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

Abstract

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<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/− 0.0188 vs. 0.0151 +/− 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/− 3.908 vs. 12.466 +/− 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.

The HIV-1 transmission bottleneck

“…Virions are sequestered into surface-accessible compartments of the DCs arising from membrane invaginations [66]. Female to male transmission is less well studied; but, the initial foci of infection appear to occur most prominently in the inner foreskin in uncircumcised [67] and urethra in circumcised and uncircumcised men [68]. Finally, although initial foci of infection in the genital tract are associated with successful transmission, they may not be a prerequisite.…”

HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles

“…It has been shown in primate models that application of HIV-1 to the surfaces of intact oropharyngeal (Joag et al, 1997), anal/rectal (Bosch et al, 1997), cervicovaginal and foreskin/penile (Carias et al, 2013; Dinh et al, 2015; Girard et al, 1998; Joag et al, 1997) epithelia can lead to systemic infection of HIV-susceptible immune cells. Application of simian immunodeficiency virus to undamaged oral and vaginal mucosal epithelia also results in the transmigration of simian immunodeficiency virus across these epithelia (Miller et al, 2005; Milush et al, 2004; Stahl-Hennig et al, 1999).…”

“…Application of simian immunodeficiency virus to undamaged oral and vaginal mucosal epithelia also results in the transmigration of simian immunodeficiency virus across these epithelia (Miller et al, 2005; Milush et al, 2004; Stahl-Hennig et al, 1999). Similarly, application of HIV-1 to human foreskin, vaginal and cervical tissue explants ex vivo leads to the transmission of HIV-1 across these epithelia (Carias et al, 2013; Dinh et al, 2015; Ganor et al; Hladik et al, 2007; Maher et al, 2005; Stoddard et al, 2009; Zhou et al, 2011). Furthermore, interaction of HIV-1 with the mucosal surface of oropharyngeal tissue explants of the fetus or infant leads to infection of CD4+ T lymphocytes, Langerhans/dendritic cells and macrophages, which is critical for HIV-1 mother-to-child transmission (MTCT) (Tugizov et al, 2012).…”

Modeling Viral Spread

“…A further improvement is the development of 3D in vitro systems to analyze viral spread in more complex environments that more closely resemble the in vivo situation (93). Live-cell imaging of viral entry on a single-cell level (94, 95); investigating viral subcellular localization and replication (74, 96); analyzing viral spread within a tissue or organism, as can be done in humanized mouse models (86); and obtaining time courses of interactions between pathogen spread and individual immune responses (97) will provide more detailed information that can be used to drive new modeling.…”