Strain and stage specific variation in Toxoplasma gondii antigens

The antigenic profile of virulent (RH, ENT, Martin) and avirulent (RRA, DEG, ME49) Toxoplasma strains was compared directly by western blotting using a panel of immune mouse sera. Dominant antigens of approximate MR 30-33, 21 and 25 x 10(3) were common to tachyzoites of all strains, however, there were significant quantitative and qualitative differences in the antigen profiles, indicating a moderate degree of strain specific polymorphism in tachyzoite antigens. We found no specific association between antigenic variation and strain virulence. Comparison of tachyzoite and bradyzoite antigens from homologous strains (RRA, DEG, ME49) confirmed the existence of stage specific antigens and demonstrated a conserved antigen profile among bradyzoites.     

Membrane antigens of the agents of glanders and melioidosis]

Double immunodiffusion in gel test was used to determine the antigenic composition of the preparations of membranes isolated from the lysozyme spheroplasts of glanders (strain No. 10230) and melioidosis (strain No. C-141) causative agents. The membranes of these microbes proved to contain antigens of cell walls, lipopolysaccharides and the thermolabile membrane antigen proper. A study of antimembrane sera in the agglutination and immunofluorescence tests demonstrated a heterogeneity of the glanders and melioidosis strains under study by the membrane thermolabile antigen.     

Sialic acid in F. tularensis and F. novicida antigens]

Sialic acid was revealed in the antigens of virulent (503/830 and Schu) and vaccine (Schu-attenuated and 15-reduced) strains of F. tularensis and also in the antigens of F. novicida isolated by the treatment with trichloracetic acid, by the thiobarbiturate method of Warren. Its content depended on the culture virulence. Sialic acid was absent in the antigens of avirulent strains of the causative agent of tularemia (503-attenuated, 15-attenuated and 21/400), but, in difference from the virulent strains, there was revealed 2-desoxyribose on account of the presence of DNA. Serological activity of F. tularensis antigen decreased only 15 times in the antibody neutralization reaction after a complete release of sialic acid as a result of hydrolysis of the preparation in 0.1 N, H2SO4 at 80 degrees C for one hour, i.e. it persisted at a sufficiently high level. On the basis of investigations carried out it can be admitted that there existed a chemical association between the sialic acid and other composites in the polysaccharide of the virulent and the vaccine strains of F. tularensis, but that this acid played no significant role in the manifestation of specific properties by the antigen.     

Isolation of non-type-specific protein antigens of Streptococcus group B

Hydrochloride extracts obtained from group B streptococcal strains of different serotypes have proved to be the source of type-nonspecific protein antigens, precipitated with ethanol and studied by gel chromatography and spectrophotometric scanning in ultraviolet rays. Thus, 2 or 3 antigens, one of them found to be common for streptococci of groups A, B and G, as well as the admixture of group-specific polysaccharide, have been detected. In extracts obtained from group B streptococcal strains of different serotypes a common protein antigen, specific only for group B, has been detected. The suitability of gel chromatography with the use Toyopearl gel HW-55F for the preparative isolation of the specific fraction of protein type-nonspecific antigen with a view to the subsequent study of immune response to group B streptococci has been shown.     

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10⁶ cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10⁷ cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.     

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.     

The C antigen of Francisella tularensis]

A new envelope antigen C, specific for virulent strains of Francisella tularensis, was revealed by immunodiffusion analysis. In contrast to antigens A and P this antigen is common for Francisella and Brucella. C-antigenic lipid fraction was obtained by chloroform-ethanol (1:1) extraction of bacterial slime. This fraction contained carbohydrates (31.6%) without proteins and detected by TLC glycolipid, which proved glycolipid nature of C-antigen. Introduction of C-fraction or alive F. tularensis resulted in accumulation of C. precipitins in blood serum.     

