To the Editor:
Ebola virus is currently causing an unprecedented outbreak in West Africa. The pathogen is classified as a biosafety level 4 (BSL-4)1 pathogen and should be handled only in high-level biosafety settings (1). Virus neutralization is considered to be the gold standard serological assay for infections, but in the case of Ebola, this test requires culturing of live virus in a BSL-4 facility. Because of these stringent biosafety requirements, options for clinical diagnosis or epidemiological studies of Ebola cases are limited. For this reason, we developed a pseudoviral particle neutralization (PPNT) assay that can detect neutralizing antibody to Ebola virus in BSL-2 containments.
Ebola glycoprotein (GP) is a major target of neutralizing antibodies in humans. The pseudoviral particle generated in this study is a lentiviral vector carrying the GP of Zaire ebolavirus detected in the current outbreak (2). Methods for the pseudoviral particle production and PPNT assay were essentially identical to those for Middle East respiratory syndrome (MERS) and influenza, as previously described (3, 4). Briefly, a codon-optimized GP sequence was chemically synthesized (Genscript) and subcloned into a protein-expressing vector, pcDNA3.1+. The resulting pseudoviral particle is capable of achieving only a single-round infection and contains a luciferase reporter gene that can be expressed in infected cells. Anti-GP neutralizing antibodies can inhibit the entry of this pseudoviral particle into cells, thereby inhibiting the expression of luciferase in the assay. For each Ebola pseudoviral particle production, the preparation was titrated and diluted to a level that could produce 10 000–50 000 counts/s in a positive infection control reaction in the luciferase assay (Steady-Glo Luciferase Assay System, Promega). In contrast, mock-infected cells (i.e., cells treated in the absence of pseudoviral particle) yielded background signals in the assay (<1% of the positive infection control) (Table 1).
Table 1.
Characterization of Ebola virus–specific neutralizing monoclonal antibodies, human sera, and animal sera in the Ebola PPNT assay.a
| Sample and dilution | Luciferase activity, %b |
|
|---|---|---|
| Mean (SD) | Range | |
| Positive infection control | 100 | |
| Mock-infection control | 0.10 (0.03) | 0.13–0.07 |
| mAb133/316 | ||
| 640 | 0.92 (0.06) | 0.88–0.99 |
| 5120 | 1.79 (1.56) | 0.88–3.60 |
| 10240 | 3.18 (2.66) | 0.86–6.09 |
| 20480 | 10.59 (5.01) | 6.31–16.10 |
| 40960 | 16.88 (9.20) | 7.11–25.36 |
| 81920 | 56.03 (35.26) | 29.97–95.61 |
| 163840 | 79.93 (46.38) | 31.92–124.50 |
| 327680 | 113.28 (14.33) | 96.78–122.61 |
| mAb266/8.1 | ||
| 640 | 0.82 (0.03) | 0.80–0.86 |
| 5120 | 1.05 (0.56) | 0.73–1.70 |
| 10240 | 7.13 (7.50) | 0.75–15.39 |
| 20480 | 11.17 (3.57) | 7.51–14.65 |
| 40960 | 13.02 (3.64) | 9.42–16.70 |
| 81920 | 49.40 (5.33) | 43.75–54.33 |
| 163840 | 110.54 (51.12) | 53.22–151.41 |
| 327680 | 84.36 (36.1) | 42.76–107.34 |
| Human (n = 61) | 55 (21) | 19–113 |
| Buffalo (n = 5) | 34 (9) | 22–46 |
| Camel (n = 5) | 30 (6) | 23–39 |
| Goat (n = 5) | 80 (18) | 52–100 |
| Sheep (n = 5) | 68 (16) | 52–90 |
Each sample was tested in triplicate, and the mean value of the triplicate reactions was used in the subsequent analysis. Dilution is expressed as fold dilution in final reaction volume. Human, buffalo, camel, goat, and sheep were all at 20-fold dilution.
Luciferase activity is expressed relative to cells treated with pseudoviral particle only (positive infection control).
For a typical PPNT assay for serological screening, 5 μL heat-treated serum sample (56 °C, 30 min) was incubated with the diluted pseudoparticles in a 100-μL reaction at 4 °C for 60 min before inoculation onto Vero cells (3, 4). The luciferase activities of infected cells were examined 72 h postinfection. Based on our previous experiences with similar assays (3, 4), the cutoff value for a positive reaction was set to be 10% of the value deduced from the positive infection control (see above). For determining the neutralization antibody titer of a serum sample, 2-fold serially diluted samples of a specimen (starting dilution 1:20) were used in the PPNT assay. Thus, a sample with a neutralizing antibody titer of 1:20 was considered a positive specimen.
To determine whether the PPNT assay was reactive to Ebola virus, we tested 2 neutralizing monoclonal antibodies specific for Z. ebolavirus GP, mAb133/316 and mAb266/8.1 (5). Both antibodies could neutralize the pseudoviral particle in a dose-dependent manner (Table 1). We also evaluated this test using serum samples collected from humans and animals (Table 1). All of these serum samples were tested at a screening dilution of 1:20 (1 part in 20 parts) and were negative in the assay (i.e., >10% of the value deduced from the positive infection control). The studied human samples were collected from healthy individuals in Hong Kong where no Ebola case has been reported in history. In addition, the studied animal species are not known to be susceptible hosts for Ebola viruses. Overall, these results suggest that this PPNT assay was specific to neutralizing antibodies against Ebola virus.
This study has some limitations, and further evaluations of the assay are needed. In particular, we were able to use only 2 Ebola virus–specific neutralizing monoclonal antibodies and negative human/animal serum samples for our evaluations. The studied monoclonal antibodies target 2 different conserved regions of Z. ebolavirus GP. One of these antibodies (mAb226/8.1) is known to bind to a conformational epitope of Z. ebolavirus GP (5), indicating that the GP of the pseudoviral particles is correctly folded. We were not able to obtain convalescent sera from Ebola patients to evaluate our assays, however; such convalescent human serum samples are difficult to obtain because of limited availability, legal issues, and biosafety concerns. Nonetheless, it is important to evaluate this assay in the future using convalescent sera known to be IgG- or IgM-positive for Ebola virus.
Our assay has potential applications in studies related to seroepidemiology, passive immunotherapy, and vaccine immunogenicity. To avoid laboratory-acquired infections, we emphasize that all serum samples from confirmed or suspected cases must be fully inactivated before downstream processing (1).
1 Nonstandard abbreviations
- BSL
biosafety level
- PPNT
pseudoviral particle neutralization
- GP
glycoprotein.
Footnotes
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Area of Excellence Scheme of the University Grants Committee (Grant AoE/M-12/06) to the University of Hong Kong; L.L.M. Poon, The Research Grant Council of Hong Kong (HKU 776213M).
Expert Testimony: None declared.
Patents: None declared.
Other Remuneration: University Medical Branch Texas has received funds from the Bill and Melinda Gates Foundation.
References
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