Jörg Timm

Hepatitis E Virus Infection as a Possible Cause of Acute Liver Failure in Europe

BACKGROUND & AIMSnIn Western countries, infection with the hepatitis E virus (HEV) is considered to be rare and imported from endemic regions. However, the prevalence of HEV infection has increased among adults in central Europe. HEV infection can cause acute liver failure (ALF), but there have been only a few confirmed cases of HEV-associated ALF in Europe. We investigated the number of cases of indeterminate ALF associated with HEV infection.nnnMETHODSnWe performed a retrospective analysis of 80 patients diagnosed with ALF or acute hepatitis at the University Hospital Essen in Germany from November 2006 through December 2013. Clinical data were collected from the hospital databases; archived sera were tested for IgG and IgM against HEV, as well as HEV RNA.nnnRESULTSnSera from 12 patients (15%) tested positive for IgG against HEV IgG; 7 of these samples did not test positive for HEV IgM or HEV RNA. Sera from 64 patients (80%) did not test positive for IgG or IgM against HEV or HEV RNA. Sera from 8 patients (10%) tested positive for HEV RNA (only 4 of these were positive for HEV IgG) and had clinical findings to support acute HEV infection.nnnCONCLUSIONSnIn a hospital in Germany, approximately 10% to 15% of patients with ALF had evidence for HEV infection. Serologic tests for IgG against HEV are insufficient to identify or exclude HEV infection; tests for HEV RNA also should be performed on patients with ALF of ambiguous etiology.

Natural prevalence of resistance-associated variants in hepatitis C virus NS5A in genotype 3a-infected people who inject drugs in Germany

BACKGROUNDnPeople who inject drugs (PWID) are the most important risk group for incident Hepatitis C virus (HCV) infection. In PWID in Europe HCV genotype 3a is highly prevalent. Unfortunately, many of the recently developed directly acting antiviral drugs against HCV (DAAs) are suboptimal for treatment of this genotype. Detection of resistance-associated variants (RAV) in genotype 3a may help to optimize treatment decisions, however, robust protocols for amplification and sequencing of HCV NS5A as an important target for treatment of genotype 3a are currently lacking.nnnOBJECTIVESnThe aim of this study was to establish a protocol for sequencing of HCV NS5A in genotype 3a and to determine the frequency of RAVs in treatment-naïve PWID living in Germany.nnnSTUDY DESIGNnThe full NS5A region was amplified and sequenced from 110 HCV genotype 3a infected PWID using an in-house PCR protocol.nnnRESULTSnWith the established protocol the complete NS5A region was successfully amplified and sequenced from 110 out of 112 (98.2%) genotype 3a infected PWID. Phylogenetic analysis of sequences from PWID together with unrelated genotype 3a sequences from a public database showed a scattered distribution without geographic clustering. Viral polymorphisms A30K and Y93H known to confer resistance in a GT3a replication model were present in 8 subjects (7.2%).nnnCONCLUSIONSnA protocol for amplification of nearly all GT3a samples was successfully established. Substitutions conferring resistance to NS5A inhibitors were detected in a few treatment-naive PWID.

Assessing the risk of CMV reactivation and reconstitution of antiviral immune response post bone marrow transplantation by the QuantiFERON-CMV-assay and real time PCR

BACKGROUNDnCMV reactivation is a major cause of severe complications in allogeneic hematopoietic stem cell transplant (HSCT) recipients. The risk of CMV reactivation depends on the serostatus (+/-) of the donor (D) and recipient (R). The reconstitution of CMV-specific T-cell responses after transplantation is crucial for the control of CMV reactivation.nnnOBJECTIVESnThe study aimed to determine the cellular immune status correlating with protection from high-level CMV viremia (>5000 copies/ml) and disease.nnnSTUDY DESIGNnWe monitored CMV-specific cellular immune responses in 9 high-risk (D-/R+), 14 intermediate risk (D+/R+) and 3 low risk individuals (D+/R-), and 8 CMV negative controls (D-/R-). Interferon- γ (IFN-γ) levels as a marker for the CD8+ T-cell response were determined by the QuantiFERON-CMV-assay and compared to viral loads determined by PCR.nnnRESULTSnEarly CMV reactivation was detected in all high-risk and 13/14 intermediate risk individuals. High-level viremia was detected in 5/7 high and 7/14 intermediate risk patients. Reconstitution of the CMV-specific cellular immune response started from 3 months after transplantation and resulted in protection against CMV reactivation. Re-establishing of CMV-specific T-cell immune responses with IFN- γ levels >8.9u202fIU/ml is crucial for protection from high-level CMV viremia.nnnCONCLUSIONSnMonitoring of HSCT-recipients with the QuantiFERON-CMV-assay might be of great benefit to optimize antiviral treatment.

