Hepatitis C Virus Peptides Research Articles

Activation-Induced Marker Assay to Identify and Isolate HCV-Specific T Cells for Single-Cell RNA-Seq Analysis.

Identification and isolation of antigen-specific T cells for downstream transcriptomic analysis is key for various immunological studies. Traditional methods using major histocompatibility complex (MHC) multimers are limited by the number of predefined immunodominant epitopes and MHC matching of the study subjects. Activation-induced markers (AIM) enable highly sensitive detection of rare antigen-specific T cells irrespective of the availability of MHC multimers. Herein, we have developed an AIM assay for the detection, sorting and subsequent single-cell RNA sequencing (scRNA-seq) analysis of hepatitis C virus (HCV)-specific T cells. We examined different combinations of the activation markers CD69, CD40L, OX40, and 4-1BB at 6, 9, 18 and 24 h post stimulation with HCV peptide pools. AIM+ CD4 T cells exhibited upregulation of CD69 and CD40L as early as 6 h post-stimulation, while OX40 and 4-1BB expression was delayed until 18 h. AIM+ CD8 T cells were characterized by the coexpression of CD69 and 4-1BB at 18 h, while the expression of CD40L and OX40 remained low throughout the stimulation period. AIM+ CD4 and CD8 T cells were successfully sorted and processed for scRNA-seq analysis examining gene expression and T cell receptor (TCR) usage. scRNA-seq analysis from this one subject revealed that AIM+ CD4 T (CD69+ CD40L+) cells predominantly represented Tfh, Th1, and Th17 profiles, whereas AIM+ CD8 T (CD69+ 4-1BB+) cells primarily exhibited effector and effector memory profiles. TCR analysis identified 1023 and 160 unique clonotypes within AIM+ CD4 and CD8 T cells, respectively. In conclusion, this approach offers highly sensitive detection of HCV-specific T cells that can be applied for cohort studies, thus facilitating the identification of specific gene signatures associated with infection outcome and vaccination.

Hepatitis C virus-specific immune responses following direct-acting antivirals administered during recent hepatitis Cvirus infection.

Individuals who spontaneously clear hepatitis C virus (HCV) infection have demonstrated evidence of partial protective immunity, whereas treatment-induced clearance provides little or no protection against reinfection. We aimed to investigate whether treatment of acute HCV infection with direct-acting antivirals (DAA) prevents establishment of, or reverses, T-cell exhaustion, leading to a virus-specific T-cell immune profile more similar to that seen in spontaneous clearance. The magnitude and breadth of HCV-specific T-cell responses before and after DAA or interferon-based therapy in acute or chronic HCV were compared to those of participants with spontaneous clearance of infection, using Enzyme-linked Immunospot (ELISPOT). PBMCs were available for 55 patients comprising 4 groups: spontaneous clearance (n= 17), acute interferon (n= 14), acute DAA (n= 13) and chronic DAA (n= 11). After controlling for sex, the magnitude of post-treatment HCV-specific responses after acute DAA treatment was greater than after chronic DAA or acute IFN treatment and similar to those found in spontaneous clearers. However, spontaneous clearers responded to more HCV peptide pools indicating greater breadth of response. In conclusion, early treatment with DAAs may prevent or reverse some degree of immune exhaustion and result in stronger HCV-specific responses post-treatment. However, individuals with spontaneous clearance had broader HCV-specific responses.

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products.

Cellular immunotherapies based on T cell receptor (TCR) transfer are promising approaches for the treatment of cancer and chronic viral infections. The discovery of novel receptors is expanding considerably; however, the clinical development of TCR-T cell therapies still lags. Here we provide a pipeline for process development and clinical-scale manufacturing of TCR-T cells in academia. We utilized two TCRs specific for hepatitis C virus (HCV) as models because of their marked differences in avidity and functional profile in TCR-redirected cells. With our clinical-scale pipeline, we reproduced the functional profile associated with each TCR. Moreover, the two TCR-T cell products demonstrated similar yield, purity, transduction efficiency as well as phenotype. The TCR-T cell products had a highly reproducible yield of over 1.4 × 109 cells, with an average viability of 93%; 97.8–99% of cells were CD3+, of which 47.66 ± 2.02% were CD8+ T cells; the phenotype was markedly associated with central memory (CD62L+CD45RO+) for CD4+ (93.70 ± 5.23%) and CD8+ (94.26 ± 4.04%). The functional assessments in 2D and 3D cell culture assays showed that TCR-T cells mounted a polyfunctional response to the cognate HCV peptide target in tumor cell lines, including killing. Collectively, we report a solid strategy for the efficient large-scale manufacturing of TCR-T cells.

