RESUMO
During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.
Assuntos
Infecções por Arenaviridae/imunologia , Linfócitos B/imunologia , Infecções por Vírus Epstein-Barr/imunologia , HIV/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Centro Germinativo/patologia , Centro Germinativo/virologia , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
HIV persistence in the brain is a barrier to cure, and potentially contributes to HIV-associated neurocognitive disorders. Whether HIV transcription persists in the brain despite viral suppression with antiretroviral therapy (ART) and is subject to the same blocks to transcription seen in other tissues and blood, is unclear. Here, we quantified the level of HIV transcripts in frontal cortex tissue from virally suppressed or non-virally suppressed people with HIV (PWH). HIV transcriptional profiling of frontal cortex brain tissue (and PBMCs where available) from virally suppressed (n = 11) and non-virally suppressed PWH (n = 13) was performed using digital polymerase chain reaction assays (dPCR). CD68+ myeloid cells or CD3+ T cells expressing HIV p24 protein present in frontal cortex tissue was detected using multiplex immunofluorescence imaging. Frontal cortex brain tissue from PWH had HIV TAR (n = 23/24) and Long-LTR (n = 20/24) transcripts. Completion of HIV transcription was evident in brain tissue from 12/13 non-virally suppressed PWH and from 5/11 virally suppressed PWH, with HIV p24+CD68+ cells detected in these individuals. While a block to proximal elongation was present in frontal cortex tissue from both PWH groups, this block was more extensive in virally suppressed PWH. These findings suggest that the brain is a transcriptionally active HIV reservoir in a subset of virally suppressed PWH.
Assuntos
Encéfalo , Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Masculino , Encéfalo/metabolismo , Encéfalo/virologia , Adulto , Pessoa de Meia-Idade , Feminino , Transcrição Gênica , Lobo Frontal/metabolismo , Lobo Frontal/virologiaRESUMO
Persistence of the rebound-competent viral reservoir (RCVR) within the CD4+ T cell compartment of people living with HIV remains a major barrier to HIV cure. Here, we determined the effects of the pan-lymphocyte-depleting monoclonal antibody (mAb) alemtuzumab on the RCVR in SIVmac239-infected rhesus macaques (RM) receiving antiretroviral therapy (ART). Alemtuzumab administered during chronic ART or at the time of ART initiation induced >95% depletion of circulating CD4+ T cells in peripheral blood and substantial CD4+ T cell depletion in lymph nodes. However, treatment was followed by proliferation and reconstitution of CD4+ T cells in blood, and despite ongoing ART, levels of cell-associated SIV DNA in blood and lymphoid tissues were not substantially different between alemtuzumab-treated and control RM after immune cell reconstitution, irrespective of the time of alemtuzumab treatment. Upon ART cessation, 19 of 22 alemtuzumab-treated RM and 13 of 13 controls rebounded with no difference in the time to rebound between treatment groups. Time to rebound and reactivation rate was associated with plasma viral loads (pVLs) at time of ART initiation, suggesting lymphocyte depletion had no durable impact on the RCVR. However, 3 alemtuzumab-treated RM that had lowest levels of pre-ART viremia, failed to rebound after ART withdrawal, in contrast to controls with similar levels of SIV replication. These observations suggest that alemtuzumab therapy has little to no ability to reduce well-established RCVRs but may facilitate RCVR destabilization when pre-ART virus levels are particularly low.
Assuntos
Alemtuzumab , Depleção Linfocítica , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Carga Viral , Animais , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Alemtuzumab/farmacologia , Depleção Linfocítica/métodos , Carga Viral/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacosAssuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Receptores CXCR5/metabolismo , Células Th17/imunologia , Antirretrovirais/uso terapêutico , Anticorpos Antivirais/imunologia , Perfilação da Expressão Gênica , Genoma/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , RNA Viral/genética , Células Th1/imunologia , Transcrição Gênica/genética , Replicação Viral/genéticaRESUMO
BACKGROUND & AIMS: New antiviral approaches that target multiple aspects of the HBV replication cycle to improve rates of functional cure are urgently required. HBV RNA represents a novel therapeutic target. Here, we programmed CRISPR-Cas13b endonuclease to specifically target the HBV pregenomic RNA and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pregenomic RNA. Mammalian cells transfected with replication competent wild-type HBV DNA of different genotypes, a HBV-expressing stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-BFP (blue fluorescent protein) and crRNA plasmids, and the impact on HBV replication and protein expression was measured. Wild-type HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and serum HBsAg was measured. PspCas13b mRNA and crRNA were also delivered to a HBsAg-expressing stable cell line via lipid nanoparticles and the impact on secreted HBsAg determined. RESULTS: Our HBV-targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p <0.0001). HBV protein expression was also reduced in a HBV-expressing stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p <0.0001) in vivo. Lipid nanoparticle-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p = 0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: Owing to the limitations of current antiviral therapies for hepatitis B, there is an urgent need for new treatments that target multiple aspects of the HBV replication cycle to improve rates of functional cure. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression, paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.
