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1.
Immunity ; 56(5): 1132-1147.e6, 2023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-37030290

RESUMO

HIV infection persists during antiretroviral therapy (ART) due to a reservoir of latently infected cells that harbor replication-competent virus and evade immunity. Previous ex vivo studies suggested that CD8+ T cells from people with HIV may suppress HIV expression via non-cytolytic mechanisms, but the mechanisms responsible for this effect remain unclear. Here, we used a primary cell-based in vitro latency model and demonstrated that co-culture of autologous activated CD8+ T cells with HIV-infected memory CD4+ T cells promoted specific changes in metabolic and/or signaling pathways resulting in increased CD4+ T cell survival, quiescence, and stemness. Collectively, these pathways negatively regulated HIV expression and ultimately promoted the establishment of latency. As shown previously, we observed that macrophages, but not B cells, promoted latency in CD4+ T cells. The identification of CD8-specific mechanisms of pro-latency activity may favor the development of approaches to eliminate the viral reservoir in people with HIV.


Assuntos
Infecções por HIV , Humanos , Linfócitos T CD8-Positivos , Latência Viral , Linfócitos T CD4-Positivos , Replicação Viral
2.
Semin Immunol ; 51: 101412, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32981836

RESUMO

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.


Assuntos
Infecções por HIV , HIV-1 , Antivirais/uso terapêutico , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Latência Viral
3.
Proc Natl Acad Sci U S A ; 119(41): e2209042119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36136978

RESUMO

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.


Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos , COVID-19 , Antígenos de Histocompatibilidade Classe I , Proteínas Virais , Aminoácidos , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Complexo Principal de Histocompatibilidade , Peptídeos , SARS-CoV-2 , Proteínas Virais/imunologia
4.
Nature ; 563(7733): E33, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30315222

RESUMO

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.

5.
J Virol ; 95(15): e0242520, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980597

RESUMO

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.


Assuntos
Benzotiazóis/farmacologia , Briostatinas/farmacologia , Reservatórios de Doenças/virologia , Isoquinolinas/farmacologia , Latência Viral/efeitos dos fármacos , Proteína bcl-X/antagonistas & inibidores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Infecções por HIV/prevenção & controle , HIV-1/crescimento & desenvolvimento , Humanos , Replicação Viral/efeitos dos fármacos , Proteína bcl-X/metabolismo
6.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32051267

RESUMO

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.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Latência Viral/efeitos dos fármacos , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Depsipeptídeos/farmacologia , Feminino , Infecções por HIV/imunologia , Soropositividade para HIV/tratamento farmacológico , HIV-1/metabolismo , HIV-1/patogenicidade , HIV-1/fisiologia , Histona Desacetilases/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Latência Viral/fisiologia , Replicação Viral/efeitos dos fármacos
7.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626677

RESUMO

Curing HIV infection has been thwarted by the persistent reservoir of latently infected CD4+ T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir depletion strategies, we developed a novel preclinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, 2 weeks after PBMC injection, was 1,000-fold higher (mean = 9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean = 6,838 HIV copies/ml) or intravenously (mean = 591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo-reactivated primary latently infected cells. The time to viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, compared to treatment with 10-1074-FcRnull, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated, or 10-1074-FcRnull-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in the plasma of 10-1074-treated mice, whereas two new clusters emerged that were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time to viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral/fisiologia , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Modelos Animais de Doenças , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Camundongos , Filogenia , Baço/imunologia , Baço/virologia , Carga Viral/imunologia , Carga Viral/fisiologia , Viremia/imunologia , Viremia/virologia , Latência Viral/imunologia , Replicação Viral/imunologia
8.
PLoS Pathog ; 14(9): e1007257, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30180214

RESUMO

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.


Assuntos
Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/patogenicidade , Antígenos HLA-C/genética , Sequência de Aminoácidos , Reservatórios de Doenças/virologia , Regulação para Baixo , Variação Genética , Genótipo , Infecções por HIV/virologia , HIV-1/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Evasão da Resposta Imune , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologia
9.
J Immunol ; 201(5): 1468-1477, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30030324

RESUMO

HIV latency occurs predominantly in long-lived resting CD4+ T cells; however, latent infection also occurs in T cell subsets, including proliferating CD4+ T cells. We compared the establishment and maintenance of latent infection in nonproliferating and proliferating human CD4+ T cells cocultured with syngeneic myeloid dendritic cells (mDC). Resting CD4+ T cells were labeled with the proliferation dye eFluor 670 and cultured alone or with mDC, plasmacytoid dendritic cells, or monocytes in the presence of staphylococcal enterotoxin B (SEB). Cells were cultured for 24 h and infected with CCR5-tropic enhanced GFP (EGFP) reporter HIV. Five days postinfection, nonproductively infected EGFP- CD4+ T cells that were either nonproliferating (eFluor 670hi) or proliferating (eFluor 670lo) were sorted and cultured for an additional 7 d (day 12) with IL-7 and antiretrovirals. At day 5 postinfection, sorted, nonproductively infected T cells were stimulated with anti-CD3/CD28, and induced expression of EGFP was measured to determine the frequency of latent infection. Integrated HIV in these cells was confirmed using quantitative PCR. By these criteria, latent infection was detected at day 5 and 12 in proliferating T cells cocultured with mDC and monocytes but not plasmacytoid dendritic cells, where CD4+ T cells at day 12 were poor. At day 5 postinfection, nonproliferating T cells expressing SEB-specific TCR Vß-17 were enriched in latent infection compared with non-SEB-specific TCR Vß-8.1. Together, these data show that both nonproliferating and proliferating CD4+ T cells can harbor latent infection during SEB-stimulated T cell proliferation and that the establishment of HIV latency in nonproliferating T cells is linked to expression of specific TCR that respond to SEB.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Monócitos/imunologia , Latência Viral/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Enterotoxinas/farmacologia , Infecções por HIV/genética , Infecções por HIV/patologia , Humanos , Monócitos/patologia , Monócitos/virologia , Latência Viral/efeitos dos fármacos , Latência Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Replicação Viral/imunologia
11.
Retrovirology ; 15(1): 36, 2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29751762

RESUMO

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.


