Candidate live, attenuated Salmonella enterica serotype Typhimurium vaccines with reduced fecal shedding are immunogenic and effective oral vaccines

Abstract

Environmental shedding of genetically manipulated microorganisms is an issue impeding the development of new live vaccines. We have investigated the immunogenicity of a number of novel Salmonella enterica serotype Typhimurium oral vaccine candidates that express the fragment C (TetC) component of tetanus toxin and harbor combinations of additional mutations in genes shdA, misL, and ratB that contribute to the persistence of serotype Typhimurium's colonization of the intestine. Serotype Typhimurium aroA (TetC) derivatives harboring additional mutations in either shdA or misL or combinations of these mutations exhibited a marked decrease in shedding of the vaccine strain in the feces of orally vaccinated mice. However, equivalent levels of anti-TetC and anti-Salmonella lipopolysaccharide immunoglobulin G (IgG), IgG1, IgG2a, and IgA were detected in sera of the vaccinated but not of the control mice. Cellular immune responses to TetC were detected in all vaccinated mice, regardless of the presence of the additional mutations in shdA or misL. Further, immunization with serotype Typhimurium aroA candidate vaccines harboring shdA and misL afforded complete protection against challenge with a virulent strain of serotype Typhimurium.

FIG. 1.

IgG response to serotype Typhimurium LPS in mice 21 days following inoculation with serovar Typhimurium vaccine strains. Anti-LPS IgG was quantified from serum from peripheral blood by ELISA. The titer for each animal (circle) and the mean titer for each group (horizontal bar) are indicated. The mutation(s) of the serotype Typhimurium strain tested in each group is indicated below the graph. All are aroA. − and + indicate the absence or presence of pTetCnirB.

FIG. 2.

IgG response to serotype Typhimurium LPS and TetC in animals 21 days following inoculation with serotype Typhimurium vaccine strains and following intranasal boost. IgG specific for TetC (A), IgG specific for TetC following intranasal boost (plasmidless MFA25 group was not boosted) (B), IgG2a specific for TetC (C), IgG2a specific for TetC following intranasal boost (plasmidless MFA25 group was not boosted) (D), IgG1 specific for TetC (E), or IgG1 specific for TetC following intranasal boost (plasmidless MFA25 group was not boosted) (F) was quantified from serum derived from peripheral blood by ELISA. The titer for each animal (circle) and the mean titer for each group (horizontal bar) are indicated. The mutation(s) of the serotype Typhimurium strain tested in each group is indicated below each graph. All are aroA. − and + indicate the absence or presence of pTetCnirB.

FIG. 3.

Production of IL-2 (A) and gamma interferon (B) by splenocytes derived from mice vaccinated with serotype Typhimurium vaccine strains in response to stimulation with recombinant TetC. Groups of five mice were vaccinated with serotype Typhimurium MFA25 aroA phoN, serotype Typhimurium MFA16(pTetCnirB15) aroA phoN, serotype Typhimurium MFA16(pTetCnirB15) aroA ratB, serotype Typhimurium MFA13(pTetCnirB15) aroA shdA, serotype Typhimurium MFA12(pTetCnirB15) aroA misL, or serotype Typhimurium MFA17(pTetCnirB15) aroA shdA misL. − and + indicate the absence or presence of pTetCnirB.

FIG. 4.

IgA response to serotype Typhimurium TetC in animals 21 days following inoculation with serotype Typhimurium vaccine strains. IgA specific for TetC (A) or IgG specific for TetC (B) was quantified from serum derived from peripheral blood by ELISA. The titer for each animal (circle) and the mean titer for each group (horizontal bar) are indicated. The mutations of the serotype Typhimurium strains tested are indicated below the graph. All groups are aroA. − and + indicate the absence or presence of pTetCnirB.

FIG. 5.

Survival curve of naïve mice and mice vaccinated with serotype Typhimurium. Approximately 1 × 10⁸ CFU of serotype Typhimurium SL1344 was inoculated orally into a group of 10 mice previously vaccinated with strain MFA26(pTetCnirB15) (ΔaroA ΔphoN::aph) (open squares), a group of 10 mice previously vaccinated with strain MFA17(pTetCnirB15) (ΔaroA ΔmisL::cat ΔshdA::aph) (solid circles), or a group of 5 naïve mice (open circles); the health of the mice was monitored twice daily, and the mice were sacrificed when they appeared moribund.