· web viewbiotinylated has were loaded onto streptavidin-coated biosensors in 1x kinetics...

Supporting Information

SI Materials and Methods

Generation of Influenza A Group 2 Neutralizing Antibodies. Fresh peripheral blood

mononuclear cells (PBMC) were obtained with written consent from four healthy donors seven

days after vaccination with 2007-2008 seasonal influenza vaccine (VAXIGRIP®, Sanofi Pasteur)

containing A/Solomon Islands/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2), and

B/Malaysia/2506/2004. B-cell receptor (BCR) positive memory B cells sorted on the

CD19+CD27+ phenotype were isolated and immortalized with Bcl-6 and Bcl-xL, essentially as

described previously (1). The immortalized memory B cells were stained with allophycocyanin

(APC)-labeled H3 hemagglutinin (HA) (A/Wisconsin/67/2005) and H3 HA reactive cells were

sorted into limiting dilution cultures. After cell recovery and expansion, binding of supernatants

to recombinant H1 (A/Brisbane/59/2007), H3 (A/Wisconsin/67/2005), and H7

(A/Netherlands/219/2003) HAs was analyzed by solid phase enzyme-linked immunosorbent

assay (ELISA). Subsequently, immunoglobulins were purified from the B cell supernatants and

in vitro neutralizing activity against A/Wisconsin/67/2005 (H3N2) was measured using a virus

neutralization assay (VNA). H3N2 neutralizing antibodies were reformatted into fully human

IgG1 antibodies by cloning the heavy and light chain variable regions (VH and VL) into a single

IgG1 expression vector. PER.C6® cells were transfected with the IgG1 expressing constructs and

expressed antibodies were purified from culture supernatants using POROS® MabCaptureTM A

Perfusion Chromatography® (Applied Biosystems). Reformatted antibodies were assessed for

binding to recombinant H1, H3, and H7 HA by solid phase ELISA, for binding to recombinant

H1, H3, and H7 HA expressed on PER.C6® cells through the use of fluorescence-activated cell

sorting (FACS), and for neutralizing activity against a panel of influenza A viruses using VNAs.

1

Preparation of Viruses and Recombinant Hemagglutinins. Wildtype influenza viruses

A/Hong Kong/1/1968 (H3N2), mouse-adapted A/Hong Kong/1/1968 (H3N2),

A/Johannesburg/33/1994 (H3N2), A/Panama/2007/1999 (H3N2), A/Hiroshima/52/2005 (H3N2),

A/Wisconsin/67/2005 (H3N2), A/Brisbane/10/2007 (H3N2), A/Perth/16/2009 (H3N2),

A/Victoria/361/2011 (H3N2), A/mallard/Netherlands/12/2000 (H7N3), mouse-adapted

A/chicken/Netherlands/621557/2003 (H7N7), A/chicken/Germany/N/1949 (H10N7), A/New

Caledonia/20/1999 (H1N1), A/Solomon Islands/3-2006 IVR-143 (H1N1), and

A/Brisbane/59/2007 (H1N1) were grown in PER.C6® cells by standard viral culture techniques.

For the challenge experiments, batches of mouse-adapted A/Hong Kong/1/1968 (H3N2) (2) and

mouse-adapted A/chicken/Netherlands/621557/2003 (H7N7) were grown in embryonated

chicken eggs. Recombinant soluble HAs of subtypes H1 (A/Brisbane/59/2007), H3

(A/Wisconsin/67/2005), and H7 (A/Netherlands/219/2003) used for ELISA were produced using

a baculovirus expression system and were purchased from Protein Sciences Corp (CT, USA).

Recombinant soluble biotinylated HAs for the Kd measurements, as well as matured HA for

crystallization and electron microscopy (EM) studies, were produced using a baculovirus

expression system as described previously (3). To produce cell surface-expressed HA, coding

regions of full-length recombinant HA of A/Brisbane/59/2007 (H1N1), A/Hong Kong/1/1968

(H3N2), A/Hong Kong/24/1985 (H3N2), A/Wisconsin/67/2005 (H3N2) or

A/Netherlands/219/2003 (H7N7) were synthesized and cloned into pcDNA-based vectors.

HEK293 or PER.C6® cells were transfected with the recombinant HA expressing constructs

using Lipofectamine (Invitrogen).

2

In vitro Virus Neutralization Assay (VNA). Madin-Darby canine kidney (MDCK) cells were

maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf

serum (FCS) at 37°C. Prior to the experiment, cells were seeded at 40,000 cells/well in 96-well

flat bottom plates, washed twice with phosphate-buffered saline (PBS) and incubated in DMEM

supplemented with 2 mM L-glutamine and 3 µg/mL trypsin-ethylenediaminetetraacetic acid

(EDTA). Two-fold serially diluted antibody containing supernatant or purified IgG1 was mixed

with an equal volume of virus, and incubated for 2 hours at 37°C. After the incubation, the

mixture (approximately 100 TCID50 [tissue culture infectious dose]) was added to confluent

MDCK monolayers in quadruplicate. Cells were cultured for 72 hours after which the

supernatant was added to an equal volume of 1% turkey red blood cells and incubated for 1 hour

at room temperature in 96-well V-bottom plates. The absence of hemagglutination was defined

as neutralization. Antibody titers required to reduce virus replication by 50% (IC50) were

determined using the Spearman-Kärber formula. As a positive control, virus-inoculated cells

were taken along and mock-inoculated cells were included as a negative control. The HA

sequences of the viruses used for VNA are available in FASTA format in Figure S6.