RESUMO
OBJECTIVE: To conduct a comprehensive, systematic review of the profile of HIV-1 reservoirs in children and adolescents with perinatally acquired HIV infection. STUDY DESIGN: Randomized and nonrandomized trials, cohort studies, and cross-sectional studies on HIV reservoirs in pediatric populations, published between 2002 and 2022, were included. Archived-drug resistance mutations (ADRMs) and the size of reservoirs were evaluated. Subgroup analyses were performed to characterize further the data, and the meta-analysis was done through random effect models. RESULTS: Overall, 49 studies from 17 countries worldwide were included, encompassing 2356 perinatally infected participants (48.83% females). There are limited data on the quantitative characterization of viral reservoirs in sub-Saharan Africa, with sensitive methodologies such as droplet digital polymerase chain reaction rarely employed. The overall prevalence of ADRMs was 37.80% (95% CI 13.89-65.17), with 48.79% (95% CI 0-100) in Africa, 42.08% (95% CI 6.68-82.71) in America, 23.88% (95% CI 14.34-34.90) in Asia, and 20.00% (95% CI 10.72-31.17) in Europe, without any difference between infants and adolescents (P = .656). Starting antiretroviral therapy (ART) before 2 months of age limited the levels of HIV-1 DNA (P = .054). Participants with long-suppressed viremia (>5 years) had lower levels of HIV-1 DNA (P = .027). Pre- and post-ART CD4 ≤29% and pre-ART viremia ≥5Log were all found associated with greater levels of HIV-1 DNA (P = .038, P = .047, and P = .041, respectively). CONCLUSIONS: The pooled prevalence of ADRMs is high in perinatally infected pediatric population, with larger proviral reservoir size driven by delayed ART initiation, a shorter period of viral suppression, and immunovirological failures. Thus, strategies for pediatric HIV functional cure should target children and adolescents with very early ART initiation, immunocompetence, and long-term viral suppression.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Lactente , Feminino , Criança , Humanos , Adolescente , Masculino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , HIV-1/genética , Estudos Transversais , Viremia , DNA , Carga ViralRESUMO
Here, we provide the first regional analysis of intact and defective HIV reservoirs within the brain. Brain tissue from both viremic and virally suppressed people with HIV (PWH) harbored HIV pol DNA in all regions tested, with lower levels present in basal ganglia and cerebellum relative to frontal white matter. Intact proviruses were primarily found in the frontal white matter but also detected in other brain regions of PWH, demonstrating frontal white matter as a major brain reservoir of intact, potentially replication competent HIV DNA that persists despite antiretroviral therapy. ANN NEUROL 2023;94:798-802.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Provírus/genética , Linfócitos T CD4-Positivos , HIV-1/genética , Carga Viral , Infecções por HIV/tratamento farmacológico , EncéfaloRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the highly contagious respiratory disease Corona Virus Disease 2019 (COVID-19) that may lead to various neurological and psychological disorders that can be acute, lasting days to weeks or months and possibly longer. The latter is known as long-COVID or more recently post-acute sequelae of COVID (PASC). During acute COVID-19 infection, a strong inflammatory response, known as the cytokine storm, occurs in some patients. The levels of interferon-γ (IFN-γ), interferon-ß (IFN-ß), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) are particularly increased. These cytokines are known to activate the enzyme indoleamine 2,3-dioxygenase 1 (IDO-1), catalysing the first step of tryptophan (Trp) catabolism through the kynurenine pathway (KP) leading to the production of several neurotoxic and immunosuppressive metabolites. There is already data showing elevation in KP metabolites both acutely and in PASC, especially regarding cognitive impairment. Thus, it is likely that KP involvement is significant in SARS-CoV-2 pathogenesis especially neurologically.
