Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera

Abstract

Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø-specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F-specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø.

Fig. 1. Serum binding activity to the pre-F and post-F conformations of the RSV F glycoprotein

(A) Human sera were analyzed for binding to pre-F and post-F conformations by kinetic ELISA (black circles, left and right panels, respectively). Binding of unadsorbed sera to pre-F and post-F binding was similar (75.8 and 75.4 mOD/min, respectively). Sera were also analyzed for binding to either F conformation after adsorption with pre-F or post-F subunit proteins. As expected, pre-F adsorption removed antibodies that could bind to pre-F, and post-F adsorption removed antibodies that could bind post-F. Post-F adsorption did not remove a substantial amount of pre-F binding antibodies (pink triangles, left panel), but pre-F adsorption removed nearly all post-F binding antibodies (light blue squares, right panel). (B) Pre-F and post-F conformations share about 50% of their surface, and about 50% of the surface is unique to each. On the basis of the binding data, about 70% of antibodies in human sera bind pre-F–specific surfaces, about 25% bind the shared surfaces, and about 5% bind post-F–specific surfaces, which is disproportionate to the available surface area.

Fig. 2. Serum NT activity against RSV after adsorption with pre-F and post-F

(A) Geometric mean reciprocal EC₅₀ NT titers for unadsorbed sera and sera adsorbed with pre-F or post-F were 511, 9, and 362, respectively. (B) Evaluation of samples by age groups showed that less than 5% of NT activity was retained after adsorption with pre-F in all age groups. (C) Adsorption with post-F removed 20 to 45% of the original NT activity, and there were no trends according to age group.

Fig. 3. Design and characterization of F constructs with mutated antigenic sites Ø and II

(A) The RSV fusion glycoprotein is shown in the stabilized pre-F (left) and post-F (right) forms in surface representation with antigenic sites Ø and II highlighted in red and dark blue, respectively. The purple surface shown on the post-F and the dark blue motavizumab epitope are common to the surface on pre-F as mentioned in Fig. 1B. (B) Close-up views of the antigenic sites Ø and II with the mutated residues are shown. The three nonnative glycans at residues 65, 205, and 209 introduced to block site Ø binding are shown in stick representation. The surface representation is set to 60% transparency with mutated residues shown in stick representation, with a single protomer shown for clarity. (C) BLI was used to measure binding by mAbs that bind pre-F: D25, motavizumab, AM14, and MPE8. Antigenic site Ø KO variant Ø-C (A2 strain) and Ø KO of the B18537 strain showed typical binding to all of the pre-F–targeting antibodies except D25, whereas antigenic site II KO variant II-E bound to all antibodies except motavizumab.

Fig. 4. Serum NT activity against RSV after competition with stabilized pre-F, post-F, and pre-F with site Ø and site II KO

(A) Protein competition neutralization assays show that pre-F removes nearly all (>90%) NT activity from human sera, in contrast to post-F, which only minimally removed NT activity (<30%). This assay also showed that antibodies specific for site Ø present on pre-F account for ~35% of NT activity. (B) Consistent with the results obtained with the RSV A2 strain, protein competition neutralization assays with pre-F of the B18537 strain removes most (>85%) NT activity from human sera, while post-F removed only ~30% of NT activity. Antibodies specific for site Ø on the B18537 pre-F are shown to account for ~45% of NT activity. (C) Results from individual samples are arranged according to increasing site Ø activity in a stacked bar graph to show the relative amount of NT activity attributed to site Ø or site II. Site Ø–specific antibodies accounted for majority of the NT activity in most subjects (red bars). However, there was significant variation between individuals, and in some subjects, most of NT activity could not be attributed to either site Ø or II (bars on the extreme left) but was clearly pre-F–specific. Although in most individuals, site II antibodies account for some NT activity (dark blue bars), it was a small fraction of the overall activity.

Fig. 5. Octet BLI competition assay using D25 and motavizumab

(A) In a BLI assay, biosensor tips were dipped in pre-F, then into analyte, and then into competing antibody. D25 does not inhibit motavizumab binding to pre-F and vice versa, making these mAbs eligible for use in serum analysis. (B) Biosensor tips were dipped into pre-F, then into D25 or motavizumab, and then into sera. The mean percent inhibition of serum antibody binding by D25 is ~40%, and that by motavizumab is ~60%. Unlike motavizumab, there is increasing inhibition by D25 with increasing NT titers. The dashed line represents background binding inhibition. (C) A moderate correlation is found between percent binding inhibited by D25 and reciprocal EC₅₀ NT titers, and a weak inverse correlation was found between percent binding blocked by motavizumab and NT titers.