Cross-reacting intraspecific antigens of Neisseria meningitidis. I. The isolation, immunochemical and serological characteristics of the cross-reacting antigens of N. meningitidis serogroup A

New data on the cross-reacting antigen of N. meningitidis, serogroup A, are presented. A complex of antigens has been isolated by treatment with tryptone X-100, ethanol precipitation and the subsequent treatment with trichloroacetic acid. The immunological analysis of the isolated preparation has shown that the proteinaceous part of the biopolymer contains 7 polypeptide fragments; of these, one fragment with a molecular weight of 31000 daltons has been found to constitute 49, 15% of all polypeptide fragments. The evaluation of the serological properties of this preparation in the precipitation test and the passive hemagglutination test has revealed that the preparation contains various cross-reactive antigenic determinants. Polyvalent erythrocyte diagnosticum obtained on the basis of this preparation permits the detection of antibodies to meningococci irrespective of their serogroup.     

Production of antibody against aflatoxin B1.

Antibody against aflatoxin B1 was obtained after one multiple-site injection of bovine serum albumin-aflatoxin B1 conjugate into rabbits. The antibody has greatest binding efficiency for aflatoxin B1, less efficiency for B2, G1, and Q1, and least for aflatoxicol, G2, and M1. Sterigmatocystin, coumarin, and 4-hydroxycoumarin did not give a cross-reaction with the antibody. The sensitivity of the binding assay for detection of aflatoxin B1 is in the range of 0.2 to 2.0 ng per 0.5-ml sample. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody titer determination are described.     

Characteristics of the B subunit of a thermolabile Escherichia coli enterotoxin produced by the A-B+ E. coli strain

The preparation and identification of B subunit of thermolabile enterotoxin produced by A-B+ gene-containing strain are described. The E. coli strain studied is shown to produce protein identical in its molecular properties and antigenic specificity to B subunit obtained from the whole thermolabile enterotoxin. Partial antigenic affinity between B subunits of thermolabile enterotoxin obtained from different sources and B subunit from cholera enterotoxin has been established in immunochemical studies. Electrophoretic and immunochemical analysis has confirmed the absence of A-subunit admixtures in B-subunit preparation obtained from /A-B+/E. coli strain.     

Antigenic and genetic characterization of a putative hybrid transferrin-binding protein B from Neisseria meningitidis

The transferrin-binding protein Bs (TbpBs) from the bacterium Neisseria meningitidis have been divided into two families according to genetic and antigenic features. TbpB from meningococcal strain B385 showed a molecular mass similar to that exhibited by TbpBs belonging to the high molecular mass family of TbpBs. TbpB was recognized by immunoassay using a specific serum directed against the TbpB of the reference strain for this family (strain M982). It was also recognized by a serum elicited against the TbpB of the reference strain for the low molecular mass family (strain B16B6). The tbpB gene from strain B385 was cloned and sequenced. The highest degree of sequence homology was found to be with the TbpBs belonging to the high molecular mass family, although a region of 14 amino acids that is only present in the TbpB from strain B16B6 was also found. This report illustrates a TbpB that shows hybrid antigenic and genetic behaviour.     

Chemical and antigenic analysis of the cell walls of Neisseria meningitidis group B

Vedros, Neylan A. (Naval Medical Research Institute, Bethesda, Md.), and Paul R. Hill. Chemical and antigenic analysis of the cell walls of Neisseria meningitidis group B. J. Bacteriol. 91:1992-1997. 1966.-Cell walls have been isolated from Neisseria meningitidis group B, by rapid freeze-thawing or treatment with sodium deoxycholate. Chemical analysis of the cell walls indicated that the amino acid composition, and content of hexosamines and of lipids, were similar to those reported for Escherichia coli, except for higher concentrations of alanine, methionine, glutamic acid, and phenylalanine. The meningococcal cell walls showed groupspecificity in the complement-fixation test, were nontoxic for rabbits, and in rabbits produced antibodies which protected mice from challenge with the homologous strain.     

