A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays.
Vaccines in general and HIV vaccines in particular are focusing ever more on the induction of cellular immunity specifically the generation of cytotoxic T cells (CTL). As progress is made towards developing a safe and effective HIV vaccine, there is a need for a robust, sensitive and reproducible assay to evaluate vaccine-induced cellular immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable and amenable to high-through-put screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay. We selected a panel of 23 8-11 mer Influenza virus (Flu), Cytomegalovirus (CMV) and Epstein Barr virus (EBV) epitopes recognized by CD8 positive T cells and presented by 11 class I HLA-A and HLA-B alleles whose cumulative frequencies represent >100% of Caucasian individuals. We examined interferon-gamma (IFN-gamma) secretion in peripheral blood mononuclear cells (PBMC) incubated with the peptides using a modified ELISPOT assay. IFN-gamma secretion was detected in 15/17 (88%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-gamma in response to the pool of peptides was CD8+ T cell mediated and HLA restricted. In vitro stimulation of PBMC with individual peptides or the pool of peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen in a CTL assay. The size, shape and appearance of the spots produced using this peptide panel provided a standard for the establishment of acceptance criteria of spots for the evaluation of ELISPOT plates using an automated reader system.
Currier
Jeffrey R
JR
The US Military HIV Research Program, Suite 200, 13 Taft Court, Rockville, MD 20851, USA.
Kuta
Ellen G
EG
Turk
Ellen
E
Earhart
Lyndsay B
LB
Loomis-Price
Larry
L
Janetzki
Sylvia
S
Ferrari
Guido
G
Birx
Deborah L
DL
Cox
Josephine H
JH
D016915:AIDS Vaccines; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D015488:HIV Antigens / Q000032:analysis* / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007163:Immunosorbent Techniques; D008213:Lymphocyte Activation; D011786:Quality Control
D016915:AIDS Vaccines; D015488:HIV Antigens; D015395:Histocompatibility Antigens Class I