Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

Ferdinard Adungo,Daisuke Hayasaka,Guillermo Posadas-Herrera,David Kamau,Matilu Mwau,Fuxun Yu,Kouichi Morita,Shingo Inoue,Rosemary Sang,R L Hodinka

doi:10.1128/cvi.00209-16

Abstract

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.

Highlights

Clinical symptoms of yellow fever (YF) range from mild febrile disease to severe forms with hemorrhagic manifestations, hepatitis, jaundice, renal failure, rapid terminal events with shock, and multiorgan failure, with case fatality rates exceeding 20%
A number of antibody (Ab)-based diagnostic tests have been developed for detection of dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) using monoclonal and polyclonal antibodies [22,23,24,25,26,27]
It is strongly believed that the current emergence and reemergence of YFV and other arboviruses is partly attributable to the increased migration of people from countries where such diseases are endemic and the expanding establishment of the vector

Summary

Introduction

Clinical symptoms of yellow fever (YF) range from mild febrile disease to severe forms with hemorrhagic manifestations, hepatitis, jaundice, renal failure, rapid terminal events with shock, and multiorgan failure, with case fatality rates exceeding 20%. Over the past 10 years, many countries in which yellow fever is endemic such as countries in West Africa and some in Central Africa such as Central African Republic, Congo, and Chad have reported YF cases to the World Health Organization (WHO) [14, 15]. Not much has been reported on the development of such diagnostic tests for YFV [28] The use of both monoclonal and polyclonal antibodies in the development of diagnostic tests for YF could help to address the lack of commercial kits and improve access to testing outside regional or reference laboratories that currently serve countries where YF is endemic. We report the development, characterization, and evaluation of YF monoclonal antibodies (MAbs) Diagnostic application of these MAbs was tested using antigen detection and IgM capture enzyme-linked immunosorbent assays (ELISA)

Methods

Results

Conclusion