Synthetic innate defense regulator peptide combination using CpG ODN as a novel adjuvant induces long­lasting and balanced immune responses.

Vaccines are critical tools for the prevention and treatment of several diseases. Adjuvants have been traditionally used to enhance immunity to vaccines and experimental antigens. In the present study, the adjuvant combination of CpG oligodeoxynucleotides (CpG ODN) and the innate defense regulator (IDR) peptide, IDR­HH2, was evaluated for its ability to enhance and modulate the immune response when formulated with alum and the recombinant hepatitis B surface antigen (HBsAg). The CpG­HH2 complex enhanced the secretions of tumor necrosis factor­α, monocyte chemotactic protein 1 and interferon­Î³ by human peripheral blood mononuclear cells and promoted murine bone marrow dentritic cell maturation. In addition, the present study demonstrated that IDR­HH2 was chemotactic for human neutrophils, THP­1 cells and RAW264.7 cells at concentrations between 2.5 and 40 µg/ml. The present study also observed that significantly higher anti­HBs antibody titers, which were sustained at high levels for as long as 35 weeks following the boost immunization, were induced by the combination adjuvant, even when co­administered with a commercial hepatitis B vaccine at a low antigen dose (0.1 µg HBsAg). Notably, the level of IgG2a was almost equal to the level of IgG1, indicating that a balanced T helper (Th)1/Th2 immune response was elicited by the novel vaccine, which was consistent with the ELISpot results. These data suggest that the CpG­HH2 complex may be a potential effective adjuvant, which facilitates a reduction in the dose of antigen and induces long­lasting, balanced immune responses.

Gene therapy using the human telomerase catalytic subunit gene promoter enables targeting of the therapeutic effects of vesicular stomatitis virus matrix protein against human lung adenocarcinoma.

The catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), is highly active in immortalized cells and more than 90% of human cancer cells, but is quiescent in the majority of normal somatic cells. Thus, the hTERT promoter has been extensively used in targeted cancer gene therapy. Vesicular stomatitis virus (VSV) matrix protein (MP) induces the apoptosis of tumor cells in the absence of other viral components. In our previous studies, we successfully constructed the pVAX-M plasmid from the pVAX plasmid, which expressed wild-type VSV MP (VSV MP is under the control of the CMV promoter) and demonstrated that pVAX-M efficiently suppresses the growth of malignant tumors via the induction of apoptosis in vitro and in vivo. The present study was designed to construct the plasmid phTERTM (VSV MP is under the control of the hTERT promoter) and investigate whether it had a targeted antitumor effect in nude mice bearing human lung adenocarcinoma. In vitro, A549 human lung adenocarcinoma cells were treated with NS, Lip-null, etoposide, Lip-pVAX-M or Lip-phTERT-M, and examined for cell viability through MTT assays or for apoptosis by flow cytometry and TUNEL assays. In vivo, A549 human lung carcinoma models in nude mice were established. Mice were treated with 10 4-weekly intravenous administrations of NS, Lip-null, etoposide (2 mg/kg), Lip-pVAX-M or Lip-phTERT-M. Subsequently, Lip-phTERT-M was found to be the most efficient inhibitor of tumor growth and inducer of tumor cell apoptosis when compared with the other groups in vivo and in vitro (P<0.05). Notably, immunohistochemical staining showed that Lip-phTERT-M significantly limited the overexpression of VSV MP to the tumor tissues and reduced VSV MP expression in other organs in comparison with Lip-pVAX-M (P<0.05). Therefore, it can be concluded that phTERT-M demonstrates a targeted antitumor effect on A549 human lung adenocarcinoma cells. These observations suggest that phTERT-M gene therapy may be a novel and potent strategy for targeting human lung adenocarcinoma.

Novel vaccine adjuvant LPS-Hydrogel for truncated basic fibroblast growth factor to induce antitumor immunity.

The need to enhance the immunogenicity of tumor-associated antigens and modulate the resulting immune responses has prompted the development of new adjuvants. We prepared a novel adjuvant, lipopolysaccharides (LPS) loaded thermosensitive hydrogel (LPS-Hydrogel), for truncated basic fibroblast growth factor (tbFGF) peptide to enhance immunological responses and improve therapeutic effects in cancer. When co-formulated with tbFGF, LPS-Hydrogel formed antigen-adjuvant complexes, which enhanced antibody and cell-mediated responses in mice, thus promoting a more balanced antibody-mediated and cytotoxic T lymphocyte (CTL)-mediated immune response to inhibit tumor growth and metastases in vivo. Furthermore, the secretion of IFN-γ and IL-4 was detected, confirming activation of the two immune responses in vivo. There were no significant systemic toxicities observed with tbFGF-LPS-Hydrogel treatment. These results suggested that the thermosensitive and biodegradable LPS-Hydrogel was a novel adjuvant and carrier for peptide vaccines in cancer immunotherapy.

Diluted povidone-iodine inhibits tumor growth through apoptosis-induction and suppression of SOD activity.

