052 cr 571 oakwood

Fourteen-week-old, male, specific pathogen-free New Zealand White (052 CR; 571 OAKWOOD) rabbits were obtained from Charles River Laboratories (Wilmington, MA). After a 2-week acclimatization period, 1 x 10⁷ lethally irradiated (100 Gy) 729.B control cells or 729 HTLV-1 producer cells (wt or mEnhancer) were inoculated into the lateral ear vein. Blood was drawn via the central auricular artery at Weeks 0 (pre-inoculation), 4, 8, 12, 16, 20, and 25 (study endpoint). Plasma was collected and rabbit PBMCs (rPBMCs) were isolated using Ficoll-Paque™ PREMIUM (Cytiva, Marlborough, MA). rPBMCs or plasma were assessed for proviral load, HTLV-1 gene expression, and HTLV-1-specific antibody response, as described below. Sanger sequencing of the viral enhancer region was performed at week 25 to monitor for viral reversions. All animal procedures were performed in accordance with a protocol approved by University Laboratory Animal Resources (ULAR) of The Ohio State University.

Fourteen-week-old, male, specific pathogen-free New Zealand White (052 CR; 571 OAKWOOD) rabbits were obtained from Charles River Laboratories (Wilmington, MA). 10⁷ lethally irradiated (100 Gy) 729 HTLV-1 producer cells were inoculated into the lateral ear vein. Blood was drawn via the central auricular artery at Weeks 0 (pre-inoculation), 2, 4, 8, and 12 (study endpoint). Plasma was collected and rabbit PBMCs (rPBMCs) were isolated using Ficoll-Paque PREMIUM (Cytiva, Marlborough, MA). rPBMCs or plasma were assessed for proviral load, HTLV-1 gene expression, and HTLV-1-specific antibody response. Sanger sequencing was performed at Week 12 to monitor for viral reversions. The HTLV-specific antibody response was measured using the Avioq HTLV-I/II Microelisa System (Avioq, Inc., Research Triangle Park, NC). The manufacturer’s protocol was modified by substituting the provided HRP-conjugated goat anti-human IgG with an HRP-conjugated goat anti-rabbit IgG (ab6721; Abcam, Cambridge, United Kingdom).