Preparation Of Novel COVID-19 Broad-spectrum Nucleic Acid Vaccine By Tandem SARS-CoV-2 RBD Strategy

COVID-19(Corona Virus Disease 2019)caused by SARS-CoV-2(severe acute respiratory syndrome coronavirus 2)is an emerging and highly contagious disease that poses a significant public health threat.The full-length Spike(S)protein of SARS-CoV-2 or its Receptor Binding Domain(RBD)is the most important target for vaccine design,and the wide application of COVID-19 vaccines based on full-length S protein or RBD in the real world plays a positive role in blocking the transmission of SARS-CoV-2.Unfortunately,SARS-CoV-2 has a strong mutation ability and thousands of mutations have occurred accumulated.The enhanced immune escape ability of the variants is one of the main reasons for the decreased efficacy of COVID-19 vaccine designed based on the Wuhan-Hu-1 strain.Secondly,the rapid decline of neutralizing antibodies has also been widely confirmed.How to overcome these two major difficulties has become a problem that must be solved in the R&D of a new generation of COVID-19 vaccine.The RBD of SARS-CoV-2 contains abundant neutralizing antibody epitopes,but only contains 223 amino acids,and the protein has a small molecular weight,which may theoretically cause weak immunogenicity.If multi-segment RBD can be expressed fusion,the vaccine antigen may have the ability to simultaneously cover the key antigen epitopes of multiple mutant strains.Moreover,a more complex protein with double protein size may be beneficial to increase the immunogenicity of the target antigen.In addition,the target protein formed after fusion will give a target antigen the potential advantage of becoming a polyvalent vaccine,which will lead to increased production and reduced cost in the process,and facilitate the rapid scale vaccination of the vaccine to block the transmission of the virus.The process of nucleic acid vaccine to play its role is to express the target protein in the host cell,so the downstream purification process of its preparation does not consider the purification characteristics of the protein,which will provide great potential for the fusion protein as the target of the vaccine.More importantly,although cross-reactivity of neutralizing antibodies between different strains may be weak,the study confirmed the broad spectrum of COVID-19 vaccine-induced T cell responses,while nucleic acid vaccines have the characteristics of T-cell immunoinduction advantage.So in the first parts,this paper explores the DNA vaccine route,which is a consideration based on the above background combined with the increase of the persistent expression characteristics of DNA vaccine itself,because the persistent expression of target antigen in the host will not only help to slow down the decline trend of SARS CoV-2 antibody,but also help the somatic mutation process in the antibody generation mechanism,so as to induce antibodies with higher affinity,Provide a theoretical basis for the broad-spectrum COVID-19 vaccine to play a more efficient role.The antigen is designed in the form of RBD(Wuhan-Hu-1)-RBD(B.1.351)-RBD(B.1.617.2)-RBD(C.37)-RBD(B.1.617.2),with a total of 5 RBD sequences.The severity of disease caused by Delta variant is relatively high among the variants,so one more Delta RBD sequence is repeated in the sequence design,to a certain extent,it can determine whether this method has the potential of specific enhancement against a certain strain.The commonly used signal peptides in vaccine design are secreted signal peptides,but considering that nucleic acid vaccines express proteins in the host cells,if carrying membrane signal peptides gives target proteins the potential on the cell membrane,it may have more contact opportunities with APCs,thus enhancing the adaptive immune response.we thus design two different signal peptides for the sequence,namely secretory signal peptide(derived from human tpa protein)and membrane signal peptide(derived from SARS-CoV-2 S protein).Finally,two DNA vaccine candidates pVAX1-WSDLD-T and pVAX1-WSDLD-S base on different signal peptides are formed,and the target gene expression is enhanced by adding Kozak sequence.Subsequently,its broad-spectrum potential is verified by in vitro expression,in vivo immune evaluation and mechanism exploration.The results show that this nucleic acid vaccine construction method has good expression potential.After immunizing Balb/C mice by intradermal injection,both of the two DNA vaccine candidates induce humoral and cellular immune responses in the mice.The Nabs show a certain broad