| Varicella-zoster virus(VZV)is a pathogenic herpesvirus that induces two distinct diseases:primary infections with the VZV result in varicella,which is a highly contagious disease characterized by a blister-like rash all over the body and systemic signs and symptoms including fever,malaise and so on,causes the greatest disease burden in children.The virus establishes latent infection in neuronal cells after primary infection,and may reactivate frequently when the immune system is senescent or compromised to cause a painful,localized vesicular rash(herpes zoster,HZ)with several complications.Both varicella and HZ can be prevented by live attenuated vaccines(LAV).The first LAV for preventing varicella was developed based on vOka strain and licensed for routine use in the United States in 1995(Varivax).Unfortunately,vOka is a mixture of quasispecies and the molecular basis for its attenuation remains unknown,suggested that vOka can infect and establish latency like a wild-type virus and reactivate to cause HZ.On the other hand,it has reported that the efficacy of the Zostavax,the herpes zoster vaccine(vOka strain),is decreased with increased age of the individual at the time of administration.There is a need for next-generation varicella/herpes zoster vaccines.Previous studies have demonstrated that glycoprotein E(gE)is the most abundant VZV glycoprotein as well as an ideal target as a subunit vaccine antigen,capable of inducing cellular and humoral immunity during natural varicella and following vOka vaccination.Recently,a recombinant HZ subunit vaccine,Shingrix(HZ/su),was approved for the prevention of herpes zoster(HZ)and postherpetic neuralgia(PHN)in adults aged≥50 years.Shingrix is composed of recombinant gE antigen expressed in Chinese hamster ovary(CHO)cells and a liposome-based adjuvant system(AS01B)containing MPL and Quillaja saponaria Molina,fraction 21(QS21).In clinical trials,Shingrix demonstrated a significantly reduced risk of HZ in these individuals independent of ages,especially in those aged 70 years and older,which is better than ZOSTAVAX.However,the high manufacturing cost and the limited supply make it not be ideal for widely used.Microbial expression systems have advantages involving rapid growth,easy culture and thus lower capital,which make it ideal for vaccine manufacturing and used in developing countries.Although the recombinant CHO cells have been the most widely used host for large-scale production of recombinant protein drugs,this system requires a long production cycle that results in higher cost of products.Therefore,antigen generated from E.coli expression system may benefit the further investigation of VZV vaccine with lower cost and great significance.In this study,we investigated whether gE protein can be expressed in E.coli.Several candidate molecules were rationally designed and screened according to their characteristics.Here,we explored the non-fusion soluble expression of gE candidate antigens for the first time.gE candidate antigens were expressed abundantly and purified from E.coli,followed by the lab scale study and structure study.Physicochemical characterization and immunogenicity analysis showed that the protein was native-like antigenic,which showed promise for vaccine candidate.First,we designed several candidate molecules according to the properties of both gE protein and E.coli expression system.The feasibility of expression was evaluated under different induction temperature.We inadvertently identified that the exogenous expression of these protein in E.coli was related to the truncation of VZV gE protein.Candidate molecules were purified by either inclusion body purification or soluble supernatant chromatography.Among them the candidate gE(31-358)protein has advantage of high soluble expression with low purification difficulty,high purity and homogeneity with excellent antigenicity,offering an ideal candidate for vaccine antigen.Furthermore,the lab scale study of gE(31-358)protein was proceed systematically.The purification of protein was efficient under the lab-scale process established here with stability and scalability.The product was used to analyze the structure for elucidating the characteristics of gE antigen,which is of great significance for a comprehensive vaccine QC system.We next evaluated the immunogenicity of gE(31-358)generated in our study compared with LAV and Shingrix,indicated that our antigen not only showed efficient humoral response but also induced antigen-specific CMI responses which plays decisive roles in the control of reactivating VZV and preventing HZ,especially when antigen combined with ASO1B adjuvant.These results providing our antigen a potency as HZ vaccine candidate.Finally,we had developed new approaches for vaccine development.A vaccine candidate harboring gE(31-358)fused with CRM197 fragment A manifested higher immunogenicity in mice even when administrated with lower dose.Besides,we developed a new bacterial surface display system based on OmpF as a carrier protein,which can be used to decorate the surface of E.coli with viral epitopes,provides an amenable way to engineer bacteria for BG-based vaccines design.Overall,we demonstrated that the gE(31-358)protein derived from the bacterial expression system could induce both humoral and cellular immune responses when formulated with appropriate adjuvants,which may offer a much more economical strategy for the development of HZ vaccines. |
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