Evidence for a pathogenic role of extrafollicular, IL-10-producing CCR6+B helper T cells in systemic lupus erythematosus.

Interleukin 10 (IL-10) is an antiinflammatory cytokine, but also promotes B cell responses and plays a pathogenic role in systemic lupus erythematosus (SLE). CD4+CCR6+IL-7R+T cells from human tonsils produced IL-10 following stimulation by naïve B cells, which promoted B cell immunoglobulin G (IgG) production. These tonsillar CCR6+B helper T cells were phenotypically distinct from follicular helper T (TFH) cells and lacked BCL6 expression. In peripheral blood, a CCR6+T cell population with similar characteristics was identified, which lacked Th17- and TFH-associated gene signatures and differentiation-associated surface markers. CD4+CCR6+T cells expressing IL-10, but not IL-17, were also detectable in the spleens of cytokine reporter mice. They provided help for IgG production in vivo, and expanded systemically in pristane-induced lupus-like disease. In SLE patients, CD4+CCR6+IL-7R+T cells were associated with the presence of pathogenic anti-dsDNA (double-stranded DNA) antibodies, and provided spontaneous help for autoantibody production ex vivo. Strikingly, IL-10-producing CCR6+T cells were highly abundant in lymph nodes of SLE patients, and colocalized with B cells at the margins of follicles. In conclusion, we identified a previously uncharacterized population of extrafollicular B helper T cells, which produced IL-10 and could play a prominent pathogenic role in SLE.

IL-10 producing regulatory and helper T-cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly heterogeneous autoimmune disease characterised by the production of pathogenic autoantibodies against nuclear self-antigens. The anti-inflammatory and tolerogenic cytokine Interleukin-10 appears to play a paradoxical pathogenic role in SLE and is therefore currently therapeutically targeted in clinical trials. It is generally assumed that the pathogenic effect of IL-10 in SLE is due to its growth and differentiation factor activity on autoreactive B-cells, but effects on other cells might also play a role. To date, a unique cellular source of pathogenic IL-10 in SLE has not been identified. In this review, we focus on the contribution of different CD4+T-cell subsets to IL-10 and autoantibody production in SLE. In particular, we discuss that IL-10 produced by different subsets of adaptive regulatory T-cells, follicular helper T-cells and extra-follicular B-helper T-cells is likely to have different effects on autoreactive B-cell responses. A better understanding of the role of IL-10 in B-cell responses and lupus would allow to identify the most promising therapies for individual SLE patients in the future.

Repression of miR-31 by BCL6 stabilizes the helper function of human follicular helper T cells.

Follicular helper T cells (TFHs) are a key component of adaptive immune responses as they help antibody production by B cells. Differentiation and function of TFH cells are controlled by the master gene BCL6, but it is largely unclear how this transcription repressor specifies the TFH program. Here we asked whether BCL6 controlled helper function through down-regulation of specific microRNAs (miRNAs). We first assessed miRNA expression in TFH cells and defined a TFH-specific miRNA signature. We report that hsa-miR-31-5p (miR-31) is down-regulated in TFH; we showed that BCL6 suppresses miR-31 expression by binding to its promoter; and we demonstrated that miR-31 inhibits the expression of molecules that control T-helper function, such as CD40L and SAP. These findings identify a BCL6-initiated inhibitory circuit that stabilizes the follicular helper T cell program at least in part through the control of miRNA transcription. Although BCL6 controls TFH activity in human and mouse, the role of miR-31 is restricted to human TFH cell differentiation, reflecting a species specificity of the miR-31 action. Our findings highlight miR-31 as a possible target to modulate human T cell dependent antibody responses in the settings of infection, vaccination, or immune dysregulation.

NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase IIIα complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes.

The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex.

Why is it so difficult to develop a hepatitis C virus preventive vaccine?

With an estimated 3% of the world's population chronically infected, hepatitis C virus (HCV) represents a major health problem for which an efficient vaccination strategy would be highly desirable. Indeed, chronic hepatitis C is recognized as one of the major causes of cirrhosis, hepatocarcinoma and liver failure worldwide and it is the most common indication for liver transplantation, accounting for 40-50% of liver transplants. Much progress has been made in the prevention of HCV transmission and in therapeutic intervention. However, even if a new wave of directly acting antivirals promise to overcome the problems of low efficacy and adverse effects observed for the current standard of care, which include interferon-α and ribavirin, an effective vaccine would be the only means to definitively eradicate infection and to diminish the burden of HCV-related diseases at affordable costs. Although there is strong evidence that the goal of a prophylactic vaccine could be achieved, there are huge development issues that have impeded reaching this goal and that still have to be addressed. In this article we address the question of whether an HCV vaccine is needed, whether it will eventually be feasible, and why it is so difficult to produce.

