Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

Abstract

Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-Å X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

Fig. 1.

RSV F ectodomain structure. (A) Linear diagram. Listed residue numbers correspond to the N terminus of each segment, the furin cleavage sites (arrowheads), and the C terminus. DI–III, domains I–III; p27, excised peptide; FP, fusion peptide; HRA, -B, and -C, heptad repeats A, B, and C. (B) Ribbon representation of one subunit. Domains colored as in A. Glycans are black. (C) Surface representation of the trimer. One subunit colored by domains as in A; the other two are white and gray.

Fig. 2.

Immunization of cotton rats with the postfusion RSV F trimer elicits neutralizing antibodies and protects from RSV challenge. Cotton rats were immunized intramuscularly with 5 μg of RSV F trimer adsorbed to alum on days 0 and 21 or were not immunized. Sera for neutralization assays were obtained on day 35. The cotton rats were challenged intranasally on day 49 with 1 × 10⁵ pfu of RSV, strain Long. (A) Titers of RSV 5 d after challenge by plaque assay in the lungs of immunized (F/alum) or not immunized (none) cotton rats. Values shown are the means with SD of eight cotton rats per group. (B) Serum RSV neutralization titers. Values shown are the mean and range of two pools of four cotton rats per group.

Fig. 3.

RSV and PIV3 comparisons. (A) Ribbon diagram of RSV F domain III. (B) Ribbon diagram of the PIV3 domain III oriented as is the domain in A. (C) Detail of the RSV (green) and PIV3 (red) domain III helical bundles superimposed on the basis of domain III β sheets. Helix labels are colored by virus. (D) RSV and PIV3 F ectodomain trimers (colored as in A and B) superimposed on the basis of their six-helix bundles. (Left) Ribbon diagram viewed perpendicular to the threefold axis; (Right) surface representation viewed along the threefold axis from the top of the head.

Fig. 4.

Motavizumab epitope. (A) Superposition of the Motavizumab-binding helices, α5 and α6, from the RSV F postfusion trimer (green) and the peptide–Motavizumab complex (magenta, PDB code 3IXT) (17). RSV residues bound by Motavizumab are shown in stick representation. Asterisks denote Palivizumab escape mutations. (B) Ribbon representation modeling a Motavizumab–RSV F postfusion trimer complex. The V_H and V_L domains of the Fab are blue and orange, respectively; helices α5 and α6 from the RSV F structure and the peptide–Motavizumab structure colored green and magenta, respectively; a glycan on F is black; and the remainder of F is white.

Fig. 5.

RSV F conformational changes, antigenic structure, and Palivizumab binding. (A) Surface representation of the postfusion structure. Antigenic sites A and C are indicated by colored outlines (pink and cyan, respectively). Asterisks indicate residues selected in neutralization escape variants or forming contacts with an antibody in the determined structures of neutralizing antibody–peptide complexes. The HRA and HRB surfaces are red and blue, respectively. (B) Surface representation of a prefusion model, annotated as in A. (C) Inhibition of Palivizumab binding to postfusion RSV F by pooled sera from unimmunized mice (open boxes) or mice immunized with the RSV F antigen (filled boxes). Palivizumab binding (percentage of ELISA signal without competing sera) is plotted as a function of the dilution of competing pooled sera.