Surface-displayed viral antigens on Salmonella carrier vaccine

We have developed a recombinant live oral vaccine using the ice-nucleation protein (Inp) from Pseudomonas syringae to display viral antigens on the surface of Salmonella spp. Fusion proteins containing viral antigens were expressed in the oral vaccine strain, Salmonella typhi Ty21a. Surface localization was verified by immunoblotting and fluorescence-activated cell sorting. The immunogenicity of surface-displayed viral antigens on the recombinant live vaccine strain was assessed in mice inoculated intranasally and intraperitoneally. Inoculation resulted in significantly higher serum antibody level than those induced by viral antigens expressed intracellularly. Thus, this multivalent mucosal live vaccine may provide an effective means for inducing mucosal or systemic immune responses against multiple viral antigens.     

Flagellin expressed by live Salmonella vaccine strains induces distinct antibody responses following delivery via systemic or mucosal immunization routes

Salmonella flagellin, expressed as flagella in live attenuated vaccine strains, elicits distinct systemic (IgG) and secreted (IgA) antibody responses in mice following delivery via mucosal (nasal/oral) or parenteral (intraperitoneal (i.p.)) immunization routes. Reduced flagellin-specific antibodies were detected either systemically or locally following delivery of flagellated derivatives of aroA Salmonella enterica serovar Dublin SL1438 via the nasal route, the most effective mucosal site for activation of immune responses in mice. In contrast, flagellin represents the most potent Salmonella antigen for the generation of specific serum antibody (IgG) responses following i.p. inoculations. The distinct immunogenic properties of Salmonella flagellin could not be ascribed to deficient colonization, reduced invasive ability or loss of the flagellin expression by the flagellated vaccine strains.     

Antibody responses against flagellin in mice orally immunized with attenuated Salmonella vaccine strains

Salmonella fIagellin has been repeatedly used as a carrier for heterologous peptide epitopes either as a parenterally delivered purified antigen or as a parenterally/orally-administered, flagellated, live, attenuated vaccine. Nonetheless, the ability to induce specific antibody responses against the flagellin moiety, fused or not with heterologous peptide, has not usually been reported in mice orally inoculated with a live, attenuated, flagellated Salmonella strain. In this work we evaluated the immunogenicity of flagellin in mice following oral inoculation with an aroA Salmonella enterica serovar Dublin SL5929 strain, which expressed plasmid-encoded recombinant hybrid flagellin fused to the CTP3 epitope (amino acids 50–64) of cholera toxin B-subunit. In contrast to parenterally immunized mice, no significant CTP3- or flagellin-specific antibody responses either in sera (IgG) or feces (IgA) were detected following repeated oral delivery of the recombinant Salmonella strain to C57BL/6 mice. Similarly, flagellin-specific antibody responses were also not detected in mice immunized with strain SL5930, which expressed a nonhybrid flagellin. The lack of flagellin-specific antibody responses was not associated with deficient Peyer patch colonization or spleen invasion. Moreover, stabilization of the flagellin-coding gene by integration into the host chromosome did not significantly improve flagellin-specific antibody responses following administration by the oral route. Taken together, these results suggest that flagellin does not represent an efficient peptide carrier for activation of antibody responses in mice orally immunized with live, attenuated Salmonella strains. Received: 29 December 1998 / Accepted: 3 May 1999     

幽门螺杆菌HspA融合蛋白口服疫苗的构建

构建表达幽门螺杆菌的保护性抗原分热休克蛋白A亚单位(HspA）和霍乱毒素B亚单位（CtxB）的重组融合蛋白的生物工程菌株,以此制备幽门螺杆菌的口服疫苗。用PCR方法扩增hspA和ctxB两个目的的基因片段,将它们分别克隆至pSK（＋）质粒上,然后插入含T7启动子ET－22b（＋）的表达载体中,构建嗓基因的表达质量pET－hct,转化E.coliBL21(DE3）,经IPTG诱导表达融合蛋白HCT。经测序,hspA-ctxB(hct)融合基因片段由726bp组成,可以编码242个氨基酸残基的多肽。经SDS－PAGE和免疫印迹分析检测发现,融合基因表达的蛋白质相对分子质量约为30kD。融合蛋白经镍离子柱纯化、复性后,和HspA共同标记同位素^125I,然后给小鼠灌胃,结果观察到HCT组小鼠血清中的^125I的放射量要明显高于HspA组（P＜0．001）,且吸收峰值时间明显提前。融合蛋白中的CtxB可明显促进小鼠对HspA的吸收,HCT融合蛋白可以作为预防和治疗幽门螺杆菌感染的侯选口服疫苗。     

