Multi-segment side-chain grafting
We previously devised a computational method for the transplantation of continuous epitopes, called side-chain grafting 14,15
. To allow identification of side-chain grafting scaffolds for the discontinuous motavizumab epitope, we extended the Rosetta-implemented matching stage14
to allow searches for backbone superposition over multiple discontinuous segments. This method, called “Multi-segment side-chain grafting”, conducts the matching stage in a similar manner as the original (single-segment) side-chain grafting – by evaluating backbone RMSDs of the epitope to matched width segments of the scaffold and evaluating steric clashes between antibody and scaffold. However, to identify matches for an epitope with N segments, separate searches are conducted using each of the segments as the “primary”, and for each primary backbone superposition match to one epitope segment, the candidate scaffold is scanned again for superposition matches to the remaining epitope segments with the rigid-body orientation of the remaining segments held fixed relative to the primary matching segment. Further, two candidate rigid-body orientations of the epitope relative to the scaffold are passed on to the design stage – one assigned by the initial single-segment match and another assigned by a subsequent backbone superposition over all the segments – the determination of which orientation is superior is made during design. The design stage is carried out as previously described for (single-segment) side-chain grafting14,15
Multi-segment side-chain grafting was employed to design epitope-scaffolds for the motavizumab helical hairpin epitope. A filtered version of the PDB16
was used for matching, that included 13337 protein chains assigned as monomers and 39621 protein chains assigned as multimers. The assignment of the oligomeric state of protein structures was performed according to information available in the PDB and in the PQS (http://www.ebi.ac.uk/pdbe/pqs/
) databases. The thresholds for matching were 1 Å for backbone RMSD and 30000 Rosetta energy units for the clash check. MES1 (1LP1) and MES2 (1S2X) were selected from the non-monomeric set and MES3 (2EIA) was selected from the monomeric set. Of the three matches obtained, two (MES2 and MES3) could have been obtained with standard side-chain grafting matching for continuous epitopes (backbone rmsds to the epitope were 1.0 and 0.92 Å over 19 residues for MES2 and MES3, respectively), but identification of the highest affinity epitope-scaffold (MES1) required the new multi-segment matching method.
In the design stage, epitope side chains responsible for the key interactions were transplanted to all-glycine versions of the matched scaffolds. The side chains transferred were: S255, L258, S259, I261, N262, D263, N268, D269, K271, K272, L273, S275, N276 (RSV F residue numbering as in PDB ID:3IXT). As previously described for side-chain grafting 14
, native scaffold side-chain rotamers outside the epitope were recovered, and residues near (heavy atom distance <4 Å) the epitope or the antibody were designed, categorized as intra and inter positions, respectively. Inter residues were allowed to be ALA, GLY, SER, and THR, and intra positions were allowed to be all amino acids except CYS. Epitope-scaffolds were ranked by antibody binding energy and a final step of human-guided design was performed to revert unnecessary or potentially destabilizing mutations, eliminate unpaired cysteines and undesired functional sites, and trim scaffold termini to avoid clash with antibody.
To generate the Surf1 variant of the original MES1, multiple positions at the surface of the protein were designed using RosettaDesign23
. The residue positions allowed to mutate were: 2, 5, 9, 12, 15, 21, 24, 17, 37, 39, 40, 43, 44, 46, 47, 50, 53 and 54 in the residue numbering of the MES1-motavizumab crystal structure. Different resurfaced MES1 constructs vary in the number of surface mutations. To achieve this mutational gradient, subsets of the enumerated residues were allowed to change in different design simulations, and in the most distinct resurfaced variants all of the residues were allowed to change simultaneously. The amino-acid identities allowed in the designed residues were ALRNDEQKST. To ensure greater sequence diversity, in some of the designed molecules amino-acid identities were restricted to subsets of the initial amino-acids allowed.
