Establishment and optimization of a high-throughput mimic perfusion model in ambr® 15.

OBJECTIVES: To establish an automated high-throughput mimic perfusion scale-down model (SDM) in ambr® 15 system. RESULTS: An optimized SDM for mimic perfusion was developed in ambr® 15 system. Cell retention in ambr® 15 was realized by sedimentation and supernatant removal with a retention rate > 95%. Although the SDM couldn't reach the viable cell density (VCD) at a bench scale bioreactor (BR), it maintained VCD at approximately 30 × 106 cells/mL with a cell bleeding rate estimated theoretically and predicted the cell specific perfusion rate (CSPR). A base-feeding strategy was developed to alleviate the pH drop during sedimentation which would adversely have an impact on cell growth, and showed an apparent cell viability improvement from 79.6% (control) to 90.1% on Day 18. The optimized SDM for mimic perfusion was employed for media screening in two cell lines. CONCLUSIONS: A small-scale high-throughput perfusion model in ambr® 15 was developed, optimized to improve cell viability, and as a result, utilized for media screening in two cell lines.

[Development and optimization of perfusion process for mammalian cell culture].

In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.

Acute enterovirus infections significantly alter host cellular DNA methylation status.

Acute infections with enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) usually cause Hand, foot and mouth disease (HFMD) among infants and young children with several large outbreaks worldwide. Unfortunately, the molecular mechanisms underlying enterovirus infections remain largely unknown. In this study, we analyzed the genome-wide DNA methylation patterns of host cells in response to EV71 and CVA16 infections using the Illumina Infinium HumanMethylation450 BeadChip. Of over 480,000 loci studied, significant differential methylation was observed between EV71 infected-cells and control cells at 3957 CpG sites, out of which 2478 were hypermethylated and 1479 were hypomethylated, whereas CVA16 infection resulted in methylation level changes of 5194 CpG sites with 4288 hypermethylated and 906 hypomethylated. These differential methylated loci displayed a wide range of genomic distributions in chromosomes, inside and surrounding areas (shores and shelves) of CpG islands, as well as functional gene regions including promoter, gene body and 3'UTR. Based on methylation alterations, 1189 genes were identified to be potentially co-associated with the replication processes of two enteroviruses. GO function annotation and enrichment analysis of 1189 common differentially methylated genes reflected a broad spectrum of biological regulatory events during viral infection. KEGG pathway analysis indicated the involvement of diverse signaling pathways including viral myocarditis, Notch signaling and antigen processing and presentation. Our present study provides a novel insight into enterovirus-host interaction network at epigenetic profile, thus contributing to improved understanding of HFMD pathogenesis.

Inhibition of mouse RM-1 prostate cancer and B16F10 melanoma by the fusion protein of HSP65 & STEAP1 186-193.

The research of tumor vaccine plays a crucial role in tumor immunotherapy. This study has constructed and prepared a fusion protein vaccine of heat shock protein 65 (HSP65) and the octapeptide epitope 186-193 of the six transmembrane epithelial antigen of the prostate 1 (STEAP1 186-193), and investigated the inhibitory effect of the fusion protein on mouse RM-1 prostate cancer and B16F10 melanoma xenografts. The fusion protein His-HSP65-STEAP1 186-193 (HHST1), His-HSP65-2×STEAP1 186-193 (HHST2) and His-HSP65-6×STEAP1 186-193 (HHST6) were obtained by setting different copy number of STEAP1 186-193 and adding His purification tag before HSP65. Firstly the inhibitory effect of fusion protein on mouse RM-1 prostate cancer xenografts has been studied, which could be the basis of the study the inhibitory effect of the best fusion protein on mouse B16F10 melanoma xenografts. All studies compared with the fusion protein His-HSP65 (HHSP65), the fusion proteins HHST1, HHST2 and HHST6 all could significantly inhibit the growth of mouse RM-1 prostate cancer xenografts. In addition, the fusion protein HHST2 was proved to be the best compared with the fusion proteins HHST1 and HHST6 (P<0.05). Apart from this, compared with the fusion protein HHSP65, the fusion protein HHST2 also significantly inhibited the growth of mouse beared B16F10 melanoma. The results above indicate that HSP65 and STEAP1 186-193 can significantly inhibit the growth of mouse RM-1 prostate cancer and B16F10 melanoma xenografts, and the appropriate increase of copy number can effectively improve that the fusion protein has an excellent anti-tumor ability.

