Study On The Immune Adjuvant Effect Of Nano/Macro Particles-based Vaccine Delivery System

The adjuvant effect of nano materilals based vaccine delivery system play an important role in the immune agument of antigen and immune ehancer. Nano/macro-particles based vaccine delivery system have multi-faceted advantages over traditional vaccine vector, including encapsulation of antigens in particles to prevent antigen degradation and increase their stability, co-encapsulation of antigen and immunostimulatory agent in particles to enhance immunogenicity and potency of vaccines, easy phagocytes and process by APCs and so on. This research focuses on the adjuvant effect and immune mechanism of vaccine delivery system based on polymer materials.This thesis consists of three parts and the main contants are described as follows: First, study on the feasibility and immune response mechanism of vaccines of PLGA nano/microparticles encapsulated with pertussis toxid (PTd). The resurged pertussis has high morbidity and mortality in the United States, Australia, Britain and other countries which has point out the urgent to develop new effective and preventive pertussis vaccine formulation to prevent its spread. Herein,We made a PLGA nano/microparticles encapsulated with protective antigen pertussis detoxin (PTd NP/MP.) using water/oil/water solvent evaporation method. Notably, PLGA nano/microparticles enchanced the phagocytosis of antigen PTd by J774.2 murine monocyte/macrophage cells compared to free antigen and in vivo immunization demonestrated significant increased the secretion of cytokine IFN-? and IL-17 by splenic lymphocytes, and presentation of pertussis toxid mediated by particles showed similar antigen-specific IgG response compared to soluble antigen. Thus,the PTd NP/MP formulation may provide an alternative to conventional acellular vaccines to provide a more balanced Thl/Th17 immune response in the treatment of pertussis.Second, study on the feasibility of guanidinylated pH-sensitive nanoparticles as protein antigen delivery systems and adjuvants for promoting antigen-specific immune responses in vivo. Weak immunogenicity and transient immune responses are the two major limitions for protein vaccines. The well-designed vaccine delivery vector can significantly enhance the immune efficiency of vaccines. In this study, a type of pH-sensitive and cationic nanoparticle adjuvant based on monomethoxypoly (ethylene glycol)-block-poly (2-(diisopro-pylamino) ethylmethacrylate)-block-poly (2-(guanidyl) ethyl methacrylate) (mPEG-b-PDPA-b-PGEM, PEDG) copolymers was prepared. PEDG could self-assemble into nanoparticles in water and encapsulated the model antigen ovalbumin (OVA) to develop the protein vaccine of Nano.OVA by facile electrostatic absorption. The lysosome escape of antigen in acidic lysosomal compartment of antigen-presenting cells was observed with the nanoparticle disassembly due to the protonation of the hydrophobic PDPA segments. Nanoparticles stimulated the maturation of bone marrow-derived dendritic cells and enhanced antigen uptake and presentation compared to free OVA, meanwhile the expression of CD40, CD86, CCR7 was highly increased. The nanoparticles also can promote the secretion of IL12, which play a crucial role in the play of BMDCs function. The nanoparticles could also induce the activation of macrophage (RAW 264.7) to produce high level of cytokines TNF-a, IL-6 and IL-10. OVA-loaded PEDG nanoparticles efficiently induced superior antigen cross-presentation effect in vitro and in vivo compared to free OVA vaccination. The strategy of nanoparticle delivery prolonged the antigen duration at the injection site and enhanced its migration to draining lymph nodes as indicated by the fluorescence tracking which provide plenty of time for recognization and process of antigen by DCs. Nano.OVA elicited a much higher antigen specific antibody titre by 4 times compared with free OVA, causing a strong Thl porlatization hummoral immune response after intradermal immunization. The significant improvement of IFN-? and IL-2 cytokine secretion showed that PEDG induced a higher cellular immune response in vivo. Taken together, these data suggested that the guanidinylated acid-sensitive nanoparticles could act as an effective adjuvant and delivery system for protein antigens to elicit both strong antigen-specific cellular immune response and Th1-dominate adaptive immune response.Third, the study of immune adjuvant effect of cationic nanoparticles as vaccine delivery systems. The effect of surface charges on nanoparticles on the exertion of immune adjuvant effect was studied by adjusting the number of cationic groups of poly (ethylene glycol)-b-poly(?-caprolactone)-g-poly(2-(guanidyl)ethylmethacrylate)(PEG-PCL) copo-lymes. There was no significant difference between different nanoparticles in the uptake by BMDCs, and the main endocytosis pathway of the three nanoparticles was caveolae-mediated endocytosis. The degree of maturation of BMDCs was promoted with the increase of guanidine groups, which was significant difference between the three nanoparticles. More guanidine groups on nanoparticles can also enhanced the antigen cross presentation in vitro after encapsulating model antigen OVA (loading efficiency =50%), and the level of cross presentation was increased by 1.8 and 4 times for PECG-1, PECG-2 nanoparticles, compared with uncharged PEC nanoparticles. The incresement of cationic groups of nanoparticle prolonged the antigen duration at the injection site and enhanced its migration to draining lymph nodes as demonstrated by the fluorescence tracking, and elicited higher level of IFN-y, TNF-a, IL-10 secretion. Therefore, rational design of surface cationic groups of nanoparticles may be a promising strategy to ehance the immune adjuvant effect of nanoparticles.