmission of resistant variants. In that regard, RC-101 induces very low level of resistance even after 28 passages in cell culture49 and that can be overcome with slight increases in peptide concentration (A.M.C., unpublished data). Retrocyclin being an evolutionary conserved host protein32,50 may be responsible for induction of low resistance. An HIV-1 entry–inhibiting microbicide, such as RC-101, has distinct advantage over RT-inhibiting microbicides, in that it blocks HIV-1 transmission before the virus can infect a target cell. Therefore, probability of developing resistance is lower because of limited viral replication at the beginning of infection.
In summary, RC-101 possesses many of the properties of an ideal microbicide candidate with strong antiviral activity and low cytotoxicity in cell culture and tissues. For these reasons, combined with recent safety demonstrated in the macaque model,48 RC-101 should be considered an excellent microbicide candidate for clinical trials.
The authors thank Ms Varsha Sridhar and Dr Yue Chen for editorial assistance and Dr Yongjun Sui for technical assistance.
1. Darroch JE, Frost JJ. Women's interest in vaginal microbicides. Fam Plann Perspect. 1999;31:16–23.
2. Klasse PJ, Shattock RJ, Moore JP. Which topical microbicides for blocking HIV-1 transmission will work in the real world? PLoS Med. 2006;3:e351.
3. Abdool 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;329:1168–1174.
4. Baba M, Schols D, Pauwels R, et al.. Sulfated polysaccharides as potent inhibitors of HIV-induced syncytium formation: a new strategy towards AIDS chemotherapy. J Acquir Immune Defic Syndr. 1990;3:493–499.
5. Balzarini J, Naesens L, Verbeken E, et al.. Preclinical studies on thiocarboxanilide UC-781 as a virucidal agent. AIDS. 1998;12:1129–1138.
6. Borkow G, Barnard J, Nguyen TM, et al.. Chemical barriers to human immunodeficiency virus type 1 (HIV-1) infection: retrovirucidal activity of UC781, a thiocarboxanilide nonnucleoside inhibitor of HIV-1 reverse transcriptase. J Virol. 1997;71:3023–3030.
7. Ito M, Baba M, Sato A, et al.. Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus (HIV) in vitro. Antiviral Res. 1987;7:361–367.
8. Srinivas RV, Fridland A. Antiviral activities of 9-R-2-phosphonomethoxypropyl adenine (PMPA) and bis(isopropyloxymethylcarbonyl)PMPA against various drug-resistant human immunodeficiency virus strains. Antimicrob Agents Chemother. 1998;42:1484–1487.
9. Roth S, Monsour M, Dowland A, et al.. Effect of topical microbicides on infectious human immunodeficiency virus type 1 binding to epithelial cells. Antimicrob Agents Chemother. 2007;51:1972–1978.
10. Abner SR, Guenthner PC, Guarner J, et al.. A human colorectal explant culture to evaluate topical microbicides for the prevention of HIV infection. J Infect Dis. 2005;192:1545–1556.
11. Collins KB, Patterson BK, Naus GJ, et al.. Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract. Nat Med. 2000;6:475–479.
12. Corpa JM, Peris B, Ribes V, et al.. Hydrocephalus in a newborn bottlenosed dolphin (Tursiops truncatus). Vet Rec. 2004;155:208–210.
13. Cummins JE, Christensen L, Lennox JL, et al.. Mucosal innate immune factors in the female genital tract are associated with vaginal HIV-1 shedding independent of plasma viral load. AIDS Res Hum Retroviruses. 2006;22:788–795.
14. Cummins JE Jr, Guarner J, Flowers L, et al.. Preclinical testing of candidate topical microbicides for anti-human immunodeficiency virus type 1 activity and tissue toxicity in a human cervical explant culture. Antimicrob Agents Chemother. 2007;51:1770–1779.
15. Fletcher PS, Elliott J, Grivel JC, et al.. Ex vivo culture of human colorectal tissue for the evaluation of candidate microbicides. AIDS. 2006;20:1237–1245.
16. Greenhead P, Hayes P, Watts PS, et al.. Parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides. J Virol. 2000;74:5577–5586.
17. Palacio J, Souberbielle BE, Shattock RJ, et al.. In vitro HIV1 infection of human cervical tissue. Res Virol. 1994;145:155–161.
18. Zussman A, Lara L, Lara HH, et al.. Blocking of cell-free and cell-associated HIV-1 transmission through human cervix organ culture with UC781. AIDS. 2003;17:653–661.
19. Gupta P, Collins KB, Ratner D, et al.. Memory CD4(+) T cells are the earliest detectable human immunodeficiency virus type 1 (HIV-1)-infected cells in the female genital mucosal tissue during HIV-1 transmission in an organ culture system. J Virol. 2002;76:9868–9876.
20. Gupta P, Ratner D, Patterson BK, et al.. Use of frozen-thawed cervical tissues in the organ culture system to measure anti-HIV activities of candidate microbicides. AIDS Res Hum Retroviruses. 2006;22:419–424.
21. Hira SK, Feldblum PJ, Kamanga J, et al.. Condom and nonoxynol-9 use and the incidence of HIV infection in serodiscordant couples in Zambia. Int J STD AIDS. 1997;8:243–250.
22. Roddy RE, Zekeng L, Ryan KA, et al.. A controlled trial of nonoxynol 9 film to reduce male-to-female transmission of sexually transmitted diseases. N Engl J Med. 1998;339:504–510.
