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Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site


Many viral surface glycoproteins and cell surface receptors are homo-oligomers1,2,3,4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5,6,7,8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

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Figure 1: Design strategy.
Figure 2: Complementing HA structural diversity with designed HSB variants.
Figure 3: Trimerization of HSB to match the HA trimer.
Figure 4: Diagnostic and therapeutic applications.

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This work was funded by DTRA grants HDTRA1-16-C-0029 and HDTRA1-11-1-0041 (D.B), Life Science Discovery Fund Grant # 9598385 (D.B.), NIH 5R01AI096184-05 (P.Y.) and the NIH/NIAID R21AI119258 (D.H.F.). E.M.S. was supported by a career development award by the NW regional center of excellence (NIAID). J.D.B. and K.A.H. were supported by NIH grant R01 GM102198, K.A.H. by an NRSA training grant (T32GM007270) and I.A.W. and S.M.B. by R56 AI117675 and the Skaggs Institute for Chemical Biology at TSRI. A.J.B. was supported by a grant from the National Science Foundation (DGE-1256082). We would like to thank Erika O. Saphire for the generous gift of Ebola GP. We thank L. Carter and her team for help with protein purifications, Y. Song for help with comparative modeling and K. Godin for help with the selections and library generation. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

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Authors and Affiliations



E.-M.S., D.L. and W.S. designed proteins; D.L. designed the first version of the HSB protein, W.S. set-up the design pipeline for oligomerization, E.-M.S. designed the trimerization of the binders. E.-M.S. characterized designs, performed re-design of HSB, generated HA sequence alignments, homology models, library designs and selections; E.-M.S. and J.W.N. performed selection at different temperature and analyzed the deep-sequencing data. E.-M.S. and R.R. screened variants for higher expression and solubility. S.M.B. and E.-M.S. performed affinity measurements via biolayer interferometry. N.K.G. collected SAXS and HDX data, and analyzed it with K.K.L.; P.S.L. and S.M.B. expressed, purified and biotinylated HA proteins, P.S.L. crystallized HSB.2, S.M.B. crystallized the structure of the complex of HSB.2A with HA, P.S.L., S.M.B. and I.A.W. analyzed the crystallographic data. T.N. characterized the designed materials by electron microscopy; T.N. and A.B.W. analyzed electron microscopy data. K.A.H. and J.D.B. planned and performed the cell-based neutralization assay; C.E.A., C.A.H. and P.Y. designed and executed the paper-based diagnostic assays. A.J.B. and D.H.F. planned and performed the animal studies. E.-M.S. and D.B. analyzed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to David Baker.

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Competing interests

The University of Washington has filed patents on the binding proteins.

Integrated supplementary information

Supplementary Figure 1 Mimicry of sialic acid binding by broadly neutralizing antibodies.

Various antibody loops interact with the RBS using an aspartate to mimic the carboxyl of sialic acid RBS- targeted antibodies.

Supplementary Figure 2 Glycosylation sites in vicinity of Tri-HSB.

Model of Tri-HSB bound to trimeric HA. Surface representation for one of the 3 HA monomers is shown along with common glycosylation sites (positions 21, 22, 33, 38, 63, 65, 81, 95, 123, 126, 129, 130, 162/3, 165, 246 – as found in H1, H2, and H3 strains and here numbered after the H3 numbering scheme) colored in dark blue. Commonly glycosylated sites close to the binding domain are labeled. None of the sites clash with the trimerization domain. Position 133 shows the highest potential for interference with HA binding, however, the design binds tightly to the glycosylated Victoria 2011 strain.

Supplementary Figure 3 Screening of random mutagenesis libraries of HSB

a) Sort 2 against 1000 nM H2 Ada HA (left) and 500 nM H3 HK68 HA (right) under avid conditions (by adding SAPE in a 1:4 concentration to enable tetramer formation through association of the biotinylated HA with tetrameric SAPE, see Methods). No variants were identified 1000 nM H1 SI HA. (b) Consensus sequences after a non-avid sort 3 for either 500 nM H2 Ada (green shaded) or 500 nM H3 HK68 (blue shaded). (c) Titration of an isolate selected for binding to H2 Ada HA. (d) Substitutions (orange) identified to enable binding to H2 Ada HA (green: H2 Ada, blue: H3 HK68, grey: HSB).

Supplementary Figure 4 Selections of the combinatorial library for binding to different HA strains

Selections for H1 SI and NewCal HAs and alternating selections for of the combinatorial library for binding to H2 Ada or H3 HK68 and H3 Vic11 HA molecules were performed using a BD Influx. Sort 1 was performed under avid conditions (by adding SAPE in a 1:4 concentration to enable tetramer formation through association of the biotinylated HA with tetrameric SAPE). All subsequent sorts were performed as described under non-avid incubation conditions (Supp. Methods) using indicated concentrations.

