Rescue and characterization of the first West African Marburg virus 2021 from Guinea.

Marburg virus (MARV) is a causative agent of a severe hemorrhagic fever with high fatality rates endemic in central Africa. Current outbreaks of MARV in Equatorial Guinea and Tanzania underline the relevance of MARV as a public health emergency pathogen. In 2021, the first known human MARV case was confirmed in Guinea, West Africa. Since no infectious virus could be isolated from that fatal case in 2021, we generated recombinant (rec) MARV Guinea by reverse genetics in order to study and characterize this new MARV, which occurred in West Africa for the first time, in terms of its growth properties, detection by antibodies, and therapeutic potential compared to known MARV strains. Our results showed a solid viral replication of recMARV Guinea in human, bat, and monkey cell lines in comparison to other known MARV strains. We further demonstrated that replication of recMARV Guinea in cells can be inhibited by the nucleoside analogue remdesivir. Taken together, we could successfully reconstitute de novo the first West African MARV from Guinea showing similar replication kinetics in cells compared to other central African MARV strains. Our reverse genetics approach has proven successful in characterizing emerging viruses, especially when virus isolates are missing and viral genome sequences are incomplete.

Short- and Long-Interval Prime-Boost Vaccination with the Candidate Vaccines MVA-SARS-2-ST and MVA-SARS-2-S Induces Comparable Humoral and Cell-Mediated Immunity in Mice.

The COVID-19 pandemic caused significant human health and economic consequences. Due to the ability of SARS-CoV-2 to spread rapidly and to cause severe disease and mortality in certain population groups, vaccines are essential for controlling the pandemic in the future. Several licensed vaccines have shown improved protection against SARS-CoV-2 after extended-interval prime-boost immunizations in humans. Therefore, in this study, we aimed to compare the immunogenicity of our two Modified Vaccinia virus Ankara (MVA) based COVID-19 candidate vaccines MVA-SARS-2-S and MVA-SARS-2-ST after short- and long-interval prime-boost immunization schedules in mice. We immunized BALB/c mice using 21-day (short-interval) or 56-day (long-interval) prime-boost vaccination protocols and analyzed spike (S)-specific CD8 T cell immunity and humoral immunity. The two schedules induced robust CD8 T cell responses with no significant differences in their magnitude. Furthermore, both candidate vaccines induced comparable levels of total S, and S2-specific IgG binding antibodies. However, MVA-SARS-2-ST consistently elicited higher amounts of S1-, S receptor binding domain (RBD), and SARS-CoV-2 neutralizing antibodies in both vaccination protocols. Overall, we found very comparable immune responses following short- or long-interval immunization. Thus, our results suggest that the chosen time intervals may not be suitable to observe potential differences in antigen-specific immunity when testing different prime-boost intervals with our candidate vaccines in the mouse model. Despite this, our data clearly showed that MVA-SARS-2-ST induced superior humoral immune responses relative to MVA-SARS-2-S after both immunization schedules.

Inhibition of Rab1B Impairs Trafficking and Maturation of SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes cellular trafficking pathways to process its structural proteins and move them to the site of assembly. Nevertheless, the exact process of assembly and subcellular trafficking of SARS-CoV-2 proteins remains largely unknown. Here, we have identified and characterized Rab1B as an important host factor for the trafficking and maturation of the spike protein (S) after synthesis at the endoplasmic reticulum (ER). Using confocal microscopy, we showed that S and Rab1B substantially colocalized in compartments of the early secretory pathway. Co-expression of dominant-negative (DN) Rab1B N121I leads to an aberrant distribution of S into perinuclear spots after ectopic expression and in SARS-CoV-2-infected cells caused by either structural rearrangement of the ERGIC or Golgi or missing interaction between Rab1B and S. Western blot analyses revealed a complete loss of the mature, cleaved S2 subunit in cell lysates and culture supernatants upon co-expression of DN Rab1B N121I. In sum, our studies indicate that Rab1B is an important regulator of trafficking and maturation of SARS-CoV-2 S, which not only improves our understanding of the coronavirus replication cycle but also may have implications for the development of antiviral strategies.

Self-amplifying RNA vaccine protects mice against lethal Ebola virus infection.

