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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.The online version contains supplementary material available at 10.1134/S0003683822070067.

Influenza viruses cause an acute respiratory infection, especially in the autumn-winter period. They are characterized by a high mutation frequency and cause annual seasonal epidemics. The detection of virus-neutralizing antibodies 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 measurement of a bioluminescent signal as a detection method. The influenza A reporter viruses of various subtypes were constructed that encode the nanoluciferase protein in the non-structural NS1 protein reading frame. The developed method was used to compare paired sera of volunteers before and after their immunization with a seasonal influenza vaccine. The proposed method was also compared with certified antibody assay methods: neutralization reaction and hemagglutination inhibition reaction. The tests showed a high correlation, while the luciferase neutralization assay reduced the time and simplified the detection procedure. microneutralization, reporter virus, influenza virus, bioluminescence, nanoluciferase The study was supported by the grant of the President of the Russian Federation for young PhDs (no. 075-15-2019-226); and also by the grant of the Government of Saint-Petersburg for undergraduate and graduate students (September 25, 2018, no 124).

The paper reports on the production of a panel of seven new monoclonal antibodies (MAbs) capable of specifically binding to the NS11–124 fragment of the influenza virus A/Puerto Rico/8/1934 (H1N1) NS1 protein in indirect enzyme-linked immunosorbent assay (ELISA), as well as to the full-size native NS1 protein in cells infected with different subtypes of the influenza A virus (A(H1N1), A(H1N1)pdm09, A(H2N2), A(H3N2), and A(H9N2)). A sensitive variant of the microcultural ELISA based on the use of the generated MAbs is proposed; it may be utilized to detect NS1 protein in infected cells and to study its antigenic variability. The results of the performed microcultural ELISA demonstrate type- and subtype-specific antigenic variability of the influenza virus NS1 protein.

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.Использование рекомбинантных вирусных векторов является одним из наиболее перспективных подходов к созданию вакцин нового поколения против туберкулёза. Нами получен вакцинный кандидат на основе холодоадаптированного гриппозного вектора с укороченным белком NS1, содержащий вставку микобактериальных антигенов ESAT-6 и Ag85А. Рекомбинантный гриппозный вектор обладал холодо-адаптированным и температурочувствительным фенотипом и был аттенуирован для мышей при интраназальном введении. Методами иммунофлюоресцентного окрашивания и Вестерн-блот показана экспрессия белка ESAT-6 при заражении клеток MDCK. При интраназальном введении мышам рекомбинантный вирус стимулировал специфический противотуберкулёзный CD4+-Т-клеточный ответ Th-1-типа с образованием полифункциональных антигенспецифических Т-клеток.

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 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.

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.

Background: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. Methodology/Principal Findings: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K -> I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. Conclusion/Significance: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

// Franco Venanzi 1,* , Victor Shifrin 2,* , Michael Y. Sherman 3 , Vladimir Gabai 2 , Oleg Kiselev 4 , Andrey Komissarov 4 , Mikhail Grudinin 4 , Maria Shartukova 4 , Ekaterina A. Romanovskaya-Romanko 4 , Yuri Kudryavets 5 , Natalya Bezdenezhnykh 5 , Oleksandra Lykhova 5 , Nadiia Semesyuk 5 , Antonio Concetti 1 , Anatoly Tsyb 6 , Marina Filimonova 6 , Victoria Makarchuk 6 , Raisa Yakubovsky 7 , Andrey Chursov 2 , Vita Shcherbinina 2 and Alexander Shneider 2 1. Laboratory of Translational Biology, Department of Biology MCA, University of Camerino, 62032, Italy 2. CureLab Oncology, Inc, Needham, MA, 02492, USA 3. Dept Biochem, Boston University School of Medicine, Boston MA, 02118, USA 4. Research Institute of Influenza, St-Petersburg, 197376, Russia 5. R.E.Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine, Kiev, 03022, Ukraine 6. Medical Radiology Reseach Center, Obninsk, 249036, Russia 7. Gertzen Research Oncology Institute, Moscow, 125284, Russia * These authors contributed equally Correspondence: Alexander Shneider, email: // Keywords : cancer immunotherapy, DNA vaccine, autophagy, metastases Received : September 10, 2013 Accepted : September 30, 2013 Published : October 2, 2013 Abstract Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors – B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.