The low frequency of latently HIV-infected cells in vivo limits the testing of potential HIV cure strategies using cells from successfully suppressed individuals. To date, primary cell models of latency use cells infected in vitro Primary CD4⁺ T cell models carrying an individual's endogenous HIV reservoir that recapitulate in vivo conditions of HIV latency are still outstanding. We developed a primary CD4⁺ T cell model of HIV latency derived from memory CD4⁺ T cells isolated from virally suppressed HIV-infected individuals that recapitulates HIV-1 latency and viral reactivation events. This model is based on the expansion of primary CD4⁺ T cells up to 300-fold in cell number. These cells reestablish a resting state without active virus production after extended culture and maintain a stable number of total HIV proviruses. The ability of these cells to respond to various classes of latency-reversing agents is similar to that of ex vivo CD4⁺ T cells directly isolated from blood. Importantly, viral outgrowth assays confirmed the ability of these expanded cells to produce replication-competent endogenous virus. In sum, this model recapitulates ex vivo viral reactivation conditions, captures the variability between individuals with different HIV reservoirs, and provides large numbers of cells for testing multiple agents from a single donor. The use of this novel model will allow accurate exploration of novel cure approaches aimed either at promoting viral reactivation or maintaining sustained latency.IMPORTANCE Primary cell models of HIV latency have been very useful to identify mechanisms contributing to HIV latency and to evaluate potential HIV cure strategies. However, the current models utilize in vitro infection with exogenous virus that does not fully recapitulate virus reactivation profiles of endogenous HIV in in vivo-infected CD4⁺ T cells. In contrast, obtaining sufficient amounts of CD4⁺ T cells from HIV-infected individuals to interrogate the HIV reservoir in vitro requires leukapheresis. In the model we propose here, in vitro expansion and extended culture of primary CD4⁺ T cells isolated from virally suppressed HIV-infected individuals enable obtaining large numbers of cells harboring endogenous latent HIV reservoirs without performing leukapheresis. This model captures the variability of HIV reservoirs seeded in different individuals and should be useful to evaluate future HIV cure strategies.

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