Yun Xing

Vaccination of non-obese diabetic mice with a fragment of peptide P277 attenuates insulin-dependent diabetes mellitus.

P277 is a peptide derived from the HSP60 regions, have potent immunological effect on insulin-dependent diabetes mellitus (IDDM) and its phase III clinical trials are currently under investigation. However, we recently discovered a positive correlation between anti-P277 autoantibodies and the presence of endothelial cells damage in inducing vascular leak syndrome. Therefore, the aim of our study was to demonstrate the critical peptide epitope of P277 to IDDM and to highlight the effects of this peptide therapy on inflammation of the islets. Groups of 4-week old female non-obese diabetic (NOD) mice were immunized one time every three weeks for three times with a residue of P277, showing a significant effect of down-regulating immunity to P277 protein and preventing the development of IDDM. Immunologic results including the suppression of T-cell proliferation, the increase of interleukin-10 (IL-10) production and reduction of interferon-γ (IFN-γ) production caused immune tolerance to P277. Hence, a functional role of the key epitope in P277 peptide capable of preventing IDDM is suggested, which could be modified to develop a novel safe and effective peptide vaccine against IDDM by reconstructing P277 in the further studies.

Microbial HSP70 peptide epitope 407–426 as adjuvant in tumor-derived autophagosome vaccine therapy of mouse lung cancer

Tumor-derived autophagome (DRibble) is an effective therapeutic cancer vaccine inducing T cell recognition and death of tumor cells in mice. However, the potential for improved anti-tumor response still remains. Our previous study demonstrated that two repeats of a mycobacterial HSP70407–426 (M2) peptide acted as adjuvant in improving anti-tumor efficacy of human umbilical vein endothelial cell (HUVEC) vaccine. Here, a DRibble vaccine conjugated with M2 (DRibble-M2) was designed as a novel vaccine to enhance anti-tumor activity. Compared with DRibble alone, DRibble-M2 vaccination more significantly inhibited the growth of mouse Lewis lung cancer both in a subcutaneous tumor model and in a lung metastasis model. Higher expression of antigen-specific CTL was induced by DRibble-M2. DRibble-M2 induced higher CD83 and CD86 expression in DC2.4 and also improved the internalization of DRibble antigen into DC2.4. Our data indicated that DRibble-M2 is a potential vaccine for clinical cancer therapy.

Improved efficacy of therapeutic vaccination with viable human umbilical vein endothelial cells against murine melanoma by introduction of OK432 as adjuvant.

Vaccination with xenogeneic or syngeneic endothelial cells targeting tumor angiogenesis is effective for inhibiting tumor growth. OK432, an effective adjuvant, was mixed with viable human umbilical vein endothelial cells (HUVECs) to prepare a novel HUVECs-OK432 vaccine, which could have an improved therapeutic efficacy. In this study, HUVECs-OK432 was administrated in mice by subcutaneous injection in a therapeutic procedure. The results showed that a stronger HUVEC-specific Abs and cytotoxic T lymphocyte immune response were elicited, which resulted in significant inhibition on the growth of B16F10 melanoma and remarkably prolonged survival of B16F10 melanoma-bearing mice compared with HUVECs. Besides, parallel results were obtained in vitro showing a stronger inhibition of HUVEC proliferation by immune sera of HUVECs-OK432 than that of HUVECs. Moreover, histochemistry and immunohistochemistry analysis showed that HUVECs-OK432 induced large areas of continuous necrosis within tumors and significantly reduced the vessel density, correlating well with the extent of tumor inhibition. Our present results suggest that OK432 could be employed as an effective adjuvant for HUVEC vaccines and therefore should be useful for adjuvant immunotherapy of cancer.

Enhanced antitumor efficacy by combination treatment with a human umbilical vein endothelial cell vaccine and a tumor cell lysate-based vaccine.

