A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies

Abstract

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.

Elicitation of broadly protective immunity to influenza by multivalent hemagglutinin nanoparticle vaccines

“…We demonstrated the apparent epitope and angle of approach of a common class of vaccine-elicited, cross-reactive, polyclonal NHP antibodies are remarkably similar to human multi-donor V H 6-1+D H 3-3-class antibodies 56,57 , the broadest and most potent stem-directed human bnAbs 51 . Precursors of this class of bnAb are in theory found in~99% of the human population 67 and can be amplified by heterologous stimuli such as pandemic influenza vaccines 57,68 .…”

“…All reporter viruses were prepared as described previously 51 . Briefly, all H1N1 and H3N2 viruses were made with a modified PB1 segment expressing the TdKatushka reporter gene (R3ΔPB1) and propagated in MDCK-SIAT-PB1 cells, while H5N1 reporter virus was made with a modified HA segment expressing the reporter (R3ΔHA) and produced in cells stably expressing H5 HA.…”

“…Immune sera or mAbs were serially diluted and incubated for 1 h at 37°C with pre-titrated viruses ( Supplementary Table 2 ). Serum-virus mixtures were then transferred to 96-well plates (PerkinElmer), and 1.0×10 4 MDCK-SIAT1-PB1 cells 51,92 were added into each well. After overnight incubation at 37°C, the number of fluorescent cells in each well was counted automatically using a Celigo image cytometer (Nexcelom Biosciences).…”

Using Neutralization Landscapes to enumerate Antibody Behavior and Decompose Antibody Mixtures

“…We utilized a panel of influenza viruses containing 15 H1N1 strains collected between 1933-2008 and 21 H3N2 strains collected from 1968-2016 ( Figure S2) (11). The concentration of each antibody needed to neutralize every virus by 50% (the half maximal inhibitory concentration, IC50) was determined for 6 HA head-binding and 12 HA stem-binding antibodies representing major lineages of broadly neutralizing antibodies elicited by vaccination (12,13).…”

“…Antibodies targeting the HA head or stem were used to map these two regions. (B) Neutralization maps were generated using neutralization data from monoclonal antibodies (gray) against a panel of viruses (hues of green/blue) (11). The axes show the units of the grid, and the distance d between each antibody-virus pair corresponds to neutralization, where positive distance d represents an IC50=10 -10+d M while 0 distance represents any IC50≤10 -10 M. A complete list of virus strains is given in Figure S2.…”