Kinetics of the mucosal antibody secreting cell response and evidence of specific lymphocyte migration to the lung after oral immunisation with attenuated S. enterica var. typhimurium

The kinetic of mucosal secretory responses elicited by the vaccine vector Salmonella enterica var. typhimurium (S. typhimurium) was examined by enzyme linked immunospot (ELISPOT) and compared with serum responses. Mice immunised orally with BRD509, the aroA, aroD mutant of virulent S. typhimurium SL1344 expressing the C Fragment of tetanus toxin (TT), simultaneously developed an IgA antibody secreting cells (ASC) response in the gastrointestinal lamina propria, the spleen and the lung, against both S. typhimurium lipopolysaccharide (LPS) and TT. The magnitude of the ASC response was greatest in the gut, was boosted by a secondary immunisation at day 25, and the kinetic of the response did not correlate with the appearance of serum antibodies. This study suggests that S. typhimurium can engage the common mucosal immune system to effect mucosal secretory responses at distal sites, however, the magnitude of the responses is both greatest in the gut and antigen-specific. The ASC origin of the serum antibodies specific for S. typhimurium and antigens expressed by the bacterium is yet to be elucidated.     

A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken

Salmonella typhimurium vaccine strains were used as antigen delivery system for oral immunisation of chickens against two antigens of the coccidian parasite Eimeria tenella. The cDNAs of the known E. tenella proteins, SO7 and TA4, were isolated from total RNA and subcloned into the expression vectors pQE30 and pTECH2. Subcutaneous immunisation of chickens with Escherichia coli-expressed SO7 and TA4 revealed that both proteins were immunogenic. Both cDNAs were subcloned into plasmids of the pTECH2 vector system, which allows them to be expressed as fusion proteins with the highly immunogenic fragment C of the tetanus toxin under control of the anaerobically inducible nirB promoter. Plasmids were introduced into the S. typhimurium vaccine strains SL3261, C5aroD and C5htrA. SDS-PAGE and Western blot analysis revealed expression of both fusion proteins in all strains under anaerobic culture conditions. Three-week-old white leghorn chickens were orally immunised with 10(9) CFU per animal. The stability of the recombinant bacteria was revealed by recovery of viable Salmonella containing the respective plasmids from the liver of the immunised chickens at day 3 after inoculation. Specific serum IgG antibodies against the SO7-or TA4-antigens were detectable by ELISA 2 weeks after oral immunisation and remained for at least 6 weeks, while specific IgA antibodies were restricted to the bile of the birds. All chickens produced serum IgG and IgA to S. typhimurium lipopolysaccharides. Our data show that a single oral inoculation with recombinant S. typhimurium SL3261, C5aroD and C5htrA can induce specific antibody responses to heterologous Eimeria antigens in chickens, suggesting that recombinant Salmonella are a suitable delivery system for vaccines against Eimeria infections.     

Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice

Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host's immune system. The N-terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In the present study, vaccine plasmids for antigen delivery in which the non-toxic tetanus toxin fragment C (TTFC), which contains a T cell epitope, is fused to the N-terminal 160 amino acids of SipB were developed. It was found that the recombinant proteins are secreted into the culture media and localized to the bacterial surface. TTFC-specific antibody responses are significantly increased in mice orally immunized with attenuated S. Typhimurium BRD509 strains carrying TTFC delivery plasmids. When the TTFC delivery cassettes were introduced into a low copy vector, the plasmid was stably maintained in the BRD509 strain and induced an immune response to the TTFC antigen in mice. These results suggest that expression and delivery of heterologous antigens fused to the N-terminus of SipB enhance the induction of antigen-specific immune responses, and that the N-terminal domain of SipB can be used as a versatile delivery system for foreign antigens.     

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection.Due to its ubiquitous ability to invade host cells,Salmonella typhimurium might be a good candidate for displaying viral antigens.We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S.typhimurium BRD509 using the ice nucleation protein.The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated.The results showed that display motifs in the protein can target integral foreign protein on the surface of S.typhimurium BRD509.Moreover,recombinant strains with surface displayed viral proteins retained their invasiveness,suggesting that the recombinant S.typhimurium can be used as live vaccine vector for eliciting complete immunogenicity.The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.     

