100 - A Comprehensive Study of Influenza in a Rural Community

Ok, here's another study that doesn't look at vaccines specifically, but rather the background of the disease they prevent, and how our bodies respond to it. In this case, influenza.

The researchers followed a small community of people in New York over the course of a flu season, looking at their antibody levels. The tests they used were neutralization (mixing serum with virus sample and seeing if the serum neutralized the virus so it didn't make mice sick) and complement fixation, though of the two tests the former seemed more useful.

In the period before the flu season, teenagers had the highest titers in the neutralization test, which were lower at younger and older ages. For complement fixation, the highest levels were in those 40 to 60 years old. Both tests observed a decrease above 60, in the elderly.

Then in the flu season, they made sure to distinguish clinical flu from the common cold or other flu-like illnesses: the flu was defined by a fever over 99 degrees F, headache, pains, general malaise, and respiratory symptoms. About 85% of the disease in the community was actually colds, which peaked in December, while the flu didn't peak until February. About 62% of the flu tested was found to be Influenza A, the rest other kinds.

So regarding the neutralization test results, having a cold didn't increase one's titers at all, but having the flu did (makes sense). Some had an increase without observing symptoms, possibly subclinical infections, too mild to be noticed. There was a definite correlation between having lower antibodies before the flu season and being likely to get the flu during the season, so titers correlated with protection. Though even those with the highest titers occasionally got sick.

One year after the season, some people's titers had returned to the same level they had been before the season (even if they got the flu), some stayed the same, the rest fell partway back. The average was 5 times more 1 year after than before.

So overall, it seems like higher titers correlate with more protection, but there's no really safe level that's 100% protective. And correlation isn't causation, so it's not clear the antibodies are doing the protecting, though neutralizing the virus seems like a good sign. Also I'm not sure how specific these tests are for specific antibodies, so they could show high levels of antibodies that aren't actually that useful, or something like that. Needs further study.

Reference:

Rickard, E. R., Lennette, E. H. & Horsfall, F. L., Jr. A Comprehensive Study of Influenza in a Rural Community. Public Health Reports (1896-1970) 55, 2146–2167 (1940).

097 - An Experiment in Immunization Against Influenza with a Formaldehyde-Inactivated Virus

There had been some good results in animals with vaccines against influenza, and possibly some good trials in humans too, though not in others; influenza is tricky though, due to antigenic drift.

So this was another study, in Hungary during an epidemic in 1937. They used formaldehyde-inactivated virus taken from infected mouse lungs, and vaccinated 306 nurses and children in Budapest institutions with a single dose. 336 were controls.

They tested some subjects before and after the vaccination, and found that antibody levels rose a decent amount after the vaccination, 24x on average. One subject got tested before, after the vaccine, and then again after getting the flu, and the disease hadn't increased the levels any higher than the vaccine had (though obviously it wasn't a protective level somehow).

The epidemic was pretty small, so there were only 34 cases total in the study: 20 in the unvaccinated, and 14 in the vaccinated. This wasn't a significant difference, so it was negative.

But they isolated virus from some of the cases and found that it was a different serotype, so the vaccine might not've been good at targeting it anyway. Oh well.

Reference:

Taylor, R. M. & Dreguss, M. An Experiment in Immunization Against Influenza with a Formaldehyde-Inactivated Virus. Am. J. Epidemiol. 31-SectionB, 31–35 (1940).

O953 - Prophylactic immunization against measles, scarlet fever, diphtheria, whooping cough, and influenza

This paper was a speech given by Sir Ledingham, director of the Lister Institute in London. He says some interesting things. Speaking on the safety of vaccines:

"The safety of any method to be applied to man must always be a prime consideration. There must be no risk to life, and even the minor inconveniences, local and general, incidental to the introduction of a prophylactic through the skin must also, so far as possible, be reduced to a minimum consonant with the effectiveness of the product."

The first topic is diphtheria, a bacterial disease that can be prevented simply by inducing an immune response toward the toxin the bacteria produce (diphtheria toxin).

"Diphtheria undoubtedly merits chief consideration because we now know quite definitely from field trials...that intelligent and persistent immunization of the child population, starting with the pre-school child one year old, can bring the diphtheria morbidity down to zero."

