Fears of a possible avian flu pandemic spurred industry and government agencies to develop potential vaccines and work on updating production techniques to improve yields.
Patrick McGee, Senior Editor
Over the last 300 years, there have been 10 pandemics of influenza A. It has been estimated that the infamous 1918 pandemic, the worst in recent history, killed 20 to 50 million people. Today, the world’s population is 6.5 billion, three times what it was in 1918, but one thing has not changed: the risk of a pandemic is still very real.
That fact was underscored in 1997 when the H5N1 strain of influenza A became the first known bird flu strain to jump from birds to humans. H5N1 emerged in Hong Kong and infected 18 people, six of whom died, and it is now endemic in birds in large parts of Asia. As of November, 62 of the 122 cases confirmed in humans have been fatal, according to the World Health Organization (WHO).
The good news is that in nearly all those cases, illness resulted from direct contact with an infected bird. But infectious disease researchers are worried that H5N1 could mutate in a way that would make this particularly virulent strain more easily transmissible between humans. Also, the lack of prior human exposure to H5N1 means that humans have very little immunity to it. All of these factors make H5N1 a prime candidate to cause a flu pandemic.
Since the emergence of H5N1 and other avian flu strains such as H9N2, researchers around the world have been working constantly to isolate the strains, clone them, and develop vaccines that could be used for inoculation. Two vaccines under development by Sanofi Pasteur, Swiftwater, Pa., and Chiron Corp., Emeryville, Calif., demonstrated promise in early clinical trials, but more studies are needed.
Production circa 1950s
But even after vaccines are developed, there is still the problem of production to contend with. The vast majority of flu vaccines are produced the same way they were 50
years ago, a proven but painstaking process that takes a great deal of patience over several months. In an effort to hasten the process and increase vaccine yields, companies and government agencies like the National Institutes of Health (NIH), Bethesda, Md., are investigating the use of cell culture techniques. Other companies are investigating novel methods such as a DNA approach that proponents say could produce vaccine quickly and in large quantities.
“I am encouraged by the work that we’ve been doing with the current vaccine manufacturers using their technology, the egg-based technology. We’re developing better ideas of what the challenges are going to be, we are getting encouraging results, and we’re getting a good feel for what will be necessary to produce avian vaccine,” says David Cho, PhD, the influenza program officer at NIH’s National Institute for Allergies and Infectious Diseases (NIAID). “We definitely would like to see the cell-based vaccine technology come up to speed, not only for the avian flu, but also for the regular circulating strains. Having that technology up to speed would really enhance vaccine development.”
NIH is working with companies such as Sanofi Pasteur and Chiron because they are licensed by the US Food and Drug Administration (FDA) to produce influenza vaccines and have the technology and facilities to do it, says Cho. He adds that the Sanofi Pasteur vaccine is farthest along in development. Researchers recently concluded a study testing the safety of Sanofi Pasteur’s H5N1 vaccine in a population of 452 healthy adults.
The vaccine produced a strong immune response but required 90 µg of vaccine, compared to the 45 µg in a typical trivalent flu vaccine. In addition, because people have never been exposed to the H5N1 virus, they would require a primer vaccination followed by a booster a month later, for a total dosage of 180 µg.
The version of the vaccine used in that trial did not contain an adjuvant, a substance which can boost the body’s immune response to the vaccine’s antigen. Other studies are being conducted by Sanofi Pasteur at sites in France to evaluate formulations with aluminum hydroxide, an adjuvant, says James Matthews, PhD, the company’s senior director of health and science policy. He says the final data from the study with 452 adults should be announced soon.
An adjuvanted vaccine
Chiron also has a H5N1 vaccine in development and hopes to deliver it to NIH early this year for testing. Because the H5N1 strain is so virulent, it tended to kill the chicken eggs used to grow the vaccine against it, so Chiron opted to work with a less pathogenic strain and developed an H5N3 virus vaccine for testing against H5N1. Preliminary studies of this vaccine included MF59, Chiron’s proprietary adjuvant.
