Preparation Of PLA-based Uniformly Sized Microspheres And Its Application For New Generation Of Hepatitis B Vaccine

Hepatitis B is an infectious disease caused by hepatitis B virus. Chronic infection caused by hepatitis B virus can lead to cirrhosis and hepatic carcinoma which is a serious threat to human health. Currently there is still no effective anti-virus drug to hepatitis B. Vaccination remains the most effective method for the prevention of hepatitis B infection. The safety of recombinant hepatitis B vaccine has been greatly improved. While the immunogenicity of recombinant antigens decreased significantly which rely on the addition of adjuvant to induce an effective immune response. Aluminum salt is still the most widely used vaccine adjuvant for humans. Though it may enhance the antibody response, the cellular immune response generated by alum-based vaccine is limited, which play the critical role during the clearance of intracellular infectious pathogens such as HBV. Thus the development of hepatitis B vaccine and therapeutic vaccine rely on the development of novel adjuvants that can enhance cellular immune responses.Nano-microparticles-based adjuvants have obtained special attention due to their pathogen like diameter to activate the immune system, making microparticles the most promising antigen delivery system. Biodegradable polymer polylactic acid (PLA) microsphere is one of the most valuable particles adjuvant. Microparticles-antigen system can promote antigen uptake by antigen presenting cell and cross-presentation via MHCI way, inducing potent cellular and humoral immune responses. Preparation of traditional micro-embedded microparticles vaccine formuations always involved vigorously conditions such as oil-water interface, ultrasound, mechanical stirring and so on, easy to cause antigen inactivation and degeneration. Microparticles prepared by these methods always showed wide size distribution ultimately affect the pharmacodynamics in vivo. So many factors that affect the the embedding efficiency and antigen activity which greatly restrict the amplification producing process. Thus the desigen of novel adjuvant need the balance of producibility and highly efficient.This research focus on the applicability and improvement of PLA-based microspheres in new generation of hepatitis B vaccine. On the basis of early studies, uniformly sized microparticles were produced by membrane emulsification techniques developed by our lab. Antigen adsorption by microparticles was applied so as to avoid vigorously conditions.In chapter Ⅱ we demonstrated potent IgG response was induced by simply mixing HBsAg with PLA microparticles which is comparable with alum-adjuvanted vaccine. Also microparticles adjuvant induced effective T cell response as showned by enhanced secretion of related cytokines.While analysis indicated that antigen adsorption efficiency in HBsAg and PLA particles formulation is less than 5%. In order to improve the antigen adsorption by microparticles, we desigen and prepared the calcium phosphate (CaP) surface modified CaP-PLA particles in chapter Ⅲ. HBsAg adsorption capacity of CaP-PLA particles increased to 65%. Immunization results confirmed that CaP-PLA particles adsorbed with HBsAg enhanced antigen uptake and dendritic cells activation, generating a higher level of antigen-specific immune responses.CaP-PLA microparticles were neutral charged and became negatively charged after antigen adsorbtion which was unfavorable for the interaction between microparticles and antigen-presenting cells. In order to systematically investigate the relationship among microparticles antigen adsorption, surface charge and related immune responses, in chapter Ⅳ we prepared different positively charged cationic PLA microparticles by surface coating modification of PLA particles with cationic polymer chitosan (CS), chitosan hydrochloride (CSC) and polyethyleneimine (PEI) respectively. Antigen adsorption capacity increased with the increasing positive charge of modified microparticles. In macrophages, with the increasing of microparticles positive charge and antigen adsorption, cell surface costimulatory molecules expression and activation related cytokines release were both enhanced. After immunization, cationic microsparticles-based vaccine formulations generated a rapid and efficient humoral immune response and cellular immunity related cytokine release which correlate with antigen adsorption. Specifically cationic microparticles with increased antigen adsorption induced significantly high level Thl cytokines IFN-γ and IL-12. These results strongly suggested that increasing antigen adsorption by cationic PLA microspheres is efficient to augment the adjuvant performance of PLA microparticles especially the stimulation of the Thl type cellular immune response.In order to better performance the adjuvant potential of cationic PLA microparticles, in chapter Ⅴ we compared the type and strength of immune responses induced by cationic PLA microsphere or alum adjuvanted formulations after administration via subcutaneous, intramuscular and intraperitoneal routes respectively. Results confirmed that cationic microparticles adjuvanted vaccine could induce potent protective immune responses via intramuscular administration than alum-adjuvanted vaccine especially the cellular immune response.Taking the above factors into consideration, in chapter Ⅵ we prepared the safer cationic lipid-PLA complexed DDAB-PLA microparticles. DDAB-PLA particles showed the positive charge about+30 mV with an antigen adsorption efficiency more than 75%. Intramuscular immunization demonstrated that antigen adsorbed DDAB-PLA microparticles can induce significantly higher IgG level (1.3 times) than alum adjuvanted formulation. Also remarkable cellular immune response related cytokines secretion and effector T cells activation were generated by DDAB-PLA adjuvanted vaccine which reflects the excellent adjuvant capacity of these particles. Over all, superior adjuvant performance, ease to preparation and amplification make DDAB-PLA microparticles the most promising adjuvant for the new generation of hepatitis B vaccine and therapeutic vaccines.