Recombinant Attenuated Salmonella Vaccine Research Articles

Oral Immunization with Attenuated Salmonella Choleraesuis Expressing the FedF Antigens Protects Mice against the Shiga-Toxin-Producing Escherichia coli Challenge.

Edema disease (ED) is a severe and lethal infectious ailment in swine, stemming from Shiga-toxin-producing Escherichia coli (STEC). An efficient, user-friendly, and safe vaccine against ED is urgently required to improve animal welfare and decrease antibiotic consumption. Recombinant attenuated Salmonella vaccines (RASV) administered orally induce both humoral and mucosal immune responses to the immunizing antigen. Their potential for inducing protective immunity against ED is significant through the delivery of STEC antigens. rSC0016 represents an enhanced recombinant attenuated vaccine vector designed for Salmonella enterica serotype Choleraesuis. It combines sopB mutations with a regulated delay system to strike a well-balanced equilibrium between host safety and immunogenicity. We generated recombinant vaccine strains, namely rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA), and assessed their safety and immunogenicity in vivo. The findings demonstrated that the mouse models immunized with rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA) generated substantial IgG antibody responses to FedF and rStx2eA, while also provoking robust mucosal and cellular immune responses against both FedF and rStx2eA. The protective impact of rSC0016 (pS-FedF) against Shiga-toxin-producing Escherichia coli surpassed that of rSC0016 (pS-rStx2eA), with percentages of 83.3%. These findings underscore that FedF has greater suitability for vaccine delivery via recombinant attenuated Salmonella vaccines (RASVs). Overall, this study provides a promising candidate vaccine for infection with STEC.

Reducing the endotoxic activity or enhancing the vaccine immunogenicity by altering the length of lipid A acyl chain in Salmonella

The balance of the attenuation and reactogenicity is an issue in the development of recombinant attenuated Salmonella vaccines (RASV). Some reactogenic strains produced side effects are partially induced by lipid A. As reported, the number of lipid A acyl chains influence the strength and outcome of immune responses. However, there is rarely any study to investigate the modifications of acyl chain length on the effect of the toxicity and immunogenicity in Salmonella. In this study, foreign acyltransferase genes lpxA and lpxD were introduced into S. Typhimurium, which produced the S006 (ΔaraBAD::PlppCtlpxAC10) or S007 (ΔproBA::PlppSslpxDC16) strains with C10 or C16 acyl chains respectively. The results showed that the increased polymyxin B susceptibility, reduced swimming and invasion capabilities were observed in the S006. In addition, it also exhibited a lower endotoxicity and colonization ability compared to the parent strain. The result indicated the introduction of C10 acyl chains could be as a candidate choice for lipid A detoxifying strategy in engineering bacteria. However, the longer acyl chain modification didn't obviously change these abilities. Parallelly, these modifications were introduced into a Salmonella vaccine strain to determine their influences on the immune responses against Pneumonia. After inoculation by the strain V003 (ΔaraBAD ΔproBA::PlppSslpxDC16 χ9241), the mice produced robust levels of anti-PspA IgG, and a balanced Th1/Th2 immunity, which resulted in a significant survival improvement of mice with challenging against Streptococcus pneumonia. Therefore, the combination of lipid A modification with C16 acyl chain may be a better strategy for the development of ideal RASVs.

Regulated delayed attenuation improves vaccine efficacy in preventing infection from avian pathogenic Escherichia coli O78 and Salmonella typhimurium

Avian pathogenic Escherichia coli (APEC) O78 and Salmonella typhimurium (S. Typhimurium) are two leading bacterial pathogens that cause significant economic loss in the poultry industry. O-antigen is an important immunogen of these two bacteria to induce host protective immune responses during infection. To develop a bivalent vaccine against APEC O78 and S. Typhimurium, the attenuated Salmonella ST01 (Δasd ΔrfbP Δcrp) was genetically constructed to deliver APEC O78 O-antigen polysaccharide (OPS), which stably expresses OPS with asd+ balanced-lethal system in vitro and in vivo. After oral immunization, the recombinant attenuated Salmonella vaccine (RASV) strain ST01 (pSS26-O78) provided insufficient protection against the APEC O78 challenge. Therefore, the regulated delayed attenuation strain ST02 (Δasd ΔrfbP ΔPcrp::TTaraC PBADcrp) was further constructed by regulating cyclic AMP receptor protein (crp) with araC PBAD cassette to better present the heterologous O-antigen to the host immune system. The innovative recombinant strain ST02 (pSS26-O78) stimulated robust antibody responses against APEC O78 and S. Typhimurium OPS, with serum titers over 1:800 for both IgG and IgA, thereby providing the complement-mediated bactericidal activity and stronger protection against APEC O78 and S. Typhimurium infection. Collectively, this study demonstrates a biologically-conjugated polysaccharide vaccine candidate that can enhance homologous protection against APEC O78 and S. Typhimurium.

