Vaccinomics to design a chimeric vaccine candidate against four bordetella species

Abstract

Bordetella species are an important group of Gram-negative respiratory pathogens that affects both humans and animals. While B. pertussis is the most extensively studied species due to its role in whooping cough, non-pertussis Bordetella species such as B. bronchiseptica, B. avium, B. hinzii, and B. holmesii have importance as significant pathogens, especially in veterinary field and immunocompromised hosts. The limited availability of broadly protective vaccines against these species highlights the need for novel vaccination strategies capable of providing cross-protective immunity. In recent years, advances in immunoinformatics and vaccinomics have enabled the design of vaccine candidates through computational prediction of antigenic determinants. Multi-epitope chimeric vaccines give a promising alternative to conventional whole-cell or subunit vaccines because they allow the selective inclusion of conserved and immunogenic regions while minimizing non-essential or potentially reactogenic components. These kinds of approaches offer improved safety profiles, enhanced immune coverage, and flexibility in targeting multiple species simultaneously. In this study, a multi-epitope chimeric vaccine candidate targeting four Bordetella species (B. avium, B. bronchiseptica, B. hinzii, and B. holmesii) was designed using a comprehensive immunoinformatics-based workflow and subsequently optimized for recombinant production. Initially, conserved antigenic proteins shared among the selected species were identified through comparative sequence analysis. Candidate proteins were further evaluated based on subcellular localization and immunological accessibility. Then, B-cell and MHC class I T-cell epitopes were predicted and screened for antigenicity, non-allergenicity, and non-toxicity. T-cell epitopes were predicted against MHC alleles of Canine spp., and due to the absence of validated canine MHC class II alleles in public databases, MHC class II epitope prediction was not included in the present study. Selected epitopes were assembled into a single chimeric construct by using appropriate linker sequences to preserve epitope integrity and enhance immunogenic presentation. Structural modeling of the designed vaccine construct was performed to evaluate its tertiary structure and stability. Three-dimensional models generated using prediction tools were refined and validated, demonstrating favorable stereochemical properties. Molecular docking analyses with Toll-like receptors (TLR 2 and TLR 4) suggested stable interactions between the vaccine construct and immune receptors. This supports the potential of the designed vaccine to activate both innate and adaptive immune responses. To assess the feasibility of experimental production, the amino acid sequence of the chimeric vaccine was reverse-translated and codon-optimized for expression in Escherichia coli by using the JCAT tool. The optimized gene was cloned into a pET-based expression vector and recombinant expression was evaluated in multiple E. coli strains. Between the tested strains, E. coli T7 gave the most reliable transformation and expression performance and was selected for further optimization studies. Recombinant protein expression was systematically optimized by evaluating induction temperature and IPTG concentration. SDS-PAGE and Western blot analyses confirmed IPTG-dependent expression of the recombinant protein at the expected molecular weight. Comparative analyses revealed that both low and high-temperature induction conditions supported expression. However, induction at 37°C for 4 hours resulted in robust and reproducible protein production. Importantly, fractionation of cell lysates into soluble and insoluble fractions demonstrated that the recombinant protein was also found in the supernatant, indicating favorable solubility. Overall, this study demonstrates the successful integration of immunoinformatics vaccine design with experimental expression. The results confirm that the designed multi-epitope chimeric vaccine candidate can be efficiently produced as a recombinant protein in E. coli. This provides an important experimental foundation for future purification and immunological evaluation studies. This work highlights the potential of vaccine design strategies for developing next-generation vaccines against complex bacterial pathogens. Also, contributes valuable insights toward cross-protective vaccine development for Bordetella species.