Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure.

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e tandem copies (Flg-4M2e-HA2) provides partial α-helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.

CROSS-PROTECTIVE PROPERTIES OF AN INFLUENZA VACCINE BASED ON HBC4M2E RECOMBINANT PROTEIN.

One of the main problems in the area of influenza prophylaxis and pandemic prevention is the development of cross-reactive vaccines, i.e. vaccines directed against all subtypes of human influenza viruses. Such vaccines are being developed in many countries for more than 10 years. A number of vaccines are presently undergoing clinical trials. We created Uniflu candidate vaccine based on recombinant HBc4M2e protein consisting of 4 tandem-connected copies of the highly conserved ectodomain of M2 protein of the influenza A virus. These 4 copies were genetically fused to the carrier protein, namely hepatitis B core antigen. Commercially available Derinat was used as adjuvant in the candidate vaccine. Preclinical studies on laboratory animals (mice, ferrets) demonstrated that immunization with Uniflu leads to significantly higher level of specific immunoglobulins in the blood and bronchoalveolar lavages. Moreover, it produces immunoglobulins belonging to subtype IgG2a that is the most important mediator of antibody-dependent cytotoxicity. The vaccine under review stimulates the proliferation of T-lymphocytes, as well as the formation of CD4+ and CD8+ T-cells synthesizing ɣ-IFN. When infected with the lethal doses (5 LD50) of influenza A viruses of the subtypes H1N1, H2N2, H3N2, and H1N1pdm09, immunized animals typically developed mild form of illness. This kept them alive in 90-100% of cases, which demonstrated almost complete protection from death. Replication of the virus in the lungs of immunized mice was reduced by 1.8-4.8 log10. High immunogenicity of the vaccine, and reduced clinical symptoms following experimental infection, were demonstrated in ferrets as well. The developed recombinant vaccine Uniflu has high specific activity and cross-protection. Uniflu can be proposed as pre-pandemic vaccine, provided that it passes clinical trials.

A Fusion Protein Based on the Second Subunit of Hemagglutinin of Influenza A/H2N2 Viruses Provides Cross Immunity.

Conserved fragments of the second subunit of hemagglutinin (HA2) are of great interest for the design of vaccine constructs that can provide protective immunity against influenza A viruses of different subtypes. A recombinant fusion protein, FlgMH, was constructed on the basis of flagellin and a highly conserved HA2 fragment (35-107) of influenza viruses of the subtype A/H2N2, containing B cell, CD4+ T cell, and CD8+ T cell epitopes. The native conformation of the HA2 fragment was partially preserved upon its attachment to the C-terminus of flagellin within the recombinant fusion protein FlgMH. FlgMH was shown to stimulate a mixed Th1/Th2 response of cross-reactive antibodies, which bind to influenza viruses of the first phylogenetic group (H1, H2, H5), to the target sequence as well as the induction of specific cytotoxic T cells (CD3+CD8+IFNγ+). Immunization with the recombinant protein protected animals from a lethal influenza infection. The developed FlgMH protein is a promising agent that may be included in an influenza vaccine with a wide spectrum of action which will be able to stimulate the T and B cell immune responses.

[Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents].

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

Ebola hemorrhagic fever: Properties of the pathogen and development of vaccines and chemotherapeutic agents.

Ebola hemorrhagic fever (EHF) epidemic currently ongoing in West Africa is not the first among numerous epidemics in the continent. Yet it seems to be the worst EHF epidemic outbreak caused by Ebola virus Zaire since 1976 as regards its extremely large scale and rapid spread in the population. Experiments to study the agent have continued for more than 20 years. The EHF virus has a relatively simple genome with seven genes and additional reading frame resulting from RNA editing. While being of a relatively low genetic capacity, the virus can be ranked as a standard for pathogenicity with the ability to evade the host immune response in uttermost perfection. The EHF virus has similarities with retroviruses, but belongs to (-)RNA viruses of a nonretroviral origin. Genetic elements of the virus, NIRV, were detected in animal and human genomes. EHF virus glycoprotein (GP) is a class I fusion protein and shows more similarities than distinctions in tertiary structure with SIV and HIV gp41 proteins and even influenza virus hemagglutinin. EHF is an unusual infectious disease, and studying the molecular basis of its pathogenesis may contribute to new findings in therapy of severe conditions leading to a fatal outcome.

[The role of adenosine Al receptors and mitochondrial K+ATP channels in the mechanism of increasing the resistance to acute hypoxia in the combined effects of hypoxia and hypercapnia].

