Monoclonal antibodies (mAbs) of the IgG1 isotype obtained to the full-length recombinant pneumolysin (rPly) did not recognize or poorly recognized conformational epitopes of native pneumolysin in ELISA. At the same time, polyclonal antibodies (pAbs(rPly)) detected native pneumolysin in sandwich ELISA when they were used as capture and detecting antibodies simultaneously. All mAbs(rPly) inhibited erythrocyte hemolysis induced by native pneumolysin. The combined use of mAbs(rPly) did not reveal an increase in optical density in ELISA and an increase in inhibition hemolytic activity, which suggests that mAbs(rPly) are directed to the same or spatially closely located epitopes. pAbs(rPly) more effectively inhibited erythrocyte hemolysis than mAbs(rPly).

Pneumolysin (Ply) is a target for the development of serotype-independent pneumococcal vaccines, an important condition for the efficacy of which is their ability to activate innate immunity with the subsequent formation of adaptive immunity. In this study, the ability of recombinant full-length Ply (rPly) of pneumococci to induce TLR expression and maturation of dendritic cells generated from mouse bone marrow was evaluated. It was shown that rPly in vitro increased the number of dendritic cells expressing Toll-like receptor 4 (TLR4) on the membrane. rPly caused maturation of dendritic cells generated from mouse bone marrow, which manifested in a decrease in the number of progenitor cells (CD34), an increase in the number of cells expressing the adhesion molecule CD38, costimulatory molecules CD80 and CD86, molecules of terminal differentiation of dendritic cells CD83, as well as molecules of antigenic presentation of the major histocompatibility complex class II.

We compared the immunogenicity of recombinant S. pneumoniae pneumolysin (rPly) when administered with and without Al(OH)3 adjuvant, and evaluated the protective properties of recombinant protein in the active defense experiment. It was shown that double immunization with rPly+Al(OH)3 increases the levels of IgG antibodies in comparison with the control (p<0.01), while triple immunization results in a more significant increase in IgG antibody levels (p<0.001). Double immunization with rPly without Al(OH)3 does not induce a significant increase in antibody levels in comparison with the control, while triple immunization results in a slight but significant increase in antibody levels (p<0.05). The active defense test proved the protective activity of rPly against S. pneumoniae serotype 3 at intranasal infection.

We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

Backgraund . Pneumolysin (Ply) is a hemolytic toxin of Streptococcus pneumoniae ( S. pneumoniae ) expressed by all strains of pneumococci. The use of sandwich enzyme-linked immunosorbent assay (ELISA) can be a simple, fast and effective way of its qualitative and quantitative determination in biological fluids. Aim. To develop and evaluate the specificity of sandwich ELISA test system for qualitative and quantitative determination of recombinant Ply (rPly) of S. pneumoniae . Materials and methods. Immobilized on the solid phase rabbit’s polyclonal antibodies (pAbs) to rPly were used as recognition antibodies in sandwich ELISA. The studied antigens were added to the pAbs (rPly). The reaction was manifested by using detecting mouse monoclonal IgG1 (rPly) – antibodies conjugated with horseradish root peroxidase. The specificity of the test system was evaluated when using recombinant α-hemolysin (rα-Hly) and water-soluble S. aureus antigens as reference preparations. Results. Using sandwich ELISA, rPly was detected at a concentration of 0.15 µ / ml. The test system was characterized by specificity, which was confirmed by the absence of reaction with recombinant rα-Hly of Staphylococcus aureus ( S. aureus ). Reference preparations of water-soluble surface antigens of S. aureus strains No 209, 1986,1991 and Cowan I gave a false positive reaction due to the presence of protein A (SpA) in their composition, a thermostable surface protein expressed by many strains of staphylococci capable of binding immunoglobulins via Fc-fragment or Fab fragments of the V3H domain of the B cells receptor. A negative reaction was obtained with antigens from the S. aureus wood 46 strain, which does not have the spa gene encoding SpA expression. The presence of protein A in preparations of water-soluble S. aureus antigens was confirmed in the ELISA inhibition assay. Conclusion. Sandwich ELISA has been developed for qualitative and quantitative determination of S. pneumoniae Ply. The conducted studies have confirmed the specificity of the test system.

