Expression and immunogenicity of a recombinant diphtheria toxin fragment A in Streptococcus gordonii

Abstract

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA(2)) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA(2) was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.

FIG. 1.

Schematic representation of SpaP-DTA fusion proteins. The sizes of SpaP (open bar) and DTA (solid bar) are indicated by the number of amino acids (a.a.). Hatched bars indicate the C-terminal cell wall-anchoring domain of SpaP.

FIG. 2.

Expression of recombinant DTA by S. gordonii. (A) Recombinant DTA proteins on the cell surface (solid bars) and in culture supernatants (open bars) were detected by ELISA. A₄₀₅ readings were determined from diluted samples equivalent to culture optical densities at 600 nm of 0.25. (B) Recombinant DTA proteins were detected in cell extracts and culture supernatants by Western immunoblotting. Lane 1, S. gordonii DTA; lane 2, S. gordonii DTA₂; lane 3, S. gordonii DTA₃; lane 4, cell extracts of S. gordonii DL-1 (negative control). (C) Proteins were extracted from cells by previously described methods (13). S, proteins from culture supernatant fluids. Prestained protein size markers are shown on the left. In both panels, the proteins were detected with a mouse anti-DT antibody.

FIG. 3.

Western immunoblots showing recognition of native DT by antiserum obtained from mice immunized with heat-killed recombinant S. gordonii. DT (1 μg) was separated on an SDS-10% PAGE gel. Mouse antisera: lane 1, S. gordonii DTA; lane 2, S. gordonii DTA₂; lane 3, S. gordonii DTA₃. P, preimmune serum; I, immune serum; +, anti-DT antiserum; M, prestained protein size markers (sizes shown in kilodaltons).

FIG. 4.

Immune responses to S. gordonii DTA₂ in BALB/c mice. (A) Anti-DT and anti-CTB antibodies elicited by intranasal immunization with live S. gordonii DTA₂ in the presence of CTB or with CTB alone. (B) Anti-DT antibodies induced by oral colonization with S. gordonii DTA₂. The values shown are mean titers ± standard error for individual animal samples. In the CTB-alone and SL3 groups, the absence of a bar indicates that an immune response was not detected. VW, vaginal wash.