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A standardized protocol was used to compare cellular toxicities and anti-human immunodeficiency virus type 1 (HIV-1) activities of candidate microbicides formulated for human use. The microbicides evaluated were cellulose acetate phthalate (CAP), Carraguard, K-Y plus nonoxynol-9 (KY-N9), PRO 2000 (0.5 and 4%), SPL7013 (5%), UC781 (0.1 and 1%), and Vena Gel, along with their accompanying placebos. Products were evaluated for toxicity on cervical and colorectal epithelial cell lines, peripheral blood mononuclear cells (PBMCs), and macrophages (MΦ) by using an ATP release assay, and they were tested for their effect on transepithelial resistance (TER) of polarized epithelial monolayers. Anti-HIV-1 activity was evaluated in assays for transfer of infectious HIV-1 from epithelial cells to activated PBMCs and for PBMC and MΦ infection. CAP, Carraguard, PRO 2000, SPL7013, and UC781 along with their placebos were 20- to 50-fold less toxic than KY-N9 and Vena Gel. None of the nontoxic product concentrations disrupted the TER. Transfer of HIV-1Ba-L from epithelial cells to PBMCs and PBMC and MΦ infection with laboratory-adapted HIV-1Ba-L and HIV-1LAI isolates were inhibited by all products except Carraguard, KY-N9, and Vena Gel. KY-N9, Vena Gel, and Carraguard were not effective in blocking PBMC infection with primary HIV-1A, HIV-1C, and HIV-1CRF01-AE isolates. The concordance of these toxicity results with those previously reported indicates that our protocol may be useful for predicting toxicity in vivo. Moreover, our systematic anti-HIV-1 testing provides a rational basis for making better informed decisions about which products to consider for clinical trials.
The majority of new human immunodeficiency virus type 1 (HIV-1) infections occur in sub-Saharan Africa and Asia (51), and of the people infected, the majority are women. Heterosexual contact is, and has been, the primary mode of HIV-1 transmission worldwide. Despite evidence showing that proper use of male condoms is effective at decreasing sexual transmission of HIV-1 (9), they are not used consistently. Consequently, women are at a risk for acquiring HIV-1 due to exposure to infectious seminal fluid during consensual or nonconsensual intercourse with high-risk sex partners (16, 47). Effective topical microbicides, products with anti-HIV-1 activity inserted into the vagina or rectum prior to sexual intercourse, would offer an alternative to condom use that would be controlled by the receptive partner.
The major efforts to develop effective microbicides have focused on products for vaginal use. However, mucosal surfaces of both the urogenital and the gastrointestinal tracts provide portals of entry for sexually transmitted pathogens, including HIV-1 (12). Moreover, the highest risk for sexual transmission of HIV-1 is receptive anal intercourse (55). Because any product made available for couples engaging in penile-vaginal sex will potentially be used by couples engaging in penile-anal sex, it is essential that potential microbicides be evaluated for safety and efficacy in both urogenital and rectal tissues. Products must be nontoxic since disruption of the mucosal tissues and normal flora has been associated with increased rates of HIV-1 acquisition (50). Ideally, anti-HIV-1 microbicides should have activity against other sexually transmitted pathogens associated with increased rates of HIV-1 acquisition and shedding (10, 20).
To date, a majority of the efforts to develop and test microbicides with anti-HIV-1 activity include products that (i) maintain or enhance normal vaginal defense mechanisms (e.g., antimicrobial peptides [D2A21]), (ii) disrupt or inactivate the pathogen (e.g., nonoxynol-9, cellulose acetate phthalate [CAP]), (iii) block binding and fusion of pathogens (e.g., carrageenan, CAP, dendrimers [SPL7013], and naphthalene sulfonate polymers), and (iv) affect the pathogen life cycle (e.g., dendrimers and reverse transcriptase inhibitors).
