ABSTRACT
BACKGROUND: Detection of viral ribo-nucleic acid (RNA) via real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of Ebola virus (EBOV) during acute infection. However, the earliest window for viral RNA detection in blood samples is 48-72 h post-onset of symptoms. Therefore, efforts to develop additional orthogonal assays using complementary immunological and serological technologies are still needed to provide simplified methodology for field diagnostics. Furthermore, unlike RT-PCR tests, immunoassays that target viral proteins and/or early host responses are less susceptible to sequence erosion due to viral genetic drift. Although virus is shed into the bloodstream from infected cells, the wide dynamic range of proteins in blood plasma makes this a difficult sample matrix for the detection of low-abundant viral proteins. We hypothesized that the isolation of peripheral blood mononuclear cells (PBMCs), which are the first cellular targets of the Ebola virus (EBOV), may provide an enriched source of viral proteins. METHODS: A mouse infection model that employs a mouse-adapted EBOV (MaEBOV) was chosen as a proof-of-principal experimental paradigm to determine if viral proteins present in PBMCs can help diagnose EBOV infection pre-symptomatically. We employed a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platform to provide both high sensitivity and specificity for the detection and relative quantitation of viral proteins in PBMCs collected during MaEBOV infection. Blood samples pooled from animals at the post-infection time-points were used to determine the viral load by RT-PCR and purify PBMCs. RESULTS: Using quantitative LC-MS/MS, we detected two EBOV proteins (vp40 and nucleoprotein) in samples collected on Day 2 post-infection, which was also the first day of detectable viremia via RT-PCR. These results were confirmed via western blot which was performed on identical PBMC lysates from each post-infection time point. CONCLUSIONS: While mass spectrometry is not currently amenable to field diagnostics, these results suggest that viral protein enrichment in PBMCs in tandem with highly sensitive immunoassays platforms, could lead to the development of a rapid, high-throughput diagnostic platform for pre-symptomatic detection of EBOV infection.
ABSTRACT
BACKGROUND: Post-translational modifications (PTMs) on proteins can be targeted by antibodies associated with autoimmunity. Despite a growing appreciation for their intrinsic role in disease, there is a lack of highly multiplexed serological assays to characterize the fine specificities of PTM-directed autoantibodies. METHODS: In this study, we used the programmable phage display technology, Phage ImmunoPrecipitation Sequencing (PhIP-Seq), to profile rheumatoid arthritis (RA) associated anti-citrullinated protein antibody (ACPA) reactivities. FINDINGS: Using both unmodified and peptidylarginine deiminase (PAD)-modified phage display libraries consisting of ~250,000 overlapping 90 amino acid peptide tiles spanning the human proteome, PTM PhIP-Seq robustly identified antibodies to citrulline-dependent epitopes. INTERPRETATION: PTM PhIP-Seq was used to quantify key differences among RA patients, including PAD isoform specific ACPA profiles, and thus represents a powerful tool for proteome-scale antibody-binding analyses. FUNDING: This research is based upon work supported in part by the Office of the Director of National Intelligence (ODNI), Intelligence Advanced Research Projects Activity (IARPA). The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of ODNI, IARPA, or the US Government. The US Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein. This study was made possible by a National Institute of General Medical Sciences (NIGMS) grant R01 GM136724 (HBL). MFK was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) grant T32AR048522. ED was supported by the Rheumatology Research Foundation.