Oral immunization of rat with chromosomal expression LipL32 in attenuated Salmonella vaccine induces immune respond against pathogenic Leptospira.

PurposeLeptospirosis caused by Leptospira spp. remains a global health problem. Available commercial leptospiral vaccines have shown an ineffective prevention for leptospiral infection. The aim of this study was to develop leptospirosis vaccine using recombinant attenuated Salmonella vaccine (RASV) as a platform. We expected that this vaccine has ability to continuous and strongly stimulate immune systems including protective mucosal, humoral, and cell mediated immunity in rat model.Materials and MethodsIn this study, we engineered RASV, NRSL32 strain containing chromosomal fusion between nucleotides encoding secretion signal of SPI-2 effector protein, SspH2 and gene encoding major pathogenic leptospiral outer membrane lipoprotein, LipL32. Subsequently, our modified RASV was oral vaccination to rat and blood samples were taken for assessment of immune responses.ResultsOur Salmonella NRSL32 strain showed expression and secretion of SspH21-215-LipL32 recombinant protein via SPI-2 T3SS. After oral administration of NRSL32 strain to rats, significant titers of total immunoglobulin G (IgG) and immunoglobulin A against rLipL32 were observed in long period up to 77 days after vaccination. The stimulated antibody showed ability to specific bind with LipL32 protein on surface of pathogenic Leptospira spp. Additionally, the balance level of IgG2a/IgG1 ratio and level of interferon-γ and interleukin-4 secretion were detected.ConclusionThe results showed that our RASV platform with chromosomal expression elicited effective immune responses to leptospiral antigen. Moreover, this platform was capable for simultaneous stimulation of Th1 and Th2-biased responses. Further investigation is necessary study of protective efficacy against leptospiral infection in animal models.

Synthesis and delivery of Streptococcus pneumoniae capsular polysaccharides by recombinant attenuated Salmonella vaccines

Streptococcus pneumoniae capsular polysaccharides (CPSs) are major determinants of bacterial pathogenicity. CPSs of different serotypes form the main components of the pneumococcal vaccines Pneumovax, Prevnar7, and Prevnar13, which substantially reduced the S. pneumoniae disease burden in developed countries. However, the laborious production processes of traditional polysaccharide-based vaccines have raised the cost of the vaccines and limited their impact in developing countries. The aim of this study is to develop a kind of low-cost live vaccine based on using the recombinant attenuated Salmonella vaccine (RASV) system to protect against pneumococcal infections. We cloned genes for seven different serotypes of CPSs to be expressed by the RASV strain. Oral immunization of mice with the RASV-CPS strains elicited robust Th1 biased adaptive immune responses. All the CPS-specific antisera mediated opsonophagocytic killing of the corresponding serotype of S. pneumoniae in vitro. The RASV-CPS2 and RASV-CPS3 strains provided efficient protection of mice against challenge infections with either S. pneumoniae strain D39 or WU2. Synthesis and delivery of S. pneumoniae CPSs using the RASV strains provide an innovative strategy for low-cost pneumococcal vaccine development, production, and use.

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

Mucosal Delivery of a Self-destructing Salmonella-Based Vaccine Inducing Immunity Against Eimeria.

