036 - Effect of Calmette's BCG Vaccine on Experimental Animals

Back in 1929, there wasn't a very good vaccine against tuberculosis, though many people (including famous ones) had tried to make one. The one that seemed to have the most potential was called BCG, or Bacillus Calmette-Guérin, discovered by Calmette and others. (Modernity note: the situation hasn't really changed much since then. BCG is still the TB vaccine of choice, and doesn't work very well. Used sometimes in countries with endemic TB, not in the USA.) This is made from a strain isolated from a cow, and is the bovine version of tuberculosis, so isn't very virulent for other species (or for cows either, apparently). And after Calmette grew it in the lab for a while, it was even less virulent.

Calmette had tried it in thousands of infants since 1921, but hadn't kept good records, so the results are unavailable, unfortunately. But it seemed to produce mild TB consistently in lab rodents, thus protecting them from more serious disease.

This study tried to clarify some of the methods and results using the BCG vaccine in several kinds of animals.  Specifically: guinea pigs, calves, and monkeys, inoculated orally, subcutaneously, or intraperitoneally (near the organs).

Feeding BCG to animals generally didn't work very well: pigs and calves didn't show any tubercles indicative of tuberculosis response, though almost half the monkeys they tested did. And animals challenged with a virulent strain of TB after oral BCG vaccine got TB almost as much as controls, especially pigs and monkeys.

Subcutaneous vaccination worked a bit better. Many pigs showed tubercles and one may even have died from it; most monkeys and calves also showed an effect. Generally it was mild though. When challenged with a virulent strain, vaccinated guinea pigs generally had milder TB (or none at all) compared to controls; similar with monkeys, and I couldn't really tell with calves. So I guess that's a positive result, if it's the best you can get.

They got similar results with intraperitoneal vaccination in pigs, though the vaccine seemed rougher for them. But they seemed to have a little protection.

Overall, it didn't seem like great results, but it wasn't a great study.

Citation: King, M. J. & Park, W. H. Effect of Calmette’s BCG Vaccine on Experimental Animals. Am J Public Health Nations Health 19, 179–192 (1929).

035 - Antirabic Immunization with Desiccated Vaccine

People had been vaccinating against rabies for a while (at least since 1893), using nervous tissue from animals infected with an attenuated strain of the virus, but apparently this wasn't always perfect, and could be difficult to obtain.

Some people had been experimenting with preserving rabies virus by freeze-drying it. They found that this method could preserve the virus's infectivity for months or even years. But in the current study, D.L. Harris found that even after the freeze-dried virus lost its infectivity, it still retained some of its immunogenicity, the ability to induce an immune response. This disappeared after a while too, though, so there was a range in which it could be used as a vaccine. However, since there was a chance that it was still slightly infectious, it had to be given in conjunction with the regular vaccine to make sure there was no chance of contracting rabies from it; a sort of vaccine against the vaccine.

This doesn't seem all that useful to me, but apparently Harris went ahead and tried it on about 3500 people and a bunch of dogs and other animals. The people had all been bitten by a potentially rabid animal, so they didn't have much choice for treatment, and apparently the vaccine worked just fine in all except one, who had received a nasty bite but refused the full course of treatment so he could get back to work. The side effects in people were limited to short-lived local redness and swelling.

It seemed to work well prophylactically in animals too: of hundreds of dogs and some rats, only one rat died in the study but it wasn't clear that it was from rabies, and it seemed to be 100% effective in protecting rats against infection (of 16 vaccinated and 8 controls, zero got rabies of the former and 4 of the latter died of it).

So according to Harris, this vaccine gives better protection, but it seems like the disadvantage of requiring an extra dose of differently prepared vaccine makes it not quite perfect. More improvement needed.

Citation: Harris, D. L. Antirabic Immunization with Desiccated Vaccine. Am J Public Health Nations Health 19, 980–985 (1929).

