DOI: 10.5061/dryad.63xsj3v44
title: Data from: Genome-wide epitope mapping reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection

description: 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 vaccination and/or infection in cattle, goats, humans,
 and sheep through genome-wide linear epitope mapping to identify candidate
 vaccine and diagnostic antigens within the predicted bacterial proteome.
 Using high-density peptide microarrays, we analyzed the seroreactivity in
 156 serum samples from vaccinated and infected individuals to peptides
 derived from 2,092 ORFs in the C. burnetii genome. We
 found significant diversity in the antibody responses within and between
 species and across different C. burnetii exposure
 statuses. However, C. burnetii exposure did result in
 more uniform seroreactivity across species. Through the implementation of
 three different vaccine candidate methods, we identified 493 candidate
 protein antigens for protein subunit vaccine design or serodiagnostic, out
 of which 65 have been previously described. This is the first study to
 investigate seroreactivity against the entire C.
 burnetii genome presented as overlapping linear peptides and
 provides the basis for selection of antigen targets for next generation Q
 fever vaccines and diagnostic tests.
description: <strong>Peptide microarray design</strong>
 In this study, peptide microarrays were designed using the genome
 sequence of the <em>C. burnetii</em> 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 <em>Clostridium
 tetani</em>. 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. <strong>Peptide microarray synthesis
 and probing</strong> The microarrays were
 synthesized using a Nimble Therapeutics Maskless Array Synthesizer via
 light-directed solid-phase peptide synthesis. Prior to incubation on the
 microarray, serum samples were diluted as follows: human/sheep - 1:100,
 goat - 1:250, and bovine - 1:50. These diluted serum samples were then
 bound to the microarrays overnight at 4°C. The bound primary antibodies
 were detected using secondary antibodies that were diluted as follows:
 human - 1:10000, goat/sheep - 1:20000, and bovine - 1:1000. The
 fluorescent signal of the secondary antibody was detected by scanning the
 microarray using an Innopsys 910AL microarray scanner. The scanned array
 images were analyzed using proprietary Nimble Therapeutics software to
 extract fluorescence intensity values for each peptide.
description: <strong>Aggregated microarray data for all
 samples:</strong>
 <em>microarray_data_aggregated.txt</em>
 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 <em>EXPR_MAIN_REP1/2: C.
 burnetii</em> 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).
 <strong>Peptide to protein mapper
 file:</strong>
 <em>peptide_map.txt</em> 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 their parent protein.
 <strong>Serum sample metadata:</strong>
 <em>sample_metadata.txt</em>
 Information regarding the species and treatment/status of the
 individuals when sera were sampled are found in a tab-seperated file
 format. The first column contains the treatment/status of the
 individuals: NEG: <em>C.
 burnetii</em> negative POS: <em>C.
 burnetii</em> positive PC: post <em>C.
 burnetii</em> challenge PV1: post <em>C.
 burnetii</em> phase I vaccination PV2: post
 <em>C. burnetii</em> phase II vaccination
 UV + C: unvaccinated <em>C. burnetii</em>
 challenged PV1/2 + C: <em>C.
 burnetii</em> phase I/II vaccinee challenged 
 Second column are numeric identifiers for the individuals, third
 column the dates sera were sampled if recorded (missing dates are denoted
 by "n/a"), fourth column the species of the individuals, and
 fifth column the ID of the microarray the individual sera were incubated
 with (corresponds to the column names in
 <em>microarray_data_aggregated.txt</em>).