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Pseudotyping of VSV with Ebola Virus Glycoprotein Is Superior to HIV-1 For www.nature.com/scientificreports OPEN Pseudotyping of VSV with Ebola virus glycoprotein is superior to HIV‑1 for the assessment of neutralising antibodies Kimberley Steeds1, Yper Hall1, Gillian S. Slack1, Stephanie Longet1, Thomas Strecker2, Sarah Katharina Fehling2, Edward Wright3, Joseph Akoi Bore4, Fara Raymond Koundouno5, Mandy Kader Konde6, Roger Hewson1, Julian A. Hiscox7, Georgios Pollakis7 & Miles W. Carroll1* Ebola virus (EBOV) is an enveloped, single-stranded RNA virus that can cause Ebola virus disease (EVD). It is thought that EVD survivors are protected against subsequent infection with EBOV and that neutralising antibodies to the viral surface glycoprotein (GP) are potential correlates of protection. Serological studies are vital to assess neutralising antibodies targeted to EBOV GP; however, handling of EBOV is limited to containment level 4 laboratories. Pseudotyped viruses can be used as alternatives to live viruses, which require high levels of bio‑containment, in serological and viral entry assays. However, neutralisation capacity can difer among pseudotyped virus platforms. We evaluated the suitability of EBOV GP pseudotyped human immunodefciency virus type 1 (HIV‑1) and vesicular stomatitis virus (VSV) to measure the neutralising ability of plasma from EVD survivors, when compared to results from a live EBOV neutralisation assay. The sensitivity, specifcity and correlation with live EBOV neutralisation were greater for the VSV‑based pseudotyped virus system, which is particularly important when evaluating EBOV vaccine responses and immuno‑therapeutics. Therefore, the EBOV GP pseudotyped VSV neutralisation assay reported here could be used to provide a better understanding of the putative correlates of protection against EBOV. Ebola virus (EBOV), a member of the family Filoviridae, is an enveloped, single-stranded RNA virus that can cause Ebola virus disease (EVD), a highly lethal illness with up to 90% mortality 1. Since its discovery in 1976, EBOV has caused sporadic outbreaks across Central Africa and was responsible for the 2013–2016 EVD epidemic in West Africa 2, which was the largest EBOV outbreak on record and resulted in more than 28,600 cases and over 11,300 deaths3. Tis outbreak constituted a public health emergency of international concern and highlighted the urgent need for vaccines and therapeutics against EBOV. Te EBOV RNA genome is approximately 19 kb in length and encodes seven main proteins. Te envelope gly- coprotein (GP) of EBOV forms homotrimeric spikes that project from the surface of the viral particles4. Surface GP is critical for host cell attachment and fusion5,6 and is a target for neutralising antibodies7. Survivors of EVD are thought to protected against subsequent EBOV infection and neutralising antibodies to the viral surface GP are possible correlates of protection 8,9. Serological assays, such as the plaque reduction neutralisation test (PRNT), are central to evaluate neutralising antibodies against EBOV GP. However, because of its severe pathogenicity, potential transmission from person-to-person and lack of approved vaccines or antiviral treatments, handling of EBOV is restricted to containment level (CL) 4 laboratories. High containment facilities are expensive and are not readily available, especially in countries and organisations with limited resources. Furthermore, the assay format 1Public Health England (PHE), Porton Down, Salisbury, Wiltshire, UK. 2Institute of Virology, Philipps University Marburg, Marburg, Germany. 3School of Life Sciences, University of Sussex, Brighton, UK. 4Institut National de Santé Publique, Conakry, Republic of Guinea. 5University Julius Nyerere of Kankan, Conakry, Republic of Guinea. 6Centre d’Excellence de Formation et Recherche sur le Paludisme et les Maladies Prioritaires en Guinée (CEFORPAG), Ratoma, Conakry, Republic of Guinea. 7Institute of Infection and Global Health (IGH), University of Liverpool, Liverpool, UK. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:14289 | https://doi.org/10.1038/s41598-020-71225-1 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Titration of EBOV (Mayinga) GP pseudotyped (a) HIV-1 and (b) VSV using diferent cell lines. Relative luminescence units per well (RLU/well) were measured. Error bars are one standard error above and below the mean, n = 4. EBOV GP pseudotyped (c) HIV-1 and (d) VSV titres expressed as 50% tissue culture infectious dose per ml (TCID50/ml), n = 1. and time required for plaque development, which can take approximately nine days, makes it time-consuming and restricts high-throughput sample processing. Development of novel serological assays that utilise geneti- cally modifed recombinant or chimeric viruses with