Multi-functional HCV-specific CD4+ T cells associated with viral control during the postpartum period express Th1- and Tfh-biasing transcription factors

Abstract Chronic hepatitis C virus (HCV) infection is characterized by dysfunctional HCV-specific CD8+ T cells, rare CD4+ T responses, and stable high-level viremia. Recovered CD4+ T cell function and unusual declines in viremia have been observed in some women after childbirth. Whether recovered CD4+ T cells are of lineages to help B cells or CD8 T cells is unknown. Preliminary studies suggest both may be true. In 7 women with postpartum viral suppression (>= 1 log10 by 3 months postpartum, “controllers”), and 7 women with more stable viral levels, “non-controllers”, circulating HCV-tetramer+ (tet+) CD4+ T cells expressed chemokine receptors associated with two predominate populations, Th1-biased Tfh (cTfh1) cells and Th1 cells – with a higher cTfh1/Th1 ratio linked to viral control. Here we assessed HCV-specific cTfh1 and Th1 functional responses by combined surface chemokine receptor and intracellular cytokine staining (ICS) after HCV peptide stimulation, and we also assessed expression of lineage-defining transcription factors on HCV-tet+ cells. HCV-specific cytokine+ cells were predominantly cTfh1 or Th1 populations, and multi-functionality correlated with viral control. HCV-tet+ CD4+ T cell Tbet and BCL6 expression levels did not differ by degree of viral control, but both were enhanced in tet+cTfh1 cells relative to bulk cTfh1 cells, and tet+ Th1 cells expressed significantly more BCL6 than bulk Th1 cells. Together, these data point to a postpartum CD4+ T cell recovery driven by transcription factors favoring cTfh1/Th1-biased phenotypes. Understanding how these cells interact with CD8+ T cells and B cell will be vital for understanding this unique instance of natural control of a chronic viral infection. Supported by a grant from the NIH (R01 AI096882).

Trans-ancestral fine-mapping of MHC reveals key amino acids associated with spontaneous clearance of hepatitis C in HLA-DQβ1

Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQβ1Leu26 (p valueMeta = 1.24 × 10−14) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQβ1Pro55 (p valueMeta = 8.23 × 10−11) located in the peptide binding region was positively associated, independently of HLA-DQβ1Leu26. These two amino acids are not in linkage disequilibrium (r2 < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB1∗03:01, and HLA-DRB1∗01:01. Additionally, HCV persistence classical alleles tagged by HLA-DQβ1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC50 nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10−38). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQβ1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.

Chronic hepatitis C virus infection impairs natural killer cells-dendritic cells cross-talk: An in vitro culture study.

To examine the cross-talk between NK cells and DCs in hepatitis C virus (HCV) infection, we isolated monocytes and NK cells from 20 chronic HCV patients and 20 healthy controls. Monocytes were used to generate immature DCs which were pulsed with HCV peptides (core, NS3-NS4, and NS5). Four different cocultures were carried out: E1, both DCs and NK cells were from a chronic HCV patient; E2, NK cells from a healthy control cocultured with DCs from a chronic HCV patient; E3, NK cells from a chronic HCV patient cocultured with DCs from a healthy control; and E4, both DCs and NK cells were from a healthy control. Using flow cytometry, we assessed the effect of these different cocultures on levels of maturation markers on DCs and levels of activation/inhibition markers on NK cells. Results showed that peptide-pulsed HCV DCs showed a maturation defect in the form of decreased HLA-DR, decreased CD86, and increased CD83 expression especially when cocultured with HCV NK. This was mainly due to core peptide pulsing and to a lesser extent due to NS5 pulsing, whereas there was no effect with NS3-NS4 pulsing. Alternatively, HCV NK cells upregulated both activation and inhibition markers especially when cocultured with healthy DCs. Compared with E2, E1 resulted in higher apoptosis of both NK cells and DCs with the percentage of NK apoptosis higher than that of DCs. Taken together, the data indicate that HCV infection impairs NK-DC cross-talk which may be a leading cause in viral persistence and chronicity.

