HIV latency in the humanized BLT mouse

Abstract

Even after extended treatment with powerful antiretroviral drugs, HIV is not completely eliminated from infected individuals. Latently infected CD4(+) T cells constitute one reservoir of replication-competent HIV that needs to be eliminated to completely purge virus from antiretroviral drug-treated patients. However, a major limitation in the development of therapies to eliminate this latent reservoir is the lack of relevant in vivo models that can be used to test purging strategies. Here, we show that the humanized BLT (bone marrow-liver-thymus) mouse can be used as both an abundant source of primary latently infected cells for ex vivo latency analysis and also as an in vivo system for the study of latency. We demonstrate that over 2% of human cells recovered from the spleens of HIV-infected BLT mice can be latently infected and that this virus is integrated, activation inducible, and replication competent. The non-tumor-inducing phorbol esters prostratin and 12-deoxyphorbol-13-phenylacetate can each induce HIV ex vivo from these latently infected cells, indicating that this model can be used as a source of primary cells for testing latency activators. Finally, we show activation-inducible virus is still present following suppression of plasma viral loads to undetectable levels by using the antiretroviral drugs zidovudine, indinavir sulfate, and didanosine, demonstrating that this model can also be used to assess the in vivo efficacy of latency-purging strategies. Therefore, the HIV-infected BLT mouse should provide a useful model for assessment of HIV latency activators and approaches to eliminate persistent in vivo HIV reservoirs.

Fig 1

Reconstitution of human immune cells in BLT mice. Representative flow cytometry analysis of peripheral blood mononuclear cells obtained from BLT mice following staining for human CD45, CD3, CD4, and CD8.

Fig 2

Generation of in vivo latency using the BLT humanized mouse model. BLT mice were infected with a modified version of HIV NL4-3 that encodes the murine heat-stable antigen (HSA) selectable marker in place of vpr (NLHSAs). At week 5 postinfection, splenocytes were obtained and immunomagnetically depleted of HSA⁺ (productively infected) cells. Cells were either left unstimulated or costimulated via CD3 and CD28 ligation in the presence of IL-2. Raltegravir was added to all cultures to prevent new integration events and inhibit virus spread during the assay. (A) At day 2 poststimulation, some cells were harvested and analyzed for intracellular Gag expression by flow cytometry. Boxed numbers represent the percentages of human cells that are HIV Gag⁺. (B) At day 5 poststimulation, supernatants were harvested and HIV p24 levels were analyzed by ELISA. CS, costimulated; US, unstimulated. Mean values from duplicate wells for each condition are presented.

Fig 3

Stimulation of latent virus with protein kinase C activators. (A) BLT mice were infected with wild-type HIV, and then the Thy/Liv implants were removed at week 7 postinfection. Thymocytes from three BLT mice were pooled and then stimulated in triplicate with the indicated concentration of prostratin for 2 days before quantitation of Gag⁺ cells by intracellular flow cytometry. Raltegravir (1 μM) was present in all cultures throughout the assay period. Prostratin activated HIV from latency in a dose-dependent manner in this assay. *, P < 0.05 (paired t test) (B) BLT mice infected with strain NLHSAs were sacrificed at week 7 postinfection, and then splenocytes were either cultured unstimulated or stimulated with costimulation (CS), 1 μM prostratin (Pro), or 1 μM 12-deoxyphorbol-13-phenylacetate (DPP). Raltegravir (1 μM) was added to all cultures. At day 4 poststimulation, cells were assayed for intracellular Gag expression. Prostratin and DPP each activated HIV from latency in splenocytes under these conditions.

Fig 4

Latency in BLT splenocytes is predominantly in the postintegration form. (A) Splenocytes were isolated from infected BLT mice and then costimulated for 4 days in the presence of the indicated drug. Levels of viral spread were measured under culture conditions without any antiretroviral drugs (No drug), and an increase in Gag⁺ cells was observed, demonstrating the presence of replication-competent HIV. Addition of the integrase inhibitor raltegravir (Ral) or the protease inhibitor saquinavir (Sqv) prevented this spread but still allowed expression of virus from latently infected cells. The frequency of Gag⁺ cells in raltegravir-treated cultures was similar to that in saquinavir-treated cultures, demonstrating that preintegration latency is not present at high levels in these cells. (B) Cells isolated from the Thy/Liv implant were treated in the same way as described for panel A. Control unstimulated cell cultures were included for all spleen and Thy/Liv implant conditions, and these were uniformly negative for Gag expression (not shown).

Fig 5

Suppression of viremia and protection of CD4⁺ T cells with combination antiretroviral therapy. BLT mice were infected with wild-type NL4-3 for 3 weeks and then treated for 2 weeks with antiretroviral therapy (ART) consisting of AZT, indinavir sulfate, and ddI. (A) HIV plasma viral loads were quantified for treated and control mice. HIV RNA copies per 100 μl of plasma are presented. Shaded areas indicate treatment with ART. (B) Mice were bled at the indicated postinfection time points, and PBMC were isolated for cell subset quantitation by flow cytometry. The CD4/CD8 ratios within the human T cell population (CD45⁺ CD3⁺) are presented. A significant decline in CD4/CD8 ratio was observed in the infected untreated animals, indicating that the ART was protective against CD4⁺ T cell depletion.

Fig 6

Presence of activation-inducible virus in the spleens of ART-treated mice with undetectable viral loads. Splenocytes were obtained from the mice described in the legend to Fig. 5 and either left unstimulated (US) or costimulated (CS) for 4 days in the presence of raltegravir. The level of p24 protein present in the supernatants was quantified to detect release of viral particles. (A) HIV p24 levels in cultures of splenocytes from mice that were not treated with antiretrovirals. (B) Levels of p24 protein in splenocyte cultures from ART-treated animals. Statistical comparisons were performed using the Wilcoxon rank-sum test.