Novel anti-idiotype antibody therapy for lipooligosaccharide-induced experimental autoimmune neuritis: use relevant to Guillain-Barré syndrome

Abstract

Campylobacteriosis is a frequent antecedent event in Guillain-Barré syndrome (GBS), inducing high-titer serum antibodies for ganglioside antigens in the peripheral nervous system (PNS). Molecular mimicry between the lipooligosaccharide (LOS) component of Campylobacter jejuni and human peripheral nerve gangliosides is believed to play an important role in the pathogenesis of GBS. Conventional treatment strategies for patients with GBS include plasmapheresis, intravenous immunoglobulin (IVIG), and immunosuppression, which are invasive or relatively ineffective. In this study, we used our animal model of GBS, in which Lewis rats were immunized with GD3-like LOS isolated from C.jejuni. The animals developed anti-GD3 ganglioside antibodies and manifested neuromuscular dysfunction. To develop novel therapeutic strategies, we treated the animals by intraperitoneal administration of an anti-GD3 antiidiotype monoclonal antibody (BEC2) that specifically interacts with the pathogenic antibody. The treated animals had a remarkable reduction of anti-GD3 antibody titers and improvement of motor nerve functions. The results suggest that ganglioside mimics, such as antiidiotype antibodies, may be powerful reagents for therapeutic intervention in GBS by neutralizing specific pathogenic antiganglioside antibodies.

Fig. 1

Isolation scheme for LOS_GD3 from crude LOS fraction. The steps involved in the isolation and purification of LOS_GD3 are shown. For details of each step, see Materials and Methods. LS, a major LOS component characterized as a slow-migrating band based on the TLC of CTxb overlay; LF, a minor LOS component characterized as a fast-migrating band based on the TLC of CTxb overlay. Fractions 1–4: Stepwise elution from a silica gel column with the following solvents: 1) n-propanol, 2) n-propanol:H₂O (75:20 v/v), 3) n-propanol: H₂O:triethylamine (75:20:5 v/v/v), and 4) n-propanol: H₂O:triethylamine (60:20:20 v/v/v). Fractions 5–8: Stepwise eluting from an mAb R24 affinity column with the following solvents: 5) washing solution 1 (10 mol of 1 and 10 mM PBS ), 6) washing solution 2 (50 mM PBS), 7) eluting solution 1 (5 ml of 50 mM diethylamine/HCl buffer, pH 8.6, containing 0.1 M NaCl), and 8) eluting solution 2 (5 ml of 50 mM diethylamine/HCl buffer, pH 10.5, containing 0.1 M NaCl).

Fig. 2

Experimental schedule of animal treatments. The timing of treatment and performance is shown between 0 and 18 weeks in the experimental period. Sixteen experimental animals were divided into three inoculum-treated groups (n = 4, each): 1) LOS_GD3, 2) LOS_GD3/BEC2, 3) vehicle, and 4) vehicle/BEC2. The remaining four animals were included as the untreated control group (n = 4).

Fig. 3

TLC of purified LOS_GD3 from silica gel column and mAb R24 affinity column chromatography. A: LOS_GD3 was detected by TLC overlay at 10 μg/ml of mAb R24 IgG; lane 1, GD3 standard; lane 2, fraction 1; lane 3, fraction 2; lane 4, fraction 3; lane 5, fraction 4, lane 6, GD3 standard; lane 7, fraction 5; lane 8, fraction 6; lane 9, fraction 7; lane 10, fraction 8; lane 11, GD3 standard; lane 12, purified LOS_GD3. B: LS and LF were detected by TLC overlay at 1:1,000 dilution of CTxb; lane 1, GM1 standard; lane 2, fraction 1; lane 3, fraction 2; lane 4, fraction 3; lane 5, fraction 4, lane 6, GD3 standard; lane 7, fraction 5; lane 8, fraction 6; lane 9, fraction 7; lane 10, fraction 8; lane 11, GM1 standard; lane 12, purified LOS_GD3. Fractions 1–8 are shown in Figure 1.

Fig. 4

Dose-dependent inhibition of binding of mAb R24 to GD3 by BEC2. A: BEC2 was first incubated with MAb R24 at room temperature for 30 min at various concentrations as indicated on the horizontal axis. Subsequently, an aliquot (100 μl) was added to each well, and each well of the ELISA plate was coated with 0.1 μg GD3. The remaining binding activity of mAb R24 was determined by ELISA, as shown in Materials and Methods. Values are means ± SD, n = 6 individual experiments. B: Mean plasma concentration–time profiles following i.p. single dosing with 1 mg kg^–1, 5 mg kg^–1, or 10 mg kg^–1 bBEC2. The experiment at each dose was performed on three rats, and values are means ± SD. C: Simulation of concentration–time course of 5 mg kg^–1 bBEC2 once per week for 8 weeks. The simulation was performed with values of t_1/2β and AUC for 5 mg kg^–1 bBEC2, as shown in Table I.

Fig. 5

A–D: Changes of serum level of the anti-GD3 antibody and the anti-LOS_GD3 antibody of rats. According to the experimental schedule shown in Figure 2, animal serum samples were obtained at the specified times to test activities of anti-GD3 antibody (solid circles) and anti-LOS GD3 antibody (open circles) using ELISA. Values are means ± SD for four animals.

Fig. 6

Effect of BEC2 on NCV and rotarod performance of animals undergoing treatment. A: Effect of BEC2 on NCV at week 0 and week 16 after primary immunization by LOS_GD3. NCVs were measured at week 0 and at week 16 for each group of animals; LOS_GD3, LOS_GD3/BEC2, vehicle, vehicle/BEC2, and control (nontreatment). Values are means ± SD for four animals. The values were analyzed by one-way ANOVA followed by Tukey's multiple-comparison tests (<0.01). The asterisk denotes statistical significance (*P < 0.01) estimated by Dunnet's multiple-comparisons test. B: Effect of BEC2 on motor performance on the rotarod test. At 17 weeks after primary immunization, the rotarod test was performed for five experimental animal groups: LOS_GD3, LOS_GD3/BEC2, vehicle, vehicle/BEC2, and control (nontreatment). Values are means ± SD for four animals. The data values were analyzed by one-way ANOVA followed by Tukey's multiple-comparisons test (<0.01). The asterisk denotes statistical significance by Dunnet's multiple-comparisons test (*P < 0.01).

Fig. 7

A: Examples of toluidine blue-stained lumbar motor neurons (a,b) and sciatic nerves (c,d). a And c are from a control rat (non-treatment); b and d are from an LOS_GD3-treated rat. EM is from sciatic nerves from a control rat (e) and an LOS_GD3-treated rat (f). Arrows show the large cells in the anterior horn. B: Immunofluorescence imaging of NMJ was performed by following primary antibodies and BTx. mAb R24 (a), BTx (b), or NF (c), each diluted appropriately by 1% BSA/PBS buffer containing 0.1% Triton X-100 at 4°C, and (d) merging of R24 mAb and BTx images. Scale bars = 100 μm in Aa,b; 10 μm in Ac,d; 2 μm in Ae,f.

Fig. 8

Time course of inhibitory effect on spontaneous muscle action potential frequencies in spinal cord–muscle cocultured cells. The frequencies were recorded every 5 sec. Arrows show addition of 10 μl rat serum and washing out. Each serum sample was obtained from four rats, in three groups. A: LOS_GD3. B: LOS_GD3 serum as shown in A was subjected to immunoabsorption by GD3. C: LOS_GD3/BEC2 at the endpoint of the experiment. One of the four sera was used for addition to the assay system for spontaneous muscle action potential activity. This absorbed serum (B) was examined in the assay system for spontaneous muscle action potential activity.