045 - The Known and Unknown of Bacillus Pertussis Vaccine

Whooping cough, or pertussis, is caused by the bacterium Bordetella pertussis, named after a researcher named Bordet. In this study it was called Bacillus pertussis. Louis Sauer reviews many aspects of the current status (at that time) of vaccines to treat or prevent this disease.

The way the vaccines had been prepared for two decades, the organism had been grown with fresh blood in culture, then killed, preserved, and standardized to a particular number of bacteria. This was given in 3 injections, 3-4 weeks apart. But the value of this formulation was not very clear; it seemed not to prevent the disease and definitely couldn't cure it, though immunization with it might make the course of the disease more mild and less deadly, which is something.

But the disease was so serious that people couldn't give up hope in creating an effective prevention. It is most deadly in infants up to 2 years of age, and it is difficult to watch them suffer.

What they had figured out, at least, was that when studying the effectiveness, it was best to study children from 8 months to 3 years of age. Younger might be too young to work well, and in older children, the standard dose seemed to be too small (assuming they hadn't already encountered the disease).

They also knew that if 100 people were exposed to the disease, only 75 of them would catch it. So 25% should stay well. It's always hard to tell whether someone really was exposed to a disease, unless you're exposing them to it yourself, which in this case would be highly unethical. So Sauer recommends studying siblings, some vaccinated and some controls, to be extra sure of exposure. But it's still important to confirm the diagnosis in the lab.

Sauer reports that over four years, some 394 children were vaccinated with a new, differently prepared pertussis vaccine, with a much higher dose than previously. These were 14 months old, on average. The researchers observed 360 exposures of 169 of the subjects to the disease over 7 years, but the only sign of pertussis in the subjects was a mild cough in a girl that had just recovered from measles. That's good results.

So Northwestern University Medical School permitted two commercial laboratories to make this new kind of vaccines, with fresh isolates of the pathogen each month, grown in fresh human blood, and refrigerated after preparation. Sounds difficult.

But it seemed to give good results: of 127 known exposures in vaccinated subjects, only 10 resulted in pertussis; this is a protection rate of 92%. Pretty good. And most of the cases were in children over 3 who might've gotten too small a dose.

Other physicians reported around 80% efficacy (21 failures out of 130 exposures), with failures again mostly in children over 3. So it seems that the dosing for older toddlers needs modifying. It's also possible that some failures took place before the immunity had a chance to establish itself; this process seems to take three months or so after injection.

Seeing that pertussis is so dangerous for young infants, some researchers tried immunizing infants under six weeks of age. These infants were able to "withstand the injections remarkably well," whatever that means. They observed 9 exposures to the disease, and five of them got the disease. So they were probably too young to be vaccinated effectively, unfortunately.

So from this, it sounds like the newer method of preparation and dosing can be 80-100% effective, and better if given at the right age. I'd like to read the actual studies reporting these experiments before accepting these numbers though, so stay tuned.

Citation: Sauer, L. The Known and Unknown of Bacillus Pertussis Vaccine. Am J Public Health Nations Health 25, 1226–1230 (1935).

044 - Cutaneous Reactions and Antibody Response to Intracutaneous Injections of Pneumococcus Polysaccharides

This study wasn’t as exciting as I expected, but I didn’t find that out until I was mostly finished with it, so I figured I’d go ahead with it and try to do better next time.

For vaccines against pneumonia caused by bacteria called pneumococcus, people tried whole bacterial cells, killed and injected, which worked somewhat, but wasn’t great. Then, over time, they realized they might not need to use whole cells, but only components of the cells’ capsule (see 028 and 039).

In today’s study, Maxwell Finland and Harry F. Dowling tested different kind of polysaccharides from pneumococcus capsules in volunteers with different histories of infection.¹

There were four groups of patients: 1) hospital patients with no recent infection, 2) hospital staff, 3) patients with recent pneumonia, and 4) patients with recent infections other than pneumonia (like typhoid or scarlet fever).

