Liver-directed gamma interferon gene delivery in chronic hepatitis C

Abstract

Gamma interferon (IFN-gamma) has been shown to inhibit replication of subgenomic and genomic hepatitis C virus (HCV) RNAs in vitro and to noncytolytically suppress hepatitis B virus (HBV) replication in vivo. IFN-gamma is also known for its immunomodulatory effects and as a marker of a successful cellular immune response to HCV. Therapeutic expression of IFN-gamma in the liver may therefore facilitate resolution of chronic hepatitis C, an infection that is rarely resolved spontaneously. To analyze immunomodulatory and antiviral effects of liver-specific IFN-gamma expression in vivo, we intravenously injected two persistently HCV-infected chimpanzees twice with a recombinant, replication-deficient HBV vector and subsequently with a recombinant adenoviral vector. These vectors expressed human IFN-gamma under control of HBV- and liver-specific promoters, respectively. Gene transfer resulted in a transient increase of intrahepatic IFN-gamma mRNA, without increase in serum alanine aminotransferase levels. Ex vivo analysis of peripheral blood lymphocytes demonstrated enhanced CD16 expression on T cells and upregulation of the liver-homing marker CXCR3. Moreover, an increased frequency of HCV-specific T cells was detected ex vivo in the peripheral blood and in vitro in liver biopsy-derived, antigen-nonspecifically expanded T-cell lines. None of these immunologic effects were observed in the third chimpanzee injected with an HBV control vector. Despite these immunologic effects of the experimental vector, however, IFN-gamma gene transfer did not result in a significant and long-lasting decrease of HCV titers. In conclusion, liver-directed IFN-gamma gene delivery resulted in HCV-specific and nonspecific activation of cellular immune responses but did not result in effective control of HCV replication.

FIG. 1.

Intrahepatic expression of IFN-γ and serum ALT values. (A) Relative IFN-γ mRNA levels were determined by real-time PCR with cDNA synthesized from RNA of total liver biopsy samples and normalized against GAPDH levels as internal controls. Data are presented as fold increases relative to the mean IFN-γ mRNA levels in nine preinfection liver biopsy samples from five other chimpanzees, because preinfection biopsies from Ch1535 and Ch1536 were not available. (B) ALT levels in chimpanzees' sera were measured. Dotted vertical lines indicate the time points of intravenous injection of 10¹¹ viral particles of rHBV-IFN-γ (week 0), 2 × 10¹¹ viral particles of rHBV-IFN-γ (week 8), and 10¹¹ infectious units of rAd-IFN-γ (week 15) in Ch1535 and Ch1536 and 2 × 10¹¹ viral particles of rHBV-Luc (week 0) in CB0561. GE, genome equivalent.

FIG. 2.

Phenotypic changes in peripheral blood and intrahepatic lymphocyte populations. (A) Frequency of CXCR3⁺ cells in peripheral blood (upper panels) and intrahepatic (lower panels) CD3⁺ lymphocytes. (B) Representative dot plots demonstrating CXCR3⁺ expression on intrahepatic CD3⁺ lymphocytes. (C) Frequency of CD16⁺ cells in CD3⁺ peripheral blood lymphocytes. (D) Representative dot plots demonstrating CD16 expression on peripheral blood lymphocytes. The dotted vertical lines in panels A and C indicate the time points of intravenous injection of 10¹¹ viral particles of rHBV-IFN-γ (week 0), 2 × 10¹¹ viral particles of rHBV-IFN-γ (week 8), and 10¹¹ infectious units of rAd-IFN-γ (week 15) in Ch1535 and Ch1536 and 2 × 10¹¹ viral particles of rHBV-Luc (week 0) in CB0561.

FIG. 3.

IFN-γ production and proliferation of circulating and intrahepatic T cells. (A-B) Ex vivo IFN-γ ELISPOT analysis of purified peripheral blood CD8⁺ (A) and CD4⁺ T cells (B) using pools of overlapping HCV peptides spanning the complete amino acid sequence of the infecting HCV. (C) Ex vivo IFN-γ ELISPOT analysis of PBLs using pools of overlapping peptides spanning the entire HBc and HBs amino acid sequence. (D) Antigen-nonspecific proliferation of intrahepatic lymphocytes in response to anti-CD3 and IL-2. The proliferation index reflects the cell number after 6 weeks of culture divided by the cell number at the start of culture. (E) The frequency of HCV-specific, IFN-γ-producing T cells in intrahepatic T-cell lines was determined in IFN-γ ELISPOT assays. CD8⁺ T cells were isolated from antigen-nonspecifically expanded intrahepatic T-cell lines and tested in IFN-γ ELISPOT assays with pools of overlapping peptides spanning the complete amino acid sequence of the infecting HCV. NT, not tested due to poor expansion of intrahepatic lymphocytes. Dotted vertical lines indicate the time points of intravenous injection of 10¹¹ viral particles of rHBV-IFN-γ (week 0), 2 × 10¹¹ viral particles of rHBV-IFN-γ (week 8), and 10¹¹ infectious units of rAd-IFN-γ (week 15) in Ch1535 and Ch1536 and 2 × 10¹¹ viral particles of rHBV-Luc (week 0) in CB0561. GE, genome equivalent.

FIG. 4.

Fine mapping of CD8⁺ T-cell responses to E1 and NS5B. (A) CD8⁺ T cells were isolated from T-cell lines established from liver biopsy samples taken at week 9 (1 week after the second rHBV-IFN-γ injection) (Fig. 3D and E) and tested in IFN-γ ELISPOT assays against individual overlapping peptides spanning the E1 sequence and the amino-terminal third of NS5B. (B) IFN-γ secretion assays confirm T-cell epitopes. After 12 h of stimulation with the selected peptide, secreted IFN-γ was captured on the surfaces of lymphocytes of Ch1536 and stained with secondary antibodies. Stimulation with DMSO was performed as a negative control. FSC, forward scatter.

FIG. 5.

Serum HCV RNA titers. Serum HCV RNA was quantified by real-time RT-PCR. Dotted vertical lines indicate the time points of intravenous injection of 10¹¹ viral particles of rHBV-IFN-γ (week 0), 2 × 10¹¹ viral particles of rHBV-IFN-γ (week 8), and 10¹¹ infectious units of rAd-IFN-γ (week 15) in Ch1535 and Ch1536 and 2 × 10¹¹ viral particles of rHBV-Luc (week 0) in CB0561. GE, genome equivalent.