Development Of MERS-CoV And SARS-CoV-2 Subunit And Nuclei Acid Vaccines

Coronavirus is a class of single-stranded positive RNA viruses,which are widely distributed in a variety of mammalian hosts with interspecies transmission ability.Seven coronaviruses were found in human.Severe acute respiratory syndrome coronavirus(SARS-CoV),Middle East respiratory syndrome coronavirus(MERS-CoV)and 2019-novel coronavirus,i.e.severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),are three highly pathogenic coronaviruses,which caused epidemics in different regions.MERS-CoV was first found in the Middle East in 2012,causing serious epidemic in South Korea in 2015.Some sporadic cases still reported in Middle East recently.In December 2019,the coronavirus disease 2019(COVID-19)pandemic caused by SARS-CoV-2 had rapidly spread,still without effective management.However,effective drugs for highly pathogenic coronaviruses are still under research.Therefore,safety and effective vaccines to control the coronavirus diseases are crucial need.The subunit vaccines and nucleic acid vaccines are showing high safety profile and are easy to develop.A MERS-CoV receptor-binding domain(RBD)recombinant protein with C-terminal trimeric modification was design based on previous research.The immunogenicity of recombinant RBD was evaluated in mouse model.Based on recombinant RBD,the messenger RNA(mRNA)vaccine and self-amplification mRNA(saRNA)vaccines were designed and the immunogenicity of MERS-CoV mRNA vaccines was evaluated in mice.In the early state of the COVID-19 pandemic,the SARS-COV-2 S protein sequence was optimized and the SARS-CoV-2 DNA and mRNA vaccines were designed.Immunogenicity and protection efficacy were evaluated in two mice strains.Our main findings are showed as follows:1.Preparation and immunogenicity research of subunit and mRNA vaccines based on recombinant MERS-CoV RBD.MERS-CoV RBD were modified with different C-terminal trimeric motifs and produced in CHO expression system.RBD with T4f modification shows an ideal multimeric structure.The immunogenicity of recombinant RBD-T4f by intramuscular vaccination with aluminum hydroxide alone or aluminum CpG ODN complex adjuvant and intranasal vaccination with CTA1-DD or CpG ODN adjuvant in BALB/c mice were next evaluated.RBD-T4f has excellent immunogenicity.Vaccination with ether adjuvant can effectively induce high level of neutralizing antibodies and certain level of antigen specific cellar immune response.High neutralizing activity systematic IgG and respiratory tract mucosal IgA response were found in intranasal vaccination groups.In addition,a strong antigen specific IFN-y secreted cellular immune response can be established in lung tissue.Based on recombinant RBD-T4f protein,the MERS-CoV mRNA and saRNA vaccines were designed.The in vitro transcription system of SFV4 backbone saRNA was optimized to prepare complete long-chain saRNA molecules.Report gene expression of optimized saRNA can sustain at least 11 days in mice.RBD-T4f mRNA or saRNA vaccines were packaged by lipopolyplex nanoparticles(LPP)or in vivo-jetPEI separately.Immunogenicity of two mRNA vaccines was determined in BALB/c mice.The mRNA-LPP vaccine can induce antigen-specific IgM response after prime and high level of specific IFN-y secreted cellular response can be detected after first boost.After third dose,a certain level of neutralizing antibody can be induced.RBD-T4f saRNA vaccine can effectively induced high level cellular immune response but unable to induce antigen specific IgG even after three doses.2.Immunogenicity and protection efficacy of SARS-CoV-2 nucleic acid vaccines.The SARS-CoV-2 DNA and mRNA vaccines were designed based on the codon-optimized S protein sequence.In vitro transcription molecular modified mRNA prepared using LPP techniques.DNA and mRNA vaccines of 3 ?g or 30 ?g dosage were used to immunize C57BL/6 and BALB/c mice.Both nucleic acid vaccines can effectively induce S protein-specific antibodies and Th1 polarized cellular immune response.In mRNA vaccinate group,a strong humoral response and cellular immune memories can be maintained in high level for at least 13 weeks.mRNA-LPP vaccine induced antibodies also shows a broad neutralizing activity.Mice serum sample can neutralize a panel of SARS-CoV-2 variants.Both DNA and mRNA-LPP vaccines with suitable dosage and times can bring good immune protective effect.Lung tissue virus titre and inflammation lesion were effectively reduced and alleviated in vaccinate mice,after SARS-CoV-2 challenge.In addition,mRNA-LPP vaccine also induces high-titre,broad-spectrum neutralizing antibodies and produce protection efficacy in rhesus macaque.In summary,recombinant MERS-CoV RBD-T4f protein prepared by the CHO expression system have great immunogenicity.It is an effective vaccine candidate both in conventional and mucosal vaccines.The humoral response of mRNA and saRNA vaccines based on recombinant RBD-T4f is relatively limited.However,higher levels of specific IFN-y cellar responses can be induced.SARS-CoV-2 S protein DNA vaccine and molecularly modified mRNA vaccine have excellent immunogenic,induces high titres,broad-spectrum neutralizing antibodies and high levels of Th1 basis cellular immune response.mRNA-LPP vaccine with suitable vaccination strategy can induce long-term protection efficacy in mice and rhesus macaque.Our research paves the way for effective coronavirus vaccine development and applications.