www.sciencemag.org/content/342/6158/592/suppl/DC1

Supplementary Materials for

Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus

Jason S. McLellan, Man Chen, M. Gordon Joyce, Mallika Sastry, Guillaume B. E.

Stewart-Jones, Yongping Yang, Baoshan Zhang, Lei Chen, Sanjay Srivatsan, Anqi Zheng, Tongqing Zhou, Kevin W. Graepel, Azad Kumar, Syed Moin, Jeffrey C.

Boyington, Gwo-Yu Chuang, Cinque Soto, Ulrich Baxa, Arjen Q. Bakker, Hergen Spits, Tim Beaumont, Zizheng Zheng, Ningshao Xia, Sung-Youl Ko, John-Paul Todd,

Srinivas Rao, Barney S. Graham,* Peter D. Kwong*

*Corresponding author. E-mail: bgraham@nih.gov (B.S.G.); pdkwong@nih.gov (P.D.K.)

Published 1 November 2013, Science 342, 592 (2013) DOI: 10.1126/science.1243283

This PDF file includes

Materials and Methods Figs. S1 to S12 Tables S1 to S4 References

Materials and Methods

Viruses and cells. Viral stocks were prepared and maintained as previously described

(50). Recombinant mKate-RSV expressing prototypic subtype A (strain A2) and subtype B

(strain 18537) F genes and the Katushka fluorescent protein were constructed as reported by

Hotard et al. (51). HEp-2 cells were maintained in Eagle's minimal essential medium containing

10% fetal bovine serum (10% EMEM) and were supplemented with glutamine, penicillin and

streptomycin.

Expression and purification of antibodies and antigen binding fragments (Fabs).

Antibodies were expressed by transient co-transfection of heavy and light chain plasmids into

HEK293F cells grown in suspension at 37 ºC for 4-5 days (7, 18, 52). The cell supernatants were

passed over Protein A agarose, and bound antibodies were washed with PBS and eluted with IgG

elution buffer (Pierce) into 1/10th volume of 1 M Tris-HCl pH 8.0. Fabs were created by

digesting the IgG with Lys-C or HRV3C protease (53), and the Fab and Fc mixtures were passed

over Protein A agarose to remove Fc fragments. Fabs were further purified by size-exclusion

chromatography.

Screening of prefusion-stabilized RSV F constructs. Prefusion RSV F variants were

derived from the RSV F (+) Fd construct (7), which consists of RSV F residues 1-513 with a C-

terminal T4 fibritin trimerization motif (54), thrombin site, 6x His-tag, and StreptagII. A 96-well

microplate-formatted transient gene expression approach was used to achieve high-throughput

expression of various RSV F proteins as described previously (28). Briefly, 24 h prior to

transfection HEK 293T cells were seeded in each well of a 96-well microplate at a density of

2.5x105 cells/ml in expression medium (high glucose DMEM supplemented with 10 % ultra-low

IgG fetal bovine serum and 1x-non-essential amino acids), and incubated at 37 °C, 5% CO2 for

20 h. Plasmid DNA and TrueFect-Max (United BioSystems, MD) were mixed and added to the

growing cells, and the 96-well plate incubated at 37 °C, 5% CO2. One day post transfection,

enriched medium (high glucose DMEM plus 25% ultra-low IgG fetal bovine serum, 2x non-

essential amino acids, 1x glutamine) was added to each well, and the 96-well plate was returned

to the incubator for continuous culture. On day five post transfection, supernatants with the

expressed RSV F variants were harvested and tested by ELISA for binding to D25 and

2

motavizumab antibodies using Ni2+-NTA microplates. After incubating the harvested

supernatants at 4º C for one week, ELISAs were repeated.

Large-scale expression and purification of RSV F constructs. Soluble postfusion RSV

F was expressed and purified as described previously (5). Prefusion variants were expressed by

transient transfection in Expi293F cells using TrueFect-Max (United BioSystems, MD). The

culture supernatants were harvested 5 days post transfection and centrifuged at 10,000 g to

remove cell debris. The culture supernatants were sterile-filtered prior to buffer exchange and

concentrated using tangential flow filtration (55). RSV F glycoproteins were purified by nickel-

and streptactin-affinity chromatography, and relevant fractions containing the RSV F variants

were pooled, concentrated, and subjected to size-exclusion chromatography (7). Affinity tags

were removed by digestion with thrombin followed by size-exclusion chromatography.

Glycoproteins used in the non-human primate immunizations were tested for endotoxins using

the limulus amebocyte lysate assay, and if necessary proteins were passed over an EndoTrap Red

(BioVendor, NC) column to remove endotoxins prior to immunizations. Endotoxin level was < 5

EU/kg body weight/h, as measured by the Endpoint Chromogenic Limulus Amebocyte Lysate

(LAL) test kit (Lonza, Basel, Switzerland).

Stabilized RSV F antigenic characterization. A fortéBio Octet Red384 instrument was

used to measure binding kinetics of RSV F to antibodies that target antigenic site Ø (D25,

AM22, 5C4), site I (131-2a), site II (palivizumab, motavizumab) and site IV (101F). All assays

were performed with agitation set to 1,000 rpm in phosphate-buffered saline (PBS)

supplemented with 1% bovine serum albumin (BSA) to minimize nonspecific interactions. The

final volume for all solutions was 100 μl/well. Assays were performed at 30 °C in solid black 96-

well plates (Greiner Bio-One). StrepMAB-Immo (35 μg/ml) in PBS buffer was used to load anti-

mouse Fc probes for 300 s, which were then used to capture relevant RSV F variant proteins that

contained a C-terminal StreptagII. Typical capture levels for each loading step were between 0.7

and 1 nm, and variability within a row of eight tips did not exceed 0.1 nm for each of these steps.

Biosensor tips were then equilibrated for 300 s in PBS + 1% BSA prior to measuring association

with Fabs in solution (0.002 μM to 1 μM) for 300 s; Fabs were then allowed to dissociate for

400-1200 s depending on the observed dissociation rate. Dissociation wells were used only once

to prevent contamination. Parallel corrections to subtract systematic baseline drift were carried

out by subtracting measurements recorded with loaded sensors incubated in PBS + 1% BSA. To

3

remove nonspecific binding responses, a HIV-1 gp120 molecule with a C-terminal StreptagII

was loaded onto the anti-mouse Fc probes and incubated with RSV Fabs, and the nonspecific

gp120 responses were subtracted from RSV F variant response data. Data analysis and curve

fitting were carried out using Octet software, version 7.0. Experimental data were fitted with the

binding equations describing a 1:1 interaction. Global analyzes of the complete data sets

assuming reversible binding (full dissociation) were carried out using nonlinear least-squares

fitting allowing a single set of binding parameters to be obtained simultaneously for all

concentrations used in each experiment.

Physical stability of RSV F variants. To assess the physical stability of designed RSV F

proteins under various stress conditions, we treated the proteins with a variety of

pharmaceutically relevant stresses such as extreme pH, high temperature, low and high

osmolality as well as repeated freeze/thaw cycles. The physical stability of treated RSV F

proteins was evaluated by their degree of preservation of antigenic site Ø after treatment, a

critical parameter assessed by binding of the site Ø-specific antibody D25.

