Cytokine profile and viral diversity in the early seronegative stage of community-acquired hepatitis C virus (HCV) infection.

Most Hepatitis C virus (HCV)-infected subjects develop chronic infection, whereas a minority clear the virus in the early phase of infection. We analyzed factors associated with outcome (chronicity vs clearance) during the preclinical seronegative phase of community-acquired HCV infection. Among 17.5 million blood donations in the years 2000-2016, 124 blood donors were found to be HCV RNA-positive/anti-HCV-negative. All were contacted after 0.5-12.7 years and 40 responded and provided blood sample. Hypervariable region 1 was analyzed by ultradeep pyrosequencing and cytokines in serum were quantified by Luminex (R&D Systems) multiplex immunoassay. Twenty-one (52.5%) donors were found to be HCV-RNA-positive, while 19 (47.5%) were HCV RNA negative (none received antiviral treatment). All but one seroconverted to anti-HCV. Donors with resolving hepatitis did not differ significantly from donors with chronic infection with respect to age, genotypes, IL28B polymorphisms, number of viral variants, nucleotide diversity per site or the overall number of nucleotide substitutions. However, the former group had significantly higher levels of IL-1beta, IL-1RA, IL-6, IFN-gamma and FGF-2 in serum. In our study of community-acquired acute hepatitis C approximately half of all subjects eliminated the virus spontaneously, and this clearance was associated with marked cytokine response in the early seronegative stage of infection.

T-Cell Exhaustion in HIV-1/Hepatitis C Virus Coinfection Is Reduced After Successful Treatment of Chronic Hepatitis C.

Background: T-cell responses during chronic viral infections become exhausted, which is reflected by upregulation of inhibitory receptors (iRs) and increased interleukin 10 (IL-10). We assessed 2 iRs-PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing protein 3)-and IL-10 mRNAs in peripheral blood mononuclear cells (PBMCs) and their soluble analogs (sPD-1, sTim-3, and IL-10) in plasma in chronic HIV-1/hepatitis C virus (HCV) coinfection and explored the effect of HCV treatment on these markers. We also aimed to establish whether iR expression may be determined by the HCV CD8+ T-cell immunodominant epitope sequence. Methods: Plasma and PBMCs from 31 persons with chronic HIV-1/HCV coinfection from the Swiss HIV Cohort Study were collected before and after HCV treatment. As controls, 45 persons who were HIV-1 negative with chronic HCV infection were recruited. Exhaustion markers were assessed by enzyme-linked immunosorbent assay in plasma and by quantitative reverse transcription polymerase chain reaction in PBMCs. Analysis of an HCV epitope sequence was conducted by next-generation sequencing: HLA-A*02-restricted NS31073-1081 and NS31406-1415 and HLA-A*01-restricted NS31436-1444. Results: The study revealed higher plasma sPD-1 (P = .0235) and IL-10 (P = .002) levels and higher IL-10 mRNA in PBMCs (P = .0149) in HIV-1/HCV coinfection. A decrease in plasma sPD-1 (P = .0006), sTim-3 (P = .0136), and IL-10 (P = .0003) and Tim-3 mRNA in PBMCs (P = .0210) was observed following successful HCV treatment. Infection with the HLA-A*01-restricted NS31436-1444 ATDALMTGY prototype variant was related to higher sTim-3 levels than infection with the ATDALMTGF escape variant (P = .0326). Conclusions: The results underscore the synergistic effect of coinfection on expression of exhaustion markers, their reduction following successful HCV treatment and imply that iR levels may operate on an epitope-specific manner.

Double Positive CD4+CD8+ (DP) T-Cells Display Distinct Exhaustion Phenotype in Chronic Hepatitis C.

In chronic hepatitis C (CHC), characterized by exhaustion of T-cell function, increased frequencies of double-positive (DP) (CD4+CD8+) cells are present in peripheral blood. We compared the exhaustion phenotype between DP and single positive (SP) T-cells, including HCV-specific cells, and assessed the effect of successful HCV treatment on inhibitory receptors expression. Blood samples from 97 CHC patients were collected before and six months post-treatment. PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule-3) expression was assessed by flow cytometry. DP T-cells displayed significantly higher PD-1 expression, lower Tim-3 expression than CD8+ SP T-cells and lower percentages of PD-1-Tim-3- cells than CD4+ SP T-cells, both before and after treatment. PD-1+Tim-3+ DP T-cells decreased following treatment. HCV-specific cells were more frequent among DP than SP T-cells, both before and after treatment. HCV-specific DP T-cells were characterized by lower PD-1 expression, higher PD-1 and Tim-3 co-expression, and lower percentages of PD-1-Tim-3- cells (both before and after treatment) and higher post-treatment Tim-3 than HCV-specific SP T-cells. Their percentages decreased following treatment, but the exhaustion phenotype remained unchanged. DP T-cells in CHC exhibit a distinct exhaustion phenotype from SP T-cells, and these changes mostly persist following successful treatment.

