LAUSR

&NA; Immunization routes and number of doses remain largely unexplored in therapeutic vaccination. The aim of the present work is to evaluate their impact on immune responses in naïve and hepatitis B virus (HBV)‐carrier mouse models following immunization with a non‐adjuvanted recombinant vaccine comprising the hepatitis B surface (HBsAg) and core (HBcAg) antigens. Mice were immunized either by intranasal (i.n.), subcutaneous (s.c.) or simultaneous (i.n. + s.c.) routes. Humoral immunity was detected in all the animal models with the induction of a potent antibody (Ab) response against HBcAg, which was stronger than the anti‐HBs response. In the HBV‐carrier mouse model, the anti‐HBs response was predominantly subtype‐specific and preferentially induced by the i.n. route. However, the Ab titers were not sufficient to clear the high concentration of HBsAg present in the sera of these mice. The i.n. route was the most efficacious at inducing cellular immune responses, in particular CD4+ T cells. In naïve mice, cellular responses in spleen were strong and mainly due to CD4+ T cells whereas the CD8+ T‐cell response was low. In HBV‐carrier mice, high frequencies of HBs‐specific CD4+ T cells secreting interferon (IFN)‐&ggr;, interleukin (IL)‐2 and tumor necrosis factor (TNF)‐&agr; were found in liver only after i.n. immunization. Increased frequencies of CD4+ T cells expressing the integrin CD49a in liver suggest a role of nasal route in the cellular homing process. Multiple dose schedules appear to be a prerequisite for protein‐based immunization in order to overcome immunotolerance in HBV‐carrier mice. These findings provide new avenues for further preclinical and clinical development.