Table_2_Chitosan Microsphere Used as an Effective System to Deliver a Linked Antigenic Peptides Vaccine Protect Mice Against Acute and Chronic Toxoplasmosis.DOC

Multiple antigenic peptide (MAP) vaccines have advantages over traditional Toxoplasma gondii vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both in vitro, via cell culture, and through in vivo mouse immunization. In vitro, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). In vivo, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against T. gondii.

多抗原肽（Multiple Antigenic Peptide，MAP）疫苗相较于传统刚地弓形虫（Toxoplasma gondii）疫苗具备显著优势，但更易受到酶解降解。壳聚糖微球（chitosan microspheres，CS）作为一种高效递送系统，可克服这一短板，同时作为天然佐剂，能够促进辅助性T细胞1（T helper 1，Th1）型细胞免疫应答。本研究采用壳聚糖微粒递送来自GRA10蛋白的多抗原表位（G10E），该表位包含3个优势表位。当G10E被包埋于壳聚糖微粒中形成G10E-CS时，足够引发免疫应答的肽段被负载于微球核心，且该复合物可稳定释放G10E肽段。研究通过体外细胞培养实验与体内小鼠免疫实验，对G10E-CS的免疫效能进行了检测。体外实验中，G10E-CS通过上调共刺激分子（CD40与CD86）的分泌水平，激活树突状细胞（Dendritic Cells，DC）与T淋巴细胞。体内实验中，经G10E-CS免疫的小鼠可激活偏向Th1型的细胞免疫与体液免疫应答，伴随IFN-γ、IL-2及IgG的分泌量显著升高，同时IL-4、IL-10及IgG1的水平显著降低。经G10E-CS免疫可发挥显著保护作用：在急性弓形虫感染小鼠模型中显著延长小鼠存活时间，在慢性弓形虫感染小鼠模型中显著降低囊孢负荷量。本研究结果表明，以壳聚糖微球作为高效递送系统来递送串联抗原肽段，是开发高效抗刚地弓形虫疫苗的极具前景的策略。