Protective immunogenicity of group A streptococcal M-related proteins

Abstract

Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines.

FIG 1

Alignment of N-terminal amino acid sequences of 33 Mrps from different M types of GAS (A) and alignment of the three Mrp sequences representing the three structurally related families (B). Numbers indicate the M type from which the Mrp sequence was derived. Highlighting indicates identical or similar amino acids.

FIG 2

Phylogenetic tree analysis of Mrp sequences, indicating three related families based on the amino acid sequences of the N-terminal semiconserved regions.

FIG 3

Bactericidal (OPK) activity of rabbit MrpN antisera against different emm types of GAS expressing Mrps in the homologous family. Black and gray bars represent the percent killing observed in two independent assays with blood from different donors. GAS of M types 3, 18, and 24 served as Mrp-negative controls.

FIG 4

Kaplan-Meier survival plot of mice (n = 15 per group) that were passively immunized i.p. with rabbit antiserum against Mrp4N (circles) or normal rabbit serum (triangles) and then challenged 24 h later with M28 GAS via the i.p. route. Survival after 14 days was 67% in the Mrp4N group versus 26% in the control group: P = 0.046, log rank chi-square test on Kaplan-Meier survival data.

FIG 5

Mrp antibody levels in sera from children ages 2 to 16. Serum samples were collected randomly from the clinical laboratories of a pediatric hospital in Memphis, TN. All sera were diluted 1:200 and assayed in duplicate by ELISA against Mrp2N, Mrp4N, and Mrp49N. The average OD values from each serum sample against all three Mrps were added and plotted against the age of the subject. Pearson's correlation coefficient was used to determine the significance of the relationship between the age of the subject and the OD value: r = 0.24, P < 0.001, n = 281.

FIG 6

Binding to the surface of intact GAS and bactericidal activity of affinity-purified human Mrp4N (MrpII family) antibodies against different emm types expressing homologous (MrpII) or heterologous (MrpIII) Mrps (A) and the relationship between Mrp antibody binding to GAS and observed bactericidal activity (B). Pearson's correlation coefficient was used to determine the significance of the relationship between Mrp antibody binding and bactericidal activity: r = 0.86, P < 0.05, n = 6.