Variants of Concern (VOCs), and variant under monitoring (VUM) of SARS-CoV-2 have emerged with heightened infectivity, antibody resistance, and vaccine breakthrough. In light of this, it is imperative to establish efficient and streamlined testing models for the prompt detection of newly emerging mutant strains. The 5-Helix Bundle (5-HB) protein was engineered as potent inhibitors predicated on the S2 subunit of the SARS-CoV-2 spike protein, exerting a broad-spectrum inhibitory influence against SARS-CoV-2 infections. Positron emission tomography (PET) quantifications revealed significantly reduced radioactive uptake in infected tissue of 5-HB protein-treated mice. The reduction was much lower than that of untreated infected mice imaged at 4 h (48.6 % reduction), 12 h (45.2 % reduction), and 24 h (36.9 % reduction) (p < 0.01). The newly engineered 5-HB protein features with the capability of binding to conservative pre-fusion spike of SARS-CoV-2 in live animals, highlighting its entry-inhibition capacity for a wide spectrum of SARS-CoV-2 variants.