Cao Rongyue

Nanjing Medical University, Nanjing, Jiangsu Sheng, China

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Publications (15)44.08 Total impact

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    ABSTRACT: Previous investigations have demonstrated that anti-inflammatory or lipid-lowering treatments could be useful for alleviating morbidity and mortality of atherosclerotic cardiovascular diseases. However, whether a vaccine designed to target inflammation and lipid simultaneously is more powerful to control the process of atherosclerosis remain to be unknown. Here, a vaccine was designed to target heat shock protein-65(Hsp65) and cholesteryl ester transfer protein (CETP) simultaneously and the effects of nasal immunization of multi-target vaccine on high-cholesterol-diet-driven rabbit atherosclerosis lesions were evaluated. Sera, nasal lavages and lung washes were used to ELISA assay for the analysis of IgG and IgA against Hsp65 and CETP. Sera were also used to the analysis of the avidity of combination of anti-Hsp65 and anti-CETP IgG antibodies with corresponding antigen, cytokines IL-10 and IFN-γ, and lipoproteins. In addition, aortas were harvested for analysis of atherosclerotic lesions. The results showed that lower and lasting specific anti-Hsp65 IgG and high anti-CETP IgG in sera and protective anti-Hsp65 and anti-CETP IgA in nasal cavity and lung were induced, the avidity of combination of anti-Hsp65 and anti-CETP IgG with antigen were higher, and more protective IL-10 and less adverse IFN-γ were produced. In addition, sera TC, and LDL-C were decreased. As a result, the size of aorta atherosclerotic plaques was significantly reduced. We conclude that multifaceted vaccine combining lipid-regulating with anti-inflammation was a potential remedy, especially for atherosclerosis with complicated etiology.
    Vaccine 12/2011; 30(6):1029-37. · 3.77 Impact Factor
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    ABSTRACT: Lactococcus is a genus of Gram positive food-grade bacteria that has been widely used as a vaccine platform for the safe delivery of heterologous antigens. Many reports support the involvement of inflammation and immunity in atherosclerosis as well as the role of autoimmunity to heat shock proteins (HSPs) in the progression of atherogenesis. In this study, experiments were specifically designed to investigate the effect of oral administration of mycobacterial heat shock protein 65 (HSP65) delivered by Lactococcus lactis (L. lactis) on atherogenesis. Two types of HSP65-encoding plasmids for intracellular expression or secretion were constructed, and then transformed into L. lactis NZ9000. Oral administration of two recombinant L. lactis strains both induced suppression of HSP65-specific proliferation, accompanied by elevation of Interleukin-10 (IL-10) production and reduction of interferon-gamma (IFN-γ) level. Inducible HSP65-specific tolerance exerted a protective effect on atherosclerotic lesion formation and endothelial damage in low-density lipoprotein receptor-deficient (LDL-RD) mice model, while no obvious pathological abnormalities were observed. In conclusion, delivery of HSP65 at the intestinal mucosa by recombinant L. lactis provides a novel approach for the prevention of atherosclerosis. The results further illustrate the potential of using genetically modified L. lactis as a safe and effective vaccine delivery to elicit antigen-specific tolerance for treatment of autoimmune diseases.
    Vaccine 05/2011; 29(24):4102-9. · 3.77 Impact Factor
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    ABSTRACT: Vaccination with xenogeneic and syngeneic endothelial cells is effective for inhibiting tumor growth. Nontoxic diphtheria toxin (CRM197), as an immunogen or as a specific inhibitor of heparin-binding EGF-like growth factor, has shown promising antitumor activity. Therefore, immunization with or administration of viable human umbilical vein endothelial cells (HUVECs) combined with CRM197 could have an enhanced antitumor effect. Six-week-old C57BL/6J male mice were vaccinated with viable HUVECs, 1 x 10(6) viable HUVECs combined with 100 μg CRM197, or 100 μg CRM197 alone by ip injections once a week for 4 consecutive weeks. RM-1 cells (5 x 10(5)) were inoculated by sc injection as a preventive procedure. During the therapeutic procedure, 6-week-old male C57BL/6J mice were challenged with 1 x 10(5) RM-1 cells, then injected sc with 1 x 10(6) viable HUVECs, 1 x 10(6) viable HUVECs + 100 μg CRM197, and 100 μg CRM197 alone twice a week for 4 consecutive weeks. Tumor volume and life span were monitored. We also investigated the effects of immunization with HUVECs on the aortic arch wall and on wound healing. Vaccination with or administration of viable HUVECs+CRM197 enhanced the inhibition of RM-1 prostatic carcinoma by 24 and 29%, respectively, and prolonged the life span for 3 and 4 days, respectively, compared with those of only vaccination or administration with viable HUVECs of tumor-bearing C57BL/6J mice. Furthermore, HUVEC immunization caused some damage to the aortic arch wall but did not have remarkable effects on the rate of wound healing; the wounds healed in approximately 13 days. Treatment with CRM197 in combination with viable HUVECs resulted in a marked enhancement of the antitumor effect in the preventive or therapeutic treatment for prostatic carcinoma in vivo, suggesting a novel combination for anti-cancer therapy.
    Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 02/2011; 44(2):140-8. · 1.08 Impact Factor
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    ABSTRACT: Gastrin-releasing peptide (GRP), a bombesin-like peptide, is an autocrine or paracrine growth factor that can stimulate the growth of various cancer cells, making it an ideal target antigen to develop vaccines against cancer. In this study, we developed a novel DNA vaccine that encodes six tandem repeats of B-cell epitope GRP(18-27) (GRP6) flanked by HSP65 as carrier and four tandem repeats of mycobacterial HSP70(407-426) (M4) as helper T-cell epitopes for enhancement of immunogenicity. When intramuscularly immunized to mice, this anti-GRP DNA vaccine-induced GRP-specific antibody (Ab) responses that were at least 10-fold higher in magnitude compared with HSP65-GRP6 protein vaccine. Both prophylactic and therapeutic antitumor immunities induced by vaccination significantly suppressed the growth of GRP-dependent prostate carcinoma RM-1 in vivo and prolonged the survival of tumor-inoculated mice. Out results also showed that the immune sera with high titer of GRP-specific Abs effectively inhibited the growth of tumor in mice and dose dependently inhibited proliferation of cultured RM-1 cells in vitro, suggesting that the GRP neutralizing Ab is responsible for the protective and therapeutic antitumor activity of vaccination. These findings may be of great importance in the further exploration of the applications of growth factors identified in human in cancer therapy.
    Gene therapy 04/2010; 17(4):459-68. · 4.75 Impact Factor
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    ABSTRACT: It has been demonstrated that the beta-subunit of human chorionic gonadotropin (beta-hCG) is ectopically expressed on a variety of human cancers of different histological types and has been used as an antigenic target in anti-cancer vaccines. We engineered a fusion protein by fusing 10 tandemly repeated copies of the 10-residue sequence of beta-hCG (109-118) (in CTP37) combined with beta-hCG C-terminal 37 peptides to mycobacterial heat-shock protein 65 and immunized mice via subcutaneous injection. Humoral immune and cellular immune responses were effectively elicited. High titer of anti-beta-hCG antibody was detected in immunized mice sera by ELISA and verified by Western blot analyses. The fusion protein of HSP65-X10-beta-hCGCTP37 effectively inhibited the growth of tumor both protective and therapeutic anti-tumor immunity in hepatocellular carcinoma tumor models in mice. Meanwhile, it also attenuated tumor-induced angiogenesis in intradermal tumor model in mice. Taken together, these results demonstrate that immune responses are effectively induced by a novel fusion protein vaccine targeting beta-hCG, suppressing the growth of hepatocellular carcinoma in mice. The beta-hCG-targeted vaccine holds promise for the treatment of a number of cancers and merits further study.
    International immunopharmacology 11/2009; 10(2):230-8. · 2.21 Impact Factor
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    ABSTRACT: A novel insulin analog, PIns, with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at human regular insulin B-chain was acquired through gene engineering. Preproinsulin for PIns was cloned and expressed using a bacterial expression system at a high level (72.1%) as fusion protein carrying a modified thioredoxin N-terminal region (1-21) linked to N-terminus of proinsulin by a lysine residue. Purified fusion protein was refolded and converted into PIns by a single enzymatic reaction. After PIns was purified, the homogeneity of it was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectronic focusing electrophoresis, amino acid composition analysis and mass spectrometry methods. A decreased tendency of self-association of PIns as compared with regular insulin was demonstrated by the size exclusion HPLC analysis. When subcutaneously administrated into normal rats, the PIns showed a faster rate of onset of action and a shorter duration of action compared with regular insulin, similar to the pharmacokinetic characteristics of insulin Lispro. These results showed that PIns is a rapid insulin analog. Furthermore, the N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain can be excised by DPPIV and recombinant peptidase with DPPIV-like activities. It is suggested that PIns serves as an artificial insulin precursor and can be transformed to regular insulin in vivo due to the truncation of N-terminal sequence of PIns B-chain by DPPIV.
