Fig 1.
Immunogenicity of recombinant protein/EM048 formulation in comparison to commercially available adjuvants for mice.
Groups of five BALB/c mice were immunized three times in three week intervals with 20 μg of rMUL2232 (A) or rMUL3720 (B) in PBS or formulated with Alum, Sigma Adjuvant or EM048. Serum three weeks after every immunization (I1, I2 and I3) was analysed in ELISA on the respective recombinant protein. Depicted are individual endpoint IgG titers as determined in one single ELISA, the mean (line) ± standard deviation. Values that are zero are not depicted but were included for statistical analysis. Overall statistical significance was calculated by Kruskal-Wallis test per immunization time point (A: PI1 = 0.0018, PI2 = 0.0372 and PI3 = ns.; B: PI1 = 0.0095, PI2 = 0.0011, PI3 = 0.0041.). Individual statistical differences between groups were assessed by the Dunn procedure and are depicted if detected (* p ≤ 0.05; ** p ≤ 0.01).
Fig 2.
Assessment of Immunoglobulin G subclasses and the longevity of antibody responses induced by immunization.
Immunoglobulin G (IgG) subclasses were determined in serum collected three weeks after the third immunization with rMUL2232 (A1) and the indicated adjuvant or rMUL3720 (B1) and the indicated adjuvant. ELISA on recombinant protein was performed with secondary antibodies specific for four IgG subclasses: IgG1 (black), IgG2a (dark grey), IgG2b (light grey), IgG3 (very light grey). Depicted are the mean individual endpoint IgG titers determined in one single ELISA (bar), and the standard deviation (error bar). Overall statistical significance was calculated by Kruskal-Wallis test (A1: PIgG1 = 0.0265., PIgG2a = 0.0205, PIgG2b = ns., PIgG3 = 0.0276; B1: PIgG1 = 0.0034., PIgG2a = 0.0017, PIgG2b = 0.0048, PIgG3 = ns.). Individual statistical differences between groups were assessed by the Dunn procedure and are depicted if detected (* p ≤ 0.05; ** p ≤ 0.01). The ratio of IgG2a to IgG1 was determined for individual animals accordingly (A2 and B2). Depicted are the individual values, the mean IgG2a to IgG1 ratio (bar) and the standard deviation (error bar). Overall statistical significance was calculated by Kruskal-Wallis test (A2: P = ns.; B2: P = 0.0024). Individual statistical differences between groups were assessed by the Dunn procedure and are depicted if detected (** p ≤ 0.01). Total IgG titers in individual immunized mice three weeks (t1) and six months (t2) after the third immunization were compared by Western blotting on M. ulcerans lysate. Depicted are individual Western blot endpoint titers and the median per group (line) for rMUL2232 immunized animals (A3) and rMUL3720 immunized animals (B3). Statistical significance was assessed by Mann-Whitney test and is depicted if detected (* p ≤ 0.05; ** p ≤ 0.01).
Fig 3.
Cross reactivity of the immune sera with M. ulcerans.
(A) Groups of five BALB/c mice (m1 –m5) were immunized three times in three week intervals with 20 μg of rMUL2232 formulated with Alum, Sigma Adjuvant or EM048. Serum three weeks after every immunization (I1, I2 and I3) was analysed by Western blotting on M. ulcerans lysate. Monoclonal anti-MUL_2232 antibody (mAb) served as positive control, pre-bleed (pb) serum or no primary antibody (nc) as negative controls. (B) Sera from three weeks after the third immunization with rMUL2232 and indicated adjuvant were used for indirect immunofluorescence staining on paraffin embedded M. ulcerans bacteria with an Alexa488 labelled secondary antibody. Pre-bleed serum did not stain the bacteria. Sera of immunized mice (a mix of sera from five individual mice per group) did reveal surface staining similar to the staining achieved with anti-MUL_2232 monoclonal antibody (mAb).
Fig 4.
Evaluation of the protective potential of immunization with rMUL3720/EM048 formulation in a M. ulcerans infection mouse model.
Groups of eight BALB/c mice were immunized twice with 20 μg of rMUL3720/EM048 or PBS alone as infection control. Three weeks after the last immunization mice were challenged with a high dose or a low dose of M. ulcerans (inoculum) into the left hind foot pad. Infection was followed by measuring foot pad thickness with a caliper (A) until mice were euthanized at day 63 after infection (high dose, H1) or at day 87 after infection (low dose, H2). Depicted is the mean foot pad thickness (diamond/circle) ± standard deviation of the differently immunized groups. (B) Bacterial load in infected foot pads was determined by qPCR for six mice per group. Depicted are individual measurements as genome copies per foot pad, the mean (line) ± standard deviation.
Fig 5.
Histopathological evaluation of mouse footpads following M. ulcerans infection.
Histological sections of foot pads from M. ulcerans-infected mice were stained with Haematoxylin/Eosin (A, B1—B7) or Ziehl-Neelsen/Methylene blue (ZN) (C1—C4, D1—D4). Mice challenged with a high dose of M. ulcerans developed typical signs of infection in the mouse foot pad model until day 63 after infection. Compared to a control foot pad (A) the infected foot pad of a representative immunized mouse (B1) showed necrosis and infiltration interspersed with AFB at the ankle (B2, B3) as well as at the base of the foot (B6, B7). Oedema was marked on top of the foot pad (B4) where bacteria were also found (B5). Immunized mice (C1) as well as control mice (D1) challenged with a lower dose of M. ulcerans developed strong infection foci until day 87 after infection. AFB appeared as big, dense clumps (C2) or in close association with infiltrating cells (D2) towards the heel of the infected foot pads as well as in the middle of the foot (C1, D1). AFB were also present in oedematous tissue in the upper half of the foot (D3) and appeared in filamentous organization (C3).
Fig 6.
Evaluation of the booster effect of M. ulcerans infection on the pre-existing antibody response in rMUL3720/EM048 immunized and subsequently infected mice.
(A1) Serum of six BALB/c mice immunized with rMUL2232/EM048 prior to infection (before infection) with M. ulcerans was compared to serum of the same animals after 42 days of infection (after infection) in ELISA on rMUL2232. Control immunized animals had received PBS/EM048 or only PBS prior to infection. Depicted are individual endpoint IgG titers as determined in one single ELISA, the group mean (line) ± standard deviation. Statistical significance was calculated by the Student’s t- test (** p ≤ 0.01). (A2) Serum of eight BALB/c mice immunized with rMUL3720/EM048 prior to infection with M. ulcerans was compared to serum of the same animals 63 days after infection with M. ulcerans bacteria in ELISA on rMUL3720. Control immunized animals (control) had only received PBS prior to infection. Depicted are individual endpoint IgG titers as determined in one single ELISA, the mean (line) ± standard deviation. Statistical significance was calculated by the Student’s t- test (**** p ≤ 0.0001). (B) Serum of six BALB/c mice (m1 –m6) immunized with rMUL3720/EM048 prior to infection with M. ulcerans (a) was compared to serum of the same animals after 42 days of infection (b) by Western blotting on M. ulcerans whole cell lysate. Monoclonal anti-MUL_3720 antibody (mAb) served as positive control, pre-bleed (pb) serum or no primary antibody (nc) as negative controls.