Article Figures & Data
Figures
- FIG 1
Schematic representation of CD20ECD-6. Each of the six CD20 components represents amino acids 155 to 176 of the murine CD20 ECD. A single glycine spacer is interposed between the CD20 component sequences, and the repeats are covalently linked to the C terminus of maltose-binding protein. Shown below the diagram is the single-letter amino acid sequence for mouse CD20 in the ECD region. The diagram is not to scale.
- FIG 2
Specific antibody responses in mice elicited through immunization with P. acnes. BALB/c mice were randomly assigned to seven groups (n = 15 per group). Mice in groups 2 to 7 were immunized subcutaneously (s.c.) with viable P. acnes 14 (2 × 107 CFU, 2 × 108 CFU, and 2 × 109 CFU) and inactivated P. acnes 14 (2 × 107 CFU, 2 × 108 CFU, and 2 × 109 CFU). The control group (group 1) received 0.2 ml PBS only. One week later, all mice received the same dose of the respective immunogens. Thirty-five days after the first immunization, blood was drawn from the tail to collect serum for enzyme-linked immunosorbent assays (ELISAs). (A) Serum anti-P. acnes antibody titers. (B) Serum anti-A. pleuropneumoniae antibody titers. (C) Geometric mean of anti-P. acnes titers and anti-A. pleuropneumoniae serotype 1 titers. *, P < 0.05; **, P < 0.01; #, P > 0.05.
- FIG 3
Survival rates of mice challenged with A. pleuropneumoniae after immunization with P. acnes. BALB/c mice were randomly assigned to seven groups (n = 10 per group). Mice in groups 2 to 7 were immunized subcutaneously (s.c.) with viable P. acnes 14 (2 × 107 CFU, 2 × 108 CFU, and 2 × 109 CFU) and inactivated P. acnes 14 (2 × 107 CFU, 2 × 108 CFU, and 2 × 109 CFU). The control group (group 1) received 0.2 ml PBS only. At 35 days after the first immunization, the mice in all groups were challenged intraperitoneally (i.p.) with 0.2 ml of A. pleuropneumoniae bacterial suspension containing 4.0 × 107 CFU (equivalent to 5× LD50). The survival rates of all groups were calculated at day 7 postchallenge. Group 1, control group, solid triangles; group 2, viable P. acnes 14 (2 × 107 CFU), open triangles; group 3, viable P. acnes 14 (2 × 108 CFU), hexagons; group 4, viable P. acnes 14 (2 × 109 CFU), circles; group 5, inactivated P. acnes 14 (2 × 107 CFU), crosses; group 6, inactivated P. acnes 14 (2 × 108 CFU), squares; group 7, inactivated P. acnes 14 (2 × 109 CFU), stars. **, P < 0.01.
- FIG 4
Dynamic relationship between level of anti-P. acnes antibody and survival rates. Based on the different IgG titers at the indicated time points, all mice immunized with P. acnes with astragalus polysaccharide adjuvant were divided into eight groups (group 1, PBS control group, solid triangles; group 2, astragalus polysaccharide control group, solid circles; group 3, IgG titer of <100 at day 35, crosses; group 4, IgG titer of 400, open triangles; group 5, IgG titer of 800, open circles; group 6, IgG titer of 1,600, squares; group 7, IgG titer of 3,200, diamonds; group 8, IgG titer of 6,400, stars). Selected mice in each group were challenged with A. pleuropneumoniae serotype 1 (5× LD50). The survival rates of all groups were calculated at day 7 postchallenge. There were no significant differences among groups 3 to 8 (P > 0.05). Also, the survival rates of groups 3 to 8 were significantly higher than those of control groups 1 and 2. *, P < 0.05; **, P < 0.01; #, P > 0.05.
- FIG 5
Indirect immunofluorescence. Rabbit anti-P. acnes hyperimmune serum was prepared (titer, ≈1:40,000). No immunofluorescence was detected in the absence of anti-P. acnes hyperimmune serum (A and B). Positive indirect immunofluorescence signals indicated binding of anti-P. acnes hyperimmune serum with A. pleuropneumoniae serotype 1 (C). This was consistent with the positive control (D).
- FIG 6
Opsonization with anti-P. acnes serum enhances the phagocytosis of A. pleuropneumoniae by J774 murine macrophages. Opsonization with P. acnes hyperimmune rabbit serum increased the efficiency of uptake of A. pleuropneumoniae by J774 macrophages compared to control serum. In contrast, the fold intracellular CFU of group anti-P. acnes hyperimmune rabbit serum (10%) was significantly more than that of negative-control groups, including the preimmune rabbit serum, PBS, and unrelated (GST) hyperimmune rabbit serum groups (P < 0.01). Also, there were no significant differences in the uptake of A. pleuropneumoniae following opsonization with either P. acnes hyperimmune rabbit serum or A. pleuropneumoniae hyperimmune rabbit serum. Results are expressed as fold CFU of intracellular bacteria over the PBS treatment and are representative of three independent experiments performed in triplicate. Bars represent means ± standard deviations. Statistical analysis was carried out using the unpaired two-tailed Student t test. **, P < 0.01; #, P > 0.05.
