Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model

Abstract

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R₄Pam₂Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of M. ulcerans Mice receiving either the experimental ER vaccine or Mycobacterium bovis bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (P < 0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

Keywords: Buruli ulcer; Mycobacterium ulcerans; machine learning; mycolactone; polyketide synthase; vaccine.

FIG 1

Analysis of purified recombinant ER protein antigen characteristics and formulation with R₄Pam₂Cys. (A) The presence of recombinant ER protein (∼37 kDa) was monitored by SDS-PAGE at each stage of the purification process. Lane 1, whole-cell lysate (WCL); lane 2, wash 1; lane 3, wash 7; lane 4, ER protein elution (containing 10 μg protein). (B) Western blot using an anti-6×His tag antibody to detect the presence of a single band corresponding to the correct molecular weight of the ER protein in the final eluate. (C) To analyze the formation of antigen-lipopeptide complexes, a constant amount of antigen was mixed with lipopeptide at different protein/lipopeptide molecular ratios in 50 μl of PBS. The size distribution of particles was then analyzed by DLS, with each profile depicting the hydrodynamic radius (in nanometers) of the complexes in each solution. The average radius of each formulation is highlighted in the accompanying table. (D) BALB/c mice (n = 5/group) were vaccinated on days 0 and 21 with R₄Pam₂Cys alone, ER antigen alone, or antigen formulated with R₄Pam₂Cys or vaccinated with BCG on day 0 only. Total serum (IgG) antibody against recombinant ER protein was measured by ELISA after the primary dose (day 20) and 2 weeks after the secondary dose (day 35). Statistical tests were conducted at the 5% significance level. The null hypothesis was rejected if there was a significant difference in mean antibody responses between treatment groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant. The error bars represent the standard deviation (n = 5).

FIG 2

Characterization of infection using a low-dose bioluminescent M. ulcerans strain. (A and B) Representative light camera images of tails from an uninfected BALB/c mouse (A) or at the point of ulceration (16 weeks) following intradermal inoculation with 20 CFU of bioluminescent M. ulcerans (B). (C and D) The same tails were visualized under an IVIS camera to detect and quantify bioluminescence intensity (as photons [p]/s). (E to H) Histological cross-section of an uninfected (E and F) or infected tail tissue (G and H) following hematoxylin & eosin (H&E) and Ziehl-Neelsen (ZN) staining. (I and J) Zoomed images of the regions indicated within the denoted boxes of panel H depict the presence of polymorphonuclear cells (PMNs) and acid-fast bacilli (ZN staining) within tissue.

FIG 3

Development of BU over time after vaccination. (A) Tails of mice were intradermally infected with 20 CFU of bioluminescent M. ulcerans and imaged weekly by IVIS. Representative panels depict the weekly progression of bioluminescent M. ulcerans burden in the tail of an unvaccinated mouse over the course of 16 weeks expressed as photons/s. (B to F) BALB/c mice (n = 10/group) were left unvaccinated (B) or vaccinated on days 0 and 21 with ER antigen alone (C), R₄Pam₂Cys alone (D), ER antigen formulated with R₄Pam₂Cys (E), or on day 0 with BCG (F), followed by challenge on week 5 with bioluminescent M. ulcerans. Threshold bioluminescence (threshold lux) for disease was defined as ≥5 × 10⁵ photons/s, as mice that reached this level typically progressed to the clinical (ethical) endpoint. Mice were classified as diseased if they reached this endpoint within the 24 weeks (vertical lines) following challenge or if their bioluminescence value at week 24 was ≥5 × 10⁵ p/s. Mice were classified as protected if they did not reach this clinical endpoint and their bioluminescence value was <5 × 10⁵ photons/s. The data point depicting when an infected mouse first exhibited bioluminescence at ≥5 × 10⁵ lb/in² is represented with a yellow symbol. The data point denoting when a mouse reached clinical endpoint is represented with a blue symbol. Protected mice with detectable bioluminescence are depicted as gray symbols. (G) Time to bioluminescence measured by IVIS. A survival curve was utilized to analyze the time (in weeks) taken for each BU diseased mouse to first reach threshold bioluminescence of ≥5 × 10⁵ photons/s. The BCG group (upside-down triangle) is labeled in blue, ER plus R₄Pam₂Cys (circle) is red, and ER alone (square), R₄Pam₂Cys alone (triangle), and no-vaccine (diamond) groups are depicted in gray. Statistical tests were conducted at the 5% significance level. The null hypothesis was rejected if there was a significant difference in survival between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