Isolation of a Herpes Simplex Virus-Specific Antigenic Fraction Which Stimulates the Production of Neutralizing Antibody

Infection of mammalian cells with herpes simplex virus (HSV) results in the production of a number of virus-induced soluble antigens. Immunodiffusion analyses of the soluble antigen mixture (SAM) obtained from HSV-infected KB or BHK cells revealed at least six well-defined immunoprecipitin bands. Calcium phosphate chromatography (Brushite) was employed to separate one immunoprecipitin (designated CP-1) from the remaining viral and host antigens. We conclude that CP-1 is a viral-specific antigen because (i) specific antiserum, which had been repeatedly absorbed with uninfected cell extracts or serum components, still retained the capacity to react in gel diffusion with CP-1 antigen; (ii) anti-CP-1 serum reacted in gel diffusion with SAM, yielding one precipitin band in identity with the band formed against human gamma globulin; (iii) the CP-1 fraction stimulated the production of HSV-neutralizing antibody of high capacity. The last observation suggests that fraction CP-1 contains a biologically active structural component of the virus which is associated with the envelope. The CP-1 immunoprecipitin was separated from SAM by an alternative method by using a cyanogen bromide-linked immunosorbent prepared from anti-CP-1 gamma globulin. The observation that the CP-1 antigen isolated from the immunosorbent effectively blocked serum-neutralizing activity provided further evidence that neutralizing antibody was directed against CP-1. Acrylamide gel electrophoresis and immunological experiments suggest that the CP-1 antigen is in part a glycoprotein. The finding that CP-1 contains only one antigenic component of the virus will permit future biological studies to be made with a monoprecipitin antiserum. In addition, the techniques described in this paper represent initial steps in the purification of HSV antigens.     

Immunochemical findings about the high molecular weight surface antigens of Shigella sonnei]

The authors studied immunochemical properties of the high molecular fraction of surface soluble antigens obtained by extraction with salt solutions from Sh. sonnei (virulent strain 1041) dried with acetone. The high molecular fraction was isolated by gel-filtration on Sepharose-4B. Along with the O-somatic antigen, this fraction contained thermostable and thermolabile antigens resistant to trypsin and RNA-ase treatment, and also protein-containing antigens disintegrated by trypsin. In difference from the O-somatic antigen, one of the thermostable components was completely precipitated with 50% alcohol.     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

Immune complex antigens as a tool in serodiagnosis of kala-azar

The 63 kDa surface antigen of Leishmania promastigotes is one of the most important virulent factors in establishing the host parasite relationship. This glycoprotein is revealed by surface iodination study as well as by metabolic labeling and immunoblot methods. In search of this specific antigen for serodiagnosis, immune complexes (ICs) were isolated from kala-azar patient sera and analysed by SDS-PAGE and Western immunoblotting. The immunoblot of kala-azar IC with patient sera, antipromastigote sera and anti gp63 sera detected the major antigen of 55 kDa. This recognition suggests that 55 kDa antigen and gp63 have common antigenic epitope(s). Normal IC did not react with anti gp63 sera indicating absence of this antigen in normal IC. To confirm the parasitic origin of the 55 kDa antigen of kala-azar IC, in vitro IC was formed with parasite antigen and acid dissociated kala-azar IC antibody. This indicated the antigenic similarity of the 55 kDa antigen and gp63 antigen of the parasite. This also suggested that the former antigen may have been processed from gp63. In summary, identification of parasite antigen (55 kDa) in IC of kala-azar patients' sera may be useful in developing a serodiagnostic assay of visceral leishmaniasis. Several other antigens are visualized in kala-azar IC when developed with patient sera. But specificity and efficacy of these antigens have not yet been evaluated in serodiagnosis of the disease.     

