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Its unique lifestyle and ease of genetic manipulation make Salmonella an attractive vehicle for antigen delivery. The fact that it can be administered orally, allowing needle-free vaccination, makes it an attractive option for vaccines for the developing world or for anyone with a fear of needles. Since the late 1980s, orally administered Salmonella has been investigated for its potential to deliver antigens from bacterial, viral, and parasite pathogens. The ability of Salmonella to colonize mucosal surfaces and generate strong mucosal T cell and B cell responses makes it an ideal vector for vaccines against mucosal pathogens. Salmonella also colonizes host spleen, liver, and lymph nodes, generating robust serum and cellular responses. Detailed analyses of the molecular mechanisms driving Salmonella pathogenesis and immunogenicity have provided a better understanding of the interplay between Salmonella and the host immune system. This information has led to innovations in vaccine strain design that include modifying Salmonella genes to enhance immunogenicity and safety, regulating the attenuation phenotype, regulating antigen production, novel mechanisms for antigen delivery within the host, modification of surface structures, and membrane remodeling of the Salmonella vectors. These advances constitute new paradigms in Salmonella vaccinology and highlight the usefulness of Salmonella as a vaccine.

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.

BackgroundNecrotic enteritis (NE) is caused by type A strains of the bacteriumClostridium perfringens. Total global economic losses to the poultry industry due to NE is estimated to be over two billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains ofC. perfringensproduce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE.MethodsWe have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized forNicotiana benthamianato enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized inN. benthamianausing a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds.ResultsImmunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulentC. perfringenswhen treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry.

Salmonella Typhi is the causative agent of typhoid fever in humans, responsible for approximately 21 million infections and 222,000 deaths globally each year. The current licensed vaccines provide moderate protection to recipients aged >2 years. Prior work on typhoid vaccines has focused on injectable Vi capsular polysaccharide or Vi–protein conjugates and live, oral attenuated S. Typhi vaccines to induce humoral anti-Vi antibodies, while the value and importance of anti-O9 antibodies is less well established. In this study, we constructed a S. Typhimurium strain that synthesizes Vi capsular antigen in vivo and produces the immunodominant O9-antigen polysaccharide instead of its native O4-antigen. The live recombinant attenuated S. Typhimurium mutants were effective in stimulating anti-Vi and anti-O9 antibodies in a mouse model, and the surface Vi capsular expression did not affect the immune responses against the O9 O-antigen polysaccharide. Moreover, the resulting anti-Vi and anti-O9 antibodies were effective at killing S. Typhi and other Salmonella spp. expressing Vi or O9 antigen polysaccharides and provided efficient protection against lethal challenge by S. Typhimurium and S. Enteritidis. Our work highlights the strategy of developing live attenuated S. Typhimurium vaccines to prevent typhoid fever by targeting the both Vi capsular and O9 O-polysaccharide antigens simultaneously.

Salmonella Typhi is the causative agent of typhoid fever in humans, responsible for approximately 21 million infections and 222,000 deaths globally each year. The current licensed vaccines provide moderate protection to recipients aged >2 years. Prior work on typhoid vaccines has focused on injectable Vi capsular polysaccharide or Vi–protein conjugates and live, oral attenuated S. Typhi vaccines to induce humoral anti-Vi antibodies, while the value and importance of anti-O9 antibodies is less well established. In this study, we constructed a S. Typhimurium strain that synthesizes Vi capsular antigen in vivo and produces the immunodominant O9-antigen polysaccharide instead of its native O4-antigen. The live recombinant attenuated S. Typhimurium mutants were effective in stimulating anti-Vi and anti-O9 antibodies in a mouse model, and the surface Vi capsular expression did not affect the immune responses against the O9 O-antigen polysaccharide. Moreover, the resulting anti-Vi and anti-O9 antibodies were effective at killing S. Typhi and other Salmonella spp. expressing Vi or O9 antigen polysaccharides and provided efficient protection against lethal challenge by S. Typhimurium and S. Enteritidis. Our work highlights the strategy of developing live attenuated S. Typhimurium vaccines to prevent typhoid fever by targeting the both Vi capsular and O9 O-polysaccharide antigens simultaneously., Typhoid fever: Turning Salmonella bacteria against each other An attenuated strain of modified Salmonella Typhimurium bacteria could answer calls for a more effective typhoid fever vaccine. Current vaccines against typhoid-causing Salmonella Typhi are only moderately effective and potentially ineffective in children under 5 years. Qingke Kong and Roy Curtiss, leading a team of US and Chinese researchers, developed an attenuated version of the less-pathogenic S. Typhimurium that, when orally dosed in mice, expresses bacterial sugar-chain molecules known to elicit a strong immune response. In an in vitro assay, the antibodies produced by the mice in response to these molecules killed S. Typhi and related Salmonella bacteria with similar surface molecules, indicating a potential cross-protective ability. Further research would reveal whether this two-pronged live vaccine has the potential to protect in vivo, in live animals and in humans.

