RESUMEN
BACKGROUND: Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral replication. Numerous studies have described HIV-1 as a poor activator of innate immunity in vitro. The exact role that the viral capsid plays in this immune evasion is not fully understood. RESULTS: To better understand the role of the HIV-1 capsid in sensing we tested the effect of making HIV-1 by co-expressing a truncated Gag that encodes the first 107 amino acids of capsid fused with luciferase or GFP, alongside wild type Gag-pol. We found that unlike wild type HIV-1, viral particles produced with a mixture of wild type and truncated Gag fused to luciferase or GFP induced a potent IFN response in THP-1 cells and macrophages. Innate immune activation by Gag-fusion HIV-1 was dependent on reverse transcription and DNA sensor cGAS, suggesting activation of an IFN response by viral DNA. Further investigation revealed incorporation of the Gag-luciferase/GFP fusion proteins into viral particles that correlated with subtle defects in wild type Gag cleavage and a diminished capacity to saturate restriction factor TRIM5α, likely due to aberrant particle formation. We propose that expression of the Gag fusion protein disturbs the correct cleavage and maturation of wild type Gag, yielding viral particles that are unable to effectively shield viral DNA from detection by innate sensors including cGAS. CONCLUSIONS: These data highlight the crucial role of capsid in innate evasion and support growing literature that disruption of Gag cleavage and capsid formation induces a viral DNA- and cGAS-dependent innate immune response. Together these data demonstrate a protective role for capsid and suggest that antiviral activity of capsid-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.
Asunto(s)
VIH-1 , Inmunidad Innata , Nucleotidiltransferasas , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , VIH-1/inmunología , VIH-1/genética , VIH-1/fisiología , Humanos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Factores de Restricción Antivirales , Macrófagos/inmunología , Macrófagos/virología , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Células THP-1 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/inmunología , Evasión Inmune , Cápside/metabolismo , Cápside/inmunología , Replicación Viral , Virión/metabolismo , Virión/genética , Virión/inmunología , Interacciones Huésped-Patógeno/inmunología , ADN Viral/genética , Línea CelularRESUMEN
Escape from cytotoxic T lymphocyte (CTL) responses toward HIV-1 Gag and Nef has been associated with reduced control of HIV-1 replication in adults. However, less is known about CTL-driven immune selection in infants as longitudinal studies of infants are limited. Here, 1,210 gag and 1,264 nef sequences longitudinally collected within 15 months after birth from 14 HIV-1 perinatally infected infants and their mothers were analyzed. The number of transmitted founder (T/F) viruses and associations between virus evolution, selection, CTL escape, and disease progression were determined. The analyses indicated that a paraphyletic-monophyletic relationship between the mother-infant sequences was common (80%), and that the HIV-1 infection was established by a single T/F virus in 10 of the 12 analyzed infants (83%). Furthermore, most HIV-1 CTL escape mutations among infants were transmitted from the mothers and did not revert during the first year of infection. Still, immune-driven selection was observed at approximately 3 months after HIV-1 infection in infants. Moreover, virus populations with CTL escape mutations in gag evolved faster than those without, independently of disease progression rate. These findings expand the current knowledge of HIV-1 transmission, evolution, and CTL escape in infant HIV-1 infection and are relevant for the development of immune-directed interventions in infants.IMPORTANCEDespite increased coverage in antiretroviral therapy for the prevention of perinatal transmission, paediatric HIV-1 infection remains a significant public health concern, especially in areas of high HIV-1 prevalence. Understanding HIV-1 transmission and the subsequent virus adaptation from the mother to the infant's host environment, as well as the viral factors that affect disease outcome, is important for the development of early immune-directed interventions for infants. This study advances our understanding of vertical HIV-1 transmission, and how infant immune selection pressure is shaping the intra-host evolutionary dynamics of HIV-1.
