Vaccination is a proven, safe and cost-effective way to protect against infectious disease1,2, but potentially vaccine-preventable illnesses continue to place a heavy burden on the human population. Data from recent epidemiological studies indicate that in 2010, infectious diseases caused 18.5% of all human deaths and 23% of disability-adjusted life years3,4. This burden could be reduced by broader deployment and use of existing vaccines or by other prevention modalities or treatment regimens. However, for maximal, affordable and sustainable gains in global health, new or improved vaccines are needed for several major pathogens including human immunodeficiency virus (HIV)-1 (ref. 5), malaria6, Mycobacterium tuberculosis7, influenza virus8, dengue virus9 and respiratory syncytial virus (RSV)10. One likely impediment to vaccine development in these cases is the limited set of antigen design or presentation methods available to vaccine engineers. For example, current licensed vaccines in the United States11 derive from strategies that have been available for many years: viral vaccines are composed of recombinant virus-like particles or live, live-attenuated or whole inactivated viruses or subunit vaccines, and bacterial vaccines are composed of bacterial surface proteins, detoxified toxins or polysaccharides with or without conjugation to a carrier protein.

Epitope-focused vaccine design is a conceptually appealing but unproven method in which immunogens are designed to elicit protective antibody responses against structural epitopes that are defined by protective antibodies isolated from infected patients or animal models12. This strategy, if validated, could offer a potential route to vaccines for many pathogens that have resisted traditional vaccine development, including highly antigenically variable viruses such as HIV, influenza and hepatitis C virus, for which broadly neutralizing antibodies have been discovered and characterized structurally with their target epitopes13. We tested the feasibility of this strategy using an epitope from RSV, a virus that causes lower respiratory tract infections in children and the elderly. In 2010 RSV was estimated to be responsible for 6.7% of all deaths in children of ages 1 month to 1 year3. We focused on the epitope targeted by the licensed, prophylactic neutralizing antibody palivizumab (also known as Synagis, pali) and an affinity-matured variant, motavizumab (mota)14. A crystal structure of mota in complex with its epitope from the RSV Fusion (F) glycoprotein revealed that the antibody-bound epitope attains a helix-turn-helix conformation15.

We previously developed ‘side-chain grafting’ and ‘backbone grafting’ methods to transplant continuous or discontinuous epitopes to scaffold proteins of known structure, for epitope conformational stabilization and immune presentation16,17,18,19,20. Epitope scaffold immunogens designed by these methods for epitopes from HIV or RSV (including the mota epitope) have in some cases induced structure-specific antibodies but have failed to induce neutralizing antibodies16,17,18. Because these methods are restricted to scaffold proteins of predetermined structure, we have developed a new computational method to design scaffold proteins with full backbone flexibility, to allow greater precision in tailoring scaffold structures for particular epitope structures. We used this method to design scaffolds for the mota epitope, and we found that the scaffolds had favourable biophysical and structural properties and that scaffold immunization of rhesus macaques induced RSV-neutralizing activity (Fig. 1).

Figure 1: A new computational method to design epitope-focused vaccines, illustrated with a neutralization epitope from RSV.
figure 1

Stages of computational design and immunological evaluation are shown; biophysical and structural evaluation are also important (see text). mAbs, monoclonal antibodies.

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Computational method

Great strides have been made in developing de novo methods to design arbitrary, idealized protein structures21,22, but the resulting proteins have lacked functional activity. We devised a computational method to allow de novo folding and design of scaffold proteins stabilizing functional motifs (Extended Data Fig. 1). This procedure, called Fold From Loops (FFL), has four stages: (1) selection of the functional motif and target topology to be folded around the motif; (2) ab initio folding to build diverse backbone conformations consistent with the target topology; (3) iterative sequence design and structural relaxation to select low-energy amino-acid sequences for the given backbone conformations; (4) filtering and human-guided optimization, in which the best designs are identified by structural metrics and then subjected to optional human-guided sequence design to correct remaining flaws.

Design of epitope scaffolds

To design scaffolds for the helix-turn-helix conformation of the mota epitope (PDB accession 3IXT, chain P), we selected a three-helix bundle (PDB 3LHP, chain S) as the template topology. Knowing that the template protein folds into thermally stable, soluble monomers16,23, we designed scaffolds of similar length and position-dependent secondary structure. We produced 40,000 designs using FFL stages 1–3 and then used multiple structural filters to select eight designs for human-guided optimization. Additional modifications were made to those designs as follows: first, to optimize solubility, nearly all surface residues outside the epitope were replaced with those from the template protein; and second, to optimize side-chain packing in the buried protein core, computational design was used to design larger hydrophobic residues at selected buried positions of most designs (Extended Data Fig. 2). The final eight FFL designs had similar but non-identical backbone conformations (pairwise root mean squared deviation (r.m.s.d.) ranging from 0.5 to 3.0 Å) with correspondingly diverse core packing solutions differing from each other by 8 to 42 mutations and from the template by 56 mutations on average (Extended Data Fig. 2). All eight FFL designs had identical surface residues (including non-epitope residues taken from the template, as well as the epitope itself). To create fully artificial scaffolds with different antigenic surfaces that could be used in heterologous prime-boost regimens with FFL scaffolds or to map immune responses to FFL scaffolds, we resurfaced23 the FFL_001 design; this produced the ‘FFL_surf’ designs (Extended Data Fig. 2) that differed from FFL_001 by 36 mutations on average and had no significant sequence similarity (BLAST E value < 10−3) to any known protein except the RSV F protein.