In the pH treatment, RSV F protein was diluted to an initial concentration of 50 μg/ml,

adjusted to pH 3.5 and pH 10 with appropriate buffers and incubated at room temperature for 1 h

before being neutralized back to pH7.5 and concentrated to 40 μg/ml. In the temperature

treatment, RSV protein at 40 μg/ml was incubated at 50 ºC, 70 ºC and 90 ºC for 1 h in a PCR

cycler with heated lid to prevent evaporation. In the osmolality treatment, 100 μl of RSV F

protein solutions (40 μg/ml) originally containing 350 mM NaCl were either diluted with 2.5

mM Tris buffer (pH 7.5) to an osmolality of 10 mM NaCl or adjusted with 4.5 M MgCl2 to a

final concentration of 3.0 M. The protein solutions were incubated for 1 h at room temperature

and then brought back to 350 mM NaCl by adding 5M NaCl or diluting with the Tris buffer,

respectively, before concentration to 100 μl. The freeze/thaw treatment was carried out 10 times

by repeated liquid nitrogen freezing and thawing at 37 ºC. Binding of antibody D25 to the treated

RSV F proteins was measured with an Octet instrument with protocols described above. The

degree of physical stability was calculated from the ratio of steady state D25-binding level before

and after stress treatment.

Crystallization and X-ray data collection of prefusion-stabilized RSV F proteins.

Crystals of RSV F DS, Cav1, DS-Cav1, and DS-Cav1-TriC were grown by the vapor diffusion

4

method in hanging drops at 20 ºC by mixing 1 µl of RSV F with 1 µl of reservoir solution (1.4 M

K/Na tartrate, 0.1M CHES pH 9.5, 0.2 M Li2SO4). Crystals were flash frozen in liquid nitrogen

or a liquid nitrogen cryostat set at 100 oK. Crystals of RSV F Cav1 and DS-Cav1 were also

grown by the vapor diffusion method in hanging drops at 20 ºC by mixing 1 µl of RSV F with

0.5 µl of reservoir solution (1.7 M ammonium sulfate, 0.1 M citrate pH 5.5). Crystals were

transferred to a solution of 3.2 M ammonium sulfate, 0.1 M citrate pH 5.5, and flash frozen. All

X-ray diffraction data were collected at a wavelength of 1.00 Å at the SER-CAT beamline ID-

22.

Structure determination, refinement and analysis of prefusion-stabilized RSV F.

X-ray diffraction data were integrated and scaled with the HKL2000 suite (56), and molecular

replacement solutions were obtained by PHASER (57) using the D25-bound RSV F structure

(PDB ID: 4JHW, (7)) as a search model. Manual model building was carried out using COOT

(58), and refinement was performed in PHENIX (59). Final data collection and refinement

statistics are presented in Table S2. Superimpositions of RSV F structures were performed using

residues 225-455 which showed high levels of structural similarity. Antigenic site Ø rmsd

calculations were based on residues 61-71 and 194-219 which were within 10 Å of the D25

antibody in the RSV F-D25 complex structure.

Negative stain electron microscopy analysis. Samples were adsorbed to freshly glow-

discharged carbon-film grids, rinsed twice with buffer, and stained with freshly made 0.75%

uranyl formate. Images were recorded on an FEI T20 microscope with a 2k x 2k Eagle CCD

camera at a pixel size of 1.5 Å. Image analysis and 2D averaging was performed with Bsoft (60)

and EMAN (61).

NHP immunizations. All animal experiments were reviewed and approved by the

Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH, and all animals

were housed and cared for in accordance with local, state, federal, and institute policies in an

American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited

facility at the NIH. Macaca mulatta animals of Indian origin weighing 8.76-14.68 kg were

intramuscularly injected with immunogens at week 0 and week 4. The frozen RSV F variant

immunogen proteins were thawed on ice and mixed with poly ICLC (Oncovir, DC) (62,63), with

5

injections taking place within 1 h of immunogen:adjuvant preparation. Blood was collected

every other week for 14 weeks.

RSV neutralization assays. Sera were distributed as four-fold dilutions from 1:10 to

1:40960, mixed with an equal volume of recombinant mKate-RSV expressing prototypic F genes

from subtype A (strain A2) or subtype B (strain 18537) and the Katushka fluorescent protein,

and incubated at 37 ºC for 1 h. Next, 50 μl of each serum dilution/virus mixture was added to

HEp-2 cells that had been seeded at a density of 1.5x104 in 30 μl MEM (minimal essential

medium) in each well of 384-well black optical bottom plates, and incubated for 20-22 h before

spectrophotometric analysis at 588 nm excitation and 635 nm emission (SpectraMax Paradigm,

Molecular Devices, CA). The IC50 for each sample was calculated by curve fitting and non-linear

regression using GraphPad Prism (GraphPad Software Inc., CA). P-values were determined by

Student's t-test.

Sera antigenicity analysis. A fortéBio Octet Red384 instrument was used to measure

sera reactivity to RSV F variant proteins with agitation, temperature, 96-well plates, buffer and

volumes identical to those used for kinetic measurements. RSV F DSCav1 and postfusion F were

immobilized by amine coupling to biosensors via activation in an EDC/NHS activation mixture

for 300 s in 10 mM acetate pH 5. The biosensor reactivity was quenched using 10 mM

ethanolamine pH 8.5. Typical capture levels were between 0.7 and 1 nm, and variability within a

row of eight tips did not exceed 0.1 nm for each of these steps. Biosensor tips were then

equilibrated for 300 s in PBS + 1% BSA buffer prior to binding measurements. Sera were diluted

to a 1/50 and 1/100 dilution in PBS + 1% BSA and binding was assessed for 300 s. Sera

depletion was carried out by using 1 μg of DS-Cav1 or postfusion F glycoprotein per 1 μl of

animal sera. Parallel corrections to subtract non-specific sera binding were carried out by

subtracting binding levels for unloaded probes incubated with sera. Site-specific antigenicities

were assessed by incubating RSV F variant-loaded probes with 1 or 2 μM D25 Fab for site Ø

assessment and motavizumab Fab for site II assessment or both antibodies to assess the

remaining non-site Ø/II reactivity.

6

Supplemental figures

Figure S1. Structure-based vaccine design for RSV: a “neutralization-sensitive site” paradigm. (A) Natural infection by RSV elicits diverse antibodies, with a range of viral neutralization potencies. (B) Antigen-binding fragments of the potently neutralizing antibody D25 are shown recognizing an epitope at the apex of the RSV F trimer. Spatially overlapping epitopes at the trimer apex are also recognized by the AM22 and 5C4 antibodies, which share the same desired neutralization characteristics as D25. These overlapping epitopes, recognized by antibodies of extremely high potency, define antigenic site Ø as a neutralization-sensitive site of RSV vulnerability. (C) After selection of a target neutralization-sensitive site, an iterative process of design, characterization of antigenic and physical properties, atomic-level structure determination, and assessment of immunogenicity allows for the structure-based optimization of vaccine antigens encoding the target site. (D) Because the neutralization-sensitive site of viral vulnerability naturally elicits highly protective antibodies, immunization with “neutralization-sensitive site immunogens” more easily elicits protective response than immunogens based on viral regions recognized by subdominant or non-potent neutralizing antibodies.

