Mice expressing the VH1-2 rearranging germline and the rearranged gl-VRC01 light chain were engineered as previously described45 (link), with the additional substitution of mouse JH1-4 with human JH2 segment45 (link). For immunization studies, mice were immunized sequentially with 25 µg of protein per animal in 60 µg of Poly I:C adjuvant (Invivogen) at two sites intramuscularly. For antibody coadministration 250 µg of anti-CTLA-4(BioXCell #BE0131), anti-OX-40 (BioXCell #BE0031) or a combination of isotype controls (Syrian Hamster IgG (BioXCell #BE0087) + Rat IgG2a (BioXCell #BE0089)). Mice were harvested at week 11 and spleen, lymph node and blood were isolated. All mice used in this study were housed in the Medical Sciences Research Building II Vivarium at Duke University Medical center in a pathogen-free environment with 12-h light/datk cycles at 20–25 ˚C, in accordance with Duke University Institutional Animal Care and Use Committee-approved animal protocols. All aspects of the procurement, conditioning/quarantine, housing, colony management, veterinary care, and carcass disposal programs comply with the guidelines set forth by the NIH guide for the care and use of laboratory animals, the Animal Welfare Act, and all applicable federal, state and institutional laws.
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