Comparative immunosecretome analysis of prevalent Streptococcus suis serotypes.

Streptococcus suis is a major Gram-positive swine pathogen associated with a wide variety of diseases in pigs. The efforts made to develop vaccines against this pathogen have failed because of lack of common cross-reactive antigens against different serotypes. Nowadays the interest has moved to surface and secreted proteins, as they have the highest chances to raise an effective immune response because they are in direct contact with host cells and are really exposed and accessible to antibodies. In this work, we have performed a comparative immunosecretomic approach to identify a set of immunoreactive secreted proteins common to the most prevalent serotypes of S. suis. Among the 67 proteins identified, three (SSU0020, SSU0934, and SSU0215) were those predicted extracellular proteins most widely found within the studied serotypes. These immunoreactive proteins may be interesting targets for future vaccine development as they could provide possible cross-reactivity among different serotypes of this pathogen.

Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis.

The bacillus Calmette Guérin (BCG) vaccine, the only licensed vaccine against TB, displays partial and variable efficacy, thus making the exploitation of novel vaccination strategies a major priority. Most of the current vaccines in pre-clinical or clinical development are based on the induction of T cells recognizing protein antigens. However, a large number of T cells specific for mycobacterial lipids are induced during infection, suggesting that lipid-based vaccines might represent an important component of novel sub-unit vaccines. Here, we investigated whether immunization with defined mycobacterial lipid antigens induces protection in guinea pigs challenged with M. tuberculosis. Two purified mycobacterial lipid antigens, the diacylated sulfoglycolipids (Ac2SGL) and the phosphatidyl-myo-inositol dimannosides (PIM2) were formulated in biophysically characterized liposomes made of dimethyl-dioctadecyl-ammonium (DDA) and synthetic trehalose 6,6'-dibehenate (TDB). In three protection trials, a reduction of bacterial load in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Moreover, a reduction in the number of lesions and severity of pathology was detected in the lungs and spleen of the lipid vaccine group compared to unvaccinated controls. As the degree of protection achieved is similar to that observed using protein antigens in the same guinea pig model, these promising results pave the way to future investigations of lipid antigens as subunit vaccines.

Evaluation of a vaccine formulation against Streptococcus pneumoniae based on choline-binding proteins.

Streptococcus pneumoniae has proteins that are attached to its surface by binding to phosphorylcholine of teichoic and lipoteichoic acids. These proteins are known as choline-binding proteins (CBPs). CBPs are an interesting alternative for the development of a cost-effective vaccine, and PspA (pneumococcal surface protein A) is believed to be the most important protective component among the different CBPs. We sought to use CBPs eluted from pneumococci as an experimental vaccine. Since PspA shows variability between isolates, we constructed strains producing different PspAs. We used the nonencapsulated Rx1 strain, which produces PspA from clade 2 (PspA2), to generate a pspA-knockout strain (Rx1 ΔpspA) and strains expressing PspA from clade 1 (Rx1 pspA1) and clade 4 (Rx1 pspA4). We grew Rx1, Rx1 ΔpspA, Rx1 pspA1, and Rx1 pspA4 in Todd-Hewitt medium containing 0.5% yeast extract and washed cells in 2% choline chloride (CC). SDS-PAGE analysis of the proteins recovered by a CC wash showed few bands, and the CBPs PspA and PspC (pneumococcal surface protein C) were identified by mass spectrometry analysis. Subcutaneous immunization of mice with these full-length native proteins without adjuvant led to significantly higher rates of survival than immunization with diluent after an intranasal lethal challenge with two pneumococcal strains and also after a colonization challenge with one strain. Importantly, immunization with recombinant PspA4 (rPspA4) without adjuvant did not elicit significant protection.

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague.

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.

Association between immunogenicity and adsorption of a recombinant Streptococcus pneumoniae vaccine antigen by an aluminum adjuvant.

The impact on immunogenicity of the degree of adsorption of three Streptococcus pneumoniae (Sp) vaccine antigens to aluminum adjuvants was studied. The three antigens evaluated (Sp1, Sp2 and Sp3) were highly adsorbed by aluminum hydroxide adjuvant, but not adsorbed by aluminum phosphate adjuvant. All of the Sp antigens adjuvanted with aluminum hydroxide elicited higher antibody responses in mice than formulations prepared with aluminum phosphate or non-adjuvanted antigen. Varying the percent aluminum-bound Sp antigen in the formulated vaccine affected the observed antibody responses. These observations suggest that the antibody response observed for Sp antigens in this study is stimulated by a depot effect of the antigen bound to an aluminum adjuvant.