Inhibition of RM-1 prostate carcinoma and eliciting robust immune responses in the mouse model by using VEGF-M2-GnRH3-hinge-MVP vaccine.

GnRH and VEGF have been investigated as prostate carcinoma enhancers that support tumor spread and progression. Although both have documented roles in prostate carcinoma and many cancer types, the weak immunogenicity of these peptides has remained a major challenge for use in immunotherapy. Here, we describe a novel strategy to inhibit GnRH and VEGF production and assess the effect on the immune responses against these hormones using the RM-1 prostate cancer model. We designed a novel recombinant fusion protein which combined GnRH and VEGF as a vaccine against this tumor. The newly constructed fusion protein hVEGF121-M2-GnRH3-hinge-MVP contains the human vascular endothelial growth factor (hVEGF121) and three copies of GnRH in sequential linear alignment and T helper epitope MVP as an immunogenic vaccine. The effectiveness of the vaccine in eliciting an immune response and attenuating the prostate tumor growth was evaluated. Results showed that administration of a new vaccine effectively elicited humoral and cellular immune responses. We found that, a novel fusion protein, hVEGF121-M2-GnRH3-hinge-MVP, effectively inhibited growth of RM-1 prostate model and effectively promoted immune response. In conclusion, hVEGF121-M2-GnRH3-hinge-MVP is an effective dual mechanism tumor vaccine that limits RM-1 prostate growth. This vaccine may be a promising strategy for the treatment of hormone refractory prostate malignancies.

Comparison of fusion protein and DC vaccine in inhibition of mouse B16F10 melanoma tumor.

Dendritic cell (DC) vaccine and fusion protein vaccine have been put into clinical use in cancer immunotherapy. This study compared DC vaccine and fusion protein vaccine directly in their capability of inducing specific immune response. We used mouse Granulocyte Macrophage-Colony Stimulating Factor (mGM-CSF) fused with gastrin-releasing peptide (GRP) and Gonadotrophin Releasing Hormone (GnRH) respectively to obtain mGM-CSF/GRP6 (mG6) and mGM-CSF/mGGn (mGGn) fusion proteins. We prepared fusion protein vaccine and DC vaccine including mG6 protein vaccine (6P), mGGn protein vaccine (nP), mG6 DC vaccine (6D) and mGGn DC vaccine (nD), then the two proteins were mixed to prepare combination proteins vaccine (6nP) and DC vaccine (6nD). After that, C57BL/6 mice were injected with B16F10 cell line to build melanoma tumor model, and were immunized with vaccines to produce antibodies to inhibit and destruct melanoma tumor cells. The discoveries showed that anti- mGM-CSF-GRP6 and anti- mGM-CSF-mGGn antibody vaccines were successfully created as expected; this was deduced from significant inhibition of melanoma tumor in vivo and significant reduction of tumor weight and volume. The effects of DC groups were better than that of the protein groups and the combination of vaccines were more effective than vaccine given separately. Our results indicate that using combination vaccine provides a new strategy to inhibit melanoma tumor growth but a complete cure of melanoma needs further investigations.

In vitro anti-tumor effect of mGM-CSF-GnRH3 and mGM-CSF-GRP6 recombinant fusion protein and theirbioinformatics prediction / 中国肿瘤生物治疗杂志

