Stability and Inactivation of Monkeypox Virus on Inanimate Surfaces.

The spread of nonzoonotic monkeypox virus (MPXV) infections necessitates the reevaluation of hygiene measures. To date, only limited data are available on MPXV surface stability. Here, we evaluate the stability of infectious MPXV on stainless steel stored at different temperatures, while using different interfering substances to mimic environmental contamination. MPXV persistence increased with decreasing temperature. Additionally, we were able to show that MPXV could efficiently be inactivated by alcohol- and aldehyde-based surface disinfectants. These findings underline the stability of MPXV on inanimate surfaces and support the recommendations to use alcohol-based disinfectants as prevention measures or in outbreak situations.

In-depth virological and immunological characterization of HIV-1 cure after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation.

Despite scientific evidence originating from two patients published to date that CCR5Δ32/Δ32 hematopoietic stem cell transplantation (HSCT) can cure human immunodeficiency virus type 1 (HIV-1), the knowledge of immunological and virological correlates of cure is limited. Here we characterize a case of long-term HIV-1 remission of a 53-year-old male who was carefully monitored for more than 9 years after allogeneic CCR5Δ32/Δ32 HSCT performed for acute myeloid leukemia. Despite sporadic traces of HIV-1 DNA detected by droplet digital PCR and in situ hybridization assays in peripheral T cell subsets and tissue-derived samples, repeated ex vivo quantitative and in vivo outgrowth assays in humanized mice did not reveal replication-competent virus. Low levels of immune activation and waning HIV-1-specific humoral and cellular immune responses indicated a lack of ongoing antigen production. Four years after analytical treatment interruption, the absence of a viral rebound and the lack of immunological correlates of HIV-1 antigen persistence are strong evidence for HIV-1 cure after CCR5Δ32/Δ32 HSCT.

Efficient Inactivation of Monkeypox Virus by World Health Organization‒Recommended Hand Rub Formulations and Alcohols.

Increasing nonzoonotic human monkeypox virus (MPXV) infections urge reevaluation of inactivation strategies. We demonstrate efficient inactivation of MPXV by 2 World Health Organization‒recommended alcohol-based hand rub solutions. When compared with other (re)emerging enveloped viruses, MPXV displayed the greatest stability. Our results support rigorous adherence to use of alcohol-based disinfectants.

Hepatitis B virus-specific CD4 T cell responses differentiate functional cure from chronic surface antigen+ infection.

BACKGROUND & AIMS: With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control. METHODS: We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining, and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly ex vivo. RESULTS: ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection. CONCLUSIONS: Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses. LAY SUMMARY: Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell).

Transcriptional Pattern Analysis of Virus-Specific CD8+ T Cells in Hepatitis C Infection: Increased Expression of TOX and Eomesodermin During and After Persistent Antigen Recognition.

Thymocyte selection-associated high mobility group box (TOX) has been described to be a key regulator in the formation of CD8+ T cell exhaustion. Hepatitis C virus (HCV) infection with different lengths of antigen exposure in acute, chronic, and after resolution of HCV infection is the ideal immunological model to study the expression of TOX in HCV-specific CD8+ T cells with different exposure to antigen. HCV-specific CD8+ T cells from 35 HLA-A*01:01, HLA-A*02:01, and HLA-A*24:02 positive patients were analyzed with a 16-color FACS-panel evaluating the surface expression of lineage markers (CD3, CD8), ectoenzymes (CD39, CD73), markers of differentiation (CD45RO, CCR7, CD127), and markers of exhaustion and activation (TIGIT, PD-1, KLRG1, CD226) and transcription factors (TOX, Eomesodermin, T-bet). Here, we defined on-target T cells as T cells against epitopes without escape mutations and off-target T cells as those against a "historical" antigen mutated in the autologous sequence. TOX+HCV-specific CD8+ T cells from patients with chronic HCV and on-target T cells displayed co-expression of Eomesodermin and were associated with the formation of terminally exhausted CD127-PD1hi, CD39hi, CD73low CD8+ T cells. In contrast, TOX+HCV-specific CD8+ T cells in patients with off-target T cells represented a progenitor memory Tex phenotype characterized by CD127hi expression and a CD39low and CD73hi phenotype. TOX+HCV-specified CD8+ T cells in patients with a sustained virologic response were characterized by a memory phenotype (CD127+, CD73hi) and co-expression of immune checkpoints and Eomesodermin, indicating a key structure in priming of HCV-specific CD8+ T cells in the chronic stage, which persisted as a residual after therapy. Overall, the occurrence of TOX+HCV-specific CD8+ T cells was revealed at each disease stage, which impacted the development of progenitor Tex, intermediate Tex, and terminally exhausted T cell through an individual molecular footprint. In sum, TOX is induced early during acute infection but is modulated by changes in viral sequence and antigen recognition. In the case of antigen persistence, the interaction with Eomesodermin leads to the formation of terminally exhausted virus-specific CD8+ T cells, and there was a direct correlation of the co-expression of TOX and Eomes and terminally exhausted phenotype of virus-specific CD8+ T cells.

