Skin vaccination via fractional infrared laser ablation - Optimization of laser-parameters and adjuvantation. 
Methods to deliver an antigen into the skin in a painless, defined, and reproducible manner are essential for transcutaneous immunization (TCI).  Here, we employed an ablative fractional infrared laser (P.L.E.A.S.E. Professional) to introduce clinically relevant vaccines into the skin.  To elicit the highest possible antibody titers with this system, we optimized different laser parameters, such as fluence and pore number per area, and tested various adjuvants.  BALB/c mice were immunized with Hepatitis B surface antigen (HBsAg) by laser-microporation.  Adjuvants used were alum, CRM197, monophosphoryl lipid A, heat-labile enterotoxin subunit B of E. coli (LT-B), and CpG ODN1826.  The influence of different fluences (2.1 to 16.8J/cm2) and pore densities (5-15%) was investigated.  Furthermore, immunogenicity of HBsAg and the commercially available conjugate vaccines ActHIB® and Menveo® applied via TCI was compared to standard i.m.  injection.  Antigen-specific antibody titers were assessed by luminometric ELISA.  Antibody titers against HBsAg were dependent on pore depth and peaked at a fluence of 8.4J/cm2.  Immunogenicity was independent of pore density.  Adjuvantation with alum significantly reduced antibody titers after TCI, whereas other adjuvants only induced marginal changes in total IgG titers.  LT-B and CpG shifted the polarization of the immune response as indicated by decreased IgG1/IgG2a ratios.  HBsAg/LT-B applied via TCI induced similar antibody titers compared to i.m.  injection of HBsAg/alum.  In contrast to i.m.  injection, we observed a dose response from 5 to 20μg after TCI.  Both, ActHIB® and Menveo® induced high antibody titers after TCI, which were comparable to i.m.  injection.  Alum, the most commonly used adjuvant, is contraindicated for transcutaneous vaccination via laser-generated micropores.  TCI with optimized laser parameters induces high antibody titers, which cannot be significantly increased by the tested adjuvants.  Commercially available vaccines formulated without alum have the potential for successful TCI via laser-generated micropores, without the need for reformulation.