This study is a comparative evaluation of the formulated candidate microbicides CAP, Carraguard, KY-N9, PRO 2000, SPL7013, UC781, and Vena Gel. Although these products were previously tested by using unformulated product and different experimental protocols, our intent was to comparatively evaluate products from each microbicide category for toxicity and anti-HIV-1 activity in a standardized protocol with relevant cell culture models. Because urogenital and colorectal epithelial cells are the primary cell types exposed to microbicide formulations, we tested the cellular toxicity of these products and their ability to prevent virus transfer from epithelial cells to activated immune cells. During sexual intercourse, breaches can occur in the epithelial layer, resulting in direct contact of the product with the underlying immune cells which are targets for HIV-1 infection. Therefore, the products also were tested for toxicity and anti-HIV-1 activity against laboratory-adapted and primary HIV-1 isolates with peripheral blood mononuclear cells (PBMCs) and macrophage (MΦ) cultures.
Epithelial cell lines were obtained from the American Type Culture Collection (Manassas, Va.). Urogenital epithelial cell lines, ME-180 (49) and HEC-1-A (26), were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 μg of streptomycin per ml, 100 U of penicillin per ml, and 100 mM l-glutamine. Colorectal epithelial cell lines, Caco-2 (21) and SW837 (27), were grown in complete medium consisting of Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 μg of streptomycin per ml, 100 U of penicillin per ml, and 100 mM l-glutamine. The epithelial cell lines were maintained at 37°C in 7% CO2. Normal human PBMCs were obtained by leukophoresis from HIV-1-negative blood donors, purified by differential centrifugation, and stored in the gas phase of liquid nitrogen until needed (54). PBMCs were depleted of CD8 T cells by using anti-CD8-conjugated magnetic beads (Dynal, Lake Success, N.Y.) according to the manufacturer's instructions. The CD8-depleted PBMCs were stimulated for 3 days at 37°C in 7% CO2 in complete RPMI medium supplemented with 10% interleukin-2 and 0.5 or 1 μg of phytohemagglutinin-P per ml. After 3 days, the PBMCs were washed and suspended in complete RPMI with 10% interleukin-2. Human monocytes (Advanced Biotechnologies, Inc., Columbia, Md.) were allowed to differentiate to MΦs and were maintained in Dulbecco's modified Eagle medium supplemented with 20% heat-inactivated fetal calf serum, 10% human AB serum, 100 μg of streptomycin per ml, 100 U of penicillin per ml, and 100 mM l-glutamine at 37°C in 7% CO2 (56). MΦs were maintained under the same media and culture conditions.
Several isolates of HIV-1 were used to assess the efficacy of prospective microbicides in blocking infection. The laboratory-adapted subtype B isolates HIV-1Ba-L and HIV-189.6 were obtained from Advanced Biotechnologies, Inc., and HIV-1LAI was obtained from stocks of the Centers for Disease Control and Prevention (CDC). Subtype C (CCR5; 96USNG58) and subtype A (CCR5; 97USSN54) isolates were provided by D. Ellenberger (48). The subtype CRF01-AE (CXCR4; CMU08) isolate was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Stocks of the primary HIV-1 isolates and HIV-1LAI were prepared and the 50% tissue culture infectious dose (TCID)50 of each isolate was determined by using the method of Reed and Muench (43).