Identification of surface proteins of group B streptococci: Serotyping versus genotyping

We compared serotyping to genotyping of group B streptococcal (GBS) surface proteins in 147 Australasian isolates. Results were concordant for the two methods in 73.8% of 122 isolates, discordant for three and partially discordant for 29 isolates. For the purpose of epidemiological typing of GBS, genotyping is superior to serotyping.     

A comparison of four methods for the serotyping of group B streptococci.

Group B streptococci are implicated in a wide range of clinical conditions in human adults and neonates. The Group is subdivided into five serotypes Ia, Ib, Ic, II and III, which are differentiated on the basis of capsular polysaccharides. In the interests of epidemiology and efficiency a cheap, rapid method which is easily interpreted would be advantageous. In this study four methods of serotyping, namely, counter-immunoelectrophoresis (CIEP), microimmunodiffusion (MID), coagglutination (COA), and the Lancefield capillary precipitin (CP) test were compared in terms of ease of operation and interpretation, accuracy and rapidity. Todd Hewitt Broth (THB) cultures and acid extracts of the group B streptococcal strains were used as antigens for these methods. It was concluded that COA using THB cultures allows cheap and rapid screening for presumptive serotyping, having a 93-96% correlation with the CP test. MID gives an accurate (100% correlation with the CP test) and unambiguous confirmatory diagnosis of serotype.     

The panmictic nature of Neisseria meningitidis serogroup B during a period of endemic disease in Canada

Three hundred and one (301) strains of Neisseria meningitidis serogroup B, isolated from patients with meningococcal disease during the years 1994-1996, were subjected to multilocus enzyme electrophoresis, serotyping, and serosubtyping. Based on the analyses of 14 enzyme loci, 177 electrophoretic types (ETs) were identified. Of these, 136 were represented by single isolates and 41 were represented by multiple isolates (range 2-31). The mean genetic diversity for isolates was 0.444 and for ETs was 0.440. The index of association (I(A)) between loci was 0.530 +/- 0.08 for isolates and 0.256 +/- 0.10 for ETs. Cluster analysis revealed the presence of 39 lineages each represented by a single ET or clusters of ETs. The most common serotypes were 4, 15, and 14 and accounted for 84 (28.0%), 53 (17.6%), and 32 (10.6%) of the isolates, respectively, and were dispersed amongst 46 ETs (1-122), 35 ETs (3-165), and 26 ETs (18-76), respectively. The 109 (36.6%) nontypable (NT) isolates were amongst 74 ETs (6-177). The mean genetic diversity for serotypes 4, 15, and 14 and NT isolates was 0.368, 0.371, 0.343, and 0.442, respectively, and for ETs was 0.363, 0.354, 0.397, and 0.440, respectively. Combinations of serotypes and serosubtypes (number of isolates) that occurred most frequently were 4:P1.14 (17), 14:P1.16 (16), NT:P1.16 (16), 15:P1.16 (13), and NT:P1.13 (13). The majority of group B disease in Canada during 1994-1996 was caused by meningococci of considerable genetic diversity, and reflects a situation of endemic disease. However, the results also indicate that organisms belonging to the ET-5 complex, which has been responsible for outbreaks of group B disease globally for several decades, have been introduced into the country.     

Immunological studies of the activity of Aspergillus flavus Link. I. Antigen analysis and evaluation of its chemical composition and toxicity

The aim of this study was to get insight into chemical structure and toxigenicity of antigenic preparations obtained from A. flavus Link strain isolated from industrial environment. A microbiol multiplication and an antigenic fractions preparation scheme is presented. The method proposed allows to obtain antigens from culture filtrate (APP), mycelial extract (AEM), and two subfractions obtained from AEM: supernatant--AS and precipitated--API. The strain tested did not show aflatoxigenic activity and antigenic fractions obtained were free of aflatoxin B1, B2, G1, G2 in concentrations detectable by thin-layer chromatography. A chemical composition of the antigenic fractions was tested. A content, depending on fractions, of proteins ranged from 32.0 to 74.5 micrograms/ml, of sugars from 15.0-44.5 micrograms/ml, phosphorus 0.5-1.5 micrograms/ml, and nitrogen 2.5-4.9 micrograms/ml. Toxicity of APP and AEM antigens designated for laboratory animals immunisation was also determined. The values of LD50 for APP preparation was 2.00 mg/mouse and for AEM - 2.75 mg/mouse. These data give evidence of moderate toxicity of these preparations.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Isolation of split and whole-virion tick-borne encephalitis vaccines in an experiment and the characteristics of their immunogenic and antigenic properties