Kd Determination. Kd values were determined by bio-layer interferometry using an Octet RED

instrument (ForteBio, Inc.), as described previously (3). Biotinylated HAs were loaded onto

streptavidin-coated biosensors in 1x kinetics buffer (1x PBS, pH 7.4, 0.01% bovine serum

albumin [BSA], and 0.002% Tween 20) for 600 sec. For the measurement of kon, association of

CR8043 Fab was measured for 180-600 sec by exposing the sensors to four to six concentrations

of Fab in 1x kinetics buffer. For the measurement of koff, dissociation of CR8043 Fab was

measured for 180-600 sec in 1x kinetics buffer. Experiments were performed at 30°C. The ratio

3

of koff to kon determines the Kd. The sequences of all proteins used for Kd determination are

available in FASTA format in Figure S6. All binding traces and curves used for fitting are

reported in Figure S7.

Prophylactic Efficacy Studies in Mice. All experiments were approved prior to commencement

by the ethical review committee of the Central Veterinary Institute (Lelystad, the Netherlands) in

accordance with Dutch law. Female specific pathogen free (SPF) 129 X1/SvJ (Jackson

Laboratories) and female BALB/c (Charles River Laboratories) mice aged six to eight weeks

were used in the H3N2 and H7N7 experiments, respectively. Groups of eight mice were injected

intravenously with 30, 10, 3, or 1 mg/kg CR8043, or 30 mg/kg CR3014 (4) in a volume of 200

µL one day prior to intranasal lethal challenge with 25 LD50 (lethal dose) of either mouse-

adapted A/Hong Kong/1/1968 (H3N2) or A/chicken/Netherlands/621557/2003 (H7N7). Animals

were monitored for survival and weighed daily.

Crystallization and X-ray Structure Determination and Refinement. CR8043 Fab was

generated by LysC cleavage (Roche) of CR8043 IgG. The Fab was purified by protein A and

protein G chromatography, followed by cation exchange and finally size exclusion

chromatography using a Superdex 200 16/60 column (GE Healthcare). Apo CR8043 Fab crystals

were grown using the automated Rigaku Crystalmation robotic system at the Joint Center for

Structural Genomics (JCSG) by sitting drop vapor diffusion. Crystals grew at 4ºC by mixing

concentrated Fab (15 mg/mL) with 20% (w/v) polyethylene glycol (PEG) 6000, 0.1 M 2-[4-(2-

hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) pH 6.5. Crystals were cryo-protected

in mother liquor supplemented with 20% (w/v) PEG 400, flash cooled, and stored in liquid

4

nitrogen until data collection. X-ray diffraction data for the apo CR8043 Fab were collected to

2.65 Å resolution at beamline 11-1 at the Stanford Synchrotron Radiation Lightsource (SSRL).

The data were processed in spacegroup P1 using XDS (5). The structure was determined by

molecular replacement with Phaser (6) using the variable and constant domains of the TR1.9 Fab

(PDB ID code 1VGE (7)) as search models; two Fab copies were found in the asymmetric unit.

The model was iteratively rebuilt using Coot (8) and refined in Phenix (9). Refinement

parameters included rigid body refinement (set for each Ig domain), simulated annealing, and

restrained refinement including TLS refinement (set for each Ig domain).

The CR8043-HK68/H3 HA complex was prepared by adding recombinant CR8043 Fab

to HA in a 3.2:1 molar ratio and the saturated complex was purified by gel filtration. Crystals of

the complex were grown by sitting drop vapor diffusion at 4ºC by mixing concentrated complex

(6.8 mg/mL) with 2.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5, 3% (w/v) PEG 400.

Crystals were cryo-protected in mother liquor supplemented with increasing concentrations of

glycerol (5% steps to a final concentration of 20%), flash cooled, and stored in liquid nitrogen

until data collection. X-ray diffraction data for the CR8043-HK68/H3 HA complex were

collected to 4.0 Å resolution at beamline 12-2 at SSRL and the data were processed in

spacegroup C2 using XDS (5). The structure was determined by molecular replacement with

Phaser (6) first using the HA trimer from A/Hong Kong/1/1968 (H3N2) (PDB ID code 4FNK

(10)) as the search model. Next, three copies of the high-resolution CR8043 Fab variable domain

were used as search models after fixing the position and orientation of the HA trimer. The

CR8043 Fab constant domains were manually placed after refinement. The model was iteratively

rebuilt using Coot (8) and refined in Phenix (9). Refinement parameters included rigid body

refinement (set for the HA trimer and the variable and constant domains of each Fab), restrained

5

refinement including TLS refinement (set for the HA trimer and the variable and constant

domains of each Fab), using the high-resolution HA and Fab as reference models.

Structural Analyses. Hydrogen bonds and van der Waals contacts were calculated using

HBPLUS and CONTACSYM, respectively (11, 12). Surface area upon Fab binding was

calculated using MS (13). MacPyMOL (DeLano Scientific) was used to render figures. Kabat

numbering was applied to the coordinate files using the AbNum server (14). The final

coordinates were validated using the JCSG quality control server (v2.8), which includes

MolProbity (15).

EM Structures of CR8043-HA Complexes. Complexes were prepared by mixing the HA with

saturating amounts of CR8043 Fab at room temperature for two hours. The complex was purified

by size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare) to

remove excess Fab. Four hundred mesh copper EM grids were coated with nitrocellulose and a

thin layer of carbon. Samples were applied (~8 µg/mL of Fab-HA complex diluted in TBS) to

freshly glow-discharged grids and stained with 2% uranyl formate. Micrographs were acquired

on a Tecnai Spirit transmission electron microscopy (TEM) (accelerating voltage of 120 kV)

fitted with a Tietz charge-coupled device camera (4096 x 4096 pixel images). A magnification of

52,000 x and a defocus of approximately -1.1 µm was used. The pixel size was previously

calibrated to be 2.05 Å using a two-dimensional catalase crystal. The stage was tilted from 0º to

55° (5° increments) to increase the number of observed orientations. The Leginon software

package (16, 17) was used to automate some steps of data acquisition.

6

Particles were selected using a difference-of-Gaussian algorithm provided in the Appion

package (18). Particle boxing was performed using Eman1.9 (19) and Spider (20); Xmipp (21)

was used to normalize the boxed images. The contrast transfer function was not corrected. Initial

classification was performed with Xmipp CL2D (22). Following classification, noisy class

averages were manually excluded.