All products tested were in formulations designed for use in humans (Table (Table1).1). Micronized CAP was provided in the form of a formulation of Aquateric (containing approximately 67% CAP; FMC Corp., Philadelphia, Pa.) by the Lindsley F. Kimball Research Institute (New York, N.Y.) and Dow Pharmaceutical Sciences (Petaluma, Calif.) (36). Carraguard (3% λ- and κ-carrageenan) was provided by the Population Council (New York, N.Y.) (59). A 2.5% methylcellulose gel was used as a placebo for Carraguard. PRO 2000 (0.5 and 4% formulations) and its placebo were provided by Indevus Pharmaceuticals, Inc. (Lexington,Mass.) (34). SPL7013, a polylysine dendrimer formulated into a 5% gel, and its placebo were provided by Starpharma Pty Ltd. (Melbourne, Australia) (3, 6, 58). UC781 (0.1 and 1% formulations), a formulated reverse transcriptase inhibitor, and its placebo were provided by Biosyn, Inc. (Huntingdon Valley, Pa.) (1). Vena Gel, a formulation containing a synthetic antimicrobial peptide (D2A21), and a hydroxyethyl cellulose placebo were provided by Demegen, Inc. (Pittsburgh, Pa.) (28). K-Y plus nonoxynol-9 (KY-N9; Ortho-McNeil Pharmaceuticals, Inc., Raritan, N.J.) and K-Y Jelly (McNeil-PPC, Inc., Skillman, N.J.) are approved over-the-counter products. CAP, SPL7013, UC781, and Vena Gel were provided in response to a solicitation placed in the Federal Register by the Division of HIV/AIDS Prevention, CDC, which requested proposals for collaboration in the evaluation of potential microbicide agents (17, 19).
Fifty thousand cells were added per well in 96-well plates. After an overnight incubation to allow the cells to adhere to the well, dilutions of the products or their placebos were added to the wells in triplicate for either 24 h or 2 h per day for 5 days. For the 5-day exposure, the products or placebos were diluted in the appropriate epithelial cell medium and added to the culture for 2 h and then washed twice with phosphate-buffered saline. Fresh medium was reapplied. Wells containing cells alone were assayed in triplicate and served as controls. To determine culture viability, cellular ATP concentrations were measured by using the CellTiter-Glo assay (Promega Corp., Madison, Wis.) according to the manufacturer's instructions. Culture ATP-dependent luminescence was measured on a DYNEX MLX instrument (Franklin, Mass.) and reported as relative light units. The ratios of the test culture relative light units to the values of the control cultures were used to assess cellular viability. No interference of products with the assay readout occurred when the products were added to the lysis buffer in luciferase control experiments. For cultures testing Carraguard toxicity, trypan blue exclusion was used instead of the ATPase assay to determine cellular viability (54).
The effect of the products and placebos on their ability to maintain an intact epithelium was determined by measuring transepithelial resistance (TER). Due to insufficient quantities of Carraguard, this product was not tested in this assay. The epithelial cell lines that form tight junctions, Caco-2 and HEC-1-A, were plated at 5 × 105 cells/well in 10-mm transwell plates (0.4-μm membrane) (Corning Incorporated, Corning, N.Y.). Apical and basolateral media were replaced, and TER was measured daily with a Millicell-ERS (electrical resistance system) instrument (Millipore Corporation, Bedford, Mass.). When plateau TER was reached, products, placebos, or media alone (control) were added in duplicate wells, and the TER was measured at 30 min and 1, 2, 4, 8, and 24 h. The epithelial resistance was expressed as follows: epithelial resistance = (Ω × cm2) − the resistance of transwells without cells.
The epithelial cell lines were plated at 5 × 105 cells per well of 12-well plates and cultured overnight. The anti-HIV-1 efficacy was tested by adding the epithelial nontoxic concentration of the product while or after the cells were exposed to virus. For testing during exposure, HIV-1Ba-L (100 TCID50/well) was mixed with the product or placebo and added to the epithelial cultures in duplicate for 18 h. The cultures were washed three times with phosphate-buffered saline before 2.5 × 106 activated PBMCs were added. For testing after exposure, HIV-1Ba-L (100 TCID50/well) was added to epithelial cultures in duplicate for 18 h. The cultures were washed three times before 2.5 × 106 activated PBMCs with product or placebo were added. For both procedures, half-volume medium changes were done every other day, and the supernatant was stored at −80°C to test for HIV-1 infection by using an HIV-1 p24gag protein enzyme-linked immunosorbent assay (ELISA) (Coulter Immunology, Hialeah, Fla.).
