Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.

Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity-suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants.

Breakthrough infection elicits hypermutated IGHV3-53/3-66 public antibodies with broad and potent neutralizing activity against SARS-CoV-2 variants including the emerging EG.5 lineages.

The rapid emergence of SARS-CoV-2 variants of concern (VOCs) calls for efforts to study broadly neutralizing antibodies elicited by infection or vaccination so as to inform the development of vaccines and antibody therapeutics with broad protection. Here, we identified two convalescents of breakthrough infection with relatively high neutralizing titers against all tested viruses. Among 50 spike-specific monoclonal antibodies (mAbs) cloned from their B cells, the top 6 neutralizing mAbs (KXD01-06) belong to previously defined IGHV3-53/3-66 public antibodies. Although most antibodies in this class are dramatically escaped by VOCs, KXD01-06 all exhibit broad neutralizing capacity, particularly KXD01-03, which neutralize SARS-CoV-2 from prototype to the emerging EG.5.1 and FL.1.5.1. Deep mutational scanning reveals that KXD01-06 can be escaped by current and prospective variants with mutations on D420, Y421, L455, F456, N460, A475 and N487. Genetic and functional analysis further indicates that the extent of somatic hypermutation is critical for the breadth of KXD01-06 and other IGHV3-53/3-66 public antibodies. Overall, the prevalence of broadly neutralizing IGHV3-53/3-66 public antibodies in these two convalescents provides rationale for novel vaccines based on this class of antibodies. Meanwhile, KXD01-06 can be developed as candidates of therapeutics against SARS-CoV-2 through further affinity maturation.

Amyloid pathology mediates the associations between plasma fibrinogen and cognition in non-demented adults.

Though previous studies revealed the potential associations of elevated levels of plasma fibrinogen with dementia, there is still limited understanding regarding the influence of Alzheimer's disease (AD) biomarkers on these associations. We sought to investigate the interrelationships among fibrinogen, cerebrospinal fluid (CSF) AD biomarkers, and cognition in non-demented adults. We included 1996 non-demented adults from the Chinese Alzheimer's Biomarker and LifestylE (CABLE) study and 337 from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. The associations of fibrinogen with AD biomarkers and cognition were explored using multiple linear regression models. The mediation analyses with 10 000 bootstrapped iterations were conducted to explore the mediating effects of AD biomarkers on cognition. In addition, interaction analyses and subgroup analyses were conducted to assess the influence of covariates on the relationships between fibrinogen and AD biomarkers. Participants exhibiting low Aß42 were designated as A+, while those demonstrating high phosphorylated tau (P-tau) and total tau (Tau) were labeled as T+ and N+, respectively. Individuals with normal measures of Aß42 and P-tau were categorized as the A-T- group, and those with abnormal levels of both Aß42 and P-tau were grouped under A+T+. Fibrinogen was higher in the A+ subgroup compared to that in the A- subgroup (p = 0.026). Fibrinogen was higher in the A+T+ subgroup compared to that in the A-T- subgroup (p = 0.011). Higher fibrinogen was associated with worse cognition and Aß pathology (all p < 0.05). Additionally, the associations between fibrinogen and cognition were partially mediated by Aß pathology (mediation proportion range 8%-28%). Interaction analyses and subgroup analyses showed that age and ApoE ε4 affect the relationships between fibrinogen and Aß pathology. Fibrinogen was associated with both cognition and Aß pathology. Aß pathology may be a critical mediator for impacts of fibrinogen on cognition.

Association between Kidney Function and the Burden of Cerebral Small Vessel Disease: An Updated Meta-Analysis and Systematic Review.

