RESUMO
Live Salmonella vaccines are limited in use by the inherent toxicity of the lipopolysaccharide. The waaN gene encodes a myristyl transferase required for the secondary acylation of lipid A in lipopolysaccharide. A waaN mutant exhibits reduced induction of the inflammatory cytokines associated with lipopolysaccharide toxicity. Here the characteristics of a Salmonella enterica serovar Typhimurium aroA waaN mutant (SK100) in vitro and in vivo compared with its parent aroA strain (SL3261) were described. Phenotypic analysis of purified lipopolysaccharide obtained from SK100 confirmed that the physical and biological activities of the lipopolysaccharide had been altered. Nevertheless both strains had similar patterns of colonization and persistence in mice and significantly the aroA waaN mutant was equally as effective as the parent at protecting against challenge with wild-type S. Typhimurium. Furthermore, a SK100 strain was constructed expressing both tetanus toxin fragment C and the circumsporozoite protein of a malaria parasite. In marked contrast to its isogenic parent, the new attenuated strain induces significantly enhanced immune responses against the circumsporozoite protein. The waaN mutation enhances the ability of this strain to elicit immune responses towards guest antigens. This study provides important insights into the development of safe and effective multivalent Salmonella vaccines.
Assuntos
Vacinas Antimaláricas/imunologia , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antiprotozoários/sangue , Proteínas de Bactérias/genética , Linhagem Celular , Contagem de Colônia Microbiana , Feminino , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/toxicidade , Fígado/microbiologia , Macrófagos/microbiologia , Vacinas Antimaláricas/genética , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/toxicidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/prevenção & controle , Vacinas contra Salmonella/genética , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Baço/microbiologia , Toxina Tetânica/genética , Toxina Tetânica/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Vacinas Tíficas-Paratíficas/genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
Novel candidate live oral vaccines based on a Salmonella enterica serovar Typhi ZH9 (Ty2 DeltaaroC DeltassaV) derivative that directed the expression of either the B subunit of Escherichia coli heat-labile toxin or hepatitis B virus core antigen from the bacterial chromosome using the in vivo inducible ssaG promoter were constructed. The levels of attenuation of the two S. enterica serovar Typhi ZH9 derivatives were similar to that of the parent as assessed by measuring the replication of bacteria within human macrophage-like U937 cells. The expression of heterologous antigen in the respective S. enterica serovar Typhi ZH9 derivatives was up-regulated significantly within U937 cells compared to similar S. enterica serovar Typhi ZH9 derivative bacteria grown in modified Luria-Bertani broth supplemented with aromatic amino acids. Immunization of mice with these S. enterica serovar Typhi ZH9 derivatives stimulated potent antigen-specific serum immunoglobulin G responses to the heterologous antigens.
Assuntos
Toxinas Bacterianas/genética , Cromossomos Bacterianos , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B/genética , Regiões Promotoras Genéticas , Salmonella typhi/genética , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Feminino , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Salmonella typhi/imunologia , Células U937RESUMO
DNA derived from regions upstream of the Salmonella enterica serovar Typhimurium ssaG gene were used to drive expression of different reporter genes in putative Salmonella vaccine strains. Expression from ssaG was shown to be significantly upregulated once Salmonella had entered murine or human macrophages, and levels of expression were dependent on the length of the ssaG 5' sequence incorporated. S. Typhimurium derivatives harbouring the Escherichia coli heat labile toxin B subunit (LT-B) fused to various lengths of the ssaG promoter region were also constructed as single copy chromosomal integrations. Expression of LT-B by these Salmonella derivatives was detected at significant levels after intra-macrophage survival and mice immunised with these derivatives mounted marked anti-LT-B humoral antibody responses.