Immunological Response of the Rabbit to Vi Antigen

Vi antibody response of rabbits varied depending on whether Vi antigen was administered in particulate or soluble state. Vi antigen in particulate form induced hemagglutinins, bacterial agglutinins, and passive cutaneous anaphylaxis (PCA) antibodies, whereas soluble Vi antigen induced only hemagglutinins. Guinea pigs passively sensitized with antisera against particulate Vi antigen gave PCA reactions when challenged with either soluble or cellular Vi antigen; antisera against soluble Vi antigen were negative for PCA. The specificity of PCA was demonstrated by its dependence on the Vi concentration and by absorption of PCA activity from antisera with V-form cells of Salmonella typhosa.     

Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii.

Certain strains of the genus Citrobacter exhibit a variable expression of the Vi surface antigen that appears to involve a special mechanism for regulation of gene expression. Two nonlinked chromosomal loci, viaA and viaB, are known to determine nonvariable Vi antigen expression in strains of Salmonella. To confirm the presence of analogous loci in Citrobacter and to ascertain whether either of them is involved in variable Vi antigen expression in this organism, donor strains were constructed from Citrobacter freundii WR7004 and used to transfer their Vi antigen-determining genes to ViaA- and ViaB- Salmonella typhi recipient strains. Vi antigen expression in C. freundii was found to be controlled by loci analogous to the Salmonella via genes. S. typhi recipients of the C. freundii viaA+ genes were restored to the full, continuous expression of the Vi antigen normally seen in S. typhi. Thus, the C. freundii viaA genes appeared to play no role in the variable expression of the Vi antigen. In contrast, S. typhi recipients of the C. freundii viaB+ genes exhibited the rapid, reversible alternation between full Vi antigen expression and markedly reduced Vi antigen expression that was seen to occur in the C. freundii parent. The C. freundii viaB locus was thus identified as the one whose genes are regulated so as to produce variable Vi antigen expression. Genes determining another C. freundii surface antigen, the synthesis of which is not affected by the mechanism regulating Vi expression, were coinherited with the C. freundii viaB+ genes. An invertible, insertion sequence element located within the C. freundii viaB locus is proposed to account for the regulation of variable Vi antigen expression.     

Interaction of Vi antigen with proteins

Whiteside, Roberta E. (Boston University School of Medicine, Boston, Mass.), and Edgar E. Baker. Interaction of Vi antigen with proteins. J. Bacteriol. 92:1597-1603. 1966.-Purified Vi antigen (Vi) mixed in equal amounts with bovine serum albumin (BSA) or human gamma globulin (HGG) at pH values above 4.7 formed a complex which was not precipitated by trichloroacetic acid or tungstic acid. At pH values below 4.7, the interaction between Vi and either BSA or HGG produced insoluble complexes except when excess Vi antigen was present. When sufficient Vi was present at the lower pH values, the soluble complex was not precipitated by trichloroacetic acid. Other acid polysaccharides tested did not form trichloroacetic acid-soluble complexes with BSA. When subjected to immunoelectrophoresis, the Vi-BSA complex migrated in agar at a rate different from that of either BSA or Vi alone. The complex reacted with both Vi and BSA antiserum. The addition of either BSA or Vi antiserum to a Vi-BSA complex resulted in dissociation of the complex and precipitation of either Vi or BSA, depending upon the antiserum used. Vi antigen mixed with purified O antigen from Salmonella typhosa formed a complex which migrated in agar at a rate different from that of either component alone when subjected to immunoelectrophoresis.     

伤寒─鼠伤寒重组株Vi4072的效力试验研究

伤寒─鼠伤寒重组株Ｖｉ４０７２所产生Ｖｉ抗原以伤寒Ｔｙ２株所产Ｖｉ抗原作对照,通过ＥＤ５０测定和小白鼠被动保护试验作了比较,结果表明Ｖｉ４０７２株Ｖｉ抗原的半数有效剂量为０．０１３６μｇ,Ｔｙ２株的半数有效剂量为０．０１８３μｇ,说明Ｖｉ４０７２─Ｖｉ对小白鼠的保护作用不低于Ｔｙ２─Ｖｉ。被动保护试验证明,在同等条件下,两种Ｖｉ抗原的免疫血清对小白鼠提供的保护作用相同。     