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 .

Researchers have iterated that the future of synthetic biology and biotechnology lies in novel consumer applications of crossing biology with engineering. However, if the new biology’s future is to be sustainable, early and serious efforts must be made towards social sustainability. Therefore, the crux of new applications of synthetic biology and biotechnology is public understanding and acceptance. The RASVaccine is a novel recombinant design not found in nature that re-engineers a common bacteria (Salmonella) to produce a strong immune response in humans. Synthesis of the RASVaccine has the potential to improve public health as an inexpensive, non-injectable product. But how can scientists move forward to create a dialogue of creating a ‘common sense’ of this new technology in order to promote social sustainability? This paper delves into public issues raised around these novel technologies and uses the RASVaccine as an example of meeting the public with a common sense of its possibilities and limitations. publishedVersion

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.

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively . All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection. Using spaceflight analog bioreactor technology, Cheryl Nickerson at Arizona State University and collaborators developed and validated a new three-dimensional (3-D) intestinal co-culture model containing multiple differentiated epithelial cell types and phagocytic macrophages with antibacterial function to study infection by multiple pathovars of Salmonella. This study is the first to show that these pathovars (known to possess different host adaptations, antibiotic resistance profiles and disease phenotypes), display markedly different colonization and intracellular co-localization patterns using this physiologically relevant new 3-D intestinal co-culture model. This advanced model, that integrates a key immune cell type important for Salmonella infection, offers a powerful new tool in understanding enteric pathogenesis and may lead to unexpected pathogenesis mechanisms and therapeutic targets that have been previously unobserved or unappreciated using other intestinal cell culture models.

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively . All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection., Modeling intestinal infection with NASA biotechnology: A new 3-D intestinal co-culture model with macrophages to study enteric infection Using spaceflight analog bioreactor technology, Cheryl Nickerson at Arizona State University and collaborators developed and validated a new three-dimensional (3-D) intestinal co-culture model containing multiple differentiated epithelial cell types and phagocytic macrophages with antibacterial function to study infection by multiple pathovars of Salmonella. This study is the first to show that these pathovars (known to possess different host adaptations, antibiotic resistance profiles and disease phenotypes), display markedly different colonization and intracellular co-localization patterns using this physiologically relevant new 3-D intestinal co-culture model. This advanced model, that integrates a key immune cell type important for Salmonella infection, offers a powerful new tool in understanding enteric pathogenesis and may lead to unexpected pathogenesis mechanisms and therapeutic targets that have been previously unobserved or unappreciated using other intestinal cell culture models.

Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Δpmi, ΔrfbK or ΔcpsG) and a double mutation (ΔrfbK ΔcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Δcya Δcrp vaccine strain. The Δpmi, ΔrfbK and ΔcpsG ΔrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Δcrp Δcya ΔcpsG ΔrfbK) and its parent S738 (Δcrp Δcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity.

Salmonella enterica serovar Typhimurium strains belonging to sequence type ST313 are a major cause of fatal bacteremia among HIV-infected adults and children in sub-Saharan Africa. Unlike “classical” non-typhoidal Salmonella (NTS), gastroenteritis is often absent during ST313 infections and isolates are most commonly recovered from blood, rather than from stool. This is consistent with observations in animals, in which ST313 strains displayed lower levels of intestinal colonization and higher recovery from deeper tissues relative to classic NTS isolates. A better understanding of the key environmental factors regulating these systemic infections is urgently needed. Our previous studies using dynamic Rotating Wall Vessel (RWV) bioreactor technology demonstrated that physiological levels of fluid shear regulate virulence, gene expression, and stress response profiles of classic S. Typhimurium. Here we provide the first demonstration that fluid shear alters the virulence potential and pathogenesis-related stress responses of ST313 strain D23580 in a manner that differs from classic NTS.

