.......Generation of tailored CTL targets with VSV-pseudotyped HIV-1
Yoshiyuki Yokomaku, Koya Ariyoshi, Hideka Miura, Sachiko Tateishi, Ai Tachikawa, Aikichi Iwamoto, Wataru Sugiura, Yoshiyuki Nagai, Zene Matsuda
............1) AIDS Research Center, National Institute of Infectious Diseases
............2) Department of Infectious Diesease, Institute of Medical Science, Tokyo University

.............Cytotoxic T lymphocytes (CTLs) play an important role in the immune response to HIV-1 infection. They are responsible for the clearance of HIV-1 after primary infection and also make a contribution to elongation of the disease-free period of HIV-1 infected individual.
.............In order to determine whether CTLs have clinically relevant potency for restricting the proliferation of HIV-1 in an individual, it is required to evaluate the specific CTL response using the HIV-1 derived from the individual of interest. Therefore, a tailored procedure is required to make antigen-presenting cells which express epitopes of the HIV-1 isolated from the patient.
.............In general, antigen presenting cells are prepared by either the peptide-pulsing or infection with the recombinant vaccinia virus expressing the specific antigens.
.............In the peptide based assay, specific synthetic peptides are extracellularly administrated to be loaded on the cell surface MHC class I molecules. Usually, the background signal is low and it can identify the new CTL epitopes by utilizing overlapping candidate peptides. However, the peptide-based assay cannot address the questions of regarding intracellular antigen processing as the peptide entirely bypasses this pathway.
.............In the recombinant vaccinia virus system, prospective antigens are expressed intracellularly and target proteins are processed naturally. However, this method suffers from its long preparation period of the recombinant vaccinia virus and the high background signal in CTL assays.
..............Furthermore, the assay has to be performed in the limited window period before the cytopathic effect (CPE) by vaccinia virus infection becomes apparent.
..............To overcome these problems, we have established a new system for making tailored CTL target cells by using a well-established VSV-pseudotyped HIV-1 method.
.............. VSV-pseudotyped HIV-1 can transduce a wide variety of target cells in high efficiency and is much less cytopathic than vaccinia virus. The system is more physiological than peptide pulsing.
...............Once the PCR fragments encoding the corresponding antigen regions are obtained, more than 4X107 target cells can be generated within a month. We performed assays for HLA-A*0201-restricted HIV-1 Gag epitopes (SLYNTVATL) specific-CTLs using these VSV pseudotyped HIV-1 transduced target cells and compared the results with those obtained by peptide pulsed or recombinant vaccinia virus transduced target cells. Specific MHC-class I restricted lysis of target cells are observed as efficiently as by the peptide-pulse method. The background signal of this system was lower than that of vaccinia virus transduced targets cells.
...............We have established a novel system allowing the generation of many tailored target cells within a short period using VSV pseudotyped HIV-1. This method facilitates the analysis of epitopes in several HIV-1 proteins derived from multiple clinical samples. Furthermore, as the antigen proteins are processed in a physiological fashion, it may also facilitate the studies on the mechanism of antigen processing.