Physicochemical Properties of Neisseria meningitidis Group X Polysaccharide Antigen

Neisseria meningitidis group X occurs in human carrier populations and is rarely implicated in serious disease. This organism possesses a capsular group antigen which is an acidic polysaccharide. It is composed of the amino sugars, glucosamine, glucosamine-6-phosphate, galactosamine, and the simple hexose, glucose. The group X capsular antigen has an S_20,w⁰ of 3.6, and the acidic nature of the polysaccharide is reflected in an isoelectric point of 3.65. The meningococcal A, B, C, and Y polysaccharide group antigens are also composed primarily of amino sugars. The chemical composition of the group X antigen most closely resembles the capsular antigen of N. meningitidis group Y, which is also predominately a carrier organism.     

Cell surface antigens of Trypanosoma cruzi: Use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein

A monoclonal antibody was produced by cell fusion from mice immunised with Trypanosoma cruzi epimastigotes. The antibody was epimastigote specific but not strain specific; the antibody bound to Y, Peru and Tulahuén epimastigotes but did not bind to Y amastigotes or trypomastigotes. The antigen recognised by the monoclonal antibody was a 72 000 molecular weight cell surface glycoprotein which represented only 0.04% of the whole cell protein. Analysis of the glycoprotein purified by antibody affinity chromatography revealed a carbohydrate content of 52% by weight which was composed of glucosamine, mannose, galactose, glucose and three different pentoses: fucose, xylose and ribose. Immunisation with the purified glycoprotein did not protect mice from a lethal infection of T. cruzi.     

Isolation and Characterization of Neisseria meningitidis Groups A, C, X, and Y Polysaccharide Antigens

To prepare Neisseria meningitidis groups A, C, X, and Y polysaccharide antigens, culture supernatant fluids were subjected to serial processes of salt precipitation, alkaline hydrolysis, ethyl alcohol precipitation, and Sephadex G-200 chromatography. This method resulted in the isolation of large quantities of group antigens. All are acidic polysaccharides, the group C antigen being a polymer of n-acetyl neuraminic acid. Thiobarbituric acid assay failed to reveal sialic acids in the other group antigens. Protein was undetectable by absorption at 280 nm or by Folin analysis. These antigens are of similar molecular size, the majority of which are excluded by Sephadex G-200. They migrate in the upper one-third of sucrose density gradients and are retained by 5% acrylamide gel. All are highly group-specific and react only with homologous hyperimmune antisera in hemagglutination, complement fixation, and immunodiffusion systems. As little as 0.03 μmoles of n-acetyl neuraminic acid in group C antigen inhibits the hemagglutination of group C-sensitized red cells. All antigens are immunogenic in rabbits. These techniques afford a simplified method for the production of relatively large yields of highly specific group antigens which participate in multiple immunologic systems.     

Structural Properties of Lipopolysaccharides from Rickettsia typhi and Rickettsia prowazekii and Their Chemical Similarity to the Lipopolysaccharide from Proteus vulgaris OX19 Used in the Weil-Felix Test

The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-d-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (β-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae and P. vulgaris OX19.The typhus group (TG) rickettsiae possess at least two different types of antigens. One type of antigen is sensitive to sodium metaperiodate, resistant to trypsin, stable in 0.2 M NaOH, and thermostable and includes erythrocyte-sensitizing substance (24), lipopolysaccharide (LPS) (26, 27), and OX19-like antigens (20). These antigens appear to be the group-specific antigens of TG rickettsiae. On the other hand, the species-specific antigens of both Rickettsia typhi and Rickettsia prowazekii are destroyed by incubation at 56°C for 45 min (15), suggesting that they are proteins.LPS from Coxiella burnetii (3, 10, 14, 28, 29), a related species of rickettsia, has been identified and chemically described, but the exact structure of this LPS is unknown. Previously, Amano et al. also reported the chemical properties of the spotted fever group (SFG) rickettsial LPS, which contains 3-deoxy-d-manno-octulosonic acid (KDO), glucosamine, 6-deoxyglucosamine (quinovosamine), ribose, glucose, phosphate, and palmitic acid (2). On the other hand, endotoxic activity analogous to LPS endotoxin was found in R. prowazekii by Olitzki et al. (22, 23), and Schramek et al. (26, 27) extracted a hydrophobic LPS-like endotoxin from R. typhi and R. prowazekii. Smith and Winkler (30) have provided evidence that R. prowazekii contains KDO, a marker for LPS. Although these data suggest the presence of LPS in TG rickettsia, the structure of this LPS has remained obscure.In attempt to ascertain the structure of LPS from TG rickettsiae, we extracted LPSs from R. typhi and R. prowazekii and analyzed their chemical components in terms of the heterogeneity of their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, glucose, KDO, fatty acids, glucosamine, quinovosamine, and phosphate were all identified as components of the LPS from TG rickettsiae.     

