Rapid Assessment of Neutralizing Antibodies Using Influenza Viruses with a Luciferase Reporter.

Influenza viruses cause acute respiratory infections, especially in the autumn-winter period. They are characterized by a high mutation frequency and cause annual seasonal epidemics. The detection of antibodies that neutralize the virus is an important criterion in the assessment of population immunity and the influenza vaccine effectiveness. In this study, a method for determining the titer of virus-neutralizing antibodies in blood serum has been developed. A new test called the luciferase neutralization assay uses a bioluminescent signal for detection. The assay is based on engineered influenza reporter viruses with various surface antigens and a nanoluciferase reporter protein in the NS1 reading frame. Using the developed method, we studied paired sera of volunteers obtained before and after vaccination. The proposed assay was compared with the conventional antibody assessment methods (microneutralization and hemagglutination inhibition assay); a high degree of correlation was observed. At the same time, the use of the luciferase neutralization assay made it possible to reduce the time required for the analysis and to simplify the detection procedure. Supplementary Information: The online version contains supplementary material available at 10.1134/S0003683822070067.

Old dog, new tricks: Influenza A virus NS1 and in vitro fibrillogenesis.

The influenza NS1 protein is involved in suppression of the host immune response. Recently, there is growing evidence that prion-like protein aggregation plays an important role in cellular signaling and immune responses. In this work, we obtained a recombinant, influenza A NS1 protein and showed that it is able to form amyloid-like fibrils in vitro. Using proteolysis and subsequent mass spectrometry, we showed that regions resistant to protease hydrolysis highly differ between the native NS1 form (NS1-N) and fibrillar form (NS1-F); this indicates that significant structural changes occur during fibril formation. We also found a protein fragment that is capable of inducing the process of fibrillogenesis at 37 °C. The discovery of the ability of NS1 to form amyloid-like fibrils may be relevant to uncovering relationships between influenza A infection and modulation of the immune response.

Cold and distant: structural features of the nucleoprotein complex of a cold-adapted influenza A virus strain.

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDFEREGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDFGREGYS). It was also shown that the SGYDFEREGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction's equilibrium dissociation constant is five orders of magnitude lower than for the SGYDFGREGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins were studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.Communicated by Ramaswamy H. Sarma.

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.

[In vitro Antiviral Activity of Recombinant Antibodies of IgG and IgA Isotypes to Hemagglutinin of the Influenza A Virus].

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

[Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures].

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.

SAFETY AND IMMUNOGENICITY OF COLD-ADAPTED RECOMBINANT INFLUENZA VECTOR EXPRESSING ESAT-6 AND AG85А ANTIGENS OF M. TUBERCULOSIS.

Recombinant viral vectors represent one of the most promising platforms for creating a new generation of vaccines against tuberculosis. We constructed a vaccine candidate based on a cold-adapted influenza vector with a truncated NS1 protein containing an insert of tuberculosis ESAT-6 and Ag85A antigens. The recombinant virus possessed a cold-adapted and temperature-sensitive phenotype and was attenuated for mice when administered intranasally. Immunofluorescent staining and Western blot showed the expression of ESAT-6 protein in MDCK cells infected by recombinant virus. After intranasal administration to mice, the recombinant virus stimulated a specific anti-tuberculosis CD4 + Th1-type response with the formation of polyfunctional antigen-specific T cells.

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.

[Vaccines against highly pathogenic influenza viruses of the avian origin].

Worldwide spreading of H5 and H7 highly pathogenic influenza viruses of the avian origin, which periodically infect and kill humans without prior adaptation, poses a constant threat of the new pandemic. The effectiveness of the pandemic prevention completely depends on the quality of the existing influenza vaccines. Typical methods of the vaccine production from the antigenically relevant strains are problematic in case of high virulent H5 and H7 viruses. Therefore, new approaches to the construction of the vaccine strains and production technologies are required in order to protect the population.

[Immunization with live attenuated A/H5N1 vaccine protects guinea pigs from reinfection].