Higher Immunological Response after BNT162b2 Vaccination among COVID-19 Convalescents-The Data from the Study among Healthcare Workers in an Infectious Diseases Center.

Introduction: The BNT162b2 vaccination studies did not specifically focus on groups that were heavily exposed to SARS-CoV-2 infection. Therefore, we aimed to assess the safety and efficacy of the BNT162b2 vaccine among healthcare workers (HCWs). Methods: Study participants were recruited from hospital employees who received BNT162b2 vaccination at the Hospital for Infectious Diseases in Warsaw. Blood samples were collected before and after each vaccination dose. At each timepoint, the levels of anti-SARS CoV-2 IgM, anti-n SARS-CoV-2 IgG, and S-RBD antibodies were measured. Data on concomitant diseases and the vaccine's adverse events (VAE) were collected after each vaccination dose. In the statistical analyses, non-parametric tests were used. Results: In total, 170 healthcare workers were included in the analysis. Their median age was 51 years (interquartile range (IQR): 41−60 years); most of them were women (n = 137, 80.6%) working in direct contact with patients (n = 137, 73.2%); and 46 (27.0%) had concomitant diseases. More than one fifth of subjects had COVID-19 before their first dose of vaccination (n = 38, 22.6%). In terms of immunological responses, our investigations showed a high level of efficacy for the BNT162b2 mRNA vaccination as measured by S-RBD antibody concentrations: these were positive in 100% of participants 14 days after the second dose of the vaccine. It was also observed that employees with high S-RBD antibodies (>=433 BAU/mL) were more likely to be COVID-19 convalescents before receiving the first vaccine dose (p < 0.001). Conclusion: The BNT162b2 vaccine is safe and effective among HCWs. Vaccine adverse events occurred, but serious events were not observed. Moreover, the BNT162b2 vaccine is effective against symptomatic and severe COVID-19­none of the workers that acquired a SARS-CoV-2 infection after vaccination required hospitalization or medical care. We also observed higher immunological responses among COVID-19 convalescents.

CD8+ T-Cell Exhaustion Phenotype in Chronic Hepatitis C Virus Infection Is Associated With Epitope Sequence Variation.

Background and Aims: During chronic hepatitis C virus (HCV) infection, CD8+ T-cells become functionally exhausted, undergoing progressive phenotypic changes, i.e., overexpression of "inhibitory" molecules such as PD-1 (programmed cell death protein 1) and/or Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule-3). The extreme intrahost genetic diversity of HCV is a major mechanism of immune system evasion, facilitating epitope escape. The aim of the present study was to determine whether T-cell exhaustion phenotype in chronic HCV infection is related to the sequence repertoire of NS3 viral immunodominant epitopes. Methods: The study population was ninety prospective patients with chronic HCV genotype 1b infection. Populations of peripheral blood CD8+ T-cells expressing PD-1/Tim-3 were assessed by multiparametric flow cytometry, including HCV-specific T-cells after magnetic-based enrichment using MHC-pentamer. Autologous epitope sequences were inferred from next-generation sequencing. The correction of sequencing errors and genetic variants reconstruction was performed using Quasirecomb. Results: There was an interplay between the analyzed epitopes sequences and exhaustion phenotype of CD8+ T-cells. A predominance of NS31406 epitope sequence, representing neither prototype KLSGLGLNAV nor cross-reactive variants (KLSSLGLNAV, KLSGLGINAV or KLSALGLNAV), was associated with higher percentage of HCV-specific CD8+PD-1+Tim-3+ T-cells, P=0.0102. Variability (at least two variants) of NS31406 epitope sequence was associated with increased frequencies of global CD8+PD-1+Tim-3+ T-cells (P=0.0197) and lower frequencies of CD8+PD-1-Tim-3- T-cells (P=0.0079). In contrast, infection with NS31073 dominant variant epitope (other than prototype CVNGVCWTV) was associated with lower frequency of global CD8+PD-1+Tim-3+ T-cells (P=0.0054). Conclusions: Our results indicate that PD-1/Tim-3 receptor expression is largely determined by viral epitope sequence and is evident for both HCV-specific and global CD8+ T-cells, pointing to the importance of evaluating autologous viral epitope sequences in the investigation of CD8+ T-cell exhaustion in HCV infection.

The Beneficial Effect of the COVID-19 Vaccine Booster Dose among Healthcare Workers in an Infectious Diseases Center.