They tested types I, II, and III of pneumococcus polysaccharide, prepared in different ways, injected into the volunteers, and they tested the antibody responses by observing skin reactions and various other ways.

What they found, essentially, were the following: group 3 (recent pneumonia) showed immediate reactions to the skin test, specific to the strain with which they were infected. The different preparations of polysaccharide gave mostly similar results, except for those of type I pneumococcus. Sometimes, in the skin test, people in group 4 (who had some infection that wasn’t pneumonia) would show a delayed reaction to pneumococcal antigens while they had a fever, but after the fever passed, frequently they would no longer show a reaction.

But the most important (and interesting) finding of all was when they tested antibodies and reactions before and after immunization in volunteers that had not recently had pneumonia. The antibodies they found in blood samples before immunization were almost all low against all pneumococci, but after immunization with polysaccharides from specific types, the antibodies from the volunteers were almost all strong and specific to the type against which they were immunized.

How long the antibodies lasted depended on the type somewhat. Type I antibodies lasted the longest, at least eight weeks after injection; most seemed to peak around the second week. 

Later reviews and publications (including from the authors themselves) summarize this work well:

"Skin reactions were also elicited with varying frequency, using polysaccharides prepared by the same and by other methods, in normal subjects and in hospital subjects without pneumonia or recent intracutaneous injections. In such subjects, positive reactions to the initial injections were not correlated with the presence of circulating antibodies but the development of antibodies for the homologous pneumococcus type was stimulated by a single or by multiple intracutaneous injections.
     "[Finland and Dowling] found only minor differences in the antibody response elicited by the polysaccharides derived by different methods from the same type of pneumococcus. Delayed cutaneous reactions occurred only with the cellular carbohydrates."²

"Drs. Finland and Dowling also in the early 1930s, showed that the purified pneumococcal polysaccharide vaccine gave rise to type-specific antibodies."³

"Vaccines prepared from heat killed bacterial cultures were used for prophylactic inoculation in the early 1900s. However, due in part to poorly controlled clinical studies, their efficacy was questionable, which together with unacceptable reactogenicity meant this vaccination approach was not pursued. A new approach utilizing purified meningococcal polysaccharides was inspired by the successful use of pneumococcal polysaccharides to immunise humans."⁴

"Following on the heels of Avery and Heidelberger's revelations [that pneumococcal polysaccharides could be targeted by the immune system], Oscar Schiemann and Wolfgang Casper in Berlin demonstrated that purified pneumococcal capsular polysaccharides were immunogenic and engendered type-specific protection in mice. Finland et al. at Boston City Hospital's Thorndike Laboratory extended these findings through a series of experiments in the 1930s that paved the way for initial clinical trials of polysaccharide vaccines."⁵

 References:

1.  Finland, M. & Dowling, H. F. Cutaneous Reactions and Antibody Response to Intracutaneous Injections of Pneumococcus Polysaccharides. J. Immunol. 29, 285–299 (1935).

2.  Finland, M. & Brown, J. W. Reactions of Human Subjects to the Injection of Purified Type Specific Pneumococcus Polysaccharides. J. Clin. Invest. 17, 479–488 (1938).

3.  Krause, R. M. Prevention through Immunization: New Opportunities or End of the Road? J. Infect. Dis. 135, 318–329 (1977).

4.  Vipond, C., Care, R. & Feavers, I. M. History of meningococcal vaccines and their serological correlates of protection. Vaccine 30, Supplement 2, B10–B17 (2012).

5.  Artenstein, A. W. & LaForce, F. M. Critical episodes in the understanding and control of epidemic meningococcal meningitis. Vaccine 30, 4701–4707 (2012).

043 - The Antibody Response of Rabbits to Injections of Emulsions and Extracts of Homologous Brain

Along the lines of 040, here is an update (in relative time) about researchers' attempts to produce encephalomyelitis in lab animals. To recap: some people a while ago, when given a vaccine against rabies containing brain material, came down with a serious brain disorder that may have been due to an immune reaction against their own nervous tissue. Thomas Rivers and colleagues managed to induce a similar condition in a few monkeys by injecting them with rabbit brain tissue.