FIG 4

Immune responses after M. ulcerans infection. BALB/c mice (n = 10/group) were left unvaccinated or vaccinated on day 0 and day 21 with ER antigen alone, ER antigen formulated with R₄Pam₂Cys, R₄Pam₂Cys alone, or on day 0 with BCG followed by challenge on day 36 with bioluminescent M. ulcerans. (A) Total serum (IgG) antibody against recombinant ER protein was measured by ELISA after the experimental endpoint. All data points for diseased mice (bioluminescence ≥5 × 10⁵ p/s) are depicted with white symbols. Statistical tests were conducted at the 5% significance level. The null hypothesis was rejected if there was a significant difference in mean antibody responses between treatment groups. The error bars represent the standard deviation. (B) After experimental endpoint was reached, CD4⁺ IFN-γ⁺ T cells were enumerated from the spleen of mice in response to ER protein. The null hypothesis was rejected if there was a significant difference in mean CD4⁺ IFN-γ⁺ T cells between treatment groups. Once the experimental endpoint was reached, cytokines from draining lymph nodes and spleens of M. ulcerans challenged mice were also measured in response to in vitro cell stimulation with recombinant ER protein (Table S1). (C and D) Shown here are TNF produced from immune cells in the spleen (C) and cytokine titers of IFN-γ produced from immune cells in the draining lymph nodes (D). The null hypothesis was rejected if there was no difference in mean cytokine titers between treatment groups. The black bars represent the mean. (E and F) The fold changes of mean cytokine titers from protected mice (bioluminescence, <5 × 10⁵ p/s) and diseased mice (bioluminescence, ≥5 × 10⁵ p/s) over naive mice were compared in the spleen (E) and draining lymph nodes (F). The null hypothesis was rejected if there was a significant difference in mean cytokine titers between treatment groups. All statistical tests were conducted at the 5% significance level. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

FIG 5

Statistical modeling to identify immune parameters (features) associated with vaccine protection. Univariate Cox proportional hazards models were specified for each of the 28 immunological features to test their association with the response variable (time to bioluminescence measured in weeks). The resulting concordance index (CI) scores were obtained, and the six features with a CI of >0.7 were retained. (A and B) The inverse associations of the top two features IFN-γ (A) and IL-2 (B) produced in murine splenocytes at the experimental endpoint are shown. (C) Plot depicting a two-dimensional representation of the top six features that associate with time to bioluminescence from the unsupervised t-SNE. The shapes/colors indicate the two groups identified through K-means clustering of bioluminescence by 8 to 17 weeks or at 18 weeks and beyond (up to 24 weeks). (D) Receiver operating characteristic curve (and corresponding area under the curve) displaying the trade-off between sensitivity and specificity across all thresholds for 1,000 random train-test splits of a logistic regression classifier (90% of observations used for training). The red dotted line depicts the expectation of a random classifier, and the blue line depicts the model performance. (E) Proportion of observations (mice) across treatment groups for each of the classes, both 8 to 17 weeks and 18 to 24 weeks, and those with no detection. (F) Group-specific univariate logistic regression analyses for each of the five treatment groups. Model coefficients were used to determine both the strength and direction of association of each feature with that of each treatment group. Depicted are those features with a corresponding P value of <0.05 (Table S2).