伤寒─鼠伤寒重组株Vi4072的效力试验研究

伤寒─鼠伤寒重组株Ｖｉ４０７２所产生Ｖｉ抗原以伤寒Ｔｙ２株所产Ｖｉ抗原作对照,通过ＥＤ５０测定和小白鼠被动保护试验作了比较,结果表明Ｖｉ４０７２株Ｖｉ抗原的半数有效剂量为０．０１３６μｇ,Ｔｙ２株的半数有效剂量为０．０１８３μｇ,说明Ｖｉ４０７２─Ｖｉ对小白鼠的保护作用不低于Ｔｙ２─Ｖｉ。被动保护试验证明,在同等条件下,两种Ｖｉ抗原的免疫血清对小白鼠提供的保护作用相同。     

Factors Affecting Formation of Incomplete Vi Antibody in Mice.

Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Factors affecting formation of incomplete Vi antibody in mice. J. Bacteriol. 90:635-642. 1965.-Single immunizing doses of purified Vi antigen elicited complete and incomplete Vi antibodies in BALB/c mice, but only incomplete antibody in Cinnamon mice. Three of six other mouse strains tested responded like BALB/c mice; the remaining three, like Cinnamon mice. Varying the quantity of antigen injected or the route of administration failed to stimulate the production of detectable complete Vi antibody in Cinnamon mice. Such antibody was evoked in these animals by multiple injections of Vi antigen or by inoculating them with Vi-containing bacilli or Vi-coated erythrocytes. The early protection afforded by serum from Vi-immunized BALB/c mice coincided with the appearance of incomplete Vi antibody, 1 day prior to the advent of complete antibody. Persistence of incomplete as well as complete antibody in the serum of immunized mice was demonstrated for at least 56 days after injection of 10 mug of Vi antigen. Incomplete Vi antibody was shown to have blocking ability, in vitro bactericidal activity, and the capability of protecting mice against intracerebral as well as intraperitoneal challenge with virulent typhoid bacilli. Production of incomplete and complete Vi antibodies was adversely affected by immunization with partially depolymerized Vi antigens.     

Immunological response of three mouse strains to typhoid vaccine and Vi antigen

Vi-agglutinin, active cutaneous anaphylaxis and protective responses (ed(50)) of three mouse strains (CFW, NIH, and Balb/cAnN) to acetone-inactivated typhoid vaccine and soluble Vi antigen were compared. Seven days after immunization with either typhoid vaccine or Vi antigen the three strains of mice differed with respect to Vi-antibody titers. Significant differences were observed in the protective responses. Each mouse strain was significantly better protected by the intraperitoneal than by subcutaneous route of immunization. Active cutaneous anaphylaxis was more pronounced in showing strain differences in response to Vi antigen. The serological responses to Vi antigen of the strains of mice did not correlate with their protective response.     

Genetic Basis of Vi Antigen Expression in Salmonella paratyphi C

Analysis of hybrids formed in a cross between a Salmonella paratyphi C Hfr and an S. typhimurium recipient indicated that the structural genetic determinants of the S. paratyphi C Vi antigen are located closely adjacent to the mel determinant, between this marker and purA. A similar location was indicated for the structural genetic determinants of the S. typhi Vi antigen (the viaB locus) by the results of a mating in which a hybrid S. typhimurium Hfr bearing the S. typhi viaB determinants was used to transfer these genes to an S. typhimurium recipient. Mating experiments with a Vi-antigen-expressing S. typhi Hfr and an S. typhimurium hybrid recipient expressing the Vi antigen of S. paratyphi C yielded no recombinants in which loss of Vi antigen expression occurred, indicating that the chromosomal locus occupied by the genetic determinants of the S. paratyphi C Vi antigen is the same one at which, in S. typhi, the viaB genes reside. Introduction of a mutant S. typhi viaA gene into an S. typhimurium hybrid expressing the Vi antigen, as the consequence of prior receipt of the S. paratyphi C viaB determinants, resulted in that hybrid's loss of Vi antigen expression, demonstrating that the viaA determinant plays a role in Vi antigen expression in S. paratyphi C, as well as in S. typhi. Although the percentages of coinheritance of the viaB and mel determinants in the mating experiments suggested that their linkage is sufficiently close to allow cotransduction by P22, attempts to accomplish this with lysates prepared on S. typhimurium hybrids expressing either S. typhi or S. paratyphi C viaB determinants were not successful.     