Activated PBMCs were mixed with serial dilutions of product or placebo in medium or medium alone (control), and 106 cells were placed in 24-well plates. Test and control cultures were set up in triplicate. After 24 h, cultures were gently mixed, and 100 μl was removed for testing. Cell viability was determined as described above. MΦs were incubated with serial dilutions of a product or placebo in medium or medium alone (control) and cultured in 24-well plates. Test and control cultures were set up in duplicate. After 24 h, media or product dilutions were removed, and the wells were washed twice with fresh medium before 500 μl of reconstituted CellTiter-Glo reagent was added to each well. The plate was placed on a horizontal rocker for 30 min. Viability was determined as described above.
PBMCs (106 cells/well) were incubated in medium containing HIV-1 (100 TCID50/well) and a nontoxic concentration of product or placebo or medium alone (controls) for 4 h. The PBMCs were then washed three times with medium and cultured in medium alone at 106 cells/ml/well in 24-well plates for 7 to 14 days. Each product, placebo, and control was tested in triplicate. Culture supernatants were collected every 3 days and replaced with fresh medium without product or placebo. The culture supernatants were stored at −80°C until they were assayed for HIV-1 by using an HIV-1 p24gag protein ELISA (Coulter Immunology).
MΦ cultures were incubated in medium containing HIV-189.6 or HIV-1Ba-L (100 TCID50) with a nontoxic concentration of product or placebo or medium alone (control) for 4 h. Cultures were then washed three times with medium and 1 ml of fresh medium was added. Cultures were maintained for 9 to 12 days, and culture supernatants were collected every 2 to 3 days and replaced with fresh medium without product or placebo. The culture supernatants were stored at −80°C until they were assayed for HIV-1 by using an HIV-1 p24gag protein ELISA (Coulter Immunology).
The effects of microbicide products on the TER were analyzed by using a partial F test to determine significant differences in the slope of the regression coefficients. To determine if the efficacies of the PRO 2000, SPL7013, and UC781 microbicide products were significantly different compared to their placebos, a one-way analysis of variance followed by a Tukey-Kramer multiple comparisons posttest was performed.
Any topical microbicide approved for human use will need to be first evaluated for epithelial toxicity, because the product will come in direct contact with vaginal or rectal mucosal surfaces. Nontoxic concentrations of the products and placebos were determined by culturing the epithelial cell lines for 24 h in diluted products or placebos. Product and placebo concentrations that gave culture viabilities of ≥60% compared to control cultures were considered to be nontoxic and were evaluated further. Overall, the four epithelial cell lines had similar toxicity levels to the products (Table (Table2).2). Nontoxic concentrations of CAP, Carraguard, PRO 2000 (0.5 and 4%), SPL7013 (5%), and UC781 (0.1 and 1%) ranged from 1:10 to 1:50 of the original formulation. In contrast, nontoxic concentrations of Vena Gel and KY-N9 were 1:1000, or 20- to 100-fold more toxic than the other four products. Nontoxic concentrations of the five placebos ranged from 1:5 for methylcellulose and the Vena Gel placebo to 1:100 for KY Jelly, relative to their original formulations. Nontoxic concentrations of PRO 2000, SPL7013, and UC781 formulations were equal to or 2.5-fold greater than those of their respective placebos, suggesting that most of the toxicities of these products were due to the excipients. In contrast, nontoxic concentrations of KY-N9 and Vena Gel were 10- and 200-fold greater, respectively, than those of their placebo formulations, suggesting that most of the toxicities of these products were due to the products rather than their excipients. The toxicities of nontoxic concentrations of products and placebos were also tested in epithelial cell cultures for 2 h each day for 5 days (Table (Table3).3). Only Carraguard and SPL7013 cultures had viabilities that were <60% after the 5-day period (50 and 58%, respectively).
