Molecular Sciences Bldg 21, James Cook University,
Townsville, 4811, Queensland, Australia
Telephone: 61-7-4781 6265 Fax: 61-7-4781 6078
Beware of brains. I don't mean beware of brainy people, but be very careful when handling bits of brains taken from humans or animals.
There's a disease as you may know called Kuru, for instance, once common in Papua New Guinea, which killed women and children in various mysterious ways, until, that is, Dr Carlton Gajdusek worked out the origins of it: mothers preparing meals from dead ancestors in a ritual that spread disease. Dr Gajdusek won the Nobel Prize for his detective work and for the insights the work gave to many other diseases of the nervous system, from Alzheimer's to multiple sclerosis.
Then there was mad cow disease, spread by feeding the remains of other animals to cattle, and what a disaster that turned out to be, which brings us to rabies and Pasteur and Professor Alan Baxter in Northern Queensland, where he's an immunologist at James Cook University.
While a second year medical student at Johns Hopkins University in Baltimore, Thomas Rivers had been incapacitated by a progressive muscular disease. Twenty-four years later, and now a senior scientist at the Rockefeller Institute in New York, his experience led him to investigate an epidemic of ascending paralysis in people treated with Louis Pasteur's rabies vaccine.
Rabies spreads from its site of introduction, usually a bite or scratch of an infected animal, towards the brain through the nervous system. Early signs of infection include restlessness, headache, fever and tiredness. Within days, patients become agitated, confused and disoriented and may fit of hallucinate.
Hydrophobia, a fear of water so intense that painful throat spasms occur upon seeing or trying to swallow fluids, is the diagnostic hallmark of the disease. Rabies is almost always fatal and without intensive care treatment, patients die within a few days.
Pasteur's vaccine was first used clinically in 1885 when it was administered to Joseph Meister, a nine-year old schoolboy severely mauled by a rabid dog. In Pasteur's laboratory, rabies virus was propagated by the inoculation of the virus into the brains of live rabbits. The spinal cords of infected rabbits were removed and dried by being suspended in sterile glass columns containing potassium hydroxide. Treatment consisted of a ten-day course of injections, starting with emulsions of cords dried for 14 days, and gradually progressing to those containing more virulent cord, dried for only 5 days. Early success brought general acceptance, and between 1885 and 1887, nearly 5,000 people were treated in 21 institutes without reported complications.
As cases of vaccine failure subsequently accumulated Pasteur and his disciples intensified treatment, applying more virulent cord earlier. At its most extreme, cords dried for only a single day were injected. Soon published reports described sporadic cases of paralysis affecting a proportion of patients vaccinated. In mild cases, one or more muscle groups were affected, resulting in facial palsy or paralysis of limbs.
Recovery was usually rapid, and persistence exceptional. In its most severe form, affecting over one third of cases, pain and paralysis ascended from lower extremities to the upper limbs and neck, affecting swallowing and breathing, and resulting in a mortality of up to 30%.
Significantly, the illness was never associated with hydrophobia nor the damage to nerve fibres typically seen in rabies. Although the incidence of this neurological disease varied widely, it appeared to rise dramatically after the First World War.
From 1894 to 1914, no cases of paralysis were reported following treatment of 7,632 people, whereas between 1915 ane 1923, 39 cases were reported following vaccination of 6,764 patients. In a single month in Kharkow, Russia, 25 patients developed paralysis after treatment with a rabbit spinal cord dried for only one day.
Thomas Rivers saw a parallel between the neurological complications of viral diseases that could affect the brain and spinal chord, such as smallpox, vaccinia and measles, and those that developed following rabies vaccination.
A series of experiments in Rhesus Macaques and rabbits tested his suspicion of a viral origin for the paralysis. In these experiments he included a control group that received repeated injections of emulsions of normal rabbit brain that did not contain virus. Two of the eight monkeys in the control group developed clumsy gaits and weakness; one had received 52 injections, the other 84.
Microscopic analysis of their brains revealed localised areas of inflammation and damage to myelin, the white matter of the nervous system. Because myelin damage can occasionally occur spontaneously in Rhesus monkeys, Rivers attempted a second experiment, in which eight uninjected monkeys provided a control group for a further eight that received up to 85 repeated injections of rabbit brain emulsions or ethanol and ether extracts of brain.
Six of the eight treated monkeys developed clumsy gaits and muscle weakness and, in some cases, became severely disabled. On post mortem examination, seven of the eight were found to have regions of myelin degeneration in the brain and spinal cord, often associated with infiltrating white blood cells. No neurological signs or microscopic evidence of brain pathology was found in any of the uninjected animals.
In other experiments, rabbits receiving up to 26 injections of emulsified rabbit brain developed varying levels of anti-brain antibody, depending on the nature of the emulsion.
