# Resistance Mutations to Broadly Neutralizing Antibodies Destabilize Hemagglutinin and Attenuate H1N1 Influenza Virus

## TL;DR

This study shows that mutations in influenza virus hemagglutinin reduce antibody resistance but also weaken the virus, making it less harmful.

## Contribution

The study reveals how resistance mutations in hemagglutinin destabilize the virus and attenuate H1N1 influenza.

## Key findings

- A44V and A44T mutations in HA2 reduce HA and virus stability and bNAb binding in vitro.
- The mutations decrease pathogenicity and lung replication in mice and reduce virus titers in ferrets.
- Restabilizing mutations can restore virus growth and transmission after A44V.

## Abstract

Because antigenic drift primarily generates amino-acid changes in the membrane-distal hemagglutinin (HA) head, broadly neutralizing antibodies (bNAbs) are being developed to target conserved epitopes in the membrane-proximal stem. Mutations to HA2 residue A44, a buried residue beneath the central stem epitope, in 2009 H1N1 viruses have been shown to cause resistance to stem-binding bNAbs. Here, we introduced A44V and A44T mutations into A/Tennessee/1-560/2009 (TN09) and A/Puerto Rico/15/2018 (PR18) and investigated their effects in cell culture, mice, and ferrets. In both virus strains, the mutations decreased HA and virus stability and decreased bNAb binding and neutralization in vitro. The mutations reduced pathogenicity and lung replication in DBA/2J mice. Ferrets were inoculated with PR18 wild-type (WT) or A44V virus, and the A44V mutation reduced day-1 and peak nasal virus titers. Airborne transmission in the A44V group occurred only after genotypic reversion (HA2-V44A) or acquisition of a distal re-stabilizing mutation (HA2-I77M). Compared to WT, an engineered PR18 virus containing both HA2 mutations (A44V and I77M) had similar growth and pathogenicity in mice in addition to decreased binding and neutralization by bNAbs. Overall, this work provides insight into the role of HA stability during HA stem-epitope remodeling that results in virus resistance to stem-binding bNAbs.