Design

Structure

Elicitation of humoral response

Immunogenicity

Antigenic &physical properties

Potently neutralizing antibodies

Moderately neutralizing antibodies

Weakly neutralizing antibodies

A Characterization of protective responses elicited by natural infection

B Structural definition of a neutralization-sensitive site of viral vulnerability

C Information matrix for structure-based vaccine design

Potent D25 antibodies recognize

antigenic site Ø

RSV F glycoprotein

trimer

D Elicitation of protective responses with a neutralization-sensitive site immunogen

Cavity filling Cavity filling

Disulfide

Natural RSV 

infection

Neutralization-sensitive site-directed

potently neutralizing antibodies

Immunization with RSV F optimized to present the

neutralization-sensitive site

Antibody recognition of homogeneous

trimer

Disulfide Cavity filling

7

155

290

Postfusion

155-290

Prefusion

Figure S2. Location of S155 and S290 in the prefusion and postfusion RSV F structures. The β-carbons of serine residues 155 and 290 are 4.4 Å apart in the D25-bound RSV F structure and 124.2 Å apart in the postfusion structure. The mutations S155C and S290C (called “DS”) restrain the structure in the prefusion conformation.  

8

      

Figure S3. Negative stain-electron microscopy of site Ø-stabilized RSV F glycoproteins. (A) and (B) show representative fields of negatively stained specimens for DS and DS-Cav1. The proteins were highly homogenous with <1% and <0.1% of postfusion F conformations observed in DS and DS-Cav1, respectively. Examples of postfusion F conformations are indicated by black arrows. Bar = 50 nm. 2D particle averages are shown as insets in the top right corner at twice the magnification. Bar = 5 nm. (C) Comparison of the 2D averages with the average of F+D25 complex (3). Bar = 5 nm.

Figure S4antigenicto DNA trfollowed band 0.4 µleach welltransferreincubationimmunog

4. Immunogec analysis. 10ransfection (dby mixing wil of TrueFect-. Five days pod to 4° C. Supn at 4° C for oens.

en screen by 0 µl of 2.5 x

day 0), cultureith 20 µl of th-Max) per weost-transfectiopernatants weone week (da

96-well micr104 HEK 293e medium in ehe transfectionell. One day pon (day 5), there assessed bay 12) to asses

9

roplate-form3T cells/well weach well wasn complex (0.post-transfectihe 96-well micby ELISA immss the antigen

matted transiewere incubates replaced wi.2 µg of DNAion 20 µl of ecroplate supemediately upo

nic characteris

ent transfectied for one dayth 65 µl of fre

A encoding thenriched mediernatant was hon harvestingstics of the ex

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prior

ns ed to after

Figure S5trimer. EELISA of(Spearman

5. D25 antiboEngineered RSf the crude cun R = 0.7572

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of culture supotein variant ptants at 4 °C with the yiel

10

pernatants coproduction byone week afted of pure RSV

orrelates wity 293 Expi ceer harvesting V F trimer gly

th yield of puells was determand found to ycoprotein (T

urified RSV Fmined by D2correlate

Table 1).

F 5

11

Figure S6. Motavizumab antibody ELISA of culture supernatant and yield of purified RSV F trimer. (A) RSV F glycoprotein production by 293 Expi cells was determined by motavizumab ELISA of the crude culture supernatants at 4 °C immediately upon harvest and (B) one week after harvesting. ELISA data are plotted versus the yield of RSV F glycoprotein variants after streptactin affinity. Interestingly, three proteins, RSV F(+) Fd and two variants F137W-F140W and T357C-N371C were detected as being highly expressed by motavizumab ELISA, but low yields were obtained after large scale purification (variants displayed on the ordinate).

S190F,V296F Cav1Postfusion

TriC

S403C,T420C

K87F,V90L I506K

V185E V178N

D486H,E487Q,D489H F137W,F140W,F488W

V207L,V220L

B

RSV F glycoprotein (mg/L)

OD 450nm

Motavizumab ELISA

Cav1

PostfusionDS

TriC

S190F,V296F

S403C,T420C

K87F,V90L

I506K

V207L,V220L V185E

V178N

D486H,E487Q,D489H

F137W,F140W,F488W

A

RSV F glycoprotein (mg/L)

OD 450n

Motavizumab ELISA

12

Figure S7. Characteristics of engineered RSV F glycoproteins on size-exclusion chromatography. RSV F variants, a: Cav1, b: Cav1-TriC, c: DS-Cav1-TriC, d: F488W, e: DS-Cav1, f: TriC, g: DS-TriC, h: DS, exhibit elution profiles characteristic of a globular trimeric protein, whereas RSV F variants i: S190F,V296F, j: K87F,V90L, k: V207L,V220L, l: V178N, m: S403C,T420C, n: I506K, o: V185E, p: F137W,F140W, F488W, q: D486H,E487Q,D489H exhibit elution profiles characteristic of higher oligomeric species. Protein standards of known molecular weight are labeled on the base of the chromatogram.

A

Atomic mo Figure S8protomer protomer red (scalediagram rregions of

20

20

obility or B-fact

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8 (continued)he six RSV F of F1 are bothfor each of the137-155 are sh

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14

refusion RSVresidue numbee RSV F-trimlored tan, pintations. A sph

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15

Figure S9. Antigenicity of immunogen-adjuvant injectates concurrent with non-human primate (rhesus macaque) immunizations. RSV F postfusion, DS and DS-Cav1 injectate reactivity were assessed with Octet by capturing RSV F variant immunogens in the presence of adjuvant using motavizumab by assessing binding to 1 μM D25 Fab less than 3 h following immunogen formulation with poly ICLC and NHP immunizations at (A) day 0 and (B) week 4.

A B

A B Figure S10. Non-human primate (rhesus macaque) RSV subtype A and B neutralization titers, weeks 2-10. (A) and (B) Neutralization titers of sera from rhesus macaques immunized with 50 µg of RSV F variants at weeks 0 and 4 formulated with 500 µg poly ICLC (indicated by arrows). Notably, mean geometric neutralizing titers were maintained at over 1000 EC50 against the homologous subtype A virus and greater than 500 EC50 against heterologous subtype B (strain 18537) beyond week 10 following a boost at week 4 in animals immunized with the DS-Cav1 variant of stabilized RSV F. Dotted lines indicate the EC50 in this assay that corresponds to ~40 μg/ml of palivizumab (Synagis®), which is the serum level at trough that provides protection in infants from severe disease when dosed at 15 mg/kg.

Figure S1Comparis(B) DS-C

A

B

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Unique 16,533 Å

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DS-Cav1

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17

for DS-Cav1the calculated) comparison

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(A) dicated.

18

Figure S12. Antigenic analysis of sera from immunized mice and rhesus macaques. (A) Sera from mice immunized with multiple stabilized RSV F variants was assessed for binding to immobilized DS-Cav1 or immobilized DS-Cav1 bound by D25 or motavizumab Fabs to assess specific site Ø or specific site II responses, respectively. (B) Sera from rhesus macaques were assessed for binding to immobilized DS-Cav1 or immobilized postfusion RSV F variants bound by D25 or motavizumab Fabs. The mean response of the animal sera is shown, with error bars for the standard deviation.

A

B

19

Table S1. Antigenic characteristics of engineered RSV F glycoprotein variants.

