HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

IMPORTANCE: Characterizing the human immunodeficiency virus (HIV) reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. We observed that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) dated to nearer ART initiation. This helps explain why studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled replication-competent HIV found almost none. Together with our findings that intact proviruses were more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that (i) on-ART and rebound viremia can have distinct within-host origins, (ii) intact proviruses have shorter lifespans than grossly defective ones and thus depend more heavily on clonal expansion for persistence, and (iii) an HIV reservoir predominantly "dating" to near ART initiation will be substantially adapted to within-host pressures, complicating immune-based cure strategies.

HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

In order to cure HIV, we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persists within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics however did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were genetically heterogeneous and sometimes ancestral, where viremia may have originated from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally-enriched and genetically younger sequences that date to the untreated infection period when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination.

Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins.

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins.

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8+ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual SARS-CoV-2 viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately 5-fold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I downregulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I downregulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single-amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8+ T-cells. Speficially, ORF7a prevented the assembly of the MHC-I peptide loading complex and causing retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary-Cell Model of HIV Latency but Not from Ex Vivo Reservoirs.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

A participant-derived xenograft model of HIV enables long-term evaluation of autologous immunotherapies.

HIV-specific CD8+ T cells partially control viral replication and delay disease progression, but they rarely provide lasting protection, largely due to immune escape. Here, we show that engrafting mice with memory CD4+ T cells from HIV+ donors uniquely allows for the in vivo evaluation of autologous T cell responses while avoiding graft-versus-host disease and the need for human fetal tissues that limit other models. Treating HIV-infected mice with clinically relevant HIV-specific T cell products resulted in substantial reductions in viremia. In vivo activity was significantly enhanced when T cells were engineered with surface-conjugated nanogels carrying an IL-15 superagonist, but it was ultimately limited by the pervasive selection of a diverse array of escape mutations, recapitulating patterns seen in humans. By applying mathematical modeling, we show that the kinetics of the CD8+ T cell response have a profound impact on the emergence and persistence of escape mutations. This "participant-derived xenograft" model of HIV provides a powerful tool for studying HIV-specific immunological responses and facilitating the development of effective cell-based therapies.

HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA).

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

HIV-specific T cell responses reflect substantive in vivo interactions with antigen despite long-term therapy.

Antiretroviral therapies (ARTs) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which reseed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.

Immunological approaches to HIV cure.

Combination antiretroviral therapy (ART) to treat human immunodeficiency virus (HIV) infection has proven remarkably successful - for those who can access and afford it - yet HIV infection persists indefinitely in a reservoir of cells, despite effective ART and despite host antiviral immune responses. An HIV cure is therefore the next aspirational goal and challenge, though approaches differ in their objectives - with 'functional cures' aiming for durable viral control in the absence of ART, and 'sterilizing cures' aiming for the more difficult to realize objective of complete viral eradication. Mechanisms of HIV persistence, including viral latency, anatomical sequestration, suboptimal immune functioning, reservoir replenishment, target cell-intrinsic immune resistance, and, potentially, target cell distraction of immune effectors, likely need to be overcome in order to achieve a cure. A small fraction of people living with HIV (PLWH) naturally control infection via immune-mediated mechanisms, however, providing both sound rationale and optimism that an immunological approach to cure is possible. Herein we review up to date knowledge and emerging evidence on: the mechanisms contributing to HIV persistence, as well as potential strategies to overcome these barriers; promising immunological approaches to achieve viral control and elimination of reservoir-harboring cells, including harnessing adaptive immune responses to HIV and engineered therapies, as well as enhancers of their functions and of complementary innate immune functioning; and combination strategies that are most likely to succeed. Ultimately, a cure must be safe, effective, durable, and, eventually, scalable in order to be widely acceptable and available.

BCL-2 antagonism sensitizes cytotoxic T cell-resistant HIV reservoirs to elimination ex vivo.

