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.

Erratum to: Experimental Search for New Means of Pathogenetic Therapy COVID-19: Inhibitor of H2-Receptors Famotidine Increases the Effect of Oseltamivir on Survival and Immune Status of Mice Infected by A/PR/8/34 (H1N1).

[This corrects the article DOI: 10.1134/S0022093022010203.].

Experimental Search for New Means of Pathogenetic Therapy COVID-19: Inhibitor of H2-Receptors Famotidine Increases the Effect of Oseltamivir on Survival and Immune Status of Mice Infected by A/PR/8/34 (H1N1).

The development of drugs for the therapy of COVID-19 is one of the main problems of modern physiology, biochemistry and pharmacology. Taking into account the available information on the participation of mast cells and the role of histamine in the pathogenesis of COVID-19, as well as information on the positive role of famotidine in the prevention and treatment of coronavirus infection, an experiment was carried out using famotidine in a mouse model. We used a type A/PR/8/34 (H1N1) virus adapted to mice. The antiviral drug oseltamivir (Tamiflu), which belongs to the group of neuraminidase inhibitors, was used as a reference drug. The use of famotidine in combination with oseltamivir can increase survival, improve the dynamics of animal weight, reduce the level of NKT cells and increase the level of naive T-helpers. Further studies of famotidine in vivo should be aimed at optimizing the regimen of drug use at a higher viral load, as well as with a longer use of famotidine.

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.

[Genetic diversity and molecular evolution of the influenza A viruses in Russia during 2006-2012].

The results of molecular genetic analysis of more than 280 strains of influenza A virus subtypes H1N1 and H3N2 circulating in Russia in 2006-2012 are presented. The genetic changes underlying the evolution of the virus strains and sensitivity to antiviral drugs were analyzed. Significant changes in the genetic structure of influenza A viruses circulating in the Russian Federation and their phylogenetic affiliation are shown to occur within the studied period. The studies identifying codons under the positive selection in silico in the genes encoding surface proteins of the influenza virus were demonstrated to be efficient for the analysis of the antigenic drift and direction of evolutionary variability of the influenza viruses.

[Prevention of tuberculosis: current approaches to development of vaccines].

This review is focused on recent advances in development of new vaccines for the prevention of tuberculosis. The main reasons for lack of BCG vaccine efficacy in different populations and geographic regions are presented. Design of new vaccines based on live modified strains of Mycobacterium bovis BCG, attenuated strains of Mycobacterium tuberculosis, recombinant proteins and viral vectors is considered in the specific examples. The usage of the heterologous "prime-boost" vaccination strategy against tuberculosis is discussed.

[The 2009 pandemic influenza in Russia. I. Diagnosis and molecular biological characteristics of the virus].

The analysis of 1558 clinical samples revealed influenza virus A(H1N1v) RNA in 339 patients with influenza and 163 fatal cases,which was made in May to December 2009. Data on the antigenic properties of more than 250 of pandemic virus strains isolated at the Research Institute of Influenza and the molecular genetic characteristics of 31 strains are presented. All the test isolates were found to have the S203 substitution in hemagglutinin, which was characteristic of one of 5 minor genome A(H1N1v) virus variants found in the United States and Mexico in 2009. All the test strains contain the S31N substitution in the M2 protein, which determines viral resistance to adamantine, and have no H275Y substitution in neuraminidase, which determines oseltamivir resistance. The substitution of amino acid residue of Asp to Gly at position 222 of HA was found in 8 (73%) of 11 isolates from postmortem lung and trachea samples and in 2 (10%) of 20 isolates from nasopharyngeal swabs. The determination of the pathogenic role of this substitution calls for further investigations.

[Pathogenic aspects of influenza during the epidemics caused by A/H1N1v virus in 2009-2010 according autopsy data].

The paper is based upon the results of clinic-pathological and virological correlations in 29 lethal cases of influenza in Saint-Petersburg and Leningrad region during the epidemics 2009/2010. Immunohistochemical analysis of lungs, heart and brain using monoclonal sera to HA and HP proteins of influenza virus, virological and morphological analysis of experimental influenza in mice infected by A/WSN/33 (HIN1) and A/California/07/09 (H1N1) viruses had been carried out. In the majority of investigated strains was proved the amino acid mutation with replacement D222G. The replication of virus was demonstrated at the late stages of diseases, but the desquamation of respiratory epithelium and cytoproliferative weren't found out. Besides the "influenza cells", previously described by A. V Zinserling the cells with enlarge light nuclei were observed. Patients with influenza died from respiratory distress syndrome with minimal bacterial infection. We've established that H1N1 virus not only damages the cells of respiratory epithelium and alveolar macrophages but it can injure endothelium of different organs and neuroglia. The questions which have to be discussed are listed.

[Study of tolerability and reactogenicity of pandemic vaccines against influenza type A/H1N1].

AIM: To study tolerability and safety of pandemic vaccines against influenza A/H1N1 "INFLUVIR" and "PANDEFLU" on limited group of volunteers during phase I clinical trial. MATERIALS AND METHODS: Thirty healthy volunteers were participated in phase I clinical trial. Clinical and laboratory tests of volunteers randomized to 2 groups (20 persons received vaccine and 10 persons - placebo) were performed. "INFLUVIR" vaccine was administered by intranasal route. Volunteers were hospitalized and followed up for development of local and systemic adverse events during 7 days after vaccination. "PANDEFLU" was administered intramuscularly; vaccinees were followed for 7 days in outpatient settings. RESULTS. Phase I clinical trial showed good tolerability and low reactogenicity of "INFLUVIR" and "PANDEFLU" vaccines. There were no moderate and severe local and systemic adverse events registered. CONCLUSION: On the basis of performed phase I clinical trial, phase II trial was recommended to perform in order to assess the reactogenicity, safety and immunogenicity of studied vaccines.

