Clinical data of the GBS and MFS patients are presented in Table . Sera from all patients reacted with several glycolipid antigens, although the fine specificity and isotype distribution of the antibodies differed between patients (Table ). In addition, all patients had antibodies to C. jejuni LPS (Table ) and protein antigens (data not shown). Detailed electrophysiological data were available only for patients GB17 and MF8. Patient GB17 had an axonal neuropathy, with predominant distal weakness. Motor nerve conduction studies in patient MF8 revealed no abnormalities.
The presence of GM1 and GQ1b-like epitopes on the GBS- and MFS-associated strains was studied with ganglioside-binding ligands. GB18 LPS reacted strongly with cholera toxin, indicating a GM1-like epitope, while MF6 and MF8 LPS showed reactivity with the antidisialosyl antibody, demonstrating a GQ1b-like epitope (data not shown). In addition, both GB17 LPS and MF6 LPS reacted with sera with anti-GM1 and anti-GA1 reactivity and GB17 LPS reacted strongly with peanut agglutinin, a ligand specific for GA1, indicating the presence of GA1-like epitopes in these strains. In contrast, Penner O:3 LPS did not show reactivity to any of the ganglioside-binding ligands (data not shown).
To exclude the possibility that the observed antibody responses resulted from an intercurrent infection of the animals with C. jejuni and not from the immunization procedure, stool cultures for C. jejuni were performed at the start of the immunization procedure and when the animals were bled. Stool cultures remained negative during the immunization period. Furthermore, antiganglioside and anti-LPS reactivity could not be detected in preimmune serum samples. Immunization of rabbits with C. jejuni LPS from neuropathy patients resulted in high-titer anti-LPS and antiglycolipid antibodies, while serum from animals that were immunized with LPS from the O:3 serostrain contained only anti-LPS but no antibodies against any of the purified glycolipids (Table ). The adjuvant controls did not shown any reactivity to LPS and only a slight elevation of anti-GA1 reactivity (Table ). All antiglycolipid responses were confirmed by TLC (data not shown).
Serum antiglycolipid and anti-LPS antibody titers in rabbits immunized with C. jejuni LPSa
The specificity of antiganglioside antibodies in the rabbits resembled the observed specificity in the patients from whom the strains were cultured. Serum from patient GB17 mainly exhibited antibody reactivity to GA1, as did the rabbits that were immunized with LPS from strain GB17. The other GBS patient, GB18, had a higher anti-GM1 titer than anti-GA1 titer. Sera from the two animals that were immunized with LPS from GB18 displayed the same pattern of antiglycolipid reactivity as that of patient GB18. Both rabbits that were immunized with LPS from MF6 mounted an immune response against GQ1b. One animal had only anti-GQ1b IgM antibodies, while the other had only anti-GQ1b IgG (Table ). In addition, both animals had high titers of anti-GA1 antibodies, which was not found in the patient from whom MF6 was isolated but was in accordance with the presence of a GA1-like structure in MF6 LPS. Immunization with MF8 LPS did not result in an anti-GQ1b response, although one of the animals had an anti-GM2 IgG response. Anti-LPS reactivity in these two rabbits was also much weaker than that in animals immunized with LPS from other C. jejuni strains.
The kinetics of antiglycolipid and anti-LPS antibody responses differed in animals, depending on the source of the LPS. In both animals that were immunized with LPS from GB17, a strong IgG response against GA1 and GB17 LPS could already be observed 2 weeks after the first immunization (Fig. A and B). In contrast, animals that were immunized with GB18 and MF6 LPS showed a gradual increase of the antiganglioside and anti-LPS IgG antibody reactivity during the immunization period (Fig. C to F). Remarkably, in one animal that was immunized with GB18, the anti-GM1 and anti-GA1 IgG responses showed different kinetics. After an initial increase, the anti-asialo-GM1 reactivity decreased, while the anti-GM1 reactivity continued to increase until the end of the immunization period (Fig. D).
FIG. 1 Kinetics of anti-LPS and antiglycolipid reactivity in rabbits immunized with C. jejuni LPS. Sequential serum samples were tested for IgM and IgG reactivity against C. jejuni LPS and glycolipids. (A and B) For rabbits 1 and 2, anti-GM1, anti-GA1, and anti-GB17 (more ...)
On Western blots, the sera from all LPS-immunized rabbits reacted with the LPS they had been injected with. Silver staining revealed a band that comigrated between 11 and 15 kDa (Fig. ), consistent with earlier observations for C. jejuni
). Serum samples predominantly reacted with this low-molecular-weight fraction of the LPS. We did not observe ladder-like patterns as described for C. jejuni
), although in some instances single bands with higher molecular weights were present. Sera from some rabbits reacted not only with the LPS with which they were immunized but also with LPS from other C. jejuni
strains. Serum from animals immunized with MF6 also reacted strongly with LPS from GB17 and vice versa (Fig. A, lanes 2 and 4, and C, lanes 2 and 4). Both strains have Penner serotype O:4. Only serum from animals that were immunized with Penner O:3 had a strong response to Penner O:3 LPS (Fig. E).
FIG. 2 Strain-specific anti-LPS responses detected by Western blotting. C. jejuni LPS were subjected to electrophoresis and silver staining (lanes 1) or transferred to nitrocellulose and overlaid with sera from immunized rabbits (lanes 2 to 6). (A) GB17 LPS (more ...)
To prove that the anti-LPS antibodies and antiganglioside antibodies are cross-reactive, we conducted a series of depletion experiments with C. jejuni LPS conjugated to Sepharose CL4B beads. The results from experiments on cross-reactive anti-GQ1b and anti-MF6 LPS antibodies are summarized in Fig. . Anti-GQ1b and anti-MF6 LPS reactivity in an MF6-immunized rabbit could be inhibited only by incubation of serum with Sepharose beads conjugated to MF6 LPS and not with LPS from GB18 and Penner O:3 (Fig. A and C). The observed specific pattern of cross-reactivity was identical to that found in the patient from which the MF6 strain was isolated (Fig. B and D). In a similar fashion, cross-reactivity of anti-GA1 and anti-GM1 antibodies in patients GB17 and GB18 and rabbits immunized with corresponding LPS was demonstrated. In the GB17-immunized rabbits, anti-GA1 reactivity could be decreased only by incubation with GB17 LPS and not GB18 LPS, indicating that the anti-GA1 antibodies are specifically induced by the GA1-like GB17 LPS.
FIG. 3 Identical pattern of cross-reactivity of antiganglioside and anti-LPS antibodies in patients and immunized rabbits. Sera from patient MF6 and from rabbit 5, immunized with MF6 LPS, were incubated with Sepharose CL4B beads that were coated with C. jejuni (more ...)
Almost all rabbits tolerated the injections well, did not show any overt signs of weakness or ataxia, and did not lose weight. However, one animal that was immunized with GB18 LPS started losing weight 4 weeks after the first injection. In addition, this animal had fewer spontaneous movements, although there were no signs of muscle weakness. After a few weeks, the animal recovered and gained weight again. Histological examination of the sciatic nerve of this animal, taken 56 days after the first injection, did not show any signs of neuropathy or inflammation.