Curing HIV infection will require the elimination of a reservoir of infected CD4+ T cells that persists despite HIV-specific cytotoxic T cell (CTL) responses. Although viral latency is a critical factor in this persistence, recent evidence also suggests a role for intrinsic resistance of reservoir-harboring cells to CTL killing. This resistance may have contributed to negative outcomes of clinical trials, where pharmacologic latency reversal has thus far failed to drive reductions in HIV reservoirs. Through transcriptional profiling, we herein identified overexpression of the prosurvival factor B cell lymphoma 2 (BCL-2) as a distinguishing feature of CD4+ T cells that survived CTL killing. We show that the inducible HIV reservoir was disproportionately present in BCL-2hi subsets in ex vivo CD4+ T cells. Treatment with the BCL-2 antagonist ABT-199 was not sufficient to drive reductions in ex vivo viral reservoirs when tested either alone or with a latency-reversing agent (LRA). However, the triple combination of strong LRAs, HIV-specific T cells, and a BCL-2 antagonist uniquely enabled the depletion of ex vivo viral reservoirs. Our results provide rationale for novel therapeutic approaches targeting HIV cure and, more generally, suggest consideration of BCL-2 antagonism as a means of enhancing CTL immunotherapy in other settings, such as cancer.

Integrated Assessment of Viral Transcription, Antigen Presentation, and CD8+ T Cell Function Reveals Multiple Limitations of Class I-Selective Histone Deacetylase Inhibitors during HIV-1 Latency Reversal.

Clinical trials investigating histone deacetylase inhibitors (HDACi) to reverse HIV-1 latency aim to expose reservoirs in antiretroviral (ARV)-treated individuals to clearance by immune effectors, yet have not driven measurable reductions in the frequencies of infected cells. We therefore investigated the effects of the class I-selective HDACi nanatinostat and romidepsin on various blocks to latency reversal and elimination, including viral splicing, antigen presentation, and CD8+ T cell function. In ex vivo CD4+ T cells from ARV-suppressed individuals, both HDACi significantly induced viral transcription, but not splicing nor supernatant HIV-1 RNA. In an HIV-1 latency model using autologous CD8+ T cell clones as biosensors of antigen presentation, neither HDACi-treated CD4+ T cell condition induced clone degranulation. Both HDACi also impaired the function of primary CD8+ T cells in viral inhibition assays, with nanatinostat causing less impairment. These findings suggest that spliced or cell-free HIV-1 RNAs are more indicative of antigen expression than unspliced HIV-RNAs and may help to explain the limited abilities of HDACi to generate CD8+ T cell targets in vivoIMPORTANCE Antiretroviral (ARV) drug regimens suppress HIV-1 replication but are unable to cure infection. This leaves people living with HIV-1 burdened by a lifelong commitment to expensive daily medication. Furthermore, it has become clear that ARV therapy does not fully restore health, leaving individuals at elevated risk for cardiovascular disease, certain types of cancers, and neurocognitive disorders, as well as leaving them exposed to stigma. Efforts are therefore under way to develop therapies capable of curing infection. A key focus of these efforts has been on a class of drugs called histone deacetylase inhibitors (HDACi), which have the potential of exposing hidden reservoirs of HIV-1 to elimination by the immune system. Unfortunately, clinical trial results with HDACi have thus far been disappointing. In the current study, we integrate a number of experimental approaches to build a model that provides insights into the limited activity of HDACi in clinical trials and offers direction for future approaches.

HTLV-1 as a Model for Virus and Host Coordinated Immunoediting.

Immunoediting is a process that occurs in cancer, whereby the immune system acts to initially repress, and subsequently promote the outgrowth of tumor cells through the stages of elimination, equilibrium, and escape. Here we present a model for a virus that causes cancer where immunoediting is coordinated through synergistic viral- and host-mediated events. We argue that the initial viral replication process of the Human T cell leukemia virus type I (HTLV-1), which causes adult T cell leukemia/lymphoma (ATL) in ~5% of individuals after decades of latency, harmonizes with the host immune system to create a population of cells destined for malignancy. Furthermore, we explore the possibility for HIV to fit into this model of immunoediting, and propose a non-malignant escape phase for HIV-infected cells that persist beyond equilibrium.

Have Cells Harboring the HIV Reservoir Been Immunoedited?

