Enhanced protective immune responses against Salmonella Enteritidis infection by Salmonella secreting an Escherichia coli heat-labile enterotoxin B subunit protein

Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent mucosal adjuvant. In this study, the effect of an attenuated Salmonella secreting LTB protein as an adjuvant strain (JOL1228) for a live Salmonella Enteritidis (SE) vaccine candidate (JOL919) was evaluated. In a single immunization experiment, chickens immunized with a mixture of JOL919 (5 parts) and JOL1228 (1 part) showed enhanced mucosal and cellular immune responses and efficient protection against salmonellosis as compared to those unimmunized control chickens. In further analysis, chickens were primed at one day of age and were boosted at the fifth week of age to prolong immune responses and to maximize the protection efficacy against salmonellosis. The immunized groups B (prime and booster with JOL919), C (prime with JOL919-JOL1228 mixture and booster with JOL919), and D (prime and booster with JOL919-JOL1228 mixture) showed significantly higher humoral and cellular immune responses as compared to those in the unimmunized control group A. In addition, immunized groups C and D showed fewer gross lesions in the liver and spleen and a lower number of SE-positive organs, with the lowest bacterial counts in the SE challenge strain as compared to the control group. These results indicate that SE vaccination with the LTB strain can have an adjuvant effect on the vaccine candidate by enhancing immune responses, and that a prime-boost strategy with the addition of the adjuvant strain can efficiently protect birds against salmonellosis.     

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12 weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.     

携带新城疫病毒F基因DNA疫苗的减毒鼠伤寒沙门氏菌的构建及其免疫原性

根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计1对引物,通过RT-PCR扩增出鹅源NDV分离株JS5F基因(约1700bp),测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1-F。pVAX1-F经脂质体转染COS-7细胞,间接免疫荧光试验检测出F基因在COS-7细胞中的表达产物。将pVAX1-F转化减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pVAX1-F)。重组菌以109CFU/只的剂量2次免疫BALB/c小鼠,免疫小鼠可以检测到特异性针对NDVF蛋白的血清抗体和小肠粘膜抗体应答,SL7207(pVAX1-F)免疫组抗体水平显著高于SL7207(pVAX1)组(P<0.05)。将SL7207(pVAX1-F)以109CFU/只剂量口服免疫1日龄雏鸡,免疫保护试验结果显示,SL7207(pVAX1-F)免疫组对鸡具有良好的保护率(77.27%),与空白对照组和SL7207(pVAX1)空载体组之间存在显著性差异(P<0.05)。结果表明,该运送DNA疫苗的减毒沙门氏菌系统在体内能成功释放所携带的质粒,并能刺激机体产生免疫应答,可对NDV强毒攻击提供良好的免疫保护作用,提示该疫苗候选株对新城疫的控制有重要应用前景。     

Replication kinetics of duck virus enteritis vaccine virus in ducklings immunized by the mucosal or systemic route using real-time quantitative PCR

A chicken embryo-adapted duck enteritis virus (DEV) strain is the most widely used vaccine against duck virus enteritis (DVE) infection. The kinetics of attenuated DEV vaccine was examined in tissues of ducklings vaccinated by the mucosal or systemic route at 20 days of age and sampled regularly up to 60 days post-vaccination (p.v.). Significant numbers of virus genomes in the lymphoid and other parenchymatous organs were first detected at 60 min p.v., and subsequently rose to peak levels during 90 min to 1 day p.v. independent of the route of vaccine administration. The peak level of vaccine virus in the individual parenchymatous organs of subcutaneously immunized ducklings was significantly higher than that of orally or nasally immunized ducklings. The route of vaccine administration had significant effect on the initial tissue distribution of vaccine virus in respiratory and digestive tracts. Vaccine viruses spread to digestive tract and trachea tissues by mucosal route, i.e. oral and nasal administration, early than that by subcutaneous route. The rapid early increase of vaccine virus levels in all samples examined followed by a steady decline from 90 min to 6 days p.v. The real-time PCR analysis of a variety of tissues is significant for further investigation of the mechanism of vaccinal protection, and the optimization of vaccination regimes.     

