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MOESM2 of Vaccination with recombinant adenovirus expressing peste des petits ruminants virus-F or -H proteins elicits T cell responses to epitopes that arises during PPRV infection

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posted on 2017-11-21, 05:00 authored by José Rojas, Miguel Avia, Elena Pascual, Noemí Sevilla, Verónica Martín
Additional file 2. PPRV T cell repertoire in mice: identification of immunoreactive PPRV-T cell epitopes in H-2 b context. To determine whether recombinant adenovirus vaccination elicits T cell responses to determinants that are also targeted during PPRV infection, we first set out to identify T cell epitopes in mice. Since few PPRV T cell epitopes have been reported [11–14], we attempted to describe new determinants in our experimental settings. We focused our approach on the F, H and NP proteins as T cell determinants involved in morbillivirus responses are usually mapped to these. Peptides predicted to bind to murine H-2b molecules (Db, Kb or I-Ab) were selected using algorithms available online (Table 1) [34–37] and synthesized. Using the TAP-deficient cell line RMA/s, we performed binding assays for MHC class I predicted binders. Most peptides bound their predicted MHC class I molecules. Only peptide NP5 did not bind to Db or Kb molecules. All 3 algorithms employed predicted Db binders quite accurately. The NetMHC prediction was nonetheless more accurate for Kb binding than ProPred-I or SYFPEITHI. PPRV-F, -H and -NP peptide immunogenicity data in C57BL/6 mice are presented in the figure of Additional file 2. PPRV peptide immunogenicity was tested on splenocytes from C57BL/6 PPRV-infected mice (IC’89; 1 × 106 PFU) using (A–C) IFN-γ ELISPOT and (D–F) proliferation assays. Responses to predicted peptides from PPRV (A and D) -F, (B and E) -H and (C and F) -NP proteins were measured in 8 mice per group. ELISPOT data are presented as average spots counted for 2 × 105 cells and proliferation as stimulation index (cpm ratio in test vs control). One-way ANOVA (Dunnett’s post-test: peptides vs control); *p < 0.05; **p < 0.01; ***p < 0.001. (A–C) Significant IFN-γ production was detected to peptides F2, F3, F7, F8, F9, F10, H2, H5, H6, H9, NP5, NP8, NP9 and NP10. (D–F) Significant splenocyte proliferation was detected to peptides F2, F7, H2, H5 and H9. Peptides F9 and F10 only tended to induce higher proliferation.


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