Immunoelectrophoretic analysis of the antigenic structure of Sh. sonnei]

The authors studied the antigenic composition of 105 Sh. sonnei strains freshly isolated from patients suffering from acute dysentery and carriers. Immunophoregrams of pure S-and R-forms species were obtained. Up to 13 antigens differing by electrophoretic and diffusion mobility and immunological specificity were revealed among soluble Sh. sonnei antigens The position of common and specific antigens was determined on the immunophoregram. Along with the thermostable somatic O-antigen detected at the I phase of the S-forms, and two thermolabile O-antigen components at the II phase, and the R-forms, there was revealed a surface, relatively thermolabile, K-antigen of A-type capable of agglutinating live bacteria in the O-antiserum; position of the latter on the immunophoregram was also determined.     

Immunologic study of the cellular components of bacillus pyocyaneus. V. Antigenic analysis of slime and its fractions]

Various slime fractions were obtained from newly isolated mucoid strains of P. aeruginosa by the method of ultrafiltration or differential centrifugation with subsequent gel chromatography. Purified slime was found to react with a broader spectrum of typing O sera than the corresponding cell wall lipopolysaccharides. Erythrocytic diagnostic preparations produced on the basis of slime antigens allowed to reveale the presence of circulating antibodies against P. aeruginosa. The slime components with molecular weight of 30,000--100,000 daltons and greater contained common antigenic determinants, and the slime components with molecular weight of 10,000--30,000 daltons contained both specific antigenic determinants and those also common to the high molecular components.     

Immunochemical analysis of water-soluble antigen complexes isolated from typing strains of Pseudomonas aeruginosa of different O-serogroups

Cross immunoelectrophoresis in agarose and immunodiffusion in agar gel were used to carry out the immunochemical analysis of water-soluble antigenic components isolated from P. aeruginosa of different O-serogroups (according to Lanyi's classification). Immunodiffusion revealed the presence of 1--3 common antigens and 1 specific O-antigen in aqueous extracts. Experiments with the use of cross immunoelectrophoresis indicated that 1--12 common antigens could be detected in aqueous extracts. The reference preparation, produced on the basis of the cell mixture of 9 P. aeruginosa strains, contained up to 47 antigenic components, many of them being common to the strains of different O-serogroups (immunotypes).     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Structure of the C-terminal domain of Neisseria heparin binding antigen (NHBA), one of the main antigens of a novel vaccine against Neisseria meningitidis

Neisseria heparin binding antigen (NHBA), also known as GNA2132 (genome-derived Neisseria antigen 2132), is a surface-exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology. It is one the three main antigens of a multicomponent vaccine against serogroup B meningitis (4CMenB), which has just completed phase III clinical trials in infants. In contrast to the other two main vaccine components, little is known about the origin of the immunogenicity of this antigen, and about its ability to induce a strong cross-bactericidal response in animals and humans. To characterize NHBA in terms of its structural/immunogenic properties, we have analyzed its sequence and identified a C-terminal region that is highly conserved in all strains. We demonstrate experimentally that this region is independently folded, and solved its three-dimensional structure by nuclear magnetic resonance. Notably, we need detergents to observe a single species in solution. The NHBA domain fold consists of an 8-strand β-barrel that closely resembles the C-terminal domains of N. meningitidis factor H-binding protein and transferrin-binding protein B. This common fold together with more subtle structural similarities suggest a common ancestor for these important antigens and a role of the β-barrel fold in inducing immunogenicity against N. meningitidis. Our data represent the first step toward understanding the relationship between structural, functional, and immunological properties of this important vaccine component.     

