Tropical Medicine and Infectious Disease

ArticleArticle Cross-NeutralisationCross-Neutralisation of ofNovel Novel Bombali Bombali Virus by by Ebola Virus Virus and Convalescent Plasma Using an Optimised AntibodiesPseudotype-Based and Convalescent Neutralisation Plasma Assay Using an Optimised Pseudotype-Based Neutralisation Assay Emma M. Bentley 1,* , Samuel Richardson 1,†, Mariliza Derveni 2, Pramila Rijal 3, Alain R. Townsend 3, 4 1 2 EmmaJonathan M. Bentley L. Heeney 1,*, Samuel, Giada Richardson Mattiuzzo 1,†, Marilizaand Edward Derveni Wright 2, Pramila Rijal 3, Alain R. Townsend 3, Jonathan L. Heeney 4, Giada Mattiuzzo 1 and Edward Wright 3 1 Division of , National Institute for Biological Standards and Control-MHRA, Blanche Lane, South Mimms EN6 3QG, UK; [email protected] (S.R.); 1 Division of Virology, National Institute for Biological Standards and Control-MHRA, Blanche Lane, [email protected] (G.M.) South Mimms EN6 3QG, UK; [email protected] (S.R.); [email protected] (G.M.) 2 School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK; [email protected] (M.D.); 2 School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK; [email protected] [email protected] (E.W.) 3 MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of 3 MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department Medicine, University of Oxford, Oxford OX3 9DS, UK; [email protected] (P.R.); of Medicine, University of Oxford, Oxford OX3 9DS, UK; [email protected] (P.R.); [email protected] (A.R.T.); [email protected] (E.W.) [email protected] (A.R.T.) 4 Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 OES, UK; [email protected] 4 Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 OES, UK; [email protected] * Correspondence: [email protected] * Correspondence: [email protected] † Current Address: The Pirbright Institute, Woking GU24 0NF, UK. † Current Address: The Pirbright Institute, Woking GU24 0NF, UK.   Abstract: continue to pose a significant outbreak threat, and while Ebola virus (EBOV)- Abstract: Ebolaviruses continue to pose a significant outbreak threat, and while Ebola virus (EBOV)- specific vaccines and antivirals have been licensed, efforts to develop candidates offering broad spe- Citation: Bentley, E.M.; Richardson, specific vaccines and antivirals have been licensed, efforts to develop candidates offering broad cies cross-protection are continuing. The use of pseudotyped virus in place of live virus is recognised S.; Derveni, M.; Rijal, P.; Townsend, species cross-protection are continuing. The use of pseudotyped virus in place of live virus is as an alternative, safer, high-throughput platform to evaluate anti- antibodies towards A.R.; Heeney, J.L.; Mattiuzzo, G.; recognised as an alternative, safer, high-throughput platform to evaluate anti-ebolavirus antibodies their development, yet it requires optimisation. Here, we have shown that the target cell line im- Citation:Wright, Bentley, E. Cross-Neutralisation E.M.; Richardson, of towards their development, yet it requires optimisation. Here, we have shown that the target cell S.; Derveni, M.; Rijal, P.; Townsend, pacts neutralisation assay results and cannot be selected purely based on permissiveness. In ex- Novel Bombali Virus by Ebola Virus line impacts neutralisation assay results and cannot be selected purely based on permissiveness. In A.R.; Heeney,Antibodies J.L.; andMattiuzzo, Convalescent G.; Plasmapanding the platform to incorporate each of the ebolavirus species envelope glycoprotein, allowing expanding the platform to incorporate each of the ebolavirus species envelope glycoprotein, allowing Wright,Using E. Cross-Neutralisation an Optimised Pseudotype- of a comprehensive assessment of cross-neutralisation, we found that the recently discovered Bombali a comprehensive assessment of cross-neutralisation, we found that the recently discovered Bombali Novel BombaliBased Neutralisation Virus by Ebola Assay. VirusTrop. virus has a point mutation in the receptor-binding domain which prevents entry into a hamster cell virus has a point mutation in the receptor-binding domain which prevents entry into a hamster AntibodiesMed. and Infect. Convalescent Dis. 2021, 6 , 155. https://line and, importantly, shows that this virus can be cross-neutralised by EBOV antibodies and con- cell line and, importantly, shows that this virus can be cross-neutralised by EBOV antibodies and Plasmadoi.org/10.3390/tropicalmed6030155 Using an Optimised valescent plasma. Pseudotype-Based Neutralisation convalescent plasma. Assay. AcademicTrop. Med. Editors:Infect. Dis. Abhishek 2021, 6,Pandey Keywords: ebolavirus; pseudotyped virus; cross-neutralisation x. https://doi.org/10.3390/xxxxxand Laura Skirp Keywords: ebolavirus; pseudotyped virus; cross-neutralisation

AcademicReceived: Editors: 30 Abhishek July 2021 Pandey and LauraAccepted: Skirp 21 August 2021 Published: 25 August 2021 1. Introduction1. Introduction Received: 30 July 2021 The 2013–2016The 2013–2016 outbreak outbreak of Ebola of Ebola virus virus in West in WestAfrica, Africa, which which was declared was declared a public a public Accepted:Publisher’s 21 August Note: 2021MDPI stays neutralhealthhealth emergency emergency of international of international concern concern (PHEIC) (PHEIC) and caused and caused over over28,000 28,000 cases cases and and Published: 24 August 2021 with regard to jurisdictional claims11,000 in 11,000 deaths deaths [1,2], [1 re-focused,2], re-focused the theworld’s world’s attention attention on on the the public public health health threat thethe EbolavirusEbo- published maps and institutional affil-lavirusgenus genus represents. represents. This This included included the the establishmentestablishment andand inclusion of Ebola Ebola virus virus on on the Publisher’siations. Note: MDPI stays neu- the WorldWorld Health Health Organization’s Organization’s research research an andd development development blueprint blueprint [3], [3], which which priori- prioritizes tral with regard to jurisdictional tizes fundamentalfundamental researchresearch andand supports supports the the development development of of vaccines vaccines and and therapeutics therapeutics along claims in published maps and institu- along with preparedness activities to mitigate future outbreaks. tional affiliations. with preparedness activities to mitigate future outbreaks. The firstThe documented first documented cases casesof viral of haemorrh viral haemorrhagicagic fever caused fever causedby an ebolavirus by an ebolavirus oc- Copyright: © 2021 by the authors.curredoccurred in 1976, in with 1976, nearly with nearlysimultaneous simultaneous outbreaks outbreaks of Ebola of Ebolavirus virus(EBOV) (EBOV) [4] and [4] Sudan and Sudan Licensee MDPI, Basel, Switzerland. virusvirus (SUDV) (SUDV) [5] in [5 ]Central in Central Africa. Africa. Up Up until until 2013, 2013, they they had had each each caused sporadicsporadic outbreaksout- This article is an open access article Copyright: © 2021 by the authors. breakslimited limited to to this this region region [6, 7[6,7]] and and this this trend trend has has continued continued since since the the PHEIC PHEIC with with a further a distributed under the terms and Submitted for possible open access further5 outbreaks 5 outbreaks of of EBOV, EBOV, as as well well as as an an outbreak outbreak in in West West Africa Africa [ 8[8].]. HumanHuman casescases have also conditions of the Creative Commons publication under the terms and con- also occurredoccurred with with two two further further species species identified; identified; these these are are the the Bundibugyo Bundibugyo virus virus (BUDV) (BUDV) and Attribution (CC BY) license (https:// ditions of the Creative Commons At- and thethe Taϊ Forest virus (TAFV).(TAFV). Distinctly,Distinctly, Reston virus (RESTV), (RESTV), which which has has been been identified iden- in creativecommons.org/licenses/by/ tribution (CC BY) license (http://crea- tifiedprimate in primate and and swine swine populations populations within within the Philippines, the Philippines, is non-pathogenic is non-pathogenic in humans in hu- [9,10]. 4.0/). tivecommons.org/licenses/by/4.0/). mans [9,10]. In 2018, as part of the PREDICT project to detect known and unknown

Trop. Med.Trop. Infect. Med. Infect.Dis. 2021 Dis., 62021, x. https://doi.org/10.3390/xxxxx, 6, 155. https://doi.org/10.3390/tropicalmed6030155 https://www.mdpi.com/journal/tropicalmedwww.mdpi.com/journal/tropicalmed Trop. Med. Infect. Dis. 2021, 6, 155 2 of 12

In 2018, as part of the PREDICT project to detect known and unknown viruses within wildlife reservoirs [11–13], the genome sequence of a further ebolavirus, Bombali virus (BOMV), was isolated from free-tailed bats in [14]. Subsequent surveys have also found it to be present in bats in Kenya and [15–17]. Through in situ sequence analysis and the use of a recombinant vesicular stomatitis virus (rVSV) encoding the BOMV glycoprotein (GP), it was determined that entry into human cells occurs via the Niemann– Pick C1 (NPC1) receptor, similar to other ebolaviruses [14]. However, the potential to cause disease in humans remains unknown. Currently, EBOV-specific vaccines and antivirals have been licensed for use following expedited development [18]. Prior to this, supportive care and convalescent plasma had been applied as an empirical treatment during filovirus outbreaks [19,20]. Yet in light of the disease potential (i.e., documented human cases or ability to infect human cells in vitro) posed by all 6 Ebolavirus species, there are efforts to develop cross-protective candidates [21]. To evaluate their effectiveness and further study serological responses against existing and newly discovered ebolaviruses, species-specific neutralisation assays are required. The use of live ebolavirus is restricted to 4 (BSL4) laboratories [22]; therefore, EBOV GP-pseudotyped virus (PV) has been used as an alternative, showing good correlation in the results between the live virus assay and the pseudotype system [23–25]. Previous work has suggested PV systems based on vesicular stomatitis virus (VSV) correlate better with the gold standard live neutralisation assay [26,27]. However, a caveat in these analyses is the variability of target cell lines used in the different reports. Here, we have optimised a lentiviral-vector based (LVV) neutralisation assay for EBOV antibodies and applied it in the evaluation of cross-neutralising antibodies against the other ebolavirus species, including BOMV.