Supplementary Figure 5 Electrostatic surfaces of the head region of HA variants

Views are from the side with the RBS groove top middle (top views) and looking down into the RBS (bottom views). Poisson-Boltzmann distribution calculated using APBS28 using a dielectric constant of 4.0 and contoured at -3.0 (red) to + 3.0 (blue) kT/e. The same structures and models as described in main text were used.

Supplementary Figure 6 Selection at increasing temperatures to identify stabilizing mutations

(a) FSC-compensated FITC fluorescence histogram was used to gate on displaying yeast cells. Histogram of compensated PE shows difference in fluorescence at indicated temperatures using 5 nM H1 SI HA; the top 1% of the PE positive population was selected for each sort. Legend contains the percentage of displaying population of the pool after incubation at indicated temperature (Methods). The mean in the PE signal of the displaying population decreases with an increase of the temperature. (b) Heat map of substitutions that increase or decrease in their frequencies compared to the lowest temperature sort: Frequencies of each gene at each temperature were computed based on counting using deep sequencing. Values used for this heatmap reflect the slope after linear regression of each substitution at temperatures between 22°, 30°, 37° and 42°C.

Supplementary Figure 7 Comparison of crystal structures and design

(a) Single chains of the HSB.2 dimer crystal structure are colored dark green and wheat colored. Helix α1 and strand β2 interact with β-strands 2-4 of another protomer to form a domain-swapped dimer. This type of domain swapping is observed in crystal structures of several other cystatins. Trp78 is shown to indicate the interface with sialic acid binding site. (b) Superposition of bound (orange) HSB.2A and the original designed HSB (grey). To compare to the unbound structure, we superpositioned a protomer unit of the swapped dimer (dark green).

Supplementary Figure 8 Biolayer interferometry trace showing extended association time and saturation of Tri-HSB.2 binding to H3 HK68

Concentrations used: 120, 40, 13.3, 4.4, 1.5, 0.5 nM)

Supplementary Figure 9 Electron microscopy of HSB trimer variants

(a) Reference free 2D class averages of HSB.1C trimer bound to HK68 HA via the original Gly-Ser linker. (b) Fourier shell correlation (FSC) of HSB.1C trimer bound to HK68 HA 3D reconstruction. At 0.5 FSC cutoff, the resolution is ~20 Å. (c) Reference free 2D class averages of HSB.1C trimer (with an additional glycine residue within the linker region) bound to HK68 HA. (d) Reference free 2D class averages of HSB.1C trimer bound to HK68 HA with a 4 gly-ser linker. (E) Reference free 2D class averages of HSB.2A trimer bound to HK68 HA. All complexes are similar, with the HSB trimer designs appearing as round densities positioned at the apex of the HA trimer.

Supplementary Figure 10 SAXS profile of the trimeric head binder Tri-HSB.2 bound to H3N2 HA ectodomain

(A) Scattering profile of HA bound to Tri-HSB.2 (black circles) plotted as the log scattering intensity as a function of the scattering vector (q). 50 all atom modeling ensembles of either 3 head binder lobes (cyan), 2 head binder lobes (red) or 1 head binder lobes (yellow) bound to the glycosylated HA ectodomain provided very similar theoretical fits to the experimental data with a χ of 1.30, 1.20 and 1.26, respectively. (B) Structures of the single best fit models of 3 head binder lobes (left), 2 head binder lobes (center) or 1 head binder lobes (right) bound to glycosylated HA. Head binder trimerization domain is in dark green, flexible Gly-linkers are red and binding lobes are light green, glycans are yellow, and HA ectodomain is grey.

Supplementary Figure 11 HDX-MS profile of H3 HK68 BHA bound to Tri-HSB.2

Modeled structure of BHA 3HMG.pdb bound to a model of Tri-HSB.2 in green. Colors on BHA correspond to percent exchange difference between unliganded BHA and bound BHA. Regions more protected from exchange when bound to head binder are shown as a blue gradient while regions more flexible when bound to head binder are shown as a red gradient. Individual BHA peptide exchange plots when unliganded (open circles) versus bound (open triangles) are presented.

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Supplementary Figures 1–11 and Supplementary Tables 1–6 (PDF 1585 kb)

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Strauch, EM., Bernard, S., La, D. et al. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site. Nat Biotechnol 35, 667–671 (2017).

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