Generation of HIV latency in humanized BLT mice

Abstract

Here we demonstrate that a combination of tenofovir, emtricitabine, and raltegravir effectively suppresses peripheral and systemic HIV replication in humanized BLT mice. We also demonstrate that antiretroviral therapy (ART)-treated humanized BLT mice harbor latently infected resting human CD4+ T cells that can be induced ex vivo to produce HIV. We observed that the levels of infected resting human CD4+ T cells present in BLT mice are within the range of those observed circulating in patients undergoing suppressive ART. These results demonstrate the potential of humanized BLT mice as an attractive model for testing the in vivo efficacy of novel HIV eradication strategies.

Fig 1

Human therapeutic antiretroviral drugs suppress HIV replication in BLT mice. (A) The in vivo assessment of ART efficacy in BLT mice is depicted. HIV-positive BLT mice were treated daily with ART consisting of FTC, TDF, and raltegravir. Viral RNA was measured in plasma throughout the experiment and in tissues at harvest. (B through D) Day 0 corresponds to the time of virus exposure. Days during ART are shaded gray. (B) Plasma viral loads from three infected BLT mice were followed in the absence of ART to demonstrate sustained viremia without treatment. The three mice in this experiment were followed for 33, 38, and 68 days postexposure. (C) Plasma viral loads from three infected BLT mice were followed pre- and post-ART initiation to demonstrate the ability of ART to rapidly suppress viremia below detection. (D) Plasma viral load from an infected BLT mouse rapidly dropped below detection and remained undetectable for the duration of ART. As seen in humans, viremia rebounded to a level similar to that observed pretreatment following a structured treatment interruption. (E) Cell-associated HIV-1 RNA levels at harvest were determined in multiple tissues of an infected BLT mouse that received ART for 58 days (followed for a total of 77 days postexposure) as well as in a pair of untreated, infected BLT mice (followed for a total of 69 and 70 days postexposure), and triplicate RNA determinations for each are presented with standard deviations. RNA was isolated from the total cell population, and the data are presented as RNA copies per 1 × 10⁵ mononuclear cells on a logarithmic scale to facilitate visualization of the low levels of HIV RNA present in the tissues of the treated animal.

Fig 2

Resting human CD4⁺ T cells are present in BLT mice. (A) Flow cytometry analysis of pooled BLT mouse mononuclear cells prior to negative selection showed the presence of CD4⁺ and CD8⁺ T cells. Within the CD4⁺ T cell population, a heterogeneous population of cells expressing variable levels of CD27, CD25, HLA-DR, and CD11b was observed. (B) Following negative selection, flow cytometry analysis showed a homogeneous population of human CD4⁺ CCR7⁺ CD27⁺ CD8⁻ CD25⁻ HLA-DR⁻ CD11b⁻ resting T cells. Results for one representative BLT mouse of two are shown.

Fig 3

Latent HIV infection occurs in BLT mouse resting human CD4⁺ T cells. (A) The ex vivo assessment of HIV-1 latency in humanized BLT mice is depicted. Daily ART dosing consisted of FTC, TDF, and raltegravir. PHA, phytohemagglutinin; IL-2, interleukin 2; PBMC, peripheral blood mononuclear cells. (B) Plasma viral loads from eight infected BLT mice were followed pre- and post-ART initiation, and then mice were harvested between days 20 and 27 of ART. Day 0 corresponds to the time at which ART was initiated, and days during ART are shaded gray. (C) At harvest, resting CD4⁺ T cells were isolated from the pooled cells from individual mice and then placed into coculture according to clinical protocols to determine the frequency of latently infected cells in each animal during ART. Outgrowth assay determinations of IUPM resting CD4⁺ T cells from six animals with nondetectable viral loads at harvest are plotted as open circles, while one mouse with detectable viral load at harvest is represented with a filled circle. The second mouse with detectable viral loads at harvest is not represented in the graph, due to an unusually low yield of resting CD4⁺ T cells.