    Regulatory Peptides 07/2009; 157(1-3):92-8. · 2.06 Impact Factor
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    ABSTRACT: Gastrin-releasing peptide (GRP), a bombesin-like peptide, is an autocrine growth factor that can stimulate the growth of various cancer cells. We developed a novel protein vaccine HSP65-(GRP-10)(6) (HG6) that consists of six copies of a 10-amino acid residue epitope of GRP C-terminal fragment carried by mycobacterial 65 kDa HSP65 and then immunized mice via subcutaneous injection. Strong humoral and cell-mediated immune responses were induced. High titer of anti-GRP antibodies was detected in immunized mice sera by ELISA and verified by Western blot analysis. Activity of CD4+T lymphocytes, especially high levels of interferon (INF)-gamma, were developed in mice immunized with HG6 when compared with HSP65 or PBS. We found that immunogene tumor therapy with a vaccine based on GRP was effective at both protective and therapeutic antitumor immunity in breast tumor models in mice. The purified GRP monoclonal antibody (McAb) was proved to be potential in inhibiting EMT-6 tumor cell proliferation in vitro. The attenuation induced by active immune responses on tumor-induced angiogenesis was observed with an intradermal tumor model in mice. Taken together, we demonstrate for the first time that immune responses that are elicited by a novel chimeric protein vaccine targeting GRP can suppress the proliferation of breast tumor cell EMT-6 in mice, and it may be of importance in the further exploration of the applications of other autocrine growth factor identified in human and other animal in cancer therapy.
    Endocrine Related Cancer 04/2008; 15(1):149-59. · 5.26 Impact Factor
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    ABSTRACT: In order to prevent atherosclerosis, a chimeric enzyme vaccine of AnsB-TTP-PADRE-CETPC was successfully constructed, expressed and purified to immunize New Zealand white rabbits for inducing high titers of anti-CETP antibodies to improve lipid abnormality. The protein was expressed as soluble protein in Escherichia coli and purified by anion exchange column and Sephadex G-100 size-exclusion chromatography. After immunizing rabbits with the purified protein, high titer anti-CETP antibodies were induced and lasted more than nineteen weeks in vivo; High density lipoprotein cholesterol (HDL-C) content in the serum was elevated to 61% while decreased low density lipoprotein cholesterol (LDL-C) to 37.2% compared with control rabbits in the presence of Al(OH) (3).
    Protein and Peptide Letters 02/2008; 15(7):745-52. · 1.99 Impact Factor
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    ABSTRACT: The beta-subunit of human chorionic gonadotropin (beta-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of beta-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with beta-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-betahCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residue sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-betahCGCTP37 and HSP65-betahCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-betahCGCTP37 elicited much higher levels of specific anti-beta-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-betahCGCTP37, which should suggest that HSP65-X10-betahCGCTP37 may be an effective protein vaccine for the treatment of beta-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.
    Biochemical and Biophysical Research Communications 08/2006; 345(4):1365-71. · 2.28 Impact Factor
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    ABSTRACT: Rabbits were intramuscularly immunized with the plasmid pCR-X8-HBc-CETP encoding a B-cell epitope of cholesteryl ester transfer protein (CETP) C-terminal fragment (CETPC) displayed by Hepatitis B virus core (HBc) particle. This plasmid also contained immunostimulatory sequences (ISS) which included eight CpG motifs 5'-GACGTT-3', functioning as immunomodulators. After anti-CETP antibodies were successfully produced, rabbits were fed with a high-cholesterol diet for 15 weeks, and then the antiatherogenic effects of this DNA immunization were evaluated. The results showed that the fraction of plasma cholesterol in HDL significantly increased and the fraction of plasma cholesterol in LDL decreased in the pCR-X8-HBc-CETP immunized rabbits compared with those in the saline control group and one group treated with the plasmid pCR-X8-HBc containing ISS but lacking CETP epitope. More importantly, DNA immunization with pCR-X8-HBc-CETP markedly reduced the average percentage of aortic lesions in the entire aorta area by 80.60% compared with the saline control (3.78% versus 19.48%) and the average thickness of hyperplastic coronary artery in this group was also significantly less than in the saline control group (146+/-11 microm versus 248+/-18 microm). Our data also showed that CpG DNA alone could be antiatherogenic in this model because the average percentage of aortic lesions in pCR-X8-HBc immunized rabbits was 16.53% lower than that of the saline control group and the average thickness of hyperplastic coronary artery was also substantially lower than saline control group (155+/-13 microm versus 248+/-18 microm). Thus, plasmid pCR-X8-HBc-CETP could significantly inhibit the progression of atherosclerosis and be potentially developed as a suitable DNA vaccine against atherosclerosis.