Emergence of the E484K mutation in SARS-COV-2-infected immunocompromised patients treated with bamlanivimab in Germany.

BACKGROUND: Monoclonal antibodies (mAb) have been introduced as a promising new therapeutic approach against SARS-CoV-2. At present, there is little experience regarding their clinical effects in patient populations underrepresented in clinical trials, e.g. immunocompromised patients. Additionally, it is not well known to what extent SARS-CoV-2 treatment with monoclonal antibodies could trigger the selection of immune escape viral variants. METHODS: After identifying immunocompromised patients with viral rebound under treatment with bamlanivimab, we characterized the SARS-CoV-2-isolates by whole genome sequencing. Viral load measurements and sequence analysis were performed consecutively before and after bamlanivimab administration. FINDINGS: After initial decrease of viral load, viral clearance was not achieved in five of six immunocompromised patients treated with bamlanivimab. Instead, viral replication increased again over the course of the following one to two weeks. In these five patients, the E484K substitution - known to confer immune escape - was detected at the time of viral rebound but not before bamlanivimab treatment. INTERPRETATION: Treatment of SARS-CoV-2 with bamlanivimab in immunocompromised patients results in the rapid development of immune escape variants in a significant proportion of cases. Given that the E484K mutation can hamper natural immunity, the effectiveness of vaccination as well as antibody-based therapies, these findings may have important implications not only for individual treatment decisions but may also pose a risk to general prevention and treatment strategies. FUNDING: All authors are employed and all expenses covered by governmental, federal state, or other publicly funded institutions.

Analysis of the Long-Term Impact on Cellular Immunity in COVID-19-Recovered Individuals Reveals a Profound NKT Cell Impairment.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected over 120 million people and killed over 2.7 million individuals by March 2021. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remain to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19-convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2-unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of annexin V and 7-aminoactinomycin D (7-AAD) double-positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies and TIM-3 expression on CD4 and CD8 T cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by granzyme B (GzmB) expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully able to proliferate and produce effector cytokines upon T cell receptor (TCR) stimulation. Collectively, we provide a comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.IMPORTANCE Wuhan was the very first city hit by SARS-CoV-2. Accordingly, the patients who experienced the longest phase of convalescence following COVID-19 reside here. This enabled us to investigate the "immunological scar" left by SARS-CoV-2 on cellular immunity after recovery from the disease. In this study, we characterized the long-term impact of SARS-CoV-2 infection on the immune system and provide a comprehensive picture of cellular immunity of a convalescent COVID-19 patient cohort with the longest recovery time. We revealed that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease; in particular, a profound NKT cell impairment was found in the convalescent phase of COVID-19.

Corrigendum: TLR2 Expression in Peripheral CD4+ T Cells Promotes Th17 Response and Is Associated with Disease Aggravation of Hepatitis B Virus-Related Acute-On-Chronic Liver Failure.

[This corrects the article DOI: 10.3389/fimmu.2017.01609.].

MMP2/MMP9-mediated CD100 shedding is crucial for inducing intrahepatic anti-HBV CD8 T cell responses and HBV clearance.

BACKGROUND & AIMS: CD100 is constitutively expressed on T cells and can be cleaved from the cell surface by matrix metalloproteases (MMPs) to become soluble CD100 (sCD100). Both membrane-bound CD100 (mCD100) and sCD100 have important immune regulatory functions that promote immune cell activation and responses. This study investigated the expression and role of mCD100 and sCD100 in regulating antiviral immune responses during HBV infection. METHODS: mCD100 expression on T cells, sCD100 levels in the serum, and MMP expression in the liver and serum were analysed in patients with chronic HBV (CHB) and in HBV-replicating mice. The ability of sCD100 to mediate antigen-presenting cell maturation, HBV-specific T cell activation, and HBV clearance were analysed in HBV-replicating mice and patients with CHB. RESULTS: Patients with CHB had higher mCD100 expression on T cells and lower serum sCD100 levels compared with healthy controls. Therapeutic sCD100 treatment resulted in the activation of DCs and liver sinusoidal endothelial cells, enhanced HBV-specific CD8 T cell responses, and accelerated HBV clearance, whereas blockade of its receptor CD72 attenuated the intrahepatic anti-HBV CD8 T cell response. Together with MMP9, MMP2 mediated mCD100 shedding from the T cell surface. Patients with CHB had significantly lower serum MMP2 levels, which positively correlated with serum sCD100 levels, compared with healthy controls. Inhibition of MMP2/9 activity resulted in an attenuated anti-HBV T cell response and delayed HBV clearance in mice. CONCLUSIONS: MMP2/9-mediated sCD100 release has an important role in regulating intrahepatic anti-HBV CD8 T cell responses, thus mediating subsequent viral clearance during HBV infection. LAY SUMMARY: Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. The clearance of HBV relies largely on an effective T cell immune response, which usually becomes dysregulated in chronic HBV infection. Our study provides a new mechanism to elucidate HBV persistence and a new target for developing immunotherapy strategies in patients chronically infected with HBV.

A Single-Nucleotide Polymorphism Upstream of the HLA-C Locus Is Associated With an Anti-Hepatitis C Virus-Seronegative State in a High-Risk Exposed Cohort.

In this study, we examined the impact of the rs9264942 single-nucleotide polymorphism, previously shown to be associated with human immunodeficiency virus infection status and HLA-C expression, on the hepatitis C virus status in 359 people who inject drugs (PWID). The linkage of rs9264942 alleles to HLA-C antigens assigned to different expression levels was confirmed. Multivariate analysis revealed the age (P = .003) and the rs9264942 genotype (P = .006) to be independent factors for the classification to the PWID groups. Our study showed that the presence of the rs9264942 C/C genotype was associated with persistent seronegativity.

TLR2 Expression in Peripheral CD4+ T Cells Promotes Th17 Response and Is Associated with Disease Aggravation of Hepatitis B Virus-Related Acute-On-Chronic Liver Failure.

Th17 responses have been shown to play crucial roles in the pathogenesis of hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF). The mechanism underlying the enhanced Th17 responses in these patients remains largely unclear. Here we investigated toll-like receptors (TLRs) expression in peripheral T cells and their roles in Th17 cell differentiation and disease aggravation in ACLF patients. 18 healthy subjects (HS), 20 chronic HBV-infected (CHB) patients, and 26 ACLF patients were enrolled and examined for TLRs expression in peripheral blood mononuclear cells (PBMCs). The correlations of T cell TLR2 expression with the antigen non-specific Th17 responses and disease aggravation, as well as the Th17 response to TLR2 ligand stimulation were evaluated in ACLF patients. Compared to HS and CHB patients, ACLF patients showed a distinct TLRs expression pattern in PBMCs. Significantly increased TLR2 expression in T cells was observed in ACLF patients. The TLR2 expression in CD4+ T cells was correlated with the Th17 responses and the clinical markers for disease aggravation in ACLF patients. Moreover, TLR2 ligands stimulation promoted Th17 cell differentiation and response in PBMCs of ACLF patients. These findings implicate that TLR2 signaling plays critical roles in Th17 cell differentiation and disease aggravation of HBV-related ACLF.

Nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis C virus infection.

Viruses lack the basic machinery needed to replicate and therefore must hijack the host's metabolism to propagate. Virus-induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host-pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)-responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4α, ketogenesis by PPARα and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4α reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARα or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a host's metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host.

Distinct Escape Pathway by Hepatitis C Virus Genotype 1a from a Dominant CD8+ T Cell Response by Selection of Altered Epitope Processing.

UNLABELLED: Antiviral CD8(+) T cells are a key component of the adaptive immune response against HCV, but their impact on viral control is influenced by preexisting viral variants in important target epitopes and the development of viral escape mutations. Immunodominant epitopes highly conserved across genotypes therefore are attractive for T cell based prophylactic vaccines. Here, we characterized the CD8(+) T cell response against the highly conserved HLA-B*51-restricted epitope IPFYGKAI1373-1380 located in the helicase domain of NS3 in people who inject drugs (PWID) exposed predominantly to HCV genotypes 1a and 3a. Despite this epitope being conserved in both genotypes, the corresponding CD8(+) T cell response was detected only in PWID infected with genotype 3a and HCV-RNA negative PWID, but not in PWID infected with genotype 1a. In genotype 3a, the detection of strong CD8(+) T cell responses was associated with epitope variants in the autologous virus consistent with immune escape. Analysis of viral sequences from multiple cohorts confirmed HLA-B*51-associated escape mutations inside the epitope in genotype 3a, but not in genotype 1a. Here, a distinct substitution in the N-terminal flanking region located 5 residues upstream of the epitope (S1368P; P = 0.00002) was selected in HLA-B*51-positive individuals. Functional assays revealed that the S1368P substitution impaired recognition of target cells presenting the endogenously processed epitope. The results highlight that, despite an epitope being highly conserved between two genotypes, there are major differences in the selected viral escape pathways and the corresponding T cell responses. IMPORTANCE: HCV is able to evolutionary adapt to CD8(+) T cell immune pressure in multiple ways. Beyond selection of mutations inside targeted epitopes, this study demonstrates that HCV inhibits epitope processing by modification of the epitope flanking region under T cell immune pressure. Selection of a substitution five amino acids upstream of the epitope underlines that efficient antigen presentation strongly depends on its larger sequence context and that blocking of the multistep process of antigen processing by mutation is exploited also by HCV. The pathways to mutational escape of HCV are to some extent predictable but are distinct in different genotypes. Importantly, the selected escape pathway of HCV may have consequences for the destiny of antigen-specific CD8(+) T cells.

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7) hepatocytes, 1.8 ± 0.5 × 10(6 )Kupffer cells, 4.3 ± 1.9 × 10(5) liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5) stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2%) and exhibited phagocytic activity, as determined with 1 µm latex beads. Endothelial cells were CD146(+) (97.8 ± 1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Adaptation of the hepatitis B virus core protein to CD8(+) T-cell selection pressure.

UNLABELLED: Activation of hepatitis B virus (HBV)-specific CD8 T cells by therapeutic vaccination may promote sustained control of viral replication by clearance of covalently closed circular DNA from infected hepatocytes. However, little is known about the exact targets of the CD8 T-cell response and whether HBV reproducibly evades CD8 T-cell immune pressure by mutation. The aim of this study was to address if HBV reproducibly selects substitutions in CD8 T-cell epitopes that functionally act as immune escape mutations. The HBV core gene was amplified and sequenced from 148 patients with chronic HBV infection, and the human leukocyte antigen (HLA) class I genotype (A and B loci) was determined. Residues under selection pressure in the presence of particular HLA class I alleles were identified by a statistical approach utilizing the novel analysis package SeqFeatR. With this approach we identified nine residues in HBV core under selection pressure in the presence of 10 different HLA class I alleles. Additional immunological experiments confirmed that seven of the residues were located inside epitopes targeted by patients with chronic HBV infection carrying the relevant HLA class I allele. Consistent with viral escape, the selected substitutions reproducibly impaired recognition by HBV-specific CD8 T cells. CONCLUSION: Viral sequence analysis allows identification of HLA class I-restricted epitopes under reproducible selection pressure in HBV core; the possibility of viral escape from CD8 T-cell immune pressure needs attention in the context of therapeutic vaccination against HBV.

