RESUMO
Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.
Assuntos
Infecções por HIV , HIV-1 , Animais , Linfócitos T CD8-Positivos , Linfócitos T CD4-Positivos , Latência Viral , Anticorpos Anti-HIVRESUMO
The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.
Assuntos
Soropositividade para HIV , Vacinas , Humanos , Linfócitos B , Anticorpos Neutralizantes , Linhagem Celular , Células ClonaisRESUMO
Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1-3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml-1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.
Assuntos
Infecções por HIV , HIV-1 , Adulto , Anticorpos Neutralizantes , Anticorpos Amplamente Neutralizantes , Dependovirus/genética , Anticorpos Anti-HIV , Infecções por HIV/tratamento farmacológico , HumanosRESUMO
Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here, a trispecific bNAb reduces viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounds transiently and returns to low levels, through CD8-mediated immune control. These viruses remain sensitive to the trispecific antibody, despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppresses the emergence of resistance in viruses derived from HIV-1-infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 neutralizing antibodies, therefore, mediate potent antiviral activity in vivo and may minimize the potential for immune escape.
Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/uso terapêutico , Anticorpos Anti-HIV/uso terapêutico , Evasão da Resposta Imune/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Vírus da Imunodeficiência Símia/imunologia , Animais , Antivirais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Humanos , Imunoterapia/métodos , Macaca mulatta , Células THP-1 , Viremia/prevenção & controle , Viremia/terapiaRESUMO
A population at low census might go extinct or instead transition into exponential growth to become firmly established. Whether this pivotal event occurs for a within-host pathogen can be the difference between health and illness. Here, we define the principles governing whether HIV-1 spread among cells fails or becomes established by coupling stochastic modeling with laboratory experiments. Following ex vivo activation of latently infected CD4 T cells without de novo infection, stochastic cell division and death contributes to high variability in the magnitude of initial virus release. Transition to exponential HIV-1 spread often fails due to release of an insufficient amount of replication-competent virus. Establishment of exponential growth occurs when virus produced from multiple infected cells exceeds a critical population size. We quantitatively define the crucial transition to exponential viral spread. Thwarting this process would prevent HIV transmission or rebound from the latent reservoir.
Assuntos
Infecções por HIV/epidemiologia , HIV-1 , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/transmissão , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Humanos , Modelos Biológicos , Dinâmica Populacional/estatística & dados numéricos , Carga Viral , Ativação Viral , Latência Viral , Replicação ViralRESUMO
Cytolytic CD8 T cells play a crucial role in the control and elimination of virus-infected cells and are a major focus of HIV cure efforts. However, it has been shown that HIV-specific CD8 T cells are infrequently found within germinal centers (GCs), a predominant site of active and latent HIV infection. We demonstrate that HIV infection induces marked changes in the phenotype, frequency, and localization of CD8 T cells within the lymph node (LN). Significantly increased frequencies of CD8 T cells in the B cell follicles and GCs were found in LNs from treated and untreated HIV-infected individuals. This profile was associated with persistent local immune activation but did not appear to be directly related to local viral replication. Follicular CD8 (fCD8) T cells, despite compromised cytokine polyfunctionality, showed good cytolytic potential characterized by high ex vivo expression of granzyme B and perforin. We used an anti-HIV/anti-CD3 bispecific antibody in a redirected killing assay and found that fCD8 T cells had better killing activity than did non-fCD8 T cells. Our results indicate that CD8 T cells with potent cytolytic activity are recruited to GCs during HIV infection and, if appropriately redirected to kill HIV-infected cells, could be an effective component of an HIV cure strategy.
Assuntos
Anticorpos Biespecíficos/imunologia , Linfócitos T CD8-Positivos/citologia , Granzimas/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Adulto , Idoso , Citocinas/imunologia , Feminino , Humanos , Linfonodos/citologia , Masculino , Pessoa de Meia-Idade , Tonsila Palatina/citologia , Perforina/imunologia , FenótipoRESUMO
Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.
