051 - Canadian Experience with BCG Vaccine

The BCG (or Bacillus Calmette-Guérin) vaccine (mentioned in 036) is an attenuated strain of Mycobacterium that seemed to work to immunize against tuberculosis, so it's pretty important, but people have always wondered about its effectiveness.

This publication was a short report about BCG usage in Canada, regarding the conclusions of a subcommittee evaluating the value of the vaccine. It claimed that in general, English-speakers (as opposed to, I suppose, French-speakers such as Dr. Calmette) were less optimistic about the vaccine.

However, there had been an 8-year study with 5,126 subjects in Montreal using the BCG vaccine. 582 of the vaccinated subjects had been exposed to tuberculosis from their family situations (not intentionally, of course), and another 500 unvaccinated controls in similar conditions had been exposed. These subjects were 1 month up to 7 years of age; those who died before reaching a month old were excluded because there were too many other causes of death in those, I think.

So overall, about 10 from every 100 of the vaccinated subjects died from any cause, while almost 20 from every 100 died of the unvaccinated. In the vaccinated, about 2 of the 10 deaths were from tuberculosis; in unvaccinated, the number was about 7 of the 20. These seem like good numbers (a 71% reduction in deaths from TB), though it's odd that all-cause mortality was so much different between the groups also. Seems like perhaps the two groups were not equivalent, but there aren't enough details to tell for sure.

The second question is whether the vaccine is safe, especially since it is a live organism (though attenuated). Some people had criticized it previously, but this article claims that they had mostly changed their minds, and all agreed it was safe. They don't show data, though, so who can say.

Overall they seem optimistic, but still have questions about how reliable and long-lasting the immunity is. Further studies are required.

Citation: Canadian Experience with BCG Vaccine. Can Med Assoc J 35, 196–197 (1936).

050 - Propagation of Rabies Virus in Tissue Culture and the Successful Use of Culture Virus as an Antirabic Vaccine

This was a short report about some researchers, Leslie Webster and A.D. Clow, who found a way to grow rabies virus in tissue culture. Previously, it had only been propagated in lab animals or isolated from wild/feral animals. Being able to grow it in a lab, one way or another, is important because that's how to make the vaccine to prevent or treat this almost-always-fatal disease.

As I have discussed in previous posts (040 and 043), injecting someone with animal brain tissue as a vaccine against rabies often worked, but the downside was that occasionally it induced an immune response against the vaccinee's own central nervous system, especially the myelin component, which could cause serious problems.

Webster and Clow claim that their method, described in this study, should be less risky in this regard, because it doesn't contain so much extra brain tissue. They didn't test this, though, at least not here, so I'm not sure how much to believe it, and here's part of why:

The method for growing rabies in culture that they devised involves keeping cells from mouse brains alive in monkey serum (the liquid components of blood), and inoculating that with virus. So it still involves mouse brain material. Probably not nearly as much though, or as many different kinds, so they could be right.

Anyway, it does seem like a good alternative to propagating the virus in whole animals. They report that they passed the virus through this culture over a series of 16 transfers, and it still worked fine as a vaccine or as a pathogen in animals at the end. If the virus weren't growing, each transfer would have diluted it more and more, until there was hardly any left after six transfers. So it seems clear that it's actually growing.

And they tried immunizing mice and ferrets with it, injecting it into their body cavities, and it protected all of them from infection with virulent forms of the virus even when injected directly into the brain. Immunization by virus under the skin didn't really work though, at least in ferrets. But the immunizing power of this tissue culture virus seemed about as strong as that taken from animal brains. So it's pretty promising, perhaps.

Citation: Webster, L. T. & Clow, A. D. Propagation of Rabies Virus in Tissue Culture and the Successful Use of Culture Virus as an Antirabic Vaccine. Science 84, 487–488 (1936).

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).

048 - Poliomyelitis Following Vaccination Against This Disease

This one is pretty short, but important. And closely related to the previous two posts (046 and 047). In this short article, J.P. Leake of the U.S. Public Health Service lists twelve cases of paralytic polio in children, but not just any polio.¹

As you may recall from the previous posts, there were a couple of experimental polio vaccines being tested around this time, and more than 11,000 people had received them. Brodie’s was allegedly completely inactivated, and Kolmer’s was still alive, but chemically attenuated enough that it rarely caused problems in monkeys. But when it is uncertain whether a given person will ever encounter the virus, let alone have a serious reaction to it (such as paralysis or death), any vaccine must be extremely safe in order to be worth giving to the whole population.

