A project of an experimental recombinant vector vaccine for prevention of diseases caused by pathogenic streptococci based on ScaAB lipoprotein of Streptococcus agalactiae and a coldadapted strain of live influenza vaccine as a vector was developed. The sequence of ScaAB lipoprotein was analyzed and fragments forming immunodominant epitopes were determined. Chimeric molecules of influenza virus hemagglutinin H7 carrying insertions of bacterial origin were constructed. Based on the results of simulation, the most promising variants were selected; they represented fragments of lipoprotein ScaAB lacking N-terminal domain bound to hemagglutinin via a flexible linker. These insertions should minimally modulate the properties of the influenza strain, while retaining potential immunogenicity to a wide group of pathogenic streptococci.

Developing vaccines against group B streptococci (GBS), which take a significant share in the prevalence of human infections, is a priority for medical biology. The objective of the present work was to assess the immunogenic and protective properties of four original chimeric recombinant peptide vaccines designed by combing the immunodominant fragments of GBS surface proteins Bac, C5a peptidase, ScaAB, CspA, and SspB1. The hybrid molecules were constructed using several technologies including the chemical synthesis of full-size DNA molecules and their cloning in expression vectors followed by protein expression in effective producer cells. Mice were used to compare the immunogenicity and post-vaccination protection afforded by the vaccines. All protein constructs were found to be immunogenic; however, they differed in their abilities to evoke immune responses against GBS and to protect against model infection.

In the present work, the immunoadjuvant properties of the influenza deltaNS1 vaccine virus after intranasal administration in combination with recombinant GBS polypeptides was tested in mice. According to our data, co-administration of recombinant GBS polypeptides and influenza deltaNS1 vaccine resulted in the increase in the immunogenicity and protective efficacy of bacterial proteins. Combined vaccination with the GBS polypeptides and influenza deltaNS1 vaccine has a potential to be used not only for prophylaxis infections caused by SGB, but also for prevention of the bacterial complications of influenza.

In the present work, the immunoadjuvant properties of the influenza ANSI vaccine virus after intranasal administration in combination with recombinant GBS polypeptides was tested in mice. According to our data, co-administration of recombinant GBS polypeptides and influenza ANSI vaccine resulted in the increase in the immunogenicity and protective efficacy of bacterial proteins. Combined vaccination with the GBS polypeptides and influenza ANSI vaccine has a potential to be used not only for prophylaxis infections caused by SGB, but also for prevention of the bacterial complications of influenza.

AIM:The study devoted to problem of using of recombinant fragments of group B streptococci (GBS) conservative proteins for induction of immune response against streptococcal infections. Two recombinant polypeptides (ScaAB and-ScpB1) corresponding to immunogenic epitopes of two surface GBS proteins ScaAB and C5a-peptidase, which are presented in other streptococcal species, were studied. The objective of the study was to assess specificity and protective activity of mentioned polypeptides against homologous and heterologous strains of pathogenic streptococci from different groups.MATERIALS AND METHODS:Strains of Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae were used in the study. Array of used methods included opsonophagocytic test as well as active and passive protection of experimental animals against streptococcal infection.RESULTS:It was shown that antibodies specific to studied polypeptides opsonized several strains of group A and B streptococci as well as pneumococci. Immunization of mice with ScpB1 polypeptide resulted in more rapid recovery of animals from challenge systemic group B streptococcal infection. Antisera specific to both polypeptides provided passive protection of animals from infection caused either GBS or GAS.CONCLUSION:Obtained data confirm the feasibility to use recombinant fragments of several GBS conservative proteins in vaccine for induction of protection against infections caused by different species of pathogenic streptococci.

Immunogenic properties of recombinant protein ScaAB mixed with polypeptide P6 as well as conjugate of the former with capsular polysaccharide type III (PSIII) were assessed. Protein-polysaccharide conjugate was synthesized by reductive amination method. Immunogenicity was studied on the mouse model using aluminium hydroxide as adjuvant. Antibody titers in antisera were measured by immunoenzyme assay. Functional activity of antibodies was evaluated by opsonophagocytic test. Immunization with ScaAB-PSIII conjugate resulted in increased immune response to both ScaAB and polysaccharide. Administration of ScaAB and P6 proteins mixture compared to their separated administration led to increased of antibody titer and prolonged circulation of specific antibodies. Injection of studied vaccines increased opsonizing activity of antisera compared to immunization with uncombined components.

