Selecting Nanobodies Specific for the Epidermal Growth Factor from a Synthetic Nanobody Library.

The epidermal growth factor (EGF) is one of the most critical ligands of the EGF receptor (EGFR), a well-known oncogene frequently overexpressed in cancerous cells and an important therapeutic target in cancer. The EGF is the target of a therapeutic vaccine aimed at inducing an anti-EGF antibody response to sequester this molecule from serum. However, strikingly, very few investigations have focused on EGF immunotargeting. Since the use of nanobodies (Nbs) for EGF neutralization may be an effective therapeutic strategy in several types of cancer, in this study, we decided to generate anti-EGF Nbs from a recently constructed, phage-displaying synthetic nanobody library. To our knowledge, this is the first attempt to obtain anti-EGF Nbs from a synthetic library. By applying a selection strategy that uses four different sequential elution steps along with three rounds of selection, we obtained four different EGF-specific Nb clones, and also tested their binding capabilities as recombinant proteins. The obtained results are very encouraging and demonstrate the feasibility of selecting nanobodies against small antigens, such as the EGF, from synthetic libraries.

Design and Construction of a Synthetic Nanobody Library: Testing Its Potential with a Single Selection Round Strategy.

Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 108 and an amino acid variability that matches the codon distribution set by design at each randomized position. We have explored the capabilities of the new library by selecting nanobodies specific for three antigens: vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and the glycoprotein complex (GnGc) of Andes virus. To test the potential of the library to yield a variety of antigen-specific Nbs, we introduced a biopanning strategy consisting of a single selection round using stringent conditions. Using this approach, we obtained several binders for each of the target antigens. The constructed library represents a promising nanobody source for different applications.

Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

Structure-based design and construction of a synthetic phage display nanobody library.

OBJECTIVE: To design and construct a new synthetic nanobody library using a structure-based approach that seeks to maintain high protein stability and increase the number of functional variants within the combinatorial space of mutations. RESULTS: Synthetic nanobody (Nb) libraries are emerging as an attractive alternative to animal immunization for the selection of stable, high affinity Nbs. Two key features define a synthetic Nb library: framework selection and CDR design. We selected the universal VHH framework from the cAbBCII10 Nb. CDR1 and CDR2 were designed with the same fixed length as in cAbBCII10, while for CDR3 we chose a 14-long loop, which creates a convex binding site topology. Based on the analysis of the cAbBCII10 crystal structure, we carefully selected the positions to be randomized and tailored the codon usage at each position, keeping at particular places amino acids that guarantee stability, favoring properties like polarity at solvent-exposed positions and avoiding destabilizing amino acids. Gene synthesis and library construction were carried out by GenScript, using our own phagemid vector. The constructed library has an estimated size of 1.75 × 108. NGS showed that the amino acid diversity and frequency at each randomized position are the expected from the codon usage.

Characterisation of a New Molecule Based on Two E2 Sequences from Bovine Viral Diarrhoea-mucosal Disease Virus Fused To the Human Immunoglobulin Fc Fragment.

INTRODUCTION: Proper conformational arrangement of the E2 molecules of bovine viral diarrhoea-mucosal disease virus (BVD-MDV) is crucial to obtain an effective recombinant vaccine candidate against the disease. In this study, we characterised a new molecule composed of two distinct sequences of the E2 glycoprotein of BVD-MDV and the Fc fragment of human immunoglobulin (BVDE2Fc). MATERIALS AND METHODS: The chimaeric protein was expressed in mammalian cell lines of different species by adenoviral transduction and purified by immobilised metal-affinity chromatography. The N-glycans were profiled by HPLC, and the BVDE2Fc immunogenicity was assessed in male mice. The antigen-antibody reactions were evaluated by ELISA. RESULTS: The MDBK cell line was selected from among five for the final production of BVDE2Fc. After purification to over 90%, the N-glycan profile showed neutral and complex oligosaccharides. The mouse immunisation induced a strong humoral response, which produced antibodies able to attach to conformational epitopes on E2 molecules, while the Fc fragment barely contributed to the immune response. Additionally, BVDE2Fc attached to antibodies from bovine sera positive to distinct BVD-MDV subtypes, whereas the loss of BVDE2Fc structure during the deglycosylation process considerably diminished those interactions. CONCLUSION: These results demonstrate that the structure of E2 molecules arranged in tandem and attached to an Fc fragment could represent a viable design for future vaccine candidates against BVD-MD.

