Structure, immunogenicity, and conformation-dependent receptor binding of the post-fusion human metapneumovirus F protein

Abstract

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (mAbs). While X-ray structures of trimeric pre-fusion and post-fusion hMPV F proteins from genotype A, and monomeric pre-fusion hMPV F protein from genotype B have been determined, structural data for the post-fusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of post-fusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparan sulfate. A library of heparan sulfate (HS) oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly pre-fusion hMPV F, but had limited binding to post-fusion hMPV F. Furthermore, mAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparan binding. In addition, we evaluated the efficacy of post-fusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 post-fusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on hMPV F. Overall, this study provides new characteristics of the hMPV F protein informative for vaccine and therapy development.

A Pan-Pneumovirus vaccine based on immunodominant epitopes of the fusion protein

“…RHMS-1 was expressed in HEK293F cells and size exclusion chromatography (SEC) showed RHMS-1 was mainly expressed as a trimeric protein, but the size is slightly bigger than RSV F trimers (Figure 2A) . For hMPV F, trimers and monomers were observed after trypsin treatment as previously described (32), but the size of trimeric hMPV F is smaller than RHMS-1 and RSV F, likely due to trypsinization (Figure 2A) . Like RSV F, RHMS-1 was deduced to be cleaved after expression, as the F2 domain was observed on the gel under reducing conditions (Figure 2B) , and the sizes of RHMS-1 and RSV F bands are consistent with the peaks shown in Figure 2A .…”

“…Plasmids encoding cDNAs of Pneumovirus proteins were synthesized (GenScript) and cloned into the pcDNA3.1 + vector (30, 32, 42). The stable cell line that expresses the hMPV B2 F protein was utilized as previously described (32). The rest of the F proteins and monoclonal antibodies were transiently expressed in Expi293F cells.…”

“…Data were analyzed in GraphPad Prism using a nonlinear regression curve fit and the log(agonist)-versus-response function to calculate the binding EC 50 values. Mouse serum IgG titers were calculated from the highest dilution of a serum sample that produced OD 405 readings of >0.3 above the background readings and were shown in a log 10 scale as previously described (32).…”

“…Three weeks after the boost, mice were bled and then intranasally challenged with RSV A2 (2.8×10 6 PFU per mouse) or hMPV TN/93-32 (3×10 5 PFU per mouse). Mice were sacrificed 5 days post-challenge, and lungs were collected and homogenized for virus titration as previously described (32). Briefly, RSV challenged lung homogenates were plated on HEp-2 cells (EMEM+2% FBS) while hMPV challenged lung homogenates were plated on LLC-MK2 cells (EMEM + 5 μg/ml trypsin-EDTA and 100 μg/ml CaCl 2 ) in 24 well plates.…”

“…Previous attempts with formalin-inactivated RSV and hMPV vaccines revealed that low affinity, non-neutralizing F-specific antibodies induced by denatured fusion proteins cannot provide protection, and lead to vaccine enhanced disease (25–29). By stabilizing the F proteins in the pre-fusion conformation, several studies have demonstrated improvement in neutralizing antibody titers (30, 31), while for hMPV, pre-fusion and post-fusion F proteins induced comparable neutralizing antibodies in mice (24) and immunization with post-fusion F completely protected mice from hMPV challenge (32). Several epitope-focused vaccine designs have been tested for RSV and hMPV F. A head-only RSV F protein boosted titers of neutralizing Abs targeting antigenic sites Ø and II (33).…”