RESUMO
BACKGROUND: Efficacious vaccines are urgently needed to contain the ongoing coronavirus disease 2019 (Covid-19) pandemic of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A candidate vaccine, Ad26.COV2.S, is a recombinant, replication-incompetent adenovirus serotype 26 (Ad26) vector encoding a full-length and stabilized SARS-CoV-2 spike protein. METHODS: In this multicenter, placebo-controlled, phase 1-2a trial, we randomly assigned healthy adults between the ages of 18 and 55 years (cohort 1) and those 65 years of age or older (cohort 3) to receive the Ad26.COV2.S vaccine at a dose of 5×1010 viral particles (low dose) or 1×1011 viral particles (high dose) per milliliter or placebo in a single-dose or two-dose schedule. Longer-term data comparing a single-dose regimen with a two-dose regimen are being collected in cohort 2; those results are not reported here. The primary end points were the safety and reactogenicity of each dose schedule. RESULTS: After the administration of the first vaccine dose in 805 participants in cohorts 1 and 3 and after the second dose in cohort 1, the most frequent solicited adverse events were fatigue, headache, myalgia, and injection-site pain. The most frequent systemic adverse event was fever. Systemic adverse events were less common in cohort 3 than in cohort 1 and in those who received the low vaccine dose than in those who received the high dose. Reactogenicity was lower after the second dose. Neutralizing-antibody titers against wild-type virus were detected in 90% or more of all participants on day 29 after the first vaccine dose (geometric mean titer [GMT], 212 to 354), regardless of vaccine dose or age group, and reached 96% by day 57 with a further increase in titers (GMT, 288 to 488) in cohort 1a. Titers remained stable until at least day 71. A second dose provided an increase in the titer by a factor of 2.6 to 2.9 (GMT, 827 to 1266). Spike-binding antibody responses were similar to neutralizing-antibody responses. On day 15, CD4+ T-cell responses were detected in 76 to 83% of the participants in cohort 1 and in 60 to 67% of those in cohort 3, with a clear skewing toward type 1 helper T cells. CD8+ T-cell responses were robust overall but lower in cohort 3. CONCLUSIONS: The safety and immunogenicity profiles of Ad26.COV2.S support further development of this vaccine candidate. (Funded by Johnson & Johnson and the Biomedical Advanced Research and Development Authority of the Department of Health and Human Services; COV1001 ClinicalTrials.gov number, NCT04436276.).
Assuntos
Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Imunogenicidade da Vacina , SARS-CoV-2/imunologia , Ad26COVS1 , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/efeitos adversos , Estudos de Coortes , Método Duplo-Cego , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/founder (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro, while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.
Assuntos
Aptidão Genética , Infecções por HIV/diagnóstico , HIV-1/genética , HIV-1/imunologia , Interações Hospedeiro-Patógeno/imunologia , Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Evolução Molecular , Feminino , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/transmissão , Interações Hospedeiro-Patógeno/genética , Humanos , Evasão da Resposta Imune/genética , Masculino , Dados de Sequência Molecular , Filogenia , Prognóstico , Carga Viral/genética , Replicação Viral/genéticaRESUMO
Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Evasão da Resposta Imune/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Reações Cruzadas/imunologia , Feminino , Infecções por HIV/virologia , Soropositividade para HIV/imunologia , Soropositividade para HIV/virologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Masculino , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Two human leukocyte antigen (HLA) variants, HLA-B*57 and -B*81, are consistently known as favorable host factors in human immunodeficiency virus type 1 (HIV-1)-infected Africans and African-Americans. In our analyses of prospective data from 538 recent HIV-1 seroconverters and cross-sectional data from 292 subjects with unknown duration of infection, HLA-B*57 (mostly B*57:03) and -B*81 (exclusively B*81:01) had mostly discordant associations with virologic and immunologic manifestations before antiretroviral therapy. Specifically, relatively low viral load (VL) in HLA-B*57-positive subjects (P ≤ 0.03 in various models) did not translate to early advantage in CD4(+) T-cell (CD4) counts (P ≥ 0.37). In contrast, individuals with HLA-B*81 showed little deviation from the normal set point VL (P > 0.18) while maintaining high CD4 count during early and chronic infection (P = 0.01). These observations suggest that discordance between VL and CD4 count can occur in the presence of certain HLA alleles and that effective control of HIV-1 viremia is not always a prerequisite for favorable prognosis (delayed immunodeficiency). Of note, steady CD4 count associated with HLA-B*81 in HIV-1-infected Africans may depend on the country of origin, as observations differed slightly between subgroups enrolled in southern Africa (Zambia) and eastern Africa (Kenya, Rwanda, and Uganda).