Introduction: Healthcare workers in Poland received a booster dose of the BNT162b2 mRNA vaccine (Pfizer-BioNTech, Manufacturer: Pfizer, Inc., and BioNTech; Moguncja, Germany) at the beginning of October 2021. Here, we report on the preliminary results of an ongoing clinical study into the antibody response to SARS-CoV-2 of healthcare workers previously exposed to the virus, with or without evidence of past infection, in the Hospital for Infectious Diseases in Warsaw before and after the vaccine booster dose. Methods: Blood samples were collected on the day the vaccine booster dose was administered and again 14 days later. The levels of SARS-CoV-2 IgG antibodies (against the n-protein, indicative of disease) and S-RBD (indicative of a response to vaccination) were measured. Results: One hundred and ten health care workers from the Hospital for Infectious Diseases were included in the study. The percentage of subjects with a positive test for anti-n-protein IgG antibodies at both time points remained unchanged (16, 14%), while a statistically significant increase in the percentage of subjects producing high levels of S-RBD antibodies (i.e., >433 BAU/mL) was observed (from 23, 21% to 109, 99%; p = 0.00001). Conclusions: The results of the study indicate that the booster dose of the vaccine significantly increases the percentage of people with high levels of S-RBD antibodies, regardless of previous contact with the virus, which may indicate greater protection against both the disease and a severe course of COVID-19.

Next-generation sequencing in the diagnosis of viral encephalitis: sensitivity and clinical limitations.

Identification of pathogens causing viral encephalitis remains challenging, and in over 50% of cases the etiologic factor remains undetermined. Next-generation sequencing (NGS) based metagenomics has been successfully used to detect novel and rare infections, but its value for routine diagnosis of encephalitis remains unclear. The aim of the present study was to determine the sensitivity of shotgun metagenomic sequencing protocols, which include preamplification, and testing it against cerebrospinal fluid (CSF) samples from encephalitis patients. For sensitivity testing HIV and HBV positive sera were serially diluted in CSF from an uninfected patient. NGS repeatedly detected HIV and HBV sequences present at concentrations from 105 to 102 and from 105 to 10 viral copies/reaction, respectively. However, when the same protocols were applied to RT-PCR/PCR positive CSF samples from 6 patients with enteroviral encephalitis (median viral load 47 copies/ml) and 15 patients with HSV, CMV or VZV encephalitis (median viral load 148 copies/ml), only 7 (28.6%) were identified as positive. In conclusions, while NGS has the advantage of being able to identify a wide range of potential pathogens it seems to be less sensitive compared to the standard amplification-based assays in the diagnosis of encephalitis, where low viral loads are common.

Decrease of T-cells exhaustion markers programmed cell death-1 and T-cell immunoglobulin and mucin domain-containing protein 3 and plasma IL-10 levels after successful treatment of chronic hepatitis C.

During chronic hepatitis C virus (HCV) infection, both CD4+ and CD8+ T-cells become functionally exhausted, which is reflected by increased expression of programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and elevated anti-inflammatory interleukin 10 (IL-10) plasma levels. We studied 76 DAA-treated HCV-positive patients and 18 non-infected controls. Flow cytometry measured pretreatment frequencies of CD4+PD-1+, CD4+PD-1+Tim-3+ and CD8+PD-1+Tim-3+ T-cells and IL-10 levels measured by ELISA were significantly higher and CD4+PD-1-Tim-3- and CD8+PD-1-Tim-3- T-cells were significantly lower in patients than in controls. Treatment resulted in significant decrease of CD4+Tim-3+, CD8+Tim-3+, CD4+PD-1+Tim-3+ and CD8+PD-1+Tim-3+ T-cell frequencies as well as IL-10 levels and increase in CD4+PD-1-Tim-3- and CD8+PD-1-Tim-3- T-cells. There were no significant changes in the frequencies of CD4+PD-1+ T-cells, while CD8+PD-1+ T-cells increased. Patients with advanced liver fibrosis had higher PD-1 and lower Tim-3 expression on CD4+T-cells and treatment had little or no effect on the exhaustion markers. HCV-specific CD8+T-cells frequency has declined significantly after treatment, but their PD-1 and Tim-3 expression did not change. Successful treatment of chronic hepatitis C with DAA is associated with reversal of immune exhaustion phenotype, but this effect is absent in patients with advanced liver fibrosis.

Reversal of T Cell Exhaustion in Chronic HCV Infection.

The long-term consequences of T cell responses' impairment in chronic HCV infection are not entirely characterized, although they may be essential in the context of the clinical course of infection, re-infection, treatment-mediated viral clearance and vaccine design. Furthermore, it is unclear whether a complete reinvigoration of HCV-specific T cell response may be feasible. In most studies, attempting to reverse the effects of compromised immune response quality by specific blockades of negative immune regulators, a restoration of functional competence of HCV-specific T cells was shown. This implies that HCV-induced immune dysfunction may be reversible. The advent of highly successful, direct-acting antiviral treatment (DAA) for chronic HCV infection instigated investigation whether the treatment-driven elimination of viral antigens restores T cell function. Most of studies demonstrated that DAA treatment may result in at least partial restoration of T cell immune function. They also suggest that a complete restoration comparable to that seen after spontaneous viral clearance may not be attained, pointing out that long-term antigenic stimulation imprints an irreversible change on the T cell compartment. Understanding the mechanisms of HCV-induced immune dysfunction and barriers to immune restoration following viral clearance is of utmost importance to diminish the possible long-term consequences of chronic HCV infection.

