[show abstract][hide abstract] ABSTRACT: Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.
Archives of Virology 01/2014; · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints and results in their progressive destruction. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4+ T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis, anergy and bystander suppression mediated by regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4+ T-cell epitope of human heat-shock protein of 60kDa, an autoantigen involved in various aspects of the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4+ CD25(high)Foxp3+ Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive RA patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4+ CD25+ T cells but not resting CD4+ CD25- T cells were identified as targets of APL-1, which suggests its high specificity as a possible therapeutic drug. Furthermore, CD4+ T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4+ T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4+ CD25(high)Foxp3+ Tregs and apoptosis in activated CD4+ T cells. These results support further investigation of this candidate drug for the treatment of RA.
International immunopharmacology 10/2013; · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract
Recombinant virus-like particles (VLPs) are attractive candidates for vaccine design since
they resemble native viroids in size and morphology, but they are non-infectious due to the
absence of a viral genome. The visualization of surface morphologies and structures can be
used to deepen the understanding of physical, chemical, and biological phenomena. Atomic
force microscopy (AFM) is a useful tool for the visualization of soft biological samples in a
nanoscale resolution. In this work we have investigated the morphology of recombinant
surface antigens of hepatitis B (rHBsAg) VLPs from Cuban vaccine against hepatitis B. The
rHBsAg VLPs sizes estimated by AFM between 15 and 30 nm are similar to those reported on
previous transmission electron microscopy (TEM) studies.
Keywords: hepatitis B, AFM, rHBsAg, virus-like particles
ADVANCES IN NATURAL SCIENCES: NANOSCIENCE AND NANOTECHNOLOGY. 04/2013; 4(2).
[show abstract][hide abstract] ABSTRACT: The existing vaccines are mainly limited to the microorganisms we are able to culture and produce and/or to those whose killing is mediated by humoral response (antibody mediated). It has been more difficult to develop vaccines capable of inducing a functional cellular response needed to prevent or cure chronic diseases. New strategies should be taken into account in the improvement of cell-based immune responses in order to prevent and control the infections and eventually clear the virus. Preclinical and clinical results with vaccine candidates developed as a vaccine platform based on virus-like particles (VLPs) evidenced their ability to stimulate mucosal as well as systemic immunity. Particles based on envelope, membrane or nucleocapsid microbial proteins induce a strong immune response after nasal or parenteral administration in mice, non-human primates and humans. In addition, the immune response obtained was modulated in a Th1 sense. The VLPs were also able to immunoenhance the humoral and cellular immune responses against several viral pathogens. Studies in animals and humans with nasal and systemic formulations evidenced that it is possible to induce functional immune response against HBV, HCV, HIV and dengue virus.
Keywords: VLPs, hepatitis, HBV, HCV, human immunodeficiency virus, Th1
Classification numbers: 2.05, 5.08
ADVANCES IN NATURAL SCIENCES: NANOSCIENCE AND NANOTECHNOLOGY. 01/2013; 4:1-4.
[show abstract][hide abstract] ABSTRACT: A fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus polyphemus protein linked to human papillomavirus (HPV)-16 E7 antigen (LALF32-51-E7) was expressed in E. coli BL21 (DE3) cells. The recombinant protein in E. coli accounted for approximately 18% of the total cellular protein and purified with a single affinity chromatographic step. Yields of approximately 38 mg purified LALF32-51-E7 per liter of induced culture was obtained with an overall 52% recovery and constitutes a promising setting for the future production and scaling-up. Purified protein was characterized as soluble aggregates with molecular weight larger than 670 kDa, which is considered an important property to increase the immunogenicity of an antigen preparation. The recombinant fusion protein LALF32-51-E7 will be a promising vaccine candidate for the treatment of HPV-16 related malignancies.
[show abstract][hide abstract] ABSTRACT: Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.
[show abstract][hide abstract] ABSTRACT: 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.
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 μg, 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.
These results support the use of anti-sulfated glycosaminoglycan antibody-based immunotherapy as a potential tool to prevent atherosclerosis.
[show abstract][hide abstract] ABSTRACT: In this study, we evaluate in mice a novel formulation containing nucleocapsid-like particles of dengue-2 virus (recNLP) co-immunized with a chimeric protein composed of the dengue-4 envelope domain III fused twice within the meningococcal P64k protein of Neisseria meningitidis (PD24). The animals receiving the PD24-recNLP mixture showed the highest levels of antiviral antibodies. Similar results were obtained for IFNγ secretion levels, indicating a functional Th1 cellular response. Consistently, the percentage of mice surviving after viral challenge was significantly higher for those immunized with the mixture than for those inoculated with PD24 protein alone. In addition, in vivo depletion experiments demonstrated the decisive role of CD4(+) and CD8(+) cells in the protection conferred by immunization with PD24-recNLP. In conclusion, this report demonstrates for the first time the adjuvant capacity of dengue-2 virus recNLP. Additionally, the evidence presented highlights the potential of these particles for enhancing the immune response against heterologous recombinant proteins.
