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Oscar R Burrone

Publications and source records attributed to Oscar R Burrone.

17 recordsLinked to original sources
PubMed2007

Recombinant dimeric small immunoproteins neutralize transmissible gastroenteritis virus infectivity efficiently in vitro and confer passive immunity in vivo.

Small immunoproteins (SIPs) are single-chain molecules comprising the variable regions of an antibody assembled in a single polypeptide (scFv) and joined to the immunoglobulin heavy-chain dimerizing domain. To investigate the potential of these molecules to provide protection against enteric infections when supplied orally, SIPs were generated against Transmissible gastroenteritis virus (TGEV), a highly pathogenic porcine virus. Different variants of TGEV-specific SIPs were created, of epsilon and alpha isotypes, by exploiting the dimerizing domains epsilonCH4 and alphaCH3 of human and swine origin. Transfected cells secreted these recombinant mini-antibodies efficiently, mainly as dimers stabilized covalently by inter-chain disulphide bridges. The specificity and functionality of the recombinant TGEV-specific SIPs were determined by in vitro binding, neutralization and infection-interference assays. The neutralization indices of the TGEV-specific SIPs were all very similar to that of the original TGEV-specific mAb, thus confirming that the immunological properties have been preserved in the recombinant SIPs. In vivo protection experiments on newborn piglets have, in addition, demonstrated a strong reduction of virus titre in infected tissues of animals treated orally with TGEV-specific SIPs. It has therefore been demonstrated that it is possible to confer passive immunization to newborn pigs by feeding them with recombinant SIPs.

Animals↗
PubMed2006

The extracellular membrane-proximal domain of human membrane IgE controls apoptotic signaling of the B cell receptor in the mature B cell line A20.

Ag engagement of BCR in mature B cells can deliver specific signals, which decide cell survival or cell death. Circulating membrane IgE+ (mIgE+) cells are found in extremely low numbers. We hypothesized that engagement of an epsilonBCR in a mature isotype-switched B cell could induce apoptosis. We studied the role of the extracellular membrane-proximal domain (EMPD) of human mIgE upon BCR engagement with anti-Id Abs. Using mutants lacking the EMPD, we show that this domain is involved in controlling Ca2+ mobilization in immunoreceptors of both gamma and epsilon isotypes, as well as apoptosis in signaling originated only from the epsilonBCR. We mapped to the epsilonCH4 ectodomain the region responsible for apoptosis in EMPD-deleted receptors. Ca2+ mobilization was not related to apoptotic signaling. This apoptotic pathway was caspase independent, involved ERK1/2 phosphorylation and was partially rescued by CD40 costimulation. We therefore conclude that the EMPD of human mIgE is a key control element of apoptotic signaling delivered through engagement of epsilonBCR within the context of a mature B cell.

Animals↗
PubMed2006

Use of virus vectors for the expression in plants of active full-length and single chain anti-coronavirus antibodies.

To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full-length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The alphaSIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the alpha-CH3 domain from human IgA. To express the full-length IgA, the individual light and heavy chains from the TGEV-specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co-infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant-expressed alphaSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody-expressing plant tissue to 2-day-old piglets showed that both the alphaSIP and full-length IgA molecules can provide in vivo protection against TGEV.

Animals↗
PubMed2005

Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner.

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.

Animals↗
PubMed2005

Design and selection of an intrabody library produced de-novo for the non-structural protein NSP5 of rotavirus.

Intracellular antibodies or intrabodies have great potential in protein knockout strategies for intracellular antigens. We applied the Intracellular Antibody Capture Technology for the direct selection in yeast of a mouse scFv library (V(L)-V(H) format) constructed from animals immunised with recombinant non-structural protein NSP5 of Rotavirus. We selected five different intracellular antibodies (ICAbs), which specifically recognize Delta2, an NSP5 deletion mutant used as bait. The anti-NSP5 ICAbs were well expressed both in yeast and mammalian cells as cytoplasmic or nuclear-tagged forms. By immunofluorescence and co-immunoprecipitation assays we characterised the intracellular interaction of the five anti-NSP5 ICAbs with the co-expressed antigens.

Amino Acid Sequence↗
PubMed2005

RNA interference of rotavirus segment 11 mRNA reveals the essential role of NSP5 in the virus replicative cycle.

Rotavirus genomes contain 11 double-stranded (ds) RNA segments. Genome segment 11 encodes the non-structural protein NSP5 and, in some strains, also NSP6. NSP5 is produced soon after viral infection and localizes in cytoplasmic viroplasms, where virus replication takes place. RNA interference by small interfering (si) RNAs targeted to genome segment 11 mRNA of two different strains blocked production of NSP5 in a strain-specific manner, with a strong effect on the overall replicative cycle: inhibition of viroplasm formation, decreased production of other structural and non-structural proteins, synthesis of viral genomic dsRNA and production of infectious particles. These effects were shown not to be due to inhibition of NSP6. The results obtained strengthen the importance of secondary transcription/translation in rotavirus replication and demonstrate that NSP5 is essential for the assembly of viroplasms and virus replication.

