DNA Vaccine Encoding a Modified Hemagglutinin Trimer of Avian Influenza A Virus H5N8 Protects Mice from Viral Challenge.

The development of a safe and effective vaccine against avian influenza A virus (AIV) H5N8 is relevant due to the widespread distribution of this virus in the bird population and the existing potential risk of human infection, which can lead to significant public health concerns. Here, we developed an experimental pVAX-H5 DNA vaccine encoding a modified trimer of AIV H5N8 hemagglutinin. Immunization of BALB/c mice with pVAX-H5 using jet injection elicited high titer antibody response (the average titer in ELISA was 1 × 105), and generated a high level of neutralizing antibodies against H5N8 and T-cell response, as determined by ELISpot analysis. Both liquid and lyophilized forms of pVAX-H5 DNA vaccine provided 100% protection of immunized mice against lethal challenge with influenza A virus A/turkey/Stavropol/320-01/2020 (H5N8). The results obtained indicate that pVAX-H5 has good opportunities as a vaccine candidate against the influenza A virus (H5N8).

Immunogenic and Protective Properties of Recombinant Hemagglutinin of Influenza A (H5N8) Virus.

In this study, we characterized recombinant hemagglutinin (HA) of influenza A (H5N8) virus produced in Chinese hamster ovary cells (CHO-K1s). Immunochemical analysis showed that the recombinant hemagglutinin was recognized by the serum of ferrets infected with influenza A (H5N8) virus, indicating that its antigenic properties were retained. Two groups of Balb/c mice were immunized with intramuscular injection of recombinant hemagglutinin or propiolactone inactivated A/Astrakhan/3212/2020 (H5N8) influenza virus. The results demonstrated that both immunogens induced a specific antibody response as determined by ELISA. Virus neutralization assay revealed that sera of immunized animals were able to neutralize A/turkey/Stavropol/320-01/2020 (H5N8) influenza virus-the average neutralizing titer was 2560. Immunization with both recombinant HA/H5 hemagglutinin and inactivated virus gave 100% protection against lethal H5N8 virus challenge. This study shows that recombinant HA (H5N8) protein may be a useful antigen candidate for developing subunit vaccines against influenza A (H5N8) virus with suitable immunogenicity and protective efficacy.

Quantitative measurement of influenza virus transmission in animal model: an overview of current state.

Influenza virus transmission is a crucial factor in understanding the spread of the virus within populations and developing effective control strategies. Studying the transmission patterns of influenza virus allows for better risk assessment and prediction of disease outbreaks. By monitoring the spread of the virus and identifying high-risk populations and geographic areas, it is possible to allocate resources more effectively, implement timely interventions, and provide targeted healthcare interventions to diminish the burden of influenza virus on vulnerable populations. Theoretical models of virus transmission are used to study and simulate of influenza virus spread within populations. These models aim to capture the complex dynamics of transmission, including factors such as population size, contact patterns, infectiousness, and susceptibility. Animal models serve as valuable tools for studying the dynamics of influenza virus transmission. This article presents a brief overview of existing research on the qualitative and quantitative study of influenza virus transmission in animal models. We discuss the methodologies employed, key insights gained from these studies, and their relevance.

Re-Emergence of Circulation of Seasonal Influenza during COVID-19 Pandemic in Russia and Receptor Specificity of New and Dominant Clade 3C.2a1b.2a.2 A(H3N2) Viruses in 2021-2022.

The circulation of seasonal influenza in 2020-2021 around the world was drastically reduced after the start of the COVID-19 pandemic and the implementation of mitigation strategies. The influenza virus circulation reemerged in 2021-2022 with the global spread of the new genetic clade 3C.2a1b.2a.2 of A(H3N2) viruses. The purpose of this study was to characterize influenza viruses in the 2021-2022 season in Russia and to analyze the receptor specificity properties of the 3C.2a1b.2a.2 A(H3N2) viruses. Clinical influenza samples were collected at the local Sanitary-and-Epidemiological Centers of Rospotrebnadzor. Whole genome sequencing was performed using NGS. The receptor specificity of hemagglutinin was evaluated using molecular modeling and bio-layer interferometry. Clinical samples from 854 cases of influenza A and B were studied; A(H3N2) viruses were in the majority of the samples. All genetically studied A(H3N2) viruses belonged to the new genetic clade 3C.2a1b.2a.2. Molecular modeling analysis suggested a higher affinity of hemagglutinin of 3C.2a1b.2a.2. A(H3N2) viruses to the α2,6 human receptor. In vitro analysis using a trisaccharide 6'-Sialyl-N-acetyllactosamine receptor analog did not resolve the differences in the receptor specificity of 3C.2a1b.2a.2 clade viruses from viruses belonging to the 3C.2a1b.2a.1 clade. Further investigation of the A(H3N2) viruses is required for the evaluation of their possible adaptive advantages. Constant monitoring and characterization of influenza are critical for epidemiological analysis.

