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Antibodies to human myelin proteins and gangliosides in patients with acute neuroparalytic accidents induced by brain-derived rabies vaccine
Journal of Neuroimmunology 91 Ž1998. 63–72
Antibodies to human myelin proteins and gangliosides in patients with acute neuroparalytic accidents induced by brain-derived rabies vaccine Dh. Laouini, M.F. Kennou, S. Khoufi, K. Dellagi
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Laboratory of Immunology, Institut Pasteur de Tunis, 13, Place Pasteur, BP 74 1002 Tunis-BelÕedere, ` Tunisia Received 31 December 1997; revised 2 June 1998; accepted 2 June 1998
Abstract Antibody responses to myelin antigens were analysed in 15 patients who developed acute neuroparalytic accidents ŽANPA. during post-exposure rabies vaccination using a rabies vaccine prepared on brain tissues and in 30 individuals who were uneventfully vaccinated. High titers ŽG 100. of IgG and IgM antibodies to GM1 or GD1a gangliosides were detected by enzyme linked immunosorbent-assay ŽELISA. in plasmas from ANPA patients but not in controls. These data suggest that antibodies to GM1 and GD1a gangliosides may play a pathogenic role in the demyelinating andror inflammatory processes characteristic of rabies vaccine-induced acute neurologic complications. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Rabies vaccine; Encephalomyelitis; Antibodies; Myelin proteins; Gangliosides; Myelin basic protein
1. Introduction Rabies is a fatal disease occurring mainly in developing countries. Prevention of disease development after a contaminating contact with a rabid animal relies on the rapid administration of post-exposure vaccination therapy using an inactivated rabies vaccine. Modern vaccines prepared on cellular substrates Žhuman diploid cells, Vero cells or chick embryo cells. are effective and innocuous ŽPlotkin and Koprowski, 1994.. However, for economic reasons, several developing countries still rely, on the classic and cheaper Semple-type vaccine prepared with virus inoculated animal brains. These vaccines contain variable amounts of neural antigens and expose occasionally recipients to the development of acute neuroparalytic accidents ŽANPA., featuring typically an acute disseminated encephalomyelitis and less frequently, a polyradiculonevritis of the Guillain–Barre´ type ŽAppelbaum et al., 1953; Held and Lopez-Adaros, 1972; Cabrera et al., 1987; Swaddiwuthipong et al., 1988.. Symptoms appear between the 5th and 10th day of the vaccination therapy and, after a monophasic progression, usually regress ŽLaplane et al., 1960; Huguenin and Bianchi, 1963.. However sequellas of
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variable severity and in rare cases, death can occur ŽSwamy et al., 1984.. The risk of post rabies vaccine ANPA has been variably estimated in the literature and considering our own experience, is likely between 0.02% and 0.1%. In Tunisia, 15 patients were reported to us as presenting post-vaccine encephalomyelitis over a period of 6 years. All patients Žexcept one. were adults, although 42% of vaccinated individuals in Tunisia are children, suggesting that adults are at higher risk to develop ANPA than children. Post-rabies vaccine ANPA is usually considered to be the human counterpart of Experimental Allergic Encephalomyelitis ŽEAE. induced by injection of various neural antigens, especially Myelin Basic Protein ŽMBP. for which a cellular autoimmune mechanism was demonstrated to be responsible of the induction of neurologic damage. However, it was reported that development of severe ANPA is closely linked to the development of antibodies to MBP ŽHemachudha et al., 1987b.. In this report, we analyse the antibody responses to myelin proteins and glycolipids in patients with post-rabies vaccine ANPA, compared to uneventfully vaccinated individuals. We show that the development of antibodies to GM1 and GD1a gangliosides, but not to myelin proteins, including MBP, is associated with the development of ANPA.
0165-5728r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 1 5 3 - 2
Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72
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2. Materials and methods 2.1. Patients and controls Blood samples were collected on admission day from 15 Tunisian patients who developed ANPA after receiving rabies vaccine post-exposure therapy. The latter was based on the Semple-type vaccine which was used in Tunisia till June 1996. The vaccine is 5% suspension of an homogenate of rabies virus-inoculated lamb brain, inactivated with b-propiolactone and freeze dried. The treatment consisted of 14 daily subcutaneous injections of 1 ml vaccine followed by two booster injections at day 28 and day 90. In patients who developed ANPA, symptoms appeared between the 4th and the 18th day of the therapy and were clinically classified as encephalitis, myelitis, polyradiculonevritis or meningitis ŽTable 1.. Thirty individuals uneventfully vaccinated using the same vaccine with the same protocol, were included in the study as controls and bled before treatment Žsample C0. and on days 7, 14 and 28 of the vaccination therapy Žsamples C7, C14 and C28, respectively.. Plasma samples were aliquoted and stored at y208C until use.
Briefly, SDS-PAGE was carried out in 12.5% gel with 1% stacking gel as described by Laemmli Ž1970., starting with 800 mg of purified human myelin and run 3–4 h at 30 mA. Two vertical strips were cut from both sides of the gel and were stained with Coomassie brilliant blue Ž0.1% wrv. to localize the different antigenic fractions. Twelve contiguous horizontal strips were cut out from the gel and placed into a dialysis bag filled with 1 ml of phosphate-buffered saline ŽPBS: 0.15 M NaCl, 0.01 M phosphate, pH 7.. Electroelution was performed at 48C and eluted fractions were extensively dialysed against PBS and aliquoted at y208C until use. The protein concentration was determined by the Folin method ŽLowry et al., 1951.. Human MBP was extracted from myelin according to the method of Deibler et al. Ž1972.. The purity was assessed by immunodetection, after 12.5% SDS-PAGE, using a rabbit polyclonal anti-MBP antibody Ža generous gift of Dr. H. Villarroya, Hopital de la Pitie´ Salpetriere, ˆ ˆ ` Paris, France.. Total glycolipids were extracted from human purified central myelin according to the method of Folch et al. Ž1957.. Purified GM1, GM2, GM3, GD1a, GD1B, GD3 and GT1b were purchased from Sigma ŽSt. Louis, MO..
2.2. Preparation of central nerÕous antigens Myelin was extracted Žby flotation on sucrose gradient. from human brain obtained post-mortem from an adult who died of non-neurological cause ŽNorton and Poduslo, 1973.. Twelve antigenic fractions were prepared by electroelution after separation of myelin proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ŽSDS-PAGE..
2.3. Immuno-blot analysis Following electrophoresis, myelin components were electrophoretically transferred onto nitrocellulose sheet Ž0.45-mm pore size, Millipore, MA. using 25 mM Tris, 192 mM Glycine, 20% Žvrv. methanol, pH 8.3 buffer ŽTowbin et al., 1979.. Antibody reactivity was tested at a
Table 1 Clinical features in 15 patients who developed major neurological symptoms following post-exposure Semple rabies vaccination Case no.
Age Žyear.rsex
No. of vaccine doses received
Day of onset of symptoms
Symptoms )
Clinical diagnosis
Outcome ) )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
44rM 48rM 43rM 43rM 29rM 35rM 48rM 19rM 60rM 37rM 45rM 12rM 30rM 38rM 22rM
7 10 14 6 14 12 10 14 4 14 13 14 5 4 10
7 10 14 7 14 12 20 18 15 5 13 15 8 18 12
HA, PP HA, F, DP, M MC, F, PP, BR, C, M PP, MC, BR F, AT, PP, BR, M HA, PP, BR, DP F, PP, MC, M F, BR, PP, M PP, FP HA, M PP, MC HA, F, M, MC F, HA, FP, PP, DP HA, HMP, FP HA, PP, BR, RF
Polyradiculonevritis Meningoradiculitis Meningoencephalomyelitis Encephalomyelitis Meningoencephalomyelitis Encephalomyelitis Meningoencephalomyelitis Meningomycloradiculitis Polyradiculonevritis Meningitis Encephalomyelitis Meningo-encephalomyelitis Mycloradiculitis Encephalomyelitis Polyradiculonevritis
CR CR Da CR CR CR CR CR CR CR CR CR CR PRb CR
)
AT, Ataxia; BR, Bladder retention; C, Coma; DP, Diplopia; F, Fever; FP, Facial paralysis; HA, Headache; HMP, Hemiplegia; M, Meningitis; M.C., Mental confusion; PP, Paraplegia or paraparesia; RF, Respiratory failure. )) CR, Complete recovery; D, Death; PR, Partial recovery. a Perforation of gastric ulcer due to corticotherapy. b Facial diplegia.
Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72
plasma dilution of 1:100 in PBS, 0.02% Tween-20 and 1% bovine serum albumin ŽBSA.. After overnight incubation at 48C, blots were washed, treated with peroxidase-conjugated rabbit immunoglobulin to human IgM or IgG ŽSigma. for 90 min at room temperature and developed with diaminobenzidine hydrochloride and H 2 O 2 ŽSigma..
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2.4. Detection of antibodies to gangliosides by ELISA Reactivity of plasmas from patients and controls with GM1, GM2, GD1a, GD1b, GD3 or GT1b gangliosides was assessed by ELISA as described elsewhere ŽGarcia-Guijo et al., 1995.. Briefly, flat bottom 96-well microtiter plates
Fig. 1. Immunoblot analysis of purified human central myelin with plasmas from patients and controls. Panels A and C: Plasmas from five controls tested at day 0 Ž). or day 28 Ž Ø . of the vaccination therapy. Panels B and D: Plasmas from five patients with post-vaccination ANPA. Panels A and B: IgM detection. Panels C and D: IgG detection.
Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72
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Table 2 ELISA reactivity for IgM and IgG classes, expressed as OD units Žmean " SD. of plasmas collected from 30 control vaccinees before ŽC0. or at day 28 ŽC28. of the vaccination therapy or from 15 patients developing ANPA. Controls C0
C28
IgM ŽA. Electroeluted fractions 1 0.102 " 0.112 2 0.082 " 0.069 3 0.122 " 0.182 4 0.092 " 0.021 5 0.092 " 0.181 6 0.082 " 0.102 7 0.029 " 0.060 8 0.160 " 0.108 9 0.128 " 0.099 10 0.021 " 0.128 11 0.069 " 0.106 12 0.082 " 0.098 ŽB. MBP 0.150 " 0.035
ANPA
IgG
IgM
IgG
IgM
IgG
0.063 " 0.071 0.037 " 0.093 0.036 " 0.073 0.001 " 0.010 0.069 " 0.106 0.037 " 0.093 0.131 " 0.091 0.113 " 0.061 0.067 " 0.039 0.011 " 0.091 0.171 " 0.036 0,049 " 0,107 0,115 " 0.075
0.037 " 0.101 0.061 " 0.057 0.049 " 0.031 0.048 " 0.104 0.052 " 0.063 0.087 " 0.107 0.024 " 0.060 0.160 " 0.103 0.060 " 0.093 0,038 " 0,071 0,087 " 0,113 0,049 " 0,036 0.140 " 0.147
0.098 " 0.154 0.056 " 0.098 0.099 " 0.103 0.039 " 0.063 0.109 " 0.065 0.047 " 0.082 0.071 " 0.073 0.123 " 0.099 0.071 " 0.123 0,057 " 0,077 0,096 " 0,089 0,024 " 0,047 0.132 " 0.137
0.045 " 0.078 0.057 " 0.093 0.056 " 0.075 0.008 " 0.010 0.069 " 0.104 0.057 " 0.095 0.151 " 0.098 0.113 " 0.061 0.042 " 0.059 0.088 " 0.091 0.121 " 0.034 0,049 " 0,102 0.185 " 0.073
0.102 " 0.157 0.087 " 0.064 0.122 " 0.132 0.042 " 0.075 0.092 " 0.131 0.031 " 0.062 0.069 " 0.104 0.097 " 0.108 0.062 " 0.097 0.069 " 0.098 0.101 " 0.124 0.036 " 0.042 0.132 " 0.136
Plasmas were tested at 1r100 dilution with 12 fractions electroeluted from SDS-PAGE of human central myelin Žpanel A. or with purified human MBP Žpanel B..
ŽCostar, MA. were filled with 100 ml per well of ethanol containing 5 mgrml of the relevant ganglioside and left to air dry. After saturation for 4 h with 1% BSA in PBS at 378C, 100 ml of serially diluted plasmas in PBS–BSA, Žstarting from 1:100. were added in duplicate and kept overnight. This incubation and the following steps were carried out at 48C. After six washes in cold PBS solution, plates were incubated with 100 ml of peroxidase-conjugated affinity purified goat anti-human IgM or IgG antibodies ŽSigma. diluted 1:2000. After six washes, the reaction was developed Žfor 10 min to 30 min depending on the antigen used and following previous standardization of the developing-time of the reaction for each antigen by a blinded-way kinetic study on five samples from each group. with 200 ml 0.004% O-phenylenediamine, 0.1 M citrate, 0.2 M NaH 2 PO4 ŽpH 5.0. and 0.012% H 2 O 2 and read at 492 nm in Titertek reader. Results were expressed as reported by Sadiq et al. Ž1990.. Briefly, the optical density ŽOD. obtained with each plasma dilution tested on
BSA-coated wells was subtracted from those obtained with the same dilution tested on wells coated with the relevant ganglioside. Antiganglioside antibody titers for each patient was defined as a highest dilution factor at which the mean OD of the duplicate wells exceeded the cut-off value. The cut-off value corresponded to the mean OD q 3 SD obtained with the plasmas of the 30 control individuals before vaccination Žsamples C0. and tested at 1:100 dilution. 2.5. High performance thin-layer chromatography (HPTLC) immunostaining Immunoreactivity with myelin gangliosides of plasmas from patients and controls, was studied according to the procedure of Magnani et al. Ž1982. by an immunostaining assay after HPTLC of total glycolipids extracted from human central myelin. Specific gangliosides in the whole preparation of myelin glycolipids were localized by comigrating, in parallel, reference purified gangliosides which
Table 3 ELISA reactivity for IgM and IgG classes expressed as OD units Žmean " SD. of plasmas collected from 30 control vaccinees, before ŽC0. or at day 28 ŽC28. of the vaccination therapy or from 15 patients developing ANPA C0
Ganglioside GD1a GD1b GD3 GM1 GM2 GM3 GT1b
C28
ANPA
IgM
IgG
IgM
IgG
IgM
IgG
0.092 " 0.052 0.067 " 0.086 0.031 " 0.092 0.158 " 0.160 0.026 " 0.037 0.072 " 0.072 0.042 " 0.083
0.079 " 0.171 0.126 " 0.221 0.166 " 0.073 0.126 " 0.121 0.088 " 0.156 0.034 " 0.102 0.275 " 0.319
0.073 " 0.093 0.061 " 0.057 0.079 " 0.031 0.395 " 0.204 0.076 " 0.055 0.174 " 0.380 0.046 " 0.053
0.098 " 0.076 0.162 " 0.293 0.372 " 0.216 0.113 " 0.204 0.160 " 0.180 0.037 " 0.097 0.137 " 0.237
0.071 " 0.083 0.082 " 0.070 0.076 " 0.093 0.846 " 0.452 0.129 " 0.104 0.176 " 0.155 0.170 " 0.310
0.455 " 0.563 0.164 " 0.296 0.362 " 0.408 0.360 " 0.541 0.102 " 0.121 0.061 " 0.161 0.258 " 0.238
Plasmas were tested individually at 1r100 dilution with a panel of seven gangliosides.
Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72
were then stained with resorcinol. Immunodetection was performed on aluminium-backed thin-layer chromatography ŽTLC. plates Žsilica gel 60, Merck, Germany. loaded with 10 mg of total glycolipids determined by the method of Svennerholm Ž1957.. Plasmas were used at a dilution of 1:100 Žin PBS solution containing 1% gelatin and 10% inactivated horse serum. and incubated for 2 h at 378C. The second antibody was a peroxidase-conjugated anti-human IgM ŽSigma.. After 1 h incubation at room tempera-
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ture, the plates were washed and bound antibodies were detected with 4-chloro-1-naphthol and 0.003% H 2 O 2 reagent. 2.6. ELISA detection of antibodies to myelin fractions and MBP Reactivity of plasmas from patients and controls with myelin fractions or purified MBP was tested by ELISA.
Fig. 2. IgM and IgG antibodies to GD3 Ža and b., GD1a Žc and d. and GM1 Že and f.. Plasmas were obtained from patients who developed post-vaccination acute disseminated encephalomyelitis. C0, C7, C14 and C28 correspond to plasma samples obtained from uneventfully vaccinated individuals bled at day 0, 7, 14 and 28, respectively.
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Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72
Briefly, each antigen was dissolved at optimal concentration Ž0.5 mg to 1.25 mgrml for the different fractions and at 4 mgrml for human purified MBP. in carbonate–bicarbonate buffer ŽpH 9.6. and 100 ml were used to coat 96-well polystyrene plates ŽCostar. and incubated overnight at 48C. The plates were blocked with PBS–BSA 1% for 1 h at 378C before adding serially diluted plasma samples. The following steps were performed as described above for antiganglioside ELISA detection ŽSection 2.4.. 2.7. Statistical analysis Statistical analysis were performed with a ‘Stat-view’ software using ‘Student’s t-test’ and ‘Fisher’s x-test’.
3. Results 3.1. Antibodies to myelin proteins To determine whether specific antibodies to human myelin antigens develop during vaccination, plasmas from patients and controls were tested at 1:100 dilution by immuno-blot with purified myelin. Although some variability in the background reactivity was noticed from sample to sample, there was no significant difference in the pattern of reactivity of the same individual before ŽC0. or at the end of the vaccination course ŽC28.. This holds
true for both IgM and IgG antibodies ŽFig. 1A and C.. Similar results were obtained with sera from patients with post-vaccination ANPA ŽFig. 1B and D.. Of particular interest was the absence of reactivity with the major components of the myelin sheets Ži.e., MBP, cyclic nucleotide phosphodiesterase ŽCNPase. and proteolipid protein ŽPLP. as well as with the myelin associated glycoprotein ŽMAG... In order to enhance the sensitivity of the assay and allow quantitative comparison between data obtained with plasmas from patients and controls, ELISAs were performed using twelve different fractions electroeluted from SDS-PAGE of human central myelin. Reactivities with all fractions including those containing MBP, PLP, WP as well as MAG did not exceed 0.3 OD units ŽTable 2A. and no significant difference could be detected between patients and controls. The presence of antibodies to human MBP, was more specifically assessed. All plasmas from patients and controls ŽC0 and C28. were tested by ELISA with MBP extracted from myelin. IgM and IgG reactivity of plasmas from patients who developed neurologic complications were constantly weak and not significantly different from pre or post-vaccination control plasmas ŽTable 2B.. These results indicate that administration of Semple rabies vaccine containing 5% lamb brain extract does not induce detectable levels of antibodies against human central myelin proteins including MBP, CNPase, MAG and
Fig. 3. IgM anti-GM1 absorbances ŽELISA. of serially diluted plasmas from 15 ANPA patients Ž`. or controls Ž Ø s day 0; I s day 7; ' s day 14; ls day 28. The absorbances of control plasmas are expressed as the mean reactivity of 30 individual plasmas" SD.. Dotted line: cut-off value of the ELISA defined as 3 SD of the mean OD of the control group ŽC0. tested at 1:100 dilution.
Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72 Table 4 Plasmas reactivity with GD1a or GM1 Žtiters G100. in 15 patients with ANPA and 30 uneventfully vaccinated controls ŽNb positiverNb tested. GD1a
ANPA Controlsa p- b
GM1
IgM
IgG
IgM
IgG
8r15 0r30 0.005
4r15 0r30 0.025
10r15 0r30 0.005
7r15 0r30 0.025
a
All plasmas obtained at days 0, 7, 14 and 28 were tested. p Values for titer comparison between ANPA patients and each control group.
b
PLP, either in asymptomatic vaccine recipients or in those who developed post-vaccination acute neuroparalytic accidents. 3.2. Antibody responses to gangliosides In order to analyse the antibody responses to brain gangliosides that could have been induced in patients and controls by the vaccination protocol, ELISA with purified gangliosides and immuno-HPTLC detection were performed. IgM and IgG reactivities with gangliosides, of plasmas from control samples and from ANPA group are shown in Table 3 Žtested at 1:100 dilution. and in Fig. 2 Žfull titration.. Three different patterns of antiganglioside responses could be observed. The first pattern includes GD1b, GM2, GM3 and GT1b gangliosides for which plasmas from controls ŽC7, C14 and C28. from ANPA patients showed no reactivity Žtiters
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- 100., as plasmas collected before the vaccination therapy ŽC0. Ždata not shown.. The second pattern concerned GD3 ganglioside: four patients Ž26% of ANPA group. and three controls Ž10% of plasmas collected at days 14 and 28. developed significant IgG anti GD3 reactivity Žantibody titers G 200.. These responses were restricted to the IgG class ŽFig. 2a and b.. The third pattern encompassed antibody responses to GD1a and GM1 gangliosides: ANPA patients but not controls developed high IgM and IgG reactivity with GD1a and GM1 Žantibody titers: 100–800.. IgM antibodies to GD1a and GM1 were detected in 8 patients Ž53%. and 10 patients Ž66%., respectively ŽFig. 2a,c,d,fFig. 3.. When antibody titers to GD1a and GM1 gangliosides in ANPA patients are compared to those of controls Žsamples C0, C7, C14 and C28., high significant statistical differences were found Ž p - 0.005 for IgM and p - 0.025 for IgG. ŽFig. 3 and Table 4.. Plasmas from four patients Žno. 5, 7, 12 and 15. had high titers to both GM1 and GD1a; in six patients Žno. 1, 3, 6, 9, 11 and 14. plasmas had high titers only to GM1. Plasmas from four patients Žno. 2, 4, 8 and 14. had high titers only to GD1a and only one patient Žno. 13. did not respond either to GM1 or to GD1a. The three patients with polyradiculonevritis Žno. 1, 9 and 15, Table 1. developed high antibody titers to GM1. In addition, one of them Žno. 15. had antibodies to GD1a. Plasma samples from 13 ANPA patients were tested by HPTLC immunostaining of human central myelin glycolipids ŽFig. 4A.. All plasmas immunostained electively GM1 ganglioside. Two plasmas showed, in addition, a dim staining of the GD1b spot. None of the 30 control plasmas obtained at day 28 showed immunostaining of HPTLC plates Ždata not shown..
Fig. 4. Immunodetection by plasmas from six ANPA ŽA., of glycolipids on thin-layer chromatography. Arrows in panel ŽA. show positive immunostaining of GM1 or GD1b gangliosides. Panel ŽB. shows standard gangliosides ŽGM1, GD1a, GD1b and GT1b. stained by resorcinol.
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Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72
4. Discussion We show that rabies vaccine post-exposure therapy using brain-derived vaccine, whether or not associated to the subsequent development of neuroparalytic accidents, is not associated with any significant antibody response to human central myelin proteins. No specific immunostaining of the major myelin proteins ŽMBP, MAG, PLP and CNPase. could be detected by immunoblot analysis, and there was no significant ELISA reactivity with 12 fractions electroeluted from SDS-PAGE of human central myelin. Moreover, we could not detect any reactivity by ELISA with human purified MBP in plasmas from either ANPA patients, or from controls. This result is in contradiction with that reported by Hemachudha et al. Ž1987b. who found antibodies to human MBP in patients who develop post-vaccine ANPA. These antibodies reacted specifically with peptides 45–89 and 90–170 of MBP ŽUbol et al., 1990.. Considering that sheep MBP has approximately 90% homology with human MBP, these investigators concluded that vaccination had presumably disturbed normal immunoregulatory mechanisms and led to the development of an autoimmune response ŽUbol et al., 1990.. The reasons which could account for discrepancies between our results and those reported by Hemachudha are unclear. Variation in vaccine composition may explain differences in the immune response of vaccine recipients. The Thai rabies vaccine was prepared on adult sheep brain while the Tunisian one used a lamb brain which is poorly myelinated and contains lower amounts of MBP. We noted previously that protein content of myelin extracted from the same quantity of crude material is 5- to 10-fold lower in lamb brain than in sheep brain Ždata not shown.. These differences in the composition of the nervous substrate and the way of preparation of the vaccine could also explain the high rate of neurologic complications induced by Thai vaccine compared to those induced by the Tunisian one ŽHemachudha et al., 1987a; Swaddiwuthipong et al., 1988.. Moreover, the Thai vaccine was inactivated by phenol whereas the Tunisian one was inactivated by b-propiolactone. These chemicals may have different effects on B and T cell epitopes of myelin proteins and hence their immunogenicity. A direct relationship exists between the content of nervous tissue in a particular vaccine and the frequency or the clinical expression of neuroparalytic accidents. Thus, Semple-type vaccine induces electively encephalomyelitis in about 90% of cases, whereas suckling mouse brain vaccine induces preferentially polyradiculonevritis of the Guillain–Barre´ type ŽHeld and Lopez-Adaros, 1972; Toro et al., 1977; Hemachudha et al., 1987a.. The neural antigens, targets of the immunopathogenic mechanisms underlying these complications are most likely different. Our results suggest that antibodies to myelin proteins are unlikely to play a significant role in the pathogenesis of rabies vaccine-induced encephalomyelitis in Tunisian patients. One cannot exclude however that MBP andror
other protein components of the human central nervous myelin ŽMAG, myelin-oligodendrocyteglycoprotein MOG., may be target antigens for a T cell response, as clearly demonstrated in experimental autoimmune demyelinating diseases Žsee Zamvill and Steinman, 1990 and Martin et al., 1992 for review.. With regard to the antibody response against gangliosides, we found that the levels of IgM and IgG antibodies to GD1b, GM2, GM3, and GT1b were not affected, either by the number of rabies vaccine doses received by the vaccinees or by the development of the post-vaccine neurologic complications. On the opposite, the vaccination therapy induced a significant increase of IgG anti-GD3 antibodies which correlated with the number of vaccine doses received by the vaccinees and most likely represent the humoral response to administration of exogenous gangliosides contained in the vaccine substrate. Interestingly, we observed high titers of IgG and IgM antibodies to GM1 and GD1a only in those vaccinees who developed neurologic symptoms, which suggest that these autoantibodies may play a role in the development of ANPA. Gangliosides have been recently identified as antibody targets in several neurological diseases. Several authors have described the presence of antiganglioside antibodies in the sera of patients with Guillain–Barre´ syndrome ŽIlyas et al., 1988. and amyotrophic lateral sclerosis ŽPestronk et al., 1988b.. High titers of IgM anti-GM1 antibodies have been associated with lower motor neuron syndrome ŽPestronk et al., 1990. and less frequently with chronic sensory multifocal neuropathy ŽPestronk et al., 1988a.. However, Hemachudha reported that after Semple rabies vaccination, antibodies to GM1 fraction were present in individuals with or without post-vaccine complications and that the mean levels were similar in both groups ŽHemachudha et al., 1987b.. The variation between our results and those of Hemachudha et al., could be explained by the difficulty in ELISA standardization for the detection of antibodies to gangliosides and the use, in our hands, of a more discriminative way to express the results Ža prior standardization of the assay and a very high cut-off value.. Moreover, several lines of evidence suggest that naturally occurring anti-GM1 antibodies are part of the antibody repertoire of normal individuals and could therefore be detected in normal plasma ŽMizutamari et al., 1994.. The level of these antibodies is not influenced by the administration of exogenous gangliosides ŽDoria and Tettamanti, 1993; Ala et al., 1994; Mizutamari et al., 1994., although, some authors claimed the opposite ŽFigeuras et al., 1992; Schneider and Roeltgen, 1993.. Interestingly, Deisenhammer et al. Ž1996. recently reported that anti-GM1 antibodies may vary in their affinity and fine specificity and that pathogenic antiGM1 in Guillain–Barre´ syndrome may belong to a subgroup of antibodies, while low affinity anti-GM1 seem to be non-specific and are not associated with any specific disease. More recently, it was reported that low affinity IgM antibodies are devoid of complement mediated lytic
Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72
activity on GM1 liposomes in contrast to high affinity anti GM1 antibodies ŽMizutamari et al., 1998.. With regards to anti-GM1 antibodies detected in our study, it is noteworthy that using a technique of lower sensitivity than ELISA, i.e., HPTLC immunodetection, we were able to detect anti-GM1 antibodies constantly and only in ANPA patients. This result suggests that this technique is the most suitable to detect high affinity andror pathogenic anti-GM1 antibodies associated to ANPA. It is tempting to speculate that GM1 ganglioside present in the substratum of rabies vaccines may trigger the appearance of pathogenic antiGM1 antibodies which will react with their respective antigen exposed on the outer surface of neuronal membrane and participate to the demyelinating process. Alternatively, these antibodies may have been induced, secondary to the demyelination process. Whatever the mechanism of demyelination in ANPA, cell-mediated or antibody-mediated, these responses may act synergistically: autoantibodies can contribute to the triggering or the aggravation of the demyelinating process, as recently demonstrated in the Experimental Autoimmune Neuritis ŽEAN., an animal model of Guillain–Barre´ syndrome ŽSpies et al., 1995.. In this context, our results show that antibodies to gangliosides ŽGM1 and GD1a. are more likely implicated in the pathogenesis of post-rabies vaccine ANPA than antibodies to myelin protein.
Acknowledgements We are grateful to Pr. N. Baumann ŽHopital la Pitie´ ˆ Salpetriere, ˆ ` Paris, France. for helpful discussion, to Dr. H. Villarroya ŽHopital la Pitie´ Salpetriere, ˆ ˆ ` Paris, France. for generous gift of anti-MBP antibody, to Pr. M. Hamdoun ŽHopital Charles Nicolle, Tunis, Tunisia. for providing ˆ human tissues and to Mrs. M. Gadhab for collecting blood samples.
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Younger, D.S., Lovelace, R.E., Trojaborg, W., Sherman, W.H., Miller, J.R., Minuk, J., Fehr, M.A., Roelofs, R.I., Hollander, D., Nichols, F.T., Mitsumoto, H., Kelley, J.J., Swift, T.R., Munsat, T.L., Latov, N., 1990. The spectrum of neurological disease associated with anti-GM1 antibodies. Neurology 40, 1067–1072. Schneider, J.S., Roeltgen, D.P., 1993. Gangliosides and neurological diseases Žletter.. Br. Med. J. 306, 205–205. Spies, J.M., Pollard, J.D., Bonner, J.G., Westland, K.W., McLoed, J.G., 1995. Synergy between antibody and P2-reactive T cells in experimental allergic neuritis. J. Neuroimmunol. 57, 77–84. Svennerholm, M.L., 1957. Quantitative estimation of sialic acids. A colorimetric resorcinol–hydrochloric acid method. Biochem. Biophys. Acta 24, 604–609. Swaddiwuthipong, W., Weniger, B.G., Wattanasri, S., Warell, M.J., 1988. A high rate of neurological complications following Semple anti-rabies vaccine. Trans. R. Soc. Trop. Med. Hyg. 82, 472–475.
Swamy, H.S., Shanker, S.K., Chandra, P.S., 1984. Neurological complications due to b-propiolactone inactivated antirabies vaccination: clinical electrophysiological and therapeutic aspects. J. Neurol. Sci. 63, 111–128. Toro, G., Vergara, I., Roman, G., 1977. Neuroparalytic accidents of antirabies vaccination with suckling mouse brain vaccine: clinical and pathologic study of 21 cases. Arch. Neurol. 34, 694–700. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Ubol, S., Hemachudha, T., Whitetaker, J.N., Griffin, D.E., 1990. Antibody to peptides of human myelin basic protein in post-rabies vaccine encephalomyelitis sera. J. Neuroimmunol. 26, 107–111. Zamvill, S.S., Steinman, L., 1990. The T lymphocyte in experimental allergic encephalomyelitis. Annu. Rev. Immunol. 8, 579–621.
Antibodies to human myelin proteins and gangliosides in patients with acute neuroparalytic accidents induced by brain-derived rabies vaccine Dh. Laouini, M.F. Kennou, S. Khoufi, K. Dellagi
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Laboratory of Immunology, Institut Pasteur de Tunis, 13, Place Pasteur, BP 74 1002 Tunis-BelÕedere, ` Tunisia Received 31 December 1997; revised 2 June 1998; accepted 2 June 1998
Abstract Antibody responses to myelin antigens were analysed in 15 patients who developed acute neuroparalytic accidents ŽANPA. during post-exposure rabies vaccination using a rabies vaccine prepared on brain tissues and in 30 individuals who were uneventfully vaccinated. High titers ŽG 100. of IgG and IgM antibodies to GM1 or GD1a gangliosides were detected by enzyme linked immunosorbent-assay ŽELISA. in plasmas from ANPA patients but not in controls. These data suggest that antibodies to GM1 and GD1a gangliosides may play a pathogenic role in the demyelinating andror inflammatory processes characteristic of rabies vaccine-induced acute neurologic complications. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Rabies vaccine; Encephalomyelitis; Antibodies; Myelin proteins; Gangliosides; Myelin basic protein
1. Introduction Rabies is a fatal disease occurring mainly in developing countries. Prevention of disease development after a contaminating contact with a rabid animal relies on the rapid administration of post-exposure vaccination therapy using an inactivated rabies vaccine. Modern vaccines prepared on cellular substrates Žhuman diploid cells, Vero cells or chick embryo cells. are effective and innocuous ŽPlotkin and Koprowski, 1994.. However, for economic reasons, several developing countries still rely, on the classic and cheaper Semple-type vaccine prepared with virus inoculated animal brains. These vaccines contain variable amounts of neural antigens and expose occasionally recipients to the development of acute neuroparalytic accidents ŽANPA., featuring typically an acute disseminated encephalomyelitis and less frequently, a polyradiculonevritis of the Guillain–Barre´ type ŽAppelbaum et al., 1953; Held and Lopez-Adaros, 1972; Cabrera et al., 1987; Swaddiwuthipong et al., 1988.. Symptoms appear between the 5th and 10th day of the vaccination therapy and, after a monophasic progression, usually regress ŽLaplane et al., 1960; Huguenin and Bianchi, 1963.. However sequellas of
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Corresponding author. Tel.: q216 1 783022; fax: q216 1 791833
variable severity and in rare cases, death can occur ŽSwamy et al., 1984.. The risk of post rabies vaccine ANPA has been variably estimated in the literature and considering our own experience, is likely between 0.02% and 0.1%. In Tunisia, 15 patients were reported to us as presenting post-vaccine encephalomyelitis over a period of 6 years. All patients Žexcept one. were adults, although 42% of vaccinated individuals in Tunisia are children, suggesting that adults are at higher risk to develop ANPA than children. Post-rabies vaccine ANPA is usually considered to be the human counterpart of Experimental Allergic Encephalomyelitis ŽEAE. induced by injection of various neural antigens, especially Myelin Basic Protein ŽMBP. for which a cellular autoimmune mechanism was demonstrated to be responsible of the induction of neurologic damage. However, it was reported that development of severe ANPA is closely linked to the development of antibodies to MBP ŽHemachudha et al., 1987b.. In this report, we analyse the antibody responses to myelin proteins and glycolipids in patients with post-rabies vaccine ANPA, compared to uneventfully vaccinated individuals. We show that the development of antibodies to GM1 and GD1a gangliosides, but not to myelin proteins, including MBP, is associated with the development of ANPA.
0165-5728r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 1 5 3 - 2
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2. Materials and methods 2.1. Patients and controls Blood samples were collected on admission day from 15 Tunisian patients who developed ANPA after receiving rabies vaccine post-exposure therapy. The latter was based on the Semple-type vaccine which was used in Tunisia till June 1996. The vaccine is 5% suspension of an homogenate of rabies virus-inoculated lamb brain, inactivated with b-propiolactone and freeze dried. The treatment consisted of 14 daily subcutaneous injections of 1 ml vaccine followed by two booster injections at day 28 and day 90. In patients who developed ANPA, symptoms appeared between the 4th and the 18th day of the therapy and were clinically classified as encephalitis, myelitis, polyradiculonevritis or meningitis ŽTable 1.. Thirty individuals uneventfully vaccinated using the same vaccine with the same protocol, were included in the study as controls and bled before treatment Žsample C0. and on days 7, 14 and 28 of the vaccination therapy Žsamples C7, C14 and C28, respectively.. Plasma samples were aliquoted and stored at y208C until use.
Briefly, SDS-PAGE was carried out in 12.5% gel with 1% stacking gel as described by Laemmli Ž1970., starting with 800 mg of purified human myelin and run 3–4 h at 30 mA. Two vertical strips were cut from both sides of the gel and were stained with Coomassie brilliant blue Ž0.1% wrv. to localize the different antigenic fractions. Twelve contiguous horizontal strips were cut out from the gel and placed into a dialysis bag filled with 1 ml of phosphate-buffered saline ŽPBS: 0.15 M NaCl, 0.01 M phosphate, pH 7.. Electroelution was performed at 48C and eluted fractions were extensively dialysed against PBS and aliquoted at y208C until use. The protein concentration was determined by the Folin method ŽLowry et al., 1951.. Human MBP was extracted from myelin according to the method of Deibler et al. Ž1972.. The purity was assessed by immunodetection, after 12.5% SDS-PAGE, using a rabbit polyclonal anti-MBP antibody Ža generous gift of Dr. H. Villarroya, Hopital de la Pitie´ Salpetriere, ˆ ˆ ` Paris, France.. Total glycolipids were extracted from human purified central myelin according to the method of Folch et al. Ž1957.. Purified GM1, GM2, GM3, GD1a, GD1B, GD3 and GT1b were purchased from Sigma ŽSt. Louis, MO..
2.2. Preparation of central nerÕous antigens Myelin was extracted Žby flotation on sucrose gradient. from human brain obtained post-mortem from an adult who died of non-neurological cause ŽNorton and Poduslo, 1973.. Twelve antigenic fractions were prepared by electroelution after separation of myelin proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ŽSDS-PAGE..
2.3. Immuno-blot analysis Following electrophoresis, myelin components were electrophoretically transferred onto nitrocellulose sheet Ž0.45-mm pore size, Millipore, MA. using 25 mM Tris, 192 mM Glycine, 20% Žvrv. methanol, pH 8.3 buffer ŽTowbin et al., 1979.. Antibody reactivity was tested at a
Table 1 Clinical features in 15 patients who developed major neurological symptoms following post-exposure Semple rabies vaccination Case no.
Age Žyear.rsex
No. of vaccine doses received
Day of onset of symptoms
Symptoms )
Clinical diagnosis
Outcome ) )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
44rM 48rM 43rM 43rM 29rM 35rM 48rM 19rM 60rM 37rM 45rM 12rM 30rM 38rM 22rM
7 10 14 6 14 12 10 14 4 14 13 14 5 4 10
7 10 14 7 14 12 20 18 15 5 13 15 8 18 12
HA, PP HA, F, DP, M MC, F, PP, BR, C, M PP, MC, BR F, AT, PP, BR, M HA, PP, BR, DP F, PP, MC, M F, BR, PP, M PP, FP HA, M PP, MC HA, F, M, MC F, HA, FP, PP, DP HA, HMP, FP HA, PP, BR, RF
Polyradiculonevritis Meningoradiculitis Meningoencephalomyelitis Encephalomyelitis Meningoencephalomyelitis Encephalomyelitis Meningoencephalomyelitis Meningomycloradiculitis Polyradiculonevritis Meningitis Encephalomyelitis Meningo-encephalomyelitis Mycloradiculitis Encephalomyelitis Polyradiculonevritis
CR CR Da CR CR CR CR CR CR CR CR CR CR PRb CR
)
AT, Ataxia; BR, Bladder retention; C, Coma; DP, Diplopia; F, Fever; FP, Facial paralysis; HA, Headache; HMP, Hemiplegia; M, Meningitis; M.C., Mental confusion; PP, Paraplegia or paraparesia; RF, Respiratory failure. )) CR, Complete recovery; D, Death; PR, Partial recovery. a Perforation of gastric ulcer due to corticotherapy. b Facial diplegia.
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plasma dilution of 1:100 in PBS, 0.02% Tween-20 and 1% bovine serum albumin ŽBSA.. After overnight incubation at 48C, blots were washed, treated with peroxidase-conjugated rabbit immunoglobulin to human IgM or IgG ŽSigma. for 90 min at room temperature and developed with diaminobenzidine hydrochloride and H 2 O 2 ŽSigma..
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2.4. Detection of antibodies to gangliosides by ELISA Reactivity of plasmas from patients and controls with GM1, GM2, GD1a, GD1b, GD3 or GT1b gangliosides was assessed by ELISA as described elsewhere ŽGarcia-Guijo et al., 1995.. Briefly, flat bottom 96-well microtiter plates
Fig. 1. Immunoblot analysis of purified human central myelin with plasmas from patients and controls. Panels A and C: Plasmas from five controls tested at day 0 Ž). or day 28 Ž Ø . of the vaccination therapy. Panels B and D: Plasmas from five patients with post-vaccination ANPA. Panels A and B: IgM detection. Panels C and D: IgG detection.
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Table 2 ELISA reactivity for IgM and IgG classes, expressed as OD units Žmean " SD. of plasmas collected from 30 control vaccinees before ŽC0. or at day 28 ŽC28. of the vaccination therapy or from 15 patients developing ANPA. Controls C0
C28
IgM ŽA. Electroeluted fractions 1 0.102 " 0.112 2 0.082 " 0.069 3 0.122 " 0.182 4 0.092 " 0.021 5 0.092 " 0.181 6 0.082 " 0.102 7 0.029 " 0.060 8 0.160 " 0.108 9 0.128 " 0.099 10 0.021 " 0.128 11 0.069 " 0.106 12 0.082 " 0.098 ŽB. MBP 0.150 " 0.035
ANPA
IgG
IgM
IgG
IgM
IgG
0.063 " 0.071 0.037 " 0.093 0.036 " 0.073 0.001 " 0.010 0.069 " 0.106 0.037 " 0.093 0.131 " 0.091 0.113 " 0.061 0.067 " 0.039 0.011 " 0.091 0.171 " 0.036 0,049 " 0,107 0,115 " 0.075
0.037 " 0.101 0.061 " 0.057 0.049 " 0.031 0.048 " 0.104 0.052 " 0.063 0.087 " 0.107 0.024 " 0.060 0.160 " 0.103 0.060 " 0.093 0,038 " 0,071 0,087 " 0,113 0,049 " 0,036 0.140 " 0.147
0.098 " 0.154 0.056 " 0.098 0.099 " 0.103 0.039 " 0.063 0.109 " 0.065 0.047 " 0.082 0.071 " 0.073 0.123 " 0.099 0.071 " 0.123 0,057 " 0,077 0,096 " 0,089 0,024 " 0,047 0.132 " 0.137
0.045 " 0.078 0.057 " 0.093 0.056 " 0.075 0.008 " 0.010 0.069 " 0.104 0.057 " 0.095 0.151 " 0.098 0.113 " 0.061 0.042 " 0.059 0.088 " 0.091 0.121 " 0.034 0,049 " 0,102 0.185 " 0.073
0.102 " 0.157 0.087 " 0.064 0.122 " 0.132 0.042 " 0.075 0.092 " 0.131 0.031 " 0.062 0.069 " 0.104 0.097 " 0.108 0.062 " 0.097 0.069 " 0.098 0.101 " 0.124 0.036 " 0.042 0.132 " 0.136
Plasmas were tested at 1r100 dilution with 12 fractions electroeluted from SDS-PAGE of human central myelin Žpanel A. or with purified human MBP Žpanel B..
ŽCostar, MA. were filled with 100 ml per well of ethanol containing 5 mgrml of the relevant ganglioside and left to air dry. After saturation for 4 h with 1% BSA in PBS at 378C, 100 ml of serially diluted plasmas in PBS–BSA, Žstarting from 1:100. were added in duplicate and kept overnight. This incubation and the following steps were carried out at 48C. After six washes in cold PBS solution, plates were incubated with 100 ml of peroxidase-conjugated affinity purified goat anti-human IgM or IgG antibodies ŽSigma. diluted 1:2000. After six washes, the reaction was developed Žfor 10 min to 30 min depending on the antigen used and following previous standardization of the developing-time of the reaction for each antigen by a blinded-way kinetic study on five samples from each group. with 200 ml 0.004% O-phenylenediamine, 0.1 M citrate, 0.2 M NaH 2 PO4 ŽpH 5.0. and 0.012% H 2 O 2 and read at 492 nm in Titertek reader. Results were expressed as reported by Sadiq et al. Ž1990.. Briefly, the optical density ŽOD. obtained with each plasma dilution tested on
BSA-coated wells was subtracted from those obtained with the same dilution tested on wells coated with the relevant ganglioside. Antiganglioside antibody titers for each patient was defined as a highest dilution factor at which the mean OD of the duplicate wells exceeded the cut-off value. The cut-off value corresponded to the mean OD q 3 SD obtained with the plasmas of the 30 control individuals before vaccination Žsamples C0. and tested at 1:100 dilution. 2.5. High performance thin-layer chromatography (HPTLC) immunostaining Immunoreactivity with myelin gangliosides of plasmas from patients and controls, was studied according to the procedure of Magnani et al. Ž1982. by an immunostaining assay after HPTLC of total glycolipids extracted from human central myelin. Specific gangliosides in the whole preparation of myelin glycolipids were localized by comigrating, in parallel, reference purified gangliosides which
Table 3 ELISA reactivity for IgM and IgG classes expressed as OD units Žmean " SD. of plasmas collected from 30 control vaccinees, before ŽC0. or at day 28 ŽC28. of the vaccination therapy or from 15 patients developing ANPA C0
Ganglioside GD1a GD1b GD3 GM1 GM2 GM3 GT1b
C28
ANPA
IgM
IgG
IgM
IgG
IgM
IgG
0.092 " 0.052 0.067 " 0.086 0.031 " 0.092 0.158 " 0.160 0.026 " 0.037 0.072 " 0.072 0.042 " 0.083
0.079 " 0.171 0.126 " 0.221 0.166 " 0.073 0.126 " 0.121 0.088 " 0.156 0.034 " 0.102 0.275 " 0.319
0.073 " 0.093 0.061 " 0.057 0.079 " 0.031 0.395 " 0.204 0.076 " 0.055 0.174 " 0.380 0.046 " 0.053
0.098 " 0.076 0.162 " 0.293 0.372 " 0.216 0.113 " 0.204 0.160 " 0.180 0.037 " 0.097 0.137 " 0.237
0.071 " 0.083 0.082 " 0.070 0.076 " 0.093 0.846 " 0.452 0.129 " 0.104 0.176 " 0.155 0.170 " 0.310
0.455 " 0.563 0.164 " 0.296 0.362 " 0.408 0.360 " 0.541 0.102 " 0.121 0.061 " 0.161 0.258 " 0.238
Plasmas were tested individually at 1r100 dilution with a panel of seven gangliosides.
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were then stained with resorcinol. Immunodetection was performed on aluminium-backed thin-layer chromatography ŽTLC. plates Žsilica gel 60, Merck, Germany. loaded with 10 mg of total glycolipids determined by the method of Svennerholm Ž1957.. Plasmas were used at a dilution of 1:100 Žin PBS solution containing 1% gelatin and 10% inactivated horse serum. and incubated for 2 h at 378C. The second antibody was a peroxidase-conjugated anti-human IgM ŽSigma.. After 1 h incubation at room tempera-
67
ture, the plates were washed and bound antibodies were detected with 4-chloro-1-naphthol and 0.003% H 2 O 2 reagent. 2.6. ELISA detection of antibodies to myelin fractions and MBP Reactivity of plasmas from patients and controls with myelin fractions or purified MBP was tested by ELISA.
Fig. 2. IgM and IgG antibodies to GD3 Ža and b., GD1a Žc and d. and GM1 Že and f.. Plasmas were obtained from patients who developed post-vaccination acute disseminated encephalomyelitis. C0, C7, C14 and C28 correspond to plasma samples obtained from uneventfully vaccinated individuals bled at day 0, 7, 14 and 28, respectively.
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Briefly, each antigen was dissolved at optimal concentration Ž0.5 mg to 1.25 mgrml for the different fractions and at 4 mgrml for human purified MBP. in carbonate–bicarbonate buffer ŽpH 9.6. and 100 ml were used to coat 96-well polystyrene plates ŽCostar. and incubated overnight at 48C. The plates were blocked with PBS–BSA 1% for 1 h at 378C before adding serially diluted plasma samples. The following steps were performed as described above for antiganglioside ELISA detection ŽSection 2.4.. 2.7. Statistical analysis Statistical analysis were performed with a ‘Stat-view’ software using ‘Student’s t-test’ and ‘Fisher’s x-test’.
3. Results 3.1. Antibodies to myelin proteins To determine whether specific antibodies to human myelin antigens develop during vaccination, plasmas from patients and controls were tested at 1:100 dilution by immuno-blot with purified myelin. Although some variability in the background reactivity was noticed from sample to sample, there was no significant difference in the pattern of reactivity of the same individual before ŽC0. or at the end of the vaccination course ŽC28.. This holds
true for both IgM and IgG antibodies ŽFig. 1A and C.. Similar results were obtained with sera from patients with post-vaccination ANPA ŽFig. 1B and D.. Of particular interest was the absence of reactivity with the major components of the myelin sheets Ži.e., MBP, cyclic nucleotide phosphodiesterase ŽCNPase. and proteolipid protein ŽPLP. as well as with the myelin associated glycoprotein ŽMAG... In order to enhance the sensitivity of the assay and allow quantitative comparison between data obtained with plasmas from patients and controls, ELISAs were performed using twelve different fractions electroeluted from SDS-PAGE of human central myelin. Reactivities with all fractions including those containing MBP, PLP, WP as well as MAG did not exceed 0.3 OD units ŽTable 2A. and no significant difference could be detected between patients and controls. The presence of antibodies to human MBP, was more specifically assessed. All plasmas from patients and controls ŽC0 and C28. were tested by ELISA with MBP extracted from myelin. IgM and IgG reactivity of plasmas from patients who developed neurologic complications were constantly weak and not significantly different from pre or post-vaccination control plasmas ŽTable 2B.. These results indicate that administration of Semple rabies vaccine containing 5% lamb brain extract does not induce detectable levels of antibodies against human central myelin proteins including MBP, CNPase, MAG and
Fig. 3. IgM anti-GM1 absorbances ŽELISA. of serially diluted plasmas from 15 ANPA patients Ž`. or controls Ž Ø s day 0; I s day 7; ' s day 14; ls day 28. The absorbances of control plasmas are expressed as the mean reactivity of 30 individual plasmas" SD.. Dotted line: cut-off value of the ELISA defined as 3 SD of the mean OD of the control group ŽC0. tested at 1:100 dilution.
Dh. Laouini et al.r Journal of Neuroimmunology 91 (1998) 63–72 Table 4 Plasmas reactivity with GD1a or GM1 Žtiters G100. in 15 patients with ANPA and 30 uneventfully vaccinated controls ŽNb positiverNb tested. GD1a
ANPA Controlsa p- b
GM1
IgM
IgG
IgM
IgG
8r15 0r30 0.005
4r15 0r30 0.025
10r15 0r30 0.005
7r15 0r30 0.025
a
All plasmas obtained at days 0, 7, 14 and 28 were tested. p Values for titer comparison between ANPA patients and each control group.
b
PLP, either in asymptomatic vaccine recipients or in those who developed post-vaccination acute neuroparalytic accidents. 3.2. Antibody responses to gangliosides In order to analyse the antibody responses to brain gangliosides that could have been induced in patients and controls by the vaccination protocol, ELISA with purified gangliosides and immuno-HPTLC detection were performed. IgM and IgG reactivities with gangliosides, of plasmas from control samples and from ANPA group are shown in Table 3 Žtested at 1:100 dilution. and in Fig. 2 Žfull titration.. Three different patterns of antiganglioside responses could be observed. The first pattern includes GD1b, GM2, GM3 and GT1b gangliosides for which plasmas from controls ŽC7, C14 and C28. from ANPA patients showed no reactivity Žtiters
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- 100., as plasmas collected before the vaccination therapy ŽC0. Ždata not shown.. The second pattern concerned GD3 ganglioside: four patients Ž26% of ANPA group. and three controls Ž10% of plasmas collected at days 14 and 28. developed significant IgG anti GD3 reactivity Žantibody titers G 200.. These responses were restricted to the IgG class ŽFig. 2a and b.. The third pattern encompassed antibody responses to GD1a and GM1 gangliosides: ANPA patients but not controls developed high IgM and IgG reactivity with GD1a and GM1 Žantibody titers: 100–800.. IgM antibodies to GD1a and GM1 were detected in 8 patients Ž53%. and 10 patients Ž66%., respectively ŽFig. 2a,c,d,fFig. 3.. When antibody titers to GD1a and GM1 gangliosides in ANPA patients are compared to those of controls Žsamples C0, C7, C14 and C28., high significant statistical differences were found Ž p - 0.005 for IgM and p - 0.025 for IgG. ŽFig. 3 and Table 4.. Plasmas from four patients Žno. 5, 7, 12 and 15. had high titers to both GM1 and GD1a; in six patients Žno. 1, 3, 6, 9, 11 and 14. plasmas had high titers only to GM1. Plasmas from four patients Žno. 2, 4, 8 and 14. had high titers only to GD1a and only one patient Žno. 13. did not respond either to GM1 or to GD1a. The three patients with polyradiculonevritis Žno. 1, 9 and 15, Table 1. developed high antibody titers to GM1. In addition, one of them Žno. 15. had antibodies to GD1a. Plasma samples from 13 ANPA patients were tested by HPTLC immunostaining of human central myelin glycolipids ŽFig. 4A.. All plasmas immunostained electively GM1 ganglioside. Two plasmas showed, in addition, a dim staining of the GD1b spot. None of the 30 control plasmas obtained at day 28 showed immunostaining of HPTLC plates Ždata not shown..
Fig. 4. Immunodetection by plasmas from six ANPA ŽA., of glycolipids on thin-layer chromatography. Arrows in panel ŽA. show positive immunostaining of GM1 or GD1b gangliosides. Panel ŽB. shows standard gangliosides ŽGM1, GD1a, GD1b and GT1b. stained by resorcinol.
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4. Discussion We show that rabies vaccine post-exposure therapy using brain-derived vaccine, whether or not associated to the subsequent development of neuroparalytic accidents, is not associated with any significant antibody response to human central myelin proteins. No specific immunostaining of the major myelin proteins ŽMBP, MAG, PLP and CNPase. could be detected by immunoblot analysis, and there was no significant ELISA reactivity with 12 fractions electroeluted from SDS-PAGE of human central myelin. Moreover, we could not detect any reactivity by ELISA with human purified MBP in plasmas from either ANPA patients, or from controls. This result is in contradiction with that reported by Hemachudha et al. Ž1987b. who found antibodies to human MBP in patients who develop post-vaccine ANPA. These antibodies reacted specifically with peptides 45–89 and 90–170 of MBP ŽUbol et al., 1990.. Considering that sheep MBP has approximately 90% homology with human MBP, these investigators concluded that vaccination had presumably disturbed normal immunoregulatory mechanisms and led to the development of an autoimmune response ŽUbol et al., 1990.. The reasons which could account for discrepancies between our results and those reported by Hemachudha are unclear. Variation in vaccine composition may explain differences in the immune response of vaccine recipients. The Thai rabies vaccine was prepared on adult sheep brain while the Tunisian one used a lamb brain which is poorly myelinated and contains lower amounts of MBP. We noted previously that protein content of myelin extracted from the same quantity of crude material is 5- to 10-fold lower in lamb brain than in sheep brain Ždata not shown.. These differences in the composition of the nervous substrate and the way of preparation of the vaccine could also explain the high rate of neurologic complications induced by Thai vaccine compared to those induced by the Tunisian one ŽHemachudha et al., 1987a; Swaddiwuthipong et al., 1988.. Moreover, the Thai vaccine was inactivated by phenol whereas the Tunisian one was inactivated by b-propiolactone. These chemicals may have different effects on B and T cell epitopes of myelin proteins and hence their immunogenicity. A direct relationship exists between the content of nervous tissue in a particular vaccine and the frequency or the clinical expression of neuroparalytic accidents. Thus, Semple-type vaccine induces electively encephalomyelitis in about 90% of cases, whereas suckling mouse brain vaccine induces preferentially polyradiculonevritis of the Guillain–Barre´ type ŽHeld and Lopez-Adaros, 1972; Toro et al., 1977; Hemachudha et al., 1987a.. The neural antigens, targets of the immunopathogenic mechanisms underlying these complications are most likely different. Our results suggest that antibodies to myelin proteins are unlikely to play a significant role in the pathogenesis of rabies vaccine-induced encephalomyelitis in Tunisian patients. One cannot exclude however that MBP andror
other protein components of the human central nervous myelin ŽMAG, myelin-oligodendrocyteglycoprotein MOG., may be target antigens for a T cell response, as clearly demonstrated in experimental autoimmune demyelinating diseases Žsee Zamvill and Steinman, 1990 and Martin et al., 1992 for review.. With regard to the antibody response against gangliosides, we found that the levels of IgM and IgG antibodies to GD1b, GM2, GM3, and GT1b were not affected, either by the number of rabies vaccine doses received by the vaccinees or by the development of the post-vaccine neurologic complications. On the opposite, the vaccination therapy induced a significant increase of IgG anti-GD3 antibodies which correlated with the number of vaccine doses received by the vaccinees and most likely represent the humoral response to administration of exogenous gangliosides contained in the vaccine substrate. Interestingly, we observed high titers of IgG and IgM antibodies to GM1 and GD1a only in those vaccinees who developed neurologic symptoms, which suggest that these autoantibodies may play a role in the development of ANPA. Gangliosides have been recently identified as antibody targets in several neurological diseases. Several authors have described the presence of antiganglioside antibodies in the sera of patients with Guillain–Barre´ syndrome ŽIlyas et al., 1988. and amyotrophic lateral sclerosis ŽPestronk et al., 1988b.. High titers of IgM anti-GM1 antibodies have been associated with lower motor neuron syndrome ŽPestronk et al., 1990. and less frequently with chronic sensory multifocal neuropathy ŽPestronk et al., 1988a.. However, Hemachudha reported that after Semple rabies vaccination, antibodies to GM1 fraction were present in individuals with or without post-vaccine complications and that the mean levels were similar in both groups ŽHemachudha et al., 1987b.. The variation between our results and those of Hemachudha et al., could be explained by the difficulty in ELISA standardization for the detection of antibodies to gangliosides and the use, in our hands, of a more discriminative way to express the results Ža prior standardization of the assay and a very high cut-off value.. Moreover, several lines of evidence suggest that naturally occurring anti-GM1 antibodies are part of the antibody repertoire of normal individuals and could therefore be detected in normal plasma ŽMizutamari et al., 1994.. The level of these antibodies is not influenced by the administration of exogenous gangliosides ŽDoria and Tettamanti, 1993; Ala et al., 1994; Mizutamari et al., 1994., although, some authors claimed the opposite ŽFigeuras et al., 1992; Schneider and Roeltgen, 1993.. Interestingly, Deisenhammer et al. Ž1996. recently reported that anti-GM1 antibodies may vary in their affinity and fine specificity and that pathogenic antiGM1 in Guillain–Barre´ syndrome may belong to a subgroup of antibodies, while low affinity anti-GM1 seem to be non-specific and are not associated with any specific disease. More recently, it was reported that low affinity IgM antibodies are devoid of complement mediated lytic
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activity on GM1 liposomes in contrast to high affinity anti GM1 antibodies ŽMizutamari et al., 1998.. With regards to anti-GM1 antibodies detected in our study, it is noteworthy that using a technique of lower sensitivity than ELISA, i.e., HPTLC immunodetection, we were able to detect anti-GM1 antibodies constantly and only in ANPA patients. This result suggests that this technique is the most suitable to detect high affinity andror pathogenic anti-GM1 antibodies associated to ANPA. It is tempting to speculate that GM1 ganglioside present in the substratum of rabies vaccines may trigger the appearance of pathogenic antiGM1 antibodies which will react with their respective antigen exposed on the outer surface of neuronal membrane and participate to the demyelinating process. Alternatively, these antibodies may have been induced, secondary to the demyelination process. Whatever the mechanism of demyelination in ANPA, cell-mediated or antibody-mediated, these responses may act synergistically: autoantibodies can contribute to the triggering or the aggravation of the demyelinating process, as recently demonstrated in the Experimental Autoimmune Neuritis ŽEAN., an animal model of Guillain–Barre´ syndrome ŽSpies et al., 1995.. In this context, our results show that antibodies to gangliosides ŽGM1 and GD1a. are more likely implicated in the pathogenesis of post-rabies vaccine ANPA than antibodies to myelin protein.
Acknowledgements We are grateful to Pr. N. Baumann ŽHopital la Pitie´ ˆ Salpetriere, ˆ ` Paris, France. for helpful discussion, to Dr. H. Villarroya ŽHopital la Pitie´ Salpetriere, ˆ ˆ ` Paris, France. for generous gift of anti-MBP antibody, to Pr. M. Hamdoun ŽHopital Charles Nicolle, Tunis, Tunisia. for providing ˆ human tissues and to Mrs. M. Gadhab for collecting blood samples.
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