MF59 is an oil-water emulsion that is used in Chiron’s Fluad, the first approved adjuvanted flu vaccine in Europe. Fluad was developed primarily for use in elderly
populations, says Bruce Scharschmidt, MD, the company’s vice president of corporate scientific affairs. “It’s been approved since about 1997 or so in Europe and it’s been in widespread use elsewhere, so there’s a great deal of human experience with it.” According to Chiron, more than 20 million doses of MF59-adjuvanted vaccine have been distributed outside the United States for seasonal vaccination, and its safety profile has been very favorable.
The adjuvant proved to be very effective in the H5N3 vaccine Chiron is developing, says Scharschmidt. “Without MF59, that vaccine didn’t generate much of an immune response or one that we would judge likely to be protective. But with the MF59, it did. It required a comparatively small amount of antigen, as low as 7.5 µg, and with additional testing, what we hoped would be protective response extended to drifted H5N1 strains that occurred subsequently in Hong Kong as well as in Vietnam and Thailand . . . It seems to not only boost immune response. It seems to broaden it in a way that could enhance the effectiveness of the vaccine.”
In October, the company reported promising results for an investigational vaccine against an H9N2 avian influenza A strain. The 96-patient study investigated the safety and immunogenicity of four different doses of the vaccine with and without MF59 in doses ranging up to 30 µg. All formulations with MF59 proved to be highly immunogenic by inducing antibody levels believed to confer protection against the strain. The lowest dose, 3.75 µg, is a quarter of the single-strain dose used in seasonal influenza vaccines.
While development is promising, there is a limitation that must be overcome once a vaccine is approved, and that is in production. Simply put, it is a question of capacity. Every year, 300 million doses of influenza vaccine containing 15 µg of antigen per strain, or 45 µg total for three strains, are produced worldwide. In the event of a pandemic, 20 times that amount would be needed to inoculate the world’s population.
But even doubling or tripling production capacity will be very difficult, even assuming that a vaccine could be developed that would only require a single 45 µg or lower dose.
click the image to enlarge
Source: National Institute of Allergy and Infectious Diseases
“Assuming you have a monovalent prep at a standard concentration and formulation, optimistically you would probably have less than a billion doses of vaccine for a pandemic. So obviously, people are looking at ways to try to extend that supply by using adjuvants or lower doses or alternate delivery,” says Matthews.
In November 2004, the US Department of Health and Human Services (HHS) awarded a contract to Sanofi Pasteur to establish and maintain flocks of egg-laying hens to allow the company to manufacture avian flu vaccine at full capacity on a year-round basis. But adding capacity alone may not be enough. Cho says that compared with regular flu strains, H5N1 does not grow as readily within eggs or cell culture, something that could affect yields. Because H5N1 is so virulent, it can also kill the eggs being used to grow the antigen against it. Scharschmidt says that problem can be addressed by genetically engineering the virus to remove parts of the genome that are lethal to the chicken cells, while leaving in the parts that encode for the antigen. But that process can take weeks to months.
Another issue is literally a chicken-and-egg problem. If avian flu begins circulating, it could kill the chickens that produce eggs used to grow vaccine. “Although these flocks are quite well protected and we like to think they’re secure in the event that there’s really a pandemic virus out there, it’s conceivable that if the flocks were affected, the availability of those eggs would be affected,” says Scharschmidt. Matthews says Sanofi Pasteur’s site in Swiftwater has a “very extensive” biosecurity program and is supported by veterinary institutions in the state as well as by the Pennsylvania Department of Agriculture.
Cell culture promise
To get around the challenges of egg-based production, many companies are investigating the use of cell-culture techniques to simplify and speed up vaccine production, as well as to provide flexibility to handle surges in demand. “It offers tremendous advantages in the event of a pandemic, because we wouldn’t be ordering eggs six to nine months in advance,” says Scharschmidt. “We would be scaling up to growth of cells from a period of days to weeks in a way that would much more quickly configure a manufacturing effort that would approach the kind of demand that’s needed and that’s feasible for eggs.”
