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Study on Multisubunit-derived Th Epitope Vaccine against Helicobacter pylori

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Tutor: ZouQuanMing
School: Third Military Medical University
Course: Biotechnology
Keywords: H. pylori,Th epitope,vaccine,Th1-biased immune response
CLC: R392
Type: PhD thesis
Year:  2013
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Abstract:
Helicobacter pylori (H. pylori) is a common bacterium, and approximately50percentof the world¡¯s population has been estimated to be infected. H. pylori infection is a keyetiological factor in chronic gastritis, peptic ulcer disease, and was listed as a class Icarcinogen by World Health Organization (WHO).Antigens are known to display their specificity mainly through epitopes. Consisting ofepitopes from versatile antigens, epitope-based vaccines are capable of inducing morespecific and potent immune responses than whole antigens. H. pylori infection leads to thebody¡¯s immunological tolerance to natural antigen. Rational combination ofimmunodominant epitopes selected from protective antigens is possible to break theimmune tolerance, so as to confer protection against H. pylori infection. Previous studiesindicates that specific CD4+T cell response is critical to the protective immunity. In thisstudy, we constructed a H. pylori Th epitope-based vaccine, and the prophylactic andtherapeutic potential was examined. Research methods, results and conclusions are asfollows:1. H. pylori Th epitopes screen and vaccine designBy means of bioinformatic algorithms, potential immunodominant CD4+T cellepitopes were screened from HpaA, UreB and CagA genes. Kinetic and thermodynamicstability of the various combinations of epitopes was analyzed by dynamics simulation, andthe theoretic optimal combination was HpaAepi-CagAepi-UreBepi.2. Construction, expression and purification of the vaccineSynthetic oligonucleotides encoding Epivac were ligated into the Xho¢ñ-Nde¢ñ sitesof the pET30a vector. Then the recombinant plasmid was transformed into E.coliBL21(DE3). After induction and purification, Epivac was obtained and was more than90%pure as determined by HPLC. LTB-epivac was obtained by the same method.3. Evaluation of protective effect of the vaccine and underlying mechanismsIn the presence of several different adjuvants, BALB/c mice were intranasally and subcutaneously immunized with Epivac and subsequently challenged with H. pylori. Theprotective effect was achieved4weeks post challenge. The results show Epivac alone orwith an adjuvant significantly reduced H. pylori colonization and better protection wasobserved when an adjuvant was used. In addition, the protective effect was observed earlierwhen immunized intranasally. Epivac-specific IgG, IgG1, IgG2a and sIgA were measuredby ELISA, and systemic and gastric cytokine expression was also measured by Real-timeRT-PCR and ELISA. Quantitation and characterization of Epivac-specific CD4+T cellswere determined by flow cytometry. Epivac-mediated protective immunity against H. pyloriinfection may occur by antibody-independent mechanisms, and Epivac-mediated local andsystemic Th1-biased immune response may contribute to the protective immunity.4. Evaluation of therapeutic effect of the vaccine and underlying mechanismsTherapeutic effect of the vaccine was evaluated by BALB/c mice. Compared tonon-immunized mice, immunization with Epivac alone or with an adjuvant significantlyreduced H. pylori colonization. By analysis of the characteristics of the antibody response inmice, we concluded that the Th1-biased response may correlate with the eradication of H.pylori.5. Safety evaluation of the vaccineThe endotoxin level of the vaccine was determined by endotoxin measure kit, andAcute Toxicity Test (ATT) and Guinea pig maximization test (GPMT) was used to evaluatethe safety of the vaccine. Results showed that endotoxin level is below the limit, andintranasally and subcutaneously immunization would not cause body injury and skinsensitization. This study indicates that the vaccine is safe and non-toxic.
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