- FIG 7
Splenic B cell depletion in BALB/c mice. Two groups of BALB/c mice (13/group) were immunized s.c. five times at 2-week intervals with 40 μg CD20ECD-6 immunogen in complete Freund's adjuvant (CFA) for priming immunizations and incomplete Freund's adjuvant (IFA) for boosting immunizations. Mice in the normal control group were immunized with the same dose of adjuvant alone by the same protocol. Ten days after the final immunization, erythrocyte-depleted single-cell suspensions of individual mouse spleens were assessed using flow cytometry to enumerate CD19+ B cell numbers as described in Materials and Methods. Bar graphs represent group means ± standard deviations of B cell numbers (%). **, P < 0.01.
- FIG 8
Antibody responses in B cell depletion mice elicited through immunization with P. acnes. Mice in one B cell depletion model group and one normal control group (n = 10 per group) were immunized s.c. with viable P. acnes (2 × 108 CFU) on two occasions at 1-week intervals. Mice in the other B cell depletion model group were immunized with PBS. At day 35 after the first immunization, blood was drawn from the tail to collect serum for ELISAs. Immune serum reactivity was compared with that of preimmune serum of normal mice. The anti-P. acnes serum titers of B cell depletion mice vaccinated with P. acnes were significantly lower than those of normal mice vaccinated with P. acnes (P < 0.01). **, P < 0.01.
- FIG 9
Survival rates of B cell depletion mice challenged with A. pleuropneumoniae after immunization with P. acnes. Mice in one B cell depletion model group and one normal control group were immunized s.c. with viable P. acnes (2 × 108 CFU) on two occasions at 1-week intervals. Mice in the other B cell depletion model group were immunized with PBS. At day 35 after the first immunization, the mice in all groups were challenged i.p. with 0.2 ml of A. pleuropneumoniae bacterial suspension containing 4.0 × 107 CFU (equivalent to 5× LD50). The survival rates of all groups were calculated on day 7 postchallenge. *, P < 0.05; #, P > 0.05.
Tables
- TABLE 1
Frequencies of CD3, CD4, CD8, and CD19 cells determined by flow cytometry
Group Immunogen Immunization dose (CFU/ml in 0.2-ml suspension) Frequency (%) Ratio CD3+ CD3+ CD4+ CD3+ CD8+ CD3− CD19+ CD3+/CD19+ CD4+/CD8+ 1 PBS 0.2 ml 39.41 ± 0.66 25.36 ± 0.33 11.71 ± 0.38 45.38 ± 0.85 0.87 ± 0.01 2.17 ± 0.06 2 Viable P. acnes 108 39.95 ± 0.33 27.27 ± 0.21a 9.86 ± 0.16a 48.57 ± 0.67a 0.82 ± 0.01a 2.77 ± 0.07a 3 Viable P. acnes 109 40.76 ± 0.40a 28.12 ± 0.23a 9.81 ± 0.20a 49.33 ± 0.43a 0.83 ± 0.01a 2.87 ± 0.03a 4 Viable P. acnes 1010 32.5 ± 0.65a 22.63 ± 0.48a 6.95 ± 0.63a 55.07 ± 0.50a 0.59 ± 0.01a 3.27 ± 0.23a 5 Inactivated P. acnes 108 49.76 ± 0.71a 35.29 ± 0.36a 14.28 ± 0.33a 42.46 ± 0.91a 1.17 ± 0.03a 2.47 ± 0.04a 6 Inactivated P. acnes 109 51.28 ± 0.71a 39.01 ± 0.31a 12.49 ± 0.44a 45.04 ± 0.49 1.14 ± 0.01a 3.13 ± 0.10a 7 Inactivated P. acnes 1010 52.74 ± 0.59a 38.46 ± 0.38a 9.64 ± 0.20a 40.35 ± 0.75a 1.31 ± 0.01a 4.02 ± 0.05a ↵a The mean difference between the immunization group and the control group is significant at the 0.05 level.
- TABLE 2
IgA and IgM titers at different IgG titers
Group Immunogen Anti-P. acnes IgG titer Mean concn (μg/ml) IgA IgM 1 PBS 0 139.142 ± 2.558 6.434 ± 0.150 2 Astragalus polysaccharides 0 140.482 ± 1.258 6.276 ± 0.492 3 P. acnes plus astragalus polysaccharides <100 at 35 days 141.887 ± 4.104 6.205 ± 0.260 4 P. acnes plus astragalus polysaccharides 400 135.798 ± 0.547 5.761 ± 0.126 5 P. acnes plus astragalus polysaccharides 800 139.091 ± 1.531 5.671 ± 0.233 6 P. acnes plus astragalus polysaccharides 1,600 142.240 ± 2.912 6.391 ± 0.458 7 P. acnes plus astragalus polysaccharides 3,200 146.418 ± 2.900a 6.350 ± 0.138 8 P. acnes plus astragalus polysaccharides 6,400 146.597 ± 0.785a 6.338 ± 0.443 ↵a The concentrations of IgA in groups 7 and 8 were significantly higher than those in the two control groups (P < 0.01).