034 - A Yellow Fever Vaccine

Back in 1928, yellow fever was causing problems in West Africa, causing epidemics and killing people who went there to investigate it. It’s caused by a virus that’s in the Flavivirus family, related to dengue and West Nile, and all these are transmitted by mosquitoes. The symptoms of yellow fever are usually fever, chills, nausea, muscle pain, and headache (among a few others), but sometimes it can go toxic and cause fatal liver damage. It’s classified as a hemorrhagic fever because it increases bleeding . Usually the mortality rate is 3% of cases, but can get as high as 50% in some outbreaks.

So, at a time in medical history when people were just learning that some infectious agents are smaller than bacteria, Edward Hindle isolated the yellow fever virus and attempted to make a vaccine against it.¹

He got the virus from a case of yellow fever in Dakar, Senegal. It was maintained by passing it through rhesus monkeys via mosquitoes; not a simple routine procedure, sounds like, not exactly streaking a Petri dish. But it worked: when he inoculated 16 other monkeys with only 1 μg of infected monkey liver, they uniformly died after a few days. It seems a lot more severe in these monkeys than in humans, but it makes for a good animal for vaccine tests I suppose.

So Hindle tried two different but similar ways to make a vaccine: the first used formaldehyde in saline to inactivate the virus, and the second used phenol in glycerin and water. In each case, some liver and spleen from an infected monkey was mixed with these ingredients, then filtered through a cloth and inoculated into test animals.

Hindle tested the formaldehyde version in one subject. It had a slight, short fever after immunization, but when inoculated with a dose of yellow fever that was about 2000 times more than a dose that would be lethal in most cases, the monkey only had a mild fever for 4 days. Two controls that received 1- or 10-times lethal doses both died as usual. Even a 10000-times lethal dose in the vaccinated monkey didn’t make it very sick.

So that’s pretty good, but the other was even better. The first monkey to get it had barely any reaction, if any, but resisted a 2000-times lethal dose with no symptoms, and then another 10000-20000-times dose.

Hindle was so pleased with these results that he tested another 10 monkeys (6 vaccinated, 4 controls). These 6 didn’t show any adverse reactions either. A second, blinded researcher inoculated the 10 with about a 10000-times lethal dose. All controls died as usual, but only one vaccinated died (who had actually received about 20000-times lethal dose); the other five were fine.

These seem like promising results, essentially a “yes or no” answer of whether the vaccine worked. There were controls involved and some blinding, though no placebo (not so important when doing an animal study though, perhaps). The author said human trials were worthwhile, though some tests should be done to determine the duration of immunity and the vaccine’s shelf-life.

However, future articles citing this study didn’t take such a bright view of it:

"Both preparations appeared to protect monkeys against yellow fever, but subsequent investigations of efficacy were inconclusive."²

"Overall, these preparations were problematic due to residual live virus or were ineffective due to inadequate antigenic potency, reflecting the rudimentary virological methods available at the time."³

"Results with this vaccine in human subjects were considered promising, though immunogenicity was described as irregular. Further attempts at refining and optimizing an inactivated YFV (yellow fever virus) vaccine however, were abandoned following the development of cost-effetive live attenuated yellow fever strains that were highly effective at protecting against natural YFV infection. These early attempts at developing an inactivated YFV vaccine were left behind as failures and this perception has persisted despite signs of early success."⁴

"The problems described with these vaccines were the presence of residual infectivity in viral preparations or the loss of immunogenicity."⁵

So I guess there are better YFV vaccines coming up in future posts.

Citations:

1. Hindle, E. A Yellow Fever Vaccine. Br. Med. J. 1, 976–977 (1928).

2. Hayes, E. B. Is it time for a new yellow fever vaccine? Vaccine 28, 8073–8076 (2010).

3. Monath, T. P. et al. Inactivated yellow fever 17D vaccine: Development and nonclinical safety, immunogenicity and protective activity. Vaccine 28, 3827–3840 (2010).

