Construction And Immune Research Of SARS-Cov-2 DNA Vaccine

Severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）belongs to theβ-type coronavirus in the genus coronavirus of coronaviridae.It is the seventh coronavirus found to infect humans.In late December 2019,the COVID-19 pandemic caused by SARS-CoV-2 had rapidly spread,still without effective management.As of March 31,2022,there are 224 countries and regions in the world,only North Korea and Turkmenistan have not reported cases of new coronary pneumonia,with a total of 485,893,787 confirmed cases and 6,149,828 deaths,with a mortality rate of 1.3%.Effective methods for prevention and control of epidemic include public health measures,clinical drug treatment and large-Scale vaccination.In the early stages of the outbreak,timely public health measures can be effective control,but only a few countries can effectively control.Therefore,the priority at this stage is the development of vaccines against SARS-CoV-2.The vaccine is an effective global measure to control the spread of an outbreak.In this study,three DNA candidate vaccines were prepared based on SARS-CoV-2 S protein,and the DNA vaccine and its optimal adjuvant were screened.The immunogenicity of the prepared DNA vaccine was studied.Finally,the changes of immune related proteins of DNA vaccine were explored by proteomics.The main findings are showed as follows:Spike protein as the main immunogen of SARS-CoV-2 has become the main target of SARS-CoV-2 vaccine.In the early stage of COVID-19 epidemic,we optimized SARS-CoV-2 S protein in two ways.One is to optimize the source code separately to obtain S-OP;the other is to obtain S-MUT by mutating the Furin cleavage site on the basis of human codon optimization.We constructed three DNA candidate vaccines for two optimized S genes:p VAX-S-OP、p VAX-SΔCD-MUT、p VAX-S1.Three recombinant plasmids and p VAX1 empty vector plasmids were transfected into HEK-293 cells.The transient expression of the target protein in mammalian cells was verified by Western blot assay.The three recombinant plasmids could be successfully expressed in mammalian cells was evaluated Western Blot assay,and the expression level of SΔCD protein was the highest.Based on the previous data support of our laboratory,p VAX-SΔCD-MUT was selected in this study DNA candidate vaccine to screen the optimal adjuvant.Three different types of adjuvants（MF-59,Liposome and Poly IC）were used to compare with p VAX-SΔCD-MUT combined immunization,and a separate immunization group and PBS group were established.The results of specific Ig G antibody showed that p VAX-SΔCD-MUT+poly IC group>p VAX-SΔCD-MUT+MF59 group>p VAX-SΔCD-MUT alone group>p VAX-SΔCD-MUT+liposome group showed that poly IC adjuvant had the best immune enhancement effect on recombinant DNA candidate vaccine.We also screened three DNA candidate vaccines,p VAX-S-OP and p VAX-SΔCD-MUT,p VAX-S1 and p VAX1 empty vector were immunized with poly IC adjuvant,and PBS group was established.According to the results of specific Ig G antibody,p VAX-SΔCD-MUT and p VAX-S-OP produced specific Ig G antibodies with the highest titer of 1:12 800,but p VAX-S1 did not produce high specific Ig G antibodies;And the Ig G subtypes of p VAX-SΔCD-MUT and p VAX-S-OP groups were analyzed.The results showed that two DNA candidate vaccines,p VAX-SΔCD-MUT and p VAX-S-OP,induce Th1 type immune response（cellular immunity）.On the basis of preliminary experiments,the immune dose was optimized,and Balb/c and h ACE2 mice were used to evaluate the immunogenicity of p VAX-SΔCD-MUT and p VAX-S-OP DNA candidate vaccines;protective experiment with h ACE2 mice.The results of protective experiment with h ACE2 mice showed that in Balb/c mice,the specific Ig G antibody titer induced by p VAX-SΔCD-MUT+Poly-IC was up to 1:52 100;p VAX-S-OP induces immune responses mediated by CD4⁺and CD8⁺T cells to produce IL-4 and IFN-γ.In h ACE2 mice,the specific Ig G antibody titers induced by p VAX-S-OP+Poly-IC group and p VAX-SΔCD-MUT+Poly-IC group were the highest（1:102 400）;Two DNA candidate vaccines induce Th1 type immune response（cellular immunity）;Both DNA vaccines could induce neutralizing antibodies,and p VAX-S-OP+Poly-IC group>p VAX-SΔCD-MUT+Poly-IC group.In the challenge and protection experiment of h ACE2 mice,the viral load in the lung of mice in the p VAX-S-OP group was reduced by 10⁴ times,and that in the p VAX-SΔCD-MUT group was reduced by 10^4.4 times.There was no obvious inflammatory lesion in the immunized lung tissue of the two groups,indicating that both p VAX-S-OP and p VAX-SΔCD-MUT had a good protective effect on the lung under the attack of SARS-CoV-2,effectively reducing the viral load of lung tissue and reducing tissue inflammatory damage.The immune related proteins of DNA vaccine were studied by proteomics technology.the spleen of immunized mice was taken,splenic lymphocytes were isolated and sent to the company for sequencing.The analysis of the results shows that:A total of 3377 proteins were identified that were significantly altered,and we used GO protein analysis,of which 83 differential proteins were classified as involved in immune system processes,of which 58 proteins were up-regulated and25 proteins were down-regulated.In the comparison group,MAVS and My D88 proteins were found in the enriched Toll-like receptor signal transduction pathway,nod-like receptor signal transduction pathway and COVID-19,and MAVS and My D88 proteins were up-regulated.These results may indicate that p VAX-SΔCD-MUT,a candidate vaccine for DNA,acts on the Toll-like receptor signal transduction pathway and NOD-like receptor signal transduction pathway,stimulating the expression of MAVS and My D88 proteins,thus exerting the antiviral immune mechanism.In conclusion,this study provides an experimental and theoretical basis for the development of SARS-CoV-2 DNA vaccine and a reference for the study of the immune mechanism of DNA vaccine.