Single-chain antibodies to block HIV transcription and prevent reactivation from latently infected resting CD4+ T cells

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PROJECT SUMMARY Despite decades of successful antiretroviral therapy (ART), persistent HIV-infected resting CD4+ T cells can remain undetected in tissue reservoirs. HIV replication is cytopathic in activated CD4+ T cells; however, a fraction of HIV-infected activated CD4+ T cells revert to the resting G0 phenotype in tissue reservoirs. These persistent HIV-infected resting CD4+ T cells undergo intermittent activation and produce fully infectious virus. HIV is invariably detected in the plasma when patients discontinue ART because plasma viremia is reseeded by intermittent activation of persistent HIV-infected resting CD4+ T cells in the tissue reservoirs. In response to RFA-AI-19-072: Novel Therapeutics Directed to Intracellular HIV Targets, we propose an innovative analytic bioassay to identify next-generation single-chain antibodies that sustain HIV remission. Our research is focused on the essential role of heat shock protein 90 (Hsp90) in HIV replication, and we have shown that mild heat shock (39.5°C) accelerates HIV transcription. Increased Hsp90 activity at 39.5°C was critical for full-length HIV RNA synthesis by host transcription factors, and we have shown that 39.5°C reactivates latent HIV replication in ART-suppressed aviremic HIV-infected patient samples, in human resting CD4+ T cells isolated from fully suppressed humanized mice, and in three distinct in vitro models of HIV latency. Reactivation of latent HIV depends on multiple host proteins interacting with their HIV counterpart, and the recently developed QUECEL (quiescent effector cell latency) model of HIV latency is an ideal in vitro system to identify single-chain antibodies that disrupt HIV-host protein interactions. The QUECEL carries a modified HIV provirus that depends on all the HIV-host protein interactions to reactivate latent HIV RNA synthesis. The latent QUECEL depends on host protein interaction for both the HIV Tat-mediated transcription and HIV Rev-mediated export of the full-length viral genome, and we anticipate that single-chain antibodies will disrupt these critical HIV-host protein interactions required for reactivation of HIV latency. We have proof that the QUECEL can be reactivated at 39.5°C and designed a high-complexity single-chain antibody library to selectively enrich for candidates that prevent QUECEL reactivation. The prospective antiviral candidates will then be further enriched in resting CD4+ T cells isolated from ART-treated aviremic patients, and we anticipate identifying potent single- chain antibodies that disrupt the HIV-host protein interactions required to reactivate HIV from latency. Our analytic bioassay is innovative because 1) mild heat shock activation is physiologically similar to the intermittent cellular activation occurring in persistent HIV-infected tissue reservoirs and 2) the small size of single- chain antibodies will increase the target range to the surface area of HIV proteins, effectively blocking critical host-protein interactions. We propose to use mild heat shock to identify potent antiviral single-chain antibodies in the following Specific Aims: 1) Selectively enrich for single-chain antibodies that prevent reactivation of latent QUECEL, and 2) Identify potent single-chain antibodies that block HIV transcription.