Despite the ability of antiretroviral therapy (ART) to inhibit HIV replication in infected patients, preventing disease progression, the virus persists in a stable reservoir and rebounds to pretherapy levels if ART is interrupted. Lifelong therapy is expensive and risks the emergence of drug resistance and toxic side effects. Consequently, identification of strategies to eliminate the reservoir is a major priority of HIV research. Reports showing that the virus can reemerge months to years after treatment interruption in patients hoped to have been cured by bone marrow transplantation (Henrich et al., Ann. Intern. Med. 161:319-327, 2014) or early treatment (Luzuriaga et al., N. Engl. J. Med. 372:786-788, 2015) support the idea that HIV rebounds from a pool of latently infected cells, as we and others have proposed, which we will refer to as the HIV reservoir. It has been suggested that the reservoir largely comprises a small number of resting, memory CD4+ T cells carrying transcriptionally silent HIV proviruses (Finzi et al., Science 278:1295-1300, 1997; Chun et al., PNAS 94:13193-13197, 1997; Wong et al., Science 278:1291-1295, 1997). Recently, we and others showed that HIV-infected cells can clonally expand and persist despite ART, and that the proviral integration site may influence this phenomenon (Maldarelli et al., Science 345:179-183, 2014; Wagner et al., Science 345:570-573, 2014). In one case, an expanded provirus was shown to match the single viral variant present at detectable levels in the persistent viremia during ART; furthermore, we showed that the virus particles produced by the clonally expanded cells were replication competent (Simonetti et al., PNAS 113:1883-1888, 2016). Hereafter, we will call such proviruses replication-competent proviruses, indicating that they are intact and capable of giving rise to virus particles that can result in full cycles of viral replication. This is one example where the source of infectious virus in blood has been traced to a clone of infected cells carrying a mostly latent provirus. A major focus of this project is to determine how commonly such clonally expanded, infected cells that persist during ART carry replication-competent proviruses and in which tissues and cell types they persist. ____Although there is considerable patient-to-patient variation, the frequency of resting CD4+ T cells that harbor HIV proviruses detectable by PCR has been very roughly estimated to average about 1 cell in 1000; however, the number of latently infected cells carrying replication-competent proviruses has been reported to be much lower (Ho et al., Cell 155:540-551, 2013). The difference is due to the presence of a large number of defective proviruses. Ya-Chi Ho and colleagues described the proviruses in resting CD4+ T cells that were not induced to produce replication-competent virus after a single round of maximal T-cell activation (Ho et al., Cell 155:540-551, 2013). Almost half of these proviruses had large internal deletions that preclude replication, while another third were lethally hypermutated by the host restriction factor APOBEC3G. Other defects and further analyses brought the fraction of defective proviruses up to more than 98% (Bruner et al., presentation at 2016 Conference on Retroviruses and Opportunistic Infections). Additionally, Ho et al. found that some of the intact proviruses were capable of producing infectious virions following a second round of activation (Cell 155:540-551, 2013), even though they had not been induced by the prior activation. This result leads to the question of how commonly the expanding clones described by Maldarelli et al. and Wagner et al. carry intact and replication-competent proviruses. To answer this question, we developed a novel method called the full-length integrated proviral single-genome sequencing (FLIP-SGS) assay, which allows us to analyze the proviruses in highly expanded clones like those identified by Drs. Frank Maldarelli and Stephen Hughes (HIV Dynamics and Replication Program) using the integration sites assay (ISA). The FLIP-SGS assay will determine if HIV proviruses in expanding clones that persist during ART have intact sequences or contain lethal mutations or deletions. We will further determine if, when activated, such clones are capable of producing infectious virions. If we show that latent, intact proviruses in infected cells commonly undergo clonal expansion, it will mean that strategies intended to cure patients will have to not only block viral replication, but also cope with the proliferation of these latently infected cells. ____ACCOMPLISHMENTS: We found (Kearney et al., PLoS Pathog. 10:e1004010, 2014) that the initially diverse pre-ART plasma virus population becomes more homogeneous with time, with the appearance, in many patients, of rakes of identical sequence, as originally reported by others (Bailey et al., J. Virol. 80:6441-6457, 2006). At about the same time, a study conducted at the University of Pittsburgh showed that the number of HIV-infected (DNA+) PBMCs declines with biphasic kinetics after the initiation of ART but achieves steady-state levels after 6-8 years with a median of about 300-400 HIV DNA-positive cells/106 CD4+ cells (Besson et al., Clin. Infect. Dis. 59:1312-1321, 2014). Studies described below strongly imply that the homogeneous sequences arise from clones expanded from a single cell infected before ART initiation (Kearney et al., PLoS Pathog. 10:e1004010, 2014). Taken together, these observations suggest that there is a dynamic balance between the clonal expansion and death of infected cells during ART. It is not yet known how commonly expanded clones carry replication-competent proviruses or if they carry HIV proviruses with CTL escape mutations that may give them a survival advantage. However, we identified one such case (Simonetti et al., PNAS 113:1883-1888, 2016). Our current studies aim to determine if expanded clones sustain the HIV-1 reservoir in other infected individuals. ____To identify the possible sources of persistent viremia and to investigate the effect of ART on viral replication, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low (van Zyl et al., J. Clin. Invest. 127:3827-3834, 2017). Eight children started ART at less than or equal to 10 months of age and had plasma viremia suppressed for 7-9 years. Two children had uncontrolled viremia for 15 and 30 months respectively before viremia suppression. The latter two children showed clear evidence of virus evolution, whereas none of the 8 children suppressed on ART showed virus evolution by multiple methods of analysis. These suggest that ongoing HIV replication in the blood is not common on ART and is, therefore, not a significant barrier to curing HIV-1 infection. Current studies aim to determine if viral replication persists in lymph nodes during ART.
| Van Zyl, Gert U; Katusiime, Mary Grace; Wiegand, Ann et al. (2017) No evidence of HIV replication in children on antiretroviral therapy. J Clin Invest 127:3827-3834 |
| Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F et al. (2017) Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART. Proc Natl Acad Sci U S A 114:E3659-E3668 |
