Development And Immunological Evaluation Of DNA-Based COVID-19 Vaccines Against SARS-CoV-2

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2（SARS-CoV-2）,the cause of coronavirus disease 2019（COVID-19）had infected more than 613 million people,resulting in more than 4.8 million deaths worldwide as of March 31^th 2022.Thus,combatting the pandemic requires effective vaccines.Recombinant DNA vaccine induces immune response similar to natural infection,which has the advantages of the quick design of multiple candidates for preclinical testing and cold-chain free for transportation and storage.Construction of SARS-CoV-2 DNA vaccine can further investigate the immune effect of different structural proteins as vaccine targets.ObjectiveIn this study,we constructed synthetic DNA vaccines expressing four structural proteins,and evaluated structural protein-specific humoral immunity,cellular immune responses,and protection post SARS-CoV-2 challenge in BALB/c mice to provide scientific reference for the optimization of target antigen in COVID-19 vaccine design.MethodThe SARS-CoV-2 structure protein gene（derived from strain WH01）was codon-optimized for expression in human cells,and cloned in the pc DNA3.1（+）,via Hind III and Xba I digestion and named as p-SARS-CoV-2-S/N/E/M.All DNA vaccine sequences were confirmed by Sanger DNA sequencing,and plamids were extracted using endotoxin-free Maxiprep kits（Qiagen,Beijing,China）.After the plasmid DNA was obtained,293T cells were transiently transfected in vitro,and the anti-6?His labeled antibody/S protein specific antibody/N protein specific antibody was used for indirect immunofluorescence assay（IFA）and western blotting（WB）.The level of specific humoral immunity and specific cellular immunity induced by vaccines were evaluated in 6-week-old female BALB/c mice:the titer of antigen-specific Ig G antibody was detected by ELISA,and the titer of antigen-specific neutralizing antibody was detected by pseudovirus neutralization assay and live virus microneutralization assay.The level of cellular immune response were detected by Interferon gamma（IFN-γ）enzyme-linked immune absorbent spot（ELISpot）assay.Four mice from each group were randomly selected for the challenge experiment on the 125th day after the first immunization.Three days of post-challenge,mice were euthanized,and necropsy was performed.The lungs of mice were harvested after sacrifice.Half of the tissues were used for nucleic acid extraction and real-time fluorescence RT-PCR reaction.Median tissue culture infective dose（TCID50）of the virus in the sample was calculated according to the CT value of the sample and standard curve.And the other half were fixed in a 4%formalin solution,for the preparation of haematoxylin and eosin-stained sections（four mice per group）for pathological evaluation indicated by the International Harmonisation of Nomenclature and Diagnostic Criteria（INHAND）scores.Result1.The expression of the SARS-CoV-2-S/N/E/M protein in vitro was verified by IFA and WB;2.Six novel H-2d-restricted T-cell epitopes specific to E/M protein were identified;After the immunization of p-SARS-CoV-2-E/M,significant cellular immune responses were elicited,whereas no robust humoral immunity was detected.The results of SARS-CoV-2challenge show that no drop in lung tissue virus titer was detected in DNA vaccinated mice,immunization with either p-SARSCoV-2-E or p-SARS-CoV-2-M provided minor protection and co-immunization with p SARS-CoV-2-E+M increased protection,demonstrating as milder histopathological changes,lower INHAND scores;3.p-SARS-CoV-2-S/S+N/S+E+M vaccine immunization induced humoral immune response of specific S protein.Compared with other groups,S+N group induced higher titer of antibody specific to S protein.Pseudovirus neutralization assay demonstrated that the immune response trend of neutralizing antibody in S+N group was better than that in S group.Live virus microneutralization assay showed that median effective concentration(EC₅₀)of S+N group was higher than that of S group after three immunizations,and the difference was statistically significant（P=0.043）.S/S+N/S+E+M vaccine immunization induced S protein specific cellular immune response,and there was no significant difference between groups.The multiple comparison results showed that compared with the second immunization,after the third immunization,there was no significant difference in cellular immunity specific to S protein in S group,but the cellular immunity specific to S protein in S+N,S+E+M groups increased statistically（P<0.001）.The results of SARS-CoV-2challenge show that S,S+N and S+E+M vaccines had protective effects on weight loss.No live virus was detected in the lungs of all immunization groups,and the RNA copies in the lungs of S+N group decreased by nearly 10,000 times,which was statistically significant（P=0.023）.Compared with the S group,the scores of INHAND in the S+N and S+E+M groups were lower,and the scores of INHAND in the S+E+M groups were the lowest.Conclusion1.E/M protein provided partial protection after challenge without obvious humoral immunity,demonstrating milder pathological changes and lower INHAND score;2.S+N group induced higher neutralizing antibody level,and reduced the viral load in lung effectively.S+N group and S+E+M group induced higher levels of S protein-specific cellular immunity after three immunizations,and exhibited milder histopathological changes post challenge.