Mouse immunization protocol

Six- to eight-week-old female BALB/c mice were purchased from Charles River (Charles River, Calco, Como, Italy) and housed under specific pathogen-free conditions in the animal facility of the Istituto Superiore di Sanità (ISS, Rome, Italy). All animal procedures have been performed in accordance with European Union guidelines and Italian legislation for animal care. All animal studies were authorized by the Italian Ministry of Healthy and reviewed by the Service for Animal Welfare at ISS (Authorization n. 314/2015-PR of 30/04/2015)., Groups of five mice, were immunized once intramuscularly (i.m.) with escalating doses (0.4 × 10⁶ − 2 × 10⁶ − 10 × 10⁶ RT units/mice) of SIV-based IDLV-UFO.664 or IDLV-UFO.750. Naïve mice were used as negative controls. Blood retro orbital sampling was performed prior to immunization, two weeks post-immunization and at monthly intervals with glass Pasteur pipettes and sera were tested for the presence of anti-UFO antibodies by ELISA assay (described below).