Optimizing vaccine design against B. bigemina: multi-antigen and multi-expression platforms for better immune responses.

VALERIA MONTENEGRO, ; JOSÉ JARAMILLO ORTIZ; DÉBORA GARANZINI, MARTINA PAOLETTA; GABRIELA CALAMANTE ; PAULA DEL MÉDICO ZAJAC; SILVINA WILCOWSKY

CABA

Congreso; International Congress on Tropical Veterinary Medicine - 2nd Joint AITVM STVM Meeting?; 2018

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Bovine babesiosis is a disease caused by protozoan parasites that cause substantial cattle morbidity and mortality in vast tropical and subtropical world areas. Current view about the mechanism of immunity to babesial parasites sustains the hypothesis that both innate and adaptive responses that include CD4 + T cells and neutralizing antibodies are required. In this sense, the rational design of new candidate vaccines displayed in adequate expression systems should include strain-conserved antigens that induce these type of responses.The aim of this work was the development of a set of Babesia bigemina vaccine candidates containing B and T cell epitopes for its future evaluation in an heterologous prime-boost vaccination scheme. Previous results from our group have shown that this scheme had induced strong humoral and cellular antigen-specific responses in B. bovis.Two chimeric multi-antigens (MABbi and MABbi) of 1661 bp and 1519 bp respectively were designed for expression in MVA viral vectors. Both multi-antigens contained linear immunodominant epitopes of 3 previously identified B. bigemina and B. bovis antigens. These proteins were the Apical Membrane Antigen 1 (AMA-1), the rhoptry -associated protein 1 (RAP-1) and the thrombospondin-related anonymous protein (TRAP-1). The first two antigens were already characterized in B. bigemina but the latter has not been reported yet, although its presence was previously described in Plasmodium sp. and Babesia bovis. The TRAP-1 gene was found by bioinformatics search in the genome of Babesia bigemina using the B. bovis sequence as query. Prediction of B cell epitopes of the three antigens was performed using software available online that uses physico-chemical properties of aminoacids, artificial neural networks and algorithms trained on epitopes and non-epitope amino acids determined from crystal structures. For the prediction of T cell epitopes, human HLA alleles were used to approximate the binding of epitope-containing peptides to the bovine MHC class II molecule BoLA-DRB3. In all cases, sequence information was retrieved from GenBank and EuPathDB and predictions were based on a consensus sequence of geographically distant Babesia sp. strains.All predicted B and T cell epitopes were concatenated sequentially as two separate single open-reading frames and synthetized in cloning vectors for further subcloning in the viral plasmids. Besides, AMA-1, RAP-1 and TRAP-1 antigens were expressed separately as recombinant proteins in E. coli using the pET28a vector with high expression levels. The three purified proteins were used to obtain polyclonal antisera in mice to further confirm expression in MVA-infected cells. Collectively, in this work, a set of new B. bigemina vaccine candidates were developed using a rational approach that combines bioinformatics prediction, strain-sequence conservation and multiple expression systems. The set comprises 2 recombinant MVA viruses coding chimeric genes which contain putative B and T cell epitopes and 3 recombinant proteins that will be used as a subunit cocktail. Future immunization studies with these candidates will provide information about their immunogenicity and the feasibility of using combinations of these immunogens as alternatives to attenuated vaccines.