Fig 1.
Grafting of the RSV palavizumab/motavizumab epitope onto the HMPV F protein.
(a) Structures of pre- and post-fusion RSV F highlighting the helix-turn-helix motif for palivizumab and motavizumab. The protein surface and ribbon are colored cyan with the epitope region in each subunit of the trimer colored blue (arrows). (b) Structure of the motavizumab:RSV peptide complex (RCSB code: 3IXT) superimposed on the partial HMPV F structure (RCSB code: 4DAG). Residues from RSV are labeled in black and those of HMPV F are in parentheses and labeled in blue. Residues highlighted with the yellow background were substituted in the RPM-1 mutant. (c) Sequence alignment of RSV and HMPV F proteins, spanning the motavizumab epitope, along with the RPM-1 mutant sequence. Yellow and red boxes highlight amino acids that contact motavizumab, with red indicating less accessible residues of the epitope. Letters above (H/L) the sequences represent contacts with the heavy of light chain CDRs of the motavizumab Fab. Letters below the RPM-1 mutant represent contact residues that were either changed (C), left unchanged (-) or are identical (.) in RSV and HMPV F. (d) Contact map of motavizumab:RSV F peptide contacts from the crystal structure, with RSV residues highlighted in yellow and antibody residues highlighted in green (heavy chain) or blue (light chain). Three residues (L258, N262, N268) are highlighted as making the most contacts per residue across the interface.
Fig 2.
Purification and comparative analysis of wt and mutant MPV F protein.
(a) SDS-PAGE analysis of purified wild-type HMPV F and the RPM-1 mutant. (b) Comparison of gel filtration traces of purified wild-type and RMP-1 MPV F proteins, demonstrating that they migrate similarly as trimers. (c, d) Negative-stain EM analysis of wild-type (c) and RPM-1 (d) proteins demonstrating that they form similar distributions of pre- and post-fusion conformations. (e, f) EM analysis of heat-treated wild-type (e) and RPM-1 (f) F proteins demonstrating that both proteins convert to a golf-tee like post-fusion conformation. In panels c-f, insets show higher magnification of selected F trimers along with schematic representations.
Fig 3.
RPM-1, but not wild-type HMPV F, binds neutralizing antibodies to both RSV and HMPV.
(a, b) Binding of the anti-HMPV F DS-7 antibody to wild-type (a) and RPM-1 (b) proteins. Both HMPV F proteins bound with similar affinity to the antibody demonstrating that the DS-7 epitope is not affected by the RPM-1 mutations. (c, d) Binding of palivizumab to wild-type (c) and RPM-1 (d) proteins. The wild-type HMPV F protein showed very weak potential binding palivizumab, while the RPM-1 mutant showed clear high affinity interactions with the antibody.
Table 1.
Binding of antibodies to wt and RMP-1 MPV F.
Fig 4.
Heat treatment of wild-type and mutant RSV F does not affect binding of palivizumab and DS-7 neutralizing antibodies.
(a, b) Binding of the anti-HMPV F DS-7 antibody to wild-type (a) and RPM-1 (b) proteins after heating to 50°C for 30 minutes. Both proteins bound with similar affinity to the antibody as the unheated F (Fig 3), demonstrating that the DS-7 epitope is not affected by conversion to the post-fusion conformation. (c, d) Binding of palivizumab to heat-treated wild-type (c) and RPM-1 (d) proteins. The wild-type HMPV F protein and RPM-1 mutant showed similar binding interactions with palivizumab as with the unheated F samples, indicating that concversion of pre- to post-fusion conformation had little effect on palivizumab antibody binding.
Fig 5.
The HMPV F mutant bound motavizumab with higher affinity than palivizumab.
Binding of the DS-7 Fab to wild-type (a) and motavizumab Fab RPM-1 (b) proteins using a BiOptix 404pi instrument. The wild-type HMPV F protein bound DS-7 with similar affinity as measured with the Biorad Proteon (Table 1). The RPM-1 mutant bound motavizumab with higher affinity than palivizumab. (c, d) Gel filtration analysis of the formation of RPM-1 complexes with palivizumab (c) and motavizumab (d) Fabs. The palivizumab complexes were unstable and dissociated during the gel filtration analysis, while motavizumab formed stable complexes consistent with three Fabs engaging a single RPM-1 trimer.
Fig 6.
RSV and MPV neutralization assays.
Data for each of 5 mice immunized with one of the three antigens (pre-fusion MPV F, post-fusion MPV F and RMP-1) is shown as the serum dilution required to obtain 60% neutralization. None of the mice showed neutralization of RSV, 4/5 immunized with pre-fusion F or RPM-1 showed MPV neutralization and 2/5 showed MPV neutralization with post-fusion MPV F.