23 research outputs found
Specific antibodies induced by immunization with hepatitis B virus-like particles carrying hepatitis C virus envelope glycoprotein 2 epitopes show differential neutralization efficiency
HighâTiter Hepatitis C Virus Production in a Scalable SingleâUse High Cell Density Bioreactor
Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradleâ˘) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log(10) focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 Ă 10(10) FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradleâ˘. CelCradleâ˘-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production
The effect of glycan shift on antibodies against HCV E2 412-425 epitope elicited by chimeric sHBsAg-based virus-like particles
Two of the most important mechanisms of hepatitis C virus (HCV) immune evasion are the high variability of the amino acid sequence and epitope shielding via heavy glycosylation of the envelope (E) proteins. Previously, we showed that chimeric sHBsAg (hepatitis B virus [HBV] small surface antigen)-based virus-like particles (VLPs) carrying highly conserved epitope I from the HCV E2 glycoprotein (sHBsAg_412â425) elicit broadly neutralizing antibodies (bnAbs). However, many reports have identified escape mutations for such bnAbs that shift the N-glycosylation site from N417 to N415. This shift effectively masks the recognition of epitope I by antibodies raised against the wild-type glycoprotein. To investigate if glycan-shift-mediated immune evasion could be overcome by targeted vaccination strategies, we designed sHBsAg-based VLPs carrying epitope I with an N417S change (sHBsAg_N417S). Studies in BALB/c mice revealed that both sHBsAg_412â425 and sHBsAg_N417S VLPs were immunogenic, eliciting antibodies that recognized peptides encompassing epitope I regardless of the N417S change. However, we observed substantial differences in E1E2 glycoprotein binding and cell culture-derived HCV (HCVcc) neutralization between the sera elicited by sHBsAg_412â425 and those elicited by sHBsAg_N417S VLPs. Our results suggest a complex interplay among antibodies targeting epitope I, the E1E2 glycosylation status, and the epitope or global E1E2 conformation. Additionally, we observed striking similarities in the E1E2 glycoprotein binding patterns and HCVcc neutralization between sHBsAg_412â425 sera and AP33, suggesting that the immunization of mice with sHBsAg_412â425 VLPs can elicit AP33-like antibodies. This study emphasizes the role of antibodies against epitope I and represents an initial effort toward designing an antigen that elicits an immune response against epitope I with a glycan shift change
Inactivated whole hepatitis C virus vaccine employing a licensed adjuvant elicits cross-genotype neutralizing antibodies in mice
SARS-COV-2 production in a single-use scalable high cell density bioreactor
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the fast development of first-generation vaccines have demonstrated the value of applying a variety of vaccine technology platforms (1). Inactivated vaccines represent a well-known approach, and their manufacturing depends on high-yield virus production in appropriate biosafety level facilities. This study (2) aimed to establish efficient Vero (WHO) cell-based and animal component-free SARS-CoV-2 production in the CelCradle bioreactor (Esco Aster Pte Ltd.). The single-use culture vessels pre-packed with 0.1 L BioNOCII carriers are highly useful for small scale cultivation of adherent cell lines. Cultures were seeded with 1.5 Ă 10⸠cells and total cell numbers peaked at 9 days post cell seeding (dpcs) with 2.7â2.8 Ă 10âš cells/vessel in non-infected cultures. To produce virus, cultures were infected at 7 dpcs at total cell numbers of 2.2â2.5 Ă 10âš cells/vessel at a multiplicity of infection of 0.006. Harvest of virus-containing supernatant twice instead of once per day improved the virus yield by 2â7 fold. Introducing a temperature shift from 37°C to 33°C upon the time of infection improved virus yield by 2â9 fold with a considerable decline of infectious titer only after 72 h post infection (hpi). Infectious titers peaked at 7.3 logââ 50% tissue culture infectious dose (TCIDâ
â)/mL at 72 hpi, and a total of 10.5 logââ TCIDâ
â were produced in ~5 L (11 harvests). While trypsin has been reported to enhance SARS-CoV-2 spread in cell culture, addition of 0.5% recombinant trypsin from the time of infection did not affect virus yield. Overall, animal component-free production of SARS-CoV-2 in Vero (WHO) cells was successfully established in a single-use packed-bed bioreactor. β-propiolactone inactivated SARS-CoV-2 from this study was immunogenic and induced neutralizing antibodies in mice with mean 50% neutralization titers of 1/150 or 1/580 after three immunizations with doses containing 0.1 Âľg or 0.5 Âľg S1 protein, respectively. The CelCradle represents a scalable technology and is a small version of the TideXCell system applying single-use culture vessels with packed-bed volumes of up to 100 L. The potential rapid response to outbreaks with inactivated vaccines has been demonstrated in the SARS-CoV-2 pandemic (1). Vero cells are susceptible to a wide range of viral pathogens (3), and these scalable single-use bioreactors provide a high level of flexibility and potentially decreased response time for production of future emerging viruses for vaccine purposes.
References: (1) Poland 2020 Lancet [PMID: 33065034], (2) Offersgaard 2021 Vaccines [PMID: 34209694], (3) Barret 2009 Expert Rev. Vaccines [PMID: 19397417
Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants
Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants
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 resistanceâassociated 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 1â4, 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 156âRASs were not maintained. For genotypes 1 and 2, persistence of 156âRASs depended on genomeâwide substitution networks, coâselected under continued PI treatment and identified by nextâgeneration 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 156âRASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and preâexisting 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 156âRASs, we observed genomeâwide selection of substitutions under treatment. Conclusion : Comprehensive PI resistance profiling for HCV genotypes 1â6 revealed 156âRASs as key determinants of highâlevel resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1â3 156âvariants, which might pose a threat to clinically relevant combination treatments