Figure 1.
Development of CpG-ODN for activation of rabTLR9.
Activation of rabTLR9 by (A) CpG-ODN derived from CpG-2007 and CpG-1826, (B) CpG-ODN derived from CpG-1826C, (C) CpG-ODN derived from CpG-C4, and (D) CpG-ODN derived from CpG-C43. HEK 293 cells were co-transfected with expression vector for rabTLR9 and an NF-κB drivened luciferase-reporter gene, and treated with 2µM of different CpG-ODN as indicated for 7h. Relative luciferase activities were then determined. Data shown represent mean ± SD (n = 3 independent experiments). The sequences of CpG-ODN used in this study are shown at the bottom of the plots.
Figure 2.
Activation of TLR9s by CpG-ODN developed for rabbits.
HEK 293 cells were transfected with expression vector for (A, C) rabTLR9 and for (B) rabbit, mouse, and human TLR9, as indicated, along with an NF-κB controlled luciferase-reporter gene, and treated with 2µM of CpG-ODN (A, B) or with different concentrations of CpG-ODN (C) for 7h. Relative luciferase activities were then determined. Data shown represent mean ± SD (n = 3 independent experiments). The sequences of CpG-ODN used in this study are shown on the right of panel A.
Figure 3.
Activation of antigen-nonspecific immune responses by CpG-ODN developed for rabbits.
Rabbit splenocytes were stimulated with 2µM of different CpG-ODN. (A) Induction of cytokines was analyzed by RT-PCR. (B) Production of IgM was determined by ELISA. (C) Proliferation of splenocytes was measured by MTS assay. Data shown represent mean ± SD (n = 3 independent experiments).*P<0.05 vs. the induction by CpG-C4609.
Figure 4.
Activation of antigen-specific immune responses by CpG-ODN developed for rabbits.
Rabbits were immunized with 100µg of RHDV NJ85 peptide mixed with or without 50µg of CpG-ODN, as indicated. (A) The cycle of immunization and serum collection followed a 14-day-interval schedule, as shown. (B) Inductions of IFN-γ from CD8 T cells by NJ85 were measured by ELISA. (C) Titers of anti-NJ85 antibody in serum samples were measured by ELISA. Data shown represent mean ± SD (n = 3 independent experiments). *P<0.05 vs. the induction by CpG-C4609.
Figure 5.
Activation of mouse splenocytes by CpG-ODN developed for rabbits.
Mouse splenocytes were stimulated with 2µM of different CpG-ODN: CpG-2006 and CpG-2007, optimized for humans; CpG-1826, optimized for mice; and CpG-C46 and CpG-C4609, developed for rabbits. Induction of (A) IL-6 and (B) IFN-γ, and production of (C) IgM, were determined by ELISA. Data shown represent mean ± SD (n = 3 independent experiments). *P<0.05 vs. the induction by CpG-1826.
Figure 6.
Efficiency of the developed CpG-ODN in boosting antibody production in mice.
(A) A 10-day-interval schedule, as shown, was adopted for each cycle of immunization and serum collection from C57/B6J mice. (B) 1µg of OVA was formulated with/without 5µg of CpG-ODN in PBS for each mouse. Anti-OVA antibody titers were measured by ELISA. Data shown represent mean ± SD (n = 3 independent experiments). *P<0.05 vs. the induction by CpG-1826.
Figure 7.
Efficacy and safety of the developed CpG-ODN in boosting antibody production in rabbits.
(A) A three-week-interval schedule, as shown, was adopted for each cycle of immunization and serum collection. (B) 10µg of OVA was formulated with 50µg of CpG-ODN in PBS or CFA/IFA. (C) 3µg of OVA was formulated with 50µg of CpG-ODN, 1% of aluminum hydroxide gel (alum), in PBS or CFA/IFA, to determine the efficacy and safety of CpG-C4609 as an adjuvant to boost antibody production. Anti-OVA antibody titers were measured by ELISA. Data shown represent mean ± SD (n = 3 independent experiments). *P<0.05 vs. the induction by CpG-C4609 or the induction by alum plus CpG-C4609. (D) Upper panels: subcutaneous tissues at the injection sites were examined on the second day after injection of different combinations of antigens and adjuvants, as indicated. Lower panels: HE staining of sections taken from these tissues. Photos show a set of representative results of three independent experiments.(E) Upper panels: representative photos of granuloma appeared at CFA/IFA injection sites (right), and skin at the alum plus CpG-C4609 injection sites (left). Lower panels: HE staining of sections taken from the granuloma (right), and tissue from the alum plus CpG-C4609 injection sites (left). The right panels show a representative granuloma developed in 30% of CFA/IFA injected rabbits (n = 20). The left panels show injection sites of an alum plus CpG-C4609 injected rabbit which is representative for rabbits (n = 20) injected without adjuvant or with adjuvant other than CFA/IFA.