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Gene Therapy Nov 2015Achieving persistent expression is a prerequisite for effective genetic therapies for inherited disorders. These proof-of-concept studies focused on adeno-associated...
Achieving persistent expression is a prerequisite for effective genetic therapies for inherited disorders. These proof-of-concept studies focused on adeno-associated virus (AAV) administration to newborn monkeys. Serotype rh10 AAV expressing ovalbumin and green fluorescent protein (GFP) was administered intravenously at birth and compared with vehicle controls. At 4 months postnatal age, a second injection was administered intramuscularly, followed by vaccination at 1 year of age with ovalbumin and GFP. Ovalbumin was highest 2 weeks post administration in the treated monkey, which declined but remained detectable thereafter; controls demonstrated no expression. Long-term AAV genome copies were present in myocytes. At 4 weeks, neutralizing antibodies to rh10 were present in the experimental animal only. With AAV9 administration at 4 months, controls showed transient ovalbumin expression that disappeared with the development of strong anti-ovalbumin and anti-GFP antibodies. In contrast, increased and maintained ovalbumin expression was noted in the monkey administered AAV at birth, without antibody development. After vaccination, the experimental monkey maintained levels of ovalbumin without antibodies, whereas controls demonstrated high levels of antibodies. These preliminary studies suggest that newborn AAV administration expressing secreted and intracellular xenogenic proteins may result in persistent expression in muscle, and subsequent vector administration can result in augmented expression without humoral immune responses.
Topics: Animals; Animals, Newborn; Antibodies, Heterophile; Antibodies, Neutralizing; Dependovirus; Female; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Immune Tolerance; Immunity, Humoral; Macaca mulatta; Ovalbumin; Pilot Projects
PubMed: 26333349
DOI: 10.1038/gt.2015.65 -
Journal of the American Society For... Jun 2000Analysis of commercial samples of chicken ovalbumin by reversed-phase high performance liquid chromatography and matrix-assisted laser desorption/ionization mass...
Analysis of commercial samples of chicken ovalbumin by reversed-phase high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) showed the presence of several other co-purifying glycoproteins. These were isolated, subjected to tryptic digestion, and two of them were identified as ovomucoid and chicken riboflavin binding-protein following database matching of the peptide masses obtained by MALDI. The N-linked glycans were released from the glycoproteins and their structures were examined by MALDI-MS in combination with exoglycosidase digestion. Ovalbumin was found to be glycosylated mainly with high-mannose and hybrid structures, consistent with profiles obtained on the intact glycoprotein by electrospray. The other glycoproteins contained mainly larger, complex glycans with up to five antennae, many of which had earlier been associated with ovalbumin.
Topics: Carbohydrates; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Glycoside Hydrolases; Hydrazines; Hydrolysis; Mass Spectrometry; Ovalbumin; Peptides; Polysaccharides; Trypsin
PubMed: 10833030
DOI: 10.1016/S1044-0305(00)00122-7 -
PloS One 2015IL-25, IL-33 and TSLP are produced predominantly by epithelial cells and are known to induce Th2-type cytokines. Th2-type cytokines are involved not only in host defense...
BACKGROUND
IL-25, IL-33 and TSLP are produced predominantly by epithelial cells and are known to induce Th2-type cytokines. Th2-type cytokines are involved not only in host defense against nematodes, but also in the development of Th2-type allergic diseases. TSLP was reported to be crucial for development of allergic airway inflammation in mice after inhalation of allergens to which they had been sensitized epicutaneously (EC) beforehand. However, the roles of IL-25 and IL-33 in the setting remain unclear.
METHODS
Mice deficient in IL-25 and IL-33 were sensitized EC with ovalbumin (OVA) and then challenged intranasally with OVA. Airway inflammation, the number of inflammatory cells in bronchoalveolar lavage fluids (BALFs) and airway hyperresponsiveness (AHR) in the mice were determined, respectively, by histological analysis, with a hemocytometer, and by using plethysmograph chambers with a ventilator. Expression of mRNA in the skin and lungs was determined by quantitative PCR, while the BALF levels of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) and the serum levels of IgE were determined by ELISA.