A programmed self-destructive Salmonella vaccine delivery system was developed to facilitate efficient colonization in host tissues that allows release of the bacterial cell contents after lysis to stimulate mucosal, systemic, and cellular immunities against a diversity of pathogens. Adoption and modification of these technological improvements could form part of an integrated strategy for cost-effective control and prevention of infectious diseases, including those caused by parasitic pathogens. Avian coccidiosis is a common poultry disease caused by Eimeria. Coccidiosis has been controlled by medicating feed with anticoccidial drugs or administering vaccines containing low doses of virulent or attenuated Eimeria oocysts. Problems of drug resistance and nonuniform administration of these Eimeria resulting in variable immunity are prompting efforts to develop recombinant Eimeria vaccines. In this study, we designed, constructed, and evaluated a self-destructing recombinant attenuated Salmonella vaccine (RASV) lysis strain synthesizing the Eimeria tenella SO7 antigen. We showed that the RASV lysis strain χ11791(pYA5293) with a ΔsifA mutation enabling escape from the Salmonella-containing vesicle (or endosome) successfully colonized chicken lymphoid tissues and induced strong mucosal and cell-mediated immunities, which are critically important for protection against Eimeria challenge. The results from animal clinical trials show that this vaccine strain significantly increased food conversion efficiency and protection against weight gain depression after challenge with 105E. tenella oocysts with concomitant decreased oocyst output. More importantly, the programmed regulated lysis feature designed into this RASV strain promotes bacterial self-clearance from the host, lessening persistence of vaccine strains in vivo and survival if excreted, which is a critically important advantage in a vaccine for livestock animals. Our approach should provide a safe, cost-effective, and efficacious vaccine to control coccidiosis upon addition of additional protective Eimeria antigens. These improved RASVs can also be modified for use to control other parasitic diseases infecting other animal species.

Protection against avian pathogenic Escherichia coli and Salmonella Kentucky exhibited in chickens given both probiotics and live Salmonella vaccine

Commercial poultry farms are increasingly threatened by bacterial infections from avian pathogenic Escherichia coli (APEC) and broad-host Salmonella serovars. Recombinant attenuated Salmonella vaccines (RASV) elicit cross-reactive immune responses against APEC in chickens; however, assessment of broad protection is lacking. Probiotics boost chicken immunity and improve vaccination responses. The objective of this study was to determine whether the RASV, the probiotics, or their combination had protection against APEC and Salmonella. White Leghorn chicks were randomly placed into 4 groups: no treatment (CON), probiotics (PRO), RASV (VAX), or both prophylactics (P + V). Chicks in the PRO and P + V groups were fed probiotics daily, beginning at the age of 1-day-old. Chicks in the P + V and VAX groups were orally inoculated with RASV at the age of 4 D and boosted 2 wks later. Total and antigen-specific IgY responses to Salmonella (lipolysaccharide [LPS]) and E. coli (IroN and IutA) were measured in serum samples via ELISA. Bactericidal potential of both serum and blood against 42 APEC isolates comprising 25 serotypes was assessed in vitro. In vivo protection against APEC was evaluated by air sac challenge with APEC χ7122 (O78:K80), gross pathological lesions were scored, and bacterial loads were enumerated. In a second similar study, birds were orally challenged with S. Kentucky (CVM29188), and feces were enumerated for Salmonella at multiple time points. Vaccination elicited significant LPS-specific antibodies regardless of probiotics (P < 0.0001). Chicks in the P + V group demonstrated increased blood and serum bactericidal abilities against multiple APEC strains in vitro compared with the CON group. Following χ7122 challenge, P+V birds had less APEC in their blood (P < 0.001) and lower signs of airsacculitis (P < 0.01) and pericarditis/perihepatitis (P < 0.05) than CON birds. Finally, only P + V birds were negative for fecal Salmonella at all time points. This study shows this combination treatment may be a feasible method to reduce infection by APEC and Salmonella in chickens.

I\u03baB\u03b6 facilitates protective immunity against Salmonella infection via Th1 differentiation and IgG production

Inhibitor of kappa B (IκB)-ζ transcription is rapidly induced by stimulation with TLR ligands and IL-1. Despite high IκBζ expression in inflammation sites, the association of IκBζ with host defence via systemic immune responses against bacterial infection remains unclear. Oral immunisation with a recombinant attenuated Salmonella vaccine (RASV) strain did not protect IκBζ-deficient mice against a lethal Salmonella challenge. IκBζ-deficient mice failed to produce Salmonella LPS-specific IgG, especially IgG2a, although inflammatory cytokine production and immune cell infiltration into the liver increased after oral RASV administration. Moreover, IκBζ-deficient mice exhibited enhanced splenic germinal centre reactions followed by increased total IgG production, despite IκBζ-deficient B cells having an intrinsic antibody class switching defect. IκBζ-deficient CD4+ T cells poorly differentiated into Th1 cells. IFN-γ production by CD4+ T cells from IκBζ-deficient mice immunised with RASV significantly decreased after restimulation with heat-killed RASV in vitro, suggesting that IκBζ-deficient mice failed to mount protective immune responses against Salmonella infection because of insufficient Th1 and IgG production. Therefore, IκBζ is crucial in protecting against Salmonella infection by inducing Th1 differentiation followed by IgG production.

