Immunity to Cholera: Relation of Fraction II of Type 2 Cholera Toxin to Vibriocidal Antibody

The nontoxic protein component in supernatant fluids of young cultures of the cholera vibrio in peptone dialysate broth contains an antigen identical in specificity to vibrio lipopolysaccharide. This material was heterogeneous after elution from diethylaminoethyl A50 Sephadex, and it contained at least five additional minor antigens. Identity was demonstrated by immunodiffusion methods, by the induction of specific vibriocidal antibody formation, and by specific interference in the vibriocidal reaction. The minor antigens appeared to be unrelated to the vibriocidal reaction. The major antigen was more highly immunogenic than lipopolysaccharide, giving higher and longer-persisting antibody titers in the rabbit, but lipopolysaccharide was the more effective interfering antigen per unit weight in the vibriocidal reaction. The nontoxicity and high immunogenic potency of the protein antigen suggest that it may be useful as an immunizing agent for the production of the antibacterial component of an effective immunity.     

Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin

Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.     

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.     

Characterization of crystal prototoxin and an activated toxin from Bacillus thuringiensis var. entomocidus

Toxin crystals from Bacillus thuringiensis var. entomocidus were lysed by proteases present in gut juice from larval Philosamia ricini (Lepidoptera) with the release of a prototoxin and an activated toxin. Some of the toxic activity of the lysate was complexed with a pheophytinlike pigment and this complex was retarded on filtration through Sephadex gels. A method is described for the removal of the pheophytin from larval protease preparations. The prototoxin has a molecular weight greater than 200,000, determined by its exclusion from Sephadex G-200 and on activation produces a toxin of molecular weight about 50,000. Isoelectric focusing of crystal lysates gave pI values of 4.5 and 6.4 for the prototoxin and toxin, respectively. The antigenic composition of the prototoxin and of the toxin are compared and the significance of antigen h as an indicator of activation is discussed.     

Genetically engineered diphtheria toxin fusion proteins carrying the hepatitis B surface antigen

Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and beta-galactosidase were obtained by expression of hybrid genes from the pR promoter of bacteriophage lambda and purification by affinity chromatography. The antigenicity and immunogenicity of the individual protein constituents were analyzed. A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment. The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles. This suggests a novel approach towards the use of DT mutants as immunogenic carriers of heterologous antigens.     

Persistence of specific antigenic protein in the serum of chickens given intravenously botulinum toxin type B, C, D, E or F

A sublethal dose of Clostridium botulinum progenitor toxin of each of types B, C, D, E, and F was injected once intravenously into chickens. Blood samples were withdrawn periodically from the chickens to determine the toxin remaining in the serum by the mouse injection test and by enzyme-linked immunosorbent assay (ELISA) for both toxic and nontoxic components composing the progenitor toxin. Both components were detected by ELISA for at least a few days after the serum had became innocuous to mice, indicating a higher stability of the antigenicities of both components than the lethal toxicity in the chicken serum. For the diagnosis of botulism, it seems justified to recommend detection of the antigen (toxic component or nontoxic component or both) by ELISA even if no toxin is detected by the mouse test. Such immunological tests would no doubt contribute to an increase in the rate of diagnosis of human and animal botulism cases, particularly when blood sampling is delayed.     

Characterization of a Cl. Perfringens type D strain, isolated in the field and optimization of epsilon toxin biosynthesis in a cell culture

A field strain of cl. perfringens, named Dt001, was isolated from kidney of ovine enterotoemia case. The isolate characterized as Cl. perfringens, type D was based on its cultural and biochemical characters and its factors of virulence. The strain was very toxinogenic and well adapted to culture conditions of biofermentation when the parameters related to ptt, incubation time, substrat ... were optimized. Thus, the use of carbon source as polymer (destrine), the continuous control of pH allowed improvement of the rate of biosynthesis of Epsilon toxine by 10 times. The study of the immunogenicity of the isolate showed that preparations of anacultures were more immunogenic then those of anatoxine type. The fact that the two forms of epsilon antigens (protoxin and active toxin) show similar immune response in rabbits, indicates that the proteolytic action of trypsin is limited only to the toxic sites and does not affect the immunogenic epsitopes of the toxin. It also suggests a molecular organization of epsilon toxin in which the immunogenic epsitopes and the toxin sites are apart. The biotechnological performances and the immunogenicity and toxinogenical of the Dt001 isolate are in favor of its possible use as a component of an inactivated vaccine against enterotoxenia.     

