Publications by authors named "Alexandra Kupke"

15 Publications

  • Page 1 of 1

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

J Immunol Methods 2021 03 4;490:112958. Epub 2021 Jan 4.

Institute of Virology, Philipps University Marburg, Marburg, Germany; German Center for Infection Research (DZIF), Partner Site Gießen-Marburg-Langen, Marburg, Germany.

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.
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http://dx.doi.org/10.1016/j.jim.2021.112958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831464PMC
March 2021

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

Cell 2020 08 13;182(4):843-854.e12. Epub 2020 Jul 13.

Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany; German Center for Infection Research, Partner Site Bonn-Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany. Electronic address:

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 IC 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.
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http://dx.doi.org/10.1016/j.cell.2020.06.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355337PMC
August 2020

Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirus interferon-λ and in mice.

Eur Respir J 2020 Nov 26;56(5). Epub 2020 Nov 26.

Dept of Internal Medicine II, University of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.

While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication , correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication and upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.
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http://dx.doi.org/10.1183/13993003.01826-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331652PMC
November 2020

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.

Lancet Infect Dis 2020 07 21;20(7):816-826. Epub 2020 Apr 21.

The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. Electronic address:

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 × 10 viral particles, the intermediate-dose group received 2·5 × 10 viral particles, and the high-dose group received 5 × 10 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 × 10 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.
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http://dx.doi.org/10.1016/S1473-3099(20)30160-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7172901PMC
July 2020

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

Lancet Infect Dis 2020 07 21;20(7):827-838. Epub 2020 Apr 21.

First Department of Medicine, Division of Infectious Diseases, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department for Clinical Immunology of Infectious Diseases, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; German Center for Infection Research, Hamburg-Lubeck-Borstel-Riems, Germany. Electronic address:

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/m 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 × 10 plaque-forming unit (PFU; low-dose group) or 1 × 10 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.
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http://dx.doi.org/10.1016/S1473-3099(20)30248-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7172913PMC
July 2020

Osteogenesis imperfecta in a male holstein calf associated with a possible oligogenic origin.

Vet Q 2020 Dec;40(1):58-67

Institute of Veterinary Medicine, University of Goettingen, Göttingen, Germany.

Neuromusculoskeletal anomalies generally in combination with severe clinical symptoms, comprise a heterogeneous group of fairly common and mostly fatal disorders in man and animals. Osteogenesis imperfecta (OI), also known as brittle bone disease, causes bone fragility and deformity. Prominent extra-skeletal accessory manifestations of OI comprise blue/gray sclerae, hearing impairment, lung abnormalities and hypercalciuria. Cases of OI in cattle have been reported. However, no causative mutations have been identified in cattle so far. To report a possible oligogenic origin identified in a calf from clinically healthy parents suffering from OI. A neonatal embryo transfer male Holstein calf developing multiple fractures with bone tissue showing marked osteopenia was used for whole genome re-sequencing as well as its parents. In addition, 2,612 randomly chosen healthy Holstein cattle were genotyped as well as controls. Sixteen candidate genes with potential protein-altering variants were selected revealing non-synonymous variants only within and genes. However, in-depth gene analysis did not result in the identification of a single causative mutation in the OI calf. The analysis of the OI case revealed a possible oligogenic origin of the disease attributable to additive effects of three candidate genes, i.e., , , and . Most OI cases in humans and domestic animals reported so far are caused by distinct dominant or recessive monogenic mutations, therefore a potential oligogenic additive genetic effect is a novel finding. Furthermore, the case presented here demonstrates that cross-species genetic analyses might not always be straightforward.
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http://dx.doi.org/10.1080/01652176.2020.1721611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034473PMC
December 2020

Humoral Immunogenicity and Efficacy of a Single Dose of ChAdOx1 MERS Vaccine Candidate in Dromedary Camels.

Sci Rep 2019 11 8;9(1):16292. Epub 2019 Nov 8.

Department of Infectious Disease Research, King Abdullah International Medical Research Center, Riyadh, Saudi Arabia.

