Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1(-/-) Mice

Zhang, Yongli; Zhang, Hangjie; Ma, Wenqiang; Liu, Kefang; Zhao, Min; Zhao, Yingze; Lu, Xuancheng; Zhang, Fuping; Li, Xiangdong; Gao, George F.; Liu, William J.

In response to the urgent need for vaccination to prevent ZIKV infection, several vaccines are in the preclinical stages of development, including RNA vaccines, recombinant vector-based vaccines, and purified protein subunit vaccines. The plasmid DNA vaccine is in phase 1 clinical trials 12 . The evaluation of safety and efficacy of ZIKV vaccines is, therefore, important. One advantage of the vaccines is their ability to elicit specific T-cell responses, which may be important for protection against the ZIKV. By using ZIKV-derived T-cell epitope-related tetramers, the T-cell 5. Use sharp Iris scissors to carefully cut from the same cavity, up the midline, toward the nose. Try to keep the end of the scissors as superficial as possible to avoid injuring the brain. 6. Lift the brain with forceps and use sharp Iris scissors to carefully dissect the cranial nerve fibers. Remove the brain with forceps and place it in a 15 mL tube containing 5 mL of ice-cold RPMI/10% FBS medium. 7. Grab the abdominal skin with forceps and use sharp Iris scissors to make a longitudinal incision through the integument and abdominal wall and expose the lowermost part of the abdomen. Push the testes up to the incision. Gently pull the fat layer with tweezers and expose a globular testis on both sides. 8. Use sharp Iris scissors to carefully dissect the fat layer and epididymis. Place the testes in a 15 mL tube containing 5 mL of ice-cold RPMI/10% FBS medium with forceps. 9. To generate a single-cell suspension from the brain or testes, place the organ on a sterile cell strainer with a 100 µm mesh on top of a 50 mL tube and add 2 mL of ice-cold RPMI/10% FBS medium. Using the plunger of a 5 mL syringe, mash the organ and add medium until the organ has been fully ground through the mesh. 10. Transfer the cell suspension to a 15 mL tube and centrifuge it at 600 x g for 5 min at 4 °C. Remove the supernatant. 11. Resuspend the cells with 5 mL of 30% density gradient medium and, then, add them very slowly to 2 mL of 70% density gradient medium in a 15 mL tube. 12. Switch off the brake and centrifuge the tubes at 4 °C at 800 x g for 30 min. Obtain the lymphocytes from the middle layer. 13. Transfer the interphase to a fresh 15-mL tube, add 10 mL of cold RPMI/10% FBS medium, and centrifuge the tube at 300 x g for 10 min at 4°C