Cold-adapted influenza virus A/HK/1/68/162/35(H3N2) was developed as unified donor of attenuation and high reproductive capacity forvaccine strains. The reassortant of this donor with surface antigens of highly pathogenic strain Alchicken/Astana/6/05 (H5N1) was tested in guinea pigs as a live or inactivated preparation. Immunization with both formulations induced equal levels of serum virus specific antibodies, while the level of mucosal antibodies was significantly higher in animals immunized with live virus. The challenge with the homologous virus demonstrated complete virus clearance only in this group, thereby indicating a direct correlation of the protection level with the level of mucosal antibodies.

[Characterization of cold-adapted influenza strain A/HongKong/1/68/162/35 as a potential donor of attenuation and high reproduction].

Live and inactivated vaccines are currently produced using virus reassortants originating from various gene donors of internal proteins. Based on the pandemic virus A/Hong Kong/1/68 (H3N2), a cold-adapted thermo-sensitive strain A/Hong Kong/1/68/162/35 was generated. It is distinguished for its high reproductive capacity (9-9.5 lg EID50), and hemagglutinating activity (1:1024-1:2048). The strain has ts and ca phenotype: reproductive capacity at t = 39 degrees C is 1.0 lg EID50; at t = 26 degrees C, 8.5 lg EID50. A total of 16 mutations have emerged from comprehensive sequencing of the virus genome. Among them 10 mutations were located in the genes of polymerase complex and NP, with respective amino-acid substitutions. The stability of strain characteristics, such as attenuation to humans and high reproductive capacity, were confirmed by repeated sequencing of the genome after tenfold passing of the virus in chicken embryos. Reassortants of the strain A/Hong Kong/1/68/162/35 with the wild-type viruses have inherited useful features of donor virus.

[Characterization of influenza virus reassortants based on new donor strain A/HK/1/68/162/35(H3N2)].

Influenza reassortant viruses A/SPb/HK/09(H1N1), A/Astana/HK/2009 (H5N1), A/Otar/HK/2010(H3N8), and A/Perth/ HK/2011(H3N2), carrying surface antigens of different subtypes, were constructed on the basis of new potential unified donor strain A/HK/1/68/162/35(H3N2). The virulence and reproduction activity of the obtained reassortants were tested. The safety of the candidate live and inactivated influenza vaccines produced from the reassortant viruses was demonstrated. The study demonstrates that A/HK/1/68/162/35 can be used as a unified donor for attenuated and high-yield vaccine reassortants.

[Human health risk assessment of environmental pollution at the municipal level].

The established tense environmental situation in Krasnouralsk, Sverdlovsk Region, presents a serious threat to human health. Development of a medium-term municipal environmental program for a Krasnouralsk urban district provides solutions of environmental problems. The human health status and carcinogenic and non-carcinogenic risks from exposure to chemical substances polluting ambient air, drinking water, and soil have been assessed within the framework of the program. The findings have served as a basis for elaborating technological and sanitary-and-hygienic measures of the environmental program to assure human environmental safety.

[Study of biological properties of cold-adapted reassortant strain of influenza virus subtype H7N3].

For the development of live attenuated influenza vaccine (LAIV) against influenza virus strains with pandemic potential, method of classic genetic reassortment of donor of attenuation A/Leningrad/134/17/57 (H2N2) [Len/17] with avian apathogenic influenza viruses of different subtypes was used. Strain with genome formula 6:2, which contains HA and NA genes from avian apathogenic virus A/wild duck/Netherlands/12/00 (H7N3) [N7N3-wt] and 6 other genes--from Len/17, was studied. Reassortant strain A/17/ wild duck/Netherlands/00/84 (H7N3) [Lenl7/ H7] exhibited ts- and ca- phenotype specific for cold-adapted strains. Reassortant was identical on antigenic profile to parent avian virus H7N3-wt. Like cold-adapted donor strain Len/17, Len17/H7 was attenuated for chickens, whereas wild-type parent strain was lethal in 60% of birds after its intravenous challenge. Reassortant strain Len17/H7 was attenuated during intranasal inoculation of 6 EID50 to white mice, which was confirmed by absence of its isolation from the lungs, actively reproduced on nasal mucosa and stimulated specific systemic and local antibody response.

[Assessment of human health risk due to environmental pollution in the city of Orsk]. / Otsenka riska, obuslovlennogo zagriazneniem okruzhaiushchei sredy, zdorov'iu zaseleniia v gorode Orske.