Volumes were reconstructed using the ‘ali3d.py’ function in the Sparx package (23). An

initial volume comprised of the coordinates of the HA from PDB ID code 4GMS (24), which

were low-pass filtered to 30 Å and inputted into the reconstruction. Initial reconstructions used

particle stacks binned by 3 pixels and were refined for 20 rounds as the translational and angular

search increments were decreased. The resulting volume clearly localized the Fabs, showing

densities for the constant and variable domains. The volume was scaled to the unbinned box size

of 192 pixels and refined for 25 rounds. For the A/Bangkok/1/1979 reconstruction, 7,009

particles were included in the final reconstruction; for the A/Hong Kong/1/1968 reconstruction,

10,033 particles were included in the final reconstruction. Fourier shell correlation curves and

resolution estimates are included in Figure S2.

Volumes were visualized and interpreted using UCSF Chimera 1.8 (25). HA from PDB

ID code 4FNK (10) was docked into the volume maps as was a single copy of the crystal

structure of CR8043. This initial docking was refined with Situs (collage) (26). Symmetry-

related Fabs were then generated within Chimera. The corresponding crystal structure of HA in

complex with CR8043 was also docked into the EM map. Both models were very similar and

agreed with the EM data.

7

Sequence Analysis of the Antibody Epitopes. The full-length and non-redundant influenza A

HA sequences were downloaded from the Influenza Virus Resource at the National Center for

Biotechnology Information (NCBI) database (27). At the time of download (July 10, 2013), the

dataset includes 5,403 HA sequences from group 2 influenza A viruses. The sequences were

aligned using MUSCLE (28) and analyzed using GCG (Accelrys) and custom shell scripts

(available from the authors upon request). The sequence identity and conservation of the CR8043

epitope are reported in Table S4 and Table S6, respectively.

Antibody Binding Competition. Antibody binding competition was measured on an Octet QK

system (ForteBio, Inc.). Recombinant soluble HA was biotinylated at room temperature for 40

min using EZ-link sulfo-NHS-LC-LC-biotin (Pierce). A buffer exchange step to PBS was

performed using Amicon Ultra 0.5 mL centrifugal filters (Millipore). Biotinylated HA was

immobilized onto streptavidin biosensors at 37°C for 1200 sec. For analysis of antibody binding

to cleaved versus uncleaved H3 and H7 HA, the bound HA was either cleaved with 1:5 diluted

TrypLE™ Select (Gibco) for 600 seconds or untreated. Association of IgGs was measured for

900 sec at 37°C by exposing the biosensors to antibody in 1x kinetics buffer (ForteBio). The

degree of additional antibody binding was assessed by exposing the biosensors to the second

antibody (in 1x kinetics buffer) in the presence of the first antibody for 900 sec at 37°C.

Generation of Neutralization-Resistant Virus Variants. A/Hong Kong/1/1968 (H3N2) virus

was cultured in the presence of CR8043 IgG1 for multiple passages. As a reference, control

experiments without antibodies were performed in parallel. Serially diluted virus was first

incubated for 1 hour at 37°C with a concentration of antibody known to reduce the viral

8

infectious titer by 3 log units. The incubated mixture was absorbed by MDCK cells for 1 hour.

Infected cells were washed twice with PBS and replenished with infection medium

complemented with the same concentration of antibody. Cytopathic effect (CPE) and

hemagglutinating units (HAU) were determined 72 hours after infection and supernatants from

CPE and HAU positive wells infected with the highest virus dilution were harvested for a

subsequent round of infection. HA sequences of viruses obtained after multiple passages were

examined.

Conformational Change FACS Assay. Full-length recombinant influenza A subtype H3

(A/Hong Kong/1/1968, A/Hong Kong/24/1985, or A/Wisconsin/67/2005) HAs were expressed

on the surface of PER.C6® cells. To measure mAb binding to different HA structural forms and

conformations, cells were detached from the plastic support using PBS-EDTA and subsequently

treated with trypsin (TrypLETMSelect, Gibco) for 5 min at room temperature, washed (1% BSA

in PBS) and incubated for 15 min in citric acid-sodium phosphate buffer pH 4.9, washed, and

then incubated for 20 min in the presence of 50 mM dithiothreitol (DTT) in PBS at room

temperature. Cell samples collected after each processing step were incubated with CR8043 or

CR8057 IgG for 1 hour. Cells were then incubated for 30 min with phycoerythrin-conjugated

anti-human IgG (Southern Biotech) in 1% BSA. Alternatively, CR8043 was added before the

low pH step. Samples of consecutive treatments were split and stained with either phycoerythrin-

conjugated anti-human IgG or HA1 specific AlexaFluro488-conjugated CR8057. Stained cells

were analyzed using a FACS Canto with FACS Diva software (Beckton Dickinson).

9

Trypsin Cleavage Inhibition Assay. Recombinant, soluble A/Wisconsin/67/2005 (H3N2) HA

(0.4 µg) was incubated in the presence of 2.5 µg CR8043 or CR8057 IgG, or in the absence of

antibody in 4 mM Tris-HCl buffer at pH 8.0 containing 6.7 µg/mL Trypsin-EDTA (Gibco) and

1% N-dodecyl-β-maltoside (Sigma). Trypsin digestion was stopped at several time-points by

addition of 1% BSA. Samples were run on sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) (reduced) and blotted according to standard methods. HA0 bands

were detected using a rabbit anti-H3 HA polyclonal antibody (Protein Sciences Corp, CT, USA).