Emerging and re-emerging viruses, such as Zaire Ebola virus (EBOV), pose a global threat and require immediate countermeasures, including the rapid development of effective vaccines that are easy to manufacture. Synthetic self-amplifying RNAs (saRNAs) attend to these needs, being safe and strong immune stimulators that can be inexpensively produced in large quantities, using cell-free systems and good manufacturing practice. Here, the first goal was to develop and optimize an anti-EBOV saRNA-based vaccine in terms of its antigen composition and route of administration. Vaccinating mice with saRNAs expressing the EBOV glycoprotein (GP) alone or in combination with the nucleoprotein (NP) elicited antigen-specific immune responses. GP-specific antibodies showed neutralizing activity against EBOV. Strong CD4+ T cell response against NP and GP and CD8+ T cell response against NP were detected by ELISpot assays. Intramuscular vaccination with saRNAs conferred better immune response than intradermal. Finally, mice vaccinated in a prime-boost regimen with saRNAs encoding both GP and NP or with GP alone survived an EBOV infection. In addition, a single dose of GP and NP saRNAs was also protective against fatal EBOV infection. Overall, saRNAs expressing viral antigens represent a promising vaccine platform.

Stabilized recombinant SARS-CoV-2 spike antigen enhances vaccine immunogenicity and protective capacity.

The SARS-CoV-2 spike (S) glycoprotein is synthesized as a large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as a candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S-infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/S2 cleavage site and K986P and V987P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of broadly reactive anti-S antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.

Ebola Virus Activates IRE1α-Dependent XBP1u Splicing.

Ebola (EBOV) and Marburg virus (MARV) are highly pathogenic filoviruses that influence cellular signaling according to their own needs. MARV has been shown to regulate the IRE1α-dependent unfolded protein response (UPR) to ensure optimal virus replication. It was not known whether EBOV affects this signaling cascade, which can be beneficial or detrimental for viruses. Activation of IRE1α leads to the expression of the transcription factor XBP1s, which binds to cis-acting UPR elements (UPRE), resulting in the expression of genes aimed at restoring homeostasis in the endoplasmic reticulum. We observed that EBOV infection, in contrast to MARV infection, led to UPR activation by IRE1α-dependent but not ATF6-dependent signaling. We showed an activation of IRE1α, XBP1s and UPRE target genes upon EBOV infection. ATF6, another UPRE transcription factor, was not activated. UPRE activation was mainly attributed to the EBOV nucleoprotein NP and the soluble glycoprotein sGP. Finally, activation of UPR by thapsigargin, a potent ER-stress inducer, in parallel to infection as well as knock-out of XBP1 had no effect on EBOV growth, while MARV proliferation was affected by thapsigargin-dependent UPR activation. Taken together EBOV and MARV differ in their strategy of balancing IRE1α-dependent signaling for their own needs.

Intranasal Administration of a Monoclonal Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection.

Despite the recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of the SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2, retains full activity against the variant of concern (VOC) B.1.1.7 and still neutralizes the VOC B.1.351, although with reduced potency. Importantly, not only systemic but also intranasal application of DZIF-10c abolished the presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology when administered prophylactically. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8+ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Development and characterization of an indirect ELISA to detect SARS-CoV-2 spike protein-specific antibodies.

The current Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic is a public health emergency of international concern. Sensitive and precise diagnostic tools are urgently needed. In this study, we developed a SARS-CoV-2 spike (S1) protein enzyme-linked immunosorbent assay (ELISA) to detect SARS-CoV-2-specific antibodies. The SARS-CoV-2 S1 ELISA was found to be specific [97.8% (95% CI, 96.7% - 98.5%)], reproducible and precise (intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%). A standard curve and the interpolation of arbitrary ELISA units per milliliter served to reduce the variability between different tests and operators. Cross-reactivity to other human coronaviruses was addressed by using sera positive for MERS-CoV- and hCoV HKU1-specific antibodies. Monitoring antibody development in various samples of twenty-three and single samples of twenty-nine coronavirus disease 2019 (COVID-19) patients revealed seroconversion and neutralizing antibodies against authentic SARS-CoV-2 in all cases. The comparison of the SARS-CoV-2 (S1) ELISA with a commercially available assay showed a better sensitivity for the in-house ELISA. The results demonstrate a high reproducibility, specificity and sensitivity of the newly developed ELISA, which is suitable for the detection of SARS-CoV-2 S1 protein-specific antibody responses.

Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients.

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and the world economy. Because approved drugs and vaccines are limited or not available, new options for COVID-19 treatment and prevention are in high demand. To identify SARS-CoV-2-neutralizing antibodies, we analyzed the antibody response of 12 COVID-19 patients from 8 to 69 days after diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days after diagnosis. Of these, 28 potently neutralized authentic SARS-CoV-2 with IC100 as low as 0.04 µg/mL, showing a broad spectrum of variable (V) genes and low levels of somatic mutations. Interestingly, potential precursor sequences were identified in naive B cell repertoires from 48 healthy individuals who were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2-neutralizing antibodies are readily generated from a diverse pool of precursors, fostering hope for rapid induction of a protective immune response upon vaccination.

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells.

The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

Safety and immunogenicity of a modified vaccinia virus Ankara vector vaccine candidate for Middle East respiratory syndrome: an open-label, phase 1 trial.

BACKGROUND: The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a respiratory disease with a case fatality rate of up to 35%. Given its potential to cause a public health emergency and the absence of efficacious drugs or vaccines, MERS is one of the WHO priority diseases warranting urgent research and development of countermeasures. We aimed to assess safety and tolerability of an anti-MERS-CoV modified vaccinia virus Ankara (MVA)-based vaccine candidate that expresses the MERS-CoV spike glycoprotein, MVA-MERS-S, in healthy adults. METHODS: This open-label, phase 1 trial was done at the University Medical Center Hamburg-Eppendorf (Hamburg, Germany). Participants were healthy men and women aged 18-55 years with no clinically significant health problems as determined during medical history and physical examination, a body-mass index of 18·5-30·0 kg/m2 and weight of more than 50 kg at screening, and a negative pregnancy test for women. A key exclusion criterion was a previous MVA vaccination. For the prime immunisation, participants received doses of 1 × 107 plaque-forming unit (PFU; low-dose group) or 1 × 108 PFU (high-dose group) MVA-MERS-S intramuscularly. A second identical dose was administered intramuscularly as a booster immunisation 28 days after first injection. As a control group for immunogenicity analyses, blood samples were drawn at identical study timepoints from six healthy adults, who did not receive any injections. The primary objectives of the study were safety and tolerability of the two dosage levels and reactogenicity after administration. Immunogenicity was assessed as a secondary endpoint by ELISA and neutralisation tests. T-cell immunity was evaluated by interferon-γ-linked enzyme-linked immune absorbent spot assay. All participants who were vaccinated at least once were included in the safety analysis. Immunogenicity was analysed in the participants who completed 6 months of follow-up. This trial is registered with ClinicalTrials.gov, NCT03615911, and EudraCT, 2014-003195-23 FINDINGS: From Dec 17, 2017, to June 5, 2018, 26 participants (14 in the low-dose group and 12 in the high-dose group) were enrolled and received the first dose of the vaccine according to their group allocation. Of these, 23 participants (12 in the low-dose group and 11 in the high-dose group) received a second dose of MVA-MERS-S according to their group allocation after a 28-day interval and completed follow-up. Homologous prime-boost immunisation with MVA-MERS-S revealed a benign safety profile with only transient mild-to-moderate reactogenicity. Participants had no severe or serious adverse events. 67 vaccine-related adverse events were reported in ten (71%) of 14 participants in the low-dose group, and 111 were reported in ten (83%) of 12 participants in the high-dose group. Solicited local reactions were the most common adverse events: pain was observed in 17 (65%; seven in the low-dose group vs ten in the high-dose group) participants, swelling in ten (38%; two vs eight) participants, and induration in ten (38%; one vs nine) participants. Headaches (observed in seven participants in the low-dose group vs nine in the high-dose group) and fatigue or malaise (ten vs seven participants) were the most common solicited systemic adverse events. All adverse events resolved swiftly (within 1-3 days) and without sequelae. Following booster immunisation, nine (75%) of 12 participants in the low-dose group and 11 (100%) participants in the high-dose group showed seroconversion using a MERS-CoV S1 ELISA at any timepoint during the study. Binding antibody titres correlated with MERS-CoV-specific neutralising antibodies (Spearman's correlation r=0·86 [95% CI 0·6960-0·9427], p=0·0001). MERS-CoV spike-specific T-cell responses were detected in ten (83%) of 12 immunised participants in the low-dose group and ten (91%) of 11 immunised participants in the high-dose group. INTERPRETATION: Vaccination with MVA-MERS-S had a favourable safety profile without serious or severe adverse events. Homologous prime-boost immunisation induced humoral and cell-mediated responses against MERS-CoV. A dose-effect relationship was demonstrated for reactogenicity, but not for vaccine-induced immune responses. The data presented here support further clinical testing of MVA-MERS-S in larger cohorts to advance MERS vaccine development. FUNDING: German Center for Infection Research.

