Publications by authors named "Judith Margarete Gottwein"

4 Publications

  • Page 1 of 1

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.
View Article and Find Full Text PDF

Download full-text PDF

Source Listing
January 2022

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.
View Article and Find Full Text PDF

Download full-text PDF

Source Listing
June 2021

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.
View Article and Find Full Text PDF

Download full-text PDF

Source Listing
June 2020

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants.

Hepatology 2019 09 5;70(3):771-787. Epub 2019 Jun 5.

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

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance-associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1-4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156-RASs were not maintained. For genotypes 1 and 2, persistence of 156-RASs depended on genome-wide substitution networks, co-selected under continued PI treatment and identified by next-generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156-RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre-existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156-RASs, we observed genome-wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1-6 revealed 156-RASs as key determinants of high-level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1-3 156-variants, which might pose a threat to clinically relevant combination treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source Listing
September 2019