鼠伤寒沙门菌pagC启动子的克隆与活性分析

从鼠伤寒沙门菌中克隆出pagC启动子(PpagC),构建体内激活的表达质粒pZW,插入庚型肝炎病毒（HGV）NS3基因,构建出表达质粒pZWNS3。以其转化减毒鼠伤寒沙门菌SL7207,观察PpagC的启动活性。结果表明：Mg2+可抑制PpagC的启动活性,Mg2+浓度小于50mmol/L时培养的重组菌经SDSPAGE和Western blot检测能高水平表达HGV NS3蛋白。Mg2+浓度升至50mmol/L时,NS3蛋白表达量明显降低。收集以50mmol/L Mg2+的培养基扩增的重组菌,灌胃接种C57小鼠,检测小鼠的血清抗体、T细胞增殖和CTL反应。结果显示PpagC是一个强的宿主体内激活的启动子,为构建以伤寒沙门氏菌为载体的高效免疫口服疫苗提供了一个新的途径。     

细菌芽胞—— 一种新型的疫苗载体

杆菌属的芽胞作为益生菌已经应用于人和动物的食品生产和细菌疗法.目前,芽胞作为一种新型的疫苗载体,开始用于破伤风、炭疽等疫苗的研究.与目前的第二代疫苗相比,细菌芽胞热稳定性好,遗传操作方便,是一种理想的疫苗载体.本文就其作为疫苗载体的相关研究进行综述.     

Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein

A green fluorescent protein (gfp) gene was ligated to the Lactobacillus reuteri-specific nisin-inducible expression-secretion vector pNIES, generating a pNIES-GFP vector capable of secreting the cloned gene as a GFP-fusion protein with fluorescent activity. To develop this system as a live vehicle carrying the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LT(B)) of the enterotoxigenic Escherichia coli (ETEC), a recombinant 5'-ST-LT(B)-3' DNA fragment was cloned into pNIES-GFP. The resulting L. reuteri/pNIES-GFP:STLT(B) system was found to possess the capability of adhering to the mice gut, secreting GFP:STLT(B) product at 0.14 and 0.026 pgcell(-1) under induced and noninduced conditions, respectively. Further analysis of the GFP:STLT(B) product confirmed its ganglioside-binding ability, LT(B) antigenicity and relative freedom from the ST-associated toxicity, making it suitable for use as an oral vaccine in mice. Oral inoculation of the L. reuteri/pNIES-GFP:STLT(B) culture in mice elicited significant (P<0.01) serum IgG and mucosal IgA antibodies against the STLT(B) antigen. These immunized mice were subsequently challenged with ETEC and showed full protection against the fluid influx response in the gut. This is the first report of using L. reuteri as a vaccine carrier to induce complete immunologic protection against ETEC.     

New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development.