MERS-CoV seronegative and seropositive camels received a single intramuscular dose of ChAdOx1 MERS, a replication-deficient adenoviral vectored vaccine expressing MERS-CoV spike protein, with further groups receiving control vaccinations. Infectious camels with active naturally acquired MERS-CoV infection, were co-housed with the vaccinated camels at a ratio of 1:2 (infected:vaccinated); nasal discharge and virus titres were monitored for 14 days. Overall, the vaccination reduced virus shedding and nasal discharge (p = 0.0059 and p = 0.0274, respectively). Antibody responses in seropositive camels were enhancedby the vaccine; these camels had a higher average age than seronegative. Older seronegative camels responded more strongly to vaccination than younger animals; and neutralising antibodies were detected in nasal swabs. Further work is required to optimise vaccine regimens for younger seronegative camels.
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http://dx.doi.org/10.1038/s41598-019-52730-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6841732PMC
November 2019

Intranasal Borna Disease Virus (BoDV-1) Infection: Insights into Initial Steps and Potential Contagiosity.

Int J Mol Sci 2019 Mar 15;20(6). Epub 2019 Mar 15.

Institute of Veterinary Pathology, Justus Liebig University, 35392 Giessen, Germany.

Mammalian Bornavirus (BoDV-1) typically causes a fatal neurologic disorder in horses and sheep, and was recently shown to cause fatal encephalitis in humans with and without transplant reception. It has been suggested that BoDV-1 enters the central nervous system (CNS) via the olfactory pathway. However, (I) susceptible cell types that replicate the virus for successful spread, and (II) the role of olfactory ensheathing cells (OECs), remained unclear. To address this, we studied the intranasal infection of adult rats with BoDV-1 in vivo and in vitro, using olfactory mucosal (OM) cell cultures and the cultures of purified OECs. Strikingly, in vitro and in vivo, viral antigen and mRNA were present from four days post infection (dpi) onwards in the olfactory receptor neurons (ORNs), but also in all other cell types of the OM, and constantly in the OECs. In contrast, in vivo, BoDV-1 genomic RNA was only detectable in adult and juvenile ORNs, nerve fibers, and in OECs from 7 dpi on. In vitro, the rate of infection of OECs was significantly higher than that of the OM cells, pointing to a crucial role of OECs for infection via the olfactory pathway. Thus, this study provides important insights into the transmission of neurotropic viral infections with a zoonotic potential.
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http://dx.doi.org/10.3390/ijms20061318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470550PMC
March 2019

The molecular tweezer CLR01 inhibits Ebola and Zika virus infection.

Antiviral Res 2018 04 8;152:26-35. Epub 2018 Feb 8.

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany; U-PEP and Core Facility Functional Peptidomics, Ulm University, 89081 Ulm, Germany. Electronic address:

Ebola (EBOV) and Zika viruses (ZIKV) are responsible for recent global health threats. As no preventive vaccines or antiviral drugs against these two re-emerging pathogens are available, we evaluated whether the molecular tweezer CLR01 may inhibit EBOV and ZIKV infection. This small molecule has previously been shown to inactivate HIV-1 and herpes viruses through a selective interaction with lipid-raft-rich regions in the viral envelope, which results in membrane disruption and loss of infectivity. We found that CLR01 indeed blocked infection of EBOV and ZIKV in a dose-dependent manner. The tweezer inhibited infection of epidemic ZIKV strains in cells derived from the anogenital tract and the central nervous system, and remained antivirally active in the presence of semen, saliva, urine and cerebrospinal fluid. Our findings show that CLR01 is a broad-spectrum inhibitor of enveloped viruses with prospects as a preventative microbicide or antiviral agent.
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http://dx.doi.org/10.1016/j.antiviral.2018.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113745PMC
April 2018

Spectrum of pathogen- and model-specific histopathologies in mouse models of acute pneumonia.

PLoS One 2017 20;12(11):e0188251. Epub 2017 Nov 20.

Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.

Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188251PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695780PMC
December 2017

ChAdOx1 and MVA based vaccine candidates against MERS-CoV elicit neutralising antibodies and cellular immune responses in mice.

Vaccine 2017 06 1;35(30):3780-3788. Epub 2017 Jun 1.

The Jenner Institute, University of Oxford, Oxford OX3 7DQ, UK.

