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.

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.