Abstract
Among the vaccines have been developed thus far against SARS-CoV-2, the mRNA-based ones have demonstrated more promising results regarding both safety and efficacy. Two remarkable features of the mRNA vaccines introduced by the Pfizer/BioNTech and Moderna companies are the use of (N1-methyl-pseudouridine-) modified mRNA and the microfluidics-based production of lipid nanoparticles (LNPs) as the carrier. In the present study, except Anti-Reverse Cap Analog (ARCA), no other nucleoside analogs were employed to synthesize Spike-encoding mRNA using the in vitro transcription (IVT) method. Furthermore, LNPs were prepared via the ethanol injection method commonly used for liposome formation as an alternative for microfluidics-based approaches. The produced mRNA-LNP vaccine was evaluated for nanoparticles characteristics, encapsulation and transfection efficiencies, in vitro cytotoxicity as well as stability and storability. The safety of vaccine was assessed in Balb/c mice injected with mRNA-LNPs containing 10 µg of spike-encoding mRNA. Eventually, the vaccine efficacy in inducing an immune response against SARS-CoV-2 was studied in Balb/c and C57BL/6 mice (received either 1 or 10 µg of mRNA) as well as in rhesus macaque monkeys (infused with mRNA-LNPs containing 100 µg of mRNA). The ELISA and virus neutralizing test (VNT) results showed a significant augmentation in the level of neutralizing antibodies against SARS-CoV-2. Moreover, the ELISA assay showed virus-specific IFN-γ secretion in immunized mice as a marker of TH1 cell-based immune response, whereas favorably no change in the production of IL-4 was detected.
Highlights
Since the outbreak of COVID-19, various research programs have been launched to produce prophylactic vaccines, some of which have succeeded in completing clinical trials and entering the market
It is well documented that SARS-CoV2 attaches to the human target cell through the interaction between the receptor binding domain (RBD) located in the Spike protein and its principal receptor, known as human Angiotensin Converting Enzyme 2, which is overexpressed in some organs, especially the lungs [3]
Among the anti-SARS-CoV-2 vaccine platforms licensed by the regulatory organizations for public vaccination, the mRNA vaccines developed by the Pfizer-BioNTech (i.e., BNT162b) [6] and Moderna [7] companies have demonstrated interesting prosperity in both efficacy and fewer side effects concerns
Summary
Since the outbreak of COVID-19, various research programs have been launched to produce prophylactic vaccines, some of which have succeeded in completing clinical trials and entering the market. Except the inactivated/attenuated virus vaccines, in other types of introduced or developing ones (e.g., non-replicating viral vector, protein subunit, and mRNA vaccines), the SARS-CoV-2 Spike glycoprotein (abbreviated as S-protein) has been considered as the main antigenic candidate for vaccine design and production [1,2]. It is well documented that SARS-CoV2 attaches to the human target cell through the interaction between the receptor binding domain (RBD) located in the Spike protein and its principal receptor, known as human Angiotensin Converting Enzyme 2 (hACE2), which is overexpressed in some organs, especially the lungs [3]. Since the chemical structures of mRNAs encoding different antigens resemble each other, the design and development of new mRNA vaccines needs similar steps [10]. Even in mass production, manufacturing the mRNA vaccines follows an entirely virus- and cell-free procedure. T-junction (mixer) microfluidic chips can be considered as the most prevalent tools for LNP preparation
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