Protection against Streptococcus pneumoniae elicited by immunization with pneumolysin and CbpA.

The need for the development of cheap and effective vaccines against pneumococcal disease has necessitated the evaluation of common virulence-associated proteins of Streptococcus pneumoniae as potential vaccine antigens. In this study, we examined the capacity of active immunization with a genetic toxoid derivative of pneumolysin (PdB) and/or a fragment of choline binding protein A (CbpA; also known as PspC, Hic, and SpsA) to protect mice from intraperitoneal challenge with medium to very high doses of a highly virulent capsular type 2 pneumococcal strain, D39. The median survival times for mice immunized with the individual protein antigens in different adjuvant combinations were significantly longer than those for mice that received the respective adjuvants alone. Mice immunized with CbpA alone were significantly better protected than mice immunized with PdB alone. Correspondingly, the median survival times for mice that were immunized with a combination of PdB and CbpA were significantly longer than those for mice that received PdB alone but not significantly different from those that received CbpA alone. Mice immunized with the protein antigens in a mixture of monophospholipid A (MPL) and aluminium phosphate (AlPO4) adjuvants had higher antibody titers than mice that received the antigens in AlPO4 alone. Mice immunized with PdB in MPL plus AlPO4 were also significantly better protected than mice that received PdB in AlPO4 alone.

Antibodies to Escherichia coli and Shigella flexneri in milk from undernourished mothers: studies on sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated antigens.

Analysis of IgA, IgM, and IgG antibodies against Escherichia coli O6, its lipopolysaccharide (LPS), and Shigella flexneri were performed in the milk of moderately undernourished Guatemalan women receiving either a low or a high calorie supplement, using SDS-PAGE. As expected, the immunostaining analysis of milk antibodies showed that IgA was the predominant isotype in both groups. Concerning the other Igs, antibodies against O6 LPS were mainly of the IgM isotype, whereas IgG antibodies were more prominent than IgM against the bacterial whole cell preparations. Seven to nine distinct bands, ranging in molecular mass from 13.5 to 109 kD were selected for each antigen to compare the milk antibodies between the two groups of women. After a 20-wk supplementation period, the IgA and IgG antibodies to the E. coli, O6 LPS, and S. flexneri were not found negatively affected by a low calorie intake. A significantly lower immunostaining intensity was, however, obtained for the low calorie intake group regarding the IgM antibody activity against four high molecular mass bands of the E. coli whole cell preparation. A decreased immunostaining intensity was also found in the same group for IgM antibodies against two bands of E. coli O6 LPS when analyzing paired samples collected at d 0 and wk 20. No differences were found for IgM antibodies against any of the S. flexneri antigens. In conclusion, the results suggest that low calorie intake does not significantly affect the production of milk IgA antibodies to E. coli and S. flexneri antigens in these women. Still, IgM antibodies against certain proteins and LPS molecules of E. coli may be decreased. IgG antibodies, although also present in milk, seemed to be directed mainly against bacterial proteins and not to LPS.

[The isolation of the antigenic substances of Mycobacterium tuberculosis and the characteristics of their basic chemical components]. / Vydilennia antyhennykh substantsii mikobakterii tuberkul'ozu ta kharakterystyka ïkh osnovnykh khimichnykh komponentiv.

Different methods of isolation were used when obtaining the antigen determinants of the tuberculosis mycobacteria (tuberculins, polysaccharide, phosphatide). Main chemical components of the antigens were investigated. Most effective and economic methods of obtaining the M. tuberculosis antigens substances were chosen. The using of the obtained antigens in the test systems for diagnostics of tuberculosis discussed.

[The isolation of the surface antigen from vegetative cells of Bacillus anthracis STI-1 and study of its protective properties]. / Vydelenie poverkhnostnogo antigena vegetativnykh kletok Bacillus anthracis STI-1 i izuchnie ego protektivnykh svoistv.

The method for the extraction of native surface protein antigen with a mol. wt. of 92 kD from vegetative cells of B.anthracis STI-1 and its purification was developed. The antigen was extracted with 3% sodium lauryl sarcosylate at 4 degrees C from bacterial mass previously treated with 0.1 M tris-HCl buffer solution, pH 8.0 Purification was carried out by adsorption chromatography on hydroxylapatite. The isolated protein antigen with a mol. wt. of 92 kD was electrophoretically and immunochemically homogeneous. The study of the protective properties of this protein in combination with Freund's adjuvant, made on white mice, revealed that it had good prospects for use in the creation of chemical vaccines against the causative agent of anthrax.

[The protective activity of Yersinia pseudotuberculosis antigens]. / Protektivnaia aktivnost' antigenov Yersinia pseudotuberculosis.

