The involvement of tumor necrosis factor in immunity to Salmonella infection

The role of TNF in immunity to Salmonella in mice was studied. Antiserum specific for murine TNF was raised and used to neutralize TNF activity in vivo. Injection of this serum into mice infected with the moderately mouse virulent Salmonella typhimurium strain M525 caused exacerbation of disease. Such treatment had no effect on the course of an infection with an attenuated S. typhimurium aroA (strain SL3261) mutant. However, the protection afforded by immunisation with live SL3261 against challenge with the virulent parent strain (SL1344) was abolished by anti-TNF antiserum. Interestingly both early (3 wk) immunity and late (10 wk) immunity was neutralized by such treatment. Inasmuch as early immunity is considered to be nonspecific and macrophage-mediated while late immunity is considered to be serotype-specific and T cell mediated, this suggests that TNF plays a role in protection from Salmonellosis in both cases.     

HCV多表位抗原基因重组减毒口服活菌苗的研究

把丙型肝炎病毒(hepatitis C virus,HCV)复合多表位抗原基因PCX与β半乳糖苷酶基因(GZ)融合后,构建重组减毒鼠伤寒沙门氏菌口服活菌苗SL3261(pWR/PCX),免疫小鼠及家兔后,于第6周始可检测到低水平的抗GZPCX IgG(1∶200),至3月时最高滴度分别达1∶800及1∶25 600,均显著高于宿主菌SL3261组及空白对照组。在免疫小鼠的肠道灌洗液中可检测到抗GZPCX sIgA\.GZPCX抗原可促进免疫小鼠及家兔淋巴细胞增殖,诱发明显的迟发性超敏反应(DTH)。口服免疫后小鼠体重出现一过性下降,但未见其它明显的毒性作用,安全性较好。本研究从新的角度探讨了HCV复合多表位重组口服活菌苗的可行性,为HCV疫苗的研究提供新的实验依据。     

HCV/HBV复合抗原基因的克隆、表达及免疫应答研究

为探讨ＨＣＶ／ＨＢＶ 复合疫苗的可行性,将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ与ＨＢｓＡｇ 基因连接成ＰＣＸＳ基因,与β－半乳糖苷酶（ＧＺ）基因融合后在大肠杆菌及减毒鼠伤寒沙门氏菌中获得表达．目的蛋白ＧＺ－ＰＣＸＳ可被抗－ＨＢｓ 及抗－ＨＣＶ 抗体所特异识别．ＧＺ－ＰＣＸＳ抗原皮下注射免疫ＩＣＲ小鼠后,诱发了较高水平的抗－ＧＺ－ＰＣＸＳＩｇＧ反应．构建的重组减毒鼠伤寒沙门氏菌ＳＬ３２６１（ｐＷＲ／ＰＣＸＳ）口服免疫小鼠后,诱发了高水平的ＣＤ８＋ Ｔ细胞增殖反应及抗ＧＺ－ＰＣＸＳＩｇＧ反应．所有免疫小鼠均未见明显的毒副作用．该研究揭示,ＨＣＶ／ＨＢＶ 复合抗原可诱发特异性体液免疫及细胞免疫应答,而活菌苗口服可能是理想的免疫途径,为ＨＣＶ／ＨＢＶ 双价疫苗研究提供了一定的理论及实验依据．     

Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice

Aim. To evaluate the protective effect of live attenuated Salmonella typhimurium expressing catalase against gastric Helicobacter pylori infection in mice, and to explore the underlying mechanisms of the protective immune reaction. Materials and Methods The H. pylori catalase gene was introduced into attenuated S. typhimurium strain SL3261. C₅₇BL/6 mice were orally immunized with the SL3261 vaccine strain expressing catalase or with SL3261 alone or phosphate‐buffered saline (PBS). Mice were sacrificed 4 weeks after immunization and 5 weeks after H. pylori challenge, respectively. Results. All PBS control mice were infected. Eight of 13 (61.5%) mice immunized with the SL3261 vaccine strain and three of 14 (21%) mice immunized with SL3261 alone showed protection against H. pylori infection. Serum anti‐H. pylori IgG2a levels of S. typhimurium‐immunized mice were higher than those of PBS controls, both before and after H. pylori challenge, while there were no differences for IgG1 and IgA. Similarly, mRNA expression of interleukin (IL)‐2, IL‐12 and interferon‐γ in the gastric mucosa of S. typhimurium‐immunized mice was significantly higher than that of PBS controls both before and after challenge. Moreover, S. typhimurium‐immunized mice were characterized by marked infiltration of lymphocyte and mononuclear cells in the gastric mucosa after challenge. IL‐4 and IL‐10 were not detected in any of the three groups. IL‐6 expression was increased in the PBS group compared with the S. typhimurium‐immunized groups after challenge. Conclusions. This study demonstrates that oral immunization of mice with catalase delivered by an attenuated S. typhimurium strain offers protection against H. pylori infection. This protective immunity was mediated through a predominantly Th1‐type response and was associated with post‐immunization gastritis.     

Oral Salmonella typhimurium (AroA-) vaccine expressing a major leishmanial surface protein (gp63) preferentially induces T helper 1 cells and protective immunity against leishmaniasis

The gp63 gene of Leishmania major was transformed into the AroA- vaccine strain of Salmonella typhimurium (SL3261). The construct (SL3261-gp63), which stably expresses the gp63 Ag in vitro, was used to immunize CBA mice by the oral route. Spleen cells from mice inoculated with SL3261-gp63 developed antibody and proliferative T cell response to L. major. They did not express detectable delayed-type hypersensitivity reactivity. The activated T cells are mainly CD4+ and secrete IL-2 and IFN-gamma but no IL-4. The orally immunized mice developed significant resistance against a challenge L. major infection. We have, therefore, demonstrated the feasibility of oral vaccination against leishmaniasis and that the oral route of antigen delivery via the heterologous carrier may preferentially induce Th1 subsets of CD4+ cells.     

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.     

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.     

染色体整合系统在多价疫苗构建中的应用研究

通过同源重组将编码异源抗原的DNA整合到减毒的鼠伤寒沙门氏菌的染色体上,获得了表达霍乱毒素B亚单位（CTB）的双价活疫苗候选株。该系统包括两个步骤：首先将GisOG缺失突变的DNA片段整合进鼠伤寒沙门氏菌疫苗候选株SL3261的染色体上,得到His营养缺陷型。然后,用带有CTB抗原基因的完整HisOGDNA片段置换HisOG缺失的DNA片段,获得表达CTB的His回复的SL3261菌株（命名为TT201）。Southern杂交证明,TT201菌株的染色体带有CTB抗原基因。Westernblot分析表明,TT201菌株能表达CTB,且具有很好的稳定性。用重组菌株口服免疫接种小鼠,能够激发抗CTB抗体的产生。TT201菌株是一种潜在的双价疫苗候选株。     

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…     

以减毒沙门氏菌为载体的口服胞嘧啶脱氨酶基因治疗小鼠肿瘤

通过电转化法将真核表达载体EGFPN1和pLCDSN导入减毒鼠伤寒沙门氏菌SL3261中,经由胃管饲于C57BL／6和BALB／c小鼠。6周后接种Lewis和4T1肿瘤细胞,待种瘤增至直径为10mm左右,辅以‘腹腔注射5－氟胞嘧啶治疗。通过流式细胞仪、共聚焦显微镜和PCR等方法,在小鼠的肝脏、脾脏、小肠、肾脏、肿瘤等组织器官中均可检测到胞嘧啶脱氨酶基因的整合,绿色荧光蛋白在小鼠的脾脏和肿瘤中表达最强,其他组织表达甚弱,利用胞嘧啶脱氨酶／5－氟胞嘧啶系统进行治疗的小鼠肿瘤生长较其他组织显著受抑（P＜0.01),小鼠的生存期明显延长（P＜0.01),未观察到明显的毒副作用。     

Recombinant salmonella-based 4-1BBL vaccine enhances T cell immunity and inhibits the development of colorectal cancer in rats: in vivo effects of vaccine containing 4-1BBL

Background

Immunotherapy with vaccines is attractive for the treatment of cancer. This study is aimed at determining the effect of recombinant Salmonella (SL3261)-based 4-1BB ligand (4-1BBL) vaccine on the development of colorectal cancers and the potential immune mechanisms in rats.

