Mouse immunization

The 6 to 8 weeks-old female C57BL/6N mice were purchased from Envigo (Gannet, France; animal permit G20/17, Regierungspräsidium Karlsruhe, Germany) and the 6 to 8 weeks-old female BALB/c mice were obtained from Charles River Laboratories (Sulzfeld, Germany; animal permit G248/16, Regierungspräsidium Karlsruhe, Germany). Mice were kept at the German Cancer Research Center under specific-pathogen-free conditions. C57BL/6N mice were used for assessment of cellular immune responses. Twenty μg of the protein adjuvanted with 50% (vol/vol) AddaVax (InvivoGen; 50 μl) or Incomplete Freund’s adjuvant (Sigma-Aldrich; 50 μl), or 50 μg aluminum hydroxide (Brenntag) and 10 μg synthetic monophosphoryl lipid A (Avanti Lipids) (5 μl aluminum hydroxide, 10 μl monophosphoryl lipid A, in a total volume of 50 μl) was injected subcutaneously (s.c.) into the base of a mouse tail. For peptide administration, the adjuvant formulation and the immunization route were the same as protein vaccination, but the injected dose was different. A peptide mixture containing 100 μg MHC-I restricted peptide and 140 μg MHC-II restricted peptide was injected s.c. in a total volume of 50 μl. Mice received a single injection and were sacrificed 7 days later for ex vivo analysis of T-cell responses by IFN-γ ELISpot assay. Alternatively, mice were immunized twice at weekly intervals and splenocytes of sacrificed mice were analyzed by multimer staining 7 days later after the last immunization. Female BALB/c mice were employed for evaluation of humoral immune responses. Twenty μg of protein adjuvanted with 50% (vol/vol) AddaVax (25 μl) were injected intramuscularly (i.m.) into the caudal thigh muscle. Mice were immunized 4 times at biweekly intervals. One month after the last immunization, blood was collected by cardiac puncture and serum was analyzed against HPV16 and HPV18 pseudovirions using L1-PBNA.