HCV E1E2-MF59 vaccine in chronic hepatitis C patients treated with PEG-IFNα2a and Ribavirin: a randomized controlled trial.

Hepatitis C virus (HCV) vaccines may be able to increase viral clearance in combination with antiviral therapy. We analysed viral dynamics and HCV-specific immune response during retreatment for experienced patients in a phase Ib study with E1E2MF59 vaccine. Seventy-eight genotype 1a/1b patients [relapsers (30), partial responders (16) and nonresponders (32) to interferon-(IFN)/ribavirin-(RBV)] were randomly assigned to vaccine (V:23), Peg-IFNα2a-180-ug/qw and ribavirin 1000-1200-mg/qd for 48 weeks (P/R:25), or their combination (P/R + V:30). Vaccine (100 µg/0.5 mL) was administered intramuscularly at week 0-4-8-12-24-28-32-36. Neutralizing of binding (NOB) antibodies and lymphocyte proliferation assay (LPA) for E1E2-specific-CD4 + T cells were performed at week 0-12-16-48. Viral kinetics were analysed up to week 16. The vaccine was safe, and a sustained virological response (SVR) was achieved in 4 P/R + V and 2 P/R patients. Higher SVR rates were observed in prior relapsers (P/R + V = 27.3%; P/R = 12.5%). Higher NOB titres and LPA indexes were found at week 12 and 16 in P/R + V as compared to P/R patients (P = 0.023 and 0.025, P = 0.019 and <0.001, respectively). Among the 22 patients with the strongest direct antiviral effects of IFN (ε ≥ 0.800), those treated with P/R + V (10) reached lower HCV-RNA levels (P = 0.026) at week 16. HCV E1E2MF59 vaccine in combination with Peg-IFNα2a + RBV was safe and elicited E1E2 neutralizing antibodies and specific CD4 + T cell proliferation. Upon early response to IFN, vaccinations were associated with an enhanced second phase viral load decline. These results prompt phase II trials in combination with new antiviral therapies.

The CD4-centered universe of human T cell subsets.

Humans are continuously exposed to a high number of diverse pathogens that induce different types of immune responses. Primary pathogen-specific immune responses generate multiple subsets of memory T cells, which provide protection against secondary infections. In recent years, several novel T cell subsets have been identified and have significantly broadened our knowledge about T cell differentiation and the regulation of immune responses. At the same time the rapidly growing number of incompletely characterized T cell subsets has also generated some controversies. We therefore review here the current knowledge on features and functions of human α/ß T cell subsets, focusing on CD4(+) T cells classified according to cytokine production and tissue localization. The principal helper and regulatory T cell subsets can be identified by a limited number of relevant surface markers, which are an integral part of the T cell differentiation programs because they are directly induced by the relevant lineage-defining transcription factors. In vivo occurring human T cell subsets can thus be purified directly ex vivo from relevant tissues for molecular and functional studies, and represent not only an ideal model to study T cell differentiation, but they also offer important clinical opportunities.

Molecular analysis of V(H)I+ B lymphocytes in hepatitis C patients.

BACKGROUND AND AIMS: Hepatitis C virus infection is often associated with lymphoproliferative disorders such as essential mixed cryoglobulinemia and B-cell non-Hodgkin lymphoma, which show preferential expression of VHI family products. By analyzing immunoglobulin heavy chain usage, we addressed the question of whether or not clonal B-cell expansion occurrs in patients free of essential mixed cryoglobulinemia or non-Hodgkin lymphoma. PATIENTS AND METHODS: Four hepatitis C virus-positive patients, all undergoing liver transplantation, were studied. Peripheral blood, intra-hepatic, and lymph node lymphocytes were used as a source of B cells. A patient with hepatocellular carcinoma and fresh blood from four healthy donors were used as negative controls. VHI family sequences were cloned and analyzed by reverse transcription-polymerase chain reaction. RESULTS: Immunoglobulin heavy chain sequences from clonally expanded B lymphocytes were identified in three out of four hepatitis C virus-infected patients. The clonally expanded B lymphocyte populations showed a broad spectra of immunoglobulin heavy chain gene usage. CONCLUSIONS: HCV infection can induce B-cell expansion with larger clonal variation. The restricted V gene usage in hepatitis C virus-associated non-Hodgkin lymphoma suggests that there may be selection mechanisms to develop non-Hodgkin lymphoma from non-malignant, clonally expanded B-cell populations in hepatitis C virus-infected patients.

Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

The small extracellular loop of CD81 is necessary for optimal surface expression of the large loop, a putative HCV receptor.