Immunopathogenesis of Liver Injury During Hepatitis C Virus Infection.

The present report describes current concepts about the mechanism of liver cell injury caused by host immune response against hepatitis C virus (HCV) infection in human beings. This report is based on the observations from experimental studies and follow-up actions on human liver diseases. The results from different investigations suggest that liver injury depends on the presentation of viral antigen and the level of host immune response raised against HCV-related peptides. Both innate and adaptive immunity are triggered to counter the viral onset. During development of host immunity, the cell-mediated immune response involving CD4+ Th1 cells and CD8+ cytotoxic T-lymphocyte (CTL) cells were found to play a major role in causing liver damage. The hepatic Innate lymphoid cells (ILCs) subsets are involved in the immune regulation of different liver diseases: viral hepatitis, mechanical liver injury, and fibrosis. Humoral immunity and natural killer (NK) cell action also contributed in liver cell injury by antibody-dependent cellular cytotoxicity (ADCC). In fact, immunopathogenesis of HCV infection is a complex phenomenon where regulation of immune response at several steps decides the possibility of viral elimination or persistence. Regulation of immune response was noted starting from viral-host interaction to immune reaction cascade engaged in cell damage. The activation or suppression of interferon-stimulated genes, NK cell action, CTL inducement by regulatory T cells (Treg), B cell proliferation, and so on was demonstrated during HCV infection. Involvement of HLA in antigen presentation, as well as types of viral genotypes, also influenced host immune response against HCV peptides. The combined effect of all these effector mechanisms ultimately decides the progression of viral onset to acute or chronic infection. In conclusion, immunopathogenesis of liver injury after HCV infection may be ascribed mainly to host immune response. Second, it is cell-mediated immunity that plays a predominant role in liver cell damage.

Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

CD8+ T cells of chronic HCV-infected patients express multiple negative immune checkpoints following stimulation with HCV peptides

Hepatitis C virus (HCV)-specific CD4+ and CD8+ T cells are key to successful viral clearance in HCV disease. Accumulation of exhausted HCV-specific T cells during chronic infection results in considerable loss of protective functional immune responses. The role of T-cell exhaustion in chronic HCV disease remains poorly understood. Here, we studied the frequency of HCV peptide-stimulated T cells expressing negative immune checkpoints (PD-1, CTLA-4, TRAIL, TIM-3 and BTLA) by flow cytometry, and measured the levels of Th1/Th2/Th17 cytokines secreted by T cells by a commercial Multi-Analyte ELISArray™ following in vitro stimulation of T cells using HCV peptides and phytohemagglutinin (PHA). HCV peptide-stimulated CD4+ and CD8+ T cells of chronic HCV (CHC) patients showed significant increase of CTLA-4. Furthermore, HCV peptide-stimulated CD4+ T cells of CHC patients also displayed relatively higher levels of PD-1 and TRAIL, whereas TIM-3 was up-regulated on HCV peptide-stimulated CD8+ T cells. Whereas the levels of IL-10 and TGF-β1 were significantly increased, the levels of pro-inflammatory cytokines IL-2, TNF-α, IL-17A and IL-6 were markedly decreased in the T cell cultures of CHC patients. Chronic HCV infection results in functional exhaustion of CD4+ and CD8+ T cells likely contributing to viral persistence.

Characterization of differential antibody production against hepatitis C virus in different HCV infection status.