Mechanism of stabilization RSV F variant* ELISA binding†

upon expression

ELISA binding† after 1 week at 4 °C

Mota‡ D25 Mota‡ D25 None RSV F wild type with C-terminal foldon* 2.33 1.72 2.33 0.24

Cavity filling/repacking V56L, T58I, L158W, V164L, I167V, L171I, V179L, L181F, V187I, I291V, V296L 0.05 0.05 0.08 0.05

V56L, T58I, L158Y, V164L, I167V, V187I, T189F, I291V, V296L 0.06 0.06 0.10 0.04

V56I, T58I, V164I, V179L, T189F, I291V, V296I, A298I 0.15 0.08 0.10 0.04

V56I, T58I, V164I, L171I, V179L, L181F, V187I, I291V, V296I, A298I 0.06 0.05 0.08 0.06

V56I, T58W, V164I, I167F, L171I, V179L, L181V, V187I, I291V, V296I 0.05 0.04 0.06 0.05

V56I, T58I, I64L, I79V, Y86W, V164I, V179L, T189F, L193V, L195F, Y198F, I199F, L203F, V207L, I214L, I291V, V296I, A298I 0.10 0.05 0.10 0.05

V56I, T58W, I64L, I79V, Y86W, V164I, I167F, L171I, V179L, L181V, V187I, L193V, L195F, Y198F, I199F, L203F, V207L, I214L, I291V, V296I 0.06 0.05

0.07 0.05

V56I, T58I, I79V, Y86F, V164I, V179L, T189F, L193V, L195F, Y198F, I199F, L203F, V207L, I214L, I291V, V296I, A298I 0.10 0.05 0.11 0.05

V56I, T58W, I79V, Y86F, V164I, I167F, L171I, V179L, L181V, V187I, L193V, L195F, Y198F, I199F, L203F, V207L, I214L, I291V, V296I 0.06 0.05

0.09 0.05

T58W, A298L 0.37 0.18 0.29 0.07

I64L, I79L, Y86W, L193V, L195F, Y198F, I199F, L203F, I214L 0.08 0.05 0.09 0.04

I64V, I79V, Y86W, L193V, L195F, Y198F, I199Y, L203F, V207L, I214L 0.05 0.04 0.06 0.05

I64F, I79V, Y86W, L193V, L195F, Y198F, I199F, L203F, V207L, I214L 0.08 0.04 0.11 0.05

I64L, I79V, Y86W, L193V, L195F, I199F, L203F, V207L, I214L 0.06 0.05 0.09 0.05

I64L, I79V, Y86W, L193V, L195F, Y198F, I199F, L203F, V207L, I214L 0.08 0.05 0.10 0.04

I64W, I79V, Y86W, L193V, L195F, Y198F, I199F, L203F, V207L, I214L 0.07 0.05 0.09 0.04

I79V, Y86F, L193V, L195F, Y198F, I199F, L203F, V207L, I214L 0.26 0.06 0.23 0.05

K87F, V90L 1.55 1.70 2.17 0.27

F137W, F140W 2.93 1.31 1.47 0.13

F137W, F140W, F488W 3.44 2.56 1.77 0.18

F137W, R339M 0.76 0.10 0.58 0.10

F137W, F140W, R339M , L375W, Y391F, K394M 0.06 0.05 0.06 0.05

F137W, F140W, L375W, Y391F, K394M 0.08 0.05 0.06 0.05

F137W, F140W, T400V, D486L, E487L, D489L 0.27 0.13 0.17 0.09

F137W, F140W, D486H, E487Q, D489H 2.85 1.82 0.61 0.15

F137W, F140W, D486N, E487Q, D489N, S491A 0.31 0.12 0.18 0.07

S190F, V207L 1.95 2.30 3.63 4.00

S190F, V296F 1.72 2.47 3.42 2.33

V207L, V220L 1.61 1.60 3.12 0.67

V220L, A153W 0.12 0.08 0.10 0.06

L260W, L83W 0.06 0.08 0.08 0.05

L375W, Y391F, K394M 0.08 0.05 0.07 0.05

L375W, Y391F, K394W 0.07 0.05 0.07 0.05

L375W, Y391F, K394M, D486N, E487Q, D489N, S491A 0.06 0.05 0.09 0.05

L375W, Y391F, K394M, D486H, E487Q, D489H 0.06 0.05 0.07 0.05

L375W, Y391F, K394W, D486N, E487Q, D489N, S491A 0.06 0.05 0.07 0.04

L375W, Y391F, K394W, D486H, E487Q, D489H 0.06 0.05 0.06 0.05

L375W, Y391F, K394M, T400V, D486L, E487L, D489L, Q494L, K498M 0.06 0.04 0.05 0.05

L375W, Y391F, K394M, T400V, D486I, E487L, D489I, Q494L, K498M 0.06 0.05 0.06 0.06

L375W, Y391F, K394W, T400V, D486L, E487L, D489L, Q494L, K498M 0.07 0.05 0.05 0.05

L375W, Y391F, K394W, T400V, D486I, E487L, D489I, Q494L, K498M 0.08 0.05 0.06 0.06

K399I, T400V, S485I, D486L, E487L, D489L, Q494L, E497L, K498L 0.05 0.04 NA NA

K399I, T400V, S485I, D486I, E487L, D489I, Q494L, E497L, K498L 0.05 0.04 NA NA

T400V, D486L, E487L, D489L 0.16 0.09 0.13 0.07

T400V, D486I, E487L, D489I 0.27 0.12 NA NA

T400V, D486L, E487L, F488W, D489L 0.44 0.10 0.28 0.12

T400V, D486I, E487L, F488W, D489I 0.27 0.17 0.16 0.12

* All mutations were assessed on wild type RSV F with a C-terminal foldon trimerization domain. † Optical density at 450 nm assessed in a 96-well format as described in Figure S4. ‡ Motavizumab (Mota).

20

Table S1 (continued). Antigenic characteristics of engineered RSV F glycoprotein variants.

Mechanism of stabilization RSV F variant* ELISA binding†

upon expression

ELISA binding† after 1 week at 4 °C

Mota‡ D25 Mota‡ D25 Cavity filling/repacking T400V, S485I, D486L, E487L, D489L, Q494L, K498L 0.05 0.04 NA NA

T400V, S485I, D486I, E487L, D489I, Q494L, K498L 0.05 0.04 NA NA

D486H, E487Q, D489H 1.46 1.68 1.21 0.48

D486H, E487Q, F488W, D489H 3.33 2.17 2.31 1.27

D486N, E487Q, D489N, S491A 0.08 0.07 0.11 0.05

F488W, D486N, E487Q, D489N, S491A 0.54 0.31 0.26 0.15

Disulfide (intrachain) E30C, L410C 0.06 0.05 0.08 0.05

Y33C, V469C 0.07 0.06 0.07 0.05

S46C, T311C 0.07 0.05 0.10 0.05

L48C, V308C 0.06 0.05 0.07 0.05

T54C, V154C 1.88 0.71 1.76 0.45

I59C, V192C 0.06 0.05 0.07 0.05

E60C, D194C 0.07 0.06 0.07 0.05

F137C, T337C 0.11 0.10 0.12 0.06

L138C, P353C 0.05 0.06 0.06 0.05

G151C, I288C 0.86 0.10 2.31 0.13

V154C, S290C 0.05 0.06 0.06 0.05

S155C, S290C 2.37 2.33 3.33 3.06

K156C, S290C 0.05 0.06 0.08 0.05

I288C, V300C 0.06 0.05 0.08 0.05

S319C, I413C 0.07 0.05 0.06 0.05

S319C, S415C 0.10 0.06 0.09 0.06

P320C, T335C 0.07 0.05 0.08 0.04

P320C, S415C 0.08 0.06 0.09 0.06

L321C, L334C 0.06 0.05 0.07 0.06

N331C, D401C 0.74 0.10 0.60 0.07

D338C, K394C 0.06 0.05 0.06 0.05

W341C, F352C 0.06 0.05 0.06 0.04

T357C, N371C 2.64 1.78 1.07 0.16

L381C, N388C 0.48 0.26 0.18 0.05

L381C, Y391C 0.09 0.05 0.09 0.05

T397C, E487C 0.34 0.19 0.43 0.11

D401C, Y417C 0.91 0.22 0.71 0.08

S403C, Y417C 0.06 0.05 0.07 0.05

S403C, T420C 2.43 1.60 0.67 0.16

V406C, I413C 0.07 0.05 0.07 0.05 Disulfide (intrachain) with insertions S430C, Cys-Ala-Ala inserted between 329 and 330 0.34 0.07 0.11 0.05