Human tetraspanin CD81 is a putative receptor for hepatitis C virus (HCV), because it has been shown to bind 'bona fide' HCV particles. CD81, as all tetraspanins, spans the membrane four times forming two extracellular loops: a small (SEL) and a large one (LEL). We have shown previously that a recombinant form of LEL is sufficient for binding HCV through the major envelope glycoprotein E2. The role of SEL in the CD81-HCV interaction was questioned. We found that transfectants expressing LEL alone bind the recombinant HCV-E2 protein at much lower levels than cells expressing the wild type CD81. And therefore whether SEL contributes to the CD81-HCV interaction or whether it influences the expression of LEL was examined. We have found that in the absence of SEL, LEL is expressed at significantly reduced levels on the cell surface because it is retained intracellularly, while HCV-E2 still binds LEL. Our data suggest that SEL of CD81 does not mediate interaction with HCV, but contributes to optimal cell surface expression of LEL by mediating translocation of the whole CD81 molecule to the cell surface.

Folding of hepatitis C virus E1 glycoprotein in a cell-free system.

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.

Nonneutralizing human antibody fragments against hepatitis C virus E2 glycoprotein modulate neutralization of binding activity of human recombinant Fabs.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 (HCV/E2) glycoprotein is the major target of a putatively protective immune response. However, even in the presence of a vigorous production of anti-HCV/E2 antibodies, reinfection can occur. Dissection of the human immune response against HCV/E2 indicated that blocking of binding of HCV/E2 to target cells [neutralization of binding (NOB) activity] varies widely among antibody clones. Moreover, in vivo, simultaneous binding of antibodies to distinct epitopes can induce conformational changes and synergies that may be relevant to understanding the anti-HCV immune response. In this study, human recombinant Fabs were generated by affinity-selecting a phage display repertoire library with antibody-coated HCV/E2. These Fabs, which share the same complementarity-determining region DNA sequences, had higher affinity than other anti-HCV/E2 Fabs but showed no NOB activity even at the highest concentrations. Binding of Fabs to HCV/E2 caused conformational changes modifying Fab-binding patterns and reducing, with a negative synergistic effect, Fab-mediated NOB activity. These data suggest that some antibody clones have the potential to modify HCV/E2 conformation and that, in this state, binding of this glycoprotein to its cellular target is less prone to inhibition by some antibody clones. This can explain why high anti-HCV/E2 antibody titers do not directly correlate with protection from infection. Information on the interactions among different antibody clones can contribute to understanding virus-host interplay and developing more effective vaccines.

Vdelta1 T lymphocytes expressing a Th1 phenotype are the major gammadelta T cell subset infiltrating the liver of HCV-infected persons.

BACKGROUND: Hepatitis C infection induces an acute and chronic liver inflammation that may lead to cirrhosis, liver failure, or hepatocarcinoma. Since the role of alphabeta T lymphocytes in hepatitis C virus (HCV) immunopathology has been analyzed extensively, we investigated the distribution and functional activation of gammadelta T cell subsets in chronically HCV-infected patients. MATERIALS AND METHODS: Blood samples and liver biopsies from 35 patients with compensated chronic HCV infection were compared in terms of T cell subset distribution, expression of activation markers, gammadelta T cell receptor (TCR) repertoire, and pattern of cytokine production. Moreover, we analyzed whether these immunological parameters were associated with other clinical observations (plasma viremia, ALT levels, Ishak index). RESULTS: Differing from peripheral blood distribution, a specific compartmentalization of Vdelta1 T cells (p < 0.001) was observed in the liver of HCV patients. These cells represented a relevant fraction of intrahepatic T lymphocytes (1.8-8.7%) and expressed the memory/effector phenotype (CD62-L- CD45-RO+CD95+). This phenotype was consistent with selective homing upon antigen recognition. Mitogenic stimulation of Vdelta1 + T lymphocytes recruited in the liver revealed the T helper cell type 1 (Th1) pattern of cytokine secretion. Interestingly, the frequency of interferon-gamma (IFN-gamma)-producing Vdelta1 T cells was associated with an higher degree of liver necroinflammation, measured by the Ishak index. Finally, the T-cell repertoire analysis revealed the absence of Vgamma selection in the TCR repertoire of intrahepatic Vdelta1 T cells. CONCLUSIONS: gammadelta T cell distribution in the peripheral blood differs from the Vdelta1 T cell subset because it is policlonally activated and recruited in the liver of chronic HCV-infected patients. During HCV-infection, this T cell subset may release Th1 cytokines and contribute to the necroinflammatory liver disease.

Virus-specific CD8(+) T cells with type 1 or type 2 cytokine profile are related to different disease activity in chronic hepatitis C virus infection.

