Viviana Falcón

Large-scale production in Pichia pastoris of the recombinant vaccine Gavac™ against cattle tick

A gene coding for the Bm86 tick protein was recently cloned, expressed in Pichia pastoris and shown to induce an inmunological response in cattle against ticks. Moreover, the Gavac vaccine (Heber Biotec S.A., Havana, Cuba), which contains this recombinant protein, has proved to control the Boophilus microplus populations under field conditions. This paper reviews the development and large-scale production of this vaccine, the efficacy of the resulting product and the strategy followed in designing its production plant. The production plant fulfills biosafety requirements and GMP.

Antitumor Effect of a Novel Proapoptotic Peptide that Impairs the Phosphorylation by the Protein Kinase 2 (Casein Kinase 2)

Protein Kinase (casein kinase 2, CK2) is a serine-threonine kinase that is frequently dysregulated in many human tumors. Therefore we hypothesized that peptides capable of binding to the CK2 acidic domain may exhibit potential anticancer properties. By screening a random cyclic peptide phage display library, we have identified a novel peptide, P15, that abrogated CK2 phosphorylation by blocking the substrate in vitro. To verify its potential antineoplastic effect, P15 was fused to the cell-penetrating peptide derived from the HIV-Tat protein. Interestingly, P15-Tat induced apoptosis as evidenced by rapid caspase activation and cellular cytotoxicity in a variety of tumor cell lines. Furthermore, direct injection of P15-Tat into C57BL6 mice bearing day 7-established solid tumors, resulted in substantial regression of the tumor mass. Our findings describe a new proapoptotic cyclic peptide that blocks the CK2 phosphorylation and exhibits antitumor effect in vivo, indicating that the P15 peptide may potentially be used clinically to treat solid tumors or as an adjuvant for cancer therapy.

Regional conformational change involving phosphorylation of tau protein at the Thr231, precedes the structural change detected by Alz-50 antibody in Alzheimer's disease

Neurofibrillary tangles (NFTs) are the neuropathological hallmarks in Alzheimers disease (AD). Densities of NFTs correlate with the dementia status. NFTs reflect the intracellular accumulation of abnormal paired helical filaments (PHFs) composed of the microtubule-associated protein tau. Hyperphosphorylation and truncation have been proposed as key events leading to the genesis of PHFs. A recent hypothesis involving conformational changes has been emerging. These structural modifications of the tau protein were detected by monoclonal antibodies (mAbs) recognizing discontinuous epitopes along the tau molecule such as Alz-50, Tau-66 and MC1. A new mAb, TG-3, detects an early pathology in AD. The epitope of mAb TG-3 maps to phosphorylated Thr231 when the tau molecule is conformationally altered. In the present study, we used confocal microscopy to analyze the state of tau molecule adopting the TG-3 conformation during tangle formation. We also compared mAb TG-3 immunoreactivity with that of mAb Alz-50. Immunoelectronmicroscopy was also performed. N- and C- termini markers evidenced that the tau molecule is intact when it adopts the TG-3 conformation. In addition to NFT, mAb TG-3 also recognized NFT-not bearing-neurons suggesting an early processing of tau prior to NFT formation. Ultrastructural analysis evidenced the presence of TG-3 and Alz-50 immunoreactive products on organelles including mitochondria and endoplasmic reticulum. Nuclear heterochromatin was densely immunolabelled. These results together with the fact that TG-3 immunoreactivity is related to intact tau suggest that the conformation recognized by TG-3 is early staged in the neuronal pathology of AD. In addition, we document that the earliest changes in tau occur closely associated with organelles and heterochromatin.

A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice.

Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4(+) and CD8(+) cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

Ultrastructural evidences of HCV infection in hepatocytes of chronically HCV-infected patients

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.

In vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue-2 virus

The capsid protein is one of the three structural proteins of flaviviruses and is the building block of the nucleocapsid. It has also a predominant role in the replication of dengue virus. To obtain nucleocapsid-like particles from recombinant dengue-2 capsid protein produced in E. coli, a purification process using cation exchange chromatography was established. The purified protein exhibited a molecular mass corresponding to a dimer; therefore, similar to that reported for alphaviruses, an in vitro assembly reaction using single-stranded DNA was performed. In all cases, particles were obtained independently of the specificity and the length of the oligonucleotides used. The present work is the first report of in vitro assembly of the recombinant dengue capsid protein, which could constitute a powerful tool in the development of vaccine candidates.

