Visualization of HIV-1 Interactions with Penile and Foreskin Epithelia: Clues for Female-to-Male HIV Transmission

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/− 0.0154 and 0.0171 +/− 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/− 0.0079 virions/image) than glans tissue (0.0167 +/− 0.0033 virions/image, p
Author Summary Although several clinical trials have demonstrated that male circumcision can protect men from becoming infected with HIV, we know very little about how men get infected through sex and how circumcision changes this. In this study, we explored possible sites of virus transmission across the penis by looking at how HIV interacts with adult male foreskins, penile tissues from circumcised and uncircumcised cadavers, and male rhesus macaques. Using epifluorescent microscopy, we captured images of individual HIV particles entering the penile skin, sometimes to depths where CD4+ (potential target) cells could be found. We found more virus in and on the inner aspect of the foreskin than the outer aspect of the foreskin after culturing for 24 hours. Additionally, there was more virus entering the glans penis as compared to foreskin tissues from uncircumcised cadaveric donors, and to greater depths in these tissues. We made similar observations of virus entering the tissue in living rhesus macaques, strengthening the results obtained from human tissues. This information should help us better understand how the virus moves into uncircumcised penile tissue placing uncircumcised men at higher risk for HIV infection during sex.