Emulsions containing foreign serum, prepared from rabbits infected with the smallpox vaccine, or from emulsions stored at room temperature for weeks, developed higher amounts of antibody.
A variable proportion of the rabbits also developed an ascending paralysis, which started in the hind limbs and progressed to the forelimbs, and heck, before finally paralysing the respiratory muscles. The more anti-brain antibodies the emulsion caused, the higher the proportion of rabbits affected by paralysis.
The presence of white blood cells in the brain lesions and the association between anti-brain antibodies and pathology suggested the possibility that the myelin damage was caused by a specific immune response.
To test this possibility, several research teams tried boosting the production of antibodies by administering brain emulsions mixed with paraffin oil and killed tuberculosis bacteria, applying a strategy to boost vaccine responses invented by Jules Freund of the Public Health Research Institute in New York.
This treatment resulted in paralysis after only a few injections in the vast majority of recipient animals. The disease they suffered, termed an 'autoimmune encephalomyelitis', bore a strong resemblance to multiple sclerosis and the brain diseases that can occur in humans after some viral infections or vaccines.
As in multiple sclerosis, Rhesus monkeys receiving three injections of rabbit brain in Freund's mixture suffered a remitting, relapsing course of the illness. An animal apparently blind might recover its vision within a few days, only to lose it again. Weakness in one limb might recede to be replaced by paralysis of another.
The experimentally induced disease was also similar to multiple sclerosis and post-infectious brain disease at the microscopic level. Features in common include the destruction of the myelin sheaths of the nerve fibres; the presence of multiple lesions distributed in time and position; and the tendency for the lesions to occur near blood vessels. It seems likely that these sorts of pathological changes represent a stereotypic immune response triggered by damage to, or injection of, brain and spinal cord tissues.
Evidence that this is the cause of the brain damage associated with Pasteur's vaccine includes the finding that the severity of neurological complications in humans following vaccination with rabies vaccines, correlated with the vigour of anti-brain immune responses induced.
This work raised the question: in what way was the Pasteur rabies vaccine like a mixture of emulsified brain and tuberculosis bacteria? It turned out that the vaccine was contaminated by the brain tissues from which it had been prepared. In fact, there was so much brain protein present in the vaccine that if it was added to Freund's mixture instead of brain extract, it could induce experimental paralysis in guinea pigs.
But what about the tuberculosis bacteria? It was extremely unlikely that either of the animals infected, or the vaccines prepared from them, would be contaminated with any bacteria, let alone those that cause tuberculosis.
We now know that the effect of killed tuberculosis bacteria on the immune system is mediated through a family of specialised detector molecules, called Toll-like receptors. It is the reaction trigged by the activation of Toll-like receptors that is responsible for the immune-boosting effect of Freund's mixture. Other members of this same family of molecules recognise viruses instead of bacteria, but trigger very similar changes in the immune system.
In 1979 it was confirmed that an emulsion of brain extract and virus could be substituted for Freund's mixture in the induction of experimental paralysis.
In order to avoid causing paralysis in humans, the rabies vaccine that is administered in Australia is no longer isolated from the brains of infected animals. The propagation of rabies virus in cultured cells was achieved in 1937, and many vaccine manufacturers have adopted this system for the production of the rabies vaccine. As a consequence, these vaccines do not contain proteins from brain or spinal cord, and do not cause brain damage.
It is these vaccines that meet the World Health Organisation standards for safety, potency and efficacy when used for post-exposure immunisation against rabies.
Unfortunately, it is quite a different story in the developing world. More than 99% of human deaths from rabies occur in Africa, Asia and South America, and each year over 10-million people in these countries receive post-exposure vaccination against the disease.
It is a sad indictment on the vaccine industry, that throughout many of the countries in which rabies exposure most commonly occurs, the rabies vaccines used are still produced in the brains of live animals. And the incidence of neurological complications in those treated is as high as one would expect from this archaic technique.
The problem persists. Cheap drugs for the undeserving poor. Alan Baxter is Professor of Biochemistry at James Cook University in Townsville, Northern Queensland, and President of the Australasian Society for Immunology.
Next week, we go to Adelaide, to hear from Bill Hall about clocks.
I'm Robyn Williams.
'A piece of my mind' is © Alan G Baxter 2008
Autoimmunity Research Group, Centenary Institute of Cancer Medicine and Cell Biology, Key words: Autoimmune diabetes, Type 1 diabetes mellitus, childhood diabetes, lupus, systemic lupus erythematosus, hemolytic anaemia, hemolytic anemia, Coombs' test, antinuclear antibodies, renal failure, glomerulonephritis, gastritis, type A gastritis, pernicious anemia, immunology, popular science, biology.