Utilización de aprendizaje basado en equipos, como metodología activa de enseñanza de farmacología para estudiantes de Enfermería / The use of equipment-based learning as an active methodology teaching for pharmacology to Nursing studen

Introducción: el cambio de la sociedad desde una economía basada en la industria a una sustentada en el conocimiento, genera un cambio de paradigma en educación. Esto lleva a las universidades a modificar sus modelos educativos y formar profesionales capaces de responder a las necesidades del entorno. Esta propuesta exige cambiar las estrategias tradicionales de enseñanza-aprendizaje a metodologías activas e innovadoras. Team based learning es un alternativa innovadora que mezcla aspectos de docencia tradicional con los beneficios del trabajo en grupos pequeños dentro de cursos numerosos. Objetivo: dar a conocer la experiencia del uso de Team based learning como metodología activa de aprendizaje en la asignatura de farmacología para estudiantes de enfermería. Métodos: se utilizó un diseño de estudio experimental en una población de 96 estudiantes de enfermería, los cuales fueron divididos en tres grupos de 32 estudiantes cada uno. Se consideró dos grupos control y un grupo experimental. El análisis del efecto del uso de Team based learning se evaluó de forma cuantitativa y cualitativa. Resultados: los estudiantes que realizaron Team based learning obtuvieron mejores calificaciones al ser comparados con los estudiantes que utilizaron metodología tradicional. Los estudiantes, del grupo experimental, manifestaron su alto grado de satisfacción por el uso de Team based learning, ya que estimuló su aprendizaje y además, favoreció el trabajo en equipo. Conclusiones: Team based learning es una metodología de enseñanza aprendizaje que promueve el autoaprendizaje y el trabajo en equipo, ello se traduce en mejores resultados académicos, es una herramienta bien aceptada por los alumnos y considerada una forma divertida y dinámica de aprender(AU)

Introduction: The change of society from an industry-based to a knowledge-based economy generates a paradigm shift in education. This leads universities to modify their educational models and train professionals able to respond to the needs of the environment. This proposal requires changing traditional teaching-learning strategies to active and innovative methodologies. Team based learning is an innovative alternative that mixes aspects of traditional teaching with the benefits of working in small groups within numerous courses. Objective: To present the experience of the use of Team based learning as an active learning methodology in the subject of pharmacology for nursing students. Methods: An experimental study design was used in a population of 96 nursing students, who were divided into three groups of 32 students each. Two control groups and one experimental group were considered. The analysis of the effect of using Team based learning was quantitatively and qualitatively assessed. Results: The students who performed Team based learning obtained better marks when compared to students who used the traditional methodology. The students of the experimental group expressed their high satisfaction with the use of Team based learning, since it stimulated their learning and also favored teamwork. Conclusions: Team based learning is a teaching-learning methodology that promotes self-learning and teamwork, which is translated into better academic outcomes, is a tool well accepted by students and considered a fun and dynamic way to learn(AU)

Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens.

Recombinant adenoviral vectors have emerged as an attractive system for veterinary vaccines development. However, for poultry vaccination a very important criterion for an ideal vaccine is its low cost. The objective of this study was to test the ability of chicken CD154 to enhance the immunogenicity of an adenoviral vector-based vaccine against avian influenza virus in order to reduce the amount of antigen required to induce an effective immune response in avian. Chickens were vaccinated with three different doses of adenoviral vectors encoding either HA (AdHA), or HA fused to extracellular domain chicken's CD154 (AdHACD). Hemagglutination inhibition (HI) assay and relative quantification of IFN-γ showed that the adenoviral vector encoding for the chimeric antigen is able to elicit an improved humoral and cellular immune response, which demonstrated that CD154 can be used as a molecular adjuvant allowing to reduce in about 50-fold the amount of adenoviral vector vaccine required to induce an effective immune response.