Asunto(s)
Evolución Molecular , Infecciones por VIH , VIH-1 , Transmisión Vertical de Enfermedad Infecciosa , Mutación , Linfocitos T Citotóxicos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/genética , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Infecciones por VIH/virología , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , Lactante , Femenino , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Evasión Inmune/genética , Recién Nacido , Filogenia , Masculino , Estudios Longitudinales , Embarazo , AdultoRESUMEN
Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccines have been long overdue. Structure-based vaccine design created a new momentum in the last decade, and the first RSV vaccines have finally been approved in older adults and pregnant individuals. These vaccines are based on recombinant stabilized pre-fusion F glycoproteins administered as soluble proteins. Multimeric antigenic display could markedly improve immunogenicity and should be evaluated in the next generations of vaccines. Here we tested a new virus like particles-based vaccine platform which utilizes the direct fusion of an immunogen of interest to the structural human immunodeficient virus (HIV) protein Gag to increase its surface density and immunogenicity. We compared, in mice, the immunogenicity of RSV-F or hMPV-F based immunogens delivered either as soluble proteins or displayed on the surface of our VLPs. VLP associated F-proteins showed better immunogenicity and induced superior neutralizing responses. Moreover, when combining both VLP associated and soluble immunogens in a heterologous regimen, VLP-associated immunogens provided added benefits when administered as the prime immunization.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Metapneumovirus , Ratones Endogámicos BALB C , Vacunas de Partículas Similares a Virus , Proteínas Virales de Fusión , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ratones , Metapneumovirus/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Femenino , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Virus Sincitial Respiratorio Humano/inmunología , Inmunogenicidad Vacunal , Humanos , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
HIV-1 CRF07_BC-p6Δ7, a strain with a seven amino acid deletion in the p6 region of the Gag protein, is becoming the dominant strain of HIV transmission among men who have sex with men (MSM) in China. Previous studies demonstrated that HIV-1 patients infected by CRF07_BC-p6Δ7 strain had lower viral load and slower disease progression than those patients infected with CRF07_BC wild-type strain. However, the underlying mechanism for this observation is not fully clarified yet. In this study, we constructed the recombinant DNA plasmid and adenovirus type 2 (Ad2) vector-based constructs to express the HIV-1 CRF07_BC Gag antigen with or without p6Δ7 mutation and then investigated their immunogenicity in mice. Our results showed that HIV-1 CRF07_BC Gag antigen with p6Δ7 mutation induced a comparable level of Gag-specific antibodies but stronger CD4+ and CD8+ T-cell immune responses than that of CRF07_BC Gag (07_BC-wt). Furthermore, we identified a series of T-cell epitopes, which induced strong T-cell immune response and cross-immunity with CRF01_AE Gag. These findings implied that the p6Gag protein with a seven amino acid deletion might enhance the Gag immunogenicity in particular cellular immunity, which provides valuable information to clarify the pathogenic mechanism of HIV-1 CRF07_BC-p6Δ7 and to develop precise vaccine strategies against HIV-1 infection.
Asunto(s)
Epítopos de Linfocito T , VIH-1 , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Aminoácidos , Animales , Antígenos Virales , Infecciones por VIH/virología , VIH-1/genética , Homosexualidad Masculina , Humanos , Masculino , Ratones , Minorías Sexuales y de Género , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
HIV-specific CD8+ T cells play a central role in immune control of adult HIV, but their contribution in pediatric infection is less well characterized. Previously, we identified a group of ART-naive children with persistently undetectable plasma viremia, termed "elite controllers," and a second group who achieved aviremia only transiently. To investigate the mechanisms of failure to maintain aviremia, we characterized in three transient aviremic individuals (TAs), each of whom expressed the disease-protective HLA-B*81:01, longitudinal HIV-specific T-cell activity, and viral sequences. In two TAs, a CD8+ T-cell response targeting the immunodominant epitope TPQDLNTML (Gag-TL9) was associated with viral control, followed by viral rebound and the emergence of escape variants with lower replicative capacity. Both TAs mounted variant-specific responses, but only at low functional avidity, resulting in immunological progression. In contrast, in TA-3, intermittent viremic episodes followed aviremia without virus escape or a diminished CD4+ T-cell count. High quality and magnitude of the CD8+ T-cell response were associated with aviremia. We therefore identify two distinct mechanisms of loss of viral control. In one scenario, CD8+ T-cell responses initially cornered low-replicative-capacity escape variants, but with insufficient avidity to prevent viremia and disease progression. In the other, loss of viral control was associated with neither virus escape nor progression but with a decrease in the quality of the CD8+ T-cell response, followed by recovery of viral control in association with improved antiviral response. These data suggest the potential for a consistently strong and polyfunctional antiviral response to achieve long-term viral control without escape. IMPORTANCE Very early initiation of antiretroviral therapy (ART) in pediatric HIV infection offers a unique opportunity to limit the size and diversity of the viral reservoir. However, only rarely is ART alone sufficient to achieve remission. Additional interventions that likely include contributions from host immunity are therefore required. The HIV-specific T-cell response plays a central role in immune control of adult HIV, often mediated through protective alleles such as HLA-B*57/58:01/81:01. However, due to the tolerogenic and type 2 biased immune response in early life, HLA-I-mediated immune suppression of viremia is seldom observed in children. We assessed a rare group of HLA-B*81:01-positive, ART-naive children who achieved aviremia, albeit only transiently, and investigated the role of the CD8+ T-cell response in the establishment and loss of viral control. We identified a mechanism by which the HIV-specific response can achieve viremic control without viral escape that can be explored in strategies to achieve remission.
