Head interface antibodies bind H5 hemagglutinins

We produced a recombinantly expressed panel of divergent soluble HA ectodomains (rHA) that samples H5 spatial, temporal, and genetic diversity (n = 14) alongside human H1 and H3 rHA controls. We also produced a panel of 12 recombinantly expressed monoclonal human head interface antibodies. These include four from the germline biased, broadly binding, IG_kV1-39*01 class [9–12] that were isolated from three donors: subjects 1 and 5 (S1 & S5) [9,10] and Donor 1 (D1) [13]. We also included eight antibodies of diverse gene utilization from subjects 1, 5, and 8 (S1, S5, and S8) [9,10], and donor EI-13 [14]. We evaluated binding by enzyme-linked immunosorbent assay (ELISA) and included broadly binding stem antibody FI6v3 [18] and SARS-CoV-1 antibody CR3022 [19] as positive and negative binding controls, respectively (Fig 1C and S2A Fig). Ten of twelve bound at least one H5 rHA, with eight binding to the majority of rHAs. Antibodies from the IG_kV1-39*01 class bound the most H5 rHAs. The pattern of H1 and H3 rHA binding matched previous reports [9,10]. The two antibodies that did not bind to an H5 rHA were reported to have limited-to-no breadth of binding to group 1 HAs [9,10].

Most head interface antibodies failed to bind a common subset of rHAs. These are descendants of the GsGd lineage that were isolated after 1997. All of these rHAs are from human infections, except A/dairy cow/Texas/24-008749-001/2024(H5N1) (similar H5N1 viruses from dairy cattle have infected humans). These HAs share a mutation at an otherwise conserved position, P221S. Among the tested antibodies, only S8V2-18 [9,10] bound A/Viet Nam/1203/2004(H5N1) rHA, while S1V2-37 weakly bound A/Egypt/N03072/2010(H5N1) rHA, and S1V2-58 [9,10] weakly bound A/dairy cow/Texas/24-008749-001/2024(H5N1) rHA. Notably, most of the head interface antibodies bound A/Cambodia/i0125001G/2024(H5N1) rHA, which has a proline at position 221.

The P221S mutation disrupts head interface antibody binding

We introduced S221 into A/Hong Kong/156/1997(H5N1) (H5-HK-97) and P221 into A/Viet Nam/1203/2004(H5N1) (H5-VN-04) rHAs to determine the impact of P221S on head interface antibody binding. Most antibodies in our panel bound H5-HK-97 rHA (except S8V2-37 [9,10], S1V2-83 [9,10] and S1V2-51 9,10) and only S8V2-18 [9,10] bound H5-VN-04 rHA (Fig 1D and S2B Fig). The H5-HK-97-P221S mutant was not bound by any head interface antibody, while six of the 12 head interface antibodies bound the H5-VN-04-S221P mutant. This mutation did not disrupt S8V2-18 [9,10] binding. For these H5 rHAs, the presence of a serine or proline at position 221 is a strong determinant of head interface antibody binding. Introduction of the P221S mutation in A/California/04/2009(H1N1) HA also diminished or abrogated the binding of many of these antibodies (S3 Fig).

Origin of H5 S221 viruses

To determine the origins of the S221 H5s, we surveyed all reported full-length H5NX HA sequences in GISAID [20]. We established that S221 is prevalent in the GsGd lineage (98.2% of sequenced isolates) (S4 Fig). In contrast, P221 is found in 99.4% of American and 98.9% of non-GsGd Eurasian lineage viruses. Phylogenetics and sequence metadata indicate that GsGd viruses acquired the P221S mutation by the year 2000 in an avian reservoir (Figs 2, S5 Fig, and S1 Table). In the year 2000, these viruses were widely distributed among ducks and geese in Eastern and Southeastern China (Hong Kong, Guangdong, Shanghai, Huadong, and Zhejiang) (Fig 2). Descendants of these viruses have largely retained S221 and are responsible for the current global panzootic.

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Fig 2. Phylogenetic analysis of H5NX isolates from 1995-2000 and HAs used in this study.

