| Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a Corona Virus Disease that seriously affects human health and causes a global public crisis 2019,COVID19)is a major pathogen and is highly contagious.A number of different types of vaccines have been developed to suppress the spread of SARS-CoV-2.The primary design target of these vaccines is spike protein S.However,a comparative analysis of immunology between recovered and uninfected populations showed significant differences in cellspecific immunity against viral Nucleocapsid protein N.This suggests that other structural proteins of the virus may play a role in the body’s immunity.Studies have shown that individuals with strong Cytotoxic T lymphocytes(CTL)for N antigen have a relatively good clinical prognosis in COVID-19 patients.Meanwhile,in previous vaccine-related experiments,we found that inactivated vaccines containing a variety of structural proteins seem to have a more durable protective effect.Because N protein is more conserved than other proteins such as S and M,it can induce cellular immune response.Therefore,exploring the immunogenicity of the active antigenic components in COVID-19 preventive vaccines,especially the N antigen and its relationship with the S antigen,which can induce neutralizing antibodies,provides a new direction for the development of COVID-19 vaccines.Based on the above basis,the recombinant protein vaccine is characterized by simple background,easy evaluation,high safety,low biosafety level requirements,and high purity.In this study,the in vitro eukaryotic expression of S1 and N antigen polypeptides is used,and the intradermal and muscular pathways,individual and mixed immunization protein vaccines are used.The Syrian golden ground mouse model sensitive to SARS-CoV-2 was immunized with two injections at an interval of 14 days,and the ability of different antigens to induce humoral immune response was observed.In order to evaluate the protective effect of the above immune response on Syrian golden ground squirrels,they were infected with the original SARS-CoV-2 live virus after 14 days of enhanced immunization(i.e.,28 days of initial immunization).Clinical symptoms and detoxification were observed daily,viral load of each tissue and pathological damage of each organ were observed regularly,and serum antibody titers were detected.The results showed that antibody response could be induced by inoculation of SARS-CoV-2 structural protein S alone or mixed with S+N protein,and there was no significant difference between intradermal and intradermal injection.Although neither vaccine was effective in inhibiting the proliferation and replication of the virus,immunized Syrian golden Gophers showed partial relief when attacked by the virus.Interestingly,the presence of the two proteins(N,S1)alone or in combination did not protect the Syrian Golden Ground squirrels from infection with the virus,only reducing their weight loss after infection,whereas similar levels of NAb induced by inactivated virus vaccine protected the animals from infection with the same dose of virus.We continue to explore the possible reasons why the two types of experimental vaccines immunize the same animal models and induce similar levels of neutralizing antibodies,but produce vastly different effects against viral infection.However,due to the limited commercial detection reagents for Syrian golden Gophers,it is difficult to detect many immunological indicators except humoral immunity,which makes it difficult to comprehensively analyze and evaluate various characteristics and related causes of experimental vaccines.Therefore,follow-up experiments were conducted in Balb/c mouse model.The same dose of protein is immunized by intramuscular injection,using the same immunization procedure as the Syrian golden Gophers.14 days after the second immunization,the third dose of stimulation was administered,and 12h,24h,36h after the third dose of stimulation,a series of immune indicators including various cellular immunity were detected.The results showed that Balb/c mice,like Syrian golden ground mouse,were inoculated with SARS-CoV-2 structural protein S alone or mixed with S+N protein to induce antibody response.Balb/c mice could also induce specific T cell immune response and activate specific CD4+and CD8+T cells after 3 injections of stimulation.In addition,the combined immunity of N protein and S protein may synergistically enhance the role of natural immune cell signaling molecules to a certain extent.In conclusion,it has great significance for us to further understand the function of N protein and its related mechanism in natural immunity,and provide a theoretical basis for the development of more effective SARS-CoV-2 vaccine,which is of great significance for the prevention and treatment of COVID-19. |
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