    Vaccine 07/2006; 24(23):4942-50. · 3.49 Impact Factor
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    ABSTRACT: Studies have demonstrated that active-specific immunotherapy has potential for controlling mammary tumor progression. Human chorionic gonadotropin (hCG) is expressed and extremely sensitive, easily detectable and highly correlated with breast cancer. We developed a gene vaccine using a plasmid vector to deliver the six copies of 10-amino acid residues of beta-hCG 109-118 and beta hCG C-terminal 37-amino acid (CTP37). BALB/c female mice were immunized with a combination of pCR-HBc-X6-betahCGCTP37 DNA vaccine and HSP-X6-betahCGCTP37 protein vaccine. pCR-HBc-X6-betahCGCTP37 DNA vaccine were injected intramuscularly three times, on days -46,-25 and -11 and HSP-X6-betahCGCTP37 protein were applied two times, 21 and 14 days before tumor cell challenge. We assessed a combined DNA and protein vaccine for its effect of against murine EMT6 mammary tumor cells. In this study, animals vaccinated DNA vaccination boosting with the repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65 induced higher avidity antibodies and effectively inhibited the growth of tumor, compared with treatment using DNA alone or BCG priming HSP-X6-betahCGCTP37 protein boosting. The data presented demonstrate that improve immunogenicity of DNA vaccination by boosting with the repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65, which should prove useful in the development of new DNA vaccine against growth factors for cancer immunotherapy.
    Vaccine 04/2006; 24(14):2575-84. · 3.49 Impact Factor
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    ABSTRACT: The recombinant chimeric enzyme, AnsB-TTP-CETPC, comprising asparaginase, tetanus toxin helper T cell epitope and human CETP B cell epitope was expressed as a soluble protein in Escherichia coli. The purified chimeric enzyme exhibited approximate 83% activity of the native asparaginase. After immunization with three doses of chimeric enzyme, high titers of anti-CETP antibodies were induced and lasted more than eighteen weeks in mice, and could even be detected at a dilution of 1:12800 by normal ELISA assay. The specificity of anti-CETP antibody was verified by Western blot assay. After displaying on the surface of asparaginase, the weak antigenicity of CETP epitope was effectively overcome, there after a strong CETP-specific immune response was evoked in mice immunized with the chimeric enzyme. Histochemical analysis of mice kidney tissue showed that immunization with the chimeric enzyme did not cause any pathological changes in mice. Collectively, the chimeric enzyme may be further developed as a vaccine against atherosclerosis in the future.
    Protein and Peptide Letters 02/2006; 13(2):149-54. · 1.99 Impact Factor
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    ABSTRACT: Active immunization against self-peptides have gained widespread acceptance inspite of their low immunogenicity. Recent applications involving multiple copies of self-peptides in linear alignment and conjugation with carrier proteins appear to increase the immune response against self-peptides. As with most vaccines, however, immunogens require supplementation with adjuvants to elicit an optimum immune response. In the present study, we prepared a double-chain mini-protein with each chain containing three linear repeats of the self-peptide gonadotropin-releasing hormone (GnRH3), the hinge region of human IgG1 (hinge), and a T-helper epitope from the measles virus protein (MVP). The GnRH3-hinge-MVP mini-protein was conjugated to purified recombinant heat shock protein 65 (Hsp 65) of Mycobacterium bovis and used to immunize rats primed with subcutaneous injections of Bacillus Calmette-Guerin (BCG) in the absence of adjuvants. The GnRH3-hinge-MVP-Hsp 65 stimulated the production of specific anti-GnRH antibodies in the absence of adjuvants and the antibody titer was comparable to that produced in rats immunized with the dimeric mini-protein in the presence of Freund's adjuvant. Moreover, immunization with the adjuvant-free GnRH3-hinge-MVP-Hsp 65 induced degeneration of the reproductive organs in both male and female rats unlike those immunized in the absence of Hsp 65 or in control animals inoculated with the vehicle only. Histological examination of the affected organs showed atrophy of the seminiferous tubules with diminished spermatogenesis in the testes of male rats. In female rats, the uteri were much smaller in size and the ovaries exhibited reduced follicular development. These findings demonstrated that GnRH3-hinge-MVP-Hsp 65 mounted a strong immune response in the absence of conventional adjuvants, and could prove useful in control of fertility and the treatment of conditions/diseases where GnRH ablation is required.