Relevance. Type-specific immunity does not protect against infection with other pneumococcal serotypes. The phenomenon of the change of serotypes dominating the population of Streptococcus pneumoniae is known, in part due to the intensive recombination process and the phenomenon of «capsule switching». Therefore, the development of a serotype-independent pneumococcal vaccine is an important global public health priority. Ams. Investigation of immunobiological properties of candidate components of a future vaccine with serotype-independent activity. Materials and methods. For immunization of mice, preparations of the capsular polysaccharide of pneumococcus serotype 3 (CPS) were used; protein-containing fraction (PCF) obtained from an aqueous extract of S. pneumoniae 6B cells; recombinant pneumolysin (Ply); mixtures of drugs (CPS + Plу; CPS + PCF; PCF + Plу); conjugate vaccine Prevnar 13 (manufactured by PFIZER Inc. USA). Mice were immunized intraperitoneally, 2 times with an interval of 14 days. Intact mice were used as a control group. To assess the humoral immune IgG response, the method of solid-phase ELISA was used. Phagocytic activity was studied at 7, 14, 21 and 28 days after the second immunization. The cytokine level was determined in the blood sera of mice after the second immunization 2, 4, 8, and 24 hours later on a NovoCyte flow cytometer (ACEA Biosciences, USA) using the MACSPIex CytoKine 10 Kit mouse (Miltenyi Biotec Inc., USA) according to the manufacturer's instructions. Results. Immunization of mice with Ply as well as mixtures with CPS and PCF caused a significant increase in the level of antibodies to Ply. It was found that there was no apparent decrease in the level of antigen-specific antibodies when antigens were administered in combination with others. Pneumolysin, used alone or in combination with PCF and CPS, induces the production of antiinflammatory cytokines IL-4, IL-10, and IL-5 detected throughout the study. This is confirmed by a study of the opsonophagocytic activity of neutrophils from immunized CPS + Ply, Ply + PCF and Ply mice; a significant increase in the number of eosinophils is observed in their blood due to the stimulation of their production of IL-5. Conclusions. As a result of the studies, it was shown that Ply, used alone or in combination with CPS and PCF, has the highest immunogenicity: it stimulates a significant increase in the level of specific antibodies, stimulates Th-2, and induces the production of anti-inflammatory cytokines.

Background. The development of a pneumococcal vaccine with serotype-independent activity is relevant for the whole world, as this is due to the high prevalence of Streptococcus pneumoniae (S. pneumoniae), the constant change of pathogen serotypes and the growth of antibiotic-resistant strains. In previous experiments, we revealed a protective effect of immunization with recombinant pneumolysin (rPly) in a model of systemic infection caused by S. pneumoniae serotype 3.Aim. Study of serotype-independent protective activity of rPly.Materials and methods. Strains of S. pneumoniae serotypes 4 and 6B were used. Mice were immunized intraperi-toneally two or three times with an interval of 14 days with rPly. To assess the protective activity of rPly, animals after double or triple immunization were infected intraperitoneally with S. pneumoniae .Results. rPly at a triple immunization in a dose of 25 μg protected mice from intraperitoneal infection with S. pneu- moniae serotype 4, the efficiency index increased by 2 times compared with the control. with a double immunization, rPly protected mice from intraperitoneal infection with S. pneumoniae serotype 6B, efficiency index increased by 6.61 times compared with the control.Conclusion. rPly protects animals from infection with different serotypes of S. pneumoniae, which allows us to consider it a promising drug for the development of pneumococcal vaccines with serotype-independent activity.

Introduction. Pneumococcal diseases remain relevant for the whole world. On the one hand, this is due to the high prevalence of pneumococcus and the other hand, the growth of antibiotic-resistant strains and the constant change of clinically significant serotypes of the pathogen.The aim of the research was to study of the protective activity of a mixture of pneumococcal antigens.Material and methods. we used preparations of a capsular polysaccharide (CPS) obtained from Streptococcus pneumoniae serotype 3; protein-containing fraction (PCF) obtained from an aqueous extract of cells of S. pneumoniae serotype 6B; recombinant pneumolysin (rPly). Mice were immunized intraperitoneally twice with an interval of 14 days with mixtures of bacterial antigens: CPS + PCF; CPS + rPly; PCF + rPly. To assess the protective activity of the studied drugs after double immunization animals were infected intraperitoneally with S. pneumoniae serotype 3. To study the effect of mixtures of bacterial preparations on the infectious process in the lungs immunized mice were infected with S. pneumoniae serotype 3. The humoral immune response was studied with IgG using the method of ELISA.Results. The CPS + rPly mixture protected mice from intraperitoneal infection with S. pneumoniae serotype 3 regardless of the infecting dose. Immunization with CPS + PCF or CPS + rPly mixtures influenced a significant decrease the number of seeded bacterial cells from lungs during the entire observation period (72 h) compared to the control. Administration of mixtures of bacterial antigens of CPS + PCF, CPS + rPly or PCF + rPly to animals led to a significant increase of the level of antibodies to all antigens, however, the highest levels of IgG were determined to PCF and rPly.Conclusion. The results obtained suggest that different antigenic drugs in mixtures affect different mechanisms of immunity activation.