INTRODUCTION: Due to anatomical and functional similarities in microvascular beds, the brain and kidney share distinctive susceptibilities to vascular injury and common risk factors of small vessel disease. The aim of this updated meta-analysis is to explore the association between kidney function and the burden of cerebral small vessel disease (CSVD). METHODS: PubMed, EMBASE, and Cochrane Library were systematically searched for observational studies that explored the association between the indicators of kidney function and CSVD neuroimaging markers. The highest-adjusted risk estimates and their 95% confidence intervals (CIs) were aggregated using random-effect models. RESULTS: Twelve longitudinal studies and 51 cross-sectional studies with 57,030 subjects met the inclusion criteria of systematic review, of which 52 were included in quantitative synthesis. According to the pooled results, we found that low estimated glomerular filtration rate (eGFR <60 mL/min/1.73 m2) was associated with cerebral microbleeds (odds ratio (OR) = 1.55, 95% CI = 1.26-1.90), white matter hyperintensities (OR = 1.40, 95% CI = 1.05-1.86), and lacunar infarctions (OR = 1.50, 95% CI = 1.18-1.92), but not with severe perivascular spaces (OR = 1.20, 95% CI = 0.77-1.88). Likewise, patients with proteinuria (OR = 1.75, 95% CI = 1.47-2.09) or elevated serum cystatin C (OR = 1.51, 95% CI = 1.25-1.83) also had an increased risk of CSVD. CONCLUSION: The association between kidney function and CSVD has been comprehensively updated through this study, that kidney insufficiency manifested as low eGFR, proteinuria, and elevated serum cystatin C was independently associated with CSVD burden.

Omics-based biomarkers discovery for Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorders presenting with the pathological hallmarks of amyloid plaques and tau tangles. Over the past few years, great efforts have been made to explore reliable biomarkers of AD. High-throughput omics are a technology driven by multiple levels of unbiased data to detect the complex etiology of AD, and it provides us with new opportunities to better understand the pathophysiology of AD and thereby identify potential biomarkers. Through revealing the interaction networks between different molecular levels, the ultimate goal of multi-omics is to improve the diagnosis and treatment of AD. In this review, based on the current AD pathology and the current status of AD diagnostic biomarkers, we summarize how genomics, transcriptomics, proteomics and metabolomics are all conducing to the discovery of reliable AD biomarkers that could be developed and used in clinical AD management.

Caspase-1 variant influencing CSF tau and FDG PET levels in non-demented elders from the ADNI cohort.

BACKGROUND: Genetic variations in the inflammatory Caspase-1 gene have been shown associated with cognitive function in elderly individuals and in predisposition to Alzheimer's disease (AD), but its detailed mechanism before the typical AD onset was still unclear. Our current study evaluated the impact of Caspase-1 common variant rs554344 on the pathological processes of brain amyloidosis, tauopathy, and neurodegeneration. METHODS: Data used in our study were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. We examined the relationship between Caspase-1 rs554344 allele carrier status with AD-related cerebrospinal fluid (CSF), PET, and MRI measures at baseline by using a multiple linear regression model. We also analyzed the longitudinal effects of this variant on the change rates of CSF biomarkers and imaging data using a mixed effect model. RESULTS: We found that Caspase-1 variant was significantly associated with FDG PET levels and CSF t-tau levels at baseline in total non-demented elderly group, and especially in mild cognitive impairment (MCI) subgroup. In addition, this variant was also detected associated with CSF p-tau levels in MCI subgroup. The mediation analysis showed that CSF p-tau partially mediated the association between Caspase-1 variant and CSF t-tau levels, accounting for 80% of the total effect. CONCLUSIONS: Our study indicated a potential role of Caspase-1 variant in influencing cognitive function might through changing tau related-neurodegeneration process.

KPNA2 promotes renal cell carcinoma proliferation and metastasis via NPM.

Karyopherin α2 (KPNA2), involved in nucleocytoplasmic transport, has been reported to be up-regulated in tumorigenesis. However, comprehensive studies of KPNA2 functions in renal cell carcinoma (RCC) are still lacking. In this study, we aim to investigate the roles of KPNA2 in kidney tumour development. Our results showed that down-regulation of KPNA2 inhibited the proliferation and invasion of kidney tumour cell cells in vitro, while the cell cycle arrest and cellular apoptosis were induced once KPNA2 was silenced. Repression of KPNA2 was proved to be efficient to repress tumorigenesis and development of kidney tumour in in nude mice. Furthermore, one related participator, NPM, was identified based on Co-IP/MS and bioinformatics analyses. The up-regulation of NPM attenuates the efficiency of knockdown KPNA2. These results indicated that KPNA2 may regulate NPM to play a crucial role for kidney tumour development.

Associations of the cerebrospinal fluid hepatocyte growth factor with Alzheimer's disease pathology and cognitive function.