Asunto(s)
Infecciones por VIH/inmunología , Sobrevivientes de VIH a Largo Plazo , Viremia/inmunología , Adolescente , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/inmunología , Niño , Preescolar , Femenino , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Antígenos HLA-B/inmunología , Humanos , Evasión Inmune , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Lactante , Masculino , Carga Viral , Viremia/virología , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Introduction: Low HIV viral load is associated with delayed disease progression and reduced HIV transmission. HIV controllers suppress viral load to low levels in the absence of antiretroviral treatment (ART). We used an antibody profiling system, VirScan, to compare antibody reactivity and specificity in HIV controllers, non-controllers with treatment-induced viral suppression, and viremic non-controllers. Methods: The VirScan library contains 3,384 phage-displayed peptides spanning the HIV proteome. Antibody reactivity to these peptides was measured in plasma from a Discovery Cohort that included 13 elite controllers, 27 viremic controllers, 12 viremic non-controllers, and 21 non-controllers who were virally suppressed on ART. Antibody reactivity to selected peptides was also assessed in an independent cohort of 29 elite controllers and 37 non-controllers who were virally suppressed on ART (Validation Cohort) and in a longitudinal cohort of non-controllers. Results: In the Discovery Cohort, 62 peptides were preferentially targeted in HIV controllers compared to non-controllers who were virally suppressed on ART. These specificities were not significantly different when comparing controllers versus viremic non-controllers. Aggregate reactivity to these peptides was also high in elite controllers from the independent Validation Cohort. The 62 peptides formed seven clusters of homologous epitopes in env, gag, integrase, and vpu. Reactivity to one of these clusters located in gag p17 was inversely correlated with viral load set point in an independent cohort of non-controllers. Conclusions: Antibody reactivity was low in non-controllers suppressed on ART, but remained high in viremic controllers despite viral suppression. Antibodies in controllers and viremic non-controllers were directed against epitopes in diverse HIV proteins; higher reactivity against p17 peptides was associated with lower viral load set point. Further studies are needed to determine if these antibodies play a role in regulation of HIV viral load.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH no-Progresivos , VIH-1/fisiología , Adulto , Antirretrovirales/uso terapéutico , Mapeo Epitopo , Epítopos/genética , Epítopos/inmunología , Femenino , Antígenos VIH/genética , Antígenos VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Humanos , Masculino , Biblioteca de Péptidos , Carga Viral , Adulto Joven , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The dynamics of T-cell receptor (TCR)selection in chronic HIV-1 infection, and its association with clinical outcome, is well documented for an array of MHC-peptide complexes and disease stages. However, the factors that may contribute to the selection and expansion of CD8+ T-cells in chronic HIV-2 infection, especially at the clonal level remain unclear. To address this question, we undertook a detailed molecular characterization of the clonotypic architecture of an HLA-B*3501 restricted Gag-specific CD8+ T-cell response in donors chronically infected with HIV-2 using a combination of flow cytometry, tetramer-specific CD8+ TCR clonotyping, and in vitro assays. We show that the response to the NY9 epitope is hierarchical and narrow in terms of T-cell receptor-alpha (TCRA) and -beta (TCRB) gene usage yet clonotypically diverse. Furthermore, clonotypic dominance in shared origin CTL clones was associated with a greater magnitude of cytokine production and antigen sensitivity at limiting antigen dilution as well as enhanced cross-reactivity for known HIV-2 variants. Hence, our data suggest that effector mobilization and expansion in human chronic HIV-2 infection may be linked to the qualitative features of specific CD8+ T-cell clonotypes, which could have implications for viral control and disease outcome.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-2/fisiología , Especificidad del Receptor de Antígeno de Linfocitos T , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Secuencias de Aminoácidos , Linfocitos T CD8-positivos/metabolismo , Enfermedad Crónica , Secuencia Conservada , Epítopos de Linfocito T/inmunología , Infecciones por VIH/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
