Publications by authors named "Anna Offersgaard"

8 Publications

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Inactivated whole hepatitis C virus vaccine employing a licensed adjuvant elicits cross-genotype neutralizing antibodies in mice.

J Hepatol 2022 Jan 3. Epub 2022 Jan 3.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital - Amager and Hvidovre, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Background And Aims: A prophylactic vaccine is required to eliminate hepatitis C virus (HCV) as a global public health threat, causing 400,000 deaths annually due to HCV-related liver diseases. We developed whole virus inactivated HCV vaccine candidates employing a licensed adjuvant and inducing broadly neutralizing antibodies in mice. Further, we investigated effects of HCV envelope protein modifications increasing neutralization epitope exposure on immunogenicity.

Methods: Whole virus vaccine antigen was produced in Huh7.5 hepatoma cells, processed using a multistep protocol and formulated with adjuvant (MF-59 analogue AddaVax or aluminium hydroxide). For IgG purified from serum of immunized BALB/c mice, we investigated capacity to neutralize genotype 1-6 HCV by virus neutralization assays and to bind homologous envelope proteins by ELISA. Viruses used for immunizations were (i) HCV5aHi with strain SA13 envelope proteins and modification of an O-linked glycosylation site in E2 (T385P), (ii) HCV5aHi(T385) with reversion of T385P to T385, featuring the original E2 sequence determined in vivo and (iii) HCV5aHi(ΔHVR1) with deletion of HVR1. For these viruses, epitope exposure was investigated using human monoclonal (AR3A and AR4A) and polyclonal (C211 and H06) antibodies in neutralization assays.

Results: Processed HCV5aHi formulated with AddaVax induced antibodies, efficiently binding homologous envelope proteins and broadly neutralizing cultured genotype 1-6 HCV with IC50 between 14 and 192μg/mL; mean IC50 against the homologous virus was 36μg/mL. Vaccination with aluminium hydroxide was less immunogenic. Compared to HCV5aHi(T385) with the original E2 sequence, HCV5aHi with a modified glycosylation site and HCV5aHi(ΔHVR1) without HVR1 showed increased neutralization epitope exposure but similar immunogenicity.

Conclusion: Using an adjuvant suitable for human use, we developed inactivated whole HCV vaccine candidates inducing broadly neutralizing antibodies for further pre-clinical development.

Lay Summary: A vaccine against hepatitis C virus is needed to prevent an estimated 2 million new infections and 400,000 deaths caused by this virus each year. We developed inactivated whole hepatitis C virus vaccine candidates using adjuvants licensed for human use, which, following immunization of mice, induced antibodies that efficiently neutralized all HCV genotypes with recognized epidemiological importance. Hepatitis C virus variants with modified envelope proteins with increased exposure of neutralization epitopes showed similar immunogenicity as the virus with the original envelope proteins.
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http://dx.doi.org/10.1016/j.jhep.2021.12.026DOI Listing
January 2022

Efficacy of Ion-Channel Inhibitors Amantadine, Memantine and Rimantadine for the Treatment of SARS-CoV-2 In Vitro.

Viruses 2021 10 15;13(10). Epub 2021 Oct 15.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, Kettegård Alle 30, 2650 Hvidovre, Denmark.

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.
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http://dx.doi.org/10.3390/v13102082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8537953PMC
October 2021

In vitro efficacy of artemisinin-based treatments against SARS-CoV-2.

Sci Rep 2021 07 16;11(1):14571. Epub 2021 Jul 16.

Max Planck Institute for Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.
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http://dx.doi.org/10.1038/s41598-021-93361-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285423PMC
July 2021

SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor.

Vaccines (Basel) 2021 Jun 29;9(7). Epub 2021 Jun 29.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, 2650 Hvidovre, Denmark.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradle 500-AP bioreactor. CelCradle 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 10 Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2-2.5 × 10 cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log 50% tissue culture infectious dose (TCID)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log TCID/mL, and a total of 10.5 log TCID were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.
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http://dx.doi.org/10.3390/vaccines9070706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310283PMC
June 2021

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 .

Antimicrob Agents Chemother 2021 08 17;65(9):e0268020. Epub 2021 Aug 17.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovregrid.5254.6, Hvidovre, Denmark.

