Mapping of CD8 T cell epitopes in human respiratory syncytial virus L protein.

OBJECTIVES: Since it has been reported that in humans there is a relationship between human respiratory syncytial virus (hRSV)-specific cytotoxic T lymphocytes and symptom reduction, and that the polymerase (structural L protein) is highly conserved among different strains, this work aimed to identify the CD8 T cell epitopes H-2(d) restricted within the L sequence for immunization purposes. METHODS: We screened the hRSV strain A2 L protein sequence using two independent algorithms, SYFPEITHI and PRED/(BALB/c), to predict CD8 T cell epitopes. The selected peptides were synthesized and used to immunize BALB/c mice for the evaluation of T cell response. The production of IFN-γ from splenocytes of hRSV-infected animals stimulated by these peptides was assayed by ELISPOT. RESULTS: Nine peptides showing the best binding scores to the BALB/c MHC-I molecules (H-2K(d), L(d) and D(d)) were selected. Sequence homology analysis showed that these sequences are conserved among different hRSV strains. Two of these peptides induced significant IFN-γ production by ex vivo-stimulated T cells. CONCLUSIONS: Our results indicate that the hRSV L protein contains H-2(d)-restricted epitopes.

Sequence similarity and structural homologies are involved in the autoimmune response elicited by mouse hepatitis virus A59.

The features of autoantibodies (autoAb) to liver fumarylacetoacetate hydrolase (FAH) elicited in mice infected with mouse hepatitis virus (MHV) were studied by ELISA and western-blot competition assays. All sera tested contained Ab to cryptic FAH epitopes according with results from western-blot tests, whereas ELISA data indicated that some of these same sera did recognize native epitopes of the autoantigen (autoAg). Such differences were detected in individual sera from various mouse strains, and were ascribed to the fact that proteins insolubilized on solid supports expose a variety of conformational and cryptic antigenic determinants. On the other hand, whereas results from both experimental protocols showed that anti-MHV Ab did not cross-react with the soluble autoAg, the opposite situation did not show analogous results. Thus, binding of autoAb to insolubilized FAH could be inhibited by MHV depending on the mouse serum or the experimental protocol used. Additionally, a set of synthetic homologous peptides from mouse FAH and various viral proteins was employed to analyze the Ab repertoire of MHV-infected mice. Results indicated that two homologous peptides were recognized by most Ab: the N-terminal sequences (1-10) from FAH and the nucleocapside, both sharing 50% of identity, and sequence 2317-2326 of the RNA polymerase, a peptide showing 30% of identity with FAH 11-20. Results indicated that MHV-infection triggers at least three distinct Ab populations: anti-MHV, anti-FAH and cross-reacting Ab. This cross-reaction implies either sequential or conformational epitopes from both the viral proteins and the autoAg and may differ between individuals.

Detection of virus infection-associated antigen and 3D antibodies in cattle vaccinated against foot and mouth disease.

The analysis of sera obtained from animals vaccinated or revaccinated with inactivated vaccines against foot and mouth disease (FMD) virus showed that these vaccines induced antibodies against the virus infection-associated (VIA) antigen, detectable by agar gel immunodiffusion (AGID). The present study evaluates the antibody response to protein 3D and the VIA antigen (VIAA) of FMD virus induced by different vaccines in a group of 51 calves. This response was detected using AGID and a liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA) for anti-3D antibodies (ELISA-3D). No anti-VIAA or anti-3D antibodies were detected after the initial vaccination. Following revaccination, animals giving positive results were detected by both methods. This immune response disappeared 60-120 days post-revaccination (dprv) according to the AGID method, and 90-180 dprv when ELISA-3D was used. Samples of oesophageal-pharyngeal fluid obtained from animals that remained positive for anti-VIAA antibodies at 90-120 dprv gave negative results for viral isolation, indicating that the transitional antibody response induced by the vaccine was due to the presence of non-structural antigens in the vaccine and not to viral infection. These results indicate that the ELISA-3D method could be used as a complementary method for sero-epidemiological studies as an indirect indicator of viral activity, as long as the age and vaccination status of the animals being sampled are taken into consideration.

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid