Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

<div><p>Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.</p></div

Antibodies compete CD81 binding to E2.

<p>Cells transfected E1E2 from isolate H77 were pre-incubated with different concentrations of the indicated antibodies before CD81-LEL at 0.05 μg/mL was added. (A) D25 was used as isotype negative control. The y-axis indicates the percentage inhibition of CD81 and the errors bars represent one SD. (B) Antibody concentration to reach 50% inhibition of CD81 binding (IC₅₀) in μg/mL. The IC₅₀ was determined using non-linear regression analysis. The assay was performed in duplicate and at least repeated once.</p

Antibody epitope mapping.

<p>To determine the more exact antibody-binding epitope single alanine amino acid substitutions were introduced in the E1E2 sequence from isolate H77 and antibody binding was tested by ELISA. E1E2 containing cell lysates were incubated on GN lectin pre-coated wells before adding an antibody (1 to 0.0002 μg/mL). Antibody concentrations needed to get 50% binding (EC₅₀) was determined using non-linear regression analysis. The relative binding was calculated by dividing the EC₅₀ obtained on wild-type protein versus alanine-mutant protein times 100. The gray scale indicates the relative binding level: Black 0% -25%, grey 25% -50% and white 50% -150%. The E2 mutants (rows) were designated X123Y where 123 is the residue position, X indicates the wild-type amino acid residue in H77 and Y indicates the replacing amino acid. The EC₅₀ which could not be calculated because of very weak binding were indicated by <10%. The data are the mean values of two experiments performed in duplicate.</p

Affinity of the antibodies for HCV E2.

<p>Affinity of the antibodies for HCV E2.</p

Breadth of cross-reactive antibodies against E1E2 glycoproteins.

<p>Antibodies were tested for binding to E1E2 protein derived from different genotypes by ELISA. E1E2 containing cell lysates were incubated on GNA pre-coated wells before antibodies were added at 1 μg/mL. A lysate from non-transfected cells and the RSV F protein specific antibody D25 were used as negative controls. The y-axis indicates the mean optical density (OD) at 450 nm and the errors bars represent one standard deviation (SD). E1E2 sequences are indicated between brackets and assays were performed in duplicate.</p

Antibodies recognize non-linear epitopes on soluble E2.

<p>(A) Antibody binding to denatured E1E2 was tested by ELISA. A cell lysate of 293T cells transfected with E1E2 (H77) was treated with 20 mM Dithiothreitol and 0.4% Sodium dodecyl sulfate (denatured E1E2) or directly added on GNA pre-coated plate (native E1E2). Subsequently the antibodies were added at 1 μg/mL. A lysate of non-transfected cells, a native E1E2 cell lysate, AP33 an antibody specific for a linear epitope on E2 and D25 were used as negative and positive controls. (B) The binding of antibodies to H77 derived E2 was tested by ELISA. The antibodies (1 μg/mL) were added to wells pre-coated with His6 tagged E2 (E2-his). Phosphate buffered saline (PBS) coated wells and D25 were used as negative controls. In A) and B) the y-axis indicates the mean Optical density (OD) at 450nm and the errors bars represent one SD. The assay was performed in duplicate wells and repeated in at least one separated experiment.</p

Antibody competition.

<p>(A) Antibody competition assay was performed by SPR using H77 derived E2-his. When the antibodies (indicated in rows) were immobilized on the chip and had captured E2, a second antibody (indicated in columns) was injected. Absence of binding after injection of the second antibody indicated the antibodies competed for E2 binding (grey); if the injected antibody bound antibody-captured E2, this was considered to be not competing (white). The assay was performed in triplicate and repeat in one separated experiment. (B) Antibody competition assay was performed by flow cytometry using cells transfected with E1E2 from isolate H77. Cells were pre-incubated with 20 μg/mL of the indicated antibodies before Alexa Fluor 647 conjugated antibodies at their EC₅₀ were added. The percentage of binding was calculated using the percentage of Alexa Fluor 647 positive cells from wells incubated without antibodies. The gray scale indicates the relative binding level: white 100% -75%, light grey 75% -50%, dark grey 50% -25% and black 25%–0%. D25 was used as isotype negative control. The mean value of three duplicate experiments is shown.</p

Characteristics of participant D18926.

<p>Characteristics of participant D18926.</p

Structures of AM14 in complex with RSV F.

<p>(A) Crystal structure of three AM14 Fabs bound to the prefusion RSV F trimer, viewed from the side and the top of RSV F. Negatively stained EM class averages that may correspond to each view are shown in the upper right. AM14 heavy chain is blue, light chain is white, and RSV F protomers are tan, light green and pink (B) Close-up of the side view, colored as in (A). Prefusion RSV F residues with Cα atoms within 8 Å of AM14 Fab Cα atoms are colored red, and Cα atoms of resistance mutations are shown as spheres. (C) Ninety-degree rotation of the view in (B), showing a molecular surface representation of RSV F and the location of the three AM14 CDR loops that contact F. The binding surface spans the two neighboring protomers (tan and pink).</p

AM14 is specific for cleaved, trimeric RSV F.

<p>Binding of antibodies (A) AM14, (B) D25, (C) MPE8, or (D) palivizumab to uncleaved monomeric RSV F (open black triangles), cleaved monomeric RSV F (black circles), uncleaved postfusion RSV F (open blue triangles), cleaved postfusion RSV F (blue circles), uncleaved prefusion RSV F (open red triangles) and cleaved prefusion RSV F (red circles) was measured by ELISA.</p