029 - The Preparation and Testing of Diphtheria Toxoid (Anatoxine-Ramon)

Diphtheria is an infection with Corynebacterium diphtheriae, which is harmful almost entirely because of the toxin the bacteria produce, called diphtheria toxin. This means that a vaccine need only induce an immune response against the toxin in order to prevent the disease, not against the bacteria themselves.

Before any vaccine was developed, people used something called antitoxin to treat serious diphtheria cases. Antitoxin was made by inoculating an animal with diphtheria toxin and extracting the antibodies that animal produced, then using the antibodies to treat the disease in people. Its effectiveness was somewhat questionable, depending somewhat on whether the illness was caught and treated early enough, but it was the best people had at the time. Actually, the Iditarod dogsled race in Alaska is in memory of a dog named Balto leading a dogsled loaded with antitoxin to Nome, Alaska to treat a serious outbreak there.

Some of the first attempts at vaccine were combining diphtheria toxin with antitoxin and injecting them both into people; the antitoxin would prevent the toxin from causing problems, but the toxin would still be present to induce an immune response against further exposure. This was risky though, since the actual toxin was present and could cause problems if the ratio was wrong; and antitoxin could cause sensitivity problems of its own in some people.

All this makes the current study exciting, as well as the fact that it's the first example of a vaccine in this blog that's close to a kind that's still in use today. It's called a toxoid vaccine. Gaston Ramon was the first to develop it, though he called it anatoxine (being French and all). A toxoid is a toxin that has been detoxified chemically, so it doesn't cause disease, but can still induce an immune response that works against both toxoid and original whole toxin. This makes an ideal vaccine: simple, non-toxic, effective.

The current study describes methods of preparing, testing, and dosing a diphtheria toxoid vaccine. It's not too hard to make, though it takes a while. First, grow the bacteria up in broth, then filter-sterilize the broth (removing all the bacteria), leaving only a solution of toxin. The authors tested the amount of toxin by determining how much of a dose it took to kill half of a number of test animals (the median lethal dose/MLD, also called LD₅₀). Too little toxin would make it ineffective.

Then, to inactivate the toxin, they added a certain amount of formaldehyde, not too much or too little, and heated to body temperature for 1-4 weeks. A pretty long time. They knew it was done by testing it weekly in guinea pigs, injecting a little into the skin and seeing how much skin reaction they got. Once the reaction was smaller than a certain diameter, it was ready. Then they tested it again in guinea pigs for overall toxicity, and for potency (how much immune response it induced in the pigs, measured by protection against whole toxin).

The exact dosing for humans had not been fully determined. They recommended two 0.5mL injections, a month apart, for young adults. It worked better for some people than others, especially well for those that exhibited a skin reaction. Children less than 8 years old rarely had such a reaction; over 8 years, about 25% had a reaction. Older subjects seemed more likely to have a more serious general reaction (headache, fever), but whether this was likely could be tested by injecting a little bit of diluted toxoid right under the skin and seeing if that induced any local reaction. But rarely did anyone have a very serious reaction lasting more than a couple days.

So that's cool, not a clinical trial or anything, but the authors claim they were doing another study of about 50,000 children in Canada, so hopefully I will report on that soon.

Citation: Moloney, P. J. The Preparation and Testing of Diphtheria Toxoid (Anatoxine-Ramon). Am J Public Health (N Y) 16, 1208–1210 (1926).