Publications (12)40.43 Total impact
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Article: First report of bovine herpesvirus 5 in bull semen.
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ABSTRACT: BoHV-5 was detected in one of several extended semen samples from a healthy donor bull during routine virus screening. This was achieved by polymerase chain reaction assay (PCR) and virus isolation, with primary identification by the fluorescent antibody test. The isolated virus, B4180, was characterized by sequencing a cloned fragment of the gC gene and by restriction enzyme analysis (REA). The nucleotide sequence shared 99 % similarity with published sequences of BoHV-5, and the REA showed that the isolate was of the BoHV-5a subtype. This study provides the first evidence of intermittent BoHV-5 shedding in bull semen as well as information about its geographic distribution.Archives of Virology 05/2012; 157(9):1775-8. · 2.11 Impact Factor -
Article: Human αIFN co-formulated with milk derived E2-CSFV protein induce early full protection in vaccinated pigs.
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ABSTRACT: Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, type I interferon is used as an immunostimulating molecule in order to increase the immunogenicity of a vaccine candidate based on the E2-CSFV antigen produced in goat milk. Pigs vaccinated with E2-CSFV antigen co-formulated with recombinant human alpha interferon were protected against clinical signs and viremia as early as 7 days post-vaccination. It was also demonstrated that interferon stimulates a response of specific anti-CSFV neutralizing antibodies. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after vaccination. These results suggest that the E2-CSFV antigen combined with type I interferons could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.Vaccine 10/2010; 28(50):7907-14. · 3.77 Impact Factor -
Article: Nimotuzumab, an antitumor antibody that targets the epidermal growth factor receptor, blocks ligand binding while permitting the active receptor conformation.
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ABSTRACT: Overexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis for cancer patients. For this reason, the EGFR has become one of the main targets of anticancer therapies. Structural data obtained in the last few years have revealed the molecular mechanism for ligand-induced EGFR dimerization and subsequent signal transduction, and also how this signal is blocked by either monoclonal antibodies or small molecules. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has been successful in the clinics. In this work, we report the crystal structure of the Fab fragment of Nimotuzumab, revealing some unique structural features in the heavy variable domain. Furthermore, competition assays show that Nimotuzumab binds to domain III of the extracellular region of the EGFR, within an area that overlaps with both the surface patch recognized by Cetuximab (another anti-EGFR antibody) and the binding site for EGF. A computer model of the Nimotuzumab-EGFR complex, constructed by docking and molecular dynamics simulations and supported by mutagenesis studies, unveils a novel mechanism of action, with Nimotuzumab blocking EGF binding while still allowing the receptor to adopt its active conformation, hence warranting a basal level of signaling.Cancer Research 08/2009; 69(14):5851-9. · 7.86 Impact Factor -
Article: Early onset and long lasting protection in pigs provided by a classical swine fever E2-vaccine candidate produced in the milk of goats.
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ABSTRACT: For vaccination against classical swine fever virus (CSFV), it is strongly desirable to induce a rapid and long lasting protection. At present, only live attenuated CSFV vaccines have shown early onset of protection, differing with the recombinant subunit-based vaccines reported so far. Recently, a new vaccine formulation based on E2 envelope viral glycoprotein produced in the milk of goats (E2his) has been shown to induce a highly protective response in pigs against CSFV infection. Pigs immunized with a single dose of this vaccine candidate, formulated as a water-in oil emulsion, elicited an effective response against CSF as early as 7 days post-vaccination. No severe CSF clinical signs were observed and no animals died although the challenge dose was 10(5)PDL(50) of a highly pathogenic CSFV strain. Noticeably, this response completely prevented CSFV infection in pigs when they were challenged under the same conditions 2 weeks after a single dose of the recombinant E2his vaccine formulation. A schedule consisting of a primary immunization with the same vaccine candidate, followed by a booster dose 2 weeks later induced a highly protective response against CSFV infection for as long as 9 months post-vaccination. These promising results demonstrate by far the feasibility of using the E2his-based vaccine in regional programs for preventing and controlling CSF.Veterinary Immunology and Immunopathology 07/2009; 133(1):25-32. · 2.08 Impact Factor -
Article: Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.
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ABSTRACT: HIV-1-derived lentiviral vectors (LvV) are within the most attractive gene delivery vehicles in the context of both dividing and quiescent cells. LvV is currently produced by the conventional calcium phosphate precipitation method. Nevertheless, this procedure is highly susceptible to variations in pH and impurities, which lead to inconsistencies in LvV production. Here, we present a simple and robust procedure for LvV production using branched 25 kDa polyethylenimine, with a transfection efficiency of over 90% and viral titer yields of about 1 x 10(7) infective lentiviral particles per milliliter. The procedure outlined is simple, consistent, and as inexpensive as the CaPO(4)-based method.Applied biochemistry and biotechnology 01/2009; 157(3):538-44. · 1.94 Impact Factor -
Article: N-glycosylation pattern of E2 glycoprotein from classical swine fever virus.
