# Preparation and Immunogenicity Prediction of Brucella melitensis mRNA Vaccine Candidate Based on omp16 and omp19 Genes

## TL;DR

This study explores the development of an mRNA vaccine candidate for brucellosis using the Brucella melitensis omp16 and omp19 genes, showing promising immunogenic properties.

## Contribution

The novel contribution is the synthesis and evaluation of mRNA vaccines based on omp16 and omp19 genes for brucellosis.

## Key findings

- Recombinant plasmids pIVTRup-omp16 and pIVTRup-omp19 were successfully constructed and used to synthesize mRNA.
- Omp16 showed stronger immunogenicity than Omp19 and could activate both CD4+ and CD8+ T cells.
- Epitope prediction identified seven and six epitopes for Omp16 and Omp19, respectively.

## Abstract

Background: Brucella outer membrane proteins (Omps) are an important part of its cell wall and major virulence-related factors. Omp16 and Omp19 proteins are the advantageous antigens of Brucella and have been widely used in research on vaccines against brucellosis. As an emerging vaccine, the mRNA vaccine has unique advantages in the fight against intracellular parasitic bacteria. Methods: In this study, mRNA encoding the omp16 and omp19 genes of Brucella. melitensis (B. melitensis) was synthesized using in vitro transcription. The target mRNA was transfected into HEK 293T cells to evaluate protein expression levels and assess its immunogenicity. Finally, bioinformatic approaches were employed to analyze potential antigenic epitopes. Results: In this study, the successfully constructed recombinant plasmids pIVTRup-omp16 and pIVTRup-omp19 were utilized to synthesize omp16-mRNA and omp19-mRNA, each approximately 600 nt in length. Western blot analysis detected the expression of proteins with molecular weights of 16 kDa and 19 kDa in HEK 293T cells at 24 h post-transfection with mRNA. Purified rOmp16 and rOmp19 had good immunogenicity, which could specifically bind to serum antibodies of brucellosis patients. rOmp16 had stronger immunogenicity than rOmp19. Epitope prediction showed that Omp16 contained seven epitopes and Omp19 contained six epitopes. In addition, Omp16 and Omp19 could form stable complexes with target receptors. Simulated immunization with Omp16 and Omp19 proteins significantly activated both CD4+ and CD8+ T cells. Conclusions: The immunogenic proteins were successfully expressed in cells based on the mRNA fragments synthesized from omp16 and omp19 genes of B. melitensis, which was a preliminary exploration for the preparation of B. melitensis mRNA vaccine.