Purification of a protein from Bacillus thuringiensis toxic to larvae of lepidoptera

The protein toxin of the parasporal body or crystal of Bacillus thuringiensis (Mattés isolate) has been purified severalfold by a combination of Sephadex G-200 gel filtration and ammonium sulphate precipitation. It has been shown that the use of highly alkaline conditions for dissolution of the crystals does not lead to serious artifacts. The crystal toxin has been shown to be quantitatively related to the crystal antigen. It is possible that there is a second distinct toxin present in the crystal and this too can be detected by its antigenic reaction. Purified toxic protein has been hydrolysed in vitro by regurgitated Pieris brassicae gut enzymes, chymotrypsin, trypsin and subtilisin. In each case the digest contained a product that was still antigenic, had mol.wt. about 40000 and was toxic to P. brassicae larvae. Smaller toxic molecules (mol.wt. approx. 10000) that did not react as antigens were also produced by proteolysis. It is possible that these smaller molecules were hydrolytic products of the larger digestion product.     

Simplified purification method for Clostridium botulinum type E toxin

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Discriminating antigen and non-antigen using proteome dissimilarity III: tumour and parasite antigens

Computational genome analysis enables systematic identification of potential immunogenic proteins within a pathogen. Immunogenicity is a system property that arises through the interaction of host and pathogen as mediated through the medium of a immunogenic protein. The overt dissimilarity of pathogenic proteins when compared to the host proteome is conjectured by some to be the determining principal of immunogenicity. Previously, we explored this idea in the context of Bacterial, Viral, and Fungal antigen. In this paper, we broaden and extend our analysis to include complex antigens of eukaryotic origin, arising from tumours and from parasite pathogens. For both types of antigen, known antigenic and non-antigenic protein sequences were compared to human and mouse proteomes. In contrast to our previous results, both visual inspection and statistical evaluation indicate a much wider range of homologues and a significant level of discrimination; but, as before, we could not determine a viable threshold capable of properly separating non-antigen from antigen. In concert with our previous work, we conclude that global proteome dissimilarity is not a useful metric for immunogenicity for presently available antigens arising from Bacteria, viruses, fungi, parasites, and tumours. While we see some signal for certain antigen types, using dissimilarity is not a useful approach to identifying antigenic molecules within pathogen genomes.     

Isolation and immunochemical study of the toxic complex of Cl. botulinum type F]

The toxic comples of Cl. botulinum, type F, was separated into the toxic and nontoxic protein fractions by the methods of ion exchange chromatography and gel filtration in accordance with a specially devised purification scheme. Highly purified, electrophoretically and serologically homogeneous toxin with a molecular weight of 150,000 and potency equal to 10 X 10(6) DLM per 1 mg of protein was isolated from the toxic fraction. The nontoxic protein component had faintly pronounced hemagglutinating properties and was essentially different from type A and B hemagglutinins. The toxic complex of Cl. botulinum, type F, was shown to contain a proteolytically active fraction.     