spectrum,and the cellular immunity is well stimulated,but the overall intensity is not strong.In the current research and approval of COVID-19 vaccines,neutralizing antibodies are still the most recognized evaluation indicators,and therefore,certain ways are needed to enhance the immunogenicity and application potential of these two DNA vaccines.We then explored how to enhance the immunogenicity of the two DNA vaccine candidates.Based on the background of COVID-19 vaccine prevention and control,a variety of vaccines have widely completed basic immunization worldwide and inactivated vaccines account for the largest proportion in China,and inactivated vaccine is the whole pathogen vaccine containing more abundant T cell epitopes information.We therefore selected prime/boost immunization strategies to explore the possibility of improving the immunogenicity of DNA vaccines with inactivated vaccines.The results show that highest levels of Nabs and cellular immunity are induced when DNA vaccine is used as a booster dose in mice that had completed basal immunization with two inactivated vaccines,and the GMT of the binding antibody induced by pVAX1-WSDLD-S and pVAX1-WSDLD-T were 2.30×106 and 2.34×106,respectively,which are more than 11 times higher than the control of the two inactivated vaccines.The levels of Nabs against Beta(B.1.351),Delta(B.1.617.2)and Lambda(C.37)are also relatively high in all groups,especially against Delta strain,which have the most significant improvement,with GMT 3420 and 2430,respectively.Using DNA vaccine as a boost dose shows a significant enhancement of T cell response,and pVAX1-WSDLD-S based on membrane signal peptide induces higher levels of Th1 cytokines(IL-2 and IFN-γ values,local lymph nodes,detected by flow cytometry).The mode of action of DNA vaccine is that the vector needs to enter the nucleus to start the process of expressing the target protein,so can the mRNA vaccine route,which is more efficient and can be expressed in the cytoplasm,form an effective broad-spectrum COVID-19 vaccine by tandem RBD in this way of antigen fusion?Therefore,in the end of this article,the feasibility of designing a broad-spectrum COVID-19 vaccine with tandem RBD genes using mRNA vaccine routes is studied.Based on the analysis of new research progress,the COVID-19 vaccine designed with the the more primitive strain(Wuhan strain)shows excellent broad-spectrum neutralization characteristics,which leads us to speculate that the vaccine designed with the ancestor strain may have relatively good broad-spectrum coverage of its offspring variants.Based on this,we used the strategy of tandem different RBD to fuse the RBD gene of SARS-CoV,the RBD gene of SARS-CoV-2 Wuhan strain and the RBD gene of omicron BA.5 as antigens,the former two strains are early strains,and BA.5 is also the early strains of BA.5.2,B.7 and BQ.1.1 that once caused widespread epidemics in China.According to in vitro expression,humoral and cellular immune responses,immunological processes are explored.The results of DNA linearization in vitro,detection of target protein expression in transfected cells in vitro,and biochemical characteristics of the LNP-mRNA complex indicate that the mRNA vaccine prepared in this article has good quality.In the animal experiments,the specific SARS-CoV,SARS-CoV-2,and BA.5 IgG binding antibodies induced by the mRNA vaccine obtain extremely significant positive results,and induced a higher level of total IgG2a.The detection of Nabs against pseudovirus showed that the serum of mice immunized with mRNA vaccine had excellent cross-neutralizing activity against pseudovirus D614G,Delta,BA.4/5 and BQ.1.1,especially the GMT of Nabs against BA.4/5 was up to 2561 and against Omicron BQ.1.1 strain was 889.4 GMT.ELISPOT show that the spleen cells were stimulated by the specific short peptide library to secret the corresponding IFN-γ and IL-4(p<0.0001).In conclusion,the strategy of covalently tandem RBD genes of different SARS-CoV-2 variants to construct broad-spectrum COVID-19 nucleic acid vaccine has great potential.In this paper,DNA vaccines constructed by concatenating RBD genes of different variant strains and mRNA vaccines constructed by tandem RBD genes of different strains have been proved to have good broad-spectrum potential to a certain extent,and the intermediate process of their role is explained by a series of immunological characteristic studies.The results in this paper may provide value for the research of a new generation of COVID-19 vaccine with stronger broad spectrum against multiple variants.