Posted: Wed May 06, 2009 11:50 am Post subject: Creating Seed Stocks
Creating the seed stock is an essential first step for any vaccine. So the C.D.C. has sent samples of the new strain to about 10 other government and academic laboratories in this country, Australia, Britain, Hungary and Russia. For the past five years, Dr. Bucher's laboratory has provided seed stock for one of the virus strains included in the seasonal flu vaccine used all over the world.
A vial containing millions of swine flu viruses in a milliliter of fluid (about a fifth of a teaspoon) arrived at her lab on April 28, packed with dry ice in a plastic foam box inside a cardboard carton stamped "infectious substance affecting humans."
The viruses had been grown from a cotton swab rubbed in the nose and throat of a child in California who received one of the first diagnoses of the flu in this country.
In one lab, members of the team amplify virus genes, cut them up with enzymes and analyze their origins. In others, their colleagues candle eggs, mark the shells with a pencil, pierce them with a drill bought at Sears and shoot them full of swine flu viruses.
Basically, the process involves repeated rounds of injecting the two types of virus into eggs, and sorting and purifying what grows. Each round of virus growth takes about 42 hours. The ultimate goal is to create a uniform seed stock from a single virus, and to produce 80 vials of it, each containing millions of viruses, that will be sent to drug companies, the C.D.C. and the Food and Drug Administration. Dr. Bucher said she expected to ship out those 80 vials by May 25.
Bucher told CNN Thursday that she had just packaged four viruses that she considers vaccine candidates and sent the samples to the CDC.
The virus used in vaccines is not the actual virus that infects people, but rather a hybrid that's been genetically modified to make it safer, and to give it the ability to multiply more quickly -- a crucial factor when it comes to manufacturing large quantities.
Bucher first injects a sample of "wild virus"-- in this case, H1N1 virus she got from the CDC, originally culled from a child who was infected in California this spring -- and then injects a sample of another flu strain that's known for its ability to rapidly multiply in eggs. For that, Bucher is using a strain with the exotic sounding name of NYMC X-157. (That's NYMC as in "New York Medical Center;" it's a hybrid of an H3N2 seasonal virus and the so-called "Puerto Rico strain," A/PR/8/34, that's used to speed the growth of seasonal flu vaccine).
Together in the egg, the viruses swap genes. In a laborious series of steps, Bucher's team guides the changes by adding antibodies that eliminate the surface proteins of the H3N2 virus. The end result is a virus with the exterior proteins of the H1N1 swine flu -- so the immune system recognizes it -- but with the inner mechanics -- the fast-growth ability -- of X-157.
Another method of creating a candidate virus also mixes wild virus with a second, fast-growing strain, but instead of growing them in eggs, lets the viruses mingle in a special solution while scientists manipulate them through a complex technique called reverse genetics.
Webby has been working to produce a candidate virus against H1N1 as well, but told CNN the virus is growing slower than expected, and that it would likely be a few weeks before a suitable strain is ready to be sent to the CDC.
With any candidate strain, CDC virologists will perform tests to see if the new virus can induce an immune response against the wild H1N1 strain. That's done by using a solution derived from the blood of ferrets, whose susceptibility to flu is similar to that of humans, and also by deliberately trying to infect live ferrets.
If test results are acceptable, the CDC will forward samples of the virus to various manufacturers. Those companies will fine-tune the virus for their own production methods, and produce small "pilot lots" of vaccine that can be tested in animals, and possibly people, for effectiveness and safety.
Sanofi Pasteur said Wednesday it received the new influenza A(H1N1) swine flu seed virus, enabling the company to begin the production process for a vaccine.
A vaccine will take several months to produce.
With the seed virus in hand, Sanofi will begin the development process for the vaccine. The first step involves "passaging" or sub-culturing the seed virus in eggs. That will tell the company the best conditions to grow the virus and produce efficient yields. The passaging process is expected to take approximately two weeks. The working seed must be certified by the FDA.
Sanofi Pasteur said it will then be prepared to begin industrial production as soon as directed by public health agencies. Clinical trials of the vaccine could take place during the manufacturing process.
Sanofi estimates it will have the first bulk concentrate vaccine in a few months following the certification of a working seed. "However, the company will be better able to determine the timing once the development of working seed is completed," company spokesperson Ellyn Schindler said.
The final formulation, filling and distribution of the vaccine have not been agreed to yet, and Sanofi will need further direction from the Department of Health and Human Services.
Posted: Thu Aug 06, 2009 11:57 pm Post subject: Good news about growing stock
And as recently as two weeks ago, heads of labs that make seed strains - including Wood - were pessimistic they'd be able to find a way to increase the virus yield, which was between 1/4 and 1/2 of the yield with the pandemic virus seed strains as compared to seasonal flu vaccine production.
John Wood of the U.K.'s National Institute for Biological Standards and Control said an improved version of the seed strain his lab produced in May seems to generate a virus yield that is on a par with what manufacturers get when they make seasonal flu vaccine.
Wood said the NIBSC team grew up quantities in 100 eggs, simulating on a minor scale what happens with vaccine production. The yield looked good at that scale, he said, suggesting that is reason to hope the new seed strain will perform well in the hands of manufacturers.
Eggs are inoculated with viruses, which grow, are harvested and then placed into a new batch of eggs for additional growth.
The process, called passaging, is generally done only a couple of times to allow a seed strain to adapt to growing in eggs.
"Normally we send these viruses out with the minimum of passaging, because we need to get it out quickly, really," Wood said.
But where NIBSC's original seed strain was passaged twice, the new one went through 13 rounds of growth in eggs.
The lab has never passaged a seed strain so many times, Wood admitted. "It was really because of the unusual circumstances that we really didn't have anywhere else to turn."
Another lab has tried a similar approach and it too may have some results as a consequence.
Doris Bucher, head of the laboratory at the New York Medical College in Valhalla, N.Y., confirmed her team has produced three new variants of their original seed strain, all of which appear to produce higher yields than the original. (The team's first seed strain was the best of the original batch made available to manufacturers.)
They were produced with six additional passages through eggs, said Bucher. She suggested in an email that the additional passaging probably contributed to the better yield of hemagglutinin, the major surface protein on a flu virus that is used in vaccine to induce development of virus-specific antibodies.
One potential drawback for manufacturers is the fact that the NIBSC seed strain was created using reverse genetics, a patented process that lets scientists create a hybrid virus made of whatever constellation of genes is desired. In this case, the seed strain has two external genes from the target virus and six internal genes from a flu virus that grows well in eggs.
Bucher's team produces its seed strains using what's called the classical method, in which the target virus and the high-growth virus are allowed to naturally swap genes. Offspring containing the desired gene constellations are then harvested.
Seed strains produced by the classical method are provided free to manufacturers. But royalties must be paid for any vaccine doses made from seed strains created using reverse genetics. The intellectual property (IP) rights are held by MedImmune Inc.. of Gaithersburg, Md., which has said it will waive payment for vaccine that is donated to the WHO for use in low-income countries.
All things being equal, vaccine makers would likely prefer to use a classically produced seed strain. But a significantly improved yield "may be persuasive," Wood said.
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