303. A Surrogate Rodent Model for Studying Hepatitis C Virus-specific CD8 T-cell Impairment and Vaccine Prevention

Abstract Background Virus-specific CD8 T cells are essential for control of acute hepatitis C virus (HCV) infections, yet spontaneously fail in most patients leading to lifelong chronicity and increased risk for severe liver diseases. Efforts to study HCV-specific CD8 T-cell impairment have been hampered by a lack of small animal models. Recently, we established a rat model of chronic HCV-like infection using a hepacivirus homolog identified in Rattus norvegicus. The nature of virus-specific CD8 T-cell immunity in this model has yet to be determined. Methods Using two MHC class I tetramers against epitopes located in the E1 and NS5B proteins, we tracked the induction and phenotype of virus-specific CD8 T cells during chronic infection. Responses to infection were similarly analyzed in immune rats that had been vaccinated against the NS3-5B proteins, a strategy that is effective in this experimental setting. Results Virus-specific CD8 T cells expanded vigorously in liver shortly after infection but did not develop into functional effectors based upon failure to produce cytokines (IFNγ, TNFα, IL-2, IL-4, IL-10, IL-17A) following peptide stimulation. Notably, subversion of responses was not due to viral escape from T-cell recognition, but rather an intrinsic defect in the antiviral response. Indeed, these populations expressed the inhibitory receptor programed cell death-1 and other markers consistent with an arrested effector-like state precluded from long-term memory formation (CD127-CD27+CD28+CD62L-GranzymeB+). In contrast, adenoviral immunization of naïve rats protected virus-specific T cells from functional impairment after infection and supported memory response development, including against the E1 epitope not encoded by vaccine. Conclusion Together, our findings reveal a spontaneous failure of virus-specific CD8 T cells following rat hepacivirus challenge that is highly reminiscent of human HCV infections. Furthermore, these results highlight the utility and significance of this model for understanding mechanisms of HCV persistence and protective immunity necessary for the development of effective vaccines and immune interventions. Disclosures All authors: No reported disclosures.

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Priming of Antiviral CD8 T Cells without Effector Function by a Persistently Replicating Hepatitis C-Like Virus

Journal of Virology ◽

10.1128/jvi.00035-20 ◽

2020 ◽

Vol 94 (10) ◽

Cited By ~ 1

Author(s):

Alex S. Hartlage ◽

Christopher M. Walker ◽

Amit Kapoor

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Animal Models ◽

Cd8 T Cells ◽

Persistent Infection ◽

Cd8 T Cell ◽

Effector Function ◽

Viral Escape

ABSTRACT Immune-competent animal models for the hepatitis C virus (HCV) are nonexistent, impeding studies of host-virus interactions and vaccine development. Experimental infection of laboratory rats with a rodent hepacivirus isolated from Rattus norvegicus (RHV) is a promising surrogate model due to its recapitulation of HCV-like chronicity. However, several aspects of rat RHV infection remain unclear, for instance, how RHV evades host adaptive immunity to establish persistent infection. Here, we analyzed the induction, differentiation, and functionality of RHV-specific CD8 T cell responses that are essential for protection against viral persistence. Virus-specific CD8 T cells targeting dominant and subdominant major histocompatibility complex class I epitopes proliferated considerably in liver after RHV infection. These populations endured long term yet never acquired antiviral effector functions or selected for viral escape mutations. This was accompanied by the persistent upregulation of programmed cell death-1 and absent memory cell formation, consistent with a dysfunctional phenotype. Remarkably, transient suppression of RHV viremia with a direct-acting antiviral led to the priming of CD8 T cells with partial effector function, driving the selection of a viral escape variant. These data demonstrate an intrinsic abnormality within CD8 T cells primed by rat RHV infection, an effect that is governed at least partially by the magnitude of early virus replication. Thus, this model could be useful in investigating mechanisms of CD8 T cell subversion, leading to the persistence of hepatotropic pathogens such as HCV. IMPORTANCE Development of vaccines against hepatitis C virus (HCV), a major cause of cirrhosis and cancer, has been stymied by a lack of animal models. The recent discovery of an HCV-like rodent hepacivirus (RHV) enabled the development of such a model in rats. This platform recapitulates HCV hepatotropism and viral chronicity necessary for vaccine testing. Currently, there are few descriptions of RHV-specific responses and why they fail to prevent persistent infection in this model. Here, we show that RHV-specific CD8 T cells, while induced early at high magnitude, do not develop into functional effectors capable of controlling virus. This defect was partially alleviated by short-term treatment with an HCV antiviral. Thus, like HCV, RHV triggers dysfunction of virus-specific CD8 T cells that are vital for infection resolution. Additional study of this evasion strategy and how to mitigate it could enhance our understanding of hepatotropic viral infections and lead to improved vaccines and therapeutics.

