Table_2_Molecular and Antigenic Properties of Mammalian Cell-Expressed Theileria parva Antigen Tp9.pdf

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, is a leading cause of morbidity and mortality in cattle of sub-Saharan Africa. The infection and treatment method (ITM) is currently the only vaccine available to control T. parva. Although ITM elicits levels of protection, its widespread adoption is limited by costs, laborious production process, and antibiotic co-treatment requirement, necessitating the development of a more sustainable vaccine. To this end, efforts have been concentrated in the identification of new T. parva vaccine antigens and in the development of suitable platforms for antigen expression. In this study, we investigated the molecular and antigenic properties of T. parva antigen Tp9 expressed by mammalian cells. Data indicate that Tp9 contains a signal peptide that is weakly functional in mammalian cells. Thus, Tp9 secretion from mammalian cells increased 10-fold after the native signal peptide was replaced with the human tissue plasminogen activator signal peptide (tPA). Sera from all T. parva-immune cattle recognized this recombinant, secreted Tp9. Additionally, PBMC from ITM-immunized cattle produced significant (p < 0.05) amounts of IFNγ following ex vivo exposure to Tp9, but this response varied between cattle of different MHC class I and class II genotypes. In addition, depletion experiments demonstrated that IFNγ to Tp9 was primarily produced by CD4+ T cells. Molecular analysis demonstrated that Tp9 presents a signal peptide that is weakly functional in mammalian cells, suggesting that it remains within lymphocytes during infection. Tp9 secretion from mammalian cells was substantially increased when the tPA secretion signal sequence was substituted for the native secretion signal sequence. Using full-length, recombinant Tp9 secreted from mammalian cells, we demonstrated that T. parva-immune cattle develop both humoral and cellular immune responses to this antigen. Collectively, these results provide rationale for further evaluation of Tp9 as a component of a T. parva subunit vaccine.

东海岸热（East Coast Fever, ECF）由蜱传顶复门寄生虫小泰勒虫（Theileria parva）引发，是撒哈拉以南非洲地区牛只发病与死亡的首要诱因。目前，感染与治疗法（Infection and Treatment Method, ITM）是唯一可用于防控T. parva的疫苗手段。尽管ITM可诱导一定水平的保护力，但其广泛应用受到成本高昂、生产流程繁琐以及需联合抗生素进行辅助治疗等因素的限制，因此亟需开发更为可持续的疫苗。
为此，相关研究工作重点集中于筛选新型T. parva疫苗抗原，以及搭建适配的抗原表达平台。本研究针对哺乳动物细胞表达的T. parva抗原Tp9的分子与抗原特性展开了系统探究。实验数据表明，Tp9携带一段在哺乳动物细胞中功能较弱的信号肽。将其天然信号肽替换为人类组织型纤溶酶原激活物信号肽（tissue plasminogen activator signal peptide, tPA）后，哺乳动物细胞分泌Tp9的水平提升了10倍。
所有感染T. parva后获得免疫的牛只血清均可识别该重组分泌型Tp9。此外，经ITM免疫的牛只外周血单个核细胞（peripheral blood mononuclear cell, PBMC）在体外暴露于Tp9后，可产生显著（p < 0.05）的干扰素γ（interferon gamma, IFNγ），但该免疫应答因牛只的主要组织相容性复合体I类（major histocompatibility complex class I, MHC I）与II类（major histocompatibility complex class II, MHC II）基因型不同而存在差异。同时，细胞耗竭实验证实，针对Tp9的IFNγ主要由CD4阳性T细胞产生。
分子分析结果显示，Tp9携带的信号肽在哺乳动物细胞中功能较弱，提示其在感染过程中仍留存于淋巴细胞内。将Tp9的天然分泌信号肽替换为tPA分泌信号序列后，哺乳动物细胞对Tp9的分泌水平得到显著提升。利用哺乳动物细胞分泌的全长重组Tp9，我们证实感染T. parva后免疫的牛只可针对该抗原同时产生体液免疫与细胞免疫应答。
综上，本研究结果为将Tp9作为T. parva亚单位疫苗（subunit vaccine）的组分之一进行进一步评估提供了坚实的理论依据。