MSACL 2016 US Abstract

Detection and Discovery of Early Markers of Ebola Virus Infection Using Serum Proteomic Analysis

Lisa Cazares (Presenter)
USAMRIID

Bio: Lisa Cazares Ph.D.-Lisa Cazares did her undergraduate work in Medical Microbiology and received her Ph.D. in Biomedical Sciences from Old Dominion University in Norfolk, Virginia. She is currently a Senior Scientist in the laboratory of Dr. Sina Bavari in the Molecular and Translational Sciences Division of US Army Research Institute of Infectious Disease (USAMRIID). Dr. Cazares has over 15 years of experience in mass spectrometry, and is the lead scientist for proteomic studies at USAMRIID where her research interests are to develop mass spectrometry approaches for therapeutic monitoring and discovery of biomarkers of infection. Dr. Cazares has over 50 peer reviewed publications in microbiology, prostate cancer protein expression, mass spectrometry tissue imaging and clinical proteomics.

Authorship: Lisa H. Cazares (1)(2), Michael D. Ward (1), Ernst Brueggemann (1) Tara Kenny (1), Cary Retterer (1), Travis Warren (1) and Sina Bavari (1)
(1) Molecular and Translational Sciences, USAMRIID Frederick, MD (2) DoD Biotechnology High Performance Computing Software Applications Institute (BHSAI)

Short Abstract

For the discovery of early markers of Ebola virus infection we have interrogated plasma from non-human primates (NHP) collected at multiple time-points during infection. Our experimental strategy employed 6-plex TMT labels for the quantitation of host proteins at pre-infection levels and 5 post-infection time-points. We discovered plasma proteins that change expression during Ebola infection in 7 NHP sample sets. Several acute phase proteins were induced systematically prior to the detection of serum viremia. Comparison of the Ebola NHP host response to that observed in Burkholderia pseudomallei infected NHP resulted in the discovery of unique differentially expressed proteins between these two infection types.

Long Abstract

Introduction:

Ebola virus (EBOV) and Marburg virus (MARV), two species within the family Filoviridae, are among the most pathogenic human viruses known and are the causative agents of hemorrhagic fever in humans and non-human primates (NHP). A confounding issue for physicians and point of care personnel is that, the initial symptoms of filovirus are so general that they are readily mistaken for other diseases. Current laboratory diagnostic tests for Ebola virus infections are based primarily on reverse-transcription polymerase chain reaction (RT-PCR) technology (most commonly used in the 2014 outbreak) and antigen-capture or antibody-detection using enzyme-linked immunosorbent assay (ELISA). These assays tend to give false-positive results, if applied prior to Day 3 of infection, and they are often only reliable within 3-10 days after the onset of symptoms. Since patients with EBOV infection usually succumb to the disease before initiating a productive, neutralizing antibody response, an acute-phase response to EBOV infection is an early, recognizable event that reflects the disease status, including the presence of viral antigen and host-response serological markers. The rapid and accurate identification of filovirus infection is critical to begin appropriate outbreak management and provide crucial antiviral treatment to infected individuals.

Methods:

Due to the scarcity of human data and access to human samples, non-human primate (NHP) models of Filovirus infection have been developed and are well established. Our experimental strategy employs a non-targeted analysis of host-response proteins in serial serum samples from an NHP EBOV infection model using LC-MS/MS (relative quantitation). Infected plasma was inactivated using SDS buffer and heat, followed by filter assisted sample preparation (FASP) after removal from BSL-4 containment. FASP captures the serum proteins onto a membrane where they are digested with trypsin, and the resulting peptides are labelled with reporter ion tags (TMT) to allow for the relative quantitation of protein abundance. In addition, the use of 6-plex TMT allows for the quantitation of proteins in 6 conditions simultaneously (i.e. pre-infection, and 5 post-infection time-points). This means that the samples for each time point (Days 0,3,5,7,9,11 post-infection) can be combined into a single LC-MS/MS run, greatly reducing instrument time. After digestion, the serum peptides are reconstituted in 0.5% formic acid and 10┬Ál is used for LC-MS/MS analysis on a Dionex Ultimate 3000 nLC coupled to a LTQ Orbitrap Elite. We have completed a preliminary interrogation of NHP host response proteins over the course of EBOV infection in 7 NHP sample sets, each with 6 time-points. In addition, for comparison to the EBOV host response, plasma samples from 6 NHP infected with the bacterial pathogen Burkholderia pseudomallei collected at similar post-infection time-points were also evaluated using the same 6-plex TMT relative quantitation method.

Results:

The protein abundance levels were determined for each time-point in each NHP using the Day 0 values as the baseline. For this analysis, in order to be considered for quantitation, a protein was required to have at least 2 peptide identifications. Proteins were considered to have a significant abundance change if there was at least a 2 fold increase or a 1.7 fold decrease from the baseline value. Analysis of the complete EBOV sample cohort was performed and 29 host response proteins had significant fold change values (15 up-regulated and 11 down-regulated) in least 4/7 NHP sample sets. We also discovered that several acute phase proteins were induced systematically prior to the first detection of serum viremia. Viral proteins were detected using our non-targeted approach, but targeted analysis will be necessary for early detection. Comparison of the EBOV NHP host response to the response observed in 5 Burkholderia pseudomallei (B.p.) infected NHP resulted in the discovery of common and differentially expressed proteins between these two infection types. We observed protein abundance changes of common acute phase reactants at earlier post-infection time-points in the B.p. infected animals compared to EBOV infected animals. The kinetic difference in the response may be attributed to the longer time necessary for viral replication in hosts cells compared to bacterial replication as well as the route of infection.

Conclusion:

The host response proteins found to be differentially expressed between EBOV and B.p. infection may represent markers that can be exploited for the discrimination of bacterial versus viral infection. We are currently developing targeted MRM assays for the unique differentially expressed proteins observed during EBOV infection to obtain absolute quantitation of these proteins and establish cut-off values. A method involving affinity capture followed by MRM (multiple reaction monitoring) MS analysis will increase the sensitivity of detection beyond what is possible with our untargeted FASP-TMT method, and will allow us to detect changes in protein abundance at earlier time-points.


References & Acknowledgements:


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