Genes, Essential↗
PubMed2004

Uncoupling substrate and activation functions of rotavirus NSP5: phosphorylation of Ser-67 by casein kinase 1 is essential for hyperphosphorylation.

Rotavirus NSP5 is a nonstructural protein that localizes in viroplasms of virus-infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced PAGE mobility. Here we show that NSP5 operates as an autoregulator of its own phosphorylation as a consequence of two distinct activities of the protein: substrate and activator. We developed an in vivo hyperphosphorylation assay in which two NSP5 mutant constructs are cotransfected. One of them, fused to an 11-aa tag, served as substrate whereas the other was used to map NSP5 domains required for activation. The activation and substrate activity could be uncoupled, demonstrating a hyperphosphorylation process in trans between the activator and substratum. This process involved dimerization of the two components through the 18-aa C-terminal tail. Phosphorylation of Ser-67 within the SDSAS motif (amino acids 63-67) was required to trigger hyperphosphorylation by promoting the activation function. We present evidence of casein kinase 1alpha being the protein kinase responsible for this key step as well as for the consecutive ones leading to NSP5 hyperphosphorylation.

Amino Acid Motifs↗
PubMed2004

BCL1 lymphoma protection induced by idiotype DNA vaccination is entirely dependent on anti-idiotypic antibodies.

DNA vaccination with the idiotype (Id) of tumour B-cell membrane immunoglobulins (Ig) is a validated strategy to induce tumour protection to several mouse lymphomas. The relative contribution of anti-Id antibodies and T lymphocytes to tumour rejection is still debated. Previous studies in the BCL1 lymphoma model showed that scFv DNA immunisation induces a polyclonal antibody response restricted to conformational epitopes formed by the parental V(L)/V(H) association. We implemented a system based on this specificity to investigate the mechanism of BCL1 lymphoma protection induced by DNA immunisation. Antibody response and survival of mice immunised with the tumour Id scFv were compared with those of mice immunised simultaneously with two chimeric scFvs, containing either the tumour-derived V(L) or V(H) paired to an irrelevant V(H) or V(L) domain, respectively. Animals vaccinated with one or both chimeric constructs were not protected, despite the exposure to all putative tumour Id-derived MHC class I and class II T-cell epitopes. In addition, conformational antibodies induced by DNA vaccination caused tumour cells apoptosis and cell cycle arrest in vitro and transferred protection in vivo. Therefore, lymphoma rejection appears to be completely dependent on the induction of anti-Id antibodies.

Animals↗
PubMed2004

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation.

Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an in vivo binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131-198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.

Animals↗
PubMed2004

Effects of intrabodies specific for rotavirus NSP5 during the virus replicative cycle.

Intracellular antibodies or intrabodies (ICAbs) have great potential in protein knockout strategies for intracellular antigens. In this study, they have been used to investigate the role of the rotavirus non-structural protein NSP5 in the virus replication cycle. Intracellular antibody-capture technology was used to select single-chain Fv format (scFv) ICAbs against an NSP5 mutant. Five different specific ICAbs were selected and expressed in MA104 cells, in the scFv format, as cytoplasmic- and nuclear-tagged forms. By confocal microscopy, it was found that three of these ICAbs recognized the full-length wild-type NSP5 specifically, forming antigen-specific aggresomes in the cytoplasm of cotransfected cells. Expression of the ICAbs in rotavirus-infected cells largely reduced the assembly of viroplasms and cellular cytopathic effect. Replication of dsRNA was partially inhibited, despite there being no reduction in virus titre. These results demonstrate for the first time a key role for NSP5 during the virus replicative cycle.

Antibodies, Viral↗
PubMed2004

Specific recognition of a dsDNA sequence motif by an immunoglobulin VH homodimer.

Anti-DNA antibodies have the potential to be applied in vast fields of fundamental as well as medical research. They are found in autoimmune diseases, such as systemic lupus erythemotosus. In most cases, anti-dsDNA antibodies do not present sequence specificity and are of low affinity. The dominant role of VH domains in DNA recognition induced us to search for binders based on VH dimers (VHD), previously reported to bind different protein antigens. We screened a phage displayed homo-VHD library against a 19-bp dsDNA sequence. A sequence-specific binder was selected, which recognizes the terminal located CTGC motif with a Kd of 250 nM. Association of the two identical VH domains of the molecule was shown to be essential for binding.

Amino Acid Sequence↗
PubMed2003

Selection and characterisation of binders based on homodimerisation of immunoglobulin V(H) domains.