The influence of autophagy on mouse inflammatory responses caused by Salmonella enterica serovar Typhimurium with spv genes

An investigation into the effects of Salmonella plasmid virulence genes (spv) on autophagy, apoptosis, and inflammation was carried out in mice, using a strain of Salmonella enterica serovar Typhimurium (S. typhimurium) SR-11 carrying spv. Strain BRD509 without spv was used as a control. Results showed that the expression of autophagy protein Beclin-1 in the livers and spleens in the SR-11 group was lower than that in the BRD509 group, while the apoptosis protein, Caspase-3, was higher in the SR-11 group. Inflammatory cytokine levels [interleukin 12 (IL-12) and interferon γ (IFN-γ)] were higher in the SR-11 group compared with those in the BRD509 group since 4 d post-infection. In addition, we found an increase in severe pathological changes and larger viable bacterial amounts in livers and spleens in the SR-11 group. After intervention with autophagy agonist rapamycin (RAPA), Beclin-1 expression increased in both groups, while Caspase-3 expression was different between the two groups: Caspase-3 decreased in the SR-11 group but increased in the BRD509 group. Moreover, RAPA decreased cytokine levels, bacterial quantity and organ-related injury in the SR-11 group whereas RAPA increased cytokine levels and aggravated organ injury in the BRD509 group. Results from these studies suggest that S. typhimurium with spv genes may exacerbate infection by inhibiting autophagy and affecting the production of inflammatory cytokines. RAPA-enhanced autophagy may improve the secretion of cytokines in order to protect the host from damaging by Salmonella infection. Our study suggests that the regulation of cellular autophagy may play a role in the prevention and control of certain infectious diseases.     

A Salmonella typhimurium htrA live vaccine expressing multiple copies of a peptide comprising amino acids 8–23 of herpes simplex virus glycoprotein D as a genetic fusion to tetanus toxin fragment C protects mice from herpes simplex virus infection

Multiple tandem copies of an immunogenic epitope comprising amino acids 8–23 of glycoprotein D of herpes simplex virus (HSV) were expressed as C-terminal fusions to tetanus toxin fragment C (TetC) in different Salmonella typhimurium live vaccine strains. Expression of the longer fusions was best in strains harbouring a lesion in htrA , a stress protein gene. SL3261, an aroA strain, did not effectively express the longer fusions. Mice immunised with an S. typhimurium C5 htrA mutant expressing fusions with two or four copies of the peptide made an antibody response to both the peptide and TetC, whereas constructs expressing one copy of the peptide only elicited antibody to TetC. A non-immunogenic octameric fusion underwent rearrangements in vivo resulting in a predominantly monomeric fusion. In contrast, the S. typhimurium SL3261 aroA vaccine expressing the TetC-tetrameric fusion did not elicit antibody to the peptide. Sera from mice immunised with a single dose of the dimer and tetramer fusions in the htrA strain neutralised HSV in vitro , and the mice were protected from HSV infection as measured by a reduction in virus load in the ear pinna. We have previously shown that mice vaccinated with salmonella expressing TetC are protected against tetanus toxin and virulent salmonella challenge. These results suggest that it may be possible to develop a multivalent vaccine against salmonellosis, tetanus and HSV.     

Salmonella-mediated mucosal cell-mediated immunity.

Oral immunization with the recombinant Salmonella typhimurium strain (BRD 847) expressing the C fragment of tetanus toxin (TT) induces brisk Ag-specific mucosal S-IgA and serum Ab responses characterized by strong IgG2a Abs to the encoded antigen. We have constructed an attenuated Salmonella typhimurium (aroA- aroD-) strain that expresses chicken egg albumin (OVA) to further elucidate the role of Salmonella-induced Th1 cell phenotype on mucosal cell-mediated immunity (CMI). Peyer's patches and spleen lymphocytes from mice that received the oral Salmonella-OVA vaccine showed dramatic increases in the percent cell lysis of the H-2b restricted EG7.OVA tumor cell line. These results indicate that a single dose of rSalmonella vaccine antigen vector is required to illicit systemic and mucosal Th1-type responses and CTLs. These results also support the existence of a highly regulated relationship between specific cell-mediated immunity and a branch of the humoral immune system, i.e. mucosal IgA responses.     