In the above-named study, Rivers and his colleague Francis Schwentker attempted to learn more about this process.¹

Considering the difficulty they had before in inducing the reaction in monkeys (dozens of injections over a long period), they tried to find ways to increase the ability of the brain tissue to cause a reaction. The methods: adding pig serum, letting the brain tissue sit around until it broke down on its own, and infecting it with vaccinia. The brains were dissolved either in water or in alcohol.

They tested a total of 79 rabbits in groups of four or five. After injecting them with the brain tissue, they tested their blood to see what kinds of antibodies they had.

Fresh rabbit brain with nothing added didn't induce any antibody responses, at least not in this study. In contrast, autolyzed brain (the one that sat around until it broke itself down) induced a lot. The other mixtures were intermediate. So apparently the additions could help form antigen somewhat, but the brain could form its own antigen too.

Schwentker and Rivers wanted to see if the antibodies formed were specific to the brain, so they tested their ability to bind to the brain and some other organs both of rabbits and of guinea pigs. Turned out that the antibodies formed against brain dissolved in water also bound to liver, kidney, and spleen, and all the same organs in guinea pigs, though the activity was less than against the brain itself. Antibodies from brain dissolved in alcohol only targeted the brain though.

It seemed that there were at least two kinds of antibody: one specific for a component of the brain that is soluble in alcohol, and one that was present in many different organs of the body.

To confirm this, the scientists removed each type of antibody by mixing the antibody-containing serum with organs. First they mixed the serum with kidney emulsions, and then tested what remained in the same way as before. The remaining antibodies bound only to the brain, not to any other organs. So antibodies to the non-specific component had been removed.

Next they mixed serum with brain dissolved in water. The remaining antibodies, if there were any, did not bind to any of the organs they tested.

Finally they mixed serum with brain dissolved in alcohol. The remaining antibodies only bound to the non-brain organs, not to the brain. This was somewhat surprising (if the antibodies bind to a common component that is also present in the brain, shouldn't they bind just as much to the brain?), but the authors explain that it is because the solution of brain tested was so dilute (so as not to overwhelm the other tests) and the antibodies present didn't bind to it very much, so it didn't show activity. Not sure why they didn't just make a less dilute solution of brain to test that, but oh well.

To figure out which component of the brain was inducing the most antibodies, Schwentker and Rivers tested the rabbit serum with antibodies against brain tissue using different proportions of white matter and grey matter from the brain. They found that as the relative amount of white matter increased, the antibodies bound to it better and better, so it seemed that something in the white matter was the antibodies' target.

One thing that's more common in white matter than in grey matter is myelin, which surrounds the sheaths of neurons. To confirm that myelin is the component of interest, they tested the anti-brain antibodies on emulsions of brain from different ages of rabbits. Newborn rabbits have hardly any myelin in their brains, and the amount increases with age, so if the antibodies primarily target myelin, they would expect more and more to bind to older and older rabbits, and that is indeed what they found.

So it seemed that injecting rabbits with solutions of rabbit brain could, in some circumstances, induce the rabbits to form antibodies against their own myelin. This could indeed be the mechanism by which encephalomyelitis occurs (in which myelin is broken down) after rabies vaccination.

Finally, the authors analyzed the health of the rabbits they used in their experiments. Most of them were fine, but a number did develop fatal paralysis. Looking at their brain tissue after they died, they noticed increased immune cell activity and inflammation in these animals, but they didn't see any breakdown of the myelin, so they were unwilling to conclude that there was a direct relationship between the antibodies against myelin and the paralysis they observed. It was interesting that the highest number of paralyzed animals (32%) was found in the group injected with autolyzed brain, which was also the group with the most anti-myelin antibodies.