Brodie and Kolmer claimed their vaccines were so safe, but Leake presents evidence to the contrary (though without naming names). His report of 12 cases come from children aged 5 months up to 20 years. Six of these children died from their polio, and at least four were still paralyzed at the time of this study. And more importantly, they all came down with symptoms one to two weeks after receiving a dose of experimental vaccine.

By itself, this wouldn’t mean much, since they could’ve been exposed to the virus soon before (or soon after) receiving the vaccine, such that they came down with symptoms before immunity set in. But in these cases, they weren’t in areas where polio epidemics were happening, and they hadn’t been exposed to people from known epidemic areas. Even worse, their paralysis tended to begin in the same area of the spine where they had received the dose of the vaccine (which in itself was pretty interesting, as it implied that the virus travels through nerves rather than other bodily networks).

So it seems like these unfortunate children were infected by an improperly-prepared product. As Leake concludes:

"Although any one of these cases may have been entirely unconnected with the vaccine, the implication of the series as a whole is clear."¹

Of the studies that cite this paper, almost none disagree with its conclusion, though a few try to propose an alternative explanation (that the vaccine interfered with an immune response to an already-ongoing polio infection, for example). But a couple reviews summarize the history well:

"The Kolmer vaccine was known to contain a small amount of live virus capable of infecting monkeys, but it was presumed to be safe for humans on the basis of the unproved assumption that serial passage in monkeys had reduced its pathogenicity for man. This vaccine was clearly implicated as the cause of a number of cases of poliomyelitis. The Brodie vaccine, believed to be completely inactivated, was also suspected of causing several cases of poliomyelitis, but the evidence is much less convincing. At a meeting of the American Public Health Association in November 1935, reports were given on both vaccines. During the discussion of these reports, both vaccines were roundly condemned, particularly by Rivers of the Rockefeller Foundation and by Leake of the U.S. Public Health Service. Shortly thereafter, Leake published a list of vaccine-associated cases, and the vaccines were withdrawn from use."²

"Undaunted, Brodie fooled himself and convinced Park that the vaccine was safe, attempting to prove it by inoculating himself, Park, and a few laboratory technicians, after he had done a group of monkeys. He presented and published papers, invoking Park's name and cajoling him to serve as cosponsor. The Brodie-Park vaccine was thereby launched and the press began to take notice. Their enthusiastic coverage of events aroused the hopes of parents and physicians anxious to believe that protection from the crippling scourge might at last be at hand.

"Meanwhile, a competitor, John A. Kolmer of Philadelpha, called a press conference to announce that he had successfully tested a vaccine, different from Brodie's in that the virus in his vaccine was "live but devitalized," the attenuation achieved by the addition of sodium ricinoleate. In addition, Kolmer's vaccine was carried one step further, having been tested not only in monkeys and on himself and his two children but also on 22 other children. Reporters asked Park whether his vaccine also was ready for human use. Brodie had assured him it was and that tests on children were planned. The press whipped up the rivalry to the point where newspapers carried frequent progress reports resulting in antics in which each tried to outdo the other.

"The pressure of public expectation, the lack of restraint, and poor judgment soon forced both Brodie and Kolmer to undertake the inoculation of several thousand children under the poorest of circumstances for such an experiemnt. The experiment resulted in at least 12 vaccine-associated cases and six deaths. Another tragic incident, another immediacy, and the U.S. Public Health Service stepped in, ordering that both of these vaccines be withdrawn and destroyed. With this were sown the seeds for the stringent legal requirements for vaccine safety and efficacy which were to sprout some years in the future."³

 Citations:

1. Leake, J. P. Poliomyelitis Following Vaccination Against This Disease. Cal West Med 44, 141–142 (1936).

2. Meier, P. Safety Testing of Poliomyelitis Vaccine. Science 125, 1067–1071 (1957).

3. Schaeffer, M. William H. Park (1863-1939): His Laboratory and His Legacy. American Journal of Public Health 75, 1296 (1985).

