Influenza: How Its Biology Affects Vaccine Production
I have received lots of email about avian influenza (thanks!), so I will try to answer at least a few of your questions by telling you about influenza’s basic biology and how that impinges upon vaccine design.
The cutaway cartoon of an individual influenza virus particle (shown below) reveals several important features. First, it shows the eight different segments of the viral genome. These segments encode all essential viral proteins and therefore, define its behavior after it invades cells within the host (you). These genomic segments are comprised of single stranded ribonucleic acids (RNA) that act as templates encoding viral proteins. As soon as the virus particle invades a host cell, it sheds its outer layer of proteins similar to a person shedding a winter coat upon entering a warm room. The “uncoated” RNA templates are immediately decoded and translated into viral proteins by the host cell’s protein-making “machinery”. These all-important RNA segments are also copied and packaged into new viral protein coats that were constructed by the host cell.
The second important feature in this picture is the two proteins on the surface of the viral particle. These proteins are often referred to as the “protein coat”. These proteins are what your body’s immune system sees and responds to. If the structures of either or both of these two proteins are distinctive enough that if your body’s immune system has seen them before, it will readily recognize them again. As a result of this recognition, your body immediately mounts an effective immune response by specifically destroying infected host cells that have been invaded, thereby protecting the host from infection.
These two proteins allow the virus to invade host cells. The first protein, hemagglutinin (abbreviated as HA. Note that it is misspelled in the accompanying cartoon), initiates infection by attaching the virus particle to specific proteins located on the outside of the host cell. These cellular proteins are referred to as “receptors”. Sticking to these receptor proteins is almost like ringing a doorbell because after HA binds, the unwitting cell invites the virus particle inside and, by doing so, signs its own death warrant by initiating viral infection. This cellular receptor protein is shared with red blood cells, too, so when HA binds to red blood cells (which contain hemoglobin, thus the word, “heme”), it causes them to clump together (agglutinate), a quality noticed by scientists many decades ago, hence this protein’s name.
The other viral surface protein, neuraminidase (NA), is an enzyme that destroys the host cell’s receptor proteins that HA binds to. By doing this immediately after the mature virus has been manufactured and assembled, NA allows these new viruses to be released from the host cell so they are free to infect new cells.
There are several types of influenza viruses; A, B and C. Types B and C have a very restricted host range: they only infect humans. In fact, type C viruses cause a very mild illness and never trigger pandemics or epidemics so health officials never subtype them. Influenza type B viruses are generally not serious and only occasionally cause epidemics so they also are not subtyped. But unlike these other two virus types, Influenza type A has a broad host range. Ducks are thought to be the natural hosts for Influenza A because they rarely develop fatal infections. But Influenza A can infect other birds as well as mammals (humans, pigs, horses, seals and whales, to name a few species) with devastating results. Due to its lethality and potential to trigger pandemics, Influenza type A viruses are carefully subtyped based upon which HA and NA proteins they carry on their surface. For example, an “H2N3” virus designates an Influenza type A virus with the HA 2 and NA 3 surface protein subtypes, and the current version of the “bird flu” is an H5N1 virus that has HA 5 and NA 1 surface proteins. Influenza A viruses are sometimes referred to by their numerical subtype alone; H1N1, H2N3, etc.
There are 16 distinct HA proteins (a new one was reported last week) and 9 different NA proteins, and hundreds of combinations of these two coat proteins are possible due to viral genomic rearrangements. Recombinations occur when two different virus particles infect the same host cell. During the virus manufacture and assembly process, individual surface proteins and RNA segments from the two different viral genomes can be mixed up as they are packaged and assembled. As a result, the new virus particles can have characters of both “parent viruses” when they have new and very different HA and NA proteins on their surface and when they contain genome segments that encode different HA and NA protein combinations. These changes radically alter the viral surface appearance such that the host’s immune system cannot recognize these viruses. Dramatic genomic changes such as these are called “antigenic shift”. When this occurs in viruses, a pandemic (worldwide spread) results. A pandemic results when the human population has little or no innate immunity against the virus, and when it can be easily and rapidly transmitted from person to person. Influenza viruses, which are primarily airborne, as especially transmissable.
Influenza viruses can also undergo smaller changes when mistakes are made while copying genomic segments. These mutations accumulate gradually over time and give rise to minor changes in viral coat proteins so they are poorly recognized by the host’s immune system. When this occurs, an epidemic (localized spread) results. These gradual changes are referred to as “antigenic drift”. Both Influenza type A and B viruses experience antigenic drift. In fact, antigenic drift is the reason that the same individual can get flu infections repeatedly, and also is the reason that people should get influenza vaccines every year.
