Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

Viruses co-opt host proteins to carry out replication of the virus. Host proteins thus repurposed may become functionally compromised, a situation analogous to a loss-of-function allele, which we term a viral-induced hypomorph. Tumor-specific loss-of-function alleles have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of these cancer-specific mutations. Synthetic lethal interactions with viral-induced hypomorphs have yet to be demonstrated, an important first step in the development of antiviral therapeutic strategies based on the principle of synthetic lethality. We focused on Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a proviral host factor for many RNA viruses. A chemogenomic screen using the GBF1-specific inhibitor, golgicide A, identified 57 putative synthetic lethal partners of GBF1, including ADP-ribosylation factor 1 (ARF1). Using a poliovirus protein, 3A, to induce a GBF1-hypomorph, we observed synthetic lethality in the context of a shRNA-mediated knockdown of ARF1. These results provide a proof-of-concept that virus-induced hypomorphs are sensitive to perturbation by synthetic lethal interaction and open up a new avenue for antiviral therapeutic development.