This study gives some good hints at the problem, though it isn't entirely conclusive. A later study speculated that the antibody response might be protective somehow, rather than damaging:

"It is conceivable that a component of brain which is both toxic and antigenic could be released under conditions favoring autolysis, such as might exist locally in the injected brain-adjuvant mass. In this case, the antibody might represent a protective reaction...It might also account for the observation by Schwentker and Rivers that autolyzed brain extracts produced paralysis and antibody formation in rabbits, while fresh brain had no effect. Although no direct evidence for a toxin has been obtained in the present study, the matter warrants further exploration."²

And another cautioned about the interpretation of these results:

"While these studies have indeed confirmed the existence of antibodies specific for nervous tissue, the use of placenta has shown that certain 'antibodies to brain' may be antibodies to some element of the brain other than specifically nervous tissue, possibly vascular endothelium. Caution is therefore needed in interpreting results obtained with crude brain tissue or tissue extracts as due to antibodies specific for brain tissue."³

And I must reiterate that, while encephalomyelitis seemed to be an unfortunate reaction for some recipients of the rabies vaccine, it was the best they had at the time (we have better and safer now) and the alternative was often almost certainly dying of rabies, compared to a very rare side effect. So this story is, at worst, an interesting lesson from the past to inform medical efforts in the future.

Citations:
1. Schwentker, F. F. & Rivers, T. M. The Antibody Response of Rabbits to Injections of Emulsions and Extracts of Homologous Brain. J Exp Med 60, 559–574 (1934). 2. Thomas, L., Paterson, P. Y. & Smithwick, B. Acute Disseminated Encephalomyelitis Following Immunization with Homologous Brain Extracts I. Studies on the Role of a Circulating Antibody in the Production of the Condition in Dogs. J Exp Med 92, 133–152 (1950). 3. Lumsden, C. E., Kabat, E. A., Wolf, A. & Bezer, A. E. Studies on Acute Disseminated Encephalomyelitis Produced Experimentally in Rhesus Monkeys V. Complement-Fixing Antibodies. J Exp Med 92, 253–270 (1950).

042 - Rabies Vaccine Protection Tests

When studying rabies vaccines (or any vaccine, I expect) in animals, it is important to challenge the animal subjects with the pathogen in such a way as can reliably infect all of them, without being so much exposure that it overwhelms any immunity or other defenses they might have (and is way more than they would encounter outside the lab anyway).

The challenge is known as the infectious dose, and people were having trouble getting it right with rabies at that time. Since the rabies virus travels slowly but inevitably (in non-immune animals) up the nerves to the brain, the location of infection is important; farther from the brain will give the animal a better chance of developing immunity before the virus becomes fatal. So the question for researchers was where to inoculate: too far away and the animals would often survive; too close and they would die.

Reichel and Schneider, in today's study, proposed a solution: the tongue. They tried it on a number of rabbits after anesthetizing them, and it seemed to work well without bothering the animals much. Compared to injection into the cranium, injection into the tongue gave the animals a couple extra days before they showed symptoms.

So they tried a protection test of a couple vaccines, prepared different ways; in one, they used chloroform to deactivate the virus, and in the other, they used phenol. Both protected all 8 rabbits tested from infection, while 80% of the 5 controls died (one survived somehow, as some older rabbits occasionally did, possibly because the virus was not as virulent as it could've been).

Then they wanted to test the vaccines' shelf life; they had some of different ages, up to a few years old, that had been sitting at room temperature. The two oldest batches didn't protect any of the test animals; they were expired. The third oldest protected 75% of rabbits, and the newest 100%. 83% of the control animals died. Just to see, they tried "vaccinating" with brain tissue not containing any virus, and that didn't protect rabbits either, obviously.

Lastly, they tried testing the minimum effective dose, and the immunity duration (how long before a vaccinated rabbit lost its immunity). So they gave three rabbits four small doses of vaccine, then challenged them; all died.

