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.Many childhood vaccines, such as the diphtheria-pertussistetanus vaccine, require multiple doses to achieve protection, which has led to reduced coverage of immunization. Live viral or bacterial vectors have been suggested as a solution to reduce the number of doses required for immunization. In addition, live vectors have the ability to induce a mucosal immune response following oral or nasal administration, which is typically difficult to obtain from conventional parenteral immunization. Among these bacteria, S. gordonii is a very attractive vector for childhood vaccines because it is one of the pioneer organisms in the human oral cavity. The organism can be detected in the oral cavity by 6 months of age and remains as a normal inhabitant of the oral mucosa and dental plaque in adults. Therefore, the organism has the potential of providing lifelong protection following a single oral inoculation. In addition, this bacterium is relatively easy to manipulate genetically, and the expression of a number of heterologous antigens has been reported (18,20,22,23,24,30). In the context of a live oral diphtheria-pertussis-tetanus vaccine, Medaglini et al. (24) showed that immunization with recombinant S. gordonii expressing tetanus toxin fragment C on its surface conferred protection from lethal tetanus toxin challenge in mice. In a previous work, we showed that the surface-expressed pertussis toxin S1 fragment (18, 19) and a secreted fusion protein consisting of pertussis toxin S1S3 fragments and filamentous hemagglutinin type I domain (20) produced by S. gordonii were immunogenic. These findings show that tetanus and pertussis antigens can be expressed by S. gordonii and that the antigens were immunogenic. However, the expression of diphtheria antigens in S. gordonii has not yet been reported.Diphtheria toxin (DT) is a secreted 535-amino-acid protein which is proteolytically cleaved into two fragments, A and B (4, 9). The catalytic domain is located on fragment A (amino acids 1 to 193), and the receptor and translocation domains are on fragment B (amino acids 194 to 535). Fragment B is responsible for DT binding to specific...

<i>Mycobacterium bovis</i> BCG as a Delivery System for the <i>dtb</i> Gene Antigen from Diphtheria Toxin

“…Although the expression of DT antigens in different bacterial species has been previously done [8] [37]- [41], in BCG the expressed DT antigens came from the mutant CRM 197 [27] and the diphtheria toxin dtb gene of the PW8 vaccine strain [42]. It is well known that DTB is crucial for the adherence of diphtheria toxin to target cell receptors, a fundamental step in the internalization of the DT toxic fragment A.…”

Expression of a Functional Single-Chain Variable-Fragment Antibody against Complement Receptor 1 inStreptococcus gordonii

“…The two fragments were joined together via overlapping PCR, and the resulting 1.8-kb PCR product was cloned into the SphI and EcoRI sites on the E. coli-Streptococcus shuttle vector pDL276, creating pSecCR1. pSecCR1 was transformed into S. gordonii (hppG::Tet r ) via natural transformation (13). Isolation of scFvs from E. coli.…”

“…The organism is considered to be an attractive vector as a live-oral-vaccine vehicle (14,23). A number of heterologous antigens have been expressed in this organism as either secreted proteins (15,27) or cell wall-anchored proteins (16,17,19,20,25,26). In the murine oralcolonization model, the recombinant S. gordonii was able to establish long-term colonization (16,20).…”

Mucosal Immunization with a Genetically Engineered Pertussis Toxin S1 Fragment-Cholera Toxin Subunit B Chimeric Protein

“…In the assay, microtiter plates were coated with 230 ng of G M1 ganglioside (CalBiochem, San Diego, Calif.) and blocked with 1% gelatin in phosphate-buffered saline (PBS) with 0.1% Tween 20 and 5 mM MgCl 2 , and samples were added. The chimeric protein bound to G M1 was detected with a rabbit anti-PT (1/300; [11]) or a goat anti-CtxB antibody followed by the goat anti-rabbit immunoglobulin G (IgG) (1/30,000; Sigma) or rabbit anti-goat IgG (1/20,000; Sigma) alkaline phosphatase conjugates, respectively. As shown in Fig.…”

“…Construction of the MBPS1S1CtxA2B chimera. Two copies of the DNA coding for the N-terminal 179-amino-acid sequence of the PT S1 subunit were cloned in tandem into the middle portion of spaP by ligating a 1.4-kb SmaI-KpnI fragment from pPTS1 into the NruI-KpnI sites of pRJMI (11). The DNA coding for the CtxA2 motif and CtxB was then cloned downstream to the SpaPS1S1 construct using the NruI-KpnI sites.…”