Results

In comparison with that in the PBS group, similar levels of 4-1BBL expression, the frequency of T cells, IFN-γ responses, and comparable numbers of tumors were detected in the SL3261 and SL3261C groups of rats. In contrast, significantly fewer numbers of tumors, increased levels of 4-1BBL expression in the spleens and colorectal tissues, higher frequency of peripheral blood and splenic CD3⁺CD25⁺ T cells, and stronger splenic T cell IFN-γ responses were detected in the SL3261R group of rats.

Conclusion

Our results indicated that vaccination with recombinant attenuated Salmonella harboring the 4-1BBL gene efficiently enhanced T cell immunity and inhibited the development of carcinogen-induced colorectal cancers in rats.     

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.     

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.     

红曲多糖抑瘤作用初步研究

以昆明种小鼠为试验动物,通过皮下注射建立小鼠移植性S180肿瘤模型,红曲多糖灌胃2周后,摘取瘤体和脾脏,计算抑瘤率和脾指数,并用SPSS软件进行统计分析。结果显示,红曲多糖低、中、高剂量组均能抑制荷瘤小鼠的肿瘤生长,抑瘤率分别达24.70%、31.03%、39.82%,呈量效关系,对S180肉瘤生长有明显的抑制作用;红曲多糖能提高小鼠的免疫力,高剂量组免疫器官质量显著增加。研究表明红曲多糖有抗肿瘤作用,具有一定的开发价值。     

Structural and immunochemical studies of the lipopolysaccharides of Salmonella strains with both antigen O4 and antigen O9.

Two Salmonella hybrid strains, SL5313 (Salmonella typhimurium with a D.rfb+ gene cluster) and SL5396 (S. enteritidis with a B.rfb+ gene cluster), each expressing both O-antigen 4 (of serogroup B) and O-antigen 9 (of serogroup D) were studied by immunofluorescence using a mixture of O4-specific mouse monoclonal and O9-specific rabbit polyclonal antibodies. Bound antibodies, detected by anti-mouse antibody labelled with fluorescein isothiocyanate and anti-rabbit antibody labelled with tetramethylrhodamine isothiocyanate showed that more than 98% of the bacteria expressed both the O4 and O9 epitopes. Phenol-water-extracted lipopolysaccharide from batch-grown cultures subjected to sugar and methylation analyses by gas-liquid chromatography and mass spectrometry were shown to contain abequose (of the O4 epitope) and tyvelose (of the O9 epitope) in ratios of 1:1.5 and 1:2.5 for SL5313 and SL5396, respectively. Isolated polysaccharide chains, obtained by weak-acid hydrolysis of the lipopolysaccharides, were found to contain both O4 and O9 specificities in the same molecule, since polysaccharide bound to O4 antibody attached to a solid-phase-adsorbed O9-specific antibody and vice versa. This demonstrates that in strains SL5313 and SL5396 O chains containing both O4 repeating units (from S. typhimurium) and O9 units (from S. enteritidis) are present.     

Type I IL-1 receptor blockade exacerbates murine listeriosis.