047 - An Improved Method of Preparing the Kolmer Poliomyelitis Vaccine

Now for another attempt at a polio vaccine: first there was Brodie’s (046), and now Kolmer’s.¹ It is prepared from monkey spinal cords infected with the virus, but the virus is attenuated with glycerin and sodium ricinoleate (a fatty acid from castor oil that has some antimicrobial activity), which reduces its infectivity enough to make it relatively safe but good for immunizing.

But this method isn’t good for keeping bacteria from growing in the preparation, so this study looks into potential preservatives that could keep bacterial contamination down without reducing the effectiveness. The candidates were formalin (diluted formaldehyde), phenol, mercurophen, merthiolate (aka thiomersal or thimerosal) and phenyl-mercuri-nitrate.

John Kolmer tested some vaccine intentionally contaminated with a few different kinds of Gram-positive or Gram-negative bacteria (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, etc), to see what concentrations of those chemicals would work. He found that phenyl-mercuri-nitrate (PMN) worked at the lowest concentrations for all of them, followed by the other mercury-containing compounds, then formalin, then phenol. E. coli was more resistant than Gram-positives. So PMN seemed best.

Then Kolmer tried making vaccines the normal way except with these preservatives at twice the minimum killing concentration, to see if they damaged the immunizing ability of the virus. He tested this by injecting rhesus monkeys and then infecting them with polio, six monkeys for each compound plus six with no preservative and four with no vaccine at all, just virus. He also tried three different doses in each group.

Results:

• The non-immune monkeys all became paralyzed within 7-8 days.
• None of them got sick from the vaccine alone.
• The preservative-free vaccine was the most effective, protecting four of the six monkeys completely, though the lowest-dose recipients got paralyzed after 11-14 days.
• PMN was second-best, with 3 of them completely protected. The others were paralyzed after 8-21 days.
• The worst was formalin, which was pretty much useless even at the highest dose; all monkeys were paralyzed after 7-8 days.
• The others were intermediate.

So Kolmer decided to start using PMN as a preservative. He also found that it didn’t cause any extra reactions in the monkeys; with or without, there were only slight local reactions. Actually it was better, because there was less chance of a bacterial infection from the injection.

The other thing he did was test his vaccine preparations for the presence of something called lymphocytic choriomeningitis virus, which others had found could be present in the monkeys, and could cause problems in humans injected with monkey spinal cords. The way to test for this was to inject some vaccine into mice and guinea pigs, in which the LCV also causes problems (makes sense since apparently it’s primarily a rodent virus anyway). But he didn’t see any sign of its presence in his vaccines.

So this all sounds pretty promising, though those preservatives do sound intimidating and would be harmful in large amounts at least. But apparently this vaccine was not a good one, as history bears out: 

"In the United States during 1935, cases of poliomyelitis followed the use of two experimental vaccines, developed by Kolmer and Brodie, respectively. These preparations were subsequently withdrawn from human use.
"Kolmer explicitly states that the virus was not killed, and his papers document the highly paralytogenic activity of the vaccine when given to monkeys by the intracerebral route. Subcutaneous injection of vaccine paralyzed 3 of 124 monkeys, while untreated virus brought down 1 of 20 animals by this route."²

 "None of these necessarily implies provocation linked to virus contained in the vaccine. The vaccine contained three components which could have provoked: 4% monkey spinal cord, 1% sodium ricinoleate, and 1:80,000 phenyl-mercuri-nitrate and in addition, some batches were contaminated with bacteria."³

So it’s not surprising we don’t use this version of the vaccine today either.

Citations:

1. Kolmer, J. A. An Improved Method of Preparing the Kolmer Poliomyelitis Vaccine. Am J Public Health Nations Health 26, 149–157 (1936).

2. Nathanson, N. & Langmuir, A. D. The Cutter Incident: Poliomyelitis Following Formaldehyde-Inactivated Poliovirus Vaccination in the United States During the Spring of 1955: I. Background. American Journal of Hygiene 78, 16–28 (1963).

3. Wyatt, H. V. Provocation poliomyelitis: neglected clinical observations from 1914 to 1950. Bull Hist Med 55, 543–557 (1981).

