Durantez, M. (Maika)
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- Combination of a TLR4 ligand and anaphylatoxin C5a for the induction of antigen-specific cytotoxic T cell responses(Elsevier, 2012) Leclerc, C. (Claude); Fayolle, C. (Catherine); Pio, R. (Rubén); Lozano-Moreda, T. (Teresa); Rudilla, F. (Francesc); Durantez, M. (Maika); Casares, N. (Noelia); Prieto, J. (Jesús); Sarobe, P. (Pablo); Villanueva, L. (Lorea); Arribillaga, L. (Laura); Lasarte, J.J. (Juan José)The complement system and Toll-like receptors (TLR) are key innate defense systems which might interact synergistically on dendritic cells (DC) to reinforce adaptive immunity. In a previous work, we found that the extra domain A from fibronectin EDA (an endogenous ligand for TLR4) can favour antigen delivery to DC and induce their maturation. Given the potential of anaphylatoxins to cause inflammation and activation of myeloid cells, we hypothesized that a fusion protein between EDA, and anaphylatoxins C3a, C4a or C5a together with an antigen might improve the immunogenicity of the antigen. Naked DNA immunization with a construct expressing the fusion protein between C5a, EDA and the cytotoxic T cell epitope SIINFEKL from ovalbumin, induced strong antigen specific T cell responses. The purified recombinant fusion protein EDA-SIINFEKL-C5a induced activation of dendritic cells, the production of proinflammatory cytokines/chemokines and stimulated antigen presenting cell migration and NK cell activation. As compared to EDA-SIINFEKL, the fusion protein EDA-SIINFEKL-C5a did not induce the production of the immunosuppressive molecules IL-10, CCL17, CCL1, CXCL12 or XCL1 by DC. Moreover, EDA-SIINFEKL-C5a induced strong specific T cell responses in vivo and protected mice against E.G7-OVA tumor growth more efficiently than EDA-SIINFEKL or SIINFEKL-C5a recombinant proteins. Our results suggest that fusion proteins containing EDA, the anaphylatoxin C5a and the antigen may serve as a suitable strategy for the development of anti-tumor or anti-viral vaccines.
- Inhibition of FOXP3/NFAT Interaction Enhances T Cell Function after TCR Stimulation(American Association of Immunologists, 2015) Riezu-Boj, J.I. (José Ignacio); Lozano-Moreda, T. (Teresa); Ruiz, M. (Marta); Coffer, P.J. (Paul J.); Oyarzabal, J. (Julen); Lourenço, A.R. (Ana R.); Hervas-Stubbs, S. (Sandra); Durantez, M. (Maika); Casares, N. (Noelia); Bandukwala, H. (Hozefa); Prieto, J. (Jesús); Sarobe, P. (Pablo); Villanueva, L. (Lorea); Gorraiz, M. (Marta); Belsue, V. (Virginia); Lasarte, J.J. (Juan José)Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4(+) T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-γ, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-β. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.
- Adjuvant combination and antigen targeting as a strategy to induce polyfunctional and high-avidity T-cell responses against poorly immunogenic tumors(American association for cancer research, 2011) Riezu-Boj, J.I. (José Ignacio); Diaz-Valdes, N. (Nancy); Mansilla, C. (Cristina); Borras-Cuesta, F. (Francisco); Ruiz, M. (Marta); Martinez, M. (Marta); Durantez, M. (Maika); Aranda, F. (Fernando); Prieto, J. (Jesús); Bezunartea, J. (Jaione); Llopiz, D. (Diana); Sarobe, P. (Pablo); Lasarte, J.J. (Juan José)Low antigen expression and an absence of coimmunostimulatory signals may be partly responsible for the low immunogenicity of many tumors. It may be possible to overcome this situation by defining a combination of adjuvants and antigens that can activate a high-avidity antitumor response. Using the poorly immunogenic B16-OVA melanoma cells as tumor model, we tested different combinations of adjuvants and antigens to treat established tumors. In the absence of exogenous antigens, repeated administration of the TLR7 ligand Imiquimod together with anti-CD40 agonistic antibodies activated only innate immunity, which was insufficient to reject intradermal tumors. Administering this adjuvant combination together with OVA as a tumor antigen induced T-cell responses that delayed tumor growth. However, administering a combination of anti-CD40 plus TLR3 and TLR7 ligands, together with antigen targeting to dendritic cells through TLR4, was sufficient to induce tumor rejection in 50% of mice. This response was associated with a greater activation of innate immunity and induction of high-avidity polyfunctional CD8(+) T-cell responses, which each contributed to tumor rejection. This therapy activated T-cell responses not only against OVA, which conferred protection against a rechallenge with B16-OVA cells, but also activated T-cell responses against other melanoma-associated antigens. Our findings support the concept that multiple adjuvant combination and antigen targeting may be a useful immunotherapeutic strategy against poorly immunogenic tumors.
