New Insights into the Roles of Long Polar Fimbriae and Stg Fimbriae in Salmonella Interactions with Enterocytes and M Cells.

Salmonella enterica serovar Typhi causes the systemic disease typhoid fever. After ingestion, it adheres to and invades the host epithelium while evading the host innate immune response, causing little if any inflammation. Conversely, Salmonella enterica serovar Typhimurium causes gastroenteritis in humans and thrives in the inflamed gut. Upon entering the host, S Typhimurium preferentially colonizes Peyer's patches, a lymphoid organ in which microfold cells (M cells) overlay an arrangement of B cells, T cells, and antigen-presenting cells. Both serovars can adhere to and invade M cells and enterocytes, and it has been assumed that S Typhi also preferentially targets M cells. In this study, we present data supporting the alternative hypothesis that S Typhi preferentially targets enterocytes. Using a tissue culture M cell model, we examined S Typhi strains with a deletion in the stg fimbriae. The stg deletion resulted in increased adherence to M cells and, as expected, decreased adherence to Caco-2 cells. Adherence to M cells could be further enhanced by introduction of the long polar fimbriae (Lpf), which facilitate adherence of S Typhimurium to M cells. Deletion of stg and/or introduction of lpf enhanced M cell invasion as well, leading to significant increases in secretion of interleukin 8. These results suggest that S Typhi may preferentially target enterocytes in vivo.

Outer membrane vesicles derived from Salmonella Typhimurium mutants with truncated LPS induce cross-protective immune responses against infection of Salmonella enterica serovars in the mouse model.

Salmonella enterica cause diarrheal and systemic diseases and are of considerable concern worldwide. Vaccines that are cross-protective against multiple serovars could provide effective control of Salmonella-mediated diseases. Bacteria-derived outer membrane vesicles (OMVs) are highly immunogenic and are capable of eliciting protective immune responses. Alterations in lipopolysaccharide (LPS) length can result in outer membrane remodeling and composition of outer membrane proteins (OMPs) changing. In this study, we investigated the impact of truncated LPS on both the production and immunogenicity of Salmonella OMVs, including the ability of OMVs to elicit cross-protection against challenge by heterologous Salmonella strains. We found that mutations in waaJ and rfbP enhanced vesiculation, while mutations in waaC, waaF and waaG inhibited this process. Animal experiments indicated that OMVs from waaC, rfaH and rfbP mutants induced stronger serum immune responses compared to OMVs from the parent strain, while all elicited protective responses against the wild-type S. Typhimurium challenge. Furthermore, intranasal or intraperitoneal immunization with OMVs derived from the waaC and rfbP mutants elicited significantly higher cross-reactive IgG responses and provided enhanced cross-protection against S. Choleraesuis and S. Enteritidis challenge than the wild-type OMVs. These results indicate that truncated-LPS OMVs are capable of conferring cross protection against multiple serotypes of Salmonella infection.

Use of Ensure® nutrition shakes as an alternative formulation method for live recombinant Attenuated Salmonella Typhi vaccines.

BACKGROUND: To be effective, orally administered live Salmonella vaccines must first survive their encounter with the low pH environment of the stomach. To enhance survival, an antacid is often given to neutralize the acidic environment of the stomach just prior to or concomitant with administration of the vaccine. One drawback of this approach, from the perspective of the clinical trial volunteer, is that the taste of a bicarbonate-based acid neutralization system can be unpleasant. Thus, we explored an alternative method that would be at least as effective as bicarbonate and with a potentially more acceptable taste. Because ingestion of protein can rapidly buffer stomach pH, we examined the possibility that the protein-rich Ensure® Nutrition shakes would be effective alternatives to bicarbonate. RESULTS: We tested one Salmonella enterica serovar Typhimurium and three Salmonella Typhi vaccine strains and found that all strains survived equally well when incubated in either Ensure® or bicarbonate. In a low gastric pH mouse model, Ensure® worked as well or better than bicarbonate to enhance survival through the intestinal tract, although neither agent enhanced the survival of the S. Typhi test strain possessing a rpoS mutation. CONCLUSIONS: Our data show that a protein-rich drink such as Ensure® Nutrition shakes can serve as an alternative to bicarbonate for reducing gastric pH prior to administration of a live Salmonella vaccine.

