Preparation of Nanoparticulate Alum Adjuvant and Study on the Immune Effect of Adsorbing HBsAg
|School||Third Military Medical University|
|Keywords||Nano - aluminum adjuvant Conventional aluminum adjuvant Hepatitis B virus surface antigen Antibody titers Cellular immunity|
The vaccine adjuvant to selectively stimulate the immune response and improve the immune effect. Aluminum salts, nearly 80 years of history as a vaccine adjuvant. Aluminum adjuvants of commercial vaccines include partial aluminum hydroxide the (crystal type AlOOH, i.e. commonly referred to aluminum hydroxide) and hydrogen phosphate of alumina (Al (OH) x (PO4) y, commonly referred to as aluminum phosphate ). Aluminum hydroxide adjuvant due to the high adsorption capacity of some antigens having good adsorption and superior to the aluminum phosphate adjuvant, it is the only by the U.S. FDA approved for human use vaccine adjuvant, and its isoelectric point iep (isoelectric point, iep) = 11.4, and with a positive charge in the body pH = 7.4 between interstitial fluid. Although aluminum hydroxide adjuvant application has a long history, but its exact mechanism is unknown, the repository effect \Aluminum adjuvant adsorbed antigen, the formation of antigen in the inoculated area repository, on the one hand, at these strongly attract dendritic cells (DC), macrophages (Mφ) and other antigen presenting cells (APC) identification and endocytosis: Another On the one hand, the injection of local slow release of antigen to extend the time of the APC and T lymphocyte interactions, thereby enhancing the antibody response, enhanced humoral immunity. Aluminum hydroxide adjuvant adsorbed antigens, mainly depends on the physical chemical characteristics of the antigen, the other adsorption conditions, including pH, temperature, colloidal particle size, and the reaction mixture ionic strength, etc., the influence of aluminum hydroxide adsorption. In the preparation process, these conditions are often ignored and rarely clarify, thus affecting the best effect of the adjuvant play. The micelle diameter concerned affecting the adsorption capacity of the study showed that: with respect to the larger particles, the smaller particles have a larger surface area, which can adsorb more antigens. For example, the aluminum hydroxide adjuvant adsorption amount of the diphtheria toxin, is inversely proportional to its diameter. We choose the HBsAg antigen as aluminum adjuvant adsorbed mode, the main consideration is as follows: HBsAg is constituted by a protein, sugar, and lipid complex macromolecular aggregates, and its molecular weight is 3500kDa, lipids phospholipid accounts for about 70%: of HBsAg adsorption with aluminum hydroxide than by hydrophobic interaction, charge attraction, but mainly of HBsAg phosphate and aluminum hydroxide adjuvant hydroxide, the exchange between the two ligands, provides the strongest adsorption mechanism; little change in vivo and in vitro adsorption, desorption by its tiny. Therefore, when the aluminum hydroxide adjuvant nanoscale particles adsorbed HBsAg, mainly related to the specific surface area increases sharply. Also, because the nanoparticles have a high surface reactivity, surface active centers adsorption ability of these excellent properties, which the adsorption degree greater impact adsorbs more of HBsAg. The nanoparticles have a close relationship with the organism, the and Nami Zuo agent to avoid the occurrence of the carrier effect, phagocytic macrophages, dendritic cells preferred target. It is based on nanoparticles that is different from the conventional particle characteristics, the Third Military Medical University, a master's degree thesis w / O microemulsion preparation of nano-aluminum adjuvant, and preliminary evaluation of the biological behavior of adjuvant HBsAg vaccine adjuvant. First, we investigated the maximum saturated water solution of a cationic surfactant (benzalkonium Australia money) and cosurfactant (n-octanol), i.e. both the top complex with a mass ratio of l: l, is slightly larger than the theoretical value of 5 8 with the theoretical value more consistent; then discussion the best complex ratio of the mixed surfactant and oil (cyclohexane) and Alcl3 solution to form a microemulsion phase behavior and pseudoternary phase diagram is drawn W / O type microemulsion pseudoternary phase diagram at the bottom. Constant temperature in the region the proportion of each component is the preparation of nano-aluminum adjuvants microemulsion formulation. Under the to ensure product for nanoscale premise, while considering the production costs, when the oil phase is constant, the water solution the more the yield more suitable as the preparation of the reaction phase of the W / 0 type microemulsion formulations, in this condition, it is determined prepared nano-aluminum adjuvant. The photos of the product of transmission electron microscopy (TEM) analysis: the particles are spherical particles of the average particle diameter of 72.62nm, the shape more regular and uniform distribution. X-ray diffraction (XRD) analysis further, the peak top XRD pattern characteristic value is mainly 4.7262,4.3710,3.2019,2.2213,1.7215,1.4577,1.3317. With JCPDS card control, to determine the product prepared by the present method for the AI ??(OH) 3. Differential calorimetry scan (DSC) analysis of the phase transition temperature, compared to conventional Al (O Qi 3 particles transition temperature greater advance, is mainly the sake of smaller particle size, indirect proof of the product was prepared as a nanometer. In order to study the biological effects of nano-aluminum adjuvant, we are not adjuvanted HBsAg antigen, ELisA difficult position given the adsorption capacity of the of nano aluminum adjuvant vaccine former supernatant OD45. values ??about conventional aluminum adjuvants 10 times from the immunological point of view, the most concerned about is whether we can enhance the immune response to viral antigens, and the level of protection against viruses nano-aluminum hydroxide adjuvant. further study nano aluminum adjuvant physical adsorption HBsAg The animal immune effects. of lmg of aluminum adjuvants adsorption IOpgHBsAg the standard, the preparation of a new type of hepatitis B vaccine for the control of conventional aluminum adjuvant, subcutaneous injection in guinea pigs, ELISA assay serum antibody levels to determine the humoral immune response. nano aluminum adjuvant in one week after the first immunization and two weeks can produce anti-HBs antibody GMT respectively 22.974 ± 1.3612 and 26.389 disabilities 1.4214, while conventional aluminum adjuvant group 2 Guests 0.073 and 13.195 ± 1.1204 Tip: Nano earlier stimulation of aluminum adjuvants can produce anti-HBs antibodies. mechanism may be after the first immunization burst-release effect the pseudo urst released) due; but its role is limited, and immunostimulatory effects subsequent The mechanism of action, both in the role time precedence relationship. May be precisely because of the explosive release of the reason, the nano-aluminum adjuvant effect rapid effects disappeared, and immunostimulatory effects are not immediately fully onset, leading to the observed in this study, the first three weeks of antibody titers declined slightly, while the the rest of the time the same as the antibody titer. Balb / c mice with bilateral inguinal subcutaneous injection, after l or third immunization, the determination of the indicators of the cellular immune response, including: 3H a TdR incorporation determination of lymphocyte proliferation ability, either subcutaneously sequence of the immune process, Third Military Medical University, a master's degree thesis, subcutaneous and intraperitoneal injection three times immunization program, the stimulating effect of the of nano aluminum adjuvant group of mouse spleen lymphocytes, are stronger than conventional aluminum adjuvant group, but both There was no significant difference. With previous sub?