Construction of an ASD+ Expression-Cloning Vector: Stable Maintenance and High Level Expression of Cloned Genes in a Salmonella Vaccine Strain

Nakayama, Koji; Kelly, Sandra M.; Curtiss, Roy

doi:10.1038/nbt0688-693

Cited by 296 publications

(261 citation statements)

References 32 publications

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“…Chrome Azurol S (CAS) plates were used to confirm the constitutive synthesis of siderophores characteristic of fur mutants (28). The presence of the ⌬asdA33 and ⌬asdA16 mutations in Salmonella was confirmed by inability of the strain to grow on media without DAP (27). LPS profiles of Salmonella strains were examined as described (29).…”

Section: Methodsmentioning

confidence: 99%

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Evaluation of new generationSalmonella entericaserovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA

Wang

Scarpellini

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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112

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Increasing the immunogenicity to delivered antigens by recombinant attenuated Salmonella vaccines (RASV) has been the subject of intensive study. With this goal in mind, we have designed and constructed a new generation of RASV that exhibit regulated delayed attenuation. These vaccine strains are phenotypically wild type at the time of immunization and become attenuated after colonization of host tissues. The vaccine strains are grown under conditions that allow expression of genes required for optimal invasion and colonization of host tissues. Once established in the host, these virulence genes are turned off, fully attenuating the vaccine strain. In this study, we compared 2 of our newly developed regulated delayed attenuation Salmonella enterica serovar Typhimurium strains 9088 and 9558 with the ⌬cya ⌬crp ⌬asd strain 8133, for their abilities to express and present a secreted form of the ␣-helical region of pneumococcal surface protein A (PspA) to the mouse immune system. All 3 strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens in orally immunized mice. However, both RASVs expressing delayed attenuation elicited significantly greater anti-PspA immune responses, including serum IgG and T cell secretion of IL-4 and IFN-␥, than other groups. Also, vaccination with delayed attenuation strains resulted in a greater degree of protection against Streptococcus pneumoniae challenge than in mice vaccinated with 8133 (71-86% vs. 21% survival, P < 0.006). Together, the results demonstrate that the regulated attenuation strategy results in highly immunogenic antigen delivery vectors for oral vaccination.Streptococcus pneumoniae ͉ bacterial vectors G enerating a safe and immunogenic vaccine strain is the biggest challenge in the development of live Salmonella vaccines (1). An ideal Salmonella vaccine strain should exhibit wild-type abilities to withstand stresses (enzymatic, acid, osmotic, ionic, etc.) and host defenses (bile, antibacterial peptides, etc.) encountered after oral or intranasal immunization, and should exhibit wild-type ability to colonize and invade host lymphoid tissues while remaining avirulent. Various attenuated Salmonella strains have been used as live vaccines to induce mucosal and systemic immunity against either the carrier itself or to a vectored antigen (2). Salmonella vaccine strains typically carry defined deletion mutations affecting either metabolic functions or virulence factors (3). Various attenuating mutations have been investigated in the pursuit to develop optimal immune responses (4, 5). Many attenuating mutations were found to either reduce Salmonella survival due to host-induced stresses and/or reduce colonization of lymphoid effector tissues leading to less than ideal immunogenicity (6, 7). To circumvent these problems, we explored ways to achieve regulated delayed attenuation in vivo (8, 9) to create vaccine strains that are phenotypically wild-type at the time of immunization and become attenuated after colonization of host tissues.O...

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Section: Methodsmentioning

confidence: 99%

“…No additions were made to the media for growing plasmid-containing 8133 cultures. DAP was added (50 g/mL) for the growth of Asd Ϫ strains (27). S. pneumoniae WU2 was cultured on brain heart infusion (BHI) agar containing 5% sheep blood or in Todd-Hewitt broth plus 0.5% yeast extract (25).…”

Section: Methodsmentioning

confidence: 99%

Evaluation of new generationSalmonella entericaserovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA

Wang

Scarpellini

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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112

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“…When pOVA was used, carbenicillin was substituted for kanamycin. 4550 pYA3259rOVA (called 4550 rOVA) and 4550 pYA3259rOVA-GFP (called 4550 rOVAGFP), which contain the cloned inserts in a balanced lethal vector system to allow plasmid maintenance in the absence of antibiotic selection (30), were grown in LB broth without antibiotics.…”

Section: Bacterial Strains and Infection Of Micementioning

confidence: 99%

Antigen Presentation Capacity and Cytokine Production by Murine Splenic Dendritic Cell Subsets upon Salmonella Encounter

Yrlid

Wick

2002

The Journal of Immunology

104

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Salmonella typhimurium is an intracellular bacterium that replicates in the spleen and mesenteric lymph nodes (MLN) of orally infected mice. However, little is known about the Ag presentation and cytokine production capacity of dendritic cells (DC), particularly CD8α+, CD8α−CD4−, and CD8α−CD4+ DC, from these organs in response to Salmonella. Infection of purified splenic DC with S. typhimiurium expressing green fluorescent protein (GFP) and OVA revealed that all three splenic DC subsets internalize bacteria, and splenic as well as MLN DC process Salmonella for peptide presentation. Furthermore, presentation of Salmonella Ags on MHC-I and MHC-II was evident in both CD8α+ and CD8α− splenic DC subsets. Direct ex vivo analysis of splenic DC from mice infected with GFP-expressing Salmonella showed that all three subsets harbored bacteria, and splenic DC purified from mice given Salmonella-expressing OVA presented OVA-derived peptides on MHC-I and MHC-II. Cytokine production analyzed by intracellular staining of splenic DC infected with GFP-expressing Salmonella revealed that TNF-α was produced by a large percentage of CD8α− DC, while only a minor proportion of CD8α+ DC produced this cytokine following bacterial exposure. In contrast, the greatest number of IL-12p40-producing DC were among CD8α+ DC. Experiments inhibiting bacterial uptake by cytochalasin D as well as use of a Transwell system revealed that bacterial contact, but not internalization, was required for cytokine production. Thus, DC in sites of Salmonella replication and T cell activation, spleen and MLN, respond to bacterial encounter by Ag presentation and produce cytokines in a subset-specific fashion.

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“…An alternative for increasing the stability of expression at higher copy numbers is to use a balanced^lethal system in which a lethal chromosomal mutation is complemented by a plasmid that carries both a functional copy of the same gene and an expression cassette for the foreign antigen. In one such system, a mutation in the chromosomal aspartate L-semialdehyde dehydrogenase (asd) gene leads to an obligate requirement for diaminopimelic acid, which can be complemented by a plasmid carrying a functional copy of the Salmonella asd gene [56,57]. In another balanced^lethal system, thymidine requirement caused by a mutation in the chromosomal thymidylate synthetase (thyA) gene is complemented by a plasmid carrying a functional thyA gene [58].…”

Section: Antigen Expressionmentioning

confidence: 99%

Recombinant live Salmonella spp. for human vaccination against heterologous pathogens

Bumann

Hueck²,

Aebischer

et al. 2000

FEMS Immunology and Medical Microbiology

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Construction of an ASD+ Expression-Cloning Vector: Stable Maintenance and High Level Expression of Cloned Genes in a Salmonella Vaccine Strain

Cited by 296 publications

References 32 publications

Evaluation of new generationSalmonella entericaserovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA

Evaluation of new generationSalmonella entericaserovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA

Antigen Presentation Capacity and Cytokine Production by Murine Splenic Dendritic Cell Subsets upon Salmonella Encounter

Recombinant live Salmonella spp. for human vaccination against heterologous pathogens

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