scholarly journals DNA Vaccination Protects Mice against Challenge with Listeria monocytogenes Expressing the Hepatitis C Virus NS3 Protein

2003 ◽  
Vol 71 (11) ◽  
pp. 6372-6380 ◽  
Author(s):  
Benjamin E. Simon ◽  
Kenneth A. Cornell ◽  
Tina R. Clark ◽  
Sunwen Chou ◽  
Hugo R. Rosen ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Listeria Monocytogenes ◽  
Immune Responses ◽  
Bacterial Pathogen ◽  
Cell Responses ◽  
Ns3 Protein ◽  
Plasmid Dna Vaccine ◽  
In Vivo Induction

ABSTRACT The goal of this study was to develop a new surrogate challenge model for use in evaluating protective cell-mediated immune responses against hepatitis C virus (HCV) antigens. The use of recombinant Listeria monocytogenes organisms which express HCV antigens provides novel tools with which to assay such in vivo protection, as expression of immunity against this hepatotropic bacterial pathogen is dependent on antigen-specific CD8+ T lymphocytes. A plasmid DNA vaccine encoding a ubiquitin-NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma interferon-secreting T cells following vaccination of BALB/c mice. These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant L. monocytogenes strain (TC-LNS3) expressing the NS3 protein. Notably, sublethal infection of naive mice with strain TC-LNS3 induced similar NS3-specific T-cell responses. These findings suggest that recombinant strains of L. monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens.

scholarly journals Humoral and CD4+ T helper (Th) cell responses to the hepatitis C virus non-structural 3 (NS3) protein: NS3 primes Th1-like responses more effectively as a DNA-based immunogen than as a recombinant protein

2001 ◽  
Vol 82 (6) ◽  
pp. 1299-1308 ◽  
Author(s):  
Una Lazdina ◽  
Catharina Hultgren ◽  
Lars Frelin ◽  
Margaret Chen ◽  
Karin Lodin ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Responses ◽  
T Helper ◽  
Cell Responses ◽  
Ns3 Protein ◽  
Antibody Titres ◽  
Ifn Γ

The non-structural 3 (NS3) protein is one of the most conserved proteins of hepatitis C virus, and T helper 1 (Th1)-like responses to NS3 in humans correlate with clearance of infection. Several studies have proposed that DNA-based immunizations are highly immunogenic and prime Th1-like responses, although few head-to-head comparisons with exogenous protein immunizations have been described. A full-length NS3/NS4A gene was cloned in eukaryotic vectors with expression directed to different subcellular compartments. Inbred mice were immunized twice in regenerating tibialis anterior (TA) muscles with either plasmid DNA or recombinant NS3 (rNS3). After two 100 μg DNA immunizations, specific antibody titres of up to 12960 were detected at week 5, dominated by IgG2a and IgG2b. NS3-specific CD4+ T cell responses in DNA-immunized mice peaked at day 13, as measured by proliferation and IL-2 and IFN-γ production. Mice immunized with 1–10 μg rNS3 without adjuvant developed antibody titres comparable to those of the DNA-immunized mice, but dominated instead by IgG1. CD4+ T cell responses in these mice showed peaks of IL-2 response at day 3 and IL-6 and IFN-γ responses at day 6. With adjuvant, rNS3 was around 10-fold more immunogenic with respect to speed and magnitude of the immune responses. Thus, immunization with rNS3 in adjuvant is superior to DNA immunization with respect to kinetics and quantity in priming specific antibodies and CD4+ T cells. However, as a DNA immunogen, NS3 elicits stronger Th1-like immune responses, whereas rNS3 primes a mixed Th1/Th2-like response regardless of the route, dose or adjuvant.

scholarly journals Hepatitis C Virus Structural Proteins Impair Dendritic Cell Maturation and Inhibit In Vivo Induction of Cellular Immune Responses

2003 ◽  
Vol 77 (20) ◽  
pp. 10862-10871 ◽  
Author(s):  
Pablo Sarobe ◽  
Juan José Lasarte ◽  
Aintzane Zabaleta ◽  
Laura Arribillaga ◽  
Ainhoa Arina ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Responses ◽  
Cell Responses ◽  
Cellular Immune Responses ◽  
Cell Immunity ◽  
Cellular Immune

ABSTRACT Hepatitis C virus (HCV) chronic infection is characterized by low or undetectable cellular immune responses against HCV antigens. Some studies have suggested that HCV proteins manipulate the immune system by suppressing the specific antiviral T-cell immunity. We have previously reported that the expression of HCV core and E1 proteins (CE1) in dendritic cells (DC) impairs their ability to prime T cells in vitro. We show here that immunization of mice with immature DC transduced with an adenovirus encoding HCV core and E1 antigens (AdCE1) induced lower CD4+- and CD8+-T-cell responses than immunization with DC transduced with an adenovirus encoding NS3 (AdNS3). However, no differences in the strength of the immune response were detected when animals were immunized with mature DC subsequently transduced with AdCE1 or AdNS3. According to these findings, we observed that the expression of CE1 in DC inhibited the maturation caused by tumor necrosis factor alpha or CD40L but not that induced by lipopolysaccharide. Blockade of DC maturation by CE1 was manifested by a lower expression of maturation surface markers and was associated with a reduced ability of AdCE1-transduced DC to activate CD4+- and CD8+-T-cell responses in vivo. Our results suggest that HCV CE1 proteins modulate T-cell responses by decreasing the stimulatory ability of DC in vivo via inhibition of their physiological maturation pathways. These findings are relevant for the design of therapeutic vaccination strategies in HCV-infected patients.

scholarly journals Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice

2018 ◽  
Vol 92 (8) ◽  
pp. e02133-17 ◽  
Author(s):  
Danushka K. Wijesundara ◽  
Jason Gummow ◽  
Yanrui Li ◽  
Wenbo Yu ◽  
Benjamin J. Quah ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Dna Vaccine ◽  
Target Cells ◽  
Cell Mediated Immunity ◽  
Specific Cell ◽  
Universal Vaccine ◽  
Cell Responses ◽  
Single Genotype

ABSTRACTA universal hepatitis C virus (HCV) vaccine should elicit multiantigenic, multigenotypic responses, which are more likely to protect against challenge with the range of genotypes and subtypes circulating in the community. A vaccine cocktail and vaccines encoding consensus HCV sequences are attractive approaches to achieve this goal. Consequently, in a series of mouse vaccination studies, we compared the immunogenicity of a DNA vaccine encoding a consensus HCV nonstructural 5B (NS5B) protein to that of a cocktail of DNA plasmids encoding the genotype 1b (Gt1b) and Gt3a NS5B proteins. To complement this study, we assessed responses to a multiantigenic cocktail regimen by comparing a DNA vaccine cocktail encoding Gt1b and Gt3a NS3, NS4, and NS5B proteins to a single-genotype NS3/4/5B DNA vaccine. To thoroughly evaluatein vivocytotoxic T lymphocyte (CTL) and T helper (Th) cell responses against Gt1b and Gt3a HCV peptide-pulsed target cells, we exploited a novel fluorescent-target array (FTA). FTA and enzyme-linked immunosorbent spot (ELISpot) analyses collectively indicated that the cocktail regimens elicited higher responses to Gt1b and Gt3a NS5B proteins than those with the consensus vaccine, while the multiantigenic DNA cocktail significantly increased the responses to NS3 and NS5B compared to those elicited by the single-genotype vaccines. Thus, a DNA cocktail vaccination regimen is more effective than a consensus vaccine or a monovalent vaccine at increasing the breadth of multigenotypic T cell responses, which has implications for the development of vaccines for communities where multiple HCV genotypes circulate.IMPORTANCEDespite the development of highly effective direct-acting antivirals (DAA), infections with hepatitis C virus (HCV) continue, particularly in countries where the supply of DAA is limited. Furthermore, patients who eliminate the virus as a result of DAA therapy can still be reinfected. Thus, a vaccine for HCV is urgently required, but the heterogeneity of HCV strains makes the development of a universal vaccine difficult. To address this, we developed a novel cytolytic DNA vaccine which elicits robust cell-mediated immunity (CMI) to the nonstructural (NS) proteins in vaccinated animals. We compared the immune responses against genotypes 1 and 3 that were elicited by a consensus DNA vaccine or a DNA vaccine cocktail and showed that the cocktail induced higher levels of CMI to the NS proteins of both genotypes. This study suggests that a universal HCV vaccine can most readily be achieved by use of a DNA vaccine cocktail.

scholarly journals Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
María Q. Marín ◽  
Patricia Pérez ◽  
Karl Ljungberg ◽  
Carlos Óscar S. Sorzano ◽  
Carmen E. Gómez ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Immune Responses ◽  
Vaccinia Virus ◽  
Cell Responses ◽  
Immunization Protocol ◽  
Modified Vaccinia Virus Ankara ◽  
Boost Immunization ◽  
T Cell Immune Responses

ABSTRACTHepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Immunization of mice with the DREP vaccines as the priming immunization followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV) induced potent and long-lasting HCV-specific CD4+and CD8+T cell immune responses that were significantly stronger than those of a homologous MVA-HCV prime/boost immunization, with the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4+and CD8+T cell responses were highly polyfunctional, had an effector memory phenotype, and were mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV by inducing high levels of HCV-specific T cell responses as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV.IMPORTANCEHCV represents a global health problem as more than 71 million people are chronically infected worldwide. Direct-acting antiviral agents can cure HCV infection in most patients, but due to the high cost of these agents and the emergence of resistant mutants, they do not represent a feasible and affordable strategy to eradicate the virus. Therefore, a vaccine is an urgent goal that requires efforts to understand the correlates of protection for HCV clearance. Here, we describe for the first time the generation of novel vaccines against HCV based on alphavirus DNA replicons expressing HCV antigens. We demonstrate that potent T cell immune responses, as well as humoral immune responses, against HCV can be achieved in mice by using a combined heterologous prime/boost immunization protocol consisting of the administration of alphavirus replicon DNA vectors as the priming immunization followed by a boost with a recombinant modified vaccinia virus Ankara vector expressing HCV antigens.