A Live Oral Fowl Typhoid Vaccine with Reversible O-Antigen Production.

Salmonella enterica serovar Gallinarum causes fowl typhoid, recognized worldwide as an economically important disease. The current vaccine, 9R, lacks a complete O antigen, which is a Salmonella virulence factor, and, in addition, has a number of other less well characterized chromosomal mutations. For optimal efficacy, 9R is administered by injection. In an effort to develop a vaccine suitable for oral administration, we constructed Salmonella Gallinarum strains with a reversible O-antigen phenotype. In this scenario, the vaccine strain produces full-length O antigen at the time it is administered to birds. After the vaccine has had time to colonize internal lymphoid tissues, the O-antigen is gradually lost, resulting in an attenuated strain. We found that strains carrying single mutations conferring this phenotype, Apmi and arabinose-regulated rfc, retained virulence. However, a mutant strain carrying both of these mutations was completely attenuated and immunogenic in chickens. This work demonstrates a novel approach for developing live Salmonella vaccines for poultry.

Protection Against Necrotic Enteritis in Broiler Chickens by Regulated Delayed Lysis Salmonella Vaccines.

Necrotic enteritis (NE), caused by Gram-positive Clostridium perfringens type A strains, has gained more attention in the broiler industry due to governmental restrictions affecting the use of growth-promoting antibiotics in feed. To date, there is only one commercial NE vaccine available, based on the C. perfringens alpha toxin. However, recent work has suggested that the NetB toxin, not alpha toxin, is the most critical virulence factor for causing NE. These findings notwithstanding, it is clear from prior research that immune responses against both toxins can provide some protection against NE. In this study, we delivered a carboxyl-terminal fragment of alpha toxin and a GST-NetB fusion protein using a novel attenuated Salmonella vaccine strain designed to lyse after 6-10 rounds of replication in the chicken host. We immunized birds with vaccine strains producing each protein individually, a mixture of the two strains, or with a single vaccine strain that produced both proteins. Immunization with strains producing either of the single proteins was not protective, but immunization with a mixture of the two or with a single strain producing both proteins resulted in protective immunity. The vaccine strain synthesizing both PlcC and GST-NetB was able to elicit strong production of intestinal IgA, IgY, and IgM antibodies and significantly protect broilers against C. perfringens challenge against both mild and severe challenges. Although not part of our experimental plan, the broiler chicks we obtained for these studies were apparently contaminated during transit from the hatchery with group D Salmonella. Despite this drawback, the vaccines worked well, indicating applicability to real-world conditions.

Membrane vesicles of Clostridium perfringens type A strains induce innate and adaptive immunity.

Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection.

Low-pH rescue of acid-sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-regulated arginine decarboxylase system.

For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the ParaBAD or PrhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter PrhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter ParaBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ΔaroD and ΔphoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.

Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity.

The development of safe live, attenuated Salmonella vaccines may be facilitated by detoxification of its LPS. Recent characterization of the lipid A 1-phosphatase, LpxE, from Francisella tularensis allowed us to construct recombinant, plasmid-free strains of Salmonella that produce predominantly 1-dephosphorylated lipid A, similar to the adjuvant approved for human use. Complete lipid A 1-dephosphorylation was also confirmed under low pH, low Mg(2+) culture conditions, which induce lipid A modifications. LpxE expression in Salmonella reduced its virulence in mice by five orders of magnitude. Moreover, mice inoculated with these detoxified strains were protected against wild-type challenge. Candidate Salmonella vaccine strains synthesizing pneumococcal surface protein A (PspA) were also confirmed to possess nearly complete lipid A 1-dephosphorylation. After inoculation by the LpxE/PspA strains, mice produced robust levels of anti-PspA Abs and showed significantly improved survival against challenge with wild-type Streptococcus pneumoniae WU2 compared with vector-only-immunized mice, validating Salmonella synthesizing 1-dephosphorylated lipid A as an Ag-delivery system.