Biophysical and structural characterization

Six out of eight FFL designs and three out of four FFL_surf designs could be expressed in Escherichia coli and purified, with yields ranging from 3 to 5 mg l−1. These nine scaffolds were monomeric in solution, showed circular dichroism spectra typical for properly folded helical proteins, and all but one were highly thermally stable with melting temperatures (Tm) greater than 75 °C (Fig. 2a, b, Table 1 and Extended Data Fig. 3). 15N heteronuclear single quantum coherence (HSQC) spectra were collected for four FFL designs, and these data showed reasonable to good peak dispersion, typical of well-behaved, globular proteins with high α-helical content in solution (Fig. 2c, Table 1 and Extended Data Fig. 3).

Figure 2: Biophysical and structural characterization of scaffold FFL_001.
figure 2

a, Size-exclusion chromatography coupled in-line with multi-angle light scattering measured a molecular weight in solution of 15 kDa, corresponding to a monomer. LS, light scattering; UV, ultraviolet absorbance. b, Circular dichroism data fit with a two-state model showed that the protein had a melting temperature of 74 °C. Inset, the wavelength scan at 25 °C exhibited two minima characteristic of an all-helical protein. Deg, degrees; MRE, mean residue ellipticity. c, Two-dimensional 1H-15N HSQC spectrum at 25 °C and 600 MHz showed good peak dispersion typical of well-folded, α-helical proteins. d, SPR data and model fits (red lines) of the interaction with mota Fab analyte, from which the dissociation constant (KD) was measured to be 29.9 pM. Similar results were obtained with scaffold analyte and mota IgG ligand. RU, response units. e, Crystal structure of unliganded FFL_005 (blue, green and salmon helices, with yellow epitope), superimposed with the design model (grey with yellow epitope). f, Crystal structure of FFL_001 bound to mota Fab, superimposed with the design model. Colouring as in e, but with the Fab light and heavy chains in grey and purple, respectively. g, Superposition of the epitope structure from unliganded FFL_005 (yellow) and the complex of peptide (green) bound to mota (PDB 3IXT). FFL_005 is coloured salmon outside the epitope. The positions of escape mutations for pali (262 and 272, RSV numbering) or mota (272) are noted. h, Superposition of the mota-liganded structures of FFL_001 and peptide (PDB 3IXT). The antibody chains of 3IXT are coloured in wheat, and the interfacial side chains of both epitope and antibody are shown in stick representation.

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Table 1 Biophysical properties of scaffolds and scaffold–antibody interactions

The nine purifiable FFL and FFL_surf scaffolds all had high affinity for mota, as assessed by surface plasmon resonance (SPR) (KD < 800 pM, Fig. 2d, Table 1 and Extended Data Fig. 4). In particular, six out of nine scaffolds had very high mota affinities (KD = 6–94 pM) and slow dissociation rates (koff 10−4 s−1) comparable to those of the mota interaction with the RSV F glycoprotein (KD = 35 pM and koff = 0.31 × 10−4 s−1)14. The mota–scaffold interaction was also specific—the point mutation K82E on scaffold FFL_001, analogous to the K272E mota escape mutation on RSV F24, reduced mota binding by more than a factor of 1,000 (Extended Data Fig. 4). These results suggested that the conformation of the native epitope was reproduced accurately on the scaffolds. The mota affinities for FFL scaffolds were three to four orders of magnitude higher than the mota affinities for the free epitope peptide (KD = 210–240 nM18) or for the best side-chain-grafting epitope scaffold previously reported (KD = 90–125 nM18).

To evaluate the degree to which high-resolution structures of the FFL designs recapitulated the design models and the mota epitope, we solved two crystal structures: unliganded FFL_005 and the complex of FFL_001 bound to mota Fab (Fig. 2e, f), to resolutions of 2.0 and 2.7 Å, respectively (Extended Data Fig. 5). The crystal structures showed good overall agreement with the design models—the backbone r.m.s.d. over all residues was 1.7 Å for FFL_005 and 1.2 Å for FFL_001 (Fig. 2e, f), and the all-atom r.m.s.d. for the core side chains was 2.5 Å for FFL_005 and 1.8 Å for FFL_001. Consistent with the biophysical data, both unliganded and mota-bound structures revealed a high degree of epitope mimicry. Compared to the structure of peptide in complex with mota (PDB 3IXT), the epitope backbone r.m.s.d. was 0.5 Å for FFL_005 (Fig. 2g) and 0.4 Å for FFL_001. Compared to structures of pre- and post-fusion RSV F trimer (PDB 4JHW and 3RRR), which were not available at the time the designs were carried out, epitope backbone r.m.s.d. was 0.3 and 0.4 Å for FFL_005, respectively. Furthermore, the interaction of FFL_001 with mota accurately recapitulated the interaction of mota with peptide; superposition of the epitope and paratope of both complexes gave an all-atom r.m.s.d. of 0.8 Å (Fig. 2h).

