International Center for Genetic Engineering and Biotechnology, Cape Town Component, and Institute of Infectious Diseases and Molecular Medicine, Medical Research Council, Division of Immunology, Faculty of Health Science, University of Cape Town, Cape Town, South Africa.
PLoS Negl Trop Dis. 2013 Aug 29;7(8):e2395. doi: 10.1371/journal.pntd.0002395. eCollection 2013.
Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection.
METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.
The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.
线虫分泌的血红蛋白对氧气具有异常高的亲和力，并且具有一氧化氮去氧酶和过氧化氢酶活性，这些被认为在保护寄生虫免受宿主免疫反应方面很重要。在这项研究中，我们生成了一种针对寄生虫线虫 Anisakis pegreffii 血红蛋白的单克隆抗体（48Eg），并旨在研究 4E8g 与不同线虫血红蛋白的交叉反应性及其介导对鼠钩虫感染的保护性免疫的潜力。
方法/主要发现：免疫沉淀用于分离 Anisakis 和 Ascaris 提取物中与 4E8g 结合的抗原，这些蛋白通过肽质量指纹图谱和 MS/MS 鉴定为血红蛋白。用啮齿动物钩虫 N. brasiliensis 的血红蛋白也证明了免疫交叉反应性。用免疫印迹法测试了线虫血红蛋白在小鼠和人中的免疫原性。感染了 Anisakis 的小鼠的 IgG 和 IgE 抗体识别了 Anisakis 血红蛋白，而小鼠和人血清中的 IgG 抗体识别了 Ascaris 血红蛋白，但不识别 IgE 抗体。Anisakis 血红蛋白的测序显示与相关海洋线虫 Pseudoterranova decipiens 的血红蛋白高度相似，后者缺乏 Ascaris 血红蛋白中对八聚体组装很重要的四个-HKEE 重复序列。通过免疫组织化学检查了不同寄生虫中血红蛋白的定位，并将其与 Anisakis、Ascaris 和 N. brasiliensis 的排泄-分泌管相关联。与据报道主要在成虫中表达的 Ascaris 血红蛋白不同，Anisakis 血红蛋白在 L3 期强烈表达。用 4E8g 对感染 N. brasiliensis 的小鼠进行被动免疫可增强保护性 Th2 免疫，并导致蠕虫负担显著减少。
单克隆抗体 4E8g 靶向寄生虫线虫中广泛等同的解剖位置的血红蛋白，并增强宿主对钩虫感染的免疫力。