CD8+ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E-restricted CD8+ T cell responses in HIV infection, however, remains unknown. In this study, CD8+ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E-restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8+ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8+ T cells. Importantly, bulk CD8+ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E-restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E-restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8+ T cell responses in HIV infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Infecciones por VIH/inmunología , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Línea Celular , Infecciones por VIH/genética , Infecciones por VIH/virología , Seronegatividad para VIH/inmunología , VIH-1/genética , Antígenos HLA-B/inmunología , Humanos , Epítopos Inmunodominantes , Técnicas In Vitro , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Antígenos HLA-ERESUMEN
The capsid (CA) domain of the HIV-1 precursor Gag (PrGag) protein plays multiple roles in HIV-1 replication, and is central to the assembly of immature virions, and mature virus cores. CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs). We have investigated the interactions of CA with anti-CA nanobodies, which derive from the antigen recognition regions of camelid heavy chain-only antibodies. The one CA NTD-specific and two CTD-specific nanobodies we analyzed proved sensitive and specific HIV-1 CA detection reagents in immunoassays. When co-expressed with HIV-1 Gag proteins in cells, the NTD-specific nanobody was efficiently assembled into virions and did not perturb virus assembly. In contrast, the two CTD-specific nanobodies reduced PrGag processing, virus release and HIV-1 infectivity. Our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1.
Asunto(s)
Cápside/inmunología , VIH-1/fisiología , Anticuerpos de Dominio Único/metabolismo , Ensamble de Virus , Cápside/química , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Línea Celular , VIH-1/inmunología , Humanos , Dominios Proteicos , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Virión/metabolismo , Liberación del Virus , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 functional cure requires sustained viral suppression without antiretroviral therapy. While effector-memory CD8+ T lymphocytes are essential for viremia control, few vaccines elicit such cellular immunity that could be potently recalled upon viral infection. Here, we investigated a program death-1 (PD1)-based vaccine by fusion of simian immunodeficiency virus capsid antigen to soluble PD1. Homologous vaccinations suppressed setpoint viremia to undetectable levels in vaccinated macaques following a high-dose intravenous challenge by the pathogenic SHIVSF162P3CN. Poly-functional effector-memory CD8+ T cells were not only induced after vaccination, but were also recalled upon viral challenge for viremia control as determined by CD8 depletion. Vaccine-induced effector memory CD8+ subsets displayed high cytotoxicity-related genes by single-cell analysis. Vaccinees with sustained viremia suppression for over two years responded to boost vaccination without viral rebound. These results demonstrated that PD1-based vaccine-induced effector-memory CD8+ T cells were recalled by AIDS virus infection, providing a potential immunotherapy for functional cure.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Vacunas contra el SIDAS/inmunología , Viremia/prevención & control , Animales , Femenino , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios , Vacunas de ADN/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Untreated HIV-1 infection leads to a slow decrease in CD4+ T cell lymphocytes over time resulting in increased susceptibility to opportunistic infections (acquired immunodeficiency syndrome, AIDS) and ultimately death of the infected individual. Initially, the host's immune response controls the infection, but cannot eliminate the HIV-1 from the host. Cytotoxic lymphocytes are the key effector cells in this response and can mediate crucial antiviral responses through the release of a set of proteases called granzymes towards HIV-1-infected cells. However, little is known about the immunological molecular mechanisms by which granzymes could control HIV-1. Since we noted that HIV-1 subtype C (HIV-1C) Gag with the tetrapeptide insertion PYKE contains a putative granzyme M (GrM) cleavage site (KEPL) that overlaps with the PYKE insertion, we analyzed the proteolytic activity of GrM towards Gag. Immunoblot analysis showed that GrM could cleave Gag proteins from HIV-1B and variants from HIV-1C of which the Gag-PYKE variant was cleaved with extremely high efficiency. The main cleavage site was directly after the insertion after leucine residue 483. GrM-mediated cleavage of Gag was also observed in co-cultures using cytotoxic lymphocytes as effector cells and this cleavage could be inhibited by a GrM inhibitor peptide. Altogether, our data indicate towards a noncytotoxic immunological mechanism by which GrM-positive cytotoxic lymphocytes target the HIV-1 Gag protein within infected cells to potentially control HIV-1 infection. This mechanism could be exploited in new therapeutic strategies to treat HIV-1-infected patients to improve immunological control of the infection.