RESUMO
In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT) and lymphocyte activation gene-3 (LAG-3) alone or in combination would enhance HIV-specific CD4+ and CD8+ T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART (n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8+ and CD107a and IL-2 production in HIV-specific CD4+ and CD8+ T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4+ and CD8+ T cells in PWH on ART. These combinations should be further explored for an HIV cure.
Assuntos
Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Adulto , Antígenos CD/imunologia , Antígenos Virais/imunologia , Antígeno CTLA-4/imunologia , Células Cultivadas , Sinergismo Farmacológico , Quimioterapia Combinada , Infecções por HIV/imunologia , Sobreviventes de Longo Prazo ao HIV , Humanos , Interleucina-1/metabolismo , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores Imunológicos/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Co-infection with hepatitis B (HBV) and human immunodeficiency virus (HIV) increases overall and liver-related mortality. In order to identify interactions between these two viruses in vivo, full-length HIV proviruses were sequenced from a cohort of HIV-HBV co-infected participants and from a cohort of HIV mono-infected participants recruited from Bangkok, Thailand, both before the initiation of antiretroviral therapy (ART) and after at least 2 years of ART. The co-infected individuals were found to have higher levels of genetically-intact HIV proviruses than did mono-infected individuals pre-therapy. In these co-infected individuals, higher levels of genetically-intact HIV proviruses or proviral genetic-diversity were also associated with higher levels of sCD14 and CXCL10, suggesting that immune activation is linked to more genetically-intact HIV proviruses. Three years of ART decreased the overall level of HIV proviruses, with fewer genetically-intact proviruses being identified in co-infected versus mono-infected individuals. However, ART increased the frequency of certain genetic defects within proviruses and the expansion of identical HIV sequences. IMPORTANCE With the increased availability and efficacy of ART, co-morbidities are now one of the leading causes of death in HIV-positive individuals. One of these co-morbidities is co-infection with HBV. However, co-infections are still relatively understudied, especially in countries where such co-infections are endemic. Furthermore, these countries have different subtypes of HIV circulating than the commonly studied HIV subtype B. We believe that our study serves this understudied niche and provides a novel approach to investigating the impact of HBV co-infection on HIV infection. We examine co-infection at the molecular level in order to investigate indirect associations between the two viruses through their interactions with the immune system. We demonstrate that increased immune inflammation and activation in HBV co-infected individuals is associated with higher HIV viremia and an increased number of genetically-intact HIV proviruses in peripheral blood cells. This leads us to hypothesize that inflammation could be a driver in the increased mortality rate of HIV-HBV co-infected individuals.
Assuntos
Coinfecção , Infecções por HIV , Hepatite B , Inflamação/virologia , Coinfecção/patologia , Coinfecção/virologia , DNA Viral/genética , Infecções por HIV/complicações , Infecções por HIV/patologia , Infecções por HIV/virologia , Hepatite B/complicações , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Humanos , Provírus/genética , Tailândia/epidemiologia , Viremia/virologiaRESUMO
The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.
Assuntos
Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular , Infecções por HIV/virologia , HIV-1/imunologia , Carga Viral , Replicação Viral , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Estudos de Casos e Controles , DNA Viral/análise , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Human immunodeficiency virus (HIV) persistence in blood and tissue reservoirs, including the brain, is a major barrier to HIV cure and possible cause of comorbid disease. However, the size and replication competent nature of the central nervous system (CNS) reservoir is unclear. Here, we used the intact proviral DNA assay (IPDA) to provide the first quantitative assessment of the intact and defective HIV reservoir in the brain of people with HIV (PWH). METHODS: Total, intact, and defective HIV proviruses were measured in autopsy frontal lobe tissue from viremic (n = 18) or virologically suppressed (n = 12) PWH. Total or intact/defective proviruses were measured by detection of HIV pol or the IPDA, respectively, through use of droplet digital polymerase chain reaction (ddPCR). HIV-seronegative individuals were included as controls (n = 6). RESULTS: Total HIV DNA was present at similar levels in brain tissues from untreated viremic and antiretroviral (ART)-suppressed individuals (median = 22.3 vs 26.2 HIV pol copies/106 cells), reflecting a stable CNS reservoir of HIV that persists despite therapy. Furthermore, 8 of 10 viremic and 6 of 9 virally suppressed PWH also harbored intact proviruses in the CNS (4.63 vs 12.7 intact copies/106 cells). Viral reservoirs in CNS and matched lymphoid tissue were similar in the composition of intact and/or defective proviruses, albeit at lower levels in the brain. Importantly, CNS resident CD68+ myeloid cells in virally suppressed individuals harbored HIV DNA, directly showing the presence of a CNS resident HIV reservoir. INTERPRETATION: Our results demonstrate the first evidence for an intact, potentially replication competent HIV reservoir in the CNS of virally suppressed PWH. ANN NEUROL 2022;92:532-544.