BackgroundThe Centers for Disease Control and Prevention (CDC) issued an update on hepatitis C virus (HCV) testing approach, in which it omitted the use of recombinant immunoblot assay (RIBA) in the diagnostic algorithm and recommended that future studies are needed to evaluate the performance of HCV testing without RIBA. As Egypt has the highest prevalence of HCV worldwide, we aimed to evaluate the value of RIBA in HCV testing in a high prevalence population. Our objective was to clarify whether enzyme linked immunosorbent assay (ELISA) anti-HCV signal-to-cutoff (S/CO) ratios were able to discriminate true positive from false positive anti-HCV antibody status and to evaluate the role of RIBA in solving this problem which may lead to a redefined strategy for diagnosis of HCV infection. Our second objective was to elucidate the effects of different HCV peptides of both structural and non-structural proteins on the humoral immune response to HCV infection. MethodsThe current study drew results from 167 individuals divided into three groups: Group I: included 77 HCV antibody positive (ELISA) high risk health care workers (HCW), Group II: included 56 presumably uninfected individuals who showed normal liver enzymes, negative HCV RNA and were asymptomatic. Their ELISA HCV antibody S/C ratio ranged from 0.9 to <5. Group III: included 34 patients enrolled from outpatient clinics of Ain Shams Hospital with persistent viral replication, elevated liver enzymes, and chronic HCV related liver disease. All study participants were assessed for the presence of anti-HCV antibodies by 3rd generation ELISA which was confirmed by RIBA.ResultsInterpreting the results of both ELISA and RIBA together, false positive results were highly significantly increased in HCW when compared with the other two groups. Indeterminate and false negative results were only found in the presumably uninfected group. For differentiated antibody responses by RIBA, chronic HCV cases had the highest frequency of positive antibody response to core peptides while the presumably uninfected group had the lowest. Antibody response to E2 was found less frequently in chronic cases than Core 1, Core 2 and NS3. The specific antibody response to the different HCV peptides showed the same distribution of frequencies in both chronic HCV cases and the presumably uninfected individuals with the chronic cases having the highest frequencies. This distribution was different from the HCW. The most evident difference was the reaction towards NS3 which was the highest antibody producing peptide in chronic HCV and presumably uninfected individuals whereas in HCW Core1 was the highest.ConclusionThe HCV antibody immunoblot assay (RIBA) is still necessary for the detection of false positive cases which can occur quite frequently in countries of high prevalence as Egypt. Indeterminate RIBA results indicate a waning antibody response in elderly individuals who recovered from previous or distant HCV infection.

Sequence variations in HCV core-derived epitopes alter binding of KIR2DL3 to HLA-C∗03:04 and modulate NK cell function

Both natural killer (NK) cells and human leukocyte antigen (HLA)/killer cell immunoglobulin like receptor (KIR) interactions have been shown to play an important role in the control, clearance and progression of hepatitis C virus (HCV) disease. Here we aimed at elucidating the effects of viral peptides derived from HCV on HLA stabilization, changes in KIR binding and primary NK cell function. Transporter for antigen presentation-deficient 722.221 cells stably transfected with HLA-C∗03:04 were used to screen 200 overlapping peptides, covering the non-structural protein 3 (NS3) and core protein of HCV genotype 1, for their ability to bind and stabilize HLA-C∗03:04. Binding of KIR2DL3 to the HLA-peptide complex was assessed using a KIR2DL3-IgG fusion construct. Primary NK cells were isolated from healthy donors to investigate the effects of identified peptides on KIR2DL3(+) NK cell function. Thirty-one peptides able to stabilize HLA-C∗03:04 were identified. One 9mer peptide, YIPLVGAPL, resulted in significantly higher KIR2DL3 binding to HLA-C∗03:04(+) 722.221 cells and suppression of primary KIR2DL3(+) NK cell function. Interestingly this sequence exhibited a high frequency of mutations in different HCV genotypes. These genotype-specific peptides showed lower HLA-C∗03:04 stabilization, decreased binding of the inhibitory KIR2DL3 and lower inhibition of NK cell function. Taken together we show that a viral peptide derived from the core protein of HCV genotype 1 binding to HLA-C∗03:04 results in a sequence-dependent engagement of the inhibitory NK cell receptor KIR2DL3, while the large majority of the remaining 30 HLA-C∗03:04 binding HCV core peptides did not. These data show that sequence variations within HCV can modulate NK cell function, providing potential pathways for viral escape. We identified a HCV peptide that dampens NK cell responses, and thereby possibly prevents killing of infected cells through this part of the innate immune system. This is facilitated via presentation of the viral peptide on HLA∗03:04 to the inhibitory KIR receptor KIR2DL3 on NK cells. Naturally occurring sequence mutations in the peptide alter these interactions making the inhibition less efficient.

Lymphocyte activation gene 3 negatively regulates the function of intrahepatic hepatitis C virus-specific CD8+ T cells.