Evaluation of new generation Salmonella enterica serovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA.

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 chi9088 and chi9558 with the Deltacya Deltacrp Deltaasd strain chi8133, for their abilities to express and present a secreted form of the alpha-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-gamma, 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 chi8133 (71-86% vs. 21% survival, P

Immunization with Salmonella enterica serovar Typhimurium-derived outer membrane vesicles delivering the pneumococcal protein PspA confers protection against challenge with Streptococcus pneumoniae.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that serve a variety of functions related to survival and pathogenicity. Periplasmic and outer membrane proteins are naturally captured during vesicle formation. This property has been exploited as a method to derive immunogenic vesicle preparations for use as vaccines. In this work, we constructed a Salmonella enterica serovar Typhimurium strain that synthesized a derivative of the pneumococcal protein PspA engineered to be secreted into the periplasmic space. Vesicles isolated from this strain contained PspA in the lumen. Mice intranasally immunized with the vesicle preparation developed serum antibody responses against vesicle components that included PspA and Salmonella-derived lipopolysaccharide and outer membrane proteins, while no detectable responses developed in mice immunized with an equivalent dose of purified PspA. Mucosal IgA responses developed against the Salmonella components, while the response to PspA was less apparent in most mice. Mice immunized with the vesicle preparation were completely protected against a 10× 50% lethal dose (LD50) challenge of Streptococcus pneumoniae and significantly protected against a 200× LD50 challenge, while control mice immunized with purified PspA or empty vesicles were not protected. These results establish that vesicles can be used to mucosally deliver an antigen from a Gram-positive organism and induce a protective immune response.

Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of Salmonella enterica serovar typhimurium.

Lipopolysaccharide (LPS) is a major virulence factor of Salmonella enterica serovar Typhimurium and is composed of lipid A, core oligosaccharide (C-OS), and O-antigen polysaccharide (O-PS). While the functions of the gene products involved in synthesis of core and O-antigen have been elucidated, the effect of removing O-antigen and core sugars on the virulence and immunogenicity of Salmonella enterica serovar Typhimurium has not been systematically studied. We introduced nonpolar, defined deletion mutations in waaG (rfaG), waaI (rfaI), rfaH, waaJ (rfaJ), wbaP (rfbP), waaL (rfaL), or wzy (rfc) into wild-type S. Typhimurium. The LPS structure was confirmed, and a number of in vitro and in vivo properties of each mutant were analyzed. All mutants were significantly attenuated compared to the wild-type parent when administered orally to BALB/c mice and were less invasive in host tissues. Strains with ΔwaaG and ΔwaaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the ΔwaaI mutant when it was administered intranasally. In the context of an attenuated vaccine strain delivering the pneumococcal antigen PspA, all of the mutations tested resulted in reduced immune responses against PspA and Salmonella antigens. Our results indicate that nonreversible truncation of the outer core is not a viable option for developing a live oral Salmonella vaccine, while a wzy mutant that retains one O-antigen unit is adequate for stimulating the optimal protective immunity to homologous or heterologous antigens by oral, intranasal, or intraperitoneal routes of administration.

Comparison of a regulated delayed antigen synthesis system with in vivo-inducible promoters for antigen delivery by live attenuated Salmonella vaccines.

