ABSTRACT
SARS-CoV-2 subunit vaccines continue to be the focus of intense clinical development worldwide. Protein antigens in these vaccines most commonly consist of the spike ectodomain fused to a heterologous trimerization sequence, designed to mimic the compact, prefusion conformation of the spike on the virus surface. Since 2020, we have produced dozens of such constructs in CHO cells, consisting of spike variants with different mutations fused to different trimerization sequences. This set of constructs displayed notable conformational heterogeneity, with two distinct trimer species consistently detected by analytical size exclusion chromatography. A recent report showed that spike ectodomain fusion constructs can adopt an alternative trimer conformation consisting of loosely associated ectodomain protomers. Here, we applied multiple biophysical and immunological techniques to demonstrate that this alternative conformation is formed to a significant extent by several SARS-CoV-2 variant spike proteins. We have also examined the influence of temperature and pH, which can induce inter-conversion of the two forms. The substantial structural differences between these trimer types may impact their performance as vaccine antigens.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Cricetinae , Humans , COVID-19 Vaccines/genetics , Temperature , Cricetulus , Antigens , Mutation , Hydrogen-Ion Concentration , Antibodies, NeutralizingABSTRACT
The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.
Subject(s)
Hepacivirus/genetics , Interferon-alpha/pharmacology , Oligopeptides/pharmacology , Thiazoles/pharmacology , Viral Nonstructural Proteins/genetics , Amino Acid Substitution , Aminoisobutyric Acids , Antiviral Agents/pharmacology , Crystallography, X-Ray , Drug Resistance, Viral , Genotype , Hepacivirus/drug effects , Hepacivirus/enzymology , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/virology , Humans , Inhibitory Concentration 50 , Kinetics , Leucine/analogs & derivatives , Mutagenesis, Site-Directed , Mutation Rate , Phenotype , Proline/analogs & derivatives , Protease Inhibitors/pharmacology , Quinolines , Replicon , Viral Nonstructural Proteins/metabolismABSTRACT
Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K(i), 80 to 90 nM) compared to genotype 1 enzymes (K(i), 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, with K(i) values below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.
Subject(s)
Antiviral Agents/pharmacology , Carbamates/pharmacology , Hepacivirus/drug effects , Hepacivirus/enzymology , Macrocyclic Compounds , Quinolines , Serine Proteinase Inhibitors/pharmacology , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Amino Acid Sequence , Binding Sites , Escherichia coli/enzymology , Escherichia coli/genetics , Genotype , Hepacivirus/classification , Humans , Kinetics , Microbial Sensitivity Tests/methods , Models, Molecular , Molecular Sequence Data , Mutation , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/geneticsABSTRACT
The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.