A Listeria ivanovii balanced-lethal system may be a promising antigen carrier for vaccine construction

Abstract

Expressing heterologous antigens by plasmids may cause antibiotic resistance. Additionally, antigen expression via plasmids is unstable due to the loss of the plasmid. Here, we developed a balanced-lethal system. The Listeria monocytogenes (LM) balanced-lethal system has been previously used as an antigen carrier to induce cellular immune response. However, thus far, there has been no reports on Listeria ivanovii (LI) balanced-lethal systems. The dal and dat genes from the LI-attenuated LIΔatcAplcB (LIΔ) were deleted consecutively, resulting in a nutrient-deficient LIΔdd strain. Subsequently, an antibiotic resistance-free plasmid carrying the LM dal gene was transformed into the nutrient-deficient strain to generate the LI balanced-lethal system LIΔdd:dal. The resultant bacterial strain retains the ability to proliferate in phagocytic cells, as well as the ability to adhere and invade hepatocytes. Its genetic composition was stable, and compared to the parent strain, the balanced-lethal system was substantially attenuated. In addition, LIΔdd:dal induced specific CD4⁺ /CD8⁺ T-cell responses and protected mice against LIΔ challenge. Similarly, we constructed an LM balanced-lethal system LMΔdd:dal. Sequential immunization with different recombinant Listeria strains will significantly enhance the immunotherapeutic effect. Thus, LIΔdd:dal combined with LMΔdd:dal, or with other balanced-lethal systems will be more promising alternative for vaccine development.

FIGURE 1

The construction process of the recombinant plasmids and strains. (A) Targeting plasmid pCW619‐LM dal and pCW619‐LI dal, derived from plasmid pCW619, was transformed into LMΔ and LIΔ, respectively. Then dal gene of LMΔ and LIΔ was deleted by homologous recombination. Consequently, the targeting plasmid pCW619‐LM dat and pCW619‐LI dat, also derived from the plasmid pCW619, was transformed into dal gene‐deleted LMΔ and LIΔ, respectively. After homologous recombination, the dat gene of the strains was deleted, thus LMΔdd and LIΔdd were constructed. (B) Antibiotic resistance‐free pCW‐GFP‐LM dal plasmid, derived from plasmid pCW‐GFP, which harboured LM dal gene under the control of LLO promoter, was transformed into LMΔdd and LIΔdd, resulting in LMΔdd:dal and LIΔdd:dal.

FIGURE 2

Growth curve and genetic stability of constructed strains. (A) LIΔdd were grown in BHI broth in the presence of gradually increased concentration of D‐alanine (100, 200, 300 and 400 μg/ml) at 37°C. (B) LIΔdd and LIΔ were grown in BHI broth in the presence or absence of exogenous D‐alanine (400 μg/ml) at 37°C. LIΔdd:dal grown in ordinary BHI broth at 37°C. (C) LMΔdd and LMΔ were grown in BHI broth in the presence or absence of exogenous D‐alanine (100 μg/ml) at 37°C. LMΔdd:dal grown in ordinary BHI broth at 37°C. Three independent experiments were repeated. Data were accumulated from three experiments and expressed as means ± SEM. p values were determined by LSD method. *p < 0.05; **p < 0.01 and ***p < 0.001 were not marked in growth curve. (D) The lengths of PCR products of the 5, 10, 15, 20 and 25st generation of LIΔdd:dal amplified by primers LI‐orfBAldh‐F/R, HomoLIdal‐F/R, HomoLIdat‐F/R and LMdal‐F/R were as expected. (E) The lengths of PCR products of the 5, 10, 15, 20 and 25st generation of LMΔdd:dal amplified by primers LM‐orfBAldh‐F/R, HomoLMdal‐F/R, HomoLMdat‐F/R and LMdal‐F/R were as expected.

FIGURE 3

The abilities of proliferation, adhesion and invasion to cells. (A) Intracellular proliferation of LIΔ, LIΔdd, LIΔdd+D (400 μg/ml of D‐alanine) and LIΔdd:dal in RAW264.7 cells at an MOI of 20:1. Intracellular proliferation was expressed by fold changes (Y axis). Fold change was calculated as: number of intracellular bacteria at each time point vs. number of bacteria invaded in cells (number of intracellular bacteria at 2 h post infection). (B) LSCM imaging to show the intracellular growth of the strains in RAW264.7 cells (MOI was 20:1). Green: GFP‐expressing bacteria; Blue: DAPI‐stained cell nucleus; The scale bar in images were 20 μm. (C) The adhesion ability of LIΔ, LIΔdd, LIΔdd+D (400 μg/ml of D‐alanine) and LIΔdd:dal to Hepa1‐6 cells at a MOI of 10:1. Adhesion rate was expressed as the percentage of the bacteria adhered to Hepa1‐6 cells vs. inoculated bacteria at 1 h after infection. (D) The invasion ability of LIΔ, LIΔdd, LIΔdd+D (400 μg/ml of D‐alanine) and LIΔdd:dal to Hepa1‐6 cells at a MOI of 10:1. Invasion rate was expressed as the percentage of the intracellular bacteria 1 h post gentamycin treatment vs. initial inoculated bacteria. The box plots depict mean ± SEM and the whiskers min‐max values from more than three independent experiments. *p < 0.05; **p < 0.01 and ***p < 0.001 (Kruskal–Wallis test).

FIGURE 4

The growth curve and genetic stability of LIΔdd:dal in vivo in mice liver and spleen. Groups (36 mice in each) of C57BL/6J mice were injected in the tail vein with maximum safe dose of LIΔdd:dal (5 × 10⁸ CFU), and six mice of each group were sacrificed at 1, 2, 3, 5, 7 and 14 dpi. The CFU in the mice liver or spleen was determined by plating on both BHI and D‐BHI plates. (A) The CFU in the mice liver. (B) The CFU in the mice spleen. Results were expressed as means ± SEM per group. p values were determined by Kruskal‐Wallis test. The detection limit was 20 CFU/spleen and 50 CFU/liver. (C) The PCR products of LIΔdd:dal isolated from mouse liver that amplified by primers LI‐orfBAldh‐F/R, HomoLIdal‐F/R, HomoLIdat‐F/R and LMdal‐F/R. (D) The PCR products of LIΔdd:dal isolated from mouse spleen that amplified by primers LI‐orfBAldh‐F/R, HomoLIdal‐F/R, HomoLIdat‐F/R and LM‐F/R.

FIGURE 5

ILO‐specific T cells cytokines in the mice spleen. (A, B) Percentages of IFN‐γ‐secreting cells in the T cell. (C, D) Percentages of TNF‐α‐secreting cells in the T cell. (E, F) Percentages of IL‐2‐secreting cells in the T cell. (G) Daily change in body weight of immunized mice after LI strains infection. (H) Survival rate of immunized mice infected with LI strains. Data were expressed as means ± SEM per group of 10 mice. *p < 0.05 (Kruskal–Wallis test).