With the avian flu threatening to break out into a pandemic, and influenza and SARS continuing to threaten parts of the human population, a small number researchers are now using proteomic technologies to try to develop vaccines, therapeutics, and diagnostics for viral diseases.
The researchers are taking a variety of approaches: some are using SELDI and MALDI techniques to study the differences in the proteomes of human samples before and after vaccination. Others are using major histocompatibility-complex chips to study the immune response of organisms to viruses. Still others are using high-throughput methods to determine the 3D structure and function of every protein produced by a virus.
"Starting now is the time to do any kind of a comprehensive proteomic analysis," said Richard Drake, an associate professor in the department of microbiology and cell biology at Eastern Virginia Medical School who is studying influenza using SELDI and MALDI techniques. "Each year the flu vaccine is only 70-percent effective for the general population, and 40- to 45-percent effective for the elderly population. Layering that over the avian flu, the clock is ticking. This is the time to do [the studies], not when it's showing up in the clinics."
One of the greatest public health concerns of the moment is the avian flu virus. Since 2003, the H5N1 strain of avian influenza has killed more than 60 people and tens of millions of birds, mostly in Asia. Though the virus currently spreads only from birds to birds, and birds to humans, there is concern that it may mutate into a form that can be passed directly from human to human.
"Starting now is the time to do any kind of a comprehensive proteomic analysis … not when it's showing up in the clinics."
Drake and his colleagues are not currently studying the avian flu virus, but their work on the human influenza virus could be applicable to an avian flu pandemic if it does break out in the future, Drake said.
Drake's research team is about to begin a clinical study to analyze the serum and plasma proteomes of healthy adults and healthy elderly people before and after they are given an influenza vaccine. The work follows an earlier, preliminary study on the effect of the nasally administered FluMist vaccine on the proteomes of serum and nasal swab samples.
Beginning in November, Drake's team will collect serum and plasma samples from 50 healthy adults aged 21 to 40, and 100 healthy elderly people who are over age 55. Samples will be collected from the subjects before they are administered a flu vaccine, and also four days, seven days, 14 days and 28 days after they have been vaccinated.
By comparing the proteomic responses from people who respond to the vaccine and people who don't respond to the vaccine, Drake hopes to discover immune cell factors that are present in responders and missing in non-responders, or vice-versa. That information could potentially be used to develop adjuvants that could be added to the current vaccine to make it more effective.
In addition, Drake hopes that his research could lead to the development of an early test that could accurately determine if a person has been infected with the influenza virus.
"It could be a very rapid, dipstick-type assay that would help you figure out who has been exposed to the virus and who hasn't," said Drake.
Lawrence Stern, a professor of pathology at the University of Massachusetts Medical School, is using MHC arrays to study people's T-cell responses after they have been exposed to an infectious agent such as a virus, bacteria, or microbe.
"What we basically do is take MHC molecules loaded with different peptides, along with anti-cytokine capture antibodies, and array them onto a chip," said Christian Walker, a graduate student who works with Stern. "By putting samples onto the MHC arrays, we can look at the T-cell repertoire that a person who has been exposed to a virus or a vaccine exhibits."
The advantage of using a chip is that hundreds to thousands of different peptides can be tested in a high-throughput manner in a single experiment, thereby reducing the required sample size. By seeing whether T-cells bind to MHC loaded peptides, and whether they become activated, researchers hope to discover new epitopes that can be the basis for new viral vaccines, or novel protein therapeutics.
According to Walker, Stern's research group is currently studying smallpox, yellow fever, dengue, and malaria using funding from the National Institute of Allergy and Infectious Diseases. Researchers at UMMS are also beginning to work with influenza, Walker said.
A third proteomic approach to studying viral disease is being used by Peter Kuhn, a professor of cell biology at the Scripps Research Institute. Kuhn's research group is actively involved in the Protein Structure Initiative, a $600 million project to determine the three-dimensional shapes of every unique protein found in organisms ranging from bacteria to humans (see ProteoMonitor 2/18/2005). About a year and three months ago, Kuhn's team started employing the same high-throughput techniques being used in the initiative to determine the 3D structure and function of every protein produced by the SARS virus.
"We have the structures of about a third of the 28 SARS virus proteins, and the functions of about a third of them, though not necessarily the same third," said Kuhn. "It's an ongoing process. What we are now doing is making knockouts to test the functions, and we're trying to establish if these proteins are potential targets of intervention."
Once potential "targets of intervention" have been identified, they can be used by pharmaceutical companies to develop therapeutics to viral diseases.
In the past, Kuhn and collaborator Raymond Stevens, also of the Scripps Institute, worked with neuroaminidase, the influenza virus protein that is the main target of Roche's influenza therapeutic Tamiflu. Now Kuhn and Stevens have teamed up with Michael Buchmeier, a coronavirus expert at Scripps, to try to do the same thing for SARS that they did for influenza.
"Our long-term research approach has been structure based," said Kuhn. "With SARS, there was very little known about the virus, so it was a straightforward decision to come in with a high-throughput technology approach to address the virus in a complete fashion."
Kuhn said that the same large-scale systematic effort could be applied to other viruses, such as the H5N1 avian flu virus, but that such an approach is not necessarily the right thing to do.
"I'm not an influenza person, so I wouldn't want to make the judgment call," said Kuhn. "I can tell you what we can do from a technology perspective, but with influenza, you need to first sit down and look at what's known and what's not known. Things need to happen quickly — the issue is critical, and there's no room to screw up."
Both Drake and Kuhn said that a major reason that few proteomics researchers are using their technologies to study viruses is that the research infrastructure does not make it easy to set up clinical studies for viral diseases.
"You've got to get a proteomics group with an immunology group with a clinical trial study group," said Drake. "Pulling people together is the rate-limiting step. A clinical study is difficult to do, no matter what the disease. There are a lot of cancer centers that already have that kind of infrastructure, but how many vaccine or viral centers can pull this together?"
Another big challenge for studying viral diseases is that experiments must be done under containment.
"Depending on what virus you're looking at, the experiments have to be done at minimum in a BSL 3 laboratory," said Drake. "Are you going to put a million-dollar mass spec in a BSL 4 laboratory where you have to work in space suits? That's not realistic."
Jeremy Carver, the CEO of the International Consortium for Anti-Virals (see Proteomics Pioneer), said that there are plans in Canada to set up contained centers with mass spectrometers and other equipment for proteomic research, but those centers will be rare because they are expensive to establish and maintain.
"You've got to be certified for the different viruses that you want to look at, so it's a long and tedious process," he said. "But there are places that are going to those kinds of lengths."
— Tien-Shun Lee ([email protected])