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10

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11

Table S1. Characteristics of influenza A group 2 neutralizing monoclonal antibodies

mAb ID Donor

Clone BCR H3

HA Reactivity

Clone supernatant Recombinant IgG1VH

Gene Famil

y

Number of VH

Somatic Mutation

s

VL

Gene Family

Number of VL

Somatic Mutation

s

HA Binding ELISA

H3N2 VNA

HA Binding ELISA

HA Binding FACS VNA

CR8020 2 + H3, H7 + H3, H7 H3, H7 H3, H7, H10 1-18 12 K3-20 7

CR8021 2 + H3, H7 + H3, H7 H3 H3 3-23 8 K4-01 4

CR8038 2 + H3 + H3 ND H3 3-23 7 K4-01 3

CR8039 3 + H3 + H3 H3 H3 4-59 8 L2-08 2

CR8041 2 + H3 + H3, H7 H3, H7 H3, H7, H10 1-18 12 K3-20 3

CR8043 2 + H3, H7 + H3, H7 H3 H3, H10 1-3 14 K4-01 8

CR8049 1 + H3 + H3 H3 H3 2-26 11 L3-21 11

CR8050 1 + H3 + H3 H3 H3 4-34 14 K3-20 8

CR8052 4 + H3 + H3 H3 - 4-61 8 K1-39 11

CR8055 2 + H3 + - H3 H3 3-33 13 K6-21 9

CR8057 1 + H3 + H3 H3 H3 3-53 16 L2-14 13

ND, not determined.

12

Table S2. X-ray data collection and refinement statistics

Data collection CR8043 Fab CR8043-HK68/H3 complexBeamline SSRL 11-1 SSRL 12-2Wavelength (Å) 0.97945 0.97950Space group P1 C2Unit cell parameters a = 60.0, b = 68.6, c = 72.4 a = 241.5, b = 142.4, c = 170.7

(Å, º) α = 78.7, β = 78.9, γ = 86.2 α = γ = 90.0, β = 133.5Resolution (Å) 50 – 2.65 (2.77 – 2.65) a 50 – 4.0 (4.20 – 4.00) a

Observations 74,100 234,117Unique reflections 31,483 (4,065) a 34,622 (4,271) a

Rmerge (%) b 8.6 (51.5) a 20.1 (82.6) a

Rpim (%) b 7.1 (42.8) a 8.3 (34.3) a

I/sigma 9.0 (1.8) a 6.4 (2.2) a

Completeness (%) 97.1 (93.8) a 97.7 (90.8) a

Multiplicity 2.4 (2.3) a 6.8 (6.5) a

Refinement statisticsResolution 44.4 – 2.65 43.8 – 4.0Reflections (total) 31,427 34,531Reflections (test) 1,577 1,730Rcryst (%) c 17.5 24.0Rfree (%) d 23.2 29.2Protein atoms 6,720 21,392Carbohydrate atoms – 280Waters 9 0Other 21 0Average B-value (Å2)Overall 53 69Wilson 55 93RMSD from ideal geometryBond length (Å) 0.009 0.006Bond angles (°) 1.36 1.23Ramachandran statistics (%) e

Favored 96.9 97.3Outliers 0.2 0.2PDB ID 4NM4 4NM8

a Numbers in parenthesis refer to the highest resolution shell.b Rmerge = Σhkl Σi | Ihkl,i - <Ihkl> | / Σhkl Σi Ihkl,I and Rpim = Σhkl [1/(n-1)]1/2 Σi | Ihkl,i - <Ihkl> | / Σhkl Σi Ihkl,I, where Ihkl,i

is the scaled intensity of the ith measurement of reflection h, k, l, < Ihkl> is the average intensity for that reflection, and n is the redundancy.c Rcryst = Σ | Fo - Fc | / Σ | Fo | x 100, where Fo and Fc are the observed and calculated structure factors, respectively.d Rfree was calculated as for Rcryst, but on a random test set comprising 5% of the data excluded from refinement.e Calculated using MolProbity (15).

13

Table S3. In vitro neutralizing activity of CR8043 and CR8057 against H3N2 viruses

Isolate

IC50 (µg/mL)CR8043 CR8057

MN HAI MN HAIA/Hong Kong/1/1968 0.8 >10 >40 >10A/Johannesburg/33/1994 1.2 >10 >40 >10A/Panama/2007/1999 4.2 >10 0.01 0.028A/Hiroshima/52/2005 1.8 >10 0.003 0.055A/Wisconsin/67/2005 1.6 >10 0.005 0.005

MN, microneutralization; HAI, hemagglutination inhibition

14

Table S4. Sequence identity of CR8043 epitope by subtypeR

esid

ue

Con

sens

usa

HK

68/H

3 Se

quen

ce Group 2 Percent Identity

(Simple)b

Group 2 Percent Identity

(Weighted)c

Percent Identity by Subtype

H3 (4027)d

H4 (486)d

H7 (607)d

H10 (273)d

H14 (5)d

H15 (6)d

21 P P 77.3 29.7 95.5 45.7 3.6 (A/S)e

33.7 (S)e

0.0(E)e

0.0 (A)e

325 E E 99.5 83.0 99.6 99.6 99.8 99.3 0.0 (D/G)e 100.0

326 K K 83.6 69.1 97.1 97.5 19.8 (N/I)e

0.0 (V/I)e 100.0 100.0

15 E E 90.9 66.6 100.0 0.2 (Q)e 100.0 99.3 0.0

(Q)e 100.0

16 G G 99.9 99.8 100.0 99.8 100.0 99.3 100.0 100.0

18 V I 62.8 30.9 75.9 0.4 (I)e

10.4 (I)e 98.9 0.0

(I)e0.0 (I)e

19 D D 96.8 96.1 99.0 99.6 79.7 98.2 100.0 100.0

25 R R 99.7 99.5 100.0 99.8 97.9 99.3 100.0 100.0

32 T T 58.9 59.8 60.1 100.0 0.0 (E)e 98.5 100.0 0.0 (Q)e

34 Q Q 79.5 33.2 100.0 0.2 (T)e 0.0 (T)e 99.3 0.0

(T)e0.0 (T)e

38 L L 83.0 49.9 99.3 100.0 0.0 (Y)e

0.0 (Y)e 100.0 0.0

(Y)e

aMost common residue at position by simple majority across all group 2 sequences. HA1 residues are listed first, followed by HA2 residues.bPercent of all group 2 sequences that are identical to the consensus sequence.cPercent of all group 2 sequences that are identical to the consensus sequence, but weighted to correct for the under/over-representation of some subtypes in the dataset (the mean of the percent conservation values for each individual subtype).dNumber of sequences available for subtype at time of download.eMost common residue in this subtype.

