The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal sequence of the protein defines the more than 200 M types of Strep A and also contains epitopes that elicit opsonic antibodies, some of which cross-react with heterologous M types. Current efforts to develop broadly protective M protein-based vaccines are directed at identifying potential cross-protective epitopes located in the N-terminal regions of cluster-related M proteins for use as vaccine antigens. In this study, we have used a comprehensive approach using the recurrent neural network ABCpred and IEDB epitope conservancy analysis tools to predict 16 residue linear B-cell epitopes from 117 clinically relevant M types of Strep A (~88% of global Strep A infections). Furthermore, to examine the immunogenicity of these epitope-based vaccines, nine peptides that together shared ?60% sequence identity with 37 heterologous M proteins were incorporated into two recombinant hybrid protein vaccines, in which the epitopes were repeated 2 or 3 times, respectively. The combined immune responses of immunized rabbits showed that the vaccines elicited significant levels of antibodies against all nine vaccine epitopes present in homologous N-terminal 1?50 amino acid synthetic M peptides, as well as cross-reactive antibodies against 16 of 37 heterologous M peptides predicted to contain similar epitopes. The epitope-specificity of the cross-reactive antibodies was confirmed by ELISA inhibition assays and functional opsonic activity was assayed in HL-60-based bactericidal assays. The results provide important information for the future design of broadly protective M protein-based Strep A vaccines.