Processed endpoint and timepoint enzyme-linked immunosorbent assay, and flow cytometry data

Ticks are ectoparasites that impact livestock and transmit pathogens that cause tick-borne diseases that severely affect livestock industries. Consequently, the control of ticks and their tick-borne diseases is imperative. However, the current chemical tick control paradigm is marred by the increasing development of acaricide resistance and necessitates supplementary control strategies, such as vaccines. Subolesin is a promising tick vaccine candidate, however the mechanism through which this antigen elicits protection is still unknown. Consequently, the expression and diversity of Subolesin (and its many variants) in Rhipicephalus microplus ticks from different tissues (ovary, gut and gland), life stages (larvae, nymph and adult) and several South African locations (Mpumalanga and a pathogen-free laboratory-strain) were elucidated for the first time on a RNA (ribonucleic acid) sequencing level. This study elucidates for the first time the gene structure of Subolesin in R. microplus. Furthermore, it was found that Subolesin has four potential mRNA (messenger ribonucleic acid) variants which could translate into three protein variants. Thus far, this finding is exclusive to R. microplus, as Subolesin in other tick species have been reported as a single mRNA and protein. Additionally, vaccines simultaneously targeting both the tick vector and pathogen with a single formulation is another ambitious goal, given the challenges with traditional subunit vaccines. Consequently, this study evaluated the immune profile elicited by vaccination with antigen coated Poly D-L-lactide-co-glycolide acid (PLGA) microparticles simultaneously targeting both the tick vector (Subolesin) and the tick-borne pathogen Anaplasma phagocytophilum (MSP4) in a murine model. Sera results indicated that Subolesin antigens (soluble and adsorbed onto particles) were not immunogenic after ELISA (enzyme linked immunosorbent assay) analysis and were consequently excluded from further analysis. Nevertheless, ELISA results demonstrated that all MSP4-receiving groups elicited a Th2 biased response in sera. Furthermore, this study offers the first insights into the T and B cell profile elicited by immunization with MSP4 in any animal model. Therefore, this study offers valuable insights into the design and application of future PLGA microparticle based vaccines for the control of ticks and A. phagocytophilum.蜱是一类寄生于宿主体表的外寄生虫，可对家畜造成危害，并传播引发蜱传疾病的病原体，这类疾病会严重冲击畜牧产业。因此，对蜱及其传播的蜱传疾病进行防控至关重要。然而，当前主流的化学蜱防控策略正面临日益严重的杀螨剂抗性问题，亟需疫苗等补充防控手段。Subolesin是一种极具潜力的蜱疫苗候选抗原，但其介导宿主免疫保护的具体机制目前仍不明确。为此，本研究首次基于RNA（核糖核酸）测序技术，解析了来自南非不同地区（姆普马兰加省及无病原体实验室品系）、不同组织（卵巢、肠道和唾液腺）、不同发育阶段（幼虫、若虫和成虫）的微小牛蜱（Rhipicephalus microplus）体内Subolesin及其多种变异体的表达与多样性特征。本研究首次明确了微小牛蜱体内Subolesin的基因结构。此外，研究发现Subolesin存在四种潜在的mRNA（信使核糖核酸）变异体，可编码产生三种蛋白质变异体。截至目前，该发现仅见于微小牛蜱，已有研究报道其他蜱种的Subolesin仅存在单一种类的mRNA与蛋白质变异体。此外，鉴于传统亚单位疫苗存在的诸多挑战，开发单一制剂同时靶向蜱媒及其传播病原体的疫苗是另一项极具挑战性的目标。为此，本研究在小鼠模型中评估了以抗原包被的聚DL-乳酸-羟基乙酸共聚物（Poly D-L-lactide-co-glycolide acid，PLGA）微粒作为疫苗载体，同时靶向蜱媒（Subolesin）与蜱传病原体嗜吞噬细胞无形体（Anaplasma phagocytophilum，MSP4）所引发的免疫应答特征。酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）结果显示，Subolesin抗原（可溶性形式及吸附于微粒表面的形式）均未表现出免疫原性，因此被排除在后续分析之外。不过，ELISA结果表明，所有接受MSP4免疫的小鼠血清均引发了以Th2型免疫应答为主的免疫反应。此外，本研究首次揭示了在任意动物模型中，MSP4免疫所引发的T细胞与B细胞应答特征。综上，本研究为未来开发基于聚DL-乳酸-羟基乙酸共聚物微粒的蜱及嗜吞噬细胞无形体防控疫苗的设计与应用提供了重要参考依据。