Immunological characteristics of the protein antigens of the family Neisseriaceae. II. The importance of an immunochemical analysis of the protein complexes for a study of taxonomy problems]

The study of antigenic interrelations in the family Neisseriaceae resulted in the isolation of 2 main immunologically separated variants of protein complexes: the first variant was characteristic of nonpathogenic and pathogenic species of the genus Neisseria as well as of 7 taxonomically undefined Neisseria species (N. lactamicus, N. cuniculi, N. ellongata, N. ovis, N. animalis, N. cinerea, N. canis) and Gemella haemolysans; the second variant was represented by the genera Branhamella and Acinetobacter. N. caviae and 3 out of 14 Neisseria strains of undefined species had no common antigens with the genera Neisseria and Branhamella. The importance of the immunotyping of protein complexes for studying the problems connected with the taxonomy of the family Neisseriaceae was considered.     

Immunochemical study of the antigenic structure of microbes of the genus Bordetella. 5. Fractionation of the extracts of the parapertussis microbes and the study of the immunochemical and biochemical properties of the isolated fractions]

Fractions responsible for the main part of the serological and immunogenic activity differing by the set of antigens were isolated from the salt extracts of parapertussis microbes by gel-filtration on Sephadex G-100, ion exchange chromatography on DEAE-cellulose, and preparative electrophoresis in agar. Fractions, disclosing a sufficiently high serological activity and possessing the immunological properties, but containing the minimal set (2--3 out of 7) antigens, which were included in the initial extract, were isolated in the agar gel in the use of the preparative electrophoresis method.     

Immunogenetic and electrophoretic studies with extracts of different adult Culex pipiens strains

The results of an immunological and electrophoretical analysis of extracts of the three adult Culex pipiens strains Paris, Hamburg, and Oggelshausen are presented. The gel-diffusion technique has revealed five antigens which are common and identical in both male and female extracts of all three strains; the females have an additional antigenic component. By disk electrophoresis in acrylamide gel the extracts could be separated into 24 protein fractions common to both sexes and an additional female component. It can be assumed that the additional components found with both methods are identical.     

Immunochemical characterization of Nocardia asteroides antigens: support for a single species concept

Nocardia asteroides strains are highly heterogeneous. They show morphological, physiological, and immunological differences. In a previous study, we delineated seven immunotypes of N. asteroides. In the present study, we compared the culture filtrate antigens of these immunotypes by antigen-antibody crossed-immunoelectrophoresis and by rocket electrophoresis. We have also compared the antigen preparations by two-dimensional electrophoresis. While unique components constitute the major portion of the components, the results indicate that similar components are present in the culture filtrates of all strains. This finding supports the view of retaining all the immunotypes in the species Nocardia asteroides rather than designating different species such as N. farcinica and N. sebivorans.     

Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.     

Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes]

The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.     

Immunodiffusion studies of some Nocardia strains

Forty-three strains of Nocardia, one of Actinomadura and two of Nocardiopsis were studied using the comparative immunodiffusion technique. Three reference precipitation systems were employed: one represented Nocardia asteroides N10, one N. asteroides ATCC 19247, and one N. otitidis-caviarum ATCC 14629. One tight cluster was formed by the N. otitidis-caviarum strains and another tight cluster was formed by some of the N. asteroides strains studied. However, other strains of N. asteroides were distinct from the latter cluster. Furthermore, N. asteroides ATCC 19247, which is the type strain, differed from most ot the N. asteroides strains tested. Strains of the species N. asteroides, N. brasiliensis, N. farcinica and N. otitidis-caviarum were found to be closely related, while N. amarae strains differed slightly from this group. The strains referred to Actinomadura and Nocardiopsis were clearly distinct from the three Nocardia reference strains; nevertheless, three antigens common to these genera were revealed.     

Antigenic analysis of three strains of Mycoplasma arginini by two-dimensional immunoelectrophoresis.

