Evaluation Of Immune Effect Of Adenylate Cyclase Toxoid Antigen RTX And Monophosphate Lipid A(MPLA) Adjuvant In Acellular Pertussis Vaccine

Objective The recombinant plasmids of pertussis adenylate cyclase toxin(CyaA/ACT)and its repeat-in-toxin domain(RTX)were constructed to obtain recombinant CyaA and RTX proteins with higher purity.The mouse model challenged by aerosol infection was used to evaluate the immune protection effect of the adenylate cyclase toxoid antigen RTX and the novel adjuvant monophosphoryl lipid A(MPLA)in acellular pertussis vaccine(aP),which would be beneficial to better understand the way to continue to improve aP in a mouse infection model.Method In the first part of the study,CyaA,acyltransferase gene of CyaA-cyclolysin activating lysine acyltransferase(CyaC),and the RTX domain of CyaA-the toxoid form of CyaA were obtained by PCR from the genome of pertussis CS strain and cloned into pET-Duet-1 and pET-2 8 a expression plasmids respectively to construct pET-Duet-CyaC-CyaA and pET-28a-RTX,and then they were expressed in BL21.In 8M urea,the CyaA protein was purified by DEAE chromatography,and the RTX protein was purified by Ni+chromatography.The validity of the expressed target proteins were verified by the method of sequencing and Western blot.In the second part of the study,aP+RTX,aP+MPLA,aP+RTX+MPLA and aP+CyaA vaccines were prepared on the basis of aP,and wP and sterile physiological saline were used as control group at 1/40 of the human dose.The three-week interval,two-vaccination procedure was used to immunize Balb/c mice intraperitoneally.Antibody levels of serum were measured before immunization and after each immunization.Mice were infected by aerosol challenge one week after the immunization.Peripheral blood leukocyte levels,lung,trachea,liver and spleen clones of pertussis,peripheral serum and lung tissue homogenate cytokine levels,and spleen T cell typing were measured at indicated time points post-infection.Result In the first part,two recombinant plasmids pET-Duet-CyaC-CyaA and pET-28a-RTX were successfully constructed and expressed in BL21 in the form of inclusion bodies and secretions,respectively.In 8M urea,the protein of CyaA was obtained by DEAE chromatography.The protein of RTX was obtained by Ni+chromatography.The validity of the expressed target proteins were verified by the method of sequencing and Western blot.In the second part,except for the control group and the wP group,high levels of PT,FHA and PRN antibodies were induced after immunization of various acellular pertussis vaccine groups.Both the CyaA and RTX vaccine groups were induced to a certain level of CyaA antibody level.In addition,both MPLA and CyaA have certain adjuvant effects,and RTX has not found this effect.In the control group,the white blood cell(WBC)level began to increase on the 6th day post-infection,and attained the peak on the 14d post-challenge,while the wP group had a mild increase in WBC on the 10th day,and swiftly returned to the normal level on the 14th day.The WBC of each acellular pertussis vaccine groups were elevated on the 14d post-infection,and went back to the normal level on the 21d.In the control group,the number of bacterial colonies in the lungs and tracheas increased rapidly,attainning a peak on the 14th day;and each vaccine group also had a certain amount of bacterial colonization.In addition,the colonization of the trachea in each group was lower than that in the lungs.And the clearance rate in the tracheas was also significantly faster than the lungs.Analysis of cytokines and classification of T cells showed that natural infection caused higher Thl and Thl7 cell immunity,and wP group and MPLA group also caused higher Thl response.And the added CyaA group could significantly improve Th17 response,while adding RTX group reduced the Th17 response.Conclusion RTX,like CyaA,could induce antibodies that recognized CyaA,but has no adjuvant effect,and the cellular immune response induced was not the same as CyaA.The addition of CyaA significantly increased the Th17 response,while the addition of RTX reduced the Th17 response.In addition,the addition of CyaA significantly improved the clearance rate of B.pertussis in the respiratory tissues of mice,and the addition of RTX alone did not significantly improve the clearance rate of bacteria in the tissues.The addition of MPLA had a great adjuvant effect and helped to promote the Thl response,but there was no significant improvement in the clearance of bacteria in tissues.The combination of RTX and MPLA also had a great adjuvant effect,and it helped to improve the Thl cell response in the lung tissue.In addition,the pertussis clearance of the tracheal tissue in mice was improved to some extent.