Chemical composition and immunological specificity of the streptococcal group O cell wall polysaccharide antigen

The group O streptococcal group antigen was shown to be a polysaccharide located in the cell wall of the organism. The antigen could be extracted by one of several methods: (i) 0.5 n NaOH at 37 C, (ii) phenol-water (50:50) at 68 C, (iii) 0.2 n HCl at 100 C, or (iv) 10% trichloroacetic acid at 4 C. The last method yielded more polysaccharide with less protein contamination. The polysaccharide was purified on diethylaminoethyl-Sephadex A-25 and Sephadex G-200. It was composed of two-thirds glucosamine and galactosamine, and the remainder glucose plus galactose. Rhamnose, glycerol, ribitol, and muramic acid were absent. Total phosphorus and amino acids were each less than 0.1%. N-Acetyl-beta-d-glucosamine exerted a strong inhibition of the precipitin reaction and is considered the immunodominant sugar. Glucosamine and glucose possessed a partial inhibitory activity. Galactose and galactosamine were essentially negative. No evidence of cross-reactivity was found between the O polysaccharide and group A and L polysaccharides, and group A and Staphylococcus aureus teichoic acids, which posesss N-acetylglucosamine specificity. The release of limited quantities of N-acetyl-glucosamine from its terminal location by enzyme, and glucose by acid hydrolysis, indicates a limited number of side chains in the O antigen. The glucosamine is in acid-stable linkage in the polysaccharide. Glucose was not released by beta-glucosidase and probably does not occupy a terminal position. The O antigen is the only known streptococcal polysaccharide antigen which does not contain rhamnose. The effect of these factors on the immunological specificity is discussed. O serum, after adsorption with the purified polysaccharide, was used to demonstrate the presence of protein antigens in acid extracts of cells from each of the nine strains examined. These antigens may represent type antigens. Two of these strains, originally described as group O, did not contain the O polysaccharide.     

Glycosylation of simian virus 40 T antigen and localization of glycosylated T antigen in the nuclear matrix

Evidence has been obtained for the glycosylation of simian virus 40 (SV40) T antigen in SV40-infected TC7 cells. Both [3H]mannose and [3H]glucosamine are incorporated into T antigen in cells grown and labeled in medium containing fructose instead of glucose. In addition, T antigen is visualized by a carbohydrate stain specific for mannose and/or glucose residues. Finally, lectin binding studies suggest that T antigen contains galactose and/or galactosamine, since T antigen is specifically eluted from soybean lectin by 0.2 M galactose. When gel-purified, [3H]glucosamine-labeled T antigen is subjected to tryptic peptide mapping, label is found in only one peptide, thought to correspond to the methionine-containing peptide extending from Asn-653 to Arg-691, near the carboxy-terminal end of T antigen. Insensitivity to tunicamycin and the localization of the glycosylation site in the carboxy-terminus of T antigen, and not at Asn-153, suggest that T antigen is not N-glycosylated. Cell fractionation studies show that [3H]glucosamine-labeled T antigen is preferentially associated with the nuclear matrix of SV40-infected TC7 cells.     

Inhibition of avian sarcoma virus replication by glucosamine

The effect of glucosamine on the replication of avian sarcoma viruses has been studied. Six hours after addition of 20 μmoles/ml of glucosamine to the culture medium replication of infectious virus was inhibited by 99.9%. The production of viral particles detectable by physical techniques ceased in parallel with the synthesis of focus forming virus.Immunofluorescence studies indicate that the glucosamine block affects the production of viral envelope antigens. Accumulation of such antigens in the cytoplasm or at the cell surface was not observed in glucosamine-treated cultures, although the accumulation of a nonglycosylated polypeptide precursor cannot be excluded. Synthesis of group specific (gs1) protein was also inhibited by glucosamine, but to a lesser degree than that of viral envelope antigens.     