Identification of individuals shedding Salmonella enterica serovar Typhi in stool is imperative during clinical trial safety evaluations. Recovery of live attenuated S. Typhi vaccine strains can be difficult because the mutations necessary for safety in humans often compromise survival in stringent selective enrichment media. RapidChek® SELECT™ Salmonella is a highly sensitive detection method for S. enterica species which utilizes a bacteriophage cocktail designed to reduce the growth of competitor microbes in mildly selective enrichment medium. Detection of Salmonella is enhanced by means of a Salmonella-specific antibody strip targeted to lipopolysaccharide. The RapidChek® SELECT™ Salmonella method was compared to conventional enrichment and plating methods to determine the most sensitive method for detecting attenuated S. Typhi strains in human stool samples. Although traditional enrichment strategies were more sensitive to the presence of wild-type S. Typhi, RapidChek® SELECT™ Salmonella was superior at detecting attenuated strains of S. Typhi. Strains containing a wide variety of attenuating mutations were detected with equal sensitivity as the wild type by RapidChek® SELECT™ Salmonella. The presence of Vi capsule or mutations which affected O-antigen synthesis (Δpmi, ΔgalE) did not decrease the sensitivity of the RapidChek® SELECT™ Salmonella assay.

Preclinical studies evaluating plague vaccine candidates have demonstrated that the F1 and V protein antigens of Yersinia pestis confer protection against challenge from virulent strains. Live-attenuated ΔphoP/Q Salmonella typhimurium recombinants were constructed expressing either F1, V antigens, F1 and V antigens, or a F1-V fusion from Asd (+) balanced-lethal plasmids. To improve antigen delivery, genes encoding plague antigens were modified in order to localize antigens to specific bacterial cellular compartments which include cytoplasmic, outer membrane, or secreted. Candidate vaccine strains were evaluated for growth characteristics, full-length lipopolysaccharide (LPS), plasmid stability, and antigen expression in vitro. Plague vaccine candidate strains with favorable in vitro profiles were evaluated in murine or rabbit preclinical oral immunogenicity studies. Attenuated S. typhimurium strains expressing cytoplasmically localized F1-V and V antigen antigens were more immunogenic than strains that secreted or localized plague antigens to the outer membrane. In particular, S. typhimurium M020 and M023, which express Asd(+)-plasmid derived soluble F1-V and soluble V antigen, respectively, at high levels in the bacterial cell cytoplasm were found to induce the highest levels of plague-specific serum antibodies. To further evaluate balanced-lethal plasmid retention capacity, ΔphoP/Q S. typhimurium PurB(+) and GlnA(+) balanced-lethal plasmid systems harboring F1-V were compared with M020 in vitro and in BALB/c mice in a immunogenicity study. Although there was no detectable difference in plague antigen expression in vitro, S. typhimurium M020 was the most immunogenic plague antigen vector strain evaluated, inducing high-titer serum IgG antibodies specific against F1, V and F1-V.

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 Δ waaG and Δ waaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the Δ waaI 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.

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 Mg2+ 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.

Three great plague pandemics caused by the gram-negative bacterium Yersinia pestis have killed nearly 200 million people and it has been linked to biowarfare in the past. Plague is endemic in many parts of the world. In addition, the risk of plague as a bioweapon has prompted increased research to develop plague vaccines against this disease. Injectable subunit vaccines are being developed in the United States and United Kingdom. However, the live attenuated Y. pestis-EV NIIEG strain has been used as a vaccine for more than 70 years in the former Soviet Union and in some parts of Asia and provides a high degree of efficacy against plague. This vaccine has not gained general acceptance because of safety concerns. In recent years, modern molecular biological techniques have been applied to Y. pestis to construct strains with specific defined mutations designed to create safe, immunogenic vaccines with potential for use in humans and as bait vaccines to reduce the load of Y. pestis in the environment. In addition, a number of live, vectored vaccines have been reported using attenuated viral vectors or attenuated Salmonella strains to deliver plague antigens. Here we summarize the progress of live attenuated vaccines against plagu

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.

