Study On Preparation And Immunization Of Helicobacter Pylori Microsphere Vaccine

Helicobacter pylori (H. pylori) infects more than half of the world's population especially in the developing countries, resulting in chronic gastritis, peptic ulcer, stomach cancer etc. At present, the method used in clinic to control the infection of Hp could not eradicate it, and there are still some problems such as poor patient compliance, high cost ,antibiotic-resistant strains and so on. Hp vaccine could prevent and eradicate the infection and is direct, effective and cheap. On the basis of the theory of common mucosal immunity system (CMIS), oral route is the easiest way and the immune results is better than the others. At the same time, another problem appeared which is the complex environment would destroy the antigen. Recently, A newly agent appeared in the drug delivery system which would solve the problem on agent of protein and polypeptide vaccine and drug orally. The microsphere (MS), microcapsule and liposomes are the mainly components of it. They could not only overcome the complex environment but also slowly release drug, transport drug to target organ, tissues and cells. It is reported that the diameter is the important factor for the location in target. When the diameter is between 1 to 10 μm, the antigen could induce fierce immune on the mucosa and system immunity. The MS also could let the drug keep a high concentration in the target tissue, which is needed in the last step to induce highly immunity. Because of this, the MS could reduce the number, cycle and dosage of immune and received better results compared with other agents. The research of immunoprotecive mechanism toward Hp is not clear although a lot of works have been done which will push the advancement of Hp vaccine.In this research we took the HpWCP-CAMS as the Hp vaccine, and gave some main results toward the preparation of MS and its characteristics; the immune results of the vaccine; the research of infection test and immunoprotective mechanism. 1. the preparation of MS vaccine with HpWCP and the study of the characteristics. In this part, we use the chitosan and alginate which are high polymer materials in nature and could be degradated in body as the main material to prepare the MS. we definited the parameters the concentration of alginate sodium (AGS) is 2% and chitosan (CTS) is 1%, the matching of the oil and AGS is 2:8, the procedure is reverse titration. we analyzed the four factors which would effect the MS diameter with iso-project method and make sure that the mix speed of 800rpm, drug concentration of 2mg/ml, environment temperature of 25℃and mix time of 30min. Based on the above results we prepared the BSA-CAMS which average diameter is 3.33μm, the drug loading effective is 61.0% and the entrap effiency is 31.5%. The results of EMS showed the surface of the MS is smooth and glossy, and the diameter is well-distributed. The test of drug release proved that the MS has good character that the longest time of drug release achieved 20 days. The protein drug which encapsulated in the MS is stable after placed in the man-made gastric juice. In the target test, the results appeared that the MS would gather in the intestinal mucosa and caught by the immunologic cells which observed by the experiments of 3H-TdR and FITC labeling tests. At the past experiments, we prepared the HpWCP-CAMS vaccine using the HpWCP as the mainly antigen. The detection of the MS characters proved that the average diameter is 3.58μm, the drug loading effective is 63.5%, the entrap effiency is 30.7% and the longest time of drug release achieved 20 days. The test of toxicity appeared that the Hela cells has well-growth after the treatment of 72h with 125μl (5mg/ml). In the vivo test of toxicity, the results are qualification that the mice are well-growth. In the investigation term, the protein stored in the MS approved good stability not only in 25℃±2℃and humidity of 75%±5% but also in 6℃±2℃and humidity of 60%±10%.The lymphocytes already have transparent sensitization when they were immunized 2 weeks and could exist for 8w. 2. The study of immune effect with HpWCP-CAMS The test include 7 groups which are HpWCP group(Ⅰ), HpWCP +CTS+AGS group( Ⅱ), HpWCP-CAMS group ( Ⅲ), HpWCP +rLT group( Ⅳ), HpWCP +CTS+AGS+rLT group(Ⅴ) , HpWCP-CAMS+rLT group(Ⅵ), and PBS as the control group(Ⅶ). The results of Ⅲgroup proved that the IgG levels is 0.959±0.161, the IgA levels is 0.799±0.153, the gastric sIgA is 0.964±0.165, the intestinal sIgA is 1.036±0.324. the results increased obviously compared with Ⅳand Ⅴgroups (p<0.05), increased veryobviously compared with Ⅰ, Ⅱand Ⅶgroups (p<0.01), but not obviously compared with Ⅵgroup (p>0.05). In the tests of the immune dose, immune cycle, immune times and