Increased sensitivity of immune macrophages to the cytotoxic action of virulent shigellae

The in vitro interaction of live bacteria belonging to virulent and avirulent Shigella and Salmonella strains with peritoneal macrophages obtained from mice immunized by the intragastric administration of these bacteria has been studied. In contrast to Salmonella-activated macrophages capable of resisting the intracellular proliferation and the cytopathic action of homologous bacteria, Shigella-activated macrophages become more sensitive to the cytopathic action of virulent shigellae. The ability of shigellae to render an aggravating cytopathic effect on the activated macrophages correlates with the virulence of dysentery bacilli and is practically absent in avirulent strains, including S. flexneri 2a No. 516 M vaccine strain.     

Functional macrophage activity in infection with virulent and avirulent strains of the dysentery microbe

The effect of S. flexneri virulent and avirulent (vaccine) strains 2a on the cytoplasmic membrane of mouse macrophages has been studied by evaluating the action of these bacteria on the activity of 5-nucleotidase. The dynamics of the activity of 5-nucleotidase after the introduction of both virulent and avirulent strains has a phasic character with alternating rises and falls in the activity of this enzyme in comparison with the control. S. flexneri vaccine strain produces mainly a stimulating effect on the functional activity of peritoneal macrophages in mice, which is confirmed by a decrease in the activity of 5-nucleotidase; on the contrary, S. flexneri virulent strain- has mainly an inhibiting effect on the functional activity of peritoneal macrophages, which is confirmed by an increase in the activity of 5-nucleotidase in these cells. The comparative study of changes in the activity of 5-nucleotidase, following the introduction of S. flexneri, in mice, previously immunized with smallpox vaccine, and in intact mice has shown that the use of animals immunized with smallpox vaccine in the study of metabolic characteristics may lead to distortions in the results of the experiment.     

The role of delayed hypersensitivity in the development of an experimental dysentery infection and vaccinal process

The results of experiments, indicating the development of cell-mediated delayed hypersensitivity (DH) skin reaction, its dependence on the virulence of Shigella flexneri 2a, the dose and the character of the antigen used for testing the reaction, are presented. One of the immunologically active components of S. flexneri virulent strain 2a No. 516 is a biologically active thermolabile factor detected in filtrate and supernatant fluid. These data suggest that the immunosuppressive action of the virulent strain is probably linked with the function of T-suppressors affecting the reaction at the period of its development (the induction phase). S. flexneri avirulent strain produced no such effect, developing DH being seemingly sufficient for activating T-cell-mediated immune response.     

福氏痢疾菌膜成分与细菌抗原性和毒力的关系

应用免疫转移技术,用从感染福氏痢疾菌的病人获取的恢复期血清分析了福氏痢疾菌膜成分与细菌抗原性和毒力的关一。发现福氏痢疾菌的膜蛋白67kD和63kD均含有两种成分,一种和膜蛋白60kD都可能是保护性抗原,而另一种与膜蛋白78kD和35kD一样与福氏痢疾菌的毒力有关。采用微细胞同位素掺入示踪显示这些与病人恢复期血清反应的膜蛋白由质粒编码。     

弗氏志贺菌毒力大质粒导入大肠杆菌MG1655

目的:将弗氏2a志贺菌2457T的毒力大质粒pSF导入大肠杆菌MG1655。方法:通过诱动转移技术,将弗氏2a志贺菌2457T的毒力大质粒导入大肠杆菌MG1655。结果:构建了MG1655/pSF:pXL275-virG的毒力大质粒导入突变株,双向电泳初步比较分析表明在重组MG1655中有志贺菌毒力的表达。结论:成功地将弗氏2a志贺菌2457T毒力大质粒pSF导入了大肠杆菌MG1655。     

Development of a new guinea-pig model of shigellosis

Shigellosis is a major form of bacillary dysentery caused by Shigella spp. To date, there is no suitable animal model to evaluate the protective efficacy of vaccine candidates against this pathogen. Here, we describe a successful experimental shigellosis in the guinea-pig model, which has shown the characteristic features of human shigellosis. This model yielded reproducible results without any preparatory treatment besides cecal ligation. In this study, guinea-pigs were discretely infected with virulent Shigella dysenteriae type 1 and Shigella flexneri type 2a into the cecocolic junction after ligation of the distal cecum. All the experimental animals lost ～10% of their body weight and developed typical dysentery within 24-h postinfection. In the histological analysis, distal colon showed edema, hemorrhage, exudation and inflammatory infiltrations in the lamina propria. Orally immunized animals with heat-killed S. dysenteriae type 1 and S. flexneri type 2a strains showed high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies and conferred significant homologous protective immunity against subsequent challenges with the live strains. The direct administration of shigellae into the cecocolic junction induces acute inflammation, making this animal model useful for assessing shigellosis and evaluating the protective immunity of Shigella vaccine candidates.     

