Engineering, Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation

<div><p>Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.</p></div

Structure of hMPV F in the postfusion conformation.

<p><i>Left</i>: One protomer of the postfusion hMPV F trimer is shown as a ribbon colored as a rainbow from the N-terminus of F2 (blue) to the C-terminus of F1 (red). <i>Right</i>: The postfusion hMPV F trimer with one protomer shown as a ribbon and two protomers shown as molecular surfaces colored white and grey. The six-helix bundle (6HB) and fusion peptides are labeled.</p

Comparison of paramyxovirus postfusion F structures.

<p>Superposition of one protomer of the postfusion hMPV F structure (white) with the corresponding structures of <b>A</b>) hRSV F, <b>B</b>) hPIV3 F, and <b>C</b>) NDV F. Two neutralizing antigenic sites (II and IV of hRSV F) are magnified.</p

Crystallographic data collection and refinement statistics.

<p>Crystallographic data collection and refinement statistics.</p

Stabilization and characterization of the soluble hMPV F protein ectodomain as a postfusion trimer.

<p><b>A</b>) Diagrams of the different constructs of hMPV F used in this study. (1) Scheme of the F protein (grey rectangle, NL/1/00 strain) denoting the last amino acid of its ectodomain (489), the fusion peptide (red), the preceding cleavage site (arrow) and the C-terminal 6xHis-tag (black box). (2) Same scheme but with the Foldon sequence (blue rectangle) and TEV cleavage site (arrow) added, and the change G294E denoted by a red circle. (3) The basic residues of hRSV F cleavage site II are shown in boldface and the amino acids deleted from the fusion peptide are indicated by dashes in the amino acid sequence. (4) Scheme of the protein after TEV cleavage. <b>B</b>) Gel-filtration traces of the four proteins depicted in panel A, labelled and color-coded as in panel A. Inset shows a Coomassie-blue-stained SDS-PAGE, run under reducing conditions, of the major peak of each chromatogram. <b>C</b>) Electron microscopy of negative-stained proteins 1–4. Some cone-shaped molecules are indicated in panel 2 by black arrowheads. Scale bar: 50 nm. <b>D</b>) Proteins 2 (green) and 3 (blue) were heated stepwise at the indicated temperatures, as described in Materials and Methods, before being tested for binding in ELISA to the mAbs indicated in each panel. Results are shown as percent of binding with proteins heated for 10 minutes at 30°C.</p

Binding and neutralization of sera from mice immunized with postfusion F proteins.

<p>Soluble hMPV F postfusion trimers derived from NL/1/00 (A1 sublineage) or NL/1/99 (B1 sublineage) viruses and the equivalent hRSV F postfusion trimer were used to immunize BALB/c mice. Sera were collected 4 weeks after immunization and tested in (<b>A</b>) ELISA and (<b>B</b>) neutralization assays. ELISA titers refer to serum dilution that yielded 50% of the maximal (saturating) value and neutralization titers refer to dilution that inhibited 50% fluorescence intensity, 48 hours after infection. Mean ELISA and neutralization titers for each group are shown by horizontal bars. Horizontal red lines in each panel indicate detection limits.</p

mAb 101F ELISA binding and neutralization.

<p><b>A</b>, <b>B</b> and <b>C</b>) ELISA binding of mAb 101F, the hMPV-specific mAb MF14, and the hRSV-specific mAb 47F to the indicated postfusion proteins. <b>D</b>, <b>E</b> and <b>F</b>) Neutralization of the noted viruses with the three mAbs.</p

Binding of mAb 101F to hMPV and hRSV F proteins.

<p><b>A</b>) Binding constants of mAb 101F for postfusion F proteins from hMPV NL/1/00 (A1 sublineage) and NL/1/99 (B1 sublineage) strains and from hRSV (Long strain) determined by surface plasmon resonance. <b>B</b>) Biacore binding sensorgrams used to determine the data in panel A.</p

Structural basis of 101F cross-reactivity.

<p><b>A</b>) <i>Left</i>: Superposition of the linear 101F epitope derived from the hMPV (white) and hRSV (blue) postfusion F proteins. Side-chains are shown as sticks, with oxygen atoms colored red and nitrogen atoms colored blue. hMPV F residues are labeled and numbered. <i>Right</i>: Model of mAb 101F bound to antigenic site IV derived from the hMPV and hRSV postfusion F proteins. 101F heavy chain is colored red and light chain is yellow. <i>Bottom</i>: Sequence alignment of the antigenic site IV domain from hMPV and hRSV F. Identical residues have white text with black backgrounds, whereas residues that are similar are in bold and have a black border. Open circles denote residues with >10 Ų buried surface area and filled rectangles denote residues whose side-chains form hydrogen bonds with 101F in the hRSV F peptide-bound crystal structure (PDBID: 3O45). <b>B</b>) Negatively stained electron micrographs of 101F Fab bound to postfusion hRSV F and hMPV F. The top two panels are 2D averages whereas the other panels are examples of individual negatively stained F–Fab complexes. <b>C</b>) Models of a single 101F Fab in complex with postfusion F trimers of hRSV and hMPV. Molecular surfaces of the trimers are shown, and residues within 5.5 Å of 101F atoms are darker.</p

N332-Directed Broadly Neutralizing Antibodies Use Diverse Modes of HIV-1 Recognition: Inferences from Heavy-Light Chain Complementation of Function

<div><p>Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332) is characterized by an <i>N</i>-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type) was generally consistent (58±10 µg/ml). In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways.</p> </div