Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

• Published in: PLoS pathogens Vol. 17; no. 11; p. e1010039
• Main Authors: Shu, Qingbo, Kenny, Tara, Fan, Jia, Lyon, Christopher J, Cazares, Lisa H, Hu, Tony Y
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.11.2021; Public Library of Science (PLoS)
• Subjects: Amino acid sequence; Amino acids; Analysis; Animals; Antibodies; Antigenicity; Antigens; Assaying; Automation; Biology and life sciences; Biomarkers; Blood plasma; Candidates; Diagnostic tests; Ebola virus; Ebolavirus; Ebolavirus - immunology; Hemorrhagic Fever, Ebola - blood; Hemorrhagic Fever, Ebola - diagnosis; Hemorrhagic Fever, Ebola - immunology; Hemorrhagic Fever, Ebola - virology; Humans; Identification and classification; Immunoassay; Immunoassays; Immunoprecipitation; Infections; Laboratories; Lethality; Macaca mulatta; Mass spectrometry; Mass spectroscopy; Medicine and Health Sciences; Methods; Pathogens; Peptide Fragments - immunology; Peptides; Physical Sciences; Prevention; Proteins; Recombinant Proteins - immunology; Research and Analysis Methods; Signal monitoring; Software; Species; Species Specificity; Tryptic peptides; Viral antibodies; Viral diseases; Viral Matrix Proteins - immunology; Virus-like particles; Viruses; VP40 protein; Sudan
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1010039

Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity caused by different species, species identification and viral burden quantification are critical for treating infected patients timely and effectively. Here we developed an immunoprecipitation-coupled mass spectrometry (IP-MS) assay for VP40 antigen detection and quantification. We carefully selected two regions of VP40, designated as peptide 8 and peptide12 from the protein sequence that showed minor variations among Ebolavirus species through MS analysis of tryptic peptides and antigenicity prediction based on available bioinformatic tools, and generated high-quality capture antibodies pan-specific for these variant peptides. We applied this assay to human plasma spiked with recombinant VP40 protein from EBOV, SUDV, and BDBV and virus-like particles (VLP), as well as EBOV infected NHP plasma. Sequence substitutions between EBOV and SUDV, the two species with highest lethality, produced affinity variations of 2.6-fold for p8 and 19-fold for p12. The proposed IP-MS assay differentiates four of the six known EBV species in one assay, through a combination of p8 and p12 data. The IP-MS assay limit of detection (LOD) using multiple reaction monitoring (MRM) as signal readout was determined to be 28 ng/mL and 7 ng/mL for EBOV and SUDV respectively, equivalent to ~1.625-6.5×105 Geq/mL, and comparable to the LOD of lateral flow immunoassays currently used for Ebola surveillance. The two peptides of the IP-MS assay were also identified by their tandem MS spectra using a miniature MALDI-TOF MS instrument, greatly increasing the feasibility of high specificity assay in a decentralized laboratory.