Baxter International Inc., Deerfield, Ill., is developing an influenza vaccine called PreFluCel that would be the first flu vaccine produced using cell-culture techniques, in this case
|Global Avian Flu Vaccine Effort
Sanofi Pasteur, Swiftwater, Pa., and Chiron Corp., Emeryville, Calif., lead the pack when it comes to developing vaccines for the emerging avian flu threat, but that hasn’t stopped other companies from trying to catch them. In September, MedImmune Inc., Gaithersburg, Md., announced a deal with the National Institute of Allergy and Infectious Diseases (NIAID) to develop vaccines against avian influenza viruses.
NIAID and MedImmune will develop at least one vaccine for each of the 16 variations of hemagglutinin, a key influenza surface protein that is represented by the “H” in the name of influenza viruses such as H5N1. They will use reverse genetics and reassortment to place hemagglutinin genes with pandemic potential into an attenuated human flu virus.
Baxter International Inc., Deerfield, Ill., will help Indonesia develop a vaccine for H5N1 in collaboration with PT Bio Farma, a vaccine and sera manufacturer in Bandung, West Java. In China, researchers announced that an H5N1 vaccine developed there proved effective in preventing the disease in chickens, water fowl, and humans, the Xinhua News Agency reported.
GlaxoSmithKline, Middlesex, UK, has an H5N1prototype pandemic vaccine in development. The prototype uses an adjuvant which may boost the immune response to the vaccine, thus allowing for lower doses. It may also protect against additional strains drifted from H5N1 that could arise; clinical trials are planned for this year.
Generex Biotechnology Corp., Toronto, is putting forth a series of hybrid vaccine peptides developed by its subsidiary, Antigen Express Inc., Worcester, Mass. MultiCell ImmunoTherapeutics, a subsidiary of MultiCell Technologies Inc., Lincoln, R.I., recently elevated one of its compounds, MCT-465, to lead-drug candidate status. Studies will build on the effectiveness that MCT-465 demonstrated in an earlier mouse model infected with H1N1, where it reduced pulmonary virus titers 1,000-fold, resulting in barely detectable levels. H1N1 is genetically similar to H5N1.
using Vero cells initiated from the African green monkey. Baxter’s proprietary technology allows the company to exclude any added proteins or raw materials derived from human or animal sources in the manufacture, purification, and formulation of its vaccines. They have used the Vero cell platform to develop the next-generation smallpox vaccine with Acambis, Cambridge, Mass., for the US government stockpile as well as on behalf of several companies. Baxter used the Vero cell platform to develop a SARS candidate vaccine and is using it to develop a candidate vaccine for H5N1.
Late in 2004, Baxter voluntarily suspended enrollment in a phase II/III clinical study for PreFluCel in Europe due to a higher than expected rate of mild fever and associated symptoms in trial participants. “We are continuing to tweak the formulation. The next milestone would be for us to conduct some additional clinical trials, hopefully in 2006,” says Baxter spokesperson Deborah Spak. “We think that this platform has the potential to offer a number of benefits, particularly for new and emerging pandemics where timing is much more critical.”
In October, Chiron announced it had initiated a phase I/II study of an investigational cell culture-derived influenza vaccine in the United States. It also completed enrollment in a second phase III study of investigational flu cell-culture vaccine in Europe that follows on the heels of a 2004 study that met safety and immunogenicity endpoints. “Hopefully, we’ll have those results sometime next year, and we are working with European regulators to get that to market as quickly as possible,” says Scharschmidt. The flu cell-culture vaccine is produced from virus propagated in the Madin-Darby Canine Kidney cell line at Chiron’s flu cell-culture vaccine manufacturing facility in Marburg, Germany. “We’ve been able to adapt the cells to grow in suspension, which offers some fairly substantial capacity and logistic advantages for growing up the virus.”
Last April, Sanofi Pasteur received a $97 million contract from HHS to speed the production process for new cell-culture influenza vaccines and for the design of a cell-culture vaccine manufacturing facility in the United States. Under the five-year agreement, Sanofi Pasteur will accelerate its cell-culture influenza program, which is based on the PER.C6 cell line of Crucell NV, Leiden, The Netherlands. Sanofi Pasteur will also develop a manufacturing process to produce up to 300 million monovalent influenza vaccine doses annually. Matthews says Sanofi researchers have been working on the cell-culture process and will be making pilot lots early this year, with a phase I clinical trial to follow (for more information on cell culturing, see Policy & Projections column, “Cell Culture Breaks Some Eggs”).
