DMT Liposome Enhances The Immunogenicity And Protective Efficacy Of A DNA Vaccine Against Mycobacterium Tuberculosis Infection

?Objective? Despite Bacillus Calmette-Guérin(BCG)has been widely used to prevent tuberculosis(TB)for almost a century,TB remains one of the leading infectious cause of death worldwide.BCG has efficacy against primary TB in children,however,it's power wane as time goes on.Therefore,an effective prophylactic vaccine is urgently required.DNA vaccine with good property of thermostabilization,easy production and storage,shows both prophylactic and therapeutic effects against TB.However,weak immunogenicity significantly hinders the development of DNA vaccines.As a strong inducer of Th1-biased immune response,DMT,consisting of dimethyldioctadecylammonium(DDA)and two pattern recognition receptor agonists monophosphoryl lipid A and trehalose 6,6?-dibehenate(TDB),was a newly developed liposomal adjuvant.To elucidate the action mechanism of DMT and improve immunological effects induced by DNA vaccine,a new recombinant eukaryotic expression plasmid pCMFO that secretes the fusion of four multistage antigens(Rv2875,Rv3044,Rv2073 c,and Rv0577)of M.tuberculosis was constructed.The secretory expression of pCMFO plasmid were performed in cell level.pCMFO/DDA and pCMFO/DMT complexes were then prepared and their physicochemical properties were analyzed.The immunogenicity and protection against M.tuberculosis infection in vaccinated C57BL/6 mice were compared.?Methods? 1.Construction of recombinant eukaryotic expression plasmid pCMFO: The recombined p VAX1-CMFO(pCMFO)were constructed by inserting the CMFO nucleotide sequence into the eukaryotic expression vector p VAX1.2.Confirmation of the secretory expression of the plasmid: the secretory expression of pCMFO plasmid were performed using 293 T cell.3.Preparation pCMFO/DDA and pCMFO/DMT: DDA liposome and DMT liposome were prepared by lipid film hydration method.Then pCMFO/DDA and pCMFO/DMT were prepared by emulsifying corresponding liposome with plasmid pCMFO solution.4.Characterization of liposome and DNA-liposome: Particle size,polydispersity index(PDI)and zeta potential of different liposomes were detected.The morphology of liposomes was detected by transmission electron microscopy.5.DNA adsorption and release from DNA–liposome complexes: The adsorbed amount of the DNA to the liposome were subtracted the amount of DNA remained in the supernatant from the amount of DNA initially added to the liposome dispersion.The formulations were incubated at 37 °C and the amount of DNA in the supernatant was detected at appropriate time intervals to reflect the release effect of pCMFO/DDA or pCMFO/DMT on DNA.6.Mice and immunization: Specific-pathogen-free female C57BL/6 mice of 6 to 8 weeks age were randomly divided into different groups.DDA,DMT,pCMFO,pCMFO/DDA,or pCMFO/DMT was immunized intramuscularly(i.m.)twice at 3-week intervals.1 × 106 CFU of BCG China were vaccinated subcutaneously(s.c.)once at the time of the first vaccination and used as a positive control and PBS were used as negative controls.7.The protective effects evaluation against M.tuberculosis infection of vaccines: 9 and 18 weeks after immunization,different vaccinated mice were challenged with approximately 60 CFU of M.tuberculosis H37 Rv by aerosol.Four weeks later,mice were sacrificed for the comparison of protective efficacy,which was evaluated by biomarkers including lungs and spleens bacterial load,lung pathology.The pathological lesions of lung were analyzed by HE and acid-fast stain.8.The immune response of vaccines: 9 and 18 weeks after immunization,the levels of CMFO,Rv0577,Rv2875,Rv3044 and Rv2073 c Ig G and its subclasses in the sera of vaccinated mice were analyzed by ELISA assay.Antigen-specific cytokines Th1(IFN-?,TNF-?),Th2(IL-4)and Th17(IL-17A)secreted by splenocytes were measured by cytometric bead assay(CBA)or ELISA.CMFO-specific memory T cells were analyzed by intracellular flow cytometry.?Results? 1.The recombinant eukaryotic expression plasmid pCMFO was successfully constructed and the protein CFMO was secreted by the plasmid pCMFO-transfected 293 T cells.2.Addition of TDB and MPLA into the DDA liposome made the vesicles uniform spherical with little aggregation and resulted in a significant decrease of the surface charge(P < 0.05).The incorporation of MPLA and TDB enhanced the stability of the liposome.What's more,The DNA–DMT complex can release the plasmid pCMFO in a slower and longer lasting way,when compared to the DNA–DDA complex.3.The protection of pCMFO/DMT against M.tuberculosis H37 Rv infection: PBS control mice had the highest bacterial load and the most severe pathological lesion among all groups(P < 0.05).Although pCMFO,pCMFO/DDA and pCMFO/DMT vaccination all conferred significant protection with decreased bacterial load and lessened tissue lesion(P < 0.05)compared to the PBS control mice,the most significant inhibition of bacterial growth in organs and the fewest lesions were observed in pCMFO/DMT group.The pCMFO/DMT provided comparable short-term and long-term protection as BCG vaccine did.4.The immune protection mechanisms of pCMFO/DMT: 1)9 weeks after immunization,pCMFO/DMT vaccinated mice produced Rv0577,Rv2875,Rv3044,and Rv2073c-specific antibodies,including Ig G,Ig G1,and Ig G2 a in the sera.Correspondingly,the ratio of Ig G2a/Ig G1 to each subcomponent was higher than 1,which indicated that pCMFO/DMT induced a Th1-type biased responses to each subcomponent.2)9 weeks after immunization,Splenocytes from pCMFO/DMT groups secreted much higher levels of TNF-? than BCG vaccinated mice when stimulated with four subcomponents protein in vitro(P < 0.05).What's more,the level of INF-? response to Rv0577,Rv2875 and Rv2073 c in pCMFO/DMT group was higher than that of BCG group(P < 0.05).3)9 and 18 weeks after vaccination,compared with pCMFO/DDA,pCMFO/DMT vaccinated mice induced higher levels of antibodies against the antigen CMFO in sera(P < 0.05)and secreted more CMFO-specific IFN-? by splenocytes(P < 0.05).4)9 and 18 weeks after vaccination,pCMFO/DMT induced higher levels of CMFO-specific IL2+ TCM cells than pCMFO/DDA in the spleen of vaccinated mice(P < 0.05).?Conclusions? 1.pCMFO/DMT,a very promising TB candidate vaccine,could confer comparable protection efficacy against infection as BCG vaccine did.The delivery and controlled release effect of the DMT liposome might be related to the enhanced efficacy of DMT adjuvanted vaccines against TB.2.Our findings lay the foundation for further evaluation of DMT adjuvanted vaccines in preclinical and clinical trials.