| Despite the remarkable progress in understanding the causes of cancer and the significant advances in cancer therapy in recent years, the disease persists, and the incidence of cancer is increasing worldwide. Unlike the highly efficient prophylactic vaccines against infectious diseases, therapeutic vaccines against cancer that do not cause unacceptable autoimmune disorders have not been as effective, eliciting only incremental therapeutic effects. The simplest explanation may be that using therapeutic vaccines to treat established tumors is equivalent to the unsuccessful approach of using hepatitis B virus (HBV) or human papilloma virus (HPV) vaccines to treat chronic HBV or HPV infection. Several count-back mechanisms have been reported for the involvement of tumor escape and immune suppression, likely driven by long-term tumor development, establishment, and growth, including impaired T cell responses, immune tolerance, and the suppressive tumor microenviroment, which likely act synergistically. This underlines the need to develop a prophylactic vaccine approach for cancer that will provide low-cost and highly efficiency rationales. A major challenge for designing prophylactic cancer vaccines is to define immunogenic and safe cancer antigens that can serve as targets for effective vaccines, including tumor-specific antigens and proteins over-expressed on the tumor but not on normal tissues. At present, only a limited number of cancer antigens have been found with few successes.It is well documented that most solid tumor types express embryonic antigens to varying extents, and there is striking similarity of antigen expression between cancer and embryonic tissues, which provides the potential to target embryonic components as an effective strategy to prevent the appearance of cancers. As a member of the heat shock protein90family, gp96has the unique ability to associate with antigenic peptides, presents these loaded antigens to both MHC class I and class Ⅱ molecules, and activates specific T cells. Our previous studies show that in hepatitis B virus (HBV)-infected liver cancer, gp96binds virus-derived peptides and activates specific CTL responses by antigen presentation. Moreover, clinical trials using autologous gp96-peptide complexes as therapeutic vaccines have been initiated for treatment of a range of tumors with modest antitumor effects. Based on the observations above, the aim of this study was to investigate whether placenta-derived gp96(P-gp96) induces prophylactic anti-tumor T cell responses.1. Placenta-derived gp96induces specific T cell responses against B16-F10melanoma and TUBO mammary tumor.C57BL/6mice or BALB/c were subcutaneously immunized three times with placenta-derived gp96(P-gp96) gp96-derived from liver (L-gp96), or PBS as a control. One week after the last immunization, mice were subcutaneously challenged with B16-F10melanoma cells or TUBO mammary cells. Compared to L-gp96or PBS, immunization with P-gp96significantly inhibited tumor growth, decreasing tumor volume. P-gp96immunization also dramatically enhanced the survival of tumor burdened mice. ELISPOT assay revealed that P-gp96immunization resulted in increase of tumor-specific T cells compared to L-gp96immunization. Additionally, as assessed by the killing assay using B16-F10cells or TUBO cells as target cells, P-gp96effectively elicited CTL with higher cytotoxicity than L-gp96.2. Placenta gp96immunization protects rats from DMBA-induced mammary tumor and autochthonous breast tumor development in MMTV-neu transgenic mice.Early immunization with P-gp96isolated from rat placenta elicited total protection against7,12-dimethylbenz(a)-anthracene (DMBA)-induced mammary tumor development in rats. Vaccination with P-gp96significantly reduced the occurrence and growth of autochthonous breast tumors in FVB/N-Tg(MMTVneu)202Mul/J mice.3. Placenta gp96may associates with Her-2-and Muc1-derived peptides.Analysis by quantitative real-time PCR showed dramatically higher expression of tumor-associated genes in the placenta than those in normal liver tissues of mice, rats and human including the tumor antigens MUC1, HER2/ERBB2, MMP2, FGF2, and c-Myc. ELISPOT analysis of splenocytes from mice immunized with P-gp96showed that P-gp96significantly increased a Kd-restricted Her-2dominant epitope E63-specific T cell response. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis indicated that P-gp96binds to a large number of peptides ranging from700to1,500Da. Human PBMCs were purified from the blood of HLA-A*0201+healthy donors and ineubated with P-gp96, TFA-treated P-gp96(TFA-gp96), or PBS in vitro. IFN-y ELISPOT analysis revealed that P-gp96treatment remarkably expanded Mucl-and Her2-specific CTLs compared to TFA-gp96treatment or PBS. Moreover, increased killing was observed when PBMCs were pulsed with P-gp96, using HLA-A2-positive breast cancer MCF-7cells (MUC1positive) as target cell.Our results reveal the novel immunogenicity of placental gp96and its potential use as multivalent cancer vaccine.The heat shock protein gp96is an adjuvant that can elicit T cell responses against cancer and infectious diseases, via antigen presentation, in both rodent models and clinical trials. Its uptake and internalization into antigen presenting cells (APCs) is a critical step in gp96-mediated immune responses. This study examined strategies to improve the cell internalization and T cell activation of gp96. It was found that recombinant Fusion with the cell-penetrating peptide TAT (trans-activator of transcription) slightly decreased the aggregation level of gp96and significantly increased its internalization into macrophages. Three well-characterized Kd-restricted CTL epitopes derived from the main antigens (HBsAg, HBcAg and HBV polymerase) of HBV, HBc87-95, HBs362-371, and HBV poL140-148were selected for testing. These antigenic peptides were used to immunize HBV transgenic BALB/c mice with or without rTAT-gp96or rgp96as an adjuvant for3times. The results of the ELISPOT assay indicated that rTAT-gp96were able to effectively elicit peptides specific T cell responses. Detection of the T cell activation marker CD69by FACS showed that mice vaccinated with the rTAT-gp96-peptide complexes exhibited significant increases of CD69+CD8+and CD69+CD4+T cells compared to rgp96-peptides complexes. IFN-γ intracellular staining showed that mice immunized with rTAT-gp96-peptide complexes exhibited significant increases of IFN-y-secreting CD8+and IFN-γ-secreting CD4+T cells. Killing assay indicated that mice immunized with rTAT-gp96-peptide complexes exhibited significant increases of peptide-specific cytotoxicity of CTLs. Immunization of HBV transgenic mice with the rTAT-gp96-peptide complexes led to a greater decrease in serum HBV DNA levels, serum HBsAg levels and HBcAg expression in their hepatocytes at week8compared to immunization with the rgp96-peptide complexes.In addition, the inclusion of TAT significantly improved the antitumor T cell immune response to a gp96vaccine in the B16-F10melanoma model. These results provide evidence that the efficient transduction of gp96into APCs can significantly enhance the outcome of gp96-based immunotherapy, and therefore provide a basis for more efficient approaches to improving the immunoregulatory and adjuvant functions of this unique T cell adjuvant. |
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