Then they gave three more rabbits seven small doses, and two survived when challenged with rabies. The third died of a broken back, seemingly not connected with its infection at all, so it could be said to be a 100% protection rate. Reichel and Schneider challenged the surviving rabbits again with rabies after half a year, though, and both died. So the immunity from that dose waned after less than half a year.

The third group of three got 14 small doses. All of them survived the first challenge, and they also survived the second, half a year later. The last challenge, a year after vaccination, killed one of the three. So immunity may have waned partially after a year, at least in one of them.

The controls for each of these three challenges mostly died, by the way.

These were pretty small groups of animals, so the conclusions that can be drawn aren't huge, but the vaccines seem to work, at least for a while, and the methods seem effective, and the point of the paper was to test the methods, so good job I suppose. If anything, you could say that the preparation methods tested didn't differ much, that vaccine older than 2 years is expired, and that a course of 14 small vaccinations protects most for at least a year.

Citation: Reichel, J. & Schneider, J. E. Rabies Vaccine Protection Tests. Am J Public Health Nations Health 24, 625–628 (1934).

041 - The Protection Afforded by Vaccination Against Secondary Invaders During Colds in Infancy

Compared to the previous post, this story is nice and simple and straightforward, and positive.

Previous studies had tried to prevent the common cold, sometimes in combination with other respiratory infections (026 and 031). Obviously these attempts didn't work (especially since they contained only bacteria, and colds are viral infections), though they helped with pneumonia sometimes.

With this in mind, Yale Kneeland, Jr. observed that in the fall, respiratory infections were usually mild and viral (common colds), but that later in the winter, there were more and more complications and secondary infections with dangerous bacteria, causing pneumonia, fevers, etc. And if vaccines couldn't prevent the colds themselves, maybe they could reduce the severity of the illness overall?

A previous study in Norway, in which 500 infants were vaccinated against bacteria and 500 were not, showed a 5-fold reduction in fevers and a smaller number of serious complications of colds.

So Kneeland enrolled 46 infants, averaging half a year old, in the Home for Hebrew Infants in New York City, 23 as controls and 23 to receive vaccinations. The vaccines consisted of three species of pathogen: pneumococcus, hemolytic streptococcus (the kinds that caused scarlet fever, rheumatic fever, and some other things), and Haemophilus influenzae, which causes pneumonia. These bacteria were all grown up, killed (with heat and phenol), and injected whole.

There were two courses of weekly injections for the vaccinated group: the first in October, 9 injections, and the second the following February, 7 injections. Seems like way more than would be acceptable these days. Three of the subjects had fevers in February, so they missed much of the second course of injections. They were still included in the study though.

In terms of side effects, they were mild. Larger doses of vaccine caused local redness and such, but there were no fevers or anything more serious.

So now, the results: first, the two groups of subjects were no different in terms of the mild "common cold" infections they experienced. Which makes sense.

The real difference was in the number of days each group suffered a fever of more than 100°. Here is the graph from the paper:

Chart 1, Kneeland, 1934

Not much difference between groups until after the first course of vaccinations, but then the control group had many more days than the vaccinated (3 times more in January, almost 2 times more in February). Pretty striking.

The actual number of infections per subject was not much different: 5.4 in vaccinated vs. 5.8 in controls. But the severity was the big difference. The controls had 5 cases of pneumonia and the vaccinated only 2 (one of which started early in the first course of injections, probably before immunity had taken effect).

Overall, these are good results, but there are a number of problems. First, the number of subjects and infections are pretty small, so it's difficult to make good comparisons. There didn't seem to be any placebo for the controls, or any blinding, or much indication that the two groups were matched very well. Also, the number of injections is pretty high and the benefit not super great (2-3 days of high fever per infant in a given month? Seems like there's room for improvement. Also, the immunity from the first course seemed to drop after only a few months). Not quite ready for prime time, but seems like a step in the right direction.

Citation: Kneeland, Y. The Protection Afforded by Vaccination Against Secondary Invaders During Colds in Infancy. J Exp Med 60, 655–660 (1934).

