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StAdvInn Résumé de rapport

Project ID: 322693
Financé au titre de: FP7-IDEAS-ERC
Pays: Croatia

Mid-Term Report Summary - STADVINN (Strengthening adaptive immunity via innate immunity: enhancing the CD8 T cell response by using the NKG2D ligand expressed in a herpesvirus vector)

The main idea behind the project is based on our initial findings showing that the infection of mice with a recombinant mouse cytomegalovirus (MCMV) expressing the NKG2D ligand RAE-1gamma (RAE-1g) in place of its viral inhibitor m152 results in a profound virus attenuation. However, in spite of this, the virus-specific CD8 T cell response induced by such a virus was equal or even more robust compared to mice infected with a wild type virus strain (Slavuljica et al., J Clin Invest, 2010). The initial results obtained in frame of this project confirmed that MCMV expressing NKG2D ligand, RAE-1g (RAE-1gMCMV), can be used as an excellent vaccine vector (Trsan et al. Proc Natl Acad Sci USA, 2013). We have shown that the protection of mice vaccinated with RAE-1gMCMV vector expressing CD8 T cell epitope of listeriolysin (LLO) from Listeria monocytogenes (LM) resulted in a dramatically enhanced protection against a lethal challenge with LM, and that this protection correlates with a higher frequency of LLO-specific CD8 T cells. This study already indicated that the superior CD8 T cell response is related to a preserved function of dendritic cells (DCs) in mice immunized with a vector expressing RAE-1g. Our finding that the induction of an enhanced CD8 T cell response to vectored antigen was independent of NKG2D receptor was completely unexpected. We have therefore devised our approach using two lines of action. First, we have further characterized the immunobiology and vaccine properties of MCMV vector expressing various NKG2D ligands. Second, we have searched for a novel immune function of RAE-1g and its putative interaction partner. Related to the first line of action, during this project period we have used tumor models to further study the capacity of RAE-1gMCMV as a CD8 T cell vaccine vector. By using RAE-1gMCMV expressing SIINFEKL we have shown that the presence of RAE-1g in the context of viral vector results in an induction of a much stronger antitumor CD8 T cell response compared to a vector without RAE-1g. This correlated with a superior and long lasting protection against a challenge with B16OVA (B16 melanoma expressing ovalbumin). Moreover, it appears that such a vector can induce a superior CD8 T cell response both in prophylactic and therapeutic tumor vaccine settings. We have characterized the specific CD8 T cells and have shown that a large proportion of them express KLRG1. Apart from the tumor model we have also initiated studies with RAE-1gMCMV vector expressing influenza hemagglutinin and testing of its protective capacity against a challenge infection with the influenza virus is currently under way. We have also conducted initial studies with HCMV expressing ULBP2 using a humanized mouse model. Although initial studies were promising with regards to the induction of HLA-A2 restricted CD8 T cells, we are currently improving the reconstitution of myeloid cells in humanized mice in order to enrich the cells which are permissive for HCMV infection and at the same time serve as antigen presenting cells. We have employed RNASeq analysis and found a differential expression of numerous genes between cells infected with RAE-1gMCMV or control vectors, and these genes are currently subject to additional studies. With regards to the novel immune function of RAE-1g and its putative NKG2D independent interaction partner, we have performed several biochemical and molecular studies. Our preliminary results suggest the existence of an additional, so far uncharacterized, RAE-1g interacting partner(s), but further studies to confirm and extend our initial results are needed (see Report). Overall, the project is progressing very well and it is likely that during the second research period we will achieve all of the proposed goals and milestones.

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