Construction of genetically marked Vibrio cholerae O1 vaccine strains

Abstract Attenuated Vibrio cholerae O1 vaccine strains lacking the gene encoding the A subunit of cholera toxin have proven efficacious in preventing experimental cholera. As these strains move from closed, contained testing environment to large-scale field trials, a readily assayable phenotypic trait to distinguish a vaccine strain from wild-type V. cholerae O1 is desirable. We have constructed three derivatives of the attenuated V. cholerae strain CVD 103 which carry a mercury resistance or urease marker in the hlyA gene. CVD 103-HgR was constructed using a protracted marker-exchange procedure; this strain was found to have somewhat lowered colonisation efficiency in infant mice in comparison to its parent strain, CVD 103. The insertion of the resistance marker was repeated using a suicide vector system; CVD 103-HgR2 was found to colonise infant mice as efficiently as CVD 103. Strain CVD 103-UR, in which sequences encoding urease were inserted using a suicide vector, also colonised infant mice as well as CVD 103. The genetically marked strains CVD 103-HgR, CVD 103-HgR2 and CVD 103-UR form the basis for a generation of defined oral vaccines that may give single-dose, long-lasting protection to populations at risk from cholera.     

Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae

Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell's surface than strains from other orders or genera such as V. cholerae.     

Development and validation of a detection system for wild-type Vibrio cholerae in genetically modified cholera vaccine.

Orochol, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intact ctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol.     

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype-specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei. O polysaccharide is covalently bound to LPS cores of both the K-12 and RI types, but neither to Salmonella (Ra-type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1-type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfd_sonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri-type LPS. Co-expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase-l-like S. sonnei LPS in E. coli K-12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae, S. sonnei O antigen expressed in a V. eholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.     

宋内Ⅰ相O抗原及霍乱毒素B亚基在福氏2a痢疾菌中的表达及其免疫保护效果的观察

本文利用基因重组的方法,将宋内Ｉ相Ｏ抗原基因以及霍乱毒素Ｂ亚单位基因（ｃｔｘ－Ｂ）克隆至带链球菌的ａｓｄ基因的表达载体,然后转化至ａｓｄ－痢疾菌苗株福氏２ａＴ３２。脂多糖银染以及Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ实验证实以上两基因都能在宿主菌中稳定表达。动物（小白鼠）保护实验表明,该重组菌对福氏２ａ、宋内氏痢疾菌的保护效率达１００％,对霍乱弧菌的保护效率也达７０％。该菌具有稳定、无抗生素标记、多价的特点。     

Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD 103-HgR

Live, attenuated Vibrio cholerae vaccines can induce potent immune responses after only a single oral dose. The strategy of harnessing these strains to present antigens from heterologous pathogens to the mucosal immune system shows great promise. To fully realize this possibility, V. cholerae strains must be created that stably express antigens in vivo in sufficient quantity to generate an immune response. In vivo -induced promoters have been shown to increase the stability and immunogenicity of foreign antigens expressed from multicopy plasmids. We report the construction of a series of genetically stabilized plasmids expressing luciferase as a heterologous protein from the following in vivo -induced promoters: V. cholerae P _argC , P _fhuC and P _vca1008 , and Salmonella enterica serovar Typhi P _ompC . We demonstrate that several of these expression plasmids meet two critical criteria for V. cholerae live vector vaccine studies. First, the plasmids are highly stable in the V. cholerae vaccine strain CVD 103-HgR at low copy number, in the absence of selective pressure. Second, real-time bioluminescent imaging (BLI) demonstrates inducible in vivo expression of the promoters in the suckling mouse model of V. cholerae colonization. Moreover, the use of BLI allows for direct quantitative comparison of in vivo expression from four different promoters at various time points.     

Bactericidal properties of hemo-cytolysin from Vibrio cholerae non O1 P-11702 strain in a panel of indicator cultures for detection of vibriocins

The influence of the preparation of hemo-cytolysin, obtained from V. cholerae non O1 strain P-11702 and inducing lysis of both red blood cells and V. cholerae cultures using a panel of indicator cultures for the detection of vibriocins, was studied. The set of indicator cultures contained 2 Shigella flexneri strains, 1 S. dysenteriae strain, 3 S. sonnei strains, 3 Escherichia coli strains and 2 V. cholerae strains, one of them being atypical. Hemo-cytolysin exhibited lytic activity with respect to S. dysenteriae, S. sonnei strains and 1 V. cholerae strain. i.e. to 4 out of 11 indicator strains. V. cholerae atypical strain proved to be resistant to the preparation in contrast to 33 V. cholerae typical strains, studied previously.     

