| Novel coronavirus Disease 2019（COVID-19）is a pandemic infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2（SARS-Co V-2）infection at the end of 2019.COVID-19 has a wide range of transmission,fast transmission speed,and strong infectivity.On January 30,2020,the World Health Organization（WHO）declared COVID-19 a public health emergency.As of March 2023,the global number of confirmed COVID-19 cases exceeded 750million and the number of deaths exceeded 6.85 million.A safe and effective vaccine is one of the important means to prevent and reduce the spread of diseases.According to the data released by WHO,183 vaccines have entered clinical research,and the exploratory research of COVID-19 vaccine is one of the current key work.This study utilized a novel antigen preparation technology,GEM-PA surface display system,to prepare SARS-Co V-2 bacterium like particles,and constructed a human subunit vaccine candidate strain expressing SARS-Co V-2 receptor binding domain（RBD）protein.Adda Vax and Al（OH）3 adjuvants were used for intramuscular injection to immunize C57BL/6N mice and intranasal administration to C57BL/6N mice to evaluate immunogenicity and protective effects against the virus.1.Construction and identification of Trim-RBD-GEMThe RBD region of SARS-Co V-2 Wuhan strain was selected for codon optimization,and connected with the T4F motif to form a trimer based high aggregation state.Then,it was connected with protein anchor（PA）through flexible linker（GGGGS）,and connected to p Fastbac1 vector.The P4 generation virus was identified by indirect immunofluorescence and Western blot,and the indirect immunofluorescence results showed that Sf9 cells infected with P4 generation virus could see green fluorescence signals;The P4 generation virus supernatant and precipitate treated with reduction can detect the target protein between 55 KDa and 70 KDa,while the supernatant and precipitate treated with non reduction can detect the target protein between 100 KDa and 130 KDa,indicating successful expression of the target protein; Lactococcus lactis（L.lactis）treated with boiled trichloroacetic acid（TCA）to form Gram positive enhancer matrix（GEM）.After binding with Trim-RBD protein,Trim-RBD-GEM was successfully expressed through SDS-PAGE,Western blot,and indirect immunofluorescence identification;After ultra-thin section identification by Transmission Electron Microscopy （TEM）,it can be seen that L.lactis has cell wall and uniform content.After TCA treatment,the content of GEM particles becomes less,showing the structure of peptidoglycan,and the bound protein attaches to the peptidoglycan skeleton;After thin layer chromatography（TLC）,the purity of Trim-RBD-GEM was identified to exceed 97%.The above results indicate that Trim-RBD-GEM was successfully constructed and the target protein was successfully expressed.2.Experimental immunization study of SARS-Co V-2 bacterium-like particles vaccine Trim-RBD-GEMAfter intramuscular immunization of C57BL/6N mice with Trim-RBD-GEM combined with Adda Vax adjuvant and Al（OH）3 adjuvant on days 0,14,and 28,the serum specific Ig G,Ig G1,Ig G2a,Ig G2b,and Ig G3 antibody levels were detected on days 10,24,and 38 after immunization.The results showed that the serum specific Ig G,Ig G1,Ig G2a,Ig G2b,and Ig G3antibody levels in Trim-RBD-GEM&Adda Vax group mice were significantly higher than those in Trim-RBD-GEM&Al（OH）3 group and Trim-RBD-GEM GEM,Trim-RBD group,GEM&Adda Vax group,GEM&Al（OH）3 group and the Mock group,the highest specific Ig G antibody titer detected in the serum of Trim-RBD-GEM&Adda Vax group mice on the 38th day after initial immunization was 1:102400.Using the SARS-Co V-2 virus strain stored to detect the neutralizing antibody level in the serum of mice in each group,it was found that the neutralizing antibody level in the serum of the Trim-RBD-GEM&Adda Vax group was significantly higher than that of the other groups.On the 38th day after the first immunization,the highest titer of neutralizing antibody was 1:160 in the serum of Trim-RBD-GEM&Adda Vax group mice.After the first immunization until the fourth month,the serum of mice was tested.The results showed that the serum specific Ig G antibody level in the Trim-RBD-GEM&Adda Vax group was significantly higher than that in other groups after the 60th,90th,and 120th days of the first immunization.The highest specific Ig G antibody titer detected in the serum of Trim-RBD-GEM&Adda Vax group mice on the 120th day after initial immunization was 1:25600.In the results on the 60th and 90th days after the first immunization,it can be seen that the neutralizing antibody level in the serum of the Trim-RBD-GEM&Adda Vax group mice is significantly higher than that of the other groups.ELISpot results showed that the number of spots of IL-4 and IFN-γcytokines in spleen lymphocytes of mice in Trim-RBD-GEM&Adda Vax group was significantly higher than that in Trim-RBD-GEM,Trim-RBD group,adjuvant control group,and Mock group.Luminex results showed that the secretion levels of IL-5 and IL-13 cytokines in the spleen lymphocyte supernatant of mice in Trim-RBD-GEM&Adda Vax group were significantly higher than those in other groups.Intracellular cytokines such as IFN-γin CD4+T cells and CD8+T cells in spleen lymphocytes of mice in Trim-RBD-GEM&Adda Vax group were significantly higher than those in other groups.On the 35th day of initial immunization,mice in the Trim-RBD-GEM&Adda Vax group,GEM&Adda Vax group,and Mock group were challenged with SARS-Co V-2 mouse adaptation strain（C57MA14）.The survival rate of mice in the Trim-RBD-GEM&Adda Vax group was 100%,while the weight of mice in the GEM&Adda Vax group and Mock group decreased significantly.On the 6th day after the challenge,their weight was all lower than 75%,and the survival rate was 0%.The viral load in the lung tissue of the Trim-RBD-GEM&Adda Vax group mice on the third day after challenge was significantly lower than that of the GEM&Adda Vax and Mock groups mice.The above results indicate that intramuscular immunization induces good cellular and humoral immunity in the body,providing better immune protection.After intranasal immunization of C57BL/6N mice with Trim-RBD and Trim-RBD-GEM on days 0 and 21,the serum specific Ig G,Ig G1,and Ig G2a antibody levels were detected on days 7,14,21,28,and 35.The results showed that the serum specific Ig G antibody levels in the Trim-RBD-GEM group were significantly higher than those in the Trim-RBD and Mock groups;On the 35th day of initial immunization,mice in the Trim-RBD-GEM group and the Mock group were challenged with SARS-Co V-2 mouse adaptation strain（C57MA14）.The survival rate of mice in the Trim-RBD-GEM group was 100%,while on the 7th day after the challenge,mice in the Mock group had a body weight of less than 75%and a survival rate of 0%;The viral load in the lung tissue of mice in the Trim-RBD-GEM group on the third day after challenge was significantly lower than that of mice in the Mock group;After HE staining,necrotic cell fragments were seen in multiple bronchial lumens of mice in the Mock group;Immunohistochemical results showed that the N protein of SARS-Co V-2 could be detected in the lung sections of Mock group mice.This indicates that intranasal immunization with bacterium like particles can provide immune protection for mice.In conclusion,the research results prove that this study has successfully constructed the SARS-Co V-2 bacterium like particle Trim-RBD-GEM,which can induce the immune response of C57BL/6N mice through intramuscular injection,and can provide immune protection for mice,providing new ideas and support for the research of COVID-19 vaccine. |