Samples of Y.pseudotuberculosis (serovar I) antigens, represent a high-molecular lipopolysaccharide (LPS) fraction with a mol. wt. of 22.5 kD and fractions of outer membrane proteins isolated by the method of M. Osborn and R. Munson (1974), were tested in comparison with the activity with live cells of Y. pseudotuberculosis I attenuated mutant KV 9/2, having lost its Cad plasmid of virulence with a mol. wt. of 47 MD and carrying 2 attenuating markers: resistance to crystal violet and nalidixic acid. In experiments on guinea pigs pathomorphological studies demonstrated high protective activity of Y.pseudotuberculosis I attenuated mutant KV 9/2 and a pronounced protective effect achieved after the immunization of the animals with complex biopolymers, including a high-molecular LPS fraction and outer membrane proteins.

[The effect of the synergistic action of enterotoxin on the specific protective complex of Shigella sonnei]. / Effekt sinergentnogo deistviia énterotoksina na spetsificheskii protektivnyi kompleks Shigella sonnei.

The study has first established that enterotoxin enhances the protective potency of S. sonnei specific protective complex. This effect has been revealed both in experiments of the oral immunization of mice and in experiments of the conjunctival immunization of guinea pigs and depends on the dose of enterotoxin used in the experiment. The increase of protection has a specific character. These observations open prospects for further enhancement for the protective properties of S. sonnei specific protective complex, which should be taken into consideration in developing the vaccinal preparation.

[Polymer-metal complexes of the Mycobacterium protein antigen prevent the dissemination of postvaccinal infection in the T-deficiency state]. / Polimermetallicheskie kompleksy belkovogo antigena mikobakterii predotvrashchaiut disseminatsiiu postvaktsinal'noi infektsii pri T-defitsitnom sostoianii.

Primary obtained triple highly immunogenetic complex consists of nonimmunogenic protein antigen, isolated from the BCG, nontoxic polyelectrolyte and bivalent cuprum ions. Immunization with this complex provides high immunoprotection in the experimented B mice (T-deficit mice) from lethal dissemination BCG. Inoculation of single components of the complex has no protection. Result of these experiments suggest that antigen-polyelectrolyte complexes obtained on the basis of nonimmunogenic protein fraction of the tuberculosis bacilli show the properties of a thymus-independent immunization preparations, and provide immune defense in the T-cell deficit of the immune system.

Isolation and structural characteristics of a monoclonal antibody-defined cross-reactive phospholipid antigen from Mycobacterium tuberculosis and Mycobacterium leprae.

A low molecular weight antigen of Mycobacterium leprae and other mycobacteria was previously defined in our laboratory by means of IgG2a monoclonal antibody termed L4. The antigen had an apparent molecular mass of 4.5-6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was assumed to be a glycoprotein on the basis of its staining with periodic acid Schiff and sensitivity to periodate treatment. In the present work, the cross-reactive and phospholipidic nature of the antigen, present in Mycobacterium tuberculosis as well as in M. leprae sonicates, was demonstrated and this enabled us to undertake its purification from crude M. tuberculosis phospholipidic extracts. The L4-reactive antigen from M. tuberculosis called L4-PIM, was purified by means of silicic acid high pressure liquid chromatography. Its characterization by gas chromatography and FAB-MS showed the antigen to be the common mycobacterial dimannosylated phosphatidylinositol (PIM2), the structure of which had been previously established by others (Lee, Y. C., and Ballou, C. E. (1964) J. Biol. Chem. 239, 1316-1327; Lee, Y. C., and Ballou, C. E. (1965) J. Biochem. (Tokyo) 4, 1395-1404). Delineation of the L4 epitope on M. tuberculosis L4-PIM revealed the involvement of the axial 2-hydroxyl of the alpha-D-mannosyl residues, without any detectable contribution from the myo-inositol. Consequently, L4 was shown to react with PIM5, the structure of which contains twice the number of epitopes as does PIM2. By using both immunostained thin layer chromatography and indirect enzyme-linked immunosorbent assay, similar L4-PIM epitopes were demonstrated in M. leprae sonicate, thereby explaining the cross-reactive nature of the L4-monoclonal antibody. Antibodies of IgG class directed against M. tuberculosis L4-PIM were detectable in sera from patients with leprosy, but no evidence of T cell reactivity to L4-PIM was obtained. The demonstration of a correlation of anti-L4-PIM IgG and anti-disacharide-conjugated bovine serum albumin IgM antibody titers in the sera of leprosy patients indicates that measurement of antibodies directed against L4-PIM may have the potential to be used as a complementary assay to the disaccharide-conjugated bovine serum albumin test for diagnosis and monitoring of patients undergoing leprosy therapy.

