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

Abstract

Many viral surface glycoproteins and cell surface receptors are homo-oligomers, 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 sites. 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.

Figure 1. Design strategy

(a) Trimeric HA bound to receptor analog 6‧SLN (PDB entry 4wea). (b) Broadly neutralizing antibody loops interact with the receptor binding site either by inserting an aromatic or aliphatic residue into a pocket above the conserved Trp153, as for HCDR3 of C05 and many other RBS-targeted antibodies or by using an aspartate to mimic the carboxyl of sialic acid^, (Fig.S1). (c) Evaluation of 80 HA binder designs by yeast display followed by next-generation sequencing. The design pool was screened against H3 HK68 and two negative control targets (Ebola GP and E. coli intimin) to monitor off-target binding to HA. Enrichment values are the ratio of the frequency of a given design in FACS-selected populations for one of the three targets to the frequency in the original unselected population. A number of the designs specifically bind HA, but not Ebola GP or intimin. (d) Models of designs that specifically bind HA (#24 and HSB); the C05 derived loop is colored in purple.

Figure 2. Complementing HA structural diversity with designed HSB variants

(a) Sequence and structural diversity around the receptor binding site of HA (4wea) presents a challenge for binding of molecules larger than sialic acid (PDB entries 4fqr, 3qqi, 4o5n and homology models, see Supp. Info). (b) Design of HSB library: residues of HSB (purple spheres) that come in contact with HA (4fp8) were allowed to mutate to match the diversity of nearby segments of HA. HA is colored by Shannon Entropy over a set of ∼3700 H3 sequences (highly conserved in blue, less in red, Suppl. Info). (c) (left) Sequence alignment of HA-contacting residues for isolates identified in independent sorting trajectories against different flu strains (HSB.1A and B were selected for H1 A/Solomon Islands/3/2006 (SI), HSB.1C for H1 A/New Caledonia/20/1999 (NewCal), HSB.1D and E alternately against H3 HK68/Vic11 and H2 A/Adachi/2/1957 (Ada)). (right) mean of the apparent K_d (nM) from yeast surface titrations of two independent yeast cultures measured on different days. (d) Model of HSB.1A (grey cartoon) with H3 HK68 (colored by electrostatic potential, see Fig. S3) indicates electrostatic repulsion between K79 and the positively charged RBS of H3 HK68; H1 SI is the only strain tested with a net negative charge within Loop 140 (Supplementary Fig. 5) explaining the high preference of HSB.1A for this strain (e) Substitution E79V of HSB.1E (grey cartoon) promotes binding to H2 strains (green) which commonly have a proline at position 145 (Fig.2a, panel IV). (f) Between loop 150 and helix 190 of HA (Fig. 2a, panel II), HSB.1B projects a positive charge (K47) which interacts with the negatively charged E156 of H2 A/Japan/305/1957 (H2 Jap). In contrast, HSB.1D has a negative charge within this loop (D45), which diminishes binding to H2 Jap.

Figure 3. Trimerization of HSB to match the HA trimer

(a) A co-crystal structure of HSB.2A bound to HK68 HA shows that the design binds to the RBS as designed. (b) Superposition of antibody C05 and the Tri-HSB.2A-HA crystal structure. (c) Close agreement is found for the contacts with HA between the tip of HCDR3 of C05 (blue), the HSB designed model (grey), and the bound crystal structure HSB.2A (orange). (d) Translational and rotational sampling of trimeric protein scaffolds (grey, magenta, blue) to identify trimers that connect the termini of three HSB (orange) molecules bound to trimeric HA (green). Termini of HSB are colored purple and those of the trimerizing unit orange. (e) (top left and middle panels) EM reconstruction of Tri-HSB.1C bound to HK68 HA in side view and top view. (top right and bottom panels) Top view and two side views of the asymmetric EM reconstruction fitted with X-ray structure of HK68 HA bound to HSB.2A monomers; a break in the EM density is indicated by the arrow. (f) BLI titrations of H3 HK68 HA binding to monomeric HSB.2 and trimer Tri-HSB.2.

Figure 4. Diagnostic and therapeutic applications

(a) Detection of immobilized HA via lateral flow on nitrocellulose paper strips using either a Tri-HSB variant for capture and the stem-region binding design HB36.6 (coupled to streptavidin-gold via biotinylation) for detection, or vice versa. Tri-HSB.2 readily binds to nitrocellulose and can be directly used as an immobilization reagent for HA. HB36.6 was immobilized through biotinylation followed by nitrocellulose-binding streptavidin. For detection via the head-region binder, Tri-HSB.2 was conjugated to HRP for signal amplification. (b) Inhibition of infection of MDCK-SIAT1-CMV-PB1 cells by H3N2 HK68 virus at indicated concentrations. Values are reported as percent infectivity compared to untreated, infected cells averaged over triplicate measurements. TriHSB.2 has an IC₅₀ of 0.15 nM for HK68 neutralization, and Tri-HSB.1C an IC₅₀ of 19 nM for neutralization of H1 NewCal; both values are close to their apparent K_d, consistent with a simple receptor blocking mechanism. (c) Mice (n=10) treated with a single dose of 3 mg/kg TriHSB.2/body weight either before (prophylactically) or after (therapeutically) intranasal challenge of H3N2 (X-31) virus show substantial protection from weight loss (P < 0.00001) following influenza challenge compared to PBS or non-binding Tri-HSB.2ko-treated controls. Mean values are plotted for each group and with s.e.m.as error bars.