Immunodiagnosis of parasitic zoonoses: purification of Toxocara canis antigens by affinity chromatography

A method of affinity chromatography developed for the purification of species-specific antigens from Toxocara canis adult worms is described. Immunochemical analyses by polyacrylamide gel electrophoresis, immunoelectrophoresis and immunodiffusion showed that ‘pure’ antigen contained fewer but more specific proteins than ‘crude’ antigen. Purified antigens and parasite sections from four parasite species (Toxocara canis, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris lumbricoides) were used in immunofluorescence tests to measure serum antibody levels in animals with natural or experimental T. canis infections and people with zoonotic toxocariasis. ‘Pure’ antigen showed higher specificity and sensitivity than ‘crude’ antigen in serological testing.     

Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres

Mature eggs of Taenia taeniaeformis hatched readily in the presence of sodium hypochlorite and no loss in infectivity of oncospheres for mice was observed after hatching. Crude and sodium deoxycholate-solubilized antigens (termed TtO-DOC) prepared from such oncospheres stimulated high levels of protection against T. taeniaeformis infection in immunized mice similar to those described previously for oncospheres prepared by other methods. Mice immunized with TtO-DOC antigens that had been exposed to potassium metaperiodate remained significantly protected against infection. Exposure of TtO-DOC antigens to pronase and thermolysin, or to trypsin, significantly reduced the ability of these antigens to protect mice against infection. These data suggest that the antigens which immunize mice against infection include protein components. ¹²⁵I-labelled TtO-DOC antigens were immunoprecipitated with sera from mice infected with T. taeniaeformis and the immunoprecipitates analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation with sera from C3H/He mice infected for 28 days revealed a single major labelled protein antigen having a relative molecular mass (M_r) of 31,000. Sera from 5-month infected C3H/He mice immunoprecipitated at least thirteen labelled antigens, including one at M_r 31,000. Attempts to use SDS-PAGE separated proteins to immunize mice showed that oncosphere antigens exposed to the reducing conditions prior to SDS-PAGE lost their ability to protect mice against infection. It was concluded that SDS-PAGE was an unsatisfactory technique for the isolation of a host protective fraction of TtO-DOC antigens. TtO-DOC proteins were resolved by PAGE performed in the presence of sodium deoxycholate (DOC-PAGE) and mice were vaccinated with cut-outs from the gel. A fraction of the DOC-polyacrylamide gel was found to be effective in immunizing mice against infection. Thus, although the characteristics of the protein antigens in this DOC-PAGE fraction have yet to be determined, an important fractionation technique has been identified. It was shown that partial removal of DOC from oncosphere antigen preparations solubilized in 1% DOC was required for the antigen to stimulate protective immunity. These findings will facilitate further antigen characterization studies towards the development of a defined-antigen vaccine in murine cysticercosis. This is particularly so as attempts to raise anti-oncospheral monoclonal antibodies capable of passively transferring protection to mice by using crude antigen preparations to immunize donor mice have not been successful.     

Identification of circulating antigens, including an immunoglobulin binding protein, from Toxoplasma gondii tissue cyst and tachyzoites in murine toxoplasmosis

Here we describe the identification of Toxoplasma gondii circulating antigens in sera of BALB/c mice experimentally infected with either the virulent RH strain, or the cystogenic WTD1 strain or with an isolate from a human patient. The circulating antigens were identified by immunoblot in tachyzoite (RH strain) and in tissue cyst (ME-49 strain) crude antigens, using antibodies produced by immunisation of BALB/c mice with homologous sera from infected animals. The most relevant tachyzoite antigen identified are in the following four clusters of 109-94, 67-57, 35-31 and 28-21 kDa. Tissue cyst-specific circulating antigens, like the 18 kDa one, were detected in sera from mice infected with the cystogenic strains. These immune sera, after depletion of tachyzoite specific antibodies, recognised three tissue cysts antigens with Mr of 120, 79 and 48 kDa, and a cluster of antigens in the range of 68-53 kDa. We produced monoclonal antibodies by fusion of myeloma cells with lymphocytes from the mouse immunised with circulating antigens from the RH strain. One of the clones (3A11/H12) obtained, secretes IgG(1) and recognises a peptide epitope from a tachyzoite 67 kDa protein. This parasite protein also binds irrelevant mouse IgG(1) as well as immunoglobulins from other species. The reactivity with non-specific antibodies was inhibited by preincubation with 2% normal mouse and goat serum, while the reaction with the monoclonal antibody 3A11/H12 was not. Furthermore, a biotinylated F(ab')(2) of an irrelevant mouse IgG(1) did not show any reactivity while the F(ab')(2) of the monoclonal antibody 3A11/H12 reacts specifically with the 67 kDa antigen suggesting that this circulating antigen is a putative Fc binding protein.     

