Synthesis And Immunological Evaluation Of COVID-19 And Anticancer Vaccines Based On Diphtheria Toxin Mutant CRM197

Cancer and COVID-19 are two significant threats to human health,but there is no way to eliminate both diseases globally.It is an effective method to prevent and control diseases by constructing a high-efficiency vaccine that combines specific antigens on viruses or tumors with immunogenic carriers to achieve active immunity.The cross-reacting material(CRM 197)is a nontoxic variant of diphtheria toxin,which provides excellent immune effects in various clinical vaccines.It has been widely used to enhance the immunogenicity of vaccines.We developed a novel SARS-CoV-2 vaccine and anti-cancer vaccine based on the diphtheria toxin mutant CRM 197 targeting SARS-CoV-2 specific epitopes and tumor-associated carbohydrate antigens in this context to seek more effective and safe methods to combat COVID-19 and cancer.SARS-COV-2 is composed of four structural proteins and internal RNA,in which the transmembrane spike glycoprotein(S protein)enters the cell by binding to the human angiotensin-converting enzyme(hACE2).COVID-19 vaccines generally have side effects,such as pain at the injection site,low-grade fever,and local thrombosis.In the early stage of the epidemic,we became interested in developing the SARS-CoV-2 B cell epitope conjugates of CRM 197,which aims to prevent the virus from infecting human cells while improving the vaccine’s safety.In this work,three B cell-based epitopes in the receptor-binding domains(RBD)of SARS-COV-2 were synthesized through solid-phase synthesis.For peptide bioconjugation,maleimide-PEG2-succinimide ester was used as a coupling molecule to conjugate CRM197 protein and peptide,generating CRM197-RBD peptide vaccines.Immunization of mice with CRM197-RBD peptide conjugates induced high peptide-specific IgG antibodies and produced a robust anti-RBD humoral immune response.More importantly,the conjugates induced antibodies can effectively inhibit RBD-hACE2 interaction and prevent the infection of host cells by SARS-COV-2 pseudovirus.In tumor genesis and development,abnormal glycosylation occurs in tumor cells.The abnormal sugar chain structures highly expressed in tumor cells are called tumor-associated sugar antigens(TACAs).Sialylgangliosides GD2 and GD3 are TACAs overexpressed in neuroblastoma,melanoma,and other tumor cells.However,normal cells can also express GD2 and GD3,and vaccines prepared with this antigen have poor specificity.The most common natural modification of sialic acid,9-OAC,is not represented on normal cells,but the compound was structurally unstable.Recent studies have shown that 9NHAc-GD2 and 9NHAc-GD3 have similar conformation with 9-OAC ganglioside.The high stability also makes them excellent candidate antigens.In our research,9-NHAcGD2 and 9-NHAcGD3 combined with CRM 197 were synthesized into a vaccine by the chemoenzymatic method.LactoseProN3 was synthesized by a 6-step chemical reaction with lactose as substrate,and 9NHAc-GD3ProN3 and 9NHAc-GD2ProN3 were synthesized by chemoenzymatic.The azido group was then converted to an isothiocyanate,which reacted with the amino group on CRM197 to synthesize vaccine CRM197-9NHAc-GD2 and CRM197-9NHAc-GD3.After immunizing mice,the results showed that all the vaccines could produce strong and lasting humoral immunity,and the induced antibodies could bind specifically to tumor cells.In summary,we reported two kinds of vaccines,which used diphtheria toxin mutant CRM 197 as the carrier protein to conjugated compounds generate peptides and gangliosides as antigens.Our results showed that the vaccines induced excellent immune effects,which played an essential role in screening major antigen regions of the COVID-19 vaccine and the clinical promotion of the ganglioside cancer vaccine.