Gustaf Ahlén

In Vivo Electroporation Enhances the Immunogenicity of Hepatitis C Virus Nonstructural 3/4A DNA by Increased Local DNA Uptake, Protein Expression, Inflammation, and Infiltration of CD3+ T Cells

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and γ-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene.

We have recently shown that the NS3-based genetic immunogens should contain also hepatitis C virus (HCV) nonstructural (NS) 4A to utilize fully the immunogenicity of NS3. The next step was to try to enhance immunogenicity by modifying translation or mRNA synthesis. To enhance translation efficiency, a synthetic NS3/4A-based DNA (coNS3/4A-DNA) vaccine was generated in which the codon usage was optimized (co) for human cells. In a second approach, expression of the wild-type (wt) NS3/4A gene was enhanced by mRNA amplification using the Semliki forest virus (SFV) replicon (wtNS3/4A-SFV). Transient tranfections of human HepG2 cells showed that the coNS3/4A gene gave 11-fold higher levels of NS3 as compared to the wtNS3/4A gene when using the CMV promoter. We have previously shown that the presence of NS4A enhances the expression by SFV. Both codon optimization and mRNA amplification resulted in an improved immunogenicity as evidenced by higher levels of NS3-specific antibodies. This improved immunogenicity also resulted in a more rapid priming of cytotoxic T lymphocytes (CTLs). Since HCV is a noncytolytic virus, the functionality of the primed CTL responses was evaluated by an in vivo challenge with NS3/4A-expressing syngeneic tumor cells. The priming of a tumor protective immunity required an endogenous production of the immunogen and CD8+ CTLs, but was independent of B and CD4+ T cells. This model confirmed the more rapid in vivo activation of an NS3/4A-specific tumor-inhibiting immunity by codon optimization and mRNA amplification. Finally, therapeutic vaccination with the coNS3/4A gene using gene gun 6–12 days after injection of tumors significantly reduced the tumor growth in vivo. Codon optimization and mRNA amplification effectively enhances the overall immunogenicity of NS3/4A. Thus, either, or both, of these approaches should be utilized in an NS3/4A-based HCV genetic vaccine.

Relation between viral fitness and immune escape within the hepatitis C virus protease

Background: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. Aims: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073–1081 of the NS3 protease was limited by viral fitness. Patients: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. Methods: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. Results: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073–1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073–1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. Conclusions: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.

The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor α mediated liver disease

Background: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. Aim: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. Methods: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl4), lipopolysaccaride (LPS), tumour necrosis factor α (TNFα), and anti-Fas antibody. Results: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl4, LPS, TNFα, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFα, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). Conclusions: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFα induced liver damage in vivo. The TNFα resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

In Vivo Clearance of Hepatitis C Virus Nonstructural 3/4A–Expressing Hepatocytes by DNA Vaccine–Primed Cytotoxic T Lymphocytes

A key question in the development of a therapeutic vaccine against hepatitis C virus (HCV) is whether vaccine-primed T cells enter the liver and eliminate HCV-expressing hepatocytes. In the absence of an infectious small-animal model, we evaluated liver homing of vaccine-primed T cells in mice with transient hepatic transgene expression of the HCV nonstructural 3/4A (NS3/4A) protein. We found that T cells primed by transdermal DNA-based vaccination entered the liver and cleared NS3/4A-expressing hepatocytes in transiently transgenic CD8(+/+) mice but not in CD8(-/-) mice. Hence, peripherally primed NS3/4A-specific CD8(+) T cells home to the liver and clear HCV protein-expressing hepatocytes.