Safety and immunogenicity of a candidate Middle East respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial.

BACKGROUND: Cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection continue to rise in the Arabian Peninsula 7 years after it was first described in Saudi Arabia. MERS-CoV poses a significant risk to public health security because of an absence of currently available effective countermeasures. We aimed to assess the safety and immunogenicity of the candidate simian adenovirus-vectored vaccine expressing the full-length spike surface glycoprotein, ChAdOx1 MERS, in humans. METHODS: This dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial was done at the Centre for Clinical Vaccinology and Tropical Medicine (Oxford, UK) and included healthy people aged 18-50 years with negative pre-vaccination tests for HIV antibodies, hepatitis B surface antigen, and hepatitis C antibodies (and a negative urinary pregnancy test for women). Participants received a single intramuscular injection of ChAdOx1 MERS at three different doses: the low-dose group received 5 × 109 viral particles, the intermediate-dose group received 2·5 × 1010 viral particles, and the high-dose group received 5 × 1010 viral particles. The primary objective was to assess safety and tolerability of ChAdOx1 MERS, measured by the occurrence of solicited, unsolicited, and serious adverse events after vaccination. The secondary objective was to assess the cellular and humoral immunogenicity of ChAdOx1 MERS, measured by interferon-γ-linked enzyme-linked immunospot, ELISA, and virus neutralising assays after vaccination. Participants were followed up for up to 12 months. This study is registered with ClinicalTrials.gov, NCT03399578. FINDINGS: Between March 14 and Aug 15, 2018, 24 participants were enrolled: six were assigned to the low-dose group, nine to the intermediate-dose group, and nine to the high-dose group. All participants were available for follow-up at 6 months, but five (one in the low-dose group, one in the intermediate-dose group, and three in the high-dose group) were lost to follow-up at 12 months. A single dose of ChAdOx1 MERS was safe at doses up to 5 × 1010 viral particles with no vaccine-related serious adverse events reported by 12 months. One serious adverse event reported was deemed to be not related to ChAdOx1 MERS. 92 (74% [95% CI 66-81]) of 124 solicited adverse events were mild, 31 (25% [18-33]) were moderate, and all were self-limiting. Unsolicited adverse events in the 28 days following vaccination considered to be possibly, probably, or definitely related to ChAdOx1 MERS were predominantly mild in nature and resolved within the follow-up period of 12 months. The proportion of moderate and severe adverse events was significantly higher in the high-dose group than in the intermediate-dose group (relative risk 5·83 [95% CI 2·11-17·42], p<0·0001) Laboratory adverse events considered to be at least possibly related to the study intervention were self-limiting and predominantly mild in severity. A significant increase from baseline in T-cell (p<0·003) and IgG (p<0·0001) responses to the MERS-CoV spike antigen was observed at all doses. Neutralising antibodies against live MERS-CoV were observed in four (44% [95% CI 19-73]) of nine participants in the high-dose group 28 days after vaccination, and 19 (79% [58-93]) of 24 participants had antibodies capable of neutralisation in a pseudotyped virus neutralisation assay. INTERPRETATION: ChAdOx1 MERS was safe and well tolerated at all tested doses. A single dose was able to elicit both humoral and cellular responses against MERS-CoV. The results of this first-in-human clinical trial support clinical development progression into field phase 1b and 2 trials. FUNDING: UK Department of Health and Social Care, using UK Aid funding, managed by the UK National Institute for Health Research.