23. van De Wijgert J, Fullem A, Kelly C, et al.. Phase 1 trial of the topical microbicide BufferGel: safety results from four international sites. J Acquir Immune Defic Syndr. 2001;26:21–27.
24. Garcia-Lerma JG, Otten RA, Qari SH, et al.. Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir. PLoS Med. 2008;5:e28.
25. Jespers VA, Van Roey JM, Beets GI, et al.. Dose-ranging phase 1 study of TMC120, a promising vaginal microbicide, in HIV-negative and HIV-positive female volunteers. J Acquir Immune Defic Syndr. 2007;44:154–158.
26. Rosen RK, Morrow KM, Carballo-Dieguez A, et al.. Acceptability of tenofovir gel as a vaginal microbicide among women in a phase I trial: a mixed-methods study. J Womens Health (Larchmt). 2008;17:383–392.
27. Woolfson AD, Malcolm RK, Morrow RJ, et al.. Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide. Int J Pharm. 2006;325:82–89.
28. Karim QA. Quarraisha Abdool Karim: investigating HIV/AIDS in South Africa. Interview by Priva Shetty. Lancet. 2009;374:871.
29. Veazey RS, Klasse PJ, Ketas TJ, et al.. Use of a small molecule CCR5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection. J Exp Med. 2003;198:1551–1562.
30. Veazey RS, Klasse PJ, Schader SM, et al.. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion. Nature. 2005;438:99–102.
31. Ketas TJ, Schader SM, Zurita J, et al.. Entry inhibitor-based microbicides are active in vitro against HIV-1 isolates from multiple genetic subtypes. Virology. 2007;364:431–440.
32. Cole AM, Hong T, Boo LM, et al.. Retrocyclin: a primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1. Proc Natl Acad Sci U S A. 2002;99:1813–1818.
33. Munk C, Wei G, Yang OO, et al.. The theta-defensin, retrocyclin, inhibits HIV-1 entry. AIDS Res Hum Retroviruses. 2003;19:875–881.
34. Owen SM, Rudolph DL, Wang W, et al.. RC-101, a retrocyclin-1 analogue with enhanced activity against primary HIV type 1 isolates. AIDS Res Hum Retroviruses. 2004;20:1157–1165.
35. Sassi AB, Cost MR, Cole AL, et al.. Formulation development of retrocyclin 1 analog RC-101 as an anti-HIV vaginal microbicide product. Antimicrob Agents Chemother. 2011;55:2282–2289.
36. Chen Y, Rinaldo C, Gupta P. A semiquantitative assay for CD8+ T-cell-mediated suppression of human immunodeficiency virus type 1 infection. Clin Diagn Lab Immunol. 1997;4:4–10.
37. Majumder B, Venkatachari NJ, O'Leary S, et al.. Infection with Vpr-positive human immunodeficiency virus type 1 impairs NK cell function indirectly through cytokine dysregulation of infected target cells. J Virol. 2008;82:7189–7200.
38. Huang XL, Fan Z, Borowski L, et al.. Multiple T-cell responses to human immunodeficiency virus type 1 are enhanced by dendritic cells. Clin Vaccine Immunol. 2009;16:1504–1516.
39. Qin S, Sui Y, Soloff AC, et al.. Chemokine and cytokine mediated loss of regulatory T cells in lymph nodes during pathogenic simian immunodeficiency virus infection. J Immunol. 2008;180:5530–5536.
40. Niruthisard S, Roddy RE, Chutivongse S. The effects of frequent nonoxynol-9 use on the vaginal and cervical mucosa. Sex Transm Dis. 1991;18:176–179.
41. Patton DL, Cosgrove Sweeney YT, Rabe LK, et al.. Rectal applications of nonoxynol-9 cause tissue disruption in a monkey model. Sex Transm Dis. 2002;29:581–587.
42. Roddy RE, Cordero M, Cordero C, et al.. A dosing study of nonoxynol-9 and genital irritation. Int J STD AIDS. 1993;4:165–170.
43. Venkataraman N, Cole AL, Ruchala P, et al.. Reawakening retrocyclins: ancestral human defensins active against HIV-1. PLoS Biol. 2009;7:e95.
44. Van Damme L, Govinden R, Mirembe FM, et al.. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med. 2008;359:463–472.
45. Patton DL, Sweeney YT, Balkus JE, et al.. Preclinical safety assessments of UC781 anti-human immunodeficiency virus topical microbicide formulations. Antimicrob Agents Chemother. 2007;51:1608–1615.
46. Pantaleo G, Graziosi C, Fauci AS. New concepts in the immunopathogenesis of human immunodeficiency virus infection. N Engl J Med. 1993;328:327–335.
47. Poli G, Fauci AS. The effect of cytokines and pharmacologic agents on chronic HIV infection. AIDS Res Hum Retroviruses. 1992;8:191–197.
48. Cole AM, Patton DL, Rohan LC, et al.. The formulated microbicide RC-101 was safe and antivirally active following intravaginal application in pigtailed macaques. PLoS One. 2010;5:e15111.
49. Cole AL, Yang OO, Warren AD, et al.. HIV-1 adapts to a retrocyclin with cationic amino acid substitutions that reduce fusion efficiency of gp41. J Immunol. 2006;176:6900–6905.
50. Nguyen TX, Cole AM, Lehrer RI. Evolution of primate theta-defensins: a serpentine path to a sweet tooth. Peptides. 2003;24:1647–1654.