All data supporting the findings of this study are available within the paper and are available from the corresponding author upon request.. a strong rationale for its evaluation as a COVID-19 therapeutic. Keywords:human VHantibody domain name, virus neutralization, electron microscopy, SARS-CoV-2, mouse and hamster models == Graphical Abstract == == Highlights == A high-affinity human antibody domain name, VHab8, specific for SARS-CoV-2 was selected VHab8 bound to all three S protomers competing with ACE2 Bivalent VH, VH-Fc PIM-1 Inhibitor 2 ab8, potently neutralized SARS-CoV-2in vitroand in animals Small size and bivalency contribute to the high ab8 SARS-CoV-2 neutralizing potency A high-affinity human antibody domain name, VHab8, specific for SARS-CoV-2, bound to all three S protomers competing with ACE2. The relatively small size and bivalency of VH-Fc ab8 contributed to its high potency in two animal models of contamination. == Introduction == The global outbreak of a severe acute respiratory distress (SARS) coronavirus 2 (SARS-CoV-2) associated disease 2019 (COVID-19) requires rapid identification of therapeutics and vaccines. While many vaccines are in clinical development, the time to market can be relatively long, and immunogenicity can be limited for high-risk groups (Amanat and Krammer, 2020). Alternatively and complementarily, antibodies can be used as safe and effective prophylactics and therapeutics PIM-1 Inhibitor 2 (Pelegrin et al., 2015). Convalescent plasma from COVID-19 patients inhibited SARS-CoV-2 contamination and alleviated symptoms of newly infected patients (Casadevall and Pirofski, 2020;Rojas et al., 2020) suggesting that potent neutralizing monoclonal antibodies (mAbs) may be even more effective. SARS-CoV-2 genome shares more than 80% homology to the SARS-CoV (Li et al., 2020b). Similar to SARS-CoV, SARS-CoV-2 uses the spike (S) envelope glycoprotein to enter into host cells. The viral entry is initiated by the receptor binding domain name (RBD) of the S protein binding to its receptor, angiotensin-converting enzyme 2 (ACE2), leading to conformational change of the S2 subunit and formation of six helical-bundle resulting in membrane fusion between viral and host cells (Jiang et al., 2020;Yan et al., 2020). The SARS-CoV RBD contains immune-dominant epitopes that can elicit neutralizing antibodies conferring protection to SARS-CoV contamination (He et al., 2005). A recent bioinformatics study showed that SARS-CoV-2 RBD has several B cell epitopes (Grifoni et al., 2020). SARS-CoV-2 RBD-based immunogens were able to elicit neutralizing sera in animals (Quinlan et al., 2020). Thus, SARS-CoV-2 RBD is a good target for developing potent neutralizing mAbs. We and others have identified such potent neutralizing Rabbit polyclonal to ZNF473 human mAbs targeting the RBD of SARS-CoV (Zhu et al., 2007) and the PIM-1 Inhibitor 2 middle east respiratory syndrome coronavirus (MERS-CoV) (Ying et al., 2014a). Recently, several groups have reported the isolation of potent neutralizing antibodies from convalescent human donors but all are in an immunoglobulin G1 (IgG1) format with a molecular mass of ~150 kDa (Cao et al., 2020;Ju et al., 2020;Rogers et al., 2020;Shi et al., 2020;Zost et al., 2020). Antibody domains and fragments such as Fab (fragment antigen binding, molecular weight of 50 kDa), scFv (singe-chain variable fragment, 30 kDa), and VH(heavy chain variable domain name, 15 kDa) are attractive antibody formats as candidate therapeutics (Nelson, 2010). For example, isotope-labeled antibody fragments are more suitable for bio-imaging due to their better tissue penetration and faster clearance compared to full-size antibodies (Freise and Wu, 2015). Single antibody domains (sAbd; e.g., camelid VHH [15 kDa]) exhibit strong antigen binding and high stability (Harmsen and De Haard, 2007). We and others have demonstrated that human IgG1 heavy chain variable domain name (VH) can be PIM-1 Inhibitor 2 engineered to achieve high stability and affinity to antigens (Nilvebrant et al., 2016), as exemplified by the VH, m36.4, targeting the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein co-receptor binding site (Chen et al., 2008a). The VHdomains small size could improve therapeutic efficacy for infectious diseases, such as COVID-19 because of greater penetration to sites of contamination. The conformation of the SARS-CoV-2 S trimer is usually dynamic with only one RBD in the up conformation presenting neutralizing epitopes while epitopes in the other two RBDs may be masked (Yan et al., 2020). Small VHs may achieve binding to the cryptic RBD epitopes during the dynamic.