@#Objective: To prepare the fusion protein mGM-CSF-GnRH3 (mGGn) of mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) combining with gonadotropin releasing hormone (GnRH) and the fusion protein mGM-CSF-GRP6 (mG6) of mGM-CSF combining with gastrin-releasing peptide (GRP), and to investigate the inhibitory effect of the above two fusion proteins on B16F10 melanoma in vitro as well as to preliminarily predict their isoelectric point, relative molecular weight, hydrophobicity, stability, subcellular localization, signal peptide, spatial structure and potential epitopes. Methods:After the successful preparation of mGGn and mG6, the effects of different concentrations of fusion proteins on tumor cell morphology, migration, proliferation and cell cycle were detected by microscopic observation, scratch test, CCK-8 method and flow cytometry, respectively. The protein online analysis systems EXPASY, GOR4, SWISS MODEL were used to predict the basic properties and secondary/tertiary structure of recombinant fusion proteins. The B cell epitopes were predicted by IEDB and ABCpred software, the CTL epitopes were comprehensively predicted by SYFPEITHI, BlMAS and NetCTL software, and the Th epitopes were predicted by NetMHCIIpan 3.1 Server and IEDB software. Results:Both mGGn and mG6 inhibited the migration and proliferation of tumor cells. mGGn could block B16F10 cell cycle at G1 phase while mG6 could block B16F10 cell cycle at S phase, all of which prevented cells entering into G2 phase to inhibit tumor cell growth. The mGGn and mG6 fusion proteins got diverse structures and had multiple potential B epitopes, CTL epitopes and Th epitopes. Conclusion: mGGn and mG6 have inhibitory effect on B16F10 melanoma in vitro, and bioinformatics predictions have laid a foundation for further study of the biological functions and immunological activities of these fusion proteins.

Oral delivery of staphylococcal nuclease by Lactococcus lactis prevents type 1 diabetes mellitus in NOD mice.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the self-destruction of insulin-producing ß cells. Recently, studies have revealed that neutrophils contribute to the early pathological injury to the pancreas, predominantly via the formation of neutrophil extracellular traps (NETs). To determine whether early intervention targeting NETs with staphylococcal nuclease (SNase) can delay the onset of T1DM, non-obese diabetic (NOD) mice were orally administered recombinant Lactococcus lactis (L. lactis) expressing SNase. The results showed that NETs were effectively disrupted by SNase both in vivo and in vitro, leading to a significant decrease in neutrophil-derived circulating free DNA (cf-DNA/NETs), neutrophil elastase (NE), and protease 3 (PR3) in the serum compared with the controls. In addition, SNase effectively regulated the blood glucose levels of NOD mice, and the onset of diabetes was postponed with reduced mortality and morbidity. Recombinant L. lactis also ameliorated inflammation in NOD mice, as evidenced by the remarkable increase in IL-4 and reductions in TNF-α and CRP. Moreover, HE staining results showed that L. lactis expressing SNase exerted protective effects on pancreatic islets and relieved inflammation of the small intestine in NOD mice. Hence, the present study indicates that the oral delivery of SNase by L. lactis can effectively prevent T1DM, ameliorate inflammation, and contribute to immunomodulatory balance in NOD mice.

A rationally designed peptide IA-2-P2 against type 1 diabetes in streptozotocin-induced diabetic mice.

Recent studies have investigated the potential of type 1 diabetes mellitus-related autoantigens, such as heat shock protein 60, to induce immunological tolerance or to suppress the immune response. A functional 24-residue peptide derived from heat shock protein 60 (P277) has shown anti-type 1 diabetes mellitus potential in experimental animals and in clinical studies, but it also carries a potential atherogenic effect. In this study, we have modified P277 to retain an anti-type 1 diabetes mellitus effect and minimize the atherogenic potential by replacing the P277 B epitope with another diabetes-associated autoantigen, insulinoma antigen-2 (IA-2), to create the fusion peptide IA-2-P2. In streptozotocin-induced diabetic C57BL/6J mice, the IA-2-P2 peptide displayed similar anti-diabetic effects to the control P277 peptide. Also, the IA-2-P2 peptide did not show atherogenic activity in a rabbit model. Our findings indicate the potential of IA-2-P2 as a promising vaccine against type 1 diabetes mellitus.

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.

Fusion protein His-Hsp65-6IA2P2 prevents type 1 diabetes through nasal immunization in NOD Mice.