The ability of mucosal epithelial cells to maintain an intact, polarized monolayer in the presence of a microbicide is a possible predictor of that product's safety on cervical and colorectal tissues. Product and placebo formulations were added to HEC-1-A (data not shown) and Caco-2 (Fig. (Fig.1)1) cell cultures after they had established confluent monolayers as determined by a constant TER (7 to 11 days in culture). The TER was followed for 24 h after product addition. The TER of the control cultures, without product or placebo, did not vary more than 10% over the 24-h period. The nontoxic concentrations of the products and placebos reduced the TER by <35% (ranging from 0 to 78 Ω × cm2) compared to the untreated controls with the exception of CAP (Fig. (Fig.1).1). The greatest differences for the nontoxic concentrations were detected by 4 h postproduct addition. After 24 h, the TER returned to preproduct levels. Conversely, the toxic concentrations of the products and placebos significantly reduced the TER by 60 to 100% by 4 h (P < 0.02). After 24 h, the toxic concentrations of the products and placebos reduced the TER by 80 to 100% except for SPL7013 (5%) and its placebo. Interestingly, the nontoxic and toxic concentrations of CAP reduced the TER by 65 and 77%, respectively, by 2 h postproduct addition. By 8 h, the monolayers exposed to the nontoxic concentration of CAP returned to preproduct TER levels, while those exposed to the toxic concentration remained at the 2-h level. Overall, neither the nontoxic nor the toxic concentration of CAP significantly affected the TER (P > 0.8).
Although urogenital epithelial cells do not become productively infected, they have the capacity to transfer infectious virus to activated PBMCs (14). To evaluate the ability of the products to block this transfer, nontoxic concentrations of products and placebos were added to epithelial cell cultures during or after they were exposed to HIV-1Ba-L. A summary of results from the four epithelial cell lines tested (Caco-2, SW837, HEC-1-A, and ME-180) showed that CAP, PRO 2000 (0.5 and 4%), SPL7013 (5%), and UC781 (0.1 and 1%) prevented the epithelial cell transfer of infectious virus to PBMCs. The HIV-1 blocking activity occurred irrespective of whether the products were added to the epithelial cells while or after they were exposed to the virus (Fig. (Fig.2).2). Carraguard, KY-N9, or Vena Gel did not completely block virus transfer. However, these products reduced p24 production when applied after the virus was added to the epithelial cells. Viral replication in this system typically peaked by day 6 in the control cultures (no product or placebo) (Fig. (Fig.2).2). Therefore, the efficacies of the products and placebos for the four epithelial and PBMC cocultures were analyzed together for log10 reduction versus the untreated control at day 6. CAP, PRO 2000 (0.5 and 4%), SPL7013 (5%), and UC781 (0.1 and 1%) blocked the transfer of infectious virus by ≥3 log10 (100% inhibition of virus infection). While Carraguard also blocked HIV-1 transfer by 2.2 log10 or greater (≥95% inhibition of virus replication), complete inhibition was not obtained. Neither KY-N9 nor Vena Gel completely blocked HIV-1 transfer to the PBMCs. Although methylcellulose, the Vena Gel placebo, and the SPL7013 placebo had little detectable anti-HIV-1 activity (<1 log10), KY Jelly and the PRO 2000 and UC781 placebos reduced the transfer of infectious HIV-1 to PBMCs after virus exposure by ≥1 log10 at day 6. While the PRO 200 and SPL7013 placebos were significantly less effective than the formulations with their active ingredients at reducing infectious HIV-1 transfer to PBMCs (P < 0.001), the UC781 placebo was as effective as the formulations with the active ingredient.
Cellular viability was determined for the PBMCs and MΦs by culturing for 24 h in the presence of serial dilutions of product or placebo. Product and placebo concentrations that gave culture viabilities of ≥60% compared to control cultures were considered to be nontoxic. Nontoxic concentrations of products and placebos in PBMC and MΦ cultures were the same or ≤2.5-fold different than those in the four epithelial cell lines with the exception of the Vena Gel placebo, which was 10-fold more toxic for the MΦs (Table (Table22).
