טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMakler Friedner Oryan
SubjectStudying Antigen Presentation of Viral T-Cells Epitopes
Using Recombinant TCR-like Antibodies
DepartmentDepartment of Biology
Supervisor Professor Yoram Reiter
Full Thesis textFull thesis text - English Version


Abstract

Of all the human herpesviruses described to date, infection with CMV is considered to be the most important cause of morbidity and mortality. Many gene products participate  in generating CTL response, but the high frequencies for the viral protein pp65 found in subsequent studies  showed that this protein is the chief target of the CTL-mediated immune response. Among all the pp65 peptides, CTL activity in HLA-A2 positive individuals was found to be mainly directed to the peptide (495-503)  NLVPMVATV.

Characterization of class I MHC-peptide presentation is essential for better understanding the acquired arm of the immune response. In recent years we and others isolated TCR-like antibodies capable of recognizing human class I MHC-peptide complexes especially for HLA-A2 the most frequent human MHC allele. These were used for phenotypic analysis of antigen presentation on APCs by direct visualization and in situ detection of the particular specific peptide-MHC complex. In this study we take advantage of this strategy and demonstrate the use of these molecules to follow the dynamics of antigen presentation in human cells infected with CMV. We describe the isolation and characterization of these human recombinant antibodies which bind with high affinity HLA-A2 complexes that display the pp65 (495-503) peptide. This was achieved by selecting a phage displayed Fab antibody library on recombinant soluble  HLA-A2 complexes containing the pp65 (495-503) peptide. The CMV-specific TCR-like antibodies were characterized for their ability to bind specifically cells presenting class I MHC-CMV peptide complexes, as well as for their affinity toward this antigen. In order  to achieve higher avidity via multipoint binding of the antibodies to the HLA-A2/pp65 complexes, recombinant Fab tetramers and whole IgG molecules were constructed from the isolated Fab antibodies. Most important, we demonstrate the experimental importance of these unique molecules in direct phenotypic analysis of the dynamics and kinetics of class I MHC complex presentation on the surface and inside human virus-infected cells. We demonstrate the generation of large intracellular pools of viral complexes and their low level of presentation on the cell surface as a function of time after infection. Also, we propose a mechanism that might explain the presentation kinetics of the HLA-A2/pp65 complexes, by using proteasome inhibitors that release the complexes from the golgi. To our knowledge the studies presented in this work are the first attempt to directly visualize and analyze the dynamics of a naturally occurring viral MHC-peptide complex in human cells after viral infection.