2. Materials and Methods 2.1. Cell Lines and Virus Envelope Plasmids The highly transfectable human embryonic kidney 293T clone-17 cells (HEK 293T/17; American Type Culture Collection (ATCC), CRL-11268) were used for pseudotyped virus production as well as subsequent titration and neutralisation assays, together with a human lung carcinoma (A549; ATCC, CCL-185), African green monkey clone-E6 kidney (Vero E6; CRL-1586), feline kidney (CRFK; ATCC, CCL-94) and Chinese hamster ovary cell line (CHO-K1; ATCC, CCL-61). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose and GlutaMAX (Gibco), except for CHO-K1 cells which were cultured in Ham’s F-12 nutrient mix (Gibco). Both were supplemented with 10% fetal calf serum (Pan-Biotech GmbH) and 1% penicillin/streptomycin (Sigma-Aldrich) and ◦ maintained at 37 C, 5% CO2. The vesicular stomatitis virus (VSV) envelope glycoprotein was cloned in the pMD2.G expression vector [28]. The envelope glycoprotein sequences of Ebolavirus species studied were each cloned within the pCAGGS expression vector [29]. This included the Ebola virus (EBOV) isolate Makona/GIN/2014/Kissidougou-C15 (GenBank accession: KJ660346), Bundibugyo virus (BDBV) isolate UGA/2007 (FJ217161) and Bombali virus (BOMV) isolates Mops condylurus/SLE/2016/PREDICT_SLAB000156 (MF319185), Chaerephon pumilus/SLE/ 2016/PREDICT_SLAB000047 (MF319186) and Mops condylurus/Kenya/B241 (MK340750), which were chemically synthesized (BioBasic, GeneWiz and GeneArt, respectively). The glycoprotein sequences of Tai Forest virus (TAFV) isolate CIV/1994 (FJ217162), Sudan virus (SUDV) isolate Boniface/SUD/1976 (FJ968794) and Reston virus (RESTV) isolate Pennsylvania/USA/1989 (AY769362) were kindly provided by Prof. Graham Simmons (Vitalant Research Institute). The S146P mutation was introduced into the BOMV envelope GP sequence (residue 142) via Q5 site-directed mutagenesis (New England Biolabs) and confirmed by Sanger sequencing.

2.2. Human Plasma and Monoclonal Samples The WHO 1st International Standard for Ebola virus antibodies–Sierra Leone Convales- cent Plasma Pool (NIBSC catalogue code: 15/262) was reconstituted as per the instructions Trop. Med. Infect. Dis. 2021, 6, 155 3 of 12

for use in 0.5 mL sterile distilled water to give a working potency of 1.5 IU/mL. Similarly, the WHO Reference Panel of anti-Ebola virus convalescent plasma (NIBSC catalogue code: 16/344) was reconstituted in 0.25 mL sterile distilled water. It comprises samples from four individuals donated through the American Red Cross (ARC; 15/280), UK National Health Service Blood and Transplant (NHSBT; 15/282), Oslo University, Norway (NOR; 15/284) and National Institute for Infectious Diseases Lazzaro Spallanzani, Italy (INMI, 15/286), as well as a negative control (NEG, 15/288). The broadly neutralising pan-ebolavirus mAb CA45 [30], isolated from an immunised cynomolgus macaque, was kindly provided by Dr. M. Javad Aman (Integrated BioTher- apeutics). Two potently neutralising mAbs were derived from a vaccinated human [31], demonstrating some ebolavirus cross-reactivity, P6, or EBOV-specific, P7.

2.3. Pseudotyped Virus Production and Titration Pseudotyped virus production was based on methods previously described [28,32,33]. HEK 293T/17 cells were seeded into a 10 cm culture dish (Nunc) to reach 60–80% conflu- ence after 24 h. The HIV-1 gag-pol plasmid, pCMV-∆8.91, and the firefly luciferase reporter plasmid, pCSFLW, were then co-transfected with the pCAGGS plasmid-containing enve- lope sequence at a ratio of 1:1.5:1 µg, respectively, using 10.5 µL Fugene-HD transfection reagent (Promega) in 175 µL serum-free Opti-MEM (Gibco). Culture supernatant con- taining pseudotyped virus was harvested at 48 and 72 hrs post-transfection, then passed through a 0.45 µm pore filter (Millipore); aliquots were prepared for storage at −80 ◦C. Titres of pseudotyped virus stocks were determined via end-point dilution on target cells. Starting from a 1:5 dilution, 5-fold serial dilutions were prepared within culture medium in triplicate across 8 points. Each dilution was transferred to a 96-well culture plate containing target cells seeded at 2 × 104 cells/well and incubated for 60 hrs at 37 ◦C, 5% CO2. Following incubation, culture medium was removed and the target cells were incubated for 5 mins with a 1:1 mix of phenol-free DMEM (Gibco) and Bright-Glo reagent (Promega), with luciferase activity detected as relative light units (RLU) using a Glomax Navigator microplate luminometer (Promega). Titres were calculated as the 50% tissue culture infective dose (TCID50)/mL following the Spearman and Kärber method [34].

2.4. Neutralisation Assays In a 96-well plate, doubling dilutions of human plasma or mAb sample, prepared in duplicate across 10 points from a 1:20 or 25 µg/mL starting dilution, respectively, ◦ were incubated with 50 TCID50 of pseudotyped virus for 1 hr at 37 C and 5% CO2. Following incubation, the antibody and pseudotyped virus dilutions were added to target cells seeded in a 96-well culture plate at 2 × 104 cells/well and incubated for a further ◦ 60 h at 37 C and 5% CO2. Luciferase activity as RLU was detected as described above. Percentage neutralisation was calculated in GraphPad Prism, as described elsewhere [35], by normalising RLU data to pseudotyped virus and cell-only controls before non-linear regression analysis, fitting a 4-parameter dose-dependent inhibition curve to interpolate 50% inhibitory concentrations (IC50).

2.5. Protein Structure Modelling and Sequence Alignment Analysis of residues at the GP-NPC1 interface was performed using the crystal struc- ture of EBOV GP bound to domain C of human NPC1 (PDB ID: 5F1B) [36], with 3D modelling and images generated in NGL viewer [37]. Sequence alignment used representative sequences of NPC1 from the species of target cell lines, including HEK293T/17—Homo sapiens (GenBank accession: AAB63982.1), CRFK— Felis catus (AAF72187.1) and CHO-K1—Cricetulus griseus (AAF31692.1). In addition to the ebolavirus GP sequences used experimentally, all publicly available Bombali ebolavirus sequences were identified from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [38] through the website at http://www.viprbrc.org/ (accessed on 15 July 2021) The NPC1 and ebolavirus GP sequences were each aligned using Clustal Omega [39] and Trop. Med. Infect. Dis. 2021, 6, x FOR PEER REVIEW 4 of 12

CRFK—Felis catus (AAF72187.1) and CHO-K1—Cricetulus griseus (AAF31692.1). In addi- tion to the ebolavirus GP sequences used experimentally, all publicly available Bombali ebolavirus sequences were identified from the NIAID Virus Pathogen Database and Anal- ysis Resource (ViPR) [38] through the website at http://www.viprbrc.org/ (accessed on 15 July 2021) The NPC1 and ebolavirus GP sequences were each aligned using Clustal Omega [39] and then input into ESPript [40] to determine amino acid similarities of resi- dues identified as involved in GP-NPC1 binding.