The primary targets for HIV-1 infection are CD4+ lymphocytes and MΦs. Therefore, the anti-HIV-1 activity of the nontoxic concentrations of the products and placebos was determined by using these cells. In addition to the standard laboratory-adapted isolates HIV-1Ba-L and HIV-1LAI, three primary isolates representative of subtype A, subtype C, and subtype CRF01-AE were used to test product efficacy in PBMCs (Fig. (Fig.3).3). CAP, PRO 2000 (0.5 and 4%), SPL7013 (5%), and UC781 (0.1 and 1%) blocked HIV-1 infection by >2.5 log10 (100% inhibition of HIV-1 infection) with laboratory-adapted and primary isolates with the exception of PRO 2000 (0.5%) and CAP. The inhibition of subtype A and C infection by PRO 2000 (0.5%) was 1.3 log10 (95% inhibition) and 1.8 log10 (100% inhibition), respectively, and inhibition of subtype C infection by CAP was 1.3 log10 (95% inhibition). KY-N9 completely inhibited HIV-1Ba-L, subtype A, and subtype CRF01-AE infection but had <1 log10 reduction against HIV-1LAI or subtype C (58 and 88% inhibition, respectively). Moreover, KY Jelly was equally as effective against HIV-1Ba-L as KY-N9 but was not effective against the other subtypes. Vena Gel placebo was twice as effective as Vena Gel at preventing subtype C infection, but neither was effective against the other isolates, with a reduction of virus infection of ≤0.25 log10. While Carraguard inhibited HIV-1LAI infection by >4 log10 (100% inhibition of virus infection), it had little to no effect against HIV-1BaL (0.6 log10 reduction) or subtypes A, C, or CRF01-AE. Methylcellulose had no detectable anti-HIV-1 activity against laboratory-adapted or primary HIV-1 isolates. The PRO 2000, SPL7013, and UC781 placebos generally had high anti-HIV-1 activity. The PRO 2000 placebo completely blocked laboratory-adapted HIV-1Ba-L and HIV-1LAI infection in PBMCs and reduced primary HIV-1 isolate infection by 63 to 93%. This was not significantly different from the formulation with the active ingredient. The SPL7013 placebo reduced primary HIV-1 isolate infection by 50 to 100% but was significantly less effective on the laboratory-adapted HIV-1Ba-L and HIV-1LAI infection (P < 0.001). While the UC781 placebo was not as effective as the PRO 2000 placebo or the SPL7013 placebo, it inhibited laboratory-adapted HIV-1Ba-L and HIV-1LAI infection in PBMCs by greater than 87%. The UC781 placebo also inhibited primary HIV-1 subtype C infection by 93% but had minimal to no inhibition of subtypes A or CRF01-AE infection (Fig. (Fig.3).3). This placebo was significantly less effective than the formulation with its active ingredient (P < 0.001).
Similar testing of the products with MΦs showed that CAP, Carraguard, PRO 2000 (0.5 and 4%), SPL7013 (5%), and UC781 (0.1 and 1%) blocked HIV-1 infection by >2 log10 (≥99% inhibition) (Fig. (Fig.4).4). No inhibition of virus infection was noted for KY-N9 or Vena Gel, nor was anti-HIV-1 activity noted for methylcellulose, KY Jelly, or Vena Gel placebo. PRO 2000, SPL7013, and UC781 placebos inhibited HIV-1 infection in MΦs by 2.5 log10 (100% inhibition), 3 log10 (100% inhibition), and 0.9 log10 (88% inhibition), respectively, and were not significantly different from the products with the active ingredient.
The only accurate evaluation of an anti-HIV-1 microbicide will be through large-scale phase III efficacy trials. However, in this study we demonstrate that in vitro testing can be used to efficiently evaluate formulated candidate products and provide comparative toxicity and efficacy data. The data presented here showed that CAP, PRO 2000 (0.5 and 4%), SPL7013 (5%), and UC781 (0.1 and 1%) were relatively nontoxic and efficacious against HIV-1 infection of primary PBMCs and MΦs as well as against transfer of virus from epithelial cell lines to activated PBMCs. The KY-N9 and Vena Gel formulations were much more toxic and, when tested at their nontoxic concentrations, were ineffective at preventing the infection of immune cells or blocking transfer of the virus from epithelial cells to activated PBMCs. Carraguard was nontoxic for all cell types tested but had poor efficacy in blocking the infection of PBMCs by primary viruses belonging to subtypes A, C, and CRF01-AE. Our data show that in vitro testing of primary HIV-1 isolates should be included in evaluations of microbicides since these viruses are more likely to represent those being sexually transmitted.
