Impact of Interventions on Clonally Expanded Proviruses and Their RNA Expression

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

BACKGROUND: Proposed curative interventions for HIV have included treating with agents, such as HDAC inhibitors, that activate latent proviruses, inducing expression of intracellular RNA, in the hopes that such expression will result in viral production and killing of infected cells by viral cytotoxicity or targeted cytotoxic T-lymphocyte (CTL) response. However, previous studies, in which we collaborated with Dr. David Margolis (University of North Carolina), showed only a limited effect on reservoir reduction in participants treated with single or multiple doses of the HDAC inhibitor vorinostat (Archin et al., J. Infect. Dis. 210:728-735, 2014; Archin et al., Nature 487:482-485, 2012), including a lack of increased viral production in vivo measured by the HIV single-copy assay (SCA). More promising data have been obtained from more potent inhibitors and from combinations of HDAC inhibitors in in vitro studies, some of which were conducted by the University of Pittsburgh directed by Dr. John Mellors (Cillo et al., PNAS 111:7078-7083; Wei et al., PLoS Pathog. 10:e1004071, 2014). Although these potent inhibitors are believed to be more effective at inducing proviral expression, it is not currently known what levels of proviral expression are necessary to induce cell killing. Furthermore, by comparing uninduced andinduced PBMCs from the same individuals, we will determine which inhibitors are capable of reactivating latent proviruses (those that are not expressing HIV RNA prior to induction) in expanded cell clones and which only increase expression of proviruses that were already expressing at low levels prior to the induction, and the heterogeneity of these effects among proviruses. While conducting the in vitro studies on the HDAC inhibitors, we also plan to assess the efficacy of curative interventions on expanded clones in vivo. We will use samples accessible through the Pitt contract from the single-dose infusion of the HDAC inhibitor romidepsin study (protocol A5315), the broadly neutralizing antibody VRC-01 study (protocol A5342), the low-dose methotrexate study (protocol A5314), and others as they are completed, by performing ISA and quantitative digital droplet PCR (ddPCR) of specific clones on samples collected before, during, and after treatment in collaboration with Drs. Frank Maldarelli and Stephen Hughes (HIV Dynamics and Replication Program). ACCOMPLISHMENTS: In addition to our contribution to studies on the effect of vorinostat on HIV viremia levels in individuals treated with antiretroviral therapy (ART), we also participated in the evaluation of two dendritic cell (DC) vaccine studies. The AGS-004 vaccine (63) consisted of matured autologous DCs coelectroporated with RNA encoding autologous HIV antigens (Gag, Vpr, Rev, and Nef) and synthetically derived CD40 ligand RNA. We found that the HIV RNA by SCA was 1 copy/ml in all participants, who started ART during acute HIV infection; despite this low level at baseline, all subjects experienced rebound viremia after treatmentinterruption (median 29 days). We also participated in evaluating the efficacy of an autologous DC-based HIV vaccine (Macatangay et al., J. Infect. Dis. 213:1400-1409, 2016) loaded with autologous infected apoptotic cells. ART-suppressed individuals received four doses of the DC-vaccine and treatment was interrupted 6 weeks after the last dose. Although vaccination did not prevent viral rebound, a modest but significant decrease in plasma viremia was observed compared with pre-ART. We performed SGS on HIV plasma virus in pre- and post-ART samples and found increases in GA hypermutants in gag and pol after vaccination. This result suggested cytolysis of infected cells, since such variants are unlikely to generate virus particles in the plasma and, therefore, must be free DNA or RNA in the plasma resulting from lysis of infected cells.