15

Table S5. In vitro neutralizing activity of CR8043 and CR8020 against H3N2 escape mutants

IsolateHA2 position IC50 (µg/mL)

19 25 33 34 CR8043 CR8020A/Hong Kong/1/1968 wildtype D R G Q 0.8 1.8Arg25Met mutant D M G Q >40 1.1Gln34Arg mutant D R G R >40 >40Asp19Asn mutant N R G Q 18.1 >40Gly33Glu mutant D R E Q 30 >40

16

Table S6. Sequence conservation of CR8043 epitope by subtypeR

esid

ue

Con

sens

usa

HK

68/H

3 Se

quen

ce Group 2 Percent

Conservation (Simple)b

Group 2 Percent

Conservation (Weighted)c

Percent Conservation by Subtype

H3 (4027)d

H4 (486)d

H7 (607)d

H10 (273)d

H14 (5)d

H15 (6)d

21 P P 77.3 29.8 95.5 45.7 3.6 (A/S)e

33.7 (S)e

0.0 (E)e

0.0 (A)e

325 E E 99.5 93.1 99.6 99.6 99.8 99.3 60.0 100.0

326 K K 85.5 69.8 99.4 99.6 19.8 (N/I)e

0.0 (V/I)e 100.0 100.0

15 E E 99.9 99.9 100.0 99.8 100.0 99.3 100.0 100.0

16 G G 99.9 99.9 100.0 99.8 100.0 99.3 100.0 100.0

18 V I 95.4 98.8 93.9 99.8 100.0 99.3 100.0 100.0

19 D D 99.9 99.8 99.9 99.8 100.0 99.3 100.0 100.0

25 R R 100.0 99.9 100.0 99.8 100.0 99.3 100.0 100.0

32 T T 58.9 59.8 60.1 100.0 0.0 (E)e 98.5 100.0 0.0

(Q)e

34 Q Q 79.5 33.3 100.0 0.2 (T)e

0.0 (T)e 99.3 0.0

(T)e0.0 (T)e

38 L L 83.4 50.0 99.7 100.0 0.0 (Y)e 0.0 (Y)e 100.0 0.0

(Y)e

aMost common residue at position by simple majority across all group 2 sequences. HA1 residues are listed first, followed by HA2 residues.bPercent of all group 2 sequences that are identical to the consensus sequence or have conservative substitutions.cPercent of all group 2 sequences that are identical to the consensus sequence or have conservative substitutions, but weighted to correct for the under/over-representation of some subtypes in the dataset (the mean of the percent conservation values for each individual subtype).dNumber of sequences available for subtype at time of download.eMost common residue in this subtype.

17

Fig. S1. Sequence alignment of variable regions of CR8020 and CR8041. CR8020 residues contacting H3 HA and corresponding CR8041 residues are depicted in red.

18

Fig. S2. Negative stain EM reconstructions of CR8043 Fab in complex with influenza H3 HA. (A) Side and top view of CR8043 with A/Bangkok/1/1979 (H3N2) HA. The crystal structure of the same complex has been docked into the EM density (gray mesh). The Fab is in red, HA1 in green and HA2 in blue. Example of (B) CR8043-HA (Bangkok79/H3) and (C) CR8043-HA (HK68/H3) CL2D reference free class averages (left) and FSC curves for the final reconstructions (right).

19

Fig. S3. Antibody binding competition of CR8020, CR8043 or CR8057 (100 nM) with immobilized A/Wisconsin/67/2005 (H3N2) HA pre-saturated with CR8020, CR8043, or CR8057 (100 nM), as measured by bio-layer interferometry.

20

Fig. S4. Representative electron density at the CR8043-HA interface. The CR8043 variable heavy and light chains are colored cyan and orange and the HA1 and HA2 are colored yellow and red. The 2Fo-Fc electron density map (blue mesh) is contoured at 1σ.

21

Fig. S5. Similar interacting residues between CR8043 and CR8020 are illustrated on the surface of the HK68/H3 HA. HA footprints of CR8043, CR8020, or the antibody overlaps are colored red, blue, or yellow, respectively. The interacting residues of CR8043 and CR8020 are colored green and orange, respectively.

22

Fig. S6. Sequences of HA proteins used in this study.

HA sequences of viruses used in neutralization studies.

>A/Hong Kong/1/1968 (H3N2)MKTIIALSYIFCLALGQDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIWSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGNYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQRGNIRCNICI

>A/Johannesburg/33/1994 (H3N2)MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSSPTGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFINENFNWTGVAQDGKSYACKRGSVNSFFSRLNWLHKLEYKYPALNVTMPNNGKFDKLYIWGVHHPSTDSDQTSLYVRASGRVTVSTKRSQQTVIPDIGYRPWVRGQSSRISIHWTIVKPGDILLINSTGNLIAPRGYFKIRNGKSSIMRSDAPIGNCSSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRLVEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTRKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQKGNIRCNICI

>A/Panama/2007/1999 (H3N2)MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVSNGTLVKTITNDQIEVTNATELVQSSSTGRICDSPHQILDGENCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVAQNGTSSACKRRSNKSFFSRLNWLHQLKYKYPALNVTMPNNEKFDKLYIWGVHHPSTDSDQISIYAQASGRVTVSTKRSQQTVIPNIGSSPWVRGVSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWACQKGNIRCNICI

>A/Hiroshima/52/2005 (H3N2)MKTIIALSYILCLAFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACKRRSNNSFFSRLNWLTQLKFKYPALKVTMPNNEKFDKLYIWGVHHPVTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDIKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