Septicemia caused by cysteine-dependent Escherichia coli.

A case of septicemia and urinary tract infection caused by cysteine-dependent Escherichia coli in a 70-year-old woman with bilateral staghorn calculi is described. This is the second report of a cysteine-dependent E. coli bacteremia. The bacterium was falsely susceptible to ampicillin and co-trimoxazole when tested on a medium without cysteine supplement.

Further novel amido sugars within the glycopeptidolipid antigens of Mycobacterium avium.

The individual serovars of the Mycobacterium avium complex, a source of serious and persistent infections in individuals with underlying immune deficiencies, also present an extraordinary set of novel sugar epitopes as part of their type-specific glycopeptidolipid surface antigens. Californium desorption-mass spectrometry has been successfully applied to the holistic glycopeptidolipid antigen of M. avium serovar 12 and its per-O-acetyl derivative, to arrive at the following structure, of molecular mass 1876: (Sequence: see text). The pentasaccharide hapten, released as the tetraglycosyl alditol, was subjected to methylation analysis, absolute configurational analysis, 1H NMR and fast atom bombardment-mass spectrometry to arrive at the structure: 4-(2'-Hydroxy) propionamido-4,6-dideoxy-3-O-Me-Glcp (beta 1----3)-4-O-Me-L-Rhap (alpha 1----3)-L-Rhap (alpha 1----3)-L-Rhap (alpha 1----2)-6-deoxytalitol. Two-dimensional proton correlation spectroscopy was also applied to determine the configuration of the unique distal segment of the oligosaccharide unit. The significance of this structure in the context of the fully elucidated structures of the antigens from 12 of the 31-member M. avium complex is discussed.

[Choleragen-anatoxin chemical cholera vaccine enriched with Ogawa O-antigen]. / Izuchenie kholernoi khimicheskoi vaktsiny "kholerogen-anatoksin", obogashchennoi O-antigenom Ogava.

The cultural fluid of Vibrio cholerae strains of serovar Ogawa, grown under the conditions of submerged cultivation, has been shown to contain a large amount of soluble O-antigen which sharply differs from all other concomitant components in its molecular weight. By enriching the commercial chemical cholera vaccine known as Choleragen Toxoid with purified Ogawa O-antigen a new preparation, consisting mainly of cholera toxoid and Ogawa and Inaba O-antigens and capable of producing pronounced immunity to V. cholerae of both serovars, has been obtained.

Immunochemical studies of Staphylococcus aureus Oeding-Haukenes antigen a5: a phosphorus-containing polysaccharide.

Antigen a5 was isolated from strain 830 of Staphylococcus aureus by autolysis in phosphate buffer followed by alcohol precipitation. Purification was principally achieved by affinity chromatography on wheat germ agglutinin ultrogel and on anti-S. aureus teichoic acid immunosorbent. The a5 antigen was weakly immunogenic in rabbits. Chemical analysis showed that a5 is a teichoic acid composed of ribitol phosphate, N-acetylglucosamine and alanine. It has similar physico-chemical properties to the wall beta-N-acetylglucosamine ribitol teichoic acid of S. aureus but is serologically distinct.

[Precipitation of the antigens of the causative agents of plague, glanders and melioidosis using aqueous saline extracts of various plant species]. / Pretsipitatsiia vodno-solevymi ékstraktami razlichnykh vidov rastenii antigenov vozbuditelei chumy, sapa, melioidoza.

The interaction of the aqueous saline extracts of 55 plant species with the antigens of the causative agents of plague, glanders and melioidosis in the reaction of immunodiffusion (RID) in gel has been studied. The aqueous saline extracts obtained from the seeds of 12 plant species have been revealed to possess precipitating capacity. At the same time these extract have been found to ensure, as a rule, RID with several antigens under test.

[Vaccine from soluble antigens for the specific therapy of Proteus infection]. / Vaktsina iz rastvorimykh abtigenov dlia spetsificheskoi terapii proteinoi infektsii.

A vaccine prepared from Proteus soluble antigens obtained by the disintegration of microbial cells with hydroxylamine is proposed. In animal experiments the vaccine has proved to be nontoxic when injected in large single doses or in smaller doses over a prolonged period and capable of protecting mice and rabbits from Proteus infection. No pronounced postvaccinal complications have been registered in humans immunized with the vaccine. The possibility of using the vaccine for immunizing donors with the aim of obtaining hyperimmune anti-Proteus plasma and for the active vaccinal therapy of patients with Proteus infection has been shown.