Chronic hepatitis C (CHC) in humans caused by persistent hepatitis C virus (HCV) infection is a global public health problem. The functional exhaustion of HCV-specific CD8(+) T cells regulated by several inhibitory receptors has been shown to contribute to chronic HCV infection. Lymphocyte activation gene 3 (LAG-3), which is an inhibitory receptor, plays an important role in several chronic viral infections. However, its effect on the function of HCV-specific CD8(+) T cells is unclear. The expression of LAG-3 on the CD8(+) T cells in intrahepatic and peripheral lymphocytes from 17 CHC patients and 15 HCV-negative patients was analyzed by flow cytometry. The LAG-3 expression in CD8(+) T cells was downregulated or upregulated by lentivirus LAG-3 shRNA or lentivirus overexpressing LAG-3. After HCV peptide stimulation, flow cytometry was used to detect cell proliferation and cytokine (γ-interferon [IFN-γ], tumor necrosis factor-α [TNF-α], granzyme B, and perforin) production of CD8(+) T cells. Cytotoxicity functions of HCV-specific CD8(+) T cells were measured using a (51) Cr release assay. The frequency of LAG-3-positive intrahepatic and peripheral CD8(+) T cells was higher in CHC patients, compared with HCV-negative patients. The cell proliferation, cytokine (IFN-γ, TNF-α, granzyme B, and perforin) expression and cytotoxicity function of HCV-specific CD8(+) T cells in CHC patients were increased by the knocking down and blockade of LAG-3. In the LAG-3 overexpressed CD8(+) T cells, cell proliferation, cytokine (IFN-γ, TNF-α, granzyme B, and perforin) expression, and cytotoxicity function were inhibited, while the LAG-3 blocking antibody reversed the inhibition. LAG-3 negatively regulated the function of HCV-specific CD8(+) T cells in CHC patients.

Increased frequency of late-senescent T cells lacking CD127 in chronic hepatitis C disease.

Hepatitis C virus (HCV) causes persistent disease in ~85% of infected individuals, where the viral replication appears to be tightly controlled by HCV-specific CD8+ T cells. Accumulation of senescent T cells during infection results in considerable loss of functional HCV-specific immune responses. We characterized the distinct T-cell phenotypes based on the expression of costimulatory molecules CD28 and CD27, senescence markers PD-1 and CD57, chronic immune activation markers CD38 and HLA-DR, and survival marker CD127 (IL-7R) by flow cytometry following activation of T cells using HCV peptides and phytohemagglutinin. HCV-specific CD4+ and CD8+ T cells from chronic HCV (CHC) patients showed increased expression of PD-1. Furthermore, virus-specific CD4+ T cells of CHC-infected subjects displayed relatively increased expression of HLA-DR and CD38 relative to HCV-specific CD8+ T cells. The CD4+ and CD8+ T cells from HCV-infected individuals showed significant increase of late-differentiated T cells suggestive of immunosenescence. In addition, we found that the plasma viral loads positively correlated with the levels of CD57 and PD-1 expressed on T cells. Chronic HCV infection results in increased turnover of late-senescent T cells that lack survival potentials, possibly contributing to viral persistence. Our findings challenge the prominence of senescent T-cell phenotypes in clinical hepatitis C infection.

C형 간염 바이러스 감염 간암 세포주와 T 림프구의 상호작용에 대한 연구

Recently, Hepatitis C virus (HCV) replication system has been established using human hepatoma cells (huh cell) and a variety of HCV clones. In this study, we established an infectious HCV replication system using huh7.5 cells and J6/JFH1 clone (genotype 2a). In addition, we investigated the antigen presentation capability of HCV-infected huh7.5 cells to HCV-specific T cells. Interestingly, HCV-infected huh7.5 cells were not capable of activating HCV-specific T cells. However, huh7.5 cells stimulated by exogenous HCV peptide were able to activate HCV-specific T cells, which was shown to produce TNF-α and IFN-γ. We further examined if HCV infection has an inhibitory effect on the expression of MHC class I molecule of huh7.5 cells. We found that HCV infection did not change the expression level of MHC class I molecule on huh7.5 cells.

Establishment of human clones producing neutralizing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus by EBV immortalization of immune CD22⁺ B cells.

