Construction and Evaluation of a Tuberculosis Multistage Vaccine Candidate Based on Recombinant Listeria ivanovii

  Objective  To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model.

  Methods  T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD₅₀) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD₅₀.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI-msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry.

  Results  A recombinant strain named LI-msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD₅₀ value of LI-msv for C57BL/6 mice (i.v.) was 3.3×10⁸ CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ⁺ CD4⁺ and IFN-γ⁺ CD8⁺ T lymphocytes were successfully induced in LI-msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4⁺ and CD8⁺ T cells was significantly higher than those of vector control group and NS control group (P<0.005). Additionally, the frequency of specific TNF-α⁺ CD4⁺ T cell in LI-msv immunized group was significantly higher than that of vector control (P<0.01) and NS control group (P<0.005), and TNF-α⁺ CD8⁺ T cell frequency obviously increased than NS control group (P<0.005).

  Conclusions  A novel multistage TB vaccine candidate expressing TB multistage antigen based on LI was successfully constructed. This vaccine candidate is safe and can induce specific cellular immune response to some extent. It is promising to be further studied as a candidate vaccine against tuberculosis.