Preparation And Experimental Immunization Of H1N1 Subtype Influenza Virus MRNA Candidate Vaccine

Influenza is an acute respiratory infectious disease caused by influenza virus(Ⅳ).The first reported case of H1N1 appeared in Mexico in February 2009,and the World Health Organization(WHO)declared H1N1 a public health emergency of global importance in April of that year.Influenza viruses often evade the host immune system through genetic recombination,resulting in viral transmission of pandemic outbreaks,a serious threat to human health and social and economic development.COVID-19 mRNA vaccine is effective in disease prevention and has a short preparation period.Currently,influenza vaccine is based on inactivated viruses or recombinant proteins,with a protection rate of only 40%-60%.Therefore,it is extremely important to prepare safe and effective mRNA vaccine for the prevention and control of influenza caused by H1N1.In this study,the seasonal H1N1 subtype influenza virus was used as a model to prepare mRNA vaccine.HA is the main antigen that induces protective immune response against Ⅳand is the key vaccine target.The length of 5’UTR and 3’UTR has a certain effect on the translation efficiency of mRNA.GC content and U content can affect mRNA stability and immunogenicity.In this study,mRNA candidate vaccines against H1N1 subtype influenza virus were prepared by gene sequence optimization,delivery system and adjuvant combination,and the immune effect of the vaccine was evaluated,providing new ideas and theoretical basis for the preparation of influenza virus mRNA vaccines.The main research contents and results include the following three parts:1.Construction of mRNA candidate vaccine vectors for H1N1 subtype influenza virusIn this study,the seasonal H1N1 subtype influenza virus was used as the model,and codon optimization was carried out based on the full length of HA gene to obtain two sequences,named JLH1HA and JLHA,respectively,and cloned into three vectors:pGEMT7-Hα(UTRs from human α-globulin),pGEM-T7-Mα(UTRs from mouse α-globulin),pGEM-n3(UTRs from human β-globulin),and 6 recombinant plasmids were obtained.The recombinant plasmids were linearized,transcribed in vitro,purified and capped,and six kinds of Cap-mRNA were obtained.Western blot and IFA confirmed that all the six Cap-mRNAs could express the target protein,and pGEM-T7-Ha vector had the highest expression efficiency.The results provided conditions for the preparation of mRNA candidate vaccines of pGEM-T7-Hα-JLHA and pGEM-T7-Hα-JLH1HA.2.Preparation of LNP/mRNA nanoparticles and selection of adjuvantsSM-102(1-octylnonyl8-[(2-hydroxyethyl)[6-oxy-6-(undecanoxy)hexyl]amino]octanoate)was used as an ionizing lipid,and the optimal N/P molar ratio between LNP and mRNA was determined to be 5.8:1 by reference to literature and previous research results.LNP/mRNA complex nanoparticles were prepared by microflow control.TEM showed that the nanoparticles were homogeneous spherules about 220 nm,and the zeta potential value was 1.47 mV.In vitro transfection test and Western blot confirmed that different forms of antigen genes can be expressed.Adjuvant R848(toll-like receptor agonist TLR8)and MK-571(a leukotriene receptor antagonist)were immunized with LNP-coated mLuc(luciferin)mice,and the results showed that the addition of adjuvant could stimulate the expression of antigen genes.The fluorescence value of MK-571 adjuvant group was higher than that of R848 group and that of no adjuvant group.It was proved that MK-571 was a better adjuvant and could improve the immune effect of the vaccine.3.Preparation of mRNA candidate vaccine of H1N1 subtype influenza virus and evaluation of immune effectpGEM-T7-Hα-JLHA and pGEM-T7-Hα-JLH1HA were selected to prepare mRNA candidate vaccines.In order to evaluate the effect of adjuvants on immune effect,MK-571 and R848 adjuvants were added,and 6 vaccine groups were set up:LNP/mJLHA,LNP/mJLHA+R848,LNP/mJLHA+MK-571,LNP/mJLH1HA,LNP/mJLH1HA+R848,LNP/mJLH1HA+MK-571,and PBS negative control group was also established.Hemagglutination inhibition test(HI)and flow cytometry were used to detect antibody production in each group.The results showed that the antibody level of mJLHlHA vaccine group was higher,and MK-571 adjuvant group>R848 adjuvant group>no adjuvant group.During the challenge,the level of inflammatory factors in lung tissue were detected by RTPCR and double antibody sandwich ELISA,and pathological sections of lung were prepared.The results showed that the inflammatory response of vaccine groups was significantly lower than that of PBS group.The weight and survival rate of mice during the challenge were recorded.The survival rate of mice in the adjuvant vaccine group was 100%.Therefore,the optimization of mJLH1HA gene improved the vaccine effect,and proved that MK-571 adjuvant enhanced the immune protective effect of mRNA vaccine.