Hepatitis C virus (HCV) genotype 1b displays higher genetic variability of hypervariable region 1 (HVR1) than genotype 3.

Hepatitis C virus (HCV) is characterized by high genetic variability, which is manifested both at the inter-host and intra-host levels. However, its role in the clinical course of infection is less obvious. The aim of the present study was to determine the genetic variability of HCV HVR1 (hypervariable region 1) of genotype 1b and 3 in plasma of blood donors in the early seronegative stage of infection (HCV-RNA+, anti-HCV-) and in samples from chronically infected patients using next-generation sequencing. Sequencing errors were corrected, and haplotypes inferred using the ShoRAH software. Genetic diversity parameters (intra-host number of variants, number of nucleotide substitutions and diversity per site) were assessed by DNA SP and MEGA. During the early infection, the number of variants were significantly lower in subjects infected with genotype 3 than with genotype 1b (p < 0.02). Similarly, intra-host number of variants, number of nucleotide substitutions and diversity per site were lower in genotype 3 chronic infection (p < 0.0005). In addition, early infection was characterized by significantly lower HVR1 variability values (p < 0.04) when compared to chronic infection for both genotypes. It seems that the observed differences in HVR1 variability represent an inherent property of particular viral genotypes.

Expression of programmed cell death protein 1 and T-cell immunoglobulin- and mucin-domain-containing molecule-3 on peripheral blood CD4+CD8+ double positive T cells in patients with chronic hepatitis C virus infection and in subjects who spontaneously cleared the virus.

Chronic hepatitis C virus (HCV) infection is characterized by increased proportion of CD4+CD8+ double positive (DP) T cells, but their role in this infection is unclear. In chronic hepatitis C, immune responses to HCV become functionally exhausted, which manifests itself by increased expression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) on T cells. The aim of our study was to determine PD-1 and Tim-3 phenotype of DP T cells in subjects with naturally resolved and chronic HCV infection. Peripheral blood mononuclear cells from 16 patients with chronic infection and 14 subjects who cleared HCV in the past were stained with anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and anti-Tim-3 antibodies and, in 12 HLA-A*02-positive subjects, MHC class I pentamer with HCV NS31406 epitope. In chronic and past HCV infection, proportions of total DP T cells and PD-1+ DP T cells were similar but significantly higher than in healthy controls. DP T cells were more likely to be PD-1+ than either CD4+ or CD8+ single positive (SP) T cells. HCV-specific cells were present in higher proportions among DP T cells than among CD8+ SP T cells in both patient groups. Furthermore, while the majority of HCV-specific DP T cells were PD-1+, the proportion of HCV-specific CD8+ T cells which were PD-1+ was 4.9 and 1.9 times lower (chronic and past infection, respectively). PD-1 and Tim-3 were predominantly expressed on CD4high CD8low and CD4low CD8high cells, respectively, and co-expression of both markers was uncommon.

Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center.

Molecular characterization of early hepatitis C virus (HCV) infection remains rare. Ten out of 78 patients of a hematology/oncology center were found to be HCV RNA positive two to four months after hospitalization. Only two of the ten patients were anti-HCV positive. HCV hypervariable region 1 (HVR1) was amplified in seven patients (including one anti-HCV positive) and analyzed by next generation sequencing (NGS). Genetic variants were reconstructed by Shorah and an empirically established 0.5% variant frequency cut-off was implemented. These sequences were compared by phylogenetic and diversity analyses. Ten unrelated blood donors with newly acquired HCV infection detected at the time of donation (HCV RNA positive and anti-HCV negative) served as controls. One to seven HVR1 variants were found in each patient. Sequences intermixed phylogenetically with no evidence of clustering in individual patients. These sequences were more similar to each other (similarity 95.4% to 100.0%) than to those of controls (similarity 64.8% to 82.6%). An identical predominant variant was present in four patients, whereas other closely related variants dominated in the remaining three patients. In five patients the HCV population was limited to a single variant or one predominant variant and minor variants of less than 10% frequency. In conclusion, NGS analysis of a cluster of HCV infections acquired in the hospital setting revealed the presence of low diversity, very closely related variants in all patients, suggesting an early-stage infection with the same virus. NGS combined with phylogenetic analysis and classical epidemiological analysis could help in tracking of HCV outbreaks.