Immunization of C57BL/6J Mice

Six- to 10-week-old female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME; catalog number 000664) were immunized with PBS, VLV, or KSHV virions in a 50-μL volume intramuscularly or a 200-μL volume intraperitoneally using insulin syringes (Becton, Dickinson, and Company [BD], Franklin Lakes, NJ; catalog number 329461) at the described time points. Immunogens were premixed with adjuvants and injected in the same volume described above. ODN2395 CpG adjuvant was purchased from Invivogen (catalog number vac-2395-1). The 21-mer poly(U) with phosphothioate linkages [poly(U)s] was custom synthesized by IDT. Lipid nanoparticles were prepared by Acuitas Therapeutics using a self-assembly process as previously described (82 (link)); the ionizable cationic lipid and LNP composition are described elsewhere (83 ). At the experimental endpoint, mice were euthanized and blood was collected by cardiac puncture with tuberculin syringes (BD catalog number 309623). Serum was collected by centrifugation in serum gel tubes (Sarstedt, Numbrecht, Germany; catalog number 41.1378.005) and heat inactivated at 56°C for 30 min before storage at −80°C. Splenocytes were harvested in RPMI (Corning catalog number 10040CV) containing 10% FBS and 1× penicillin-streptomycin. Single-cell suspensions were prepared by pushing spleens through 70-μm cell strainers (Fisher Scientific catalog number 22-363-548). Red blood cells were removed with ACK lysing buffer (Thermo Fisher Scientific catalog number A1049201). Splenocytes were resuspended in complete RPMI and stored at 4°C for no longer than overnight until stimulation.