To that end, we have developed an animal model to investigate vaginal transmission by HIV-1-infected cells in the setting of GUD. In this model, we apply 3% benzalkonium gel to the cervical and vaginal mucosa of macaques to induce epithelial disruption and acute inflammation. The irritation is visible by the second day of treatment (9, 16) and lasts 7 to 9 days before resolving spontaneously. We have recently shown that intravaginal inoculation of surprisingly few SIV-infected cells established systemic infection with a high level of viremia (9).

A major question about cell-associated viral transmission is whether infected donor cells traverse the reproductive tract mucosa or whether they remain in the vaginal lumen and release infectious virions that only later cross the epithelial barrier (22). To successfully locate those few donor cells that may migrate into and beneath the recipient's epithelium, we must pinpoint the anatomical site of entry and possibly the initial rounds of viral proliferation. Following our success in establishing the GUD-infection model, we hypothesized that genital ulceration could be used to define likely portals of entry for SIV, enabling us to dissect the earliest events in vaginal transmission. In the present study, we induced lower genital tract ulcers in female macaques and exposed the animals to cell-associated SIVmac239 as described previously (9). Subsequently, we investigated the cervix and vaginal mucosa for the presence of viral RNA (vRNA) and vRNA⁺ cells within the first 5 days of infection. We also collected the draining and distal lymph nodes (LNs) to gather information about the initial kinetics of viral dissemination.

In these experiments, we used sexually mature, cycling female cynomolgus macaques. Animals were housed at the Wisconsin National Primate Research Center (WNPRC) according to the NIH Guide to the Care and Use of Laboratory Animals. All procedures were performed according to an experimental protocol reviewed and approved by the Research Animal Resource Committee of the University of Wisconsin—Madison. Induction of transient lower female genital tract ulcers and intravaginal viral challenge were performed as described previously (9).

We inoculated animals cy0089 and cy0096 with a total of 10⁵ and animals cy0070 and cy0147 with 2.3 ×10⁶ and 3.0 × 10⁶ in vitro-infected allogeneic peripheral blood monocytes, respectively. On days 2 (cy0089, cy0096, cy0147) and 3 (cy0070) after inoculation, we inspected the lower female reproductive tract for signs of inflammation by using a rigid fiber-optic scope and then euthanized the animals and processed the cervix and three evenly separated sections encompassing the entire length of the vaginal mucosa. Additionally, we collected genital, inguinal, axillary, and mesenteric LN samples. We fixed a portion of each sample with 4% paraformaldehyde and processed them for in situ hybridization to examine them for SIVmac239 RNA⁺ cells as previously described (26). We tested the remaining portion of every sample for SIVmac239 vRNA burden by using a quantitative reverse transcription-PCR as detailed previously (2, 13).

First we confirmed in hematoxylin-and-eosin-stained sections that epithelial disruption was associated with those foci of ulceration that we had previously identified by visual inspection (Fig. ​(Fig.1A).1A). We investigated the association of initial infection with these sites of epithelial disruption by focusing on the 16 female genital mucosa samples obtained from animals euthanized 2 and 3 days after a cell-associated viral challenge, excluding the one animal (cy0155) euthanized 1 day after a cell-associated viral challenge, where detection of vRNA in samples from the female genital mucosa was likely due to residual inoculum (Table ​(Table1).1). We detected vRNA in 7 out of the 16 female genital mucosa samples obtained from these four macaques. Five of the 7 positive samples were associated with visible ulcer/inflammation versus 2 positive out of the 10 nonulcerated mucosal samples (83% versus 20%, P = 0.035 by Fisher's exact test) (Table ​(Table1).1). As additional evidence of the association of initial infection with epithelial disruption, we found that vRNA⁺ cells were concentrated in an ulcer (Fig. ​(Fig.2)2) in one animal (cy0121) euthanized 5 days after a cell-associated viral challenge, and the ulcer site also had the highest number of copies of vRNA (Table ​(Table11).

To test this issue, we inoculated animals cy0070 and cy0147 with cells that were not only infected but also labeled with bromodeoxyuridine (BrdU) in vitro (53% and 55.7% BrdU⁺ cells in the inoculum). Using anti-BrdU specific histochemistry on 6-μm paraffin-embedded tissue sections, we tracked the allogeneic donor cells. We did not detect BrdU⁺ cells in the cervical and vaginal tissues. We detected BrdU⁺ cells in peripheral LNs, most strikingly in the mesenteric LNs of both animals. Most of the BrdU⁺ cells appeared to be macrophages that had ingested apoptotic allogeneic cells (Fig. ​(Fig.3B).3B). However, we also found intact BrdU⁺ donor cells (Fig. ​(Fig.3A).3A). Despite observing a small number of intact donor cells up to 3 days postchallenge, we were unable to detect SIVmac239 vRNA⁺ BrdU⁺ cells by in situ hybridization. This we attribute to the low frequency of infected intact donor cells at these locations. Collectively, these findings are consistent with the concept that ulceration/inflammation supports the rapid entry and widespread dissemination of allogeneic donor cells, which the host immune system then eliminates.