Characterization of Spleen Transcriptome and Immunity Against Avian Colibacillosis After Immunization With Recombinant Attenuated Salmonella Vaccine Strains

Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in poultry. Vaccines targeting APEC in chickens have been partially successful, but many lack heterologous protection. Recombinant attenuated Salmonella vaccine (RASV) strains can induce broad immunity against Salmonella and be modified to deliver E. coli antigens. Along with vaccine characteristics, understanding the host response is crucial for developing improved vaccines. The objectives of this study were to evaluate host responses to vaccination with an RASV producing E. coli common pilus (ECP) and assess protection against APEC infection in chickens. Four-day-old White Leghorn chickens were unvaccinated or orally vaccinated and boosted 2 weeks later with RASV χ8025(pYA3337), RASV χ8025(pYA4428) carrying ecp operon genes, or a combination of χ8025(pYA3337) and χ8025(pYA4428) (Combo). To assess host responses, serum IgY and intestinal IgA antibody titers were measured, and spleen samples (n = 4/group) were collected from unvaccinated and Combo vaccinated 4-week-old chickens for RNA-seq. Vaccine protection potential against Salmonella and APEC was evaluated in vitro using bacterial inhibition assays. Five-week-old chickens were challenged via air sac with either an APEC O2 or O78 strain. E. coli was enumerated from internal organs, and gross colibacillosis lesions were scored at necropsy. RASV immunized chickens elicited anti-E. coli antibodies. The spleen transcriptome revealed that 93% (89/96) of differentially expressed genes (DEG) were more highly expressed in Combo vaccinated compared to unvaccinated chickens, with signal as the most significantly impacted category. RNA-seq analysis also revealed altered cellular and metabolic processes, response to stimulus after vaccination, and immune system processes. Six DEG including genes linked to transcription regulation, actin cytoskeleton, and signaling were highly positively correlated with antibody levels. Samples from RASV immunized chickens showed protection potential against Salmonella strains using in vitro assays, but a variable response was found for APEC strains. After APEC challenges, significant differences were not detected for bacterial loads or gross lesions scores, but χ8025(pYA3337) immunized and χ8025(pYA4428) immunized chickens had significantly fewer number of APEC-O2-positive samples than unvaccinated chickens. This study shows that RASVs can prime the immune system for APEC infection, and is a first step toward developing improved therapeutics for APEC infections in chickens.

Conversion of RpoS- Attenuated Salmonella enterica Serovar Typhi Vaccine Strains to RpoS+ Improves Their Resistance to Host Defense Barriers.

The vast majority of live attenuated typhoid vaccines are constructed from the Salmonella enterica serovar Typhi strain Ty2, which is devoid of a functioning alternative sigma factor, RpoS, due to the presence of a frameshift mutation. RpoS is a specialized sigma factor that plays an important role in the general stress response of a number of Gram-negative organisms, including Salmonella. Previous studies have demonstrated that this sigma factor is necessary for survival following exposure to acid, hydrogen peroxide, nutrient-limiting conditions, and starvation. In addition, studies with Salmonella enterica serovarTyphimurium and the mouse model of typhoid fever have shown that RpoS is important in colonization and survival within the infected murine host. We converted 4 clinically studied candidate typhoid vaccine strains derived from Ty2 [CVD908-htrA, Ty800, and χ9639(pYA3493)] and the licensed live typhoid vaccine Ty21a (also derived from Ty2) to RpoS+ and compared their abilities to withstand environmental stresses that may be encountered within the host to those of the RpoS- parent strains. The results of our study indicate that strains that contain a functional RpoS were better able to survive following stress and that they would be ideal for further development as safe, effective vaccines to prevent S.Typhi infections or as vectors in recombinant attenuated Salmonella vaccines (RASVs) designed to protect against other infectious disease agents in humans. The S.Typhi strains constructed and described here will be made freely available upon request, as will the suicide vector used to convert rpoS mutants to RpoS+. IMPORTANCE Recombinant attenuated Salmonella vaccines (RASVs) represent a unique prevention strategy to combating infectious disease because they utilize the ability of Salmonella to invade and colonize deep effector lymphoid tissues and deliver hetero- and homologous derived antigens at the lowest immunizing dose. Our recent clinical trial in human volunteers indicated that an RpoS+ derivative of Ty2 was better at inducing immune responses than its RpoS- counterpart. In this study, we demonstrate that a functional RpoS allele is beneficial for developing effective live attenuated vaccines against S.Typhi or in using S.Typhi as a recombinant attenuated vaccine vector to deliver other protective antigens.