Asunto(s)
Granzimas/metabolismo , Infecciones por VIH/virología , VIH-1/patogenicidad , Linfocitos T Citotóxicos/virología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Estudios de Casos y Controles , Técnicas de Cocultivo , Progresión de la Enfermedad , Células HEK293 , Infecciones por VIH/enzimología , Infecciones por VIH/inmunología , VIH-1/inmunología , VIH-1/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Proteolisis , Especificidad por Sustrato , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/inmunología , Carga Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the human immunodeficiency virus (HIV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the local tissues to suppress HIV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs elicit the antiviral response remain to be fully elucidated. In this study, we generated the functional HIV-1 Gag epitope SL9-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the suppression of SL9-specific iPSC-CTLs on viral replication and the protection of CD4+ T cells. A chimeric HIV-1, i.e., EcoHIV, was used to produce HIV replication in mice. We show that adoptive transfer of SL9-specific iPSC-CTLs greatly suppressed EcoHIV replication in the peritoneal macrophages and spleen in the animal model. Furthermore, we demonstrate that the adoptive transfer significantly reduced expression of PD-1 on CD4+ T cells in the spleen and generated persistent anti-HIV memory T cells. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the local tissues to suppress HIV replication and prevent CD4+ T cell exhaustion through reduction of PD-1 expression.
Asunto(s)
Antígenos Virales/inmunología , VIH/genética , VIH/inmunología , Receptor de Muerte Celular Programada 1/genética , Linfocitos T Citotóxicos/virología , Replicación Viral/genética , Replicación Viral/inmunología , Traslado Adoptivo , Animales , Antígenos Virales/metabolismo , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , VIH/fisiología , Infecciones por VIH/inmunología , Humanos , Células Madre Pluripotentes Inducidas , Células T de Memoria/inmunología , Ratones , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T Citotóxicos/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Biomarcadores , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Citocinas/metabolismo , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Humanos , Inmunofenotipificación , Células Jurkat , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Péptidos/química , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Antígenos HLA-ERESUMEN
The genetic diversity of circulating HIV-1 strains poses a major barrier to the design, development and evaluation of HIV-1 vaccines. The assessment of both vaccine- and natural infection-elicited T cell responses is commonly done with multivalent peptides that are designed to maximally capture the diversity of potential T cell epitopes (PTEs) observed in natural circulating sequences. However, depending on the sequence diversity of viral subtypes and number of the HIV immunogens under investigation, PTE estimates, including HLA-guided computational methods, can easily generate enormous peptide libraries. Evaluation of T cell epitope specificity using such extensive peptide libraries is usually limited by sample availability, even for high-throughput and robust epitope mapping techniques like ELISpot assays. Here we describe a novel, two-step protocol for in-vitro polyclonal expansion of CD8 T cells from a single vial of frozen PBMC, which facilitated the screening 441 HIV-1 Gag peptides for immune responses among 32 HIV-1 positive subjects and 40 HIV-1 negative subjects for peptide qualification. Using a pooled-peptide mapping strategy, epitopes were mapped in two sequential ELISpot assays; the first ELISpot screened 33 large peptide pools using CD8 T cells expanded for 7 days, while the second step tested pool-matrix peptides to identify individual peptides using CD8 T cells expanded for 10 days. This comprehensive epitope screening established the breadth and magnitude of HIV-1 Gag-specific CD8 T cells and further revealed the extent of immune responses to variable/polymorphic epitopes.
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Linfocitos T CD8-positivos/inmunología , Ensayo de Immunospot Ligado a Enzimas/métodos , Mapeo Epitopo , Biblioteca de Péptidos , Vacunas contra el SIDA/inmunología , Adulto , Anticuerpos Biespecíficos/inmunología , Epítopos de Linfocito T/inmunología , Femenino , VIH-1/inmunología , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Nutrient starvation is a common phenomenon that occurs during T cell activation. Upon pathogen infection, large amounts of immune cells migrate to infection sites, and antigen-specific T cells are activated; this is followed by rapid proliferation through clonal expansion. The dramatic expansion of cells will commonly lead to nutrient shortage. Cellular autophagy is often upregulated as a way to sustain the body's energy requirements. During infection, human immunodeficiency virus (HIV) co-opts a series of host cell metabolic pathways for replication. Several HIV proteins, such as Env, Nef, and Vpr, have already been reported as being involved in autophagy-related processes. In this report, we identified that the HIV p17 protein acts as a major factor in suppressing the autophagic process in T cells, especially under glucose starvation condition. HIV p17 interacts with Obg-like ATPase 1 (OLA1) and disrupts OLA1-glycogen synthase kinase-3 beta (GSK3ß) complex, leading to GSK3ß hyperactivation. Consequently, a prior proliferation of HIV-infected T cells under glucose starvation will occur. The inhibition of autophagy also aids HIV replication by antagonizing the antiviral effect of autophagy. Our study shows a new cellular pathway that HIV can hijack for viral spreading by a prior proliferation of HIV-loaded T cells and may provide new therapeutic targets for acquired immunodeficiency syndrome intervention.
