Ochoa, L. (Laura)
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- Treatment of chronic viral hepatitis in woodchucks by prolonged intrahepatic expression of interleukin-12(American Society for Microbiology, 2009) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Benito-Boilos, A. (Alberto); Rodriguez-Madoz, J.R. (Juan Roberto); Pañeda, A. (Astrid); Kochanek, S. (Stefan); Otano, I. (Itziar); Astudillo, A. (Aurora); Menne, S. (Stephan); Ochoa, L. (Laura); Crettaz, J. (Julien); Aurrekoetxea, I. (Igor); Prieto, J. (Jesús); Kreppel, F. (Florian); Ruiz, J. (Juan)Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.
- Intrahepatic injection of adenovirus reduces inflammation and increases gene transfer and therapeutic effect in mice(Wiley-Blackwell, 2006) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Rooijen, N. (Nico) van; Qian, C. (Cheng); Kochanek, S. (Stefan); Barajas, M. (Miguel); Shankar, V. (Vijay); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús); Hernandez-Alcoceba, R. (Rubén); Mauleon, I. (Itsaso)Recombinant adenoviruses (Ad) are among the most extensively used vectors for liver gene transfer. One of the major limitations for the clinical application of these vectors is the inflammatory immune response associated with systemic administration of high dose of virus. We evaluated the effect of Ad administration route on the inflammatory immune response and liver transgene expression. We compared direct intrahepatic injection (IH) with the systemic administration via tail vein (IV). IH injection of Ad resulted in a lower inflammatory response and a higher transgene expression. When a relatively low dose of virus was used, IV administration resulted in no detectable protein expression but production of proinflammatory cytokines. In contrast, IH administration induced high levels of transgene expression and no inflammation, although we detected a transient hypertransaminemia, which fully resolved within days. Furthermore, IH injection resulted in a faster protein expression being more intense at the site of injection, whereas IV administration caused slower but diffuse liver expression. IH injection also reduced the spreading of the virus to other organs. Independently of the route, depletion of Kupffer cells significantly enhanced the transduction efficiency of Ad. This effect was stronger when using IV injection, indicating that IH injection partially overcomes Kupffer cell phagocytic activity. Moreover, the antitumor efficacy of high-capacity-Ad encoding murine interleukin-12 (IL-12) was significantly greater when the vector was administered by IH injection than when given IV. In conclusion, IH injection of adenovirus represents a safe and efficient administration route for clinical applications of gene therapy targeting the liver.
- IFN-alpha gene therapy for woodchuck hepatitis with adeno-associated virus: differences in duration of gene expression and antiviral activity using intraportal or intramuscular routes(Nature Publishing Group, 2005) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Rotellar, F. (Fernando); Vales, A. (África); Zaratiegui, M. (Mikel); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús); Ruiz, J. (Juan); Martinez-Anso, E. (Eduardo)Gene delivery of IFN-alpha to the liver may represent an interesting strategy to maximize its antiviral efficacy and reduce side effects. We used a recombinant adeno-associated virus (AAV) encoding woodchuck IFN-alpha (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection. The vector was given by intraportal or intramuscular route. Long-term transgene expression was detected after intraportal administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN-alpha was transient (30-40 days) and was associated with a significant but transient decrease in viral load. One animal, in which hepatic production of IFN-alpha persisted at high levels, died because of bone marrow toxicity. The disappearance of IFN-alpha expression correlated with the disappearance of AAV genomes from the liver. Intramuscular administration of AAV-IFN resulted in prolonged but fluctuating expression of the cytokine with no significant antiviral effect. In summary, this report shows that long-term expression of IFN-alpha in muscle is feasible but higher interferon levels might be needed to control viral replication. On the other hand, IFN-alpha gene delivery to the liver using an AAV vector induces a significant but transient antiviral effect in the woodchuck model.
- Induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex(Elsevier, 2005) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Vales, A. (África); Rooijen, N. (Nico) van; Garzon, M. (Manolo); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús); Vera, M. (María); Ruiz, J. (Juan); Zulueta, J. (Javier); Lasarte, J.J. (Juan José)The induction of IFN-gamma-secreting CD8+ T cells and neutralizing antibodies to HIV-1 are both key requirements for prevention of viral transmission and clearance of pathogenic HIV. Although DNA vaccination has been shown to induce both humoral and cellular immune responses against HIV antigens, the magnitude of the immune responses has always been disappointing. In this report, we analyze the ability of polyethylenimine (PEI)-DNA complex expressing an HIV-glycoprotein 120 (gp120) antigen (PEI-pgp120) to induce systemic CD8+ T cell and humoral responses to the gp120 antigen. The administration of PEI-plasmid complex resulted in rapid elevation of serum levels of IL-12 and IFN-gamma. Furthermore, a single administration of PEI-pgp120 complex elicits a number of gp120-specific CD8+ T cells 20 times higher than that elicited by three intramuscular injections of naked DNA. Interestingly, we found that systemic vaccination with PEI-pgp120 induced protective immune responses against both systemic and mucosal challenges with a recombinant vaccinia virus expressing a gp120 antigen. The data also demonstrated that the depletion of macrophages with liposome-encapsulated clodronate completely abolished gp120-specific cellular response. Overall, our results showed that a single administration of PEI-pgp120 complexes, eliciting strong immune responses, is an effective vaccination approach to generate protection against systemic and mucosal viral infections.
- Production of recombinant woodchuck IFNalpha and development of monoclonal antibodies(Mary Ann Liebert, 2009) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Vales, A. (África); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús); Ruiz, J. (Juan); Martinez-Anso, E. (Eduardo)Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans.
- Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice(Elsevier, 2010) González-Aseguinolaza, G. (Gloria); Vales, A. (África); Olagüe, C. (Cristina); Otano, I. (Itziar); Menne, S. (Stephan); Ochoa, L. (Laura); Prieto, J. (Jesús); Sarobe, P. (Pablo); Lasarte, J.J. (Juan José)A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS "HLA Peptide Binding Predictions" program, we have identified and further characterized novel H-2(d)-restricted CD8+ epitopes within the WHV core (peptides C#12-21, C#18-32, C#19-27, C#61-69) and surface antigens (peptides preS2#10-18, preS2#27-35, S#76-84, S#133-140 and S#257-265), respectively. These peptides bind to H-2(d) with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-gamma producing T cells. More importantly, WHV core peptides C#19-27 and C#61-69 and WHV surface peptides S#133-140 and S#257-265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18-32, S#76-84, and S#257-265 resulted in significant production of IFN-gamma. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection.
- Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment of activated T lymphocytes(American Association for Cancer Research, 2011) Dubrot, J. (Juan); Martinez-Forero, I. (Iván); Jure-Kunkel, M. (María); Perez-Gracia, J.L. (Jose Luis); Rouzaut, A. (Ana); Peñuelas-Sanchez, I. (Ivan); Morales-Kastresana, A. (Aizea); Luque, A. (Alfonso); Hervas-Stubbs, S. (Sandra); Ochoa, L. (Laura); Ochoa, M.C. (María Carmen); Dinchuk, J. (Joseph); Melero, I. (Ignacio); Palazon, A. (Asís); Martinez, A. (Alfredo); Roncal, C. (Carmen); Teijeira, A. (Álvaro)Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag(-/-) mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies.
- Intrahepatic injection of recombinant adeno-associated virus serotype 2 overcomes gender-related differences in liver transduction(Mary Ann Liebert, 2006) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Pañeda, A. (Astrid); Troconiz, I.F. (Iñaki F.); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús)The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was associated with the fastest and strongest onset of luciferase expression. Moreover, no gender differences in liver transduction were observed and luciferase expression was confined to the site of injection. Thus, direct intrahepatic injection of rAAV offers specific advantages, which support the potential of this route of administration for future clinical applications.