the immune procedure, we found that the MS could improve the deficiency and flaw which exist in the other agents. The results of this test approved that the antibody levels of 50μg MS is higher than the results of 150μg protein (p<0.05), but there is no obviously difference between the antibody levels of 25μg MS and 150μg protein(p<0.05). When the mice were immunized on 0,14,30 and 60th day, the antibody levels is higher than the 0,14,30,90th day and 0,30,60,90 th day groups(p>0.05),but is not obviously difference than 0,7,14,21th day group(p<0.05). In the test of immune times, the project which done on the 0,14,30th day approved the same result as above, and the IgG, IgA and sIgA achieved a higher level. The levels of immune produces were not different obviously between the two groups that using the neutralization of gastric acid or not (p>0.05). This result suggested that the MS have the acid-fast characteristic which were identical to the test in vivo. We could concluded that because the character of target and slow time-release, the MS vaccine could change the deficiency and flaw such as complicated routine, large dosage and in confirmed immune results. 3. Study of the immunoprotecive mechanism and the immunoprotecive test In the research of immune protection on the immunized mice with the four methods which are tissue smear, tissue culture, extract and amplification of the Hp DNA, ELISA of the whole bacteria exist in the stool, There are four detection methods in the research of immune protection on the immunized mice, which are tissue smear, tissue culture, extract and amplification of the Hp DNA, ELISA of the whole bacteria exist in the stool. The positive result requests three of the four detections results are positive. respectively mice were immunized with global immune and infected with the Hp at 2,5 and 10th Weeks,two weeks after infection the immunoprotecive was detected .But in the control mice, all of the rate of the immunoprotecive protection were beyond 90%, This result suggested that the Hp MS vaccine could induced good immune results in mice. the results showed that the rate of the immune protection were 94.4%,89.4% and 94.7%, In the study of immunoprotecive mechanism, we used the technique including RT-PCR, ELISPOT and FACS. In the test of the IgA-ASC and IgG-ASC quantity in the Peyer's patch and spleen after immunized with the HpWCP-CAMS. The results approved in Peyer's patch,the quantity of IgA-ASC rose very obviously from (1.63±0.32)×103/105 to (11.06±2.31)×103/105 (p<0.01), the quantity of IgG-ASC rose obviously from (1.02±0.35)×103/105 to (5.69±1.06)×103/105 (p<0.05). The quantity of IgG-ASC in spleen rose very obviously from (1.22±0.21)×103/105 to (10.35±1.02)×103/105 (p<0.01), the quantity of IgA-ASC rose obviously from (0.99±0.14)×103/105 to (5.24±0.85)×103/105 (p<0.05). In the comparative research of the IL-4 and IFN-γmRNA level, we obtained that both of them had caught a higher step-up in the spleen lymphocytes after immunized with the MS vaccine which suggested that the protected immune response is induced by the subtype of TH1 and TH2 and not as some reports suggest that the immune response type is only either of them. In the FACS study, we concluded that the quantity of the CD3+cells which was thought as the maturity lymphocytes has got a rise obviously from 67.2%to73.2%(p<0.05), just as this of the CD4+T cell and CD4+CD25+T cell which are the main immune and inhibition cell from 31.8%and 10.3% to 42.9% and 14.5%(p<0.05). At the same time the quantity of CD8+T cell, the other component in CD3+cells, has got a rise but not obviously. CD4+/CD8+, the sign of immune level for body, rose also which approved that the bodies have got a higher immune response. The quantity of the CD19+cells, which was thought as the maturity B cell, also got a rise obviously from 12.3%to 18.6%, that result suggest that the immune response in the mice were induced by the body fluid and the cell immune response together. Conclusion: In this research we prepared the HpWCP-CAMS which diameter is 3.58 μm. It not only has the target characteristics to Peyer's patch but also has anti-acid and control release characteristics. The lyophilized MS has good stability, and the test results of toxicity also satisfactory. The immune effect of HpWCP-CAMS approved better results than other agents, the lower immune dose, fewer immune times and simpler immune procedure could also induce the better immune results. The immune protection effect could achieved 89.4% at least immunized with the MS. in the test of immunoprotecive mechanism, the results approved a rose obviously that not only the IgG-ASC and IgA-ASC in spleens and Peyer's patches but also the mRNA IL-4 and IFN-γin lymphocytes, the quantity of lymphocytes subgroups rose also.