Abstract Summary/Description
Respiratory Syncytial Virus (RSV) and human Metapneumovirus (hMPV) are global pathogens and two leading causes of lower respiratory tract infection (LRTI), hospitalizations and deaths in infant, elderly, and immunocompromised populations. Belonging to the Pneumoviridae family, the two viruses share a homologous membrane fusion (F) protein that has shown potential as a target for therapeutic monoclonal antibodies (mAbs). By binding to F proteins at their pre-fusion (preF) conformation rather than post-fusion (postF) conformation, mAbs can neutralize viruses and block their entry into host cells. Nanobodies are antigen-binding entities derived from the heavy chain-only antibodies of camelid animals, and they retain full antigen specificity at a very small size, creating unique potential advantages over traditional approaches. Here we sequentially immunized llama with prefusion stabilized hMPV and RSV F proteins, and identified two nanobodies, NB16 and NB28, that target a conserved epitope on preF proteins. NB16 and NB28 showed strong binding affinity with both viral preF proteins and demonstrated cross-neutralization capabilities against the viruses. To further increase neutralization efficacy, in vitro maturation was performed by site directed mutagenesis in the variable regions of the nanobody sequences, and all cross-binding mutants were isolated for comparisons with the parent sequence. Further binding analyses and neutralization assays will be performed with the goal of identifying nanobodies with increased affinity for preF proteins and high levels of viral neutralization. Our work provides a framework of developing countermeasures against LRTI caused by HMPV and RSV and establishes a generalizable strategy of nanobody-based therapies development against other viral pathogens.