Immunoediting is an important concept in oncology, delineating the mechanisms through which tumors are selected for resistance to immune-mediated elimination. The recent emergence of immunotherapies, such as checkpoint inhibitors, as pillars of cancer therapy has intensified interest in immunoediting as a constraint limiting the efficacy of these approaches. Immunoediting manifests at a number of levels for different cancers, for example through the establishment of immunosuppressive microenvironments within solid tumors. Of particular interest to the current review, selection also occurs at the cellular level; and recent studies have revealed novel mechanisms by which tumor cells acquire intrinsic resistance to immune recognition and elimination. While the selection of escape mutations in viral epitopes by HIV-specific T cells, which is a hallmark of chronic HIV infection, can be considered a form of immunoediting, few studies have considered the possibility that HIV-infected cells themselves may parallel tumors in having differential intrinsic susceptibilities to immune-mediated elimination. Such selection, on the level of an infected cell, may not play a significant role in untreated HIV, where infection is propagated by high levels of cell-free virus produced by cells that quickly succumb to viral cytopathicity. However, it may play an unappreciated role in individuals treated with effective antiretroviral therapy where viral replication is abrogated. In this context, an "HIV reservoir" persists, comprising long-lived infected cells which undergo extensive and dynamic clonal expansion. The ability of these cells to persist in infected individuals has generally been attributed to viral latency, thought to render them invisible to immune recognition, and/or to their compartmentalization in anatomical sites that are poorly accessible to immune effectors. Recent data from ex vivo studies have led us to propose that reservoir-harboring cells may additionally have been selected for intrinsic resistance to CD8+ T cells, limiting their elimination even in the context of antigen expression. Here, we draw on knowledge from tumor immunoediting to discuss potential mechanisms by which clones of HIV reservoir-harboring cells may resist elimination by CD8+ T cells. The establishment of such parallels may provide a premise for testing therapeutics designed to sensitize tumor cells to immune-mediated elimination as novel approaches aimed at curing HIV infection.

Short Communication: Expression of Host Restriction Factors by Memory CD4+ T Cells Differs Between Healthy Donors and HIV-1-Infected Individuals with Effective Antiretroviral Therapy.

Much has been learnt from the functions of host restriction factors during acute and chronic HIV-1 infection, but far less is known about their role in HIV-1-infected individuals in which viral load is stably suppressed with antiretroviral therapy (ART). In this study transcriptional expression of 42 host restriction factors was determined for memory CD4+ T cells sorted from 10 uninfected and 21 HIV-1-infected individuals, treated with suppressive ART and for which the viral reservoir was quantified. No significant associations were observed between restriction factor expression and HIV-1 reservoir size, quantified by measurement of HIV-1 Gag DNA using droplet digital polymerase chain reaction, and by measurement of replication-competent inducible virus using quantitative viral outgrowth assays. Expression of eight of the restriction factors differed significantly, and with a false discovery rate of <10%, between ART-suppressed and uninfected individuals. APOBEC3G, ISG15, LGALS3BP, RNASEL, and MX2 were upregulated in the ART-suppressed individuals, likely because of increased levels of immune activation observed in virally suppressed compared with uninfected individuals. In contrast CDKN1A, TRIM11, and BRD4 were expressed at lower levels in ART-suppressed than uninfected individuals. This suggests perturbation of the CD4+ memory T cell compartment, in which a viral reservoir persists in HIV-1-infected individuals with effective ART. Modulation of restriction factor expression, or overrepresentation of cell subsets that intrinsically express these restriction factors at lower levels could result in the distinct expression of restriction factors observed in treated infected individuals.

Publisher Correction: Evidence that CD32a does not mark the HIV-1 latent reservoir.

In this Brief Communications Arising Comment, the first three authors (Osuna, Lim and Kublin) should have been listed as equally contributing authors; this has been corrected online.

HLA-C downregulation by HIV-1 adapts to host HLA genotype.