Archives of Virology 10/2010; 155(10):1587-95. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a disease of the central nervous system characterized by loss of spinal motor neurons, for which no effective treatment exists. Epidermal growth factor (EGF) and growth hormone releasing peptide-6 (GHRP-6) have been considered as good candidates for the treatment of this disease, due to their well documented effects in eliciting pleiotrophic and cell survival mechanisms. The aim of the present work was to evaluate the separate and combined effects of both peptides in an experimental animal model of ALS, the proximal axonopathy induced by 1,2 diacetylbenzene (1,2 DAB) in mice. The evaluations were conducted by means of behavioral tests (trapeze, tail suspension, gait pattern, and open field) and by recording the complex muscle action potential (CMAP) in three different hind limb segments: proximal S1, medial S2, and distal S3. Intraperitoneal daily administration of 1,2 DAB produced significant reduction in body weight, muscle strength, extensor reflex, spontaneous activity, and changes in gait pattern parameters. In parallel 1,2 DAB produced significant prolongation of onset latency and decrease in amplitude of CMAP and in the integrated complex action potential index. Daily administration of the separate compounds did not accelerate the recovery of the affected parameters, except for the gait pattern. The combined treatment produced significant improvement in behavioral parameters, as well as in electrophysiological recovery, particularly in the proximal segment of CMAP. The latter results confirm the proximal character of 1,2 DAB neuropathy, and suggest that combined therapy with EGF and GHRP-6 might be a good therapeutic strategy for the treatment of ALS.
Neurotoxicity Research 02/2010; 19(1):195-209. · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 LALF(32-51) 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.
Journal of Peptide Science 11/2009; 16(1):40-7. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Archives of Virology 02/2009; 154(4):695-8. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-gamma producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.
Biological research 02/2009; 42(1):41-56. · 1.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein Kinase CK2 is a serine-threonine kinase frequently deregulated in many human tumors. Here, we hypothesized that a peptide binder to the CK2 phosphoacceptor site could exhibit anticancer properties in vitro, in tumor animal models, and in cancer patients. By screening a random cyclic peptide phage display library, we identified the CIGB-300 (formerly P15-Tat), a cyclic peptide which abrogates the CK2 phosphorylation by blocking recombinant substrates in vitro. Interestingly, synthetic CIGB-300 led to a dose-dependent antiproliferative effect in a variety of tumor cell lines and induced apoptosis as evidenced by rapid caspase activation. Importantly, CIGB-300 elicited significant antitumor effect both by local and systemic administration in murine syngenic tumors and human tumors xenografted in nude mice. Finally, we performed a First-in-Man trial with CIGB 300 in patients with cervical malignancies. The peptide was found to be safe and well tolerated in the dose range studied. Likewise, signs of clinical benefit were clearly identified after the CIGB-300 treatment as evidenced by significant decrease of the tumor lesion area and histological examination. Our results provide an early proof-of-principle of clinical benefit by using an anti-CK2 approach in cancer. Furthermore, this is the first clinical trial where an investigational drug has been used to target the CK2 phosphorylation domain.
Molecular and Cellular Biochemistry 07/2008; 316(1-2):163-7. · 2.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: 52 Nuevas evidencias acerca de la cápsida del virus como candidato vacunal contra el virus Dengue 2 sin la inducción de anticuerpos neutralizantes Introducción El dengue es la enfermedad viral transmitida por ar-trópodos más ampliamente difundida que afecta a la población humana. El agente causal de esta enferme-dad es el virus dengue (VD), perteneciente al género Flavivirus, familia Flaviviridae, que se transmite por la picada del mosquito Aedes aegypti . Muchas estrategias están actualmente en investi-gación para desarrollar una vacuna efectiva y segura contra el dengue, aunque hasta la fecha ninguna por sí sola ha demostrado ser completamente efectiva. Los candidatos vacunales más avanzados en el mundo están constituidos por cepas atenuadas del virus, las cuales tienen como limitantes la posibilidad de reversión a la virulencia y la reactogenicidad detectada en humanos . Teniendo en cuenta estas desventajas, la variante de una formulación basada en proteínas recombinantes constituye una estrategia atractiva para el desarrollo de un candidato vacunal. La proteína de la envoltura constituye la diana principal de la respuesta inmune hospedera, capaz de inducir altos títulos de anticuerpos neutralizantes. Sin embargo, los candidatos vacunales basados en esta proteína conllevan el riesgo potencial de inducir el fenómeno de amplificación dependien-te de anticuerpos (ADA) si no se logra una respuesta neutralizante efectiva contra los cuatro serotipos . Existen trabajos recientes que apuntan a una correla-ción entre la generación de una respuesta celular ci-totóxica y la protección frente al reto viral en diferentes modelos animales . Tomando en consideración lo anteriormente expuesto, se propuso desarrollar la pro-teína de la cápsida como candidato vacunal contra el citado virus, la cual no es capaz de inducir anticuerpos antivirales y por tanto, se