# Antigen flexibility supports the avidity of hemagglutinin-specific antibodies at low antigen densities

## TL;DR

This study shows how the flexibility and density of the influenza virus's HA protein affect how well antibodies can bind to it, which has implications for vaccine design.

## Contribution

The study reveals that HA flexibility and low-density configurations can still support strong antibody binding through bivalent interactions.

## Key findings

- Bivalent antibody binding remains effective even at HA densities as low as 10% of natural levels.
- HA flexibility allows for tilting, which enhances bivalent antibody attachment.
- Antibody occupancy increases at the lowest HA densities for certain antibodies like C05.

## Abstract

The receptor-binding protein of influenza A virus, hemagglutinin (HA), is the most abundant protein on the viral surface. While high densities of HA are thought to improve cellular attachment by increasing avidity for the viral receptor, they may also increase the avidity of neutralizing antibodies. The tradeoff between these two competing effects of avidity is not well understood. To better understand how features of the viral surface influence antibody avidity, we developed fluorescence-based assays to measure dissociation kinetics and steady-state binding of antibodies to intact virions. Focusing on two antibodies that bind to the HA head domain (S139/1 and C05), we confirm that binding orientations that favor bivalent attachment of antibodies to the viral surface can offset weak monovalent affinity by facilitating crosslinking. By modulating HA density in both engineered viruses and synthetic nanoparticles, we find that bivalent antibody binding remains resilient down to one-tenth the HA density on the viral surface and, in the case of C05, that antibody occupancy increases at these lowest densities. Finally, using a combination of structure-guided modeling and antibodies that lock HA in a tilted conformation, we identify flexibility of the HA ectodomain as an additional determinant of antibody avidity. Together, these results establish features of the viral surface that help support or suppress the binding of neutralizing antibodies.

Influenza viruses package high densities of the surface protein hemagglutinin (HA) into the viral membrane during assembly. This promotes viral attachment and entry into host cells by enabling multiple parallel interactions between viral HA and cell surface sialic acid. However, high densities of HA on the viral surface may also increase viral susceptibility to antibodies whose avidity is enhanced by the availability of multiple HA binding partners. We examined how the density of HA on the viral surface influences the binding of bivalent IgG antibodies that depend on engaging multiple HAs for robust attachment. Using fluorescence-based measurements on authentic and synthetic viruses, we find that reducing HA densities to 10% of their natural levels is not sufficient to disrupt antibody avidity. Additionally, we find that flexibility of the HA anchor may facilitate bivalent antibody binding by allowing HA to tilt relative to the membrane. These findings highlight how both the organization and dynamics of viral surface proteins can influence immune recognition and may inform strategies for antigen presentation in vaccine design.