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

Ahlén, Gustaf; Nyström, Jessica; Pult, Irmgard; Frelin, Lars; Hultgren, Catharina; Sällberg, Matti

doi:10.1086/498218

Cited by 47 publications

(54 citation statements)

References 16 publications

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“…Cells were transfected using 4 mg plasmid DNA together with 6 ml TurboFect in vitro transfection reagent (Fermentas International, Burlington, ON, Canada) and incubated for 36 h before harvesting for analysis of NS3 and HBcAg protein content by immunoprecipitation and Western blot as described (30,34). In brief, cell lysates were applied to protein A-Sepharose beads conjugated with anti-NS3 or anti-HBcAg Abs for 2 h (+4˚C).…”

Section: Transient Transfection and Detection Of Expressed Ns3/4a Andmentioning

confidence: 99%

Heterologous T Cells Can Help Restore Function in Dysfunctional Hepatitis C Virus Nonstructural 3/4A-Specific T Cells during Therapeutic Vaccination

Chen

Ahlén

Brenndörfer

et al. 2011

The Journal of Immunology

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The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting “healthy” heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2–restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.

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Section: Transient Transfection and Detection Of Expressed Ns3/4a Andmentioning

confidence: 99%

Heterologous T Cells Can Help Restore Function in Dysfunctional Hepatitis C Virus Nonstructural 3/4A-Specific T Cells during Therapeutic Vaccination

Chen

Ahlén

Brenndörfer

et al. 2011

The Journal of Immunology

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“…Histochemistry and Immunohistochemistry-Sections and staining of the paraffin or cryosections was performed as described recently (18,23,24). For detection of NS5A, a rabbitderived V5-epitope-specific serum (Invitrogen) was used.…”

Section: Infection Of Mice and Virusmentioning

confidence: 99%

The Hepatitis C Virus Non-structural NS5A Protein Impairs Both the Innate and Adaptive Hepatic Immune Response in Vivo

Kriegs

Bürckstümmer

Himmelsbach

et al. 2009

Journal of Biological Chemistry

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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 ␤, 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. Chronic hepatitis C virus (HCV)4 infection is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). The HCV genome is a single-stranded positivesense RNA molecule of ϳ9600 bp (1). The viral RNA codes for one large polyprotein of ϳ3100 amino acids that is post-translationally processed by cellular and viral proteases, leading to the structural proteins core, E1 and E2, the p7 protein, and the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2). The mature NS5A protein is generated by the action of the NS3/NS4A serine protease. NS5A is a phosphoprotein that exists in a basal or in a hyperphosphorylated state (p56 and p58) (3). Through an amphipathic ␣-helix, NS5A is associated with the cytoplasmic face of the ER (4) and is an integral part of the replication complex (5). Mutations in NS5A affect the rate of HCV replication suggesting a role of NS5A in modulating viral expression and replication (6). Moreover, NS5A is able to interfere with a variety of cellular proteins. Some of these interaction partners, such as Grb2, PI3K, p53, or Raf-1 are important key players in host cell signal transduction, enabling NS5A to deregulate important cellular check points (7-10). Recent reports even suggest that NS5A may deregulate cell cycle progression by modulating the expression of cell cycle regulatory genes (11). In light of these observations and that it has been suggested to transform murine fibroblasts (12), it is speculated that NS5A could represent an important factor for the development of HCV-associated HCC (13).Infection of transgenic mice expressing the complete HCV polyprotein with lymphocytic choriomeningitis virus (LCMV) showed a reduced IFN response and a delayed viral eli...

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“…While other studies have shown that liver transfection with HCV antigens can be used to assess the cytoxicity of the DNA vaccine induced CD8 + T cell responses in mice 19 and HCV DNA immunization studies in chimpanzees have demonstrated the presence of HCV-specific IFNγ + T cells within the liver, 21 our study is the first to suggest that the liver actively recruits certain subsets of vaccine-specific T cells following peripheral immunization. Additionally, we have shown that the liver recruits vaccine-specific T cells despite the absence of cognate antigen and that this recruitment leads to the formation of a fully functional pool of vaccine-specific T cells able to both clear HCV protein expressing hepatocytes and to upregulate expression of CCR5 and IFNγ within the liver.…”

Section: Discussionmentioning

confidence: 57%

“…[18][19][20] In order to induce liver expression of HCV NS3, C57BL/6 mice were tail vein injected with pConNS3/NS4A using a previously described method of liver transfection. 19 Both naïve and immunized animals received tail vein injections of 100 μg of pConNS3/NS4A and were sacrificed 48 h following injection (Fig. 4A).…”

Section: Resultsmentioning

confidence: 99%

Peripheral immunization induces functional intrahepatic Hepatitis C specific immunity following selective retention of vaccine-specific CD8 T cells by the liver

et al. 2011

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

Cited by 47 publications

References 16 publications

Heterologous T Cells Can Help Restore Function in Dysfunctional Hepatitis C Virus Nonstructural 3/4A-Specific T Cells during Therapeutic Vaccination

Heterologous T Cells Can Help Restore Function in Dysfunctional Hepatitis C Virus Nonstructural 3/4A-Specific T Cells during Therapeutic Vaccination

The Hepatitis C Virus Non-structural NS5A Protein Impairs Both the Innate and Adaptive Hepatic Immune Response in Vivo

Peripheral immunization induces functional intrahepatic Hepatitis C specific immunity following selective retention of vaccine-specific CD8 T cells by the liver

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