046 - Results of Field Studies With Poliomyelitis Vaccine

One of the vaccines I'm excited to learn about is polio, because apparently it works well enough that we've almost eradicated it from the world. But apparently it took a while to develop, probably because it took a while to figure out what caused it. (Something way smaller than bacteria? Whoa)

But in today's study, Gilliam and Onstott do a pretty good trial to test a vaccine against polio!¹ This version was developed by someone named Brodie, so it's not one of the ones in use today (which are Salk and Sabin), but I wonder why not. Apparently it was made of an emulsion of virus from monkey spinal cords, treated with formaldehyde long enough to inactivate the virus.² It seemed safe enough in preliminary tests, so the authors went ahead with a field trial.

They selected several communities in North Carolina and Virginia, near where polio outbreaks were occurring but not near enough to be in the midst of an outbreak (they wanted to make sure there was enough time to establish immunity before exposure). To parents of young children in these communities, they offered the vaccine as experimental and reasonably safe, and got 1452 volunteers for the trial in total.

Of these, they split them into those who would receive the vaccine, 766, and the control group, 686, based on the place of their surnames in the alphabet. Nice and random, supposedly. Each patient's physician would be the one doing the vaccination though.

Brodie cooperated with the study by providing the vaccine and also by withholding it from anyone else in the area who might want it, so that the researchers could be sure the controls really were controls.

When it came time to actually get the shots, only 458 volunteers actually showed up; a few of the controls managed to come upon a vaccine in other ways too, so they were excluded from the study.

There were two doses of vaccine, about two weeks apart. At first, they tried giving a shot of novocaine along with the vaccine to make it less painful, but it seemed just to complicate things without helping much. So in the end, 422 subjects got both doses, mostly at the right interval, and 36 got only one.

Gilliam and Onstott were closely involved in following up with the patients, asking about side effects or symptoms they may have had, since apparently the physicians missed some things. They observed that 50% of the subjects had some sort of reaction, though mostly mild and temporary: redness, swelling, etc. Most of the reactions seemed to be in patients that had received the novocaine. Hmmmm.

However, there were 15 local reactions that were more serious. Fourteen patients had an abscess or similar at the injection site, all of which healed, sometimes slowly.

Seventeen patients had general reactions (like fever or headache or nausea), which isn't bad, and these were usually associated with the abscesses. But some of the reactions were disturbing. For example, one girl fainted and stopped breathing after the shot, though she was fine after they revived her. Another had leg cramps at the injection site, though they got less frequent over time.

So it's pretty clear that this vaccine was not as safe as they thought, or at least not as safe as it should be. Better than being vulnerable to polio? Perhaps, but still not ideal.

What were the results other than safety? Well, of people who weren't involved in the study at all, about 6 in 10000 had polio, so the number of people you'd expect to have the disease in a similar group of a thousand is about 1, plus or minus 1. So between zero and two. In actuality, they didn't see any cases in either the vaccinated or controls. The authors calculated that even if the vaccine were 100% effective, they'd have to have about 10000 patients in each group to see a significant difference between them.

So that's unfortunate; the study was pretty much useless for judging effectiveness. As a later publication says,

"They were able to show, however, that Brodie's vaccine was relatively harmless, although they encountered a few very disturbing reactions."²

I had some other issues with the design of the trial as well. First, a minor issue: randomizing by alphabet? I don't think it's safe to assume that there isn't some bias inherent in people's family names, such that a group of people with names from the first half of the alphabet would be effectively identical to a group of people with names from the second half. But maybe.

Second, there's no placebo, which means there's almost no blinding (everyone knows who gets the vaccine and who doesn't), so bias in diagnosis could be introduced (like assuming people who got the vaccine are less likely to get polio, so being more likely to attribute symptoms to some other disease).
It also means that people dropping out of the study may not have been random; certain types of people might drop out more often in the vaccinated group but not in the control group (because there's no shot or other commitment involved), so the groups wouldn't be equivalent.

It didn't seem like there was much monitoring of rates of adverse events in the controls, to see if the observed side effects could be reasonably attributed to the vaccine or not, but perhaps that would need a different kind of study.

So overall, it's not great.

Citations:

1.

Gilliam, A. G. & Onstott, R. H. Results of Field Studies With Poliomyelitis Vaccine. Am J Public Health Nations Health 26, 113–118 (1936).

2.

Maxcy, K. F. Changing Conceptions of the Prevention of Acute Anterior Poliomyelitis. The American Journal of the Medical Sciences 192, 436–444 (1936).