Broadly neutralizing antibodies (bnAbs) have been developed as potential countermeasures for seasonal and pandemic influenza. Deep characterization of these bnAbs and polyclonal sera provides pivotal understanding for influenza immunity and informs effective vaccine design. However, conventional virus neutralization assays require high-containment laboratories and are difficult to standardize and roboticize. Here, we build a panel of engineered influenza viruses carrying a reporter gene to replace an essential viral gene, and develop an assay using the panel for in-depth profiling of neutralizing antibodies. Replication of these viruses is restricted to cells expressing the missing viral gene, allowing it to be manipulated in a biosafety level 2 environment. We generate the neutralization profile of 24 bnAbs using a 55-virus panel encompassing the near-complete diversity of human H1N1 and H3N2, as well as pandemic subtype viruses. Our system offers in-depth profiling of influenza immunity, including the antibodies against the hemagglutinin stem, a major target of universal influenza vaccines.

A number of broadly neutralizing antibodies (bnAbs) to influenza virus have been isolated, characterized and developed as potential countermeasures for seasonal influenza epidemic and pandemic. Deep characterization of these bnAbs and polyclonal sera is critical to our understanding of influenza immunity and for desgining universal influenza vaccines. However, conventional influenza virus neutralization assays with live viruses require high-containment laboratories and are difficult to standardize and roboticize. Here, we built a panel of engineered influenza viruses carrying a fluorescent reporter gene to replace an essential viral gene. This restricts virus replication to cells expressing the missing viral gene in trans, allowing it to be manipulated in a biosafety level 2 environment. Using this system, we characterize the neutralization profile of a set of published and new bnAbs with a panel consisting of 55 viruses that spans the near complete antigenic evolution of human H1N1 and H3N2 viruses, as well as pandemic viruses such as H5N1 and H7N9. Our system opens opportunities to systematically characterize influenza immunity in greater depth, including the response directed at the viral hemagglutinin stem, a major target of universal influenza vaccines.

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