Salmonella Vaccines: Conduits for Protective Antigens.

Vaccines afford a better and more cost-effective approach to combatting infectious diseases than continued reliance on antibiotics or antiviral or antiparasite drugs in the current era of increasing incidences of diseases caused by drug-resistant pathogens. Recombinant attenuated Salmonella vaccines (RASVs) have been significantly improved to exhibit the same or better attributes than wild-type parental strains to colonize internal lymphoid tissues and persist there to serve as factories to continuously synthesize and deliver rAgs. Encoded by codon-optimized pathogen genes, Ags are selected to induce protective immunity to infection by that pathogen. After immunization through a mucosal surface, the RASV attributes maximize their abilities to elicit mucosal and systemic Ab responses and cell-mediated immune responses. This article summarizes many of the numerous innovative technologies and discoveries that have resulted in RASV platforms that will enable development of safe efficacious RASVs to protect animals and humans against a diversity of infectious disease agents.

Antigen Localization Influences the Magnitude and Kinetics of Endogenous Adaptive Immune Response to Recombinant Salmonella Vaccines.

ABSTRACTThe use of recombinant attenuated Salmonella vaccine (RASV) strains is a promising strategy for presenting heterologous antigens to the mammalian immune system to induce both cellular and humoral immune responses. However, studies on RASV development differ on where heterologous antigens are expressed and localized within the bacterium, and it is unclear how antigen localization modulates the immune response. Previously, we exploited the plasmid-encoded toxin (Pet) autotransporter system for accumulation of heterologous antigens in cell culture supernatant. In the present study, this Pet system was used to express early secretory antigen 6 (ESAT-6), an immunodominant and diagnostic antigen from Mycobacterium tuberculosis, in Salmonella enterica serovar Typhimurium strain SL3261. Three strains were generated, whereby ESAT-6 was expressed as a cytoplasmic (SL3261/cyto), surface-bound (SL3261/surf), or secreted (SL3261/sec) antigen. Using these RASVs, the relationship between antigen localization and immunogenicity in infected C57BL/6 mice was systematically examined. Using purified antigen and specific tetramers, we showed that mice infected with the SL3261/surf or SL3261/sec strain generated large numbers of Th1 CD4+ ESAT-6+ splenic T cells compared to those of mice infected with SL3261/cyto. While all mice showed ESAT-6-specific antibody responses when infected with SL3261/surf or SL3261/sec, peak total serum IgG antibody titers were reached more rapidly in mice that received SL3261/sec. Thus, how antigen is localized after production within bacteria has a more marked effect on the antibody response than on the CD4+ T cell response, which might influence the chosen strategy to localize recombinant antigen in RASVs.

Evaluation of Recombinant Attenuated Salmonella Vaccine Strains for Broad Protection against Extraintestinal Pathogenic Escherichia coli.