The antigen-binding surface of antibodies is formed by the heterodimerisation of the two variable domains of the light (V(L)) and heavy (V(H)) chains. We have previously described the spontaneous formation of V(H) dimers (VHD) in both bacteria and mammalian cells. The self-association of a single domain produces a homo-VHD, in which the two identical V(H) domains generate a unique symmetric surface for antigen binding that is never found in the normal V(L)/V(H) antibody binding site. We developed a phagemid vector for the construction of phage display libraries in which a cysteine residue, introduced at the C-terminus of the only V(H) cloned, allowed display of homo-VHDs. Panning of the library on different proteins yielded antigen specific binders against lysozyme, glutathione S-transferase and streptavidin. A lysozyme specific homo-VHD was further characterised with an apparent affinity determined to be 216+/-6.6 nM. Importantly, the results showed that its binding activity was fully dependent on the dimerisation of both identical V(H) domains.

Amino Acid Sequence↗
PubMed2003

Binders based on dimerised immunoglobulin VH domains.

Antibody binding to antigen is mediated by the surface formed by the association of the two variable (V) regions of the L (VL) and H (VH) chains. The capacity of VL to dimerise and the high structural similarity of VL and VH domains suggested the possibility that VH could also associate. We show here that spontaneous formation of VH dimers (VHD) is in many cases permissive, producing stable molecules with antigen binding specificity. VHD were displayed on filamentous phages for the selection of antigen-specific binders. VHD were expressed and secreted efficiently from both bacteria and mammalian cells in different formats, including single-chain (VH(1)-linker-VH(2)), double chain ((VH(2)) and IgG analogues having the VL replaced by VH. The affinity (Kd,app) achieved with a VH dimer expressed in the IgG format, specific for a glutenin subunit was around 30 nM measured by two different methods, which was about 20 times higher than that corresponding to the VL/VH counterpart.

Amino Acid Sequence↗
PubMed2003

Evidence of CXC, CC and C chemokine production by lymphatic endothelial cells.

Although production of chemokines by vascular endothelial cells has been documented, there is only limited information regarding the expression of chemokines by the lymphatic endothelium. Here we used lymphatic endothelial cells (LEC) derived from experimentally induced murine lymphangiomas to investigate the pattern of chemokine expression by these cells. Histological analysis of the lymphatic hyperplasia revealed the presence of leucocytes in the tissues surrounding the lesions, suggesting the presence of chemoattractant activity. A functional chemotactic assay on human polymorphonuclear cells and on purified subpopulations of murine leucocytes using culture supernatants from LEC primary cultures confirmed the presence of chemoattractant activity. The identity of different cytokines of the CXC, CC and C subfamilies was investigated by reverse trancriptase-polymerase chain reaction on total endothelial cell RNA. Amplified fragments corresponding to KC, IP10, Mig-1, BCL, MIP-2, SLC, RANTES, MCP-1, C10, and Lptn were obtained, and confirmed by Southern blot and sequencing. In contrast, MIP-1alpha, MIP-1beta, and MIP-1gamma were not detected. Higher levels of expression were revealed by Northern blot analysis for Mig-1, MCP-1 and C10. The lymphatic endothelium-restricted production of these chemokines was also confirmed by in situ hybridization. The presence of high C10 mRNA expression levels in LEC was particularly unexpected, because the production of this molecule has been previously identified only in cells of the haematopoietic lineage. These observations represent the first detailed analysis of chemokine production by lymphatic endothelial cells and may account, in part, for the mechanism of leucocyte recruitment into the lymphatics, and of lymphocyte recirculation within the lymphatic system.

Animals↗
PubMed2002

Anti-idiotypic DNA vaccines for B-cell lymphoma therapy.

The idiotypic determinants expressed by immunoglobulin at the surface of malignant B-cells provide specific targets for vaccination strategies. However, as self-antigens they are poorly immunogenic and vaccines must include carriers to improve immune responses. Chemical cross-linking of purified idiotypic protein is so far the only method which has been employed in clinical trials while a number of second-generation vaccines have been developed in mouse models. These strategies are based on the use of recombinant DNA technology to create fusion proteins that contain the idiotype genetically linked to molecules that act as immunological adjuvants. Fusion proteins can be made available to the immune system by direct delivery of naked DNA with great advantages in terms of time and costs. The most relevant approaches are listed and discussed with particular emphasis to the mechanism by which different molecules exert their adjuvant effect. The role of cellular versus antibody mediated suppression of tumor growth following Id vaccination is examined by comparing the most recent data on the mechanism of tumor protection. In addition, an analysis of the specificity of anti-idiotypic antibodies induced by scFv-DNA vaccination as compared to protein immunization is provided.

Animals↗
PubMed2002

Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains.

Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione S-transferase-NSP5 mutants were produced in Escherichia coli and used to determine phosphoacceptor sites. These were mapped to four serines (Ser(153), Ser(155), Ser(163), and Ser(165)) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localization in the context of virus infection.

Animals↗