Immortalization and Targeted Enrichment of HIV-Infected CD4⁺ T-Cells from Patients Under Antiretroviral Therapy

Defective HIV-1 proviruses harboring mutations and/or large internal deletions represent the majority of HIV-1 sequences found in circulating peripheral blood mononuclear cells of people living with HIV with viremia suppressed by combination antiretroviral therapy; indirect evidence suggests that such sequences are transcriptionally active and may contribute to immune activation. In this study, we present a new approach allowing for high-efficiency screening, immortalization, and targeted enrichment of HIV-positive CD4⁺ T-cells isolated from people living with HIV. Using this method, we were able to isolate and expand patient-derived cells, identify mutations and deletions via sequencing, and confirm that those proviruses were transcriptionally and translationally active in vitro. Moreover, our findings indicate that the majority of proviral sequences circulating in suppressed HIV-infected patients may undergo 3'-LTR deletions, suggesting that sequence diversity reported using LTR-to-LTR amplification and sequencing approaches may indeed be underscored.