The best approach to this is using something called toxoid, which is toxin treated such that it produces an immune response but doesn't cause problems.

"By the end of 1938 some 800,000 troops had been so vaccinated [with diphtheria toxoid], and Ramon was able to state that clinical diphtheria was disappearing from the French army."

"It is only when the proportion of vaccinated children reaches 70 to 80 per cent. that, as Ramon says, one sees the real fruits of one's work in a great reduction of the diphtheria incidence, perhaps to zero."

"Among the immunized children there have been since March, 1935, seventy-three notifications of diphtheria, giving an attack rate of 26 per 10,000 children, while among the unimmunized, including some 3,000 known Schick-negatives, the attack rate was 251 per 10,000, or ten times that among the immunized."

The Schick test was a way to test immunity in a person (though not perfect): if there was no reaction (Schick-negative), that indicated some immunity.

Considering the success of immunization, Ledingham makes a statement that seems relevant still today:

"When liberty becomes anti-social and impedes the application of scientific truth to the improvement of human well-being then I think some form of compulsion is the only remedy, though one might hope it would be merely temporary, pending the wider diffusion of scientific knowledge in the community."

It was a bit more complicated than just toxoid though, for diphtheria vaccine options. There was formol-toxoid, toxin-antitoxin floccules, alum-precipitated toxoid, and toxoid-antitoxin mixture, each with different properties in terms of safety or immune stimulation. Ledingham has some opinions:

"T.A.F. and T.A.M. are relatively innocuous at all ages, while A.P.T. is well borne in young children. In older children and adults it may give rise to painless nodes, which take some time to disappear. A.P.T., by virtue of the slowness of absorption of the antigen and consequent longer stimulus to the antibody-forming mechanism, can give rise...to considerably higher antitoxin titre, given in a single dose, than any of the other antigens. For this reason it may well become the antigen of choice for young children, the group we wish above all to see in course of time fully immunized"

In terms of stimulation, alum-precipitated toxoid seemed the best (probably the alum was an adjuvant); two doses of it was equivalent to three of formol-toxoid. Ledingham also stressed that trials of the vaccine should have prevention of diphtheria as their endpoint, not Schick test results; with a good vaccine, the test shouldn't be necessary. One dose with APT followed by another of FT might be good.

Then he moved on to whooping cough:

"As a killing disease of infancy, therefore, whooping-cough closely rivals diphtheria"

He cites Kendrick and Eldering's study that showed only 3.8% of vaccinated subjects with severe disease, compared to 13.1% in the unvaccinated, and other studies giving similar results.

"The age at which a pre-school child, say 9 months, may be submitted to immunization against diphtheria is also the age at which this same child might very desirably be immunized against whooping-cough. What is to be done? I am not aware that diphtheria toxoid has so far been administered in combination with a pertussis vaccine, but I see no reason why this should be attempted if Ramon's claim holds that the potency of diphtheria toxoid in mixture with T.A.B. [typhoid] vaccine is in now way interfered with and indeed appears to be enhanced. Such mixtures would first require to be tested on animals in which, at any rate, the rise in antitoxin titre can be accurately evaluated."

Then moving on to scarlet fever, he thinks it isn't worth trying to vaccinate most people, even if there were an effective vaccine, because it wasn't a serious disease anymore. Also it seemed that there was a patent preventing good vaccine development.

He thought treating measles with immune serum seemed to help, but there hadn't been much effort to acquire or store it. For active immunization, not enough was known yet about the disease.

With influenza though, more was known and a vaccine was closer, with some animal trials.

"One finding, however, which is likely to complicate the problem of active immunization as applied to man is the multiplicity of antigenic types among the strains recovered from different outbreaks of human influenza."

But he was frustrated by public health policy in general to some extent:

"When I reflect that, owing to ignorance, vested interest, or complacency in high places, a municipality is still prevented from ordering the pasteurization of all milk that comes into it, I sometimes despair of getting preventative science across."

One final quote:

"It may sound a totalitarian policy, if you like, but in essence it seems to me quite democratic, for it involves a negligible sacrifice on the part of the individual for his own and the common good."

Reference:

Ledingham, J. C. G. Prophylactic immunization against measles, scarlet fever, diphtheria, whooping cough, and influenza. British Medical Journal 2, 841–846 (1939).