Induction of strong immune responses against a vectored antigen in hosts immunized with live attenuated Salmonella vaccines is related in part to the amount of antigen delivered and the overall fitness of the Salmonella vector in relation to its ability to stimulate the host immune system. Constitutive high-level antigen synthesis causes a metabolic burden to the vaccine vector strain that can reduce the vaccine strain's ability to interact with host lymphoid tissues, resulting in a compromised immune response. A solution to this problem is the use of systems that regulate antigen gene expression, permitting high levels of antigen synthesis only after the vaccine strain has reached its target tissues. In vivo-inducible promoters (IVIPs) are often used to accomplish this. We recently developed an alternative strategy, a regulated delayed antigen synthesis (RDAS) system, in which the LacI-repressible P(trc) promoter controls antigen gene expression by adding arabinose. In this paper, we compared the RDAS system with two commonly used IVIPs, P(ssaG) and P(pagC). Three nearly identical plasmids, differing only in the promoter used to direct transcription of the pneumococcal pspA gene, P(trc), P(ssaG), or P(pagC), were constructed and introduced into isogenic Salmonella vaccine strains with or without arabinose-inducible LacI synthesis. Mice immunized with the RDAS strain developed slightly higher titers of mucosal and serum anti-PspA antibodies than P(pagC)-immunized mice, while titers in mice immunized with the P(ssaG) strain were 100-fold lower. Both the RDAS and P(pagC) strains conferred similar levels of protection against Streptococcus pneumoniae challenge, significantly greater than those for the P(ssaG) strain or controls. Thus, RDAS provides another choice for inclusion in the live vaccine design to increase immunogenicity.

Palmitoylation state impacts induction of innate and acquired immunity by the Salmonella enterica serovar typhimurium msbB mutant.

Lipopolysaccharide (LPS), composed of lipid A, core, and O-antigen, is a major virulence factor of Salmonella enterica serovar Typhimurium, with lipid A being a major stimulator to induce the proinflammatory response via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. While Salmonella msbB mutants lacking the myristate chain in lipid A were investigated widely as an anticancer vaccine, inclusion of the msbB mutation in a Salmonella vaccine to deliver heterologous antigens has not yet been investigated. We introduced the msbB mutation alone or in combination with mutations in other lipid A acyl chain modification genes encoding PagL, PagP, and LpxR into wild-type S. enterica serovar Typhimurium. The msbB mutation reduced virulence, while the pagL, pagP, and lpxR mutations did not affect virulence in the msbB mutant background when administered orally to BALB/c mice. Also, all mutants exhibited sensitivity to polymyxin B but did not display sensitivity to deoxycholate. LPS derived from msbB mutants induced less inflammatory responses in human Mono Mac 6 and murine macrophage RAW264.7 cells in vitro. However, an msbB mutant did not decrease the induction of inflammatory responses in mice compared to the levels induced by the wild-type strain, whereas an msbB pagP mutant induced less inflammatory responses in vivo. The mutations were moved to an attenuated Salmonella vaccine strain to evaluate their effects on immunogenicity. Lipid A modification caused by the msbB mutation alone and in combination with pagL, pagP, and lpxR mutations led to higher IgA production in the vaginal tract but still retained the same IgG titer level in serum to PspA, a test antigen from Streptococcus pneumoniae, and to outer membrane proteins (OMPs) from Salmonella.

Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment.

We have devised and constructed a biological containment system designed to cause programmed bacterial cell lysis with no survivors. We have validated this system, using Salmonella enterica serovar Typhimurium vaccines for antigen delivery after colonization of host lymphoid tissues. The system is composed of two parts. The first component is Salmonella typhimurium strain chi8937, with deletions of asdA and arabinose-regulated expression of murA, two genes required for peptidoglycan synthesis and additional mutations to enhance complete lysis and antigen delivery. The second component is plasmid pYA3681, which encodes arabinose-regulated murA and asdA expression and C2-regulated synthesis of antisense asdA and murA mRNA transcribed from the P22 P(R) promoter. An arabinose-regulated c2 gene is present in the chromosome. chi8937(pYA3681) exhibits arabinose-dependent growth. Upon invasion of host tissues, an arabinose-free environment, transcription of asdA, murA, and c2 ceases, and concentrations of their gene products decrease because of cell division. The drop in C2 concentration results in activation of P(R), driving synthesis of antisense mRNA to block translation of any residual asdA and murA mRNA. A highly antigenic alpha-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with chi8937(pYA3685) developed antibody responses to PspA and Salmonella outer membrane proteins. No viable vaccine strain cells were detected in host tissues after 21 days. This system has potential applications with other Gram-negative bacteria in which biological containment would be desirable.

