RESUMO
BACKGROUND: M01ZH09 (Salmonella enterica serovar Typhi [Ty2 aroC(-) ssaV(-)] ZH9) is a live oral-dose typhoid vaccine candidate. M01ZH09 was rationally modified with 2 independently attenuating mutations, including a novel mutation in Salmonella pathogenicity island (SPI)-2. We demonstrate that M01ZH09, in a single oral dose, is well tolerated and prompts broad immune responses, regardless of whether prevaccination with a bicarbonate buffer is given. METHODS: Thirty-two healthy adult subjects were randomized and given 5x109 cfu of M01ZH09, with (presentation 1) or without (presentation 2) prevaccination with a bicarbonate buffer. Immunogenicity data included Salmonella Typhi lipopolysaccharide (LPS)-specific immunoglobulin (Ig) A antibody-secreting cells (enzyme-linked immunospot [ELISPOT] assay), IgG serologic responses to Salmonella Typhi LPS, lymphocyte proliferation, and interferon (IFN)- gamma production. RESULTS: The vaccine was well tolerated; adverse events after vaccination were mild. No fever or prolonged vaccine shedding occurred. Immunogenicity data demonstrated that 88% and 93% of subjects who received presentation 1 and presentation 2, respectively, had a positive response by ELISPOT assay; 81% of subjects in both groups underwent IgG seroconversion on day 14. Both groups had similar cellular immune responses to presentation 1 and presentation 2; lymphocyte proliferation to Salmonella Typhi flagellin occurred in 63% and 67% of subjects, respectively, and 69% and 73% of subjects, respectively, had an increase in IFN- gamma production. CONCLUSION: The oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in a single oral dose, with and without prevaccination with a bicarbonate buffer. Field studies to demonstrate protective efficacy are planned.
Assuntos
Lipopolissacarídeos/imunologia , Febre Tifoide/imunologia , Vacinas Tíficas-Paratíficas/imunologia , Administração Oral , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Ensaio de Imunoadsorção Enzimática , Feminino , Liofilização , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Salmonella enterica/imunologia , Salmonella typhi/imunologia , Vacinas Tíficas-Paratíficas/administração & dosagem , Vacinas Tíficas-Paratíficas/efeitos adversos , Vacinas Tíficas-Paratíficas/toxicidade , Vacinas AtenuadasRESUMO
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
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.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Proteínas de Escherichia coli , Vetores Genéticos/imunologia , Hidrocarbonetos Aromáticos/metabolismo , Proteínas de Membrana/imunologia , Salmonella typhi/genética , Salmonella typhi/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/imunologia , Sobrevivência Celular , Enterotoxinas/biossíntese , Enterotoxinas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunidade nas Mucosas/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Interferon gama/genética , Interferon gama/fisiologia , Intestinos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fenótipo , Vagina/imunologiaRESUMO
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.
Assuntos
Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/imunologia , Genes Bacterianos/imunologia , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Antígenos de Bactérias/genética , Sequência de Bases , Sobrevivência Celular , Células Cultivadas , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/imunologia , Meios de Cultura , Primers do DNA , Citometria de Fluxo , Genes Bacterianos/genética , Genes Reporter/genética , Humanos , Imunoglobulina G/biossíntese , Óperon Lac/genética , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas contra Salmonella/genética , Salmonella typhimurium/genética , Vacinas de DNA/biossíntese , Vacinas de DNA/imunologiaRESUMO
The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 Delta aroC Delta ssaV, designated ZH9) and S. enterica serovar Typhimurium (TML Delta aroC Delta ssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 10(7), 10(8), or 10(9) CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 10(8) and two of three receiving 10(9) CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 10(8) and 10(9) CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 10(9) CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.