Poor survival but high immunogenicity of IL-2-expressing Salmonella typhimurium in inherently resistant mice

An IL-2-expressing, attenuated strain of Salmonella typhimurium (strain GIDIL2) was previously shown to survive poorly and to have lower immunogenicity in susceptible mice than its parental, non-cytokine-expressing strain (designated BRD509). In the present study, we compared the immune responses induced by both bacterial strains in inherently Salmonella-resistant C3H/HeN mice. Analysis of the bacterial loads in the peritoneum and spleen revealed that colony-forming units (CFUs) of GIDIL2 were consistently lower than the corresponding BRD509 CFUs. As early as 48 h after inoculation, there were 60-fold lower CFUs of GIDIL2 than BRD509 organisms in the peritoneal cavity. Similarly, the differences in splenic CFUs of GIDIL2 were 20- to 50-fold lower than those of BRD509 over a period of 3-21 days post-injection. This rapid rate of clearance of the GIDIL2 organisms correlated with significantly decreased infection-induced splenomegaly and nitric oxide production by spleen cells. However, despite the poor survival of GIDIL2 organisms in vivo, they could activate peritoneal NK cells efficiently. As early as 48 h after immunization, equivalent levels of NK-mediated cellular cytotoxicity were induced by BRD509 and GIDIL2 strains. Direct evidence for priming of the immune response was shown by demonstrating increased production of IFN-gamma in a recall response by spleen memory T cells obtained 3 weeks after immunization. Finally, mice inoculated with a single dose of either BRD509 or GIDIL2 organisms were fully protected against a challenge of >100-fold the LD50 dose of virulent Salmonella. Taken together, our data demonstrate that despite their rapid clearance from the reticuloendothelial system, IL-2-expressing Salmonella are immunogenic and fully capable of affording excellent protection against virulent challenge in Salmonella-resistant C3H/HeN mice.     

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in th...     

Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains

The asd mutants of Salmonella typhimurium have an obligate requirement for diaminopimelic acid (DAP) and will undergo lysis in environments deprived of DAP. This has allowed the development of a balanced-lethal system for the expression of heterologous antigens in vaccine strains using vectors containing the wild-type asd gene from Streptococcus mutans and asd mutant Salmonella hosts [Nakayama et al., Biotechnology 6 (1988) 693-697]. We have cloned the asd gene from S. typhimurium, characterized the gene product and used this gene to construct Asd+ expression cloning vectors. In addition we have constructed an asd cassette and a transposon derived from Tn5 that allow the rapid modification of other vectors for use with delta asd vaccine strains of S. typhimurium adding versatility to the Asd+ vector/delta asd host system of plasmid maintenance.     

Development of a nonantibiotic dominant marker for positively selecting expression plasmids in multivalent Salmonella vaccines

We report the novel application of a herbicide-resistance-based dominant marker for the positive selection of expression plasmids in Salmonella serovar vaccines. The beta-lactamase gene of the plasmid pTETnir15, which expresses fragment C of tetanus toxin (TetC), has been replaced with the bar gene marker. The new plasmid pBAT1 can be positively selected in vitro within Salmonella serovars in the presence of the herbicide DL-phosphinothricin. The expression of TetC remains unaltered, and the Salmonella enterica serovar Typhimurium vaccine strain is stable and immunogenic in vivo.     

减毒鼠伤寒沙门氏菌全长hpaA基因工程菌的构建

为构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并探讨以减毒鼠伤寒沙门氏菌为载体构建H .pylori疫苗株的意义 ,应用PCR法从H .pylori基因组DNA中扩增 783bp的hpaA基因 ,经酶切 连接反应将其克隆入原核表达质粒pTrc99A的NcoⅠ SalⅠ位点 ,并进行了核苷酸序列测定。重组质粒转化减毒鼠伤寒沙门氏菌SL3 2 6 1 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌C5 7BL 6小鼠喂灌 ,分批两d和 1 0d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果表明 ,经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约3 0kDHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 3 8 9% ,Westernblot证实其有免疫反应性。小鼠重组菌喂灌两d或 1 0d后 ,脾和末段回肠均发现携目的基因的菌落。这些结果提示 ,构建了表达H .pyloriHpaA的重组减毒…     