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Differences in Hepatitis C Virus (HCV)-Specific CD8 T-Cell Phenotype during Pegylated Alpha Interferon and Ribavirin Treatment Are Related to Response to Antiviral Therapy in Patients Chronically Infected with HCV

Journal of Virology ◽

10.1128/jvi.02175-07 ◽

2008 ◽

Vol 82 (15) ◽

pp. 7567-7577 ◽

Cited By ~ 20

Author(s):

Joana Caetano ◽

António Martinho ◽

Artur Paiva ◽

Beatriz Pais ◽

Cristina Valente ◽

...

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Cd8 T Cells ◽

T Cell Responses ◽

Cd8 T Cell ◽

Alpha Interferon ◽

Response To Treatment ◽

Cell Responses

ABSTRACT CD8 T cells play a major role in antiviral immune responses. Their importance for progression to chronic hepatitis C and response to treatment are still unclear. To address these issues, hepatitis C virus (HCV)-specific CD8 T-cell responses were monitored, at the single-cell level, using HLA class I pentamers specific for HCV core and HCV NS3 epitopes, in 23 chronically infected patients during treatment with pegylated alpha interferon and ribavirin. Patients who presented a sustained-response to therapy had stronger HCV-specific CD8 T-cell responses at all time points studied. Moreover, there were clear differences in the phenotypes of these cells during therapy: in responder patients, terminally differentiated effector cells increased more rapidly, and their frequency was always higher than in nonresponder patients. Sustained-responder patients also showed a higher frequency of HCV-specific CD8 T cells producing cytotoxic factors. Overall, a late and inefficient differentiation process of HCV-specific CD8 T cells might be associated with lack of response to treatment. A better knowledge of the mechanisms underlying this impairment may be important for the development of new therapeutic strategies to maintain, restore, or increase CD8 T-cell effectiveness in chronic HCV infection.

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The magnitude and breadth of hepatitis C virus–specific CD8+ T cells depend on absolute CD4+ T-cell count in individuals coinfected with HIV-1

Blood ◽

10.1182/blood-2004-06-2336 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1170-1178 ◽

Cited By ~ 75

Author(s):

Arthur Y. Kim ◽

Georg M. Lauer ◽

Kei Ouchi ◽

Marylyn M. Addo ◽

Michaela Lucas ◽

...

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Cd8 T Cells ◽

T Cell Responses ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Cell Responses ◽

Hiv 1

AbstractCD8+ T-cell responses are an essential antiviral host defense in persistent viral infections, and their sustained effectiveness is thought to be critically dependent on CD4+ T-helper cells. To determine the relationship between HIV-1–induced CD4+ T-cell depletion and hepatitis C virus (HCV)–specific CD8+ T-cell responses during viral persistence, we studied 103 persons positive for HCV, 74 coinfected with HIV-1. CD8+ T-cell responses to the entire HCV polyprotein were determined by using an interferon-γ enzyme-linked immunospot (ELISpot) assay. Although HIV-1 infection by itself was not associated with a diminished HCV-specific response, HIV-1–associated CD4+ depletion was associated with significantly lower HCV-specific CD8+ T cells (R = 0.48, P < .0001). In contrast, declining CD4+ counts over the same range were not associated with diminished Epstein-Barr virus (EBV)– (R = 0.19, P = .31) or HIV-1–specific (R = –0.13, P = .60) CD8+ T-cell responses in persons infected with all viruses. These data indicate that frequencies of circulating HCV-specific CD8+ T-cell responses are sensitive to absolute CD4+ T-cell counts and provide a possible explanation for the accelerated HCV disease course in persons coinfected with HIV-1 and HCV.

