Effect of the infectious dose and the presence of hepatitis C virus core gene on mouse intrahepatic CD8 T cells.

AIM: Chronic hepatitis C viral (HCV) infections often result in ineffective CD8 T-cell responses due to functional exhaustion of HCV-specific T cells. However, how persisting HCV impacts CD8 T-cell effector functions remains largely unknown. The aim of this study is to examine the effect of the infectious dose and the presence of HCV core gene. METHODS: We compared responses of intrahepatic CD8 T cells during infection of wild-type or HCV core transgenic (Tg) mice with various infectious doses of HCV-NS3-expressing recombinant adenovirus (Ad-HCV-NS3). RESULTS: Using major histocompatibility complex class I tetramer and intracellular interferon (IFN)-γ staining method to track HCV-NS3-specific CD8 T cells, we found that a significant expansion of HCV-NS3-specific CD8 T cells was restricted to a very narrow dosage range. IFN-γ production by intrahepatic CD8 T cells in HCV core Tg mice was suppressed as compared with wild-type mice. Higher levels of expression of regulatory molecules, Tim-3 and PD-1, by intrahepatic CD8 T cells and PD-L1 by intrahepatic antigen-presenting cells were observed in HCV core Tg mice following Ad-HCV-NS3 infection, and the expression increased dependent on infectious dose. Furthermore, we found a significant inverse correlation between the percentages of IFN-γ-producing cells and expression of regulatory molecules in antigen-specific intrahepatic CD8 T cells. CONCLUSION: High infectious dose and the presence of HCV core gene were strongly involved in ineffective CD8 T-cell responses. We consider that HCV core Tg mouse infected with high infectious dose of Ad-HCV-NS3 is useful as a chronic infection model in the development of immunotherapy for chronic hepatitis C.

Highly efficient antiviral CD8+ T-cell induction by peptides coupled to the surfaces of liposomes.

In previous studies, we have demonstrated that liposomes with differential lipid components display differential adjuvant effects when antigens (Ags) are chemically coupled to their surfaces. When ovalbumin was coupled to liposomes made by using unsaturated fatty acids, it was found to be presented not only to CD4(+) T cells but also to CD8(+) T cells and induced cytotoxic T lymphocytes (CTLs) which effectively eradicated the tumor from mice. In this study, we coupled liposomes to immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus (LCMV) and evaluated its potency as an antiviral vaccine. The intramuscular immunization of mice with the peptide-liposome conjugates along with CpG resulted in the efficient induction of antiviral CD8(+) T-cell responses which conferred complete protection against not only LCMV Armstrong but also a highly virulent mutant strain, clone 13, that establishes persistent infections in immunocompetent mice. The intranasal vaccination induced mucosal immunity effective enough to protect mice from the virus challenge via the same route. Complete protection was achieved in mice even when the Ag dose was reduced to as low as 280 ng of liposomal peptide. This form of vaccination with a single CTL epitope induced Ag-specific memory CD8(+) T cells in the absence of CD4(+) T-cell help, which could be shown by the complete protection of CD4-knockout mice in 10 weeks as well as by the analysis of recall responses. Thus, surface-linked liposomal peptide might have a potential advantage for the induction of antiviral immunity.

Synthetic peptides coupled to the surface of liposomes effectively induce SARS coronavirus-specific cytotoxic T lymphocytes and viral clearance in HLA-A*0201 transgenic mice.

We investigated whether the surface-linked liposomal peptide was applicable to a vaccine based on cytotoxic T lymphocytes (CTLs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We first identified four HLA-A*0201-restricted CTL epitopes derived from SARS-CoV using HLA-A*0201 transgenic mice and recombinant adenovirus expressing predicted epitopes. These peptides were coupled to the surface of liposomes, and inoculated into mice. Two of the liposomal peptides were effective for peptide-specific CTL induction, and one of them was efficient for the clearance of vaccinia virus expressing epitopes of SARS-CoV, suggesting that the surface-linked liposomal peptide might offer an effective CTL-based vaccine against SARS.

IL-23 enhances host defense against vaccinia virus infection via a mechanism partly involving IL-17.

To investigate roles of IL-23 in viral infection, we have engineered recombinant vaccinia virus (VV) expressing IL-12 (VV-IL-12) and expressing IL-23 (VV-IL-23). We found VV-IL-23 was less virulent in BALB/c mice than wild-type VV (VV-WT), indicating that IL-23 enhances resistance to VV. VV-specific CTL activity in VV-IL-23-infected mice was slightly higher than activity in VV-WT-inoculated mice, although antiviral Ab production and NK activity were not increased. IL-12/23p40-deficient mice survived the infection with VV-IL-23, indicating that IL-23 promotes VV resistance independently of IL-12. The mechanism of the IL-23-mediated resistance was distinct from that of the IL-12-regulated resistance because IFN-gamma-deficient mice did not eliminate VV-IL-12, but did eradicate VV-IL-23. These data indicate that IFN-gamma is essential for the IL-12-mediated resistance, but dispensable for the IL-23-regulated resistance. Because IL-17 is a key in the IL-23-regulated resistance to bacteria, we hypothesized an involvement of IL-17 in the resistance to VV. Treatment with an anti-IL-17 mAb resulted in a significant increase of viral titers in VV-IL-23-infected IFN-gamma-deficient mice. In addition, VV-IL-17 was less virulent than VV-WT in BALB/c mice, and IL-17-deficient mice were more sensitive to VV-WT than control mice. However, the effect of neutralization with an anti-IL-17 mAb was limited, and IL-17-deficient mice survived the infection with VV-IL-23. Taken together, these data suggest that the IL-23/IL-17 axis plays a certain but subdominant role in the IL-23-mediated resistance to VV. Unveiling of an alternative pathway in the IL-23-regulated resistance might provide a novel strategy against infectious pathogens without side effects of autoimmunity.