宋内Ⅰ相抗原和霍乱CT-B共表达的免疫保护效果观察

将编码宋内氏痢疾菌(Shigella sonnei)I相O抗原的基因和霍乱弧菌(Vibrio choler-ae)的CT-B基因克隆至带asd基因的质粒PYA248,得重组质粒PMGL105。将该重组质粒转入asd基因缺失的减毒伤寒沙门氏菌X4072,构成了一个不带抗药性基因的载体-宿主平衡致死系统。一系列实验表明,该重组菌X4072(PMGL105)能稳定地表达宋内I相O抗原和霍乱弧菌的CT-B抗原。小鼠免疫保护实验表明,该重组菌对有毒的宋内氏I相痢疾杆菌及霍乱弧菌的攻击均具有良好保护作用。     

Lipopolysaccharides of Vibrio cholerae. I. Physical and chemical characterization

Vibrio cholerae is the causative organism of the disease cholera. The lipopolysaccharide (LPS) of V. cholerae plays an important role in eliciting the antibacterial immune response of the host and in classifying the vibrios into some 200 or more serogroups. This review presents an account of our up-to-date knowledge of the physical and chemical characteristics of the three constituents, lipid-A, core-polysaccharide (core-PS) and O-antigen polysaccharide (O-PS), of the LPS of V. cholerae of different serogroups including the disease-causing ones, O1 and O139. The structure and occurrence of the capsular polysaccharide (CPS) on V. cholerae O139 have been discussed as a relevant topic. Similarity and dissimilarity between the structures of LPS of different serogroups, and particularly between O22 and O139, have been analysed with a view to learning their role in the causation of the epidemic form of the disease by avoiding the host defence mechanism and in the evolution of the newer pathogenic strains in future. An idea of the emerging trends of research involving the use of immunogens prepared from synthetic oligosaccharides that mimic terminal epitopes of the O-PS of V. cholerae O1 in the development of a conjugate anti cholera vaccine is also discussed.     

Lipopolysaccharides of Vibrio cholerae II. Genetics of biosynthesis

An account of our up to date knowledge of the genetics of biosynthesis of Vibrio cholerae lipopolysaccharide (LPS) is presented in this review. While not much information is available in the literature on the genetics of biosynthesis of lipid A of V. cholerae, the available information on the characteristics and proposed functions of the corepolysaccharide (core-PS) biosynthetic genes is discussed. The genetic organizations encoding the O-antigen polysaccharides (O-PS) of V. cholerae of serogroups O1 and O139, the disease causing ones, have been described along with the putative functions of the different constituent genes. The O-PS biosynthetic genes of some non-O1, non-O139 serogroups, particularly the serogroups O37 and O22, and their putative functions have also been discussed briefly. In view of the importance of the serogroup O139, the origination of the O139 strain and the possible donor of the corresponding O-PS gene cluster have been analyzed with a view to having knowledge of (i) the mode of evolution of different serogroups and (ii) the possible emergence of pathogenic strain(s) belonging to non-O1, non-O139 serogroups. The unsolved problems in this area of research and their probable impact on the production of an effective cholera vaccine have been outlined in conclusion.     

Identification of distinct lipopolysaccharide patterns among <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> and <Emphasis Type="Italic">Y. enterocolitica</Emphasis>-like bacteria

The lipopolysaccharide (LPS) of strains representing various serotypes of Yersinia enterocolitica and Y. enterocolitica-like bacteria was studied by deoxycholate-PAGE and silver staining analysis. Four main types of LPS were detected based on the O-polysaccharide (O-PS): (i) LPS with homopolymeric O-PS, (ii) LPS with ladder-forming heteropolymeric O-PS, (iii) LPS with single-length O-PS, and (iv) semi-rough LPS without O-PS. Within the first three types, several subvariants were detected. Selected serotypes representing all above LPS types are sensitive to bacteriophage ϕR1-37 indicating that they share the phage receptor, a hexasaccharide called outer core in Y. enterocolitica O:3. Whereas phage ϕR1-37-resistant mutants of homopolymeric O-PS have lost only the outer core, those of ladder-forming or single-length O-PS have lost also the O-PS suggesting that in the latter ones the outer core is bridging between O-PS and lipid A-core. This work forms a basis of further structural, biochemical and genetic studies of these LPSs.     

Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.     

霍乱cT-B和LPs-o双价抗原工程菌苗株的构建

作者将霍乱弧菌O抗原及毒素B亚单位基因片段,经DNA体外重组技术,得到了能表达双价抗原的工程菌株1046(pMG305)。经GM1-ELISA分析表明该菌株能够表达特异的霍乱CT-B抗原,且能分泌到胞外,通过菌体凝集,全细胞O抗原酶联分析和血凝抑制试验表明在1046(pMG305)菌体表面表达了霍乱的O抗原,它的脂多糖O抗原通过SDS-PAGE电泳分析,显示它表达了霍乱LPS的特征区带。小鼠腹腔免疫后用霍乱弧菌毒株攻击表明,有良好的保护作用,因此1046(pMG305)可望成为霍乱活疫苗的候选株。     

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。     

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。     

Effect of miconazole on the structure and function of plasma membrane of Candida albicans

