Dual Immunization with Lipoprotein Tp0663 and Flagellin FlaB3 Offers Augmented Protection against Treponema pallidum in Mice

Abstract

Syphilis is a significant multistage sexually transmitted infection caused by the bacterium Treponema pallidum. The pathogenesis of this pathogen remains inadequately understood, impeding the progress of syphilis vaccine development. Our prior study has demonstrated the potential of the Tp0663 protein as a viable candidate for a vaccine against T. pallidum. In the present study, we sought to explore the protective response of dual immunization using two different antigenic entities (i.e., flagellin FlaB3 and lipoprotein Tp0663) against T. pallidum in a murine model. Our investigation revealed that FlaB3 + Tp0663 can elicit robust humoral and cellular immune responses. In addition, the FlaB3 + Tp0663 vaccine demonstrated a notable reduction in the Treponemal burden within various anatomical sites of infected mice, including the blood, brain, liver, lymph nodes, spleen, and testicles. It is worth noting that the FlaB3 + Tp0663 vaccine suppressed the dissemination of T. pallidum in C57BL/6 mice. The findings demonstrate that T. pallidum flagellin FlaB3 may augment the immunoprotection of Tp0663. This represents a valuable practical perspective and offers insights into developing a syphilis vaccine.

Figure 1

Detection of serum IgG in mice immunized with recombinant Tp0663 protein or flagellin FlaB3. C57BL/6 mice were immunized with 100 μg of recombinant Tp0663, FlaB3, or 100 μg of recombinant Tp0663 mixed with 100 μg of recombinant FlaB3. The control mice were immunized with PBS. Serum samples were collected from the mice’s tail veins at 0, 2, 4, and 6 weeks, and the IgG antibody levels were determined by indirect ELISA. Each set of data is based on measurements derived from three mice.

Figure 2

FlaB3 + Tp0663-induced specific T cell responses in the spleen. Splenocytes were isolated from three mice of each group at day 30 postimmunization and assayed for Tp0663-specific CD4⁺ T cell response. (A) Tp0663-specific CD4⁺ T cell response was determined by intracellular IL-4 and IFN-γ staining. (B) Statistical analysis of Tp0663-specific CD4⁺ T cell response of three mice in each group. Data are presented as the means ± SD. Statistical significance was determined using Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Cytokine productions by splenocytes isolated from immunized mice after restimulation with Tp0663. Splenocytes collected from immunized and nonimmunized mice were detected using mice IL-4 (A) and IFN-γ (B) ELISA kit according to the manufacturer’s instructions. The results are expressed as the mean ± SD from three individual mice in each group. Each experiment was performed in three independent experiments (ns = not significant, *P < 0. 05, **P < 0. 01, ***P < 0. 001).

Figure 4

SI of Tp0663-specific splenic T lymphocyte proliferation. Spleen cells collected from immunized and nonimmunized mice were restimulated with 10 μg of Tp0663. After culturing for 44 h, T lymphocyte proliferations were detected using a CCK8 kit according to the manufacturer’s instructions. SI is presented as the A450 value of the stimulation group/control group. The results are expressed as the mean ± SD from three individual mice in each group. Each experiment was performed in three independent experiments (*P < 0. 05, **P < 0. 01).

Figure 5

Immunization with FlaB3 + Tp0663 inhibited T. pallidum dissemination. Spirochete numbers were evaluated in PBS, FlaB3, Tp0663, or FlaB3 + Tp0663-immunized animals using qRT-PCR to measure flaA DNA concentrations in lesion biopsies. Bacterial burdens in the blood (A), brain (B), liver (C), lymph node (D), spleen (E), testicle (F), and rectum (G) at day 30 postinfection. Results were normalized within each tissue type based on the concentration of mouse gDNA and presented as the median ± interquartile range. Significance was assessed using Student’s t test (ns = not significant; P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001). Points corresponding to two separately extracted tissue samples from each mouse organ (six total points in each group) were analyzed for reproducibility.

Figure 6

Detection of T. pallidum by immunohistochemistry in tissues of FlaB3 + Tp0663-immunized mice. Liver (A), spleen (B), and testicle tissues (C) of PBS, FlaB3, Tp0663, or FlaB3 + Tp0663-immunized mice were sectioned and stained with S–P immunohistochemistry 30 days after infection with T. pallidum. The UltraSensitiveSP IHC Kit, with a rabbit anti-T. pallidum was used as the first antibody to detect T. pallidum. Mice immunized with PBS were used as controls. Those with brown granules contain T. pallidum.