鼠伤寒沙门菌spvBC基因编辑株的构建

利用λRed重组系统和pBAD原核表达载体构建鼠伤寒沙门菌spvBC质粒毒力基因修饰菌株,为深入探究沙门菌毒力基因spv的功能和致病机制及宿主抗感染免疫提供工具菌。以pKD4为模板,PCR扩增含spvBC同源臂的卡那霉素抗性基因以构建同源打靶片段,再将其电转入含有质粒pKD46的鼠伤寒沙门菌中进行同源重组,随后将质粒pCP20电转导入阳性转化子,消除卡那霉素抗性基因,PCR鉴定敲除株的构建。PCR扩增含酶切位点的spvBC基因片段,扩增产物与原核表达载体pBAD/gⅢ分别双酶切后连接构建pBAD-spvBC重组质粒,PCR筛选阳性菌落并测序鉴定。将构建成功的pBAD-spvBC重组质粒电转导入spvBC敲除株中,Western blot测定不同浓度L-阿拉伯糖诱导SpvB和SpvC蛋白表达情况。PCR结果表明鼠伤寒沙门菌spvBC基因敲除成功;PCR及测序结果表明pBAD-spvBC重组质粒构建成功,Western blot结果表明13 mmol/L L-阿拉伯糖可诱导SpvB和SpvC蛋白正常表达。λRed重组系统可用于沙门菌质粒上大片段基因的敲除,pBAD原核表达载体可用于沙门菌质粒上大片段基因的回补,丰富了细菌质粒的基因修饰和编辑策略。     

Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens

The efficacy of an oral DNA vaccine carrying the Eimeria tenella 5401 antigen gene delivered by attenuated Salmonella typhimurium was examined in an experimental challenge study. The DNA vaccine preparation was made by transforming the recombinant plasmid pcDNA3-5401 into the attenuated S. typhimurium strain (Dam(-) and PhoP(-)) (designated hereafter as ZJ111/pcDNA3-5401). The chickens were randomly divided into six groups, 50 per group. Group A were given PBS as control. Chickens in group B were fed with 10(8) colony forming units (CFU) of attenuated S. typhimurium carrying pcDNA3. Group C were immunised with 100 microg of the recombinant 5401 protein via intramuscular injection. Groups D to F orally received ZJ111/pcDNA3-5401 at doses of 10(7), 10(8) and 10(9)CFU per chicken, respectively. All immunisations were boosted 2 weeks later. The immunised chickens were challenged with 6x10(4) homologous sporulated oocysts 14 days after the second immunisation. No significant differences in body weight were detected between the groups before immunisation and at week 4 after the booster immunisation. The ZJ111/pcDNA3-5401 was eventually eliminated from the spleen and liver on week 6 post-immunisation. The plasmid pcDNA3-5401 was stably maintained in over 80% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunisation of chickens with ZJ111/pcDNA3-5401 elicited specific humoral responses and stimulated proliferation of peripheral blood lymphocytes. The lymphocyte proliferation response was significantly higher in all vaccinated groups than in the control chickens. Antibody response was significantly lower in group C than in groups immunised with strain ZJ111/pcDNA3-5401. Vaccination with the strain ZJ111/pcDNA3-5401 at 10(8) (group E) and 10(9) (group F) CFU per chicken provided 55.0 and 57.5% protection against E. tenella challenge, respectively. These results have important implications for the development of DNA vaccines against avian coccidiosis by bacteria-vectored oral delivery system.     

Introduction of protein or DNA delivered via recombinant Salmonella typhimurium into the major histocompatibility complex class I presentation pathway of macrophages

Recombinant (r) Salmonella typhimurium aroA strains which display the hen egg ovalbumin OVA(257-264) peptide SIINFEKL in secreted form were constructed. In addition, attenuated rS. typhimurium pcDNA-OVA constructs harbouring a eukaryotic expression plasmid encoding complete OVA were used to introduce the immunodominant OVA(257-264) epitope into the major histocompatibility complex (MHC) class I presentation pathway. Both modes of antigen delivery (DNA and protein) by Salmonella vaccine carriers stimulated OVA(257-264)-specific CD8 T-cell hybridomas. An in vitro infection system was established that allowed both rSalmonella carrier devices to facilitate MHC class I delivery of OVA(257-264) by coexpression of listeriolysin (Hly) or by coinfection with rS. typhimurium Hlys (Hess J., Gentschev I., Miko D., Welzel M., Ladel C., Goebel W., Kaufmann S.H.E., Proc. Natl. Acad. Sci. USA 93 (1996) 1458-1463). Coexpression of Hly and coinfection with rS. typhimurium Hlys slightly improved MHC class I processing of OVA. Our data provide further evidence for the feasibility of attenuated, Hly-expressing rS. typhimurium carriers secreting heterologous antigens or harbouring heterologous DNA as effective vaccines for stimulating CD8 T cells in addition to CD4 T cells.     

