Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis.

BACKGROUND: Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium 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 of C. perfringens produce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. METHODS: We 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 for Nicotiana benthamiana to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds. RESULTS: Immunized 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 virulent C. perfringens when 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-vectored vaccine delivering three Clostridium perfringens antigens protects poultry against necrotic enteritis.

Necrotic enteritis is an economically important poultry disease caused by the bacterium Clostridium perfringens. There are currently no necrotic enteritis vaccines commercially available for use in broiler birds, the most important target population. Salmonella-vectored vaccines represent a convenient and effective option for controlling this disease. We used a single attenuated Salmonella vaccine strain, engineered to lyse within the host, to deliver up to three C. perfringens antigens. Two of the antigens were toxoids, based on C. perfringens α-toxin and NetB toxin. The third antigen was fructose-1,6-bisphosphate aldolase (Fba), a metabolic enzyme with an unknown role in virulence. Oral immunization with a single Salmonella vaccine strain producing either Fba, α-toxoid and NetB toxoid, or all three antigens, was immunogenic, inducing serum, cellular and mucosal responses against Salmonella and the vectored C. perfringens antigens. All three vaccine strains were partially protective against virulent C. perfringens challenge. The strains delivering Fba only or all three antigens provided the best protection. We also demonstrate that both toxins and Fba are present on the C. perfringens cell surface. The presence of Fba on the cell surface suggests that Fba may function as an adhesin.

Bi-valent polysaccharides of Vi capsular and O9 O-antigen in attenuated Salmonella Typhimurium induce strong immune responses against these two antigens.

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.

Analysis of Spleen-Induced Fimbria Production in Recombinant Attenuated Salmonella enterica Serovar Typhimurium Vaccine Strains.

Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 105 CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 109 CFU of wild-type S Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.IMPORTANCESalmonella enterica is the leading cause of bacterial food-borne infection in the United States. S. Typhimurium is capable of producing up to 13 distinct surface structures called fimbriae that presumably mediate its adherence to surfaces. The roles of most of these fimbriae in disease are unknown. Identifying fimbriae produced during infection will provide important insights into how these bacterial structures contribute to disease and potentially induce protective immunity to Salmonella infection. We identified four fimbriae that are produced during infection. Deletion of all four of these fimbriae results in a significant reduction in virulence. We explored ways in which the expression of these fimbriae may be exploited for use in recombinant Salmonella vaccine strains and found that production of Saf and Stc fimbriae are important for generating a strong immune response against a vectored antigen. This work provides new insight into the role of fimbriae in disease and their potential for improving the efficacy of Salmonella-based vaccines.

Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns.

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.

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.

Reversible synthesis of colanic acid and O-antigen polysaccharides in Salmonella Typhimurium enhances induction of cross-immune responses and provides protection against heterologous Salmonella challenge.

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.

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.

Physiological fluid shear alters the virulence potential of invasive multidrug-resistant non-typhoidal Salmonella Typhimurium D23580.

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.

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.

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.

Characterization of the Invasive, Multidrug Resistant Non-typhoidal Salmonella Strain D23580 in a Murine Model of Infection.

A distinct pathovar of Salmonella enterica serovar Typhimurium, ST313, has emerged in sub-Saharan Africa as a major cause of fatal bacteremia in young children and HIV-infected adults. D23580, a multidrug resistant clinical isolate of ST313, was previously shown to have undergone genome reduction in a manner that resembles that of the more human-restricted pathogen, Salmonella enterica serovar Typhi. It has since been shown through tissue distribution studies that D23580 is able to establish an invasive infection in chickens. However, it remains unclear whether ST313 can cause lethal disease in a non-human host following a natural course of infection. Herein we report that D23580 causes lethal and invasive disease in a murine model of infection following peroral challenge. The LD50 of D23580 in female BALB/c mice was 4.7 x 10(5) CFU. Tissue distribution studies performed 3 and 5 days post-infection confirmed that D23580 was able to more rapidly colonize the spleen, mesenteric lymph nodes and gall bladder in mice when compared to the well-characterized S. Typhimurium strain SL1344. D23580 exhibited enhanced resistance to acid stress relative to SL1344, which may lend towards increased capability to survive passage through the gastrointestinal tract as well as during its intracellular lifecycle. Interestingly, D23580 also displayed higher swimming motility relative to SL1344, S. Typhi strain Ty2, and the ST313 strain A130. Biochemical tests revealed that D23580 shares many similar metabolic features with SL1344, with several notable differences in the Voges-Proskauer and catalase tests, as well alterations in melibiose, and inositol utilization. These results represent the first full duration infection study using an ST313 strain following the entire natural course of disease progression, and serve as a benchmark for ongoing and future studies into the pathogenesis of D23580.