We aimed to establish Human cell lines producing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus (HCV). Two protocols for EBV immortalization of CD22⁺ cells separated from HCV positive patients were used; 1) Immortalization with 100% EBV only, 2) immortalization by 30% EBV and CPG2006. Immortalization was checked microscopically and verified by screening the culture supernatant for antibody production using dot blot and ELISA analysis. ELISA plates were coated by HCV E1/E2 derived from cell lysate transfected by plasmid expressed HCV E1/E2. Also we tested the reactivity of human antibodies based on ELISA plates coating with one linear peptides derived from HCV E1 (a.a 315-319) and two peptides derived from HCV E2 (a.a 412-419) and (a.a 517-530). Neutralization activity was measured using H77C HCV retroviral pseudoparticles (HCVpp). Fifteen clones secreting human immunoglobulin G against HCV E1/E2 protein were isolated. Results of ELISA plates coated with HCV peptides showed that one antibody was binding to E2 peptide (a.a 517-530), and two antibodies binding to HCV E2 peptide (a.a 412-419). The three generated antibodies showed extremely neutralization activity against HCV pp. The three human antibodies were IgG3 and IgG2. These antibodies may be useful for passive immunotherapy of HCV infection.

Maintenance of Th1 hepatitis C virus (HCV)‐specific responses in individuals with acute HCV who achieve sustained virological clearance after treatment

T-cell responses against hepatitis C are believed to be critical in achieving both natural and treatment-induced clearance. However, rapid clearance of antigen with early treatment of primary infection may result in reduced or poorly sustained cellular immunity. This study longitudinally examined Th1 and Th2 hepatitis C virus (HCV)-specific cytokine production and T-cell effector function from subjects enrolled in the Australian Trial in Acute Hepatitis C comparing three groups: treatment-induced clearance (sustained virological response [SVR]), treatment non-response, and untreated spontaneous clearance. HCV-specific T-cell responses were characterized by HCV peptide ELISpot, in vitro cytokine production, and T-cell flow cytometry assays. Treated subjects with a sustained virological response (SVR) displayed a better maintenance of HCV-specific Th1 responses compared to treatment non-responders (higher interferon [IFN]-γ and interleukin (IL)-2 magnitude at week 24, broader IFN-γ responses at weeks 24 and 48, P < 0.05) and significantly increased IFN-γ responses between screening and week 48 (magnitude P = 0.026, breadth P = 0.009). Treatment-induced viral clearance was also associated with a trend toward decreased IL-10 responses (screening to week 48, P = 0.070), higher expression of CD45RO (P = 0.042) and CD38 (P = 0.088) on CD4+ T cells, and higher IFN-γR expression (CD56+ IFN-γR+ P = 0.033) compared to treatment non-responders. Untreated subjects with viral clearance also displayed high magnitude and broad HCV-specific IFN-γ and IL-2 responses early in infection; however, IFN-γ responses were not as well maintained compared to treated subjects with a SVR (week 48 magnitude, breadth P = 0.064). Treatment-induced viral clearance of recent HCV infection is associated with maintenance of HCV-specific Th1 responses.

Hepatitis C virus‐specific cellular immune responses in sustained virological responders with viral persistence in peripheral blood mononuclear cells

Hepatitis C virus (HCV)-RNA detection in peripheral blood mononuclear cells (PBMCs) after recovery from HCV infection, is a type of occult HCV infection although is unclear how the viral persistence in PBMCs affects HCV-specific T-cell responses. The aim of this study was to investigate if cellular immune responses are modified by HCV persistence in PBMCs. HCV-specific CD4(+) and CD8(+) T-cell responses against six HCV peptides, situated within the non-structural (NS) proteins NS3, NS4b and NS5b, were measured by flow cytometry-through intracellular detection of gamma interferon (IFN-γ) or interleukin 4 (IL-4) and CD69 expression- in 27 sustained virological responders (SVR): 13 with and 14 without occult HCV infection in PBMCs, detected by strand-specific real-time PCR. Ten healthy individuals and 14 chronically infected patients with viraemia, were included as controls. SVR without occult infection showed a higher percentage of activated CD4(+) cells against peptides belonging to NS3 (p124, p153) and NS5b (p257, p294), activated CD8(+) cells against NS3 (p124, p153, p158) and NS5b-p294, as well as an elevated percentage of CD4(+) cells releasing IFN-γ+IL-4 against NS3-p153, and by CD8(+) cells against NS3 (p124, p153). SVR without occult infection showed a higher percentage of activation and release of IFN-γ+IL-4 by both cell subpopulations than the two group of controls, in contrast to SVR with occult infection. The lower HCV-specific T-cell response found in SVR with occult infection indicates that the immune response may be impaired when the virus persists in PBMCs.