HLA-Bw4 80(T) and multiple HLA-Bw4 copies combined with KIR3DL1 associate with spontaneous clearance of HCV infection in people who inject drugs

BACKGROUND & AIMSnNatural killer (NK) cell function is regulated by inhibitory and activating receptors including killer cell immunoglobulin-like receptors (KIRs). Here, we analyzed the impact of different KIR/KIR-ligand genotypes on the outcome of hepatitis C virus (HCV) infection in people who inject drugs (PWID).nnnMETHODSnKIR/KIR-ligand genotypes associated with spontaneous clearance of HCV infection were identified in a cohort of PWID from Germany (n=266) and further validated in a second anti-HCV positive cohort of PWID recruited in North America (n=342). NK cells of PWID and healthy donors were functionally characterized according to their KIR/KIR-ligand genotype by flow cytometry.nnnRESULTSnMultivariate logistic regression analysis revealed that KIR3DL1/HLA-Bw4 80(T) was associated with spontaneous clearance of HCV infection in PWID, which was confirmed in the PWID cohort from North America. Compared with PWID with detectable HCV RNA, the frequency of individuals with multiple HLA-Bw4 alleles was significantly higher in anti-HCV positive PWID with resolved HCV infection (29.7% vs. 15.2%; p=0.0229) and in anti-HCV seronegative PWID (39.2%; p=0.0006). KIR3DL1+ NK cells from HLA-Bw4 80(T)-positive PWID showed superior functionality compared to HLA-Bw4 80(I)-positive PWID. This differential impact was not observed in healthy donors; however, the HLA-Bw4 copy number strongly correlated with the functionality of KIR3DL1+ NK cells.nnnCONCLUSIONSnHLA-Bw4-80(T) and multiple HLA-Bw4 copies in combination with KIR3DL1 are associated with protection against chronic hepatitis C in PWID by distinct mechanisms. Better licensing of KIR3DL1+ NK cells in the presence of multiple HLA-Bw4 copies is beneficial prior to seroconversion whereas HLA-Bw4 80(T) may be beneficial during acute hepatitis C. Lay summary: Natural killer (NK) cells are part of the innate immune system and are regulated by a complex network of activating and inhibiting receptors. The regulating receptor-ligand pairs of an individual are genetically determined. Here, we identified a particular set of ligand and receptor genes that are associated with better functionality of NK cells and better outcome upon exposure to HCV in a high-risk group.

Acute hepatitis C virus infection induces anti‐host cell receptor antibodies with virus‐neutralizing properties

Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The mechanisms of persistence and clearance are only partially understood. Antibodies (Abs) against host cell entry receptors have been shown to inhibit HCV infection in cell culture and animal models. In this study, we aimed to investigate whether anti‐receptor Abs are induced during infection in humans in vivo and whether their presence is associated with outcome of infection. We established an enzyme‐linked immunosorbant assay using a recombinant CD81‐claudin‐1 (CLDN1) fusion protein to detect and quantify Abs directed against extracellular epitopes of the HCV CD81‐CLDN1 coreceptor complex. The presence of anti‐receptor Abs was studied in serum of patients from a well‐defined cohort of a single‐source HCV outbreak of pregnant women and several control groups, including uninfected pregnant women, patients with chronic hepatitis B and D virus (HBV/HDV) infection, and healthy individuals. Virus‐neutralizing activity of Abs was determined using recombinant cell culture–derived HCV (HCVcc). Our results demonstrate that HCV‐infected patients have statistically significantly higher anti‐CD81/CLDN1 Ab titers during the early phase of infection than controls. The titers were significantly higher in resolvers compared to persisters. Functional studies using immunoadsorption and HCV cell culture models demonstrate that HCV‐neutralizing anti‐receptor Abs are induced in the early phase of HCV infection, but not in control groups. Conclusion: The virus‐neutralizing properties of these Abs suggest a role for control of viral infection in conjunction with antiviral responses. Characterization of these anti‐receptor Abs opens new avenues to prevent and treat HCV infection. (Hepatology 2015;62:726–736)

CEACAM1 promotes CD8 + T cell responses and improves control of a chronic viral infection

