New technologies in using recombinant attenuated Salmonella vaccine vectors

Abstract

Recombinant attenuated Salmonella vaccines (RASVs) have been constructed to deliver antigens from other pathogens to induce immunity to those pathogens in vaccinated hosts. The attenuation means should ensure that the vaccine survives following vaccination to colonize lymphoid tissues without causing disease symptoms. This necessitates that attenuation and synthesis of recombinant gene encoded protective antigens do not diminish the ability of orally administered vaccines to survive stresses encountered in the gastrointestinal tract. We have eliminated these problems by using RASVs with regulated delayed expression of attenuation and regulated delayed synthesis of recombinant antigens. These changes result in RASVs that colonize effector lymphoid tissues efficiently to serve as "factories" to synthesize protective antigens that induce higher protective immune responses than achieved when using previously constructed RASVs. We have devised a biological containment system with regulated delayed lysis to preclude RASV persistence in vivo and survival if excreted. Attributes were added to reduce the mild diarrhea sometimes experienced with oral live RASVs and to ensure complete safety in newborns. These collective technologies have been used to develop a novel, low-cost, RASV-synthesizing, multiple-protective Streptococcus pneumoniae antigens that will be safe for newborns/infants and will induce protective immunity to diverse S. pneumoniae serotypes after oral immunization.

FIGURE 1

The regulated programmed lysis system. A, Map of plasmid pYA3681. Plasmid sequences include the trpA, rrfG, and 5S ribosomal RNA transcriptional terminators; the P_BAD, P_trc, and P22 P_R promoters; the araC gene; and the start codon-modified murA and asdA genes preceded by their SD sequences. B, Diagram of model illustrating the regulatory interactions in the programmed lysis system. Details of this system relative to expression of murA and asdA genes and repression of anti-mRNA and protective antigen gene transcription in vitro when arabinose is present and the opposite activities in vivo when arabinose is absent are detailed more fully in the text. (Slight modification of Figure 2 from Kong et al., copyright 2008, National Academy of Sciences. Used with permission.)

FIGURE 2

Asd⁺ secretion vectors pYA4102 and pYA4106 with omission of P_asdA but with retention of the SD sequence preceding the asdA gene start codon. The −35, −10, and SD sequences associated with P_trc are indicated, and the translation start codon is in boldface. An arrow within the sequence indicates the signal peptidase cleavage site. Unique restriction enzyme sites in the multi-cloning site are indicated. 5ST1T2 is a transcriptional terminator. Both plasmids contain the pBR ori. A, ompA SS vector pYA4102. The map of pYA4102 and the nucleotide sequences of the P_trc promoter region and multi-cloning sites are shown. B, phoA SS vector pYA4106. The map of pYA4106 and the nucleotide sequences of the P_trc promoter region and multi-cloning sites are shown. (Slight modification of Figure 1 from Xin et al. and republished with permission from the American Society for Microbiology. Copyright 2008, American Society for Microbiology.)