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Kutscher, Sarah (2010): Immunomonitoring technologies for the evaluation of Modified Vaccinia Virus Ankara expressing HIV-1 nef as a vaccine against AIDS. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The WHO/UNAIDS “Global summary of the AIDS epidemic” released in December 2009, estimates that 33 million of people are living with HIV, 2.7 million were newly infected and 2.0 million of people died of AIDS in the last year. After more than two decades of research an effective preventive or therapeutic vaccine against HIV remains elusive and immunological correlates of protection remain unknown. T-cell mediated immunity is considered to play an important role in controlling HIV infection and progression to AIDS. Several candidate vaccines against HIV aiming to stimulate cellular immune responses are investigated in phase I to phase III clinical trials and assays enabling for a reliable, informative and sensitive measurement of CD4 and CD8 T-cell need to be implemented. Among the most promising vaccine candidates is recombinant modified vaccinia virus Ankara (MVA), a live viral vector system for the delivery of HIV-derived antigens. Several vaccination trials have made use of the modified vaccinia virus Ankara (MVA) as delivery vector. At present, the IFN-γ-based ELISPOT assay is considered as a gold standard and preferred primary assay in vaccine trials. However, despite its high sensitivity the measurement of the sole IFN-γ production provides limited information on the quality of the immune response. Polychromatic flow-cytometry-based assays as intracellular cytokine staining (ICS) offer the possibility to detect several markers on the same cell. Several findings from cross-sectional and longitudinal studies investigating different grades of HIV control highlight the importance of developing assays able to simultaneously measure several parameters on a single-cell level and strongly suggest the use of flow cytometry to monitor immune responses. In this work polychromatic flow-cytometry based assays were developed, optimized, and standardized for T-cell immunomonitoring purposes. In addition, these ICS based methods were compared with ELISPOT assays performed in two different experienced laboratories. The comparative study provided evidence that by the use of a special analysis system, the sensitivity of the ICS could be increased up to levels comparable to the sensitivity of the ELISPOT assay. The established polychromatic ICS together with a polychromatic CFSE-based proliferation assay were applied to a re-evaluation study of a vaccination trial using recombinant MVA expressing HIV-1-Nef (MVA-nef) in HIV-1 infected HAART treated individuals. In this study, the impact of the immunologic intervention with MVA-nef on the specific anti-Nef T-cell immune response was investigated in regard to cytokine production (IFN-γ and IL-2), chemokine production (MIP-1β), activation and differentiation marker expression (CD154 and CD45RA, respectively) and proliferative potential. Vaccine-induced polyfunctionality and proliferative capacity, which were associated with nonprogressive HIV-1 infection in several studies, were detectable by combining the two immune assays. By means of short-term ICS, a significant increase of polyfunctional Nef-specific CD4 T cells expressing IFN-γ, IL-2 and CD154 was observed following vaccination, whereas changes in the quality of the CD8 T-cell response could not be observed. Only the additional use of a long-term polychromatic CFSE-based proliferation assay revealed vaccine-induced Nef-specific CD8 as well as CD4 T cells with proliferative capacity. The correlation between the vaccine-induced IL-2 production by CD4 T cells and the increase of proliferating Nef-specific CD8 T cells suggests a causal link between these two functions. The insight gathered in this reevaluation study exceeded by far the information obtained in the original work using a simple IFN-γ-based immune assay. These results highlight the importance of combining sophisticated immunomonitoring tools to unravel concealed effects of immunologic interventions and support the use of the poxvirus-derived MVA vector to stimulate effective HIV-specific T-cell responses. From a technical point of view, these findings are important to guide the choice for suitable immune assays able to characterize the phenotype and function of specific T-cells in a highly sensitive way.