MF59 formulated with CpG ODN as a potent adjuvant of recombinant HSP65-MUC1 for inducing anti-MUC1+ tumor immunity in mice

Abstract

MF59 is an oil-in-water emulsion adjuvant approved for influenza vaccines for human use in Europe. Due to its Th2 inducing properties, MF59 is seldom tested for cancer vaccines. In this study, MF59 formulated with a C-type CpG oligodeoxynucleotide (YW002) was tested for its Th1 adjuvant activity to induce immune responses to HSP65-MUC1, a recombinant fusion protein incorporating a mycobacterial heat shock protein (HSP65) and mucin 1, cell surface associated (MUC1) derived peptide. Combination of YW002 with MF59 (MF59-YW002) could confer a potent Th1 biasing property to the adjuvant, which enhanced the immunogenicity of HSP65-MUC1 to induce significantly higher levels of specific IgG2c, increased IFN-γ mRNA expression in splenocytes and the generation of antigen-specific cytotoxic T lymphocytes in mice. When prophylactically applied, MF59-YW002 adjuvant containing HSP65-MUC1 inhibited the growth of MUC1+ B16 melanoma and prolonged the survival of tumor-bearing mice. In contrast, adjuvant containing MF59 with HSP65-MUC1 in the absence of YW002, promoted the growth of MUC1+ B16 melanoma in mice. These results suggest that MF59 plus CpG oligodeoxynucleotide might be developed as an efficient adjuvant for tumor vaccines against melanoma, and possibly other tumors.

Fig. 1

Identification of HSP65-MUC1 and MUC1⁺ B16 melanoma cells. (A) SDS-PAGE analysis of purified HSP65-MUC1. (B) Western blots of HSP65-MUC1 using anti-MUC1 VNTR mAb or anti-HSP65 mAb. (C) MUC1⁺ melanoma B16 cells.

Fig. 2

Anti-melanoma effects induced by prophylactic immunization of MF59-YW002/HSP65-MUC1 in mice. C57BL/6 mice (n = 10) were injected sub-cutaneously (s.c.) with HSP65-MUC1 alone, MF59/HSP65-MUC1, or MF59-YW002/HSP65-MUC1 four times and inoculated s.c. with 1.2 × 10⁵ MUC1⁺ B16 melanoma cells on day 0. Tumor volume was measured every 2 days, and survival of mice was calculated. PBS-injected mice were used as a negative control. Statistical significance (p < 0.05) compared with other groups was represented as follows: * vs. PBS; Δ vs. HSP65-MUC1 + MF59; # vs. HSP65-MUC1; & vs. HSP65-MUC1 + YW002. (A) Experimental procedure. (B) Tumor growth curves. Each line represents tumor growth kinetics in each mouse. (C) Survival of mice. (D) Tumor incidence. Each line represents the tumor incidence in each group.

Fig. 3

Therapeutic anti-melanoma effect induced by immunization of MF59-YW002/HSP65-MUC1 in mice. C57BL/6 mice (n = 8) were inoculated 1.2 × 10⁵ MUC1⁺ B16 melanoma cells on day 0 and injected sub-cutaneously (s.c.) with HSP65-MUC1 alone, or combined with MF59-YW002/HSP65-MUC1, or MF59-YW002 on days 1, 8, 15 and 23. Tumor volume was measured every 2 days, and the survival of mice was calculated. PBS-injected mice were used as a negative control. *p < 0.05 compared with the PBS group. (A) Experimental procedure. (B) Tumor growth curves. Each line represents tumor growth kinetics in each mouse. (C) Survival of mice.

Fig. 4

HSP65-MUC1 specific antibody levels in sera of immunized mice. HSP65-MUC1 specific IgG1 and IgG2c levels in sera of immunized mice were determined by ELISA on day 7 after the last immunization. (A) Serum IgG1 and IgG2c levels. (B) IgG2c/IgG1 ratio.

Fig. 5

Relative expression of IFN-γ and IL-4 mRNA in splenocytes from immunized mice. The IFN-γ and IL-4 mRNA expression in splenocytes of immunized mice was determined on day 1 after the last immunization. All results, normalized to the IFN-γ and IL-4 mRNA expression in splenocytes of HSP65-MUC1-immunized mice, were expressed as 2^−ΔΔCt (ΔCt = Ct_IFN-γ/IL-4 − Ct_GAPDH, ΔΔCt = ΔCt_Purpose − ΔCt_HSP65-MUC1).

Fig. 6

Induction of MUC1-specific CD69⁺CD8⁺ T cells and cytotoxic T lymphocytes in immunized mice. C57BL/6 mice (n = 3) were immunized with HSP65-MUC1 alone, MF59/HSP65-MUC1 or MF59-YW002/HSP65-MUC1 on days 1 and 8. On day 18, mice were sacrificed for isolating splenocytes. After stimulation with HSP65-MUC1 for 12 h, the splenocytes were stained with FITC-labeled anti-CD8 mAb and PE-labeled anti-CD69 mAb and analyzed by flow cytometry. (A) Fluorescence analysis of CD8⁺CD69⁺ T cells in splenocytes. (B) Average ratio of CD8⁺CD69⁺ T cells in splenocytes from three mice. (C) Splenocyte cytotoxicity assay. Splenocytes were stimulated with HSP65-MUC1 for 5 days, and then co-cultured with the MUC1⁺ B16 melanoma cells at an effector/target (E:T) ratio of 5:1, 15:1, and 45:1, for 6 h. Statistical significance (p < 0.05) compared with other groups was represented as follows: * vs. PBS; # vs. HSP65-MUC1; Δ vs. MF59/HSP65-MUC1.

Fig. 7

Immunological memory is induced by immunization with MF59-YW002/HSP65-MUC1 in mice. Mice (n = 5) from the group receiving prophylactic immunization with MF59-YW002/HSP65-MUC1 followed by transfer of MUC1⁺ B16 melanoma cells were rechallenged sub-cutaneously (s.c.) with 1.2 × 10⁵ MUC1⁺ B16 melanoma cells. Naïve mice (n = 5) were inoculated s.c. with 1.2 × 10⁵ MUC1⁺ B16 melanoma cells as controls. Survival of mice was recorded after re-challenge. *p < 0.05 compared with naïve mice.