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 attenuatedSalmonellavaccine (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 withStreptococcus pneumoniaeWU2 strain (family 1), intravenous challenge withS. pneumoniae3JYP2670 strain (family 2), and intranasal challenge withS. pneumoniaeA66.1 (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 ofS. pneumoniae.

The use of live recombinant attenuated Salmonella vaccines (RASV) synthesizing Yersinia proteins is a promising approach for controlling infection by Yersinia species. In this study, we constructed attenuated Salmonella strains which synthesize a truncated form of LcrV, LcrV196 and evaluated the immune response and protective efficacy elicited by these strains in mice against two other major species of Yersinia: Yersinia pseudotuberculosis and Yersinia enterocolitica. Surprisingly, we found that the RASV strain alone was sufficient to afford nearly full protection against challenge with Y. pseudotuberculosis, indicating the likelihood that Salmonella produces immunogenic cross-protective antigens. In contrast, lcrV196 expression was required for protection against challenge with Y. enterocolitica strain 8081, but was not sufficient to achieve significant protection against challenge with Y. enterocolitica strain WA, which expressed a divergent form of lcrV. Nevertheless, we are encouraged by these findings to continue pursuing our long-term goal of developing a single vaccine to protect against all three human pathogenic species of Yersinia.

Increasing the immunogenicity to delivered antigens by recombinant attenuatedSalmonellavaccines (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 attenuationSalmonella entericaserovar Typhimurium strains χ9088 and χ9558 with the ΔcyaΔcrpΔasd strain χ8133, for their abilities to express and present a secreted form of the α-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 toSalmonellaantigens 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-γ, than other groups. Also, vaccination with delayed attenuation strains resulted in a greater degree of protection againstStreptococcus pneumoniaechallenge than in mice vaccinated with χ8133 (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.

The synthesis of Vi polysaccharide, a major virulence determinant in Salmonella enterica serotype Typhi (S. Typhi), is under the control of two regulatory systems, ompR-envZ and rscB-rscC, which respond to changes in osmolarity. Some S. Typhi isolates exhibit over-expression of Vi polysaccharide, which masks clinical detection of LPS O-antigen. This variation in Vi polysaccharide and O-antigen display (VW variation) has been observed since the initial studies of S. Typhi. We have reported that the status of the rpoS gene is responsible for this phenomenon. We review the regulatory network of the Vi polysaccharide, linking osmolarity and RpoS expression. Also, we discuss how this may impact live attenuated Salmonella vaccine development.