The present study demonstrates that the quality of the virus-specific CD8(+) T cell responses, as detected by both enzyme-linked immunospot assay and specific MHC-peptide tetramers, changed in relation to the different disease activity in chronically hepatitis C virus-infected patients. Indeed, both the serum alanine transaminase and the hepatic flogosis levels were related directly to the frequencies of peripheral memory effector CD8(+) T cells producing IFN-gamma (Tc1), but inversely to the frequencies of those producing both IL-4 and IL-10 (Tc2). Longitudinal studies highlighted that Tc1 or Tc2 responses fluctuate in relation to the different phases of the disease in the same individual. Furthermore, the Tc1 or Tc2 phenotype correlates with tetramer-positive cells expressing either CXCR3 or CCR3, promoting differential tissue localization of these cells and the maintenance of T cell homeostasis. Finally, studies at the level of liver-infiltrating lymphocytes indicated that they produced both IFN-gamma and IL-4 with an evident bias towards the Tc1-like phenotype. Our studies suggest that the progressive fluctuation of Tc1 and Tc2 responses may play a fundamental role in maintaining a long-lasting low-level liver inflammation, and may constitute the basis for new therapeutic strategies of immune regulation.

CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs.

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

Binding of the hepatitis C virus envelope protein E2 to CD81 provides a co-stimulatory signal for human T cells.

Chronic hepatitis C virus (HCV) infection frequently develops into liver disease and is accompanied by extra-hepatic autoimmune manifestations. The tetraspanin CD81 is a putative HCV receptor as it binds the E2 envelope glycoprotein of HCV and bona fide HCV particles. Here we show that HCV E2 binding to CD81 on human cells in vitro lowers the threshold for IL-2 receptor alpha expression and IL-2 production, resulting in strongly increased T cell proliferation. HCV E2-induced co-stimulation also enhances the production of IFN-gamma and IL-4 and causes increased TCR down-regulation. This suggests that binding of HCV particles to CD81 on T cells in vivo may lead to activation by otherwise suboptimal stimuli. Therefore, co-stimulation of autoreactive T cells by HCV may contribute to liver damage and autoimmune phenomena observed in HCV infection.

Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes.

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.

Neonatal invariant Valpha24+ NKT lymphocytes are activated memory cells.

NKT cells are a small subset of T lymphocytes which express an invariant V(alpha24JalphaQ TCR and recognize glycolipids presented by CD1d. In adults, NKT cells have a memory phenotype, frequently associated with oligoclonal expansion, express NK cell markers, and produce TO cytokines upon primary stimulation. Because of these features, NKT cells are regarded as lymphocytes of innate immunity. We investigated NKT cells from cord blood to see how these cells appear in the absence of exogenous stimuli. We found that NKT cells are present at comparable frequencies in cord blood and adult peripheral blood mononuclear cells and in both cases display a memory (CD45RO+CD62L-) phenotype. However, neonatal NKT cells differ from their adult counterparts by the following characteristics: (1) they express markers of activation, such as CD25; (2) they are polyclonal; (3) they do not produce cytokines in response to primary stimulation. Together, our data show that human NKT cells arise in the newborn with an activated memory phenotype, probably due to recognition of an endogenous ligand(s). The absence of oligoclonal expansion and primary effector functions also suggest that neonatal NKT cells, despite their activated memory phenotype, require a further priming/differentiation event to behave as fully functional cells of innate immunity.

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates.

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Life, activation and death of intrahepatic lymphocytes in chronic hepatitis C.

The healthy liver of adult humans has little or no lymphocyte component and the histological finding of intrahepatic lymphocytes (IHL) is evidence of liver pathology. In a liver injured by chronic hepatitis C, the most common chronic liver disease, most IHL are activated/pro-inflammatory cells, which are particularly enriched for effectors of innate immunity (natural killer (NK), natural T, and other NK-like T cells). IHL do not undergo clonal expansion in the liver but migrate from extrahepatic sites to the chronically infected liver, where they display effector function and subsequently die, suggesting that maintenance of the IHL pool depends on continuous lymphocyte migration. The cytotoxic and inflammatory functions of these IHL have three potential outcomes: 1) they could be helpful in clearing the virus (a rare case in hepatitis C virus (HCV) infection); 2) they could be useless and have no effect on the infection; or 3) they could be harmful, whereby overaggressive lymphocyte responses destroy the liver in a continuous and unsuccessful attempt to clear the virus. Unfortunately, we do not know as of yet which of these possibilities is the case and, therefore, a more complete picture of the intrahepatic immune response will be relevant to the development of new therapeutic strategies against HCV. Additionally and from a more general perspective, due to the availability of biopsied material and the high prevalence (approximately 3%) of HCV infection worldwide, studying the chronically inflamed liver of hepatitis C patients is an ideal model to investigate the poorly understood processes of lymphocyte trafficking, activation and death to non-lymphoid sites of chronic inflammation in man.