A potently neutralizing and protective human antibody targeting antigenic site V on RSV and hMPV fusion glycoprotein

Abstract

Human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are frequent drivers of morbidity and mortality in susceptible populations, most often infantile, older adults, and immunocompromised. The primary target of neutralizing antibodies is the fusion (F) glycoprotein on the surface of the RSV and hMPV virion. As a result of the structural conservation between RSV and hMPV F, three antigenic regions are known to induce cross-neutralizing responses: sites III, IV, and V. Leveraging LIBRA-seq, we identify five RSV/hMPV cross-reactive human antibodies. One antibody, 5-1, potently neutralizes all tested viruses from the major subgroups of RSV and hMPV and provides protection against RSV and hMPV in a mouse challenge model. Structural analysis reveals that 5-1 utilizes an uncommon genetic signature to bind an epitope that spans sites Ø, II and V, defining a new mode of antibody cross-reactivity between RSV and hMPV F. These findings highlight the molecular and structural elements influencing RSV and hMPV cross-reactivity as well as the potential of antibody 5-1 for translational development.

Figure 1:. Identification and characterization of RSV/hMPV cross-reactive antibodies

A: LIBRA-seq predicted RSV and hMPV specific B cells. Each dot indicates an individual B cell. Max RSV A / RSV B LIBRA-seq score on the x-axis, max hMPV A / hMPV B LIBRA-seq score on the y-axis. Dots colored in purple were selected for further characterization. B: Sequence characteristics of RSV/hMPV cross-reactive antibodies. Percent identity is calculated at the nucleotide level and sequences and VDJ/VJ length are displayed at the amino acid level. C: ELISA binding of recombinantly produced antibodies against RSV and hMPV prefusion F trimer, calculated as absorbance at 450 nm. Experiments were performed in technical and biological duplicate.

Figure 2:. Binding characteristics of RSV/hMPV cross-reactive mAbs

A: Antibody-antibody competition binding to RSV and hMPV prefusion F trimer against control site specific antibodies. Percentage of binding of biotinylated antibody is shown as a heatmap from 0% (black) to 100% (white). Non-biotinylated competitor antibodies were coated first, and then biotinylated control mAbs were added to detect competition. Competition is calculated as the signal obtained for binding of the biotin-labelled reference antibody in the presence of the unlabeled antibody, expressed as a percentage of the binding of the reference antibody alone. B: Epitope binning via BLI for binding of mAbs 20 and 5–1 to RSV and hMPV prefusion F trimer. Data indicate the percent binding of the second antibody in the presence of the first antibody, as compared to the second antibody alone. Percentage of binding is shown as a heatmap from 0% (black) to 100% (white). C: ELISA binding of germline reverted, recombinantly produced antibodies against RSV A and B and hMPV A and B prefusion F trimer, calculated as absorbance at 450 nm. ELISA area under the curve (AUC) shown as a heatmap from minimum (white) to maximum binding (purple).

Figure 3:. Neutralization potency of RSV/hMPV cross-reactive mAbs.

A: Antibody neutralization against RSV A2, RSV B1, hMPV A2, and hMPV B2 via PRNT. B: IC₅₀ values, expressed as a heatmap with strong neutralization (<0.1 μg/mL) shown in purple and weak/non neutralizing (>10 μg/mL) shown in light purple. Calculated by non-linear regression analysis by GraphPad Prism software. Neutralization assays were performed in technical triplicate; data are represented as mean ± SD.

Figure 4:. 5–1 Fab binds to the prefusion hMPV F at site II, V and the glycan at Asn172.

A: Front view and side view of the fit of hMPV F complex into a DeepEMhanced EM map at the contour level of 0.432. The global DeepEMhanced EM map was show as a white transparent map with a single hMPV F protomer and Fab variable domain colored (hMPV F, blue; heavy chain variable domain, red; light chain variable domain, orange). B: Overlay of the 5–1 epitope onto the defined antigenic sites of hMPV F revealing that 5–1 primarily interacts with residues in site II and V, with additional contacts within site Ø. C: Atomic model of 5–1 and hMPV F interface with key residues highlighted as sticks. 5–1 and one hMPV F protomer are shown as cartoons. Oxygen atoms are colored red and Nitrogen atoms are colored blue. Partially modeled Asn-172 glycan is shown as deep color sticks. D: Sequence conservation of the 5–1 epitope between hMPV F and RSV F with the epitope of 5–1 delineated in white. E: Sequence alignment of the 5–1 epitope with four representative hMPV F sequences from A1, A2, B1, B2 subgroup and two representative RSV sequences from A2 and B subgroup. The conservation of each residue is described underneath and the 5–1 interacting residues are highlighted in red. The glycosylation site at Asn-172 is shown as a branch.

Figure 5:. 5–1 Prophylaxis of 5–1 against RSV and hMPV challenge.

Protective efficacy of 5–1 against A) RSV and B) hMPV replication in vivo. BALB/c mice were treated intraperitoneally with 10 mg/kg, 1 mg/kg, and 0.1 mg/kg of mAb 5–1 6h prior to intranasal RSV and hMPV infection. Viral titers in the lung homogenates of BALB/c mice in each treatment group (n = 5 mice per group, 5 females) were determined by plaque assay. n.s., not significant, Limit of detection (LOD) is indicated with a dashed line.