|
As of June 2, 2009, the highly pathogenic H5N1 avian influenza has spread to 15 countries and regions, the cumulative infections 433 people, 262 people died, a mortality rate of up to 60%. 38 cases in China, the confirmed cases, 25 people died, a mortality rate of about 65.8%. In addition, along with changes in the geographical distribution of poultry and wild birds, the highly pathogenic H5N1 avian influenza virus has spread in parts of Asia and the Middle East, Europe, North America, Africa, the genotype and antigenicity changing the mammalian host The range is also expanding. The visible human avian influenza prevention and control of the situation has been unusually severe. To this end, WHO proposal to accelerate the research and development process of human avian influenza vaccine, do a good job of technical reserves. The most effective means of prevention and control of highly pathogenic H5N1 avian influenza vaccine. Highly pathogenic H5N1 avian influenza vaccine inactivated whole virus vaccine, cracking vaccine, attenuated vaccine research. Has prepared pandemic mode vaccine is inactivated whole virus and lysis of virus vaccine, experiments to prove its safety, effective and stable. Such vaccines, however, mostly were immunized, the vaccine can induce relatively high titers of specific anti-virus serum neutralizing antibodies, but poor immune protective effect for other subtypes of the virus, and in special populations (elderly and children) immune effects are not very satisfied with the vaccination cumbersome, is not conducive to the mass vaccination of the population, these challenges its vaccine. The recent development of transdermal immunization undoubtedly provides a new approach to the solution of the above problem. Transdermal the immune immune pathway as a needle-free injection has a great advantage in the prevention of influenza pandemic: the vaccination rate increase vaccination safety and compliance, no professionals. In addition, the effectiveness of transdermal immunization has been confirmed, but its mechanism is not very clear, and was asked the following theory: because of the presence of a large number of antigen-presenting cells, especially Langerhans cells in the skin, the uptake and processing of antigen, presented to the T and B cells, to stimulate the systemic and mucosal immune responses. Langerhans cells accounted for only 2% of the total number of human skin cells, but its surface area accounts for about 25% of the total area of ??the skin. The density of Langerhans cells and antigen-presenting function undoubtedly provide the ideal target for vaccine transdermal delivery. In this study the WHO recommended H5N1 the vaccine strains -----A/Vietnam/H5N1-PR8/CDC-RG prepared inactivated whole virus vaccine antigen to cholera toxin (CT), cholera toxin B sub-unit (CTB) E. coli heat-labile the intestinal toxins (LT), MF59, CpG1826 CpG2006 adjuvant, ethanol, propylene glycol, dimethyl sulfoxide, retinoic acid, oleic acid as penetration enhancers, transdermal immune Balb / c mice, to evaluate the safety, efficacy, which screened effective transdermal immunoadjuvant and penetration agents, and the effect of the transdermal vaccine immune response were observed. In addition, in view of the E. coli heat-labile enterotoxin, difficult to obtain, this study also its preparation exploration. The study is divided into three parts: 1. The transdermal vaccine adjuvant initial screening of the highly pathogenic H5N1 avian influenza: optional A/Vietnam/H5N1-PR8/CDC-RG inactivated whole virus vaccine antigen design two experiments: (1) Screening of adjuvants: CT, CTB, LT, MF59, CpG1826, CpG2006 as adjuvant, with 50μg antigen sufficiently mixed to adjust the concentration of antigen to 1.0μg/μl, using oleic acid as a penetration agent, transdermal immune Balb / c mice, serum IgG subtype antibody and its antibody titer of blood suppression, lung lavage fluid, nasal lavage fluid, vaginal lavage IgG, sIgA antibodies titer, which preliminary screened two effective transdermal immunoadjuvant -------- CT, LT. (2) rLT Preparation and biological activity of verification: pull to the genome of strains of E. coli E24377A mode to construct the expression vector pET28a-LTA after pET28a-LTB and pET22b-LTB, of the protein induced the expression and purification of proportion of 1:5 (n / n) LTA and LTB mixing, ultimately obtained rLT