Abstract
The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development. The prefusion conformation is thought to be the optimal vaccine antigen, but previously described prefusion F proteins expressed poorly and were not well stabilized. Here, we use structures of hMPV F to guide the design of 42 variants containing stabilizing substitutions. Through combinatorial addition of disulfide bonds, cavity-filling substitutions, and improved electrostatic interactions, we describe a prefusion-stabilized F protein (DS-CavEs2) that expresses at 15 mg/L and has a melting temperature of 71.9 °C. Crystal structures of two prefusion-stabilized hMPV F variants reveal that antigenic surfaces are largely unperturbed. Importantly, immunization of mice with DS-CavEs2 elicits significantly higher neutralizing antibody titers against hMPV A1 and B1 viruses than postfusion F. The improved properties of DS-CavEs2 will advance the development of hMPV vaccines and the isolation of therapeutic antibodies.
Highlights
The human metapneumovirus fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development
HMPV F0 is cleaved by trypsin-like extracellular serine proteases, such as TMPRSS2, the extent to which this occurs in the producing cells versus target cells is not well defined[6]
We set out to design variants based on the prefusion (PDB ID: 5WB0) and postfusion (PDB ID: 5L1X) structures of human metapneumovirus (hMPV) F, and characterize their expression yield, monodispersity, thermostability, and antigenicity
Summary
The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development. Three F2/F1 heterodimers associate into a metastable prefusion trimer that constitutes the active form of the protein In cell culture, this proteolytic activation can be accomplished by the addition of trypsin, which cleaves the protein at a monobasic cleavage site[1,4,5]. The metastable prefusion F protein undergoes a substantial conformational change, liberating and extending the fusion peptide into the host-cell membrane. This unstable pre-hairpin intermediate collapses back onto itself to form a highly stable sixhelix bundle composed of a trimer of the N-terminal and. Given its critical role in viral entry, vaccine candidates for hMPV generally include the F protein
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