Selection of T-cell epitopes from foot-and-mouth disease virus reflects the binding affinity to different cattle MHC class II molecules

Abstract

The major histocompatibility complex (MHC)-restricted selection of T-cell epitopes of foot-and-mouth disease virus (FMDV) by individual cattle MHC class II DR (BoLA-DR) molecules was studied in a direct MHC-peptide binding assay. By in vitro priming of T lymphocytes derived from animals homozygous for both MHC class I and II, five T-cell epitopes were analyzed in the context of three MHC class II haplotypes. We found that the presentation of these T-cell epitopes was mediated by DR molecules, since blocking this pathway of antigen presentation using monoclonal antibody TH14B completely abolished the proliferative responses against the peptides. To study the DR-restricted presentation of these T-cell epitopes, a direct MHC-peptide binding assay on isolated cattle DR molecules was developed. Purified cattle MHC class II DR molecules of the BoLA-DRB3*0201, BoLA-DRB3*1101, and BoLA-DRB3*1201 alleles were isolated from peripheral blood mononuclear cells. For each allele, one of the identified T-cell epitopes was biotinylated, and used as a marker peptide for the development of a competitive MHC-peptide binding assay. Subsequently, the T-cell epitopes of FMDV with functionally defined MHC class II specificity were analyzed in this binding assay. The affinity of the epitopes to bind to certain DR molecules was significantly correlated to the capacity to induce T-cell proliferation. This demonstrated at the molecular level that the selection of individual T-cell epitopes found at the functional level was indeed the result of MHC restriction.

Biological effect of varying peptide binding affinity to the BoLA-DRB3*2703 allele

“…FMD-VP2 and VP4, for example, were both demonstrated to bind with high affinity to BoLA-DRB3*1101 and BoLA-DRB3*0201, respectively. Moreover, their high binding affinity was consistent with their ability to stimulate lymphocyte proliferation [7]. In the present study, OVA peptides were demonstrated to have binding affinity for BoLA-DRB3*2703 ranging from 26.92 to > 320 µM.…”

“…Various OVA and FMD-V peptides have previously been used as immunogenic epitopes to define antigen presentation, the kinetics of peptide-MHC binding, and assess antibody responses of cattle and mice [3,4,7,11,17,22,31]. FMD-VP2 and VP4, for example, were both demonstrated to bind with high affinity to BoLA-DRB3*1101 and BoLA-DRB3*0201, respectively.…”

“…Several self-peptides (BoLA-DQ and fibrinogen fragments), and non-self peptides (sperm whale myoglobin and mycobacterium heat shock protein fragments) previously eluted from BoLA-DRB3*2703 molecules were selected for initial study based on sequences derived from mass spectrometry analysis of pooled peptides [26]. The assumption being that one or more of these peptides would bind with high affinity to [17,22] and two viral peptides from the foot and mouth disease virus (FMDV), including VP2 74-88 and VP4 20-34 [7] were synthesized as competitor peptides (Tab. I).…”

“…The BoLA class II-peptide binding assay was carried out as previously described [7]. Briefly, purified BoLA-DRB3*2703 molecules were incubated with 10 µM of either biotinylated self-peptide DQ or fibrinogen [23], and a non-labeled synthetic competitor peptide (Tab.…”

“…supplemented with FCS, penicillin, streptomycin, and β-mercaptoethanol. BMC were cultured as previously described with slight modification [7]. Briefly, BMC were primed in vitro with various concentrations (0, 2.5, 5.0, 10.0 and 20.0 µg · mL −1 ) of synthetic peptides (Tab.…”