BACKGROUND: Hepatocyte growth factor (HGF) plays a role in neuronal survival and development, and has been implicated in neurodegenerative diseases. We sought to examine the associations of the CSF HGF with Alzheimer's disease (AD) pathology and cognitive function. METHODS: A total of 238 participants (including 90 cognitively normal (CN) and 148 mild cognitive impairment (MCI)) who had measurements of CSF HGF were included from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. Multiple linear regression models were utilized to explore the cross-sectional associations of CSF HGF with AD biomarkers (including Aß42, pTau, and tTau proteins) in non-demented participants. Moreover, linear mixed-effects regression models were utilized to explore the longitudinal associations of HGF subgroups with cognitive function. Mediation analyses were utilized to explore the mediation effects of AD markers. RESULTS: MCI individuals had significantly increased CSF HGF compared with the CN individuals. Results of multiple linear regressions showed significant correlations of CSF HGF with CSF Aß42, pTau, and tTau in non-demented participants. Higher level of baseline CSF HGF was associated with faster cognitive decline. Influences of the baseline CSF HGF on cognition were partially mediated by Aß42, pTau, and tTau pathologies. CONCLUSIONS: High concentrations of HGF in CSF may be related to faster cognitive decline. The cognitive consequences of higher CSF HGF partly stem from AD pathology, which suggests that the CSF HGF may be an attractive biomarker candidate to track AD progression.

Structural and functional definition of a vulnerable site on the hemagglutinin of highly pathogenic avian influenza A virus H5N1.

Most neutralizing antibodies against highly pathogenic avian influenza A virus H5N1 recognize the receptor-binding site (RBS) on the globular head domain and the stem of H5N1 hemagglutinin (HA). Through comprehensive analysis of multiple human protective antibodies, we previously identified four vulnerable sites (VS1-VS4) on the globular head domain. Among them, the VS1, occupying the opposite side of the RBS on the same HA, was defined by the epitope of antibody 65C6. In this study, we report the crystal structures of two additional human H5N1 antibodies isolated from H5N1-infected individuals, 3C11 and AVFluIgG01, bound to the head at 2.33- and 2.30-Å resolution, respectively. These two new antibody epitopes have large overlap with and extend beyond the original VS1. Site-directed mutagenesis experiments identified eight pivotal residues (Ser-126b, Lys-165, Arg-166, Ser-167, Tyr-168, Asn-169, Thr-171, and Asn-172) critical for 65C6-, 3C11-, and AVFluIgG01-binding and neutralization activities. These residues formed a unique "Y"-shaped surface on H5N1 globular head and are highly conserved among H5N1 viruses. Our results further support the existence of a vulnerable site distinct from the RBS and the stem region of H5N1 HA, and future design of immunogens should take this particular site into consideration.

IgH isotype-specific B cell receptor expression influences B cell fate.

Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighε/ε mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighε/ε mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgµ > mIgγ1 > mIgε, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighε/ε mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE+ memory B cells-which essentially do not exist under normal conditions-to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.

Cloning of hepatic lipase and the effects of dietary nutrition on hepatic lipase expression in genetically improved farmed tilapia (Oreochromis niloticus).

Hepatic lipase is an important gene in lipid metabolism, which is crucial in the growth of fish. In this study, the cDNA sequence of genetically improved farmed tilapia (GIFT) HL gene was cloned by aimed rapid amplification of cDNA ends (RACE) method. Then, the characteristics of HL were analyzed with bioinformatics methods, and the expression of HL was assessed by the quantitative real-time PCR. To study the regulation of HL expression, GIFT were fed with diets containing different contents of lipid (40, 80, and 120 g kg-1) and choline (500, 750, and 1000 mg kg-1) and fed with different frequencies (2 or 3 times/day) and amounts (20, 40, 60, 80, and g kg-1 of body weight). Our results revealed that the GIFT HL gene has a full length of 1872 bp, encoding 493 amino acids. Consistent with the study in other species, GIFT HL was specially expressed in the liver. The HL gene of GIFT shared identity of 60.9-96.6% with other species. The expression of HL in 120 g kg-1 dietary lipid and 1000 mg kg-1 dietary choline group was the highest in all groups (P < 0.01). The expression of HL was increased gradually with 3 times/day frequency. All these results revealed the cDNA sequence of GIFT HL, and the expression of HL was affected by dietary choline and lipid levels, feeding frequency, and amount. This would guide the aquaculture of GIFT in the future.

Complementary recognition of the receptor-binding site of highly pathogenic H5N1 influenza viruses by two human neutralizing antibodies.