4. Amanna, I. J. & Slifka, M. K. Wanted, dead or alive: New viral vaccines. Antiviral Res. 84, 119–130 (2009).

5. Gaspar, L. P. et al. Pressure-inactivated yellow fever 17DD virus: Implications for vaccine development. J. Virol. Methods 150, 57–62 (2008).

033 - Immunization with Diphtheria Toxoid (Anatoxine Ramon)

So far we've seen the development of a promising diphtheria vaccine made of toxoid (inactivated toxin, 029 and 030), but haven't seen any trials to show that this vaccine actually protects its recipients from diphtheria. Unfortunately, today's study is not such a trial either.¹

The authors were comparing the Schick test for immunity to diphtheria toxin to a test that they had developed (in the future called the Moloney test). Both involved injecting a small amount of toxin under the skin of a patient to see if they reacted to it; if so, they were not immune. The main difference was the substance used as a control: in the Schick test, heat-inactivated toxin was used as a negative control, but Moloney and Fraser here used toxoid. This approach also helped determine whether an individual would have an adverse reaction to immunization with the toxoid at full strength.

So they did this test on 141 girls, ages 10-18 years. 47% were Schick-positive, reacting to the toxin but not toxoid control; 13% were Schick-negative, reacting to neither, and thus considered to be immune; 20% reacted to the toxoid control but not the toxin; and the last 20% reacted to both. Of this last group, it's hard to tell whether they're immune or not, but could be dangerous to immunize them.

So then the authors vaccinated the 47% Schick-positive subjects with toxoid, two doses of 0.5 mL each, a month apart. They did the Moloney test again after 1 1/2 months. Of those immunized, 65% became Schick-negative, 28% didn't, and 7% reacted to both toxin and control. They tested for antibodies in the latter group and found that half of them were immune. So the vaccine converted about 70% of Schick-positive to Schick-negative; thus 70% effectiveness, according to this test.

The authors concluded that their test was reliable, though the 70% effectiveness was not great for a vaccine. Perhaps three doses would work better than two.

Papers citing this study generally acknowledged the Moloney test as a useful thing. Here are some things they say:

"The use of diluted toxoid as a control interferes with the reading of the [Schick] test by producing more marked pseudo reactions, but is therefore of great value in indicating those persons susceptible to toxic symptoms following immunizing injections of antigen."²

"The writer is of opinion that, if one test has to be omitted, that test should certainly be the Schick-control and not the Moloney injection. It has been shown that the one test cannot be used as a substitute for the other, and of the two the information given by the Moloney test is much the more important."³

Citations:
1. Moloney, P. J. & Fraser, C. J. Immunization with Diphtheria Toxoid (Anatoxine Ramon). Am J Public Health (N Y) 17, 1027–1030 (1927).
2. Loeffel, E. & Massie, E. Relative Value of Heated Toxin and Toxoid as Controls in the Schick Test. Am J Public Health Nations Health 25, 1018–1022 (1935).
3. Underwood, E. A. The Diphtheria Toxoid-Reaction (Moloney) Test: Its Applications and Significance. The Journal of Hygiene 35, 449–475 (1935).

032 - Duration of Immunity Following Modern Smallpox Vaccine Inoculation

Safety and effectiveness are two important things with vaccines, but something else that is important to consider (that is also maybe part of effectiveness) is duration of immunity: how long someone is immune to a disease after being vaccinated against it. This varies considerably between different vaccines and diseases, depending on the strength and type of the immune response and on the pathogen itself, which may shift around its antigens so that the immune system no longer recognizes it (a good example of this is influenza).

So the study today is an attempt to get some handle on the duration of immunity to smallpox after inoculation with cowpox (aka vaccinia). Even in the days of Edward Jenner, some observed that people lost their immunity to smallpox sometime after being vaccinated. But the most specific they could say about that was that immunity lasted several years after one vaccination. It did seem that repeated exposure/inoculation led to more long-term immunity, such that if one were immunized three times about 5-6 years apart, immunity was life-long.

