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

Ekaterina V Starostina,Natalia V Danilchenko,Vasiliy Yu Marchenko,Sergei I Bazhan,Mariya B Borgoyakova,Anton N Chikaev,Andrey P Rudometov,Nadezhda B Rudometova,Anna E Lemza,Georgiy Yu Shevelev,Alexander A Ilyichev,Grigory A Stepanov,Denis N Antropov,Larisa I Karpenko,Sergei V Sharabrin

doi:10.3390/vaccines9050452

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.

Highlights

It is important to construct vaccines against influenza viruses
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]
We initially carried out a comparative analysis of different variants of mRNA modification to detect a combination of modified nucleotides that provided efficient translation in human cells (Figure 1B)

Summary

Introduction

It is important to construct vaccines against influenza viruses. To protect the population against influenza, healthcare systems use attenuated (live) or inactivated vaccines (approved by the World Health Organization) after genetically analyzing circulating seasonal influenza A and B virus strains. Influenza virus composition should be changed annually, yet the process of producing an influenza vaccine is time-consuming. A number of research teams and pharma companies have carried out studies aimed at developing a universal vaccine against the influenza virus. There are different approaches to constructing a universal influenza vaccine, e.g., developing recombinant protein immunogens, using conserved influenza virus antigens (hemagglutinin stem, and proteins NP, PB1, M1, and M2), or developing new types of vaccines [1,2,3,4,5,6]

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Conclusion