Antibiotic-resistant bacterial infections are difficult to treat, producing a burden on healthcare and the economy. Extraintestinal pathogenic Escherichia coli (ExPEC) strains frequently carry antibiotic resistance genes, cause infections outside of the intestine, and are causative agents of hospital-acquired infections. Developing a prevention strategy against this pathogen is challenging due to its antibiotic resistance and antigenic diversity. E. coli common pilus (ECP) is frequently found in ExPEC strains and may serve as a common antigen to induce protection against several ExPEC serotypes. In addition, live recombinant attenuated Salmonella vaccine (RASV) strains have been used to prevent Salmonella infection and can also be modified to deliver foreign antigens. Thus, the objective of this study was to design a RASV to produce ECP on its surface and assess its ability to provide protection against ExPEC infections. To constitutively display ECP in a RASV strain, we genetically engineered a vector (pYA4428) containing aspartate-β-semialdehyde dehydrogenase and E. coli ecp genes and introduced it into RASV χ9558. RASV χ9558 containing an empty vector (pYA3337) was used as a control to assess protection conferred by the RASV strain without ECP. We assessed vaccine efficacy in in vitro bacterial inhibition assays and mouse models of ExPEC-associated human infections. We found that RASV χ9558(pYA4428) synthesized the major pilin (EcpA) and tip pilus adhesin (EcpD) on the bacterial surface. Mice orally vaccinated with RASV χ9558(pYA3337) without ECP or χ9558(pYA4428) with ECP, produced anti-Salmonella LPS and anti-E. coli EcpA and EcpD IgG and IgA antibodies. RASV strains showed protective potential against some E. coli and Salmonella strains as assessed using in vitro assays. In mouse sepsis and urinary tract infection challenge models, both vaccines had significant protection in some internal organs. Overall, this work showed that RASVs can elicit an immune response to E. coli and Salmonella antigens in some mice, provide significant protection in some internal organs during ExPEC challenge, and thus this study is a promising initial step toward developing a vaccine for prevention of ExPEC infections. Future studies should optimize the ExPEC antigens displayed by the RASV strain for a more robust immune response and enhanced protection against ExPEC infection.

Efficacy of an oral recombinant attennated Salmonella vaccine encoding cjaA in turkeys colonized with Campylobacter jejuni

Abstract Campylobacter jejuni spp. jejuni, (C. jejuni) is a commensal bacterium in the lower gastrointestinal tract of poultry (e.g., chickens and turkeys), where it causes no clinical disease. Consumption of C. jejuni-contaminated poultry products is the most likely route of transmission to humans. Strategies previously attempted in chickens to reduce intestinal C. jejuni colonization include administration of probiotics, non-antibiotic feed additives, vaccines and bacteriophages. These interventions have not been evaluated in turkeys. Recombinant attenuated Salmonella vaccines (RASV) encoding C. jejuni cjaA are reported to reduce cecal C. jejuni colonization in chickens. The goal of this study was to evaluate the efficacy of a RASV encoding cjaA in turkey poults experimentally colonized with C. jejuni. The cjaA gene from C. jejuni was cloned into RASV strain χ8133 (RASV-cjaA). A control RASV χ8133 (RASV-control) was generated lacking a cjaA insert. At different dates of age, poults received either one or two doses of RASV-cjaA. Other poults received 2 doses of RASV-control. Intestinal colonization by RASV-cjaA was evaluated and was detectable up to 6 days after oral inoculation. Poults were orally challenged with C. jejuni and euthanized at days 3, 7, 14 and 28 days post-challenge to evaluate cecal colonization (cfu/g content) and histopathological changes. Poults vaccinated with 2 doses of RASV-cjaA showed significant reduction (P&amp;lt;0.01) in C. jejuni cecal colonization and histopathological lesion scoring, as compared to those receiving a single dose of RASV-cjaA or RASV-control. These data demonstrate that oral RASV-cjaA significantly reduced C. jejuni colonization in turkeys, which may enhance food safety.

Salmonella Typhimurium strain expressing OprF-OprI protects mice against fatal infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa poses a major threat to human health and to the mink industry. Thus, development of vaccines that elicit robust humoral and cellular immunity against P. aeruginosa is greatly needed. In this study, a recombinant attenuated Salmonella vaccine (RASV) that expresses the outer membrane proteins fusion OprF190-342 -OprI21-83 (F1I2) from P. aeruginosa was constructed and the potency of this vaccine candidate assessed by measuring F1I2-specific humoral immune responses upon vaccination through s.c. or oral routes. S.C. administration achieved higher serum IgG titers and IgA titers in the intestine and induced stronger F1I2-specific IgG and IgA titers in lung homogenate than did oral administration, which resulted in low IgG titers and no local IgA production. High titers of IFN-γ, IL-4, and T-lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized s.c., indicating elicitation of cellular immunity. Importantly, when immunized mice were challenged with P. aeruginosa by the intranasal route 30 days after the initial immunization, s.c. vaccination achieved 77.78% protection, in contrast to 41.18% via oral administration and 66.67% via Escherichia coli-expressed F1I2 (His-F1I2) vaccination. These results indicate that s.c. vaccination provides a better protective response against P. aeruginosa infection than do oral administration and the His-F1I2 vaccine.

