DISCUSSION AND CONCLUSION THE ANTIGENICITY OF VP1 PEPTIDES EXPRESSED AS A FUSION TO A PATHOGEN ASSOCIATED MOLECULAR PATTERN MOLECULE MV Mamabolo, & M Crampton CSIR Biosciences, PO Box 395, Pretoria, 0001, South Africa1 E-mail: mcrampton@csir.co.za, www.csir.co.za Protein Expression and Antigenicity Genes encoding the PALP fused to each of the antigenic peptides were cloned into B. halodurans respectively and secreted as sandwich fusions (Figure 2A). When a western blot membrane was probed with serum from a guinea pig which was vaccinated with the FMDV SAT2 Zim/7/83 antigen, PALP-EpiGH/C had a reactive band (Figure 2B), as shown with an arrow, indicating that the fusion protein was antigenic. INTRODUCTION Peptides can be used as immunogens in vaccines to elicit specific antibody immune response. However, simple peptides often lack the structural conformation found in their native environments. They also often do not possess sufficient T-cell epitopes to induce a strong cell-mediated response (Li et al., 2014). This can limit their biological activity and ability to induce a solid protective immune response. In addition, short peptides have a short half-life in vivo, especially if they lack structural modifications such as acetylation, amidation, glycosylation or carbonation. Some of these limitations can be overcome by linking the peptides to other proteins that act as carriers and molecular adjuvants. M 1 2 3 15 40 35 25 70 55 kDa 100 M 1 2 3 4 15 40 35 25 55 kDa 100 70 A protein with adjuvant like properties, here referred to as PALP was shown in several studies to enhance the immunogenicity of proteins and peptides. PALP is a ligand of one of the members of the toll-like receptor (TLR) family. Members this family are capable of activating the adaptive immune response through the recognition of pathogen associated molecular patterns and mediate the production of cytokines, such as IL-8, which are necessary for the development of effective immunity. In this project antigenic peptides of the foot-and-mouth disease (FMD) virus were fused to PALP and their antigenicity evaluated. Figure 2A: An image of a 15% SDS-PAGE gel. Loaded in lane 1 is PALP; lane 2, PALP-EpiGH and lane 3, PALP-EpiGH/C. Figure 2B: An image of a western blot membrane with proteins that were probed with serum from a guinea pig that was vaccinated with the FMDV SAT2 Zim/7/83 antigen. Loaded in lane 1 is the entire VP1 protein; lane 2, PALP; lane 3, PALP-EpiGH and lane 4, PALP-EpiGH/C. METHODOLOGY The IEDB Analysis Resource program was used to define the antigenic peptides on the VP1 protein of the FMD virus. Based on the generated profile, complementary peptides were synthesized, annealed, then cloned for expression as sandwich fusions into the variable region of the PALP. The antigenicity of the fusion proteins was determined by western blot where the membrane was probed with serum from a guinea pig which was vaccinated with the FMDV SAT2 Zim/7/83 antigen. The bioactivity of the fusion proteins was measured by incubating different concentrations of the proteins with HEK-293 cells that were transfected with the toll-like receptor (TLR) gene for which PALP is a ligand. Once activated the cells release IL-8 which was measured by ELISA. Bioactivity HEK-293 cells that were incubated with PALP, PALP-GH and PALP-GH/C secreted high levels of IL-8. The PALP in the fusion protein bound to the TLR on the HEK cells and induced the production of IL-8. RESULTS Antigenicity profile The antigenicity profile of the VP1 protein of FMDV SAT2 Zim/7/83 was generated using the IEDB program. The position of the selected antigenic peptides and amino acid sequence is indicated in Table 1 and in Figure 1. Table 1: FMDV VP1 protein antigenic peptides that were fused to PAMP. DISCUSSION AND CONCLUSION Name Position Amino acid sequence EpiGH 127- 139 YTQQSTAIRGDRA EpiGH/C 127- 139/ 184- 199 YTQQSTAIRGDRA/ LLPGYDHADRDRFDSP The magnitude and quality of the adaptive immune response depends on signals derived from the innate immune response (Medzhitov and Janeway, 1997). The PALP-EpiGH/C was found to be antigenic and able to activate the innate immune response when using the HEK-293 cells that were transfected with the specific TLR gene. The PALP-EpiGH did not react with positive serum. This could be due to the fact that only the N-terminal half (aa 127- 139) of the GH-loop was fused to PALP instead of the entire loop (aa 126- 150) which makes up the B-cell epitope (Opperman et al., 2012). The antigenicity of both the Epi-GH and Epi-GH/C will be improved by incorporating the full GH-loop and then tested for immunogenicity. Once this data has been obtained can the fused antigenic peptides of the FMDV VP1 protein fused to the PALP be evaluated for protection of livestock against FMD infection. EpiGH EpiC Figure 1: The antigenicity profile of the VP1 protein of the FMDV REFERENCES Li W, Joshi MD, Singhania S, Ramsey KH, Murthy AK. 2014. Peptide vaccine: progress and challenges. Vaccines 2: 515- 536. Medzhitov R, Janeway CA. 1997. Innate immunity: impact on the adaptive immune response. Current Opinion in Immunology 9: 4- 9 Opperman PA, Maree FF, Van Wyngaart W, Vosloo W, Theron J. 2012. Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments. Virus Research 167: 370- 379. HANOI , VIETNAM GFRA 2015 OCTOBER 20-22, 2015