>A/Wisconsin/67/2005 (H3N2)MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGGICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNDESFNWTGVTQNGTSSSCKRRSNNSFFSRLNWLTQLKFKYPALNVTMPNNEKFDKLYIWGVHHPVTDNDQIFLYAQASGRITVSTKRSQQTVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAINQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTK

23

KQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI>A/Brisbane/10/2007 (H3N2)MKTIIALSYILCLVFTQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFPYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

>A/Perth/16/2009 (H3N2)MKTIIALSYILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEICDSPHQILDGKNCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSKNSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQASGRITVSTKRSQQTVSPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

>A/Victoria/361/2011 (H3N2)MKTIIALSHILCLVFAQKLPGNDNSTATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTQLNFKYPALNVTMPNNEQFDKLYIWGVHHPVTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGYRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

>A/mallard/Netherlands/12/2000 (H7N3)MNTQILVFALMAIIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNVPRICSKGKRTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKETMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDNAAFPQMTKSYKNTRKDPALIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNGQSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEGDCYHSGGTIISNLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEIPKGRGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFTEVEKQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAMQNRIQIDPVKLSSGYKDVILWFSFGASCFILLAIAMGLVFICVKNGNMRCTICI

>A/chicken/Netherlands/621557/2003 (H7N7)MNTQILVFALVAIIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNVPRICSKGKRTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKETMGFTYSGIRTNGATSACRRSGSSFYAEMKWLLSNTDNAAFPQMTKSYKNTRKDPALIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNGQSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSEVQVDANCEGDCYHSGGTIISNLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEIPKRRRRGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFTEVEKQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAIQNRIQIDPVKLSSGYKDVILWFSFGASCFILLAIAMGLVFICVKNGNMRCTICI

24

>A/chicken/Germany/N/1949 (H10N7)MYKVVVIIALLGAVKGLDRICLGHHAVANGTIVKTLTNEQEEVTNATETVESTNLNKLCMKGRSYKDLGNCHPVGMLIGTPVCDPHLTGTWDTLIERENAIAHCYPGATINEEALRQKIMESGGISKMSTGFTYGSSINSAGTTKACMRNGGDSFYAELKWLVSKTKGQNFPQTTNTYRNTDTAEHLIIWGIHHPSSTQEKNDLYGTQSLSISVESSTYQNNFVPVVGARPQVNGQSGRIDFHWTLVQPGDNITFSHNGGLIAPSRVSKLTGRGLGIQSEALIDNSCESKCFWRGGSINTKLPFQNLSPRTVGQCPKYVNQRSLLLATGMRNVPEVVQGRGLFGAIAGFIENGWEGMVDGWYGFRHQNAQGTGQAADYKSTQAAIDQITGKLNRLIEKTNTEFESIESEFSETEHQIGNVINWTKDSITDIWTYQAELLVAMENQHTIDMADSEMLNLYERVRKQLRQNAEEDGKGCFEIYHTCDDSCMESIRNNTYDHSQYREEALLNRLNINSVKLSSGYKDIILWFSFGASCFVLLAVVMGLVFFCLKNGNMRCTICI

>A/New Caledonia/20/1999 (H1N1)MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

>A/Solomon Islands/3/2006 (H1N1)MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISRESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTTTGVSASCSHNGESSFYKNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQRALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDREGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSRVLLVSLGAISFWMCSNGSLQCRICI

>A/Brisbane/59/2007 (H1N1)MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVEKPNPENGTCYPGHFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGESSFYRNLLWLTGKNGLYPNLSKSYANNKEKEVLVLWGVHHPPNIGDQKALYHTENAYVSVVSSHYSRKFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPRYAFALSRGFGSGIINSNAPMDKCDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFIDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

25

Sequences of HA proteins used for Kd determination. The sequences listed below represent the full-length ORF as cloned in the baculovirus transfer vector. Most of the N-terminal signal peptide (MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA) is presumably removed during secretion, leaving four non-native residues (ADPG) at the N-terminus of HA1. The C-terminal biotinylation site, thrombin cleavage site, trimerization domain, and His6 tag are retained on all proteins.

>A/duck/Ukraine/1/1963 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTIVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGRACTLIDALLGDPHCDVFQNETWDLFVERSNAFSNCYPYDIPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSSACKRGPANGFFSRLNWLTKSESAYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTDLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGQPGRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPGKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINRKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLADSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDIYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Hong Kong/1/1968 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Hong Kong/1/1968 mutant I18M (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMMDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Hong Kong/1/1968 mutant D19N (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMINGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

26

>A/Hong Kong/1/1968 mutant R25M (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFMHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Hong Kong/1/1968 mutant T32E (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGEGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Hong Kong/1/1968 mutant T32I (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Hong Kong/1/1968 mutant T32R (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Hong Kong/1/1968 mutant Q34T (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGTAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

27

>A/Hong Kong/1/1968 mutant Q34R (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGRAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTGRQLRENAEDMGNGCFKIYHKCDNACIESIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Victoria/3/1975 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSSSTGKICNNPHRILDGINCTLIDALLGDPHCDGFQNEKWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFINEGFNWTGVTQNGGSSACKRGPDSGFFSRLNWLYKSGSTYPVQNVTMPNNDNSDKLYIWGVHHPSTDKEQTNLYVQASGKVTVSTKRSQQTIIPNVGSRPWVRGLSSRISIYWTIVKPGDILVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIGTCSSECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Leningrad/360/1986 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSSSTGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNEKWDLFIERSKAFSNCYPYDVPDYASLRSLVASSGTLEFINEGFNWTGVTQSGGSYTCKRGSVNSFFSRLNWLYESEYKYPALNVTMPNNGKFDKLYIWGVHHPSTEKEQTNLYVRASGRVTVSTKRSQQTVIPNIGSRPWVRGLSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRTGKSSIMRSDAPIGTCSSECITPNGSIPNDKPFQNVNKITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRLIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQYTIDLTDSEMNKLFEKTRKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Beijing/353/1989 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSSSTGRICDSPHRILDGKNCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFINEDFNWTGVAQSGESYACKRGSVKSFFSRLNWLHESEYKYPALNVTMPNNGKFDKLYIWGVHHPSTDREQTNLYVRASGRVTVSTKRSQQTVIPNIGSRPWVRGLSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRTGKSSIMRSDAPIGTCSSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRLIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Shangdong/9/1993 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSSSTGRICGSPHRILDGKNCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFINEDFNWTGVAQDGGSYACKRGSVNSFFSRLNWLHKLEYKYPALNVTMPNNGKFDKLYIWGVHHPSTDSDQTSLYVRASGRVTVSTKRSQQTVTPNIGSRPWVRGQSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRNGKSSIMRSDAPIGNCSSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRLIEKTNEKFQQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