090 - Serologic Studies in Epidemic Influenza: With Particular Reference to the Persistence of Antibodies After Infection

Many virus infections seem to lead to long-term immunity, so you get it only once; at least, that's how it seemed. But one obvious exception is influenza, which you can get every year almost. Part of the reason for this is its ability to mutate frequently, becoming different enough each year that our immune system doesn't recognize it as well; but Fairbrother and Martin wondered whether this were really the whole story.

So they studied the antibody levels of people before and after an epidemic of flu and for about a year after. There were two kinds of tests they used: complement fixation, and neutralization where they mixed antibodies with virus and injected it into mice to see if they died.

As we saw before (O860), antibody levels seem to correlate with immunity, and definitely increase after infection. They saw the same thing here: after being infected in the epidemic, people's levels were much higher. For those that didn't get infected, levels were pretty variable, some high, some low.

But after about a year, antibodies in people who had been infected were much lower than they had been, nearly back to where they had been before the people were infected.

So it's possible that natural immunity to influenza wanes after a year, though that can't be concluded here because they didn't actually test immunity, only antibody levels.

Reference:

Fairbrother, R. W. & Martin, A. E. Serologic Studies in Epidemic Influenza: With Particular Reference to the Persistence of Antibodies After Infection. The Lancet 231, 718–720 (1938).

O860 - Investigation on Volunteers Infected with the Influenza Virus

Apparently back in the 30s, the Soviet Union was doing research on influenza, some of which used human volunteers.¹ Well, better flu research than smallpox or something, and it seems to have worked out alright.

Here, they tried infecting 72 volunteers with virus taken from infected mice, by aerosolizing it and letting them inhale it. It seemed to work in some of the volunteers, though their illness was mild; the authors speculate that the virus might have been attenuated by passage through animals.

The most interesting result was the antibody levels in the volunteers before and after inoculation. They measured antibodies by drawing blood, mixing it with live virus, and injecting it into mice. If the mice survived, the virus had been neutralized by the antibodies.

What they saw was that volunteers who did get sick had pretty low antibodies to begin with, almost none in some cases; afterward their levels were 25-100 times higher. In those that didn't get sick, the levels had started out high and risen only a little (to about the same level as the others). So antibody levels correlated well with immunity to influenza.

The authors thought this infection with somewhat-attenuated virus method might be a good strategy for immunization. The editor of this study's journal disagreed with that statement, as did later researchers:

"A paper published from the Soviet Union by Smorodintsev[sic] et al. in 1937 - frequently cited as the first paper on live virus vaccine - described the administration of a mouse-lethal strain of the...virus by protracted inhalation of atomized virus. Typical febrile influenza developed in 20% of volunteers, hardly an acceptable vaccine by present standards, and certainly not attenuated, as claimed by the authors. Remarkably, they claimed, as well, that the virus appeared not to multiply in men, but the study was a landmark in establishing unequivocally the role of the virus in the development of the disease and in demonstrating antibody response to the virus during convalescence."⁴

Others questioned whether this study could really be compared to natural infections:

"These results suggested that the level of neutralizing antibodies was of significance in determining susceptibility or immunity to influenza A in man. But the conditions under which the experimental disease was produced and the relatively large quantities of virus suspensions used seem so different from conditions encountered in the natural epidemic disease as to make comparisons between these two conditions hazardous."²

"Whether experimentally induced influenza A in human beings is entirely analogous to the naturally occurring epidemic disease may be open to some question."³

Seems like a reasonable question, but at least it's easier to control this kind of study for unwanted variables.

References:
1. Smorodintseff, A. A., Tushinsky, M. D., Drobyshevskaya, A. I., Korovin, A. A. & Osetroff, A. I. Investigation on Volunteers Infected with the Influenza Virus. The American Journal of the Medical Sciences 194, 159–170 (1937).
2. Rickard, E. R., Horsfall, F. L., Jr., Hirst, G. K. & Lennette, E. H. The Correlation between Neutralizing Antibodies in Serum against Influenza Viruses and Susceptibility to Influenza in Man. Public Health Reports (1896-1970) 56, 1819–1834 (1941).
3. Horsfall, Jr., F. L. Recent Studies in Influenza. Am J Public Health Nations Health 31, 1275–1280 (1941).
4. Kilbourne, E. D. in History of Vaccine Development (ed. Plotkin, S. A.) 137–144 (Springer New York, 2011).