Yersinia pestis with regulated delayed attenuation as a vaccine candidate to induce protective immunity against plague.

Two mutant strains of Yersinia pestis KIM5+, a Deltacrp mutant and a mutant with arabinose-dependent regulated delayed-shutoff crp expression (araC P(BAD) crp), were constructed, characterized in vitro, and evaluated for virulence, immunogenicity, and protective efficacy in mice. Both strains were highly attenuated by the subcutaneous (s.c.) route. The 50% lethal doses (LD(50)s) of the Deltacrp and araC P(BAD) crp mutants were approximately 1,000,000-fold and 10,000-fold higher than those of Y. pestis KIM5+, respectively, indicating that both strains were highly attenuated. Mice vaccinated s.c. with 3.8 x 10(7) CFU of the Deltacrp mutant developed high anti-Y. pestis and anti-LcrV serum IgG titers, both with a strong Th2 bias, and induced protective immunity against subcutaneous challenge with virulent Y. pestis (80% survival) but no protection against pulmonary challenge. Mice vaccinated with 3.0 x 10(4) CFU of the araC P(BAD) crp mutant also developed high anti-Y. pestis and anti-LcrV serum IgG titers but with a more balanced Th1/Th2 response. This strain induced complete protection against s.c. challenge and partial protection (70% survival) against pulmonary challenge. Our results demonstrate that arabinose-dependent regulated crp expression is an effective strategy to attenuate Y. pestis while retaining strong immunogenicity, leading to protection against the pneumonic and bubonic forms of plague.

Immune responses to recombinant pneumococcal PsaA antigen delivered by a live attenuated Salmonella vaccine.

Streptococcus pneumoniae is a leading cause of morbidity and mortality among children worldwide and particularly in developing countries. In this study, we evaluated PsaA, a conserved antigen important for S. pneumoniae adhesion to and invasion into nasopharynx epithelia, for its ability to induce protective immunity against S. pneumoniae challenge when delivered by recombinant attenuated Salmonella vaccine (RASVs) strains. RASVs were engineered to synthesize PsaA peptides of various lengths. Vaccination with an RASV synthesizing full-length PsaA induced high titers of anti-PsaA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes, nasal washes, and lung homogenates) sites. BALB/c (haplotype H2(d)) or C57BL/6 (haplotype H2(b)) mice vaccinated either orally or intranasally exhibited a significant reduction in colonization of nasopharyngeal tissues after intranasal challenge with S. pneumoniae strains compared to controls, although protection was not observed with all challenge strains. None of the vaccine constructs provided protection against intraperitoneal challenge with S. pneumoniae strain WU2 (serotype 3). Immunization with RASVs synthesizing truncated PsaA generated lower titers of IgA and IgG and did not provide significant protection. Our results showed that RASVs synthesizing full-length PsaA can provide protection against nasal colonization by some S. pneumoniae strains. PsaA may be a useful addition to a multivalent vaccine, providing protection against pneumonia, otitis media, and other diseases caused by S. pneumoniae.

Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity.

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 arabinose-regulated araC PBAD promoter. LacI serves to regulate expression from a plasmid promoter, Ptrc, that directs antigen synthesis. In the presence of arabinose LacI is produced, which binds to Ptrc, 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 ribosome-binding site, start codon, and/or codon content to construct RDAS strains chi9095, chi9959, and chi9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain chi9555 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 chi9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain chi9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain chi9241 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.

Leucine-responsive regulatory protein (Lrp) acts as a virulence repressor in Salmonella enterica serovar Typhimurium.