Recombinant attenuated Salmonella vaccines (RASVs) have been used to induce mucosal and systemic immunity either against itself or to a vectored heterologous antigen (30, 31). It is clear from previous studies that the mode of attenuation (10), antigen burden (12), and the expression system (11, 19, 21) can have a profound effect on the immune response to a vectored antigen. Many attenuating mutations were found to reduce Salmonella survival due to host-induced stresses and/or reduce colonization of lymphoid effector tissues leading to less than ideal immunogenicity (16, 48). An ideal Salmonella vaccine strain should exhibit wild-type abilities to withstand all stresses (enzymatic, acid, osmotic, ionic, etc.) and host defenses (bile, antibacterial peptides, etc.) encountered following oral or intranasal immunization and should exhibit wild-type abilities to colonize and invade host lymphoid tissues while remaining avirulent. Achieving maximal immune responses to a foreign antigen is directly correlated with the amount of the antigen produced, and thus, it is important that the immunizing strain produce adequate levels of antigen. However, for RASVs, this need must be weighed against the fact that high-level antigen production can be a drain on the energy resources of the cell, leading to reduced growth rates and a compromised ability to colonize and stimulate effector lymphoid tissues (55). We have addressed these problems by exporting the antigen outside of the attenuated Salmonella (19), by delaying expression of the attenuated phenotype (6; unpublished results), and by heterologous antigen synthesis (53) until the vaccine strain has colonized host lymphoid tissues. In addition, we have developed the Asd+-balanced lethal system for plasmid maintenance, obviating the need for antibiotic resistance markers (32). The SopB protein in Salmonella, encoded by SPI-5, is secreted and translocated into the cytosol through the SPI-1-encoded type III secretion system (60). SopB has an inositol phosphate phosphatase activity which affects cytoskeletal rearrangement, host-cell invasion and chloride homeostasis (34). sopB mutants of Salmonella enterica serovar Typhimurium are attenuated in their ability to cause gastroenteritis in calves (59), but not for invasion of host tissues (39). In addition, sopB mutants of both Salmonella enterica serovars Typhimurium and Dublin are similarly attenuated in their ability to induce inflammatory responses and fluid secretion in bovine ileal loops (13, 42). In the context of a vaccine, these features may be beneficial in reducing or eliminating reactogenicity and diarrhea sometimes observed in clinical trial volunteers receiving live attenuated bacterial vaccines (17, 47, 49-51). The SopB protein can also protect infected cells from apoptosis by sustained activation of Akt, which may be helpful for Salmonella survival in host tissues but will necessarily affect host immune responses (22). A recent study showed that introduction of a sopB mutation into several different attenuated Salmonella strains led to enhanced immune responses in mice to a vectored test antigen, β-galactosidase (27). Based on these observations, we evaluated the effects of a sopB deletion mutation on the performance of our Salmonella vaccine strains. Streptococcus pneumoniae is the principle cause of bacterial pneumonia in children and adults and is also a major cause of otitis media, bacterial meningitis, and septicemia (2). Developing an inexpensive, safe, and effective vaccine against this pathogen is still in urgent demand. Pneumococcal surface protein A (PspA) is an important virulence factor found on the surface of all pneumococci (29). It is a protective antigen in animal models and is immunogenic in humans (2, 58). Our previous work has demonstrated that mice immunized with an RASV expressing PspA are protected against challenge with virulent S. pneumoniae WU2 (20). In this work, we evaluated the effects of a sopB mutation on two different RASV strains with different modes of attenuation on their ability to deliver PspA. We analyzed humoral, mucosal, and cellular immune responses against both the bacterial vector and PspA and protective immunity against challenge with S. pneumoniae WU2 in immunized mice.

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 χ8937, 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. χ8937(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 α-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with χ8937(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.

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler's diarrhea. The heat-labile (LT) and heat-stable (ST) toxins mediate ETEC induced diarrhea. ETEC strains may express LT, ST, or both LT and ST, with LT-expressing strains accounting for approximately 50-60% of ETEC-related traveler's diarrhea. Cholera toxin (CT) is80% homologous to LT and vaccination with CT-B subunit (CT-B) -based vaccines elicit a protective immune response against LT-producing ETEC strains. Peru-15 is an oral, single-dose, live-attenuated cholera vaccine candidate that has been investigated in several clinical trials (n400 subjects) and was proven well tolerated, immunogenic, and efficacious. Peru-15 was genetically engineered to express and secrete high levels of CT-B by cloning ctxB onto a glnA balanced-lethal plasmid under the transcriptional control of a strong constitutive promoter, resulting in Peru-15pCTB. In vitro characterization demonstrated that Peru-15pCTB secreted approximately 30-fold more CT-B than Peru-15 and CT-B was stably produced after 40 generations of growth and throughout simulated Seed Bank and FDP (Final Drug Product) production conditions. In preclinical studies, the geometric mean anti-CT-B IgG titer in the sera of mice inoculated intranasally with two doses of Peru-15pCTB was32-fold higher than in mice inoculated with Peru-15. Similarly, rabbits orally inoculated with a single dose of Peru-15pCTB developed titers that were approximately 30-fold higher than rabbits inoculated with a single dose of Peru-15. Sera from Peru-15pCTB vaccinated mice and rabbits neutralized LT toxicity in an in vitro assay. Peru-15pCTB has several promising characteristics of an oral, single-dose, bivalent cholera/ETEC vaccine and is advancing towards a Phase 1 clinical trial.

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.

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.

