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 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.

Methods
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 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.