It was found that IL-1 is produced in livers and spleens of mice shortly after the i.v. injection of a sublethal or lethal Listeria monocytogenes inoculum. In sublethally infected mice, IL-1 was present in infected livers and spleens by the end of the first day of infection. Thereafter, the amounts of IL-1 in these organs increased and decreased in concordance with bacterial numbers. IL-1 was not present in the peripheral circulation of mice during sublethal listeriosis, but was present in the blood late in lethal infection. Evidence showing that IL-1 plays a role in antibacterial resistance early in listeriosis was obtained through the use of 35F5 mAb that binds to the murine type I IL-1R and functions to block IL-1 alpha and IL-1 beta actions. Blockade of the type I IL-1R by the 35F5 mAb results in greatly enhanced bacterial growth in the livers and spleens of mice that had received a sublethal Listeria inoculum. Consistent with the exacerbation of listeriosis caused by 35F5 mAb, but in contrast to the effect of 35F5 mAb in other murine models, 35F5 mAb-treated mice exhibit markedly elevated levels of IL-6 in their circulation and infected organs.     

减毒沙门氏菌介导的血小板第四因子活性片段的放射保护作用研究

目的:观察减毒沙门氏菌携带的血小板第四因子活性片段PF417 70 的放射保护作用。方法:通过口服途经喂饲小鼠携带PF4活性片段的减毒沙门氏菌,在第 2次喂饲后小鼠接受 70 0cGy全身照射,然后观察PIRES2 EGFP PF417 70 在小鼠体内的表达,并观察小鼠的造血恢复情况。结果:在小鼠的肝脏、脾脏、肾脏、小肠、外周血及骨髓均能检测到GFP的表达和转基因的整合。与对照组比较,实验组小鼠的生存期明显延长,照射后第 7d和 1 4d骨髓有核细胞数、骨髓培养的CFU GM和HPP CFC数量明显增加 (P <0 0 5 )。结论:首次应用减毒沙门氏菌SL32 61为载体来介导PF4活性片段的生物学作用,并证实通过口服途径可以保护小鼠免受放射损伤,并促进放射损伤后小鼠的造血恢复。     

稳定携带新城疫病毒DNA疫苗减毒沙门氏菌的构建及其免疫原性

采用PCR技术从重组质粒pVAX1-F中扩增出新城疫病毒JS5株的融合蛋白(F)基因,将其克隆入真核表达质粒pmcDNA3.1 中,获得重组表达质粒pmcDNA3.1-F.通过电穿孔转化法将重组质粒转入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(pmcDNA3.1-F).体内、体外试验结果表明,重组质粒pmcDNA3.1-F在沙门氏菌中的稳定性显著高于pcDNA3.1-F.将重组菌SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)分别以1×109 CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对新城疫病毒F蛋白的血清抗体和黏膜抗体.重组菌以5×109 CFU剂量两次口服免疫4日龄SPF鸡,免疫鸡产生的针对新城疫病毒F蛋白的血清抗体和小肠黏膜抗体效价水平与空载体组之间存在显著性差异(P＜0.05).免疫保护试验结果显示,SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)免疫组的免疫保护率均与空载体组之间存在显著性差异(P＜0.05),且SL7207(pmcDNA3.1-F)免疫组的保护率较SL7207(pcDNA3.1-F)免疫组提高了20.0%,说明稳定携带新城疫病毒DNA疫苗的减毒沙门氏菌具有良好的免疫原性和免疫保护性.     

Salmonella typhimurium induces apoptosis in human monocyte-derived macrophages

Salmonella species represent a leading cause of gastroenteritis worldwide. More recently, they have been proposed as putative vaccine delivery vehicles in humans. Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages. In this study, we investigated the fate of Salmonella typhimurium-infected human macrophages differentiated from blood monocytes by GM-CSF. Wild type S. typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30 min post-infection. Three hours later more than 80% of the macrophages in the culture were killed. In contrast, during the same period, macrophages infected with the non-invasive S. typhimurium strain BJ66 remained viable. Chromatin fragmentation is a hallmark of cells undergoing apoptosis. Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells. Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase-3, a member of the pro-apoptotic enzyme family shown to be involved in S. typhimurium-induced killing of mouse macrophages, reduced SL1344-mediated cytotoxicity by 40%. Our study provides the first evidence that invasive S. typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM-CSF, and that cell death is a caspase-dependent phenomenon.