Final Report Summary - TCDIFLU (Impact of adjuvants on T cell differentiation after Influenza Vaccination analyzed at the Single Cell Level) TCDIFLU tried to reveal the presence of protective T cell subpopulations by detailed analysis of CD4+ and CD8+ T lymphocytes at the single cell level after influenza (flu) infection with mouse-adapted PR8 flu strain or immunisation with seasonal flu vaccine, with special consideration on the effect of adjuvants (MF59, alum) or other proprietary adjuvant candidates (host).In more detail the objectives were:- selection, design and validation of suitable primers for biomarker detection;- profiling of the differentiation state of single cell T lymphocytes in mice after flu vaccination: these studies determined the differentiation status of vaccine-specific CD4+ and CD8+ T lymphocytes in mice vaccinated with the seasonal flu vaccine;- profiling of the differentiation state of single cell T lymphocytes in mice after flu infection;- comparison of the T cell differentiation state in mice after vaccination with adjuvant;- assessment of functionality of induced memory T cell;- verification of gene expression data by assessing protein biomarker expression.Suitable primers for biomarker detection have been selected, designed and validated. The selection comprised literature search to choose appropriate biomarkers. Primers were designed using software and validated in the laboratory (specificity, efficiency, simplified limit of detection, competition). Flu specific CD8+ T cells were analysed for 18 different biomarkers at the single-cell level for different immunisation regimens. Mice were immunised with flu protein vaccine (H1N1 2009 California) or flu protein vaccine plus adjuvant (MF59 or AbISCO-100) or nucleic acid-based proprietary vaccine. The use of a nucleic acid-based proprietary vaccine shifted the main interest to CD8+ T cells. This is as well the reason for the change in adjuvant (alum to AbISCO). Single flu specific CD8+ T cells from the spleen or the draining lymph nodes were analysed at different time points post immunisation.Different frequency of flu specific CD8 T cells and different gene expression profiles were detected when immunising mice with protein vaccine, protein plus adjuvants or using nucleic acid-based vaccines. Especially, the activation status of cytolytic T lymphocytes and memory T cells has been assessed.This work can be used to clarify the mode of action of vaccines and assess their immunogenicity. The results might be used to decide about the future use of vaccine candidates or their modification in order to obtain a desired immune response. This pre-clinical work might be used for translation to human studies and clinical trials.