Assuntos
Infecções por HIV/imunologia , HIV-1 , Antígenos HLA-B/metabolismo , Fatores Celulares Derivados do Hospedeiro/metabolismo , Carga Viral/imunologia , África Oriental , Análise de Variância , População Negra , Linfócitos T CD4-Positivos/imunologia , Contagem de Células , Estudos Transversais , Humanos , Imunofenotipagem , Estudos Prospectivos , Análise de Sequência de DNA , ZâmbiaRESUMO
As part of an ongoing study of early human immunodeficiency virus type 1 (HIV-1) infection in sub-Saharan African countries, we have identified 134 seroconverters (SCs) with distinct acute-phase (peak) and early chronic-phase (set-point) viremias. SCs with class I human leukocyte antigen (HLA) variants B*44 and B*57 had much lower peak viral loads (VLs) than SCs without these variants (adjusted linear regression beta values of -1.08 ± 0.26 log(10) [mean ± standard error] and -0.83 ± 0.27 log(10), respectively; P < 0.005 for both), after accounting for several nongenetic factors, including gender, age at estimated date of infection, duration of infection, and country of origin. These findings were confirmed by alternative models in which major viral subtypes (A1, C, and others) in the same SCs replaced country of origin as a covariate (P ≤ 0.03). Both B*44 and B*57 were also highly favorable (P ≤ 0.03) in analyses of set-point VLs. Moreover, B*44 was associated with relatively high CD4(+) T-cell counts during early chronic infection (P = 0.02). Thus, at least two common HLA-B variants showed strong influences on acute-phase as well as early chronic-phase VL, regardless of the infecting viral subtype. If confirmed, the identification of B*44 as another favorable marker in primary HIV-1 infection should help dissect mechanisms of early immune protection against HIV-1 infection.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Antígenos HLA-B/imunologia , Carga Viral , Adulto , África Subsaariana , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/virologia , HIV-1/patogenicidade , Antígenos HLA-B/genética , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-IdadeRESUMO
A Sendai virus (SeV) vector is being developed for delivery of an HIV immunogen. SeV is not known to cause disease in humans. Because it is genetically and antigenically related to human parainfluenza virus type 1 (hPIV-1), it is important to determine whether pre-existing hPIV-1 antibodies will affect immune responses elicited by a SeV vector-based vaccine. To quantify SeV neutralizing antibodies (NAb) in human serum, a sensitive virus neutralization assay was developed using a SeV vector encoding green fluorescent protein. Samples from 255 HIV-uninfected subjects from Africa, Europe, United States, and Japan, as well as from 12 confirmed hPIV-1-infected patients, were analyzed. SeV NAb titers did not vary significantly after serum was treated with receptor-destroying enzyme, indicating that non-specific hemagglutination inhibitors did not affect the assay sensitivity. A significant correlation was observed between hPIV-1 ELISA and SeV NAb titers. SeV NAb were detected in 92.5% subjects with a median titer of 60.6 and values ranging from 5.9- 11,324. The majority had titers < 1000 with 71.7% < 100 (< 5 considered negative). There was no significant difference in titer or prevalence by gender, age range or geographic origin. However, African males had a lower titer than non-Africans of either gender (p=0.007). Overall, the prevalence of SeV NAb is high and likely due to neutralization by cross-reactive hPIV-1 antibodies. Clinical trials will be needed to assess the influence of pre-existing SeV NAb on HIV-specific immune responses elicited by a SeV vaccine vector expressing HIV.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus Sendai/imunologia , Adolescente , Adulto , África , Reações Cruzadas , Europa (Continente) , Feminino , Vetores Genéticos , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Vírus da Parainfluenza 1 Humana/imunologia , Vírus Sendai/genética , Estados UnidosRESUMO
Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81(+) cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis.
Assuntos
Hepacivirus/fisiologia , Proteínas de Membrana/fisiologia , Receptores Virais/fisiologia , Internalização do Vírus , Linhagem Celular , Claudina-1 , Claudinas , Células Endoteliais/virologia , Inativação Gênica , Hepatócitos/virologia , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses. METHODS: In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01E or F4/AS01B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01B; or three co-administrations of Ad35-GRIN and F4/AS01B. T cell and antibody responses were measured. RESULTS: The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration. CONCLUSION: Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses. TRIAL REGISTRATION: ClinicalTrials.gov NCT01264445.
Assuntos
Vacinas contra a AIDS/imunologia , População Negra , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Voluntários Saudáveis , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/efeitos adversos , Adenoviridae/genética , Adenoviridae/imunologia , Adjuvantes Imunológicos , Adolescente , Adulto , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Imunidade Celular , Imunidade Humoral , Interferon gama/biossíntese , Interferon gama/sangue , Masculino , Proteínas Recombinantes de Fusão/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinação , Adulto JovemRESUMO
Emergence of SIV and HIV specific CD8 T cells has been shown to correlate with control of in vivo replication. Poor correlation between IFN-γ ELISPOT responses and in vivo control of the virus has triggered the development of more relevant assays to assess functional HIV-1 specific CD8 T-cell responses for the evaluation and prioritization of new HIV-1 vaccine candidates. We previously established a viral inhibition assay (VIA) that measures the ability of vaccine-induced CD8 T-cell responses to inhibit viral replication in autologous CD4 T cells. In this assay, viral replication is determined by measuring p24 in the culture supernatant. Here we describe the development of a novel VIA, referred to as IMC LucR VIA that exploits replication-competent HIV-1 infectious molecular clones (IMCs) in which the complete proviral genome is strain-specific and which express the Renilla luciferase (LucR) gene to determine viral growth and inhibition. The introduction of the luciferase readout does provide significant improvement of the read out time. In addition to switching to the LucR read out, changes made to the overall protocol resulted in the miniaturization of the assay from a 48 to a 96-well plate format, which preserved sample and allowed for the introduction of replicates. The overall assay time was reduced from 13 to 8 days. The assay has a high degree of specificity, and the previously observed non-specific background inhibition in cells from HIV-1 negative volunteers has been reduced dramatically. Importantly, we observed an increase in positive responses, indicating an improvement in sensitivity compared to the original VIA. Currently, only a limited number of "whole-genome" IMC-LucR viruses are available and our efforts will focus on expanding the panel to better evaluate anti-viral breadth. Overall, we believe the IMC LucR VIA provides a platform to assess functional CD8 T-cell responses in large-scale clinical trial testing, which will enhance the ability to select the most promising HIV-1 vaccine candidates capable of controlling HIV-1 replication in vivo.
Assuntos
Vacinas contra a AIDS/uso terapêutico , Linfócitos T CD8-Positivos/virologia , Genes Reporter , Infecções por HIV/terapia , HIV-1/crescimento & desenvolvimento , Ensaios de Triagem em Larga Escala/normas , Luciferases de Renilla/biossíntese , Replicação Viral , Automação Laboratorial/normas , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Fidelidade a Diretrizes/normas , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Luciferases de Renilla/genética , Miniaturização/normas , Variações Dependentes do Observador , Guias de Prática Clínica como Assunto/normas , Valor Preditivo dos Testes , Controle de Qualidade , Reprodutibilidade dos Testes , Fatores de Tempo , Transfecção , Resultado do TratamentoRESUMO
In HIV-1 infection, plasma viral load (VL) has dual implications for pathogenesis and public health. Based on well-known patterns of HIV-1 evolution and immune escape, we hypothesized that VL is an evolving quantitative trait that depends heavily on duration of infection (DOI), demographic features, human leukocyte antigen (HLA) genotypes and viral characteristics. Prospective data from 421 African seroconverters with at least four eligible visits did show relatively steady VL beyond 3 months of untreated infection, but host and viral factors independently associated with cross-sectional and longitudinal VL often varied by analytical approaches and sliding time windows. Specifically, the effects of age, HLA-B(â)53 and infecting HIV-1 subtypes (A1, C and others) on VL were either sporadic or highly sensitive to time windows. These observations were strengthened by the addition of 111 seroconverters with 2-3 eligible VL results, suggesting that DOI should be a critical parameter in epidemiological and clinical studies.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Viremia/virologia , Adulto , África/epidemiologia , Anticorpos Antivirais/sangue , População Negra , Estudos Transversais , Infecções por HIV/epidemiologia , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/genética , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Estudos Longitudinais , Masculino , Carga Viral , Viremia/epidemiologia , Viremia/genética , Viremia/imunologiaRESUMO
OBJECTIVE: To describe immunologic, virologic, and clinical HIV disease progression by HIV-1 subtype among Africans with well documented estimated dates of HIV infection (EDIs). DESIGN: Prospective cohort. METHODS: Adults and youth with documented HIV-1 infection in the past 12 months were recruited from seroincidence cohorts in East and Southern Africa and followed at 3-6 month intervals. Blood for lymphocyte subset and viral load determination was collected at each visit. Pol was sequenced from the first positive specimen to ascertain subtype. Preantiretroviral therapy disease progression was measured by three time-to-event endpoints: CD4 cell count 350âcells/µl or less, viral load measurement at least 1â×â10âcopies/ml, and clinical AIDS. RESULTS: From 2006 to 2011, 615 participants were enrolled at nine research centers in Kenya, Rwanda, South Africa, Uganda, and Zambia; 579 (94.1%) had viral subtyping completed. Predominant subtypes were C (256, 44.2%), A (209, 36.1%), and D (84, 14.5%). After adjustment for age, sex, and human leukocyte antigen alleles in Cox regression analyses, subtype C-infected participants progressed faster than subtype A to all three endpoints [CD4 hazard ratio 1.60, 95% (confidence interval) CI 1.16, 2.20; viral load hazard ratio 1.59, 95% CI 1.12, 2.25; and AIDS hazard ratio 1.60, 95% CI 1.11, 2.31). Subtype D-infected participants reached high viral load more rapidly (hazard ratio 1.61, 95% CI 1.01, 2.57) and progressed nearly twice as fast to AIDS compared to subtype A (hazard ratio 1.93, 95% CI 1.21, 3.09). CONCLUSION: Subtype-specific differences in HIV disease progression suggest that the local subtype distribution be considered when planning HIV programs and designing and defining clinical endpoints for HIV prevention trials.
Assuntos
Progressão da Doença , Infecções por HIV/epidemiologia , Infecções por HIV/patologia , HIV-1/isolamento & purificação , Adolescente , Adulto , África Subsaariana/epidemiologia , Contagem de Linfócito CD4 , Estudos de Coortes , Feminino , Infecções por HIV/virologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Carga Viral , Adulto JovemRESUMO
We report the molecular identification, cloning and initial biological characterization of 12 full-length HIV-1 subtype A, D and A/D recombinant transmitted/founder (T/F) genomes. T/F genomes contained intact canonical open reading frames and all T/F viruses were replication competent in primary human T-cells, although subtype D virus replication was more efficient (p<0.05). All 12 viruses utilized CCR5 but not CXCR4 as a co-receptor for entry and exhibited a neutralization profile typical of tier 2 primary virus strains, with significant differences observed between subtype A and D viruses with respect to sensitivity to monoclonal antibodies VRC01, PG9 and PG16 and polyclonal subtype C anti-HIV IgG (p<0.05 for each). The present report doubles the number of T/F HIV-1 clones available for pathogenesis and vaccine research and extends their representation to include subtypes A, B, C and D.
Assuntos
Clonagem Molecular , Genoma Viral , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Anticorpos Monoclonais/imunologia , Células Cultivadas , Feminino , Efeito Fundador , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Masculino , Fases de Leitura Aberta/genética , Receptores CCR5/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Linfócitos T/virologia , Replicação ViralRESUMO
BACKGROUND: We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults. METHODS: Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×10(9) (A), 2×10(10) (B), 2×10(11) (C), or Ad35-GRIN 1×10(10) (D) viral particles. RESULTS: No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A-D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 10(6) PBMC to any antigen was 78-139 across Groups A-C and 158-174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A-C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination. CONCLUSION/SIGNIFICANCE: Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional. TRIAL REGISTRATION: ClinicalTrials.gov NCT00851383.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Adenovírus Humanos/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Proteínas dos Retroviridae/imunologia , Vacinação , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Adenovírus Humanos/genética , Adolescente , Adulto , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta Imunológica , Método Duplo-Cego , Feminino , Infecções por HIV/sangue , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/genética , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/genética , Masculino , Pessoa de Meia-Idade , Proteínas dos Retroviridae/genéticaRESUMO
We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.
Assuntos
Ânions/farmacologia , Antivirais/farmacologia , HIV/fisiologia , Hepacivirus/fisiologia , Animais , Metabolismo dos Carboidratos , Linhagem Celular Tumoral , HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Hepacivirus/efeitos dos fármacos , Humanos , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia , Replicação ViralRESUMO
L-SIGN is a C-type lectin that is expressed on liver sinusoidal endothelial cells. Capture of Hepatitis C virus (HCV) by this receptor results in trans-infection of hepatoma cells. L-SIGN alleles have been identified that encode between three and nine tandem repeats of a 23 residue stretch in the juxtamembrane oligomerization domain. Here, it was shown that these repeat-region isoforms are expressed at the surface of mammalian cells and variably bind HCV envelope glycoprotein E2 and HCV pseudoparticles. Differences in binding were reflected in trans-infection efficiency, which was highest for isoform 7 and lowest for isoform 3. These findings provide a molecular mechanism whereby L-SIGN polymorphism could influence the establishment and progression of HCV infection.
Assuntos
Moléculas de Adesão Celular/fisiologia , Hepacivirus/patogenicidade , Hepatite C/etiologia , Lectinas Tipo C/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Virais/fisiologia , Alelos , Carcinoma Hepatocelular/virologia , Moléculas de Adesão Celular/genética , Células HeLa , Hepatite C/genética , Hepatite C/virologia , Hepatócitos/virologia , Humanos , Lectinas Tipo C/genética , Repetições Minissatélites , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Receptores de Superfície Celular/genética , Receptores Virais/genética , Transfecção , Proteínas do Envelope Viral/fisiologiaRESUMO
Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/patogenicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Deleção de Genes , Proteína gp120 do Envelope de HIV/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Peptídeos/química , Peptídeos/metabolismo , Receptores CCR5/química , Proteínas Recombinantes de Fusão , Enxofre/química , TransfecçãoRESUMO
CCR5 and CXCR4 usage has been studied extensively with a variety of clade B human immunodeficiency virus type 1 (HIV-1) isolates. The determinants of CCR5 coreceptor function are remarkably consistent, with a region critical for fusion and entry located in the CCR5 amino-terminal domain (Nt). In particular, negatively charged amino acids and sulfated tyrosines in the Nt are essential for gp120 binding to CCR5. The same types of residues are important for CXCR4-mediated viral fusion and entry, but they are dispersed throughout the extracellular domains of CXCR4, and their usage is isolate dependent. Here, we report on the determinants of CCR5 and CXCR4 coreceptor function for a panel of non-clade B isolates that are responsible for the majority of new HIV-1 infections worldwide. Consistent with clade B isolates, CXCR4 usage remains isolate dependent and is determined by the overall content of negatively charged and tyrosine residues. Residues in the Nt of CCR5 that are important for fusion and entry of clade B isolates are also important for the entry of all non-clade B HIV-1 isolates that we tested. Surprisingly, we found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects fusion and entry of all non-clade B isolates tested. This points to a different mechanism of CCR5 usage by these viruses and may have important implications for the development of HIV-1 inhibitors that target CCR5 coreceptor function.