3. Results 3.1. Target Cell Line Is Critical for Optimal Ebolavirus Pseudotype-Based Neutralisation Assay Ebola virus lentiviral-pseudotyped virus (EBOV-LVV) incorporating a firefly lucifer- ase reporter gene was titrated on different cell lines to select for those most permissive to EBOV-LVV entry (Figure 1a). EBOV-susceptible human HEK 293T/17 and A549 cells were evaluated in parallel to feline CRFK, hamster CHO-K1 and African green monkey Vero E6 cells. The EBOV-LVV reporter signal (RLU/mL) on Vero E6 was too low to be usable; this is not surprising, as monkey cells possess a restriction factor inhibiting post-entry Trop. Med. Infect. Dis. 2021, 6, 155steps of HIV-1-based particles [41]. Indeed, the VSV-G LVV control had a signal4 of over 12 100- fold lower on Vero E6 than on the other cell lines. It is more unclear why the EBOV-LVV entry signal on human A549 was lower than the envelope-less LVV control. thenTherefore, input into the ESPript 3 remaining [40] to determine cells lines, amino HE acidK 293T/17, similarities CRFK of residues and CHO-K1, identified were as then usedinvolved to evaluate in GP-NPC1 EBOV-LVV binding. neutralisation performance using a normalized input of 50 TCID50, with well-characterised anti-EBOV mAbs (P6 and CA45) and the WHO Interna- tional3. ResultsStandard (IS) comprising a pool of convalescent plasma from Ebola-recovered indi- 3.1. Target Cell Line Is Critical for Optimal Ebolavirus Pseudotype-Based Neutralisation Assay viduals. Both visual and mathematical analysis of the fit of dose-dependent inhibition Ebola virus lentiviral-pseudotyped virus (EBOV-LVV) incorporating a firefly luciferase curves showed that performance was poor across all samples on CRFK cells, with an R2 reporter gene was titrated on different cell lines to select for those most permissive to valueEBOV-LVV of 0.33 for entry each (Figure of the1a). mAbs EBOV-susceptible P6 and CA45 human and HEK 0.28 293T/17 for the andWHO A549 IS cells plasma were sample (Figureevaluated 1b). There in parallel was to a feline similarly CRFK, poor hamster fit for CHO-K1 the WHO and African IS plasma green sample monkey Veroand E6the mAb P6 oncells. HEK The 293T/17 EBOV-LVV cells reporter (R2 = 0.36 signal and (RLU/mL) 0.47, respectively). on Vero E6 wasA superior too low todose-response be usable; this profile wasis observed not surprising, using as the monkey CHO-K1 cells possesstarget acell restriction line, with factor a robust inhibiting fit post-entry(R2 ≥ 0.70) steps across all samplesof HIV-1-based tested, supporting particles [41 its]. Indeed, selection the as VSV-G the LVVoptimal control target had cell a signal line over to evaluate 100-fold cross- reactivitylower on across Vero E6the than ebolavirus-pseudotyped on the other cell lines. It isviruses. more unclear why the EBOV-LVV entry signal on human A549 was lower than the envelope-less LVV control.

Figure 1. Comparison of EBOV-pseudotyped virus infectivity and neutralisation on various target cell lines. (a) Infectivity Figure 1. Comparison of EBOV-pseudotyped virus infectivity and neutralisation on various target cell lines. (a) Infectivity of the EBOV-pseudotyped lentiviral particles measured as relative luminescent units (RLU/mL) were assessed on 5 target of the EBOV-pseudotyped lentiviral particles measured as relative luminescent units (RLU/mL) were assessed on 5 target cell lines. A vesicular stomatitis virus G protein (VSV-G) pseudotype was used as a positive control for the LVV system. LVV without an envelope protein (∆Env) was used to determine the background signal. (b) The percentage neutralisation of dilutions of monoclonal antibodies P6 or CA45 (µg/mL) and WHO IS (20/262; mIU/mL) following incubation with 2 50 TCID50 of EBOV-pseudotyped virus on each of the target cells, with the fit (R ) of the dose-response curve annotated. Error bars indicate SEM (n = 2).

Therefore, the 3 remaining cells lines, HEK 293T/17, CRFK and CHO-K1, were then used to evaluate EBOV-LVV neutralisation performance using a normalized input of 50 TCID50, with well-characterised anti-EBOV mAbs (P6 and CA45) and the WHO Inter- national Standard (IS) comprising a pool of convalescent plasma from Ebola-recovered individuals. Both visual and mathematical analysis of the fit of dose-dependent inhibition curves showed that performance was poor across all samples on CRFK cells, with an R2 value of 0.33 for each of the mAbs P6 and CA45 and 0.28 for the WHO IS plasma sample (Figure1b). There was a similarly poor fit for the WHO IS plasma sample and the mAb P6 on HEK 293T/17 cells (R2 = 0.36 and 0.47, respectively). A superior dose-response profile Trop. Med. Infect. Dis. 2021, 6, x FOR PEER REVIEW 5 of 12

cell lines. A vesicular stomatitis virus G protein (VSV-G) pseudotype was used as a positive control for the LVV system. LVV without an envelope protein (ΔEnv) was used to determine the background signal. (b) The percentage neutralisation

ofTrop. dilutions Med. Infect. of Dis. monoclonal2021, 6, 155 antibodies P6 or CA45 (μg/mL) and WHO IS (20/262; mIU/mL) following incubation5 of 12with 50 TCID50 of EBOV-pseudotyped virus on each of the target cells, with the fit (R2) of the dose-response curve annotated. Error bars indicate SEM (n = 2).

2 3.2.was CHO-K1 observed Cells using Are the CHO-K1Refractory target to Bombali cell line, withVirus a Entry robust fit (R ≥ 0.70) across all sam- ples tested, supporting its selection as the optimal target cell line to evaluate cross-reactivity acrossFollowing the ebolavirus-pseudotyped identification of viruses.the hamster cell line CHO-K1 as the optimal cell line for EBOV LVV-pseudotype neutralisation, we expanded the analysis of the neutralisation ac- tivity3.2. CHO-K1 to other Cells species Are Refractory of the to genus Bombali Ebolavir Virus Entryus, including the most recently identified BOMV.Following Pseudotyped identification LVV of titres the hamster were cellevaluated line CHO-K1 via end-point as the optimal titration cell line to for determine EBOV LVV-pseudotype neutralisation, we expanded the analysis of the neutralisation activ- TCID50/mL values on the HEK293T/17, CRFK and CHO-K1 target cell lines used for the ity to other species of the genus Ebolavirus, including the most recently identified BOMV. EBOV-LVV neutralisation assay (Figure 2). All the Ebolavirus LVV presented the same Pseudotyped LVV titres were evaluated via end-point titration to determine TCID50/mL patternvalues onwith the the HEK293T/17, highest titres CRFK (>1 and × 10 CHO-K15 TCID target50/mL) cell obtained lines used using for the HEK293T/17 EBOV-LVV cells. The titresneutralisation on the CHO-K1 assay (Figure cells,2). previously All the Ebolavirus identi LVVfied presentedas the most the suitable same pattern target with cells line for 5 thethe neutralisation highest titres (>1 assay,× 10 wereTCID lower50/mL) (3–60 obtained fold), using similar HEK293T/17 to titres cells. on CRFK The titres (8–30 fold re- duction),on the CHO-K1 but still cells, suitable previously for the identified downstream as the most neutralisation suitable target assays. cells line The for only the exception neutralisation assay, were lower (3–60 fold), similar to titres on CRFK (8–30 fold reduction), was for the BOMV-LVV, which showed a 750-fold reduction when titrated on CHO-K1 but still suitable for the downstream neutralisation assays. The only exception was for the cells,BOMV-LVV, with a whichnegligible showed titre a 750-foldof 259 TCID reduction50/mL, when which titrated is not on CHO-K1sufficient cells, for withperformance a of downstreamnegligible titre neutralisation of 259 TCID50/mL, assays. which is not sufficient for performance of downstream neutralisation assays.

