J. Richard L. Pink

Selection of T cell epitopes and vaccine engineering

Publisher Summary This chapter discusses approaches to identify and characterize epitopes recognized by human CD4 + T cells. It describes in vitro stimulation of human peripheral blood mononuclear cells (PBMC) and subsequent cloning of T cells as an assay for the immunogenicity of potential vaccine candidates. It also provides overview on methods for identifying the epitopes and restriction dements recognized by the T lymphocyte clones (TLC). The chapter describes assays for identifying peptides that bind to human major histocompatibility complex (MHC) class II antigens. These assays may be used to select peptides that bind strongly to several different MHC allelic products, and are therefore likely to be useful immunogens. The immunogenicity of vaccine candidates can be tested by using the candidate to stimulate human T cells in vitro . If the donors are already primed, a standard proliferation assay can be used.

Development and characterization of antibodies against the N terminus of the human dopamine D4 receptor

Abstract: The human dopamine D4 receptor (hD4R), which has been implicated in human diseases such as schizophrenia and in a personality trait called “novelty seeking,” has not yet been characterized at the protein level. Following epitope scanning of the hD4R, we have produced a highly specific monoclonal antibody named DFR1 raised against an amino‐terminal peptide in a predicted extracellular region of the receptor. DFR1 decorated recombinant hD4Rs on the surface of intact Chinese hamster ovary (CHO) cells by flow cytometry and fluorescence microscopy and also recognized recombinant hD4.2, hD4.4, and hD4.7 receptor isoforms by western blot analysis. When expressed stably in CHO cells, all three hD4R isoforms contained N‐linked glycosylation and showed apparent molecular masses of 48, 55, and 67 kDa for hD4.2, hD4.4, and hD4.7, respectively. DFR1 immunoreactivity representing hD4R protein or dopamine D4 receptor‐like antigens was observed in crude membrane extracts of postmortem human brain tissue by immunoblotting. The DFR1 antibody provides a new immunological tool with the potential to further our understanding of the human dopamine D4 receptor protein.

Peptide-MHC Interaction: A Rational Approach to Vaccine Design

The goal of vaccination is to induce a protective immune response without inducing the disease itself or other undesirable effects. In the past this was achieved by giving inactivated or attenuated pathogens, with notable success in some cases, but problems of safety, efficacy or production in others. A currently pursued alternative is the construction of synthetic peptide vaccines that contain pathogen-derived determinants able to induce an immune response both at the B and the T cell level. Because T cells recognize peptide fragments which are derived from the processing of soluble proteins [1], the technical problems of epitope conformation can be largely avoided in the design of synthetic T cell sites. However the constraints presented by the extensive polymorphism of MHC antigens on one hand [2], and the obligate interaction between the antigenic peptide and the MHC molecule on the other [3], create a problem for the immune system. When an antigenic determinant does not interact favorably with the MH...

Requirements for screening recombinant DNA libraries for T cell epitope expression

The possibility of screening cDNA expression libraries with T cell clones was investigated. The model system was based on human T cell clones specific for the recombinant malaria protein 190L, which was expressed fused to beta-galactosidase in lambda gt11. Several membranes were tested for their capacity to bind antigen and stimulate T cell proliferation. Pretreatment of membranes with DMSO and/or sonication to release the antigen improved the sensitivity of the assay. Under optimal conditions, T cell proliferation in response to antigen bound to a low protein binder membrane was comparable to that observed with the antigen in solution. A dot-blot type apparatus was designed for screening large numbers of plaques with T cells. The technical problems of this approach, its requirements and possible applications are discussed.