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Modulation of Hepatitis C Virus-Specific CD8 Effector T-Cell Function with Antiviral Effect in Infectious Hepatitis C Virus Coculture Model

Journal of Virology ◽

10.1128/jvi.02129-16 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 4

Author(s):

Keisuke Ojiro ◽

Xiaowang Qu ◽

Hyosun Cho ◽

Jang-June Park ◽

Annelise Vuidepot ◽

...

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Function ◽

Cd8 T Cell ◽

Hepatoma Cells ◽

Antiviral Effects

ABSTRACT The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1β, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 μM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects. IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.

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Hepatitis C virus (HCV)-specific CD8 T-cells in HCV-seronegative individuals: Frequency and T-cell receptor repertoire (TCR)

Zeitschrift für Gastroenterologie ◽

10.1055/s-0030-1269657 ◽

2011 ◽

Vol 49 (01) ◽

Author(s):

R Bakshi ◽

B Heidrich ◽

B Calle Serrano ◽

SV Pothakamuri ◽

MP Manns ◽

...

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Cell Receptor ◽

Receptor Repertoire ◽

T Cell Receptor Repertoire ◽

Cell Receptor Repertoire

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Magnitude of CD8+T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Hepatology Research ◽

10.1111/j.1872-034x.2008.00459.x ◽

2009 ◽

Vol 39 (3) ◽

pp. 256-265 ◽

Cited By ~ 4

Author(s):

Hiroyoshi Doi ◽

Kazumasa Hiroishi ◽

Tomoe Shimazaki ◽

Junichi Eguchi ◽

Toshiyuki Baba ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Virus Infection ◽

Acute Hepatitis ◽

T Cell Responses ◽

Cd8 T Cell ◽

Acute Hepatitis C ◽

Hepatitis C Virus Infection ◽

Cell Responses

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The Kinetics of Hepatitis C Virus-Specific CD8 T-Cell Responses in the Blood Mirror Those in the Liver in Acute Hepatitis C Virus Infection

Journal of Virology ◽

10.1128/jvi.00475-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9782-9788 ◽

Cited By ~ 17

Author(s):

Eui-Cheol Shin ◽

Stefania Capone ◽

Riccardo Cortese ◽

Stefano Colloca ◽

Alfredo Nicosia ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Cell Activity ◽

T Cell Responses ◽

Cd8 T Cell ◽

Functional Markers ◽

Acute Hepatitis C ◽

Cell Responses ◽

Acute Hcv

ABSTRACT Peripheral blood T-cell responses are used as biomarkers in hepatitis C virus (HCV) vaccine trials. However, it is not clear how T-cell responses in the blood correlate with those in the liver, the infection site. By studying serial liver and blood samples of five vaccinated and five mock-vaccinated control chimpanzees during acute HCV infection, we demonstrate a correlation between HCV-specific CD8 T-cell responses in the blood and molecular and functional markers of T-cell responses in the liver. Thus, HCV-specific CD8 T-cell responses in the blood are valid markers for intrahepatic T-cell activity.

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Recovery of CMV-Specific T Cells Following Alternative Donor Allogeneic Transplant with Post-Transplant Cyclophosphamide

Blood ◽

10.1182/blood.v126.23.5462.5462 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5462-5462

Author(s):