Marburg virus regulates the IRE1/XBP1-dependent unfolded protein response to ensure efficient viral replication.

Viruses regulate cellular signalling pathways to ensure optimal viral replication. During Marburg virus (MARV) infection, large quantities of the viral glycoprotein GP are produced in the ER; this may result in the activation of the unfolded protein response (UPR). The most conserved pathway to trigger UPR is initiated by IRE1. Activation of IRE1 results in auto-phosphorylation, splicing of the XBP1 mRNA and translation of the XBP1s protein. XBP1s binds cis-acting UPR elements (UPRE) which leads to the enhanced expression of genes which should restore ER homeostasis. XBP1u protein is translated, if IRE1 is not activated. Here we show that ectopic expression of MARV GP activated the IRE1-XBP1 axis of UPR as monitored by UPRE luciferase assays. However, while at 24 h of infection with MARV IRE1 was phosphorylated, expression of XBP1s was only slightly enhanced and UPRE activity was not detected. The IRE1-XBP1 axis was not active at 48 h p.i. Co-expression studies of MARV proteins demonstrated that the MARV protein VP30 suppressed UPRE activation. Co-immunoprecipitation analyses revealed an RNA-dependent interaction of VP30 with XBP1u. Knock-out of IRE1 supported MARV infection at late time points. Taken together, these results suggest that efficient MARV propagation requires specific regulation of IRE1 activity.

A Fluorescently Labeled Marburg Virus Glycoprotein as a New Tool to Study Viral Transport and Assembly.

The single surface glycoprotein (GP) of filoviruses is indispensable for recognition of its cellular receptor and infection of target cells. To study the intracellular trafficking of GP by using live-cell imaging, the mucin-like domain of Marburg virus (MARV) GP was replaced by the fluorophore mCherry (GP∆MLD_mCherry). Intracellular distribution, surface transport, and recruitment of GP∆MLD_mCherry into virus-like particles were similar to observations for wild-type GP. Using reverse genetics, we generated a recombinant MARV expressing GP∆MLD_mCherry (recMARV MARVGP∆MLD_mCherry). Time-lapse microscopy of recMARV MARVGP∆MLD_mCherry-infected cells revealed that GP∆MLD_mCherry-positive vesicles were transported to the cell surface in a tubulin-dependent manner. Moreover, dual-color live-cell imaging revealed cotransport of GPΔMLD_mCherry and VP40 and their colocalization at the plasma membrane. In this proof-of-concept study we showed that the newly developed GP∆MLD_mCherry is a promising tool to elucidate intracellular trafficking and assembly pathways of MARV.

Development of an antibody capture ELISA using inactivated Ebola Zaire Makona virus.

The 2014 Zaire Ebola virus (ZEBOV) outbreak in West Africa represents an international public health concern. Highly sensitive and precise diagnostic tools are needed. In the present study, we developed a ZEBOV-specific enzyme-linked immunosorbent assay (ELISA) using inactivated ZEBOV isolate Makona from March 2014. Mock antigen was used to address nonspecific binding. Specificity, reproducibility and precision were determined to measure assay performance. The ZEBOV ELISA proved to be specific (96 %), reproducible and precise (Intra-assay CV 8 %, Inter-assay CV 18 %). Using the human monoclonal antibody KZ52, we showed that the ELISA was able to detect conformation-specific antibodies. Monitoring antibody development in 29 PCR-positive EBOV disease (EVD) patients revealed seroconversion in all cases. In addition, the ELISA was used to detect ZEBOV glycoprotein (GP)-specific antibodies in a vaccinated volunteer from day 14 until 5 years post-vaccination with a VSV-ZEBOV candidate vaccine. The results demonstrate the high reproducibility, specificity and sensitivity of this newly developed ELISA, which is suitable for the detection of specific antibody responses directed against different ZEBOV proteins in EVD patients and against the ZEBOV surface glycoprotein GP in vaccinated individuals.