RESULTS AND DISCUSSION Evaluating Cell Surface Display as a Potential Brucellosis Antigen Delivery System 1, 2S Goolab, 2H van Heerden, 1R Roth, 1C Kenyon & 1M Crampton 1. CSIR Biosciences, PO Box 395, Pretoria, 0001, South Africa 2. Department of Veterinary Tropical Diseases, University of Pretoria, Private bag X04 Onderstepoort, 0110 E-mail: sgoolab@csir.co.za, www.csir.co.za INTRODUCTION RESULTS AND DISCUSSION Brucellosis is a one of the most widespread global zoonotic diseases, which is associated with significant morbidity that can lead to spontaneous abortion and infertility in livestock. Brucella abortus is the causal agent of bovine brucellosis and the primary source of infection for people in Sub-Saharan Africa [1]. The current vaccines against bovine brucellosis are based on live attenuated strains of Brucella. The disadvantages associated with the use of these vaccines include being infectious to humans, interference with diagnosis as they elicit similar immune profiles to infected animals, and induction of abortions in pregnant animals [2].   - Omp19 P2 L1- Omp16 P2 L3- Omp16 P1 - Omp19 P3 L2- Omp16 P3 - Omp19 P1 C- terminal N- terminal Protein modelling Omp A is used as a carrier protein and contains four surface exposed loops. These loops are used for the insertion of foreign peptides, hence natural display on the cell surface. The 3D protein model of Omp A replaced with B. abortus antigenic peptides is depicted in Figure 3. The 3D structure of Omp A after insertion remained structurally similar therefore, it would remain functioning as an integral membrane protein and cell viability would not be compromised. This study aims to develop a viable, recombinant whole cell brucellosis vaccine through the surface display of foreign Brucella antigens, outer membrane protein (Omp) 16 and 19 (derived by reverse vaccinology) on the E. coli outer membrane for the induction of antigen-specific antibody responses, as overviewed in Figure 1. Recombinant expression B. abortus B and T cell epitope Cell surface display Figure 3: E. coli OmpA mutant protein substituted with B. abortus Omp 16 and Omp 19 surface exposed peptides. Loop 1 (L1), loop 2 (L2) and loop 3 (L3) are denoted by orange, purple and green curves respectively and P1, P2 and P3 represent the Brucella peptides. Figure 1: The development of a recombinant whole cell vaccine for the treatment of B. abortus. Expression of Omp 16 and Omp 19 in E. coli The expressed proteins are shown in Figure 4a and 4b. CO and WT Omp 16 and 19 were expressed in E. coli BL21 (DE3) strain in the soluble and insoluble fractions (solubilized with Urea). Together with the C-terminal histidine tag, Omp 16 and Omp 19 are 16kDa and 19kDa in size, respectively. Based on Figure 4b Omp 19 expressed in the soluble fraction and Omp 16 in the insoluble fraction. CO Omp 16 demonstrated greater levels of expression in comparison to WT Omp 16 and expression levels were similar for both CO Omp 19 and WT Omp19 (Figure 4b). Furthermore, an additional band of marginally higher molecular mass in CO Omp19 (Figure 4b) might correspond to a precursor form of Omp19 due to protein acylation [4]. AIMS in silico epitope prediction of B. abortus Omp 16 and Omp 19 using the E. coli Omp A carrier protein for bacterial surface display Expression of wild type (control) and codon optimized Omp 16 and Omp 19 in E. coli METHODOLOGY An outline of reverse vaccinology for B. abortus biovar 1 is shown in Figure 2a. Vaxign, a web-based vaccine design system was used to predict the vaccine targets, Omp 16 and 19 based on genome sequences [3]. Thereafter, the antigenic peptides (B and T cell) were evaluated for the targets using the Immune epitope database (IEDB) and Epitopia server. These antigenic peptides were used to replace surface exposed loop regions of the carrier protein, E. coli OmpA (PDB code 1BXW). CO16 WT16 CO19 WT19 kDa CO16 W16 CO19 W19 Soluble M Insoluble 25 10 15 35 CO16 WT16 CO19 WT19 kDa CO16 W16 CO19 W19 Soluble M Insoluble 4a 4b 2a IEDB T-cell epitope prediction Vaxign vaccine target prediction: Omp 16 & Omp 19 Proteasome cleavage TAP interaction MHC I/II binders IEDB and Epitopia Protective mAb-epitope prediction Linear epitopes conformational epitopes Protein modelling Figure 4a: 15% SDS-PAGE gel showing the expressed proteins in the soluble and solubilized insoluble fractions 4b: soluble and solubilized insoluble Omp16 and Omp19 were transferred to a nitrocellulose membrane and identified with anti-His-HRP. A summary of the cloning and expression strategy for Omp 16 and Omp 19 is shown in Figure 2b. The wild type (WT) and codon optimized (CO) genes (synthesized by GenScript) encoding the Omp 16 and Omp 19 were cleaved with the relevant restriction enzymes flanking the genes and thereafter ligated into the pET20b(+) expression vector. For protein expression, the cassettes were transformed into E. coli BL21 (DE3) cells, which were induced with 1mM IPTG and grown at 37⁰C for a further 4 hours. A western blot using anti-his HRP was used to probe the immobilized proteins. CONCLUSION AND FUTURE SCOPE The protein structure of Omp A replaced with B. abortus antigenic peptides was resolved to ensure correct folding of Omp A hence surface display of the antigenic peptides in E. coli. The expression (in E. coli) and identification (Figure 4) of WT and CO Omp 16 and 19 has been demonstrated. Future studies will focus on determining the location of Omp 16 and Omp 19, defining antigenicity and immunogenicity of expressed Omp’s using SDS-PAGE, ELISA and small animal models. Immunogenicity studies PCR amplify WT genes pSK-WT Omp 16 & 19 Ligations into pET20B(+) Expression in E. coli BL21 (DE3) Antigenicity studies pUC57-CO Omp 16 & 19 REFERENCES Godfroid, J., AL Dahouk, S., Pappas, G., Roth, F., Matope, G., Muma, J., Marcotty, T., Pfeiffer, D. and Skjerve, E., 2013. A “One Health” surveillance and control of brucellosis in developing countries: moving away from improvisation. Comparative immunology, microbiology and infectious diseases, 36(3), pp. 241-248. Olsen, S.C. and Stoffregen, W., 2005. Essential role of vaccines in brucellosis control and eradication programs for livestock. He, Y. and Xiang, Z., 2010. Bioinformatics analysis of Brucella vaccines and vaccine targets using VIOLIN. Immunome research, 6 Suppl 1, pp. S5-7580-6-S1-S5. Tibor, A., Decelle, B., and Letesson, J. J., 1999. Outer membrane proteins Omp10, Omp16, and Omp19 of Brucella spp. are lipoproteins. Infect. Immun. 67:4960. 2b Figure 2a and 2b: Summary of research methodology