Ayman Saad ◽

Samantha B Langford ◽

Shin Mineishi ◽

Lawrence S. Lamb

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Response ◽

Cd8 T Cell ◽

T Cell Response ◽

Cell Recovery ◽

Post Transplant ◽

Increased Risk ◽

Cmv Reactivation

Abstract Background: Post-transplant cyclophosphamide (PTCy) is increasingly used for GVHD prophylaxis after allogeneic hematopoietic stem cell transplantation (HCT) using alternative donors. However, immune reconstitution can be delayed posing an increased risk for CMV reactivation. We evaluated the outcomes of patients who received HCT-apheresis products comparing the impact of PTCy on lymphocyte recovery, CMV reactivation and CMV-specific CD8+ T cell recovery following haplo-identical (HAPLO), matched unrelated donor (MUD), and mismatched unrelated donor (mMUD) grafts vs. with conventional matched related donor (MRD) graft recipients. Methods: We examined 26 patients (median age, 49 years; range, 20-72 years) with advanced hematologic malignancies; n=5 (HAPLO); 6 (MRD); 15 (MUD). All patients received myeloablative conditioning regimens that was either busulfan- or total body irradiation (TBI)-based. PTCy (50 mg/kg/day) was administered on days +3 and +4 following HAPLO and on day +3 following MUD/mMUD transplant. Peripheral blood lymphocyte reconstitution and frequency of circulating CMV-directed CD8+ T cells was assessed (day ± 10 days) on post-transplant days +30, +60, and +90. Circulating anti-CMV T cell frequency was assessed using a phycoerythrin-tagged MHC dextramer against HLA-specific CMV pp65, IE-1, or pp50 peptides (Immudex; Copenhagen, DK) in combination with Tru-Count¨ tubes and fluorescent-labeled monoclonal antibodies against CD3, CD8, CD4, CD16/56, and CD19 (BD Biosciences; San Jose, CA). Anti-CMV CD8+ T cell immunity was defined as a CMV-dextramer (CMV/DEX) positive count of ≥7cells/ml. CMV reactivation was defined as a serologic titer of >500IU/mL. All patients with CMV reactivation received ganciclovir therapy until CMV titer became negative. Results: Day +30 total T cell recovery was significantly faster in MRD than CY-treated recipients (p=0.015) due principally to more robust CD8+ T cell recovery. CD4 T cell recovery remained below normal range in all groups through day +100. NK cells recovered to normal numbers at day +28 in all groups. Neither PTCy nor donor source significantly impacted the percentage of patients that recovered anti-CMV CD8+ T cells at each time interval (p = 0.8232). Excluding donors (D) and recipients (R) that were both negative, CMV/DEX+ T cells recovery was >7/mL in 4/5 MRD, 7/14 MUD, and 3/5 HAPLO by day +100. Among MRD recipients either D+ or R+ (n=5), 2 patients showed CMV reactivation within 40 days of transplant that was associated with <7 CMV/DEX+ T cells on day +30. Subsequent high (>90/mL) CMV/DEX T cell response in one patient shortened the duration of viremia to 10 days (vs. 16 days with poor responder) and 3 patients showed no CMV reactivation and a high CMV/DEX+ T cell response by day +60. For MUD CMV D+ and/or R+ recipients (n=14), 3 showed CMV reactivation within 50 days of transplant. All 3 patients had suboptimal CMV/DEX T cell response on day +30. Robust CMV/DEX+ T cell response on day +60 predicted shorter duration of viremia (20 days vs. average of 32 days). For HAPLO CMV D+ and/or R+ (n=5) recipients, 4 experienced CMV reactivation within 50 days of transplant. All patients had a <7 CMV/DEX+ T cells/mL +30. Robust CMV/DEX+ T cell response by day +60 was associated with shorter duration of viremia (range 7-21 days), while one patient with <7/mL CMV/DEX+ T cells had continued CMV viremia for 36 days. Conclusion: In this preliminary analysis, neither PTCy nor donor source significantly impacted the percentage of patients that recovered anti-CMV CD8+ T cells at each time interval. A weak CMV/DEX+ response (<7 cells/mL) on day +30 was consistent with increased risk of CMV reactivation (viremia) in all groups. A CMV/DEX+ T cell count ≥7 cells/mL was not immediately protective against CMV reactivation, but higher counts were associated with a shortened duration of viremia while on antiviral therapy. Conversely, subnormal counts were associated with a longer duration of viremia. This interim analysis suggests that CMV/DEX+ T cell enumeration is a useful biologic correlate for determining clinical response to antiviral therapy, and that donor-derived CMV specific T cell immunity is not further compromised with following PTCy in alternative donor HCT. Disclosures No relevant conflicts of interest to declare.

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