Recombinant Receptor Binding Domain Protein Induces Partial Protective Immunity in Rhesus Macaques Against Middle East Respiratory Syndrome Coronavirus Challenge()

Lan, Jiaming; Yao, Yanfeng; Deng, Yao; Chen, Hong; Lu, Guangwen; Wang, Wen; Bao, Linlin; Deng, Wei; Wei, Qiang; Gao, George F.; Qin, Chuan; Tan, Wenjie

Necropsy was performed on all animals according to the standard protocol. Lung, trachea, spleen and kidney tissue samples were collected for histopathological and immunohistochemical examination. Procedures were performed using fixed 10% neutral buffered formalin, embedded in paraffin, sequential sectioned to 4-μm thickness, and stained with haematoxylin and eosin (HE) prior to examination by light microscopy. For immunohistochemistry analysis, staining methods included antigen retrieval with citrate buffer. A rabbitserum-derived polyclonal antibody against nucleoprotein (Sino Biological Inc., cat: 100213-RP02) at 1:1000 dilution, was then incubated with the sections. The secondary antibody used was goat anti-rabbit (ZSGB-Bio, cat: pv-9001) at 1:2000. Finally, results were evaluated using light microscopy. To acquire a subject result, different and multiple sites of the lung, trachea, spleen and kidney tissue samples were collected for the histopathology and immunohistochemistry analysis. Finally, quantitation of 4-5 slides per animal was used to assess the effect and impact of the immunized animals. , at intervals of 8 or 17 weeks, and inoculated intratracheally with hCoV-EMC at a dosage of 6.5 × 10 7 TCID 50 (red arrow). Monkeys were bled periodically at 2, 10, and 25 weeks (−14 days), and at −1 day, 1 day and 3 days. Additionally, nasal, oral and rectal swabs were collected at −1 day, 1 day and 3 days. Chest X-rays were performed 1 day before inoculation, and at 1 and 3 dpi. Following euthanasia, lung, trachea, spleen and kidney specimens of challenged monkeys were acquired at 3 dpi for detection.