Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication

Abstract

The HIV reverse-transcriptase inhibitor, tenofovir, was recently formulated into a vaginal gel for use as a microbicide. In human trials, a 1% tenofovir gel inhibited HIV sexual transmission by 39% and, surprisingly, herpes simplex virus-2 (HSV-2) transmission by 51%. We demonstrate that the concentration achieved intravaginally with a 1% tenofovir topical gel has direct antiherpetic activity. Tenofovir inhibits the replication of HSV clinical isolates in human embryonic fibroblasts, keratinocytes, and organotypic epithelial 3D rafts, decreases HSV replication in human lymphoid and cervicovaginal tissues ex vivo, and delays HSV-induced lesions and death in topically treated HSV-infected mice. The active tenofovir metabolite inhibits HSV DNA-polymerase and HIV reverse-transcriptase. To exert dual antiviral effects, tenofovir requires topical administration to achieve a drug concentration higher than systemic levels achieved by oral treatment. These findings indicate that a single topical treatment, like tenofovir, can inhibit the transmission of HIV and its copathogens.

Figure 1

Inhibitory activity of tenofovir against HSV-2 (strain NS and RV-124)-induced cytopathicity in HEL cell cultures using different multiplicities of infection. Virus yield was determined at 24, 48 and 72 hours post infection, and EC₉₀ and EC₉₉ values were determined from the graphical plots. Error bars represent ± SD.

Figure 2

Effects of tenofovir on organotypic epithelial rafts cultures infected with HSV-2_G at 10 days post lifting. Compounds were added to the culture media on the day of infection and remained in contact with the cells till the rafts were fixed (i.e. at 15 days after lifting). Magnification: 40X.

Figure 3

Quantification of virus yield in organotypic epithelial raft cultures infected with HSV-1 (a) or HSV-2 (b) at day 10 after initiation of the cell cultures. Compounds were added to the culture media on the day of infection (i.e. 10 days post initiation of differentiation) and remained in contact with the cells for 5 days until the rafts were frozen for determination of virus production by a plaque assay in HEL cell cultures. Two independent rafts were used for the quantification of virus production to take into account the variation of epithelial thickness among the rafts. Error bars represent ± SD.

Figure 4

Suppression of HSV-2 in singly infected and in HIV-1-coinfected human ex vivo lymphoid tissue by tenofovir. a: Blocks of human tonsillar tissue were inoculated ex vivo with HSV-2_G and treated or not with tenofovir (3, 15, 66, 240 μg/ml). HSV-2_G replication was monitored by measuring viral DNA in culture media (day 9 post-infection). Presented are means ± SEM of the results with tissues from 14 donors. For each donor, each data point represents pooled viral release from 27 tissue blocks. b: Blocks of human tonsillar tissue were co-inoculated ex vivo with HSV-2_G and HIV-1_LAI and treated or not with tenofovir (66 μg/ml). HIV-1 replication was monitored by measuring p24_gag accumulated in culture media over 3 day periods. Presented are means ± SEM of the results with tissues from 6 donors. For each donor, each data point represents pooled viral release from 27 tissue blocks. c: Blocks of human cervico-vaginal tissues were inoculated ex vivo with HSV-2_G and treated or not with tenofovir (150 μg/ml). HSV-2_G replication was monitored by measuring viral DNA accumulated in culture media at different time points throughout the culture period. Presented is a representative experiment (out of five) with a tissue from individual donor. Each data point represents pooled viral release from 16 tissue blocks. d: Blocks of human cervico-vaginal tissues were inoculated ex vivo with HSV-2_G and treated or not with tenofovir (150 μg/ml). HSV-2_G replication was monitored by measuring viral DNA accumulated in culture media at different time-points throughout the culture period. Presented are means ± SEM of the results with tissues from 5 donors. For each donor, each data point represents pooled viral release from 16 tissue blocks.

Figure 5

Effect of tenofovir, adefovir, and cidofovir treatment in DMSO formulation (a) or in gel identical to that used in the CAPRISA 004 trial (b) on development of lesions and mortality in mice inoculated with HSV-1 or HSV-2. Groups of five nu/nu mice were inoculated with HSV-1 or HSV-2 on the lumbosacral area. Each cohort was then subjected to topical treatment twice daily for 5 consecutive days, starting the day of viral infection. The placebo groups received a similar treatment with the test formulation without drug. Development of skin lesions, paralysis and mortality were recorded over a period of 20 days. Animals were euthanized when more than 30% loss in body weight or development of paralysis occurred. Survival rates were estimated according to the Kaplan-Meier method and were compared using the log-rank test (Mantel-Cox using GraphPad Prism). *Statistical significance: *denotes p < 0.05; **denotes p < 0.01.