| Background:Tuberculosis（TB）caused by（Mycobacterium tuberculosis,M.tb）is a major infectious disease worldwide,mainly due to the lack of effective vaccines.Protein subunit vaccine has the advantages of clear composition,easy large-scale production and high safety,but it has low immunogenicity and usually needs usually needs to be used in combination with adjuvants.The tuberculosis subunit vaccine of fusion protein ESAT6-Rv2626c and Mtb10.4-Hsp X constructed in our laboratory（named M4）combines with liposome DP adjuvant（DDA-poly（I:C））can stimulate protective immune response in mice.Polylactic-co-glycolic acid（PLGA）and polyethylene glycol（PEG）have good biocompatibility which are widely used in drug delivery and biomedicine.In order to develop polymer nanoparticles adjuvant with good biocompatibility,we combined them with tuberculosis fusion protein M4 to form a new protein subunit vaccine.Objectives:In this study,PLGA and PEG was condensed to form PLGA-PEG diblock copolymer,loaded with M4 protein PLGA-PEG nanoparticles as the core structure,surface modified poly（I:C）immunostimulatory molecules,trying to develop a good biocompatibility polymer nanoparticles vaccine（named NP/M4）.The adjuvant effect of nanoparticles on M4 was evaluated by studying the immunogenicity of NP/M4.Methods:1.Preparation of nanoparticlesPLGA and NH2-PEG were condensed to form PLGA-PEG diblock copolymers by amidation.The synthesis of the copolymer was verified by FTIR and 1H NMR.PLGA-PEG nanoparticles loaded with M4 protein were prepared by the double emulsion solvent evaporation method（W/O/W）.Using polydopamine as the secondary modification platform,the immunostimulatory molecule poly（I:C）was modified to the surface of nanoparticles by Michael addition reaction to form the final PLGA-PEG-poly（I:C）nanoparticles（NP）.The particle size of PLGA-PEG nanoparticles was determined by multi-angle particle size analyzer;The size and morphological structure of NP were observed by scanning electron microscope;The supernatant was collected by centrifugation,the protein concentration was determined by BCA method,and the entrapment efficiency and drug loading rate of NP were calculated;After the nanoparticles were cracked,the stability of NP coated M4 was verified by SDS-PAGE gel electrophoresis;The effect of NP/M4 on the growth of human mononuclear macrophage line THP-1 cells was detected by MTT method.2.Immunological evaluation of nanoparticlesC57BL/6 female mice of 6-8 weeks were selected as animal models and immunization strategies were grouped as follows:（1）PBS group;（2）BCG group;（3）M4 fusion protein group;（4）DP combined with fusion protein M4 group（DP/M4）;（5）Nano-encapsulated fusion protein M4 group（NP/M4）.Intranasal immunization:Intranasal instillation was performed at 0w and 8w,in which the amount of BCG immunization was 5×106CFU,and the amount of immune protein in M4 group,DP/M4 group and NP/M4 group was 10μg each time.Subcutaneous immunization group:PBS,BCG and NP/M4 groups.The mice were immunized with the same procedure as the intranasal groups.As the control group,the suitable delivery pathway of NP adjuvant was discussed.Six weeks after the last immunization,splenic lymphocytes were isolated with lymphocyte separation solution and stimulated by Hsp X and Rv2626c antigens in vitro.The expression of IL-2 and IFN-γin splenic lymphocytes was detected by flow cytometry（FCM）;The secretion level of IFN-γin splenic lymphocytes was detected by ELISA;The splenic lymphocytes were isolated and cultured in vitro for 3 days,and the proliferation of CD4+T cells stimulated by Hsp X and Rv2626c antigen was detected by Ed U;The level of s Ig A in bronchoalveolar lavage fluid（BALF）and the levels of serum Ig G,Ig G1 and Ig G2c antibodies were detected by ELISA.Results:1.Characterization of nanoparticlesThe synthesis of block copolymer PLGA-PEG was confirmed by FTIR and 1H NMR.The particle size analyzer and scanning electron microscope showed that the diameters of most of the nanoparticles ranged from 200 nm to 500 nm,and the dispersion coefficient（PDI）was less than 0.2.The surface charge of the nanoparticles was-32.97±0.42mv,the entrapment efficiency was 73.25±1.63%,and the drug loading rate was 6.81±0.96%.SDS-PAGE gel electrophoresis confirmed that M4 protein could be stably encapsulated in NP.The results of MTT assay showed that the nanoparticles had no obvious inhibitory effect on THP-1 cell growth.2.Results of immunogenicity test（1）Intranasal immunization with NP/M4 could promote the cellular immunity of M4 protein:FCM results showed that NP/M4 could promote the proliferation of CD4+T cells compared with DP/M4 and M4 group（P<0.01,P<0.001）,and the expression of IL-2 and IFN-γsecreted by CD8+T cells in NP/M4 group was significantly higher than those in M4 and BCG group（P<0.01,P<0.001）.The results of ELISA showed that the secretion of IFN-γof splenic T lymphocytes induced by NP/M4 group was higher than that of M4 and BCG group（P<0.05）.（2）Intranasal immunization with NP/M4 could promote the humoral immunity of M4 protein:the bronchoalveolar lavage fluid（BALF）and serum of mice were detected by ELISA method.Compared with M4 group,NP/M4 group significantly promoted the levels of bronchoalveolar lavage fluid-specific s Ig A,and the serum Ig G,Ig G1 and Ig G2c antibodies（P<0.05）.（3）Comparison of NP/M4 intranasal immunization with subcutaneous immunization:FCM results showed that NP/M4 intranasal immunization activated higher CD4+T cell proliferation ability（P<0.01）;Compared with subcutaneous immunization,mice immunized with NP/M4 intranasally produced higher levels of IL-2 and IFN-γexpressed by CD8+T cell in spleen（P<0.01,P<0.05）.The results of ELISA showed that the level of IFN-γsecreted by spleen lymphocytes induced by intranasal immunization with NP/M4 was higher than that induced by subcutaneous immunization（P<0.01）.NP/M4 induced higher titers of Ig G,Ig G1 and Ig G2c antibodies than M4 in both immune pathways.Conclusion:We have successfully prepared PLGA-PEG-poly（I:C）polymer nanoparticles NP,which encapsulate tuberculosis fusion protein M4 in intranasal immunization mice can effectively induce cellular and humoral immune response and is expected to be a candidate nano-adjuvant for tuberculosis subunit vaccine. |
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