The split preparation, obtained by the treatment of purified and concentrated tick-borne encephalitis virus with 0.5% Tween-80 and ether and inactivated with formalin at a minimal concentration of 1:10,000, did not contain the active virus. After the removal of the detergent, the preparation retained its antigenic and immunogenic properties.     

Serotypes and subtypes of Neisseria meningitidis serogroup B strains associated with meningococcal disease in Canada, 1977-1989

Typing of Neisseria meningitidis serogroup B disease isolates was carried out using a panel of serotype-and subtype-specific monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (ELISA). Three hundred and sixty-two strains isolated from 1977 to 1986 were typed using five serotyping and seven subtyping reagents and outer membrane vesicles as antigens. Serotype 2b accounted for 30% of the disease isolates. The most common subtype was P1.2, which occurred on 18.5% of all strains or 48.6% of the serotype 2b strains. Of the 362 strains typed, 135 (37.3%) were serotyped and 122 (33.7%) were subtyped. Overall, 185 (51.1%) of the strains could be assigned a serotype and (or) subtype. Strains (221) isolated during the years 1987-1989 were typed using a panel of 6 serotyping and 12 subtyping reagents by whole-cell ELISA. Strains of serotypes 4 (21.7%) and 15 (20.8%) were the most common and carried a wide variety of subtypes. The most common subtypes were P1.2 (11.8%) and P1.16 (9.5%). Of the 221 strains analyzed, 132 (59.7%) were assigned a serotype and 123 (55.7%) a subtype and with all 18 MAbs, 192 (86.9%) of the strains were serotyped and (or) subtyped. Two different MAbs to the four epitopes 2a, 15, P1.2, and P1.16 gave discordant reactions of 0.3, 6.6, 2.6, and 2.2%, respectively, when used to analyze over 300 strains of N. meningitidis.     

Blocking action of the soluble H-2 antigens and H-2 antibody isolated by various methods and mouse immune lymphocytes]

The treatment of tumour and lymphoid cells of mice with 3M KC1 solution having high ionic strength, nonionic detergent and by subsequent freeze-thawing resulted in obtaining serologically active H-2 antigen preparation capable of specifically blocking the cytotoxicity of H-2 antisera. The antigenic activity of the preparations thus obtained depended on the source from which they were isolated (spleen cells and their membrane fragments proved to be the best source), on the degree of maturity of tumor cells and the degree of purification of the preparation, as well as on the methods of solubilization. The blocking action of soluble H-2 antigens on the cytotoxicity of immune lymphocytes depended on the method used for isslating these antigens. The interaction of immune lymphocytes and H-2 antisera with soluble antigens was probably effected by different mechanisms.     

Regulation of glutaminase B in Escherichia coli. I. Purification, properties, and cold lability.

Escherichia coli contains two glutaminases, A and B, with pH optima below pH 5 and above pH 7, respectively. Neither glutaminase A nor B is released from E. coli by osmotic shock. Glutaminase B has been purified 6,000-fold and the purified preparation is estimated to contain about 40% glutaminase B. The enzyme has a molecular weight of 90,000 and an isoelectric point of 5.4. Glutaminase B exhibits a broad pH optimum between 7.1 and 9.0. Only L-glutamine is deamidated by glutaminase B, L-asparagine and D-glutamine are not deamidated. The substrate saturation curve for glutaminase B shows an intermediary plateau region. Like many regulatory enzymes, glutaminase B is cold-labile. The enzyme is inactivated by cooling and activated by warming; both processes are first order with respect to time. The activation energy for activation by warming was calculated to be 5900 cal/mol. Activation by warming increased the Vmax and decreased the S0.5 for L-glutamine, but did not alter the molecular weight of the catalytically active enzyme. Borate and glutamate protected glutaminase B from inactivation by cold.     