The highly pathogenic avian influenza virus H5N1 is a major threat to global public health and therefore a high-priority target of current vaccine development. The receptor-binding site (RBS) on the globular head of hemagglutinin (HA) in the viral envelope is one of the major target sites for antibody recognition against H5N1 and other influenza viruses. Here, we report the identification and characterization of a pair of human RBS-specific antibodies, designated FLD21.140 and AVFluIgG03, that are mutually complementary in their neutralizing activities against a diverse panel of H5N1 viruses. Crystallographic analysis and site-directed mutagenesis revealed that the two antibodies share a similar RBS-binding mode, and their individual specificities are governed by residues at positions 133a, 144, and 145. Specifically, FLD21.140 preferred Leu-133a/Lys-144/Ser-145, whereas AVFluIgG03 favored Ser-133a/Thr-144/Pro-145 residue triplets, both of which perfectly matched the most prevalent residues in viruses from epidemic-originating regions. Of note, according to an analysis of 3758 H5 HA sequences available in the Influenza Virus Database at the National Center for Biotechnology Information, the residues Leu-133a/Ser-133a and Ser-145/Pro-145 constituted more than 87.6 and 99.3% of all residues at these two positions, respectively. Taken together, our results provide a structural understanding for the neutralizing complementarity of these two antibodies and improve our understanding of the RBS-specific antibody response against H5N1 infection in humans.

Macrophage Migration Inhibitor Promoted the Intrahepatic Bile Duct Injury in Rats with Severe Acute Pancreatitis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is involved in many acute and chronic inflammatory diseases. However, its role in intrahepatic bile duct (IBD) cell damage associated with severe acute pancreatitis (SAP) remains unclear. AIMS: This study was aimed to identify the role of MIF and its underlying mechanisms in SAP complicated by IBD cell damage. METHODS: Forty-eight specific-pathogen-free male Wistar rats were randomly divided into four groups (N = 12): a sham operation group (SO group) and three SAP model groups (SAP-3h, SAP-6h, and SAP-12h). Immunohistochemistry was used to detect the expression of MIF and P38 in IBD cells. MIF mRNA expression in IBD cells was observed using real-time fluorescent quantitative polymerase chain reaction (real-time PCR). In addition, Western blotting was performed to detect the protein expression of P38, phosphorylated P38 (P-P38), nuclear factor-κB (NF-κB p65), and tumor necrosis factor alpha (TNF-α). Enzyme-linked immunosorbent assays were used to analyze the levels of TNF-α, IL-1ß, and IL-6 in the IBD of rats. RESULTS: Compared with the SO group, the expression of MIF in the IBD was significantly upregulated both at mRNA and at protein levels in the SAP group. Besides, the protein expression levels of P38, P-P38, NF-κB, p65, TNF-α, IL-1ß, and IL-6 in the IBD in rats were also significantly increased in the SAP group and the levels increased gradually as acute pancreatitis progressed (all P < 0.05). CONCLUSIONS: MIF may promote the IBD injury and inflammatory reaction in SAP via activating the P38-MAPK and NF-κB signaling pathways.

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

Fetal liver injury ameliorated by migration inhibitory factor inhibition in a rat model of acute pancreatitis in pregnancy.

AIM: This study was designed to investigate and assess fetal liver injury in a rat model of acute pancreatitis in pregnancy (APIP) as well as its possible mechanisms and potential therapeutic targets. METHODS: The APIP model was induced by sodium taurocholate in Sprague-Dawley rats during the third trimester. ISO-1, a macrophage migration inhibitory factor (MIF) antagonist, was given before the induction of APIP. In addition, sham-operated rats at later gestation were set as controls. Histological changes in the fetal liver and maternal pancreas were assessed. Amylase and lipase activity as well as the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß were examined. The expression of MIF in fetal liver was determined by immunochemistry and the expression of NF-κB, IκBα, high mobility group box-1 protein (HMGB1), TNF-α, and IL-1ß in fetal liver was determined by Western blot analysis. Ultrastructures of hepatic cells in fetal rats were observed under transmission electron microscopy. RESULTS: ISO-1 ameliorated the following: (i) pathological injuries in maternal pancreas and fetal liver; (ii) levels of TNF-α and IL-1ß in maternal serum; and (iii) levels of MIF, myeloperoxidase, NF-κB, HMGB1, TNF-α, and IL-1ß in fetal liver. CONCLUSION: Pathological damage and an inflammatory response in fetal liver were induced by APIP, and MIF inhibition ameliorated fetal liver injury by inhibiting the inflammatory cascade.

