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Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infection and continues to infect humans, thereby contributing to a high mortality rate (34.3% in 2019). In the absence of an available licensed vaccine and antiviral agent, therapeutic human antibodies have been suggested as candidates for treatment. In this study, human monoclonal antibodies were isolated by sorting B cells from patient's PBMC cells with prefusion stabilized spike (S) probes and a direct immunoglobulin cloning strategy. We identified six receptor-binding domain (RBD)-specific and five S1 (non-RBD)-specific antibodies, among which, only the RBD-specific antibodies showed high neutralizing potency (IC50 0.006–1.787 μg/ml) as well as high affinity to RBD. Notably, passive immunization using a highly potent antibody (KNIH90-F1) at a relatively low dose (2 mg/kg) completely protected transgenic mice expressing human DPP4 against MERS-CoV lethal challenge. These results suggested that human monoclonal antibodies isolated by using the rationally designed prefusion MERS-CoV S probe could be considered potential candidates for the development of therapeutic and/or prophylactic antiviral agents for MERS-CoV human infection. [ABSTRACT FROM AUTHOR]