Figure 2. Comparison of ebolavirus-pseudotyped virus infectivity on different target cell lines. Titres Figure 2. Comparison of ebolavirus-pseudotyped virus infectivity on different target cell lines. Ti- calculated as TCID50/mL for pseudotyped virus incorporating ebolavirus envelope glycoproteins trestitrated calculated on HEK293T/17, as TCID50 CRFK/mL andfor pseudotyped CHO-K1 target cells.virus The incorporat dotted lineing indicates ebolavirus the minimum envelope glycopro- teinstitre requiredtitrated foron downstreamHEK293T/17, neutralisation CRFK and assays. CHO-K1 target cells. The dotted line indicates the mini- mum titre required for downstream neutralisation assays. 3.3. Identification of a Critical Residue Involved in Bombali Virus Binding to the NPC1 Receptor 3.3. IdentificationTo investigate of the a Critical cause of Residue CHO-K1 Involved cells being in refractoryBombali Virus to BOMV Binding entry, to we the looked NPC1 Receptor to analyse sequence residues at the interface of ebolavirus GP binding to the NPC1 receptor (FigureTo 3investigatea). Analysis the of NPC1cause residuesof CHO-K1 in direct cells contact being withrefractory the EBOV to BOMV GP [ 36 entry,] from we looked tothe analyse different sequence target cell residues species revealedat the interface mutations of ofebolavirus two residues GP (Q421Ebinding and to F504Y)the NPC1 recep- torwithin (Figure the Cricetulus3a). Analysis griseus ofCHO-K1 NPC1 residues cell line, whichin direct are contact conserved with between the EBOV the Homo GP [36] from thesapiens differentHEK293T/17 target andcellFelis species catus CRFKrevealed cell linesmutations (Figure3 ofb). two While residues this may contribute(Q421E and F504Y) withinto species-specific the Cricetulus susceptibility griseus CHO-K1 to BOMV, sequencecell line, alignment which are of theconserved interacting between regions the Homo across the ebolavirus GP was undertaken to identify a determining factor (Figure3c). sapiensThe receptor-binding HEK293T/17 residuesand Felis are catus highly CRFK conserved cell lines across (Figure the ebolaviruses, 3b). While with this a singlemay contribute toproline species-specific to serine mutation susceptibility (P146S) unique to BOMV, to the BOMVsequence GP identified.alignment Further of the analysis interacting of regions acrossthe alignment the ebolavirus of the five GP complete was undertakenBombali ebolavirus to identifyisolate a determining sequences available factor reveals (Figure 3c). The receptor-bindingthis mutation is common residues across are fourhighly identified conserv in theed across bat species the Mopsebolaviruses, condylurus ,with yet the a single pro- lineconserved to serine proline mutation occurs (P146S) in a single unique isolate to identified the BOMV in another GP identified. bat species, FurtherChaerephon analysis of the alignmentpumilus (Figure of the S1). five Of note,complete attempts Bombali in our labebolavirus to produce isolate pseudotyped sequences virus available expressing reveals this the BOMV GP isolated from the C. pumilus bat has not yielded titres high enough for mutationdownstream is common studies, whileacross BOMV four identified GP from both in the viruses bat species isolated Mops from condylurusM. condylurus, yet the con- servedachieved proline usable occurs titres (Figure in a single S2). isolate identified in another bat species, Chaerephon pu- milus (Figure S1). Of note, attempts in our lab to produce pseudotyped virus expressing the BOMV GP isolated from the C. pumilus bat has not yielded titres high enough for

Trop. Med. Infect. Dis. 2021, 6, x FOR PEER REVIEW 6 of 12

downstream studies, while BOMV GP from both viruses isolated from M. condylurus Trop. Med. Infect. Dis. 2021, 6, x FOR PEER REVIEW 6 of 12 achieved usable titres (Figure S2). To test our theory, we performed site-directed mutagenesis to revert the serine at position 146 of the BOMV GP to the conserved proline. Pseudotyped virus was then pro- downstreamduced with the studies, mutant whileS146P alongside BOMV theGP wi fromld type both BOMV viruses GP sequence isolated and fromtitrated M. on condylurus each of the target cell lines. As expected, the S146P mutation restored entry of BOMV achieved usable titres (Figure S2). pseudotyped virus into the CHO-K1 cell line, with no impact on entry into the alternative targetTo cellstest (Figureour theory, 4). The titrewe onperformed CHO-K1 cells site-direc was 70-foldted higher,mutagenesis at 1.6 × 10 to4 TCID revert50/mL, the serine at positionthan that 146 of PVof thebearing BOMV the wildtype GP to the glycopro conserteinved and proline. was comparable Pseudotyped to the titre virus of other was then pro- ducedebolavirues with the on mutantthis target S146P cell line alongside (Figure 2). the This wi confirmedld type BOMV the S146 GP residue sequence is critical and in titrated on Trop. Med. Infect. Dis. 2021, 6, 155 eachthe ofability the oftarget BOMV cell to bindlines. the As NPC1 expected, receptor the of C.S146P griseus mutation and allowed restored entry to entrybe6 of re- 12 of BOMV pseudotypedstored for the virus performance into the of CHO-K1 neutralisation cell experimentsline, with no using impact the optimal on entry CHO-K1 into the tar- alternative get cell line. target cells (Figure 4). The titre on CHO-K1 cells was 70-fold higher, at 1.6 × 104 TCID50/mL, than that of PV bearing the wildtype glycoprotein and was comparable to the titre of other ebolavirues on this target cell line (Figure 2). This confirmed the S146 residue is critical in the ability of BOMV to bind the NPC1 receptor of C. griseus and allowed entry to be re- stored for the performance of neutralisation experiments using the optimal CHO-K1 tar- get cell line.

FigureFigure 3. Analysis3. Analysis of of NPC1 NPC1 receptor receptor binding binding to to the the ebolavirus ebolavirus glycoprotein. glycoprotein.( a(a)) The The structure structure ofof humanhuman NPC1NPC1 receptorreceptor (blue)(blue) in in complex complex with with the the EBOV EBOV glycoprotein glycoprotein (white) (white) which which highlights highlights three three NPC1-interacting NPC1-interacting regionsregions (red)(red) coveringcovering glycoprotein amino acids 79–88, 111–118 and 141–152. Alignment of the interface of (b) NPC1 sequence from HEK293T/17 glycoprotein amino acids 79–88, 111–118 and 141–152. Alignment of the interface of (b) NPC1 sequence from HEK293T/17 (Homo sapiens), CRFK (Felis catus) and CHO-K1 (Cricetulus griseus) cell lines and (c) ebolavirus glycoprotein sequences, (Homowith sapiens residues), CRFK in direct (Felis contact catus) highlighted and CHO-K1 in (squaredCricetulus boxe griseuss. Virus-specific) cell lines and polymorphisms (c) ebolavirus are glycoprotein shaded yellow sequences, and red with for residuesamino inacids direct similar contact or dissimilar highlighted to inthe squared consensus, boxes. respective Virus-specificly. The polymorphismsBOMV glycoprotein are shaded proline yellow (P) to serine and red (S) forpolymor- amino acidsphism similar (P146S) or dissimilar is outlined. to the consensus, respectively. The BOMV glycoprotein proline (P) to serine (S) polymorphism (P146S) is outlined.

To test our theory, we performed site-directed mutagenesis to revert the serine at position

146 of the BOMV GP to the conserved proline. Pseudotyped virus was then produced with the Figure 3. Analysis of NPC1 receptormutant S146P binding alongside to the the ebolavirus wild type glycoprotein. BOMV GP sequence (a) The and structure titrated of on human each of theNPC1 target receptor (blue) in complex with the EBOVcell lines. glycoprotein As expected, (white) the S146P whic mutationh highlights restored three entry NPC1-interac of BOMV pseudotypedting regions virus (red) into covering glycoprotein amino acids 79–88,the CHO-K1111–118 and cell line,141–152. with noAlignment impact on of entry the interface into the alternative of (b) NPC1 target sequence cells (Figure from4 HEK293T/17). The 4 (Homo sapiens), CRFK (Felis catustitre on) and CHO-K1 CHO-K1 cells (Cricetulus was 70-fold griseus higher,) cell at 1.6 lines× 10 andTCID (c) 50ebolavirus/mL, than glycoprotein that of PV bearing sequences, with residues in direct contactthe highlighted wildtype glycoprotein in squared andboxe wass. Virus-specific comparable to polymorphisms the titre of other ebolaviruesare shaded onyellow this target and red for amino acids similar or dissimilarcell lineto the (Figure consensus,2). This respective confirmedly. theThe S146 BOMV residue glycoprotein is critical proline in the ability (P) to ofserine BOMV (S) polymor- to

phism (P146S) is outlined. bind the NPC1 receptor of C. griseus and allowed entry to be restored for the performance of neutralisationFigure 4. Restored experiments infectivity using of BOMV the optimal lentiviral-pseud CHO-K1otyped target virus cell into line. CHO-K1 target cells after reversion of a serine at position 146 of the glycoprotein to a conserved proline (S146P). Infectivity, measured as TCID50/mL, calculated relative to a pseudotyped virus incorporating the wild type

Figure 4. Restored infectivity of BOMV lentiviral-pseudotyped virus into CHO-K1 target cells after Figure 4. Restored infectivity of BOMV lentiviral-pseudotyped virus into CHO-K1 target cells after reversion of a serine at position 146 of the glycoprotein to a conserved proline (S146P). Infectivity,measured reversion of a serine at position 146 of the glycoprotein to a conserved proline (S146P). Infectivity, as TCID50/mL, calculated relative to a pseudotyped virus incorporating the wild type BOMV glycoprotein measuredsequence on as HEK293T/17, TCID50/mL, CRFK calculated and CHO-K1 relative target to cell a lines. pseudotyped Error bars represent virus incorporating SEM (n = 2). the wild type