Assuntos
Infecções por HIV , Provírus , Antirretrovirais/uso terapêutico , Encéfalo , Linfócitos T CD4-Positivos , DNA Viral/genética , DNA Viral/uso terapêutico , Infecções por HIV/tratamento farmacológico , Humanos , Provírus/genética , Carga Viral/métodosRESUMO
BACKGROUND: A sterilizing cure of HIV-1 infection has been reported in 2 persons living with HIV-1 who underwent allogeneic hematopoietic stem cell transplantations from donors who were homozygous for the CCR5Δ32 gene polymorphism. However, this has been considered elusive during natural infection. OBJECTIVE: To evaluate persistent HIV-1 reservoir cells in an elite controller with undetectable HIV-1 viremia for more than 8 years in the absence of antiretroviral therapy. DESIGN: Detailed investigation of virologic and immunologic characteristics. SETTING: Tertiary care centers in Buenos Aires, Argentina, and Boston, Massachusetts. PATIENT: A patient with HIV-1 infection and durable drug-free suppression of HIV-1 replication. MEASUREMENTS: Analysis of genome-intact and replication-competent HIV-1 using near-full-length individual proviral sequencing and viral outgrowth assays, respectively; analysis of HIV-1 plasma RNA by ultrasensitive HIV-1 viral load testing. RESULTS: No genome-intact HIV-1 proviruses were detected in analysis of a total of 1.188 billion peripheral blood mononuclear cells and 503 million mononuclear cells from placental tissues. Seven defective proviruses, some of them derived from clonally expanded cells, were detected. A viral outgrowth assay failed to retrieve replication-competent HIV-1 from 150 million resting CD4+ T cells. No HIV-1 RNA was detected in 4.5 mL of plasma. LIMITATIONS: Absence of evidence for intact HIV-1 proviruses in large numbers of cells is not evidence of absence of intact HIV-1 proviruses. A sterilizing cure of HIV-1 can never be empirically proved. CONCLUSION: Genome-intact and replication-competent HIV-1 were not detected in an elite controller despite analysis of massive numbers of cells from blood and tissues, suggesting that this patient may have naturally achieved a sterilizing cure of HIV-1 infection. These observations raise the possibility that a sterilizing cure may be an extremely rare but possible outcome of HIV-1 infection. PRIMARY FUNDING SOURCE: National Institutes of Health and Bill & Melinda Gates Foundation.
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Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/genética , Receptores CCR5/genética , Adulto , Argentina , Linfócitos T CD4-Positivos/imunologia , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Massachusetts , Gravidez , Resultado da Gravidez , Provírus/genética , Provírus/imunologia , Carga Viral , Viremia/virologia , Replicação Viral/imunologiaRESUMO
BACKGROUND: Analytical treatment interruptions (ATI) are pauses of antiretroviral therapy (ART) in the context of human immunodeficiency virus (HIV) cure trials. They are the gold standard in determining if interventions being tested can achieve sustained virological control in the absence of ART. However, withholding ART comes with risks and discomforts to trial participant. We used mathematical models to explore how ATI study design can be improved to maximize statistical power, while minimizing risks to participants. METHODS: Using previously observed dynamics of time to viral rebound (TVR) post-ATI, we modelled estimates for optimal sample size, frequency, and ATI duration required to detect a significant difference in the TVR between control and intervention groups. Groups were compared using a log-rank test, and analytical and stochastic techniques. RESULTS: In placebo-controlled TVR studies, 120 participants are required in each arm to detect 30% difference in frequency of viral reactivation at 80% power. There was little statistical advantage to measuring viral load more frequently than weekly, or interrupting ART beyond 5 weeks in a TVR study. CONCLUSIONS: Current TVR HIV cure studies are underpowered to detect statistically significant changes in frequency of viral reactivation. Alternate study designs can improve the statistical power of ATI trials.