Vaccines-related Podcast Episode - BacterioFiles 196

This blog is not my only project, nor even my primary: I also have a podcast, called BacterioFiles, which in some ways is sorta the opposite of this blog. It is about how microbes (bacteria, viruses, archaea, fungi) are awesome and useful, whereas this blog is more about how they are deadly and unpleasant.

But sometimes, in happy coincidence, they overlap, and the latest episode was one of those times:

BacterioFiles 196 - Flagellin Facilitates Flu-shot Function

Gut bacteria are important for a good immune response to unadjuvanted influenza vaccines!

It's a much more recent study than the ones on this blog have been so far, but interesting context to keep in mind while reading (or in my case, writing) the entries for this blog, so I thought I would share. Enjoy!

075 - Epidemiological Studies in Influenza

People still hadn't worked out exactly what influenza was or wasn't. Was it a disease caused by a single virus, or a collection of symptoms that could be caused by multiple viruses? Part of that was an arbitrary definition. If just one virus, were there different strains? They also strongly suspected that bacterial secondary infections could play an important role sometimes.

So Thomas Francis attempted to define influenza as well as possible at the time, and give some other information about it.¹ He defined what flu is not (common colds, pneumonia, sore throat, diarrhea) and described a typical case. 

He discussed Shope's studies (049) distinguishing the swine flu virus from Haemophilus influenzae suis bacterial infection, and other animal studies in ferrets and mice (such as 074). Also important research growing flu virus on tissue culture or in eggs.

More importantly, he discussed the issue that some had suggested that people don't form immunity against the flu. It's understandable how people could get that impression, considering how the flu virus mutates enough pretty much yearly to be able to reinfect even people who had it the previous year, so we need a new flu vaccine every year. But the presence of at least some immunity is important; otherwise every flu infection could be as scary as avian flu is supposed to be.

Part of the problem was that the techniques for identifying and distinguishing strains of virus weren't very developed at the time. They could try to infect animal models with samples to isolate virus from them, but if the virus were a type that didn't infect such animals very well, it would give a false negative. This happened to Francis: he observed an epidemic of influenza in California in 1936 with all the usual clinical symptoms, but hardly any patient samples gave infectious virus. As others noted:

"Although it has been suggested by Stuart-Harris et al. that in the presence of an epidemic of respiratory disease a certain symptom complex may serve to differentiate influenza from similar but etiologically different diseases, the California epidemic studied by Francis makes this possibility seem unlikely."²

Later, people realized that this was the first identifiable observation of an outbreak of Influenza B.³ Previous studies apparently had focused mainly on Influenza A.

Lastly, Francis discussed attempts to immunize people against the flu. Mostly it was similar to the results in 074: they saw a rise in antibodies against flu for at least a few months, but didn't actually test if it were protective against infection. Interestingly, some tried inoculation with live virus, subcutaneously, and didn't see any evidence of respiratory infection or serious side effects. Looking at antibodies in people who had just recovered from flu (the way they did this was to inject mice with human serum and see if it protected them against viral infection; another technique with questionable assumptions), they found good antibodies in about 30-60%, and also found strong antibodies in about 30% of people who gave negative histories of flu. So, more work to be done.

References:

1.  Francis, T. Epidemiological Studies in Influenza. Am J Public Health Nations Health 27, 211–225 (1937).

2.  Horsfall, Jr., F. L., Hahn, R. G. & Rickard, E. R. Four Recent Influenza Epidemics: An Experimental Study. J Clin Invest 19, 379–392 (1940).

3.  Burnet, F. M., Stone, J. D. & Anderson, S. G. An Epidemic of Influenza B in Australia. The Lancet 247, 807–811 (1946).

074 - Influenza: Further Experiments on the Active Immunization of Mice

Andrewes and Smith were some of the researchers working on creating a flu vaccine, especially since people had discovered that influenza was caused by a virus, not bacteria.

The vaccine they were developing was made from infected mouse lungs, and mice were the model animal they focused on mostly, especially in this study. Mouse lungs produced a lot of virus, but it wasn't the cleanest, so what they were attempting in this study was to produce a cleaner version.