Purification and Chemical Composition of the Protective Slime Antigen of Pseudomonas aeruginosa

The slime obtained from Pseudomonas aeruginosa strain BI was purified by a system of ethanol precipitation, gel filtration, and ion-exchange chromatography. The slime polysaccharide was eluted as a single peak at a potassium chloride molarity of 0.30 to 0.40. The purification procedure was monitored by immunodiffusion techniques, and the number of bands was reduced from four to one, indicating the elimination of antigenic impurities that were present in the crude extracts of slime. The purified slime behaved as a homogeneous antigen, stimulating the production of a single species of antibody in rabbits. Hydrolyzed preparations of purified slime contained rhamnose, glucose, mannose, glucosamine, galactosamine, and glucuronic acid, as well as N-acetyl and O-acetyl groups. Only trace amounts of nucleic acids were detectable. A significant amount of protein was found to be associated with the carbohydrate moiety. The substrate characteristic of the slime was reaffirmed by measuring the release of hexosamines in the presence of the Pseudomonas phage 2 depolymerase PDB₂, and its activity as a protective antigen was demonstrated in passive-protection tests of mice.     

Soluble group- and type-specific antigens from type III group B Streptococcus.

Two soluble polysaccharide antigens of a type III group B Streptoccus were isolated from the culture medium after growth of strain M732 in a chemically defined broth supplemented with acid-hydrolyzed casein. The type- and group- specific antigens were isolated from the culture supernatant by anion-exchange chromatography with diethylaminoethyl-Sephacel. Two carbohydrate-containing peaks, which had serological reactivity with group B or type III antiserum, respectively, were eluted with a linear NaCl gradient and further purified by gel filtration. The type III polysaccharide was found to contain glucose, galactose, glucosamine, and sialic acid, whereas the group B polysaccharide contained galactose, glucosamine, and rhamnose. For the type III polysaccharide, sialic acid was shown to be the major immunodeterminant, and for the group B polysaccharide, rhamnose was the immunodominant sugar. Both the type III and group B polysaccharides were obtained in high yields without employing harsh physical or chemical treatment and both were immunologically distinct. By immunoelectrophoresis or counterimmunoelectrophoresis, type III antigen failed to react with group-specific antiserum and the group B antigen failed to react with type III antiserum.     

Release of O antigen polysaccharide from Salmonella newington by phage epsilon 34.

Phage ?³⁴ cleaves its own receptor site on the cell surface of its host bacterium, Salmonella newington. The receptor is an O antigen polysaccharide consisting of mannosyl-rhamnosyl-galactose repeating units joined together by β-galactosyl linkages. A series of polysaccharides produced by this cleavage contains mannose, rhamnose and galactose. The identified end-groups generated by this cleavage are galactose, indicating the cleavage is caused by the specific breakage of β-galactosyl 1–6 mannose linkages.A soluble protein found in ?³⁴-infected cells also cleaves the O antigen, liberating polyand oligosaccharide fragments. The protein was identified as the base plate parts of this phage by morphological hybrid formation with phage P22 head particles. From the results obtained we conclude that each base plate part of this phage is a kind of endogalactosidase, which specifically acts on the O antigen of S. newington. The phage or its base plate parts cleave the O antigen of S. anatum or that of S. minneapolis poorly or not at all.Free base plate parts were also found in lysate of c341-infected cells. In this case, however, no cleavage of O antigen polysaccharide was detected using either phage particles or the isolated base plate parts.     

Immunochemical analysis of distinct cellular antigens isolated from a nontypable bovine strain of group B streptococci.

Four immunologically distinct antigens were isolated from a bovine strain of group B streptococci, designated 14 Mi. Chemical analysis indicated that two of the cell surface antigens consisted of glucose, galactose, and glucosamine, whereas the other cell surface antigen, an acidic protein, contained a predominance of aspartic acid, glutamic acid, and alanine. A cell wall-associated triheteroglycan consisting of galactose, glucose, and glucosamine was isolated from strain 14 Mi by 10% trichloroacetic acid. Immunochemical studies suggested that this glycan is type specific and consists of an immunodominant alpha-linked galactosyl-glucose disaccharide.     