Trop. Med. Infect. Dis. 2021, 6, 155 7 of 12

3.4. Ebola Virus Convalescent Serum and Monoclonal Antibodies Cross-Neutralise Bombali Virus The level of cross-neutralisation afforded against BOMV in comparison to other ebolaviruses was evaluated using both convalescent plasma and existing cross-reactive mAbs. Given CHO-K1 are refractory to BOMV, BOMV P146 was used. The panel of con- valescent plasma samples collected from survivors during the 2013-2016 EBOV outbreak in West Africa included a pool of high titre samples from patients in Sierra Leone (15/262) and individual samples of mid-low titres from repatriated patients (15/280–15/286), along with a negative control (15/288) [42]. Dose-response curves plotting the level of neutralisation across the dilutions tested shows a reducing degree of cross-reactivity based on titre of the sample tested (high-low), with four out of five samples cross-reacting with BOMV (Figure5a ). The high titre pool 15/262 cross-reacts with all the ebolavirus PV, with the BOMV IC50 of 83 being the second most potently neutralised in comparison to EBOV (IC50 120), the virus of exposure (Figure5b ). Interestingly, in the case of the mid-titre sample 15/280, BOMV is the most potently neutralised PV, with an IC50 titre 1.5× higher than EBOV. Of the mAb samples tested, CA45 has been shown to have neutralising activity against EBOV, BDBV, RESTV and SUDV. This is supported by our data, with CA45 found to addi- tionally have activity against BOMV and TAFV (Figure5a), with its potency against BOMV ranked moderately (IC50 3.85 µg/mL) amongst the panel of ebolavirus PV (Figure5c ). A study to isolate P6 from a human vaccinee showed that it had cross-reactivity against BDBV but not SUDV [31]; this again is supported by our data and extended to include both BOMV and TAFV (Figure5a,c ). In the same study, P7 was found to be EBOV specific and is used here as a control, with reactivity solely detected against the EBOV PV. In all cases except with the low potency sample 15/284, BOMV was found to be neutralised at a Trop. Med. Infect. Dis. 2021, 6, x FOR PEER REVIEW 8 of 12 similar or more potent level than other ebolaviruses, which suggests likely efficacy of both existing and cross-reactive treatments under development.

Figure 5. Cont.

Figure 5. Cross-neutralisation of BOMV afforded by a panel of human EBOV convalescent serum and monoclonal anti- bodies. (a) Dose-response curves plotted as percentage neutralisation for dilutions of the WHO IS (15/262) and WHO Reference Panel members from four individuals recovered from EBOV (15/280, 15/282, 15/284, 15/286) and a negative control (15/288) along with two pan-ebolavirus mAbs (CA45, P6) and an EBOV-specific mAb negative control (P7). (n = 2) (b,c) 50% inhibitory concentrations (IC50) expressed as reciprocal of the dilution factor for plasma samples (b) or in μg/mL for mAb (c); IC50 were interpolated via non-linear regression analysis, with potency ranked good to weak based on a 50th percentile mid-point. Where the IC50 is outside the dilution range tested, values are reported as greater or less than the minimum serum (1:20) (b) and mAb (12.5 μg/mL) (c) dilution tested.

4. Discussion Current ebolavirus vaccine and therapeutic development efforts are focused on cross-neutralising candidates [43,44]. Neutralising antibodies against the envelope GP are considered both a determinant of infection control and a possible correlate of protection [45–47]. To measure the potency of neutralising antibodies, the gold standard method is a live Ebola virus neutralisation assay, requiring BSL4 facilities and specialised operators. Implementation of pseudotyped virus, which has been shown to offer good levels of cor- relation with live virus assays [23–25], provides a suitable and safer alternative to investi- gate neutralisation activity of serological samples. An additional advantage is that their production only requires the envelope GP sequence of the virus of interest. That said, it is important to take steps to optimise pseudotyped virus production and downstream as- says to ensure reliability of results [48,49].

Trop. Med. Infect. Dis. 2021, 6, x FOR PEER REVIEW 8 of 12

Trop. Med. Infect. Dis. 2021, 6, 155 8 of 12

FigureFigure 5. 5.Cross-neutralisation Cross-neutralisation of of BOMV BOMV afforded afforded by by a panela panel of humanof human EBOV EBOV convalescent convalescent serum serum and and monoclonal monoclonal antibodies. anti- bodies. (a) Dose-response curves plotted as percentage neutralisation for dilutions of the WHO IS (15/262) and WHO (a) Dose-response curves plotted as percentage neutralisation for dilutions of the WHO IS (15/262) and WHO Reference Reference Panel members from four individuals recovered from EBOV (15/280, 15/282, 15/284, 15/286) and a negative Panelcontrol members (15/288) from along four with individuals two pan-ebolavirus recovered mAbs from (CA45, EBOV P6) (15/280, and an EBOV-specific 15/282, 15/284, mAb 15/286) negative and control a negative (P7). (n control = 2) (15/288)(b,c) 50% along inhibitory with two concentrations pan-ebolavirus (IC50 mAbs) expressed (CA45, as P6) reciprocal and an EBOV-specificof the dilution factor mAb negativefor plasma control samples (P7). (b ()n or= in 2) μ (bg/mL,c) 50% inhibitoryfor mAb concentrations(c); IC50 were interpolated (IC50) expressed via non-linear as reciprocal regression of the analysis, dilution with factor potency for plasma ranked samples good to (b weak) or in basedµg/mL on a for 50th mAb (c);percentile IC50 were mid-point. interpolated Where via non-linearthe IC50 is outside regression the analysis,dilution withrange potency tested, values ranked are good reported to weak as basedgreater on or a less 50th than percentile the minimum serum (1:20) (b) and mAb (12.5 μg/mL) (c) dilution tested. mid-point. Where the IC50 is outside the dilution range tested, values are reported as greater or less than the minimum serum (1:20) (b) and mAb (12.5 µg/mL) (c) dilution tested. 4. Discussion 4. DiscussionCurrent ebolavirus vaccine and therapeutic development efforts are focused on cross-neutralisingCurrent ebolavirus candidates vaccine [43,44]. and therapeuticNeutralising development antibodies against efforts the are envelope focused GP on are cross- neutralisingconsidered both candidates a determinant [43,44]. Neutralisingof infection co antibodiesntrol and againsta possible the correlate envelope of GP protection are consid- ered[45–47]. both To a measure determinant the potency of infection of neutralising control and antibodies, a possible the correlate gold standard of protection method [45 is– a47 ]. Tolive measure Ebola virus the potency neutralisation of neutralising assay, requiring antibodies, BSL4 the facilities gold standard and specialised method is operators. a live Ebola virusImplementation neutralisation of pseudotyped assay, requiring virus, BSL4 which facilities has been and shown specialised to offer operators. good levels Implementa- of cor- tionrelation of pseudotyped with live virus virus, assays which [23–25], has provides been shown a suitable to offer and good safer levels alternative of correlation to investi- with livegate virus neutralisation assays [23 activity–25], provides of serological a suitable samples. and saferAn additional alternative advantage to investigate is that neutral-their production only requires the envelope GP sequence of the virus of interest. That said, it is isation activity of serological samples. An additional advantage is that their production Trop. Med. Infect. Dis. 2021, 6, x FOR importantPEER REVIEW to take steps to optimise pseudotyped virus production and downstream as-9 of 12 only requires the envelope GP sequence of the virus of interest. That said, it is important to says to ensure reliability of results [48,49]. take steps to optimise pseudotyped virus production and downstream assays to ensure reliability of results [48,49]. ItIt is is well-understood well-understood that that the the choice choice of of target target cell cell line line influences influences the the titre titre of of the the pseudotypedpseudotyped virus virus stock, stock, due due to to differences differences such such as as the the cell cell surface surface receptor receptor density density or or availabilityavailability of of requiredrequired protease. Ebolaviruses Ebolaviruses are are known known to tohave have a broad a broad host host range, range, with withthe Niemann–Pick the Niemann–Pick C1 (NPC1) C1 (NPC1) entry entry receptor receptor being being mostly mostly ubiquitously ubiquitously expressed expressed across acrossnon-lymphoid non-lymphoid cells [50–53]. cells [50 –However,53]. However, there thereis variability is variability in the in activity the activity of cathepsin of cathepsin B and BL and proteases L proteases required required for GP for priming GP priming [54,55]. [54 This,55]. likely This likelycontributes contributes towards towards the differ- the differencesences in entry in entry efficiency efficiency observed observed within within this and this other and otherstudies studies [26,56–58]. [26,56 While–58]. WhileAfrican Africangreen monkey green monkey Vero E6 Vero cells E6 are cells commonly are commonly used for used the for propagation the propagation and assaying and assaying of live ofebolavirus, live ebolavirus, they theyare refractory are refractory to LVV-based to LVV-based pseudotypes pseudotypes due due to the to the presence presence of ofthe the in- intrinsictrinsic restriction restriction factor, factor, TRIM5ɑ,, inhibitinginhibiting HIV-1HIV-1 post-entrypost-entry steps steps [ 41[41].]. This This presents presents a a caveatcaveat to to studies studies reporting reporting that that pseudotypes pseudotypes based based on on a a VSV VSV core core system, system, using using Vero Vero E6 E6 targettarget cells, cells, offer offer better better correlation correlation with with live live ebolavirus ebolavirus neutralisation neutralisation than than that that achieved achieved withwith LVV LVV pseudotypes pseudotypes on on human human HEK HEK 293T/17 293T/17 cells cells [26 [26,27].,27]. Data Data presented presented here here showed showed thatthat the the hamster hamster cell cell line line CHO-K1, CHO-K1, despite despite sustaining sustaining lower lower EBOV-LVV EBOV-LVV infectivity infectivity titres titres thanthan the the most most commonly commonly usedused HEKHEK 293T/17, a allowedllowed for for a amore more consis consistenttent neutralisation neutralisa- tionassay assay producing producing better better fit dose-response fit dose-response curves curves both both for formonoclonal monoclonal antibodies antibodies and and con- convalescentvalescent plasma. plasma. TheThe use use ofof CHO-K1CHO-K1 as target cells cells for for other other species species of of ebolaviruses ebolaviruses revealed revealed that that the therecently recently discovered discovered BOMV BOMV was was the theonly only pseudotype pseudotype with with impaired impaired entry. entry. Further Further inves- investigationtigation highlighted highlighted that thatone mutated one mutated residu residuee in the in BOMV the BOMV GP S142 GP S142(P146 (P146 in the in EBOV the EBOVsequence) sequence) is primarily is primarily responsible responsible for the for inab theility inability of BOMV-pseudotyped of BOMV-pseudotyped virus virus to infect to infecthamster hamster cell lines. cell lines.It is possible It is possible that the that mutation the mutation of this ofproline this proline residue residue impacts impacts binding bindingby causing by causing structural structural changes changes to the receptor to the receptor-binding-binding domain, domain, as is proposed as is proposed to be tothe becase the for case other for otherebolavirus ebolavirus polymorphisms, polymorphisms, including including a proline a proline at nearby at nearbyposition position 148 [59]. 148Other [59]. studies Other studiesalso show also that show single that singleamino amino acid changes acid changes within within the EBOV the EBOV GP GP[60] [ 60and] andNPC1 NPC1 receptor receptor [61,62] [61, 62can] canhave have marked marked effects effects on species-specific on species-specific virus virus tropism. tropism. Of note, Of S142 is not present in the single BOMV sequence isolated from C. pumilus. It has not been possible to produce viable pseudotyped virus with this envelope, which may be attributed to mutations within the highly conserved coiled-coil domain shown to impact GP binding [63] (Figure S1); this warrants further analysis. Yet, it is conceivable that S142 may repre- sent an adaptation of BOMV to M. condylurus NPC1. It has previously been suggested that each ebolavirus is adapted to a specific bat host [62], with variation occurring across the NPC1 binding domain, although a partial M. condylurus NPC1 sequence shows F504 is conserved [64]. To include the BOMV-pseudotyped virus in our analysis of cross-neutralising activ- ity of EBOV antibodies and convalescent plasma, the S146 in the BOMV GP was mutated to a proline. We tested three monoclonal antibodies, two with known cross-ebolavirus species neutralising activity which target the fusion loop (CA45) and glycan cap (P6) and one which is EBOV-specific and binds the receptor binding domain (P7), and a panel of convalescent plasma from Ebola virus disease survivors collected following the EBOV outbreak in 2013-2016. For the monoclonal antibodies, our results confirm what has been previously reported in the literature for CA45 [30] and P6 [31] and expands their efficacies to BOMV and TAFV. Interestingly, BOMV was also neutralised by most of the EBOV con- valescent plasma samples. This builds upon studies that have shown a pan-ebolavirus neutralising response can be mounted from natural infection [65] and existing therapeutic mAb candidates can neutralise BOMV [66], supporting the feasibility of developing cross- protective vaccines and therapeutics able to target all species within the ebolavirus genus.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1: Supple- mentary Figure S1. Sequence alignment of the EBOV GP sequence to four BOMV isolates GP; Sup- plementary Figure S2. Mean titres of lentiviral pseudotypes bearing different Bombali ebolavirus (BOMV) isolate GP. Author Contributions: Conceptualization E.M.B., G.M. and E.W.; methodology, E.M.B., S.R., M.D. and E.W.; validation, E.M.B., S.R. and E.W.; formal analysis, E.M.B., S.R. and E.W.; investigation,