attenuated pathogenicity have enabled more widespread investigation of neutralising antibodies against highly pathogenic viruses including EBOV10,11. Pseudotyped viruses are replication-defective chimeric virions that comprise the structural and enzymatic core of one virus, bearing the envelope protein or glycoprotein of another, and encode a quantifable reporter gene. Retroviruses, including lentiviruses and gammaretroviruses such as human immunodefciency virus (HIV) and murine leukaemia virus (MLV), respectively, and rhabdoviruses, such as vesicular stomatitis virus (VSV), have been used extensively as cores for pseudotyped viruses12,13, including for EBOV 14,15. A number of EBOV GP pseudotyped virus neutralisation assays have been developed to investigate immune responses to EBOV infection and vaccination16–18, as well as for evaluation of monoclonal antibody (mAb) therapies19–21. Tere are many factors that need to be considered when developing and optimising pseudotyped virus neu- tralisation assays, to assess experimental parameters that can afect assay performance and to ensure accuracy and reproducibility. Tese include, choice of core virus and reporter gene, determination of target cell line and amount of pseudotyped virus input, as well as correlation with live virus neutralisation 22. Te aim of this study was to assess the suitability of EBOV GP pseudotyped HIV-1 and VSV systems to measure neutralisation by EVD survivor plasma, in comparison with results from a live EBOV neutralisation assay. Results Cell tropism of EBOV GP pseudotyped viruses. Pseudotyped HIV-1 and VSV bearing the envelope GP from EBOV (Mayinga) were generated and quantifed by measuring luminescence in a range of target cell lines, in order to determine the optimum cell line to use in neutralisation assays. Cells only controls were used to determine background levels of luminescence (Supplementary Fig. S1). Reporter activity was detected in all cell lines infected with EBOV GP pseudotyped HIV-1 and VSV, demonstrating the broad tissue range conferred by EBOV GP, although diferences in luminescence were observed (Fig. 1a,b). For EBOV GP pseudotyped HIV-1, highest TCID50/ml values were observed in 293T/17 cells, followed by Huh-7 cells (Fig. 1c). Titres generated by infection of 293T/17 cells were approximately 3, 33 and 73 times greater than those produced by infection of Huh-7, HeLa and Vero E6 cells, respectively. For EBOV GP pseudotyped VSV, highest titres were obtained in SCIENTIFIC REPORTS | (2020) 10:14289 | https://doi.org/10.1038/s41598-020-71225-1 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 2. Neutralisation of EBOV (Mayinga) GP pseudotyped (a) HIV-1, n = 2, and (b) VSV, n = 4, by human anti-EBOV GP mAb, KZ52. Percentage infectivity was calculated relative to pseudotyped virus only controls. Data are shown for mean with log (inhibitor) vs. normalised response curves. Error bars are 1 standard error above and below the mean. Dotted lines represent 50% infectivity. Vero E6 cells. Te TCID50/ml values generated by infection of Vero E6 cells were approximately 1.5, 22 and 30 times greater than those produced by infection of 293T/17, Huh-7 and HeLa cells, respectively (Fig. 1d). Based on these results, the 293T/17 and Vero E6 target cell lines were selected for use in all subsequent neutralisation assays using EBOV GP pseudotyped HIV-1 and VSV, respectively. Neutralisation of EBOV GP pseudotyped viruses by anti‑EBOV GP mAb. During the initial stages of assay development, it is important to evaluate neutralisation of pseudotyped viruses using well characterised antibodies in order to demonstrate the validity and accuracy of the assay. Te EBOV GP pseudotyped viruses were assessed for neutralisation by the human anti-EBOV GP mAb, KZ52. KZ52 is an antibody isolated from a human survivor of the 1995 outbreak in Kikwit that neutralises EBOV in vitro and recognises a conformational epitope at the base of the GP23–25. Human anti-EBOV GP mAb, KZ52 was unable to neutralise the EBOV GP pseudotyped HIV-1 (Fig. 2a) within the range tested, however it was able to neutralise the EBOV GP pseudo- typed VSV (Fig. 2b), suggesting that VSV-based pseudotyped viruses are more sensitive to neutralisation then lentiviral-based, possibly the density of EBOV GP on the pseudotyped HIV-1 may difer from that on the pseu- dotyped VSV or live EBOV. Assessment of EBOV GP pseudotyped virus input for neutralisation. To determine the optimal pseudotyped virus input to use in the HIV- and VSV-based assays, neutralisation of diferent amounts of the EBOV GP pseudotyped viruses by plasma from a Guinean EVD survivor donor or human anti-EBOV GP mAb KZ52 was assessed. KZ52 was selected as it is commercially available and there is accompanying information regarding its neutralisation activity
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