Enterotoxigenic Escherichia coli (ETEC) is recognized as one of the major causes of infectious diarrhea in developing countries. Worldwide, the incidence of ETEC infections is estimated to result in 650 million cases of diarrhea and 380,000 deaths in children under 5 years of age. ETEC is also an important cause of travelers' diarrhea in people traveling to endemic regions of the world. Although ETEC is an uncommon cause of infections in the USA, there have been 14 reported outbreaks of ETEC in the USA and seven on cruise ships over the 20-year period between 1975 and 1995. ETEC strains are comprised of a large number of serotypes that produce a variety of colonization factors and enterotoxins. On infection, ETEC first establishes itself by adhering to the epithelium of the small intestine via one or more colonization factor antigens or coli surface proteins. Once established, ETEC expresses one or more enterotoxin(s), which results in the production of secretory diarrhea. While the need for an efficacious, easily administered vaccine is great, there are currently no licensed ETEC vaccines available for use in endemic countries or for US travelers.

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli-derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.

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.

Salmonella entericaserovar Gallinarum is the etiological agent of fowl typhoid, which constitutes a considerable economic problem for poultry growers in developing countries. The vaccination of chickens seems to be the most effective strategy to control the disease in those areas. We constructedS. Gallinarum strains with a deletion of the global regulatory genefurand evaluated their virulence and protective efficacy in Rhode Island Red chicks and Brown Leghorn layers. Thefurdeletion mutant was avirulent and, when delivered orally to chicks, elicited excellent protection against lethalS. Gallinarum challenge. It was not as effective when given orally to older birds, although it was highly immunogenic when delivered by intramuscular injection. We also examined the effect of apmimutant and a combination offurdeletions with mutations in thepmiandrfaHgenes, which affect O-antigen synthesis, andansB, whose product inhibits host T-cell responses. TheS. Gallinarum Δpmimutant was only partially attenuated, and the ΔansBmutant was fully virulent. The ΔfurΔpmiand ΔfurΔansBdouble mutants were attenuated but not protective when delivered orally to the chicks. However, a ΔpmiΔfurstrain was highly immunogenic when administered intramuscularly. All together, our results show that thefurgene is essential for the virulence ofS. Gallinarum, and thefurmutant is effective as a live recombinant vaccine against fowl typhoid.

Clostridium perfringens is an important Gram-positive pathogen responsible for food poisoning, necrotic enteritis, gas gangrene, and even death. Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic strain with demonstrated benefits. In this study, we evaluated the effects of EcN on growth, toxin production, biofilm formation, and inflammatory cytokine responses of C. perfringens. In vitro co-culture experiments demonstrated that EcN inhibited growth, gas production, and toxin production (α-toxin and NetB) of C. perfringens in a dose-dependent manner. The growth inhibition effect was not observed when C. perfringens was incubated with EcN cell-free supernatants (CFSE), suggesting that growth inhibition was caused by nutrition competition during co-incubation. In vitro studies demonstrated that pre-incubation with EcN did not inhibit C. perfringens attachment to Caco-2 cells, but did reduce C. perfringens total number, toxin production, and cytotoxicity after 24 h. The similar growth inhibition results were also observed during the formation of C. perfringens biofilm. Finally, pre-incubation of EcN with RAW264.7 cells significantly decreased the production of inflammatory cytokines caused by the introduction of C. perfringens. Our results indicate that EcN can inhibit many of the pathological effects of C. perfringens in vitro conditions.

Attenuated Salmonella vaccines can be administered orally to deliver recombinant antigens to mucosal surfaces inducing a protective immune response against a variety of targeted pathogens. A number of exciting new approaches and technologies for attenuated Salmonella vaccines have been developed recently. However, a disconnect remains between results obtained with mice in preclinical studies and results obtained in human clinical trials. This is due to an incomplete understanding of Salmonella Typhi interactions with human hosts and inadequate animal models available for study. In this review, the authors describe recent progress in identifying important differences underlying S. Typhi-host interactions, the development of novel approaches to vaccine design and six recent clinical trials evaluating Salmonella-vectored vaccines.

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials.

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 P araBAD or P rhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter P rhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter P araBAD 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 Δ aroD and Δ phoPQ 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.

In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni . Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 10 5 CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.

Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, an important systemic disease of poultry with economic consequences in developing nations. A live attenuated orally applied S. Gallinarum vaccine could provide a low cost method for controlling this disease. We constructed S. Gallinarum strains in which the expression of the crp, rfc and rfaH genes, important for virulence of Salmonella Typhimurium in mice, were under the control of an arabinose-regulated promoter. We evaluated the virulence of these strains compared to wild-type S. Gallinarum and to mutants carrying deletions in these genes. We found that rfc mutants were fully virulent, indicating that, unlike the S. Typhimurium mouse model, the rfc gene is dispensable in S. Gallinarum for virulence in birds. In the case of rfaH, the deletion mutant was attenuated and protective, while the strain with arabinose-regulated rfaH expression retained full virulence. The strain exhibiting arabinose-regulated crp expression was attenuated. Its virulence was not affected by the inclusion of 0.2% arabinose in the drinking water. Birds immunized with this strain were protected against a lethal S. Gallinarum challenge and against colonization with the human pathogen Salmonella Enteritidis. This work shows that an arabinose-regulated crp strain provides a basis for further development of a fowl typhoid vaccine.

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 .

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines. Published version

Background Salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in E. coli by mutating several genes including the recA, recE, recF and recJ. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in Salmonella enterica. Results The effect of recA, recF and recJ deletions on DNA recombination was examined in three serotypes of Salmonella enterica. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a ΔrecA or ΔrecF mutation; (2) in all three Salmonella serotypes, both ΔrecA and ΔrecF mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) ΔrecA and ΔrecF mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a ΔrecJ mutation could reduce plasmid recombination but was less effective than ΔrecA and ΔrecF mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec+ strains. A ΔrecA mutation reduced both intrachromosomal recombination and plasmid integration frequencies. Conclusions The ΔrecA and ΔrecF mutations can reduce plasmid recombination frequencies in Salmonella enterica, but the effect can vary between serovars. This information will be useful for developing Salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrp(C)) mutant strains. In this work, we used chromosomal P(fimA)-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to P(fimA) was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in P(fimA) play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to P(fimA) without competitive exclusion. In addition, both Lrp and FimZ binding to P(fimA) caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.

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 (LD 50 ) challenge of Streptococcus pneumoniae and significantly protected against a 200× LD 50 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.

Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2'-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC P(BAD) promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::P(lpxL)lpxL ΔP(crp21)::TT araC P(BAD)crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD(50) of the mutant in mice, when administered subcutaneously or intranasally was >10(7)-times and >10(4)-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6×10(7) wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2×10(4) live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.

We evaluated the ability of Yersinia pestis antigens HmuR, Psn and modified forms of LcrV delivered by live attenuated Salmonella strains to stimulate a protective immune response against subcutaneous or intranasal challenge with Y. pestis CO92. LcrV196 is a previously described truncated protein that includes aa 131–326 of LcrV and LcrV5214 has been modified to replace five key amino acids required for interaction with the TLR2 receptor. Psn is the outer membrane receptor for the siderophore, yersiniabactin, and the bacteriocin, pesticin. Mice immunized with Salmonella synthesizing Psn, LcrV196 or LcrV5214 developed serum IgG responses to the respective Yersinia antigen and were protected against pneumonic challenge with Y. pestis. Immunization with Salmonella synthesizing Psn or LcrV196 was sufficient to afford nearly full protection against bubonic challenge, while immunization with the strain synthesizing LcrV5214 was not protective. Immunization with Salmonella synthesizing HmuR, an outer membrane protein involved in heme acquisition in Y. pestis, was poorly immunogenic and did not elicit a protective response against either challenge route. These findings indicate that both Psn and LcrV196 delivered by Salmonella provide protection against both bubonic and pneumonic plague.

We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuatedSalmonellavaccine (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-regulatedaraCPBADpromoter. 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 thelacIribosome-binding site, start codon, and/or codon content to construct RDAS strains χ9095, χ9959, and χ9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain χ9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initialin vitroassessment and theStreptococcus pneumoniaePspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression oflacIdid not interfere with the capacity to induce an immune response. Strain χ9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain χ9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain χ9241 also induced significantly greater protective efficacy against challenge with virulentS. pneumoniae. These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (chi9639 and chi9640) were derived from the rpoS mutant strain Ty2 and one (chi9633) from the RpoS(+) strain ISP1820. In chi9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS(+) vaccines induced a balanced Th1/Th2 immune response while the RpoS(-) strain chi9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS(+) strain chi9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, chi9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts.