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Letter
Contamination with gangliosides in brain-derived rabies vaccine may trigger Guillain–Barré syndrome
  1. Hiromichi Sakai1,
  2. Faqeehah Mohamed Harun1,
  3. Naoki Yamamoto1,2,
  4. Nobuhiro Yuki1,2
  1. 1Department of Microbiology, National University of Singapore, Singapore
  2. 2Department of Medicine, National University of Singapore, Singapore
  1. Correspondence to Professor Nobuhiro Yuki, Department of Microbiology, National University of Singapore, Block MD4A, Level 5, 5 Science Drive 2, Singapore 117597, Singapore; micyuki{at}nus.edu.sg

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Guillain–Barré syndrome (GBS) is an autoimmune-mediated peripheral neuropathy typically occurring after microbial infections such as Campylobacter jejuni enteritis. It can also occur following vaccinations such as the 1976 swine flu vaccine in the USA.1 GBS is divided into demyelinating and axonal subtypes. There is now good evidence that gangliosides or similar components trigger the development of axonal GBS.2 Axonal GBS associated with IgG anti-GM1 or anti-GD1a antibodies after bovine brain ganglioside administration have been recorded in several patients. Sensitisation of rabbits with bovine brain gangliosides or isolated GM1 produced a replica of axonal GBS. Based on these findings, it has been suggested that C jejuni components mimic human gangliosides GM1 and GD1a, and C jejuni infection induces the production of autoantibodies against the gangliosides that are expressed in the peripheral nerves, resulting in the limb weakness seen in GBS. By contrast, the mechanism by which certain vaccines elicit the development of GBS remains unresolved, although there have been studies to suggest that the 1976 swine flu vaccine could elicit anti-GM1 antibodies in mice and that the GM1 epitope was present in the influenza haemagglutinin.3

It is important to understand the pathogenesis of postvaccination GBS to allow safer vaccines to be developed. A number of cases of GBS developing in patients who received the rabies vaccine have been reported.1 GBS has been associated with two forms of rabies vaccines: the Semple rabies vaccine and the suckling mouse brain vaccine. The former is produced by inoculating the rabies virus into mature sheep or goat brain and then inactivating the virus with phenol. The newer formulations of rabies vaccine, derived from chick embryo cells, do not appear to be associated with GBS. We, therefore, hypothesised that the older formulations of the vaccines were contaminated with gangliosides.

A sheep brain, a sheep brain-derived rabies vaccine and two chick embryo cell culture vaccines (Chiron Behring Vaccines Private Ltd., Ankleshwar, India, and Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan) were separated in 10% or 14% SDS-polyacrylamide gel. The latter was blotted on a polyvinylidene difluoride membrane and incubated with antimyelin basic protein antibodies (1:500; Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), followed by peroxidase-conjugated antisheep IgG antibodies. Binding was made visible with an enzyme chemiluminometric reagent. Lipids were extracted from a sheep brain, a sheep brain-derived rabies vaccine and chick embryo cell culture vaccines using a mixture of chloroform, methanol and 12 mM magnesium chloride in water in the ratio of 30:60:8. Acidic glycolipids were prepared and each sialic acid was quantified with a resorcinol reagent. Thin-layer chromatogram plates were developed and stained with the resorcinol reagent, monoclonal anti-GM1 antibody (GB2) and sera from patients with GBS subsequent to C jejuni enteritis. Informed written consent was obtained from each subject.

Figure 1 shows the biochemical results. The sheep brain-derived vaccine contained myelin basic protein, a candidate antigen that induces encephalomyelitis,4 indicating contamination of the vaccine with nerve tissue. One hundred microlitres of the brain tissue-derived vaccine included 2.5 μg of sialic acids, whereas the sialic acids were not detected in the same amount of the cell culture vaccines. The resorcinol staining along with the immunostaining revealed that the brain-derived rabies vaccine was contaminated with gangliosides such as GM1, GD1a, GD1b and GT1b, but the cell culture vaccines were not. Serum from a GBS patient reacted with GalNAc-GD1a, as well as with GM1, or GD1b in the nerve tissue-derived vaccine. These findings lend further support to the hypothesis that contamination with nerve tissues in brain-derived rabies vaccine leads to the frequent occurrence of GBS following vaccination, and the rare occurrence of GBS following the chick embryo cell vaccination.1

Figure 1

(A) Myelin basic protein and other brain-derived proteins in a sheep brain-derived vaccine. A sheep brain (lane 1, 15 μg of protein), 10 μl of the brain-derived vaccine (lane 2, 7.7 μg of protein) and 10 μl of two chick embryo cell culture vaccines (lanes 3 and 4, 2.8 and 1.5 μg of proteins, respectively) were separated on (a) 10% or (b) 14% SDS-polyacrylamide gel. (a) A gel stained with Coomassie blue and (b) western blot immunostained with antibodies against myelin basic protein. (B) Gangliosides in a sheep brain-derived vaccine. Acidic glycolipid fractions prepared from a sheep brain (lane 1), a sheep brain-derived vaccine (lane 2) and the cell culture vaccines (lanes 3 and 4) were developed by a solvent system comprising chloroform:methanol:12 mM magnesium chloride in water (5:4:1, by volume). (a) A thin-layer chromatogram plate stained with the resorcinol reagent. The brain-derived vaccine, as well as the sheep brain, contains GM1, GD1a, GD1b and GT1b. (b) A chromatogram immunostained with monoclonal anti-GM1 antibody (GB2). (c, d) Chromatograms stained with sera from patients with Guillain–Barré syndrome subsequent to Campylobacter jejuni enteritis. The serum from a patient reacts with GM1 in the brain-derived vaccine, and the serum from another reacts with GM1, GalNAc-GD1a and GD1b.

A group from Tunisia tested plasma anti-ganglioside antibodies in 15 patients who developed an acute paralytic event following exposure to rabies vaccine prepared in brain tissue.5 Antibodies to GM1 or GD1a were detected in plasma from 14 patients, which included 3 cases of GBS and 8 cases of encephalomyelitis, but not in 30 individuals who were uneventfully vaccinated with the same vaccine using the same protocol. Neurophysiological features were not specified in the reported cases of GBS following brain-derived vaccination. However, we postulate that the neurophysiological changes are likely to be axonal because anti-GM1 or anti-GD1a antibodies were detected in the three patients.

Taking into account the findings from the clinical and experimental studies in GBS following ganglioside administration,2 we postulate that brain-derived rabies vaccine induces the production of anti-GM1, anti-GD1a or anti-GalNAc-GD1a antibodies, and the development of axonal GBS in certain patients. Our results, however, do not rule out the traditional explanation that GBS following brain-derived vaccination is due to the protein content of the vaccine. To prove our hypothesis, reproduction of a GBS model by sensitisation with the brain-derived vaccine as well as neurophysiological data on patients supporting an axonal pattern of GBS is required.

The WHO currently recommends the use of cell culture vaccines following its recommendation for cessation of the production of nerve tissue vaccines in 2003, 2006 and 2009. However, several countries continue to use the older form of vaccine due to its low cost. As a result, there continue to be reported cases of GBS following some vaccinations.1 Our biochemical results support the recommendation against the use of nerve tissue vaccines to avoid the occurrence of GBS. In addition, we would also suggest that biochemical analyses of vaccines should be performed prior to their use to reduce the risk of GBS following vaccination.

Acknowledgments

We thank Nortina Shahrizaila (University of Malaya) for her help in editing the manuscript.

References

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Footnotes

  • Funding This work was supported by Yong Loo Lin School of MedicineStart-up Grant (Naoki Yamamoto).

  • Competing interests None.

  • Ethics approval National University of Singapore.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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