Development And Protection Efficacy Of An MRNA Vaccine Against SARS-CoV-2 Beta Variant

The COVID-19 outbreak caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has rapidly affected more than 200 countries and regions worldwide since its outbreak in late 2019,and causing a global pandemic.In January2020,the World Health Organization declared the COVID-19 outbreak a “Public Health Emergency of International concern”.Therefore,the development of safe and effective COVID-19 vaccine and the realization of herd immunity through large-scale vaccination is the hope of effective prevention and control of the disease.At present,inactivated vaccines,protein subunit vaccines,adenovirus vector vaccines and m RNA vaccines have been approved for marketing,making a great contribution to epidemic prevention and control.However,with the continuous evolution and mutation of SARSCoV-2,a variety of mutant strains appeared successively.Among them,The SARSCoV-2 Beta variant has attracted close attention because of its increased transmission ability and multiple mutation sites of spike protein,which has a great impact on the current antibody therapy and the protective efficacy of existing vaccines.Therefore,to respond effectively to the rapid mutation of the SARS-CoV-2,we need rapid development and large-scale deployment of new vaccines against mutant strains.And m RNA vaccines have many advantages over traditional vaccines,firstly,m RNA vaccine will not be integrated with human DNA,no risk of exogenous virus infection,and high safety;Secondly,it can present a variety of antigens and has the potential of developing multi-conjugate and multi-valent vaccines,which can cause strong humoral and cellular immunity.In addition,the m RNA vaccine has a short development cycle,which enables the rapid development of new candidate vaccines,and the manufacturing process is simple and easy to mass production.Therefore,in this study,we used mature m RNA vaccine technology to rapidly develop the SARS-CoV-2Beta variants m RNA vaccine(ARCoV-Beta),then in vitro antigenicity,and in mice immunogenicity and immune protection effect were systematically evaluated,providing technical reserve and material guarantee for the rapid prevention and control of SARS-CoV-2 Beta variants.This study is divided into four parts:In the first part of the study,the main work is the construction and in vitro expression identification of ARCoV-Beta.Based on the early research and development of SARS-CoV-2 m RNA vaccine(ARCoV),the RBD nucleotide sequence of SARSCoV-2 Beta variants was selected to replace the RBD sequence of its wild type strain,and codon optimization was conducted to obtain transcriptional RNA through in vitro transcription.After transfection,western blotting and immunofluorescence were used to identify the expression of RBD protein in Beta variants.The results showed that transcriptional RNA can highly express RBD protein specific to Beta variants in cells.On the basis of the successful preparation of SARS-CoV-2 Beta variants m RNA stock solution,used the m RNA-LNP platform to prepare ARCoV-Beta,and its particle size,PDI,encapsulation rate and concentration were determined.The results showed that the average particle size of the vaccine preparation was 73.08 nm,the PDI was 0.041,the purity was 84.3%,and the concentration was 240.3 ug/m L,indicating that the prepared ARCoV-Beta nanoparticles had uniform particle size,good dispersion and high purity.In the second part of the study,we evaluated the immunogenicity of ARCoV-Beta in mice.First,BALB/c mice were immunized with two doses of the prepared ARCoVBeta at 10 μg dose.Ig G antibody titers were measured by ELISA,neutralizing antibody titers were measured by pseudovirus neutralization and Live virus neutralization.IFN-γ,TNF-α and IL-2 cytokines by T cell immunity were detected by ELISpot and flow cytometry.The results showed that ARCoV-Beta successfully induced high levels of Ig G and neutralizing antibodies,as well as Th1-biased specific cellular immune responses in mice.In the third part of the study,we evaluated the immune protection of ARCoV-Beta against SARS-CoV-2 Beta variants infection in mice.Mice immunized with ARCoVBeta were intranasal(i.n.)challenged with 12000 PFU SARS-CoV-2 Beta via,and viral RNA,lung lesions and viral protein were detected by q RT-PCR,histopathology,RNA in situ hybridization and tissue immunofluorescence,respectively.Results Showed that high levels of viral RNA and viral protein were detected in lung tissue and lung lesions were observed in mice inoculated with LNP placebo,while immunization with two doses of ARCoV-Beta completely blocked replication of the SARS-CoV-2 Beta variants in the lower respiratory tract and protected mice from lung lesions.In the fourth part of the study,we evaluated the antibody titer and immune protection effect of ARCoV-Beta heterologous enhanced immunization against SARSCoV-2 Beta variants in mice.BALB/c mice were immunized with 2 doses of 10 μg ARCoV and then booster with 10 μg ARCoV-Beta 300 days after immunization.Neutralizing antibody titers were detected before and after booster immunization by pseudovirus neutralization and live virus neutralization.Then,the booster immunized mice were attacked with SARS-CoV-2 Beta variants,and the viral load and pathological damage of lung tissues were detected by q RT-PCR,H&E staining,RNA scope and immunofluorescence.The results showed that after booster immunization with ARCoV-Beta,mice could not only produce a higher level of neutralizing antibodies against wild-type and Beta variants,but also completely prevent the Beta variants from replicating in lung tissue,thus avoiding lung lesions in mice.In conclusion,in this study,the RBD nucleotide sequence of the SARS-CoV-2Beta variants was selected to optimize the codon,and the SARS-CoV-2 Beta variants m RNA vaccine(ARCoV-Beta)was constructed by using the successfully established m RNA-LNP preparation platform,and completed the preparation of the m RNA stock solution,encapsulation and quality test.On this basis,immunogenicity and immunoprotection of ARCoV-Beta in mice was further evaluated.Finally,the immunoprotection effect of booster ARCoV-Beta vaccine on Beta variants was confirmed.This study provides an important experimental basis and technical support for the effective prevention and control of SARS-CoV-2 Beta variants.