Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma are associated with nonprogressive HIV-1 infection.

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.

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT.

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.

Longitudinal changes in HIV-1-specific T-cell quality associated with viral load dynamic.

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.

A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses.

Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database information. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the standard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting individual responses (Tat VOPSD vs. Tat 15mers or 20mers: p≤0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p<0.001) than standard sets, enhancing both CD4 and CD8 T-cell responses. Moreover, this peptide design allowed a marked reduction of the peptides number, representing a powerful tool for investigating novel HIV-1 candidate vaccine antigens in cohorts of HIV-seronegative and seropositive individuals.