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Ensaios Clínicos como Assunto , Infecções por HIV , Suspensão de Tratamento , Antirretrovirais/uso terapêutico , Ensaios Clínicos como Assunto/métodos , Infecções por HIV/tratamento farmacológico , Humanos , Projetos de Pesquisa , Medição de Risco , Carga Viral/estatística & dados numéricosRESUMO
BACKGROUND: Circadian transcription factors that regulate cell-autonomous circadian clocks can also increase human immunodeficiency virus (HIV) transcription in vitro. We aimed to determine whether circadian variation in HIV transcription exists in people with HIV (PWH) on antiretroviral therapy (ART). METHODS: We performed a prospective observational study of male PWH on ART, sampling blood every 4 hours for 24 hours. Using quantitative polymerase chain reaction, we quantified expression of circadian-associated genes, HIV deoxyribonucleic acid (DNA), and cell-associated unspliced (CA-US) ribonucleic acid (RNA) in peripheral blood CD4+â T cells. Plasma sex hormones were quantified alongside plasma and salivary cortisol. The primary outcome was to identify temporal variations in CA-US HIV RNA using a linear mixed-effect regression framework and maximum likelihood estimation. RESULTS: Salivary and plasma cortisol, and circadian genes including Clock, Bmal1, and Per3, varied with a circadian rhythm. Cell-associated unspliced HIV RNA and the ratio of CA-US HIV RNA/DNA in CD4+â T cells also demonstrated circadian variations, with no variation in HIV DNA. Circulating estradiol was highly predictive of CA-US HIV RNA variation in vivo. CONCLUSIONS: Cell-associated unspliced HIV RNA in PWH on ART varies temporally with a circadian rhythm. These findings have implications for the design of clinical trials and biomarkers to assess HIV cure interventions.
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Infecções por HIV , Hidrocortisona , Linfócitos T CD4-Positivos , HIV/genética , Infecções por HIV/tratamento farmacológico , Humanos , Hidrocortisona/uso terapêutico , Masculino , RNA Viral/genéticaRESUMO
BACKGROUND: Identifying factors that determine the frequency of latently infected CD4+â T cells on antiretroviral therapy (ART) may inform strategies for human immunodeficiency virus (HIV) cure. We investigated the role of CD4+ count at ART initiation for HIV persistence on ART. METHODS: Among participants of the Strategic Timing of Antiretroviral Treatment Study, we enrolled people with HIV (PWH) who initiated ART with CD4+â T-cell counts of 500-599, 600-799, orâ ≥â 800 cells/mm3. After 36-44 months on ART, the levels of total HIV-DNA, cell-associated unspliced HIV-RNA (CA-US HIV-RNA), and two-long terminal repeat HIV-DNA in CD4+â T cells were quantified and plasma HIV-RNA was measured by single-copy assay. We measured T-cell expression of Human Leucocyte Antigen-DR Isotype (HLA-DR), programmed death-1, and phosphorylated signal transducer and activator of transcription-5 (pSTAT5). Virological and immunological measures were compared across CD4+â strata. RESULTS: We enrolled 146 PWH, 36 in the 500-599, 60 in the 600-799, and 50 in theâ ≥â 800 CD4 strata. After 36-44 months of ART, total HIV-DNA, plasma HIV-RNA, and HLA-DR expression were significantly lower in PWH with CD4+â T-cell countâ ≥â 800 cells/mm3 at ART initiation compared with 600-799 or 500-599 cells/mm3. The median level of HIV-DNA after 36-44 months of ART was lower by 75% in participants initiating ART withâ ≥â 800 vs 500-599 cells/mm3 (median [interquartile range]: 16.3 [7.0-117.6] vs 68.4 [13.7-213.1] copies/million cells, respectively). Higher pSTAT5 expression significantly correlated with lower levels of HIV-DNA and CA-US HIV-RNA. Virological measures were significantly lower in females. CONCLUSIONS: Initiating ART with a CD4+â countâ ≥â 800 cells/mm3 compared with 600-799 or 500-599 cells/mm3 was associated with achieving a substantially smaller HIV reservoir on ART.