Abstract Fructose, a rarely occurring sugar constituent of Gram-negative bacterial lipopolysaccharides (LPS), is distributed ubiquitously in LPS of 01 Vibrio cholerae so far examined, but its location in LPS has not hitherto been elucidated. It was found that hydrazinolysis of LPS successfully affords a derivative retaining virtually all the fructose of intact LPS, but no ester-bound phosphate. Structural analysis carried out on the LPS derivative prepared by the hydrazinolysis of R-type LPS isolated from a rough mutant strain (NIH 41R) of 01 V. cholerae NIH 41 (Ogawa) revealed that the fructose is present as a non-reducing terminal residue bound to position C-6 of a glucose residue in the core region. This finding is considered to exclude the possibility that, in the LPS of 01 V. cholerae , the fructose is present in the region of the inner core in place of 2-keto-3-deoxyoctonate.     

The binding of synthetic analogs of the upstream,terminal residue of the O-polysaccharides (O-PS) of Vibrio cholerae O:1 serotypes Ogawa and Inaba to two murine monoclonal antibodies (MAbs) specific for the Ogawa lipopolysaccharide (LPS)

The binding of nineteen analogues of the upstream, terminal, monosaccharide residue of each of the O-polysaccharide (O-PS) of Vibrio cholerae O:1, serotype Ogawa and Inaba, with two murine monoclonal IgG antibodies both specific for the Ogawa LPS were measured using fluorescence spectroscopy. The use of the deoxy and the deoxyfluoro analogs allowed further refinement of the hydrogen-bonding pattern involved in the binding. Based on the binding characteristics observed for some of the ligands in the Inaba series, the binding of the monosaccharide that represents the upstream, terminal unit of the O-PS of V. cholerae O:1 serotype Inaba was redefined. We show for the first time that the upstream, terminal monosaccharide of the Inaba O-PS shows weak binding with these two anti-Ogawa antibodies. The results obtained allow further rationalization of the structural basis for the binding of V. cholerae O:1 antigens to their homologous antibodies.     

Study of immunochemical heterogeneity of Azospirillum brasilense lipopolysaccharides

A comparative immunochemical analysis of lipopolysaccharides (LPS) in Azospirillum brasilense model strains Sp7 and Sp245 and in mutants with transformed somatic antigens has been performed. According to the results of a complex of various immunochemical methods, including studies with polyclonal antibodies against the LPS these bacteria, their LPS consist of an assembly of macromolecules with different antigenic characteristics. Two types of O-specific polysaccharides (O-PS) are present in the LPS of every strain of A. brasilense under study. The major difference between the two O-PS is the antigenic heterogeneity of one of them. This heterogeneous O-PS has been shown to possess at least two O-factors (antigenic determinants) different in their structure. Meanwhile, according to all the tests performed, the other O-PS in every strain is immunochemically homogeneous and identical to one of the determinants revealed in the more diversified O-PS. The LPS heterogeneity among the given strains may be due to the pattern of O-specific polysaccharide synthesis, one of the O-PS being an intermediate in the synthesis of the other.     

Evaluation of the immunological activity of a vaccine from mutant Salmonella minnesota R 595 on a model of experimental Pseudomonas aeruginosa infection in mice

The comparative study of heated corpuscular vaccines prepared from S. minnesota mutant R 595 with defective lipopolysaccharide (LPS), chemotype Re, derived from S. minnesota strain SF 1111 with unchanged LPS, and from P. aeruginosa strain PA 103, was carried out. In contrast to the vaccine from S. minnesota strain SF 1111, the vaccine prepared from the mutant with chemotype Re induced the development of cell-mediated and humoral immunity to P. aeruginosa, and its immunogenicity was close to that of the vaccine from P. aeruginosa strain PA 103.     

The effect of Shigella sonnei strains opposite in their virulence on different links in the immune system]

For the first time different action of S. sonnei strains, opposite in their virulence, on hematopoiesis and the functional activity of T- and B-lymphocytes has been shown. The hematopoiesis-disturbing action of virulent shigellae is manifested by their capacity, more pronounced than similar capacity of an avirulent (vaccine) strain, for stimulating the processes of endo- and exocolony formation, the proliferation of hematopoietic stem cells and their migration to the blood. The effect produced by shigellae on T-cell-mediated immune response is manifested by the suppression of macrophage migration and its subsequent activation, whose manifestations and duration depend on the virulence of S. sonnei strains under study. The modulating effect of S. sonnei on B-cell-mediated immune reactions is manifested by the inhibiting action of S. sonnei virulent strain and the stimulating action of S. sonnei vaccine strain on the formation of antibody-producing cells synthesizing S. sonnei lipopolysaccharide antibodies shortly after the injection of shigellae. The results of this study indicate that S. sonnei virulent and avirulent (vaccine) produce multifunctional and differing effects on cell-mediated immune reactions, these processes being dependent on the virulence of shigellae and their individual specific antigens.