Study Of The COVID-19 Recombinant Subunit Vaccine Based On Spike Glycoprotein And Novel Adjuvant

The COVID-19 epidemic caused by SARS-CoV-2 infection has had a huge impact on human economy and society.As of June 15,2021,more than 175 million people worldwide have been diagnosed with SARS-CoV-2,resulting in approximately 3.8 million deaths,and the number of infected people is still increasing rapidly.Although there are multiple strategies that may be useful for slowing the continued spread of the virus.However,it is generally believed that the control of this pandemic ultimately depends on safe and effective vaccines to achieve herd immunity.Therefore,this study aims to develop a COVID-19 subunit vaccine that can efficiently induce a balanced humoral and cellular immune response.Part 1:We designed a COVID-19 subunit vaccine StriFK-FH002C based on the trimeric spike protein.First,in the vaccine antigen module,5 antigen candidates(RBD,RBDTfd,S1,S2,StriFK)derived from SARS-CoV-2 Spike protein were designed and expressed based on CHO cell expression system.The immunogenicity data in mice showed that the StriFK(Trimeric Spike)has more advantages than the other four candidate antigens in terms of induced neutralizing antibody titers and antibody composition.In adjuvant module,traditional aluminum adjuvant(Al001)and nitrogen bisphosphonates-modified zinc-aluminum hybrid adjuvant(FH002C)were systematically evaluated in mice and hamsters.This result shows that the level of neutralizing antibody induced by FH002C is 12-17 times higher than that of Al001.StriFK-FH002C rapidly induces a strong germinal center response in mice,including a significantly higher frequency of GCB cells,Tfh cells and plasma cells that promoting the rapid production of antibodies.IFN-γ ELISPOT assay and intracellular cytokine staining show StriFK-FH002C induced a stronger Th1 immune response than Al001 adjuvant in mice,which indicated that StriFK-FH002C induced a more balanced TH1/TH2 immune response in mice.In cynomolgus monkeys,StriFK-FH002C also exhibits robust immunogenicity.Compared with Al001 adjuvanted or RBD containing vaccine,StriFK-FH002C induces neutralizing antibody production faster.Besides,preliminary safety data demonstrated the favorable safety profile of StriFK-FH002C in cynomolgus monkeys.Furthermore,the efficacy of StriFK-FH002C against SARS-CoV-2 in hamster under various challenge modes were evaluated.In nasal challenge,StriFK-FH002C confered hamsters effective protection regardless of two-dose or three-dose immunization,significantly reducing the viral RNA load in the hamster’s respiratory tract,and no infectious virus was detected in the lung tissues.More importantly,lung tissue pathology demonstrated the effectiveness of StriFK-FH002C in the hamster infection model.Although there is no significant difference between StriFK-FH002C and StriFK-Al001 in terms of body weight,infectious virus titer and pathology of lung tissue.However,the StriFK-FH002C showed a significantly better protective effect than the StriFK-Al001 in terms of viral RNA load in lung,nasal turbinate and trachea.In addition,contact transmission and aerosol transmission challenge demonstrated that StriFK-FH002C vaccination could not only protect the vaccinated animals from SARSCoV-2 infection,but also had effects in reducing virus transmission from vaccinated to unvaccinated subjects.The comparison of antibody titer before and after challenge shows that,at least in some animals,StriFK-FH002C vaccine immunization can confer sterilizing immunity against SARS-CoV-2.Part 2:Although the trimerized spike protein shows favorable immunogenicity and protective effects,the introduction of exogenous trimerization sequences may bring potential safety risks.Moreover,there may be a variety of polymer forms in the largescale preparation of trimerized spike,which further reduces the purification yield.Because of the continuity of the functional domains of the spike of the coronavirus,and we proved in the first part that the monomeric S1 subunit can also elicit neutralizing antibodies.Therefore,we explored COVID-19 subunit vaccine(STFK vaccine)based on monomeric spike protein in this part.On the basis of the antigen candidate StriFK in Part 1,the monomeric immunogen STFK was designed by removing the trimerization domain of T4 fibrin and His tag,and further truncating and mutating at the C-terminus.STFK was stable and homogeneous in monomer form,and is superior to the trimeric StriFK in terms of expression yield,reactivity and immunogenicity.STFK vaccine based on the monomer antigen STFK and the adjuvant FH002C showed favorable immunogenicity in mice,rhesus monkeys and SD rats.In rhesus monkeys,the neutralizing antibodies against SARS-CoV-2 pseudovirus and authentic virus induced by STFK Vaccine were 12 and 14 times higher than that of the aluminum adjuvant vaccine(STFK-Al Vaccine),respectively.In terms of cellular immunity,the IFN-γ ELISPOT assay showed that the STFK Vaccine can induce a significantly stronger cellular immune response than the STFK-Al Vaccine in mice.RNA-Seq of mouse lymphocytes showed that compared with STFK-Al vaccine,STFK vaccine significantly up-regulated a variety of cytokines and chemokines related to TH1-type responses,and also up-regulated chemokines related to humoral immune responses.These results indicated that STFK vaccine also induced a more balanced TH1/TH2 immune response in mice.Furthermore,the protective efficacy of the STFK Vaccine was evaluated in hamsters and hACE2 mice.In hamsters,a single dose of STFK Vaccine can rapidly induce protective antibodies,and two doses immunization can induce high titers of protective antibodies.The antibody titer is significantly higher than that of STFK-Al Vaccine.Importantly,STFK Vaccine significantly relieved weight loss,reduced the viral RNA load in respiratory tissues,and dramatically reduced lung inflammation atfer nasal challenge,regardless of single-dose or two-dose immunization.The neutralizing antibody response and protective efficacy provided by STFK Vaccine is better than that of the aluminum adjuvant vaccine containing the same antigen.In hACE2 mouse model,the STFK Vaccine can also induce high titer of neutralizing antibodies and provided a promising protection against SARS-CoV-2.Prove the effectiveness of STFK vaccine in a variety of in vivo challenge models.In summary,the STFK vaccine provides protection in a variety of animal models.In addition,we also evaluated the neutralizing activity of the STFK Vaccine serum against a variety of SARS-CoV-2 variants.The data shows that the serum of rhesus monkeys immunized with STFK vaccine has favorable neutralizing activity against a variety of SARS-CoV-2 variants,and showing 100%neutralization positive against variants.In summary,this study has developed two COVID-19 subunit vaccine based on CHO cell,which are composed of the trimeric spike protein StriFK or monomeric spike protein STFK and an innovative adjuvant FH002C.Both vaccines induced a potent humoral and cellular immune responses.It shows favorable immunogenicity in mice,rats,hamsters and non-human primates.In the hamster and hACE2 mouse challenge models,the STFK Vaccine showed a better immune protection effect than the STFKAl Vaccine.More importantly,in hamster animal model that simulates severe new coronary pneumonia in humans,a single dose of STFK Vaccine induced neutralizing antibody about 5.2 times higher than individuals recovered from COVID-19,and provided a potent protected from SARS-CoV-2 challenge.