Trop. Med. Infect. Dis. 2021, 6, 155 9 of 12

note, S142 is not present in the single BOMV sequence isolated from C. pumilus. It has not been possible to produce viable pseudotyped virus with this envelope, which may be attributed to mutations within the highly conserved coiled-coil domain shown to impact GP binding [63] (Figure S1); this warrants further analysis. Yet, it is conceivable that S142 may represent an adaptation of BOMV to M. condylurus NPC1. It has previously been suggested that each ebolavirus is adapted to a specific bat host [62], with variation occurring across the NPC1 binding domain, although a partial M. condylurus NPC1 sequence shows F504 is conserved [64]. To include the BOMV-pseudotyped virus in our analysis of cross-neutralising activity of EBOV antibodies and convalescent plasma, the S146 in the BOMV GP was mutated to a proline. We tested three monoclonal antibodies, two with known cross-ebolavirus species neutralising activity which target the fusion loop (CA45) and glycan cap (P6) and one which is EBOV-specific and binds the receptor binding domain (P7), and a panel of convalescent plasma from Ebola virus disease survivors collected following the EBOV outbreak in 2013-2016. For the monoclonal antibodies, our results confirm what has been previously reported in the literature for CA45 [30] and P6 [31] and expands their efficacies to BOMV and TAFV. Interestingly, BOMV was also neutralised by most of the EBOV convalescent plasma samples. This builds upon studies that have shown a pan-ebolavirus neutralising response can be mounted from natural infection [65] and existing therapeutic mAb candidates can neutralise BOMV [66], supporting the feasibility of developing cross- protective vaccines and therapeutics able to target all species within the ebolavirus genus.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/tropicalmed6030155/s1: Supplementary Figure S1. Sequence alignment of the EBOV GP sequence to four BOMV isolates GP; Supplementary Figure S2. Mean titres of lentiviral pseudotypes bearing different Bombali ebolavirus (BOMV) isolate GP. Author Contributions: Conceptualization E.M.B., G.M. and E.W.; methodology, E.M.B., S.R., M.D. and E.W.; validation, E.M.B., S.R. and E.W.; formal analysis, E.M.B., S.R. and E.W.; investigation, E.M.B., S.R., M.D. and E.W.; resources, P.R., A.R.T. and J.L.H.; writing—original draft preparation, E.M.B., S.R. and G.M.; writing—review and editing, E.M.B., S.R., M.D., P.R., A.R.T., J.L.H., G.M. and E.W.; visualization, E.M.B.; supervision, G.M. and E.W.; project administration, E.M.B. and E.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by BBSRC, grant number BB/R020116/1. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest.

References 1. World Health Organisation WHO Situation Report: Ebola Virus Disease. 10 June 2016. Available online: http://apps.who.int/ iris/bitstream/10665/208883/1/ebolasitrep_10Jun2016_eng.pdf?ua=1 (accessed on 27 July 2021). 2. Baize, S.; Pannetier, D.; Oestereich, L.; Rieger, T.; Koivogui, L.; Magassouba, N.; Soropogui, B.; Sow, M.S.; Keïta, S.; De Clerck, H.; et al. Emergence of Zaire Ebola Virus Disease in Guinea - Preliminary Report. N. Engl. J. Med. 2014, 371, 1418–1425. [CrossRef] 3. WHO List of Blueprint Priority Diseases. Available online: http://www.who.int/blueprint/priority-diseases/en/ (accessed on 19 July 2017). 4. Johnson, K. Ebola haemorrhagic fever in Zaire, 1976. Bull. World Health Organ. 1978, 56, 271–293. 5. Smith, D. Ebola haemorrhagic fever in Sudan, 1976. Report of a WHO/International Study Team. Bull. World Health Organ. 1978, 56, 247–270. 6. Leroy, E.M.; Gonzalez, J.-P.; Baize, S. Ebola and haemorrhagic fever viruses: Major scientific advances, but a relatively minor public health threat for Africa. Clin. Microbiol. Infect. 2011, 17, 964–976. [CrossRef] 7. To, K.K.W.; Chan, J.F.W.; Tsang, A.K.L.; Cheng, V.C.C.; Yuen, K.-Y. Ebola virus disease: A highly fatal infectious disease reemerging in West Africa. Microbes Infect. 2015, 17, 84–97. [CrossRef] Trop. Med. Infect. Dis. 2021, 6, 155 10 of 12