Immunological evaluation

To assess whether humans can make antibodies specific for the RSV epitope structure stabilized on the scaffolds, we tested the binding of sera from six RSV-seropositive humans to RSV F, FFL_001 and FFL_001 variants with two different epitope mutations (N72Y and K82E) corresponding to RSV escape mutations for pali (N262Y and K272E) and mota (K272E) (Fig. 2g and Extended Data Fig. 6). Although all sera reacted with RSV F and none reacted to the scaffold escape mutants, three sera displayed reactivity to FFL_001. These data confirmed that the FFL scaffolds presented a clinically relevant epitope conformation and illustrated that epitope scaffolds have promise as reagents to assay levels of epitope-structure-specific antibodies in polyclonal sera.

We tested whether the FFL scaffolds could induce RSV-neutralizing antibodies by vaccination in both BALB/c mice and rhesus macaques. Four immunogens were tested: monomeric scaffolds FFL_001, FFL_005 and FFL_007, and a virus-like particle consisting of hepatitis B core antigen (HBcAg) particles conjugated with multiple copies of FFL_001 (refs 25, 26). Mice produced robust binding antibody responses against the autologous antigens, but binding antibody responses against RSV F protein or RSV viral lysate were detected in only a few animals (Extended Data Fig. 6), and neutralizing activity as judged by a plaque reduction assay was not detected (not shown). In contrast to the mouse results, after three immunizations all macaques produced robust binding responses not only against the autologous antigens (Fig. 3a) but also recombinant RSV F protein (Extended Data Fig. 6), and most animals responded to RSV viral lysate (Fig. 3a and Supplementary Table 1). Neutralizing activity was detected by the plaque assay in 7 out of 16 macaques after three immunizations and in 12 of 16 macaques after five immunizations (Figs 1 and 3a and Supplementary Table 2). Neutralizing activities were confirmed at selected time points using two different assays (microneutralization and a flow cytometry-based assay) in different laboratories, and included measurement of neutralizing activity against RSV subtype B27 as well as subtype A (Extended Data Figs 6, 7 and Supplementary Table 2). To benchmark the neutralization potency, selected macaque sera were tested side by side with sera from seropositive human adults, in both the plaque reduction and flow cytometry assays (Fig. 3b, c). The results in both assays demonstrate that the best-responding macaques, including two out of four animals in the particle group at week 20 and one animal in that group at week 12, have neutralization titres comparable to those induced by natural human infection. This is noteworthy given that natural infection exposes multiple epitopes on the RSV F and G glycoproteins, whereas the scaffolds exposed only one epitope.

Figure 3: Serological analysis of immunized macaques.
figure 3

a, ELISA end point titres measured against the autologous immunogen (left) or against RSV whole viral lysate (middle), and 50% neutralization titres as determined by the plaque reduction assay (right). The immunization groups are shown on the far left, and the schedule is indicated at the bottom. Small symbols connected with dashed lines indicate individual animals. Large symbols connected with solid lines report group averages, with error bars showing standard deviations, measured over the four animals in each group at each time point. b, Comparison of 50% neutralization titres for sera from six RSV-seropositive humans and sera from eight macaques from weeks 12 and 20, measured side by side in the plaque reduction assay. Mean ± standard deviation for the human data is 218 ± 145. Two macaque data points at both week 12 and week 20 are not visible in the graph because no neutralizing activity was detected. c, Comparison of 50% neutralization titres for sera from 20 RSV-seropositive humans and sera from five macaques from week 20, measured side by side in the flow cytometry assay. Mean ± standard deviation for the human data is 462 ± 792.

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Monoclonal antibody characterization

To study the molecular basis for the vaccine-induced neutralizing activity, we used single-B-cell sorting to isolate epitope-specific monoclonal antibodies28 from memory B cells of one animal from the particle group with potent serum neutralizing activity. We isolated B cells that bound strongly to FFL_001 but not to a double mutant of FFL_001 (FFL_001_N72Y_K82E) containing both pali escape mutations. Following DNA sequencing of antibody variable genes in those cells, we produced 11 recombinant monoclonal antibodies, of which eight bound with high avidity to FFL_001 and two (17-HD9 and 31-HG7) bound with high avidity to RSV F protein (Fig. 4a and Extended Data Fig. 8). SPR revealed that these two monoclonal antibodies, which are clonal relatives, have extremely high affinities (KD ≈ 3 pM) for the scaffold FFL_001 that elicited them when mounted on the particle (Extended Data Fig. 8). Concomitant with high affinities, these two monoclonal antibodies have neutralization potencies similar to mota and higher than pali by nearly an order of magnitude (Fig. 4b and Extended Data Fig. 8).

