Microb Pathog. 2013 May;58:17-28.

New technologies in developing recombinant attenuated Salmonella vaccine vectors.

Wang S, Kong Q, Curtiss R 3rd.

Center for Infectious Diseases and Vaccinology, The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401, USA.

 

Abstract

Recombinant attenuated Salmonella vaccine (RASV) vectors producing recombinant gene-encoded protective antigens should have special traits. These features ensure that the vaccines survive stresses encountered in the gastrointestinal tract following oral vaccination to colonize lymphoid tissues without causing disease symptoms and to result in induction of long-lasting protective immune responses. We recently described ways to achieve these goals by using regulated delayed in vivo attenuation and regulated delayed in vivo antigen synthesis, enabling RASVs to efficiently colonize effector lymphoid tissues and to serve as factories to synthesize protective antigens that induce higher protective immune responses. We also developed some additional new strategies to increase vaccine safety and efficiency. Modification of lipid A can reduce the inflammatory responses without compromising the vaccine efficiency. Outer membrane vesicles (OMVs) from Salmonella-containing heterologous protective antigens can be used to increase vaccine efficiency. A dual-plasmid system, possessing Asd+ and DadB+ selection markers, each specifying a different protective antigen, can be used to develop multivalent live vaccines. These new technologies have been adopted to develop a novel, low-cost RASV synthesizing multiple protective pneumococcal protein antigens that could be safe for newborns/infants and induce protective immunity to diverse Streptococcus pneumoniae serotypes after oral immunization. Copyright © 2012 Elsevier Ltd.

PMID: 23142647

 

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Figure 1 – Three stages in the delivery of a recombinant attenuated Salmonella vaccine with regulated delayed attenuation, regulated delayed antigen synthesis and regulated delayed lysis in vivo. (In Vitro) The first panel shows a Salmonella bacterium as it is grown in vitro in medium supplemented with the sugars arabinose and mannose. LPS O-antigen is synthesized with exogenously provided mannose and is attached to the LPS core. Several key proteins are shown being produced with the exogenously provided arabinose (colored dots). Fur (iron uptake regulatory protein) and Crp (cAMP receptor protein) enable bacteria to display wild-type virulence. MurA and Asd are enzymes made in the presence of arabinose that are required for synthesis of the essential peptidoglycan cell wall components muramic acid and diaminopimelic acid, respectively, to maintain the integrity of the cell wall. The LacI repressor protein is also synthesized in the presence of arabinose and acts to repress protective antigen production. (In Vivo Initial) The middle panel shows the consequences of the vaccine entering the in vivo environment with the unavailability of the sugars arabinose and mannose. The absense of arabinose causes a reduction in the Fur and Crp proteins that are diluted out as a consequence of cell division, which gradually attenuates the vaccine. Reduced Fur protein results in increased synthesis of iron-regulated outer membrane proteins (IROMPs), which are immunodominant protective antigens and also result in iron uptake to toxic levels. Proteins MurA and Asd are also reduced which results in decreased bacterial cell wall peptidoglycan synthesis. The absence of the arabinose also causes a reduction in LacI concentration due to cell division and this initiates the production of the vaccine protective antigen (black dots). The absence of mannose ceases synthesis of new LPS O-antigen such that LPS cores with LPS O-antigen attached decrease as a consequence of cell division. (In Vivo Late) The final panel shows the complete lysis of the bacterium following some 5 to 10 cell divisions with the absence of Fur, Crp, LacI, MurA and Asd proteins resulting in the release of the protective antigen which has been synthesized in large amount prior to cell lysis. There is also release of IROMPS and LPS cores that are highly immunogenic and cross-protective against all Salmonella serotypes. Lysing of the bacterial vaccine strain also ensures no persistance of vaccine cells in vivo, and no survival if released into the environment through shedding.

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