Immunogenic variation between multiple HLA-A*0201-restricted, Hepatitis C Virus-derived epitopes for cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in the immune control of Hepatitis C Virus (HCV) infection. In the current study, a number of HLA-A*0201-restricted CTL epitopes derived from HCV were evaluated by examining the peptide-binding affinity for major histocompatibility complex (MHC) class I molecules, the stability of peptide-MHC complexes, killing activities of peptide-induced CTLs, and frequencies of intracellular interferon (IFN)-gamma-positive CD8+ T cells. Among 24 peptides tested, 15 peptides induced high or medium killing activities of peptide-specific CTLs. Thirteen of the 15 peptides exhibited high or medium binding affinities for HLA-A*0201 molecules, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. In contrast, the stability of peptide-MHC class I complexes was not correlated with killing activities of peptide-induced CTLs. Furthermore, only a limited number of peptides could induce high or medium frequencies of IFN-gamma-producing CD8+ T cells, which were generally considered to play a crucial role for the clearance of HCV. Analyses of the immunogenicity of CTL epitopes such as in the current study should provide important information about the design of an efficient HCV vaccine that induces vigorous, sustained, and broad HCV-specific CTL responses.

Adjuvant activities of novel cytokines, interleukin-23 (IL-23) and IL-27, for induction of hepatitis C virus-specific cytotoxic T lymphocytes in HLA-A*0201 transgenic mice.

Searching the sequence databases has revealed two novel cytokines: interleukin-23 (IL-23) and IL-27. These cytokines are quite similar to, but clearly distinct from IL-12 in their structures and T-cell stimulatory fashions. In contrast to IL-12, however, little is known about the roles of IL-23 and IL-27 in the immune regulation. Previously, we evaluated the prime-boost immunization consisting of priming and the first boosting with the hepatitis C virus (HCV)-core expression plasmid, followed by a second boosting with recombinant adenovirus expressing HCV core for induction of HCV core-specific cytotoxic T lymphocytes (CTLs) in BALB/c mice. The present study demonstrates that HCV-specific CTL induction was greatly enhanced by coinoculation of an IL-12 expression plasmid in the prime-boost immunization, indicating the potent adjuvant activity of IL-12. We investigated whether similar adjuvant effects could be exerted by either IL-23 or IL-27 in a prime-boost immunization with HLA-A*0201 transgenic mice. Coadministration of either an IL-23 or an IL-27 expression plasmid, as well as an IL-12 expression plasmid, in a prime-boost immunization enhanced induction of HCV-specific CTLs and led to dramatic increases in the numbers of gamma interferon (IFN-gamma)-producing, HCV-specific CD8+ cells. Further, preinjections of IL-12, IL-23, or IL-27 expression plasmids before immunization resulted in great increases in the number of IFN-gamma-producing, HCV-specific CD8+ cells in response to immunization with recombinant adenovirus. These data revealed that both IL-23 and IL-27, as well as IL-12, are potent adjuvants for epitope-specific CTL induction. The two novel cytokines might offer new prophylactic and therapeutic strategies against infectious pathogens such as HCV.

Enhanced induction of hepatitis C virus-specific cytotoxic T lymphocytes and protective efficacy in mice by DNA vaccination followed by adenovirus boosting in combination with the interleukin-12 expression plasmid.

We evaluated the prime-boost immunization consisting of hepatitis C virus (HCV)-core expression plasmid (pCEP4-core) and replication-defective adenovirus expressing HCV-core (Adex1SR3ST) for core-specific CTL induction in mice. Compared to a single booster, double boosters after priming enhance CTL induction. The prime-double boosts immunization involving pCEP4-core priming followed by pCEP4-core and Adex1SR3ST boostings (pC/pC/aC) can induce core-specific CTLs as well as other combinations: pC/aC/aC; aC/pC/pC; aC/aC/aC, whereas pC/pC/pC does not induce CTLs. Furthermore, co-administration of interleukin-12 (IL-12) expression plasmid leads to the highly efficient CTL induction and clearance of HCV-core expressing vaccinia virus challenged. Thus, the prime-double boosts immunization together with IL-12 may be promising for HCV vaccine.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells transduced with replication-defective recombinant adenovirus.

We studied the potential of dendritic cells (DCs) in priming hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) in mice. Recombinant adenovirus expressing HCV core (Adex1SR3ST) was employed to express core in DCs. Core-specific CTLs are effectively elicited by injecting Adex1SR3ST-transduced DCs, whereas injection of Adex1SR3ST does not result in effective priming. Further, Adex1SR3ST-transduced DCs more efficiently prime core-specific CTLs than Adex1SR3ST-transduced macrophages, or DCs treated with an anthrax toxin fusion protein reported previously. Upon challenge with recombinant HCV-core-expressing vaccinia virus, vaccinia titers are significantly reduced in mice immunized with Adex1SR3ST-transduced DCs. Thus, adenovirus-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.