8. World Health Organisation Ebola Virus Disease. Available online: https://www.who.int/health-topics/ebola/#tab=tab_1 (accessed on 15 July 2021). 9. Miranda, M.E.; Ksiazek, T.G.; Retuya, T.J.; Khan, A.S.; Sanchez, A.; Fulhorst, C.F.; Rollin, P.E.; Calaor, A.B.; Manalo, D.L.; Roces, M.C.; et al. Epidemiology of Ebola (subtype Reston) Virus in the Philippines, 1996. J. Infect. Dis. 1999, 179, S115–S119. [CrossRef][PubMed] 10. Barrette, R.W.; Metwally, S.A.; Rowland, J.M.; Xu, L.; Zaki, S.R.; Nichol, S.T.; Rollin, P.E.; Towner, J.S.; Shieh, W.; Batten, B.; et al. Discovery of Swine as a host for the Reston ebolavirus. Science 2009, 325, 204–206. [CrossRef][PubMed] 11. Kelly, T.R.; Karesh, W.B.; Johnson, C.K.; Gilardi, K.V.K.; Anthony, S.J.; Goldstein, T.; Olson, S.H.; Machalaba, C.; Mazet, J.A.K. One Health proof of concept: Bringing a transdisciplinary approach to surveillance for zoonotic viruses at the human-wild animal interface. Prev. Vet. Med. 2017, 137, 112–118. [CrossRef] 12. Morse, S.S.; Mazet, J.A.; Woolhouse, M.; Parrish, C.R.; Carroll, D.; Karesh, W.B.; Zambrana-Torrelio, C.; Lipkin, W.I.; Daszak, P. Prediction and prevention of the next pandemic zoonosis. Lancet 2012, 380, 1956–1965. [CrossRef] 13. Predict 2017 Semi-Annual Report. Available online: https://ohi.sf.ucdavis.edu/sites/g/files/dgvnsk5251/files/files/page/ predict-2017-semiannual-report.pdf (accessed on 27 July 2021). 14. Goldstein, T.; Anthony, S.J.; Gbakima, A.; Bird, B.H.; Bangura, J.; Tremeau-Bravard, A.; Belaganahalli, M.N.; Wells, H.L.; Dhanota, J.K.; Liang, E.; et al. The discovery of Bombali virus adds further support for bats as hosts of ebolaviruses. Nat. Microbiol. 2018, 3, 1084–1089. [CrossRef] 15. Forbes, K.M.; Webala, P.W.; Jääskeläinen, A.J.; Abdurahman, S.; Ogola, J.; Masika, M.M.; Kivistö, I.; Alburkat, H.; Plyusnin, I.; Levanov, L.; et al. Bombali Virus in Mops condylurus Bat, Kenya. Emerg. Infect. Dis. 2019, 25, 955–957. [CrossRef] 16. Karan, L.S.; Makenov, M.T.; Korneev, M.G.; Sacko, N.; Boumbaly, S.; Yakovlev, S.A.; Kourouma, K.; Bayandin, R.B.; Glady- sheva, A.V.; Shipovalov, A.V.; et al. Bombali Virus in Mops condylurus Bats, Guinea. Emerg. Infect. Dis. 2019, 25, 955–957. [CrossRef][PubMed] 17. Kareinen, L.; Ogola, J.; Kivistö, I.; Smura, T.; Aaltonen, K.; Jääskeläinen, A.J.; Kibiwot, S.; Masika, M.M.; Nyaga, P.; Mwaengo, D.; et al. Range expansion of bombali virus in mops condylurus Bats, Kenya, 2019. Emerg. Infect. Dis. 2020, 26, 3007–3010. [CrossRef] 18. Tomori, O.; Kolawole, M.O. Ebola virus disease: Current vaccine solutions. Curr. Opin. Immunol. 2021, 71, 27–33. [CrossRef][PubMed] 19. World Health Organisation Use of Convalescent Whole Blood or Plasma Collected from Patients Recovered from Ebola Virus Disease for Transfusion, as an Empirical Treatment during Outbreaks. Available online: https://www.who.int/publications/i/ item/WHO-HIS-SDS-2014.8 (accessed on 27 July 2021). 20. Winkler, A.M.; Koepsell, S.A. The use of convalescent plasma to treat emerging infectious diseases. Curr. Opin. Hematol. 2015, 22, 521–526. [CrossRef][PubMed] 21. King, L.B.; Milligan, J.C.; West, B.R.; Schendel, S.L.; Ollmann Saphire, E. Achieving cross-reactivity with pan-ebolavirus antibodies. Curr. Opin. Virol. 2019, 34, 140–148. [CrossRef] 22. Bayot, M.L.; King, K.C. Biohazard Levels. Available online: http://www.ncbi.nlm.nih.gov/pubmed/30570972 (accessed on 27 July 2021). 23. Konduru, K.; Shurtleff, A.C.; Bavari, S.; Kaplan, G. High degree of correlation between Ebola virus BSL-4 neutralization assays and pseudotyped VSV BSL-2 fluorescence reduction neutralization test. J. Virol. Methods 2018, 254, 1–7. [CrossRef] 24. Ewer, K.; Rampling, T.; Venkatraman, N.; Bowyer, G.; Wright, D.; Lambe, T.; Imoukhuede, E.B.; Payne, R.; Fehling, S.K.; Strecker, T.; et al. A Monovalent Chimpanzee Adenovirus Boosted with MVA. N. Engl. J. Med. 2016, 374, 1635–1646. [CrossRef] 25. Xiao, J.H.; Rijal, P.; Schimanski, L.; Tharkeshwar, A.K.; Wright, E.; Annaert, W.; Townsend, A. Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein. J. Virol. 2018, 92, 1–18. [CrossRef] 26. Steeds, K.; Hall, Y.; Slack, G.S.; Longet, S.; Strecker, T.; Fehling, S.K.; Wright, E.; Bore, J.A.; Koundouno, F.R.; Konde, M.K.; et al. Pseudotyping of VSV with Ebola virus glycoprotein is superior to HIV-1 for the assessment of neutralising antibodies. Sci. Rep. 2020, 10, 14289. [CrossRef] 27. Wilkinson, D.E.; Page, M.; Mattiuzzo, G.; Hassall, M.; Dougall, T.; Rigsby, P.; Stone, L.; Minor, P. Comparison of platform technologies for assaying antibody to Ebola virus. Vaccine 2017, 35, 1347–1352. [CrossRef][PubMed] 28. Wright, E.; Temperton, N.J.; Marston, D.A.; McElhinney, L.M.; Fooks, A.R.; Weiss, R.A. Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: A cross-species comparison. J. Gen. Virol. 2008, 89, 2204–2213. [CrossRef] 29. Niwa, H.; Yamamura, K.; Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991, 108, 193–199. [CrossRef] 30. Zhao, X.; Howell, K.A.; He, S.; Brannan, J.M.; Wec, A.Z.; Davidson, E.; Turner, H.L.; Chiang, C.; Lei, L.; Fels, J.M.; et al. Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability. Cell 2017, 169, 891–904.e15. [CrossRef] 31. Rijal, P.; Elias, S.C.; Machado, S.R.; Xiao, J.; Schimanski, L.; O’Dowd, V.; Baker, T.; Barry, E.; Mendelsohn, S.C.; Cherry, C.J.; et al. Therapeutic Monoclonal Antibodies for Ebola Virus Infection Derived from Vaccinated Humans. Cell Rep. 2019, 27, 172–186.e7. [CrossRef] Trop. Med. Infect. Dis. 2021, 6, 155 11 of 12

