Es (lower panels) from animals immunised three times with gp140 intranasally. Asterisks indicate significant differences between the different adjuvant/antigen groups and the PBS control group. doi:10.1371/journal.pone.0050529.gMucosal TLR ASP-015K biological activity adjuvants for HIV-gplowered mean IgG1/IgG2a ratios although this did not reach a level of statistical significance (Figure S2A). When TT was given alone intra-nasally, appreciable systemic IgG responses were elicited, with an average titre of 26103. All the adjuvants significantly increased specific IgG and IgA titres in sera (p,0.01), up to an IgG titre of 26106 for CpG-B and an IgA titre of 3.66104 for FSL-1. The effect mediated by R848 was again significantly lower than that of the other candidates (Fig. 4A and B). For the vaginal antibody responses, TT in combination with the different adjuvants elicited good IgG titres, significantly higher than the group with TT alone (p,0.01) where titres were very low or undetectable. The only exception was the group with R848 that, although showing a positive trend, did not reach statistical significance (Figure 4C). Higher titres were also observed for specific vaginal IgA with all adjuvant candidates showing significantly increased titres 
(Fig. 4D), with a maximum average titre of 7.46103 for FSL-1 IgG subclass analysis of sera indicated that all candidates significantly increased IgG2a and IgG1 titres (data not shown) relative to antigen alone. The administration of TT alone intranasally gave an IgG1/IgG2a ratio of 20 suggesting a Th2 biasing of responses. This ratio was significantly increased when the antigen was used in combination with chitosan (Figure S2B), whilst poly I:C, Pam3CSK4, R848 and CpG-B gave lower average ratios although differences were not statistically significant.responsiveness to gp140 was also observed for both IgG and IgA in all the groups tested (data not shown). In contrast, when TT was administered via the same route in a pilot experiment, 
the antigen alone gave low but Dimethylenastron chemical information detectable systemic IgG responses, with an average titre of 46103. Furthermore some TLR ligands such as FSL-1, poly I:C, LPS and R848 increased systemic IgG titres up to a maximum value of 106 for FSL-1 (Figure 5A). Systemic IgA titres on the other hand were low or not detectable (Figure 5B). In the genital mucosal compartment, both FSL-1 and poly I:C increased specific IgG and IgA titres (Figure 5C and D). However, the TT specific antibody titres observed were overall lower than those obtained with the other routes of immunisation.Parenteral immunisation with gp140 and TTTo compare mucosal and parenteral immunisation routes, gp140 and TT administered by the subcutaneous route. gp140 alone induced very strong systemic IgG responses, with an average titre of 6.06104. Of the adjuvants tested, Pam3CSK4 and chitosan significantly enhanced antibody titres up to 10 fold (p = 0.016 and 0.03 respectively) (Figure 6A). Conversely, specific serum IgA 12926553 responses were barely above background in the antigen-alone group and none of the adjuvants increased specific IgA titres. A similar pattern was observed in vaginal wash samples, with detectable IgG titres and very poor or no specific IgA responses. FSL-1, poly I:C and Pam3CSK4 significantly increased mucosal IgG titres giving titres up to 4.26102 (p,0.01) (Fig. 6C). IgG subclass analysis of sera, indicated that gp140 alone induced a very high average IgG1/IgG2a ratio of above 50 (Figure S3A) that was similar to responses induc.Es (lower panels) from animals immunised three times with gp140 intranasally. Asterisks indicate significant differences between the different adjuvant/antigen groups and the PBS control group. doi:10.1371/journal.pone.0050529.gMucosal TLR Adjuvants for HIV-gplowered mean IgG1/IgG2a ratios although this did not reach a level of statistical significance (Figure S2A). When TT was given alone intra-nasally, appreciable systemic IgG responses were elicited, with an average titre of 26103. All the adjuvants significantly increased specific IgG and IgA titres in sera (p,0.01), up to an IgG titre of 26106 for CpG-B and an IgA titre of 3.66104 for FSL-1. The effect mediated by R848 was again significantly lower than that of the other candidates (Fig. 4A and B). For the vaginal antibody responses, TT in combination with the different adjuvants elicited good IgG titres, significantly higher than the group with TT alone (p,0.01) where titres were very low or undetectable. The only exception was the group with R848 that, although showing a positive trend, did not reach statistical significance (Figure 4C). Higher titres were also observed for specific vaginal IgA with all adjuvant candidates showing significantly increased titres (Fig. 4D), with a maximum average titre of 7.46103 for FSL-1 IgG subclass analysis of sera indicated that all candidates significantly increased IgG2a and IgG1 titres (data not shown) relative to antigen alone. The administration of TT alone intranasally gave an IgG1/IgG2a ratio of 20 suggesting a Th2 biasing of responses. This ratio was significantly increased when the antigen was used in combination with chitosan (Figure S2B), whilst poly I:C, Pam3CSK4, R848 and CpG-B gave lower average ratios although differences were not statistically significant.responsiveness to gp140 was also observed for both IgG and IgA in all the groups tested (data not shown). In contrast, when TT was administered via the same route in a pilot experiment, the antigen alone gave low but detectable systemic IgG responses, with an average titre of 46103. Furthermore some TLR ligands such as FSL-1, poly I:C, LPS and R848 increased systemic IgG titres up to a maximum value of 106 for FSL-1 (Figure 5A). Systemic IgA titres on the other hand were low or not detectable (Figure 5B). In the genital mucosal compartment, both FSL-1 and poly I:C increased specific IgG and IgA titres (Figure 5C and D). However, the TT specific antibody titres observed were overall lower than those obtained with the other routes of immunisation.Parenteral immunisation with gp140 and TTTo compare mucosal and parenteral immunisation routes, gp140 and TT administered by the subcutaneous route. gp140 alone induced very strong systemic IgG responses, with an average titre of 6.06104. Of the adjuvants tested, Pam3CSK4 and chitosan significantly enhanced antibody titres up to 10 fold (p = 0.016 and 0.03 respectively) (Figure 6A). Conversely, specific serum IgA 12926553 responses were barely above background in the antigen-alone group and none of the adjuvants increased specific IgA titres. A similar pattern was observed in vaginal wash samples, with detectable IgG titres and very poor or no specific IgA responses. FSL-1, poly I:C and Pam3CSK4 significantly increased mucosal IgG titres giving titres up to 4.26102 (p,0.01) (Fig. 6C). IgG subclass analysis of sera, indicated that gp140 alone induced a very high average IgG1/IgG2a ratio of above 50 (Figure S3A) that was similar to responses induc.