Figure 4: Analysis of monoclonal antibodies isolated from an immunized macaque.
figure 4

a, Enzyme-linked immunosorbent assay (ELISA) binding of the macaque monoclonal antibodies (17-HD9 and 31-HG7) and pali to RSV F. b, Neutralization of RSV by the macaque monoclonal antibodies and pali, measured by a microneutralization assay. The half-maximum inhibitory concentrations (IC50) for pali, 17-HD9 and 31-HG7 were 0.08, 0.005 and 0.007 μg ml−1, respectively. c, Molecular replacement model of 31-HG7 bound to FFL_001 (left), a crystal structure of 17-HD9 bound to a 35-residue helix-turn-helix peptide from FFL_001 (middle) and the crystal structure of mota (PDB: 3IXT) bound to peptide. The three structures are aligned with respect to the helix-turn-helix epitope. d, Structural alignment of the helix-turn-helix epitopes bound to mota (blue) and 17-HD9 (white), in which side chains are coloured orange if at least 15% of the total area (backbone plus side chain) of that residue is buried by the respective antibody. Nine positions are buried by both antibodies, two positions in the turn are buried only by 17-HD9 (P265 and T267, RSV numbering), and two positions near the peptide termini are buried only by mota (S255 and N276). e, Close-up view of the interface between 17-HD9 and helix-turn-helix epitope. Interaction residues are shown in stick, and the complementary determining region H3 (CDRH3) is coloured red. K82/K272 (scaffold numbering/RSV numbering), at the edge of the interface, is coloured grey.

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To map the epitopes for 17-HD9 and 31-HG7, we assessed binding to several scaffold variants (Extended Data Fig. 9). Both monoclonal antibodies: (1) bound with very high affinity (KD = 40–50 pM) to FFL_001_surf1, which has an antigenically distinct surface from FFL_001 outside the RSV epitope; (2) retained high affinity (KD = 180–330 pM) for the FFL_001_K82E mota escape mutant; (3) retained modest affinity (KD = 60–140 nM) for the FFL_001_N72Y_K82E double escape mutant; and (4) lacked detectable affinity for FFL_MPV_001, which swaps RSV residues on FFL_001 to those at the analogous positions on human metapneumovirus, which has a similar helix-turn-helix conformation (r.m.s.d. 0.9 Å, PDB 4DAG29) but very different amino acid sequence. These results indicate that the two macaque monoclonal antibodies target the same helix-turn-helix epitope as mota and pali but have different fine specificities.

To understand the structural basis for the binding and neutralizing potency of these macaque monoclonal antibodies, we pursued crystallography of 17-HD9 and 31-HG7 complexes with FFL_001. We obtained crystals of the 31-HG7–FFL_001 complex that diffracted to 3.8 Å, which was sufficient to determine a molecular replacement solution using the FFL_001 crystal structure and a composite Fab model, but insufficient to perform detailed rebuilding and refinement. The molecular replacement solution allowed determination of the rigid-body orientation of 31-HG7 relative to FFL_001 and demonstrated that 31-HG7 approaches the helix-turn-helix from a different angle than mota (angle difference 56°, Fig. 4c). We also obtained crystals and determined the structure of the 17-HD9–FFL_001 complex (resolution = 2.5 Å), which contained four complexes of 17-HD9 bound to a 35-residue helix-turn-helix peptide (scaffold substructure) in the asymmetric unit (Fig. 4c and Extended Data Fig. 10). The 17-HD9 complex structures demonstrated that 17-HD9 recognizes essentially the same helix-turn-helix epitope as mota and pali—the conformation of the epitope in the 17-HD9 complexes is very similar to that in the structures of mota–FFL_001 (r.m.s.d. 0.5–0.7 Å), RSV F pre-fusion (r.m.s.d. 0.3–0.4 Å) and RSV F post-fusion (r.m.s.d. 0.5 Å), and 85% of the epitope residues buried by either mota or 17-HD9 are also buried by the other (Fig. 4d and Supplementary Table 3). Although 17-HD9 and mota bury a similar amount of area on the epitope (690 Å2 versus 683 Å2), 17-HD9 uses a different paratope to make more hydrogen bonds (15–18 versus 7) that plausibly contribute to its higher scaffold affinity and higher neutralization potency (Fig. 4e and Supplementary Tables 4, 5). The 17-HD9 complexes are also consistent with the ability of 17-HD9 to bind to the K82E mota escape mutant: density for the K82 side chain is absent in two out of four 17-HD9 complexes, and K82 is only 37% buried by 17-HD9 in the other two complexes (Fig. 4e); by contrast, K82 is 65% buried by mota and makes a buried salt bridge to mota light-chain residue D50. Taken together, these results demonstrate that epitope scaffold immunization can ‘re-elicit’ neutralizing antibodies that target with high precision an epitope predefined by a protective antibody.


We have demonstrated that small, thermally and conformationally stable protein scaffolds that accurately mimic the structure of a viral neutralization epitope can induce neutralizing activity in a majority of vaccinated macaques. The results establish the feasibility of epitope-focused and scaffold-based vaccine design, and encourage the application of these strategies for a variety of vaccine targets. The biophysical, structural and functional data on the mota scaffolds validate the computational design method (FFL), and support its continued development and application to other vaccine epitopes and other types of functional sites. Indeed, the data should encourage the general use of methods using protein backbone flexibility to design novel functional proteins.