Disulfide (inter-protomer) A74C, E218C 0.06 0.05 0.08 0.05

G143C, S404S 0.05 0.05 0.06 0.05

V144C, V406C 0.06 0.05 0.06 0.05

S146C, I407C 0.07 0.05 0.08 0.04

A149C, Y458C 0.05 0.05 0.07 0.05

A153C, K461C 0.06 0.05 0.11 0.05

A346C, N454C 0.06 0.05 0.06 0.05

T369C, T455C 0.06 0.05 0.06 0.04

T374C, N454C 0.05 0.05 0.07 0.04

K399C, Q494C 0.08 0.05 0.07 0.05

T400C, D489C 0.35 0.10 0.25 0.06

V402C L141C 0.12 0.07 0.12 0.12

* All mutations were assessed on wild type RSV F with a C-terminal foldon trimerization domain. † Optical density at 450 nm assessed in a 96-well format as described in Figure S4. ‡ Motavizumab (Mota).

21

Table S1 (continued). Antigenic characteristics of engineered RSV F glycoprotein variants.

Mechanism of stabilization RSV F variant* ELISA binding†

upon expression

ELISA binding† after 1 week at 4 °C

Mota‡ D25 Mota‡ D25 Disulfide (inter-protomer) with insertions S146C, N460C, Ala-Ala inserted between 146 and 147 0.16 0.06 0.12 0.08

N183C, N428C, Gly inserted between 182 and 183 0.05 0.05 0.08 0.05

N183C, K427G, Cys inserted between 427 and 428 0.05 0.05 0.07 0.04

N183C, K423C, Ala-Ala-Ala inserted between 182 and 183 0.06 0.05 0.10 0.05

Destabilization of postfusion form L160K 1.73 1.21 3.32 0.43

V178K 1.76 1.06 2.61 0.17

V185E 1.70 1.57 3.21 0.52

V192S 0.07 0.06 0.09 0.05

L305S, I64S, S155A 0.25 0.08 NA NA

L305S, I64S, S155A, D486V, E487L, D489V 0.05 0.04 0.06 0.05

D486V, E487L, D489V 0.53 0.25 0.40 0.16

L503E 1.33 0.39 1.26 0.15

I506K 1.93 1.80 3.17 0.40 Destabilization of postfusion form by glycan addition V157N 0.76 0.11 0.47 0.06

L160N, G162S 1.57 0.27 2.89 0.10

V178N 2.04 1.73 3.37 0.42

A177S 1.65 0.41 2.85 0.14

V185N, V187T 0.06 0.07 0.07 0.05

Y478T 0.08 0.09 0.09 0.06

L503N, F505S 0.99 0.18 1.73 0.13

I506N, K508T 1.78 0.50 3.31 0.24

* All mutations were assessed on wild type RSV F with a C-terminal foldon trimerization domain. † Optical density at 450 nm assessed in a 96-well format as described in Figure S4. ‡ Motavizumab (Mota).

22

Table S2. Crystallographic data collection and refinement statistics. DS (pH9.5) Cav1 (pH9.5) Cav1 (pH5.5) DS-Cav1

(pH9.5) DS-Cav1 (pH5.5)

DS-Cav1-TriC (pH9.5)

PDB ID 4MMQ 4MMR 4MMS 4MMT 4MMU 4MMV Data collection Space group P4132 P4132 P41212 P4132 P4132 P4132 Cell constants a, b, c (Å) α, β, γ (°)

168.4, 168.4, 168.4

90.0, 90.0, 90.0

170.9, 170.9, 170.9

90.0, 90.0, 90.0

170.7, 170.7, 163.9

90.0, 90.0, 90.0

170.7, 170.7, 170.7

90.0, 90.0, 90.0

168.6, 168.6, 168.6

90.0, 90.0, 90.0

170.4, 170.4, 170.4

90.0, 90.0, 90.0 Wavelength (Å) 1.00 1.00 1.00 1.00 1.00 1.00 Resolution (Å) 50-3.25

(3.31-3.25) 50-3.1

(3.21-3.1) 50-2.40

(2.49-2.40) 50-3.05

(3.16-3.05) 50-3.0

(3.11-3.0) 50-2.8

(2.90-2.80) Rmerge 14.8 (64.3) 20.3 (70.7) 11.1 (59.8) 14.0 (76.3) 13.0 (79.3) 13.0 (92.6) I / σI 14.8 (2.2) 11.8 (1.5) 12.6 (2.1) 11.4 (2.1) 22.4 (2.2) 42.9 (1.8) Completeness (%) 99.6 (96.4) 99.1 (91.7) 95.6 (97.7) 96.9 (97.6) 99.8 (98.1) 99.9 (99.4) Redundancy 8.3 (4.1) 11.7 (3.8) 4.4 (4.0) 6.6 (4.6) 16.3 (7.5) 12.6 (6.2) Refinement Resolution (Å) 3.25 3.10 2.40 3.05 3.00 2.80 Unique reflections 13,345 15,925 90,779 16,252 16,999 21,005 Rwork ,Rfree (%) 23.7, 27.4 20.6, 23.7 18.7, 21.4 20.1, 23.5 18.6, 23.9 22.5, 25.9 No. atoms Protein 3033 3546 10,421 3510 3526 3771 Ligand/ion 30 - 70 - 156 - Water - 39 522 - 108 69 B-factors (Å2) Protein 75.1 114.9 56.4 103.8 76.3 106.9 Ligand/ion 136.4 - 101.5 - 155.8 - Water - 87.6 48.5 - 63.4 80.0 R.m.s. deviations Bond lengths (Å) 0.008 0.012 0.003 0.003 0.010 0.005 Bond angles (°) 1.35 1.24 0.84 1.32 1.42 0.93 Ramachandran Favored (%) 95.8 95.3 95.1 95.3 93.3 96.2 Allowed (%) 3.9 4.3 4.5 4.3 6.0 3.3 Disallowed (%) 0.3 0.4 0.4 0.4 0.7 0.5 Values in parentheses are for highest-resolution shell.

23

Table S3. Antigenic analysis of NHP sera (week 6) and calculated immunogen-surface area.

