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

Abstract

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.

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

“…To synthesize mRNA vaccine constructs, we used a template from DNA plasmids encoding the previously designed antigens AgH1, AgH3, and AgM2, which were constructed based on conserved fragments of the hemagglutinin stem of two influenza virus subtypes: H1N1 and H3N2, as well as conservative virus protein M2 [ 13 ]. Briefly, the design of the AgH1 (H1N1) antigen structure was carried out based on the hemagglutinin of influenza A virus A/Puerto Rico/8/1934(H1N1) [ 24 ], according to the algorithm described by Squires et al [ 25 ] with modifications described by Bazhan et al [ 13 ].…”

“…To synthesize mRNA vaccine constructs, we used a template from DNA plasmids encoding the previously designed antigens AgH1, AgH3, and AgM2, which were constructed based on conserved fragments of the hemagglutinin stem of two influenza virus subtypes: H1N1 and H3N2, as well as conservative virus protein M2 [ 13 ]. Briefly, the design of the AgH1 (H1N1) antigen structure was carried out based on the hemagglutinin of influenza A virus A/Puerto Rico/8/1934(H1N1) [ 24 ], according to the algorithm described by Squires et al [ 25 ] with modifications described by Bazhan et al [ 13 ]. A common structure of the constructed immunogen (a) and relevant amino acid sequence (b) appears as follows: leader peptide–HA1 18-41 –gsa–HA1 290-323 –gsagsa–HA2 541-613 –transmembrane and cytosolic fragments MKANLLVLLCALAAADA —DTVDTVLEKNVTVTHSVNLLEDSHgsaNSSLPYQNTHPTTNGESPKYVRSAKLRMVTGLRNgsagsaTQNAINGITNKVNTVIEKMNIQDTATGKEFNKDEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDAHDS—NVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI …”

“…Previously, we demonstrated one possible approach for developing a universal influenza vaccine based on constructing artificial antigens containing conserved fragments of hemagglutinin and conserved protein M2 [ 13 ]. Eukaryotic plasmid vectors were used to obtain DNA vaccines encoding genes of relevant immunogens.…”

“…Eukaryotic plasmid vectors were used to obtain DNA vaccines encoding genes of relevant immunogens. We showed that these DNA vaccine constructs induced responses for specific antibodies and cytotoxic T-lymphocytes, providing cross-protection of mice against fatal infection with two influenza virus strains: A/California 4/09 (H1N1pdm09) and A/Aichi/2/68 (H3N2) [ 13 ].…”

Research Updates for Influenza Virus and Vaccine Development

“…Four other manuscripts in this Special Issue report how novel vaccines can increase the strength and breadth of immune responses beyond the traditional HA response in animal or clinical studies. Bazhan et al [ 3 ] describe a DNA vaccine that combines epitopes from the highly conserved M2 influenza protein with H1 and H3 epitopes. After intramuscular injection, immunized mice had a humoral response and better survival against lethal challenge using either H1N1 or H3N2 viruses.…”