Assuntos
Vacinas contra a AIDS/imunologia , Adenoviridae/genética , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunização Secundária/métodos , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Adenoviridae/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos , Feminino , Anticorpos Anti-HIV/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Vacinas de DNA/administração & dosagemRESUMO
Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Sangue/virologia , Linfócitos T CD4-Positivos/imunologia , Doença Crônica , DNA Viral/genética , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Leucócitos Mononucleares , Linfonodos/virologia , Provírus/imunologia , Análise de Sequência de DNA , Fenômenos Fisiológicos Virais , Replicação ViralRESUMO
Passive immunization with HIV-1-neutralizing monoclonal antibodies (mAbs) is being considered for prevention and treatment of HIV-1 infection. As therapeutic agents, mAbs could be used to suppress active virus replication, maintain suppression induced by antiretroviral therapy (ART), and/or decrease the size of the persistent virus reservoir. We assessed the impact of VRC01, a potent human mAb targeting the HIV-1 CD4 binding site, on ART-treated and untreated HIV-1-infected subjects. Among six ART-treated individuals with undetectable plasma viremia, two infusions of VRC01 did not reduce the peripheral blood cell-associated virus reservoir measured 4 weeks after the second infusion. In contrast, six of eight ART-untreated, viremic subjects infused with a single dose of VRC01 experienced a 1.1 to 1.8 log10 reduction in plasma viremia. The two subjects with minimal responses to VRC01 were found to have predominantly VRC01-resistant virus before treatment. Notably, two subjects with plasma virus load <1000 copies/ml demonstrated virus suppression to undetectable levels for over 20 days until VRC01 levels declined. Among the remaining four subjects with baseline virus loads between 3000 and 30,000 copies, viremia was only partially suppressed by mAb infusion, and we observed strong selection pressure for the outgrowth of less neutralization-sensitive viruses. In summary, a single infusion of mAb VRC01 significantly decreased plasma viremia and preferentially suppressed neutralization-sensitive virus strains. These data demonstrate the virological effect of this neutralizing antibody and highlight the need for combination strategies to maintain virus suppression.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Doença Crônica , Anticorpos Anti-HIV/sangue , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Humanos , Cinética , Pessoa de Meia-Idade , Carga Viral/imunologia , Adulto JovemRESUMO
The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127high CD4 T cells within the early/intermediate differentiated (EI) (CD27highCD45RAlow) T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load). A significant loss of circulating PD-1highCTLA-4low CD4 T cells was found specifically in the CD127highCD27highCD45RAlow compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1high compared to PD-1low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1low EI CD4 T cells. In line with our previous findings, PD-1high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Infecções por HIV/fisiopatologia , Receptor de Morte Celular Programada 1/fisiologia , Fármacos Anti-HIV/uso terapêutico , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/efeitos dos fármacos , Citocinas/biossíntese , Progressão da Doença , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1 , Humanos , Reação em Cadeia da Polimerase , Receptor de Morte Celular Programada 1/imunologia , Receptores CCR5/metabolismo , Carga ViralRESUMO
The treatment of AIDS with combination antiretroviral therapy (cART) remains lifelong largely because the virus persists in latent reservoirs. Elimination of latently infected cells could therefore reduce treatment duration and facilitate immune reconstitution. Here we report an approach to reduce the viral reservoir by activating dormant viral gene expression and directing T lymphocytes to lyse previously latent, HIV-1-infected cells. An immunomodulatory protein was created that combines the specificity of a HIV-1 broadly neutralizing antibody with that of an antibody to the CD3 component of the T-cell receptor. CD3 engagement by the protein can stimulate T-cell activation that induces proviral gene expression in latently infected T cells. It further stimulates CD8 T-cell effector function and redirects T cells to lyse these previously latent-infected cells through recognition of newly expressed Env. This immunomodulatory protein could potentially help to eliminate latently infected cells and deplete the viral reservoir in HIV-1-infected individuals.