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ABSTRACT: The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantennary N-glycans with variable number of alpha1-3-Gal-Gal antennae terminals. The most abundant neutral N-glycan has a composition of Hex(7)HexNAc(4)dHex(1), Negative ion ESI-MS/MS confirmed the presence of the alpha1-3-Gal-Gal motif on each arm of the fucosylated biantennary N-glycan. The most abundant monosialylated glycan was Hex(6)HexNAc(4)dHex(1)Neu(5)Ac(1), with the sialic acid linked to the terminal beta1-4-Gal-GlcNAc. Sialic acid on the antenna capping position was predominantly of the N-acetyl form.Journal of Proteome Research 01/2009; 8(2):546-55. · 5.11 Impact Factor -
Article: Classical swine fever virus E2 glycoprotein antigen produced in adenovirally transduced PK-15 cells confers complete protection in pigs upon viral challenge.
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ABSTRACT: E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3' terminus. The recombinant antigen was produced at a range of 120-150 microg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 microg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 10(5) PLD(50) of homologous CSFV "Margarita" strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.Vaccine 03/2008; 26(7):988-97. · 3.77 Impact Factor -
Article: Highly protective E2-CSFV vaccine candidate produced in the mammary gland of adenoviral transduced goats.
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ABSTRACT: Classical swine fever virus is the etiological agent of the most economically important highly contagious disease of swine worldwide. E2 is the major envelope glycoprotein present as a homodimer on the outer surface of the virus and represents an important target for the induction of neutralizing immune response against the viral infection. The E2 extracellular domain was expressed in the milk of adenoviral transduced goats at the highest level about 1.2g/L. The recombinant glycoprotein was purified from clarified serum milk by a single metal chelate affinity chromatography step, as a homodimer of approximately 100kDa and purity over 98%. Glycosylation analysis showed the presence of oligomannoside, hybrid and complex type N-glycans, attached to the recombinant E2. The capacity of goat milk-derived E2 antigen to protect pigs from both classical swine fever clinical signs and viral infection was assessed in a vaccination and challenge trial. The immunized pigs became protected after challenge with 10(5) LD(50) of a highly pathogenic CSFV strain. In the context of veterinary vaccines, this expression system has the advantages that the recombinant antigen could be harvested in about 48h after adenoviral transduction with expression levels in the range of g/L. This approach may turn into a scalable expression system for the assessment and production of veterinary vaccines.Journal of Biotechnology 03/2008; 133(3):370-6. · 3.05 Impact Factor -
Article: High expression level of recombinant human erythropoietin in the milk of non-transgenic goats.
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ABSTRACT: The high degree of structural conservation of erythropoietin between species, make it, especially, difficult to produce this protein growth factor in the milk of transgenic animals. Here, we show that through the direct transduction of the mammary epithelium, it is possible to produce high levels of recombinant human erythropoietin in the milk of non-transgenic goats without causing harm to the animals. The efficiency of viral transduction was improved through a temporal disruption of tight-junctions with EGTA allowing for the expression of human erythropoietin at levels of up to 2g/L in milk. The human erythropoietin was purified from the milk using a multi-step protocol involving milk clarification, two precipitation steps and two affinity chromatographies, with a yield of about 70% and purity over 98%. However, the human erythropoietin expressed in milk was underglycosylated, which seems to be the main cause for its low in vivo hematopoietic activity. Nonetheless, these results demonstrate that through the direct transduction of the mammary epithelium it is possible to produce potentially toxic proteins in milk, at levels high enough for their purification and biological characterization.Journal of Biotechnology 06/2006; 123(2):225-35. · 3.05 Impact Factor -
Article: Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells.
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ABSTRACT: The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal.Biochimica et Biophysica Acta 11/2005; 1726(1):48-56. · 4.66 Impact Factor -
Article: New procedure for the production of biopharmaceutical proteins in the milk of non-transgenic animals
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ABSTRACT: The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that through the direct transduction of the mammary glandular epithelium by means of adenoviral vectors it is possible to promote high expression levels of recombinant proteins in the milk of non-transgenic animals. Through this approach we were able to express high levels of human growth hormone and human erythropoietin in the milk of both mice and goats. We found that the expression levels were closely dependent on both the degree of differentiation of the secretory epithelium and the adenoviral dose used. Direct transduction of the mammary gland seems to be a suitable alternative to express high levels of complex proteins in milk and it may eventually constitute a platform for the development of new biotechnological processes.Biotecnologia Aplicada 01/2005; 22. -
Article: Adenoviral vector mediates high expression levels of human growth hormone in the milk of mice and goats.
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ABSTRACT: The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that a direct instillation of a recombinant adenoviral vector containing an expression cassette for the human growth hormone gene into the mammary gland of mice and goats allowed for the efficient secretion of human growth hormone in the milk. Through this approach we were able to express human growth hormone at maximal levels of 2.8 mg/ml in the milk of mice and up to 0.3 mg/ml in goat milk. We found that the expression levels were closely dependent on both the degree of differentiation of the secretory epithelium and on the adenoviral dose used. Here we demonstrated that the direct transduction of mammary epithelial cells by means of a recombinant adenovirus could be a suitable alternative to transgenic technology for the production of recombinant proteins of biopharmaceutical interest.Journal of Biotechnology 11/2004; 114(1-2):89-97. · 3.05 Impact Factor
Top co-authors
Top Journals
Institutions
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2010
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University of Concepción
Concepción, Region del Biobio, Chile
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2004–2009
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Center for Genetic Engineering and Biotechnology
Havana, Provincia de La Habana, Cuba
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