As Pam3CSK4 (TLR2) and R848 (TLR7/8) are under investigation and proven to be safe in different clinical trials [16]. In this study we have evaluated the potential of several TLR ligands as adjuvants for mucosal immunisations in mice via three different routes of mucosal administration: intranasal (IN), intravaginal (IVag), sublingual (SL); and a parenteral route, subcutaneous (SC), as a control. We compared the responses induced against CN54gp140, a recombinant clade C envelope protein [17], versus those against the potent immunogen Tetanus toxoid (TT). In our study we also included chitosan, a polysaccharide widely used in vaccine formulations that can enhance immune responses, as control adjuvant [18]. Our approach focused on the evaluation of candidate adjuvants’ ability to induce specific genital and systemic humoral responses, both IgG and IgA through different mucosal routes of immunisation. Moreover, IgG subclasses, IgG2a and IgG1, were investigated in order to address the influence of adjuvant and route of administration on the balance between Th1 and Th2-type immune responses.weeks in between immunisations. Blood samples were collected two weeks after the last immunisation by tail vein puncture and vaginal washes were collected, under anaesthesia, flushing the mouse vagina with 75 ml of PBS. For all immunisations and vaginal sampling mice were anaesthetised using Isoflurane-Vet (50-14-6 biological activity Merial).Mouse samplesSera were collected 2 hours after bleeding, spinning the blood samples for 10 min at 23,000 g and collecting clear supernatants. Vaginal washes were treated with a protease inhibitor cocktail (SIGMA) for 30 min at 4uC then spun for 10 min at 23,000 g to remove cell debris. All samples were stored at 280uC.Detection of specific IgG and IgASerum and vaginal samples were tested for the presence of specific (gp140 or Tetanus toxoid) IgG and IgA using an in-house ELISA protocol. Plates were coated with 5 mg/ml antigen overnight at 4uC and blocked for 1 hour at 37uC in PBS containing 1 BSA (SIGMA). Samples were diluted in assay buffer (PBS containing 1 BSA and 0.05 Tween 20) and incubated for 1 hour at 37uC. Specific IgG was detected using a goat anti-mouse HRP (Serotec) antibody whilst IgA was detected indirectly using a goat anti-mouse biotin antibody (SouthernBiotec) and then adding streptavidin (R D). Plates were read at 450 nm after addition of Peptide M biological activity SureBlue TMB substrate (KPL) followed by 1N H2SO4 to stop the colorimetric reaction. Endpoint titres were calculated using GraphPad Prism version 4 as the reciprocal of the highest dilution giving an absorbance value equal or higher ?to the background (naive mouse serum) plus two standard deviations. Cut-off value was set at 0.1.Materials and Methods ReagentsTetanus toxoid was obtained from Statens Serum 1531364 Institute and CN54gp140 was obtained from Polymun Scientific. The TLR ligands FSL-1 (TLR2/6), Poly I:C (TLR3), Pam3CSK4 (TLR1/2), R848 (TLR7/8) were purchased from Invivogen, monophosphoryl Lipid A (MPLA, TLR4) from SIGMA and CpGB (TLR9) from MWG. Chitosan was provided by Novamatrix.Detection of IgG subtypesSpecific IgG subclasses were detected as described above, using anti-mouse IgG1 HRP and anti-mouse IgG2a HRP (Serotec).Statistical analysisThe statistical difference between groups was determined by Mann-Whitney test and one way ANOVA. All analyses were performed using GraphPad Prism v 4. Significant differences between the different antigen/adjuvant groups and the no adjuvant control g.As Pam3CSK4 (TLR2) and R848 (TLR7/8) are under investigation and proven to be safe in different clinical trials [16]. In this study we have evaluated the potential of several TLR ligands as adjuvants for mucosal immunisations in mice via three different routes of mucosal administration: intranasal (IN), intravaginal (IVag), sublingual (SL); and a parenteral route, subcutaneous (SC), as a control. We compared the responses induced against CN54gp140, a recombinant clade C envelope protein [17], versus those against the potent immunogen Tetanus toxoid (TT). In our study we also included chitosan, a polysaccharide widely used in vaccine formulations that can enhance immune responses, as control adjuvant [18]. Our approach focused on the evaluation of candidate adjuvants’ ability to induce specific genital and systemic humoral responses, both IgG and IgA through different mucosal routes of immunisation. Moreover, IgG subclasses, IgG2a and IgG1, were investigated in order to address the influence of adjuvant and route of administration on the balance between Th1 and Th2-type immune responses.weeks in between immunisations. Blood samples were collected two weeks after the last immunisation by tail vein puncture and vaginal washes were collected, under anaesthesia, flushing the mouse vagina with 75 ml of PBS. For all immunisations and vaginal sampling mice were anaesthetised using Isoflurane-Vet (Merial).Mouse samplesSera were collected 2 hours after bleeding, spinning the blood samples for 10 min at 23,000 g and collecting clear supernatants. Vaginal washes were treated with a protease inhibitor cocktail (SIGMA) for 30 min at 4uC then spun for 10 min at 23,000 g to remove cell debris. All samples were stored at 280uC.Detection of specific IgG and IgASerum and vaginal samples were tested for the presence of specific (gp140 or Tetanus toxoid) IgG and IgA using an in-house ELISA protocol. Plates were coated with 5 mg/ml antigen overnight at 4uC and blocked for 1 hour at 37uC in PBS containing 1 BSA (SIGMA). Samples were diluted in assay buffer (PBS containing 1 BSA and 0.05 Tween 20) and incubated for 1 hour at 37uC. Specific IgG was detected using a goat anti-mouse HRP (Serotec) antibody whilst IgA was detected indirectly using a goat anti-mouse biotin antibody (SouthernBiotec) and then adding streptavidin (R D). Plates were read at 450 nm after addition of SureBlue TMB substrate (KPL) followed by 1N H2SO4 to stop the colorimetric reaction. Endpoint titres were calculated using GraphPad Prism version 4 as the reciprocal of the highest dilution giving an absorbance value equal or higher ?to the background (naive mouse serum) plus two standard deviations. Cut-off value was set at 0.1.Materials and Methods ReagentsTetanus toxoid was obtained from Statens Serum 1531364 Institute and CN54gp140 was obtained from Polymun Scientific. The TLR ligands FSL-1 (TLR2/6), Poly I:C (TLR3), Pam3CSK4 (TLR1/2), R848 (TLR7/8) were purchased from Invivogen, monophosphoryl Lipid A (MPLA, TLR4) from SIGMA and CpGB (TLR9) from MWG. Chitosan was provided by Novamatrix.Detection of IgG subtypesSpecific IgG subclasses were detected as described above, using anti-mouse IgG1 HRP and anti-mouse IgG2a HRP (Serotec).Statistical analysisThe statistical difference between groups was determined by Mann-Whitney test and one way ANOVA. All analyses were performed using GraphPad Prism v 4. Significant differences between the different antigen/adjuvant groups and the no adjuvant control g.