Originally developed as a spermicide, N9 was shown in initial reports to have in vitro activity against sexually transmitted pathogens including HIV-1 (24, 31). Despite this promising early work, Baurinbaiar and Fruhstorfer (5) reported that the N9 antiviral activity only occurred at doses that were cytotoxic. Several clinical studies have reported epithelial disruption and inflammation of the female genital tract (44, 46, 52), and two additional studies showed toxicity of the rectal mucosa in humans and nonhuman primates (40, 42). Since that time, the recommendation has been that products containing N9 not be used for HIV-1 prevention, especially rectally (8). Our data were consistent with these results, showing that KY-N9 was highly toxic to the cells tested and had reduced efficacy in blocking HIV-1 infection at its nontoxic concentration. Likewise, the 1% Vena Gel preparation was highly toxic at its original concentration. This was unexpected since other antibiotic peptides, such as defensins, are used at concentrations approximately 1,000-fold higher (13, 29). Moreover, animal studies showed mild vaginal irritation at the 1% concentration and greater vaginal irritation at higher concentrations (E. Spencer, unpublished data). Both CAP and Carraguard are used in the pharmaceutical industry and are classified as compounds that are “generally recognized as safe.” The animal vaginal irritation studies showed that CAP did not have any deleterious effects (A. R. Neurath, unpublished data). Preliminary safety and acceptability trials have shown carrageenan products to be well tolerated in humans (11, 15). SPL7013 and UC781 have shown low toxicity in preclinical studies (2, 3, 6), and PRO 2000 has shown low toxicity in preclinical (45) and phase I clinical studies (33, 53). The comparison evaluation in this study for toxicity of the microbicide formulations was consistent with the findings from these published reports and suggests that our evaluation was predictive of in vivo toxicity.
Most sexual transmission of HIV-1 is due to CCR5 coreceptor-using isolates being preferentially transmitted over CXCR4 coreceptor-using isolates. The reasons behind the preferential spread are unknown since both viruses can be found in mucosal secretions and can infect resident mucosal immune cells (23, 61). Many of the products being considered for use as topical microbicides have been tested against CCR5- and CXCR4-using HIV-1 isolates (2, 37, 38, 45). However, these data have been primarily restricted to subtype B laboratory-adapted isolates. CAP, PRO 2000, and UC781 have been shown to be highly effective in preventing subtype B infection in this and other studies (2, 36, 38, 45). With the exception of CAP, SPL7013, and UC781, limited data are available on microbicide effectiveness against primary isolates belonging to HIV-1 subtypes which are geographically distinct (7; S. Jiang and A. R. Neurath, unpublished data; T. McCarthy, unpublished data). This is important since the majority of infections occur outside subtype B regions. Therefore, we used three primary isolates that are representative of subtypes A (Central Africa and Asia), C (Central and South Africa), and CRF01-AE (Asia). Our data showed that CAP, PRO 2000 (0.5 and 4%), SPL7013 (5%), and UC781 (0.1 and 1%) are highly effective (≥95% inhibition) at preventing primary HIV-1 isolate infection of PBMCs. These data are in agreement with published preclinical efficacy data for these products (2, 4, 36, 38, 45). KY-N9 and Vena Gel had equivocal or poor results at protecting against infection, presumably due to the need to dilute each product to reach a nontoxic concentration. While Vena Gel was not effective in this study, other compounds that maintain or restore the normal vaginal pH are being pursued and appear effective in animal models (25, 60). Unexpectedly, Carraguard did not block infection of PBMCs by the primary isolates. Since Carraguard activity is associated with its negative charge, the reduced anti-HIV-1 activity against the primary isolates may be due to the greater number of glycosylation moieties on the gp120 envelope of primary isolates compared with those on laboratory-adapted isolates. It has been shown recently that an increased “glycan shield” on gp120 can block neutralizing antibodies (57), and this shield may have a dampening effect on microbicide activity that relies on charge as a mechanism of HIV-1 inactivation.