040 - Observations on Attempts to Produce Acute Disseminated Encephalomyelitis in Monkeys, and related studies

This entry will address a few issues relating to vaccines that were very serious, at least in their time. Not so much anymore, fortunately, but I’ll get into that later. A number of studies form the basis of this topic^1–9. Our understanding of the topic has changed over the years, but the fundamental issue is something called encephalomyelitis.

Encephalomyelitis comes in a number of forms, caused by different things, but it’s almost always a disorder of the brain and central nervous system in which the sheaths surrounding neurons/nerve cells, which are made of myelin, get broken down somehow, so the neurons stop working. In the worst cases, this leads to paralysis and sometimes death. One example of this is multiple sclerosis. It’s not pleasant.

How does this relate to vaccines though? Well, for a while it was suspected that the rabies vaccine, which was made from nervous system material of animals that had been infected with rabies, occasionally induced acute encephalomyelitis as a rare side effect⁹. But then in the 1920s, Turnbull and McIntosh observed several cases of encephalomyelitis after vaccinating people against smallpox⁴.

Post-Infectious Encephalomyelitis

The vaccine itself was a live virus, called vaccinia, or cowpox. This was inoculated into people’s arms, where it usually caused very little disease but induced enough of an immune response to protect the person from the much deadlier smallpox.

An article in the Lancet describes Turnbull’s and McIntosh’s observations. They saw 7 cases of post-vaccinal encephalomyelitis (or PVE) in 14 years of vaccinating people at their hospitals in London and Middlesex. The appearance of the disease, especially in autopsy, was distinct from poliomyelitis or other similar things². After this, others observed similar cases of encephalomyelitis in Holland, 35 cases of which 15 were fatal. The researchers didn’t find any vaccinia in the brain tissue though, but they all happened 10-13 days after vaccination. They speculated that there might be some latent infection activated by the vaccinia³.

Vaccinia wasn’t the only infection that could induce such a condition; smallpox itself could too, as well as other fever disorders, especially measles^6,7. Others included chickenpox, influenza, mumps, rubella, and possibly diphtheria, pertussis, and scarlet fever⁸.

According to Ricardo Jorge in the Lancet, the most common cause of encephalomyelitis was measles: one in 250 cases got it, though only 10% of the time was it fatal. More often than that, though, there were long-lasting sequelae, which could include partial paralysis or other impairments. Smallpox caused it in about 1 in 400 cases. Chickenpox-induced encephalomyelitis was relatively mild, while that induced by influenza (largely present in the 1918 pandemic) was variable in its severity. Encephalomyelitis from vaccinia was the most often fatal though⁸.

According to the CDC’s website, the vaccine for smallpox is still capable of inducing such reactions (though only in fewer than 14 to 52 out of a million people vaccinated); it’s not an entirely risk-free treatment. So it’s no wonder that it is not recommended for general use anymore; fortunately, smallpox has been essentially eliminated from the world, so it is no longer needed.

Somewhat puzzling was the frequency of post-vaccinal encephalomyelitis in the 1920s, which seemed to reach epidemic proportions, and then faded away to some extent, for no apparent reason:

"Attention may next be directed to the occurrence of encephalitis after vaccination (ordinary cow-pox), a sequel that has aroused some concern in the course of the last three years, over one hundred cases having been recorded in that time. There can be little doubt that such cases were formerly extremely infrequent, and the conclusion seems justifiable that they are somehow connected with the times through which we are passing."¹⁰

But this mystery, I think, remains unsolved.

Post-Rabies Vaccine Encephalomyelitis

Returning to the issue of encephalomyelitis induced by the rabies vaccine, that too was an unfortunate side effect, but better understood now. According to a paper from 2008,

"The susceptibility of humans to the induction of experimental allergic encephalomyelitis was discovered accidentally when patients were vaccinated against rabies with spinal cords from rabbits that were infected with the rabies virus."¹¹

What this means is that the injection of nervous system material induced an immune response against the myelin that surrounds the nerves. The patient’s own immune system then attacked the myelin, breaking it down, thus causing serious damage to the nervous system itself.