Purification and partial characterization of a toxin produced by Ganoderma lucidum,the coconut Ganoderma disease pathogen

Abstract

An extracellular, hydrophilic, thermostable phytotoxin was purified to homogeneity from culture fluids of Ganoderma lucidum. The phytotoxin was purified by solvent extraction, gel filtration on Sephadex G-75. Toxicity was evaluated with detached leaf sheath and electrolyte leakage bioassays. Purified phytotoxin induced visible symptoms of the disease, when applied to coconut leaves, fronds and roots even at a low concentration. The toxin is a glycoprotein with carbohydrate as the major component. The importance of the carbohydrate moiety for toxic activity was indicated by inactivation of toxic compounds after periodate oxidation. The toxin caused lesions on a number of other monocots and dicots and proved to be non-host specific.     

Immunologic response to the influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. I. Studies in human vaccinees

Analysis of an earlier study of H3N2 and H7N2 inactivated influenza vaccines in schoolchildren demonstrated a greater viral neuraminidase (NA) immunogenicity of the vaccine containing the H7 hemagglutinin (HA) antigen to which they had not been primed, despite the lesser NA antigen content of that vaccine. Thus, prior experience with the influenza viral HA appeared to have a negative influence on immune response to NA, the associated external glycoprotein, presumably on the basis of intermolecular antigenic competition. In a second study, sequential immunologic response to influenza viral NA was compared in college students who were immunized with either conventional commercial vaccine or an antigenic reassortant H7N1 vaccine, and who subsequently experienced natural infection with an H1N1 influenza virus. Although both vaccines were only marginally immunogenic in inducing NA antibody response in seronegative subjects, in vaccinees initially seropositive for HA antibody significant NA antibody titer increases occurred with H7N1 vaccine. Subsequent natural infection boosted NA antibody less effectively in the population previously primed by natural infection than in initially seronegative subjects primed by H7N1 vaccination. It is suggested that primary immunization monospecific for influenza viral NA may alter the subsequent pattern of immune response to one more favorable to the induction of NA antibody when virus is encountered.     

Comparative study on the immunogenic properties of Clostridium perfringens type A toxoid

The paper presents the results of a study on the immunogenic properties of toxoid preparations from Cl. perfringens type A obtained using the routine method of detoxifying alpha = toxin in the culture medium (commercial preparations) and by means of detoxifying a previously purified alpha = toxin (experimental preparations). When tested in immunized guinea pigs, the immunogenicity of experimental preparations was found to be 4.5 to 6 times that of commercial preparations. In mice, there was no difference in the immunogenic properties of the two types of preparations as determined by the ED30 of the antigen and the serum levels of Cl. perfringens antitoxin. The possibility is discussed of using the guinea pig as a laboratory animal model due to its ability to reflect most clearly the differences in the immunogenicity of Cl. perfringens type A toxoid preparations.     

Purification and chemical characterization of the major neurotoxin from the venom of Pelamis plautrus.

A major toxin was isolated from the venom of the sea snake Pelamis platurus (yellow-bellied sea snake) by Sephadex G-50 and carboxymethylcellulose column chromatography. The LD50 of the pure toxin (Pelamis toxin a) was 0.044 mug/g in mice representing a tenfold increase in toxicity after purification. The toxin was homogeneous in acrylamide disc gel electrophoresis and eluted as a single peak after isoelectric focusing in a sucrose density gradient column. The isoelectric point was 9.69; thus it is a highly basic protein. The toxin contained 55 amino acid residues with four disulfide linkages. When all disulfide linkages were reduced and alkylated, the toxic action of the pure toxin disappeared leading to the conclusion that the disulfide bonds of the neurotoxin were essential for toxic action.     

A one dose experimental cholera vaccine

The number of cholera vaccine doses required for immunity is a constraint during epidemic cholera. Protective immunity following one dose of multiple Vibrio cholerae (Vc) colonization factors (Inaba LPS El Tor, TcpA, TcpF, and CBP-A) has not been directly tested even though individual Vc colonization factors are the protective antigens. Inaba LPS consistently induced vibriocidal and protective antibodies at low doses. A LPS booster, regardless of dose, induced highly protective secondary sera. Vc protein immunogens emulsified in adjuvant were variably immunogenic. CBP-A was proficient at inducing high IgG serum titers compared with TcpA or TcpF. After one immunization, TcpA or TcpF antisera protected only when the toxin co-regulated pilus operon of the challenge Vc was induced by AKI culture conditions. CBP-A was not consistently able to induce protection independent of the challenge Vc culture conditions. These results reveal the need to understand how best to leverage the 'right' Vc immunogens to obtain durable immunity after one dose of a cholera subunit vaccine. The dominance of the protective anti-LPS antibody response over other Vc antigen antibody response needs to be controlled to find other protective antigens that can add to anti-LPS antibody-based immunity.     