Antigen-specific natural killer cell cytokine production in chronic hepatitis C infection (P6062)

Abstract Chronic hepatitis C virus (HCV) infection results in an inflammatory liver disease leading to fibrosis and cirrhosis. The progression of liver disease is thought to be immune-mediated because HCV itself is non-cytopathic. Given that HCV-specific T cells are diminished in number and functionally exhausted in chronic HCV infection, it remains unclear which cell population drives disease pathogenesis. Here, we investigated the function of natural killer (NK) cells, the major innate immune cell population in the liver. PBMCs from 15 patients with chronic HCV infection were stimulated for 8 hours in a whole blood activation assay with pools of overlapping 18-mer peptides comprising HCV structural (E1, E2) and nonstructural (NS3) proteins. Cytokine production by NK cells and T cells was assessed by multicolor flow cytometry. The frequency of IFN-γ+ NK cells was 5-10 fold greater than the frequency of IFN-γ+ T cells. A minority of IFN-γ+ NK cells co-produced TNF-α. NK cell responses to HCV peptides varied between subjects, but did not correlate with T cell responses or viremia. This study demonstrates that NK cells are activated in an antigen-specific manner in chronic HCV infection and respond to both structural and nonstructural HCV proteins. The mechanism of antigen-specific NK cell activation is currently under investigation.

Impaired Hepatitis C Virus (HCV)–Specific Interferon-γ Responses in Individuals With HIV Who Acquire HCV Infection: Correlation With CD4+ T-Cell Counts

Studies examining the effect of coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) on the HCV-specific immune response in acute HCV infection are limited. This study directly compared acute HCV-specific T-cell responses and cytokine profiles between 20 HIV/HCV-coinfected and 20 HCV-monoinfected subjects, enrolled in the Australian Trial in Acute Hepatitis C (ATAHC), using HCV peptide enzyme-linked immunospot (ELISPOT) and multiplex in vitro cytokine production assays. HIV/HCV coinfection had a detrimental effect on the HCV-specific cytokine production in acute HCV infection, particularly on HCV-specific interferon γ (IFN-γ) production (magnitude P=.004; breadth P=.046), which correlated with peripheral CD4(+) T-cell counts (ρ=0.605; P=.005) but not with detectable HIV viremia (ρ=0.152; P=.534).

Development of multiplex serological assays to detect oncoviral infections

Serological markers of infection (antibodies or antigens) of viruses that cause cancer are most often detected using ELISA-based methodologies. In many cases, multiple markers of infection must be assessed to determine a final sero-status. Volume requirements and costs of reagents for single analyte ELISAs are high and studies which include multiple viruses can require milliliters of plasma, often not available from archived cohorts. Thus, we sought to develop a Luminex® bead-based customizable panel initially including Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Epstein-Barr Virus (EBV) and often the concomitant infection, Human Immunodeficiency Virus (HIV) to reduce sample volume requirements, overall cost and increase flexibility. Peptides, antigens and antibodies were sourced from multiple manufacturers and tested for suitability in this platform. Where necessary, suitable reagents were designed and produced in-house. The HCV assay multiplexes four HCV peptides designed to detect antibodies raised to HCV Core (2), HCV NS4, HCV NS5 gene regions. The HBV assay multiplexes a HBV early antigen peptide, a recombinant HBV core protein and either a recombinant HBV surface antigen or an antibody specific for HBV surface antigen to assess HBV infection. The EBV assay multiplexes peptides specific to viral capsid antigen, EBNA-1 and early antigen (Cyto-Barr, Zuidhorn, The Netherlands). HIV-1 assay development is ongoing and the list of antigens to be included in the assay has not been finalized. These assays can be run singly or with any combination (multi-plex) of the above listed targets. Each target has been independently validated using samples of known molecular and serological status to determine specificity (false positive versus false negative) and re-evaluated under multiplex conditions to confirm assay performance. In addition, where possible, samples were assayed on commercial testing platforms as well as our multiplex assay to assess concordance (94-99%). Dependent on the panel selected and the expected antibody titers in a particular population, plasma or serum volumes in the range of 10 µL to 125 µL per subject would be required to determine the HBV, HCV, EBV and/or HIV serostatus of a subject. This assay platform is inherently flexible and the benefits include amenability to expansion to include other oncogenic viruses as well as screening large epidemiological cohorts or smaller subsets of samples in an economical and high throughput manner.