Molecular mimetics of polysaccharide epitopes as vaccine candidates for prevention of Neisseria meningitidis serogroup B disease

Neisseria meningitidis is a major cause of meningitis and sepsis. Despite nearly 25 years of work, there is no promising vaccine candidate for prevention of disease caused by meningococcal B strains. This review summarizes newer approaches for eliciting protective meningococcal B immune responses, including the use of molecular mimetics of group B polysaccharide and conserved membrane proteins as immunogens. The capsular polysaccharide of this organism is conserved and serum antibody to this capsule confers protection against disease. However, the immunogenicity of meningococcal B polysaccharide-based vaccines is poor. Further, a portion of the antibody elicited has autoantibody activity. Recently, our laboratory produced a panel of murine monoclonal antibodies (Mabs) that react specifically with capsular polysaccharide epitopes on meningococcal B that are distinct from host polysialic acid. These Mabs elicit complement-mediated bactericidal activity and confer passive protection in animal models. The anti-capsular Mabs were used to identify molecular mimetics from phage display peptide libraries. The resulting peptides were antigenic mimetics as defined by binding to the Mabs used to select them but, to date, are poor immunogenic mimetics in failing to elicit anti-capsular antibodies.     

Thermal stability of hepatitis B surface antigen S proteins.

Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.     

Isolation of different variants of Pseudomonas aeruginosa anatoxin and their characteristics

The conditions for the detoxification of the crude preparations of P. aeruginosa exotoxin A, obtained by the cultivation of strain PA-7 in Martin's broth, have been studied, and the schemes for obtaining nontoxic, stable, specifically antigenic preparations of toxoid from exotoxins A with different degrees of purification have been developed. Toxoid obtained by formalin treatment on the level of a crude preparation with its subsequent purification and additional detoxification with formalin in the presence of lysin has been shown to possess high immunogenic potency. The preparation has been found to induce immune response and to ensure the protection of experimental animals challenged not only with the lethal dose of exotoxin A, but also with P. aeruginosa toxigenic and protease-producing strains.     

Intragroup serotyping of meningococci

The method for obtaining antisera to meningococci of different serotypes are described and the scheme for the preparation of serotyping is presented, as well as the method for the preparation of the determinate fraction of serotype 2. Antisera to typing antigens 1, 2, 2-7, 2-10, 4, 5, 6, 8 (1) have been obtained, their specificity tested in parallel experiments with American and French typing sera. When typing meningococci, the use of antisera to purified protein antigen 2 is recommended.     

Evaluation of methods for the identification of Salmonella enterica serotype Typhimurium DT104 from poultry environmental samples

An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide. This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104. A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5%. Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates. These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104. Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively. Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete.     

Study of the antigenic relationships between strains of Bacteroides, intermedius, B. melaninogenicus, B. corporis, and B. denticola revealed by immunoblotting with rabbit antisera

Antigen profiles of saccharolytic oral black-pigmented Bacteroides have been developed by Western blotting. Visual comparisons indicated extensive cross-reactions between B. intermedius, B. melaninogenicus, B. denticola, and B. corporis. Porphyromonas gingivalis, P. asaccharolyticus, and B. buccae showed less cross-reaction. Quantitation of antigenic similarity was made from densitometric scans. Calculation of the Jackard coefficient gave results of 33-72% similarity among the saccharolytic pigmented species, with the two homology groups of B. intermedius separated at 53%. Species were separated below 70%. Subtraction of the profile of a cross-reacting strain from that of the homologous strain also allowed quantitation of similarities. These similarities were lower; the range between species was 4-62%, although the two homology groups of B. intermedius still separated at 50 and 58%. Species were separated below 63%. Sera absorbed with a cross-reacting strain gave reduced reactions with the homologous strain and cross-reacting strains, indicating several common antigens among the four species. The species-specific antigens demonstrated by sera absorbed with cells of cross-reacting species were relatively few (3-6) compared with cross-reacting antigens detected by non-absorbed sera (18-28). The method appears useful to quantitate antigenic similarities among Bacteroides species and strains and allows analysis and quantitation of individual humoral responses in animals to these bacteria.