EK1 is a broad-spectrum human coronavirus fusion inhibitor for combating infection of current and emerging coronaviruses.

The majority of neutralizing antibodies (NAbs) against SARS-CoV-2 recognize the receptor-binding domain (RBD) of the spike (S) protein. As an escaping strategy, the RBD of the virus is highly variable, evolving mutations to thwart a natural immune response or vaccination. Targeting non-RBD regions of the S protein thus provides a viable alternative to generating potential, robust NAbs. Using a pre-pandemic combinatorial antibody library of 10¹¹, through an alternate negative and positive screening strategy, 11 non-RBD-targeting antibodies are identified. Amongst one NAb that binds specifically to the N-terminal domain of the S protein, SA3, shows mutually non-exclusive binding of the angiotensin-converting enzyme 2 receptor with the S protein. SA3 appears to be insensitive to the conformational change and to interact with both the “open” and “closed” configurations of the trimeric S protein. SA3 shows compatible neutralization as S-E6, an RBD-targeting NAb, against the wild type and variant of concern (VOC) B.1.351 (Beta) of the SARS-CoV-2 pseudo virus. More importantly, the combination of SA3 with S-E6 is synergistic and recovers from the 10-fold loss in neutralization efficacy against the VOC B.1.351 pseudo virus.

Cross-reactive antibodies targeting multiple epitopes have been identified in Sarbecoviruses, but the precise molecular mechanism(s) behind the crossreactivity remain poorly understood. Here, we isolate 3D1, a broadly neutralizing antibody (bnAb) derived from a human combinatorial antibody library targeting the conserved HR1 domain. 3D1 uniquely recognizes a β-turn fold comprising a 6-mer peptide (pep^DVVNQN/Q) that forms during a pre-hairpin transition state, occurring exclusively before membrane fusion during viral infection. 3D1 effectively neutralizes a wide range of live SARS-CoV-2 wild-type strains except for Omicron, which evades neutralization due to a detrimental point mutation (Q954H). Notably, this cryptic epitope reveals a signature motif that extends throughout the core region of coronaviruses and is also present in various RNA viruses, including HIV and Marburgvirus. 3D1 functions as a natural or background antibody capable of binding to a diverse array of non-self antigens. 3D1's cross-reactivity underscores the effectiveness of the library approach, which encompasses the entire antibody repertoire.

Cross-species spillover to humans of coronaviruses (CoVs) from wildlife animal reservoirs poses marked and global threats to human and animal health. Recently, sporadic infection of canine coronavirus–human pneumonia-2018 (CCoV-HuPn-2018) in hospitalized patients with pneumonia genetically related to canine and feline coronavirus were identified. In addition, swine acute diarrhea syndrome coronavirus (SADS-CoV) had the capability of broad tropism to cultured cells including from humans. Together, the transmission of Alphacoronaviruses that originated in wildlife to humans via intermediate hosts was responsible for the high-impact emerging zoonosis. Entry of CoV is mainly mediated by Spike and formation of a typical six helix bundle (6-HB) structure in the postfusion state of Spike is pivotal. Here, we present the complete fusion core structures of CCoV-HuPn-2018 and SADS-CoV from Alphacoronavirus at 2.10 and 2.59 Å, respectively. The overall structure of the CCoV-HuPn-2018 fusion core is similar to Alphacoronavirus like HCoV-229E, while SADS-CoV is analogous to Betacoronavirus like SARS-CoV-2. Collectively, we provide a structural basis for the development of pan-CoV small molecules and polypeptides based on the HR1-HR2 complex, concerning CCoV-HuPn-2018 and SADS-CoV.

Human coronavirus OC43 (HCoV-OC43) is usually associated with common colds, but also related to severe disease in the frail. Its envelope glycoproteins spike (S) is responsible for host-cell attachment and membrane fusion. To understand the molecular basis of membrane fusion of HCoV-OC43, we solved the 3.34 Å crystal structure of the post-fusion state formed by two heptad repeat domains (HR1P and HR2P) of OC43-S. This fusion core comprises a parallel trimeric coiled coil of three HR1 helices with 61 Å at length, around which three HR2 helices are entwined in an antiparallel manner, as anticipated. Moreover, a pan-CoV fusion inhibitor EK1 derived from OC43-HR2P was also crystalized with OC43-HR1P in the resolution of 2.71 Å. Parallel comparisons rationalize the design of EK1, maintaining various hydrophobic and charged or hydrophilic interactions formed in the initial fusion core to stabilize the overall conformation. Together, our results not only reveal the critical intrahelical and interhelical interactions underlying the mechanism of action of OC43-S fusion, but also help our understanding on the mechanism of HCoV-OC43 inhibition by analogue HR2 mimic peptide.