Isolation from Pasteurella multocida of a Lipopolysaccharide Antigen with Immunizing and Toxic Properties

A heat-stable, particulate, lipopolysaccharide-protein antigenic complex has been isolated from a virulent, encapsulated strain of Pasteurella multocida by extraction with cold, formalinized saline, and centrifugation at 105,000 x g. The original bacterial culture had been obtained from a bison that died of hemorrhagic septicemia, an infectious disease of cattle and buffalo. Injection of fractional milligram amounts of the antigen into mice, rabbits, and calves produced toxic reactions which frequently resulted in death of the host. The surviving animals demonstrated a high degree of immunity to challenge with live, virulent organisms. Two injections with 15 mug of the antigen produced a high degree of immunity in mice without the development of any signs of toxicity. The gross chemical composition and toxicity of the antigen were similar to those reported for endotoxins obtained by the Boivin or Westphal procedure. Although strong serological cross-reactions were obtained in Ouchterlony plates between the 105,000 x g antigens from the bison strain and an avian strain with antisera to these strains, these antisera agglutinated only the bacterial cells of the homologous strain. The active immunity obtained in mice by the injection of the 105,000 x g antigens of each strain was specific and could be correlated with the agglutination tests.     

Changes in antigenic properties of lipopolysaccharides of Shigella sonnei phase I having lost the virulence due to transposon integration into the invasiveness plasmid PSS120

A nonvirulent strain of Shigella sonnei phase I has been obtained by integration of the transposon Tn5 into the invasiveness plasmid pSS120 in the virulent strain and designated NR18. The presence of the plasmid pSS120 in both strains results in the similar morphology and bacterial ability to agglutinate in the presence of antiserum to Shigella sonnei phase I antigen. The lipopolysaccharide preparations from the virulent and nonvirulent strains give the similar reactions with the antiserum in the reaction of hemagglutination. However, in the reaction of passive local hemolysis in the gel (Jerne reaction) the significant difference is revealed in the immunogenicity of the preparations, with the preparations from the virulent strain being 4-5 fold more immunogenic. In crossreaction, the antibodies secreted by the mouse spleen cells immunized by LPS from the virulent strain show a weak reaction with the ram erythrocytes sensitized by the LPS of the nonvirulent strain. Thus, the biological changes in the LPS of the nonvirulent strains that are, evidently, the consequence of the structural changes, are identified only by the most sensitive immunological techniques.     

The MT3 specificity resides on a novel human class II antigen distinct from the HLA-DR antigen and DC-like antigen

The MT3 specificity is closely associated with the HLA-DR4, DR7, and DRw9, and is a supertypic specificity. To determine whether the MT3 specificity resides on a novel class II antigen, the MT3 antigen, DR antigen and the DC-like antigen from the DR4-, DR7- and DRw9-homozygous B lymphoid cell lines were identified and compared with one another by two-dimensional gel electrophoresis using alloantisera. The analysis revealed that each of the three antigens exists as a structurally distinct class II antigen in each cell line. The light chains of the MT3, DR and DC-like antigens are different in charge from one another. The molecular weight of the heavy chains of the MT3 and DR antigens is higher than that of the DC-like antigen. On the other hand, no electrophoretic differences are observed between the heavy chains of the MT3 and DR antigens. These results strongly suggest that the MT3 specificity resides on a light chain of a novel class II antigen distinct from the DR antigen and the DC-like antigen. These observations also support our previous proposition that the MT3 antigen belongs to the fourth group of the human class II antigens.     

Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide

A crucial feature of peptide antigen presentation by major histocompatibilty complex (MHC) class I and II molecules is their differential ability to sample cytosolic and extracellular antigens. Intracellular viral infections and bacteria that are taken up in phagosomes, but then escape from the endocytic compartment efficiently, enter the class I pathway via the cytosol. In contrast, phagosome-resident bacteria yield protein antigens that are sampled deep in the endocytic compartment and presented in a vacuolar acidification-dependent pathway mediated by MHC class II molecules. Despite this potential for antigen sampling, microbes have evolved a variety of evasive mechanisms that affect peptide transport in the MHC class I pathway or blockade of endosomal acidification and inhibition of phagosome–lysosome fusion that may compromise the MHC class II pathway of antigen presentation. Thus, besides MHC class I and II, a third lineage of antigen-presenting molecules that bind lipid and glycolipid antigens rather than peptides exists and is mediated by the family of CD1 proteins. CD1 isoforms (CD1a, b, c, and d) differentially sample both recycling endosomes of the early endocytic system and late endosomes and lysosomes to which lipid antigens are differentially delivered. These CD1 pathways include vacuolar acidification-independent pathways for lipid antigen presentation. These features of presenting lipid antigens, independently monitoring various antigen-containing intracellular compartments and avoiding certain evasive techniques employed by microbes, enable CD1 molecules to provide distinct opportunities to function in host defense against the microbial world.     

Investigation of the antigenic composition of the cells and the exotoxin of Sh. dysenteriae (Shiga) by agar immunoelectrophoresis and polyacrylamide gel disc electrophoresis.

Using the method of immunoelectrophoresis in agar, we established the presence of 12 soluble antigens in Sh. dysenteriae (Shiga). We differentiated among them 3 specific antigens situated in the cathodic region. One of them is the somatic O antigen while the other two are thermolabile surface K antigens of these bacteria. K antigens of Grigoryev-Shiga bacteria differ from the earlier described K antigens of the Newcastle and Boyd Shigellae in their positive electric charge, in greater lability in relation to temperature and chemical effects and evidently also in their chemical character. The remaining protein antigens are common for either the genus of Shigella or for the family of enterobacteria. The exotoxin preparation isolated from the R strain of Sh. dysenteriae (Voile 30) by precipitation with trichloroacetic acid is represented in the immunophoreogram by five precipitation lines. Disc electrophoresis in polyacrylamide gel made possible the investigation of the composition of the exotoxin and isolation of the two fractions responsible for its toxic effect. The method of preparative electrophoresis in polyacrylamide gel can evidently be used for obtaining purified Sh. dysenteriae exotoxin preparations.     

A mouse partially protective glycoprotein antigen inStreptococcus agalactiae serotype Ia

The immunological properties of a glycoprotein antigen (antigen 2) ofStreptococcus agalactiae serotype Ia were investigated. A specific antiserum was prepared by immunizing rabbits with antigen 2 immunoprecipitates excised from Crossed immunoelectrophoresis (Crossed IEP) gels. This antiserum produced a single peak representing antigen 2 when reacted with a Triton X-100 sonicate of heat-killed whole serotype Ia cells in Crossed IEP analysis. With polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent immunoelectroblotting, three strongly reacting polypeptides were detected at 60, 56, and 35 kilo-Daltons. Many faintly reacting polypeptides were detected between 67 and 30 kilodalton. The specific anti-antigen 2 serum used in Crossed immunoisoelectric focusing (XIEF) detected three immunoprecipitates, two with a pI of 8.4 and one with a pI of 6.7. Identification of the antigens detected in XIEF with the polypeptides detected by immunoelectroblotting was not attempted. The specific anti-antigen 2 serum partially protected mice against lethal serotype Ia infection.