Opportunity to increase of immunogenicity of recombinant polypeptide P6 constructed on the basis of surface protective Bac protein by its chemical conjugation with dextran (D) 40 was studied. 3 preparations with different quantity of protein and polysaccharide components were obtained. Their testing with standard serum showed that antigenic determinants of the polypeptide were preserved although partly enclosed and structure of antigenic determinants did not significantly changed. On the model of subcutaneous immunization of mice it has been shown that two preparations--P6D2 and P6D3--have improved immunological characteristics. Conjugation of polypeptide P6 with dextran let to increase of immune response to P6 and affinity of P6-specific antibodies. Injection of nonconjugated P6 and dextran mixture showed that free dextran is not immunogenic and it suppress synthesis of P6-specific antibodies without effect on their affinity. Intranasal administration of nonconjugated P6 did not lead to P6-specific IgG in serum. After conjugation with dextran polypeptide P6 was recognized as an antigen and stimulated production of small quantity of antibodies. Technological process of chemical binding of protein antigen with polysaccharides, which let to regulate protein and polysaccharide components ratio, can be the effective method to increase immunogenicity of recombinant polypeptides.

On the basis of genes, which control synthesis of externally localized proteins of group B streptococci (bac and scaAB), recombinant polypeptides P6 and ScaAB were obtained. Data on protective activity of these polypeptides during experimental infection of immunized mice as well as in opsonophagocytic test on cultivated peritoneal macrophages are presented. It has been shown that protective effect of specific antibodies to P6 was dependent from intensity of immune response. Titer of specific IgG to P6 equal 1:25000 was protective for mice during challenge with LD50. During sublethal challenge level of humoral immunity determined both rate of microorganism elimination and degree of decrease of concentration of streptococci in the spleen. Recombinant polypeptide ScaAB also had marked protective activity and protective titer ScaAB-specific IgG was significantly lower compared with the first polypeptide (1:1600). It has been established that both types of antibodies have opsonizing activity against different strains of group B streptococci. Opsonizing properties of antibodies to P6 were restricted to Bac protein-producing streptococci whereas specificity of antibodies to ScaAB was not restricted by type and group borders. Opsonization of both group B and group A streptococci was revealed. It has been established that protective efficacy mediated by antibodies was dependent not only from their opsonizing characteristics but also from availability of protein antigens, which under certain conditions can be shielded by capsular polysaccharide. It has been assumed that vaccine preparation developed on the basis of polypeptides P6 and ScaAB is promising for further research.

Recombinant polypeptides corresponding to the conservative N-terminal area of Bac surface protein of group B streptococci (GBS) and having the human IgA binding-site were obtained and evaluated in terms of Immunogenic and protective properties. GBS strain 219, serotype 1 bc, served as the source of chromosomal DNA used for cloning. Three polypeptides P1, P5 and P6 were constructed. Polypeptides P1 and P5 with a molecular weight of 22 kD were coded by practically identical DNA fragments (685 nucleotide pairs) and differed in two aminoacids In their central part. Polypeptide P6 had a molecular weight of 35 kD, The fragment of recombinant Bac protein with a molecular weight of 35 kD was found to have protective properties: when used for the subcutaneous immunization of animals, it stimulated the development of immune response capable, in case of challenge, to induce accelerated elimination of streptococci and the prevention of the mice death. Recombinant fragment P6 of GBS protein Bac may be regarded as a candidate for inclusion into GBS-specific vaccine.

