Impact of Interventions on Clonally Expanded Proviruses and Their RNA Expression

BACKGROUND: The persistence of HIV during antiretroviral therapy (ART) has been shown to be driven by the proliferation of cells that were infected prior to initiating treatment and not from ongoing cycles of HIV replication in the peripheral blood or in tissues such as lymph nodes or gut (McManus et al., J. Clin. Invest. 129:4629-4642, 2019; Bozzi et al., Sci. Adv. 5:eaav5045, 2019). However, little is known about the drivers of cellular proliferation or about their selective maintenance during ART or during treatment with new interventions aimed at controlling HIV without ART. New interventions aimed at achieving HIV remission without ART include latency-reversing agents, broadly neutralizing antibodies, genetic modifications of HIV proviruses, enhancement of immune recognition of infected cells, and more. Our current studies are aimed at understanding the selection and persistence of cell clones during ART, the drivers of clonal proliferation before and during ART, and the effect of new interventions on clonal expansion of infected cells and on the expression of proviruses that persist during ART. Firstly, we investigated the distribution of HIV integration sites in cell clones prior to ART and compared it to the distribution of cell clones after years on ART to identify the selection pressures that allow HIV to persist despite treatment (Coffin et al., JCI Insight 4:e128432, 2019; Coffin et al,. PLoS Pathog. 17:e1009141, 2021; Bale, Katusiime et al., mBio 12:e00568-21, 2021). We found that HIV proviruses that persist on ART are more frequently integrated in regions of the host genome that are not highly expressed and that they are often integrated in the opposite orientation of the host gene. We also found that infected cell clones arise in acute infection and persist for at least 9 years on ART. We are now investigating the effect of the latency-reserving agents on the persistence and dynamics of infected cell clones and on the expression of HIV within infected cell clones. _____One proposed curative intervention for HIV has 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., Nature 487:482-485, 2012; Archin et al., J. Infect. Dis. 210:728-735, 2014), 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, 2014; 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. By comparing uninduced and induced 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 are using samples accessible through our collaboration with Dr. Eli Boritz at NIAID from before and after a single-dose infusion of anti-PD-1 to measure the effects on clonal proliferation of infected and uninfected cell clones. ____ACCOMPLISHMENTS: Our current research is focused on the effect of anti-PD-1 on the HIV reservoir in vivo. In collaboration with Dr. Boritz, we obtained samples from HIV-1-infected patients who received anti-PD-1 infusions to determine the effect of this checkpoint inhibitor on levels of HIV-infected cells during ART (Perez-Rodrigo et al., Conference on Retroviruses and Opportunistic Infections, 2021). Using our MDA-SGS assay (described in Project ZIA BC 011681), we are characterizing the sites of integration into the host genome and the proviral structure in infected cells obtained before and after treatment with anti-PD-1. Thus far, we have demonstrated the expansion of infected cells with defective proviruses in response to anti-PD-1. We are currently evaluating the effect of anti-PD-1 on cells with intact proviruses. ____Because children have immune systems that are different from adults, including a much higher fraction of naive T cells that have been reported to be more resistant to HIV infection, we investigated the number of infected cells clones in children, the timing of their emergence, and their persistence on ART (Bale, Katusiime et al., mBio 12:e00568-21, 2021). We found that infected cell clones in children arise in early infection and persist for at least 9 years on ART. We also found that there is a selection for proviruses that are in the opposite orientation of the host gene and are in genes that are not highly expressed. We are currently investigating the levels of infected naive and memory T cells in children and the degree to which they form cell clones. To date, we have found that 8/8 children studied carry populations of infected naive cells and that some infected naive cells can form cell clones. We are in the process of determining if these cell clones can differentiate and persist despite infection creating a renewable source of infected cells during ART. ____To investigate the drivers of clonal expansion of infected cells before and during ART, we compared the integration site profile in ex vivo infected PBMCs to in vivo infected PBMCs collected from donors on short- and long-term ART (Coffin et al., PLoS Pathog. 17:e1009141, 2021). We found that integration into oncogenes is only a minor driver of clonal expansion of infected cells, suggesting that homeostatic and/or antigenic-driven clonal expansion are the main drivers for persistence of HIV during ART. ____We developed a new technology to investigate the dynamics of infected cell clones before and during ART with higher accuracy and sensitivity than the integration sites assay (Brandt et al., Viruses 13:1235, 2021). Using this technology, we showed that clones with intact or defective proviruses can increase or decrease in size during treatment.