Construction and characterization of bivalent Shigella flexneri 2a vaccine strains SC608(pCFAI) and SC608(pCFAI/LTB) that express antigens from enterotoxigenic Escherichia coli.

An invasive strain of Shigella flexneri 2a (SC608) has been developed as a vector for the expression and delivery of heterologous antigens. SC608 is an aspartate semialdehyde dehydrogenase (asd) derivative of SC602 (icsA iuc), a well-characterized live attenuated vaccine strain which has undergone several clinical trials in human volunteers. When administered orally at a single 10(4) (CFU) dose, SC602 is both immunogenic and efficacious against shigellosis. Using asd-based plasmid vectors, we designed SC608 to express the enterotoxigenic Escherichia coli (ETEC) fimbrial subunit CfaB (CFA/I structural subunit) alone or in combination with the E. coli B subunit of heat-labile enterotoxin (LTB). The expression of each heterologous protein in SC608 was verified by immunoblot analysis. Each strain was comparable to the parent strain, SC602, in a HeLa cell invasion assay. After intranasal immunizations of guinea pigs, serum and mucosal immune responses were detected against both Shigella lipopolysaccharide and heterologous ETEC antigens by enzyme-linked immunosorbent assay and ELISPOT analysis. All immunized animals were subsequently protected against a challenge with wild-type S. flexneri 2a in a keratoconjunctivitis Sereny test. Serum antibodies generated against LTB and CfaB demonstrated antitoxin and agglutination activities, respectively. These results suggest that CfaB and LTB expressed in SC608 retain important conformational epitopes that are required for the generation of antibodies that have functional activities. These initial experiments demonstrate that a fully invasive Shigella vaccine strain can be engineered to deliver antigens from other diarrheal pathogens.

Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine.

Attenuated Salmonella enterica serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and Salmonella antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the Salmonella vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic alpha-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the beta-lactamase signal sequence in the multicopy Asd(+) pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd(+) vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The S. enterica serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations Delta crp-28 and Delta asdA16. Approximately 50% of the recombinant PspA (rPspA) expressed in a Salmonella strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant Salmonella. After a single oral immunization in BALB/c mice with 10(9) CFU of the recombinant Salmonella vaccine strain carrying pYA3494, immunoglobulin G (IgG) antibody responses were stimulated to both the heterologous antigen rPspA and Salmonella lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were IgG1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were IgG2a (indicating a Th1-type response). A sublethal infection with Streptococcus pneumoniae WU2 boosted PspA antibody levels and maintained IgG2a/IgG1 ratios similar to those seen before the challenge. Oral immunization with Salmonella-PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent S. pneumoniae WU2.