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

Adrian Creanga,Tyler Stephens,Julie E Ledgerwood,Michelle C Crank,Brian E Fisher,Masaru Kanekiyo,Barney S Graham,Liam Hatch,Adrian B Mcdermott,John R Mascola,Christina Yap,Sarah F Andrews,Julia Lederhofer,Rebecca A Gillespie,Yaroslav Tsybovsky

doi:10.1038/s41467-021-21954-2

Abstract

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.

Highlights

Neutralizing antibodies have been developed as potential countermeasures for seasonal and pandemic influenza
Each segment consists of either one or two protein-coding sequences flanked by short noncoding regions (NCRs)
For individuals vaccinated with the H5 prime-boost regimen[38], we found that neutralization titers against the vaccine strain H5N1 A/Indonesia/05/2005 increased at 2 weeks post-boost from a baseline reciprocal IC80 GMT of 727.8 [range 206.6–1603.5] to a final reciprocal IC80 GMT of 33,014.2 [range 3235.7–223,905.4] (p < 0.0001)

Summary

Introduction

Neutralizing antibodies (bnAbs) have been developed as potential countermeasures for seasonal and pandemic influenza. 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. The discovery of broadly neutralizing antibodies (bnAbs) capable of neutralizing multiple influenza virus subtypes in humans opens an opportunity for developing a universal influenza vaccine, which elicits such antibodies[4,5,6] Many of these antibodies target conserved epitopes in the HA stem and neutralize virus by inhibiting the viral fusion machinery, the activity is not detectable by traditional hemagglutination inhibition (HAI) assay, which measures the ability of antibody to inhibit virus–receptor interaction. The reporter virus assay provides a more robust method for probing the breadth of anti-influenza immunity needed for developing universal influenza vaccines

Objectives

Methods

Results

Conclusion