Povidone-iodine (PVP-I) is widely used in clinical practice as an antiseptic and flushing agent after surgery to remove a tumor. Our present study was designed to determine whether diluted PVP-I is cytotoxic to colon cancer cells and ascetic tumor cells in vitro and to examine its antitumor effects in vivo. In vitro, CT26 and H22 cells treated with different concentrations of diluted PVP-I (0-1.56 µg/ml) were analyzed using the mononuclear cell direct cytotoxicity assay (MTT) and a flow cytometry assay. In vivo, Balb/c mice injected in the abdominal cavity with CT26 cells or H22 cells were treated intraperitoneally with different concentrations of PVP-I (0-312.5 µg/mouse), cisplatin (40 mg/kg) or 5'-FU (30 mg/kg) or left untreated. In vitro, the studies demonstrated the antiproliferative and significant apoptosis-inducing effects of PVP-I in a dose- and time-dependent manner. In vivo, PVP-I significantly repressed the growth of H22 and CT26 cells in Balb/c mice compared to controls. To explore the mechanism of the antitumor effect of PVP-I, the superoxide dismutase (SOD) activity of ascites extracted from a mouse model and the supernatant of CT26 cells was detected by an SOD kit. The activity of SOD was significantly inhibited in the experimental groups. Taken together, our data suggest that PVP-I exhibits a strong inhibitory effect on tumor growth in colon cancer (CT26) and hepatoma (H22) resulting from apoptosis, both in vitro and in vivo, suggesting a new potential therapeutic approach after tumor excision surgery to colon cancer and hepatoma.

bFGF peptide combined with the pVAX-8CpG plasmid as adjuvant is a novel anticancer vaccine inducing effective immune responses against Lewis lung carcinoma.

Due to the poor immunogenicity of subunit protein antigens, there is a need to use adjuvants in order to generate effective immune responses. Basic fibroblast growth factor (bFGF) is one of the best characterized pro-angiogenic cytokine and is a candidate target for anticancer therapy. We used truncated bFGF (tbFGF) combined with engineered pVAX-nCpG as novel adjuvant to immunize mice in order to inhibit tumor angiogenesis and suppress tumor growth. In our study, the results demonstrated that the mice immunized with tbFGF-alum-pVAX-8CpG produced a better tumor-suppression effect compared with the other groups, apart from the group treated with tbFGF-alum-CpG. In addition, the function of immune modulation of pVAX-8CpG was similar to CpG ODNs. The vaccine composed of tbFGF, alum and pVAX-8CpG effectively inhibited tumor angiogenesis and induced strong antitumor immune responses. The antitumor activity induced by the vaccine tbFGF-alum-pVAX-8CpG was not only associated with the antigen-specific antibody, but also with the killing activity of cytotoxic cells. This indicates that alum-pVAX-8CpG may be an innovative adjuvant for cancer vaccines.

A novel combined conjugate vaccine: enhanced immunogenicity of bFGF with CRM197 as a carrier protein.

Tumor growth is partly dependent on tumor-associated angiogenesis, which is regulated by angiogenic growth factors. As the first angiogenic growth factor to be identified, basic fibroblast growth factor (bFGF) plays a major role in angiogensis and tumor growth and has been an effective target for anti-tumor therapy. However, due to its low immunogenicity, injection with bFGF alone cannot stimulate the body to produce a strong immune response. In this study, we investigated the role of CF (containing bFGF and CRM197) assisted by CpG and alum in enhancing antigen-specific immune response and suppressing the growth of murine colon carcinoma. The results revealed that compared to bFGF, CF could not stimulate NIH-3T3 fibroblast proliferation even at a concentration of 10 µg/ml in vitro. In vivo, the CF-CpG-alum produced a stronger antigen-specific immune response and inhibited tumor growth. The anti-tumor activity was associated with generating antigen-specific antibody, suppressing angiogenesis, promoting the apoptosis of tumor cells and inducing the mixed Th1 and Th2 responses. This indicates that CRM197 may be an innovative intramolecular adjuvant and provides a rational preservation for mouse CT26 colon carcinoma.

Combination of vesicular stomatitis virus matrix protein gene therapy with low-dose cisplatin improves therapeutic efficacy against murine melonoma.

Vesicular stomatitis virus (VSV) matrix protein (MP) can directly induce apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. Our previous studies have demonstrated that MP gene therapy efficiently suppressed the growth of malignant tumor in vitro and in vivo. The present study was designed to determine the possibility that the combination of MP gene therapy with low-dose cisplatin would improve therapeutic efficacy against murine melanoma. Immunocompetent C57BL/6 mice bearing B16-F10 melanoma were established. Mice were treated once every 5 days with i.v. administration of 10 microg pVAX-MP/30 microg liposome complex per mouse for 16 days and i.p. delivery of cisplatin at 4 mg/kg/mouse on days 6 and 12 after the initiation of MP treatment. We found that MP + cisplatin treatment resulted in significant inhibition of tumor growth and improved the survival time of melanoma-bearing mice. MP successfully inhibited angiogenesis as assessed by CD31. Histological examination revealed that the combination therapy led to significant increased induction of apoptosis, tumor necrosis, and elevated CD8(+) lymphocyte infiltration. Furthermore, the induction efficacy of the CTL response was dramatically enhanced by the combination therapy. Our findings may prove useful in further explorations of the application of these combinational approaches to the treatment of malignant melanoma.