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.

Making Common Sense of Vaccines: An Example of Discussing the Recombinant Attenuated Salmonella Vaccine with the Public.

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.

Evaluation of protective efficacy of live attenuated Salmonella enterica serovar Gallinarum vaccine strains against fowl typhoid in chickens.

Salmonella enterica serovar 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 constructed S. Gallinarum strains with a deletion of the global regulatory gene fur and evaluated their virulence and protective efficacy in Rhode Island Red chicks and Brown Leghorn layers. The fur deletion mutant was avirulent and, when delivered orally to chicks, elicited excellent protection against lethal S. 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 a pmi mutant and a combination of fur deletions with mutations in the pmi and rfaH genes, which affect O-antigen synthesis, and ansB, whose product inhibits host T-cell responses. The S. Gallinarum Δpmi mutant was only partially attenuated, and the ΔansB mutant was fully virulent. The Δfur Δpmi and Δfur ΔansB double mutants were attenuated but not protective when delivered orally to the chicks. However, a Δpmi Δfur strain was highly immunogenic when administered intramuscularly. All together, our results show that the fur gene is essential for the virulence of S. Gallinarum, and the fur mutant is effective as a live recombinant vaccine against fowl typhoid.

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.

Multiple effects of Escherichia coli Nissle 1917 on growth, biofilm formation, and inflammation cytokines profile of Clostridium perfringens type A strain CP4.

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.

A low gastric pH mouse model to evaluate live attenuated bacterial 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.

Assessment of attenuated Salmonella vaccine strains in controlling experimental Salmonella Typhimurium infection in chickens.

Salmonella hold considerable promise as vaccine delivery vectors for heterologous antigens in chickens. Such vaccines have the potential additional benefit of also controlling Salmonella infection in immunized birds. As a way of selecting attenuated strains with optimal immunogenic potential as antigen delivery vectors, this study screened 20 novel Salmonella Typhimurium vaccine strains, differing in mutations associated with delayed antigen synthesis and delayed attenuation, for their efficacy in controlling colonization by virulent Salmonella Typhimurium, as well as for their persistence in the intestine and the spleen. Marked differences were observed between strains in these characteristics, which provide the basis for selection for further study as vaccine vectors.

La bactérie Salmonella est considérée comme un vecteur vaccinal prometteur pour la livraison d'antigènes hétérologues chez les poulets. De tels vaccins ont le potentiel bénéfique supplémentaire de limiter les infections par Salmonella chez les oiseaux immunisés. Comme moyen de sélectionner les souches atténuées avec le potentiel immunogène optimal comme vecteur de livraison d'antigènes, la présente étude a examiné 20 souches vaccinales nouvelles de Salmonella Typhimurium, qui différaient en mutation associées avec une synthèse antigénique retardée et une atténuation retardée, pour leur efficacité à limiter la colonisation par du Salmonella Typhimurium virulent, ainsi que pour leur persistance dans l'intestin et la rate. Des différences marquées furent observées entre les souches pour ces caractéristiques, fournissant ainsi des éléments de sélection pour des études ultérieures comme vecteurs vaccinal.(Traduit par Docteur Serge Messier).

Salmonella as a vaccine delivery vehicle.

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.