Pasteurella multocida PlpE Protein Polytope as a Potential Subunit Vaccine Candidate.

Abstract

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope (PlpE1 + 2 + 3) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway.