Project Title

Reciever Operator Characterization of Novel EBOV/MARV-GP Epitopes using 2014-2015 Sierra Leonian Ebola Patient-Samples at the NICD BSL-IV (EBOV-RDT-ROC)

EDCTP Project Call

Career Development Fellowship (CDF)

Project Objectives

The overall goal of this project is to determine receiver operator characteristics of novel EBOV/MARV-GP epitopes using Sierra Leonean patient-samples obtained from the 2014-2015 Ebola outbreak stored at the National Institute for Communicable Diseases (NICD) Center for Emerging Zoonotic Diseases (CEZD) BSL-IV. Two specific aims are contingent to this goal i.e to (i) Pre-characterize the patient sample by clinical picture and outcome, ELISA, RT-PCR, and viral culture, and (ii) Determine the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and receiver operator curves (ROCs) of our in-vitro pre-validated novel epitopes. Approach: (i) Pre-characterization of the patient samples will be done by retrospectively reviewing available clinical notes; and conducting in-vitro tests including ELISA, RT-PCR and viral culture within vero-cells, (ii) Direct and sandwich Ag capture ELISAs will be assembled to detect native GP in patient sample. Data obtained from (i) and (ii) will be analyzed by GraphPAD and PRISM software to generate sensitivity, specificity, PPV, NPV and RCOs.

Study Design

Our team, with funding from Grand Challenges Canada and the Medical Research Council (MRC) of South Africa has established a network with the team at the Biosafety level IV Laboratory of National Institute for Communicable Diseases NICD) Center for Emerging Zoonotic Diseases (CEZD) in Johannesburg. We have already initiated testing of the 12 panel of Mabs above with vero-cell derived EBOV zaire virus. Nonetheless, the signal obtained from this testing is low—intimating that the affinity and purity of the Mabs is low when directed at native EBOV-Gp, relative to purified recombinant Gp cloned and expressed in HEK mammalian cells (0.6ug/uL). Our team has already established protocols with the BSL-IV for this testing, and trained two staff in the use of the BSL-IV facility. The reason we are seeking EDCTP2 funding is to re-purify the same Mabs from hybridomas stored at GenSCRIPT., USA, to higher concentrations for re-testing against patient samples. (i) Pre-characterization of the patient samples will be done by retrospectively reviewing available clinical notes; and conducting in-vitro tests including ELISA, RT-PCR and viral culture within vero-cells as follows: -Method 1: Following waiver IRB approval from the Sierra Leonean government, we will conduct a retrospective review of patient files linked to the samples stored at the NICD-CEZD-P4. Specifically, we will look out for method of initial diagnosis, duration of admission in quarantine, and outcome. -Method 2: Further validation laboratory testing will be conducted to confirm diagnosis of EBOV. Specifically, ELISA, RT-PCR and viral culture within vero-cells will be carried out on samples within the BSL-IV as described elsewhere by Saijo M et al [3]. Overall, results of viral culture will be used as the gold standard test for evaluating the receiver operator characteristics of our epitopes. (ii) Direct and sandwich Ag capture ELISAs will be assembled to detect native GP in patie

Project Summary

Background: Ebola and Marburg viruses (EBOV and MARV) form the two generic members of the filoviridae family of viruses. Filoviruses cause rare but highly fatal viral hemorrhagic fevers (VHFs) within rural villages of equatorial Africa. The 2014-to-2015 ebola outbreak in West Africa turned a public health emergency of international concern (PHEIC). Filoviruses are class A pathogens of potential bioterror. There is absence of easy to use, affordable rapid diagnostic tests for early detection of filoviruses at the point of care (POC). Preliminary data: In Jan 2013, Grand Challenges Canada through its rising Stars in Global Health funded us to test novel conserved epitopes of EBOV/MARV glycoprotein (Gp) as potential diagnostic biomarkers (Grant # S4- 0280-01). During the 2014-to 2015 outbreak in West Africa, we were transitioned to scale. Over this period, we generated and validated a panel of over 12 mice-derived hybridomas and monoclonal antibodies (MAbs- targeting 4 unique epitopes inclusive of the extra-cellular domain of EBOV/MARV Gp) for their reactivity with recombinant Gp cloned and expressed within HEK mammalian cells. Objective: The overall goal of this project is to determine receiver operator characteristics of novel EBOV/MARVGP epitopes using Sierra Leonean patient-samples obtained from the 2014-2015 Ebola outbreak stored at the National Institute for Communicable Diseases (NICD) Center for Emerging Zoonotic Diseases (CEZD) BSL-IV. Two specific aims are contingent to this goal i.e to (i) Pre-characterize the patient sample by clinical picture and outcome, ELISA, RT-PCR, and viral culture, and (ii) Determine the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and receiver operator curves (ROCs) of our in-vitro pre-validated novel epitopes. Methods: (i) Pre-characterization of the patient samples will be done by retrospectively reviewing available clinical notes; and conducting in-vitro tests including ELISA, RT-PCR and viral culture within vero-cells, (ii) Direct and sandwich Ag capture ELISAs will be assembled to detect native GP in patient sample. Data obtained from (i) and (ii) will be analyzed by GraphPAD and PRISM software to generate sensitivity, specificity, PPV, NPV and RCOs. In addition, the Career Fellow will train 2 Masters level students in P4 Laboratory Practice and Standards. Potential impact: These conserved EBOV/MARV-Gp epitopes and their derivative MAbs are potential biomarkers to mount on a lateral flow immunochromatographic strip-test (LFT) for use at the point of care (POC) within remote village settings of equatorial Africa, or during a bioterror attack. Our team already initiated testing at the NICDCEZD- BSL-IV in collaboration with LifeAssay Diagnostics, but require more funding to reproduce high-affinity purified MAbs required for clinical testing.

Host Organisation

Current Organisation

Makerere University

Current Job Title

Snr Lecturer

Biography

Publications