Profiling of hMPV F-specific antibodies isolated from human memory B cells

Abstract

Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.

Fig. 1. CDRH3 lengths and somatic hypermutations (SHM) of hMPV F-specific mAbs.

a Germline frequency of VH and VK/VL. b CDRH3 lengths. c Nucleotide substitutions of VH (excluding CDRH3), and VK/VL. Red bars indicate the median. The analysis was based on the Kabat delineation system. Source data are provided as a Source Data file.

Fig. 2. Characterization of an ultra-potent neutralizing mAb M4B06.

a Neutralization of M4B06 IgG to hMPV A and B strains. Data were presented as mean values ± SD of three replicates. b BLI binding of M4B06 IgG to unprocessed PreF trimer, processed PreF trimer, and PostF trimer. c Neutralization of M4B06 IgG and Fab, as well as an irrelevant control antibody (DD1L), to hMPV PreF and three types of MARM viruses. Data were presented as mean values ± SD of three replicates. d Binding of M4B06 IgG to unprocessed hMPV PreF WT and PreF carrying MARM mutants, determined by ELISA with Expi293 cell culture supernatants containing expressed antigens. Data were presented as mean values ± SD of two replicates. e M4B06 epitopes identified by MARM and HDX-MS were mapped on an hMPV PreF structure, with epitope residues colored in red in the ribbon diagram. f Cryo-EM map of three M4B06 Fabs in complex with a processed PreF trimer at 3.75 Å. The hMPV PreF model with the same coloring as (E) fit in the density. Source data are provided as a Source Data file.

Fig. 3. Epitope mapping of selected site II and V hMPV F-specific mAbs by binding to mutant F proteins.

a Contact residues for different site II (yellow) and V (orange) mAbs were mapped on an hMPV PreF trimer structure (PDB: 5WB0, subtype A). b Binding of selected site II and V mAbs to unprocessed hMPV PreF and mutants, determined by ELISA with Expi293 cell culture supernatants containing expressed antigens. Dots indicate two replicates. Source data are provided as a Source Data file.

Fig. 4. Epitope mapping of hMPV F-specific mAbs by BLI binning.

a Epitope binning of selected mAbs with the unprocessed hMPV PreF antigen. Seventy-three isolated mAbs which showed apparent BLI binding response (>0.2 nm) with the tested antigen were shown. The heatmap shows epitope binning from the sandwich-based BLI binding assay, with darker green color indicating more competition between the first and the second antibodies. Top side-bars showed the neutralization potency (IC50) of mAbs to hMPV A, hMPV B, RSV A, and RSV B virus strains. b Major antigenic sites II, III, III′, IV, IV′, V were mapped on the hMPV PreF trimer protein structure. DS7-site was also labeled as a reference. c Percentage of antibodies targeting each antigenic site, grouped by neutralization potency. For each mAb, the stronger neutralization potency of hMPV A and B were chosen for grouping. Source data are provided as a Source Data file.

Fig. 5. Correlations among neutralization potency, antigenic sites, and binding-specificity to different hMPV F conformations.

a, b BLI binding response of hMPV F-specific mAbs to unprocessed hMPV PreF trimer and PostF trimer. Each dot represents an isolated hMPV F-specific antibody, colored by neutralization potency (a) or mapped antigenic site (b). c, d BLI binding response of hMPV F-specific mAbs to unprocessed hMPV PreF and processed stabilized hMPV PreF (115BV) antigens. Each dot represents an isolated hMPV F-specific antibody, colored by neutralization potency (c) or mapped antigenic site (d). e Correlations between neutralization potency (after Log₁₀ transformation) and the ratio of processed PreF/unprocessed PreF BLI binding. The dashed line and R-square number indicate the linear regression. Each dot represents an isolated hMPV F-specific antibody, colored by the mapped antigenic site. For each antibody, the higher neutralization potency between hMPV A and B was chosen to plot. Source data are provided as a Source Data file.