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SRI Scientists Develop HTS Assay, Search for Bird Flu Vaccine Takes Flight

Currently, no vaccine exists to protect humans against the highly pathogenic avian influenza H5N1 virus that is spreading among birds across Europe, Asia, and Africa.
William Severson, a research virologist specializing in emerging and infectious diseases, recently won a one year, $25,000 R03 grant from the NIH to access the Molecular Libraries Screening Center Network’ HTS resources to identify new molecular probes for the inhibition of the H5N1 virus.
Severson and his team at the Southern Research Institute in Birmingham, Ala., have developed and validated a 384-well cell-based assay that measures cytopathic effects that an avian influenza virus infection has on Madin Darby canine kidney cells. They did this by using a luminescence-based detection system for signal endpoint.  
The investigators will assist the MLSCN in transferring the validated HTS assay to the appropriate screening center and provide technical support for follow-up assays on single-dose hits from the primary assay in an HTS dose-response format.
Severson talked with CBA News this week about the project.
Can you give me some background on this project?
This is an influenza assay that is being funded by the NIH through their Molecular Library Screening program, which is part of the NIH Roadmap.
I joined SRI about 18 months ago, in the drug-discovery section. I am collaborating with other researchers here on the development of HTS assays for biosafety level 3 conditions. This was one of the first assays to come up.
We adapted it from a SARS assay where we screened 100,000 compounds in the BSL 3 facility as part of a government contract. Colleen Jonsson is the PI of that contract, and she introduced me to another researcher here, Jim Noah. Jim’s wife, Diana, is also a researcher at SRI. She is an influenza biology specialist, so she propagates the virus.
We have a series of eight plasmids that are transfected into Madin Darby canine kidney cells. We use a reverse genetics system to actually transfect these plasmids into MDCK cells and amplify the virus.
We then re-infect these cells with this amplified virus, choose a single plaque from that infection, and use virus isolated from that plaque to infect chicken eggs.
We then purify the virus from the allantoic fluid of chicken embryos. Since the avian bird flu is a select agent, we have to be select-agent cleared. This means the select agent is regulated by the CDC, and it takes two people to access the agent: the person with the key to the freezer where the agent is kept, and the person with the combination to the padlock on the freezer.
The fact that avian influenza is a federally regulated agent makes this a very difficult assay to run. So we initially set up this assay under BSL-2 conditions using a regular strain [H3N2] of influenza. Jim Noah was the first author on that paper, which was published earlier this year in Antiviral Research. We have already used that assay to screen 1.3 million compounds, looking for new inhibitors of the influenza virus.
I later took the assay and brought it into the BSL-3 lab, and we wrote to the MLSCN and got funding through them.
I got this grant awarded at the end of April, we started to screen in May, and we have now screened 95,000 compounds in about four weeks with the BSL-3 assay. That data has been uploaded to PubChem, and we have our chemists looking at the structures to perform structure/activity relationship analysis on the compound.
They will choose compounds that they feel are novel scaffolds and could potentially be a hit. Then we will do follow-up dose response and toxicity studies using the same HTS assay.
What is the set-up of this assay? How is it run?
It’s a three-day assay. We seed about 6,000 MDCK cells per well into a 384-well plate. We incubate them overnight for about 12 hours. The next day our HTS center adds the test compounds to the plate by automation. The plates are then taken down to the BSL-3 lab, where I go in and infect the cells with the H5N1 virus.
We then incubate them at 37° Celsius for 72 hours. We then do an endpoint to determine if the compound is effective against the virus.
On our plates, we have ribavirin as a control. We have been asked why we did not use Tamiflu. We validated the assay with ribavirin because it was cheaper and easier to obtain compared to Tamiflu. It also has good IC50 and EC50 values.
For our endpoint, we use the Promega CellTiter-Glo platform. We read these plates using an Invision plate reader. The dynamic range is very good for this assay. The signal-to-background ratio is something like 20 to 1, and the signal-to-noise ratio is very high, too.
What is the next step in this project?
The next step is looking at targeted approaches that previous approaches have not yet looked at. We have another researcher on staff here developing those assays, and he’s got an NIH grant to do that.
This is a whole-cell based assay that will look at all different sorts of targets within the cell. We plan to follow up with dose-response and toxicity assays, and we have also developed a microplaque assay, which is in a 96-well format.
We can determine if the compounds are protecting the virus with this plaque reduction assay. We also have the same controls on these plates, including ribavirin, Tamiflu, and rimantadine, I believe. We also intend to do time-of-addition experiments to determine where the compounds affect the virus during its life cycle.
After the primary screen, we will run the dose response and toxicity assays. We had contacted the project officer to see if we couldn’t get some more supporting funds to run the plaque assays also. 
What will the grant money actually be used for?
This funding is basically for more assay development. We want to do targeted assays, so some of the money will be used for those. We would also like to fund some other things.
As a team, we are working on respiratory viruses and emerging pathogens. We are using the BSL-3 facility as our strength. 
We are looking at a number of respiratory viruses, including the respiratory syncytial virus, influenza, hantaviruses, and the human metapneumovirus.
These assays were developed as tools to screen our relatively diverse proprietary library for potential inhibitors.
Is there potential for these assays to be commercialized? 
We did have a service contract for a commercial client last year, and we screened about one million compounds for them using this assay.
If the contracts are very large, such as the one we had last year, then we would be very interested in that.
Is this where you see antiviral research going? Is the avian flu still a major concern?
You have to remember, everyone has kind of forgotten about SARS. But what if that re-emerges? There are no drugs for that on the market. Other viruses, such as hantaviruses, are remerging in places like the Ukraine and South America.
So I don’t necessarily see avian flu viruses as the wave of the future, but it is a cause for concern. We do not have any drugs for avian flu — what if it crosses the species barrier? What if the virus develops a resistance to drugs such as Tamiflu?
I think there is always room for continued research. In addition, the NIH is short on funding now. Everyone is scrambling trying to figure out a way to get money into their organizations.
Do you think that this assay is adaptable to viruses other than the avian flu?
This assay is very adaptable to things such as the West Nile virus, the respiratory syncytial virus, and the human metapneumovirus.
It is not a novel assay, in that everything has been done before with a lot of CPE-based assays, but we are using it in the BSL-3 capacity. That is what makes it unique to SRI.

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