New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies

It has been difficult to evaluate the protective efficacy of vaccine candidates against shigellosis, a major form of bacillary dysentery caused by Shigella spp. infection, because of the lack of suitable animal models. To develop a proper animal model representing human bacillary dysentery, guinea pigs were challenged with virulent Shigella flexneri serotype 2a (strains 2457T or YSH6000) or S. flexneri 5a (strain M90T) by the intrarectal (i.r.) route. Interestingly, all guinea pigs administered these Shigella strains developed severe and acute rectocolitis. They lost approximately 20% of their body weight and developed tenesmus by 24 h after Shigella infection. Shigella invasion and colonization of the distal colon were seen at 24 h but disappeared by 48 h following i.r. infection. Histopathological approaches demonstrated significant damage and destruction of mucosal and submucosal layers, thickened intestinal wall, edema, erosion, infiltration of neutrophils, and depletion of goblet cells in the distal colon. Furthermore, robust expression of IL-8, IL-1beta, and inducible NO synthase mRNA was detected in the colon from 6 to 24 h following Shigella infection. Most importantly, in our new shigellosis model, guinea pigs vaccinated with an attenuated S. flexneri 2a SC602 strain possessing high levels of mucosal IgA Abs showed milder symptoms of bacillary dysentery than did animals receiving PBS alone after Shigella infection. In the guinea pig, administration of Shigella by i.r. route induces acute inflammation, making this animal model useful for assessing the protective efficacy of Shigella vaccine candidates.     

Fluorescent-Antibody and Histological Study of Vaccinated and Control Monkeys Challenged with Shigella flexneri

Formal, Samuel B., (Walter Reed Army Institute of Research, Washington, D.C.), T. H. Kent, S. Austin, and E. H. LaBrec. Fluorescent-antibody and histological study of vaccinated and control monkeys challenged with Shigella flexneri. J. Bacteriol. 91:2368-2376. 1966.-Groups of monkeys were fed four doses of a living Escherichia coli-Shigella flexneri 2a hybrid strain, and, together with control animals, were challenged with virulent S. flexneri 2a. Two experiments were carried out; in the first, the animals were challenged 10 days after and in the second, 1 month after the last vaccine dose was administered. At 48 hr after challenge, tissues were removed from the vaccinated and control animals, and examined by use of histological and fluorescent-antibody techniques. The results of this study demonstrate that the animals receiving the vaccine were protected from the tissue damage ordinarily observed after experimental challenge with virulent dysentery bacilli. The virulent challenge strain appeared to be unable to penetrate into the intestinal mucosa of immunized animals.     

Protection of monkeys against experimental shigellosis with a living attenuated oral polyvalent dysentery vaccine

Formal, Samuel B. (Walter Reed Army Institute of Research, Washington, D.C.), T. H. Kent, H. C. May, A. Palmer, and E. H. LaBrec. Protection of monkeys against experimental challenge with a living attenuated oral polyvalent dysentery vaccine. J. Bacteriol. 91:17-22. 1966.-Virulent strains of Shigella flexneri 1b, S. flexneri 3, and S. sonnei I were mated with an Hfr strain of Escherichia coli K-12, and hybrids were selected for the xylose marker. One hybrid strain of each of the serotypes was chosen for study of their biological characteristics. Their capacity to cause a fatal enteric infection in starved guinea pigs was reduced, they failed to cause dysentery when fed to monkeys, they caused keratoconjunctivitis in the guinea pig eye, and they penetrated HeLa cells. Two doses of a polyvalent oral vaccine composed of S. flexneri 1b, 2a, and 3, and S. sonnei I hybrid strains were fed to groups of monkeys at an interval of 4 to 7 days, and they, together with controls, were challenged 10 days after the last dose with one or another of the virulent parent dysentery strains. A significant degree of protection was afforded in all vaccinated groups with the exception of one group challenged with S. flexneri 6, a component not included in the vaccine. When animals were challenged with virulent S. flexneri 2a 1 month after oral vaccination, they were also protected. The vaccine produced a rise in serum antibody, but we were not able to detect coproantibody in saline extracts of feces from animals which received the vaccine.     

Reaction of the hematopoietic system in mice to the administration of virulent and avirulent strains of Shigella flexneri 2A

The influence of live bacteria and antigenic preparations of virulent and avirulent stains of Shigella flexneri 2a on the endogenous splenic colony formation in murine hematopoietic cells has been studied. The different stimulating activity of live microbial cells of virulent and avirulent strains Shigella flexneri 2a and their antigenic preparations on the endogenous colony formation has been shown. The effect observed depends on the preparation doses and time of their administration before irradiation of the animals. The stimulating influence on the development of hemopoiesis endogenous foci may be conditioned by the action of heat-labile products of the live microbial cells and closely correlates with the virulence of strains studied.     