表达乙型脑炎病毒E蛋白重组减毒沙门菌活载体疫苗株的构建

目的:构建表达乙型脑炎病毒(JEV)E蛋白的口服重组减毒鼠伤寒沙门菌活载体疫苗株。方法:克隆JEV E基因,将其插入表达载体pYA3341中,构建重组质粒pYA3341-E,将重组质粒电转入鼠伤寒沙门菌疫苗株X4550(缺失asd、cya、crp基因),获得重组疫苗菌株X4550(pYA3341-E);鉴定重组菌E蛋白的表达,测定重组菌的稳定性、生长曲线、安全性,以及小鼠的免疫试验和血清中和试验。结果:酶切鉴定和序列测定证实重组质粒构建成功;SDS-PAGE检测有目的蛋白条带;Western印迹证实表达的E蛋白能与猪抗JEV阳性血清特异性结合;重组菌株在体外营养选择压力下,可稳定地携带重组质粒传代繁殖,在体内可较稳定地定居于肠系膜淋巴结和脾脏;小鼠口服试验证实重组菌无毒性作用,安全可靠;小鼠口服重组菌免疫,ELISA检测产生了抗JEV抗体;中和试验表明产生的抗体具有中和活性。结论:构建了能稳定表达JEV E蛋白的口服减毒鼠伤寒沙门菌疫苗株X4550(pYA3341-E),为研究乙型脑炎口服基因工程疫苗奠定基础。     

Expression of human IL-1 beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo.

The feasibility of using Salmonella typhimurium aroA mutant (SL3261) to deliver protein therapeutic agents was investigated in a murine model system. We have constructed an Escherichia coli expression plasmid designed to express the human protein IL-1 beta. This plasmid expresses IL-1 beta to high levels (greater than 30% total cell protein) in E. coli. In Salmonella the IL-1 beta is expressed constitutively to about 10% total cell protein, as verified by Western blotting analysis using polyclonal rabbit anti-IL-1 beta antibody. The protein is produced in a soluble and biologically active form. BALB/c mice administered orally or i.v. with S. typhimurium aroA mutants carrying the plasmid produced highly significant antibody responses against human IL-1 beta as determined by a solid-phase RIA. Furthermore, mice injected with the construct were significantly protected against lethal gamma-irradiation (850 rad). This study therefore demonstrates that the vaccine strain of Salmonella mutants can also be used effectively to deliver therapeutic proteins in vivo.     

鼠伤寒沙门氏菌asd基因的克隆及序列分析

以鼠伤寒沙门氏茵标准株基因组DNA作为模板,用PCR的方法扩增鼠伤寒沙门氏菌的asd基因并克隆入质粒pUCl9,并对其进行测序,序列与献报道一致。同时将质粒pYA248上的链球菌asd基因进行了置换,观察了分别含有链球菌asd基因与鼠伤寒沙门氏菌asd基因的质粒在减毒鼠伤寒沙门氏菌X4072中的生长情况,结果表明含有鼠伤寒沙门氏菌的asd基因的高拷贝质粒pUCl9的菌株生长情况更好。为完善染色体／质粒平衡致死系统,构建减毒鼠伤寒沙门氏活菌疫苗奠定了基础。     

In vitro and in vivo stability of plasmids in attenuated Salmonella enterica serovar typhimurium used as a carrier of DNA vaccine is associated with its replication origin.

The ability of live attenuated Salmonella enterica serovar typhimurium (S. typhimurium) as a carrier of DNA vaccine was evaluated using model plasmid encoding beta-galactosidase (beta-Gal) and BALB/c mice. We constructed pBRCMVbeta, beta-Gal expression apparatus having a replication origin from low copy pBR322. Comparison of the plasmid stability showed that pBRCMVbeta remained stable in Salmonella even after oral administration, while pUC-based pCMVbeta tended to be lost quickly. However, titers for beta-Gal specific IgG in sera did not significantly increase in mice orally administered S. typhimurium harboring pBRCMVbeta. These data suggest that the stability of plasmid in S. typhimurium is associated with its replication origin. Further studies are required to scientifically establish this methodology.     

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.