Decades after recovery from hepatitis B and HBsAg clearance the CD8+ T cell response against HBV core is nearly undetectable.

BACKGROUND & AIMS: CD8(+) T cells are an essential component of a successful immune response against hepatitis B virus (HBV). Patients who spontaneously clear HBsAg after acute HBV infection have a strong CD8(+) T cell immune response, predominantly directed against the HBV core protein (HBcAg). However, the fate and phenotype of HBcAg-specific CD8(+) T cells after immune control are unclear. METHODS: The CD8(+) T cell immune response against HBV core was determined in 65 patients with chronic HBV infection, 16 patients after recovery from acute HBV infection, and four patients with acute HBV infection utilizing overlapping peptides and HLA class I/peptide-multimers. RESULTS: Patients who had cleared HBsAg >30 years ago had significantly weaker CD8(+) T cell responses after antigen-specific expansion compared to patients who had cleared the virus <10 years ago and patients with HBeAg negative chronic infection and low viral load (<2000 IU/ml; p<0.01). Also directly ex vivo, patients who had cleared the HBsAg >30 years ago had less HBV-specific CD8(+) T cells compared to patients with HBeAg negative chronic infection (p=0.0025). In patients with acute HBV infection, the frequency of HBc-specific CD8(+) T cells continued to decline after clearance of HBV-DNA and HBsAg even at a time when ALT levels had already normalized (p=0.0313). CONCLUSIONS: The frequency of HBcAg-specific CD8(+) T cells continuously declines after HBsAg clearance. In line with clinical observations, this suggests that humoral and not CD8(+) T cell immune responses mainly contribute to prevention of HBV reactivation decades after HBsAg clearance.

Mutational escape of CD8+ T cell epitopes: implications for prevention and therapy of persistent hepatitis virus infections.

Over the past two decades, much has been learned about how human viruses evade T cell immunity to establish persistent infection. The lessons are particularly relevant to two hepatotropic viruses, HBV and HCV, that are very significant global public health problems. Although HCV and HBV are very different, the natural history of persistent infections with these viruses in humans shares some common features including failure of T cell immunity. During recent years, large sequence studies of HCV have characterized intra-host evolution as well as sequence diversity between hosts in great detail. Combined with studies of CD8+ T cell phenotype and function, it is now apparent that the T cell response shapes viral evolution. In turn, HCV sequence diversity influences the quality of the CD8+ T cell response and thus infection outcome. Here, we review published studies of CD8+ T cell selection pressure and mutational escape of the virus. Potential consequences for therapeutic strategies to restore T cell immunity against persistent human viruses, most notably HBV, are discussed.

KIR2DL3⁺NKG2A⁻ natural killer cells are associated with protection from productive hepatitis C virus infection in people who inject drugs.