This review attempts to synthesize the new knowledge of pathogenesis of bacterial enteric infections and relate this information to vaccine development. Discussion focuses on human infections and to those in which significant strides have been made. As a general theme in the pathogenesis of bacterial enteric infections, pathogens can be characterized into 5 groups on the basis of their degree of ultimate invasiveness after ingestion by a susceptible hose: mucosal adherence and enterotoxin production; mucosal adherence and brush border dissolution -- enteropathogenic E. coli (EPEC) of "classical" serotypes; mucosal invasion and intraepithelial cell proliferation; mucosal translocation followed by bacterial proliferation in the lamina propria and mesenteric lymph nodes; and mucosal translocation followed by generalized infection. The review covers cholera (motility and chemotaxis, mucosal adhesion, flagellar sheath protein, hemagglutinins, outer membrane proteins, enterotoxin production, quality and duration of infection derived immunity, immune response in humans, LPS, flagellar sheath protein, cholera lectin, other cholera hemagglutinins, outer membrane protein, previous cholera vaccines, killer whole cell vaccines, toxoids, combination vaccines, attenuated versus cholerae vaccines): enterotoxigenic Escherichia coli (ETEC) (entertoxins, O:H serotypes and enterotoxin phenotypes, colonization factors, immune response in humans, vaccines against ETEC, and toxiods); EPEC (vaccines against EPEC); Shigella (smooth LPS O antigen, epithelial cell invasiveness, Shigella toxin, and Shigella vaccines); and typhoid fever (caccines against typhoid fever). The major attraction of a nonliving oral cholera vaccine is its safety. A review of available information leads to the conclusion that an oral vaccine consisting of a combination of antigens, intending to stimulate both antibacterial and antitoxic immunity, would be most likely to succeed. Current approaches to immunoprophylaxis of ETEC infection involve vaccines that stimulate antitoxic or antiadhesion immunity or both by means of killed antigens or attenuated strains. It is likely that the most effective vaccines will contain appropriate antigens intended to simultaneously stimulate both antibacterial and antitoxic immunity, thereby leading to a synergistic protective effect. Now that the speical enteroadhesive properties of EPEC have been characterized and shown to be associated with a plasmid, it should be possible to identify the phenotypic gene products responsible for this phenomenon. It is likely that fimbriae or outer membrane proteins will prove to be the organelle of adhesion. When such information becomes available, it should be possible to prepare oral vaccines consisting of the purified antigen. Efficacy has been shown for attenuated Shigella strains utilized as oral vaccines. The major thrust in the development of new immunization agensts against typhoid fever is to identify immunizing agents at least equal in efficacy to the parenteral acetone killed vaccine but which cause no adverse reactions.     

Protective Immunity Induced by Oral Immunization with a Rotavirus DNA Vaccine Encapsulated in Microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

Intranasal immunization with a peptide conjugated to Salmonella flagellin induces both systemic and mucosal peptide‐specific antibody responses in mice

In this study, the mucosal adjuvant activity of Salmonella flagellin as a carrier in a conjugate of EXP153–rFliC was investigated. EXP153–rFliC was made by conjugation of a synthetic B‐cell epitope peptide derived from Plasmodium falciparum exported protein‐1(EXP153) to recombinant phase 1 flagellin of Salmonella enterica serovar Typhimurium expressed in Escherichia coli (rFliC), and used to immunize BALB/c mice via intranasal instillation. It was found that robust EXP153‐specific serum IgG antibodies were induced without additional adjuvant. EXP153‐specific sIgA antibodies were also induced, these being detected in bronchoalveolar, nasal, vaginal and intestinal washes. These observations demonstrate that Salmonella flagellin as a carrier is an effective mucosal adjuvant in that its conjugated peptide induces antibody responses.     

Oral immunization of mice with plant-derived fimbrial adhesin FaeG induces systemic and mucosal K88ad enterotoxigenic Escherichia coli-specific immune responses

The importance of adhesins in pathogenicity has resulted in them being useful targets in the defense against bacterial infections. To produce edible vaccines against piglet diarrhea caused by enterotoxigenic Escherichia coli (ETEC), plants were genetically engineered to produce recombinant fimbrial adhesin FaeG. To evaluate the efficacy of the edible vaccine FaeG in mice, the soluble protein extracts were examined by about 15 microg recombinant FaeG for each oral immunization dose per mouse. After four doses of vaccination, both IgG and IgA antibodies specific to K88ad fimbriae were elicited in serum, and specific IgA antibodies were also evoked in feces of the immunized mice. Moreover, visible K88ad ETEC agglutination by the specific serum from the immunized mice was observed, implying the antibody was highly specific and effective. Results from an in vitro villous-adhesion assay further confirmed that serum antibodies of the immunized mice could inhibit K88ad ETEC from adhering to pig intestinal receptors, further demonstrating the oral immune efficacy of the plant-derived FaeG. This study provides a promising, noninvasive method for vaccinating swine by feeding supplements of transgenic plant. Moreover, the low cost and ease of delivery of this edible ETEC vaccine will facilitate its application in economically disadvantaged regions.     

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…     

Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1

Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.     