The Middle East respiratory syndrome coronavirus (MERS-CoV) has infected more than 1900 humans, since 2012. The syndrome ranges from asymptomatic and mild cases to severe pneumonia and death. The virus is believed to be circulating in dromedary camels without notable symptoms since the 1980s. Therefore, dromedary camels are considered the only animal source of infection. Neither antiviral drugs nor vaccines are approved for veterinary or medical use despite active research on this area. Here, we developed four vaccine candidates against MERS-CoV based on ChAdOx1 and MVA viral vectors, two candidates per vector. All vaccines contained the full-length spike gene of MERS-CoV; ChAdOx1 MERS vaccines were produced with or without the leader sequence of the human tissue plasminogen activator gene (tPA) where MVA MERS vaccines were produced with tPA, but either the mH5 or F11 promoter driving expression of the spike gene. All vaccine candidates were evaluated in a mouse model in prime only or prime-boost regimens. ChAdOx1 MERS with tPA induced higher neutralising antibodies than ChAdOx1 MERS without tPA. A single dose of ChAdOx1 MERS with tPA elicited cellular immune responses as well as neutralising antibodies that were boosted to a significantly higher level by MVA MERS. The humoral immunogenicity of a single dose of ChAdOx1 MERS with tPA was equivalent to two doses of MVA MERS (also with tPA). MVA MERS with mH5 or F11 promoter induced similar antibody levels; however, F11 promoter enhanced the cellular immunogenicity of MVA MERS to significantly higher magnitudes. In conclusion, our study showed that MERS-CoV vaccine candidates could be optimized by utilising different viral vectors, various genetic designs of the vectors, or different regimens to increase immunogenicity. ChAdOx1 and MVA vectored vaccines have been safely evaluated in camels and humans and these MERS vaccine candidates should now be tested in camels and in clinical trials.
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http://dx.doi.org/10.1016/j.vaccine.2017.05.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516308PMC
June 2017

Intranasal Location and Immunohistochemical Characterization of the Equine Olfactory Epithelium.

Front Neuroanat 2016 13;10:97. Epub 2016 Oct 13.

Institute of Veterinary Pathology, Faculty of Veterinary Medicine, Justus Liebig University Giessen Giessen, Germany.

The olfactory epithelium (OE) is the only body site where neurons contact directly the environment and are therefore exposed to a broad variation of substances and insults. It can serve as portal of entry for neurotropic viruses which spread via the olfactory pathway to the central nervous system. For horses, it has been proposed and concluded mainly from rodent studies that different viruses, e.g., Borna disease virus, equine herpesvirus 1 (EHV-1), hendra virus, influenza virus, rabies virus, vesicular stomatitis virus can use this route. However, little is yet known about cytoarchitecture, protein expression and the intranasal location of the equine OE. Revealing differences in cytoarchitecture or protein expression pattern in comparison to rodents, canines, or humans might help to explain varying susceptibility to certain intranasal virus infections. On the other hand, disclosing similarities especially between rodents and other species, e.g., horses would help to underscore transferability of rodent models. Analysis of the complete noses of five adult horses revealed that in the equine OE two epithelial subtypes with distinct marker expression exist, designated as and which resemble those previously described in dogs. Detailed statistical analysis was carried out to confirm the results obtained on the descriptive level. The equine OE was predominantly located in caudodorsal areas of the nasal turbinates with a significant decline in rostroventral direction, especially for . Immunohistochemically, olfactory marker protein and doublecortin (DCX) expression was found in more cells of OE , whereas expression of proliferating cell nuclear antigen and tropomyosin receptor kinase A was present in more cells of . Accordingly, resembles the mature epithelium, in contrast to the more juvenile . Protein expression profile was comparable to canine and rodent OE but equine and were located differently within the nose and revealed differences in its cytoarchitecture when compared to canine OE. Equine OE closely resembles rat OE. Whether the observed differences contribute to species-specific susceptibility to intranasal insults such as virus infections has to be further investigated.
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http://dx.doi.org/10.3389/fnana.2016.00097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061740PMC
October 2016

A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform.

J Virol 2015 Nov 9;89(22):11654-67. Epub 2015 Sep 9.

Oncolytic Measles Viruses and Vaccine Vectors, Paul-Ehrlich-Institut, Langen, Germany German Center for Infection Research, Langen, Germany

Unlabelled: In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR(-/-))-CD46Ge mice with 2 × 10(5) 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice.

Importance: Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV.
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http://dx.doi.org/10.1128/JVI.01815-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645655PMC
November 2015

Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein.

J Virol 2015 Aug 27;89(16):8651-6. Epub 2015 May 27.

German Center for Infection Research (DZIF), Institute for Infectious Diseases and Zoonoses, LMU University of Munich, Munich, Germany

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8(+) T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.
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http://dx.doi.org/10.1128/JVI.00614-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524222PMC
August 2015