We studied the role of the role of mitoK+ATp channels and Al-adenosine receptor in the mechanism of increasing the resistance to acute hypoxia after hypoxic, hypercapnic and hypercapnic-hypoxic preconditioning. It is shown that mitochondrial ATP-sensitive potassium channels and Al-adenosine receptors, an important mechanism of preconditioning have a high value to increase the resistance to acute hypoxia/ischemia in the combined effect of hypoxia and hypercapnia. However, with regard to the adenosine receptor, this mechanism is realized without the participation hypercapnic component, which apparently starts neuroprotection without activation of the adenosine Al receptors.

[Immunogenicity of recombinant proteins including ectodomain of M2 influenza virus A].

Two recombinant proteins with three copies of the ectodomain of the conserved influenza protein M2 (M2e) of influenza viruses were developed: A (H1N1)pdm09, A/Kurgan/05/05 (H5N1), and M2e consensus sequence of the human influenza A virus (H1N1, H2N2, H3N2) based on flagellin and core antigen of hepatitis B (HBc). The first recombinant protein comprised flagellin fused to three tandem copies of M2e, the second preparation was based on non-covalent interaction between M2e peptides and HBc. The immunogenicity of two preparations was comparatively tested. A covalent linkage of flagellin with M2e significant increased the immunogenicity of the target antigen compared with non-covalent interaction M2e and HBc. Flagellin as a protein carrier of M2e induced mainly IgG1 subclass, whereas HBc stimulated more balanced Th1/Th2 response. Our study showed a decrease in the viral titers in lung tissues of immunized mice after lethal challenge of A/PR/8/34 (H1N1). The study revealed a possibility to obtain a vaccine preparation with equal immunogenicity both against human influenza viruses and highly pathogenic avian influenza viruses.

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein.

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1-2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.

[A new method of producing biologically active nanocomplexes by non-covalent conjugation of proteins with viral particles].

Currently, a range of biologically active molecules have been attached to plant and bacterial viras nanoscaffolds, yielding stable nanoparticles that display multiple copies of the desired molecule. In this paper we propose a new method of non-covalent attachment of peptides to the surface of virios. We have demonstrated that this method is efficient in a model system that includes tobacco mosaic virus particles, synthetic polycation (quaternized poly(4-vinylpyridine) carrying ethyl ethyl pendant radicals) and polypeptide of interest. This principle of step-by-step binding to the surface of virions was used for electrostatic association with hydrophilic fragment of influenza virus haemagglutinin.

Development of Recombinant Vaccine against A(H1N1) 2009 Influenza Based on Virus-like Nanoparticles Carrying the Extracellular Domain of M2 Protein.

The conventional vaccines currently being used to deal with influenza are based on a virus obtained in chicken embryos or its components. The high variability of the major immunogenic surface proteins - hemagglutinin and neuraminidase-require the development of strain-specific vaccines that match the antigenic specificity of a newly emerging virus. Recombinant vaccines based on single viral proteins that could be easily produced in standard expression systems are attractive alternatives to traditional influenza vaccines. We constructed recombinant nanosized virus-like particles based on a nuclear antigen of the hepatitis B virus. These particles expose on the surface the extracellular domain of the M2 protein of the highly pathogenic A(H1N1) 2009 influenza virus. The methods of production of these virus-like particles in Escherichia coli and their purification were developed. Experiments on animals show that M2sHBc particles are highly immunogenic in mice and provide complete protection against the lethal influenza challenge.

[Cowpea mosaic virus chimeric particles bearing ectodomain of matrix protein 2 (M2E) of influenza A virus: production and characteristics].

The epitope presentation system for ectodomain of M2-protein of influenza A virus (M2e) based on Cowpea Mosaic Virus (CPMV) was constructed for expression in plants Vigna unguiculata. CPMV is widely used as a vector for production of immunogenic chimeric virus particles (CVPs) bearing epitopes of different infectious human and animal pathogens. To produce chimeric CPMV virus particles in plants, two binary vectors were constructed bearing modified gene coding for S-coat protein of CPMV with insertions of M2e epitopes of human influenza and bird influenza viruses. Antigenic and immunogenic properties of CVPs obtained were investigated in mice immunization experiments and it was shown that they can induce anti-M2e IgG production and partial protection mice against challenge with low doses of flu virus. However, low infectivity and immunogenicity of CPMV chimeric particles indicate the need for further optimization of plant virus-based systems for M2e-epitopes presentation to use plants as a possible source of flu vaccines.

[An analytical evaluation of the peripheral blood charging parameters on the condition of the organism and its systems]. / Analiticheskaia otsenka vliianiia pokazatelei perifericheskoi krovi na sostoianie organizma i ego sistem.