scholarly journals In Vivo Study of the HC-TN Strain of Hepatitis C Virus Recovered from a Patient with Fulminant Hepatitis: RNA Transcripts of a Molecular Clone (pHC-TN) Are Infectious in Chimpanzees but Not in Huh7.5 Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 7208-7219 ◽  
Author(s):  
Akito Sakai ◽  
Shingo Takikawa ◽  
Robert Thimme ◽  
Jean-Christophe Meunier ◽  
Hans Christian Spangenberg ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immune Responses ◽  
Disease Severity ◽  
Neutralizing Antibodies ◽  
Viral Evolution ◽  
Acute Infection ◽  
Hcv Infection ◽  
Rna Transcripts

ABSTRACT Both viral and host factors are thought to influence the pathogenesis of hepatitis C virus (HCV) infection. We studied strain HC-TN (genotype 1a), which caused fulminant hepatic failure in a patient and, subsequently, severe hepatitis in a chimpanzee (CH1422), to analyze the relationship between disease severity, host immune response, viral evolution, and outcome. A second chimpanzee (CH1581) was infected from CH1422 plasma, and a third chimpanzee (CH1579) was infected from RNA transcripts of a consensus cDNA of HC-TN (pHC-TN). RNA transcripts of pHC-TN did not replicate in Huh7.5 cells, which were recently found to be susceptible to infection with another fulminant HCV strain (JFH1). The courses of viremia were similar in the three animals. However, CH1581 and CH1579 developed a less severe acute hepatitis than CH1422. CH1579 and CH1422 resolved the infection, whereas CH1581 became persistently infected. CH1579 and CH1581, despite their differing outcomes, both developed significant intrahepatic cellular immune responses, but not antibodies to the envelope glycoproteins or neutralizing antibodies, during the acute infection. We analyzed the polyprotein sequences of virus recovered at five and nine time points from CH1579 and CH1581, respectively, during the first year of follow-up. High mutation rates and high proportions of nonsynonymous mutations suggested immune pressure and positive selection in both animals. Changes were not selected until after the initial decrease in virus titers and after the development of immune responses and hepatitis. Subsequently, however, mutations emerged repeatedly in both animals. Overall, our results indicate that disease severity and outcome of acute HCV infection depend primarily on the host response.

Hepatitis C Virus Continuously Escapes From Neutralizing Antibody and T-Cell Responses During Chronic Infection In Vivo

2007 ◽  
Vol 132 (2) ◽  
pp. 667-678 ◽  
Author(s):  
Thomas von Hahn ◽  
Joo Chun Yoon ◽  
Harvey Alter ◽  
Charles M. Rice ◽  
Barbara Rehermann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Chronic Infection ◽  
T Cell Responses ◽  
Neutralizing Antibody ◽  
Cell Responses

scholarly journals Dendritic cells pulsed with hepatitis C virus NS3 protein induce immune responses and protection from infection with recombinant vaccinia virus expressing NS3

2006 ◽  
Vol 87 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Hong Yu ◽  
Hui Huang ◽  
Jim Xiang ◽  
Lorne A. Babiuk ◽  
Sylvia van Drunen Littel-van den Hurk
Keyword(s):  
Dendritic Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immune Responses ◽  
Vaccinia Virus ◽  
Recombinant Vaccinia Virus ◽  
Interleukin 12 ◽  
Cpg Odn ◽  
Recombinant Vaccinia ◽  
Ns3 Protein

Infections with Hepatitis C virus (HCV) pose a serious health problem worldwide. In this study, the hypothesis that adoptive transfer of dendritic cells (DCs) pulsed with HCV NS3 protein and matured with an oligodeoxynucleotide (ODN) containing CpG motifs (CpG) ex vivo would initiate potent HCV-specific protective immune responses in vivo was tested. NS3 protein was efficiently transduced into DCs and treatment of DCs with CpG ODN induced phenotypic maturation and specifically increased the expression of CD40. DCs matured with CpG ODN produced higher interleukin 12 levels and a stronger allogeneic T-cell response compared with untreated DCs. Notably, there were no differences between NS3-pulsed DCs and DCs pulsed with a control protein with respect to phenotype, cytokine production or mixed lymphocyte reaction, indicating that transduction with NS3 protein did not impair DC functions. Compared with the untreated NS3-pulsed DCs, the NS3-pulsed DCs matured with CpG ODN induced stronger cellular immune responses including enhanced cytotoxicity, higher interferon-γ production and stronger lymphocyte proliferation. Upon challenge with a recombinant vaccinia virus expressing NS3, all mice immunized with NS3-pulsed DCs showed a significant reduction in vaccinia virus titres when compared with mock-immunized mice. However, the NS3-pulsed DCs matured with CpG ODN induced higher levels of protection compared with the untreated NS3-pulsed DCs. These data are the first to show that NS3-pulsed DCs induce specific immune responses and provide protection from viral challenge, and also demonstrate that CpG ODNs, which have a proven safety profile, would be useful in the development of DC vaccines.

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