Therapeutic DNA Vaccination Using In Vivo Electroporation Followed by Standard of Care Therapy in Patients With Genotype 1 Chronic Hepatitis C

Clearance of infections caused by the hepatitis C virus (HCV) correlates with HCV-specific T cell function. We therefore evaluated therapeutic vaccination in 12 patients with chronic HCV infection. Eight patients also underwent a subsequent standard-of-care (SOC) therapy with pegylated interferon (IFN) and ribavirin. The phase I/IIa clinical trial was performed in treatment naive HCV genotype 1 patients, receiving four monthly vaccinations in the deltoid muscles with 167, 500, or 1,500 μg codon-optimized HCV nonstructural (NS) 3/4A-expressing DNA vaccine delivered by in vivo electroporation (EP). Enrollment was done with 2 weeks interval between patients for safety reasons. Treatment was safe and well tolerated. The vaccinations significantly improved IFN-γ–producing responses to HCV NS3 during the first 6 weeks of therapy. Five patients experienced 2–10 weeks 0.6–2.4 log10 reduction in serum HCV RNA. Six out of eight patients starting SOC therapy within 1–30 months after the last vaccine dose were cured. This first-in-man therapeutic HCV DNA vaccine study with the vaccine delivered by in vivo EP shows transient effects in patients with chronic HCV genotype 1 infection. The interesting result noted after SOC therapy suggests that therapeutic vaccination can be explored in a combination with SOC treatment.

The hepatitis C virus non-structural NS5A protein impairs both the innate and adaptive hepatic immune response in vivo

The role of hepatitis C virus (HCV) protein non-structural (NS) 5A in HCV-associated pathogenesis is still enigmatic. To investigate the in vivo role of NS5A for viral persistence and virus-associated pathogenesis a transgenic (Tg) mouse model was established. Mice with liver-targeted NS5A transgene expression were generated using the albumin promoter. Alterations in the hepatic immune response were determined by Western blot, infection by lymphocytic choriomeningitis virus (LCMV), and using transient NS3/4A Tg mice generated by hydrodynamic injection. Cytotoxic T lymphocyte (CTL) activity was investigated by the Cr-release assay. The stable NS5A Tg mice did not reveal signs of spontaneous liver disease. The intrahepatic immunity was disrupted in the NS5A Tg mice as determined by clearance of LCMV infection or transiently NS3/4A Tg hepatocytes in vivo. This impaired immunity was explained by a reduced induction of interferon β, 2′,5′-OAS, and PKR after LCMV infection and an impairment of the CTL-mediated elimination of NS3-expressing hepatocytes. In conclusion, these data indicate that in the present transgenic mouse model, NS5A does not cause spontaneous liver disease. However, we discovered that NS5A could impair both the innate and the adaptive immune response to promote chronic HCV infection.

A Heterologous Prime/Boost Vaccination Strategy Enhances the Immunogenicity of Therapeutic Vaccines for Hepatitis C Virus

Background. We explored the concept of heterologous prime/boost vaccination using 2 therapeutic vaccines currently in clinical development aimed at treating chronically infected hepatitis C virus (HCV) patients: prime with a DNA-based vaccine expressing HCV genotype 1a NS3/4A proteins (ChronVac-C) and boost with a modified vaccinia virus Ankara vaccine expressing genotype 1b NS3/4/5B proteins (MVATG16643). Methods. Two ChronVac-C immunizations 4 weeks apart were delivered intramuscularly in combination with in vivo electroporation and subsequently 5 or 12 weeks later boosted by 3 weekly subcutaneous injections of MVATG16643. Two mouse strains were used, and we evaluated quality, magnitude, and functionality of the T cells induced. Results. DNA prime/MVA boost regimen induced significantly higher levels of interferon γ (IFN-γ) or interleukin 2 (IL-2) ELISpot responses compared with each vaccine alone, independent of the time of analysis and the time interval between vaccinations. Both CD8+ and CD4+ T-cell responses as well as the spectrum of epitopes recognized was improved. A significant increase in polyfunctional IFN-γ/tumor necrosis factor α (TNF-α)/CD107a+ CD8+ T cells was detected following ChronVac-C/MVATG16643 vaccination (from 3% to 25%), and prime/boost was the only regimen that activated quadrifunctional T cells (IFN-γ/TNF-α/CD107a/IL-2). In vivo functional protective capacity of DNA prime/MVA boost was demonstrated in a Listeria-NS3-1a challenge model. Conclusions. We provide a proof-of-concept that immunogenicity of 2 HCV therapeutic vaccines can be improved using their combination, which merits further clinical development.

Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes

Background: Hepatitis C virus (HCV) effectively establishes persistent infection in human livers. The non-structural (NS) 3/4A complex participates in this process by cleavage of interferon β (IFNβ) promoter stimulator-1 (IPS-1; also termed Cardif/MAVS/VISA), which inhibits responses to double stranded (ds) RNA. However, it is not known whether this effect extends beyond innate responses. Aims: To test if HCV NS3/4A affects innate and adaptive immune responses in vivo. Methods: NS3 levels were semi-quantified in human liver biopsies, transfected cells, and in transgenic (Tg) mouse livers by western blot. The effect of NS3/4A on dsRNA-mediated signalling and on the integrity of IPS-1 was analysed using in vitro translation, transfected cells and Tg mice. Cytotoxic T cell (CTL)-mediated clearance of transient firefly luciferase (FLuc)- and/or NS3/4A-Tg hepatocytes was determined using in vivo imaging and western blot. Results: NS3 protein levels were in a comparable range (0.1–49 μg/g tissue) in infected human livers and Tg mouse livers. Importantly, these levels of NS3/4A reduced murine innate responses to synthetic dsRNA in vivo, supporting the possibility that this occurs also in infected humans. The likely explanation for this was the NS3/4A-mediated cleavage of mouse IPS-1, albeit less efficiently than human IPS-1. Despite this, FLuc- and/or NS3/4A-expressing murine hepatocytes were effectively eliminated by hepatic CTLs, utilising the classical molecules for virus-infected cell lysis, including CD8, IFNγ, perforin and FasL. Conclusions: Although HCV NS3/4A inhibits the innate immunity, this does not prevent CTL-mediated clearance of NS3/4A-expressing hepatocytes in vivo. Thus, other HCV proteins are most likely responsible for interfering with the adaptive immunity.

Anti–tumor necrosis factor α treatment promotes apoptosis and prevents liver regeneration in a transgenic mouse model of chronic hepatitis C

Tumor necrosis factor α (TNFα) has been implicated in a variety of inflammatory diseases, and anti‐TNFα has been shown to improve therapy when added to standard of care in chronic hepatitis C virus (HCV) infection. In addition, patients with chronic HCV have increased serum levels of TNFα and the macrophage‐attracting chemokine (C‐C motif) ligand 2 (CCL2). A mouse model of chronic HCV with hepatic nonstructural (NS) 3/4A protein expression mimics the human infection through a reduced response to double‐stranded RNA and cleavage of the T cell protein tyrosine phosphatase. The mice also display a resistance to TNFα in vivo. We therefore analyzed the relationship between NS3/4A and TNFα. Wild‐type and NS3/4A‐transgenic (Tg) mice were treated with TNFα/D‐galactosamine (D‐galN), acting through the TNF receptor 1 on hepatocytes and macrophages, or lipopolysaccharide (LPS)/D‐galN, acting through Toll‐like receptor 4 on sinusoidal endothelial cells, macrophages, and dendritic cells. Mice were analyzed for hepatic signaling, liver damage, TNFα, and CCL2. Similar to HCV‐infected humans, NS3/4A‐Tg mice displayed elevated basal levels of TNFα and CCL2. Treatment of NS3/4A‐Tg mice with TNFα/D‐galN or LPS/D‐galN led to increased hepatic nuclear factor kappa B (NFκB) activation, increased TNFα and CCL2 levels, decreased apoptosis, and increased hepatocyte regeneration. Importantly, blocking NFκB activation (bortezomib) or administering anti‐TNFα (infliximab) 4 hours after LPS/D‐galN injection reversed the resistance of NS3/4A‐Tg mice to TNFα‐induced liver injury. Conclusion: Resistance to TNFα seen in NS3/4A‐Tg mice is explained by a hepatoprotective effect of NFκB and TNFα. Hence, anti‐TNFα agents block these effects and are antiviral by promoting hepatocyte apoptosis and preventing hepatocyte regeneration. (HEPATOLOGY 2010;.)