Characterization of a Non-Type-Specific Antigen(s) Associated with Group A Streptococcal Type 12 M Protein

The sharing of one and possibly two or more non-type-specific antigens by most strains of groups A, C, and G streptococci is described. With the exception of a single strain of Proteus mirabilis, this antigen(s) was not found among strains of groups B and D streptococci, coagulase-positive staphylococci, Escherichia coli, Pseudomonas sp., and Salmonella sp. The non-type-specific antigen(s) could not be separated from M protein by fractionation with various saturations of ammonium sulfate or by column chromatography with calcium hydroxylapatite even though the latter method allowed the recovery of a fraction of M protein which was free of the cross-reactive antigen(s). The resistance of this non-type-specific antigen(s) to hot acid extraction and its sensitivity to treatment with trypsin differentiate it from the T and R antigens of group A streptococci, both of which are trypsin resistant. Co-precipitation of both type-and non-type-specific antigens occurred with type-specific antiserum and suggested that the type- and non-type-specific antigens represent either different, covalently bonded antigenic determinants on the same protein or different proteins noncovalently linked together.     

Herpes simplex virus protein synthesis in the presence of 2-deoxy-D-glucose.

The proteins and glycoproteins induced by herpes simplex virus type 1 (HSV-1) were labeled with [¹⁴C]amino acids or [¹⁴C]glucosamine in the presence or absence of 2-deoxy-d-glucose (deoxyglucose) and analyzed by slab gel electrophoresis. In the presence of 0.1% deoxyglucose (6.1 mM), the major envelope glycoprotein (VP123, MW 123,000) labeled with [¹⁴C]glucosamine was shifted to a component of an apparent lower molecular weight (VP123′). In the presence of increasing concentrations of deoxyglucose, there was a progressive decrease in the amount of [¹⁴C]amino acids incorporated into polypeptides which normally band in the VP123 region. Concomitant with this decrease was an increase in [¹⁴C]amino acids incorporated into a polypeptide(s) of greater electrophoretic mobility and of an apparently lower molecular weight. The polypeptide(s) was designated DG92 (MW 92,000) and was found to be predominantly associated with the nuclear fraction of HSV-1-infected cells cultured in the presence of deoxyglucose. The effects of deoxyglucose on HSV-1 polypeptide synthesis could be prevented by the addition of mannose.     

Immunochemical evidence for multiple serotypes of Bacteroides fragilis.

An immunochemical comparison of outer membrane antigens obtained from five select and biochemically defined strains indicated that there are several serotypes of Bacteroides fragilis. Each strain was serologically defined by individual or by combinations of determinant groups composed of carbohydrates in the form of polysaccharides or glycoproteins. The carbohydrate constituents were tentatively identified as glucose, galactose, fucose, rhamnose, glucosamine, galactosamine, and traces of mannose. Strains were observed to have minor qualitative and major quantitative variations in carbohydrate composition.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) II. Isolation and characterization of additional temperature-sensitive mutants of SV40.

Fourteen independent temperature-sensitive mutants of simian virus (SV40) were isolated following nitrous acid or hydroxylamine mutagenesis. Three mutants were assigned to the A group and seven to the BC group on the basis of standard qualitative and quantitative complementation assays. Three other mutants did not complement mutants of any complementation group well under standard conditions nor was delayed complementation observed in quantitative assays. However, these mutants were shown to complement members of the A and BC complementation groups but not members of the D group when the qualitative complementation test was modified by allowing the parental virions to uncoat at permissive temperature prior to incubation at 41°. The assignment of these mutants to the D group was substantiated by demonstrating the wild-type infectivity of DNA extracted from cells infected at 33° for growth at 41°. Thirteen of the mutants were tested for the production of tumor (T), capsid (C), virion (V), and major coat protein (VP1) antigens at permissive and nonpermissive temperature by immunofluorescence assays along with mutants which have been described previously by others for comparison. The temperature-sensitive (ts) mutants isolated in this study produced fully immunoreactive T antigen at both temperatures. None of the tsA mutants produced C, VPl, or V antigens at elevated temperature. The BC mutants isolated in this study all produced T antigen at 41°. These late mutants demonstrated two patterns of expression of virion antigens. One group synthesized C, V, and VP1 at 41° and were indistinguishable from wild type on the basis of antigenic phenotype. A second group showed cytoplasmic and nucleolar fluorescence for C and VPl antigens at the nonpermissive temperature similar to that observed for tsBCll previously. Mutants in this group did not produce V antigen at high temperature.     

Extraction of skin test activity from Coccidioides immitis mycelia by water, perchloric acid, and aqueous phenol extraction.