    Vaccine 10/2005; 23(40):4834-43. · 3.49 Impact Factor
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    ABSTRACT: Asparaginase of Escherichia coli, a tetramer of identical subunits, was tested as a vector to display linear peptides on the surface of each enzyme subunit. A recombinant gene encoding a chimeric protein composed of asparaginase, a tetanus toxin peptide (TTP) spacer (831-854 fragment), and the foreign cholesteryl ester transfer protein C-terminal fragment (CETPC) was expressed and targeted to the periplasm of E. coli. The purified chimeric enzyme exhibited approximately 83% activity of the native enzyme, allowing the rapid screening of recombinant clones. In contrast, an asparaginase-CETPC fusion protein without the TTP spacer produced only about 23% activity of the native enzyme. Rats immunized with bacterial cells containing the chimeric enzymes induced CETP-specific immunoresponse. In contrast, rats inoculated with the cells expressing asparaginase only did not generate specific anti-CETP antibodies. Our study showed that asparaginase of E. coli was an effective carrier for displaying foreign peptides or epitopes. Moreover, the use of the TTP spacer appeared to play a critical role in maintaining the catalytic activity of the chimeric enzymes by redirecting the foreign CETPC peptide to the surface of the enzyme. The chimeric enzyme constructs fusing asparaginase with foreign peptides via a TTP spacer could be utilized as a rapid pepscan technique for antigen epitope mapping. The fusion protein of asparaginase-TTP-CETPC could also be useful for the development of a vaccine against atherosclerosis.
    Journal of Immunological Methods 05/2005; 299(1-2):9-19. · 2.23 Impact Factor
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    ABSTRACT: In this study, we designed two linear peptides, GnRH-hinge-MVP, which consists of human gonadotrophin-releasing hormone (GnRH), hinge fragment 225-232/225'-232' of human IgG1 and a T helper peptide from measles virus protein (MVP), and GnRH3-hinge-MVP, which contains three copies of GnRH (so termed GnRH3). The DNA constructs encoding for the two peptides were fused to the C-terminal encoding sequence of asparaginase, encompassing residues 199-326, through an acid-labile aspartyl-prolyl linker. The chimeric genes were expressed at high levels in Escherichia coli. The fusion proteins were purified to approximate homogeneity by means of washing the inclusion bodies and by ethanol precipitation. The GnRH-hinge-MVP or the GnRH3-hinge-MVP was released from the fusion proteins by cleavage with hydrochloric acid and further oxidized into double-chain miniproteins after purification. Both dimeric constructs proved to be efficient immunogens. It was shown that rats immunized with the immunogens generated antibodies specific for GnRH. The dimeric GnRH3-hinge-MVP containing three copies of GnRH in each chain induced a higher titre of anti-GnRH antibodies than the GnRH-hinge-MVP, containing a single copy of GnRH in each chain. These results demonstrate that combining multicopies or single copies of peptide with hinge fragment of human IgG and T helper peptide from measles virus protein can induce anti-peptide immune responses. Our data also suggest that these methods of preparation and dimerization of the recombinant polypeptides may provide a useful strategy for other polypeptide vaccine developments.
    Journal of Immunological Methods 07/2004; 289(1-2):111-22. · 2.23 Impact Factor

Publication Stats

122 Citations
44.08 Total Impact Points

Institutions

  • 2011
    • Nanjing Medical University
      • Department of Pharmacy
      Nanjing, Jiangsu Sheng, China
    • Xinxiang University
      Sinsiang-hsien, Henan Sheng, China
  • 2008–2011
    • China Pharmaceutical University
      • School of Life Science and Technology
      Nanjing, Jiangxi Sheng, China