The scaffolds themselves represent promising leads for RSV vaccine research and development (particularly the scaffolds presented on virus-like particles). Non-replicating RSV vaccine candidates are not tested in RSV naive young infants, the highest priority target population, owing to vaccine-mediated disease enhancement in early clinical trials of formalin-inactivated RSV10. Scaffold immunogens that focus antibody responses to a known protective epitope but are otherwise unrelated to RSV may have a lower safety barrier to testing in human infants than other non-replicating RSV vaccine approaches. The frequency, magnitude and durability of neutralizing responses to these scaffolds remain to be optimized, by varying such parameters as adjuvant, dose, schedule, particle display system and mode of delivery. In the context of vaccinating a diverse range of humans (or nonhuman primates) with different, time-dependent B-cell repertoires, neutralizing antibody responses to a single epitope may be more variable than responses to whole antigens or pathogens containing multiple neutralization epitopes. (Indeed, our discordant results in mice compared to macaques may reflect differences in species-dependent repertoires.) Thus, epitope-focused or scaffold-based vaccines for RSV or other pathogens may also be improved by inclusion of more than one epitope.

Features of the very potent neutralizing monoclonal antibodies isolated from one vaccinated macaque offer implications for vaccine design. These monoclonal antibodies had unusually high affinity for the eliciting antigen, for example when compared to monoclonal antibodies isolated from macaques that had been immunized with different, more conformationally labile antigens using similar regimens28. This suggests that rigid epitope structures may more efficiently induce extremely high-affinity antibodies, a possibility that merits further investigation. In cases of antigenically highly variable pathogens such as HIV, influenza or hepatitis C virus, the vaccine challenge is to induce responses to conserved but immunorecessive epitopes instead of the strain-specific epitopes that dominate the response to native antigens. Such conserved epitopes—the sites of vulnerability targeted by broadly neutralizing antibodies—are typically in close physical proximity to variable residues, making precision of immuno-focusing a vaccine requirement. Our crystallographic finding that scaffold-elicited monoclonal antibodies recapitulate the mota neutralization specificity with high precision provides proof of principle that epitope-focused vaccine design can meet this immuno-focusing challenge.

Methods Summary

Details of the FFL computational design protocol are provided in Methods. Protocols for protein expression and purification, biophysical characterization, virus-like particle preparation, X-ray crystallography, NMR, animal immunization, enzyme-linked immunosorbent assays, neutralization assays and monoclonal isolation are provided in Methods and Supplementary Information.

Online Methods


The FFL protocol was implemented in the Rosetta molecular modelling platform30. The four-stage protocol is described in the main text, and we provide additional details here. In stage (1), FFL required two structural inputs in the form of atomic coordinates: a functional motif and a target topology (Extended Data Fig. 1). In stage (2), ab initio folding sampled conformational space using a fragment assembly protocol21,31. Fragments of lengths 3 and 9 were generated on the basis of sequence and secondary structure of PDB 3LHP, chain S using the program NNMAKE31, and were provided as input to FFL. The sequence location of the functional motif in the target topology was defined by a loop file (Supplementary Methods). FFL automatically appended extended polypeptide chains to the termini of the functional motif to match the total number of residues of the template topology. The ab initio simulations were performed in Rosetta centroid mode in which the backbone atoms were explicitly defined along with a pseudo-atom representing the side chain. The FFL protocol allows optional use of residue distance restraints to bias the folding trajectories towards similar structures to the target topology, using methods described previously31. Here, distance restraints between Cα atoms were extracted from the coordinates of the target topology. Restraints were defined for residue pairs that were at least six residues apart, except for the segment composed of the functional motif plus five residues upstream and downstream. The FFL restrained simulations were performed allowing standard deviations of 1.5 or 3 Å for each Cα–Cα distance. FFL allows the user to set the termini of the functional motif as moveable in order to favour smooth structural transitions between the structurally rigid functional motif and the rest of the protein. Here, two residues at each terminus of the functional motif were set as moveable, whereas the rest of the backbone dihedral angles within the functional motif were fixed. In stage (3), low-resolution models generated in the folding stage were filtered by r.m.s.d. relative to the coordinates of the target topology. Models with r.m.s.d. >5 Å were discarded, and the remaining were subjected to iterative sequence design and conformational relaxation. Side-chain conformations from the functional motif were imposed and kept fixed throughout the design and relaxation simulations. Sequence design was performed with RosettaDesign21 in which each position outside of the functional motif was allowed to mutate to any amino acid except cysteine. After each step of sequence design, a step of all-atom relaxation31 was performed, composed of several rounds of small perturbations in the backbone dihedral angles, side-chain repacking, and energy minimization. The FFL designs were generated by using three cycles of sequence design and structural relaxation, but the number of cycles is user-adjustable. FFL allows the option to perform sequence design at selected positions within the functional motif. In the case of the mota epitope as functional motif, only one face of the helix-turn-helix motif constituted the antibody-binding interface, so the side chains of the other face were allowed as designable. In stage (4) designs from four separate FFL simulations of 10,000 models each were filtered and subjected to human-guided optimization. Rosetta full-atom energy was the first filter; the 50 lowest-energy designs from each simulation were retained for further examination. Next, we applied a composite filter to select designs with the best structural features according to Ramachandran score21, counts of buried polar atoms not involved in hydrogen bonds, and core packing as assessed by RosettaHoles32. Designs that scored within the top 25 by each filter were retained; this included four to nine designs for each of the four simulations. Several of the design models were curved helical bundles, unlike the starting topology that was relatively straight, so we used Helanal33 to identify the straightest designed bundles. FFL_005, FFL_007 and FFL_008 were identified by Helanal. Human-guided computational design and subsequent relaxation was then performed to improve core packing and to modify surfaces, as described in the main text. At this stage, mutations in the core nearly always caused the Rosetta energy to increase, even after relaxation, but mutations were allowed if the post-relaxation energy was within 20% of the pre-mutation energy. The different criteria used to select each design, the number of mutations in the core, and the Rosetta energies at the different stages are summarized in Supplementary Table 1. Example command lines for FFL and other Rosetta modes are provided in Supplementary Information.

