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. 2017 Nov 30;8(1):1877.
doi: 10.1038/s41467-017-01858-w.

Structural basis of respiratory syncytial virus subtype-dependent neutralization by an antibody targeting the fusion glycoprotein

Daiyin Tian  1   2 , Michael B Battles  3 , Syed M Moin  1 , Man Chen  1 , Kayvon Modjarrad  4   5 , Azad Kumar  1 , Masaru Kanekiyo  1 , Kevin W Graepel  6 , Noor M Taher  3 , Anne L Hotard  7   8 , Martin L Moore  7   9 , Min Zhao  10   11 , Zi-Zheng Zheng  10   11 , Ning-Shao Xia  12   13 , Jason S McLellan  14 , Barney S Graham  15
Affiliations

Structural basis of respiratory syncytial virus subtype-dependent neutralization by an antibody targeting the fusion glycoprotein

Daiyin Tian et al. Nat Commun. .

Abstract

A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines.

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Conflict of interest statement

M.C., M.Z., Z.Z., N.–S.X., J.S.M. and B.S.G. are listed as co-inventors on a patent application describing the discovery and utility of 5C4 (PCT/CN2014/073505). The remaining authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
5C4 is a potent subtype A-specific neutralizing antibody. a Neutralization potency of 5C4 (closed circles) and D25 (open circles) against RSV subtype A strains A2 (left) and Line19 (right). b Neutralization potency of 5C4 and D25 against subtype B strains 18537 (left) and Tx11-54 (right), depicted as in a. The data in panels a and b are  representative of two independent experiments performed in duplicate
Fig. 2
Fig. 2
Mode of 5C4 binding to antigenic site Ø is similar to that of D25. a 5C4–RSV F complex crystal structure in ribbon and molecular surface representation. Two pre-F protomers are shown as dark and light gray surfaces, and the third protomer is shown as ribbons with F1 colored green and F2 colored blue. The 5C4 Fab bound to the F protomer is also displayed in ribbon representation, with the heavy chain colored pink and the light chain colored white. The other 5C4 Fabs are colored similarly but shown in surface representation. b RSV F protomers are shown in ribbon representation and colored as above. Fabs (upper) and antibody variable domains (lower) are shown as a molecular surface with light chains colored white and the heavy chains of 5C4 and D25 colored pink and red, respectively. The difference in vertical (upper) and horizontal (lower) angle of approach between 5C4 and D25 is represented as intersecting lines with a measured angle of ~ 60° and ~ 45°, respectively
Fig. 3
Fig. 3
5C4 forms few hydrogen bonds to RSV F main-chain atoms. a Two pre-F protomers are shown as dark and light gray surfaces, and the third protomer is shown as ribbons with F1 colored green and F2 colored blue. 5C4 heavy and light chains are shown as ribbons with the heavy and light chains colored pink and white, respectively. Antibody complementarity-determining regions (CDRs) involved in molecular recognition are labeled. Main-chain and side-chain atoms involved in molecular recognition are shown in stick representation with oxygen atoms colored red and nitrogen atoms colored blue. Hydrogen bonds and salt bridges are depicted as black dotted lines. b Linear sequence of antigenic site Ø for both RSV subtypes with the F2 β2–α1 loop on the left and the F1 α4 helix on the right. Subtype-specific residues in site Ø are colored blue in the subtype B consensus sequence. RSV F residues that make hydrogen bonds to 5C4 and D25 are denoted with symbols (side-chain hydrogen bonds, main-chain hydrogen bonds, or side-chain and main-chain hydrogen bonds are represented as open, filled, or partially filled diamonds, respectively)
Fig. 4
Fig. 4
Arg or Lys at position 201 is critical for RSV F recognition and neutralization by 5C4. a–d Fractional binding of mAbs 5C4 (black bars) and D25 (white bars) to DS-Cav1–stabilized pre-F variants relative to motavizumab. a Binding to A-chimera constructs (RSV A2 pre-F variants). b Binding to B-chimera constructs (RSV B18537 pre-F variants). c Binding to RSV A2 pre-F position 201 variants. d Binding to RSV B strain 18537 pre-F position 201 variants. Bars represent the standard relative fractional binding of three replicates with error bars indicating standard deviation. P values calculated using two-way ANOVA are shown with symbols, where ns is P > 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001. e, f Neutralization activity of 5C4 against subtype A strain A2 and subtype B strain 18537 with and without subtype-specific substitutions at position 201. e The EC50 of 5C4 against A2 wild-type and K201N-mutated virus is 0.004±0.0008 μg/mL (Mean±SD) and 0.41±0.11 μg/mL, respectively, from three independent experiments. f The EC50 of 5C4 against RSV B strain 18537 wild-type and N201K-mutated virus is 9.149±0.056 μg/mL and 0.022±0.003 μg/mL, respectively. Data are representative from three independent experiments
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