Granule-exocytosis of granulysin and granzyme B as a potential key mechanism in vaccine-induced immunity in cattle against the nematode Ostertagia ostertagi

Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative for anthelmintic treatment, an experimental host-protective vaccine was previously developed based on ASP-proteins derived from the adult worms. Intramuscular injection of this vaccine, combined with QuilA as adjuvant, significantly reduced the faecal egg counts by 59 %. However, the immunological mechanisms triggered by the vaccine are still unclear. Therefore, in this study, the differences in immune responses at the site of infection, i.e. the abomasal mucosa, between ASP/QuilA-vaccinated animals and QuilA-vaccinated control animals were investigated on a transcriptomic level, using a whole genome bovine micro-array, combined with histological analysis. Sixty nine genes were significantly impacted in animals protected by the vaccine, 48 of which were upregulated. A correlation study between the parasitological parameters and gene transcription levels showed that the transcription levels of two of the upregulated genes, granulysin (GNLY) and granzyme B (GZMB) negatively correlated to cumulative faecal egg counts and total worm counts, respectively. Both genes also positively correlated to each other, and to another upregulated gene, the IgE receptor subunit FCER1A. Surprisingly, these three genes also correlated significantly to CMA1, a mast cell marker, and to cell counts for mast cells and cells previously described as globule leukocytes. Furthermore, immunohistochemical data showed that GNLY was present in the granules of globule leukocytes and that it was secreted in the mucus. Overall, the results suggest a potential role of granule exocytosis by globule leucocytes, potentially IgE-mediated, in the vaccine induced protection against O. ostertagi Overall design: 12 helminth‐free Holstein crossbred calves at 7‐10 months of age were randomly divided in 2 groups of 6 animals. All animals were immunized three times intramuscularly in the neck with a three‐week interval. One group received 750 μg of QuilA with Tris‐buffer instead of antigen (negative control). The other group received ~30 μg of the native ASP fraction per immunization in combination with 750 μg of QuilA adjuvant. After the final immunization, the animals were challenged with a trickle infection of 30,000 infective L3 larvae (1,000 L3/day for 30 days, 5 days a week during a period of 6 weeks). 3 weeks after the last challenge infection, all animals were sacrified and total RNA from fundic abomasa was extracted. A total of 12 microarrays were used. Only biological replicates were used.奥斯特他线虫（Ostertagia ostertagi）被认为是对牛经济影响最为显著的寄生虫之一。作为抗蠕虫治疗的替代方案，此前研究人员基于成虫来源的ASP蛋白（ASP-proteins）开发了一款具有宿主保护作用的实验性疫苗。该疫苗以QuilA作为佐剂进行肌内注射后，可使粪便虫卵计数显著降低59%。但该疫苗所触发的免疫应答机制目前仍未明确。 因此，本研究采用全基因组牛微阵列（whole genome bovine micro-array）结合组织学分析，在转录组层面探究了感染部位（即皱胃黏膜）中，接种ASP/QuilA疫苗的牛与仅接种QuilA佐剂的对照牛之间的免疫应答差异。在疫苗保护的牛体内，共有69个基因的表达发生显著变化，其中48个基因呈上调表达。 寄生虫学参数与基因转录水平的相关性分析显示，两个上调基因——颗粒溶素（granulysin, GNLY）和颗粒酶B（granzyme B, GZMB）的转录水平分别与累积粪便虫卵计数和总虫体负荷呈负相关。这两个基因彼此间呈正相关，同时也与另一个上调基因——IgE受体亚基FCER1A（FCER1A）呈正相关。值得注意的是，这三个基因还与肥大细胞标志物CMA1（CMA1）以及肥大细胞和此前被称为球型白细胞的细胞的计数呈显著相关。 此外，免疫组化数据显示，颗粒溶素存在于球型白细胞的颗粒中，并可被分泌至黏液内。总体而言，本研究结果表明，球型白细胞的颗粒胞吐作用（可能由IgE介导）在该疫苗对抗奥斯特他线虫（Ostertagia ostertagi）的保护效应中发挥潜在作用。 实验设计：将12头7~10月龄无蠕虫感染的荷斯坦杂交犊牛随机分为两组，每组6头。所有牛只均于颈部进行三次肌内免疫，每次免疫间隔三周。其中一组仅接种含750μg QuilA的Tris缓冲液（不含抗原），作为阴性对照；另一组每次免疫接种约30μg天然ASP组分，同时辅以750μg QuilA佐剂。 末次免疫后，对所有牛只进行渐变感染攻虫：共接种30000条感染性三期幼虫（L3），即每周接种5天、每天接种1000条L3，持续6周共30天。末次攻虫3周后，对所有牛只实施安乐死，并提取皱胃底部组织的总RNA。本实验共使用12张微阵列芯片，且仅采用生物学重复样本。