In silico design of influenza a virus artificial epitope-based T-cell antigens and the evaluation of their immunogenicity in mice.

The polyepitope strategy is promising approach for successfully creating a broadly protective flu vaccine, which targets T-lymphocytes (both CD4+ and CD8+) to recognise the most conserved epitopes of viral proteins. In this study, we employed a computer-aided approach to develop several artificial antigens potentially capable of evoking immune responses to different virus subtypes. These antigens included conservative T-cell epitopes of different influenza A virus proteins. To design epitope-based antigens we used experimentally verified information regarding influenza virus T-cell epitopes from the Immune Epitope Database (IEDB) (http://www.iedb.org). We constructed two "human" and two "murine" variants of polyepitope antigens. Amino acid sequences of target polyepitope antigens were designed using our original TEpredict/PolyCTLDesigner software. Immunogenic and protective features of DNA constructs encoding "murine" target T-cell immunogens were studied in BALB/c mice. We showed that mice groups immunised with a combination of computer-generated "murine" DNA immunogens had a 37.5% survival rate after receiving a lethal dose of either A/California/4/2009 (H1N1) virus or A/Aichi/2/68 (H3N2) virus, while immunisation with live flu H1N1 and H3N2 vaccine strains provided protection against homologous viruses and failed to protect against heterologous viruses. These results demonstrate that mechanisms of cross-protective immunity may be associated with the stimulation of specific T-cell responses. This study demonstrates that our computer-aided approach may be successfully used for rational designing artificial polyepitope antigens capable of inducing virus-specific T-lymphocyte responses and providing partial protection against two different influenza virus subtypes.Communicated by Ramaswamy H. Sarma.

Genome Features and In Vitro Activity against Influenza A and SARS-CoV-2 Viruses of Six Probiotic Strains.

PURPOSE: The aim of this work was to analyze the complete genome of probiotic bacteria Lactobacillus plantarum 8 RA 3, Lactobacillus fermentum 90 TC-4, Lactobacillus fermentum 39, Bifidobacterium bifidum 791, Bifidobacterium bifidum 1, and Bifidobacterium longum 379 and to test their activity against influenza A and SARS-CoV-2 viruses. METHODS: To confirm the taxonomic affiliation of the bacterial strains, MALDI TOF mass spectrometry and biochemical test systems were used. Whole genome sequencing was performed on the Illumina Inc. MiSeq platform. To determine the antiviral activity, A/Lipetsk/1V/2018 (H1N1 pdm09) (EPI_ISL_332798) and A/common gull/Saratov/1676/2018 (H5N6) (EPI_ISL_336925) influenza viruses and SARS-CoV-2 virus strain Australia/VIC01/2020 (GenBank: MT007544.1) were used. RESULTS: All studied probiotic bacteria are nonpathogenic for humans and do not contain the determinants of transmission-type antibiotic resistance and integrated plasmids. Resistance to antibiotics of different classes is explained by the presence of molecular efflux pumps of the MatE and MFS families. Cultures of L. fermentum 90 TC 4, L. plantarum 8 RA 3, and B. bifidum 791 showed a pronounced activity against influenza A viruses in MDCK cells. Activity against the SARS-CoV-2 virus was demonstrated only by the L. fermentum 90 TC 4 strain in VERO cells. CONCLUSIONS: The studied probiotic bacteria are safe, have antiviral activity, and are of great importance for the prevention of diseases caused by respiratory viruses that can also infect the human intestine.

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens.

Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A-H1N1 and H3N2-and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0-M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.

An influenza A(H5N8) virus isolated during an outbreak at a poultry farm in Russia in 2017 has an N294S substitution in the neuraminidase and shows reduced susceptibility to oseltamivir.

