Summary Paragraph Long-lasting, latently-infected, resting CD4 + T cells are the greatest obstacle to cure HIV infection, as they persist despite decades of treatment with ART. Estimates indicate the need for >70 years of continuous, fully suppressive, antiretroviral therapy (ART) to eliminate the HIV reservoir 1 . Alternatively, induction of HIV from its latent state could accelerate decline of the reservoir, thereby shortening time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in peripheral blood with minimal focus on tissue reservoirs and had limited effect 2 - 9 . Here we show that activation of the non-canonical NF-κB signaling pathway via AZD5582 results in induction of HIV- and SIV-RNA expression in the blood and tissues of ART-suppressed bone marrow/liver/thymus (BLT) humanized mice and rhesus macaques. Analysis of resting CD4 + T cells from tissues after AZD5582 treatment revealed increased SIV-RNA in lymph nodes in macaques and robust induction of HIV in virtually all tissues analyzed in humanized mice including lymph nodes, thymus, bone marrow, liver, and lung. This promising new approach to latency reversal, in combination with appropriate tools for systemic clearance of persistent HIV infection, greatly increases opportunities for HIV eradication.

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

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.

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TLR expression patterns were remarkably conserved across the study population and evaluated tissues indicating a predictable responsiveness to STI. The results support cautious use of immortalized cells for genital tract modeling.