Evaluation of efficacy of a new live Salmonella Typhimurium vaccine candidate in a murine model

Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by two virulence gene deletions was evaluated in this study. Live form of the vaccine was orally administered and formalin-inactivated form was intramuscularly (IM) administered. 105 female BALB/c mice were used and divided into 5 groups, A to E, containing 21 mice per group. Serum IgG and secretory IgA titers and levels of serum IFN-γ, IL-4, TNF-α, IL-12 of the immunized groups, especially group C (oral prime-oral booster) significantly increased. In addition, all animals in groups C showed no clinical symptoms and survived the virulent challenges, whereas all or some of mice from the other groups died of the challenge. These indicate that the vaccine candidate can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and leading to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection.     

携带猪传染性胃肠炎病毒S/N融合双基因的减毒沙门氏菌的构建与鉴定

旨在构建携带猪传染性胃肠炎病毒(TGEV)S/N融合双基因的减毒沙门氏菌,并鉴定该疫苗菌株的生物学特性,为开展TGEV口服免疫研究奠定材料基础。采用PCR方法从克隆质粒19T-S和19T-N中分别扩增了TGEV的S基因(含主要抗原位点,2.1kb)和N基因(1.2kb),将S基因和N基因插入pVAX1载体,构建携带S/N融合双基因的真核表达质粒pVAX-S/N。将pVAX-S/N电转化减毒沙门氏菌SL7207,筛选获得重组菌株SL7207(pVAX-S/N),并对重组菌株SL7207(pVAX-S/N)的体外稳定性、目的基因在体内的转录、口服接种小鼠的安全性及在体内稳定性等特性进行了鉴定。结果表明,真核质粒pVAX-S/N构建成功,该质粒转染COS7中能表达2个目的蛋白,重组菌SL7207(pVAX-S/N)在Kan+抗性下体外培养稳定性好,口服接种小鼠3d可从回肠组织检测到目的基因的转录,以0.5×109、1×109和2×109 CFU口服对小鼠均具有安全性,重组菌在接种小鼠的肝、脾于4周左右逐渐被机体清除。结果表明成功构建TGEVS/N双基因疫苗SL7207(pVAX-S/N),该疫苗具有良好的稳定性与安全性等特点,为开展TGEV口服免疫研究奠定了基础。     

Immunogenicity of a DNA vaccine of Avian Reovirus orally delivered by attenuated Salmonella typhimurium

This study aimed to assess the ability of a Salmonella typhimurium-mediated Avain Reovirus DNA vaccine in eliciting antibody production. Six-day-old SPF chickens were orally immunized with SL7207 (pVAX-σC) twice at 2-week interval, detectable antibody was generated 2 weeks after immunization and was significantly higher than the control groups (P < 0.01) and ten chickens (66.7%) were considered safe in the subsequent challenge. These results show that SL7207 (pVAX-σC) can induce protective antibody in chickens and the newly-constructed vaccine is also effective in protection chickens against ARV infection.     

Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens

In order to develop a novel, safe and immunogenic fowl typhoid (FT) vaccine candidate, a Salmonella Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene E was constructed using pMMP99 plasmid in this study. The formation of the Salmonella Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h after the induction of gene E expression by an increase in temperature from 37 °C to 42 °C. The safety and protective efficacy of the Salmonella Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control), group B (orally immunized), group C (subcutaneously immunized) and group D (intramuscularly immunized). The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent Salmonella Gallinarum strain at 3 weeks post-immunization (wpi). Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the Salmonella Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4⁺ and CD8⁺ T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed Salmonella Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine candidate that protects against FT.     

