Abstract
The latest outbreak of Ebola virus disease (EVD) in West Africa has highlighted the urgent need for the development of rapid and reliable diagnostic assays. We used monoclonal antibodies specific to the ebolavirus nucleoprotein to develop an immunochromatography (IC) assay (QuickNavi-Ebola) for rapid diagnosis of EVD. The IC assay was first evaluated with tissue culture supernatants of infected Vero E6 cells and found to be capable of detecting 103–104 focus-forming units/mL of ebolaviruses. Using serum samples from experimentally infected nonhuman primates, we confirmed that the assay could detect the viral antigen shortly after disease onset. It was also noted that multiple species of ebolaviruses could be detected by the IC assay. Owing to the simplicity of the assay procedure and absence of requirements for special equipment and training, QuickNavi-Ebola is expected to be a useful tool for rapid diagnosis of EVD.
Ebolaviruses and marburgviruses are enveloped, nonsegmented, negative-stranded RNA viruses belonging to the family Filoviridae. These filoviruses are known to cause severe hemorrhagic fever in humans and nonhuman primates, with human case-fatality rates of up to 90% [1]. In contrast to the genus Marburgvirus, which contains only 1 known species, Marburg marburgvirus, consisting of Marburg virus (MARV) and Ravn virus, 5 distinct species are known in the genus Ebolavirus: Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus, represented by Ebola virus (EBOV), Sudan virus (SUDV), Taï Forest virus (TAFV), Bundibugyo virus (BDBV), and Reston virus (RESTV), respectively [2].
Ebola virus disease (EVD) poses a significant public health threat, as implicated by the increase in the incidence of EVD outbreaks in Africa over the past 2 decades, with some occurring in previously unaffected areas [3]. The most recent epidemic of EVD severely affected Sierra Leone, Guinea, and Liberia and emphasizes the need for rapid, sensitive, reliable, and virus-specific diagnostic tests to control the spread of the virus. Rapid and simple antigen-detection tests, such as immunochromatography (IC) assays using filovirus-specific monoclonal antibodies (mAbs), are likely one of the options for early diagnosis of filovirus infections in the field setting.
Ebolavirus particles consist of 7 structural proteins: nucleoprotein (NP), viral protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and polymerase [1, 4]. NP appears to be an ideal target antigen for IC assays because of its abundance in filovirus particles, its strong antigenicity, and the presence of common epitopes among ebolavirus species [5]. The average EBOV particle contains about 3200 NP molecules [6]. The NP plays an important role in the replication of the viral genome and is essential for formation of the ribonucleocapsid [7]. Coexpression of NP VP40 and GP in cultured cells leads to efficient production of virus-like particles (VLPs) containing NP in the core [6, 8].
Previously, we generated a panel of mouse mAbs recognizing the EBOV NP and identified their epitopes, some of which are shared among multiple ebolavirus species, whereas others are species specific [5]. Using these mAbs, we generated an IC assay (QuickNavi-Ebola) and evaluated its ability to detect the NP antigen in culture supernatants of infected Vero E6 cells and in sera collected from EBOV-infected nonhuman primates (NHPs).
MATERIALS AND METHODS
Viruses and Cells
EBOV (Mayinga, Kikwit, Makona C05, and C07), SUDV (Boniface), TAFV (Pauléoula), BDBV (Butalya), RESTV (Pennsylvania), and MARV (Angola) were propagated in African green monkey kidney Vero E6 cells and stored at −80°C. Virus titers were determined as focus-forming units (FFU) by immunoplaque assays. All experiments involving the use of infectious filoviruses were performed in the biosafety level 4 laboratories of the Integrated Research Facility at the Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Hamilton, Montana). Standard operating procedures for infectious work were approved by the Rocky Mountain Laboratories Biosafety Committee.
Preparation of VLPs and Purified Recombinant NPs (rNPs)
Plasmids encoding NP, VP40, and GP of EBOV (Mayinga and Makona C07), SUDV, TAFV, BDBV, and RESTV were constructed as described previously [5, 9]. VLPs were produced by transfection of 293T cells with plasmids expressing ebolavirus NP, VP40, and GP as described previously [6, 8]. Forty-eight hours after transfection, supernatants containing VLPs were harvested and used for kit evaluation assays. For the preparation of purified NPs, 293T cells were transfected with the plasmids expressing the NPs of the respective ebolaviruses. Forty-eight hours later, the cells were lysed, and the NP fraction was collected by discontinuous CsCl gradient centrifugation as described previously [6, 8].
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA was performed to determine the reactivities of mAbs with rNPs as previously described [5]. Briefly, 96-well ELISA plates (Nunc, Maxisorp) were coated with purified ebolavirus rNPs (2 µg/mL in phosphate-buffered saline; 50 µL/well) overnight at 4°C, followed by blocking with 1% bovine serum albumin, and purified mAbs (1 µg/mL or 4-fold serial dilutions from 10 µg/mL) were added. Bound antibodies were visualized with horseradish peroxidase–conjugated goat anti-mouse IgG (H + L; Jackson ImmunoResearch) and 3,3′,5,5′-tetramethylbenzidine (Sigma).
Evaluation of the IC Assay
Tissue culture supernatants from Vero E6 cells infected with filoviruses (EBOV, SUDV, TAFV, BDBV, RESTV, or MARV), sera collected from NHPs (rhesus and cynomolgus macaques) experimentally infected with EBOV, and EBOV-infected patients were used to determine the sensitivity and specificity of the IC assay. For each assay, 30 μL (for serum/plasma and culture supernatant samples) or 10–20 μL (for whole-blood samples) were used. Other human pathogens were used to test for cross-reactivity of the assay (Supplementary Table 1).
Ethical Statement
Collection of NHP serum samples [10–13] complied with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). EBOV-infected human blood samples were collected from patients during the 2014 EBOV outbreak in the Democratic Republic of the Congo. Blood collection during outbreak investigations was approved under a special-response protocol established between the World Health Organization and national authorities. The experiments involving the use of human samples were approved by the institutional ethics committee, (Denka Seiken, Niigata, Japan) in accordance with the Declaration of Helsinki.
RESULTS
Selection of mAbs for the IC Assay
We first analyzed the binding affinities of 10 different clones of NP-specific mAbs in ELISA with purified rNPs of the representative isolates of all known ebolavirus species (EBOV, SUDV, TAFV, BDBV, and RESTV), including the 2014 EBOV Makona isolate, as antigens (Table (Table11 and Supplementary Figure 1). To select 2 mAbs used in the IC assay (ie, one labeled mAb and the other capturing mAb), we produced a test device based on a lateral-flow IC assay and assessed all possible combinations of mAbs suitable for labeled and capturing mAbs to detect EBOV VLPs. We found 2 combinations of mAbs giving the highest sensitivity (ZNP105-7/ZNP108-2-5 and ZNP105-7/ZNP62-7; data not shown). Based on their cross-reactivity profiles, we selected the ZNP105-7 (labeled)/ZNP108-2-5 (capturing) mAbs because this combination was expected to have the potential to detect different species of ebolaviruses (ie, EBOV, TAFV, and BDBV), whereas the other mAb combination was EBOV specific. The binding assay with the chimeric rNPs between EBOV and SUDV indicated that both mAbs ZNP105-7 and ZNP108-2-5 recognized amino acids at positions 632–739 of the NP molecule (Supplementary Figure 2). These amino acids are located in the C-terminal region of NP, which has been reported to have strong antigenicity [14].
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