RESUMEN
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.
Asunto(s)
Vacunas contra la Malaria/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/genética , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antiprotozoarios/sangre , Proteínas Bacterianas/genética , Línea Celular , Recuento de Colonia Microbiana , Femenino , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/toxicidad , Hígado/microbiología , Macrófagos/microbiología , Vacunas contra la Malaria/genética , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Polisacáridos Bacterianos/aislamiento & purificación , Polisacáridos Bacterianos/toxicidad , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/prevención & control , Vacunas contra la Salmonella/genética , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Bazo/microbiología , Toxina Tetánica/genética , Toxina Tetánica/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Vacunas Tifoides-Paratifoides/genética , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
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.
Asunto(s)
Toxinas Bacterianas/genética , Cromosomas Bacterianos , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Vectores Genéticos , Antígenos del Núcleo de la Hepatitis B/genética , Regiones Promotoras Genéticas , Salmonella typhi/genética , Administración Intranasal , Animales , Anticuerpos Antibacterianos/sangre , Femenino , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Salmonella typhi/inmunología , Células U937RESUMEN
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.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Genes Bacterianos/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Antígenos Bacterianos/genética , Secuencia de Bases , Supervivencia Celular , Células Cultivadas , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/inmunología , Medios de Cultivo , Cartilla de ADN , Citometría de Flujo , Genes Bacterianos/genética , Genes Reporteros/genética , Humanos , Inmunoglobulina G/biosíntesis , Operón Lac/genética , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/inmunología , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas contra la Salmonella/genética , Salmonella typhimurium/genética , Vacunas de ADN/biosíntesis , Vacunas de ADN/inmunologíaRESUMEN
The S. typhimurium strain (TML deltaaroC deltassaV) WT05, harbouring defined deletions in genes involved in both the aromatic biosynthesis pathway (aroC) and the Salmonella Pathogenicity Island-2 (SPI-2) (ssaV) was shown to be significantly attenuated in C57 BL/6 interferon gamma knockout mice following oral inoculation. Similarly, the S. typhi strain (Ty2 deltaaroC deltassaV) ZH9 harbouring the aroC and ssaV mutations propagated less efficiently than wild type in human macrophages. These studies demonstrated the attractive safety profile of the aroC ssaV mutant combination. Strains S. typhimurium (TML deltaaroC deltassaV ) WT05 and S. typhi (Ty2 deltaaroC deltassaV) ZH9 were subsequently tested as vaccine vectors to deliver E. coli heat-labile toxin (LT-B) mucosally to mice. Mice inoculated orally with S. typhimurium (TML deltaaroC deltassaV) WT05 expressing LT-B (WT05/LT-B) elicited high titres of both LT-specific serum IgG and intestinal IgA, although no specific IgA was detected in the vagina. Similarly, intranasal inoculation of mice with S. typhi (Ty2 deltaaroC deltassaV) ZH9 expressing LT-B (ZH9/LT-B) elicited even higher titres of LT-specific serum antibody as well as LT-specific Ig in the vagina. We conclude that deltaaroC deltassaV strains of Salmonella are highly attenuated and are promising candidates both as human typhoid vaccines and as vaccine vectors for the delivery of heterologous antigens.