In this report we described a human cervical tissue-derived organ culture to study HIV-1 transmission. We previously provided biochemical, histological, and in situ molecular data to demonstrate that the transmission of HIV-1 observed in the organ culture system was not due to leakiness of the system. These data included the lack of transmission from blue dextran and UV-inactivated HIV-1, the maintenance of structural integrity of H&E-stained tissue as determined by low- and high-power microscopy, the detection of HIV-1 RNA-positive T cells in the submucosa, and a correlation of coreceptor utilization of HIV-1 in the tissues during viral transmission with the coreceptor properties of inoculating virus. In the present study we provided further evidence for the integrity of the organ culture by showing the physical integrity of the basal layer by transmission electron microscopy. Furthermore, we have shown the lack of transmission from a bigger particle size (7.2 μm) than blue dextran across the mucosa. An increase in the level of HIV-1 RNA-positive T cells and dendritic cells in the tissue during the 96 h of culture further demonstrates the specificity of HIV-1 transmission in the organ culture. Although the modified raft medium used in the present study contained hydrocortisone, subsequent studies have indicated similar levels of HIV-1 transmission in medium lacking hydrocortisone (data not shown).
Using the organ culture system we demonstrated HIV-1 transmission from both cell-free and cell-associated virus of different phenotypic properties across the genital mucosa. A relatively low level of viral transmission (6 to 12.5%) observed in the organ culture probably reflects the in vivo situation, wherein it is estimated that for every single vaginal exposure to infected semen the likelihood of an infection being established is only ca. 0.01% (15
). However, HIV-1 transmission from cell-free virus in our organ culture system (1 day) is faster than that observed in the monkey (3 days) after intravaginal inoculation of SIV. A comparable level of viral transmission from cell-free and cell-associated HIV-1 suggests that both forms of virus that are present in semen can be transmitted across the female genital organ. However, the high level of cell-free HIV-1 and low level of cell-associated HIV-1 often found in semen (11
) would favor transmission from cell-free HIV-1 compared to cell-associated HIV-1. By using cell-free virus, we examined the effect of phenotypes on HIV-1 transmission across the mucosa. We have demonstrated significant levels of transmission from both R5 and X4 HIV-1, although the transmission efficiency was higher from the cell-free R5 virus. Based on these data and those of Long et al. (16
), we hypothesize that at least in women there is no preferential transmission of macrophage-tropic HIV-1, although there could be a difference in transmission efficiency between strains of HIV-1 with different phenotypic properties.
Studies from a group of men acutely infected with HIV-1 led to the suggestion of a selective transmission of a homogeneous macrophage-tropic virus during sexual transmission in men (37
). Recently, this suggestion gained support from the studies of Meng et al. (18
), who demonstrated that intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+
T cells. However, since biologic and molecular analysis of HIV-1 in semen indicated that the majority of the viral population present in semen is of the R5 type (7
), the transmission of R5 viruses in men may be a reflection of the existing pool of R5 variants of HIV-1 present in donor semen and not due to selective transmission. On rare occasions when X4 viruses are transmitted, the subjects develop AIDS in less than 2 years (1
By using the in situ hybridization assay for HIV-1 RNA in the tissue of the organ culture, we demonstrated that within 6 h of exposure to HIV-1, memory CD4+
T cells and not the Langerhans cells located immediately below the epithelial cell layer were the first HIV-1-infected cells detected in this reproductive organ. Because of the detection of a few HIV-1 RNA-positive CD4+
T cells in the tissue 6 h after virus exposure, we have confirmed such an observation by examining the immune cells directly for CD4+
T cells expressing HIV-1 RNA by a flow cytometric simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization assay. HIV-1 RNA-expressing Langerhans cells were detected between 1 and 4 days after infection. The lack of HIV-1 RNA-positive Langerhans cells at early time points after virus exposure could not be due to the migration of these cells from the tissue during culture because a sufficient number of CD1a+
Langerhans cells were present during the first 24 h of the expression. However, we could not rule out the possibility that Langerhans cells first became transiently infected with HIV-1, which was then transferred to the adjacent activated CD4+
T cells. A few HIV-1-infected macrophages were detected in the first 3 days after infection, although we did not extend the culture to 7 days to address the findings of Greenhead et al. (9
), who found that the majority of HIV-1-infected cells are of macrophage lineage after 7 days in culture. These authors also could not detect any HIV-1-infected dendritic cells. However, by using an explant system, Kawamura et al. (14
) implicated Langerhans cells as the first cells to become infected during sexual transmission of HIV-1. The difference in results between our study and that of Kawamura et al. (14
) could be due to differences between the two model systems: our organ cultures are designed to study transmission, whereas explant cultures are HIV-1 infection models. Our data contrast with the studies of Hu et al. (13
) in the SIV-rhesus monkey model, in which SIV-infected dendritic cells were detected as the first infected cell types shortly after intravaginal exposure to SIV. However, it is difficult to compare our data with the transmission data from the SIV-macaque model because human cervical biopsies from sexually active women with or without known sexually transmittted diseases generally have greatly elevated numbers of activated T cells compared to non-sexually active women (22
) and macaques (B. K. Patterson, unpublished data). These data indicate that the cellular and viral factors responsible for the sexual transmission of HIV-1 could be different than those of SIV.
Identification of memory T cells as the first HIV-1-infected cells in cervical tissue after HIV exposure has an important implication on the biology of sexual transmission of HIV-1. This finding provides an alternative pathway for the dissemination of infection to the body. Since T cells support higher replication than do dendritic cells, early infection of T cells in genital mucosa would allow virus to expand rapidly and get transferred to the dendritic cells, which would then carry HIV-1 to draining lymph nodes, where the infection would be passed to CD4+ T lymphocytes that would spread the virus systemically throughout the body. These findings may have practical implications for developing strategies to block HIV sexual transmission.
The fact that PMPA, a known antiviral agent for HIV-1 and SIV (27
), was able to block HIV-1 transmission across the mucosa provides further support for the specificity of HIV-1 transmission in the organ culture system. Since our organ culture system mimics in most part the in vivo situation, this system could also be useful to screen potential topical microbicides for their antiviral activity before they are used in clinical trials. The ability of UC781, a known microbicide of HIV-1 (2
), to block HIV-1 transmission in the organ culture system supports such a contention. Studies are ongoing to test a number of such topical microbicides in this organ culture system.