Human heat shock protein 60 (Hsp60), is an endogenous ß-cells autoantigen, it could postpone the onset of insulitis and sooner type 1 diabetes mellitus. P277 is one of Hsp65 determinants at position 437-469 of amino acids cascaded. Meanwhile, it's already well-known that there were several better anti-diabetic B epitopes, such as insulinoma antigen-2 (IA-2). Currently, fusion protein IA2P2 has constructed in order to enhance its pharmacological efficacy. In addition, added homologous bacterial-derived Hsp65 and His tag were beneficial to protein immunogenicity and purification separately. So, finally we examined a fusion protein His-Hsp65-6IA2P2 could regulate Th2 immune response and reduce natural diabetic incidence in NOD mice. We constructed two express vector pET28a-His-Hsp65-6P277 and pET28a-His-Hsp65-6IA2P2. After purification, we observed that triple intranasal administration of these two fusion protein in 4-week-old NOD mice maintained normal blood glucose and weight, with a lower diabetic or insulitis incidence. Consistent with induced splenic T cells proliferation and tolerance, His-Hsp65-6IA2P2-treated mice performed reduced IFN-γ and increased IL-10 level. In conclusion, we suggested that fusion protein His-Hsp65-6IA2P2 could be reconstructed and purified successively. Furthermore, nasal administration of this fusion protein could rebalance T cells population and prevent T1DM.

Promotion of atherosclerosis in high cholesterol diet-fed rabbits by immunization with the P277 peptide.

Previous evidence has proved the ability of immunization with heat shock protein (HSP) 60/65 to induce atherosclerosis. P277, a 24-residue peptide of human HSP60, is a promising peptide vaccine against autoimmune diabetes. But as a fragment of HSP60, its potential ability of promoting atherosclerosis has never been investigated yet. In the present study, the rabbits fed with normal standard diet or high cholesterol diet were immunized with P277 or PBS emulsified in incomplete Freund's adjuvant 4 times at 4-week intervals. Atherosclerotic lesions of the rabbits receiving P277 treatment and fed with high cholesterol diet increased significantly compared with those of the rabbits receiving PBS treatment and the same diet. However, no obvious lesions were found in the two groups of rabbits fed with the normal standard diet. Significant expression of P277 was detected in the high cholesterol diet-induced atherosclerotic lesions and heat-stressed endothelial cells. Surface exposure of P277 was also observed in the stressed cells. In the subsequent assay of endothelial cells in vitro, the purified anti-P277 antibodies mediated a noticeable cytotoxicity to the stressed cells with the participation of complement. In conclusion, subcutaneous immunization with P277 emulsified in IFA can aggravate the atherosclerosis in high cholesterol diet-fed rabbits. Surface expression of P277 was observed on stressed endothelial cells, and were suggested to mediate the autoimmune attack and promote the disease.

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.

Oral administration of recombinant Lactococcus lactis expressing HSP65 and tandemly repeated P277 reduces the incidence of type I diabetes in non-obese diabetic mice.

Diabetes mellitus type 1 (DM1) is an autoimmune disease that gradually destroys insulin-producing beta-cells. We have previously reported that mucosal administration of fusion protein of HSP65 with tandem repeats of P277 (HSP65-6P277) can reduce the onset of DM1 in non-obese diabetic (NOD) mice. To deliver large amounts of the fusion protein and to enhance long-term immune tolerance effects, in the present study, we investigated the efficacy of using orally administrated L. lactis expressing HSP65-6P277 to reduce the incidence of DM1 in NOD mice. L. lactis strain NZ9000 was engineered to express HSP65-6P277 either constitutively or by nisin induction. After immunization via gavage with the recombinant L. lactis strains to groups of 4-week old female NOD mice for 36 weeks, we observed that oral administration of recombinant L. Lactis resulted in the prevention of hyperglycemia, improved glucose tolerance and reduced insulitis. Immunologic analysis showed that treatment with recombinant L. lactis induced HSP65- and P277- specific T cell immuno-tolerance, as well as antigen-specific proliferation of splenocytes. The results revealed that the DM1-preventing function was in part caused by a reduction in the pro-inflammatory cytokine IFN-γ and an increase in the anti-inflammatory cytokine IL-10. Orally administered recombinant L. lactis delivering HSP65-6P277 may be an effective therapeutic approach in preventing DM1.