- Induction of multiepitopic and long-lasting immune responses against tumour antigens by immunization with peptides, DNA and recombinant adenoviruses expressing minigenes(Blackwell, 2009) Borras-Cuesta, F. (Francisco); Lopez-Diaz-de-Cerio, A. (Ascensión); Durantez, M. (Maika); Casares, N. (Noelia); Prieto, J. (Jesús); Huarte, E. (Eduardo); Lopez-Vazquez, A.B. (Ana B.); Sarobe, P. (Pablo); Lasarte, J.J. (Juan José)The development of immunization strategies to induce strong and multiepitopic T-cell responses against tumour antigens is needed for anti-tumour immunotherapy. However, a common finding after immunization with complex antigens is the preferential induction of immune responses against immunodominant epitopes. In this study, with the aim of inducing multiepitopic responses against several common tumour antigens, we have designed a minigene construct encoding four human leucocyte antigen (HLA)-A2-restricted epitopes belonging to tumour antigens CEA (CEA-691 and CEA-571), MAGE2 (MAGE2-157) and MAGE3 (MAGE3-112), as well as the universal PADRE epitope recognized by T helper lymphocytes. To optimize the activation of immune responses against these epitopes, we have used different antigen formats (short peptides encompassing individual epitopes and DNA plasmids or adenoviral constructs expressing the minigene) in single or combined immunization schedules. A single immunization with either DNA plasmid or recombinant adenovirus induced a monospecific immune response against the immunodominant epitope CEA-571, whereas immunization with the peptide pool induced responses against all epitopes. Combination of peptide priming followed by a boost with the plasmid and the recombinant adenovirus expressing the minigene induced stronger, multi-specific and long-lasting immune responses, overcoming the immunodominance imposed by the main T-cell epitope. Moreover, these combined immunization strategies were able to induce responses that were able to recognize Mel624 HLA-A2+ tumour cells expressing MAGE2. These results suggest that heterologous immunization strategies combining peptides and DNA or recombinant adenoviruses can be useful to broaden the specificity and enhance the efficacy of subunit vaccines.
- Tumor therapy in mice by using a tumor antigen linked to modulin peptides from Staphylococcus epidermidis(Elsevier, 2010) Riezu-Boj, J.I. (José Ignacio); Leclerc, C. (Claude); Fayolle, C. (Catherine); Borras-Cuesta, F. (Francisco); Durantez, M. (Maika); Casares, N. (Noelia); Prieto, J. (Jesús); Sarobe, P. (Pablo); Belsue, V. (Virginia); Lasarte, J.J. (Juan José)Staphylococcus epidermidis releases a complex of at least four peptides, termed phenol-soluble modulins (PSM), which stimulate macrophages to produce proinflammatory cytokines via activation of TLR2 signalling pathway. We demonstrated that covalent linkage of PSM peptides to an antigen facilitate its capture by dendritic cells and, in combination with different TLR ligands, can favour the in vivo induction of strong and persistent antigen-specific immune responses. Treatment of mice grafted with HPV16-E7-expressing tumor cells (TC-1) with poly(I:C) and a peptide containing αMod linked to the H-2D(b)-restricted cytotoxic T-cell epitope E7(49-57) from HPV16-E7 protein allowed complete tumor regression in 100% of the animals. Surprisingly, this immunomodulatory property of modulin-derived peptides was TLR2 independent and partially dependent upon the EGF-receptor signalling pathway. Our results suggest that alpha or gamma modulin peptides may serve as a suitable antigen carrier for the development of anti-tumoral or anti-viral vaccines.
- A fusion protein between streptavidin and the endogenous TLR4 ligand EDA targets biotinylated antigens to dendritic cells and induces T cell responses in vivo(2013) Borras-Cuesta, F. (Francisco); Lozano-Moreda, T. (Teresa); Martinez, M. (Marta); Rudilla, F. (Francesc); Durantez, M. (Maika); Casares, N. (Noelia); Rehberger-Bescos, F. (Federico); Prieto, J. (Jesús); Sarobe, P. (Pablo); Villanueva, L. (Lorea); Gorraiz, M. (Marta); Arribillaga, L. (Laura); Lasarte, J.J. (Juan José)The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a ~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβ by TLR4-expressing cells, as well as the production of TNF-α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.
- The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo(American association of immunologists, 2007) Leclerc, C. (Claude); Mansilla, C. (Cristina); Borras-Cuesta, F. (Francisco); Hervas-Stubbs, S. (Sandra); Durantez, M. (Maika); Casares, N. (Noelia); Prieto, J. (Jesús); Llopiz, D. (Diana); Sarobe, P. (Pablo); Gorraiz, M. (Marta); Arribillaga, L. (Laura); Lasarte, J.J. (Juan José)Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.
- Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin(Wiley-Blackwell, 2011) Leclerc, C. (Claude); Berraondo, P. (Pedro); Mansilla, C. (Cristina); Borras-Cuesta, F. (Francisco); Lozano-Moreda, T. (Teresa); Martinez, M. (Marta); Rudilla, F. (Francesc); Durantez, M. (Maika); Casares, N. (Noelia); Prieto, J. (Jesús); Sarobe, P. (Pablo); Villanueva, L. (Lorea); Arribillaga, L. (Laura); Fioravanti, J. (Jessica); Lasarte, J.J. (Juan José)Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma