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1.
AIDS Res Ther ; 7: 20, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20598119

RESUMO

BACKGROUND: Long-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease. RESULTS: We analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease. CONCLUSION: The novel antigen-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.

3.
AIDS Res Ther ; 5: 22, 2008 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-18837993

RESUMO

BACKGROUND: T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-gamma-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-gamma production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level. RESULTS: The cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-gamma producing T-cells were also producing MIP-1beta whereas T-cells characterized by the sole production of IFN-gamma were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-gamma+ MIP-1beta+ T-cells was equivalent to the measurement of the total IFN-gamma+ T-cells, we adopted the IFN-gamma+ MIP-1beta+ data analysis system to evaluate IFN-gamma-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-gamma+ MIP-1beta+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay. CONCLUSION: The IFN-gamma+ MIP-1beta+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.

4.
Vaccine ; 34(7): 923-32, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26776470

RESUMO

BACKGROUND: Therapeutic vaccination is a novel treatment approach for chronic hepatitis B, but only had limited success so far. We hypothesized that optimized vaccination schemes have increased immunogenicity, and aimed at increasing therapeutic hepatitis B vaccine efficacy. METHODS: Modified Vaccinia virus Ankara (MVA) expressing hepatitis B virus (HBV) antigens was used to boost protein-prime vaccinations in wildtype and HBV-transgenic (HBVtg) mice. RESULTS: Protein-prime/MVA-boost vaccination was able to overcome HBV-specific tolerance in HBVtg mice with low and medium but not with high antigenemia. HBV-specific antibody titers, CD8+ T-cell frequencies and polyfunctionality inversely correlated with HBV antigen levels. However, optimization of the adjuvant formulation, increasing the level of antigen expression and utilization of HBsAg of heterologous subtype induced HBV-specific CD8+ and CD4+ T-cells and neutralizing antibodies even in high-antigenemic HBVtg mice. CONCLUSIONS: Our results indicate that high HBV antigen levels limit the immunological responsiveness to therapeutic vaccination but optimization of the vaccine formulation can overcome tolerance even in the presence of high antigenemia. These findings have important implications for the development of future therapeutic hepatitis B vaccination strategies and potentially also for the stratification of chronic hepatitis B patients for therapeutic vaccination.


Assuntos
Vacinas contra Hepatite B/imunologia , Hepatite B/prevenção & controle , Tolerância Imunológica , Vaccinia virus , Animais , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Anticorpos Anti-Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , Antígenos E da Hepatite B/sangue , Imunização Secundária , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes de Neutralização
5.
PLoS One ; 8(10): e76215, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146841

RESUMO

Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFNγ, TNFα, IL2 and MIP1ß production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.


Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ensaios Clínicos como Assunto , Epitopos/imunologia , Monitorização Imunológica , Adulto , Idoso , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Sobrevivência Celular , Quimiocinas/metabolismo , Criopreservação , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Espaço Intracelular/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Coloração e Rotulagem , Fatores de Tempo
6.
J Clin Virol ; 55(2): 114-20, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22795599

RESUMO

BACKGROUND: Several correlates of HIV control have been described; however their predictive values remain unclear, since most studies have been performed in cross-sectional settings. OBJECTIVES: We evaluated the cause and consequence relationship between quality of HIV-specific T-cell response and viral load dynamic in a temporal perspective. STUDY DESIGN: HIV-1-specific T-cell responses were monitored over 7 years in a patient that following treatment interruption maintained a stable/low viral set point for 3.1 years before control of viral replication was lost and antiretroviral therapy restarted. RESULTS: We observed that high frequencies of HIV-1-specific CD4 and CD8 T cells were unable to prevent loss of viral control. Gradual loss of functionality was observed in these responses, characterized by early loss of IL-2, viral load-dependent decrease of IFN-γ and CD154 expression as well as increase of MIP-1ß production. Terminally differentiated HIV-1-specific CD8 T cells expressing CD45RA were lost independently of viral load and preceded the loss-of-control phase of HIV infection. CONCLUSION: By describing qualitative changes in HIV-1-specific T-cell responses that coincide with loss of viral control, we identified specific correlates of disease progression and putative markers of viral control. Our findings suggest including the markers IL-2, IFN-γ, MIP-1ß, CD154 and CD45RA into monitoring of HIV-specific T-cell-responses to prospectively determine correlates of protection from disease-progression.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Linfócitos T/imunologia , Carga Viral , Fármacos Anti-HIV/administração & dosagem , Biomarcadores , Ligante de CD40/metabolismo , Quimiocina CCL4/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Estudos Longitudinais , Prognóstico , Suspensão de Tratamento
7.
Virology ; 395(1): 45-55, 2009 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-19800648

RESUMO

Here we report a novel strategy for the induction of CD8(+) T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef(mut)) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLP-associated Nef(mut) is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef(mut) led to an anti-E7 CD8(+) T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freund's adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef(mut)-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef(mut) VLPs a promising candidate for new vaccine strategies focused on the induction of CD8(+) T cell immunity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Proteínas Oncogênicas Virais/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Imunidade Adaptativa , Animais , Linhagem Celular , Apresentação Cruzada , HIV-1/imunologia , Papillomavirus Humano 16/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/imunologia
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