holotoxin, its molecular weight by SDS-PAGE, WB, GM-ELISA, CHO cell toxicity test, and by MALDI-TOF-MS methods such and activity was verified also been verified by animal experiments of its adjuvant activity, the results indicate that rLT prepared in this manner not only have better toxicity, and transdermal immune response is preferably adjuvants, and (1 ) used in the LT compared adjuvant effect is considerable. The highly pathogenic H5N1 avian influenza transdermal vaccine penetration enhancers initial screening: still choose A/Vietnam/H5N1-PR8/CDC-RG inactivated whole virus vaccine antigen, the choice of the 10μg rLT as adjuvants, respectively, to ethanol , propylene glycol, dimethyl sulfoxide (DMSO), retinoic acid, oleic acid as a penetration agent, transdermal immune Balb / c mice, from the safety and efficacy of these two aspects of these five kinds of penetration enhancers were preliminary evaluation and initial screening of the three security effective penetration enhancers -------- oleic acid, DMSO and retinoic acid. In addition, the pathological changes of the skin before and after the penetration enhancers processing observed experimental results show that the large differences different penetration enhancers on the degree of disorder of the lamina propria, oleic acid, DMSO, retinoic acid to remove the effect of the stratum corneum compared better. DMSO has a strong pungent odor but difficult to promote the practical application, and therefore preferred oleic acid and retinoic acid as a highly pathogenic H5N1 avian influenza transdermal penetration enhancers vaccine. Preliminary observation of the effects of highly pathogenic H5N1 avian influenza transdermal vaccine immune response: selection of oleic acid as a penetration enhancer, carried out to explore the dose-effect relationship between the antigen and adjuvant, and transdermal immune response effect preliminary observation. To different doses of the antigen-5μg, 10μg, 25μg, 50μg, 100μg with different doses of rLT 0μg 1μg, 5μg, 10μg, 25μg, 50μg, 100μg mixed adsorption, the choice of oleic acid as penetration enhancers, transdermal immunization of Balb / c mice, by the detection of serum IgG antibodies in mice before and after immunization ISOTYPE hemagglutination inhibition titer, lung lavage fluid, nasal lavage fluid, vaginal lavage IgG, sIgA antibody titer, which initially determine the best ratio of antigen dose and adjuvant doses of antigen (HA content) 50μg rLT10μg. And the group transdermal immune response effect with PBS group, single antigen group, the injection group, and intranasal group compared found that transdermal immunization group of serum IgG antibody titer and blood suppression titer was significantly higher than PBS group, single-antigen group, but lower than the injection group and intranasal group; mucosal immune response intensity and intranasal group was not statistically significant compared; the ELISPOT experimental results also show that the highly pathogenic H5N1 avian influenza transdermal vaccine induced CD4 T-cell immune response to Th2-type cellular immune response based. Thus, the highly pathogenic H5N1 avian influenza transdermal vaccine is not only capable of inducing a strong humoral immune response, but also capable of inducing mucosal immune responses and cellular immune responses. Conclusion: In this study, adjuvant, penetration enhancers, antigen, immune response of these four aspects of highly pathogenic H5N1 avian influenza transdermal vaccine research. The findings, in a mouse model of highly pathogenic H5N1 avian influenza vaccine transdermal immunization, initial screening and determine transdermal immune adjuvant CT, LT, penetration enhancer is oleic acid, retinoic acid, antigen dose The optimum ratio of the the adjuvant rLT dose antigen able to induce a better immune response effect 50μg, rLT10μg immune after three. In addition, the successful expression of Escherichia coli heat-labile enterotoxin, and proved by experiments with strong toxicity and better adjuvant effect. In addition, the results also showed that transdermal immunization can induce better humoral immune response, but also able to induce mucosal immune responses and cellular immune responses, which laid a good foundation for transdermal vaccine for the highly pathogenic H5N1 avian influenza , but also for the other transdermal vaccine research ideas.
|