The finding, published in the journal Science Express on July 7, 2011, shows the influenza subtypes neutralized with the new antibody include H3N2, strains of which killed an estimated one million people in Asia in the late 1960s.

"Together this antibody and the one we reported in 2009 have the potential to protect people against most influenza viruses," said Ian Wilson, who is the Hansen Professor of Structural Biology and a member of the Skaggs Institute for Chemical Biology at Scripps Research, as well as senior author of the new paper with Crucell's chief scientific officer Jaap Goudsmit.

Tackling a Major Shortcoming

Wilson's laboratory has been working with Crucell scientists since 2008 to help them overcome the major shortcoming of current influenza vaccines: They work only against the narrow set of flu strains that the vaccine makers predict will dominate in a given year, so their effectiveness is temporary. In addition, current influenza vaccines provide little or no protection against unforeseen strains.

These shortcomings reflect a basic flu-virus defense mechanism. The viruses come packaged in spherical or filamentous envelopes that are studded with mushroom-shaped hemagglutinin (HA) proteins, whose more accessible outer structures effectively serve as decoys for a normal antibody response. "The outer loops on the HA head seem to draw most of the antibodies, but in a given strain these loops can mutate to evade an antibody response within months," said Wilson. Antiviral drugs aimed at these and other viral targets also lose effectiveness as flu virus populations evolve.

"The major goal of this research has been to find and attack relatively unvarying and functionally important structures on flu viruses," said Damian Ekiert, a graduate student in the Scripps Research Kellogg School of Science and Technology who is working in the Wilson laboratory. Ekiert and Crucell's Vice President for Antibody Discovery Robert H. E. Friesen are co-first authors of the Science Express report.

By sifting through the blood of people who had been immunized with flu vaccines, Goudsmit and his colleagues several years ago discovered an antibody that bound to one such vulnerable structure. In mice, an injection of the antibody, CR6261, could prevent or cure an otherwise-lethal infection by about half of flu viruses, including H1 viruses such as H1N1, strains of which caused deadly global pandemics in 1918 and 2009.

The Crucell researchers approached Wilson, whose structural biology lab has world-class expertise at characterizing antibodies and their viral targets. Ekiert, Wilson, and their colleagues soon determined the three-dimensional molecular structure of CR6261 and its binding site on HA, as they reported in Science in 2009. That binding site, or "epitope," turned out to be on HA's lower, less-accessible stalk portion. The binding of CR6261 to that region apparently interferes with flu viruses' ability to deliver their genetic material into host cells and start a new infection. That antibody is about to begin tests in human volunteers.

The Missing Piece

Crucell researchers subsequently searched for an antibody that could neutralize some or all of the remaining flu viruses unaffected by CR6261, and recently found one, CR8020, that fits this description. As the team now reports in the Science Express paper, CR8020 powerfully neutralizes a range of human-affecting flu viruses in lab-dish tests and in mice. The affected viruses include H3 and H7, two subtypes of great concern for human health that have already caused a pandemic (H3) or sporadic human infections (H7).

As with the CR6261 project, Ekiert and colleagues were able to grow crystals of the new antibody bound to an HA protein from a deadly strain of H3N2, and to use X-ray crystallography techniques to determine the antibody's structure and its precise epitope on the viral HA protein.

"It's even lower on the HA stalk than the CR6261 epitope; in fact it's closer to the viral envelope than any other influenza antibody epitope we've ever seen," said Ekiert.

Crucell is about to begin initial clinical trials of CR6261 in human volunteers, and the company expects eventually to begin similar trials of CR8020. If those trials succeed, aside from a vaccine the two antibodies could be combined and used in a "passive immunotherapy" approach. "This would mainly be useful as a fast-acting therapy against epidemic or pandemic influenza viruses," said Wilson. "The ultimate goal is an active vaccine that elicits a robust, long-term antibody response against those vulnerable epitopes; but developing that is going to be a challenging task."

The research was supported by the US National Institute of Allergy and Infectious Diseases, National Institutes of Health; the US Department of Energy; and by Crucell Holland BV.