A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections

Tracking the pathogen of coronavirus disease 2019 (COVID-19) in live subjects may help estimate the spatiotemporal distribution of SARS-CoV-2 infection in vivo. This study developed a positron emission tomography (PET) tracer of the S2 subunit of spike (S) protein for imaging SARS-CoV-2. A pan-coronavirus inhibitor, EK1 peptide, was synthesized and radiolabeled with copper-64 after being conjugated with 1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid (NOTA). The in vitro stability tests indicated that [⁶⁴Cu]Cu-NOTA-EK1 was stable up to 24 h both in saline and in human serum. The binding assay showed that [⁶⁴Cu]Cu-NOTA-EK1 has a nanomolar affinity (K_i = 3.94 ± 0.51 nM) with the S-protein of SARS-CoV-2. The cell uptake evaluation used HEK293T/S⁺ and HEK293T/S^- cell lines that showed that the tracer has a high affinity with the S-protein on the cellular level. For the in vivo study, we tested [⁶⁴Cu]Cu-NOTA-EK1 in HEK293T/S⁺ cell xenograft-bearing mice (n = 3) and pseudovirus of SARS-CoV-2-infected HEK293T/ACE2 cell bearing mice (n = 3). The best radioactive xenograft-to-muscle ratio (X/N^xenograft 8.04 ± 0.99, X/N^pseudovirus 6.47 ± 0.71) was most evident 4 h postinjection. Finally, PET imaging in the surrogate mouse model of beta-coronavirus, mouse hepatic virus-A59 infection in C57BL/6 J mice showed significantly enhanced accumulation in the liver than in the uninfected mice (1.626 ± 0.136 vs 0.871 ± 0.086 %ID/g, n = 3, P < 0.05) at 4 h postinjection. In conclusion, our experimental results demonstrate that [⁶⁴Cu]Cu-NOTA-EK1 is a potential molecular imaging probe for tracking SARS-CoV-2 in extrapulmonary infections in living subjects.