Abstract
Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.
Highlights
In HIV-infected individuals, HIV RNA was detected at higher frequencies in primary [42] and secondary lymphoid tissues, such as gut-associated lymphoid tissue (GALT) [43,44] and lymph nodes [45], our experiments focused on the investigation of the cellular reservoir of HIV in the lymphoid compartments of NSG-bone marrow-liver-thymus (BLT) mice
To document the superior human leukocyte engraftment and susceptibility to HIV infection in the lymphoid compartments of NSG-BLT mice, we employed a combination of state-of-the-art RNAscope in situ hybridization (ISH) technology and IHC
We developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the highly potent HIV reverse transcriptase inhibitor ethynyl-2-fluoro-2 -deoxyadenosine (EFdA) to the level of single cells recapitulating antiretroviral therapy (ART)-suppression in HIV-infected individuals
Summary
Despite the extraordinary success of antiretroviral therapy in suppressing viremia in HIV-infected individuals [1,2,3], ART cannot eliminate all persistently infected cells enabling a stable viral reservoir that remains in the body, and HIV re-establishes active infection after ART is discontinued [4,5,6,7,8,9]. HIV most commonly enters the body through mucosal surfaces and disseminates throughout the lymphoid tissues [10,11,12,13,14], which serve as sanctuary sites where HIV may replicate at low levels despite potent ART and undetectable viremia [3,7]. Given the complexity of HIV persistence and the variety of current curative strategies for eliminating or suppressing the viral reservoir, identification of persistently infected cells and their anatomical location as well as study of the effect of tissue environment on viral gene expression and replication in vivo are critical for understanding HIV pathogenesis and for developing effective HIV eradication regimens.
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