Mutations in Influenza A Virus Neuraminidase and Hemagglutinin Confer Resistance against a Broadly Neutralizing Hemagglutinin Stem Antibody

Abstract

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure. IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function. FIG 1 Experimental design and viral amplification for passaging with the broadly neutralizing antibody F10. (A) Schematic of experiment 1 and experiment 2 trajectories. Cyan boxes indicate virus that was passaged in the absence of antibody, with the top three passages as P1, P2, and P3 and additional passages as labeled. Red and orange boxes indicate virus that was passaged in the presence of F10 broadly neutralizing antibody (experiments 1 and 2, respectively). Gray boxes indicate virus that was passaged in the presence of 80R control antibody (experiment 2). (B) Ratios of viral titers ...

High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Viruses in vitro

“…Tile scans and z-stacks of the tissue at 20x and 40x magnification were acquired. and titered by viral plaque assay using MDCK cells as previously reported [29]. Viral infection experiments using PREDICT96-ALI tissue used A/WSN/33 H1N1 at passage 1, A/California/04/09 H1N1 at passage 3, and A/Hong Kong/8/68 H3N2 at passage 2.…”

Structure-Based Modification of an Anti-neuraminidase Human Antibody Restores Protection Efficacy against the Drifted Influenza Virus

“…The relatively conserved stem region is probably a better target than the head domain, as it has a much higher adaptive evolution rate. Moreover, some escape mutations on HA can indirectly modulate antibody binding but are not located at the antibody epitope (20). In this case, modification of a single amino acid to restore the effectivity of the antibody to mutant HA may be a challenge.…”

Generation of a protective murine monoclonal antibody against the stem of influenza hemagglutinins from group 1 viruses and identification of resistance mutations against it

“…The major resistance mutations often directly reduce antibody binding affinity with the epitopes [8,9,11,14,47,55], while others enhance fusion ability of HA [47,56]. Recently, Prachanronarong and co-workers reported non-epitope mutations in HA that confer resistance to the stem MAb F10 through the changes of receptor binding, membrane fusion or budding, as well as mutations in NA that allowed virus growth in the presence of the neutralizing antibody [56]. The mutation N460S (N446S, H3 HA numbering, without signal peptide) in their report and our V444L are two residues apart, but both mutations appear to alter the fusion peptide.…”