As described, there are hundreds of possible recombinations of influenza surface proteins can result from antigenic shift, but currently, only several subgroups circulate throughout a particular species or population at any one time. For example, H3N8 and H7N7 cause illness in horses while H1N1, H1N2 and H3N2 currently cause illness in humans. Incidentally, an H1N1 virus caused the 1918 “Spanish flu” pandemic that killed an estimated 50 or more million people worldwide while the less-deadly (but still impressive) 1957 pandemic resulted from H2N2.
Viral subtypes H1, H2 and H3 are best adapted to humans and often cause epidemics and pandemics. Avian subtypes, H5, H7 and H9, have caused sporadic outbreaks of disease in the past when they jumped from birds into humans but they did not trigger epidemics because they were unable to be transmitted effectively from human to human.
How are effective and up-to-date flu vaccines designed and produced? It is a combination of science, educated guesswork and at least some luck because, as you have surmised, the flu virus is a moving target. First, because flu vaccines are effective against only a limited number of viruses, it is important to carefully choose each strain to include in the vaccine. To this end, many labs throughout the world maintain influenza virus collections. Each year, groups of these viruses are sent to one of four World Health Organization (WHO) reference labs. One of these reference labs is located in the United States at the Centers for Disease Control (CDC) in Atlanta. These labs carry out extensive testing and characterization of circulating and new flu viruses to determine how effectively human antibodies made against the current vaccine react against them. These data are combined with information about current flu activity and then a group of health officials from the Federal Drug Administration (FDA) and from WHO select two type A and one type B virus (for a total of three viruses) that will be used to manufacture the next year’s flu vaccine. Because influenza strains change every year, each year’s vaccine differs from the previous one by including one or even two new virus strains.
Even though more modern cell culture methods are now being developed, influenza vaccines are still prepared in the traditional way. Fertilized chicken eggs that are 11 days old are injected with a small amount of one of the three targeted “seed strains” of flu viruses. Each targeted flu virus is injected into the albumin (egg white) of individual eggs through a small hole in the shell. After injection, the hole is resealed and the virus then infects the lungs of the developing chicken embryo. Within several days (the specific length depends upon the virus strain), the virus has multiplied to great enough numbers to be harvested from the eggs.
Even with robotic assistance, “working with eggs is tedious,” says Samuel L. Katz of the Duke University School of Medicine, a member of the vaccine advisory committee for the U.S. Food and Drug Administration. “Opening a culture flask is a heck of a lot simpler.”
Because of the nature of flu vaccine production, vaccine companies must place their egg orders six months or more in advance before they begin producing vaccines. Further, each egg is innoculated with only one virus strain and produces enough of that strain for 1-2 doses, so approximately two to three eggs are required per vaccine. This process consumes hundreds of millions of eggs (270 million or more for the United States alone) to produce a sufficient supply of vaccine for the United States. Additionally, when a pandemic is looming, vaccine companies must manufacture as much as ten times more vaccine than they would normally produce.
“The egg method isn’t very flexible if you need to rapidly ramp up vaccine supply,” says Jonathan Seals, director of Process Development at ID Biomedical Corporation of Northborough, MA. “Vaccine manufacturers need to arrange for egg supplies months in advance — and you can’t tell a chicken to lay more eggs.”
Several days after injection and incubation, the virus particles are mechanically harvested from the eggs and purified several times to separate the virus particles from egg proteins. After purification, the viruses are chemically inactivated so they cannot cause influenza and then they are “split” using a detergent. The detergent releases the surface proteins, HA and NA, from the virus particles to increase accessibility to the immune cells of the body. Finally, the three split viruses are combined to make one “trivalent” flu vaccine. After passing safety tests, the vaccine is packaged and distributed to health care workers in time for the September vaccination campaign.
The entire process, from collecting, injecting and incubating millions of specially produced eggs through the safety tests on the vaccine, takes five to eight months. This means that health officials must decide one year in advance which flu viruses to use in its vaccines and how many batches of vaccine to purchase. This can be a risky business when trying to contain a potential pandemic of a rapidly changing virus such as the “bird flu”.
Graphic of a individual Influenza Virus particle
Weekly Report: Influenza Summary Update (Centers for Disease Control)
Background on Influenza (Centers for Disease Control)
Influenza production process graphic [PDF]
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More Essays about Avian Influenza;
Avian Influenza and 'The War on Birds', Part 2.
Avian Influenza and 'The War on Birds'.
Influenza: How Its Biology Affects Vaccine Production.
Public Confusion Surrounding Influenza.
Is Avian Influenza THAT deadly?
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