Hepatitis C Virus and the Host: A Mutual Endurance Leaving Indelible Scars in the Host's Immunity.

Hepatitis C virus (HCV) has spread worldwide, and it is responsible for potentially severe chronic liver disease and primary liver cancer. Chronic infection remains for life if not spontaneously eliminated and viral persistence profoundly impairs the efficiency of the host's immunity. Attempts have been made to develop an effective vaccine, but efficacy trials have met with failure. The availability of highly efficacious direct-acting antivirals (DAA) has created hope for the progressive elimination of chronic HCV infections; however, this approach requires a monumental global effort. HCV elicits a prompt innate immune response in the host, characterized by a robust production of interferon-α (IFN-α), although interference in IFN-α signaling by HCV proteins may curb this effect. The late appearance of largely ineffective neutralizing antibodies and the progressive exhaustion of T cells, particularly CD8 T cells, result in the inability to eradicate the virus in most infected patients. Moreover, an HCV cure resulting from DAA treatment does not completely restore the normal immunologic homeostasis. Here, we discuss the main immunological features of immune responses to HCV and the epigenetic scars that chronic viral persistence leaves behind.

High Frequencies of Functional Virus-Specific CD4+ T Cells in SARS-CoV-2 Subjects With Olfactory and Taste Disorders.

Olfactory and taste disorders (OTD) are commonly found as presenting symptoms of SARS-CoV-2 infection in patients with clinically mild COVID-19. Virus-specific T cells are thought to play an important role in the clearance of SARS-CoV-2; therefore the study of T cell specific immune responses in patients with mild symptoms may help to understand their possible role in protection from severe disease. We evaluated SARS-CoV-2-specific T cell responses to four different peptide megapools covering all SARS-CoV-2 proteins during the acute phase of the disease in 33 individuals with mild or no other symptom beside OTD and in 22 age-matched patients with severe infection. A control group of 15 outpatients with OTD and consistently negative nasopharyngeal SARS-CoV-2 RNA swabs and virus-specific IgG serology was included in the study. Increased frequencies of virus-specific CD4+ and CD8+ T cells were found in SARS-CoV-2 positive patients with OTD compared with those with severe COVID-19 and with SARS-CoV-2 negative OTD individuals. Moreover, enhanced CD4+ and CD8+ T-cell activation induced by SARS-CoV-2 peptides was associated with higher interferon (IFN)γ production. Increased frequencies of Spike (S1/S2)-specific CD4+ T cells showing enhanced IFNγ secretion and granzyme B content were associated with serum spike-specific IgG in the OTD group. In conclusion, patients with SARS-CoV-2 induced OTD develop highly functional virus-specific CD4+ and CD8+ T cells during the symptomatic phase of the disease, suggesting that robust and coordinated T-cell responses provide protection against extension of COVID-19 to the lower respiratory tract.

Unique immunological profile in patients with COVID-19.

The relationship between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host immunity is poorly understood. We performed an extensive analysis of immune responses in 32 patients with severe COVID-19, some of whom succumbed. A control population of healthy subjects was included. Patients with COVID-19 had an altered distribution of peripheral blood lymphocytes, with an increased proportion of mature natural killer (NK) cells and low T-cell numbers. NK cells and CD8+ T cells overexpressed T-cell immunoglobulin and mucin domain-3 (TIM-3) and CD69. NK cell exhaustion was attested by increased frequencies of programmed cell death protein 1 (PD-1) positive cells and reduced frequencies of natural killer group 2 member D (NKG2D)-, DNAX accessory molecule-1 (DNAM-1)- and sialic acid-binding Ig-like lectin 7 (Siglec-7)-expressing NK cells, associated with a reduced ability to secrete interferon (IFN)γ. Patients with poor outcome showed a contraction of immature CD56bright and an expansion of mature CD57+ FcεRIγneg adaptive NK cells compared to survivors. Increased serum levels of IL-6 were also more frequently identified in deceased patients compared to survivors. Of note, monocytes secreted abundant quantities of IL-6, IL-8, and IL-1ß which persisted at lower levels several weeks after recovery with concomitant normalization of CD69, PD-1 and TIM-3 expression and restoration of CD8+ T cell numbers. A hyperactivated/exhausted immune response dominate in severe SARS-CoV-2 infection, probably driven by an uncontrolled secretion of inflammatory cytokines by monocytes. These findings unveil a unique immunological profile in COVID-19 patients that will help to design effective stage-specific treatments for this potentially deadly disease.

Human Monoclonal Antibodies as Adjuvant Treatment of Chronic Hepatitis B Virus Infection.

Despite the availability of an effective prophylactic vaccine leading to sterilizing immunity, hepatitis B virus (HBV) is responsible for chronic liver disease in more than 250 million individuals, potentially leading to cirrhosis and hepatocellular carcinoma. Antiviral drugs able to completely suppress virus replication are indeed available but they are, by and large, unable to eradicate the virus. Several alternative new treatment approaches are currently being developed but none have so far captured the interest of clinicians for possible clinical development. A constant feature of chronic HBV infection is T-cell exhaustion resulting from persistent exposure to high antigen concentrations as shown by the high expression of programmed cell death protein 1 (PD-1) by HBV-specific CD8 T cells. One way of tackling this problem is to develop HBV-specific neutralizing antibodies that would clear excess envelope proteins from the circulation, allowing for nucleos(t)ide analogs or other antiviral drugs now in preclinical and early clinical development to take advantage of a reconstituted adaptive immunity. Several fully human monoclonal antibodies (mAb) have been developed from HBV-vaccinated and subjects convalescent from acute hepatitis B that show different properties and specificities. It is envisaged that such neutralizing mAb may be used as adjuvant treatment to reduce viral protein load, thus rescuing adaptive immunity in an effort to optimize the effect of antiviral drugs.

A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity.

We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common "a" determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.

Enhanced B-cell differentiation and reduced proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections.

BACKGROUND & AIMS: Chronic microbial infections are frequently associated with B-cell activation and polyclonal proliferation, potentially leading to autoimmunity and lymphoproliferative disorders. We assessed B-cell phenotype and function in chronic hepatitis B (HBV) and chronic hepatitis C (HCV) virus infection. METHODS: We studied 70 patients with chronic HCV infection, 34 with chronic HBV infection and 54 healthy controls. B-cell phenotype was assessed by flow cytometry using monoclonal antibodies specific for CD27, the CD69, CD71, and CD86 activation markers and the chemokine receptor CXCR3. Differentiation into immunoglobulin-producing cells (IPC) was analysed by ELISpot upon stimulation and with CD40 ligand±IL-10 as surrogate bystander T-cell help or CpG oligodeoxynucleotide±IL-2, as innate immunity signal. Proliferation was examined by flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) after stimulation with CpG. RESULTS: A significantly higher proportion of B cells from both HCV- and HBV-infected patients expressed activation markers compared with controls and a positive correlation was found between CXCR3(+) B cells and HCV RNA values. Memory B cells from patients with chronic HCV and HBV infections showed enhanced differentiation into IPC compared with controls, although this was restricted to IgG and at a lower level in HCV-compared with HBV-infected patients. Moreover, patients' activated B cells displayed significantly lower proliferative ability compared to healthy donors despite low expression of the FcRL4 exhaustion marker. CONCLUSIONS: B-cell activation, but not exhaustion, is common in chronic viral hepatitis. However, enhanced B-cell differentiation and deficient proliferative capacity were not associated with commitment to terminal differentiation.

PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV.

CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell-dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3- effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death-1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death-1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms.