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Adenosina Trifosfatasas/genética , Autofagia/genética , Proliferación Celular , Proteínas de Unión al GTP/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Antígenos VIH/genética , Antígenos VIH/metabolismo , VIH-1/inmunología , Linfocitos T/fisiología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Glucosa/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Antígenos VIH/inmunología , VIH-1/patogenicidad , Células HeLa , Interacciones Microbiota-Huesped , Humanos , Células Jurkat , Activación de Linfocitos , Inanición , Linfocitos T/inmunología , Linfocitos T/virología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor ß-chain (TCRß) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRß repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRß and integration site analysis showed that infection could occur early or late in the course of a clone's response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.
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Linfocitos T CD4-Positivos/inmunología , Selección Clonal Mediada por Antígenos , Infecciones por VIH/inmunología , VIH-1/fisiología , Integración Viral/inmunología , Latencia del Virus/inmunología , Adulto , Linfocitos T CD4-Positivos/patología , Femenino , Infecciones por VIH/terapia , Humanos , Masculino , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
HIV-1 transmission is associated with a severe bottleneck in which a limited number of variants from a pool of genetically diverse quasispecies establishes infection. The IAVI protocol C cohort of discordant couples, female sex workers, other heterosexuals and men who have sex with men (MSM) present varying risks of HIV infection, diverse HIV-1 subtypes and represent a unique opportunity to characterize transmitted/founder viruses (TF) where disease outcome is known. To identify the TF, the HIV-1 repertoire of 38 MSM participants' samples was sequenced close to transmission (median 21 days post infection, IQR 18-41) and assessment of multivariant infection done. Patient derived gag genes were cloned into an NL4.3 provirus to generate chimeric viruses which were characterized for replicative capacity (RC). Finally, an evaluation of how the TF virus predicted disease progression and modified the immune response at both acute and chronic HIV-1 infection was done. There was higher prevalence of multivariant infection compared with previously described heterosexual cohorts. A link was identified between multivariant infection and replicative capacity conferred by gag, whereby TF gag tended to be of lower replicative capacity in multivariant infection (p = 0.02) suggesting an overall lowering of fitness requirements during infection with multiple variants. Notwithstanding, multivariant infection was associated with rapid CD4+ T cell decline and perturbances in the CD4+ T cell and B cell compartments compared to single variant infection, which were reversible upon control of viremia. Strategies aimed at identifying and mitigating multivariant infection could contribute toward improving HIV-1 prognosis and this may involve strategies that tighten the stringency of the transmission bottleneck such as treatment of STI. Furthermore, the sequences and chimeric viruses help with TF based experimental vaccine immunogen design and can be used in functional assays to probe effective immune responses against TF.
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Linfocitos T CD4-Positivos/inmunología , Efecto Fundador , Infecciones por VIH , VIH-1/fisiología , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Enfermedad Aguda , Adolescente , Adulto , Linfocitos B/inmunología , Linfocitos B/patología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Femenino , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Humanos , Masculino , Persona de Mediana Edad , Viremia/genética , Viremia/inmunología , Viremia/patología , Replicación Viral/genética , Replicación Viral/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.
