Our goal was to validate and implement a preclinical in vivo
experimental system to evaluate the ability of topically applied inhibitors to prevent vaginal HIV-1 transmission. We first used flow cytometry to characterize the site of exposure (i.e., the female reproductive tract [FRT]) in BLT mice for the presence of human CD4+
T cells ( A to F). The human T cells expressing CD4 (primary viral receptor) and CCR5 (viral coreceptor) represent important HIV target cells found in the FRT of BLT mice. When these data were compared to human studies of T cells in cervicovaginal mucosa and cervical epithelia, we found that BLT mice exhibit a higher CD4+
T cell proportion of total CD3+
T cells than reported in the human lower reproductive tract; however, the proportions of CCR5-positive T cells in the FRT were essentially the same between humans and BLT mice (21
). Second, we determined the systemic reconstitution with HIV-1 target cells by measuring the levels of human cells in peripheral blood. Blood analysis of all mice used in the testing of microbicides showed that 52% (mean; standard deviation [SD] ±17%; n
= 64) of all circulating cells were human ( to ). Further analysis showed that 83% (mean; SD, ±7%; n
= 64) of circulating human T cells expressed CD4 ( to ). The BLT mouse blood CD4+
T cell proportion is higher than normally seen in humans but still within the range of what has been described for healthy humans (2
). These results demonstrate the suitability of the utilized BLT mice to perform the in vivo
evaluation of topical microbicides to prevent vaginal HIV-1 transmission.
Fig. 1. The female reproductive tract of humanized BLT mice is efficiently repopulated with human HIV-1 target cells rendering them susceptible to infection. (A) Flow cytometric analysis of cells from the FRT of a representative naive humanized BLT mouse demonstrates (more ...)
Description of BLT mice used to evaluate the vaginal HIV-1 transmission prevention potential of 1% tenofovira
Description of BLT mice used as no-inhibitor controls for vaginal HIV-1 transmissiona
To date, CAPRISA 004 has provided the only successful demonstration that a topical microbicide can prevent vaginal HIV-1 transmission (1
). Therefore, to validate the use of BLT mice for the evaluation of topical inhibitors of vaginal HIV-1 transmission, we utilized the same study design as in the CAPRISA 004 trial (), where study subjects were asked to apply a 1% tenofovir product twice (once before and once after sex with both applications occurring within a 24-h period) (1
). In our experiments, BLT mice received 1% tenofovir once before and once after HIV-1JR-CSF
exposure according to the clinical trial protocol. In the CAPRISA 004 study, a 39% overall reduction in HIV incidence was observed, as determined by rapid tests and confirmed by viral load analysis (1
). In BLT mice, we observed 88% protection from vaginal HIV-1 transmission, as determined by plasma viral load analysis, and protection from vaginal HIV transmission was confirmed by the lack of detectable viral DNA in peripheral blood and tissues ( A to E and ). In contrast, plasma viral RNA and cell-associated viral DNA were readily detectable in all infected control mice and the one breakthrough mouse which received 1% tenofovir treatment (; ). These results obtained using the CAPRISA 004 study design in BLT mice demonstrate the ability of 1% tenofovir to reduce vaginal HIV-1 infection in this model and strongly support the use of this model for the evaluation of candidate topical microbicides for their ability to prevent HIV transmission.
Fig. 2. Experimental design for the validation of BLT mice for preclinical efficacy evaluation of HIV prevention strategies. (A) CAPRISA 004 experimental design. The CAPRISA 004 trial design incorporated a novel strategy for dosing referred to as BAT24 and was (more ...)
Fig. 3. Effective protection from vaginal HIV-1 infection by topical 1% tenofovir. Using the experimental protocol shown in that reproduces CAPRISA 004, BLT mice were exposed vaginally to HIV with (n = 8) or without (n = 4) tenofovir treatments. Seven (more ...)
Based on these encouraging results, we proceeded to evaluate the efficacy of six additional microbicide candidates to prevent vaginal HIV-1 transmission in humanized BLT mice ( and ). Prior to selection for in vivo
efficacy evaluation here, each inhibitor was shown to efficiently inhibit HIV in vitro
). Our experimental approach for testing these six inhibitors was a single dose of the product administered prior to exposure rather than the two doses of drug administered within 24 h as used in CAPRISA 004 (1
). This experimental design is based on the more standard single-dose administration prior to exposure (19
). This approach is simpler, more cost-effective than multiple applications per sexual encounter and has been postulated to help increase adherence among women (51
). For these reasons this approach has been included as one arm in an upcoming trial by the Microbicides Development Programme (MDP 302) (51
). Furthermore, the “single dose prior to or with each sexual encounter” protocol has been the most common microbicide application protocol applied in clinical trials and animal model studies (19
). The six additional candidate microbicides that we tested using this experimental design represent a broad spectrum of inhibitors with distinct mechanisms capable of blocking HIV infection at several different steps of the viral replication cycle from preentry to postbudding ().
Fig. 4. Experimental design for the evaluation of candidate microbicides for their ability to prevent vaginal HIV transmission in humanized BLT mice. (A) Conventional trial design for evaluation in humans and NHPs. The conventional clinical trial design and NHP (more ...)
