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
). 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
), and two additional studies showed toxicity of the rectal mucosa in humans and nonhuman primates (40
). 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
). 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mild vaginal irritation at the 1% concentration and greater vaginal irritation at higher concentrations (E. Spencer, unpublished data).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
). SPL7013 and UC781 have shown low toxicity in preclinical studies (2
), and PRO 2000 has shown low toxicity in preclinical (45
) and phase I clinical studies (33
). 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
). Many of the products being considered for use as topical microbicides have been tested against CCR5- and CXCR4-using HIV-1 isolates (2
). 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
). 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
). 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
). 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
). 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
). 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
). 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
). 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