Mutagenesis and immunological evaluation of group A streptococcal C5a peptidase as an antigen for vaccine development and as a carrier protein for glycoconjugate vaccine design

Abstract

A truncated form (AA32-1032) of group A streptococcus (GAS) C5a peptidase (ScpA), an important GAS virulence factor, and its mutants were prepared and examined to find suitable GAS vaccine candidates and conjugate vaccine carriers. Enzymatic evaluation of the recombinant proteins with a MALDI-TOF MS-based method to analyze the reaction indicated that D130 and N295 were not critical for its activity and S512 was significant but not absolutely required either. Therefore, ΔScpA^D130A, ΔScpA^N295A and ΔScpA^S512A were not suitable vaccine and carrier protein candidates due to their remaining enzymatic activity. A single mutation of H193 to Ala abolished the ScpA activity completely, thereby identifying ΔScpA^H193A as a promising candidate that was subjected to immunological studies in mouse. It was shown to elicit high titers of antigen-specific IgG1 antibodies and robust T cell-mediated immunities, verifying its potential as a GAS vaccine. Moreover, conjugating the trisaccharide repeating unit of GAS polysaccharide with ΔScpA^H193A could convert the nonimmunogenic oligosaccharide into a highly active and T cell-dependent antigen, demonstrating the potential of ΔScpA^H193A as a carrier protein to help formulate robust glycoconjugate vaccines.