This study aimed to assess the antiviral susceptibility of influenza A(H5N8) viruses isolated in Russia in 2014-2018. Genetic analysis of 57 Russian isolates with full genome sequences did not find any markers of reduced susceptibility to baloxavir. Only one strain bore an amino acid substitution associated with adamantane resistance (M2-S31N). The neuraminidase of 1 strain had an NA-N293/294S (N8/N2 numbering) substitution associated with reduced inhibition by oseltamivir and normal inhibition by zanamivir, which was confirmed phenotypically. There were no other strains with reduced inhibition by oseltamivir and zanamivir in the phenotypic analysis. In order to estimate the worldwide prevalence of influenza A(H5N8) viruses bearing genetic markers of antiviral resistance, genome sequences deposited in the GISAID database were analyzed (database access: October 2020). The M2 protein of A(H5N8) viruses from the 2.3.4.4c clade had an M2-S31N substitution associated with reduced susceptibility to adamantanes. On the contrary, the majority (94%) of viruses from the 2.3.4.4b clade had the M2-S31 genotype. Fewer than 1% of analyzed viruses had amino acid substitutions associated with reduced susceptibility to baloxavir (PA-E199G, PA-E199E/G) or reduced or highly reduced inhibition by neuraminidase inhibitors (NA-R150/152K, NA-I221/222M, NA-I221/222I/M, NA-I221/222V, NA-I115/117V, NA-G145/147R, NA-R291/292R/K). An NA-N293/294S substitution was not present in sequences from the GISAID database. To the best of our knowledge, influenza A(H5N8) viruses with reduced inhibition by oseltamivir bearing an NA-N293/294S substitution have not been previously reported in epidemiological surveillance studies.

Evaluation of HA-D222G/N polymorphism using targeted NGS analysis in A(H1N1)pdm09 influenza virus in Russia in 2018-2019.

Outbreaks of influenza, which is a contagious respiratory disease, occur throughout the world annually, affecting millions of people with many fatal cases. The D222G/N mutations in the hemagglutinin (HA) gene of A(H1N1)pdm09 are associated with severe and fatal human influenza cases. These mutations lead to increased virus replication in the lower respiratory tract (LRT) and may result in life-threatening pneumonia. Targeted NGS analysis revealed the presence of mutations in major and minor variants in 57% of fatal cases, with the proportion of viral variants with mutations varying from 1% to 98% in each individual sample in the epidemic season 2018-2019 in Russia. Co-occurrence of the mutations D222G and D222N was detected in a substantial number of the studied fatal cases (41%). The D222G/N mutations were detected at a low frequency (less than 1%) in the rest of the studied samples from fatal and nonfatal cases of influenza. The presence of HA D222Y/V/A mutations was detected in a few fatal cases. The high rate of occurrence of HA D222G/N mutations in A(H1N1)pdm09 viruses, their increased ability to replicate in the LRT and their association with fatal outcomes points to the importance of monitoring the mutations in circulating A(H1N1)pdm09 viruses for the evaluation of their epidemiological significance and for the consideration of disease prevention and treatment options.

Immunogenicity and Protective Efficacy of Influenza A DNA Vaccines Encoding Artificial Antigens Based on Conservative Hemagglutinin Stem Region and M2 Protein in Mice.

BACKGROUND: Development of a universal vaccine capable to induce antibody responses against a broad range of influenza virus strains attracts growing attention. Hemagglutinin stem and the exposed fragment of influenza virus M2 protein are promising targets for induction of cross-protective humoral and cell-mediated response, since they contain conservative epitopes capable to induce antibodies and cytotoxic T lymphocytes (CTLs) to a wide range of influenza virus subtypes. METHODS: In this study, we generated DNA vaccine constructs encoding artificial antigens AgH1, AgH3, and AgM2 designed on the basis of conservative hemagglutinin stem fragments of two influenza A virus subtypes, H1N1 and H3N2, and conservative M2 protein, and evaluate their immunogenicity and protective efficacy. To obtain DNA vaccine constructs, genes encoding the designed antigens were cloned into a pcDNA3.1 vector. Expression of the target genes in 293T cells transfected with DNA vaccine constructs has been confirmed by synthesis of specific mRNA. RESULTS: Immunization of BALB/c mice with DNA vaccines encoding these antigens was shown to evoke humoral and T-cell immune responses as well as a moderated statistically significant cross-protective effect against two heterologous viruses A/California/4/2009 (H1N1pdm09) and A/Aichi/2/68 (H3N2). CONCLUSIONS: The results demonstrate a potential approach to creating a universal influenza vaccine based on artificial antigens.

Comparative thermostability analysis of zoonotic and human influenza virus A and B neuraminidase.

Neuraminidase (NA) thermostability of influenza A and B viruses isolated from birds, swine and humans was measured to evaluate its variability associated with host body temperature. The highest 50% inactivation temperature (IT50) was observed with H3N8 avian influenza virus (74 °C), and the lowest IT50 was observed with the seasonal human H3N2 virus (45.5 °C). The IT50 values of A(H1N1)pdm09 viruses 56.4-58.5 °C were statistically higher than that of the prepandemic strain A/Solomon Islands/03/06 (52.5 °C). An analysis of Ca2+ binding sites revealed the correspondence of amino acid changes to NA thermostability. This study demonstrates that changes in NA thermostability correspond to differences in host body temperature.

Correction: Severe cases of seasonal influenza in Russia in 2017-2018.

[This corrects the article DOI: 10.1371/journal.pone.0220401.].

Genetic Characterization of Avian Influenza A(H5N6) Virus Clade 2.3.4.4, Russia, 2018.

Timely identification of pandemic influenza threats depends on monitoring for highly pathogenic avian influenza viruses. We isolated highly pathogenic avian influenza A(H5N6) virus clade 2.3.4.4, genotype G1.1, in samples from a bird in southwest Russia. The virus has high homology to human H5N6 influenza strains isolated from southeast China.

Severe cases of seasonal influenza in Russia in 2017-2018.

The 2017-2018 influenza epidemic season in Russia was characterized by a relatively low morbidity and mortality. We evaluated herd immunity prior to the 2017-2018 influenza season in hemagglutination inhibition assay, and performed characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. During the 2017-2018 epidemic season, 87 influenza A and B viruses were isolated and viruses of the 75 influenza cases, including selected viral isolates and viruses analyzed directly from the original clinical material, were genetically characterized. The analyzed A(H1N1)pdm09 viruses belonged to clade 6B.1, B/Yamagata-like viruses belonged to clade 3, and B/Victoria-like viruses belonged to clade 1A and they were antigenically similar to the corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically and the analysis indicated antigenic differences from the corresponding egg-grown vaccine strain. The next generation sequencing revealed the presence of D222/G/N polymorphism in the hemagglutinin gene in 32% of the analyzed A(H1N1)pdm09 lethal cases. This study demonstrated the importance of monitoring D222G/N polymorphism, including detection of minor viral variants with the mutations, in the hemagglutinin gene of A(H1N1)pdm09 for epidemiological surveillance. One strain of influenza virus A(H1N1)pdm09 was resistant to oseltamivir and had the H275Y amino acid substitution in the NA protein. All other isolates were susceptible to NA inhibitors. Prior to the 2017-2018 epidemic season, 67.4 million people were vaccinated, which accounted for 46.6% of the country's population. Just before the epidemic season 33-47% and 24-30% of blood sera samples collected within the territory of Russia showed the presence of protective antibody titers against vaccine strains of influenza A and influenza B/Victoria-like, respectively. Mass vaccination of the population had evidently reduced the severity of the flu epidemic during the 2017-2018 influenza epidemic season in Russia.

Intranasal immunization of guinea pig with trivalent influenza antigen adjuvanted by Cyclamen europaeum tubers extract.

Cyclamen europaeum tubers extract (CTE) with concentration commonly used for human rhinosinusitis treatment was tested as mucosal adjuvant in experimental intranasal immunization of guinea pigs with concentrated commercially available influenza trivalent vaccine and subsequent infection with influenza strain A/California/04/2009 H1N1pdm. Dual intranasal immunization with vaccine compound consisting of 7.5 µg of each hemagglutinin and 500 µg of CTE in 50 µl induced reciprocal GMT on day 21 after immunization 40 (5-640) against H1N1pdm; 43.20 (5-1280) against H3N2; 10.80 (5-80) against influenza B. Animals with HI titers 1/80 against cell-derived antigen were completely protected against challenge with A/California/04/2009 H1N1pdm09.

Reintroduction of highly pathogenic avian influenza A/H5N8 virus of clade 2.3.4.4. in Russia.

In the spring of 2016, a loss of wild birds was observed during the monitoring of avian influenza virus activity in the Republic of Tyva. That outbreak was caused by influenza H5N8 virus of clade 2.3.4.4. In the fall, viruses of H5N8 clade 2.3.4.4 were propagated in European countries. This paper presents some results of analysis of the virus strains isolated during the spring and fall seasons in 2016 in the Russian Federation. The investigated strains were highly pathogenic for mice, and some of their antigenic and genetic features differed from those of an H5N8 strain that circulated in 2014 in Russia.

Influenza A(H5N8) virus isolation in Russia, 2014.

In this study, we report the isolation of influenza A(H5N8) virus from a Eurasian wigeon (Anas penelope) in Sakha Republic of the Russian Far East. The strain A/wigeon/Sakha/1/2014 (H5N8) has been shown to be pathogenic for mammals. It is similar to the strains that caused outbreaks in wild birds and poultry in Southeast Asia and Europe in 2014.

Influenza A (H15N4) virus isolation in Western Siberia, Russia.

The rarely identified influenza A viruses of the H15 hemagglutinin subtype have been isolated exclusively in Australia. Here we report the isolation of an H15N4 influenza A virus (A/teal/Chany/7119/2008) in Western Siberia, Russia. Phylogenetic analysis demonstrated that the internal genes of the A/teal/Chany/7119/2008 strain belong to the Eurasian clade and that the H15 and N4 genes were introduced into the gene pool of circulating endemic avian influenza viruses through reassortment events.