Potentialities of analyzing the health condition of human organism and of its separate systems, i.e. digestive, respiratory, cardiovascular, etc., by the peripheral-blood parameters and with the help of the suggested AKS-ENOFIT analytical system are demonstrated in the paper.

[Humoral and local immune response to the influenza vaccine in people of old and young ages]. / Gumoral'ny i mestnyi otvet na grippoznye vaktsiny u lits pozhilogo i molodogo vozrasta.

The specific features of the humoral and local immune responses to influenza vaccines were comparatively studied in people of different age groups. A total of 79 elderly people (aged 67-89) and 80 young people (aged 18-27) were immunized according to one of the four schemes: live cold-adapted reassortant trivalent influenza vaccine (LIV), administered intranasally; inactivated split trivalent influenza vaccine (IIV), administered parenterally; a combination of both above vaccines; and placebo. IIV was found, as compared to LIV, to stimulate more effectively the production of circulating antihaemagglutinins as well as of IgG,-, Ig1-, and Ig3-AT in young persons, while LIV has advantages before IIV in stimulating the synthesis of these immunoglobulins in the elderly. LIV has advantages before IIV in stimulating the synthesis of secretory IgA-AT irrespective of an age of the immunized persons. The combined immunization of the elderly by both vaccines increases the quantitative parameters of the humoral and local responses up to the level of intensity observed in young people. The obtained data are indicative of the possibility of correcting the immune response in the high-risk elderly in respect to influenza infection.

[Specific immune response to vaccination with an inactivated flue vaccine depending on prevaccine status and age of the person vaccinated]. / Spetsificheskii immunnyi otvet na vaktsinatsiiu inaktivirovannoi grippoznoi vaktsinoi v zavisimosti ot prevaktsinal'nogo statusa i vozrasta vaktsiniruemykh.

Specific antibody immune response to vaccination with commercial inactivated trivalent vaccine A(H1N1) + A(H3N2) + B (IgA, IgG, IgG, subclasses G1, G3, G4, and accumulation of antiCD8) was studied in subjects aged 20-95 years. The initial immune status before vaccination is significant for a positive immune response to the vaccine. Subjects responding to immunization by an increment in specific IgG had a much lower prevaccination level of these antibodies than subjects without these Ig conversion. Antibody immune response to vaccination depended on patient's age. All vaccinees aged 20-25 years developed an increment in IgG to at least one of influenza antigens used. Specific postvaccinal immune response to inactivated influenza vaccine included accumulation of G1, G3, and A antibodies, but not G4 or E antibodies. This latter fact suggests the absence of sensitizing effect of vaccination. In elderly subjects an increment in G1, G3, and A antibodies may not involve an increase in the total level of IgG. In part of elderly subjects secretion of specific antibodies was observed in the presence of increased concentration of antilymphocytic antibodies (antiCD8), indicating a possibility of autoimmune reactions in subjects of this age after injection of inactivated influenza vaccine.

Kinetic approach and estimation of the parameters of cellular interaction between the immunity system and a tumor.

A method is suggested to estimate multi component dynamic systems, which permits, with the help of the computer-calculated kinetic curves, to obtain information about the possible mechanisms of the system component interaction. The method is based on the structural and parametrical identification of mathematical models presented in the form of a system of nonlinear differential equations, using a multi-criterial approach. Using experimental data of studies on growth kinetics and regression of multicellular tumor EMT6 line spheroids in the mouse allogenic system and the immune system cell accumulation in spheroids a mathematical model has been developed of the cellular interaction process in a spheroid. It has been stated that the rate of macrophage and neutrophil accumulation in a spheroid depends on the amount of tumor cells and is determined by the hyperbolic law (as analogous to the Michaelis-Menthen kinetics), while the accumulation of immune lymphocytes in a tumor is determined besides that by the three-cellular cooperation of lymphocytes, macrophages and tumor cells. According to the model, the inhibition of the process of neutrophil and lymphocyte (but not of macrophages) accumulation is realized through the auto-suppression mechanism. The numerical values of the process parameters, which characterise the rates of accumulation, cellular death in a tumor and of local cellular interactions intensity are obtained.

[A method of determining leukocyte adhesion in autoplasma]. / Metod opredeleniia adgezii leikotsitov v autoplazme.

The author suggests a method for assessment of the peripheral blood leukocyte adhesive properties by making use of glass syringes and fit for clinical practice. The technique is reproducible, informative, simple. Seasonal variations of polymorphonuclear and mononuclear leukocyte adhesive capacity were detected, that should be borne in mind when estimating the peripheral blood cell functional activities in various diseases.