稳定、无抗药的痢疾福氏2a和宋内双价菌苗候选株的构建

通过体内外基因重组,将大肠杆菌粘附因子cs3基因定位整合到痢疾杆菌福氏2a疫苗株T32菌染色体的asd基因内,使asd基因灭活;将来内O抗原基因克隆至无抗药性表达载体pXL378,获得重组质粒pXL390,将其转化asd-的T32受体菌,构建成福氏2a和宋内双价苗苗株FS01。实验表明:重组质粒pXL390在不带任何抗菌素基因的情况下,在asd-的T32受体菌内是稳定的。FS01株遗传稳定,能表达两种痢疾菌的PLS-O抗原,无明显毒性作用。动物试验表明,以FS01株皮下免疫的小鼠对福氏2a和宋内有毒株的腹腔攻击有100％的保护。     

Construction of a multivalent vaccine strain of Shigella flexneri and evaluation of serotype-specific immunity

Shigella flexneri causes more fatalities by shigellosis than any other Shigella species. There are 13 different serotypes of S. flexneri and their distribution varies between endemic geographical regions. The immune response against S. flexneri is serotype-specific, so current immunization strategies have required the administration of multiple vaccine strains to provide protection against multiple serotypes. In this study, we report the construction of a multivalent S. flexneri vaccine strain, SFL1425, expressing the O-antigen structure specific for serotypes 2a and 5a. This combination of type antigens has not previously been reported for S. flexneri. The multivalent vaccine strain, SFL1425 was able to induce a specific immune response against both serotypes 2a and 5a in a mouse pulmonary model.     

Characteristics of the action of the biologically active factor from virulent Shigella flexneri strains in a study on mice and a cell culture]

The authors subjected to further study the biologically active factor revealed by them earlier in the virulent Sh. flexneri cultures by using the genetically bound triad of Sh. flexneri 5a-222 cultures and the corresponding couple of Sh. flexneri 2a-516. There was shown correlation of the strains virulence determined by the keratoconjunctival test, with the presence of genetically-determined production of the biologically active factor detectable in the culture filtrate, which produced toxic action of the continuous cell cultures in the virulen Sh. flexneri strains of different serovars (2a and 5a), and lethal action in intravenous injection to mice. Comparative study of toxicity of the preparations of the endotoxin, free endotoxin, and neurotoxin types showed the biologically active factor to resemble the neurotoxin, differing from it in the toxic action and thermolability. Filtrates of the virulent and genetically characterised avirulent strains differed in the protein and lipids content, this permitting to suggest participation of the protein and lipid complex in the toxic action of the biologically active factor.     

Differentiation of virulent strains of Shigella and entero-invasive Escherichia from their avirulent variants using modified indirect immunoenzyme analysis

The data obtained in this investigation confirm that the modified indirect enzyme immunoassay (EIA) permits the differentiation of virulent bacteria of the genus Shigella and enteroinvasive Escherichia (group 1), regularly containing virulence plasmids with a molecular weight of 120-140 MD, from their avirulent variants which have lost these plasmids (group 2). The ratio of the optic density (OD) values of the positive control samples (the OD of group 1) to the OD values of the negative ones (the OD of group 2) is significantly higher than 2.1. As revealed by EIA, the differences between groups 1 and 3 (avirulent Shigella strains and E. coli smooth strain O124, retaining high-molecular plasmids with a molecular weight of 120-140 MD or their fragments) are statistically insignificant. The ratio of the OD of group 1 to the OD of group 3 is significantly less than 2.0. Analysis of outer membrane protein (OMP) preparations isolated from S. flexneri virulent strain 2a and its isogenic avirulent plasmid-containing variant has revealed significant differences in their EIA results. The ratio of the OP of OMP preparations isolated from the virulent strain to the OD of OMP preparations from the avirulent strain exceeds 2.1.     

The isolation and immunologic study of ribosomal preparations from Shigella flexneri

S. flexneri ribosomal preparations were isolated by differential centrifugation or by fractionation with polyethylene glycol-6000. Their chemical composition and spectrophotometric properties were characteristic of ribosomes, and, as shown by the results of the serological assay, the content of O-specific component was, on the average, 1.4%. The ribosomal preparations were nontoxic for mice when injected intraperitoneally and intravenously in large doses and induced systemic O-antibody response in mice and rabbits. The parenteral administration of ribosomes to guinea pigs led to the increase of resistance to Shigella keratoconjunctivitis. The results of different tests with the use of this model greatly varied. According to the summary data of several tests, the ribosomal vaccine enhanced the resistance of the eyes from 11.3% to 48.5% and the effectiveness coefficient of immunization was 42 +/- 6. Ribosomes isolated from S. flexneri avirulent strain 2a 51.6 M (Iu. A. Belaia's vaccine) showed the same activity as those isolated from virulent strains. The results obtained in this study suggest the expediency of further experimental study of ribosomal preparations obtained from S. flexneri as potential vaccine.     