Mucosal priming of simian immunodeficiency virus-specific cytotoxic T-lymphocyte responses in rhesus macaques by the Salmonella type III secretion antigen delivery system

Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested DeltaphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01(+) macaques were inoculated with three oral doses of recombinant Salmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag(181-189) epitope were detected following each dose of SALMONELLA: After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag(181-189)-specific CD8(+) T-cell responses in peripheral blood. A significant percentage of the Gag(181-189)-specific T-cell population in each animal also expressed the intestinal homing receptor alpha4beta7. Additionally, Gag(181-189)-specific CD8(+) T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.     

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.     

Development of a mucosal complex vaccine against oral Salmonella infection in mice

We examined the immunogenicity of a Salmonella enterica complex vaccine (CV), consisting of flagellin and polysome purified from serotype Typhimurium LT2. CV plus cholera toxin (CT), in three oral doses given at 7-day intervals, conferred complete protection on C57BL/6 mice against lethal oral infection with a wild-type strain. It elicited mucosal IgA > IgG2a > IgG1 and systemic IgG2a > IgG1 > IgA antibodies to flagellin and polysome, and delayed footpad response (DFR) to both antigens. In Peyer's patches (PPs) and lamina propria (LP), IgA was produced under a Th1-dominant environment; CD4+T cells from produced interleukin (IL)-2, interferon (IFN)-gamma, and IL-10 by stimulation with salmonella extract. On the same protocol, flagellin plus CT induced flagellin-specific mucosal and systemic IgA and IgG1 antibodies, CD4+T cells producing IL-10 and IFN-gamma in PPs and LP, and only minimal levels of flagellin-specific DFR. Polysome plus CT induced polysome-specific mucosal and systemic IgG2a in addition to IgG1 and IgA antibodies, CD4+T cells producing IFN-gamma and IL-2 in PPs and LP, and polysome-specific DFR. These two vaccines, however, conferred at most 50-60% survival rates. Our results suggest that polysomes in CV provide effective adjuvant activity for the induction of both mucosal and systemic Th1-biased responses toward flagellin.     

Evaluation of Trimethoprim-sulphamethoxazole Compound in Treatment of Salmonella Infections

Fifty patients suffering from infections caused by various salmonella species were treated with trimethoprim-sulphamethoxazole compound. Twenty-three had enteric fever and two were biliary carriers of Salmonella typhi. The other 25 suffered from infections caused by salmonella species other than S. typhi or S. paratyphi B. Twenty-one of the patients with enteric fever responded clinically to the drug, one failed treatment, and one died. Two patients suffering from typhoid fever relapsed and three temporarily excreted S. typhi in stools following treatment. One of the typhoid carriers was successfully treated. All patients with infections caused by salmonella species other than S. typhi or S. paratyphi B responded to treatment but 17 continued to excrete the organism in their stools after the course of trimethoprim-sulphamethoxazole compound. Four patients developed rashes during therapy and two became anaemic.     

Systemic and mucosal immunity induced by oral somatic transgene vaccination against glycoprotein B of pseudorabies virus using live attenuated Salmonella typhimurium

Glycoprotein B mediates the absorption and penetration of the pseudorabies virus in the form of an immunodominant Ag, and represents a major target for the development of new vaccines. This study evaluated the efficiency of live attenuated Salmonella typhimurium SL7207 for the oral delivery of DNA vaccine encoding the pseudorabies virus glycoprotein B (pCI-PrVgB) in vivo, leading to the generation of both systemic and mucosal immunity against the pseudorabies virus Ag. An oral transgene vaccination of pCI-PrVgB using a Salmonella carrier produced a broad spectrum of immunity at both the systemic and mucosal sites, whereas the intramuscular administration of a naked DNA vaccine elicited no mucosal immunoglobulin (Ig)A response. Interestingly, the Salmonella-mediated oral transgene vaccination of the pseudorabies virus glycoprotein B biased the immune responses to the Th2-type, as determined by the IgG2a/IgG1 ratio and the cytokine production profile. However, oral vaccination mediated by Salmonella harbouring pCI-PrVgB showed inferior protection to systemic immunization against virulent pseudorabies virus infection. The expression of transgene delivered by Salmonella bacteria in antigen-presenting cells of both the systemic and mucosal-associated lymphoid tissues was further demonstrated. These results highlight the potential use of live attenuated S. typhimurium for an oral transgene pseudorabies virus glycoprotein B vaccination to induce broad immune responses.     

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.