Assuntos
HIV-1/patogenicidade , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , MutaçãoRESUMO
Hepatitis C virus (HCV) envelope glycoproteins E1/E2 can pseudotype retroviral particles and efficiently mediate entry into target cells. Using this experimental system, we determined HCV tropism for different cell types. Only primary hepatocytes and one hepatoma cell line were susceptible to HCV pseudovirus entry, which could be inhibited by sera from HCV-infected individuals. Furthermore, expression of the putative HCV receptor CD81 on nonpermissive human hepatic but not murine cells enabled HCV pseudovirus entry. Importantly, inhibition of viral entry by an anti-CD81 mAb occurred at a step following HCV attachment to target cells. Our results indicate that CD81 functions as a post-attachment entry coreceptor and that other cellular factors act in concert with CD81 to mediate HCV binding and entry into hepatocytes.
Assuntos
Antígenos CD/fisiologia , Hepacivirus/fisiologia , Receptores Virais/fisiologia , Linhagem Celular Tumoral , Humanos , Fusão de Membrana/fisiologia , Tetraspanina 28RESUMO
Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates exclusively in the cytoplasm of infected cells. The viral envelope glycoproteins, E1 and E2, appear to be retained in the endoplasmic reticulum, where viral budding is thought to occur. Surprisingly, we found that the expression system used to generate HCV envelope glycoproteins influences their subcellular localization and processing. These findings have important implications for optimizing novel HCV fusion and entry assays as well as for budding and virus particle formation.
Assuntos
Membrana Celular/metabolismo , Hepacivirus/patogenicidade , Íntrons , Proteínas do Envelope Viral/metabolismo , Proteínas Estruturais Virais/metabolismo , Sequência de Bases , Dimerização , Células HeLa , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Dados de Sequência Molecular , Deleção de Sequência , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/genéticaRESUMO
Target cell tropism of enveloped viruses is regulated by interactions between viral and cellular factors during transmission, dissemination, and replication within the host. Binding of viral envelope glycoproteins to specific cell-surface receptors determines susceptibility to viral entry. However, a number of cell-surface molecules bind viral envelope glycoproteins without mediating entry. Instead, they serve as capture receptors that disseminate viral particles to target organs or susceptible cells. We and others recently demonstrated that the C type lectins L-SIGN and DC-SIGN capture hepatitis C virus (HCV) by specific binding to envelope glycoprotein E2. In this study, we use an entry assay to demonstrate that HCV pseudoviruses captured by L-SIGN+ or DC-SIGN+ cells efficiently transinfect adjacent human liver cells. Virus capture and transinfection require internalization of the SIGN-HCV pseudovirus complex. In vivo, L-SIGN is largely expressed on endothelial cells in liver sinusoids, whereas DC-SIGN is expressed on dendritic cells. Capture of circulating HCV particles by these SIGN+ cells may facilitate virus infection of proximal hepatocytes and lymphocyte subpopulations and may be essential for the establishment of persistent infection.