Angiogenesis inhibitors combined with other anticancer drugs have been shown to inhibit tumor growth in animal models and some of them were recently used in clinical trials. In the present study, a whole hepatocellular carcinoma cell lysate based vaccine with diphtheria toxin (DT) and two tandem repeats of microbial HSP70 peptide epitope 407-426 (2 mHSP70407-426, M2) as adjuvant, which was called HDM, was combined with a whole human umbilical vein endothelial cell (HUVEC) vaccine to develop a combination treatment regimen. This combination treatment regimen was named HUVEC-HDM, which was supposed to enhance its antitumor efficiency. HUVEC-HDM was administrated subcutaneously in both prophylactic and therapeutic procedures. Compared to either single vaccine, HUVEC-HDM induced a more significant inhibition on the growth and metastasis of H22 hepatocellular carcinoma in mice and prolonged the survival of tumor-bearing mice. Besides, HUVEC-HDM immunization elicited strong humoral and cellular immune responses targeting tumor cell as well as tumor angiogenesis, which could be responsible for the enhanced antitumor effect. Moreover, histochemistry analysis showed that HUVEC-HDM induced large areas of continuous necrosis within tumors, correlating well with the extent of tumor inhibition. These results not only highlight the superiority of the combined HUVEC-HDM treatment regimen, but also support the translation of such approaches into the clinic for the treatment of patients with hepatocellular carcinoma.

Protective antitumor immunity induced by tumor cell lysates conjugated with diphtheria toxin and adjuvant epitope in mouse breast tumor models

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2 in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.

A Novel Multi-Epitope Vaccine Based on Urate Transporter 1 Alleviates Streptozotocin-Induced Diabetes by Producing Anti-URAT1 Antibody and an Immunomodulatory Effect in C57BL/6J Mice

Hyperuricemia (HUA) is related to diabetes. Uric acid-induced inflammation and oxidative stress are risk factors for diabetes and its complications. Human urate transporter 1 (URAT1) regulates the renal tubular reabsorption of uric acid. IA-2(5)-P2-1, a potent immunogenic carrier designed by our laboratory, can induce high-titer specific antibodies when it carries a B cell epitope, such as B cell epitopes of DPP4 (Dipeptidyl peptidase-4), xanthine oxidase. In this report, we describe a novel multi-epitope vaccine composing a peptide of URAT1, an anti-diabetic B epitope of insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in streptozotocin-induced diabetes C57BL/6J mice successfully induced specific anti-URAT1 antibody, which inhibited URAT1 action and uric acid reabsorption, and increased pancreatic insulin level with a lower insulitis incidence. Vaccination with U-IA-2(5)-P2-1 (UIP-1) significantly reduced blood glucose and uric acid level, increased Th2 cytokines interleukin (IL)-10 and IL-4, and regulated immune reactions through a balanced Th1/Th2 ratio. These results demonstrate that the URAT1-based multi-epitope peptide vaccine may be a suitable therapeutic approach for diabetes and its complications.

Immunopreventive effects against murine H22 hepatocellular carcinoma in vivo by a DNA vaccine targeting a gastrin- releasing peptide.

There is a continuing need for innovative alternative therapies for liver cancer. DNA vaccines for hormone/ growth factor immune deprivation represent a feasible and attractive approach for cancer treatment. We reported a preventive effect of a DNA vaccine based on six copies of the B cell epitope GRP18-27 with optimized adjuvants against H22 hepatocarcinoma. Vaccination with pCR3.1-VS-HSP65-TP-GRP6-M2 (vaccine) elicited much higher level of anti-GRP antibodies and proved efficacious in preventing growth of transplanted hepatocarcinoma cells. The tumor size and weight were significantly lower (p<0.05) in the vaccine subgroup than in the control pCR3.1-VS-TP-HSP65-TP-GRP6, pCR3.1-VS-TP-HSP65-TP-M2 or saline subgroups. In addition, significant reduction of tumor-induced angiogenesis associated with intradermal tumors of H22 cells was observed. These potent effects may open ways towards the development of new immunotherapeutic approaches in the treatment of liver cancer.

