Specificity of monoclonal antibodies binding to the polysaccharide antigens (Vi, O9) of Salmonella typhi

A panel of monoclonal antibodies were generated against the surface polysaccharide antigens of the cell envelope of Salmonella typhi. Four clones (IgM) were specific for the capsular Vi polysaccharide, and one clone (IgG3) reacted selectively with the S. typhi lipopolysaccharide in enzyme immunoassay. On the basis of their reactivity pattern and binding affinity, MATy-V7 (IgM) and MATy-O9 (IgG3) antibodies were selected for further characterization of their antigenic specificity. In an inhibition enzyme immunoassay with rabbit factor-specific anti-Salmonella antibodies as the competing agents, the reactivity of MATy-V7 and MATy-O9 were significantly inhibited by the anti-Vi and anti-O9 antisera, respectively. Moreover, both the Vi- and O9-specific monoclonal antibodies were shown to be useful serotyping agents by correct identification in slide agglutination tests of 32 clinical isolates of all the S. typhi and other serogroup D salmonellae among a total of 140 bacterial isolates representing eight different Enterobacteriaceae genera tested.     

Demonstration of an antigenic protein specific for Salmonella typhi

Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10(5) cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

Abstract The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10⁵ cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Detection of lymphocytes binding erythrocytes conjugated with Salmonella antigens

Immune reagents for the detection of specific antigen-binding lymphocytes (ABL) with respect to different Salmonells antigens were developed. Rabbits were immunized with killed S. typhi and other salmonellae containing cross-reacting antigens, and the dynamics of the formation of ASL of each specificity was studied. Differences in the time of the appearance of ASL with receptors to thymus-independent (09, 12 or Vi) and thymus-dependent (Hd) antigens were studied. The relative content of ASL, determined with the use of immune reagents prepared from S. typhi antigens, was higher, on the whole, in rabbits immunized with S. typhi than in rabbits immunized with salmonellae containing one of cross-reacting antigens (S. enteritidis--09, 12; S. paratyphi C--Vi; S. virginia--Hd).     

Search for the systems of DNA host specificity in Salmonella typhi

Seventeen pure lines of S. typhi bacteriophages have been obtained from mother races O and Vi; of these, three were used to study 152 S. typhi strains with a view of detecting their DNA host specificity systems. 10 S. typhi strains having the DNA host specificity system have been detected by the rough determination of the lytic spectrum and the cross titration of phage Vi IX.     

伤寒杆菌稳定L型粗糙型返祖菌的蛋白图谱和抗原研究

甲型副伤寒杆菌温和噬菌体转导伤寒杆菌稳定Ｌ型返祖形成的两株粗糙型伤寒杆菌,在ＳＤＳ－ＰＡＧＥ电泳中缺失多条高分子量蛋白带。抗原分析表明,两株粗糙型返祖菌具有同其亲代伤寒杆菌一致的Ｈ抗原,但缺乏Ｏ抗原以及伤寒杆茵和甲型副伤寒杆茵共同的某些表面抗原。     

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.     

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.     

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.     

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 antigenic characteristics of Salmonella typhi L forms

The study of the possibility of detecting the specific antigens of S. typhi L forms has revealed that out of three destructive methods under study (osmotic lysis, freezing-thawing, sonication) only ultrasonic disintegration has proved to be effective for S. typhi L forms. Three specific fractions capable of interacting only with specific antibodies to S. typhi L forms have been revealed in the course of chromatographic separation of the soluble antigenic complex of S. typhi stable L forms and the subsequent analysis of the fractions thus obtained in the enzyme immunoassay.     

Protective efficacy and immunogenicity of Vi-porin conjugate against Salmonella typhi.

A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyl dithio)-propionate (SPDP). After preparing the Vi-porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5-fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti-Vi antibody (IgG) levels (P < 0.01) than the mice immunized with Vi alone. Anti-porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate-immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.     