The adoption of cell-culture techniques promises to cut production times, but Lyle Lash believes he may have a method to save even more time. Lash, a PhD candidate in the department of chemical engineering at the University of Michigan, Ann Arbor, came up with a distributed manufacturing plan that would use existing cell culture capacity to quickly ramp up production in the event of a pandemic. Lash and his advisor, Henry Wang, PhD, unveiled their plan last March at a meeting of the American Chemical Society in San Diego.
The plan calls for a smaller facility to constantly grow the cell line at a set level, anywhere from 10 liters to 100 liters. In the event of an outbreak, those seed cells can be shipped to existing bioreactors to grow them in greater volumes. “You would be able to produce the vaccine in a large amount, but also get the time savings as well,” Lash says. “Theoretically, it would be a lot more economical, because you’re only investing in a small facility to grow up the cells and then all the other facilities would join in. There are a lot of regulatory issues as well as business issues on how you would enable such a network like this. We’re trying to advocate it from the scientific standpoint as far as how it could be done, what tools need to be developed to enable it, how you transport the cells, how you ensure sterility.”
Lash says much of his research focuses on developing ways to grow cells while transporting them. Researchers typically use cryo-preservation, but a great deal of equipment is needed to freeze and thaw cells and the process also takes time. Cryopreservation can damage cells as well. Lash is also investigating ways to make sterility testing methods capable of handling larger samples and methods to determine whether cells will grow properly after being transported. In the event of a pandemic, cells would be distributed to existing biopharmaceutical production facilities retrofitted to produce vaccine. When presenting their work at the meeting in San Diego, Wang said there are about a dozen FDA-approved facilities in the world that use mammalian cell culture to make pharmaceutical products on a large scale.
While it is promising, Lash acknowledges there are challenges inherent in his plan. For example, when a biotechnology or biopharmaceutical product such as a vaccine is approved, it is just for one site or a few sites. Having a system that would allow for the use of any available bioreactor would require stringent regulations to ensure that it is done properly according to a predetermined protocol.
“There is a certain amount of science that needs to show that this is similar, even if you produce it at this bioreactor versus that bioreactor or using this equipment versus that equipment,” Lash says. Perhaps an even more significant hurdle is convincing companies to hand over their bioreactors for such a task, but that may be feasible considering the seriousness of a pandemic.
Other researchers are taking a different approach. PowderMed Ltd., Oxford, UK, developed a DNA-based avian flu vaccine that it says could be produced quickly and in large quantities. The vaccine is not expected to enter clinical trials until the middle of this year, but the company believes it has an advantage because very small amounts of the vaccine are needed. Because only 2 µg per dose are required, roughly 1.2 kg of DNA would be enough to vaccinate the entire population of the United States twice, says John Beadle, MD, the company’s chief medical officer.
The vaccine is made by cloning the H5 gene from the H5N1 strain into PowderMed’s proprietary DNA vaccine backbone, the same backbone used for the company’s annual flu vaccine, which will enter phase IIb trials this year.
Under PowderMed’s approach, DNA is precipitated onto microscopic gold particles and propelled by pressurized helium at near-supersonic speeds into the epidermis (see diagram on page 20). The advantage of this approach, Beadle says, is that it delivers the DNA directly into cells. DNA approaches generated a great deal of excitement in the 1990s, but when research moved into nonhuman primates and humans, the immune response seen in smaller animals was not evident.
Beadle says that was because the DNA was being delivered into muscle, which does not have any immunological function. The epidermis, on the other hand, is rich in immunologically active antigen presenting cells (APCs); once the vaccine enters these cells, the DNA elutes off the gold, becomes transcriptionally active, and produces an encoded protein. When this protein is presented by the APCs to lymphocytes, it triggers a strong T cell-mediated immune response.
Filed Under: Drug Discovery