MES1 cloning, expression and purification
Mammalian codon-optimized genes encoding MES1 and its variants were synthesized by GeneArt with an N-terminal secretion signal (MGSLQPLATLYLLGMLVASVLA) and a C-terminal HRV3C cleavage site, PADRE epitope (AKFVAAWTLKAAA), Caspase-3 cleavage site, 6x His-tag and Strep-tag II. The genes were cloned into the mammalian expression vector pαH, which is a modified version of pLEXm24
. Proteins were expressed from the plasmids by transient transfection using the Free Style 293 expression system (Invitrogen). MES1 proteins were purified from the media using Ni2+
-NTA resin (Qiagen) and then Strep-Tactin resin (IBA) as per manufacturer s instructions, followed by passage over a 16/60 Superdex 75 column (GE Healthcare). For SPR, ITC and CD measurements, all tags were retained. For immunization experiments, Procaspase-3 D9A, D28A (a kind gift from A. Clay Clark25
) was added to remove the 6x His-tag and Strep-tag II. The tags and protease were removed from cleaved MES1 by passage over Ni2+
-NTA resin. MES1 F2Y/H15N used for crystallization was similarly prepared, but it lacked the HRV3C site and PADRE epitope on the C-terminus.
MES2 cloning, expression and purification
E. coli codon-optimized genes encoding MES2 and its variants were synthesized by GeneArt and cloned into a custom vector based on pMAL-c2X (New England Biolabs). The expression vectors were transformed into BL21(DE3) cells, and the cells were grown in Terrific Broth at 37ºC until OD600= 2.0. The temperature was then reduced to 22ºC, and isopropyl β-D-thiogalactoside (IPTG) was added to 1 mM. After overnight incubation at 22ºC, the cells were harvested and lysed with Bug Buster (Novagen), and MES2 proteins were purified using Ni2+-NTA resin (Qiagen). Fusion tags were removed by incubation with Procaspase-3 D9A, D28A and passage over Ni2+-NTA resin. MES2 proteins were further purified by passage over a 16/60 Superdex 75 column (GE Healthcare), and anion exchange chromatography using a MonoQ column (GE Healthcare).
MES3 cloning, expression and purification
A mammalian codon-optimized gene encoding MES3 was synthesized and cloned as described for MES1. Protein expression and purification were also performed as described for MES1.
Surface plasmon resonance
All experiments were carried out on a Biacore 3000 instrument (GE Healthcare). For the detection of motavizumab binding to MES1 and MES2, motavizumab antigen-binding fragments (Fabs) were covalently coupled to a CM5 chip at 530 RU, and a blank surface with no antigen was created under identical coupling conditions for use as a reference. Initially, epitope-scaffolds were serially diluted 2-fold, starting at 10 μM, into 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% polysorbate 20 (HBS-EP) and injected over the immobilized Fab and reference cell at 40 μl/min. MES1 measurements were repeated using lower protein concentrations, with the 2-fold dilutions starting at 500 nM. The data were processed with SCRUBBER-2 and double referenced by subtraction of the blank surface and a blank injection (no analyte). Binding curves were globally fit to a 1:1 binding model.
For the detection of motavizumab binding to peptide, motavizumab Fab was covalently coupled to a CM5 chip at high density (1,950 RU) and a blank surface with no antigen was created for use as a reference. An N-terminally acetylated peptide with the sequence NSELLSLINDMPITNDQKKLMSNNGYSGTETSQVAPA and a C-terminal biotinylated lysine residue was serially diluted 2-fold, starting at 500 nM, into HBS-EP and injected over the immobilized motavizumab Fab and reference cell at 40 μl/min. Data were processed with BIAevalution software and double referenced by subtraction of the blank surface and a blank injection. Binding curves were globally fit to a 1:1 binding model with drifting baseline and no Bulk RI.
Isothermal titration calorimetry
Experiments were carried out on an iTC200 calorimeter (MicroCal Inc) at 25ºC. Samples were dialyzed into phosphate-buffered saline (PBS) and degassed prior to titrations. MES1 or peptide at 350 μM was titrated into 14 μM motavizumab IgG in 2 μl aliquots with stirring at 1,000 rpm. MES2 at 170 μM was titrated into 7 μM motavizumab IgG in 2 μl aliquots with stirring at 1,000 rpm. Data were processed with Origin software and best fit by a single binding-site model.
To evaluate the secondary structures and thermostabilities of the epitope-scaffolds in solution, circular dichroism experiments were performed with an Aviv 62A DS spectrometer. Far-UV wavelength scans (190 nm –260 nm) at the concentration of 20 μM were collected in a 1 mm path length cuvette. Temperature-induced protein denaturation was followed by change in ellipticity at 210 nm.