Dysfunction of CD8+ T cells can lead to the development of chronic viral infection. Identifying mechanisms responsible for such T cell dysfunction is therefore of great importance to understand how to prevent persistent viral infection. Here we show using lymphocytic choriomeningitis virus (LCMV)xa0infection that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is fundamental for recruiting lymphocyte-specific protein kinase (Lck) into the T cell receptor complex to form an efficient immunological synapse. CEACAM1 is essential for activation of CD8+ T cells, and the absence of CEACAM1 on virus-specific CD8+ T cells limits the antiviral CD8+ T cell response. Treatment with anti-CEACAM1 antibody stabilizes Lck in the immunological synapse, prevents CD8+ T cell exhaustion, and improves control of virus infection in vivo. Treatment of human virus-specific CD8+ T cells with anti-CEACAM1 antibody similarly enhances their proliferation. We conclude that CEACAM1 is an important regulator of virus-specific CD8+ T cell functions in mice and humans and represents a promising therapeutic target for modulating CD8+ T cells.Chronic viral infections are frequently associated with the dysfunction of CD8+ T cells which includes loss of function and results in CD8+ T cell exhaustion. Here the authors show a role of CEACAM1 in promoting responsive CD8+ T cells in the context of a chronic lymphocytic choriomeningitis virus (LCMV) infection model.

A genotype independent, full-genome reverse-transcription protocol for HCV genotyping and resistance testing

BACKGROUNDnHCV treatment options and cure rates have tremendously increased in the last decade. Although a pan-genotype HCV treatment has recently been approved, most DAA therapies are still genotype specific. Resistance-associated variants (RAVs) can limit the efficacy of DAA therapy and are associated with increased risk for therapy failure. With the approval of DAA regimens that recommend resistance testing prior to therapy, correct assessment of the genotype and testing for viruses with RAVs is clinically relevant. However, genotyping and resistance testing is generally done in costly and laborious separate reactions.nnnOBJECTIVEnThe aim of the study was to establish a genotype-independent full-genome reverse transcription protocol to generate a template for both genotyping and resistance testing and to implement it into our routine diagnostic setup.nnnSTUDY DESIGNnThe complete HCV genome was reverse transcribed with a pan-genotype primer binding at the 3end of the viral RNA. This cDNA served as template for transcription of the genotyping amplicon in the core region as well as for the resistance testing of NS3, NS5A, and NS5B.nnnRESULTSnWith the established RT-protocol the HCV core region was successfully amplified and genotyped from 124 out of 125 (99.2%) HCV-positive samples. The amplification efficiency of RAV containing regions in NS3, NS5A, NS5B was 96.2%, 96.6% and 94.4%, respectively.nnnCONCLUSIONSnWe developed a method for HCV full-genome cDNA synthesis and implemented it into a routine diagnostic setup. This cDNA can be used as template for genotyping amplicons covering the core or NS5B region as well as for resistance testing amplicons in NS3, NS5A and NS5B.

Early Transcriptional Divergence Marks Virus-Specific Primary Human CD8+ T Cells in Chronic versus Acute Infection

&NA; Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus‐specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV‐specific CD4+ T cell populations. These early changes in HCV‐specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection. Graphical Abstract Figure. No caption available. HighlightsDisease outcome and viral escape are linked to transcriptional differences in T cellsT cells from different outcomes share a core of co‐regulated T cell identity genesMetabolic, nucleosome, and immune genes are dysregulated early in chronic infectionDysregulation correlates with sex, age, and presence of HCV‐specific CD4 T cells &NA; Wolski et. al show that transcriptional dysregulation of metabolic, nucleosomal, and immune processes in virus‐specific CD8+ T cells during early persistent HCV infection is both under tight transcriptional control and associated with differences in predictors of disease outcome, like patient sex, age, and the presence of HCV‐specific CD4+ T cells.

Lymphocytes Negatively Regulate NK Cell Activity via Qa-1b following Viral Infection

NK cells can reduce anti-viral Txa0cell immunity during chronic viral infections, including infection with the lymphocytic choriomeningitis virus (LCMV). However, regulating factors that maintain the equilibrium between productive Txa0cell and NK cell immunity are poorly understood. Here, we show that a large viral load resulted in inhibition of NK cell activation, which correlated with increased expression of Qa-1b, a ligand for inhibitory NK cell receptors. Qa-1b was predominantly upregulated on B cells following LCMV infection, and this upregulation was dependent on type I interferons. Absence of Qa-1b resulted in increased NK cell-mediated regulation of anti-viral T cells following viral infection. Consequently, anti-viral Txa0cell immunity was reduced in Qa-1b- and NKG2A-deficient mice, resulting in increased viral replication and immunopathology. NK cell depletion restored anti-viral immunity and virus control in the absence of Qa-1b. Taken together, our findings indicate that lymphocytes limit NK cell activity during viral infection in order to promote anti-viral Txa0cell immunity.

Differential escape of HCV from CD8+ T cell selection pressure between China and Germany depends on the presenting HLA class I molecule

Adaptation of hepatitis C virus (HCV) to CD8+ T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I‐associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I‐restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK1635‐1643, which was presented by HLA‐A*03 as well as HLA‐A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA‐A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA‐A*11‐positive individuals in China and associated with different patterns of CD8+ T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell‐based vaccine strategies.