076 - Effect of Preservatives on Undenatured Bacterial Antigens

A big concern among parents seems to be preservatives used in vaccines, especially mercury-containing thimerosal. This study was a short one that looked at different preservatives and their effect on the immunity-producing components of bacteria.

Often it is desirable to separate out just the part of a pathogen that an immune response targets best and make a vaccine out of that, because that way you get a good, focused immune response against the important part and don't have anything else that could produce unwanted reactions. So Krueger and Nichols had developed a method to purify bacterial antigens, removing all whole bacterial cells from them, resulting in a product called Undenatured Bacteria Antigen, or UBA.

Using preservatives in vaccine preparations was often a good idea, because a bacterial contamination could lead to pretty bad outcomes in the recipients who received vaccines by injection. So this study tested how harsh different preservative candidates were on the UBA.

The ones they tested were tricresol, phenol, and merthiolate (aka thimerosal or thiomersal). The concentrations they used of the first two were much higher than of the third, more than 10 times higher, presumably because they didn't inhibit bacterial growth very well at lower concentrations. But they tried three different concentrations of merthiolate, going as low as 20 parts per million, or 0.002%. Then they tested how much UBA was denatured over time.

Denaturation by all of them was as complete as it was going to be by one week, and didn't increase beyond that. UBA with no preservative remained mostly undenatured, 95%, while that with phenol or tricresol was nearly half denatured. Merthiolate was intermediate, the lower concentration the better, between 24 and 37% denaturation.

So it seemed that merthiolate was the best preservative to use for preserving UBA.

Citation: Krueger, A. P. & Nichols, V. C. Effect of Preservatives on Undenatured Bacterial Antigens. Exp Biol Med 34, 335–337 (1936).

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).

073 - Vaccination Against Acute Anterior Poliomyelitis

I've talked about John A. Kolmer and his polio vaccine before (047, 048, and 063), but I wanted to touch on it once more.¹

Kolmer's vaccine was a "live" but partially inactivated virus. He took infected monkey spinal cords, treated them with sodium ricinoleate, added some phenyl-mercuri-nitrate as a preservative to prevent bacterial contamination, and injected them subcutaneously. These things should prevent infectivity in humans, he thought. And it seemed to work well in monkeys, though it could still paralyze if injected into the brain. The reason he wanted it partially "alive" was that he thought completely inactivated virus was unable to immunize, for some reason.

By this point, more than 12,000 people had received Kolmer's vaccine. None seemed to have severe reactions, like encephalomyelitis, though some that received the version without preservative had abscesses temporarily.

However, there were 10 cases Kolmer knew of in which the subject seemed to get sick with polio soon after receiving the vaccine (soon meaning 1-6 days later). Usually it was after the second dose, never after the third, but five of the 10 (50%) actually died from their illness, from paralysis.

In this paper, Kolmer thought it unlikely that the polio had come from his vaccine, considering the many that received the same lot without getting sick, and how no one receiving all three doses got sick. However, he was unable to explain where the virus had come from for some of the cases, since there wasn't an outbreak in their areas. It was a mystery.

It seems like later, though, he does conclude that the vaccine is not safe enough to use in people, especially because he hadn't been able to establish its efficacy in preventing any disease.

"It was my hope that this strain of virus had lost infectivity for human beings by reason of its long adaptation to the monkey, and especially after treatment with sodium ricinoleate and when given by subcutaneous injection, but the occurrence of nine cases of poliomyelitis among 10,725 individuals given the vaccine in 1935 has indicated that the virus apparently possesses infectivity for human beings and that this vaccine as well as the formalized vaccine of Park and Brodie is too dangerous for use."²

So, I'm not sure whether it was the correct decision or not, but that's why we don't use Kolmer's vaccine these days.

References:
1. Kolmer, J. A. Vaccination Against Acute Anterior Poliomyelitis. Am J Public Health Nations Health 26, 126–135 (1936).
2. Kolmer, J. A. The Present Status of Methods for the Prophylaxis of Acute Anterior Poliomyelitis. Ann Intern Med 12, 95–105 (1938).