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Immunological evaluation of subunit vaccine prepared by combining specific antigen Rv3117 of Mycobacterium tuberculosis with DDA/MPL adjuvant

YE Juan1,2, GAO Meng-zhe3, ZHANG Shu-lin2, CHEN Li1, SUN Zhan-qiang4   

  1. 1.Key Laboratory of Medical Molecular Virology， Ministry of Education and Ministry of Health， Shanghai Medical College， Fudan University， Shanghai 200032, China； 2.Department of Immunology and Microbiology, Basic Medicine Faculty of Shanghai Jiao Tong University, Shanghai 200025, China; 3.Medical Experimental Center， Zhengzhou University， Zhengzhou 450006, China； 4.School of Life Science， Fudan University， Shanghai 200433, China
  • Online:2015-01-28 Published:2015-01-29
  • Supported by:

    National Natural Science Foundation of China, 81271794; Science and Technology Support Program of Science and Technology Commission of Shanghai Municipality, 12441903300

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Abstract:

Objective To construct a TB subunit vaccine (Rv3117/DDA/MPL) by combining specific antigen Rv3117 of Mycobacterium tuberculosis (M. tuberculosis) with the adjuvant of dimethyl dioctadecyl ammoniumbromide (DDA) /monophosphoryl lipid A (MPL) and to evaluate the immune responses after the bacillus calmetteguerin (BCG)-Prime in C57BL/6 mice. Methods The molecular cloning method was adopted to construct a recombinant plasmid pET32a-Rv3117 and the specific recombinant Rv3117 protein of M. tuberculosis expressed in Escherichia coli BL21(DE3) PLysS was obtained. Then the antigen Rv3117 was induced and purified. The endotoxin was removed by Triton X-114 phase separation method and the subunit vaccine was prepared by fully emulsifying M.tuberculosis specific antigen Rv3117 with DDA/MPL adjuvant. The C57BL/6 mice were randomly divided into the control group (without any treatment), PBS group (immunized with PBS), BCG group (only immunized with BCG), BCG+DDA/MPL group (immunized with DDA/MPL adjuvant twice after being immunized with BCG for 2 weeks), and BCG+Rv3117/DDA/MPL group (immunized with subunit vaccine Rv3117 twice after being immunized with BCG for 2 weeks) (n=5/group). The humoral immunity and cellular immunity were evaluated by the enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot Assay (ELISPOT), respectively. Results The specific antigen Rv3117 of M. tuberculosis was successfully coloned and purified. The results of ELSPOT and ELISA indicated that level of interferon γ (IFN-γ) produced by T lymphocytes of mice of the BCG+Rv3117/DDA/MPL group was significantly higher than that of the control group, PBS group, BCG group, and BCG+DDA/MPL group (P<0.05) after being stimulated by the purified protein derivative (PPD) of tuberculin (10.0 μg/mL). The levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-12p70 of culture supernatants of the BCG+DDA/MPL group were significantly higher than those of the control group and PBS group (P<0.05) and the total level of IgG and IgG1 was also higher. The levels of above indexes of the BCG+Rv3117/DDA/MPL group were significantly better than those of the BCG+DDA/MPL group (P<0.05). Conclusion The TB subunit vaccine Rv3117/DDA/MPL can strengthen the humoral immunity and cellular immunity of BCG in C57BL/6 mice. The specific antigen Rv3117 of M. tuberculosis is expected to be a novel candidate antigen for designing the TB vaccine and diagnosing the TB.

Key words: Mycobacterium tuberculosis, Rv3117, subunit vaccine, humoral immunity, cellular immunity