Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2-/- gammac -/- (RAG-hu) mice

Abstract

Studies on HIV-1 mucosal transmission to evaluate early events in pathogenesis and the development of effective preventive/prophylactic methods have thus far been hampered by the lack of a suitable animal model susceptible to HIV-1 infection by either vaginal and/or rectal routes. In this regard, while primate-SIV/SHIV and cat-FIV models provided useful surrogate platforms to derive comparative data, these viruses are distinct and different from that of HIV-1. Therefore an optimal model that permits direct study of HIV-1 transmission via mucosal routes is highly desirable. The new generation of humanized NOD/SCID BLT, NOD/SCIDgammac(-/-), and Rag2(-/-)gammac(-/-) mouse models show great promise to achieve this goal. Here, we show that humanized Rag2(-/-)gammac(-/-) mice (RAG-hu) engrafted with CD34 hematopoietic progenitor cells harbor HIV-1-susceptible human cells in the rectal and vaginal mucosa and are susceptible to HIV-1 infection when exposed to cell-free HIV-1 either via vagina or rectum. Infection could be established without any prior hormonal conditioning or mucosal abrasion. Both R5 and X4 tropic viruses were capable of mucosal infection resulting in viremia and associated helper T cell depletion. There was systemic spread of the virus with infected cells detected in different organs including the intestinal mucosa. R5 virus was highly efficient in mucosal transmission by both routes whereas X4 virus was relatively less efficient in causing infection. HIV-1 infection of RAG-hu mice by vaginal and rectal routes as shown here represents the first in vivo model of HIV-1 transmission across intact mucosal barriers and as such may prove very useful for studying early events in HIV-1 pathogenesis in vivo, as well as the testing of microbicides, anti-HIV vaccines/therapeutics, and other novel strategies to prevent HIV-1 transmission.

Figure 1. RAG-hu mouse mucosal tissues are populated by HIV-1-susceptible human cells

Engrafted and unengrafted mice were sacrificed and vagina, rectum, and large intestines were dissected and frozen. Tissue sections from engrafted mice were immunostained with fluorescent-labeled antibodies for the presence of the human cell markers CD45 (A–C), CD3 (D–F), CD68 (G–I), and CD4 (J–L). Unengrafted tissues were negative for the human cell markers CD45 (M–O), as well as CD3, CD4, and CD68 (data not shown).

Figure 2. HIV-1 viral load in peripheral blood following mucosal infection

Infected mouse plasma was collected weekly and RNA was extracted. Viral load (in RNA copies/ml plasma) was determined by Q-RT-PCR as described in methods. A. R5 virus infection. B. X4 virus infection. Mice that failed to become viremic are not included.

Figure 3. HIV-1 replication in proximal and distal organs following mucosal infection

Mucosally-infected mice were sacrificed and organs were collected and probed for HIV-1 RNA using an env riboprobe by in situ hybridization. Mouse 262 was sacrificed at 4 weeks following rectal infection with HIV-1 BaL-1, and mouse 320 was sacrificed at 8 weeks following rectal infection with HIV-1 NL4-3. (A, C, E): uninfected tissue sections from mesenteric lymph node, small intestines, and spleen, respectively. (B, D, F): R5 virus-infected tissue sections from mesenteric lymph node, small intestines, and spleen, respectively. (G, I): uninfected tissue sections from thymus and lymph node, respectively. (H, J): X4 virus-infected tissue sections from thymus and lymph node, respectively. Panels A, B, E, F: original magnification 600X. Panels C, D, G, H, I, J: original magnification 200X.

Figure 4. CD4⁺ T cell depletion following mucosal infection with HIV-1

Mice mucosally infected with HIV-1 were screened for CD4⁺ T cell depletion by FACS analysis of peripheral blood cells stained with CD3 and CD4. The CD4:CD3 ratio reflects the percentage of CD3⁺ cells that were also CD4⁺ relative to total CD3⁺ cells. Since individual mice have varying CD4:CD3 ratios, all data shown is standardized to the baseline values obtained prior to infection. A. Mucosal infection with R5 tropic virus. Vaginal infection, n=7. Rectal infection, n=9. Uninfected mice, n=2. B. FACS dot plots from representative R5 virus-infected mice infected either vaginally or rectally. C. Mucosal infection with X4 tropic virus. Vaginal infection, n=4. Rectal infection, n=2. Key: V=vaginal infection, R=rectal infection.