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The virus grower

May 14, 2009 By Declan DB Butler This article courtesy of Nature News.

Immunologist Doris Bucher talks about cultivating the swine flu virus.

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Just three labs worldwide grow seed strains for the seasonal flu vaccine. One of them is the lab of Doris Bucher, a microbiologist at New York Medical College, who is renowned for her green thumb in engineering viruses that yield high amounts of flu vaccine. She now has a new and urgent target – creating a seed strain for a vaccine against H1N1 swine flu. Nature's Declan Butler catches up with her on her progress.

How do you go about creating seed strains?

Wild-type flu viruses grow poorly. So to get a better-growing vaccine seed strain, what we do is reassort the strain that we need the vaccine against with a faster-growing, high-yield donor strain. We make the current H3N2 component of seasonal flu, and for that we reassort the H3N2 target with an H1N1 donor strain known as A/PR/8/34. The PR stands for Puerto Rico, 1934 — it's an old strain well-adapted to growing in eggs.

What you want to get from combining these are seed-strain viruses that look like the target seasonal H3N2 on the outside — which the immune system sees — but which on the inside have the proteins of the high-yield Puerto Rico donor strain. We do this by selecting strains using antibodies against the surface proteins of the Puerto Rico strain, so that we end up with strains that have the surface proteins you need for a vaccine against the target virus. It's our reassortants resulting from this selection process that vaccine makers use for the H3N2 component of seasonal flu vaccine.

And for the new H1N1?

For the H1N1, we don't want to use the Puerto Rico strain, as it itself is also an H1N1 virus, so you risk running into some cross-reactivity. Having a donor strain from a different flu subtype, which is immunologically more different, just makes the entire antibody-selection process much easier. Four or five years ago, we realized we needed a good donor strain for H1N1 targets, so we used a strain that we called, in honour of our institution, NYMC X-157, a reassortant between the PR/8/34 and an H3N2 strain. X-157 was the H3N2 component in the 2005-06 vaccine. It has six genes from the high-yield Puerto Rico strain, so although it's essentially a high-yield PR/8/34, its surface is largely H3N2. So to the immune system it looks like an H3N2 strain, making it a better donor strain when you are dealing with an H1N1 target virus. And X-157 has shown great ability to reassort with H1N1 targets.

When you reassort the target and donor, what are the steps in the lab?

We got an egg isolate of the new virus from the US Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, on 28 April. Every step we do has to be done in eggs. Almost all the flu vaccine in the world is manufactured by growing virus in eggs. Okay, some people say it's an old-fashioned way, but flu viruses grow really well in eggs.

In the first step, we co-infect an egg with the swine flu target virus and the X-157 donor; we use about 20-30 eggs in every experiment. So we inject the viruses into the allantoic cavity [a fluid-filled sac surrounding the embryo] — it's in the cells of its membrane that the virus replicates. We put in enough virus to be sure of getting both strains into the same cell, as you need that to get the gene shuffling event to take place. Then we incubate them for 42 hours. The allantoic cavity contains about 10 millilitres of fluid, and once you are done, you just slurp out the liquid and proceed to the next step.

And then it's on to antibody selection?

Yes, out of that first cycle you get 256 possible different genetic reassortants. Remember, flu has 8 genes, so as each might be from either of the two strains, the maths gives you the 256. Then we do the antibody-selection process. It works on the same principles as vaccination itself. We use antibodies against H3N2 to target and kill those virus combinations that have surface antigens from the X-157, different from the H1N1 ones we want. We want viruses that look like H1N1 on the outside but that have the genes for the 'internal proteins' of the high-yield H3N2 donor. What the strain looks like on the outside is what is really important.

So we do three cycles of 42 hours, where we add antibodies against the H3 and N2 to eliminate any reassortant viruses with those antigens on their surface, which reduces the possible reassortant combinations to 64, of the sort you want. At the end of each cycle, we harvest the allantoic fluids and carry them to the next step. As well as antibody selection, at the same time you have selection to pick out the fastest-growing, high-yield viruses in each cycle. It's very Darwinian; the virus best adapted to growth in eggs will outgrow other viruses that are less well adapted, and so win.

After that, we amplify the virus for 42 hours, clone the reassortants, and then amplify them again, at which point we have about 80 millilitres of seed virus to ship to the CDC and other labs, who will test them with immune sera to ensure that the reassortant virus looks like H1N1. They will also infect ferrets with the reassortant to evaluate immune response. The CDC also sequences the haemagglutinin to monitor any changes that may have occurred with egg adaptation. We'll usually offer about three different candidate viruses.

How is the virus growing so far?

It is proceeding quite well. Altogether we have done seven cycles of 42 hours each, to develop the seed strain - we can get through about three cycles a week. We are still on target to ship the virus around 22 May. It is growing quite well. And the antibodies that we have seen seem to be working very well. The reassortment experiments are behaving as we would hope.

What's the trick that makes your lab special?

The trick is that we have very good reagents for selecting reassortants and highly skilled and experienced researchers. We inherited the laboratory of Edwin Kilbourne. He first proposed the idea of high-yield reassortants to make vaccine in 1969, and it was first used for the Hong Kong flu vaccine in 1971. This approach has been used for the preparation of the type A components for all vaccines since then. I have 40 years of experience working on flu, and have put a lot of years into developing systems to purify the different antigens, and making antibodies. My lab's major contribution is to have made extremely good selection antibodies.

When Ed hired me, I was very attracted to the idea of working on the flu vaccine. I'm a very practical person, grew up on a farm, and like to do something that is useful. What's terrific is that what we do is produce something that is actually used that year, that you know, hopefully, saves lives.

For Nature's ongoing coverage of the H1N1 outbreak, see


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