#06-174

Complete

Date Full Report Received

06/16/2008

Date Abstract Report Received

06/16/2008

Investigation

Institution:
Primary Investigator:

Current vaccines have not met the needs of producers who want to control and eliminate PRRS. Development of vaccines that elicit protective anti-viral immune responses requires identification of common viral epitopes that stimulate cellular responses to all/most PRRSV isolates. Many antibody epitopes, especially the important serum neutralizing epitopes, are viral isolate specific. This project had one objective: to refine the methodology needed to identify potentially protective T cell epitopes in PRRSV proteins. Nursery pigs were inoculated with PRRSV VR-2332. Based on our previous results, pigs were maintained for 6-7 months post inoculation, by which time they had resolved the infection, as indicated by absence of serum antibody and absence of viral antigen in nodes at necropsy. Lymph nodes were collected, processed to single cell suspensions, and cultured in two types of assays: the ELISPOT assay to enumerate interferon-gamma secreting cells, and a very sensitive flow cytometric proliferation assay using a fluorescent dye to label cells in order to determine what proportion responded to a given stimulant. Peptides of 30 amino acids each, overlapping one another by 10 amino acids at each end, and based on the predicted amino acid sequence of GP5 of PRRSV VR-2332, were chemically synthesized and used as stimulants to evaluate the response of T lymphocytes from previously infected pigs to the peptides in a recall response. Our preliminary data had suggested that the peptide termed P6 stimulated interferon-gamma secretion and T cell proliferation from infected, but not from control, pigs. Additionally, several of the other peptides stimulated secretion and proliferation from some, but not all of the infected pigs. The simplest explanation was that the concentration used, 5 µg/ml of culture medium, was not optimal. The results from this project, however, indicated that 5 µg/ml was optimal for both proliferation and interferon-gamma secretion. Moreover, only P6 stimulated a recall response from all of the infected, but not the uninfected, pigs in this study, suggesting that this peptide is a candidate for inclusion in an efficacious vaccine against PRRSV. This information is important because an effective vaccine must include only those epitopes that stimulate protective recall responses against the virus, and P6 is the first T cell epitope identified that can stimulate potentially protective recall responses. Carol Wyatt, cwyatt@vet.k-state.edu