Currently, most products that are being formulated for use as a topical microbicide are based on a single active ingredient. While these products may have excellent activity against HIV-1 infection, such as nonnucleoside reverse transcriptase inhibitors, they may not have activity against other sexually transmitted pathogens. This is important since these pathogens have been shown to increase HIV-1 acquisition and shedding (10, 20). Combination microbicides have been proposed that act at several steps along the HIV-1 infection pathway and may overlap with other pathogen life cycles. For example, CAP is not a combination microbicide, but it has been shown to act at several steps along the HIV-1 infection pathway, including inducing gp41 six-helix bundle formation resulting in virucidal activity, and has a low pH which results in loss of HIV-1 integrity (38). Further, CAP has been shown to be effective against other sexually transmitted pathogens (22, 35, 39). Alternatively, the formulation of a product may be in an active excipient like carbopol or polycarbophil, as is the case for PRO 2000, SPL7013, and UC781. Carbopol is a weak acid, has a high buffering capacity, and has been shown to be effective at inhibiting herpes simplex virus infection in mice (30). Indeed, the placebos for PRO 2000, SPL7013, and UC781 were shown to have some anti-HIV-1 activity and in certain instances were equally as effective as the product with the active ingredient. Additive effects were seen when the active ingredients were present in the majority of testing. PRO 2000 and a compound similar to SPL7013 have been shown to interfere with HIV-1 adsorption to the cell surface (45, 58). While UC781 is a known reverse transcriptase inhibitor and is highly effective against HIV-1 infection, a compound similar to SPL7013 also was shown to inhibit later steps in the HIV-1 life cycle (58). The products which acted at multiple steps against HIV-1 were the products that performed best in our evaluation. These data support the work exploring combinational microbicides or microbicides that act at multiple sites of the HIV-1 infection pathway.
We evaluated topical microbicides that were representative of four broad categories of microbicides, including those products that maintain or enhance normal vaginal defense mechanisms, products that disrupt or inactivate the pathogen, products that block the binding and fusion of pathogens, and products that affect the pathogen life cycle. While not all of these representative products were nontoxic or effective at inhibiting HIV-1 infection and spread, other products in the same classes may be effective microbicides. On the basis of this evaluation protocol, toxicity data presented in this study were consistent with nonhuman primate studies and phase I and II safety and acceptability trials (11, 15, 33, 41, 42, 44, 46, 52, 53). Furthermore, efficacy data from this study were concordant with efficacy data from nonhuman primate studies (32; M. G. Lewis, W. Wagner, J. Yalley-Ogunro, J. Greenhouse, and A. T. Profy, Abstr. Microbicides 2002, Antwerp, Belgium, p. 84, 2002). These results indicate that a comparison made according to this protocol of candidate microbicides may be predictive of their effect on tissues and anti-HIV-1 activity and thus aid in the selection of products to advance to clinical study.
CDC remains interested in the testing of new agents through these methods. Potential agents should have demonstrated in vitro anti-HIV-1 activity and have been formulated for vaginal or rectal application. Current information regarding this program may be found in the May 17, 2002 Federal Register, available at http://www.gpoaccess.gov/fr/search.html (18).
V.N.J., A.S., and S.T.S. were supported by the Oak Ridge Institute for Science and Education postgraduate research program.
CAP was provided by A. Robert Neurath, Biochemical Virology Laboratory, The Lindsley F. Kimball Research Institute of the New York Blood Center. PRO 2000 was provided by Albert T. Profy, Indevus Pharmaceuticals, Inc. SPL7013 was provided by Tom McCarthy, Starpharma Pty Ltd. UC781 was provided by Joseph Romano, Biosyn, Inc. Vena Gel was provided by Elizabeth Spencer, Demegen, Inc.
The use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the CDC.