The established tense ecological situation in the town of Orsk presents a serious human threat. The use of methods for assessing the risk has allowed the authors to determine the values of carcinogenic and noncarcinogenic risks. Due to the influence of all environments polluted by industrial emissions, the total annual carcinogenic risk is 2.31 cases for the adult population of the town and 0.49 for its children. The greatest carcinogenic risk is associated with arsenic in water and foodstuffs, hexavalent chromium, cadmium, and formaldehyde in the air. The high concentrations of dust, phenol, nitrogen dioxide, and carbon oxide cause a major damage to human health. The established specific values of this risk are of relative significance.

Validation of A&D UA-767 device for the self-measurement of blood pressure.

BACKGROUND: The validation of self-measurement devices has been recommended. Automatic monitor A&D UA-767 (A&D Company, Ltd, Tokyo, Japan) is well known and widely used, but not tested according to the Association for Advancement of Medical Instrumentation (AAMI) and British Hypertension Society (BHS) recommendations. OBJECTIVE: To perform a clinical validation for use by adults of the A&D UA-767 device according to the criteria of the AAMI and a modified BHS protocol. METHODS: The test concerned 101 subjects (57 men and 44 women) aged 15-85 years with arm circumferences of 22-39 cm, a systolic blood pressure (SBP) range of 89-206 mmHg, and a diastolic blood pressure (DBP) range of 53-122 mmHg. For each subject, three readings of the UA-767 were compared with simultaneous auscultatory measurements by two trained independent observers who used a mercury manometer and dual stethoscope. The results were graded according to the BHS (1990 and 1993) and AAMI recommendations. RESULTS: Observers showed close agreement, with mean differences of 1.1+/-2.4 mmHg for SBP and -0.7+/-2.0 mmHg for DBP. The proportion of values agreeing to within 5, 10 and 15 mmHg were 93, 100, and 100% for SBP and 97, 100, and 100% for DBP for the two observers. The sphygmomanometer measurements were 132+/-24/79+/-14 mmHg (mean+/-SD). The average difference between the mercury sphygmomanometer and A&D UA-767 readings for SBP and DBP were, respectively, -0.4+/-5.4 and -0.4+/-4. 8 mmHg. The proportion of values agreeing to within 5,10, and 15 mmHg were 82, 94, and 98% for SBP and 80, 95, and 98% for DBP for the observers and device (A/A grade for BHS). CONCLUSIONS: For an adult population, the A&D UA-767 device for the self-measurement of blood pressure satisfied the AAMI criteria, achieved a BHS grade of A/A and can therefore be recommended for monitoring blood pressure in home and clinical conditions by patients with mild-to-moderate arterial hypertension.

The evolution of biotransformation technologies.

Biotransformation is a broad and growing field of biotechnology and encompasses both enzymatic and microbial biocatalysis. Progress has been made in research on the key drivers of biotransformations, including the isolation and characterization of microbes and their enzymes from, and their utilization in, extreme environments, the manipulation, alteration, and augmentation of metabolic pathways, and the use of combinatorial biosynthesis and biocatalytic methodologies for new compound development.

Biocatalytic plastics as active and stable materials for biotransformations.

Enzyme-containing polymeric materials have been developed that have high activity and stability in both aqueous and organic media. These biocatalytic plastics, containing alpha-chymotrypsin and subtilisin Carlsberg, can contain up to 50% (w/w) total protein in plastic materials such as poly(methyl methacrylate, styrene, vinyl acetate, and ethyl vinyl ether). The activation achieved in organic solvents by incorporating proteases in plastic matrices allows for the efficient synthesis of peptides, and sugar and nucleoside esters. The marriage of enzyme technology with polymer chemistry opens up an array of unique applications for plastic enzymes, including active and stable biocatalysts in paints, coatings, resins, foams, and beads, as well as membranes, fibers, and tubings.

[Interconnection of physico-chemical characteristics of organic solvents and their denaturing ability in relation to proteins]. / Vzaimosviaz' fiziko-khimicheskikh kharakteristik organicheskikh rastvoritelei s ikh denaturiruiushchei sposobnost'iu po otnosheniiu k belkam.

Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis.

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.