Epitope scaffold expression and purification

Epitope scaffolds were purified as described previously16. For NMR, 15N isotopically labelled samples of FFL_001, FFL_005, FFL_006 and FFL_007 were grown in minimal MOPS medium supplemented with 1 g l−1 of 15N ammonium chloride. The starter cultures were expanded to 1 l of MOPS and incubated overnight at 37 °C; 3 ml of 40% 15N glucose was added to continue growth, 250 μM of IPTG was added to the cultures to induce protein expression, and the cells were then incubated overnight at 16 °C.

Circular dichroism

Experiments were performed on Aviv 62A DS and Aviv 420 spectrometers. Far-ultraviolet wavelength scans (190–260 nm) of protein samples with concentration ranging from 15 to 25 μM were collected in a 1-mm path length cuvette. Temperature-induced denaturation was monitored by changes in ellipticity at 210 nm, over the temperature range 1–99 °C, in 2 °C increments every 3 min. The resulting data was converted to mean residue ellipticity, fitted to a two-state model, and melting temperatures (Tm) were obtained34. Chemical denaturations were performed using GuHCl in increments of 0.2 M, and the total GuHCl concentration ranged from 0 to 8 M. The protein concentrations ranged from 1 to 3 μM. The free-energy differences for the unfolding transitions (ΔG) were obtained from the denaturation curves by a nonlinear least-squares fitting using a two-state unfolding and linear extrapolation model35.

Light scattering

The monodispersity and molecular weight of purified proteins were assessed by HPLC (Agilent, 1200 series) coupled in-line to a static light scattering device (miniDAWN TREOS, Wyatt). 100 μl of 1–2 mg ml−1 protein sample was used and the collected data was analysed with the ASTRA software (Wyatt).

Preparation of FFL_001-conjugated virus-like particles

To conjugate FFL_001 to HBcAg virus-like particles we used an engineered version of the HBcAg in which a segment containing the lysine for chemical conjugation (GGKGG) was introduced between P79 and A80 (ref. 26). This places a lysine at the tip of the major immunodominant region of HBcAg. HBcAg constructs were expressed in E. coli using a published protocol16 up to the point of cell lysis. Lysed cells were pelleted and the supernatant was concentrated approximately fourfold using spin concentrators with a 100-kDa membrane cutoff (Vivaspin). Next, 1-ml aliquots of the concentrated supernatant were layered on top of 10–50% sucrose gradients with the total volume of 10.5 ml. The gradients were spun in an ultracentrifuge (Beckmann Coulter SW 41 Ti rotor) at 29,000 r.p.m. for 4 h, fractionated in 12 1-ml fractions and analysed by SDS–PAGE. Fractions containing HBcAg were dialysed overnight in 4 l of EndoFree 1 × PBS in a dialysis membrane of 100 kDa to remove the sucrose and then concentrated using concentrators with 100-kDa cutoff (Vivaspin). Conjugation of FFL_001_R33C to HBcAg particles was achieved with two heterobifunctional chemical crosslinkers (PEG4-PFB and MHPH, Solulink) that react covalently with specific residues in each of the protein counterparts (PEG4-PFB with lysine on HBcAg, MHPH with cysteine on FFL_001_R33C) and also react with each other to form a covalent bond between the formylbenzamide (FB) group of PEG4-PFB and hydrazinonicotinamide (HyNic) group of MHPH. After functionalization of HBcAg with PEG4-PFB and of FFL_001_R33C with MHPH, the HyNic–4FB conjugation was performed to obtain FFL_001_R33C covalently conjugated to HBcAg particles. Dialysed fractions of HBcAg were buffer-exchanged to modification buffer (100 mM PBS, 150 mM NaCl, pH 7.4) using Zeba desalting columns (Solulink) according to the manufacturer’s protocol. The HBcAg fractions in modification buffer were then incubated with sevenfold molar excess of PEG4–PFB (stock solution of 20 mg ml−1) and the conjugation reaction proceeded for 2 h. Upon the completion of the conjugation, the reaction product was buffer-exchanged to conjugation buffer (100 mM PBS, 150 mM NaCl, pH 6.0) using desalting columns. To conjugate MHPH to FFL_001_R33C, the scaffold was incubated under reducing conditions (2 mM dithiothreitol) for 15 min to prevent potential dimerization and to maximize the number of cysteines available to react with MHPH. After the reduction step the scaffold sample was buffer-exchanged to conjugation buffer and incubated with tenfold molar excess of MHPH (stock solution of 20 mg ml−1) during 1 h. The conjugation between the functionalized FFL_001_R33C and HBcAg particles was performed overnight in a scaffold to particle molar ratio of 3:1. The reaction product was split into 1-ml aliquots and scaffold-conjugated particles were purified using the sucrose gradient protocol described above. To confirm the conjugation of the FFL_001_R33C scaffold to the HBcAg particles, the fractions resulting from the sucrose purification were analysed by SDS–PAGE. SPR studies were also used to verify that the mota Fab had high affinity for scaffold-conjugated particles (not shown). Preparation of low-endotoxin protein for immunization studies was carried out as described previously16.