Differential JNK, p38 and ERK response to renal injury in a rat model of acute pancreatitis in pregnancy.

OBJECTIVE: The objective of this study was to determine the mechanism of acute renal injury (ARI) in acute necrotizing pancreatitis in late pregnancy (ANPIP). METHODS: Pregnant Sprague-Dawley rats in the third trimester were used for this study, and an ANPIP model was induced by injecting 5% sodium taurocholate into the biliary pancreatic duct. The rats were randomly divided into three groups: the normal, sham-operated (SO) and acute necrotizing pancreatitis (ANP) groups. Rats were killed at 3, 6, 12 h after the operation, and blood, pancreatic and renal tissue samples were harvested. Differences were detected in the physiology, pathology and cellular and molecular responses among the different groups. RESULT: Serum amylase, lipase, urea and Cr levels were increased in rats with ANPIP. Additionally, expression of phosphorylation p38 and JNK as well as TNF-α and NF-κB were increased in the renal tissues of rats with ANPIP. The expression of phosphorylation ERK was decreased in the renal tissues of rats with ANPIP. CONCLUSIONS: Mitogen-activated protein kinases may play an important role in renal injury in rat models of ANPIP.

Poly(ADP-ribose) polymerase inhibition suppresses inflammation and promotes recovery from adrenal injury in a rat model of acute necrotizing pancreatitis.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) participates in multi-organ failure in various inflammatory diseases including acute necrotizing pancreatitis (ANP). Since pancreatitis-associated adrenal insufficiency is partly caused by inflammatory damage to the adrenal cortex, we examined whether PARP antagonism could alleviate adrenal insufficiency in a rat model of ANP. METHODS: ANP was induced by retrograde infusion of sodium taurocholate into the bile-pancreatic duct. At 30 min prior to taurocholate infusion, rats were pretreated with the PARP inhibitor 3-Aminobenzamide (3-AB, 20 mg/kg) or vehicle. Pancreatic pathological injury, adrenal histology, neutrophil infiltration, cell apoptosis, and serum corticosterone level were assessed at various times points. Activities of poly(ADP-ribosyl)ated protein (PAR), nuclear factor-kappaB (NF-kB), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) in the adrenal were also examined. RESULTS: PARP overactivation in ANP rats is associated with reduced serum corticosterone level and marked cellular alterations in adrenocortical tissue. Inflammatory stress caused by ANP reduced adrenal corticosterone release. 3-AB reduced the activation of PARP and inflammatory markers, decreased myeloperoxidase activity, attenuated adrenal morphologic lesions and cells apoptosis, simultaneously improved the impaired adrenal function. CONCLUSIONS: Our data demonstrate the involvement of PARP overactivation in the pathogenesis of adrenal dysfunction after ANP. PARP inhibition may suppress inflammation and promote functional recovery from adrenal injury.

Combinatorial library-based profiling of the antibody response against hepatitis C virus in humans.

The antibody response plays a crucial role against hepatitis C virus (HCV) infection, and our understanding of this intricate progress in vivo is far from complete. We previously reported a novel and robust technique based on a large combinatorial viral antigen library displayed on the surface of the yeast Saccharomyces cerevisiae, allowing comprehensive profiling of polyclonal antibody responses in vivo in both qualitative and quantitative terms. Here, we report the generation and application of a combinatorial library of HCV strain JFH1 envelope glycoprotein to profile the antibody response in four HCV chronically infected individuals. By systematic analysis of the location and frequency of antigenic fragments along the JFH1 envelope glycoprotein, we showed that the major binding antibody response was targeted to E2 (80.9-99.8 %), whilst that against E1 was relatively small (0.3-19.0 %). A total of five major antigenic domains (D1-D5) were identified: one was within E1 and an additional four within E2, despite substantial variability among the different individuals. However, serum absorption with the yeast clones containing the antigenic domain D1 resulted in more reduction in neutralizing antibody activity against pseudotyped HCV than those in E2, suggesting that E1 contains additional neutralizing epitopes. Our results have provided additional insights into the HCV-specific antibody response in humans and should assist in a better understanding of protective antibody immunity and in guiding the development of effective vaccines and therapeutics against HCV infection.