A Colanic Acid Operon Deletion Mutation Enhances Induction of Early Antibody Responses by Live Attenuated Salmonella Vaccine Strains

Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterobacteriaceae. It is critical for biofilm formation on HEp-2 cells and on chicken intestinal tissue by Salmonella. In this study, we generated different CA synthesis gene mutants and evaluated the immune responses induced by these mutants. One of these mutations, Δ(wza-wcaM)8, which deleted the whole operon for CA synthesis, was introduced into two Salmonella vaccine strains attenuated by auxotrophic traits or by the regulated delayed attenuation strategy (RDAS). The mice immunized with the auxotrophic Salmonella vaccine strain with the deletion mutation Δ(wza-wcaM)8 developed higher vaginal IgA titers against the heterologous protective antigen and higher levels of antigen-specific IgA secretion cells in lungs. In Salmonella vaccine strains with RDAS, the strain with the Δ(wza-wcaM)8 mutation resulted in higher levels of protective antigen production during in vitro growth. Mice immunized with this strain developed higher serum IgG and mucosal IgA antibody responses at 2 weeks. This strain also resulted in better gamma interferon (IFN-γ) responses than the strain without this deletion at doses of 10(8) and 10(9) CFU. Thus, the mutation Δ(wza-wcaM)8 will be included in various recombinant attenuated Salmonella vaccine (RASV) strains with RDAS derived from Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Typhi to induce protective immunity against bacterial pathogens.

New technologies in developing recombinant attenuated Salmonella vaccine vectors

Recombinant attenuated Salmonella vaccine (RASV) vectors producing recombinant gene-encoded protective antigens should have special traits. These features ensure that the vaccines survive stresses encountered in the gastrointestinal tract following oral vaccination to colonize lymphoid tissues without causing disease symptoms and to result in induction of long-lasting protective immune responses. We recently described ways to achieve these goals by using regulated delayed in vivo attenuation and regulated delayed in vivo antigen synthesis, enabling RASVs to efficiently colonize effector lymphoid tissues and to serve as factories to synthesize protective antigens that induce higher protective immune responses. We also developed some additional new strategies to increase vaccine safety and efficiency. Modification of lipid A can reduce the inflammatory responses without compromising the vaccine efficiency. Outer membrane vesicles (OMVs) from Salmonella-containing heterologous protective antigens can be used to increase vaccine efficiency. A dual-plasmid system, possessing Asd+ and DadB+ selection markers, each specifying a different protective antigen, can be used to develop multivalent live vaccines. These new technologies have been adopted to develop a novel, low-cost RASV synthesizing multiple protective pneumococcal protein antigens that could be safe for newborns/infants and induce protective immunity to diverse Streptococcus pneumoniae serotypes after oral immunization.

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform.

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.

Effective protection against secondary pneumococcal pneumonia by oral vaccination with attenuated Salmonella delivering PspA antigen in mice

Influenza infection followed by pneumococcal infection can cause severe pneumonia and this secondary pneumococcal pneumonia is the most common cause of influenza-associated death. Therefore, vaccination against Streptococcus pneumoniae is highly desirable for reducing the disease burden caused by seasonal epidemic and pandemic influenza. In this study, mice were vaccinated orally with a recombinant attenuated Salmonella vaccine (RASV) strain that delivers pneumococcal surface protein A (PspA) in order to examine protective efficacy against secondary pneumococcal pneumonia. A single dose of oral RASV resulted in attenuated pulmonary inflammation and effective protection against secondary pneumococcal pneumonia. Additionally, oral RASV induced long-term protection against secondary pneumococcal pneumonia. Treatment with an antiviral drug (i.e., oseltamivir) after treatment with RASV further prevented pulmonary inflammation after secondary pneumococcal infection. These results imply that oral RASV can protect mice from secondary pneumococcal infection after influenza infection and that vaccination against respiratory bacterial pathogens is a promising approach for dampening the impact of annual epidemic and pandemic influenza outbreaks.