A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) ...vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.
SputnikV is a vaccine against SARS-CoV-2 developed by the Gamaleya National Research Centre for Epidemiology and Microbiology. The vaccine has been shown to induce both humoral and cellular immune ...responses, yet the mechanisms remain largely unknown. Forty SputnikV vaccinated individuals were included in this study which aimed to demonstrate the location of immunogenic domains of the SARS-CoV-2 S protein using an overlapping peptide library. Additionally, cytokines in the serum of vaccinated and convalescent COVID-19 patients were analyzed. We have found antibodies from both vaccinated and convalescent sera bind to immunogenic regions located in multiple domains of SARS-CoV-2 S protein, including Receptor Binding Domain (RBD), N-terminal Domain (NTD), Fusion Protein (FP) and Heptad Repeats (HRs). Interestingly, many peptides were recognized by immunized and convalescent serum antibodies and correspond to conserved regions in circulating variants of SARS-CoV-2. This breadth of reactivity was still evident 90 days after the first dose of the vaccine, showing that the vaccine has induced a prolonged response. As evidenced by the activation of T cells, cellular immunity strongly suggests the high potency of the SputnikV vaccine against SARS-CoV-2 infection.
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Virus infections cause diseases of different severity ranged from mild infection e.g. common cold into life threatening diseases e.g. Human Immunodeficiency virus (HIV), Hepatitis B. ...Virus infections represent 44% of newly emerging infections. Although there are many efficient antiviral agents, they still have drawbacks due to accumulation at off target organs and developing of virus resistance due to virus mutation. Therefore, developing a delivery system that can selectively target drug into affected organs and avoid off target accumulation would be a highly advantageous strategy to improve antiviral therapy. Nanoparticles (NP) can be effectively targeted to the liver, and therefore it could be used for improving therapy of hepatic virus infections including hepatitis B virus and hepatitis C virus (HCV). Many studies were performed to encapsulate antiviral agents into nano-delivery system to improve their pharmacokinetics parameters to have a better therapeutic efficacy with lower side effects. However, the effect of virus infection on the uptake of NP has not yet been studied in detail. The latter is a crucial area as modulation of endocytic uptake of nanoparticles could impact on reduce potential therapeutic usefulness of antiviral agents loaded into nano-delivery system. In this study, a fluorescently-labelled polymeric nanoparticle was prepared and used to track NP uptake into Huh7.5, human hepatoma cells transfected with replicating HCV genomes, compared with non-transfected cells as a model representing hepatocyte uptake. Confocal microscopy and flow cytometry of virus transfected Huh7.5 cells unexpectedly demonstrated two-fold increase in uptake of NP compared to non-transfected cells. Therefore, virus transfection enhanced NP uptake into Huh7.5 cells and NP could be considered as a promising delivery system for targeted treatment of hepatitis viruses.
Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with ...HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.
•Development of a novel human monoclonal antibody (mAb), designated 2A5, that targets the HCV envelope glycoprotein E2.•mAb 2A5 efficiently neutralizes HCVpp and HCVcc in a pan-genotypic manner.•mAb 2A5 protects human-liver chimeric mice from HCV challenge.•Our novel mAb could be used to protect liver transplant patients from HCV recurrence.•The 2A5 epitope represents a valuable target for the development of HCV vaccines with broad-spectrum activity.
An optimal HCV vaccine requires the induction of antibodies that neutralise the infectivity of many heterogenous viral isolates. In this study, we have focused on determining the optimal recombinant ...envelope glycoprotein component to elicit cross-neutralising antibodies against global HCV genotypes. We compared the immunoreactivity and antigenicity of the recombinant HCV genotype 1a strain H77C envelope glycoprotein heterodimer gpE1/gpE2 with that of recombinant gpE2 alone derived from an infectious molecular clone (H77C).
Characterization of the envelope glycoproteins was accomplished by determining their ability to bind to a panel of broadly cross-neutralising monoclonal antibodies (bNAbs). Immunogenicity was determined by testing the ability of vaccine antisera to neutralise the infectivity in vitro of a panel of pseudotyped HCV particles in which gpE1/gpE2 derived from representative isolates of the major global HCV genotypes were displayed.
gpE1/gpE2 binds to more diverse bNabs than gpE2 alone and elicits a broader profile of cross-neutralising antibodies in animals, especially against more heterologous, non-1a genotypes. While not all heterologous HCV strains can be potently inhibited in vitro by gpE1/gpE2 antisera derived from a single HCV strain, the breadth of heterologous cross-neutralisation is shown to be substantial.
