Humanized Mouse Model for HIV-1 Challenge

Male 6–8-week-old NOD/SCID/IL2Rγc^−/− (NSG) mice (Jackson Labs, Bar Harbor, ME, USA) were injected intramuscularly with NMCAB or CAB LAP at 45 mg CAB equivalents/kg. Eleven days after nanoformulation treatments, mice were reconstituted by intraperitoneal injection with 25 × 10⁶ human peripheral blood lymphocytes (PBL) obtained by leukapheresis and centrifugal elutriation. Eleven days after reconstitution, mice were challenged with 10⁴ 50% tissue culture infectious dose (TCID₅₀) HIV-1_ADA by intraperitoneal injections. Mice were sacrificed 10 days after viral challenge. The experimental timeline is shown in Fig. 7A. Peripheral blood was collected at days 10 (prior to PBL reconstitution), 21 (prior to HIV-1 challenge), and 32 (10 days post HIV-1 challenge) for flow cytometry analysis of human pan-CD45, CD3, CD4 and CD8 immune markers [38 (link)]. Plasma was collected via centrifugation at 2000 × g for 5 min for drug quantitation by UPLC-MS/MS. HIV-1 RNA was analyzed in day 32 plasma samples using the Roche Amplicor and Taqman-48 system with HIV-1 kit V 2.0 according to the manufacturer’s instructions (Roche Diagnostics, Indianapolis, USA). Tissues were collected for CAB concentrations by UPLC/MS/MS, viral RNA and DNA quantitation by semi-nested real-time PCR [39 (link)], and immunohistochemical staining for HIV-1p24 antigen as described previously [40 (link)].