Minimal requirements for in vitro activity of chemically defined synthetic antigens as immunogenic carrier molecules

A primary antibody response to 2,4-dinitrophenyl (Dnp)-oligolysines of defined chain length was induced in vitro. A molecular size of at least eight lysine residues was critical for the induction of antibody response to the hapten, but the location of the hapten on the carrier did not seem to play a significant role in this regard. Shorter peptides were nonimmunogenic, but did not paralyze the response to other, noncrossreacting antigens. The electric charge of the carrier molecule was found to have an effect on immunogenicity in vitro. A synthetic copolymer with a positive charge was immunogenic, whereas a similar molecule carrying a negative charge was inactive. On the other hand, changing the negative charge of carriers such as polyglutamic acid was not sufficient to render them immunogenic. Furthermore, neutralization of the negative charge of the surface of the spleen tissue by preincubation with positive polymers did not enhance the response to conjugates of the hapten with negatively charged carriers. These observations are interpreted on the basis of higher affinity of positively charged molecules for the negatively charged cell surface. Accordingly, the specific binding of the antigen to the cell surface is made more stable, and this ensures stimulation.     

Idiotype-restricted antibody response to specific immune complexes

In this report, we compared the immunogenicity of specific antigen/antibody complexes with that of free antigen. The complexes were prepared in antigen excess using the TEPC-15 myeloma protein and a phosphorylcholine-containing polysaccharide antigen (PnC), and the PnC-specific antibody response was measured using a hemolytic plaque assay 5 days after immunization. The results showed that the complexes were as immunogenic as the free antigen; however, the PnC-specific antibody response induced with the complexes was completely dominated by one particular idiotope (defined by plaque inhibition with the AB1-2 monoclonal antibody). On the other hand, the response of mice immunized with free antigen (PnC) was dominated to a lesser degree by the AB1-2 idiotope, and there was a great degree of variability in idiotope expression among individual mice. The results suggest that immunization with antigen/antibody complexes restricts the response to the expression of idiotopes that are present in the immune complex.     

Clostridium botulinum Type E Toxin: Effect of pH and Method of Purification on Molecular Weight

The toxin of Clostridium botulinum type E was isolated from intact cells and from toxic culture filtrates by column chromatography at three pH values, 4.5, 5.3, and 6.0. At pH 6.0 and 5.3, the isolated toxin was in a form with a molecular weight (MW) of 86,000. This toxin was homogeneous on polyacrylamide gel electrophoresis and gel filtration and had an optical density ratio, 280 nm/260 nm, greater than 2.0. It did not dissociate at higher pH levels, but was dissociated into nontoxic components of approximately 12,000 MW when reduced and alkylated in the presence of 6 M guanidine hydrochloride. At pH 4.5, smaller amounts of an impure toxic moiety with a MW of 12,000 were found. After storage for 6 months, the 86,000-MW moiety had lost 60% of its lethality. Gel filtration revealed that the bulk of the toxicity was associated with a component having a MW of 150,000. Toxic components with MW of 12,000 and over 200,000 were also found. The toxin appears to polymerize or aggregate when in a pure form, so that most, if not all, of the MW previously reported for the toxin may belong to different polymers of a monomer with a MW of 12,000 or less. Treatment of the 86,000-MW toxin with trypsin resulted in an 18- to 128-fold increase in lethality, but no detectable change in MW.