A comparative study of immunogenic and antigenic features of recombinant polypeptides based on the surface expressed Group В streptococcal protein Вас was accomplished. Polypeptides P6, P1, P5 with molecular mass 35, 22, 22 kDa were constructed by cloning of different DNA fragments corresponding to 3' part Вас, using expression vectors. P5 was different from PI due to substitution of two amino acids in the potential antigenic domain region. The immune response on laboratory mice demonstrated that intraperitoneal injection of the polypeptides under study induces different levels of occurrence of specific IgG. Titers of immunoglobulines reached 1 : 100 000; 1 : 50 000 and 1 : 12 000 for P6, P1 и Р5, consequently. Immune response was different by affinity levels of antibodies. Binding constant (Kc) varied from 108 M-1 to 1013 M-1 for P6 and from 106 M1 to 1012 M-1 for P1 and P5. Investigation of immunochemical cross reactions of polypeptides P6 with P1 and P5 and mutual adsorption of antisera demonstrated that P6 and P1 have identical antigenic domains. Polypeptide P5 was generating antibodies of two types one of which could not recognize P6. Comparative study of antigenic and immunogenic features of recombinant polypeptides based on Вас protein allowed concluding that polypeptide P6 expressed the highest level of antibodies with high affinity with long period of circulation. Polypeptide P6 can be considered a good potential candidate for part of the complex peptide vaccine against GBS. (Med. Immunol., 2004, vol. 6, N 6, pp 493-498)

The authors substantiated the assumptions of virus-induced modification of membranes as the central link in the pathogenesis of viral diseases. The defensive response of the body to viral inoculation was based on the basis of rapid mobilization of all existing factors of nonspecific stability and the following engagement of defense mechanisms which formulated de novo. The results obtained could be a methodological basis for the choice of strategy of viral diseases control.

The authors studied intracellular location of the viral structures in the lung tissue in the acute period of infection (on the 3rd-6th after inoculation) and on the 28th day when the infectious virus failed to be detected. It was stated that on the 3rd and 6th day after the inoculation viral antigens were equally distributed between cytoplasmic and membraneous fractions. The 28th day demonstrated the presence of the antigens in cytoplasma and their absence in the cellular membranes of the lung. The results obtained could be used for specification of mechanisms of influenza virus persistence in the body.

The authors made a comparative analysis of the contribution to the outcome of the disease by realization of information of viral genome and host responses in the time-course of simulated lethal and nonlethal mice influenza. They studied the effect of ionol, pathogenetic antioxidant, prevention and depicted absolutely new features of the diseases severity and outlined possible points of effective drug application. The suggested scheme of the time-course measurement of viral involvement of the host body and the formation of its responses could be recommended in the study of mechanisms of the viral infection development and the activity of antiviral agents.

The paper presents the results of a comparative study of changes in a thioldisulfide component of the antioxidation system in cases of sublethal and lethal patterns of influenza. The both patterns were featured with a correlation between individual values of thioldisulfide equilibrium and the severity of virus-induced pathological process. Characteristic features of SH/SS coefficient in the lethal pattern of the infection were evidently linked with a specific adaptation of the body under the effect of strong irritants. The possibility to use the parameters of the thioldisulfide system performance for the prognosis of the influenza course was discussed.

Virus-induced processes in organs and tissues of Syrian hamsters in relation to the influenza A virus strain used (HON1 or H3N2), age of the animals, and in the presence of mixed infection were compared. The infection of young hamsters with A/PR8/34 and A/Bangkok/1/79 viruses was shown to induce the synthesis of viral proteins NP and M in spleen cells lasting for up to 15 days (the observation period). In mixed influenza and respiratory syncytial virus infection the possibility of influenza virus genome expression did not change. After infection of mature hamsters, synthesis of virus-specific NP and M proteins in splenocytes was observed only in the animals infected with influenza A/PR8/34 virus but not in those infected with the less pathogenic influenza A/Bangkok/1/79 virus.

Incomplete reproduction cycle of influenza virus A/134/17/57, the attenuation donor being used for preparation of recombinant vaccine strains, hs been analyzed with the use of molecular biology methods. Virus A/134/17/57 with two mutations in P3, NP and M genes remains capable of synthesis of viral polypeptides that are devoid of ability to be inserted into cellular plasma membrane, when the virus is propagated in MDCK culture at non-permissive temperature. The process is preceded by defects in the process of formation of RNP structures connected, evidently, with deficient synthesis of viral RNA due to mutations in the gene coding for P3 protein.