32. Wright, E.; McNabb, S.; Goddard, T.; Horton, D.L.; Lembo, T.; Nel, L.H.; Weiss, R.A.; Cleaveland, S.; Fooks, A.R. A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of and lyssaviruses in Africa. Vaccine 2009, 27, 7178–7186. [CrossRef] 33. Mather, S.T.; Wright, E.; Scott, S.D.; Temperton, N.J. Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation -assay-based diagnostic kit. J. Virol. Methods 2014, 210, 51–58. [CrossRef] 34. Hierholzer, J.C.; Killington, R.A. Virus isolation and quantitation. In Virology Methods Manual; Mahy, B.M., Kangro, H.O., Eds.; Elsevier: Amsterdam, The Netherlands, 1996; pp. 25–46, ISBN 9780124653306. 35. Ferrara, F.; Temperton, N. Pseudotype Neutralization Assays: From laboratory Bench to Data Analysis. Methods Protoc. 2018, 1, 8. [CrossRef][PubMed] 36. Wang, H.; Shi, Y.; Song, J.; Qi, J.; Lu, G.; Yan, J.; Gao, G.F. Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1. Cell 2016, 164, 258–268. [CrossRef][PubMed] 37. Rose, A.S.; Bradley, A.R.; Valasatava, Y.; Duarte, J.M.; Prlic, A.; Rose, P.W. NGL viewer: Web-based molecular graphics for large complexes. Bioinformatics 2018, 34, 3755–3758. [CrossRef] 38. Pickett, B.E.; Sadat, E.L.; Zhang, Y.; Noronha, J.M.; Squires, R.B.; Hunt, V.; Liu, M.; Kumar, S.; Zaremba, S.; Gu, Z.; et al. ViPR: An open bioinformatics database and analysis resource for virology research. Nucleic Acids Res. 2012, 40, D593–D598. [CrossRef] 39. Madeira, F.; Park, Y.M.; Lee, J.; Buso, N.; Gur, T.; Madhusoodanan, N.; Basutkar, P.; Tivey, A.R.N.; Potter, S.C.; Finn, R.D.; et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019, 47, W636–W641. [CrossRef] 40. Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014, 42, W320–W324. [CrossRef] 41. Stremlau, M.; Owens, C.M.; Perron, M.J.; Kiessling, M.; Autissier, P.; Sodroski, J. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 2004, 427, 848–853. [CrossRef] 42. Wilkinson, D.E.; Hassall, M.; Mattiuzzo, G.; Stone, L.; Atkinson, E.; Hockley, J.; Rigsby, P.; Di Caro, A.; MacLennan, S.; Olaussen, R.; et al. WHO collaborative study to assess the suitability of the 1st International Standard and the 1st International Reference Panel for antibodies to Ebola virus. WHO Expert Commun. Biol. Stand. 2017. WHO/BS/2017.2316. Available online: https://apps.who.int/iris/handle/10665/260257 (accessed on 27 July 2021). 43. Feldmann, H.; Sprecher, A.; Geisbert, T.W. Ebola. N. Engl. J. Med. 2020, 382, 1832–1842. [CrossRef] 44. Suschak, J.J.; Schmaljohn, C.S. Vaccines against Ebola virus and : Recent advances and promising candidates. Hum. Vaccines Immunother. 2019, 15, 2359–2377. [CrossRef] 45. Saphire, E.O.; Schendel, S.L.; Fusco, M.L.; Gangavarapu, K.; Gunn, B.M.; Wec, A.Z.; Halfmann, P.J.; Brannan, J.M.; Herbert, A.S.; Qiu, X.; et al. Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection. Cell 2018, 174, 938–952.e13. [CrossRef] 46. Lambe, T.; Bowyer, G.; Ewer, K. A review of Phase I trials of Ebolavirus vaccines: What can we learn from the race to develop novel vaccines? Philos. Trans. R. Soc. B 2017, 372, 20160295. [CrossRef][PubMed] 47. Sullivan, N.J.; Martin, J.E.; Graham, B.S.; Nabel, G.J. Correlates of protective immunity for Ebola vaccines: Implications for regulatory approval by the animal rule. Nat. Rev. Microbiol. 2009, 7, 393–400. [CrossRef][PubMed] 48. King, B.; Temperton, N.J.; Grehan, K.; Scott, S.D.; Wright, E.; Tarr, A.W.; Daly, J.M. Technical considerations for the generation of novel pseudotyped viruses. Future Virol. 2016, 11, 47–59. [CrossRef] 49. Toon, K.; Bentley, E.M.; Mattiuzzo, G. More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation. Viruses 2021, 13, 217. [CrossRef][PubMed] 50. Côté, M.; Misasi, J.; Ren, T.; Bruchez, A.; Lee, K.; Filone, C.M.; Hensley, L.; Li, Q.; Ory, D.; Chandran, K.; et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection. Nature 2011, 477, 344–348. [CrossRef][PubMed] 51. Kuroda, M.; Fujikura, D.; Nanbo, A.; Marzi, A.; Noyori, O.; Kajihara, M.; Maruyama, J.; Matsuno, K.; Miyamoto, H.; Yoshida, R.; et al. Interaction between TIM-1 and NPC1 Is Important for Cellular Entry of Ebola Virus. J. Virol. 2015, 89, 6481–6493. [CrossRef][PubMed] 52. Miller, E.H.; Obernosterer, G.; Raaben, M.; Herbert, A.S.; Deffieu, M.S.; Krishnan, A.; Ndungo, E.; Sandesara, R.G.; Carette, J.E.; Kuehne, A.I.; et al. Ebola virus entry requires the host-programmed recognition of an intracellular receptor. EMBO J. 2012, 31, 1947–1960. [CrossRef][PubMed] 53. Wool-Lewis, R.J.; Bates, P. Characterization of Ebola virus entry by using pseudotyped viruses: Identification of receptor-deficient cell lines. J. Virol. 1998, 72, 3155–3160. [CrossRef][PubMed] 54. González-Hernández, M.; Müller, A.; Hoenen, T.; Hoffmann, M.; Pöhlmann, S. Calu-3 cells are largely resistant to entry driven by filovirus glycoproteins and the entry defect can be rescued by directed expression of DC-SIGN or cathepsin L. Virology 2019, 532, 22–29. [CrossRef] 55. Gnirß, K.; Kühl, A.; Karsten, C.; Glowacka, I.; Bertram, S.; Kaup, F.; Hofmann, H.; Pöhlmann, S. Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression. Virology 2012, 424, 3–10. [CrossRef][PubMed] 56. Chan, S.Y.; Empig, C.J.; Welte, F.J.; Speck, R.F.; Schmaljohn, A.; Kreisberg, J.F.; Goldsmith, M.A. Folate receptor-α is a cofactor for cellular entry by Marburg and Ebola viruses. Cell 2001, 106, 117–126. [CrossRef] 57. Ito, H.; Watanabe, S.; Takada, A. Ebola Virus Glycoprotein: Proteolytic Processing, Acylation, Cell Tropism, and Detection of Neutralizing Antibodies. J. Virol. 2001, 75, 1576–1580. [CrossRef] Trop. Med. Infect. Dis. 2021, 6, 155 12 of 12

58. Takada, A. Filovirus tropism: Cellular molecules for . Front. Microbiol. 2012, 3, 1–9. [CrossRef] 59. Takadate, Y.; Kondoh, T.; Igarashi, M.; Maruyama, J.; Manzoor, R.; Ogawa, H.; Kajihara, M.; Furuyama, W.; Sato, M.; Miyamoto, H.; et al. Niemann-Pick C1 Heterogeneity of Bat Cells Controls Filovirus Tropism. Cell Rep. 2020, 30, 308–319.e5. [CrossRef] 60. Urbanowicz, R.A.; McClure, C.P.; Sakuntabhai, A.; Sall, A.A.; Kobinger, G.; Müller, M.A.; Holmes, E.C.; Rey, F.A.; Simon-Loriere, E.; Ball, J.K. Human Adaptation of Ebola Virus during the West African Outbreak. Cell 2016, 167, 1079–1087.e5. [CrossRef] 61. Zhao, Y.; Ren, J.; Harlos, K.; Stuart, D.I. Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions. FEBS Lett. 2016, 590, 605–612. [CrossRef] 62. Ng, M.; Ndungo, E.; Kaczmarek, M.E.; Herbert, A.S.; Binger, T.; Kuehne, A.I.; Jangra, R.K.; Hawkins, J.A.; Gifford, R.J.; Biswas, R.; et al. Filovirus receptor NPC1 contributes to species-specific patterns of ebolavirus susceptibility in bats. Elife 2015, 4, 1–22. [CrossRef] 63. Watanabe, S.; Takada, A.; Watanabe, T.; Ito, H.; Kida, H.; Kawaoka, Y. Functional Importance of the Coiled-Coil of the Ebola Virus Glycoprotein. J. Virol. 2000, 74, 10194–10201. [CrossRef][PubMed] 64. Bokelmann, M.; Edenborough, K.; Hetzelt, N.; Kreher, P.; Lander, A.; Nitsche, A.; Vogel, U.; Feldmann, H.; Couacyhymann, E.; Kurth, A. Utility of primary cells to examine npc1 receptor expression in mops condylurus, a potential ebola virus reservoir. PLoS Negl. Trop. Dis. 2020, 14, 1–20. [CrossRef][PubMed] 65. Bramble, M.S.; Hoff, N.; Gilchuk, P.; Mukadi, P.; Lu, K.; Doshi, R.H.; Steffen, I.; Nicholson, B.P.; Lipson, A.; Vashist, N.; et al. Pan-filovirus serum neutralizing antibodies in a subset of Congolese ebolavirus infection survivors. J. Infect. Dis. 2018, 218, 1929–1936. [CrossRef][PubMed] 66. Wec, A.Z.; Bornholdt, Z.A.; He, S.; Herbert, A.S.; Goodwin, E.; Wirchnianski, A.S.; Gunn, B.M.; Zhang, Z.; Zhu, W.; Liu, G.; et al. Development of a Human Antibody Cocktail that Deploys Multiple Functions to Confer Pan-Ebolavirus Protection. Cell Host Microbe 2019, 25, 39–48.e5. [CrossRef][PubMed]