Encapsulation Of HPV L1Capsomere In Microspheres And Study Of Immune Response As Vaccine Adjuvants

Infection by certain human papillomavirus(HPV) is the major cause of benigngenital warts, cervical cancer, etc. To develop a effective vaccine against HPVinfection is a available strategy. Compared with HPV L1VLP-based vaccines onmarket, recombinant HPV L1pentamers expressed in E. coli represents a promisingand potentially cost-effective vaccine based on capsomere for preventing HPV relatedcervical cancer and genital warts. Drug delivery systems like biodegradable liposomes,polymer carriers have been widely exploited for antigen delivery and adjuvant systemin recent years. In this study uniform-sized polylactic-co-glycolic acid (PLGA)microspheres and lecithin-based PLGA microspheres were employed to evaluate theirbiological response on aspect of vaccine application.In detail, this thesis mainly included the following three issues:1. In this report uniform-sized PLGA microspheres prepared by membraneemulsification technique combined with solvent extraction method were employedas HPV vaccine delivery and adjuvant system. The effects of internal water phasepH value, the concentration of buffer solution in internal water phase, initialemulsions prepared methods, and the concentration of antigen on the proteinencapsulation efficiency and drug-loading effciency were studied. On this basis,the PLGA microspheres of different size (430nm vs.1.38μm) prepared bymembrane emulsification technique were successfully prepared. The resultsshowed the HPV L1pentamers-loaded PLGA microspheres was round andsmooth, the loading capacity and the encapsulation efficiency were66.56μg/mg,47.335μg/mg and73.78%,65.69%respectively.2. The PLGA microspheres of different size (430nm vs.1.38μm) prepared bymembrane emulsification technique were employed as HPV vaccine delivery andadjuvant system. The effect of particle size (430nm vs.1.38μm) on enhancingHPV L1specific immune responses was investigated. The result showed that HPVL1pentamers encapsulated in PLGA microspheres could elicit similar level ofIgG binding antibodies and neutralizing antibodies with alum-antigen groups, andsignificantly higher level of cellular immune responses than alum-antigen groups.In addition, The results showed that HPV L1pentamers encapsulated inlarge-sized PLGA (1.38μm) microspheres could elicit significantly higher levelof IgG binding antibodies(2.3times) and neutralizing antibodies(3.8times) than counterparts with430nm (P<0.05or P<0.01).The similar trends was also found incytokine secretion levels; and higher levels of IL-6(4.7times)and IL-4(1.5times)were observed in mice immunized with large-sized PLGA microspheres (1.38μm)encapsulated HPV L1pentamers. While small-sized PLGA microspheres (430nm)encapsulated HPV L1pentamers elicited significantly higher IFN-γ secretionlevels(2.1times) than large-sized PLGA microspheres (1.38μm). Taken together,we concluded that small-sized PLGA microspheres can enhance antigen specificcellular immune responses, which is suitable for therapeutic vaccine, whilelarge-sized PLGA microspheres can elicit higher level of humoral immuneresponses, which is favorable for prophylactic HPV vaccine.3. The lecithin-based PLGA microspheres encapsulating HPV L1pentamersprepared by membrane emulsification technique were employed as HPV vaccinedelivery and adjuvant system. The result showed that it could elicit higher level ofneutralizing antibodies than alum-antigen groups (P<0.05), and significantlyhigher level of IgG binding antibodies and neutralizing antibodies thanalum-antigen groups (P<0.01). In addition, the HPV L1pentamers-loadedlecithin-based PLGA microspheres could elicit higher IFN-γ secretion levels thanalum-antigen groups (P<0.05), but lower than HPV L1pentamers-loaded PLGAmicrospheres; It simultaneously could elicit higher level of significantly higherlevels of IL-6(P<0.05), and similar level of IL-4with alum-antigen groups, andhigher levels of IL-6and IL-4than HPV L1pentamers-loaded PLGAmicrospheres (P<0.05). We concluded that HPV L1pentamers-loadedlecithin-based PLGA microspheres was a promising vaccine delivery and adjuvantsystem.It could elicit notably higher level of IgG binding antibodies andneutralizing antibodies than alum-antigen groups and PLGA-antigen groups, andsignificantly higher level of cellular immune responses than alum-antigen groups.