Actually there were three main goals:
1) Try to get as much virus as possible,
So less volume is needed for the same dose

2) Purify the virus as much as possible without reducing its immunizing ability,
So there aren't contaminants that could cause unnecessary reactions

3) and if possible, inactivate the virus (so it can't infect) without reducing its immunizing ability.
So that it can't possibly infect and cause disease.

For objective 1, they tried filtering the virus with membranes that the viruses were too large to pass through, but that didn't really seem to help. At some point though, their virus densities increased 10 to 100 times spontaneously, maybe through some mutation, so that worked out.

For objective 2, they wanted to remove mouse proteins from the preparation, so they tried adsorption/elution, in which they could stick the virus to something and wash everything else off, but they lost a lot of virus with this method too so it wasn't great. Filtering seemed to help though.

For objective 3, they tried inactivating the virus with formaldehyde. A solution of 0.01% could inactivate almost completely in 5 days at -2°C, and 0.02% could completely. This inactivated virus couldn't infect mice when put into their nose.

Immunization Experiments
Then they tested these preps in mice, to see which gave the best immunity against flu virus challenge. What they found was that washed live virus immunized about as well as unpurified virus (when inoculated into the skin or body cavity), and inactivated virus seemed almost as good, though it seemed like the dose they gave of this was higher than the dose of live. Even 0.1% formaldehyde-inactivated gave good immunity. Virus-free filtrate didn't help at all, so the antigen is not soluble.

They did find that virus that had been washed and then inactivated (or the reverse) didn't have much immunizing power in mice. That was unfortunate.

The immunity from each vaccine seemed to fade in mice after 6 weeks. However, this was similar to how long mice had immunity when they had gotten sick with the flu and recovered, so the vaccine was as long-lasting as natural immunity (especially considering that most of the infected mice died from the disease).

Preliminary Human Trial
Finally, they tested inactivated virus in a few human volunteers. They didn't want to use live virus, considering how others had seen what seemed like flu outbreaks from live virus vaccines (049). So 5 volunteers got washed and inactivated virus, and two more got inactivated unwashed virus. They also added 0.01% merthiolate (thimerosal) to prevent bacterial contamination just in case.

The first two had some pain, maybe from excess formaldehyde, so for the others Andrewes and Smith changed the pH to convert the formaldehyde to something else, which worked better. They didn't see any serious reactions to any version, though the ones getting unpurified virus had more tenderness (possibly sensitivity to mouse proteins).

What they saw was that in all but one volunteer, levels of antibodies against the virus rose after the first dose (not much after the second dose for some reason). This was heartening, especially with the washed+inactivated virus that hadn't worked well in mice. Even better, the levels seemed higher than in other people who had recently recovered from the flu! (Though I'm not sure the flu the people had would be the same antigenically as the virus used in this study.) And the levels still seemed high after 2.5 months. So they might be on the way to a good flu vaccine, but they weren't sure yet if antibody levels correlated well with immunity. More work to be done.

Citation: Andrewes, C. H. & Smith, W. Influenza: Further Experiments on the Active Immunization of Mice. Br J Exp Pathol 18, 43–55 (1937).

058 - Immunity in Influenza: The Bearing of Recent Research Work

In 1939, people had already discovered the influenza virus, but there were still a lot of questions. For example, how can one distinguish between illness caused by this virus and very similar illnesses caused by many other things? And are all flu viruses the same? In this paper, C.H. Andrewes addressed these issues.

People had observed that major outbreaks tended to be the real flu, while minor outbreaks were typically something else. Andrewes called the former "epidemic influenza" and the latter "febrile catarrhs." Clinically, the real thing tended to be sudden in fever onset, and have more symptoms like headache and general achiness rather than the cough and sore throat characterizing other things. But there was a lot of overlap.

Mostly they tried to distinguish by infecting ferrets. The real flu tended to infect ferrets while the other stuff didn't. But it could've been possible to infect ferrets with more than one virus; it was hard to tell.

Serologically (that is, regarding the antibodies that bind to a virus), there seemed to be at least 4 different kinds of flu antigen. They hadn't found good ways to characterize these yet. Antibodies against one wouldn't bind as well, or at all, to another.