But in 1927, it was no longer legal to intentionally inoculate people with smallpox to test their immunity; at least not in Europe or the United States. No surprise. So researchers had to find other ways to determine immunity duration. Previous reports from Germany and Britain stated that immunity lasted 10 years on average, at which point it should be renewed, though a study in Algeria found a duration of only 5 years.

This article goes into some depth about proper vaccine technique and what reactions to expect when it's done right or not done right. Apparently many practitioners in the US were doing it wrong, in ways that had been banned in Europe. But when done right, the reaction observed in the patient's skin was an indication of how much immunity they had to cowpox before being inoculated, whether no immunity, partial, or full immunity. Though it wasn't clear exactly how much immunity to vaccinia paralleled immunity to smallpox; definitely a significant amount, of course.

So using these reactions, researchers tested the immunity of schoolchildren in California, inoculating them once in 1921 and then again in 1927, 6 years later. After the first round, the reactions indicated that about 55% had no immunity before inoculation (no surprise again), 12% had partial immunity, and 23% had full immunity somehow. The remainder didn't show any reaction from the vaccine, probably indicating poor vaccination technique.

Then after 6 years, they re-inoculated and observed reactions. If the vaccine worked well and immunity lasted at least 6 years, then perhaps 10% of subjects should show no or partial immunity from the re-test, while the rest should be fully immune. But actually, 1% had no immunity, 26% had partial, and 73% had full. So 15-25% of subjects had declined in their immunity after 6 years from the first inoculation.

This wasn't a great study by any means, but it was something, and should've been helpful in establishing a schedule for re-inoculation. Progress!

Citation: Gillihan, A. F. Duration of Immunity Following Modern Smallpox Vaccine Inoculation. Am J Public Health (N Y) 17, 906–911 (1927).

031 - The Value of Mixed Vaccines in the Prevention of the Common Cold

You can probably guess from the title what the conclusion of this study was.

That said, it was actually a very good study, at least for the time; future studies claimed that this one might've been the first real clinical trial. It was in the vein of post 026, using a vaccine made of multiple types of killed bacteria to immunize against the common cold.

This one took place at the University of Manchester, and contained four main groups of subjects: those that volunteered to be randomly assigned either to vaccination group or control group, those that volunteered to be vaccinated, and those that volunteered to be controls. So about half the subjects were randomized and the others weren't. This didn't make much difference in the end though. The randomized groups, at least, were about evenly matched in sex and time since last cold.

The vaccine was made with several different types of bacteria commonly found in respiratory infections, including pneumococcus. Three injections were given, a week apart. Most reactions, if any, were trivial; some had an induration lasting a couple weeks, and one had a swelling that became an ulcer that healed slowly. But nothing very serious.

However, considering the results, the risks probably weren't worthwhile: the vaccinated groups got sick more than the controls. It wasn't a significant difference, 1.47 colds per person for vaccinated compared to 1.14 per person for controls, but it was quite clear that the vaccine did not help. Severity of illness was no less for the vaccinated group either. And the pattern was the same for the randomly assigned groups as for those that volunteered for one group or the other.

The scientists kept good records of different things in this study, and considered the possibility of different kinds of bias, so it was a relatively good study overall. There was randomization and group matching and such. Not up to today's standards, of course—it wasn't single- or double-blinded and there wasn't a placebo, for two things—but it was progress.

As a side note, there was a graph of how many colds lasted how long, and most of them were around 7 days long (as expected), though a few were more than 50 days.

Finally, I liked this quote from the article:

"We would add a word of warning with regard to the utter uselessness of the reports of individual patients as evidence of efficient prophylaxis. Among any large population, some persons will experience fewer colds during any particular winter, than they have experienced in previous year. In no department of human reasoning does the argument, post hoc propter hoc hold more absolute sway, than in the lay evaluation of medical procedures."

Always a good thing to remember, regarding conventional medicine or alternative medicine alike.

Citation: Ferguson, F. R., Davey, A. F. C. & Topley, W. W. C. The Value of Mixed Vaccines in the Prevention of the Common Cold. J Hyg (Lond) 26, 98–109 (1927).