Antibody recognition of the Pneumovirus fusion protein trimer interface

Abstract

13Human metapneumovirus is a leading cause of viral respiratory infection in children, and can 14 cause severe lower respiratory infection in infants, the elderly, and immunocompromised patients. 15However, there remain no licensed vaccines or specific treatments for hMPV infection. Although 16 the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological 17 properties of hMPV F are still poorly understood. To further define the humoral immune response 18 to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and 19 MPV465. Both mAbs are neutralizing in vitro and target a unique antigenic site harbored within 20 the trimeric interface of the hMPV F protein. We determined both MPV458 and MPV465 have 21 higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with 22 hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F 23 protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV 24 F protein, suggesting significant breathing of the hMPV F protein must occur for hMPV F protein 25 recognition of the novel epitope. In addition, significant glycan interactions were observed with a 26 somatically mutated light chain framework residue. The data presented identifies a novel epitope 27 on the hMPV F protein for structure-based vaccine design, and provides a new mechanism for 28 human antibody neutralization of viral glycoproteins.Human metapneumovirus (hMPV) is a leading cause of viral respiratory infections in children, the 32 majority of which are seropositive for hMPV by five years of age 1 . Although hMPV was discovered 33 in 2001 2 , there are no vaccines or therapeutics approved to prevent or treat viral infection. Similar 34 to other respiratory pathogens, children, the elderly, and the immunocompromised are the major 35 groups for which hMPV infection may require hospitalization [3][4][5][6][7][8][9][10][11] . Several reports have 36 demonstrated hMPV infection can be lethal in both adults and children. In particular, haemopoietic 37 stem cell transplant patients are at high risk of severe hMPV infection [10][11][12][13] , and several outbreaks 38 of hMPV in nursing homes have been reported [14][15][16] . In addition, fatal hMPV has been observed in 39 one child during an outbreak of hMPV in a daycare center. 17 hMPV is also a significant cause of 40 febrile respiratory illness in HIV-infected patients 18 , and has been linked to exacerbations of 41 chronic obstructive pulmonary disease 19 . Co-circulation of hMPV was observed during the SARS 42 outbreak of 2003, suggesting interactions with other circulating respiratory viruses. 20-22 43 44 hMPV circulates as two genotypes, A and B, and based on the sequence variability of the surface 45 proteins, hMPV is further grouped into four subgroups, A1, A2, B1, and B2 23,24 , and two additional 46 subgroups, A2a and A2b, have been proposed 12 . hMPV has three surface glycoproteins, the small 47 hydrophob...

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

“…Stabilized post-fusion hMPV A1 F protein has previously been generated by expression in CV-1 cells by removal of the hMPV F fusion peptide (residues 103–111) to prevent aggregation, replacing the transmembrane domain with the fibritin trimerization domain (Foldon) from T4 bacteriophage, and altering the cleavage site with the second furin-cleavage site of hRSV F (16). We previously reported a post-fusion hMPV B2 F protein by incorporating similar genetic modifications, but this protein was expressed in HEK293F cells (29, 30). Here, we utilized this protein construct to determine the X-ray crystal structure of post-fusion hMPV F from subgroup B2.…”

“…Plasmids encoding cDNA for hMPV 130-BV F and hMPV B2 F proteins were synthesized (GenScript) and cloned into the pcDNA3.1+ vector as previously described (29, 30). The plasmids were expanded by transformation in Escherichia coli DH5α cells with 100 µg/mL of ampicillin (Thermo Fisher Scientific) for selection.…”

“…plasmid maxi kit (Omega BioTek), according to the manufacturer’s protocol. The stable cell line that expresses the hMPV B2 F protein was generated as previously described (30). For protein expression and purification, the stable cell lines were expanded in 500 mL of Freestyle293 medium supplemented with G418 at 1 × 106 cells/mL.…”

“…For recombinant mAbs, plasmids encoding cDNAs for heavy and light chain sequences of 101F (31), MPE8 (31), and DS7 (32) were synthesized (GenScript), and were cloned into vectors encoding human IgG1 and lambda or kappa light chain constant regions, respectively. mAbs were obtained by transfection of plasmids into Freestyle HEK293F cells as described previously (30). For hybridoma-derived mAbs, hybridoma cell lines were expanded in serum-free medium (Hybridoma-SFM; Thermo Fisher Scientific).…”

“…hMPV B2 strain TN/93-32 and hMPV A2 strain CAN/97-83 viruses were grown in LLC-MK2 cells (ATCC) as previously described (30). Briefly, cells were grown to 80% confluence in 225 cm 2 flasks in Opti-MEM supplemented with 2% FBS.…”