肠毒素源性定居因子CS6在减素伤寒沙门氏菌中表达及免疫原性的研究

将编码肠毒素源性大肠杆菌定居因子抗原ＣＳ６基因克隆到ｐＸＬ６７０,转化ａｓｄ基因突变的Ｅ．ｃｏｌｉ Ｘ６０９７,获得重组质粒ｐＳＳ６４,再将后者转化至减毒的△ａｒｏＡ、△ａｒｏＣ、△ａｓｄ伤寒沙门氏菌,构建了无药物抗性且稳定的大肠杆菌和伤寒双价菌苗候选株。小鼠腹腔免疫和攻击实验表明,该菌株对伤寒沙门氏菌毒株的攻击具有良好的保护作用。家兔免疫实验证明,该菌株能产生抗ＣＳ６和伤寒菌Ｖｉ抗原的血清抗体。     

Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum

An immunochromatographic strip test using gold nanoparticles was developed for the rapid detection of Salmonella typhi (S. typhi) in human serum. The strip test based on the principle of sandwich immunoassay by the specific binding of antigens from S. typhi O901 and antibody of S. typhi O901 on a nitrocellulose membrane. Antibody-gold nanoparticle conjugate was used as the label and was coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and control zone, antibody of S. typhi O901 and an anti-IgG were dotted on the nitrocellulose membrane, respectively. Positive samples were displayed as red dots at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red dot only in the control zone. The limit of detection (LOD) was found to be 1.14×10(5) cfu mL(-1), which could be visually detected by the naked eye within 15 min. This strip test provided a lower detection limit and analysis time than a dot blot immunoassay (8.88×10(6) cfu mL(-1) for LOD and 110 min for reaction time). In addition, our immunochromatographic strip test was employed to detect S. typhi in human serum effectively, with high accuracy. This strip test offers great promise for a rapid, simple and low-cost analysis of S. typhi.     

The serological specificities of Salmonella typhi antigens.

Sera prepared with two different strains of Salmonella typhi were analysed against all the soluble antigens isolated from S. typhi 0901, S. typhi Ty2 and S. typhi Vi. Agar-gel diffusion against individual sera showed that, in all the sera, antibodies were induced against somatic antigens and free proteins. Absorptions of the sera with polysaccharides, split from the somatic antigens, removed the antibodies induced against the polysaccharide and its proteinic carrier in most of the somatic antigens of S. typhi 0901. The antibodies left in the absorbed sera reacted against the proteinic moieties of more complex somatic antigens of S. typhi and against free proteins from all the analysed strains. Only the absorption with proteins removed all the precipitating antibodies from the sera. Moreover, in incomplete absorptions with proteins, the first antibodies removed are the antipolysaccharides, since antibodies are never induced against the haptenic polysaccharide but against somatic conjugates; in these the proteinic moiety eventually varies with every batch of bacteria. The sera exhausted of precipitins still agglutinate the bacteria, thus confirming the assumption that agglutinins and precipitins may be different antibodies.     

Surface K antigen in Salmonella paratyphi B.

It was established by agar immunoelectrophoresis that Salmonella paratyphi B lysate contains a large number of soluble antigens which display a varying degree of serological specifity as well as different diffusion and electrophoretic mobility. Salmonella paratyphi B was found to possess, apart from specific O and H antigens, a surface K antigen. This is a distinct antigen having strict serological specificity. Purified K antigen displayed anodic mobility in immunoelectrophoresis. A detailed study of K antigen properties in cultures treated by different methods as well as immunochemical investigations of purified K antigen showed that the surface K antigen of S. paratyphi B differs from its O, M, Vi, H and other known antigens in terms of basic characteristics.     

Detection of O-acetylated Vi polysaccharide of Salmonella enterica subspecies typhi by enzyme immunoassay.

Immunisation with capsular Vi polysaccharide (Vi PS) of Salmonella enterica serovar Typhi (S. typhi) protects against typhoid. This protection depends on the presence of O-acetyl groups on the Vi PS, which form an immunodominant epitope. An antiserum raised against conjugated Vi PS was used as the basis for an indirect Enzyme Immunoassay (EIA). The antiserum did not react with lipopolysaccharide of five gram negative bacteria including S. typhi. Vi PS from three different sources was tested, and all but one of 18 native Vi PS preparations had EIA values comparable to a standard Vi PS preparation. The sensitivity of the EIA for the detection of O-acetyl groups on Vi PS was compared to an NMR spectroscopy assay (Biologicals 28 (2000) 17-24). The EIA distinguished between O-acetylated and de-O-acetylated Vi PS preparations. However, significantly lower EIA reactivity was observed only for samples which had O-acetylation levels of 25% or less. This assay should facilitate batch control of Vi vaccines.     

Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.