Readout (nm)

Sera Quenching Readout Calculated surface

area (Å2)

Ds-Cav1 DS Postfusion

Postfusion - DS-Cav1 76742 1.80 Postfusion Postfusion DS-Cav1 0 0.10 Postfusion DS DS-Cav1 0 0.08 Postfusion DS-Cav1 DS-Cav1 0 0.07

DS - DS-Cav1 127734 3.02 DS Postfusion DS-Cav1 51392 1.21 DS DS DS-Cav1 0 0.52 DS DS-Cav1 DS-Cav1 0 0.09

DS-Cav1 - DS-Cav1 144631 3.17 DS-Cav1 Postfusion DS-Cav1 55820 1.89* DS-Cav1 DS DS-Cav1 16500 1.41* DS-Cav1

DS-Cav1

DS-Cav1

0

0.07

Postfusion - DS 76742 1.97 Postfusion Postfusion DS 0 0.09 Postfusion DS DS 0 0.07 Postfusion DS-Cav1 DS 0 0.23

DS - DS 130581 2.26 DS Postfusion DS 54239 0.84 DS DS DS 0 0.09 DS DS-Cav1 DS 2847 0.31

DS-Cav1 - DS 127734 1.51† DS-Cav1 Postfusion DS 36866 0.85 DS-Cav1 DS DS 0 0.07 DS-Cav1

DS-Cav1

DS

0

0.25

Postfusion - Postfusion 130104 2.92 Postfusion Postfusion Postfusion 0 0.07 Postfusion DS Postfusion 53366 0.86 Postfusion DS-Cav1 Postfusion 53366 0.96

DS - Postfusion 76742 1.71 DS Postfusion Postfusion 0 0.07 DS DS Postfusion 0 0.14 DS DS-Cav1 Postfusion 0 0.07

DS-Cav1 - Postfusion 76742 0.57‡ DS-Cav1 Postfusion Postfusion 0 0.07 DS-Cav1 DS Postfusion 0 0.09 DS-Cav1

DS-Cav1

Postfusion

0

0.07

*DS-Cav1 sera quenched by either postfusion or DS protein gives higher than expected binding responses indicating that the DS-Cav1 unique surface areas generate greater immunogenicity than expected. †DS-Cav1 sera binding to DS, is lower than expected (given DS and DS-Cav1 structural similarities) indicating that DS-Cav1 elicits more antibodies than expected to non-DS regions, i.e. site Ø antibodies. ‡DS-Cav1 sera binding to postfusion is significantly lower than expected (given that DS-Cav1 and postfusion have a large common surface area). This indicates that the DS-Cav1 immunogen elicits more antibodies to non-postfusion regions of RSV F. Octet response units are a measure of the change in wavelength (nm) of the measured white light interference pattern compared to an internal control and are proportional to binding.

24

Table S4. Reciprocal serum dilution associated with 50% virus neutralization (EC50) for individual animals (Rhesus macaques) at each time point (weeks 2-14). RSV subtype A Week 2 Week 4 Week 6 Week 8 Week 10 Week 14

Postfusion

5

5

666

793

619

131 10

5 5

5 5 5

355 78 49

276 36 13

193 63 52

171 12 13

DS 5

5 5 5

5 5 5 9

1418 2395

556 520

1220 2203

792 2080

739 1482

373 510

347 518 169 263

DS-Cav1 81 114 3336 5295 1792 736 41

18 19

110 10 10

2516 2160 2298

3776 7168

13549

903 1037

688

396 437 550

RSV subtype B

Week 2 Week 4 Week 6 Week 8 Week 10 Week 14

Postfusion

5

10

640

135

251

105 10

5 5

10 10 10

160 56 14

72 5 5

26 4 5

60 5 5

DS 5

5 5 5

10 10 10 10

602 1214

134 324

232 281 177 507

296 313

57 70

323 191

80 85

DS-Cav1 134 160 1375 1111 976 605 40

10 10

57 10 10

1360 1770 1914

1088 3220 3474

239 529 466

211 238 458

25

References and Notes 1. R. Lozano, M. Naghavi, K. Foreman, S. Lim, K. Shibuya, V. Aboyans, J. Abraham,

T. Adair, R. Aggarwal, S. Y. Ahn, M. Alvarado, H. R. Anderson, L. M. Anderson, K. G. Andrews, C. Atkinson, L. M. Baddour, S. Barker-Collo, D. H. Bartels, M. L. Bell, E. J. Benjamin, D. Bennett, K. Bhalla, B. Bikbov, A. Bin Abdulhak, G. Birbeck, F. Blyth, I. Bolliger, S. Boufous, C. Bucello, M. Burch, P. Burney, J. Carapetis, H. Chen, D. Chou, S. S. Chugh, L. E. Coffeng, S. D. Colan, S. Colquhoun, K. E. Colson, J. Condon, M. D. Connor, L. T. Cooper, M. Corriere, M. Cortinovis, K. C. de Vaccaro, W. Couser, B. C. Cowie, M. H. Criqui, M. Cross, K. C. Dabhadkar, N. Dahodwala, D. De Leo, L. Degenhardt, A. Delossantos, J. Denenberg, D. C. Des Jarlais, S. D. Dharmaratne, E. R. Dorsey, T. Driscoll, H. Duber, B. Ebel, P. J. Erwin, P. Espindola, M. Ezzati, V. Feigin, A. D. Flaxman, M. H. Forouzanfar, F. G. Fowkes, R. Franklin, M. Fransen, M. K. Freeman, S. E. Gabriel, E. Gakidou, F. Gaspari, R. F. Gillum, D. Gonzalez-Medina, Y. A. Halasa, D. Haring, J. E. Harrison, R. Havmoeller, R. J. Hay, B. Hoen, P. J. Hotez, D. Hoy, K. H. Jacobsen, S. L. James, R. Jasrasaria, S. Jayaraman, N. Johns, G. Karthikeyan, N. Kassebaum, A. Keren, J. P. Khoo, L. M. Knowlton, O. Kobusingye, A. Koranteng, R. Krishnamurthi, M. Lipnick, S. E. Lipshultz, S. L. Ohno, J. Mabweijano, M. F. MacIntyre, L. Mallinger, L. March, G. B. Marks, R. Marks, A. Matsumori, R. Matzopoulos, B. M. Mayosi, J. H. McAnulty, M. M. McDermott, J. McGrath, G. A. Mensah, T. R. Merriman, C. Michaud, M. Miller, T. R. Miller, C. Mock, A. O. Mocumbi, A. A. Mokdad, A. Moran, K. Mulholland, M. N. Nair, L. Naldi, K. M. Narayan, K. Nasseri, P. Norman, M. O’Donnell, S. B. Omer, K. Ortblad, R. Osborne, D. Ozgediz, B. Pahari, J. D. Pandian, A. P. Rivero, R. P. Padilla, F. Perez-Ruiz, N. Perico, D. Phillips, K. Pierce, C. A. Pope III, E. Porrini, F. Pourmalek, M. Raju, D. Ranganathan, J. T. Rehm, D. B. Rein, G. Remuzzi, F. P. Rivara, T. Roberts, F. R. De León, L. C. Rosenfeld, L. Rushton, R. L. Sacco, J. A. Salomon, U. Sampson, E. Sanman, D. C. Schwebel, M. Segui-Gomez, D. S. Shepard, D. Singh, J. Singleton, K. Sliwa, E. Smith, A. Steer, J. A. Taylor, B. Thomas, I. M. Tleyjeh, J. A. Towbin, T. Truelsen, E. A. Undurraga, N. Venketasubramanian, L. Vijayakumar, T. Vos, G. R. Wagner, M. Wang, W. Wang, K. Watt, M. A. Weinstock, R. Weintraub, J. D. Wilkinson, A. D. Woolf, S. Wulf, P. H. Yeh, P. Yip, A. Zabetian, Z.-J. Zheng, A. D. Lopez, C. J. Murray, M. A. AlMazroa, Z. A. Memish, Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: A systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2095–2128 (2012).

2. S. Johnson, C. Oliver, G. A. Prince, V. G. Hemming, D. S. Pfarr, S. C. Wang, M. Dormitzer, J. O’Grady, S. Koenig, J. K. Tamura, R. Woods, G. Bansal, D. Couchenour, E. Tsao, W. C. Hall, J. F. Young, Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J. Infect. Dis. 176, 1215–1224 (1997).

3. The IMpact-RSV Study Group, Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics 102, 531–537 (1998).

26

4. L. J. Earp, S. E. Delos, H. E. Park, J. M. White, The many mechanisms of viral membrane fusion proteins. Curr. Top. Microbiol. Immunol. 285, 25–66 (2005).