Investigation of core machinery for biosynthesis of Vi antigen capsular polysaccharides in Gram-negative bacteria

Salmonella enterica serovar Typhi causes typhoid fever. It possesses a Vi antigen capsular polysaccharide coat that is important for virulence and is the basis of a current glycoconjugate vaccine. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (such as Bordetella bronchiseptica) produce a capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The Vi antigen backbone is composed of poly-α-(1→4)-linked N-acetylgalactosaminuronic acid, modified with O-acetyl residues that are necessary for vaccine efficacy. Despite its biological and biotechnological importance, some central aspects of Vi antigen production are poorly understood. Here we demonstrate that TviE and TviD, two proteins encoded in the viaB (Vi antigen production) locus, interact and are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and site-directed mutagenesis reveal that TviE is a GT4-family glycosyltransferase. While TviD has no identifiable homologs beyond Vi antigen systems in other bacteria, structural modeling suggests that it belongs to the large SGNH hydrolase family, which contains other O-acetyltransferases. Although TviD possesses an atypical catalytic triad, its O-acetyltransferase function was verified by antibody reactivity and ¹³C NMR data for tviD-mutant polysaccharide. The B. bronchiseptica genetic locus predicts a mode of synthesis distinct from classical S. enterica Vi antigen production, but which still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. These findings provide new insight into Vi antigen production and foundational information for the glycoengineering of Vi antigen production in heterologous bacteria.     

宋内氏痢疾菌无毒株S7表达Vi抗原工程菌的构建

将含有编码Ｖｉ抗原ＶｉａＢ基因片断的质粒转导进入宋内氏痢疾菌无毒株Ｓ７中,组建了重组菌株Ｓ７Ｖｉ。质粒电泳图谱显示重组菌株Ｓ７Ｖｉ中存在被转入的外源质粒带。重组株的生化特性没有改变。菌体凝集及Ｖｉ抗血清标记的ＳＰＡ菌液凝集反应证明在重组株的菌体表面,同时表达了Ｖｉ抗原和宋内氏毒菌的Ｏ抗原。以５×１０~８ＣＦＵ、１０×１０~８ＣＦＵ的重组株免疫近交系的ＬＩＢＰ小鼠,免疫小鼠对宋内氏毒株Ｓ６３攻击的保护率为６０％至９０％,对伤寒毒株Ｔｙ２攻击的保护率为２５％至４０％。     

Expression of Vi antigen in Escherichia coli K-12: characterization of ViaB from Citrobacter freundii and identity of ViaA with RcsB.

The Vi antigen in Salmonella typhi is stably expressed and may act to protect the strain against the defensive system of the host. Citrobacter freundii, not usually a common human pathogen, also expresses the Vi antigen but expresses it unstably, exhibiting a reversible transition between the Vi+ and Vi- states. Two widely separated chromosomal regions, ViaA and ViaB, are needed for Vi synthesis. Escherichia coli K-12 harboring a functional ViaB plasmid can also express Vi antigen, but the cloned ViaB sequence can only be stably maintained and expressed in recA hosts. Vi- derivatives arise either through IS1-like insertional events occurring in ViaB sequences or by chromosomal mutations at the ViaA region. P1vir mapping indicates that the ViaA mutations are located at min 47.75 on the E. coli chromosome. All the spontaneous viaA mutants isolated from E. coli and S. typhi were identified as rcsB mutants by complementation tests using plasmid pJB100. Introduction of rcsA::Tn10 into E. coli harboring functional ViaB sequences eliminates the expression of Vi antigen. These results indicate that Vi antigen synthesis is regulated by the same regulatory proteins involved in colanic acid synthesis in E. coli.     