Assuntos
Anticorpos Neutralizantes/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Animais , Sequência de Bases , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Células HEK293 , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Humanos , Ativação Linfocitária/imunologia , Macaca mulatta , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
BACKGROUND: Achievement of a cure for HIV infection might need reactivation of latent virus and improvement of HIV-specific immunity. As an initial step, in this trial we assessed the effect of antiretroviral therapy intensification and immune modulation with a DNA prime and recombinant adenovirus 5 (rAd5) boost vaccine. METHODS: In this multicentre, randomised, open-label, non-comparative, phase 2 clinical trial, we enrolled eligible adults 18-70 years of age with chronic HIV-1 infection on suppressive antiretroviral therapy with current CD4 count of at least 350 cells per µL and HIV DNA between 10 and 1000 copies per 10(6) peripheral blood mononuclear cells. After an 8 week lead-in of antiretroviral intensification therapy (standard dose raltegravir and dose-adjusted maraviroc based on baseline antiretroviral therapy), patients were randomly assigned (1:1) to receive antiretroviral therapy intensification alone or intensification plus injections of HIV DNA prime vaccine (4 mg VRC-HIVDNA016-00-VP) at weeks 8, 12, and 16, followed by HIV rAd5 boost vaccine (10(10) particle units of VRC-HIVADV014-00-VP) at week 32. Randomisation was computer generated in permuted blocks of six and was stratified by study site. The primary endpoint was a 0·5 log10 or greater decrease in HIV DNA in peripheral blood mononuclear cells at week 56. This study is registered with ClinicalTrials.gov, number NCT00976404. FINDINGS: Between Nov 29, 2010, and Oct 28, 2011, we enrolled 28 eligible patients from three academic HIV clinics in the USA. After the 8 week lead-in of antiretroviral intensification therapy, 14 patients were randomly assigned to continue antiretroviral therapy intensification alone and 14 to intensification plus vaccine. Enrolled participants had median CD4 count of 636 cells per µL, median HIV DNA 170 copies per 10(6) peripheral blood mononuclear cells, and duration of antiretroviral therapy of 13 years. The median amount of HIV DNA did not change significantly between baseline and week 56 in the antiretroviral therapy intensification plus vaccine group. One participant in the antiretroviral therapy intensification alone group reached the primary endpoint, with 0·55 log10 decrease in HIV DNA in peripheral blood mononuclear cells. Both treatments were well tolerated. No severe or systemic reactions to vaccination occurred, and five serious adverse events were recorded during the study, most of which resolved spontaneously or were judged unrelated to study treatments. INTERPRETATION: Antiretroviral therapy intensification followed by DNA prime and rAd5 boost vaccine did not significantly increase HIV expression or reduce the latent HIV reservoir. A multifaceted approach that includes stronger activators of HIV expression and novel immune modulators will probably be needed to reduce the latent HIV reservoir and allow for long-term control in patients off antiretroviral therapy. FUNDING: Objectif Recherche Vaccin SIDA (ORVACS).
Assuntos
Vacinas contra a AIDS/administração & dosagem , Adenoviridae/imunologia , Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/imunologia , Vacinas contra a AIDS/imunologia , Adenoviridae/genética , Adolescente , Adulto , Idoso , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Humanos , Imunização Secundária , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Carga Viral , Adulto JovemRESUMO
The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Linfócitos B/imunologia , Antígenos CD4/metabolismo , Regiões Determinantes de Complementaridade , Epitopos de Linfócito B , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Persistent cytomegalovirus (CMV) infection has been suggested to be a major driving force in the immune deterioration and an underlying source of age-related diseases in the elderly. CMV antibody titers are associated with lower responses to vaccination, cardiovascular diseases, frailty, and mortality. CMV infection is also associated with shorter T-cell telomeres and replicative senescence. Although an age-related deregulation of CMV-specific T-cell responses could be an underlying cause of the relationship between CMV and immune defects, strong and polyfunctional responses are observed in elderly individuals, casting uncertainty on their direct role in age-related immune frailty. In this study, we longitudinally followed a cohort of healthy donors aged over 50 years, assessing their mortality rates and time to death during a 2-year period. Specific T-cell responses to the immunodominant antigen pp65 (IFNγ, TNFα, IL2, MIP1α, CD107a, and perforin production) were analyzed at the beginning of the 2-year observation period. A cytotoxic CD8 pp65-specific T-cell response, without cytokine or chemokine coexpression, was independently associated with all-cause mortality in these elderly individuals. This pp65-specific CD8 T-cell response could be a useful tool to identify individuals with depressed immune function and a higher risk of death.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/mortalidade , Interferon gama/imunologia , Interleucina-2/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Perforina/imunologia , Fosfoproteínas/imunologia , Fator de Necrose Tumoral alfa/imunologia , Proteínas Supressoras de Tumor/imunologia , Proteínas da Matriz Viral/imunologia , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/imunologia , Causas de Morte , Proteínas do Citoesqueleto , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Fatores de Risco , Análise de SobrevidaRESUMO
Next-generation sequencing of antibody transcripts provides a wealth of data, but the ability to identify function-specific antibodies solely on the basis of sequence has remained elusive. We previously characterized the VRC01 class of antibodies, which target the CD4-binding site on gp120, appear in multiple donors, and broadly neutralize HIV-1. Antibodies of this class have developmental commonalities, but typically share only â¼50% amino acid sequence identity among different donors. Here we apply next-generation sequencing to identify VRC01 class antibodies in a new donor, C38, directly from B cell transcript sequences. We first tested a lineage rank approach, but this was unsuccessful, likely because VRC01 class antibody sequences were not highly prevalent in this donor. We next identified VRC01 class heavy chains through a phylogenetic analysis that included thousands of sequences from C38 and a few known VRC01 class sequences from other donors. This "cross-donor analysis" yielded heavy chains with little sequence homology to previously identified VRC01 class heavy chains. Nonetheless, when reconstituted with the light chain from VRC01, half of the heavy chain chimeric antibodies showed substantial neutralization potency and breadth. We then identified VRC01 class light chains through a five-amino-acid sequence motif necessary for VRC01 light chain recognition. From over a million light chain sequences, we identified 13 candidate VRC01 class members. Pairing of these light chains with the phylogenetically identified C38 heavy chains yielded functional antibodies that effectively neutralized HIV-1. Bioinformatics analysis can thus directly identify functional HIV-1-neutralizing antibodies of the VRC01 class from a sequenced antibody repertoire.