This was quite unfortunate, though rare; Stuart and Krikorian estimated that only 28 to 130 people per 100,000 vaccinated came down with the condition⁹. When you were bitten by a rabid animal, you could be virtually 100% certain that you would die from rabies before too long unless treated with the vaccine, so it was definitely worth the risk. The authors also suggested that adequate preparation of the vaccine material to denature and dilute out the myelin could significantly reduce the potential to cause encephalomyelitis, even in 1930⁹.

Since that time, of course, people have developed rabies vaccines with no myelin contaminating them at all, incapable of inducing an immune response against the recipient’s own brain¹².

Attempts to Induce Encephalomyelitis in Monkeys

All this brings me to the study that is the primary focus of this post¹. Thomas Rivers, Sprunt, and Berry had noted others’ previous observations of encephalomyelitis after vaccine or infection (as described above), though they called it “acute disseminated encephalomyelitis” (or ADE, or sometimes ADEM) instead of post-vaccinal encephalomyelitis or other names; I think ADE in later publications is something distinct from what I’m discussing here.

Anyway, the authors decided to try to induce encephalomyelitis in the lab, in rhesus monkeys, in several different ways, using vaccinia virus and some extracts or emulsions of virus-free body tissues.

First, eight monkeys were vaccinated with vaccinia the normal way, in their skin. Five negative controls were unvaccinated. Then at various intervals after vaccination, the monkeys received injections of live virus into their brains. The unvaccinated controls all died of meningitis from vaccinia multiplying in their brains; this was expected. The monkeys injected the soonest after vaccination died the same way, and the second-soonest survived a mild meningitis, but the rest proved immune and unharmed. None of them had encephalomyelitis though.

The authors weren’t satisfied with this, because the monkeys’ immune response was too quick somehow, so they tried again with more concentrated injections of virus. The results were pretty much the same though. Apparently vaccination protects the brain as much as other parts of the body, but even when the virus does get into a susceptible brain, it doesn’t cause encephalomyelitis, or at least not often enough to be detected in 21 monkeys (not very many).

The next hypothesis was that extracts from rabbit testicles might enhance the virus’s action, so they tried this with a few more monkeys. One got just testicle extract, but had no reaction. Another was vaccinated and then received extract, but also had no reaction. A couple more, one vaccinated and the other not, got virus combined with extract injected in their brains. They both had symptoms like stiff neck, lack of activity, etc, but when sacrificed and examined, their nervous system didn’t have the characteristic encephalomyelitis appearance. So testicle extract didn’t really seem to cause or enhance it either (at least not in 2-4 monkeys).

Next, the authors tested the hypothesis that injections of virus-free nervous material from rabbits, like in the rabies vaccine, could cause encephalomyelitis. So they made extracts or emulsions of rabbit brains and injected them into the muscles of eight monkeys, between 14 and 93 injections total, three per week (so over the course of up to 8 months). Five of these monkeys, including two with the fewest injections and two others with the most, had no symptoms or problems. One suddenly died after receiving 50 injections, for no apparent reason. But two had characteristic neurological and mobility problems, and their nervous tissue showed the loss of myelin and increased immune cell activity associated with encephalomyelitis. So that seemed like a positive result!

The four monkeys that hadn’t reacted to injections of brain at all were tested further with injections of vaccinia into their brains, after vaccination or not. The results here were the same as with those that hadn’t been injected with brain material, so the brain injections didn’t seem to affect the course of the vaccinia infection.

The two positive results in this study turned out to be a big deal, cited by almost 400 later papers and reviews; they were labeled “experimental allergic (or autoimmune) encephalomyelitis,” or EAE. This has been studied a lot as a (possibly controversial) model of multiple sclerosis. It was definitely decent evidence of the link between encephalomyelitis and brain-derived rabies vaccines though.