Crystallization conditions and structure-determination protocols are provided in Supplementary Information.


Samples were prepared in 25 mM sodium phosphate, 150 mM NaCl, pH 7.0, and 90% H2O/10% D2O at a concentration of 500 μM. HSQC spectra for FFL_001, FFL_005, FFL_006 and FFL_007 were recorded on a Bruker Avance 600 MHz NMR spectrometer equipped with an actively shielded z-gradient triple-resonance cryo-probe. All spectra were recorded at 25 °C. Spectra were processed using NMRPipe36 and NMRView37.


All experiments were carried out on a Biacore 2000 (GE Healthcare) at 25 °C with HBSEP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA and 0.005% (v/v) Surfactant P20) (GE Healthcare) as running buffer. For binding analysis, 200–500 response units of IgG were captured on a CM5 sensor chip containing 8,000–9,000 response units of amine-linked mouse anti-human IgG (Human Antibody Capture kit, GE Healthcare). Samples of different protein concentrations were injected in duplicates over this surface at a flow rate of 50–100 μl min−1. If necessary, surface regeneration was performed with two 60-s injections of 3 M MgCl2 at a flow rate of 10 μl min−1. One flow cell contained anti-human IgG only and its interaction with the analyte was used as reference. Alternatively, other formats were used, as follows: (1) epitope scaffolds were amine-coupled to the sensor chip and mota Fab was flowed as analyte; (2) NHP_ D039 isolated monoclonal (17-HD9 and 31-HG7) IgGs were amine-coupled to the chip and the epitope scaffold was flowed as analyte; (3) mota Fab was amine-coupled to the chip and the epitope scaffold was flowed as analyte. The alternative formats were performed with identical flow rates as mentioned above. Data preparation and analysis were performed using Scrubber 2.0 (BioLogic Software).

Mouse immunization

A total of nine groups of four mice each were immunized three to six times on a monthly schedule, and different doses, adjuvants and routes of administration were tested in addition to the different immunogens.

Macaque immunization

Sixteen rhesus macaques (male, 4 years old) were screened for antibody to RSV by ELISA before initiation of the study. In brief, 96-well plates were coated with 100 ng of RSV whole virus lysate in carbonate buffer. Serum from NHPs (1:100 and 1:200 dilution) were added to the plates and incubated for 30 min at room temperature (≈25 °C). Serum was decanted, and plates were washed with PBS–Triton X-100. Horseradish peroxidase (HRP)-conjugated secondary antibody was added to the plates and incubated for 30 min at room temperature. The secondary antibody was decanted, plates washed with PBS–Triton X-100, and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added. Substrate incubated for 10 min was then stopped with 1 N H2SO4. Plates were read immediately on a spectrophotometer at an OD of 450 nm. Positive results were indicated by OD values of greater than 1.0. All animals were seronegative by this assay, none registering an OD value higher than 0.07. Measurements were done in triplicate and compared to positive controls using Synagis antibody. Rhesus macaques were immunized with FFL scaffold monomers and FFL_001-conjugated HBcAg particles. Immunogens were scaffold monomers FFL_001, FFL_005, FFL_007 and FFL_001-conjugated HBcAg particles, in which the latter are referred to as HBcAg-FFL_001. Four animals per immunogen were immunized by the intramuscular route at 0, 4, 8, 12 and 18 weeks. Animals were injected with 1 ml total volume, consisting of antigen and Adjuplex adjuvant diluted into PBS, with 0.5 ml injected into each arm. The first immunization included a total of 200 μg of scaffold equivalent; subsequent immunizations included a total of 100 μg scaffold equivalent. Plasma was taken at weeks 0, 2, 4, 5, 8, 9, 12, 13, 16, 18, 19, 20, 28 and 32.