Assuntos
Anticorpos Neutralizantes/genética , Linfócitos B/metabolismo , HIV-1/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Sequência de Bases , Variação Genética , Humanos , Cadeias Pesadas de Imunoglobulinas/classificação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/classificação , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
Elite controllers suppress human immunodeficiency virus (HIV) viremia to below the limit of detection in the absence of antiretroviral therapy (ART). However, precise frequencies of CD4(+) T cells carrying replication-competent HIV and/or the dynamics of the infectious viral reservoirs in response to initiation and discontinuation of ART in elite controllers are unknown. We show that the size of the pool of CD4(+) T cells harboring infectious HIV diminished significantly after initiation of ART and rebounded to baseline upon cessation of therapy. Our data provide compelling evidence that persistent viral replication occurs in untreated elite controllers even in the absence of detectable plasma viremia.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Viremia/tratamento farmacológico , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Doenças Assintomáticas , Relação CD4-CD8 , Linfócitos T CD4-Positivos/virologia , DNA Viral/sangue , DNA Viral/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Quimioterapia Combinada , Emtricitabina , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Imunidade Inata , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Pirrolidinonas/farmacologia , Pirrolidinonas/uso terapêutico , Raltegravir Potássico , Tenofovir , Viremia/imunologia , Replicação ViralRESUMO
Genetic recombination contributes to the diversity of human immunodeficiency virus (HIV-1). Productive HIV-1 recombination is, however, dependent on both the number of HIV-1 genomes per infected cell and the genetic relationship between these viral genomes. A detailed analysis of the number of proviruses and their genetic relationship in infected cells isolated from peripheral blood and tissue compartments is therefore important for understanding HIV-1 recombination, genetic diversity and the dynamics of HIV-1 infection. To address these issues, we used a previously developed single-cell sequencing technique to quantify and genetically characterize individual HIV-1 DNA molecules from single cells in lymph node tissue and peripheral blood. Analysis of memory and naïve CD4(+) T cells from paired lymph node and peripheral blood samples from five untreated chronically infected patients revealed that the majority of these HIV-1-infected cells (>90%) contain only one copy of HIV-1 DNA, implying a limited potential for productive recombination in virus produced by these cells in these two compartments. Phylogenetic analysis revealed genetic similarity of HIV-1 DNA in memory and naïve CD4(+) T-cells from lymph node, peripheral blood and HIV-1 RNA from plasma, implying exchange of virus and/or infected cells between these compartments in untreated chronic infection.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , DNA Viral/metabolismo , Genoma Viral , Infecções por HIV/metabolismo , HIV-1/metabolismo , Linfonodos/metabolismo , Adulto , Linfócitos T CD4-Positivos/virologia , Doença Crônica , DNA Viral/genética , Infecções por HIV/genética , HIV-1/genética , Humanos , Linfonodos/virologia , Masculino , FilogeniaRESUMO
BACKGROUND: Correlates of immune protection in patients with human immunodeficiency virus (HIV)-associated cryptococcal meningitis are poorly defined. A clearer understanding of these immune responses is essential to inform rational development of immunotherapies. METHODS: Cryptococcal-specific peripheral CD4(+) T-cell responses were measured in 44 patients with HIV-associated cryptococcal meningitis at baseline and during follow-up. Responses were assessed following ex vivo cryptococcal mannoprotein stimulation, using 13-color flow-cytometry. The relationships between cryptococcal-specific CD4(+) T-cell responses, clinical parameters at presentation, and outcome were investigated. RESULTS: Cryptococcal-specific CD4(+) T-cell responses were characterized by the production of macrophage inflammatory protein 1α, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α). Conversely, minimal interleukin 4 and interleukin 17 production was detected. Patients surviving to 2 weeks had significantly different functional CD4(+) T-cell responses as compared to those who died. Patients with a response predominantly consisting of IFN-γ or TNF-α production had a 2-week mortality of 0% (0/20), compared with 25% (6/24) in those without this response (P = .025). Such patients also had lower fungal burdens (10 400 vs 390 000 colony-forming units/mL; P < .001), higher cerebrospinal fluid lymphocyte counts (122 vs 8 cells/µL; P < .001), and a trend toward faster rates of clearance of infection. CONCLUSIONS: The phenotype of the peripheral CD4(+) T-cell response to Cryptococcus was associated with disease severity and outcome in HIV-associated cryptococcal meningitis. IFN-γ/TNF-α-predominant responses were associated with survival.