Here are some comments from later papers regarding this study:

 "Rivers and his associates were the first to succeed in producing, in the monkey, sterile, disseminated meningo-encephalomyelitis by often repeated injections of material from central nervous system tissue. Many of the observations made by these authors suggest that the disease induced was the result of an immunologic mechanism. Nevertheless, Rivers and his associates hesitated to express this view. Even 20 years later and after the accumulation of much new information—subsequent to the introduction of the use of adjuvants in the study of sterile experimental disseminated encephalomyelitis—conclusive evidence for its allergic nature is lacking."¹³

"Acute disseminated encephalomyelitis, which is essentially identical to the disease that occurs in patients receiving live or killed rabies virus vaccine, was produced in monkeys by Rivers and his colleagues in the 1930s by repeated injection of the animals with emulsions or extracts of normal nervous tissue from the central nervous system. These findings, together with increasing evidence that central nervous system tissue possesses organ-specific antigenic activity, provided strong evidence that the disseminated encephalomyelitis occurring in humans after rabies vaccination resulted from the host's immunologic responses. These responses, called forth by parenteral injections of nervous tissue, had the capacity to interact with antigenic components of the host's own central nervous system and thus to cause disease. It is important to note, however, that at about this same time there was increasing recognition that smallpox vaccination and some common exanthematous diseases of childhood such as rubeola were occasionally complicated by an acute encephalitic process. These cases of para- or postviral encephalitides had histopathological features that bore a striking similarity to those of the encephalomyelitis associated with rabies vaccine and the disseminated encephalomyelitis induced in monkeys by injections of nervous tissue."¹⁴

"Studies in the 1920s indicated that inoculation of rabbits with extracts of normal human spinal cord, or sheep brain, likewise resulted in occasional instances of post-vaccinal encephalomyelitis. The question was taken up by Thomas Rivers at the Rockefeller Institute, NY, perhaps because he himself had once been incapacited by progressive muscular atrophy and, being a virologist, he would be curious whether a virus could be implicated in post-vaccinal encephalomyelitis...Rivers concluded that the relation of the injections [of monkeys] to the central nervous system disease was 'not clear.'"¹⁵

"Experimental autoimmune encephalomyelitis is a well established model used to investigate the possible autoimmune etiology of multiple sclerosis. This model originated with Louis Pasteur's vaccinations with spinal cord from rabies-infected rabbits from 1885. This acute demyelinating disorder was later found to occur due to contamination of the inoculums by spinal cord components."¹⁶

Mackay and Anderson also provide a good overall history of Rivers’ and colleagues’ work¹⁷.

So overall, these were some legitimate problems with vaccines of old, though no longer. At the time, it was likely that, despite the risks, the vaccines were preferable to the diseases they prevented, on average. But for individual victims of encephalomyelitis, it probably was not worthwhile, so it’s good that those that study vaccines are constantly trying to improve them.

References:

1. Rivers, T. M., Sprunt, D. H. & Berry, G. P. Observations on Attempts to Produce Acute Disseminated Encephalomyelitis in Monkeys. J Exp Med 58, 39–53 (1933).

2. Encephalo-Myelitis Following Vaccination. The Lancet 208, 504–505 (1926).

3. Encephalo-Myelitis Following Vaccination. The Lancet 208, 764 (1926).

4. Turnbull, H. M. & McIntosh, J. Encephalo-Myelitis following Vaccination. Br J Exp Pathol 7, 181–222.19 (1926).

5. Turnbull, H. M. & Mcintosh, J. Encephalo-Myelitis Following Vaccination. The Lancet 211, 1094–1095 (1928).

6. Perdrau, J. R. Encephalo-Myelitis Following Vaccination. The Lancet 211, 1201 (1928).

7. Ward, G. Encephalomyelitis Following Vaccination. The Lancet 214, 1331–1332 (1929).

8. Jorge, R. Post-Vaccinal Encephalitis :: Its Association with Vaccination and with Post-Infectious and Acute Disseminated Encephalitis. The Lancet 219, 267–272 (1932).

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