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).