Vi antigen of <Emphasis Type="Italic">Salmonella</Emphasis> enetrica serovar Typhi — biosynthesis,regulation and its use as vaccine candidate

Vi capsular polysaccharide (Vi antigen) was first identified as the virulence antigen of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever in humans. The presence of Vi antigen differentiates S. Typhi from other serovars of Salmonella. Vi antigen is a linear polymer consisting of α-1,4-linked-N-acetyl-galactosaminuronate, whose expression is controlled by three chromosomal loci, namely viaA, viaB and ompB. Both viaA and viaB region are present on Salmonella Pathogenicity Island-7, a large, mosaic, genetic island. The viaA region encodes a positive regulator and the viaB locus is composed of 11 genes designated tviA-tviE (for Vi biosyhthesis), vexA-vexE (for Vi antigen export) and ORF 11. Vi polysaccharide is synthesized from UDP-N-acetyl glucosamine in a series of steps requiring TviB, TviC, and TviE, and regulation of Vi polysaccharide synthesis is controlled by two regulatory systems, rscB-rscC (viaA locus) and ompR-envZ (ompB locus), which respond to changes in osmolarity. This antigen is highly immunogenic and has been used for the formulation of one of the currently available vaccines against typhoid. Despite advancement in the area of vaccinology, its pace of progress needs to be accelerated and effective control programmes will be needed for proper disease management.     

A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi

A rapid and sensitive gold-nanobioprobe based immunoassay format has been presented for the detection of capsular Vi polysaccharide of Salmonella enterica serovar Typhi (surface antigen) using anti-Vi antibodies. The Vi antigen was extracted from serovar Typhi cells, under the optimised growth conditions for its over-expression. Anti-Vi antibodies were produced and conjugated with gold nanoparticles (GNPs) of definite size (~30 nm), which served as the nano-bioprobe in the detection system. A sandwich immunoassay was developed using nitrocellulose dot blot comb (8/12 wells) membranes immobilized with anti-Salmonella antibodies at the optimal concentration (43 ng spot(-1)). The Vi antigen in the clinical isolates, spiked samples and also in the standard strain (serovar Typhi Ty2) was detected by measuring the colour intensity of GNPs and correlating it with the concentration of serovar Typhi in samples. Using this developed immunoassay technique Vi positive serovar Typhi strains could be detected with a sensitivity of up to 10(2) cells mL(-1) in the clinical isolates as well as in the spiked samples. The developed immunoassay technique could be useful for the detection of typhoid fever and may be important from an epidemiological point of view.     

Molecular cloning of the ViaB region of Salmonella typhi

The ViaB region required for Vi antigen production in Salmonella typhi was cloned. The plasmid pGBM124 containing a 14-kb S. typhi chromosomal DNA fragment conferred the ability to produce Vi antigen on Escherichia coli HB101 and ViaB-deleted S. typhi GIFU10007-3. Tn5 insertion analysis showed that the 14-kb DNA was split into three regions. Region 1 and region 2 are involved in the biosynthesis of Vi polysaccharide. Region 3 is involved in translocation of the Vi polysaccharide to the cell surface. Southern blot hybridization showed that regions 2 and 3 but not region 1, were considerably homologous to the DNA of Vi-positive Citrobacter freundii.     

The Vi antigen of Salmonella typhi: molecular analysis of the viaB locus.

Strains of Salmonella typhi isolated from the blood of patients with typhoid fever invariably express a capsular polysaccharide, termed the Vi antigen. Vi antigen expression is controlled by two separate chromosomal loci, viaA and viaB. The viaA locus is commonly found in enteric bacteria. In contrast, the viaB locus appears to be specific to Vi-expressing strains of Salmonella and Citrobacter. Here the cloning, expression and analysis of viaB determinants from S. typhi Ty2 is described. Whole-cell DNA from strain Ty2 was size-fractionated and cloned into the pLA2917 cosmid vector. A recombinant cosmid, pVT1, conferring a Vi-positive phenotype upon Escherichia coli and upon the Vi-non-expressing strain Ty21a of S. typhi, was characterized and used for further studies. Transposon Tn5 insertion mutagenesis demonstrated that the Vi-antigen-encoding region on pVT1 consisted of a 15 kb fragment. A subclone, designated pVT3, which contained an 18 kb insert, was sufficient to confer Vi antigen expression upon E. coli and S. typhi Ty21a. Results of recombination experiments indicated that this DNA sequence was the viaB locus of S. typhi Ty2. In E. coli SE5000 maxicells, the viaB determinants encoded at least eight polypeptides, with molecular masses of 80, 65, 59, 48, 44, 39, 35 and 28 kDa. Functional characterization of viaB mutations in S. typhi Ty2 suggested that the 80 and 65 kDa proteins were required for cell-surface localization of the Vi antigen.     

Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi.