Assuntos
Antígenos de Fungos/imunologia , Linfócitos T CD4-Positivos/imunologia , Cryptococcus/imunologia , Infecções por HIV/imunologia , HIV/imunologia , Meningite Criptocócica/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Estudos de Coortes , Cryptococcus/classificação , Citocinas/líquido cefalorraquidiano , Feminino , Seguimentos , Infecções por HIV/complicações , Infecções por HIV/virologia , Humanos , Interferon gama/líquido cefalorraquidiano , Masculino , Meningite Criptocócica/complicações , Meningite Criptocócica/microbiologia , Meningite Criptocócica/mortalidade , Fenótipo , Índice de Gravidade de Doença , África do Sul/epidemiologia , Fator de Necrose Tumoral alfa/líquido cefalorraquidiano , Carga ViralRESUMO
BACKGROUND: The licensing of herpes zoster vaccine has demonstrated that therapeutic vaccination can help control chronic viral infection. Unfortunately, human trials of immunodeficiency virus (HIV) vaccine have shown only marginal efficacy. METHODS: In this double-blind study, 17 HIV-infected individuals with viral loads of <50 copies/mL and CD4(+) T-cell counts of >350 cells/µL were randomly assigned to the vaccine or placebo arm. Vaccine recipients received 3 intramuscular injections of HIV DNA (4 mg) coding for clade B Gag, Pol, and Nef and clade A, B, and C Env, followed by a replication-deficient adenovirus type 5 boost (10(10) particle units) encoding all DNA vaccine antigens except Nef. Humoral, total T-cell, and CD8(+) cytotoxic T-lymphocyte (CTL) responses were studied before and after vaccination. Single-copy viral loads and frequencies of latently infected CD4(+) T cells were determined. RESULTS: Vaccination was safe and well tolerated. Significantly stronger HIV-specific T-cell responses against Gag, Pol, and Env, with increased polyfunctionality and a broadened epitope-specific CTL repertoire, were observed after vaccination. No changes in single-copy viral load or the frequency of latent infection were observed. CONCLUSIONS: Vaccination of individuals with existing HIV-specific immunity improved the magnitude, breadth, and polyfunctionality of HIV-specific memory T-cell responses but did not impact markers of viral control. CLINICAL TRIALS REGISTRATION: NCT00270465.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Método Duplo-Cego , Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Seguimentos , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Imunidade Humoral , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes , Linfócitos T Citotóxicos/imunologia , Vacinação , Carga Viral , Latência ViralRESUMO
A small fraction of HIV-infected individuals (<1%), referred to as elite controllers (EC), are able to maintain undetectable viral loads indefinitely without treatment. The role of the maturational phenotype of T cells in the control of HIV infection in these individuals is not well described. We compared the maturational and functional phenotypes of Gag-specific CD4 and CD8 T cells from EC, who maintain undetectable viral loads without treatment; relative controllers (RC), who maintain viral loads of <1,000 copies/ml without treatment; and noncontrollers (NC), who fail to control viral replication. EC maintained higher frequencies of HIV-specific CD4 T cells, less mature polyfunctional Gag-specific CD4 T cells (CD27(+) CD57(-) CD45RO(+)), and Gag-specific polyfunctional CD4 T cells than those observed in NC. In EC, the frequency of polyfunctional Gag-specific CD8 T cells was higher than that observed in RC and NC. RC had a similar functional phenotype to that observed in NC, despite consistently lower viral loads. Finally, we found a direct correlation between the frequency of Gag-specific CD27(+) CD57(-) CD45RO(+) CD4(+) T cells and the frequency of mature HIV-specific CD8 T cells. Altogether, our data suggest that immature Gag-specific interleukin-2 (IL-2)-producing CD4(+) T cells may play an important role in spontaneous control of HIV viremia by effectively supporting HIV-specific CD8 T lymphocytes. This difference appears to differentiate EC from RC.