| Background Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has been prevalent since the outbreak in December 2019.SARS-CoV-2 has mutated continuously with the passage of time,and there have been a variety of mutants.Among them,WHO listed five VOC(Variants of Concern)with strong transmission,increased clinical manifestations,reduced serum neutralization and vaccine protection in recovered patients.Such as Alpha(B.1.1.7),Beta(B.1.351),Gamma(P.1),Delta(B.1.617.2)and Omicron(B.1.1.529).Among these mutants,we screened two sites worth studying.L452R enhances S-protein cleavage,and although the L452 residue is in the hydrophobic region of the receptor binding domain(RBD)of the spike-in S-protein and does not directly contact the receptor angiotensin-converting enzyme 2(ACE2),the L452 R mutation may cause structural changes that facilitate the interaction between the spike-in protein and the ACE2 receptor.This mutation could enhance the ability of the virus to bind to the ACE2 receptor and thus accelerate infection,and could also reduce the ability of vaccine-generated antibodies to attach to the spiked glycoprotein.Studies have shown that mutations in L452 R and T478 K induce an increased ability to fuse cell-cells into syncytia.t478 K is located in the region that interacts with the human ACE2 receptor and it is predicted that a change from polar but uncharged threonine(T)to charged basic lysine(K)increases the electrostatic potential of the Spike protein in the region in direct contact with ACE2 to a more positive surface,and this change in position may affect the affinity to human cells and thus the infectivity of the virus.In addition to this,larger lysine side chains are predicted to increase the spatial site resistance of the mutant,potentially further affecting Spike/ACE2 interactions.The effects of electrostatic surface of the protein,ACE2,drug or antibody interactions may also be increased if combined with other Spike mutations at the same time.As the protein that binds SARS-CoV-2 to the ACE2 receptor,the RBD plays an important role in the process of viral infection and is rich in T-and B-cell epitopes,including neutralizing epitopes,which can induce neutralizing antibody production for the prevention of viral infection,making the RBD an important target for subunit vaccines.In this study,we propose to design single and double mutant RBD proteins containing L452 R and T478 K in order to investigate the immunogenicity of the mutant proteins.The tetanus toxoid TT peptide,an antigen rich in T helper epitopes,is effective in enhancing the humoral and cellular immune response to antigenic epitopes.TT peptide has an immune enhancing effect and promotes high levels of antibody production by achieving an effective T-B cell response.In this study,a small sequence of TT peptide was intercepted and added to the S protein sequence as an intramolecular immune adjuvant,hoping to enhance the immunogenicity of the protein.Methods In this study,two single mutant plasmids and one double mutant plasmid were constructed by tetanus toxin peptide and S protein RBD region through flexible peptide tandem by PCR,based on which mutation sites L452 R and T478 K were added,respectively.The plasmids were transformed into E.coli receptor cells for prokaryotic expression,and the three recombinant RBD-TT mutant proteins were identified and expressed as inclusion bodies.The inactive proteins were purified by two ion exchange chromatographic purifications followed by tangential flow ultrafiltration dialysis to refold the inactive proteins,and finally the corresponding recombinant RBD-TT mutant proteins were purified by removing the aggregates with the aid of molecular sieve.The recombinant RBD-TT mutant protein was mixed with aluminium adjuvant and then immunized in BALB/c mice to evaluate the effect of humoral and cellular immunity.Results Two single mutant proteins containing L452 R and T478 K on the RBD-binding domain of SARS-CoV-2 were successfully constructed in the laboratory earlier and both were confirmed to be relatively good in terms of humoral immunity(Ig G antibody levels)and cellular immunity(ELISPOT detected good Th1(IL-2)cellular immune responses against RBD in splenic lymphocytes of immunised mice)effect.In the live virus neutralisation assay,the antibody was found to neutralise the Omicron mutant up to a titer of 3072 and a minimum of 384,while the Delta mutant had a titer of 6144 and a minimum of 1024.The results of the live virus neutralization assay showed that the neutralizing antibodies produced were effective against all three strains,and it can be concluded that this mutant protein has a broad spectrum neutralizing ability against different mutants of SARS-CoV-2.In addition to the effect of humoral immunity,mice immunized with the mutant protein also developed specific cellular immunity,with the mutant protein group producing(208.50±90.78)and(512.88±131.16)SFC/5×105 cells secreting IFN-γ and IL-2,respectively,after stimulation with the peptide of the prototype RBD strain,which were higher than those of the control group(14.63±12.98)and(14.75±7.5).The number of effector T cells secreting IFN-γ and IL-2 after stimulation with RBD Delta strain peptide was(243.50±12.83)and(633.75±250.47)SFC/5×105 cells,respectively,higher than that of the control group(26.88±19.58)and(12.88±12.58).The effector T cells in the control group were(243.50±12.83)and(633.75±250.47)SFC/5×105 cells,respectively,which were statistically different from those in the control group.Conclusion In this study,a single mutant RBD protein containing L452 R and T478 K was constructed,which was proved to be effective in humoral and cellular immunity.On this basis,the double mutant protein was constructed,which also proved that it had strong cellular and humoral immunity.The live virus neutralization test showed that it was effective against epidemic SARS-CoV-2Delta and Omicron(BA.1),which provided a basis for the development of broad-spectrum recombinant COVID-19 mutant protein vaccine. |
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