Immunogenic analysis of two DNA vaccines of avian reovirus mediated by attenuated Salmonella typhimurium in chickens

Avian reovirus (ARV) is an important pathogen in poultry industry and causes great economic losses. As attenuated Salmonella typhimurium is already being used as an effective vehicle for the transfer of DNA vaccines, so in this study we evaluated two DNA vaccines mediated by S. typhimurium on their ability of eliciting antibody production. SPF chickens were respectively immunized with SL7207 (pVAX-σB), SL7207 (pVAX-σC) and SL7027 (pVAX-σB-σC) three times. The results showed that the antibody production was highly dependent on the immunizing times, detectable antibodies of serum antibody IgG and small intestinal mucosal antibody IgA were generated at week 4 and were further improved at week 6 and antibody titers in group SL7207 (pVAX-σC) were higher than that in group SL7207 (pVAX-σB), demonstrating that SL7207 (pVAX-σC) was more powerful than SL7207 (pVAX-σB) in antibody production. The higher antibody titer in SL7027 (pVAX-σB-σC) than that in SL7207 (pVAX-σC) group showed that co-expressing σB and σC could improve antibody production. IFN-γ detection showed that significant higher IFN-γ was generated both in groups SL7027 (pVAX-σB-σC) and SL7207 (pVAX-σC). Subsequent challenge showed that SL7207 (pVAX-σB), SL7207 (pVAX-σC) and SL7027 (pVAX-σB-σC) conferred 50%, 75% and 87.5% respectively.     

Immunogenicity of a DNA vaccine of Avian Reovirus orally delivered by attenuated Salmonella typhimurium

This study aimed to assess the ability of a Salmonella typhimurium-mediated Avain Reovirus DNA vaccine in eliciting antibody production. Six-day-old SPF chickens were orally immunized with SL7207 (pVAX-σC) twice at 2-week interval, detectable antibody was generated 2 weeks after immunization and was significantly higher than the control groups (P<0.01) and ten chickens (66.7%) were considered safe in the subsequent challenge. These results show that SL7207 (pVAX-σC) can induce protective antibody in chickens and the newly-constructed vaccine is also effective in protection chickens against ARV infection.     

Effect of Edwardsiella tarda immunization on systemic immune response,mucosal immune response and protection in catla (Catla catla)

The effect of immunization on systemic and cutaneous mucosal immune responses of fish and their possible relation with protection has not been fully assessed. In this study, healthy catla (Catla catla) were immunized against Edwardsiella tarda using two antigenic preparations namely, whole cell bacterin (B) and bacterin mixed with Freund’s complete adjuvant in a 1:1 (v/v) ratio (B+A) followed by a booster dose after 3 weeks of first injection. Different systemic and cutaneous mucosal immune responses were measured at weekly interval upto 8th week post vaccination (pv). Fish were challenged 8 weeks pv with live E. tarda to study vaccine induced protection. The result showed that although there were strong systemic as well as mucosal immune responses, particularly after booster dose, the challenge produced low to moderate protection in terms of relative percent survival (RPS). The maximum RPS (50 %) was recorded in the adjuvanted bacterin group after 8 weeks pv. Low to moderate protection after challenge, which may be attributed to the intracellular nature of E. tarda and/or use of crude antigenic preparation, accounts for new strategy to be developed for immunization programme against such intracellular pathogen. The results collectively suggest possible involvement of systemic as well as mucosal immune responses in inducing protective immunity in catla.     

Modulation of systemic and mucosal immunity against an inactivated vaccine of Newcastle disease virus by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 and interferon-α

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines.     

