The potency of the S1B-specific mAbs to inhibit receptor binding corresponds with the ability of these antibodies to neutralize virus infection (Table 2), indicating that the inhibition of virusreceptor interaction by these antibodies is their main mechanism of neutralizationin vitro. == Anti-MERS-S1AmAb 1.10f3 blocks binding of MERS-S1Ato sialoglycoconjugates == Recently we demonstrated that the MERS-S1Adomain facilitates viral binding to cell-surface sialoglycoconjugates, which can serve as a cell attachment factor for MERS-CoV [16]. with 2298 cases as of February 2019 with an estimated 35% lethality [1]. Dromedary camels are the natural reservoir of MERS-CoV from which zoonotic transmission can occur. A vast majority of dromedary camels in the Arabian Peninsula appeared to be seropositive for MERS-CoV and MERS-CoV strains found in epidemiologically-linked humans and dromedary camels are nearly identical [25]. Human-to-human transmission is inefficient but can occur upon close contact such as in household or hospital settings among patients and from patients to health-care workers [2]. The high rate of MERS-CoV infection in the dromedary camel reservoir poses a persistent threat for reintroduction of MERS-CoV into humans. Despite its continuous threat to public health, antiviral therapies or vaccines to treat or prevent MERS-CoV infection are currently lacking. The viral spikes on the surface of the enveloped MERS-CoV virions are the primary antigenic target for the development of vaccines and antibody therapies [6]. These spikes mediate virus entry into host cells and consist of three spike (S) glycoproteins, each containing the receptor binding subunit S1 and the membrane-anchored fusion-mediating subunit S2. Single-particle cryo-electron microscopic analyses of trimeric S ectodomains of several coronaviruses, including MERS-CoV [713], revealed a multi-domain architecture of the S1 subunit, consisting of four core domains (designated A though D). For MERS-CoV, two of these spike domains engage with host molecules to ensure entry into target cells. The S1Bdomain of the MERS-CoV spike protein binds the host receptor DPP4 which is essential for cell entry [14,15], whereas the N-terminal domain S1Abinds to sialoglycans on mucins and on the host cell surface, which enhances infection of MERS-CoV on human lung cells [16]. Three S2 subunits form the stem of the spikes, which can undergo extensive conformational changes enabling membrane fusion. The surface of the S2 subunit to which antibodies bind shows a higher degree of sequence conservation than the more variable S1 subunit [7]. Several potently neutralizing human monoclonal antibodies against MERS-CoV have been developed using various approaches including single cell culturing methods of memory B cells isolated from MERS-CoV patients [17], hybridoma fusion of B-cells from immunized transgenic mice encoding human variable immunoglobulin domains [1820], and phage or yeast display screening of Fab fragments of non-immune human antibody libraries [2123]. Some of these antibodies showed protection against MERS-CoV challenge in animal models [17,1922,2427]. All of these antibodies were selected based on theirin Rabbit Polyclonal to RASD2 vitroneutralizing capacity and most of them targeted the MERS-CoV S1Breceptor binding domain (RBD). Structural and functional studies indicate that the epitopes of those S1B-specific antibodies overlap with the DPP4 binding site, explaining their potent neutralizing capacity [17,19,20,28,29]. So far only two murine monoclonal antibodies have been described that target the MERS-CoV S outside the RBD [29]. Using a combination of DNA and protein vaccination of mice, Wang et al, described S1-specific non-RBD and S2-specific neutralizing murine antibodies that showed potency for protection against MERS-CoVin vivo[30]. Although RBD-specific antibodies are undoubtly very potent in neutralizing MERS-CoV, effective antibody therapies are likely to require the combination of neutralizing and non-neutralizing antibodies, targeting multiple epitopes and exhibiting diverse mechanisms of actions, Coelenterazine including Fc-mediated antibody effector functions [3133]. In addition, such antibodies should preferably be human to avoid an immune reaction against anti-MERS antibodies of a different species. Identification of such protective antibodies, their targets and mechanisms of activity is important for developing antibody-based therapies against MERS-CoV. In this study, we developed a set of human monoclonal antibodies against MERS-CoV with diverse mechanisms of action that show protective efficacyin vivo. We used the transgenic H2L2 mice encoding the human immunoglobulin variable regions to generate antibodies targeting the MERS-CoV spike protein (MERS-S). From a large panel of MERS-S-specific H2L2 antibodies, eight fully human monoclonal antibodies were generated that bind non-overlapping epitopes on MERS-S with high affinity Coelenterazine and interfere with the three known functions of the viral protein: sialic acid binding, receptor binding and membrane fusion. These antibodies were shown to protect mice from a lethal MERS-CoV infection Coelenterazine at low dosage. These studies extend our knowledge on the protective value of monoclonal antibodies targeting non-RBD domains of MERS-S. == Results ==.