Preparation Of Genetically Engineering Mucosal Adjuvant CTA1-DD And Evaluation Of Its Immune Effects

The most efficient method to prevent and control pathogen transmission is vaccination.At present,the majority of vaccines are usually administered parenterally by intramuscular(i.m.)injection,although some pathogens enter the host via mucosal membranes.This immunization mode mainly induces the production of IgG antibody,but causes relatively weak cell-mediate immunity and little or no mucosal immunity However,mucosal immunization can not only stimulate effective mucosal immune response,induce local IgA antibody secretion to prevent infections that target the mucosa or gain access to the body through the mucosal membrane.In addition,it is more easily accepted,cheaper,simplifying vaccination procedures and avoiding cross-contamination and so on.Although mucosal immunization has many advantages,a limit number of mucosal vaccines have been approved for use in human because of the need for large antigen and antigen enter the mucosa need to overcome the mucosal tolerance and so on.Vaccine adjuvants are very important for enhancing immune response in the immune process and have attracted much attention in the field of vaccine research However,only a limited number of safe and effective adjuvants,especially mucosal adjuvants,are available for use in vaccine.The development of a practically applicable mucosal adjuvant is therefore urgently needed.In this study,a novel mucosal adjuvant fusion protein CTA1-DD was constructed,which combined the full enzymatic activity of the A1 subunit of cholera toxin(CT)with two immunoglobulin-binding domains of Staphylococcus aureus protein A(SpA),and we evaluated the effect of CTA1-DD as a mucosal adjuvantInfluenza is an acute respiratory disease caused by the influenza virus.It is often accompanied by high fever,runny nose and soreness throughout the body,causing outbreaks of influenza and season influenza.According to the statistics,influenza virus infects 10%-20%of the total worldwide population during seasonal epidemics,resulting in 3-5million cases of severe illness and 290,000-65,000 deaths annually Influenza viruses are classified into four groups:influenza A virus(A),influenza B virus(B),influenza C virus(C)and influenza D virus(D).In addition to influenza D virus,they can infect humans and cause influenza,of which H1N1,H3N2 and H5N1 in the influenza A virus are the main pathogens casing the influenza pandemic Therefore,the prevention and control of this virus is of great significance to human public healthInfluenza A virus is a single strand of negative-strand RNA virus,belonging to Orthomyxoviridae,which often has an antigenic shift in the propagation process,which in turn causes a new type of influenza outbreak.Inactivated vaccines,split vaccines and subunit vaccines are usually administered parenterally by intramuscular(i.m.)injection,although this pathogen enters the body via mucosal membranes.Therefore,it is of great significance to study a safe and effective mucousal immunization influenza vaccine.Thus,in the present study,we investigated the capacity of the nontoxic adjuvant CTA1-DD to serve as a mucosal adjuvant for HIN1 split subunit components and H3N2 split subunit components.It lays a foundation for the study of CTA1-DD as a new mucosal adjuvant of human vaccineIn this study,mucosal adjuvant CTA1-DD protein was expressed by prokaryotic expression system.The target gene was inserted into the expression vector pET30a by using the techniques of codon optimization,gene synthesis,enzyme digestion and ligation,and the pET30a-CTA1-DD was constructed successfully and then transferred to Escherichia coli(E.coli)BL21(DE3)for expression.The results showed that this protein could be expressed in both soluble and inclusion bodies,and the molecular weight was about 36KD.In order to facilitate the later experiment,the soluble expressed protein was purified.This protein was purified by DEAE ion exchange chromatography and SephacrylTM S-200 gel filtration chromatography.Finally,we obtained the target protein with high concentration and purity(2.4mg/ml and 98.43%,respectively).In addition,CTA1-DD adjuvant protein was immunized intranasally with HIN1 split subunit components and H3N2 split subunit components respectively,and compared with conventional aluminum hydroxide adjuvant combined with split subunit components by intramuscular injection.The results showed that the combination of the mucosal adjuvant CTA1-DD with influenza split subunit components significantly improved serum antigen-specific IgG titers and lavage antigen-specific IgA titers compared with alum adjuvant combined with split subunit components.These results indicated that CTA1-DD can not only stimulate the humoral immune response,but also stimulate the mucosal immune response Although high level IgG titers were detected in alum-adjuvanted split subunit components group,whereas no IgA antibody was detected in lavage,suggesting that this immunization method can effectively activate the humoral immune response,but can not stimulate mucosal immune response.Further,the data from ELISPOT and IgG subclass showed that CTA1-DD combined with influenza split subunit components could increase the numbers of IFN-y spot-forming cells and decrease the ratio of IgG1/IgG2a.These results indicated that CTA1-DD adjuvant predominantly induced a Thl-type immune response.While alum-adjuvant split subunit components triggered an increase in IL-4-secreting Th2 cells and increased the ratio of IgGl/IgG2a.These results demonstrated that the effect of alum adjuvant were skewed towards Th2-type immunity.Meanwhile,the results of Hemagglutionation inhibition(HI)assay showed that CTA1-DD as an mucosal adjuvant combined with influenza split subunit components had better effect than alum adjuvant,and could stimulate the mice to produce stronger HI titers.Finally,in order to evaluate the protective potential of CTA1-DD as mucosal immune adjuvant combined with influenza split subunit components,we challenged the immunized mice with HIN1 A/Califomia/04/2009 and H3N2 A/Hong Kong/1968 respectively.We observed that 100%protection and survival for the CTAI-DD-adjuvant subunit components group compared with other groups,and significantly reduced the morbidity.While alum combined with split subunit components had partial protection(66.67%,50%respectively),and a series of clinical symptoms were detected,including listlessness,anorexia,weight loss,and piloerection.These data illustrated that CTA1-DD adjuvant as an mucosal adjuvant combined with influenza split subunit components via i.n.rote can play a more perfect protective effect than alum adjuvant.In summary,high purity CTA1-DD protein was obtained by DEAE ion exchange chromatography and SephacrylTM-200 gel filtration chromatography using prokaryotic expression system.Meanwhile,the results presented in this study have demonstrated that intranasal immunization of mice with CTA1-DD combined with the influenza split vaccines promooted mucosal and systemic humoral and the virus-specific cell-mediated immune response.We also demonstrated that CTAI-DD-adjvant vaccine provided 100%protection against mortality and greatly reduced morbidity in mouse model.We also illustrated that addition of CTAI-DD in vaccine elicited significantly higher hemagglutination inhibition(HI)titers and IgG antibodies in sera than alum adjuvant.Meanwhile,it significantly promoted the production of mucosal sIgA in lung lavages and vaginal lavages.In addition,CTA1-DD was also superior at inducing Thl-type immunity in mice.Taken together,this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for respiratory diseases and other mucosal diseases.