5. J. S. McLellan, Y. Yang, B. S. Graham, P. D. Kwong, Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J. Virol. 85, 7788–7796 (2011).

6. K. A. Swanson, E. C. Settembre, C. A. Shaw, A. K. Dey, R. Rappuoli, C. W. Mandl, P. R. Dormitzer, A. Carfi, Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl. Acad. Sci. U.S.A. 108, 9619–9624 (2011).

7. J. S. McLellan, M. Chen, S. Leung, K. W. Graepel, X. Du, Y. Yang, T. Zhou, U. Baxa, E. Yasuda, T. Beaumont, A. Kumar, K. Modjarrad, Z. Zheng, M. Zhao, N. Xia, P. D. Kwong, B. S. Graham, Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113–1117 (2013).

8. L. Liljeroos, M. A. Krzyzaniak, A. Helenius, S. J. Butcher, Architecture of respiratory syncytial virus revealed by electron cryotomography. Proc. Natl. Acad. Sci. U.S.A. 110, 11133–11138 (2013).

9. L. J. Anderson, P. R. Dormitzer, D. J. Nokes, R. Rappuoli, A. Roca, B. S. Graham, Strategic priorities for respiratory syncytial virus (RSV) vaccine development. Vaccine 31 (Suppl. 2), B209–B215 (2013).

10. M. Magro, V. Mas, K. Chappell, M. Vázquez, O. Cano, D. Luque, M. C. Terrón, J. A. Melero, C. Palomo, Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention. Proc. Natl. Acad. Sci. U.S.A. 109, 3089–3094 (2012).

11. H. Spits, T. Beaumont, RSV-specific binding molecules and means for producing them. Patent Application 12/600,950 (2010).

12. T. Beaumont, A. Q. Bakker, E. Yasuda, RSV specific binding molecule. Patent Application 12/898,325 (2012).

13. D. R. Burton, Antibodies, viruses and vaccines. Nat. Rev. Immunol. 2, 706–713 (2002).

14. T. Zhou, L. Xu, B. Dey, A. J. Hessell, D. Van Ryk, S. H. Xiang, X. Yang, M. Y. Zhang, M. B. Zwick, J. Arthos, D. R. Burton, D. S. Dimitrov, J. Sodroski, R. Wyatt, G. J. Nabel, P. D. Kwong, Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 445, 732–737 (2007).

15. G. Ofek, F. J. Guenaga, W. R. Schief, J. Skinner, D. Baker, R. Wyatt, P. D. Kwong, Elicitation of structure-specific antibodies by epitope scaffolds. Proc. Natl. Acad. Sci. U.S.A. 107, 17880–17887 (2010).

16. J. Jardine, J. P. Julien, S. Menis, T. Ota, O. Kalyuzhniy, A. McGuire, D. Sok, P. S. Huang, S. MacPherson, M. Jones, T. Nieusma, J. Mathison, D. Baker, A. B. Ward, D. R. Burton, L. Stamatatos, D. Nemazee, I. A. Wilson, W. R. Schief, Rational HIV immunogen design to target specific germline B cell receptors. Science 340, 711–716 (2013).

27

17. A. T. McGuire, S. Hoot, A. M. Dreyer, A. Lippy, A. Stuart, K. W. Cohen, J. Jardine, S. Menis, J. F. Scheid, A. P. West, W. R. Schief, L. Stamatatos, Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies. J. Exp. Med. 210, 655–663 (2013).

18. J. S. McLellan, M. Chen, A. Kim, Y. Yang, B. S. Graham, P. D. Kwong, Structural basis of respiratory syncytial virus neutralization by motavizumab. Nat. Struct. Mol. Biol. 17, 248–250 (2010).

19. J. S. McLellan, B. E. Correia, M. Chen, Y. Yang, B. S. Graham, W. R. Schief, P. D. Kwong, Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus. J. Mol. Biol. 409, 853–866 (2011).

20. L. Kong, J. H. Lee, K. J. Doores, C. D. Murin, J. P. Julien, R. McBride, Y. Liu, A. Marozsan, A. Cupo, P. J. Klasse, S. Hoffenberg, M. Caulfield, C. R. King, Y. Hua, K. M. Le, R. Khayat, M. C. Deller, T. Clayton, H. Tien, T. Feizi, R. W. Sanders, J. C. Paulson, J. P. Moore, R. L. Stanfield, D. R. Burton, A. B. Ward, I. A. Wilson, Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120. Nat. Struct. Mol. Biol. 20, 796–803 (2013).

21. V. P. Efimov, I. V. Nepluev, B. N. Sobolev, T. G. Zurabishvili, T. Schulthess, A. Lustig, J. Engel, M. Haener, U. Aebi, S. Y. Venyaminov, S. A. Potekhin, V. V. Mesyanzhinov, Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure. J. Mol. Biol. 242, 470–486 (1994).

22. K. A. Miroshnikov, E. I. Marusich, M. E. Cerritelli, N. Cheng, C. C. Hyde, A. C. Steven, V. V. Mesyanzhinov, Engineering trimeric fibrous proteins based on bacteriophage T4 adhesins. Protein Eng. 11, 329–332 (1998).

23. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

24. Information on materials and methods is available on Science Online. 25. RSV F variants were assessed by transient expression in 96-well format (fig. S4). If

supernatants retained reactivity with antibodies motavizumab and D25 after 1 week at 4°C, they were expressed by transient transfection of 1 l of Expi293F cells, purified by use of appended His-tag and StreptagII, and analyzed by size-exclusion chromatography (fig. S7).

26. The inability to express potential inter-chain double cysteine substitutions, despite reasonable modeling in the mature prefusion F1F2 structure, may indicate that RSV F0 protomers, before cleavage and removal of peptide 27, adopt substantially different inter-protomer conformations.

27. Cavities in the D25-bound RSV F structure were visualized with PyMol using the “Cavities & Pockets Only” option for Surface settings. Amino acid substitutions designed to fill the resulting cavities were identified using the Mutagenesis wizard.

28. M. Pancera, Y. Yang, M. K. Louder, J. Gorman, G. Lu, J. S. McLellan, J. Stuckey, J. Zhu, D. R. Burton, W. C. Koff, J. R. Mascola, P. D. Kwong, N332-Directed broadly

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neutralizing antibodies use diverse modes of HIV-1 recognition: Inferences from heavy-light chain complementation of function. PLoS ONE 8, e55701 (2013).

29. The engineered RSV F variants had Cα-root mean square deviations from the D25-bound conformation of 0.7 to 1.5 Å and from the postfusion conformation of approximately 30 Å.

30. Crystallized DS retained ~20% of its D25 recognition relative to crystallized Cav1 (normalized to motavizumab recognition) after 4 months at 20°C, thereby indicating that the crystallized DS was both capable of recognizing D25 as well as converting to a conformation incompatible with D25 binding. By contrast, soluble DS lost all recognition of D25 after 2 months of incubation at 4°C in PBS. We were unable to crystallize DS, which had been heat triggered at 50°C, despite the heat-triggered DS retaining a trimeric state on size exclusion chromatography.

31. Observing Ala107 rather than Ag109 at the C terminus of F2 is not totally unexpected: Garten and Klenk (48) found that an arginine at the cleavage site of influenza hemagglutinin was trimmed by a cellular carboxypeptidase activity.

32. Cα-root mean square deviations of 0.86 Å for 447 residues between Cav1 and DS-Cav1 crystal structures obtained from ammonium sulfate; Cα-root mean square deviations of 0.47 Å for 447 residues of DS-Cav1 in cubic lattices.