Improved efficacy of therapeutic vaccination with dendritic cells pulsed with tumor cell lysate against hepatocellular carcinoma by introduction of 2 tandem repeats of microbial HSP70 peptide epitope 407-426 and OK-432.

Therapeutic vaccination with dendritic cells (DCs) pulsed with tumor cell lysate vaccine (H-D) represents an attractive approach for hepatocellular carcinoma (HCC) treatment. However, the efficacy of this approach is not most satisfactory for the low levels of T helper 1 (Th1)-type cytokines secretion and weak T cell responses. In this study, in order to increase the potency of H-D, two tandem repeats of microbial HSP70 peptide epitope 407-426 (2mHSP70(407-426), M2) which has been demonstrated to be effective in enhancing DC maturation were applied. The DC vaccine (HM-D) which was HCC tumor cell lysate pulsed with M2 was developed. Nevertheless, the immunotherapeutic effect was still not satisfactory enough even some promotion was obtained. Therefore, OK-432 (OK), which is a useful anti-cancer agent and effectively in stimulating DC maturation, was introduced to HM-D. Our results demonstrated that treatment with the improved DC vaccine which was tumor cell lysate pulsed with M2 and OK (HMO-D), compared with H-D and HM-D, significantly increased cell surface markers (MHC-I and II, CD40, CD80, CD86 and CD11c) expression on DCs, enhanced Th1-type cytokines (IL-12, TNF-α and IFN-γ) production but not Th2-type cytokine (IL-5) production, induced remarkable high levels of lymphocytes proliferation and CD8(+) cytotoxic T-lymphocyte (CTL). Furthermore, immunization with HMO-D effectively reduced tumor progression and enhanced the survival of mice with H22 tumors. Besides, we also found that the capability of M2 in inducing the Th1 cytokines was stronger than OK. In view of these results, HMO-D vaccination provided a novel immunotherapeutic approach for the treatment of HCC.

1-Methyl-D-tryptophan Reduces Tumor CD133 + cells, Wnt/β-catenin and NF-κβp65 while Enhances Lymphocytes NF-κβ2, STAT3, and STAT4 Pathways in Murine Pancreatic Adenocarcinoma

Abstract1-Methyl-D-tryptophan (1-MT) is extensively utilized in preclinical trials to deplete indoleamine 2,3-dioxigenase (IDO) activity and kynurenine pathway. Since IDO related signaling pathways aren’t well understood, some clinical reports affirmed IDO inhibiting therapeutic significance. Therefore, we did use direct tumor autologous antigens vaccination and 1-MT without chemotherapy to explore biological mechanisms and immunomodulations of 1-MT that motivate antitumor responses. However, DCs antigen-uptake capability, anti-tumor efficiency, intra-tumor and intracellular cytokines were assessed. Besides, CD133+ cells viability and tumor biomarkers were investigated. Splenocytes responses and their signaling pathways such TLRs 2 to 9, NF-κβ1-2, Wnt/β-catenin and TGF-β were dissected. Results evinced that a regimen of 1-MT and TAAs significantly reduced CSC CD133 + viability inside tumor microenvironment, besides increasing tumor cells necrosis and apoptosis. Expression of TGF-β, IDO, RANTES, and PDL-1 was also significantly reduced. Interestingly, 1-MT enhanced lymphocytes TLR2, TLR7, TLR8, and TLR9 pathways. It motivated lymphocytes’ NF-κβ2, STAT3, and STAT4 pathways, while reduced tumors’ NF-κβp65 and Wnt/β-catenin signaling pathways. We found that periphery and intra-tumor Treg cells were significantly decreased. In conclusion, depletion of indoleamine 2,3-dioxigenase activity evidenced IDO relation with tumor stem cells proliferation pathways. Furthermore, 1-MT supports immunotherapeutic vaccines susceptibility and tumor specific targeting by reducing tumorgensis signaling pathways.

α-Ketoglutarate Promotes Pancreatic Progenitor-Like Cell Proliferation

A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG), a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133+ and CD133− cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133+ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet). αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.