Regarding duration of immunity, they had found that in animals it lasted a few months, and Andrewes thought that duration seemed to correlate to body size, so it might last up to 1 year in people. Not sure if this is valid. But in any case, it declined over time, at least in ferrets.

In ferrets, subcutaneous vaccination worked for moderate immunity, not great. But moderate might be enough: humans wouldn't normally encounter very much flu at a time; and if it didn't prevent it completely, it could reduce the severity so that more people survived; and if people had some immunity already, it could increase it.

But studying immunity from vaccines is tough, because epidemics are infrequent and unpredictable. So people were trying to use antibody levels as a proxy. Some showed that antibodies and immunity correlated well, especially in ferrets, but it was unclear.

Andrewes tended to use a vaccine of virus inactivated by dilute formaldehyde. This seemed safer than a live attenuated vaccine, because a virus changed to be less virulent could always revert to become more virulent.

Lastly, he discussed the issue of when is best to vaccinate people. Epidemics in England were happening about every 4 years, but this wasn't reliable. Usually they happened in December or January, so the best time would be 1-2 months before, in October and November.

Citation: Andrewes, C. H. Immunity in Influenza: The Bearing of Recent Research Work. Proc R Soc Med 32, 145–152 (1939).

054 - The Effect of Hemophilus influenzae suis Vaccines on Swine Influenza

Richard Shope and others already knew that it was possible to vaccinate against influenza using the influenza virus, in animals at least (049). But an important part of typical swine influenza was an infection with bacteria called H. influenzae suis, which often caused a secondary infectious pneumonia that could be fatal. Immunizing with this bacterium intranasally doesn't prevent the flu, but it seems to help when combined with the virus. It seemed like the bacteria needed help from the virus to get into the body and infect. So Shope wanted to know if it would help on its own when inoculated intramuscularly, into muscle.

So he took H. influenzae suis (henceforth "H.suis" for ease of typing) cultures, killed some of them with heat, and kept the rest alive as a live vaccine. He inoculated 8 pigs with the former, heat-killed ones, and 6 with the live, 3 injections each. He didn't see any side effects from the killed vaccine, but the live consistently caused a fever after the second injection.

After a week or two, Shope tested their immunity with flu virus plus bacteria. He observed them for a few days, and then killed and autopsied them.

All of them got the regular, virus-caused flu, of course. Of those that received the killed vaccine, only one seemed completely protected from the bacterial infection, with no H.suis found anywhere in its body. Another two had bacteria only in their upper respiratory tract, not their lungs, and the remaining 4 had bacteria in the lungs, but their pneumonia was not as severe as that of the 3 unvaccinated control pigs.

The live results were a big weirder. The 6 pigs got very sick when infected with flu, but recovered remarkably after only a day and then had no more than mild illness, compared to controls that had typical flu. All the pigs had bacteria in their respiratory tract, but only one had them in the lungs.

Shope also tested the antibodies in the pigs' serum before infecting them, and none of them had inactivating antibodies against flu or bacteria. Which doesn't necessarily mean they weren't immune.

So in conclusion, intramuscular H.suis, either killed or live, seems to affect the course of the flu but doesn't prevent it. The live seems slightly better at protecting after the initial severe reaction, but the reaction does make it seem less appealing, so it's not clear which is better.

Shope speculates that the severe reaction could be due to an allergic-type reaction to H.suis naturally in the lungs being quickly cleared out, but it's not clear why the killed vaccine wouldn't induce this also.

And he says that this study is just interesting, not practically very useful because there's already a virus-based vaccine that can prevent the whole flu, not just the bacterial part. This is not quite true, as we know, because H. influenzae in humans was a common cause of secondary pneumonia after the flu, so a vaccine against it is quite useful, especially because even today our flu virus vaccine is not super-great enough to depend on.

Citation: Shope, R. E. The Effect of Hemophilus influenzae suis Vaccines on Swine Influenza. J Exp Med 66, 169–175 (1937).

O469 - Advances in Vaccine Therapy

This isn't a full post, but I really liked this quote (whole section, really) from a review written by Alexander Fleming (who you'll recall is credited with discovering penicillin, not that it's particularly relevant here).