Pharmacokinetic study of HS061, a new human insulin, in non-diabetic rat using ultra performance liquid chromatography-tandem mass spectrometry.

HS061, a new structure analogue of human insulin, was investigated for the treatment of diabetes. In this study, we developed a simple and accurate UPLC-MS/MS method for the pharmacokinetic studies of HS061 in non-diabetic rats followed by a full method validation. Following a simple protein precipitation with acetonitrile, the analyte and internal standard (Levemir, IS) were separated on a Waters XBridge™ BEH300 C4 column (100 mm × 4.6 mm i.d., 3.5 µm) with a gradient elution using acetonitrile and 0.2% aqueous formic acid. The method was operated under pseudo-multiple reaction monitoring (pseudo-MRM) in the positive electrospray ionization mode. The monitored transitions were set at m/z 1563.4→1563.4 for HS061 by pseudo-MRM and m/z 1184.7→454.5 for IS by MRM. Linear calibration curves were obtained over the concentration ranges of 10-1000 ng/mL and no interfering peaks were detected at the retention time of HS061 and IS in blank rat plasma. The mean extraction recoveries of HS061 at three concentrations of 20, 100, 800 ng/mL were greater than 95.17%. Stability was assessed under different conditions and no significant degradations were found. The validated method was then successfully applied in measuring HS061 following subcutaneous (0.5, 1.0, 3.0 U/kg) and intravenous (1.0 U/kg) injection in rat plasma to support the pre-clinical pharmacokinetic study. Maximum plasma concentration (Cmax) and area under the curve (AUC) for the subcutaneous doses of HS061 was approximately dose proportional while other pharmacokinetic parameters showed no significant differences among the three doses (p>0.05). The absolute bioavailability of HS061 after subcutaneous administration at 1.0 U/kg was estimated to be 70.40%.

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.

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.

Administration of heat shock protein 65 inhibits murine melanoma growth in vivo.

The association between heat shock protein (HSP) 65 and immune diseases has been investigated for many years. The aim of this study was to explore the antitumor effects and possible antitumor mechanism of HSP65. Mice were immunized with HSP65 via subcutaneous injection. Specific IgG antibodies against HSP65 were detected in the sera of immunized mice by enzyme­linked immunosorbent assay and verified by western blot analysis. HSP65 effectively inhibited the growth of tumors as well as both the protective and therapeutic antitumor immunities in the melanoma tumor models of mice and prolonged the survival of the tumor-bearing mice. Furthermore, HSP65 also attenuated tumor-induced angiogenesis in the intradermal model and pulmonary metastasis in the tail intravenously injected model of mice. It was demonstrated that the administration of HSP65 is able to effectively inhibit the growth, angiogenesis and metastasis of murine melanoma in vivo and provide new prospects for the immunotherapy of melanoma.

Vaccination with the repeat ß-hCG C-terminal peptide carried by heat shock protein-65 (HSP65) for inducing antitumor effects.

The ß-subunit of human chorionic gonadotropin (ß-hCG) is ectopically expressed in various types of cancer and has been utilized as an antigenic target in anti-cancer vaccines. In view of the low immunogenicity of this self-peptide, we designed a method based on the isocaudamer technique to generate 14 tandem repeats of the 10-residue sequence X of ß-hCG (109-118). These tandemly repeated copies were then combined with ß-hCG C-terminal 37 peptides (CTP37) and finally fused to mycobacterial heat-shock protein 65 (HSP65) to construct a fusion protein HSP65-X14-ßhCGCTP37 as an immunogen. In this study, BALB/c female mice were immunized via subcutaneous injection of the designed protein. Humoral immune and cellular immune responses were effectively elicited. A high titer of anti-ß-hCG antibody was detected in immunized mice sera by enzyme-linked immunosorbent assay and verified by Western blot analysis. The fusion protein, HSP65-X14-ß-hCGCTP37, effectively inhibited the growth of Ehrlich ascites carcinoma in mice. These results suggest that HSP65-X14-ßhCGCTP37 may be an effective tumor vaccine, and the use of multiple tandem repeats of a certain epitope is an effective method to overcome the low immunogenicity of self-peptide antigens.