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84887270083
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note
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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.
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24
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84855502585
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Information on materials and methods is available on
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Information on materials and methods is available on Science Online.
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Science Online
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25
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84887291765
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note
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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 liter of Expi293F cells, purified by use of appended His-tag and StreptagII, and analyzed by size-exclusion chromatography (fig. S7).
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26
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84887291963
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note
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The inability to express potential interchain 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 interprotomer conformations.
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27
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84887298275
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note
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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.
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84887314828
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note
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The engineered RSV F variants had Ca-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.
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30
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84887268417
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note
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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.
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note
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Observing Ala107 rather than Arg109 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.
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32
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84887308714
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note
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Ca-root mean square deviations of 0.86 Å for 447 residues between Cav1 and DS-Cav1 crystal structures obtained from ammonium sulfate; Ca-root mean square deviations of 0.47 Å for 447 residues of DS-Cav1 in cubic lattices.
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note
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When palivizumab (Synagis) is dosed at a concentration of 15 mg/kg, serum levels at trough are ∼40 mg/ml, which provides protection in infants from severe disease and protection in cotton rats from RSV infection. In our neutralization assay, ∼40 mg/ml of palivizumab yields an EC50 of 100.
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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.
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84887281144
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note
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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.
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For the "prefusion" form of RSV F, we used the DS-Cav1 stabilized variant of RSV F.
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39
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84887310594
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note
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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.
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Although 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.
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47
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84887277642
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note
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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.
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