Hepatitis C virus (HCV) infection may elicit neutralizing antibodies targeting epitopes conserved in all viral genotypes

Anti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection

HCV Proteins and Immunoglobulin Variable Gene (IgV) Subfamilies in HCV-Induced Type II Mixed Cryoglobulinemia: A Concurrent Pathogenetic Role

The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved

Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

Objective: The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design: Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3 zeta domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results: In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon gamma, interleukin 2 and tumour necrosis factor a. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions: Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool

A biologically-validated HCV E1E2 heterodimer structural model

The design of vaccine strategies and the development of drugs targeting the early stages of Hepatitis C virus (HCV) infection are hampered by the lack of structural information about its surface glycoproteins E1 and E2, the two constituents of HCV entry machinery. Despite the recent crystal resolution of limited versions of both proteins in truncated form, a complete picture of the E1E2 complex is still missing. Here we combined deep computational analysis of E1E2 secondary, tertiary and quaternary structure with functional and immunological mutational analysis across E1E2 in order to propose an in silico model for the ectodomain of the E1E2 heterodimer. Our model describes E1-E2 ectodomain dimerization interfaces, provides a structural explanation of E1 and E2 immunogenicity and sheds light on the molecular processes and disulfide bridges isomerization underlying the conformational changes required for fusion. Comprehensive alanine mutational analysis across 553 residues of E1E2 also resulted in identifying the epitope maps of diverse mAbs and the disulfide connectivity underlying E1E2 native conformation. The predicted structure unveils E1 and E2 structures in complex, thus representing a step towards the rational design of immunogens and drugs inhibiting HCV entry

Entry inhibition of HSV-1 and -2 protects mice from viral lethal challenge

The present study focused on inhibition of HSV-1 and -2 replication and pathogenesis in vitro and in vivo, through the selective targeting of the envelope glycoprotein D. Firstly, a human monoclonal antibody (Hu-mAb#33) was identified that could neutralise both HSV-1 and -2 at nM concentrations, including clinical isolates from patients affected by different clinical manifestations and featuring different susceptibility to acyclovir in vitro. Secondly, the potency of inhibition of both infection by cell-free viruses and cell-to-cell virus transmission was also assessed. Finally, mice receiving a single systemic injection of Hu-mAb#33 were protected from death and severe clinical manifestations following both ocular and vaginal HSV-1 and -2 lethal challenge. These results pave the way for further studies reassessing the importance of HSV entry as a novel target for therapeutic intervention and inhibition of cell-to-cell virus transmission

Towards a universal influenza vaccine: different approaches for one goal

Abstract Influenza virus infection is an ongoing health and economic burden causing epidemics with pandemic potential, affecting 5–30% of the global population annually, and is responsible for millions of hospitalizations and thousands of deaths each year. Annual influenza vaccination is the primary prophylactic countermeasure aimed at limiting influenza burden. However, the effectiveness of current influenza vaccines are limited because they only confer protective immunity when there is antigenic similarity between the selected vaccine strains and circulating influenza isolates. The major targets of the antibody response against influenza virus are the surface glycoprotein antigens hemagglutinin (HA) and neuraminidase (NA). Hypervariability of the amino acid sequences encoding HA and NA is largely responsible for epidemic and pandemic influenza outbreaks, and are the consequence of antigenic drift or shift, respectively. For this reason, if an antigenic mismatch exists between the current vaccine and circulating influenza isolates, vaccinated people may not be afforded complete protection. There is currently an unmet need to develop an effective “broadly-reactive” or “universal” influenza vaccine capable of conferring protection against both seasonal and newly emerging pre-pandemic strains. A number of novel influenza vaccine approaches are currently under evaluation. One approach is the elicitation of an immune response against the “Achille’s heel” of the virus, i.e. conserved viral proteins or protein regions shared amongst seasonal and pre-pandemic strains. Alternatively, other approaches aim toward eliciting a broader immune response capable of conferring protection against the diversity of currently circulating seasonal influenza strains. In this review, the most promising under-development universal vaccine approaches are discussed with an emphasis on those targeting the HA glycoprotein. In particular, their strengths and potential short-comings are discussed. Ultimately, the upcoming clinical evaluation of these universal vaccine approaches will be fundamental to determine their effectiveness against preventing influenza virus infection and/or reducing transmission and disease severity

The Answer Lies in the Energy: How Simple Atomistic Molecular Dynamics Simulations may Hold the Key to Epitope Prediction on the Fully Glycosylated SARS-CoV-2 Spike Protein

SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated Spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad-hoc parameter combination is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains, poised to host (several) epitopes, and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes; those with weaker coupling could themselves be prone to antibody recognition

Convergent antibody evolution and clonotype expansion following influenza virus vaccination.

Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016-2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches

The Effect of Waning on Antibody Levels and Memory B Cell Recall following SARS-CoV-2 Infection or Vaccination

In order to longitudinally track SARS-CoV-2 antibody levels after vaccination or infection, we assessed anti-RBD antibody levels in over 1000 people and found no significant decrease in antibody levels during the first 14 months after infection in unvaccinated participants, however, a significant waning of antibody levels was observed following vaccination. Participants who were pre-immune to SARS-CoV-2 prior to vaccination seroconverted to higher antibody levels, which were maintained at higher levels than in previously infected, unvaccinated participants. Older participants exhibited lower level of antibodies after vaccination, but a higher level after infection than younger people. The rate of antibody waning was not affected by pre-immunity or age. Participants who received a third dose of an mRNA vaccine not only increased their antibody levels ~14-fold, but also had ~3 times more antibodies compared to when they received their primary vaccine series. PBMC-derived memory B cells from 13 participants who lost all circulating antibodies were differentiated into antibody secreting cells (ASCs). There was a significant recall of memory B cell ASCs in the absence of serum antibodies in 5–8 of the 10 vaccinated participants, but not in any of the 3 infected participants, suggesting a strong connection between antibody levels and the effectiveness of memory B cell recall