60 research outputs found
SARS-CoV-2 spike HexaPro formulated in aluminium hydroxide and administered in an accelerated vaccination schedule partially protects Syrian Hamsters against viral challenge despite low neutralizing antibody responses
SARS-CoV-2 continues to pose a threat to human health as new variants emerge and thus a diverse vaccine pipeline is needed. We evaluated SARS-CoV-2 HexaPro spike protein formulated in AlhydrogelÂŽ (aluminium oxyhydroxide) in Syrian hamsters, using an accelerated two dose regimen (given 10 days apart) and a standard regimen (two doses given 21 days apart). Both regimens elicited spike- and RBD-specific IgG antibody responses of similar magnitude, but in vitro virus neutralization was low or undetectable. Despite this, the accelerated two dose regimen offered reduction in viral load and protected against lung pathology upon challenge with homologous SARS-CoV-2 virus (Wuhan-Hu-1). This highlights that vaccine-induced protection against SARS-CoV-2 disease can be obtained despite low neutralizing antibody levels and suggests that accelerated vaccine schedules may be used to confer rapid protection against SARS-CoV-2 disease
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
Viral genome wide association study identifies novel hepatitis C virus polymorphisms associated with sofosbuvir treatment failure
Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment
Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus:Essential tools in testing of antivirals and emerging vaccine strategies
Lipid Droplets Accumulation during Hepatitis C Virus Infection in Cell-Culture Varies among Genotype 1â3 Strains and Does Not Correlate with Virus Replication
Liver steatosis is a common complication of chronic hepatitis C virus (HCV) infection, which can result in accelerated liver fibrosis development, especially in patients infected with genotype 3a. The precise mechanisms of HCV-induced liver steatosis remain unclear, but it is often posited that increased intracellular lipid accumulation is the underlying cause of steatosis. To study experimentally how HCV infection in human liver derived cells by different genotypes and subtypes might affect lipid accumulation, we performed detailed cytofluorimetric and microscopy analyses of intracellular lipid droplets (LDs) in relation to the viral Core and to cell endoplasmic reticulum proteins. Following culture infection with HCV genotype 1a, 2a, 2b, 2c, and 3a strains, we found variable levels of intracellular LDs accumulation, associated to the infecting strain rather than to the specific genotype. Although two genotype 3a strains showed high levels of lipid accumulation, as previously observed, some strains of other genotypes displayed a similar phenotype. Moreover, the analyses of LDs size, number, and shape indicated that the apparent increase in lipid accumulation is due to an increase in the overall number rather than in the size of droplets. Finally, differences in total lipid content across genotypes did not correlate to differences in Core distribution nor Core levels. In conclusion, our study provides a quantitative in-depth analysis of the effect of HCV infection on LDs accumulation in cell-culture
Non-genotype-specific role of the hepatitis C virus 5' untranslated region in virus production and in inhibition by interferon
AbstractThe 5Ⲡuntranslated region (5â˛UTR) of hepatitis C virus (HCV) is structured into four domains (IâIV) with numerous genotype-specific nucleotides. It is unknown whether the polymorphisms confer genotype-specific functions to the 5â˛UTR. Using viable JFH1-based Core-NS2 recombinants, we developed and characterized HCV genotypes 1â7 recombinants with highly diverse 5â˛UTRs (genotypes 1a and 3a), 2a recombinants (J6/JFH1) with 5â˛UTR of genotypes 1â6 or with heterotypic chimeric (1a/3a and 3a/1a) 5â˛UTR domains I, II or III, and 1a recombinants with 5â˛UTR domain I of genotypes 1â6. All were fully functional in Huh7.5 cells; therefore, the 5â˛UTR apparently functions in a non-genotype-specific manner in HCV production in vitro. However, adenine at the 5â˛-terminus was required. We demonstrated that J6/JFH1 with 5â˛UTR of genotypes 1â6 responded similarly to interferon-Îą2b. This study provides novel insight into the role of the 5â˛UTR in the HCV life cycle and facilitates HCV basic research and testing of 5â˛UTR-targeting antivirals
Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77
The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5=UTR-NS5A (where UTR stands for untrans-lated region) and the JFH1 NS5B-3=UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/ D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced superna-tant infectivity titers of 104.0 focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 vi-ruses, and the A970T/I1312V/C2419R/A2919Tmutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5 and 104.4 FFU/ml, respectively
Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir
- âŚ