"The question whether or not an individual can be protected against epidemic influenza by means of a vaccine is one which is of the greatest importance in medical practice. This question leads to another, What is epidemic influenza? Before the great war we were quite content to think that it was infection by the influenza bacillus of Pfeiffer, and there is no doubt that in the 1918-19 pandemic this bacillus was present in practically 100 per cent of cases in England and France, and that it was responsible for much of the mortality. However, more recent work, especially at the Medical Research Council laboratires, has definitely established that the primary infective agent in epidemic influenza is a virus. This virus can be passed on in ferrets and mice, and can be cultivated in the incubator in chicken embryos still in the shell. It appears likely that mild influenza, such as the first wave of 1918 or the more recent epidemics, is to all intents and purposes a pure infection of the virus and that the serious and deadly epidemic of 1918-19 was due to a combination of this virus with a bacterium, especially the influenza bacillus.

"A very interesting influenza story comes from America. During and following the 1918 pandemic an epidemic of swine influenza appeared among the pigs in that country. This swine influenza is a serious and fatal disease, and has been investigated by Shope (1937) [054] more thoroughly than was possible in any human disease. Shope discovered that swine influenza was due to a combined infection with a virus and a bacillus practically identical with the influenza bacillus of Pfeiffer. If he infected pigs with a pure virus without the bacillus a transient fever resulted and all the pigs recovered; if he infected them with the bacillus alone practically nothing happened; but if he infected pigs with both the virus and the bacillus typical swine influenza resulted and the pigs died with pneumonia. Shope found that the pigs which had been given the pure virus and had recovered from the trifling infection that resulted were completely immune to fresh infection with the virus. He also found that inoculation of the pigs with vaccines made from the influenza bacillus gave some degree of protection against swine influenza, although it was not so complete as the protection with the virus.

"Here it will be seen that all the animals receiving the vaccine of the influenza bacillus suffered from a much milder disease than the controls, which had not been protected. This observation is of the greatest interest in view of the attempts made to prevent influenza in man with bacterial vaccines, especially those of the influenza bacillus. After the 1918 pandemic many papers appeared stating that such vaccines had conferred a degree of protection against influenza, but none of the figures given have satisfied statisticians. In view, however, of Shope's experimental results with pigs it appears that these claims were justified and that protection can be obtained by such bacterial vaccines.

"Attempts are at present being made to protect man against influenza by means of a vaccine of the influenza virus. The results on experimental animals have been successful, but the opportunity has not yet arisen for the final proof that it can protect man during an epidemic. It seems likely that in the near future it will be possible to obtain a virus vaccine which will give, either alone or in combination with the older bacterial vaccine, complete protection against epidemic influenza in the majority of the inoculated individuals."

Citation: Fleming, A. Advances in Vaccine Therapy. Br Med J 2, 99–104 (1939).

049 - Immunization Experiments with Swine Influenza Virus

Now for something different: influenza. By 1936, people knew that the disease was viral, not bacterial, though bacterial infections often complicated flu, causing even more sickness. So searching for a way to prevent the viral disease in the first place was a good idea.

Richard E. Shope took advantage of earlier studies of influenza in animals, and tested the possibility of immunizing animals against the flu by injecting them with the virus. No killing or inactivating at all, just injecting the live virus into muscle or skin or other areas of the body, apparently is enough to safely generate an immune response.

This was already known, but what Shope intended to test with this study was whether flu virus that had been cultured in one kind of animal (pig, ferret, or mouse) could provide immunity to another type. As secondary goals, he was looking at dosage and route of inoculation (either subcutaneous, under the skin, or intraperitoneal, into the body in the spaces between organs).

The flu Shope used had been isolated from swine originally, but in the lab he had passed some of it through mice and some through ferrets, so those strains were adapted to those animals. He used infected lung tissue from each animal, ground up and dissolved in saline, as vaccines and infecting doses.

First he tested seven pigs, inoculating them with virus from pigs, ferrets, or mice either subcutaneously or intramuscularly. Then, along with two non-immune controls, he infected them with virus through the nose. After 4 days, Shope killed the pigs and examined their lungs for flu lesions and their blood for anti-flu antibodies.

