Standardized Method for the Study of Antibody Neutralization of HCV Pseudoparticles (HCVpp)

Bailey, Justin R.; Urbanowicz, Richard A.; Ball, Jonathan K.; Law, Mansun; Foung, Steven K. H.

doi:10.1007/978-1-4939-8976-8_30

Cited by 19 publications

(24 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, mice sera can contain viral inhibitory components that can interfere with neutralization assays as reported for HIV-1 [ 46 ]. To test this, we first performed a small pilot HCV pseudoparticle (HCVpp) neutralization assay [ 64 ] using three serum samples from mice immunized with DREP-HCV/MVA-HCV, MVA-HCV/MVA-HCV and MVA-WT/MVA-WT [ 25 ]. We found that even a negative control serum (MVA-WT/MVA-WT) showed ~50% neutralizing activity at a 1/20 dilution, while the purified IgG samples did not ( Figure 8 B).…”

Section: Resultsmentioning

confidence: 99%

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV

et al. 2020

View full text Add to dashboard Cite

Hepatitis C virus (HCV) represents a major global health challenge and an efficient vaccine is urgently needed. Many HCV vaccination strategies employ recombinant versions of the viral E2 glycoprotein. However, recombinant E2 readily forms disulfide-bonded aggregates that might not be optimally suited for vaccines. Therefore, we have designed an E2 protein in which we strategically changed eight cysteines to alanines (E2.C8A). E2.C8A formed predominantly monomers and virtually no aggregates. Furthermore, E2.C8A also interacted more efficiently with broadly neutralizing antibodies than conventional E2. We used mice to evaluate different prime/boost immunization strategies involving a modified vaccinia virus Ankara (MVA) expressing the nearly full-length genome of HCV (MVA-HCV) in combination with either the E2 aggregates or the E2.C8A monomers. The combined MVA-HCV/E2 aggregates prime/boost strategy markedly enhanced HCV-specific effector memory CD4+ T cell responses and antibody levels compared to MVA-HCV/MVA-HCV. Moreover, the aggregated form of E2 induced higher levels of anti-E2 antibodies in vaccinated mice than E2.C8A monomers. These antibodies were cross-reactive and mainly of the IgG1 isotype. Our findings revealed how two E2 viral proteins that differ in their capacity to form aggregates are able to enhance to different extent the HCV-specific cellular and humoral immune responses, either alone or in combination with MVA-HCV. These combined protocols of MVA-HCV/E2 could serve as a basis for the development of a more effective HCV vaccine.

show abstract

Section: Resultsmentioning

confidence: 99%

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV

et al. 2020

View full text Add to dashboard Cite

show abstract

“…HCVpp neutralization assays. Neutralization assays were performed as previously described (44). Huh7.5 cells were plated on wells of sterile 96-well plates (Corning) precoated with collagen solution (0.01%, MilliporeSigma) at a density of 1 × 10 4 cells/well.…”

Section: Discussionmentioning

confidence: 99%

Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion

Salinas¹,

Boisvert²,

Upadhyay³

et al. 2021

Journal of Clinical Investigation

View full text Add to dashboard Cite

“…This is problematic for vaccine screens and antibody-neutralizing breadth studies, as only strains that recover workable infectivity are included in such analyses and they may not necessarily be representative of the diversity circulating in the population. Moreover, some infectious strains give low yields, which fail to reach a signal-to-noise threshold sufficient for neutralization analysis, a signal at least tenfold above background is favoured but can be lowered to a minimum of 5 for clinical isolates [30,51]. Perhaps owing to its flexibility, several studies have demonstrated the HCVpp system is amenable to optimization.…”

Section: Discussionmentioning

confidence: 99%

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Kalemera

Capella-Pujol

Chumbe

et al. 2021

Journal of General Virology

View full text Add to dashboard Cite

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

show abstract

Standardized Method for the Study of Antibody Neutralization of HCV Pseudoparticles (HCVpp)

Cited by 19 publications

References 19 publications

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV

Optimized Hepatitis C Virus (HCV) E2 Glycoproteins and their Immunogenicity in Combination with MVA-HCV

Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Contact Info

Product

Resources

About