Hallmark of acute hepatitis C virus (HCV) infection is a severe virus-specific effector CD8(+) T cell dysfunction that seems to be a critical factor in preventing the resolution of infection and in favoring the onset of chronic liver immunopathology. We suggest that this dysfunction is critical in the establishment of HCV persistence, unless it is compensated by multispecific responses, as found in individuals resolving infection. Analyses on purified populations indicate that central memory HCV-specific CCR7(+)/CD8(+) T cells efficiently proliferate and differentiate in vitro, although the large population of memory effector CCR7(-) cells found in the peripheral blood of acutely infected patients display poor effector functions ex vivo (semi-effectors). However, we report strong evidence in support of IL-2 being capable of pushing semi-effector CTL to complete their effector cell program. Therefore, IL-2 deficiency during T cell activation may be responsible for the dichotomy between memory CTL expansion and incomplete effector differentiation shown in patients with acute HCV infection. These data are consistent with the possible therapeutic treatment with IL-2 to rebuild the effector T cell pool in these patients.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: the role of the virus.

In a companion study, we showed a dichotomy between the expansion of central memory (CCR7(+)) hepatitis C virus (HCV)-specific CTL and the incomplete memory effector differentiation in patients with acute HCV infection. Indeed, effector cells were unable to perform immediate functions, despite expressing the tissue-homing phenotype of effector memory cells (CCR7(-); semi-effectors). However, since they promptly differentiated into full-effectors upon IL-2 contact, we suggested that the inhibitory effect by environmental (possibly viral) factors on IL-2 production may have a pivotal role in generating the large population of semi-effector CCR7(-)/IFN-gamma(-) CTL. In accord with this view, we report here strong evidence in support of circulating HCVcore protein (HCVcore) playing a central role in inhibiting effector CTL differentiation, but not memory CTL expansion. The regulatory HCVcore effect is related to inhibition of the signal transduction pathway instrumental for IL-2 production, supporting the evidence that IL-2 was capable both of pushing semi-effector CTL to complete their effector cell program and of restoring the HCVcore-dependent inhibitory effect. Therefore, the strength of CTL activation is dependent on the balance between the threshold of stimulatory signals and the viral interference capacities provided during priming.

Monitoring response to antiviral therapy for patients with chronic hepatitis C virus infection by a core-antigen assay.

A recently released immunoassay detecting total serum hepatitis C virus (HCV) core antigen was used to prospectively monitor virological responses to antiviral treatment in patients with chronic HCV infection. Sustained responders cleared core protein from serum within the first month of therapy and maintained stably negative values for the entire duration of follow-up after treatment discontinuation. However, patients who relapsed or failed to respond showed transient negative values and could not be accurately discriminated either because of the intrinsic lower sensitivity of the core-antigen assay than those of molecular assays or because of differentially regulated secretion of immunoreactive core protein from infected hepatocytes.

Variability or conservation of hepatitis C virus hypervariable region 1? Implications for immune responses.

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous in its primary sequence and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to immune escape and persistent infection. A binding site for neutralizing antibodies (Ab) has also been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from naturally-occurring HVR1 sequences showed promiscuous recognition of HVR1 variants in most patients via binding to C-terminal amino acid residues with conserved physicochemical properties. Monoclonal antibodies generated by immunization of mice with peptides derived from natural HVR1 sequences were shown to recognize several HVR1 variants in line with evidence gathered from studies using human sera. In addition, selected mAbs were able to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded sequences, and were shown to specifically capture circulating and recombinant HCV particles, suggesting that HVR1 is expressed on intact virus particles and therefore potentially able to interact with cellular receptor(s). These findings suggest that it is possible to induce a broadly reactive clonal immune response to multiple HCV variants and that this mechanism could be used in principle to induce protective immunity for a large repertoire of HCV variants.

Kinetics and significance of serum hepatitis C virus core antigen in patients with acute hepatitis C.

An immunoassay detecting hepatitis C virus core antigen was evaluated for its ability to predict clinical outcome in a series of patients with acute hepatitis C. In subjects who cleared the virus, core antigen was no longer detectable within 16 weeks of onset, whereas considerable fluctuations were noted among patients progressing to chronic hepatitis, one of whom showed consistently negative values despite the intermittent presence of viral RNA.