T adjuvants, the exception being chitosan. IN-immunisation with gp140 provided a more balanced Th1/Th2 profile than SL- or SC-routes, however responses were appreciably shifted towards a Th2 bias by FSL-1, MPLA, Pam3CSK4 and chitosan. IN-immunisation with TT alone was more skewed toward a Th2 profile (20) than SL-administration with TT and greater than that seen with IN-gp140. A more balanced Th1/Th2 response was induced with Poly I:C, R848 and CpG-B. These data demonstrate route, antigen, and adjuvant dependent effects. It is important to take into 47931-85-1 site account that the Balb/C mouse strain, used in our experiments, has a strong Th2 bias compared to other strains of mice [40],[41] and this might explain the high Th2 bias we observed for some experimental groups. Nevertheless the trend for modulating responses toward Th1 or Th2 phenotypes is likely to hold true in other mice species irrespective of their preexisting bias. Interestingly there was no correlation between the ratio of IgG1/IgG2a and the extent of mucosal IgA responses observed. This suggests that the route of immunisation may play a more important role in the induction of IgA responses than the Th1/Th2 bias of individual TLR ligands. When taken together our data indicate that IN-immunisation provided the best balance between systemic and mucosal responses with most of the adjuvants evaluated (with notable exceptions discussed above): for systemic and mucosal IgG, IN- was equivalent to SC- and better than SL-immunisation; while for systemic and mucosal IgA, IN- was equivalent to or better than SL- and both were appreciably better than SC-immunisation. Thus IN-immunisation induced comparable systemic and mucosal IgG responses to SC but appreciably better systemic and mucosal IgA responses. Further studies in other species, especially nonhuman primates and human clinical studies, are needed to assess whether similar results can be confirmed.Supporting InformationFigure SSerum IgG1/IgG2a ratios after sublingual immunisations. Specific IgG1/IgG2a ratios in sera after three SL immunisations with gp140 (A) or with Tetanus Toxoid (B). Asterisks indicate get (-)-Calyculin A significant differences between the different adjuvant/antigen groups and the PBS control group. (TIF) Serum IgG1/IgG2a ratios after nasal immunisations. Specific IgG1/IgG2a ratios in sera after three IN immunisations with gp140 (A) or with Tetanus Toxoid (B). Asterisks indicate significant differences between the different adjuvant/antigen groups and the PBS control group. (TIF)Figure SFigure SSerum IgG1/IgG2a ratios after subcutaneous immunisations. Specific IgG1/IgG2a ratios in sera after three SC immunisations with gp140 (A) or with Tetanus Toxoid (B). Asterisks indicate significant differences between the different adjuvant/antigen groups and the PBS control group. (TIF)AcknowledgmentsWe are grateful to Professors Wagner and Wolf, University 24786787 of Regensburg, Germany and GENEART AG for access to CN54 sequence, to Simon Jeffs, Imperial College for generation of CHO producer cells and Polymun for production of recombinant CN54gp140. Support for the animal work in these studies was provided by the St George’s University of London Biological Research Facility. We would also like to acknowledge Drs. Asna Siddiqui who acted as project manager for this study and Sarah Harman for preparation of the manuscript.Author ContributionsConceived and designed the experiments: RJS VB. Performed the experiments: VB KK LF. Analyzed the data: VB KK. Wrote.T adjuvants, the exception being chitosan. IN-immunisation with gp140 provided a more balanced Th1/Th2 profile than SL- or SC-routes, however responses were appreciably shifted towards a Th2 bias by FSL-1, MPLA, Pam3CSK4 and chitosan. IN-immunisation with TT alone was more skewed toward a Th2 profile (20) than SL-administration with TT and greater than that seen with IN-gp140. A more balanced Th1/Th2 response was induced with Poly I:C, R848 and CpG-B. These data demonstrate route, antigen, and adjuvant dependent effects. It is important to take into account that the Balb/C mouse strain, used in our experiments, has a strong Th2 bias compared to other strains of mice [40],[41] and this might explain the high Th2 bias we observed for some experimental groups. Nevertheless the trend for modulating responses toward Th1 or Th2 phenotypes is likely to hold true in other mice species irrespective of their preexisting bias. Interestingly there was no correlation between the ratio of IgG1/IgG2a and the extent of mucosal IgA responses observed. This suggests that the route of immunisation may play a more important role in the induction of IgA responses than the Th1/Th2 bias of individual TLR ligands. When taken together our data indicate that IN-immunisation provided the best balance between systemic and mucosal responses with most of the adjuvants evaluated (with notable exceptions discussed above): for systemic and mucosal IgG, IN- was equivalent to SC- and better than SL-immunisation; while for systemic and mucosal IgA, IN- was equivalent to or better than SL- and both were appreciably better than SC-immunisation. Thus IN-immunisation induced comparable systemic and mucosal IgG responses to SC but appreciably better systemic and mucosal IgA responses. Further studies in other species, especially nonhuman primates and human clinical studies, are needed to assess whether similar results can be confirmed.Supporting InformationFigure SSerum IgG1/IgG2a ratios after sublingual immunisations. Specific IgG1/IgG2a ratios in sera after three SL immunisations with gp140 (A) or with Tetanus Toxoid (B). Asterisks indicate significant differences between the different adjuvant/antigen groups and the PBS control group. (TIF) Serum IgG1/IgG2a ratios after nasal immunisations. Specific IgG1/IgG2a ratios in sera after three IN immunisations with gp140 (A) or with Tetanus Toxoid (B). Asterisks indicate significant differences between the different adjuvant/antigen groups and the PBS control group. (TIF)Figure SFigure SSerum IgG1/IgG2a ratios after subcutaneous immunisations. Specific IgG1/IgG2a ratios in sera after three SC immunisations with gp140 (A) or with Tetanus Toxoid (B). Asterisks indicate significant differences between the different adjuvant/antigen groups and the PBS control group. (TIF)AcknowledgmentsWe are grateful to Professors Wagner and Wolf, University 24786787 of Regensburg, Germany and GENEART AG for access to CN54 sequence, to Simon Jeffs, Imperial College for generation of CHO producer cells and Polymun for production of recombinant CN54gp140. Support for the animal work in these studies was provided by the St George’s University of London Biological Research Facility. We would also like to acknowledge Drs. Asna Siddiqui who acted as project manager for this study and Sarah Harman for preparation of the manuscript.Author ContributionsConceived and designed the experiments: RJS VB. Performed the experiments: VB KK LF. Analyzed the data: VB KK. Wrote.
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