Levels of Expression and Immunogenicity of Attenuated Salmonella enterica Serovar Typhimurium Strains Expressing Escherichia coli Mutant Heat-Labile Enterotoxin

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium Δcya Δcrp Δasd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated Δcya Δcrp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.

Enterotoxigenic Escherichia coli strains produce a plasmid-encoded heat-labile enterotoxin (LT) (15, 34) related to cholera toxin (CT) (9, 35). LT is composed of two subunits, A and B, which are exported to the periplasmic space, where they assemble into an AB₅ multimeric complex (16). Several mutants of LT-A have been constructed, and in particular, a nontoxic mutant which contains a substitution of serine 63 with lysine (LTK63) has been shown to maintain the structural and immunogenic properties of wild-type LT (21, 27, 28). LTK63 has also been found to display the strong mucosal adjuvant activity pertaining to wild-type LT. Efficient induction of mucosal immune response, specifically in the mouse vagina, has been achieved via the intranasal route of immunization (10). For the development of oral vaccines, however, it would be desirable to exploit the properties of LTK63 for enhancing antigen-specific immune response in the intestinal mucosa by means of oral delivery of the potent mucosal adjuvant.Oral delivery of antigens by live vaccines is known to lead to a more effective production of antigen-specific antibodies in mucosal secretions than oral administration of the soluble antigen (36, 39). Several antigen delivery systems which use as carriers mutant intracellular pathogens that have lost the ability to persist and produce the disease while retaining limited growth in vivo have been developed. In particular, attenuated Salmonella mutants are suitable immunological carriers for virulence determinants from other enteric bacteria in that they can induce humoral immune response selectively at the site of colonization, the gut mucosa. Vaccine strains of Salmonella have been successfully attenuated by introducing different types of mutations (5, 8, 23, 26). Notably, Salmonella strains with a galactose epimerase (galE) mutation (18) or deletions in genes for the biosynthesis of aromatic compounds (aro mutants) (11, 12, 17, 19) or in the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes (6) are the most extensively characterized.Delivery of the B subunit of the E. coli enterotoxin (LT-B) by a galE mutant of Salmonella typhimurium has been shown to elicit low levels of anti-LT-B serum and mucosal antibodies. Since the vector used for expression of LT-B was rapidly lost in vivo, i.e., in the absence of the antibiotic required for selection of the plasmid, the level of immune response could be correlated only with the amount of antigen expressed during the initial phase of invasion (3).Recently, direct comparison between the aroA aroD/pnirB and the Δcya Δcrp Δasd/asd⁺ delivery systems for the ability to induce humoral and cellular immunity after a single immunization showed that the former vaccine strain had greater potential as a carrier for antigen delivery (20). However, the balanced lethal asd system for in vivo selection of plasmids expressing heterologous antigens in the attenuated Δcya Δcrp Δasd strains is still very attractive in that asd⁺ plasmids do not require antibiotic resistance markers for selection while stably maintained in vivo (24). In addition, the Δcya Δcrp Δasd/asd⁺ delivery system has been reported to induce protective immunity against several pathogens (25, 29, 40). Most of these studies have restricted analysis of the immune response to antigens expressed from the same asd⁺ plasmid carried by Δcya Δcrp Δasd mutants usually of the same S. typhimurium serotype. In this work, we have analyzed the influence of heterologous gene dosage, and thus level of expression, as well as S. typhimurium strain variability on immune response toward both the heterologous antigen, a nontoxic mutant of E. coli LT, and the carrier Salmonella strain. Effects of a single integration into the host DNA and episomal vectors at different copy numbers were compared in S. typhimurium strains of two different Δcya Δcrp Δasd serotypes, UK-1 and SR-11.