28

>A/Moscow/10/1999 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTLVKTITNDQIEVTNATELVQSSSTGRICDSPHQILDGENCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVAQNGTSSACKRRSINSFFSRLNWLHQLKYRYPALNVTMPNNDKFDKLYIWGVHHPSTDSDQTSLYTQASGRVTVSTKRSQQTVIPNIGSRPWVRGISSRISIYWTIVKPGDILLIKSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMMDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTRKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Panama/2007/1999 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVSNGTLVKTITNDQIEVTNATELVQSSSTGRICDSPHQILDGENCTLIDALLGDPHCDGFQNKEWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVAQNGTSSACKRRSNKSFFSRLNWLHQLKYKYPALNVTMPNNEKFDKLYIWGVLHPSTDSDQISLYAQASGRVTVSTKRSQQTVIPNIGSRPWVRGVSSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGTGQAADLKSTQAAINQINGKLNRLIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFERTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Brisbane/10/2007 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLKFKYPALNVTMPNNEKFDKLYIWGVHHPGTDNDQIFPYAQASGRITVSTKRSQQTVIPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGIGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Perth/16/2009 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQSSSTGEICDSPHQILDGKNCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSKNSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQASGRITVSTKRSQQTVSPNIGSRPRVRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQNTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/Victoria/361/2011 (H3N2)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGATLCLGHHAVPNGTIVKTITNDQIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFNNESFNWTGVTQNGTSSACIRRSNNSFFSRLNWLTHLNFKYPALNVTMPNNEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRNIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCNSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNGTYDHDVYRDEALNNRFQIKGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

29

>A/duck/Czechoslovakia/1956 (H4N6)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGPVICMGHHAVANGTMVKTLADDQVEVVTAQELVESQNLPELCPSPLRLVDGQTCDIINGALGSPGCDHLNGAEWDVFIERPNAVDTCYPFDVPEYQSLRSILANNGKFEFIAEEFQWNTVKQNGKSGACKRANVNDFFNRLNWLVKSDGNAYPLQNLTKINNGDYARLYIWGVHHPSTDTEQTNLYKNNPGRVTVSTKTSQTSVVPNIGSRPLVRGQSGRVSFYWTIVEPGDLIVFNTIGNLIAPRGHYKLNNQKKSTILNTAIPIGSCVSKCHTDKGSLSTTKPFQNISRIAVGDCPRYVKQGSLKLATGMRNIPEKASRGLFGAIAGFIENGWQGLIDGWYGFRHQNAEGTGTAADLKSTQAAIDQINGKLNRLIEKTNDKYHQIEKEFEQVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERVRRQLRENAEDKGNGCFEIFHKCDNNCIESIRNGTYDHDIYRDEAINNRFQIQGVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/chicken/Netherlands/219/2003 (H7N7)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGDKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNVPRICSKGKRTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKETMGFTYSGIRTNGTTSACRRSGSSFYAEMKWLLSNTDNAAFPQMTKSYKNTRKDPALIIWGIHHSGSTTEQTKLYGSGNKLITVGSSNYQQSFVPSPGARPQVNGQSGRIDFHWLILNPNDTVTFSFNGAFIALDRASFLRGKSMGIQSEVQVDANCEGDCYHSGGTIISNLPFQNINSRAVGKCPRYVKQESLLLATGMKNVPEIPKRRRRGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIEKTNQQFELIDNEFTEVERQIGNVINWTRDSMTEVWSYNAELLVAMENQHTIDLADSEMNKLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHSKYREEAIQNRIQIDPVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/chicken/Germany/N/1949 (H10N7)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGDRICLGHHAVANGTIVKTLTNEQEEVTNATETVESTNLNKLCMKGRSYKDLGNCHPVGMLIGTPVCDPHLTGTWDTLIERENAIAHCYPGATINEEALRQKIMESGGISKMSTGFTYGSSINSAGTTKACMRNGGDSFYAELKWLVSKTKGQNFPQTTNTYRNTDTAEHLIIWGIHHPSSTQEKNDLYGTQSLSISVESSTYQNNFVPVVGARPQVNGQSGRIDFHWTLVQPGDNITFSHNGGLIAPSRVSKLTGRGLGIQSEALIDNSCESKCFWRGGSINTKLPFQNLSPRTVGQCPKYVNQRSLLLATGMRNVPEVVQGRGLFGAIAGFIENGWEGMVDGWYGFRHQNAQGTGQAADYKSTQAAIDQITGKLNRLIEKTNTEFESIESEFSETEHQIGNVINWTKDSITDIWTYQAELLVAMENQHTIDMADSEMLNLYERVRKQLRQNAEEDGKGCFEIYHTCDDSCMESIRNNTYDHSQYREEALLNRLNINSVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/mallard/Astrakhan/263/1982 (H14N5)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGPIICLGHHAVENGTSVKTLTDNHVEVVSAKELVETNHTDELCPSPLKLVDGQDCDLINGALGSPGCDRLQDTTWDVFIERPTAVDTCYPFDVPDYQSLRSILASSGSLEFIAEQFTWNGVKVDGSSSACLRGGRNSFFSRLNWLTKATNGNYGPINVTKENTGSYVRLYLWGVHHPSSDNEQTDLYKVATGRVTVSTRSDQISIVPNIGSRPRVRNQSGRISIYWTLVNPGDSIIFNSIGNLIAPRGHYKISKSTKSTVLKSDKRIGSCTSPCLTDKGSIQSDKPFQNVSRIAIGNCPKYVKQGSLMLATGMRNIPGKQAKGLFGAIAGFIENGWQGLIDGWYGFRHQNAEGTGTAADLKSTQAAIDQINGKLNRLIEKTNEKYHQIEKEFEQVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERVRRQLRENAEDQGNGCFEIFHQCDNNCIESIRNGTYDHNIYRDEAINNRIKINPVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