Protein crystallization and data collection
A five-fold molar excess of MES1 F2Y/H15N was incubated with motavizumab Fab for 1 hour at 22ºC, and the complex was concentrated to 11.2 mg/ml in an Amicon Ultra centrifugal filter with a 30 kDa molecular weight cut-off (Millipore). 192 crystallization conditions were screened using a Cartesian Honeybee crystallization robot, and initial crystals were grown by the vapor diffusion method in sitting drops at 20ºC by mixing 0.2 μl of protein complex with 0.2 μl of reservoir solution (20.5% (w/v) PEG 4000, 0.2 M lithium sulfate monohydrate, 0.1 M Tris-HCl pH 8.5, 100 mM NaCl). These crystals were manually reproduced in hanging drops by mixing 0.8 or 1.6 μl protein complex with 0.8 μl of the initial reservoir solution containing a range of PEG 4000 concentrations. Larger, single crystals were obtained by streak seeding with clusters of crystals that had been pulverized with a PTFE bead, and these crystals were flash frozen in liquid nitrogen in 24% (w/v) PEG 4000, 30% (v/v) ethylene glycol, 0.2 M lithium sulfate monohydrate, 0.1 M Tris-HCl pH 8.5. Data to 1.90 Å were collected at a wavelength of 1.00 Å at the SER-CAT beamline ID-22 (Advanced Photon Source, Argonne National Laboratory).
Structure determination, model building and refinement
Diffraction data were processed with the HKL2000 suite26
and a molecular replacement solution consisting of two motavizumab Fab molecules13
and two Protein Z molecules27
per asymmetric unit was obtained using PHASER28
. Model building was carried out using COOT29
, and refinement was performed with PHENIX30
. Final data collection and refinement statistics are presented in . The Ramachandran plot as determined by MOLPROBITY31
shows 97.7% of all residues in favored regions and 99.8% of all residues in allowed regions. All structural images were created using PyMol (The PyMOL Molecular Graphics System, Version 1.1, Schrödinger, LLC).
Mice and immunizations
6–8 week old female C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used for all experiments. Mice were immunized with scaffold proteins and 25 μg CpG per mouse intramuscularly. Mice were boosted with either the same protein or an alternative scaffold protein, and the sera were tested by kinetic ELISA for binding to RSV F protein or scaffolds and for neutralization activity.
Proteins were diluted in PBS to a concentration of 1 μg/ml and coated onto 96-well flat bottom ELISA plates overnight at 4°C. Nonspecific adsorption was prevented with 200 μL/well of blocking buffer (2% BSA in PBS) for 1 h at room temperature. Plates were washed four times on an automated plate washer (Bio-Tek Instruments, Winooski, VT) with wash buffer (0.02% Tween-20 in PBS). 100 μL of diluted test sera (1:100 in blocking buffer) and positive serum control were added to each well. Plates were incubated for one hour at room temperature, washed four times, and incubated for 1 hour at room temperature with HRP-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Plates were washed with wash buffer four times followed by distilled water. 100 μl of Super AquaBlue ELISA substrate (eBioscience, San Diego CA) was added to each well and plates were read immediately using a Dynex Technologies microplate reader (Chantilly, VA). The rate of color change in mOD/min was read at a wavelength of 405 nm every 9 s for 5 min with the plates shaken before each measurement. The mean mOD/min reading of duplicate wells was calculated, and the background mOD/min was subtracted from the corresponding control well.
Competition Kinetic ELISA
Competitive ELISA was used to determine the binding specificity of MES1-induced antibodies for the motavizumab epitope. Motavizumab IgG was used as a competitor for the binding of serum antibody from MES1-immunized mice to RSV F, MES1, or MES1 K272E. Motavizumab (50 μl, 1 μg/ml) was added to ELISA plates coated with each antigen and incubated 30 minutes before adding serum samples. Each sample was tested with and without motavizumab. The data are normalized for each sample pair as the percent of binding compared to the untreated well by dividing mOD/min of the motavizumab-treated well by the mOD/min of the control untreated well. This experiment was performed with sera from a group of 5 immunized mice. The P value was determined by Student s two-tailed T-test.
RSV neutralization assay
Antibody-mediated RSV neutralization was measured by a flow cytometry neutralization assay32
. Briefly, HEp-2 cells were infected with RSV-GFP and infection was monitored as a function of GFP expression at 18 hours post-infection by flow cytometry. Data were analyzed by curve fitting and non-linear regression (GraphPad Prism, GraphPad Software Inc., San Diego CA).