Nanobody toolkit for human coronavirus classification

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ABSTRACT The endemic human coronaviruses (HCoV) NL63, OC43, 229E and HKU1 have typically been associated with relatively mild and self-limiting respiratory disease resembling the common cold in healthy individuals. However, the newly emerging zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) can cause more severe respiratory distress, especially in those with underlying health conditions and can be fatal. Coronaviruses are diverse and spillover from zoonotic reservoirs either directly to humans or via an intermediate host. Spillover is likely to be more frequent due to the ever-expanding human population and ecosystem erosion, thereby ensuring more collisions between natural and unnatural host. Our exploratory research proposal seeks to develop a small toolkit of immunoreagents that will form the basis of a logic gate to identify and classify coronaviruses. A single immunoreagent, with broad recognition of overall shared protein architecture, will allow the identification of the coronavirus genera. Separate immunoreagents with absolute specificity for unique amino-acid variation between the human and zoonotic coronaviruses will allow the further classification to a known pathogen. Such a toolkit will quickly establish if a newly emerging virus is a coronavirus we are familiar with or is a novel genus or lineage that has never before spilled over into the human population and may demand urgent follow up for epidemic or pandemic potential. Our immunoreagents are based on llama single domain antibodies or nanobodies that are inexpensive to produce at high yields in E. coli and highly modular, allowing fusion to enzymatic reporter enzymes for one-step probing of virus infected cells and facile establishment of sandwich assays. Immortalization of nanobody sequences in silico means these are immediately available to the wider research community following deposition in Genbank unlike polyclonal sera or hybridomas secreting IgG. Single-pot library technology enables a rapid response to be mounted against any newly emerged coronavirus in the future, ensuring we are more pro-active than re-active to help safeguard human health.