Genome-wide epitope mapping reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of infection. Vaccination is a key control measure and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin preparations. Subunit vaccines based on recombinant proteins offer significant advantages, as they can be designed to reduce reactogenicity and can be co-designed with defined antigen serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccin..., Peptide microarray design In this study, peptide microarrays were designed using the genome sequence of the C. burnetii Nine-Mile strain RSA493 (Accession no. NC_002971), which included 2092 proteins. The arrays also contained internal control peptides from Human Herpes Virus 5 with 170 proteins and tetanus toxin protein from Clostridium tetani. These 2263 protein sequences were processed into 168,792 unique 15 amino acid peptide sequences tiling four amino acids (overlapping by 11) in their parent proteins. 960 random 15 amino acid peptide sequences with similar amino acid frequencies were generated from the 2263 protein sequences to assess background control reactivity. C. burnetii-derived, tetanus toxin protein, and control peptide sequences were synthesized in duplicate, leading to a total of 328,984 peptide sequences. Peptide microarray synthesis and probing The microarrays were synthesized using a Nimble Therapeutics Maskless Array Synthesizer via light-directed solid-phase peptid..., Aggregated microarray data for all samples: microarray_data_aggregated.txt Fluorescence intensity values for each peptide on the microarray microarray are found in an aggregated tab-seperated file format. First column contains the synthesized peptide sequences, second column contains the peptide group: EXPR_HHV5: HHV5-derived peptides EXPR_MAIN_REP1/2: C. burnetii and tetanus toxin protein derived peptides EXPR_NEG_CTRL_REP1/2: The random peptides The remaining columns contain the fluorescence intensity values extracted for each of the 156 serum samples. The column names correspond to the microarray identifier (ID). Peptide to protein mapper file: peptide_map.txt The peptide parent protein names and their locations are found in a tab-seperated file format. First column contains the synthesized peptide sequences, second column the name of their parent proteins, third column the length of the parent proteins, and fourth and fifth columns the start and coordinates of the peptides in the...,贝氏柯克斯体（Coxiella burnetii）是一种具有全球公共卫生重要性的重要人畜共患细菌性病原体，可在多种动物宿主中引发Q热（Q fever）。反刍家畜尤其是绵羊和山羊被认为是该病原菌的主要储存宿主。疫苗接种是关键的防控措施，目前已有两款基于福尔马林灭活贝氏柯克斯体菌体疫苗的商用疫苗上市。然而，由于在既往已被贝氏柯克斯体抗原致敏的个体中可引发显著的反应原性，这类疫苗的应用受到限制。此外，现有基于贝氏柯克斯体菌体疫苗制备的血清学诊断试验（serodiagnostic tests）会受到此类疫苗的干扰。基于重组蛋白的亚单位疫苗具备显著优势：其可通过设计降低反应原性，还可与明确的抗原血清学诊断试验联合开发，从而实现疫苗接种者与感染个体的区分。本研究旨在探究贝氏柯克斯体疫苗相关抗体应答的多样性。 肽微阵列（peptide microarray）设计 本研究基于贝氏柯克斯体九英里株RSA493的基因组序列（登录号：NC_002971）设计肽微阵列，该菌株共包含2092种蛋白质。微阵列同时包含170种人疱疹病毒5型（Human Herpes Virus 5）的内参肽段，以及破伤风梭菌（Clostridium tetani）的破伤风毒素蛋白。上述2263条蛋白质序列被切割为168792条独特的15氨基酸肽段，以每4个氨基酸为步长（重叠11个氨基酸）在其亲本蛋白质中进行铺展。从2263条蛋白质序列中随机生成960条氨基酸频率相似的15氨基酸肽段，用于评估背景反应性对照。贝氏柯克斯体来源、破伤风毒素蛋白及对照肽段序列均以双份形式合成，最终共计得到328984条肽段序列。 肽微阵列合成与杂交检测 本研究采用Nimble Therapeutics公司的无掩模阵列合成仪（Maskless Array Synthesizer），通过光导向固相肽合成法制备微阵列…… 所有样本的微阵列汇总数据： microarray_data_aggregated.txt 微阵列上所有肽段的荧光强度值以制表符分隔的文本文件格式存储。文件第一列为合成的肽段序列，第二列为肽段分组： EXPR_HHV5：人疱疹病毒5型来源肽段 EXPR_MAIN_REP1/2：贝氏柯克斯体与破伤风毒素蛋白来源肽段 EXPR_NEG_CTRL_REP1/2：随机对照肽段 剩余列包含156份血清样本各自的荧光强度值，列名对应微阵列识别号（ID）。 肽段-蛋白质映射文件： peptide_map.txt 肽段与其亲本蛋白质的名称及位置信息以制表符分隔的文本文件格式存储。文件第一列为合成的肽段序列，第二列为其亲本蛋白质名称，第三列为亲本蛋白质长度，第四、第五列为肽段在亲本蛋白质中的起始坐标……