[Clinical trial of new inactivated influenza vaccine "Grifor"].

AIM: To confirm and prove on the extended contingent of volunteers the non-reactogenicity, safety and immunogenicity of "Grifor" vaccine in comparative trial with registered in Russia commercial vaccine "Vaxigrip". MATERIALS AND METHODS: Phase II clinical trial was performed on the research bases of Mechnikov Institute of Vaccines and Sera and Institute of Influenza. In single-blind comparative prospective randomized trial 300 adult volunteers (150 volunteers on each base) aged 18 - 60 y.o. were divided on 3 equivalent groups. Assessment of antigenic characteristics of "Grifor" vaccine was performed using hemagglutination inhibition assay (HAI) with chicken erythrocytes measuring geometric mean titer (GMT), seroconversion factor as well as level of seroconversion and seroprotection. RESULTS: Previously performed studies proved non-reactogenicity, safety and high immunogenicity of "Grifor", whereas this comparative trial performed with commercial vaccine "Vaxigrip" did not reveal significant advantage in any of studied vaccine. CONCLUSION: "Grifor" vaccine meet the requirements of both EMEA CPMP and methodic guidelines MY 3.3.2. 1758-03 for inactivated influenza vaccines, which allows to register vaccine "Grifor" in Russian Federation.

[The effectiveness of influenza vectors expressing the protective mycobacterial antigen ESAT-6 in the complex therapy of generalized tuberculosis in mice].

The use of Flu/ESAT-6 in chemotherapy for experimental tuberculosis has been found to cause a significant improvement of its results, which appears as the higher clearance of lung tissue from mycobacteria, the reduced extent of specific inflammation foci, and the altered cell composition of granulomas with mainly epithelioid to mainly lymphoid one. Flu/ESAT-6 of Tx-1 immunity (spontaneous and phytohemagglutinin-induced proliferation of splenocytes, spontaneous production of gamma-IFN) and ESAT-6-specific stimulation of a cell immune response (the proliferative activity of splenic lymphocytes and the production of IL-2 in response to recESAT-6) is shown to activate. The influenza vector A/PR8/NS1-125 that contains no ESAT-6 insert shows immunoadjuvant properties, which is likely to cause some reduction in the severity of tuberculosis in the control group of the vaccine.

[Comparative clinical trial of vaccines against avian influenza].

Scientic-production association "Microgen" has finished 1st phase of clinical trials of candidate vaccines against avian influenza in order to assess their reactogenicity, safety, and immunogenicity. Two vaccines constructed from NIBRG-14 vaccine strain [A/Vietnam/1 194/2004 (H5N1)], obtained from World Health Organization, were studied: "OrniFlu" (inactivated subunit influenza vaccine adsorbed on aluminium hydroxide) and inactivated polymer-subunit influenza vaccine with polyoxydonium (IPSIV). Clinical trial of the vaccines with different quantity of antigen (15, 30, and 45 mcg of H5N1 virus hemagglutinin) was carried out in Influenza Research Institute (St. Petersburg) and in Mechnikov Research Institute of Vaccines and Sera (Moscow). Analysis of results allowed to conclude that both vaccines were safe, well tolerated and characterized by low reactogenicity. Two-doses vaccination schedule was needed to meet required seroconversion and seroprotection rates (> or =1:40 in > or =70% of vaccinated volunteers). "Orni-Flu" vaccine containing 15 mcg of hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) in one dose as well as IPSIV containing 45 mcg of hemagglutinin and 0.75 mg of polyoxydonium in one dose were most immunogenic after 2 doses - seroprotection rates in microneutralization assay were 72.2% and 77.0% respectively. Marked influence of aluminium hydroxide content on immunogenicity of the "OrniFlu" vaccine was confirmed in the study. Optimal quantity of adjuvant was 0.5 mg per dose. According to basic concept of vaccine development, preference is given to vaccine that under minimal quantity of antigen induces sufficient specific immune response and is safe in volunteers. "OrniFlu" vaccine containing 15 mcg of H5N1 virus hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) corresponded to these requirements that allowed researchers to recommend it for clinical trials of 2nd phase.

Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT-6 protein.

We generated several attenuated recombinant influenza A vectors expressing the Mycobacterium tuberculosis early secretory antigenic target (ESAT-6) protein. The ESAT-6 protein was recently identified as one of the most promising protective antigens for cell-mediated immunity. The obtained vectors appeared to be capable of inducing ESAT-6 specific Th1 immune response in mice after intranasal immunization. We found that double immunization with two influenza vectors of different subtypes provided a significant level of protection in mice, when applied as prophylactic vaccine, as well as substantial therapeutic effect in mice with pre-established tuberculosis infection. Moreover, we found a strong synergistic effect when vaccination with Flu/ESAT-6 vectors was combined with isoniazid treatment, resulting in a dramatic reduction of bacterial load in the lungs of infected mice.

[The molecular mechanism of the action of antiviral preparations in the adamantane series]. / Molekuliarnyi mekhanizm deistviia antivirusnykh preparatov adamantanovogo riada.

Regions of possible interaction between remantadine and transmembrane M2 protein are revealed by analysis of amino acid substitutions in remantadine- and deutiforin-resistant influenza viruses. The major region includes 5-6 amino acid residues at position 25-31, partially involving the premembrane region and the first position of a hydrophobic membrane-associated domain. The proposed model action of remantadine and its derivatives suggests that remantadine is included into the cell membrane lipid bimolecular layer by its adamantane share and its positively charged NH2-group is exposed to the cell surface. This allows remantadine and its analog to be regarded as molecular "hindrances" for viral particle decapsidation and budding.