Antivirals targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could improve treatment of COVID-19. We evaluated the efficacy of clinically relevant hepatitis C virus (HCV) NS3 protease inhibitors (PIs) against SARS-CoV-2 and their interactions with remdesivir, the only direct-acting antiviral approved for COVID-19 treatment. HCV PIs showed differential potency in short-term treatment assays based on the detection of SARS-CoV-2 spike protein in Vero E6 cells. Linear PIs boceprevir, telaprevir, and narlaprevir had 50% effective concentrations (EC) of ∼40 μM. Among the macrocyclic PIs, simeprevir had the highest (EC, 15 μM) and glecaprevir the lowest (EC, >178 μM) potency, with paritaprevir, grazoprevir, voxilaprevir, vaniprevir, danoprevir, and deldeprevir in between. Acyclic PIs asunaprevir and faldaprevir had ECs of 72 and 23 μM, respectively. ACH-806, inhibiting the HCV NS4A protease cofactor, had an EC of 46 μM. Similar and slightly increased PI potencies were found in human hepatoma Huh7.5 cells and human lung carcinoma A549-hACE2 cells, respectively. Selectivity indexes based on antiviral and cell viability assays were highest for linear PIs. In short-term treatments, combination of macrocyclic but not linear PIs with remdesivir showed synergism in Vero E6 and A549-hACE2 cells. Longer-term treatment of infected Vero E6 and A549-hACE2 cells with 1-fold EC PI revealed minor differences in the barrier to SARS-CoV-2 escape. Viral suppression was achieved with 3- to 8-fold EC boceprevir or 1-fold EC simeprevir or grazoprevir, but not boceprevir, in combination with 0.4- to 0.8-fold EC remdesivir; these concentrations did not lead to viral suppression in single treatments. This study could inform the development and application of protease inhibitors for optimized antiviral treatments of COVID-19.
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http://dx.doi.org/10.1128/AAC.02680-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8370243PMC
August 2021

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine.

Sci Rep 2020 10 1;10(1):16261. Epub 2020 Oct 1.

Institute of Bioprocess Engineering and Pharmaceutical Technology, Department of Life Science Engineering, University of Applied Sciences Mittelhessen (THM), Giessen, Germany.

There is a large unmet need for a prophylactic hepatitis C virus (HCV) vaccine to control the ongoing epidemic with this deadly pathogen. Many antiviral vaccines employ whole viruses as antigens. For HCV, this approach became feasible following the development of infectious cell culture systems for virus production. However, the lack of efficient downstream processes (DSP) for HCV purification poses a roadblock for the development of a whole virus vaccine. Using cell culture-derived genotype 1a HCV we developed a scalable and efficient DSP train, employing commonly used clarification and ultrafiltration techniques, followed by two membrane-based chromatography steps. For virus capture, steric exclusion chromatography using cellulose membranes was established, resulting in a virtually complete virus recovery with > 99% protein and 84% DNA depletion. Virus polishing was achieved by sulphated cellulose membrane adsorbers with ~ 50% virus recovery and > 99% protein and 90% DNA depletion. Additional nuclease digestion resulted in 99% overall DNA depletion with final DNA concentrations of 2 ng/mL. Process results were comparable for cell culture-derived HCV of another major genotype (5a). This study provides proof-of-concept for establishment of an efficient and economically attractive DSP with potential application for production of an inactivated whole virus vaccine against HCV for human use.
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http://dx.doi.org/10.1038/s41598-020-72328-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530675PMC
October 2020

Specific Antibodies Induced by Immunization with Hepatitis B Virus-Like Particles Carrying Hepatitis C Virus Envelope Glycoprotein 2 Epitopes Show Differential Neutralization Efficiency.

Vaccines (Basel) 2020 Jun 10;8(2). Epub 2020 Jun 10.

Laboratory of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology, University of Gdańsk, 80-309 Gdańsk, Poland.

Hepatitis C virus (HCV) infection with associated chronic liver diseases is a major health problem worldwide. Here, we designed hepatitis B virus (HBV) small surface antigen (sHBsAg) virus-like particles (VLPs) presenting different epitopes derived from the HCV E2 glycoprotein (residues 412-425, 434-446, 502-520, and 523-535 of isolate H77C). Epitopes were selected based on their amino acid sequence conservation and were previously reported as targets of HCV neutralizing antibodies. Chimeric VLPs obtained in the expression system, in combination with the adjuvant Addavax, were used to immunize mice. Although all VLPs induced strong humoral responses, only antibodies directed against HCV 412-425 and 523-535 epitopes were able to react with the native E1E2 glycoprotein complexes of different HCV genotypes in ELISA. Neutralization assays against genotype 1-6 cell culture infectious HCV (HCVcc), revealed that only VLPs carrying the 412-425 epitope induced efficient HCV cross-neutralizing antibodies, but with isolate specific variations in efficacy that could not necessarily be explained by differences in epitope sequences. In contrast, antibodies targeting 434-446, 502-520, and 523-535 epitopes were not neutralizing HCVcc, highlighting the importance of conformational antibodies for efficient virus neutralization. Thus, 412-425 remains the most promising linear E2 epitope for further bivalent, rationally designed vaccine research.
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http://dx.doi.org/10.3390/vaccines8020294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350033PMC
June 2020

High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals.

Sci Rep 2018 11 30;8(1):17505. Epub 2018 Nov 30.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log FFU/mL and 10.4 log IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development.
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http://dx.doi.org/10.1038/s41598-018-35010-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269495PMC
November 2018
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