33. J. Stetefeld, S. Frank, M. Jenny, T. Schulthess, R. A. Kammerer, S. Boudko, R. Landwehr, K. Okuyama, J. Engel, Collagen stabilization at atomic level: Crystal structure of designed (GlyProPro)10foldon. Structure 11, 339–346 (2003).

34. When palivizumab (Synagis) is dosed at a concentration of 15 mg/kg, serum levels at trough are ~40 μg/ml, which provides protection in infants from severe disease and protection in cotton rats from RSV infection. In our neutralization assay, ~40 ug/ml of palivizumab yields an EC50 of 100.

35. G. J. Nabel, Philosophy of science. The coordinates of truth. Science 326, 53–54 (2009).

36. By contrast, affinity of RSV F variants for D25 antibody did not show correlation with elicitation of protective titers. Yield of RSV F trimers also did not correlate with protective titers.

37. Flexibility of an antigenic site may increase its immunogenicity by allowing the site to conform to a wider diversity of antibodies. We note in this context that the atomic-mobility factors of antigenic site Ø were among the highest in the RSV F ectodomain.

38. For the “prefusion” form of RSV F, we used the DS-Cav1 stabilized variant of RSV F.

39. It should be possible to deconvolute the elicited response, by using structurally defined probes, as shown with D25 and motavizumab-bound RSV F in fig. S12.

40. P. R. Dormitzer, G. Grandi, R. Rappuoli, Structural vaccinology starts to deliver. Nat. Rev. Microbiol. 10, 807–813 (2012).

41. M. L. Azoitei, B. E. Correia, Y. E. Ban, C. Carrico, O. Kalyuzhniy, L. Chen, A. Schroeter, P. S. Huang, J. S. McLellan, P. D. Kwong, D. Baker, R. K. Strong, W. R.

29

Schief, Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold. Science 334, 373–376 (2011).

42. I. Delany, R. Rappuoli, K. L. Seib, Vaccines, reverse vaccinology, and bacterial pathogenesis. Cold Spring Harb. Perspect. Med. 3, a012476 (2013).

43. A. Sette, R. Rappuoli, Reverse vaccinology: Developing vaccines in the era of genomics. Immunity 33, 530–541 (2010).

44. D. G. Moriel, I. Bertoldi, A. Spagnuolo, S. Marchi, R. Rosini, B. Nesta, I. Pastorello, V. A. Corea, G. Torricelli, E. Cartocci, S. Savino, M. Scarselli, U. Dobrindt, J. Hacker, H. Tettelin, L. J. Tallon, S. Sullivan, L. H. Wieler, C. Ewers, D. Pickard, G. Dougan, M. R. Fontana, R. Rappuoli, M. Pizza, L. Serino, Identification of protective and broadly conserved vaccine antigens from the genome of extraintestinal pathogenic Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 107, 9072–9077 (2010).

45. M. Mora, C. Donati, D. Medini, A. Covacci, R. Rappuoli, Microbial genomes and vaccine design: Refinements to the classical reverse vaccinology approach. Curr. Opin. Microbiol. 9, 532–536 (2006).

46. While an epitope-scaffold strategy may not elicit titers as high as a neutralization-sensitive site strategy, for specific antibodies, such as antibody MPE8 (49), which is capable of neutralizing not only RSV, but other paramyxoviruses including human metapneumovirus, an epitope-specific strategy may suffice.

47. The magnitude of antibody activity is a crucial determinant of how well an individual will be protected following vaccination and how long protective responses will be maintained in infants who have passively acquired antibody from their mothers.

48. W. Garten, H. D. Klenk, Characterization of the carboxypeptidase involved in the proteolytic cleavage of the influenza haemagglutinin. J. Gen. Virol. 64, 2127–2137 (1983).

49. D. Corti, S. Bianchi, F. Vanzetta, A. Minola, L. Perez, G. Agatic, B. Guarino, C. Silacci, J. Marcandalli, B. J. Marsland, A. Piralla, E. Percivalle, F. Sallusto, F. Baldanti, A. Lanzavecchia, Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439–443 (2013).

50. B. S. Graham, M. D. Perkins, P. F. Wright, D. T. Karzon, Primary respiratory syncytial virus infection in mice. J. Med. Virol. 26, 153–162 (1988).

51. A. L. Hotard, F. Y. Shaikh, S. Lee, D. Yan, M. N. Teng, R. K. Plemper, J. E. Crowe Jr., M. L. Moore, A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis. Virology 434, 129–136 (2012).

52. J. S. McLellan, M. Chen, J. S. Chang, Y. Yang, A. Kim, B. S. Graham, P. D. Kwong, Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F. J. Virol. 84, 12236–12244 (2010).

53. J. S. McLellan, M. Pancera, C. Carrico, J. Gorman, J.-P. Julien, R. Khayat, R. Louder, R. Pejchal, M. Sastry, K. Dai, S. O’Dell, N. Patel, S. Shahzad-ul-Hussan, Y.

30

Yang, B. Zhang, T. Zhou, J. Zhu, J. C. Boyington, G. Y. Chuang, D. Diwanji, I. Georgiev, Y. D. Kwon, D. Lee, M. K. Louder, S. Moquin, S. D. Schmidt, Z.-Y. Yang, M. Bonsignori, J. A. Crump, S. H. Kapiga, N. E. Sam, B. F. Haynes, D. R. Burton, W. C. Koff, L. M. Walker, S. Phogat, R. Wyatt, J. Orwenyo, L. X. Wang, J. Arthos, C. A. Bewley, J. R. Mascola, G. J. Nabel, W. R. Schief, A. B. Ward, I. A. Wilson, P. D. Kwong, Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature 480, 336–343 (2011).

54. S. Frank, R. A. Kammerer, D. Mechling, T. Schulthess, R. Landwehr, J. Bann, Y. Guo, A. Lustig, H. P. Bächinger, J. Engel, Stabilization of short collagen-like triple helices by protein engineering. J. Mol. Biol. 308, 1081–1089 (2001).

55. R. van Reis, J. M. Brake, J. Charkoudian, D. B. Burns, A. L. Zydney, High-performance tangential flow filtration using charged membranes. J. Membr. Sci. 159, 133–142 (1999).

56. Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

57. A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni, R. J. Read, Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

58. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

59. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L. W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart, PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

60. J. B. Heymann, D. M. Belnap, Bsoft: Image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007).

61. S. J. Ludtke, P. R. Baldwin, W. Chiu, EMAN: Semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

62. B. J. Flynn, K. Kastenmüller, U. Wille-Reece, G. D. Tomaras, M. Alam, R. W. Lindsay, A. M. Salazar, B. Perdiguero, C. E. Gomez, R. Wagner, M. Esteban, C. G. Park, C. Trumpfheller, T. Keler, G. Pantaleo, R. M. Steinman, R. Seder, Immunization with HIV Gag targeted to dendritic cells followed by recombinant New York vaccinia virus induces robust T-cell immunity in nonhuman primates. Proc. Natl. Acad. Sci. U.S.A. 108, 7131 (2011).

63. M. R. Rosenfeld, M. C. Chamberlain, S. A. Grossman, D. M. Peereboom, G. J. Lesser, T. T. Batchelor, S. Desideri, A. M. Salazar, X. Ye, A multi-institution phase II study of poly-ICLC and radiotherapy with concurrent and adjuvant temozolomide in adults with newly diagnosed glioblastoma. Neuro Oncol. 12, 1071 (2010).