Recombinant protein ELISA assay

384-well plates (Nunc) were coated with antigen (2 μg ml−1 of FFL scaffolds or RSV F) and incubated overnight at 4 °C. The plate was washed three times, incubated with blocking buffer for 2 h at room temperature, and washed once more. Serum samples were serially diluted in blocking buffer at 4 °C and applied to the plate. Following overnight incubation at 4 °C, the plate was washed four times. A 1:4,000 dilution of alkaline phosphatase-conjugated anti-monkey IgG (Fitzgerald Industries) was applied to the plate and incubated for 1 h at room temperature. The plate was washed four times and then incubated with 4-nitrophenyl phosphate disodium salt hexahydrate substrate solution (Sigma) for 30 min. Absorbance was measured at 405 nm using a BioTek Power Wave HT plate reader. All washes were performed using a BioTek EL406 plate washer. For NHP sera, ELISA end point titres indicate the serum dilution that resulted in an absorbance twofold over naive sera at the same dilution. For mouse sera, ELISA end point titres indicate the serum dilution that resulted in an absorbance twofold over background. Background was assigned on the basis of absorbance of wells that received secondary antibody only, and was 0.1 absorbance units. The same assay was used to measure binding of recombinant purified monoclonal antibodies to the different antigens, but binding time of purified monoclonal antibodies to plate-bound antigen was reduced to 1 h. The concentrations of the purified monoclonal antibodies ranged from 100 mg ml−1 to 0.01 mg ml−1.

RSV F protein for ELISA

Plasmid pcDNA3.1 encoding a codon-optimized version of RSV A2 F plus a GCN4 trimerization domain and His tag was transfected via polyethyleneimine (Polysciences) into 293-F cells (Life Technologies). Four days later, recombinant RSV F was purified from 293-F culture supernatant via a HisTrap HP column (GE Healthcare) using an AKTA FPLC (Amersham Biosciences).

Whole-virus ELISA assay

Immulon 96-well ultra-high binding plates (Thermo Scientific) were coated with 100 ng per well of purified human RSV viral lysate (ABI) in BupH carbonate buffer (pH 9.5) (Pierce Thermo Scientific) in a volume of 100 μl per well for positive wells or negative wells (no antigen) with 100 μl per well of plain BupH carbonate buffer overnight at 4 °C. The next day, coating buffer was removed and plates were blotted dry before the addition of 275 μl per well of blocking solution (1 × PBS, 5% non-fat dry milk, 1% normal goat serum) for 1 h at room temperature. Sera from immunized animals were diluted 1:100–1:51,200 (twofold dilutions) in sample diluent (1 × PBS, 0.1% Triton X-100). Human anti-RSV F (Synagis/palivizumab) (MedImmune) was used as a positive control and diluted twofold from 100 mg ml−1 to 1:40,000–1:5,120,000. Samples and controls were added to the plate (100 μl per well) into two wells, one coated with the RSV viral lysate and other with no antigen, and incubated for 30 min at room temperature. Serum was removed, plates were washed by immersion and blotted dry. Secondary antibody (goat anti-monkey IgG HRP) (Vector) was diluted 1:1,000 in 1 × PBS, 5% non-fat dry milk, added at 100 μl per well and incubated at room temperature for 30 min. Upon removal of the secondary antibody, the plates were washed by immersion and blotted dry. The plates were developed with addition of 1-Step Ultra TMB ELISA substrate (Thermo Scientific) followed by 2 N sulphuric acid in equal volumes. The plates were read immediately on an ELISA plate reader at 450 nm (SpectraMax M5, Molecular Devices). ELISA titres were calculated as the last dilution for which the absorbance of a sample on the RSV viral lysate well is two times the absorbance of the same sample on the no antigen well. The procedure described above was also used to measure RSV F reactivity of the NHP_ D039 isolated monoclonal antibody samples. These samples were diluted twofold from their various concentrations to 1:100–1:51,200.

Neutralization assays

The plaque reduction assay was performed using RSV subtype A2 as described previously38. Serum dilutions (1:10, 1:40, 1:160 and 1:640) were mixed 1:1 with virus to yield final dilutions of 20, 80, 320 and 1,280. 50% and 60% plaque reduction neutralizing activity was determined by regression curve analysis. The microneutralization assay method is provided in Supplementary Information. The flow cytometry neutralization assay was performed as described previously39.

Monoclonal isolation from immunized NHP

DNA sequences for antigen-specific monoclonal antibodies were isolated by B-cell sorting, RT–PCR and DNA sequencing as described previously28. The difference in the protocol occurred on the last step of cell sorting, where epitope-specific B cells were required to be FFL_001(+) and FFL_001_N72Y_K82E(−). Both FFL scaffolds had an engineered cysteine (R33C) that was conjugated with biotin-maleimide (Solulink) according to the manufacturer’s protocol. Complete, high-quality sequences with unambiguous nucleotide identifications were obtained for the variable regions of both heavy and light chains for approximately 24 antibodies, from which 12 were recombinantly expressed and 11 were successfully purified.

IgG and Fab expression and purification

IgGs and Fabs were produced in FreeStyleTM 293F (Invitrogen) suspension cultures by co-transfection of pFUSEss (IgG, Invitrogen) or pHLsec (Fab) expression vectors containing the heavy and light chains, using 293Fectin (Invitrogen). Supernatants were collected 96 h after transfection. IgGs were purified using ProteinA Sepharose (GE Healthcare) and dialysed overnight into PBS (0.01 M sodium phosphate, pH 7.4, 0.137 M sodium chloride). Fab supernatants were concentrated to 100 ml by tangential flow concentration (Millipore), and Fabs were purified by CaptureSelect IgG-CH1 (BAC) affinity chromatography followed by dialysis in HBS (10 mM HEPES, 150 mM NaCl, pH 7.4) overnight.