Plasmid pGBM124, which contains a 14-kb Salmonella typhi chromosomal DNA fragment capable of producing the Vi antigen in Escherichia coli HB101 and ViaB-deleted S. typhi GIFU 10007-3, was studied. We determined the complete nucleotide sequence of this fragment and found 11 open reading frames. Mutagenesis, subcloning, and complementation analysis showed that three genes (vipA, vipB, and vipC) are involved in biosynthesis of the Vi polysaccharide. The putative primary amino acid sequence suggests that both vipA and vipB encode the NAD- or NADP-dependent enzymes to synthesize the nucleotide sugar for the Vi polysaccharide. Five genes (vexA, vexB, vexC, vexD, and vexE) may be involved in translocation of the Vi polysaccharide. Proteins VexA, VexB, VexC, and VexD had moderate similarities to components of group II capsule transporters, and the VexC protein had a putative ATP-binding site. These data indicate that the transport system for the Vi polysaccharide belongs to the ATP-binding cassette transporters. By using the isogenic Vi+ and Vi- strains constructed in this study, we reconfirmed that the Vi antigen is necessary for the serum resistance of S. typhi.     

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.     

火箭电泳法测定伤寒Vi多糖菌苗多糖含量

伤寒沙门氏菌Ｖｉ抗原系ａ─１,４─Ｎ─乙酰─半乳糖醛酸高度聚合体,其Ｃ２和Ｃ３位分别被Ｎ─乙酰基和０─乙酰基取代,它不能用分光光度法测定其含量,实验表明火箭电泳的火箭峰高与其样品中多糖含量的对数成直线相关关系,作者采用火箭电泳方法对全国六个生物制品研究所的１８批伤寒Ｖｉ多糖菌苗中Ｖｉ多糖含量进行了测定,结果表明多数制品符合规程要求,但部分制品多糖含量偏高,值得引起注意,各所三批制品差别不大,表明各所的生产工艺相对稳定,利用火箭电泳测定伤寒Ｖｉ多糖,操作简单,用时较短,所需样品极少,是目前较为合适的测定伤寒Ｖｉ多糖菌苗多糖含量的方法。     

Vi antigen biosynthesis in Salmonella typhi: characterization of UDP-N-acetylglucosamine C-6 dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic acid C-4 epimerase (TviC)

Vi antigen, the virulence factor of Salmonella typhi, has been used clinically as a molecular vaccine. TviB and TviC are two enzymes involved in the formation of Vi antigen, a linear polymer consisting of alpha-1,4-linked N-acetylgalactosaminuronate. Protein sequence analysis suggests that TviB is a dehydrogenase and TviC is an epimerase. Both enzymes are expected to be NAD(+) dependent. In order to verify their functions, TviB and TviC were cloned, expressed in Escherichia coli, and characterized. The C-terminal His(6)-tagged TviB protein, purified from soluble cell fractions in the presence of 10 mM DTT, shows UDP-N-acetylglucosamine 6-dehydrogenase activity and is capable of catalyzing the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylglucosaminuronic acid (UDP-GlcNAcA) with a k(cat) value of 15.5 +/- 1.0 min(-)(1). The K(m) values of TviB for UDP-GlcNAc and NAD(+) are 77 +/- 9 microM and 276 +/- 52 microM, respectively. TviC, purified as C-terminal hexahistidine-tagged protein, shows UDP-GlcNAcA 4-epimerase and UDP-N-acetylgalactosamine (UDP-GalNAc) 4-epimerase activities. The K(m) values of TviC for UDP-GlcNAcA and UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) are 20 +/- 1 microM and 42 +/- 2 microM, respectively. The k(cat) value for the conversion of UDP-GlcNAcA to UDP-GalNAcA is 56.8 +/- 0.5 min(-)(1), while that for the reverse reaction is 39.1 +/- 0.6 min(-)(1). These results show that the biosynthesis of Vi antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDP-GalNAc, followed by the TviC-catalyzed epimerization at C-4 to form UDP-GalNAcA, which serves as the building block for the formation of Vi polymer. These results set the stage for future in vitro biosynthesis of Vi antigen. These enzymes may also be drug targets to inhibit Vi antigen production.     

The hydration of the IX and Vi antigen ofBacterium Typhosum

Summary The Vi antigen as well as the IX antigen are hydrophilic colloids.