The occurrence of species-specific and strain-specific antigens in three strains of Mycoplasma arginini (G-230, leonis and 23243) was studied by two-dimensional immunoelectrophoresis. Approximately 20 antigenic components could be detected in each strain. It was possible to analyze 6 to 7 major and distinct components from each strain by two techniques: "enhancement" where antigen to an additional strain is added to the first phase of the electrophoresis which increases the size of common peaks and "suppression" where antiserum to an additional strain is incorporated in the second phase whereby peak size of components to which both sera have antibody are decreased. A total of 10 distinct antigens were recognized. Electrophoretic mobilities relative to bovine albumin ranged from 0.2 to 1.08. Three components were common to all strains; two of these represented major amounts of material. Four components represented strain-specific components. Unique fast components were found both in strains 23243 and G-230. Three antigens were distributed into only two of the three strains. The electrophoretic mobilities of some common antigens were quite different between strains.     

Humoral response to Staphylococcus aureus antigens evaluated by the western blotting method]

Cellular antigens extracted from the cells of four Staphylococcus aureus strains from different kinds of infections (sepsis, osteomyelitis, furunculosis) were analysed by the western blotting technique. Antibiotic sensitivity pattern of the strains was compared. One isolate was found to be MRSA strain. Sera samples from patients of whom strains were isolated and four sera from blood donors (as a control) were used in the investigation. IgG levels for purified staphylococcal antigens (lipase, alpha-toxin and teichoic acid) were estimated. Interaction between extracted bacterial antigens and serum antibodies of IgG class were analysed in homologous and heterologous systems. The most strong immunological reaction of the investigated sera with staphylococcal antigens was observed in the case of homologous system. Serum from sepsis patient was found to be the most reactive serum with all staphylococcal antigens mixtures.     

Molecular characterization of the capsular antigens of Pasteurella multocida isolates using multiplex PCR

The use of molecular techniques for detection and characterization of the Pasteurella multocida is very important for rapid and specific detection and characterization of the organism. During the period from 15th February, 2014 to 15th April, 2015, 425 nasopharyngeal swabs and 175 lung and spleen samples were collected and examined by conventional methods, 80 strains (18.82%) of P. multocida were isolated from the calves, sheep and goat with respiratory manifestation. Meanwhile, 77 strains (44%) were isolated from emergency slaughtered animals. All the recovered strains were positive for specific PCR for detection of P. multocida strains previously identified as P. multocida by standard microbiological techniques. Multiplex PCR for molecular typing of the capsular antigens of the recovered P. multocida revealed positive amplification of 1044 bp fragments specific to the capsular antigen type A with 105 strains (66.88%), and amplification 511 bp fragments of the capsular antigen type E with 52 strain (33.12%) and absence of B, D and F antigens. Multiplex PCR for molecular typing of the capsular antigens of P. multocida can be used as a simple, sensitive, rapid, reliable technique instead of the serological techniques for identification of the capsular antigens of P. multocida     

Characteristics of the soluble antigens of bifidobacteria

The immunological study of aqueous buffer extracts obtained from 45 strains of bifidobacteria belonging to the species B. bifidum, B. longum, B. adducens, B. breve, B. infantis and B. parvulorum was made. This study revealed 3 levels of the immunological specificity of soluble bifidobacterial proteins: common to the genus Bifidobacterium, common to a limited number of strains belonging to one or several species of bifidobacteria and strain-specific.     

Immunogenicity of a vaccine made from soluble Proteus antigens

Hemagglutinins with different specificity were determined in 270 subjects: of these, 101 were examined in the time course before and after immunization with Proteus vaccine prepared from soluble antigens. The preparation possessed pronounced immunological potency and stimulated the formation of antibodies to the vaccine strain, to heterologous Proteus strains and to the common antigens of Gram-negative bacteria. The combined scheme of the subcutaneous and local administration of the above-mentioned Proteus vaccine ensured an increase in the synthesis of IgM and IgA in patients with Proteus wound infection. Active immunization ensured an essential rise in the level of hemagglutinins to Re-glycolipid in donors and oncological patients not infected with Proteus, but did not ensure the statistically significant shifts in the antibody level to Re-glycolipids in patients with chronic Proteus infection.     

Primary alveolar echinococcosis: Course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis

We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host–parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.