福氏2a志贺氏菌△aroA突变减毒株的构建

志贺氏菌芳香族氨基酸合成酶基因缺陷能够使菌体明显减毒,并有可能成为新一代痢疾疫苗．用ＰＣＲ技术从野生型福氏２ａ志贺氏菌２４５７Ｔ中克隆出ａｒｏＡ基因,在体外进行精确的缺失突变,并通过体内同源重组,构建成△ａｒｏＡ突变体ＲＳ４２６．实验结果表明,这种突变体仍保持了侵袭能力和保护性Ｏ抗原的表达,但其毒力已明显降低,不能产生豚鼠角结膜炎,小鼠半数致死量明显提高．免疫保护试验显示,ＲＳ４２６可在小鼠中产生对福氏２ａ野生菌１００％的保护作用．     

鉴定痢疾杆菌毒力基因的SW480细胞模型构建

信号标签诱变技术（STM）是一种在体内高通量筛选病原体毒力基因的新方法,在应用时的一个先决条件是要建立合适的体内筛选系统。为将该技术应用于福氏痢疾杆菌,我们使用三个福氏痢疾杆菌菌株进行了预试验：通过同源重组构建而成的带有氯霉素抗性且aroA和virG基因失活的突变株RC426;因在侵袭质粒上自发缺失3个基因座(ipaBCDA, invA 和 virG)的另一减毒突变株T32,其曾被用作福氏痢疾杆菌的口服疫苗;还有具侵袭宿主细胞能力的野生性菌株2457T。将RC426、T32和2457T混合后侵袭结肠细胞系SW480,不同时间回收经侵袭后细胞裂解液中的菌体并统计。结果显示在侵袭12h内回收到减毒突变株的量与野生有毒株存在显著性差异,表明SW480 细胞系可用于痢疾杆菌的STM研究。Abstract: Signature-tagged mutagenesis (STM) is a novel technology with high throughput screening ability to identify virulent genes of pathogen in vivo. An appropriate animal or cell line model is one of prerequisites by exploiting this technique. In order to apply STM to Shigella flexneri, RC426 was constructed as an attenuated mutant with chloramphenicol resistance and aroA and virG genes inactivated by homologous recombination; Another attenuated strain T32 was used as an oral S. flexneri 2a vaccine due to a spontaneous deletion in three loci (ipaBCDA, invA and virG) on the virulence plasmid. The wild type strain 2457T had the invasion ability into host cells. The three strains, RC426, T32 and 2457T, were mixed together to invade colon cancer cell line SW480, and the distinct strains were recovered and counted from cell lysates of invaded SW480 in different time. The results showed that there were statistically significant differences between the amounts of two attenuated strains recovered and that of virulent strain within 12h invasion, indicating SW480 was a suitable cell model for applying STM to screen virulent genes of Shigella flexneri.     

Effect of a live enteral dysentery vaccine from spontaneous Flexner mutant 2a 516m on the immunoglobulin-producing cell count in the large intestine mucosa in dysentery

The influence of vaccinal therapy with live oral dysentery vaccine prepared from S. flexneri 2a 516M on the content of immunoglobulin-producing cells in the mucous membrane of the large intestine was studied. A considerable increase in the number of IgA- and IgM-synthetizing cells was shown to occur in the course of the infectious process in acute dysentery. In chronic dysentery the content of IgA- and IgM-synthetizing cells in patients was considerably lower than in a smooth course of acute dysentery. The use of the vaccine for the therapy of patients with chronic and especially acute dysentery resulted in a considerable rise in the number of plasma cells synthetizing IgA, IgM and IgG.     

宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷型减毒株的研究Ⅱ:生物学特性的研究

进一步对FS54—2、—3、—4、—5、—6、—7及—7b等双价减毒株的侵袭力、毒力、免疫原性与免疫保护性及遗传稳定性等进行了研究,证明双价减毒株仍保持了入侵肠上皮细胞的能力;小动物实验安全低毒;有很好的免疫原性,对小鼠有很好的双重免疫保护性。     

痢疾菌苗研究进展

痢疾是全世界范围内流行的肠道传染病。随着分子生物学技术的发展,迄今已构建了新一代预防痢疾的疫苗,其中包括:具有侵袭力和不具侵袭力的单价口服活菌苗候选株;它们与大肠杆菌、伤寒沙门氏菌和血清型不同的痢疾杆菌组成的双价和三价菌苗候选株;以及以脂多糖和核糖体为基础的不经胃肠的组分菌。猴体和人体试验证明它们安全有效。预计在未来的10年中,将会有一个或几个安全有效的痢疾疫苗投放市场。