HIV-1 can downregulate HLA-C on infected cells, using the viral protein Vpu, and the magnitude of this downregulation varies widely between primary HIV-1 variants. The selection pressures that result in viral downregulation of HLA-C in some individuals, but preservation of surface HLA-C in others are not clear. To better understand viral immune evasion targeting HLA-C, we have characterized HLA-C downregulation by a range of primary HIV-1 viruses. 128 replication competent viral isolates from 19 individuals with effective anti-retroviral therapy, show that a substantial minority of individuals harbor latent reservoir virus which strongly downregulates HLA-C. Untreated infections display no change in HLA-C downregulation during the first 6 months of infection, but variation between viral quasispecies can be detected in chronic infection. Vpu molecules cloned from plasma of 195 treatment naïve individuals in chronic infection demonstrate that downregulation of HLA-C adapts to host HLA genotype. HLA-C alleles differ in the pressure they exert for downregulation, and individuals with higher levels of HLA-C expression favor greater viral downregulation of HLA-C. Studies of primary and mutant molecules identify 5 residues in the transmembrane region of Vpu, and 4 residues in the transmembrane domain of HLA-C, which determine interactions between Vpu and HLA. The observed adaptation of Vpu-mediated downregulation to host genotype indicates that HLA-C alleles differ in likelihood of mediating a CTL response that is subverted by viral downregulation, and that preservation of HLA-C expression is favored in the absence of these responses. Finding that latent reservoir viruses can downregulate HLA-C could have implications for HIV-1 cure therapy approaches in some individuals.

HIV latency reversing agents act through Tat post translational modifications.

BACKGROUND: Different classes of latency reversing agents (LRAs) are being evaluated to measure their effects in reactivating HIV replication from latently infected cells. A limited number of studies have demonstrated additive effects of LRAs with the viral protein Tat in initiating transcription, but less is known about how LRAs interact with Tat, particularly through basic residues that may be post-translationally modified to alter the behaviour of Tat for processive transcription and co-transcriptional RNA processing. RESULTS: Here we show that various lysine and arginine mutations reduce the capacity of Tat to induce both transcription and mRNA splicing. The lysine 28 and lysine 50 residues of Tat, or the acetylation and methylation modifications of these basic amino acids, were essential for Tat transcriptional control, and also for the proviral expression effects elicited by histone deacetylase inhibitors (HDACi) or the bromodomain inhibitor JQ1. We also found that JQ1 was the only LRA tested that could induce HIV mRNA splicing in the absence of Tat, or rescue splicing for Tat lysine mutants in a BRD4-dependent manner. CONCLUSIONS: Our data provide evidence that Tat activities in both co-transcriptional RNA processing together with transcriptional initiation and processivity are crucial during reactivation of latent HIV infection. The HDACi and JQ1 LRAs act with Tat to increase transcription, but JQ1 also enables post-transcriptional mRNA splicing. Tat residues K28 and K50, or their modifications through acetylation or methylation, are critical for LRAs that function in conjunction with Tat.

No adverse safety or virological changes 2 years following vorinostat in HIV-infected individuals on antiretroviral therapy.

OBJECTIVE: To determine the long-term effects of vorinostat on safety and virological parameters in HIV-infected individuals on suppressive antiretroviral therapy (ART). DESIGN: Prospective longitudinal observational extended follow-up of 20 HIV-infected individuals on ART previously enrolled in a clinical trial of daily vorinostat 400 mg for 14 days. Extended follow-up included visits at 6, 12, 18 and 24 months postenrolment in the initial clinical trial. METHODS: Cell-associated unspliced HIV RNA, total HIV DNA and plasma HIV RNA were quantified by PCR, and CD4 and CD8 T cells quantified by flow cytometry. Changes over time in each parameter were assessed using the Wilcoxon matched pair signed-rank test and generalized estimating equations for trend modelling. RESULTS: We recorded a total of 31 adverse events (26 grade 1 and five grade 2) in all study participants (n = 20). There were no significant changes in the number of CD4 or CD8 T cells or plasma HIV RNA over time. In 12 participants for whom baseline samples were available, there were no significant changes in total HIV DNA, cell-associated unspliced HIV RNA, plasma RNA or CD4 and CD8 T cells at 6, 12, 18 or 24 months. CONCLUSION: Extended follow-up for 24 months did not reveal any long-term toxicity or changes in markers of HIV persistence or transcription in participants on ART who had received 14 days of vorinostat.