Our work supports the inclusion of gpE1/gpE2 in an HCV vaccine in order to maximise the cross-neutralisation of heterogenous HCV isolates. Our data also offers future directions in formulating a cocktail of gpE1/gpE2 antigens from a small selection of HCV genotypes to further enhance cross-neutralisation of global HCV strains and hopefully, achieving global protection.
An HCV vaccine is urgently required to prevent the high global incidence of HCV infection and disease. Since HCV is a highly heterogeneous virus, it is desirable for a vaccine to elicit antibodies that neutralise the infectivity of most global strains. To this end, we have compared the immunoreactivity and antigenicity of recombinant H77C E1E2 heterodimer with that of H77C E2 alone and show that the former exhibits more cross-neutralising epitopes and demonstrates a broader cross-neutralisation profile in vitro. In addition, our data suggests a way to further broaden cross-neutralisation using a combination of E1E2 antigens derived from a few different HCV clades. Our work provides encouragement for the development of an effective global HCV vaccine.
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•Glycoprotein-based HCV vaccines are immunogenic.•Compared the antigenicities of E1E2 and E2.•HCV E1E2-based vaccine induced a broader cross-neutralising profile.
Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels ...may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays.
We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps.
Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones.
Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.
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A panel of hepatitis C virus pseudoparticles with 4 tiers of antibody resistance was developed. These pseudoparticles can be used to measure neutralizing breadth of antibodies induced by candidate vaccines.
Understanding the impact of prior infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the response to vaccination is a priority for responding to the coronavirus disease ...2019 (COVID-19) pandemic. In particular, it is necessary to understand how prior infection plus vaccination can modulate immune responses against variants of concern. To address this, we sampled 20 individuals with and 25 individuals without confirmed previous SARS-CoV-2 infection from a large cohort of health care workers followed serologically since April 2020. All 45 individuals had received two doses of the Pfizer-BioNTech BNT162b2 vaccine with a delayed booster at 10 weeks. Absolute and neutralizing antibody titers against wild-type SARS-CoV-2 and variants were measured using enzyme immunoassays and pseudotype neutralization assays. We observed antibody reactivity against lineage A, B.1.351, and P.1 variants with increasing antigenic exposure, through either vaccination or natural infection. This improvement was further confirmed in neutralization assays using fixed dilutions of serum samples. The impact of antigenic exposure was more evident in enzyme immunoassays measuring SARS-CoV-2 spike protein–specific IgG antibody concentrations. Our data show that multiple exposures to SARS-CoV-2 spike protein in the context of a delayed booster expand the neutralizing breadth of the antibody response to neutralization-resistant SARS-CoV-2 variants. This suggests that additional vaccine boosts may be beneficial in improving immune responses against future SARS-CoV-2 variants of concern.
Human blood plasma and serum have been a source of biomarkers for the indication and progression of many diseases for a few decades now. Human blood plasma is also an excellent source material to ...enable patients to monitor their health, with a multitude of biomarkers detectable for the assessment of health status. Blood sampling kits are increasingly available for use in the home with no specialist clinical skills required to obtain good quality samples for pathology lab analysis. Many of the proteins that constitute plasma are glycosylated with both N- and O-type glycans. There is increasing interest in the scientific community to identify potential glycan biomarkers or glycan features that are indicative of disease, and in particular disease at an early stage. The quality and reproducibility of glycan analysis data is key in order to identify and utilise glycan-based blood biomarkers with sufficient specificity and sensitivity; hence, the required analytical tools need to be robust. In this chapter, we describe an analytical method for the UHPLC separation of plasma N-glycans which utilizes both glycan reducing terminus fluorophore labeling, to ensure stoichiometric analysis of relative glycan abundance, and online mass spectrometry for glycan identification. Exoglycosidase digestions were employed as example technique to aid and enable structure identification.
An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting ...HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.
Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested
and
, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.