And all seven turned out to be immune, while the controls got sick. One of the seven pigs had live virus in its nose, but none had it in their lungs, where the infection is worst. And they all had antibodies against the virus.

Next he tried ferrets, inoculating them subcutaneously or intraperitoneally. The 8 controls all had severe illness with virus easily found, but those immunized with ferret-derived virus were immune, all except one out of nine. Of those inoculated subcutaneously, all the ferrets immunized with swine or mouse virus got sick, but intraperitoneal inoculations from those animals seemed better able to protect (66% from swine, 100% from mice).

Finally was the test in mice, which apparently are a useful model because the virus kills them easily but doesn't really spread between them, so strict isolation is not necessary. The setup was pretty much the same as with the ferrets. In this case, 79 out of the 83 control mice died within 7 days after infection, as expected.

Of those immunized with mouse virus, 77 out of 99 immunized with two large doses survived, about equal proportions subcutaneous or intraperitoneal (it didn't matter), though only 29 out of 68 given one large dose survived, and 42 out of 83 given two smaller doses.

Of mice given swine or ferret virus subcutaneously, only 8% survived from swine virus and 17% from ferret. Intraperitoneal injection was better: 70% from swine, especially with a higher immunizing dose, and 67% from ferret.

Since it is a live virus vaccine, Shope monitored the inoculated mice for illness, and a few did die, but mostly from intestinal infections. A very few did die from pneumonia, but he wasn't able to find any flu virus present in these, so it could've been something else.

These results are promising, but Shope also presents some data that are more troubling. Some pig farm in Iowa had done an experiment with this sort of vaccine, immunizing more than 1500 pigs. Mostly the vaccinated were kept separate from the others, but in a few droves that was not possible. In one drove of 223 animals, 23 were vaccinated, and soon after, a flu infection broke out in the drove, infecting all but 30 (including 20 of the vaccinated). There were no other infections going on in Iowa at the time, and it was too early in the season for flu to be going around, so Shope suspected that it came from the inoculation somehow. A similar thing happened in another drove.

So it seems that live virus in body tissues can generate a protective immune response, especially if the virus originates from the same kind of animal (except in pigs, who can gain immunity from any virus apparently). And it's possible that with this type of vaccine, other susceptible individuals in the environment may be at risk of infection from the vaccinated. So it's promising, but not quite ideal.

Citation: Shope, R. E. Immunization Experiments with Swine Influenza Virus. J Exp Med 64, 47–61 (1936).

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).

9. Stuart, G. & Krikorian, K. S. A Fatal Neuro-Paralytic Accident of Antirabies Treatment. The Lancet 215, 1123–1125 (1930).

10. Wilson, S. A. K. A Lecture On Acute Cerebral Lesions At Different Ages. The British Medical Journal 1, 487–491 (1929).

11. Sampson, J. H., Archer, G. E., Mitchell, D. A., Heimberger, A. B. & Bigner, D. D. Tumor-specific immunotherapy targeting the EGFRvIII mutation in patients with malignant glioma. Seminars in Immunology 20, 267–275 (2008).

12. Glück, R., Matthieu, J. M., Wegmann, A. & Méan, F. Absence of myelin basic protein in an improved purified duck embryo rabies vaccine. Neurochem Pathol 4, 69–75 (1986).

13. Lipton, M. M., Freund, J. & Brady, E. The Transfer of Experimental Allergic Encephalomyelitis in the Rat by Means of Parabiosis. J Immunol 71, 380–384 (1953).

14. Paterson, P. Y. Neuroimmunologic Diseases of Animals and Humans. Reviews of Infectious Diseases 1, 468–482 (1979).

15. Mackay, I. R. Travels and travails of autoimmunity: A historical journey from discovery to rediscovery. Autoimmunity Reviews 9, A251–A258 (2010).

16. Mecha, M., Carrillo-Salinas, F. J., Mestre, L., Feliú, A. & Guaza, C. Viral models of multiple sclerosis: Neurodegeneration and demyelination in mice infected with Theiler’s virus. Progress in Neurobiology 101–102, 46–64 (2013).

17. Mackay, I. R. & Anderson, W. H. What’s in a name? Experimental encephalomyelitis: ‘Allergic’ or ‘autoimmune’. Journal of Neuroimmunology 223, 1–4 (2010).