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

Abstract

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.

Advances in nanoparticle-based mRNA delivery for liver cancer and liver-associated infectious diseases

“…150 Efforts are being made to further elucidate the mechanism of mRNA vaccines against influenza, such as modification of nucleosides in the mRNA and the modification of the antigen structure and function. 151,152…”

Message in a bottle: mRNA vaccination for influenza

“…Combining conserved antigens with polyvalency, possibly including internal viral protein antigens, is an avenue to improved protection via both humoral and cellular immunity [61][62][63]. Multivalent immunization using the mRNA format has been shown to confer broad cross-protection in mice even at low doses [64].…”

The infinite possibilities of RNA therapeutics

“…In the case of IVT mRNAs, the cap can be added with two different approaches: the first one is the incorporation of the cap by a recombinant viral capping enzyme after the initial synthesis (Martin et al., 1975 ); the second one is adding a synthetic cap analog during the in vitro transcription reaction (Malone et al., 1989 ). Most of the ongoing clinical trials still use the latter or recently a variation of this approach: a cap incorporated in reverse orientation (antireverse cap analog; ARCA) with a more efficient translation (Jemielity et al., 2003 ; Montanaro et al., 2002 ; Sahin et al., 2014 ; Starostina et al., 2021 ; Stepinski et al., 2001 ; Ziemniak et al., 2013 ).…”