Leucine-responsive regulatory protein (Lrp) is a global gene regulator that influences expression of a large number of genes including virulence-related genes in Escherichia coli and Salmonella. No systematic studies examining the regulation of virulence genes by Lrp have been reported in Salmonella. We report here that constitutive expression of Lrp [lrp(Con)] dramatically attenuates Salmonella virulence while an lrp deletion (Deltalrp) mutation enhances virulence. The lrp(Con) mutant caused pleiotropic effects that include defects in invasion, cytotoxicity, and colonization, whereas the Deltalrp mutant was more proficient at these activities than the wild-type strain. We present evidence that Lrp represses transcription of key virulence regulator genes--hilA, invF, and ssrA--in Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2), by binding directly to their promoter regions, P(hilA), P(invF), and P(ssrA). In addition, Western blot analysis showed that the expression of the SPI-1 effector SipA was reduced in the lrp(Con) mutant and enhanced in the Deltalrp mutant. Computational analysis revealed putative Lrp-binding consensus DNA motifs located in P(hilA), P(invF), and P(ssrA). These results suggest that Lrp binds to the consensus motifs and modulates expression of the linked genes. The presence of leucine enhanced Lrp binding to P(invF) in vitro and the addition of leucine to growth medium decreased the level of invF transcription. However, leucine had no effect on expression of hilA and ssrA or on cellular levels of Lrp. In addition, Lrp appears to be an antivirulence gene, since the deletion mutant showed enhanced cell invasion, cytotoxicity, and hypervirulence in BALB/c mice.

Regulated delayed expression of rfaH in an attenuated Salmonella enterica serovar typhimurium vaccine enhances immunogenicity of outer membrane proteins and a heterologous antigen.

RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A DeltarfaH mutant strain is attenuated in mice (50% lethal dose [LD(50)], >10(8) CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC P(BAD) promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the P(BAD) promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, DeltaP(rfaH178), was introduced into the attenuated vaccine strain chi9241 (DeltapabA DeltapabB DeltaasdA) expressing the pneumococcal surface protein PspA from an Asd(+) balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic DeltarfaH derivative or the isogenic RfaH(+) parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD(50) of virulent Streptococcus pneumoniae WU2. Immunization with DeltaP(rfaH178) mutant strains led to increased levels of protection compared to that of the parent chi9241 and of a DeltarfaH derivative of chi9241.

PspA family fusion proteins delivered by attenuated Salmonella enterica serovar Typhimurium extend and enhance protection against Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is highly immunogenic and can induce a protective immune response against pneumococcal infection. PspA is divided into two major families based on serological variability: family 1 and family 2. To provide broad protection, PspA proteins from pneumococcal strains Rx1 (family 1) and EF5668 (family 2) were combined to form two PspA fusion proteins, PspA/Rx1-EF5668 and PspA/EF5668-Rx1. Each protein was fused to a type II secretion signal and delivered by a recombinant attenuated Salmonella vaccine (RASV). Both PspA/Rx1-EF5668 and PspA/EF5668-Rx1 were synthesized in the RASV and secreted into the periplasm and supernatant. The fusion proteins reacted strongly with both anti-PspA/Rx1 and anti-PspA/EF5668 antisera. Oral immunization of BALB/c mice with RASV synthesizing either PspA fusion protein elicited serum immunoglobulin G (IgG) and mucosal IgA responses against both families of PspA. Analysis of IgG isotypes (IgG2a and IgG1) indicated a strong Th1 bias to the immune responses to both proteins. Sera from mice immunized with RASV synthesizing PspA/Rx1-EF5668 bound to the surface and directed C3 complement deposition on representative strains from all five PspA clades. Immunization with RASV synthesizing either protein protected mice against intraperitoneal challenge with Streptococcus pneumoniae WU2 strain (family 1), intravenous challenge with S. pneumoniae 3JYP2670 strain (family 2), and intranasal challenge with S. pneumoniae A66.1 (family 1). The PspA/Rx1-EF5668 protein elicited significantly greater protection than PspA/EF5668-Rx1, PspA/Rx1, or PspA/EF5668. These results indicate an RASV synthesizing a PspA fusion protein representing both PspA families constitutes an effective antipneumococcal vaccine, extending and enhancing protection against multiple strains of S. pneumoniae.