>A/shearwater/Western Australia/2576/1979 (H15N9)MVLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPGDKICLGHHAVANGTKVNTLTERGVEVVNATETVEITGIDKVCTKGKKAVDLGSCGILGTIIGPPQCDLHLEFKADLIIERRNSSDICYPGRFTNEEALRQIIRESGGIDKESMGFRYSGIRTDGATSACKRTVSSFYSEMKWLSSSMNNQVFPQLNQTYRNTRKEPALIVWGVHHSSSLDEQNKLYGTGNKLITVGSSKYQQSFSPSPGARPKVNGQAGRIDFHWMLLDPGDTVTFTFNGAFIAPDRATFLRSNAPSGIEYNGKSLGIQSDAQIDESCEGECFYSGGTINSPLPFQNIDSRAVGKCPRYVKQSSLPLALGMKNVPEKIRTRGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGQGTAADYKSTQAAIDQITGKLNRLIEKTNKQFELIDNEFTEVEQQIGNVINWTRDSLTEIWSYN

30

AELLVAMENQHTIDLADSEMNKLYERVRRQLRENAEEDGTGCFEIFHRCDDQCMESIRNNTYNHTEYRQEALQNRIMINPVSGGGGLNDIFEAQKIEWHERLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH

31

Fig. S7. Binding curves for reported Kd values for CR8043. Blue curves are the experimental trace obtained from bio-layer interferometry experiments, and red curves are the best global fits to the data used to calculate the Kd values reported in Figure 2.

A/duck/Ukraine/1/1963 (H3N2), Kd = 4.7 nM

These data were fit with a 2:1 binding model, yielding apparent Kd1 = 4.7 nM and Kd2 = 100 nM. The dominant binding process (accounting for the majority of the shift in observed wavelength) corresponds to the higher affinity process (Kd1 = 4.7 nM). The lower affinity process may reflect a non-specific interaction. Therefore, we report the affinity for this interaction as 4.7 nM in Figure 2.

A/Hong Kong/1/1968 (H3N2), Kd = 2.5 nM

A/Hong Kong/1/1968 mutant I18M, Kd = 3.0 nM

A/Hong Kong/1/1968 mutant D19N, Kd = 7.5 nM

32

A/Hong Kong/1/1968 mutant R25M, Kd = 760 nM

These data were fit with a 2:1 binding model, yielding apparent Kd1 = 780 nM and Kd2 = 740 nM (although kon and koff differ for the two binding processes). As these two binding processes have similar affinities, we report the affinity as the average of Kd1 and Kd2, ~760 nM, in Figure 2.

A/Hong Kong/1/1968 mutant T32E, Kd = 1.0 nM

A/Hong Kong/1/1968 mutant T32I, Kd = 2.3 nM

A/Hong Kong/1/1968 mutant T32R, Kd = 19 nM

A/Hong Kong/1/1968 mutant Q34T, Kd = 120 nM

33

A/Hong Kong/1/1968 mutant Q34R, Kd = 350 nM

A/Hong Kong/1/1968 mutant L38Y, Kd = 0.9 nM

A/Victoria/3/1975 (H3N2), Kd = 0.5 nM

A/Leningrad/360/1986 (H3N2), Kd = 1.3 nM

A/Beijing/353/1989 (H3N2), Kd = 2.6 nM

34

A/Shangdong/9/1993 (H3N2), Kd = 0.4 nM

A/Moscow/10/1999 (H3N2), Kd = 6.4 nM

A/Panama/2007/1999 (H3N2), Kd = 5.4 nM

These data were fit with a 2:1 binding model, yielding apparent Kd1 = 5.4 nM and Kd2 = 190 nM. The dominant binding process (accounting for the majority of the shift in observed wavelength) corresponds to the higher affinity process (Kd1 = 5.4 nM). The lower affinity process may reflect a non-specific interaction. Therefore, we report the affinity for this interaction as 5.4 nM in Figure 2.

A/Brisbane/10/2007 (H3N2), Kd = 0.7 nM

35

A/Perth/16/2009 (H3N2), Kd = 28 nM

A/Victoria/361/2011 (H3N2), Kd = 35 nM

A/duck/Czechoslovakia/1956 (H4N6), Kd = 390 nM

A/chicken/Netherlands/219/2003 (H7N7), Kd = 190 nM


A/chicken/Germany/N/1949 (H10N7), Kd = 0.7 nM

36

A/mallard/Astrakhan/263/1982 (H14N5), Kd = 480 nM


A/shearwater/West Australia/2576/1979 (H15N9), Kd = 350 nM

These data were fit with a 2:1 binding model, yielding apparent Kd1 = 350 nM and Kd2 = 11 nM. The dominant binding process (accounting for the majority of the shift in observed wavelength) corresponds to the lower affinity process (Kd1 = 350 nM). The higher affinity process may reflect a non-specific interaction. Therefore, we report the affinity for this interaction as 350 nM in Figure 2.

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· web viewbiotinylated has were loaded onto streptavidin-coated biosensors in 1x kinetics...

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