Intestinal mucosal immune response against virulent duck enteritis virus infection in ducklings

Duck virus enteritis (DVE) is an acute and contagious herpes virus infection of duck, geese and swans with high morbidity and mortality. The development of specific mucosal immune system against duck enteritis virus (DEV) infection for ducks has been hindered by a lack of knowledge concerning the purification of immunoglobulin A (IgA) of duck. In the present work, the method for purification of duck immunoglobulin A was developed, and the induction of intestinal mucosal immune responses against DEV was studied by orally infected ducklings with virulent DEV. The results showed that a continuous increased DEV DNA levels were observed in blood and various organs examined of orally infected ducklings throughout the infection, which was accompanied by the development of infection in ducklings from mild progressed to severe pathological lesions. Furthermore, a marked increased level of DEV-specific IgA and IgG antibodies in bile, serum and the intestinal tract, as well as the density of IgA⁺ cells in intestine were detected between 1 and 12 days p.i., followed by a drastic reduction of the antibody levels and the density of IgA⁺ cells at 15 days p.i. The results indicate that the DVE infection can stimulate both IgA-dominated antibody immune responses in the intestinal tract, and IgG-dominated antibody systemic immunity in the serum of ducklings orally inoculated with virulent DEV. The severe lesions of the villus epithelial cells and the lymphoid organs can suppress the intestinal mucosal immune responses.     

Cross-protection against Salmonella Typhimurium infection conferred by a live attenuated Salmonella Enteritidis vaccine

In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.     

Immune responses to oral and IM administration of M2e-Hsp70 construct

The eukaryotic expression plasmid of M2e-Hsp70 is a candidate M2e-based DNA vaccine. In order to evaluate the immunization potential of this construct, Specific Pathogen Free chickens were immunized either intramuscularly or orally. Mutant Salmonella typhimurium was used as carrier for oral delivery of the M2e-Hsp70 construct. M2e-specific humoral and cellular responses were tested by ELISA and Lymphocyte Proliferation Assay, respectively. Our results indicate that both humoral and cellular immune responses are conferred against M2e-Hsp70 plasmid in either of the intramuscular or oral routes of administration; however, these responses are significantly higher in intramuscular injection in contrast to oral administration. When it comes to mass vaccination of commercial chicken flocks oral administration is preferred due to the ease of application as well as its capability of eliciting mucosal, humoral and cellular immune responses; so measurements should be taken to improve the immunization potency of our orally delivered DNA vaccine.     

Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection

Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0174-3) contains supplementary material, which is available to authorized users.     

Passive protection of mice pups through oral or intranasal immunization of dams with recombinant Lactobacillus casei vaccine against ETEC F41

Mucosal immunization is advantageous over other routes of antigen delivery because it can induce both mucosal and systemic immune responses. In this study, we have developed fimbriae protein of enterotoxigenic Escherichia coli (ETEC) F41 was stably expressed on the surface Lactobacillus casei 525. The method of expressing vaccine antigens in L. casei induces both systemic and mucosal immunity after oral or intranasal administration. We demonstrate that an oral or intranasal vaccine based on live recombinant L. casei 525 protects infant mice from ETEC F41 infection. This platform technology can be applied to design oral or intranasal vaccine delivery vehicles against several microbial pathogens.     

Immunization of calves with live and inactivated whole-cell vaccines against Salmonella typhimurium infection

Eight calves were immunized with live auxotrophic Salmonella typhimurium mutants (aro -SL 1479, gal E 3821) and twelve calves with phenol-killed whole-cell S. typhimurium vaccine, respectively. The clinical status of the animals was followed and serial reisolation of vaccine and challenge strains from faeces was attempted. The immunization of calves with the live aro- auxotrophic S. typhimurium SL 1479 mutant proved to be unsuitable due to the death of calves after revaccination. The calves immunized with live auxotrophic gal E S. typhimurium CCM 3821 mutant proved to be protected against challenge with virulent S. typhimurium 4/5 strain administered orally at a dose of 10(6) colony forming units (CFU). The postvaccination complications showed serious shortcomings. The immunization of calves with three doses of whole-cell inactivated vaccine containing 5 strains of S. typhimurium was effective against oral challenge with virulent S. typhimurium 4/5 at a dose of 10(6) CFU.