Recombinant bacterial vaccines must be fully attenuated for animal or human hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Unfortunately, many well-studied means for attenuating Salmonella render strains more susceptible to host defense stresses encountered following oral vaccination than wild-type virulent strains and/or impair their ability to effectively colonize the gut-associated and internal lymphoid tissues. This thus impairs the ability of recombinant vaccines to serve as factories to produce recombinant antigens to induce the desired protective immunity. To address these problems, we designed strains that display features of wild-type virulent strains of Salmonella at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. We recently described one means to achieve this based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen. We report here a second means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC P BAD cassette for the promoters of the fur, crp, phoPQ, and rpoS genes such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur, Crp, PhoPQ, and/or RpoS proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Means for achieving regulated delayed attenuation can be combined with other mutations, which together may yield safe efficacious recombinant attenuated Salmonella vaccines.
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...
Recombinant attenuated Salmonella vaccines (RASVs) have been used extensively to express and deliver heterologous antigens to host mucosal tissues. Immune responses can be enhanced greatly when the antigen is secreted to the periplasm or extracellular compartment. The most common method for accomplishing this is by fusion of the antigen to a secretion signal sequence. Finding an optimal signal sequence is typically done empirically. To facilitate this process, we constructed a series of plasmid expression vectors, each containing a different type II signal sequence. We evaluated the utilities of these vectors by fusing two different antigens, the ␣-helix domains of pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC), to the signal sequences of -lactamase (bla SS), ompA, and phoA and the signal sequence and C-terminal peptide of -lactamase (bla SS؉CT) on Asd ؉ plasmids under the control of the P trc promoter. Strains were characterized for level of expression, subcellular antigen location, and the capacity to elicit antigen-specific immune responses and protection against challenge with Streptococcus pneumoniae in mice. The immune responses to each protein differed depending on the signal sequence used. Strains carrying the bla SS-pspA and bla SS؉CT-pspC fusions yielded the largest amounts of secreted PspA and PspC, respectively, and induced the highest serum IgG titers, although all fusion proteins tested induced some level of antigen-specific IgG response. Consistent with the serum antibody responses, RASVs expressing the bla SS-pspA and bla SS؉CT-pspC fusions induced the greatest protection against S. pneumoniae challenge.
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.
We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuated Salmonella vaccine (RASV) strains to enhance immune responses by reducing the adverse effects of high-level antigen synthesis. This system includes a chromosomal repressor gene, lacI, expressed from the arabinoseregulated araC P BAD promoter. LacI serves to regulate expression from a plasmid promoter, P trc , that directs antigen synthesis. In the presence of arabinose LacI is produced, which binds to P trc , blocking antigen synthesis. In vivo, an arabinose-poor environment, the concentration of LacI decreases with each cell division, allowing increased antigen synthesis. To optimize the system and for comparison, we altered the lacI ribosomebinding site, start codon, and/or codon content to construct RDAS strains 9095, 9959, and 9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain 9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initial in vitro assessment and the Streptococcus pneumoniae PspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression of lacI did not interfere with the capacity to induce an immune response. Strain 9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain 9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain 9241 also induced significantly greater protective efficacy against challenge with virulent S. pneumoniae. These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.
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