Date Published: April 18, 2019
Publisher: Public Library of Science
Author(s): Thomas L. Rudge, Karen A. Sankovich, Nancy A. Niemuth, Michael S. Anderson, Christopher S. Badorrek, Nick D. Skomrock, Chris M. Cirimotich, Carol L. Sabourin, Alexander N. Freiberg.
The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the recent and devastating 2014–2016 outbreak of Ebola virus (EBOV) disease in Guinea, Sierra Leone, and Liberia that resulted in more than 10,000 casualties. Such a vaccine would need to be vetted through a U.S. Food and Drug Administration (FDA) traditional, accelerated, or Animal Rule or similar European Medicines Agency (EMA) regulatory pathway. Under the FDA Animal Rule, vaccine-induced immune responses correlating with survival of non-human primates (NHPs), or another well-characterized animal model, following lethal EBOV challenge will need to be bridged to human immune response distributions in clinical trials. When possible, species-neutral methods are ideal for detection and bridging of these immune responses, such as methods to quantify anti-EBOV glycoprotein (GP) immunoglobulin G (IgG) antibodies. Further, any method that will be used to support advanced clinical and non-clinical trials will most likely require formal validation to assess suitability prior to use. Reported here is the development, qualification, and validation of a Filovirus Animal Nonclinical Group anti-EBOV GP IgG Enzyme-Linked Immunosorbent Assay (FANG anti-EBOV GP IgG ELISA) for testing human serum samples.
The filoviruses (family Filoviridae) from the genera Ebolavirus and Marburgvirus are etiologic agents of sporadic viral hemorrhagic fever outbreaks in humans with high mortality rates. An unprecedented outbreak of Ebola virus (EBOV; species Zaire ebolavirus) disease that began in Guinea during December 2013  subsequently spread into neighboring West African countries of Sierra Leone and Liberia, prompting the World Health Organization (WHO) to declare the epidemic a public health emergency of international concern (http://www.who.int/mediacentre/news/statements/2014/ebola-20140808/en/). Phylogenetic analysis of viral isolates from this epidemic suggests a single transmission event introduced the virus, named the EBOV Makona variant , from an undetermined natural reservoir into humans in Guinea, followed by transmission between humans to spread the virus throughout Guinea and into Sierra Leone and Liberia . Implementation of containment measures such as patient isolation and improved burial practices eventually controlled the epidemic, which resulted in 28,616 reported cases with a mortality rate of approximately 40% (http://www.who.int/csr/disease/ebola/en/).
According to the FDA document entitled, “Guidance for Industry: Bioanalytical Method Validation,” validation of a bioanalytical method for quantitative measurement of analyte in a biological matrix, such as an ELISA, includes demonstrating the method is reliable and reproducible for the intended use . In our validation, the FANG anti-EBOV GP IgG ELISA was found to be suitable for its intended purpose to measure human anti-EBOV GP IgG levels in human serum. In addition, the method is sensitive (low LOD), precise (intra- and inter-precision), dilutionally linear across its analytical range, selective (minimal matrix effects), and specific for anti-EBOV GP IgG in human serum as reported here. Two distinct assay endpoints were validated for the assay: concentration and endpoint titer. Both endpoints are valid as a final assay readout depending on the intended purpose; however, concentration is a more precise measurement due to its relative measure calculated based on the RS of the plate rather than the discrete and discontinuous nature of the endpoint titer. All parameters that were validated passed stringent criteria set for both endpoints through empirical testing and statistical analysis.