Sequences containing P221 are colored black. Sequences containing S221 are colored red. Stars indicate HAs used in ELISAs: black star indicates a sequence between 1995-2000, and open star indicates a sequence outside 1995-2000. Red and black circle marks the last common ancestor of P221 and S221 Goose Guangdong viruses. Open gray circle marks node from which post-2000 Goose Guangdong lineage members descend. A labeled tree is in S4 Fig.

https://doi.org/10.1371/journal.ppat.1014005.g002

Cells

293F cells were maintained at 37°C with 8% CO₂ in FreeStyle 293 Expression Medium (ThermoFisher) supplemented with penicillin/streptomycin (ThermoFisher).

Recombinant HA and IgG Expression and Purification

Sequences encoding HA ectodomains (FLsE) were subcloned into a modified pVRC8400 vector encoding a T4 fibritin (foldon) trimerization tag and a 6xHis tag [21,22]. The A/dairy cow/Texas/24-008749-001/2024(H5N1) rHA was further modified with stabilizing mutations (HA0: H355W, K380I and E432I) [23]. When present, polybasic sites were not modified. Antibody heavy and light chain variable domains were cloned into modified pVRC8400 expression vectors to produce full IgG1 heavy chains and light chains [21,22]. HA and antibodies were expressed by polyethylenimine (PEI) facilitated, transient transfection of 293F cells. Transfection complexes were prepared in Opti-MEM (Gibco) and added to cells. 5 days post transfection, cells were pelleted, supernatants were harvested. rHA supernatants were incubated overnight with Talon Metal Affinity Resin (Takara) at 4°C. Resin was collected in a chromatography column, washed with 10mM Tris, 150mM NaCl pH 7.5, eluted using 350mM imidazole in 10mM Tris-HCl, 150 mM NaCl pH 7.5, concentrated using a 10kDa centrifugal filter (Millipore), then purified via size exclusion chromatography on a Superdex 200 (GE Healthcare). IgG1 supernatants were incubated overnight with Protein A Agarose Resin (GoldBio) at 4°C. Resin was collected in a chromatography column and washed with 10mM Tris, 150 mM NaCl pH 7.5. mAbs were eluted in 0.1M glycine pH 2.5 which was immediately neutralized by 1M Tris (pH 8.5), concentrated in 10kDa centrifugal filter (Millipore), and dialyzed in PBS pH 7.4.

Recombinant HA ELISA

ELISAs were performed as previously described [21,22]. 500 ng of rHA FLsE were adhered to wells of a high binding 96-well plate (Corning) in PBS pH 7.4 overnight at 4°C. Plates were washed with PBS with 0.05% TWEEN-20 (PBS-T) and blocked at room temperature for 1 hour with PBS-T with 2% BSA. Blocking solution was removed, and serially 5-fold diluted mAbs in PBS-T 2% BSA were added to plates in triplicate. A positive binding control, mAb FI6v3 [18], was present on each plate. Plates were incubated for 1 hour at room temperature, then washed 3 times with PBS-T. Anti-human HRP IgG (abcam) diluted 1:10,000 in PBST 2% BSA was added to wells and incubated for 30 minutes at room temperature. Plates were washed 3 times with PBS-T, developed with 1-Step ABTS (ThermoFisher), and stopped by 1% SDS solution. Absorbances at 405nm were measured on a Molecular Devices SpectraMax 340PC384 Microplate Reader. Background subtracted data were normalized to the FI6v3 [18] standard curve present on each ELISA plate per rHA. Equilibrium dissociation constant K_d values were calculated using a nonlinear fit (One site binding, hyperbola) and graphed using GraphPad PRISM v10.

Phylogenetics

H5 phylogenetic tree was generated by first downloading all available, full length H5 HA sequences from GISAID [20]. Sequences were aligned using MAFFT [24]. Downselected alignments were produced for samples collected from January 1^st 1995 through December 31^st 2000 or using CD-HIT [25] (clustered at 95%, retaining one representative sequence per cluster). HA sequences for isolates characterized in this study were added to these alignments. Phylogenetic trees were produced using MrBayes [26] and images made in FigTree [27].

All genome sequences and associated metadata in this dataset are published in GISAID’s EpiFlu database. To view the contributors of each sequence with details such as accession number, Virus name, Collection date, Originating Lab, Submitting Lab and the list of Authors, visit EPI_SET_250807vm DOI: https://doi.org/10.55876/gis8.250807vm.