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Proteínas Aviares/inmunología , Virus del Sarcoma Aviar/inmunología , Antígeno 2 del Estroma de la Médula Ósea/inmunología , Evolución Molecular , Galliformes/inmunología , Sarcoma Aviar/inmunología , Secuencia de Aminoácidos , Animales , Proteínas Aviares/genética , Virus del Sarcoma Aviar/genética , Virus del Sarcoma Aviar/patogenicidad , Antígeno 2 del Estroma de la Médula Ósea/genética , Línea Celular , Fibroblastos/inmunología , Fibroblastos/virología , Galliformes/genética , Galliformes/virología , Regulación de la Expresión Génica , Células HEK293 , VIH-1/genética , VIH-1/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Passeriformes/genética , Passeriformes/inmunología , Passeriformes/virología , Sarcoma Aviar/genética , Sarcoma Aviar/virología , Selección Genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Liberación del Virus , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
HIV-1 particles incorporate various host transmembrane proteins in addition to viral Env glycoprotein during assembly at the plasma membrane. In polarized T cells, HIV-1 structural protein Gag localizes to the plasma membrane of uropod, a rear-end protrusion. Notably, uropod transmembrane proteins PSGL-1 and CD43 cocluster specifically with Gag assembling at the plasma membrane even in cells that do not form uropods. Recent reports have shown that expression of either PSGL-1 or CD43 in virus-producing cells reduces the infectivity of progeny virions and that HIV-1 infection reduces the cell surface expression of these proteins. However, the mechanisms for both processes remain to be determined. In this study, we found that virion incorporation of PSGL-1 and CD43 closely correlates with diminished virion infectivity. PSGL-1 and CD43 inhibited virus attachment to CD4+ cells irrespective of the presence of Env. These proteins also inhibited virion attachment to CD4- lymphoid organ fibroblastic reticular cells that mediate transinfection of CD4+ T cells. Consistent with the possibility that highly extended extracellular domains of these proteins physically block virus-cell attachment, the inhibitory effect of PSGL-1 required its full-length ectodomain. HIV-1 encoding Gag mutants that are defective in either coclustering with these host proteins or ESCRT-dependent particle release failed to reduce PSGL-1 on surface of infected cells. This study reveals an anti-HIV-1 mechanism that suppresses virus-cell attachment and a previously unappreciated process of HIV-1-mediated down-regulation of host antiviral proteins, both of which likely require virion incorporation of these proteins.
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Infecciones por VIH/inmunología , VIH-1/inmunología , Interacciones Huésped-Patógeno/genética , Leucosialina/genética , Glicoproteínas de Membrana/genética , Virión/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Capa Leucocitaria de la Sangre/citología , Regulación hacia Abajo , Técnicas de Inactivación de Genes , Células HEK293 , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Voluntarios Sanos , Interacciones Huésped-Patógeno/inmunología , Humanos , Mutación , Dominios Proteicos/genética , Linfocitos T/inmunología , Ensamble de Virus/genética , Ensamble de Virus/inmunología , Acoplamiento Viral , Replicación Viral/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Pregnancy induces alterations in peripheral T-cell populations with both changes in subset frequencies and anti-viral responses found to alter with gestation. In HIV-1 positive women anti-HIV-1 responses are associated with transmission risk, however detailed investigation into both HIV-1-specific memory responses associated with HIV-1 control and T-cell subset changes during pregnancy have not been undertaken. In this study we aimed to define pregnancy and gestation related changes to HIV-1-specific responses and T-cell phenotype in ART treated HIV-1 positive pregnant women. Eleven non-pregnant and 24 pregnant HIV-1 positive women were recruited, peripheral blood samples taken, fresh cells isolated, and compared using ELISpot assays and flow cytometry analysis. Clinical data were collected as part of standard care, and non-parametric statistics used. Alterations in induced IFNγ, IL-2, IL-10, and granzyme B secretion by peripheral blood mononuclear cells in response to HIV-1 Gag and Nef peptide pools and changes in T-cell subsets between pregnant and non-pregnant women were assessed, with data correlated with participant clinical parameters and longitudinal analysis performed. Cross-sectional comparison identified decreased IL-10 Nef response in HIV-1 positive pregnant women compared to non-pregnant, while correlations exhibited reversed Gag and Nef cytokine and protease response associations between groups. Longitudinal analysis of pregnant participants demonstrated transient increases in Gag granzyme B response and in the central memory CD4 T-cell subset frequency during their second trimester, with a decrease in CD4 effector memory T cells from their second to third trimester. Gag and Nef HIV-1-specific responses diverge with pregnancy time-point, coinciding with relevant T-cell phenotype, and gestation associated immunological adaptations. Decreased IL-10 Nef and both increased granzyme B Gag response and central memory CD4 T cells implies that amplified antigen production is occurring, which suggests a period of compromised HIV-1 control in pregnancy.