A defined syphilis vaccine candidate inhibits dissemination of Treponema pallidum subspecies pallidum

Abstract

Syphilis is a prominent disease in low- and middle-income countries, and a re-emerging public health threat in high-income countries. Syphilis elimination will require development of an effective vaccine that has thus far remained elusive. Here we assess the vaccine potential of Tp0751, a vascular adhesin from the causative agent of syphilis, Treponema pallidum subsp. pallidum. Tp0751-immunized animals exhibit a significantly reduced bacterial organ burden upon T. pallidum challenge compared with unimmunized animals. Introduction of lymph nodes from Tp0751-immunized, T. pallidum-challenged animals to naive animals fails to induce infection, confirming sterile protection. These findings provide evidence that Tp0751 is a promising syphilis vaccine candidate.

Figure 1. Experimental overview.

(a) Immunization and challenge. Recombinant Tp0751-immunized rabbits (N=3; each immunization was delivered as four 0.1 ml subcutaneous (SC) injections into the shoulders and two 0.04 ml intramuscular (IM) injections into the quadricep muscles) and control animals (N=2) were intradermally challenged with 1 × 10⁶ T. pallidum subsp. pallidum, Nichols strain, at each of 10 sites. On day 14 post challenge (PC), animals were euthanized and organs were harvested for qPCR assessment of bacterial burden; popliteal lymph nodes were extracted and processed for the lymph node transfer. (b) Lymph node transfer. Popliteal lymph nodes were collected from recombinant Tp0751-immunized and control animals and injected into the testes of naive animals. These animals were monitored for evidence of infection using serology, darkfield microscopy and orchitis development over 185 days.

Figure 2. Immunization with Tp0751 decreases severity of and bacterial load in primary lesions.

(a) Lesion ulceration and (b) lesion diameter were monitored over 14 days in control (N=2) and immunized (N=3) animals following T. pallidum challenge. (c) Bacterial load in lesions was assessed at day 10 PC by examining the presence of T. pallidum using darkfield microscopy. Results are presented as median±95% confidence interval between individual immunized (N=3) or control (N=2) animals. Significance was assessed using nonlinear regression and extra sum of squares F-test (**P<0.005 (a,b)) or a two-tailed t-test (*P<0.05 (c)).

Figure 3. Immunization with Tp0751 inhibits T. pallidum dissemination.

T. pallidum burden was evaluated in control (Ct, N=2) and immunized (Im, N=3) animals using quantitative real-time PCR to measure flaA DNA concentrations in lesion biopsies ((a); local infection site) and in disseminated infection sites, (b) bone, (c) liver and (d) spleen. Results were normalized to rabbit gDNA concentration and presented as median±interquartile range (IQR); points correspond to separately extracted samples from each animal. (e) Total disseminated burden was assessed between control animals (n=6 samples, 3 samples per animal) and immunized animals (n=9 samples) across all disseminated sites (bone, blue; liver, orange; spleen, purple) using Mann–Whitney test (**P<0.005).

Figure 4. Tp0751-immunized animals have increased cellular infiltration in lesions.

Lesion biopsies from control (Ct, N=2) and immunized (Im, N=3) animals on day 14 PC were microscopically evaluated to quantify (a) neutrophils, (b) dendritic cells, (c) lymphocytes and (d) differentiated B cells from five fields of view (FOV, × 400) using two vertical sections per biopsy. Shown is the median±interquartile range (IQR); individual points represent one FOV. (e) Total cellular infiltrate composition in lesions (mean±s.e.) was assessed between control animals (n=6 samples, 3 samples per animal) and immunized animals (n=9 samples) across all cell types (green, lymphocytes; purple, neutrophils; red, differentiated B cells; blue, dendritic cells) using a two-tailed t-test (*P<0.05).

Figure 5. Summary of key findings and proposed mechanisms.

(a) Summary of key findings. Tp0751 immunization promotes cellular infiltration into primary lesions and inhibits T. pallidum dissemination to distant organ sites. Lymph node transfer from Tp0751-immunized to naïve animals does not cause T. pallidum infection, confirming sterile immunity. (b) Proposed mechanism for the protection against T. pallidum dissemination conferred by Tp0751 immunization. We propose that Tp0751 immunization induces specific antibody production in primary lesions, facilitating T. pallidum clearance via macrophage opsonophagocytosis. Local Tp0751-specific antibodies would also inhibit T. pallidum bloodstream entry by preventing Tp0751-mediated adhesion to blood vessel components. Collectively, these reactions would prevent T. pallidum from accessing the bloodstream and secondary infection sites.