BACKGROUND & AIMS: Despite continuous high-risk behavior, a subgroup among people who inject drugs (PWID) remains seronegative for hepatitis C virus (HCV) suggesting that a state of "natural resistance" to HCV Infection may exist. Homozygosity for KIR2DL3 and its ligand HLA-C1 group alleles has been associated with control of HCV infection, however, the mechanism mediating this protective effect remained unclear. METHODS: Peripheral NK cells from PWID (n=104) were phenotypically and functionally characterized by multicolor flow cytometry. Expression levels of the NK cell receptor ligands were analysed in liver biopsies and primary human hepatocytes. RESULTS: HCV seronegative PWID (n=34) had increased levels of KIR2DL3(+)NKG2A(-) NK cells compared to healthy controls (n=10; p<0.001) and PWID with chronic (n=38; p<0.001) or resolved infection (n=37; p<0.001). There was an inverse correlation between the frequency of KIR2DL3(+) and NKG2A(+) NK cells (r=-0.53; p<0.0001). Importantly, expression of HLA-E, the ligand for NKG2A, was significantly upregulated in liver biopsies of HCV infected patients (n=51) compared to HBV infected patients (n=22; p<0.01) and correlated with HCV viral load (r=0.32; p<0.0029). In functional analyses KIR2DL3(-)NKG2A(+) NK cells but not KIR2DL3(+)NKG2A(-) NK cells were significantly inhibited by HLA-E ligation. Accordingly, interferon gamma secretion of NK cells from PWID with chronic infection but not from HCV seronegative PWID was significantly suppressed in the presence of HLA-E. CONCLUSIONS: KIR2DL3(+)NKG2A(-) NK cells are not sensitive to HLA-E-mediated inhibition and may thereby control early HCV infection prior to seroconversion and result in an apparent state of "natural resistance" to HCV in PWID.

Genetic heterogeneity of hepatitis C virus cell entry receptors seems to have no influence on selection of virus variants.

BACKGROUND: No information is available on the possible influence of the genetic heterogeneity of major hepatitis C virus (HCV) cell receptors on selection of virus variants. FINDINGS: Anti-D globulin preparations contaminated with the HCV strain AD78 caused hepatitis C infection in more than 3000 women in East Germany in 1978. Analysis of the core to NS2 gene sequences of this strain in several globulin batches revealed the presence of three closely related but distinct virus variants of the same strain. Apparently even distribution of these three virus variants was observed in 91 patients infected with the AD78 strain. None of these patients was infected with more than one virus variant, suggesting a selection mechanism of a particular virus variant in each patient. To verify the hypothesis that heterogeneity of HCV cell receptors might influence the virus variant selection, single-nucleotide polymorphisms (SNPs) in low-density lipoprotein receptor (LDLR), occludin (OCLN), and scavenger receptor B1 (SCARB1) genes in AD patients were analyzed. No evident correlation between receptor polymorphisms and presence of a particular virus variant was noted. CONCLUSION: SNPs of HCV cell entry receptors have no influence on virus selection in patients infected with an inoculum containing different virus variants.

Identification of proteins that mediate the pro-viral functions of the interferon stimulated gene 15 in hepatitis C virus replication.

In previous studies we identified the interferon stimulated gene 15 (ISG15) as a pro-viral host factor in the pathogenesis of hepatitis C virus (HCV) infection. However, the functional link between ISG15 and the HCV replication cycle is not well understood. Aim of the present study was to functionally analyze the role of ISG15 and to identify possible HCV promoting effector molecules. Isg15 suppression was investigated in the murine subgenomic HCV replicon (MH1) transfected with Isg15-specific siRNA and in C57BL/6 mice intravenously injected with lipid nanoparticles (LNP)-formulated siRNA. Interestingly, the LNP-formulated siRNA led to hepatocyte-specific knockdown of Isg15 in vivo, which mediated a hypo-responsiveness to endogenous and exogenous interferon. A label free proteome analysis accompanied by western blot and quantitative RT-PCR techniques led to identification of five candidate proteins (Heterogeneous nuclear ribonucleoprotein A3 (HnrnpA3), Heterogeneous nuclear ribonucleoprotein K (HnrnpK), Hydroxymethylglutaryl-CoA synthase (Hmgcs1), Isocitrate dehydrogenase cytoplasmic (Idh1) and Thioredoxin domain-containing protein 5 (Txndc5)) that are either involved in lipid metabolism or belong to the family of Heterogeneous nuclear ribonucleoprotein (Hnrnp). All candidate proteins are likely to be associated with the HCV replication complex. Furthermore treatment with HnrnpK-specific siRNA directly suppressed HCV replication in vitro. Taken together these data suggest that targeting Isg15 may represent an attractive novel therapeutic option for the treatment of chronic HCV infection.