Assuntos
Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Latência Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Azepinas/farmacologia , Proteínas de Ciclo Celular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Mutação , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Splicing de RNA , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Triazóis/farmacologia , Ativação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
12.
Retrovirology ; 13(1): 49, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27459960

RESUMO

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.


Assuntos
Linfócitos T CD4-Positivos/virologia , Quimiocina CCL19/farmacologia , NF-kappa B/metabolismo , Receptores CCR/genética , Integração Viral , Latência Viral , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Integrase de HIV/genética , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , NF-kappa B/genética , Receptores CCR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Integração Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
13.
mBio ; : e0241723, 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-37971267

RESUMO

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.

14.
bioRxiv ; 2023 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-37090500

RESUMO

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.

15.
Retrovirology ; 9: 54, 2012 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-22731404

RESUMO

BACKGROUND: The characterization of HIV-1 transmission strains may inform the design of an effective vaccine. Shorter variable loops with fewer predicted glycosites have been suggested as signatures enriched in envelope sequences derived during acute HIV-1 infection. Specifically, a transmission-linked lack of glycosites within the V1 and V2 loops of gp120 provides greater access to an α4ß7 binding motif, which promotes the establishment of infection. Also, a histidine at position 12 in the leader sequence of Env has been described as a transmission signature that is selected against during chronic infection. The purpose of this study is to measure the association of the presence of an α4ß7 binding motif, the number of N-linked glycosites, the length of the variable loops, and the prevalence of histidine at position 12 with HIV-1 transmission. A case-control study design was used to measure the prevalence of these variables between subtype B and C transmission sequences and frequency-matched randomly-selected sequences derived from chronically infected controls. RESULTS: Subtype B transmission strains had shorter V3 regions than chronic strains (p = 0.031); subtype C transmission strains had shorter V1 loops than chronic strains (p = 0.047); subtype B transmission strains had more V3 loop glycosites (p = 0.024) than chronic strains. Further investigation showed that these statistically significant results were unlikely to be biologically meaningful. Also, there was no difference observed in the prevalence of a histidine at position 12 among transmission strains and controls of either subtype. CONCLUSIONS: Although a genetic bottleneck is observed after HIV-1 transmission, our results indicate that summary characteristics of Env hypothesised to be important in transmission are not divergent between transmission and chronic strains of either subtype. The success of a transmission strain to initiate infection may be a random event from the divergent pool of donor viral sequences. The characteristics explored through this study are important, but may not function as genotypic signatures of transmission as previously described.


Assuntos
Infecções por HIV/transmissão , HIV-1/genética , Histidina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Estudos de Casos e Controles , Sequência Consenso , Variação Genética , Técnicas de Genotipagem/métodos , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/patogenicidade , Humanos , Integrinas/genética , Integrinas/metabolismo , Modelos Logísticos , Dados de Sequência Molecular , Razão de Chances , Sinais Direcionadores de Proteínas , Especificidade da Espécie
16.
bioRxiv ; 2022 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-35665005

RESUMO

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.

17.
J Exp Med ; 218(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33988715

RESUMO

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.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Xenoenxertos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Células HEK293 , Infecções por HIV/virologia , Xenoenxertos/virologia , Humanos , Imunoterapia/métodos , Interleucina-15/imunologia , Camundongos , Mutação/imunologia , Viremia/imunologia , Viremia/virologia , Replicação Viral/imunologia
18.
Nat Commun ; 12(1): 165, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420062

RESUMO

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.


Assuntos
Biodiversidade , DNA Viral/análise , HIV-1/genética , Provírus/genética , Sequência de Bases , Linfócitos T CD4-Positivos/virologia , DNA Viral/genética , Infecções por HIV/virologia , Humanos , Filogenia , Reação em Cadeia da Polimerase/métodos
19.
JCI Insight ; 6(3)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33400687

RESUMO

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.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Antígenos HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Adulto , Idoso , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Estudos de Coortes , Feminino , Granzimas/metabolismo , Infecções por HIV/virologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Evasão da Resposta Imune , Interferon gama/metabolismo , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Linfócitos T/efeitos dos fármacos , Carga Viral , Adulto Jovem
20.
J Clin Invest ; 130(5): 2542-2559, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32027622

RESUMO

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.


Assuntos
HIV/imunologia , HIV/patogenicidade , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Adulto , Terapia Antirretroviral de Alta Atividade , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linfócitos T CD4-Positivos/classificação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Técnicas de Cocultura , Terapia Combinada , Citotoxicidade Imunológica/genética , Reservatórios de Doenças/virologia , Feminino , Perfilação da Expressão Gênica , HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Sulfonamidas/farmacologia , Latência Viral/efeitos dos fármacos
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