PROJECT SUMMARY There is an urgent need to rapidly detect SARS-CoV-2 (CoV-2) virus in clinical and nonclinical settings, including point of care sites, workplace, and home, at sensitivity and specificity comparable or superior to RT-PCR detection of CoV-2 RNA. Much of the person-to-person CoV-2 transmission occurs before infected individuals develop symptoms. This significant pre-symptomatic/asymptomatic reservoir of CoV-2 transmission mandates efficient identification of infected individuals and their contacts at population-wide screening scale to prevent outbreaks of Covid-19 disease while allowing societies to open and economies to recover. This level of surveillance will also be needed to fully evaluate the effectiveness of countermeasures, including vaccines and therapies. We will develop a new class of diagnostic product reagents, antibody polymers, to create products that can be produced and used at population screening scale for rapid CoV-2 antigen detection at significantly improved sensitivity that approaches the sensitivity of :gold-standard” RT-PCR detection of CoV-2 genomic RNA. Our antibody polymers will be produced by engineering a novel class of small, stable, single polypeptide anti- CoV-2 antibodies termed variable lymphocyte receptors (VLRs) as polymers to achieve essentially irreversible binding to CoV-2 virus. VLR polymers are compatible with all diagnostic immunoassay forms, including lateral flow assay (LFA) “dipsticks”, which is likely to be preferred in non-laboratory settings, and with established signaling modalities, e.g., colloidal gold and horseradish peroxidase (HRP). VLRs, the antigen receptors of jawless vertebrates (lamprey and hagfish), are composed of highly diverse leucine-rich repeat domains and are the only known antigen-specific immune receptors that are not immunoglobulins (Igs). The binding site of VLRs is contained within a small single polypeptide and comprised by amino acid residues in the rigid beta-sheets that form the concave surface of the VLR structure. The over 500 million year evolutionary separation of jawed and jawless vertebrates and distinctive antigen-binding site structure of VLR antibodies have proved a source of novel specificities distinct from conventional Ig antibodies. The outer envelope of the CoV-2 virus is composed of a multivalent array of spike (S) protein that is an ideal target(s) for multivalent, essentially irreversible binding, by appropriately multivalent binding agents. We will genetically link in tandem genes encoding VLRs with binding specificity for CoV-2 S protein to create such multivalent VLR polymer binding agents that couple binding of CoV- 2 antigens to an amplifiable, visually observable result, e.g., color change. The high ratio of CoV-2 S protein 50 – 100 S protein trimers per virus particle, to the single copy CoV-2 RNA genome, and the amplification available via catalysis, e.g., HRP, provides significantly improved CoV-2 antigen detection sensitivity that ap...