C52L is a sequence-modified C-peptide version of T-20 that binds to the N-terminal heptad repeat of gp41 to block fusion between the virion and the cell membranes (10
). C5A is an amphipathic α-helical peptide HIV inhibitor derived from the hepatitis C virus NS5A anchor domain (5
). C5A acts at preentry by disrupting the integrity of the viral membrane and the mature viral core. PIE12-Trimer is a d
-peptide that binds to a conserved pocket on the surface of trimeric gp41. PIE12-Trimer blocks the final stages of the fusion process between the virion and cell membranes (54
). TC247 is a thioester compound zinc finger inhibitor that ejects zinc ions from the two HIV nucleocapsid zinc finger loops (32
). TC247 has been shown to affect HIV at multiple stages of replication but is thought to primarily target the virus at the level of Gag processing and maturation of virions, rendering progeny virions noninfectious and thus preventing viral spread. NSC23766 is a small molecular inhibitor of Rac1-guanine nucleotide exchange factors (GEFs) TrioN and Tiam1. NSC23766 prevents viral entry from the inside-out by interfering with the structural modifications that occur within the cell during fusion (16
). The combination of the reverse transcriptase inhibitors FTC-TDF, the inhibitors present in Truvada (3
), targets postentry viral DNA synthesis.
Following the vaginal administration of each inhibitor, treated BLT mice were challenged vaginally with the same dose of HIV-1JR-CSF as described above for tenofovir. Peripheral blood from BLT mice was sampled longitudinally following viral exposure for evidence of infection (). The presence of HIV-1 plasma antigenemia, PBMC-associated viral DNA, or plasma viral RNA in exposed mice was considered evidence of transmission. In the absence of HIV inhibitor, transmission was documented in 10/12 control mice ().
Fig. 5. Peripheral blood analyses show differential protection from vaginal HIV transmission by the indicated HIV inhibitors when applied topically prior to exposure. (A to C) Ten of 12 control BLT mice were positive for plasma antigenemia, for PBMC viral DNA, (more ...)
Differential levels of protection from HIV transmission were noted among the six inhibitors. Specifically, the Rac inhibitor NSC23766 did not protect any BLT mice (4/4 pretreated BLT mice were infected). The zinc finger inhibitor TC247 protected 4/7 BLT mice. The combination of FTC-TDF protected 8/9 BLT mice from infection (). Remarkably, complete protection was observed with C52L (0/7), C5A (0/8), or PIE12-Trimer (0/5) (). Protection from vaginal HIV-1 infection by C52L, C5A, PIE12-Trimer, FTC-TDF, and TC247 was verified by three additional criteria: viral rescue by coculture with allogeneic PBMC, real-time PCR analysis for the presence of viral DNA in tissues, and in situ hybridization analysis for the presence of productively infected cells. Using these techniques, there was no evidence of infection in any of the inhibitor-protected mice. In contrast, clear evidence of HIV-1 infection was obtained in the tissues analyzed from control or breakthrough animals ( to ).
Description of BLT mice used to evaluate the vaginal HIV-1 transmission prevention potential of C52L, C5A, and PIE12-Trimera
Description of BLT mice used to evaluate the vaginal HIV-1 transmission prevention potential of FTC/TDF, TC247, and NSC23766a
Importantly, protection of BLT mice pretreated with tenofovir, C52L, C5A, PIE12-Trimer, FTC-TDF, and TC247 was not attributable to a lack of human reconstitution within the FRT. Flow cytometry analysis of the FRT confirmed the presence of human CD4+ T cells in protected animals at levels that were comparable to controls (naive [n = 4] versus protected [n = 6]; Mann-Whitney test, P = 0.76) ( and A). Furthermore, in addition to human CD3+ T cells, the presence of CD11c+ dendritic cells and CD68+ monocyte/macrophages in the FRT of BLT mice was confirmed by immunofluorescence staining (). Therefore, lack of HIV-1 infection in the mice treated with these inhibitors was not due to the absence of target cells in the FRT. Rather, the lack of vaginal HIV-1 transmission is attributed to the protective effect provided by the tested inhibitors.
Fig. 6. Protection in inhibitor-pretreated BLT mice was not attributable to a lack of human reconstitution within the FRT. (A) Human CD4+ and CD8+ T cell levels in the FRT of mice protected by vaginally applied HIV inhibitors (1% tenofovir, mouse 3; C52L, mouse (more ...)
Based on the extensive molecular and cellular analyses presented above, we formulated the following summary (): the Rac inhibitor (NSC23766; n = 4) did not protect any mice from HIV infection. The zinc finger inhibitor (TC247; n = 7) protected 57% of treated animals. The postentry reverse transcriptase inhibitor combination (FTC-TDF; n = 9) and two applications of a single reverse transcriptase inhibitor (1% tenofovir; n = 8) each similarly protected 88% of treated animals. Each of the three peptide inhibitors (C52L [n = 7], C5A [n = 8], and PIE12-Trimer [n = 5]) protected 100% of treated BLT mice from vaginal HIV-1 transmission. The protection afforded by these last five inhibitors, when each was independently compared to the no-inhibitor controls, was significant as determined by log rank (Mantel-Cox) analysis ().
Fig. 7. Differential protection from vaginal HIV-1 transmission by topically applied inhibitors. Kaplan-Meyer plots representing the percentage of BLT mice that each inhibitor protected as a function of the number of weeks postexposure until the first peripheral (more ...)