Oncolytic viruses, including live attenuated measles virus (MV) vaccine strains, have recently been shown as promising therapeutic agents against human malignancies. In this study, the oncolytic potential of the attenuated vaccine strain Leningrad-16 (L-16) of MV was evaluated in a panel of human metastatic melanoma cell lines. The L-16 measles virus was shown to replicate within melanoma cells mediating direct cell killing of tumor cells, although all melanoma cell lines varied in regard to their ability to respond to L-16 MV infection, as revealed by the different pattern of the Interferon Stimulated Gene expression, cytokine release and mechanisms of cell death. Furthermore, the statistically significant L-16 measles virus related tumor growth inhibition was demonstrated in a melanoma xenograft model. Therefore, L-16 MV represents an appealing oncolytic platform for target delivery of therapeutic genes along with other attenuated measles virus strains.

Immunogenic and protective activity of recombinant pneumolysin was studied in experiments on male BALB/c mice. The mice were immunized intraperitoneally with recombinant pneumolysin sorbed on Al(OH)3 (200 μg per mouse). In 2 weeks after immunization, the isotypes of antibodies to recombinant pneumolysin in the serum of immunized mice were determined by ELISA. The animals were infected with Streptococcus pneumoniae serotype 3. Immunization with recombinant pneumolysin induced the production of anti-pneumolysin antibodies, mainly of IgG1 subisotype. On day 21 after intraperitoneal infection with S. pneumoniae serotype 3 in a dose of 106 microbial cells, the survival rate of animals immunized with recombinant pneumolysin in a dose of 25 μg/mouse was 67% vs. 0% in the control (p<0.001). Recombinant pneumolysin could be considered as a promising protective antigen for inclusion in the serotype-independent vaccine against S. pneumoniae.

Aim. The study of immunochemical and immunobiological properties of native protein-containing antigens of pneumococcus. Materials and methods. The study was carried out on the strains of the Collective Usage Center «Collection of Mechnikov Res. Inst. for Vaccine and Sera». In the work studied the chemical composition, the molecular weight of the obtained antigens in SDS-electrophoresis and antibody titers in ELISA. Protective activity of protein-containing antigens of pneumococcus was determined in experiments of active protection of mice. Results. Protein-containing antigens of pneumococcus were isolated from S. pneumoniae serotypes 3, 6B, 10A, 14, 19F, 23F and 36. The chemical composition of the preparations contained from 16 to 35% protein. In SDS-electrophoresis in polyacrylamide gel it was established that the molecular weight of protein-containing antigens of pneumococcus ranged from 14 to 116 kDa. Using ELISA shows the cross-activity of native antigens. Virtually all drugs reacted with antimicrobial rabbit serum obtained to serotype 19F (p≤0.05). Serotype serum 14 was less active and only protein-containing pneumococcal antigens obtained from 14 and 19F serotypes (p≤0.05) interacted with it. In the precipitation test according to Ouchterlony it was confirmed that preparations of serotypes 3, 6B, 14, 19F and 36 reacted with rabbit immune serum obtained for S. pneumoniae 19F serotype. In immunoblotting it was found that protein-containing antigens of pneumococcus isolated from serotypes 3, 6B, 10A, 14, 19F and 36 were associated with monoclonal antibodies to pneumococcal protein — pneumolysin. In vivo experiments it was shown that protein-containing antigens of pneumococcus protected animals from intraperitoneal infection of S. pneumoniae in homologous and heterologous systems (p≤0.05). Conclusion. The revealed immunochemical and cross-protective activity of protein-containing antigens of pneumococcus in vitro and in vivo experiments allows to select drugs derived from serotypes 6B, 10A, 19F and 36, as the most promising for further study of the intraspecific protective activity of individual native proteins of pneumococcus.

Infections caused by Streptococcus pneumoniae are relevant for Russia and the world. One of the key factors in the pathogenicity of pneumococcus is a polysaccharide capsule. The structure of polysaccharide antigens is described more than 90 serotypes of the pathogen. The experience of using polysaccharide and conjugated pneumococcal vaccines shows that these preventive drugs protect against a limited number of serotypes of the pneumococcus. It is of interest to study the protective properties of pneumococcal proteins, as they are conservative and have high homology within the species, potentially expanding serotype non-specific protection level. Thus, the efforts of researchers focus on the development of protein vaccines or conjugated vaccines based on proteins of S. pneumoniae. The review considers the biological properties of the most well-known proteins of pneumococcus and provides data on preclinical studies of the obtained recombinant proteins as experimental vaccine preparations. Immunization with various proteins of S. pneumoniae provides protection of animals from nasopharyngeal colonization, pneumonia and sepsis. Currently, clinical trials (I/II phases) are being tested with several experimental protein vaccines. In the near future it will be possible to assess the real effectiveness of such vaccines.