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. 2020 May 4;94(10):e00035-20.
doi: 10.1128/JVI.00035-20. Print 2020 May 4.

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

Alex S Hartlage  1   2 , Christopher M Walker  1   3 , Amit Kapoor  4   3
Affiliations

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

Alex S Hartlage et al. J Virol. .

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.

Keywords: T cells; animal models; antiviral agents; antivirals; hepacivirus; hepatitis C virus; vaccines.

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Figures

FIG 1
FIG 1
Identification of novel RHV class I epitopes. Seven- to 10-week-old Lewis rats were vaccinated with 5 × 108 IFU adenovirus expressing the RHV NS3-5B proteins. Two or 3 weeks later, animals were challenged with 106 genomes of RHV intravenously. On days 17 to 56 postinfection, CD8 T cell recall responses were analyzed in liver. (A) Course of viremia in three representative vaccinees that cleared RHV infection after challenge. (B, left) Percentage of CD8 T cells producing IFN-γ following 5-h stimulation with the indicated peptides (10 μg/ml). Representative results from a single animal are shown. (Right) Amino acid sequences of reactive peptides. Boldface indicates predicted RT1-Al binding nonamers.
FIG 2
FIG 2
Early expansion of intrahepatic CD8 T cells targeting RHV after infection. Seven- to 10-week-old Lewis rats were infected with 106 genomes of RHV intravenously. Infection course and CD8 T cell immunity were tracked until 90 to 97 days p.i. (A, left) Plasma viremia, ALT levels, and percentage of liver-infiltrating CD8 T cells targeting the E1191 or NS5B2511 epitope, as determined by class I tetramer staining. (Right) Representative flow plots at day 14 p.i. showing frequency of intrahepatic CD8 T cells that bind the E1191 and NS5B2511 tetramers. Combined data from two independent experiments with n = 2 to 3 rats per time point are shown (means ± standard errors of the means [SEM]). (B) Frequency of CD8 T cells targeting the E1191 and NS5B2511 epitopes in blood and spleen, as determined by class I tetramer staining. (C) Sequence evolution of RHV class I epitopes. Consensus sequences were determined by direct PCR sequencing. The frequencies of rats infected with virus containing the indicated sequences are shown.
FIG 3
FIG 3
RHV-specific CD8 T cells fail to produce effector cytokines. Seven- to 10-week-old Lewis rats were infected with 106 genomes of RHV intravenously. Virus-specific CD8 T cells were assessed for effector function by cytokine-staining assay until 90 to 97 days p.i. (A) Representative flow plots at day 14 p.i. showing percentage of intrahepatic CD8 T cells that stain positive for classical (IFN-γ, TNF-α, and IL-2) and nonclassical (IL-17A, IL-10, and IL-4) antiviral cytokines. Cells were stimulated for 5 h with the E1191 or NS5B2511 epitope (10 μg/ml), a pool of class I and II epitopes (5 μg/ml each; Table 1), or no peptide or PMA plus ionomycin (PMA/Iono) as negative and positive controls, respectively. (B) Percentage of CD8 T cells producing cytokine at the indicated days p.i. Combined data from two independent experiments of n = 2 to 3 rats per time point are shown (means ± SEM). n.d., not detected. (C) Number of ELISpot assay IFN-γ spot-forming cells (SFCs) following 40 to 48 h of stimulation with the E1191 or NS5B2511 epitope (10 μg/ml). Combined data from two independent experiments of n = 2 to 3 rats per time point are shown (means ± SEM). n.d., not detected.
FIG 4
FIG 4
RHV-specific CD4 T cell cytokine responses after infection. Seven- to 10-week-old Lewis rats were infected with 106 genomes of RHV intravenously. Virus-specific CD4 T cells were assessed for effector function by cytokine-staining assay until 90 to 97 days p.i. (A) Representative flow plots at day 14 p.i. showing percentage of intrahepatic CD4 T cells that stain positive for classical (IFN-γ, TNF-α, and IL-2) and nonclassical (IL-17A, IL-10, and IL-4) antiviral cytokines. Cells were stimulated for 5 h with a pool of class I and II epitopes (5 μg/ml each; Table 1) or with no peptide or PMA plus ionomycin (PMA/Iono) as negative and positive controls, respectively. (B) Percentage of CD4 T cells producing cytokine at the indicated days p.i. Combined data from two independent experiments of n = 2 to 3 rats per time point are shown (means ± SEM). n.d., not detected.
FIG 5
FIG 5
Phenotypic differentiation of RHV-specific CD8 T cells. Seven- to 10-week-old Lewis rats were infected with 106 genomes of RHV intravenously, and virus-specific CD8 T cells were profiled for phenotypic changes by multiparametric flow cytometry. Responses were analyzed until 90 to 97 days p.i. (A) Representative flow plots showing percentage of intrahepatic E1191-specific CD8 T cells expressing the indicated markers at 14 days p.i. Total CD8 T cells are shown underlayed in black for comparison. (B) Percentage of E1191- and NS5B2511-specific CD8 T cells expressing the indicated markers. Baseline expression values at day 0 p.i. were assessed on naive (CD62L+) CD8 T cells from uninfected rats. Combined data from two independent experiments of n = 2 to 3 rats per group are shown (means ± SEM).
FIG 6
FIG 6
Early DAA treatment partially ameliorates RHV-specific CD8 T cell dysfunction. Seven- to 10-week-old Lewis rats were infected with 106 genomes of RHV intravenously. Starting at day 2 p.i., rats were treated daily with 10 mg sofosbuvir subcutaneously for twelve days. Following treatment, half of the rats were analyzed immediately at day 14 p.i. for recovery of liver CD8 T cell immunity. The remaining half were assessed for immunity and infection outcome at day 63 p.i. (A) Serum RHV RNA and ALT levels. Shading indicates timing of sofosbuvir treatment. ALT data show results from n = 6 to 12 rats per time point (means ± SEM). (B, left) Percentage of intrahepatic CD8 T cells targeting the E1191 and NS5B2511 epitopes as determined by class I tetramer staining. (Right) Representative flow plots showing frequency of CD8 T cells that bind the E1191 or NS5B2511 tetramer at day 14 p.i. (C) Percentage of CD8 T cells producing IFN-γ or TNF-α after 5 h of stimulation of the E1191 or NS5B2511 epitope (10 μg/ml) or a pool of class I and II epitopes (5 μg/ml each; Table 1) at the indicated days p.i. (D) Percentage of CD4 T cells producing IFN-γ or TNF-α after 5 h of stimulation of a pool of class I and II epitopes (5 μg/ml each; Table 1) at the indicated days p.i. (E) Comparison of frequency of E1191-specific CD8 T cells expressing the indicated markers at day 14 p.i. between untreated control (Fig. 5B) and sofosbuvir-treated rats. (F) Sequence evolution of E1191 and NS5B2511 epitopes during RHV infection. Consensus sequences were determined by direct PCR sequencing. Frequencies of rats infected with virus containing the indicated sequences are shown. (G) Percentage of intrahepatic CD8 T cells from immune rat producing IFN-γ upon 5 h of stimulation with titrated concentrations of the E1191 epitope or peptide containing the F199L mutation. Panels B to E show data from n = 6 rats per group (means ± SEM). n.d., not detected. ***, P < 0.001; *, P < 0.05; ns, not significant as determined by Student's t test.
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