Analysis of factors affecting surface expression and immunogenicity of recombinant proteins expressed by gram-positive commensal vectors

Abstract

Several key protein structural attributes were altered in an effort to optimize expression and immunogenicity of a foreign protein (M protein from Streptococcus pyogenes) exposed on the surface of Streptococcus gordonii commensal bacterial vectors: (i) a shorter N-terminal region, (ii) the addition of a 94-amino-acid spacer, and (iii) the addition of extra C-repeat regions (CRR) from the M6 protein. A decrease in the amount of cell surface M6 was observed upon deletion of 10 or more amino acid residues at the N terminus. On the other hand, reactivity of monoclonal antibody to surface M6 increased with the addition of the spacer adjacent to the proline- and glycine-rich region, and an increase in epitope dosage was obtained by adding another CRR immediately downstream of the original CRR. The results obtained should facilitate the design of improved vaccine candidates using this antigen delivery technology.

FIG. 1.

Schematic representation of the M6 recombinants based on the L16 backbone. NTR, N-terminal region; triangle, cleavage point; CRR, C repeat region of the M6 protein; Pro/Gly, proline- and glycine-rich region.

FIG. 2.

Competition ELISA with S. gordonii recombinant strains expressing surface-anchored M protein versus purified M6 protein. The graphs show percent inhibition of binding of MAb 10F5 to recombinant M6 protein by decreasing concentrations of cells. (A) Strains used were L16, L6, L6:sA94, L6:sG184, and GP204, which is used as a negative control. (B) Strains used were L16, L16:sA94, and L16:sA94: XCRR. Undil., undiluted.

FIG. 3.

Serum IgG ELISA results for pooled sera from mice inoculated with L6, L6:sA94, and L6:sG184 (A) or L16, L16:sA94, and L16:sA94:XCRR (B) collected at 0, 4, 8, and 12 weeks postinoculation.

FIG. 4.

Salivary IgA ELISA results of pooled saliva from mice inoculated with L6, L6:sA94, and L6:sG184 (A) or L16, L16:sA94, and L16:sA94:XCRR (B) collected at 0, 4, 8, and 12 weeks postinfection.