elimina de este modo el riesgo de inducir ADA. Resultados El presente trabajo constituye el primer estudio so-bre el clonaje y la expresión en Escherichia coli de la proteína recombinante de la cápsida del VD2. Como resultado de la expresión, la proteína se obtuvo con un peso molecular de 15 kDa, y constituyó el 15% del to-tal de las proteínas celulares. Posteriormente la pro-teína fue semipurificada hasta el 60% de pureza mediante cromatografía de intercambio iónico y carac-terizada mediante cromatografía de filtración en gel en la que se detectaron agregados de alto peso molecular. Con la preparación semipurificada y agregada se rea-lizó un esquema de inmunización en ratones Balb/c para evaluar la capacidad inmunogénica y protectora de la proteína recombinante. Como resultado de este primer esquema se obtuvo el 44% de protección en ratones tras la inoculación intracraneal con VD2 y sin detección de anticuerpos neutralizantes . Similares porcentajes de protección en ratones se han correla-cionado con una respuesta protectora total en el mo-delo de monos. A partir del resultado obtenido se desarrolló un proceso de purificación, el cual permitió obtener la proteína con más del 90% de pureza. A causa de la na-turaleza dimérica de la proteína pura, se estandarizó un proceso de obtención de partículas in vitro. Se mostró mediante microscopía electrónica que la ob-tención de partículas de la proteína dependía de la neutralización de las cargas positivas de los dímeros y se obtuvo partículas semejantes a virus de 25 nm de diámetro (Figura 1). El diámetro de las partículas obtenidas in vitro concuerda con el diámetro de la nu-cleocápsida viral nativa. Con la nueva preparación pura y las partículas se realizó una nueva evaluación inmunológica en ratones,
[show abstract][hide abstract] ABSTRACT: Currently, there are no effective tools to fight multiple pathogens due to the insufficient knowledge on their components and the limited arsenal of vaccine alternatives. In the present paper, combinations of recombinant viral antigens and nucleic acids are studied using electron microscopy, electrophoresis, nuclease digestion and sucrose or cesium chloride gradients. We describe new data on morphology, size, and the increased stability of protein particles composed of viral antigens in close association with nucleic acids. We demonstrate, for the first time, that particles of recombinant HCV viral core proteins are able to interact with plasmid molecules for DNA immunization, forming complexes of particles with increased density and size. Additionally, the increased immunogenicity of these recombinant viral protein-plasmid DNA mixtures for DNA immunization was evidenced when administered in animal models, compared to the individual components. In addition, we discuss the possible mechanisms behind the enhancement of the immune response, based on the experimental evidence of the interaction between mixture components and their properties. The use of recombinant viral proteins interacting with nucleic acids as the active principle, and simultaneously as an adjuvant or molecular vehicle for DNA vaccines, has important implications for the development of new effective strategies to prevent and treat diseases caused by different pathogens.
[show abstract][hide abstract] ABSTRACT: Recently, it has been shown that a truncated HCV core (HCcAg) variant, covering the first 120 aa (HCcAg.120), interacts with plasmid DNA vaccine (pIDKE2), encoding the HCV structural proteins (HCcAg, E1 and E2). In the present work, HCcAg.120-pIDKE2 complexes, forming heterogeneous packaged structures, were visualized using a negative stain/rotary shadow technique. Interestingly, 72 hours after intramuscular injection of HCcAg.120-pIDKE2 complexes in Balb/c mice, E2 protein was immunolabeled in muscle cells. In fact, HCcAg.120-pIDKE2 complexes induced anti-HCV humoral and cellular immune responses in mice when inoculated by both, parenteral or mucosal routes, although intranasal administration generally rendered weaker results. On the other hand, data demonstrated that Alum enhanced the HCV-specific IgG antibody production. However, the analysis of the HCV-specific cellular immune response showed that HCcAg.120-pIDKE2 delivered in PBS by the intramuscular route induced the strongest HCV-specific lymphoproliferative response, especially against E1 and induced viremia control in a vaccinia virus surrogate challenge model. These results support the use of HCcAg.120-pIDKE2 complexes in the rational design of therapeutic or preventive vaccine strategies against HCV infection.
American journal of immunology 01/2006; 2(3):71-76.
[show abstract][hide abstract] ABSTRACT: The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect of two protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesis is that freezing affects cell membranes in a way that interferes with the fusion process upon cloning but without hampering normal cell development in vitro. We found that freezing cell lines without controlling the cooling rate gives lower yields in the fusion step and in the final development into blastocysts, compared with cells frozen with a controlled cooling rate of approximately 1 degrees C/min. Transmission electron microscopy of the cells subjected to different freezing procedures showed major damage to the cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning is a critical step in the procedure and should be monitored carefully using a method that allows for a step-wise, controlled cooling rate.
[show abstract][hide abstract] ABSTRACT: Neurofibrillary tangles (NFTs) are the neuropathological hallmarks in Alzheimer's 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.