Preparation And Immunogenicity Of Influenza Virus-like Particle-based Mycobacterium Tuberculosis Subunit Vaccine LV20

Tuberculosis（TB）is one of the major global health problem caused by Mycobacterium tuberculosis（M.tuberculosis）.Bacillus Calmette-Guérin（BCG）is currently the only licensed and preventive tuberculosis vaccine.It protects children from severe TB while BCG has a poor protective effect on adults.Due to the limited protection of BCG,it is urgently needed to develope novel vaccines to protect children and adults from tuberculosis.Among the novel vaccines,virus-like particles（VLPs）is a special subunit vaccine.It has been successfully developed in sevaral virus vaccines and provides effective protection for humans.Especially,chimeric VLPs fused with exogenous epitopes have also been widely used in vaccine development in recent years.Objective:The fusion protein HBHA_109-170-MTP_27-89（HM）constructed by M.tuberculosis cell wall antigen HBHA and fimbriae antigen MTP is displayed on the surface of influenza virus-like particles to construct an influenza virus-like particle-based M.tuberculosis subunit vaccine LV20.And immunogenicity of the subunit vaccine LV20 is detected in the mice model.Methods:1.Construction of shuttle vector Bacmid-HM-MS of insect baculovirus expression vector system（IBEVS）.The p Fast Bac Dual-MS plasmid was obtained by inserting genes of influenza VLPs M1 and HA（transmembrane region and extracellular signal peptide region）into P_PH and p_p10 promoter sites of p Fast Bac Dual plasmid,respectively.The plasmid p Fast Bac Dual-HM-MS was generated by inserting the genes HM into the multiple cloning sites of the vector p Fast Bac Dual-MS.The p Fast Bac Dual-HM-MS plasmid was transformed into DH10Bac^TM competent cells.Then the recombinant Bacmid-HM-MS were verified by blue-white screening and PCR analysis.2.Expression,purification and identification of the chimeric VLPs LV20.Bacmid-HM-MS was extracted and transfected into Expi Sf9?insect cells to obtain the P0baculovirus with low virus titers.Subsequent expansion culture resulted in the P1baculovirus with higher virus titers.And the enhancer reagent was added to express LV20 in large quantities.Ultracentrifugation and sucrose density gradient centrifugation was used to obtain purified LV20,and the purified VLPs were verified by Western blot and electron microscopy to verify the expression and assembly.3.Immunogenicity detection of chimeric VLPs LV20.The mice were immunized three times with PBS,BCG,LV20,LV20 combined with DDA-Poly I:C adjuvant（DP）and HBHA combined with DP adjuvant at 0,2,and 4 weeks.Six weeks after the last immunization,the frequencies of cytokines producing CD4⁺T and CD8⁺T cells following special antigen stimulation were evaluated by flow cytometry.And the production of specific antibody（Ig G,Ig G2c and Ig G1）in mouse serum was detected by ELISA.Results:1.The DNA sequence related to influenza VLPs was synthesized by Beijing Genomics institution and verified by sequencing to obtain the recombinant plasmid p Fast Bac Dual-MS.The p Fast Bac Dual-HM-MS plasmid was successfully constructed through genetic engineering after PCR verification and DNA sequencing verification.The insect baculovirus system vector Bacmid-HM-MS was successfully obtained after p Fast Bac Dual-HM-MS plasmid was transformed into DH10Bac^TM competent cells by blue-white screening and PCR analysis.2.LV20 was expressed in IBEVS and purified by sucrose density gradient centrifugation.The expression of matrix protein M1 of influenza VLPs and tuberculosis protein HM were verified by Western blot.And virus-like particles with diameters between 80-100nm were also observed by electron microscopy.The tuberculosis subunit vaccine LV20 based on virus-like particle vector was successfully prepared.3.Vaccination schedules of LV20 group and LV20 combined with DP adjuvant group induced higher level of antigen specific IL-2 and IFN-γin spleen lymphocytes than BCG group and HBHA combined with adjuvant DP group.LV20 combined with DP adjuvant can induce higher level of specific antibody（Ig G,Ig G2c and Ig G1）in serum than BCG group.Conclusion:We have successfully constructed an influenza VLPs-based M.tuberculosis subunit vaccine LV20.The chimeric VLPs immunized mice can trigger an ideal M.tuberculosis antigen-specific cellular and humoral immune response in the immune system,and is expected to become a new candidate vaccine for tuberculosis.