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Antirretrovirais , Infecções por HIV , Humanos , Feminino , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos , Antígenos HLA-DR , RNA/uso terapêutico , HIV , Carga ViralRESUMO
In HIV-hepatitis B virus (HBV) co-infection, adverse liver outcomes including liver fibrosis occur at higher frequency than in HBV-mono-infection, even following antiretroviral therapy (ART) that suppresses both HIV and HBV replication. To determine whether liver disease was associated with intrahepatic or circulating markers of inflammation or burden of HIV or HBV, liver biopsies and blood were collected from HIV-HBV co-infected individuals (n = 39) living in Bangkok, Thailand and naïve to ART. Transient elastography (TE) was performed. Intrahepatic and circulating markers of inflammation and microbial translocation were quantified by ELISA and bead arrays and HIV and HBV infection quantified by PCR. Liver fibrosis (measured by both transient elastography and liver biopsy) was statistically significantly associated with intrahepatic mRNA for CXCL10 and CXCR3 using linear and logistic regression analyses adjusted for CD4 T-cell count. There was no evidence of a relationship between liver fibrosis and circulating HBV DNA, qHBsAg, plasma HIV RNA or circulating cell-associated HIV RNA or DNA. Using immunohistochemistry of liver biopsies from this cohort, intrahepatic CXCL10 was detected in hepatocytes associated with inflammatory liver infiltrates in the portal tracts. In an in vitro model, we infected an HBV-infected hepatocyte cell line with HIV, followed by interferon-γ stimulation. HBV-infected cells lines produced significantly more CXCL10 than uninfected cells lines and this significantly increased in the presence of an increasing multiplicity of HIV infection. Conclusion: Enhanced production of CXCL10 following co-infection of hepatocytes with both HIV and HBV may contribute to accelerated liver disease in the setting of HIV-HBV co-infection.
Assuntos
Quimiocina CXCL10/metabolismo , Coinfecção/complicações , Infecções por HIV/complicações , HIV/isolamento & purificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B/complicações , Cirrose Hepática/epidemiologia , Adulto , Austrália/epidemiologia , Estudos de Coortes , Coinfecção/virologia , Feminino , Infecções por HIV/virologia , Hepatite B/virologia , Humanos , Incidência , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Masculino , Países Baixos/epidemiologia , Prognóstico , Tailândia/epidemiologiaRESUMO
HIV latency is the major barrier to a cure for people living with HIV (PLWH) on antiretroviral therapy (ART) because the virus persists in long-lived non-proliferating and proliferating latently infected CD4+ T cells. Latently infected CD4+ T cells do not express viral proteins and are therefore not visible to immune mediated clearance. Therefore, identifying interventions that can reverse latency and also enhance immune mediated clearance is of high interest. Interferons (IFNs) have multiple immune enhancing effects and can inhibit HIV replication in activated CD4+ T cells. However, the effects of IFNs on the establishment and reversal of HIV latency is not understood. Using an in vitro model of latency, we demonstrated that plasmacytoid dendritic cells (pDC) inhibit the establishment of HIV latency through secretion of type I IFNα, IFNß and IFNω but not IFNε or type III IFNλ1 and IFNλ3. However, once latency was established, IFNα but no other IFNs were able to efficiently reverse latency in both an in vitro model of latency and CD4+ T cells collected from PLWH on suppressive ART. Binding of IFNα to its receptor expressed on primary CD4+ T cells did not induce activation of the canonical or non-canonical NFκB pathway but did induce phosphorylation of STAT1, 3 and 5 proteins. STAT5 has been previously demonstrated to bind to the HIV long terminal repeat and activate HIV transcription. We demonstrate diverse effects of interferons on HIV latency with type I IFNα; inhibiting the establishment of latency but also reversing HIV latency once latency is established.
Assuntos
Linfócitos T CD4-Positivos , Repetição Terminal Longa de HIV/imunologia , HIV-1/fisiologia , Interferon-alfa/imunologia , Transcrição Gênica/imunologia , Latência Viral/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Células HEK293 , Humanos , NF-kappa B/imunologia , Fatores de Transcrição STAT/imunologiaRESUMO
One strategy to eliminate latently infected cells that persist in people with HIV on antiretroviral therapy is to activate virus transcription and virus production to induce virus or immune-mediated cell death. This is called latency reversal. Despite clear activity of multiple latency reversal agents in vitro, clinical trials of latency-reversing agents have not shown significant reduction in latently infected cells. We review new insights into the biology of HIV latency and discuss novel approaches to enhance the efficacy of latency reversal agents.