Water, perchloric acid extracts, and fractions of partially defatted whole mycelia of Coccidioides immitis contained delayed skin test activity when tested in C. immitis-infected guinea pits. Aqueous phenol extraction of these fractions resulted in partitioning of activity between aqueous-soluble and phenol-soluble fractions; activity was found to be water soluble after removal of phenol by extensive dialysis. Highest specific activity skin test antigen was invariably found in the phenol-soluble phase, water-soluble fraction. Material of equivalent activity could also be extracted directly from the defatted mycelia. Skin test active fractions contained glucose, mannose, 3-O-methylmannose, glucosamine, and amino acids.     

Isolation and characterization of a cross-reacting antigen in strains of Bacteroidaceae.

A Bacteroidaceae cross-reacting antigen (BCA) was isolated from several strains belonging to species in the genera Fusobacterium and Bacteroides. We showed that each of the 28 strains examined synthesized either BCA or the O-specific lipopolysaccharide (LPS). The structural difference between the two antigens was demonstrated by passive hemagglutination. BCA coated erythrocytes spontaneously and reacted with equal intensity to all bacterial antisera of BCA+ strains, whereas LPS was species specific and also coated erythrocytes after the antigen was treated with NaOH. BCA is an acid polysaccharide as proven by immunoelectrophoresis and by its capacity to form a salt linkage with lysozyme. Chemical analysis demonstrated the presence of galactosamine, glucosamine, galactose, glucose, mannose, and N-acetyl. BCA was nontoxic and did not contain lipid A in its molecule. The small particle size of BCA determined its hapten character. When attached to acid-treated Salmonella minnesota Re cells, it acted as an immunogen. By immunizing rabbits with this hapten-bacterial cell suspension, we obtained a highly potent antiserum that agglutinated all BCA+ strains.     

The carbohydrates of influenza virus

Summary Two carbohydrate fractions can be obtained from egg-grown influenza virus after Pronase digestion followed by gel chromatography. One fraction contains glycopeptides (MW ca. 2000–2600) which represent the carbohydrate side chains of the viral glycoproteins. The constituent sugars of this material are fucose, galactose, glucosamine, and mannose, and their position within the side chain has been partially elucidated by methylation studies. The other fraction (MW>6000) appears to be mucopolysaccharide and is composed of fucose, galactose, glucose, galactosamine, and glucosamine.With 1 Figure     

Expression of an F1/V fusion protein in attenuatedSalmonella typhimuriumand protection of mice against plague

A novel approach to making fusions of F1 and V antigens, which may be incorporated into a live recombinant vaccine for plague, was developed. The nucleotide sequences encodingYersinia pestisV antigen (lcrV) and the mature form of F1 antigen (caf1) were amplified by PCR with primers which included tails. At the 3′ end ofcaf1and the 5′ end oflcrV, the tails encoded one of three six- or eight-amino acid linkers or their complementary sequences. The DNA overlap in each linker region was used to prime a second PCR to generate three F1/V fusions, which were cloned into pUC18. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-Ile-Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Arg. As shown by Western blotting of bacterial cell lysates with anti-V and anti-F1 sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenicity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Ile-Glu-Gly-Arg linker, was electroporated into the attenuatedSalmonella typhimuriumstrain SL3261 (aroA). Mice receiving two intravenous doses of 5 × 10⁶cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG₁:IgG_2aisotype ratios, and T cell responses specific for V and F1 antigens. Six weeks after vaccination, mice were challenged subcutaneously with 7.4 × 10²or 7.4 × 10⁴LD₅₀s ofY. pestisstrain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressingY. pestisantigens as fusion proteins to develop a live recombinant vaccine against plague.     

Human antibody response to three meningococcal outer membrane antigens: comparison by specific hemagglutination assays

Three cell surface antigens, protein, lipopolysaccharide, and polysaccharide, were purified from group B and group C strains of Neisseria meningitidis representing a variety of serotypes. Chemical analysis indicated that cross-contamination was on the order of 1%. Sensitization of sheep erythrocytes with these antigens resulted in highly specific passive hemagglutination assays for the three kinds of antigens. Paired human sera from several groups of individuals were tested by hemagglutination for antibody against each of the antigens. Patients with group B or C systemic meningococcal disease showed increases in antibody titer against all three kinds of antigens, but the antibody response to B polysaccharide was low compared with the response to C polysaccharide. Nasopharyngeal carriers of group B meningococci showed significant increases in titer only against the protein antigens, and noncarriers who received a C-polysaccharide vaccine had a specific response to the C polysaccharide. A given protein or lipopolysaccharide antigen reacted on the average equally well with either group B or C convalescent sera. These results suggest that all three antigens may play a role in the broad human immunity following natural infection.