Antiatherosclerotic Effect of an Antibody That Binds to Extracellular Matrix Glycosaminoglycans

Objective—Subendothelial retention of proatherogenic lipoproteins by proteoglycans is critical in atherosclerosis. The aim of this study was to characterize the recognition and antiatherogenic properties of a chimeric monoclonal antibody (mAb) that reacts with sulfated molecules. Methods and Results—chP3R99 mAb recognized sulfated glycosaminoglycans, mainly chondroitin sulfate (CS), by ELISA. This mAb blocked ≈70% of low-density lipoprotein (LDL)–CS association and ≈80% of LDL oxidation in vitro, and when intravenously injected to Sprague-Dawley rats (n=6, 1 mg/animal), it inhibited LDL (4 mg/kg intraperitoneally, 1 hour later) retention and oxidation in the artery wall. Moreover, subcutaneous immunization of New Zealand White rabbits (n=19) with chP3R99 mAb (100 &mgr;g, 3 doses at weekly intervals) prevented Lipofundin-induced atherosclerosis (2 mL/kg, 8 days) with a 22-fold reduction in the intima-media ratio (P<0.01). Histopathologic and ultrastructural studies showed no intimal alterations or slight thickening, with preserved junctions between endothelial cells and scarce collagen fibers and glycosaminoglycans. In addition, immunization with chP3R99 mAb suppressed macrophage infiltration in aorta and preserved redox status. The atheroprotective effect was associated with the induction of anti-CS antibodies in chP3R99-immunized rabbits, capable of blocking CS-LDL binding and LDL oxidation. Conclusion—These results support the use of anti-sulfated glycosaminoglycan antibody–based immunotherapy as a potential tool to prevent atherosclerosis.

Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin‐2 N‐terminal peptide linked to the N‐terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2‐PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N‐fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C‐peptide and the N‐terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100–200 μg/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N‐terminal fused peptide and the C‐peptide and avoids the use of toxic cyanogen bromide.

Levansucrase from Acetobacter diazotrophicus SRT4 Is Secreted via Periplasm by a Signal-Peptide-Dependent Pathway

Abstract.Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization. Immunogold electron microscopical studies revealed that LsdA accumulates in the periplasm before secretion. The periplasmic and extracellular forms of the enzyme were purified to homogeneity. Both proteins exhibited similar physical and biochemical characteristics indicating that LsdA adopts its final conformation in the periplasm. The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg as determined by PSD-MALDI-TOFMS (post-source decay—matrix-assisted laser desorption/ionization—time-of-flight mass spectrometry). Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide followed by the formation of the pyroglutamic acid (pGlu) residue. Thus, in contrast with other Gram-negative bacteria, A. diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism.

Identification of a novel antitumor peptide based on the screening of an Ala-library derived from the LALF32–51 region

Novel therapeutic peptides are increasingly making their way into clinical application. The cationic and amphipathic properties of certain peptides allow them to cross biological membranes in a non‐disruptive way without apparent toxicity increasing drug bioavailability. By modifying the primary structure of the Limulus‐derived LALF32–51 peptide we designed a novel peptide, L‐2, with antineoplastic effect and cell‐penetrating capacity. Interestingly, L‐2 induced cellular cytotoxicity in a variety of tumor cell lines and systemic injection into immunocompetent and nude mice bearing established solid tumor, resulted in substantial regression of the tumor mass and apoptosis. To isolate the gene transcripts specifically regulated by L‐2 in tumor cells, we conducted suppressive subtractive hybridization (SSH) analysis and identified a set of genes involved in biological processes relevant to cancer biology. Our findings describe a novel peptide that modifies the gene expression of the tumor cells and exhibits antitumor effect in vivo, indicating that peptide L‐2 is a potential candidate for anticancer therapy. Copyright