Molekulare Quantifizierung der mCMV-Immunevasion

The infection with murine Cytomegalovirus (mCMV) is primarily controlled by virus-spe¬cific CD8 T cells. These cells recognize viral peptides presented by MHC-class I mole¬cules and lyse the infected cell. In order to subvert immune control by CD8 T cells, mCMV has developed strategies of immune evasion during virus-host co-evolution. mCMV encodes two negative viral regulators of antigen presentation (vRAPs), the Early (E)-phase proteins m152/gp40 and m06/gp48, which interfere with antigen presentation in the MHC class I pathway. The vRAPs obstruct the transport of peptide-loaded MHC molecules to the cell surface and downmodulate MHC class I surface expression in a cooperative effect As a consequence, the recognition of infected cells by CD8 T cells is inhibited. Previous functional studies demonstrated the qualitative reduction of MHC class I surface presen¬tation by the vRAPs and indicated differential vRAP effects on total cell surface class I and peptide-loaded cell surface class I. So far, the differential effects of mCMV vRAPs could not be quantitated because there exists no antibody to distinguish between all molecules of a certain MHC class-I allele and those presenting a defined mCMV peptide. However, T AG25-DL1.16 is the prototype of an antibody that can recognize a presentation complex of class I and bound pep¬tide, namely the presentation complex formed between Kb and the ovalbumin (OVA)-derived peptide SIINFEKL (OVA257-264). For studying efficacy and specificity of mCMV immune evasion using T-AG25-DL1.16, the SIINFEKL-coding sequency was integrated into the viral genome by BAC mutagenesis for an “orthotopic peptide swap” replacing the dominant m164 150-158 epitope of the mCMV protein m164/gp37.5 with SIINFEKL. The corresponding recombinant viruses mCMV-vRAP-SIINFEKL and mCMV-ΔvRAP-SIINFEKL were found to process SIINFEKL in infected fibroblasts, and vRAPs controlled its presentation just like that of an authentic viral peptide. Both viruses were found to prime an acute and memory CD8 T cell response in which the “transantigen” SIINFEKL took an intermediate position in the immunodominance hierarchy of the authentic mCMV pep¬tides.The successful integration of SIINFEKL allowed for the first time a quantitation of viral im¬mune evasion in terms of absolute numbers. vRAP expression was shown to reduce Kb-SIINFEKL complexes on the cell surface by a factor of >100, whereas the downmodulation of total cell surface Kb was only ~4-fold. Further experiments demonstrated that in the absence of vRAPs endogenous SIINFEKL presentation in infected cells is superior to an exogenous loading of MHC class I molecules with synthetic peptide.