Opsonic human antibodies from an endemic population specific for a conserved epitope on the M protein of group A streptococci

Abstract

SUMMARYThis study demonstrates the presence of epitope-specific opsonic human antibodies in a population living in an area endemic for group A streptococci (GAS) infection. Antibodies recognizing a conserved C-terminal region epitope (p145, sequence in single letter amino acids: LRRDLDASREAKKQVE-KALE) of the M protein of GAS were isolated from human patients by affinity chromatography and were shown to be of the immunoglobulin G1 (IgG1) and IgG3 subclasses. These antibodies could reduce the number of colonies of serotype 5 GAS in an in vitro opsonization assay by 71-92%, compared with an equal amount of IgG from control adult donors living in non-endemic areas and without antibodies to p145. Addition of the peptide, p145, completely inhibited this opsonization. Indirect immunofluorescence showed that p145-specific antibodies were capable of binding to the surface of M5 GAS whereas control IgG did not. Using chimeric peptides, which contain overlapping segments of p145, each 12 amino acids in length, inserted into a known helical peptide derived from the DNA binding protein of yeast, GCN4, we have been able to further define two minimal regions within p145, referred to as pJ2 and pJ7. These peptides, pJ2 and pJ7, were able to inhibit opsonization by p145 specific antibodies. Finally, we have observed an association between the age-related development of immunity to GAS and the acquisition of antibodies to the conserved epitope, p145, raising the possibility of using this epitope as a target in a prophylactic vaccine administered during early childhood.

Protective and Nonprotective Epitopes from Amino Termini of M Proteins from Australian Aboriginal Isolates and Reference Strains of Group A Streptococci

“…Fifty microliters of the bacteria dilution was mixed with 50 l of fresh heat-inactivated serum and 400 l of nonopsonic heparinized donor blood, as a source of neutrophil and complement. All donor blood was screened prior to assay to ensure that it could support the growth of the GAS strain to at least 32 times the inoculum level in a 3-h incubation at 37°C (4,5). The mixture was incubated end over end at 37°C for 3 h, 50 l from each tube was plated out, in duplicate, on THB blood agar pour plates and incubated overnight, and the number of colonies on each plate was counted.…”

“…2 A, C, and F, respectively), which correlated to ELISA cross-reactivity. Antiserum to 88/ 30 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] could also cross-opsonize two other isolates, 2034 (97.1%), which correlated with ELISA cross-reactivity, and Y530S (97.4%), which did not correlate with ELISA crossreactivity ( Fig. 2C and D, respectively).…”

Divergence in the Plasminogen-binding Group A Streptococcal M Protein Family

“…Murine sera were assayed for the ability to opsonize GAS in vitro as described previously (31). Briefly, GAS strains NS13 and NS455 were grown overnight at 37°C in 5 ml of THB, followed by serial dilution to FIGURE 1.…”

“…Following this incubation, 400 l of nonopsonic heparinized human blood was added and incubated at 37°C with end-over-end mixing. All human blood was tested prior to performing the assay to ensure that it could support the growth of the GAS strain to at least 32 times the colony-forming units (CFU) of the inoculum in a 3-h incubation at 37°C (31)…”

In VivoEfficacy of a Chimeric Peptide Derived from the Conserved Region of the M Protein against Group C and G Streptococci

“…A direct bactericidal assay was performed as previously described (3,4,24). Pooled immune sera from cohorts of mice immunized with MD605 surface protein or J8-DT were capable of the opsonization of the MD605 GCS, with 78% and 39% reductions in CFU, respectively (P values of Ͻ0.05 and Ͻ0.05, respectively, in a nonparametric t test compared to the PBS control group).…”