RESULTS
Normal OVA-specific Th2- and Th17-cell responses of lymph nodes and spleens were observed in IL-25-deficient (IL-25-/-) and IL-33-/- mice after EC sensitization with OVA. Nevertheless, the number of eosinophils, but not neutrophils, in the BALFs, and the levels of Th2 cytokines, but not Th17 cytokines, in the lungs were significantly decreased in the IL-25-/- and IL-33-/- mice pre-sensitized EC with OVA, followed by inhalation of OVA, whereas their levels of AHR and OVA-specific serum IgE were normal.
CONCLUSIONS
Both IL-25 and IL-33 are critical for induction of Th2-type cytokine-mediated allergic airway eosinophilia, but not Th17-type cytokine-mediated airway neutrophilia, at the local sites of lungs in the challenge phase of mice sensitized EC with OVA. They do not affect OVA-specific T-cell induction in the sensitization phase.
Topics: Animals; Eosinophils; Interleukin-17; Interleukin-33; Mice; Mice, Inbred BALB C; Ovalbumin; Respiratory Hypersensitivity
PubMed: 26230091
DOI: 10.1371/journal.pone.0134226 -
The Journal of Biological Chemistry May 1972
Topics: Animals; Antigen-Antibody Reactions; Carbon Isotopes; Cell Fractionation; Centrifugation, Density Gradient; Chemical Precipitation; Chickens; Electrophoresis; Estrogens; Female; Iodine Isotopes; Ovalbumin; Oviducts; Protein Binding; RNA, Messenger; Rabbits; Reticulocytes; Ribonucleases; Ribosomes; gamma-Globulins
PubMed: 4112809
DOI: No ID Found -
Respiratory Physiology & Neurobiology Dec 2011BALB/c mice received saline (SAL groups) or ovalbumin (OVA groups) intraperitoneally (days 1, 3, 5, 7, 9, 11 and 13). After 27 days, a burst of intratracheal OVA or SAL...
BALB/c mice received saline (SAL groups) or ovalbumin (OVA groups) intraperitoneally (days 1, 3, 5, 7, 9, 11 and 13). After 27 days, a burst of intratracheal OVA or SAL (days 40, 43 and 46) was performed. Animals were then divided into four groups (N=8, each) and intranasally instilled with saline (SAL-SAL and OVA-SAL) or residual oil fly ash (SAL-ROFA and OVA-ROFA). 24h later, total, initial and difference resistances (Rtot, Rinit, Rdiff) and static elastance (Est) were measured. Lung responsiveness to methacholine was assessed as slope and sensitivity of Est, Rtot, Rinit, and Rdiff. Lung morphometry (collapsed and normal areas and bronchoconstriction index) and cellularity (polymorphonuclear, mononuclear and mast cells) were determined. OVA or ROFA similarly impaired lung mechanics and increased the amount of polymorphonuclear cells and collapsed areas. OVA-ROFA showed even higher hyperresponsiveness, bronchoconstriction and mast cell infiltration. Thus, we concluded that ROFA exposure may add an extra burden to hyperresponsive lungs.
Topics: Air Pollutants; Air Pollution; Animals; Bronchial Hyperreactivity; Coal Ash; Hypersensitivity; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Respiratory Function Tests
PubMed: 21816235
DOI: 10.1016/j.resp.2011.07.011 -
Colloids and Surfaces. B, Biointerfaces Feb 2023This study aimed to develop surface modified PLGA nanocarriers protecting a protein-based antigen in the stomach to enable potential release of the antigen at target...
This study aimed to develop surface modified PLGA nanocarriers protecting a protein-based antigen in the stomach to enable potential release of the antigen at target intestinal sites. PLGA nanoparticles (NPs) were prepared by double emulsion and solvent evaporation techniques while surface functionalization was performed using polyethylene glycol (PEG), sodium alginate (ALG) and Eudragit L100 (EUD) with ovalbumin (OVA) as a model protein antigen. Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), and stability in simulated gastric fluid (SGF)/simulated intestinal fluid (SIF). Structural integrity of released OVA was analyzed by circular dichroism (CD) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while cytotoxicity against Jurkat cells was determined using MTT assay. Surface functionalized PLGA NPs protected the protein in SGF and SIF better than the non-functionalized NPs. Average size of OVA encapsulated NPs was between 235 and 326 nm and were spherical. FTIR band change was observed after surface modification and the surface modified NPs showed sustained OVA release compared with the uncoated NPs. The secondary structure of OVA released after 96 h remained intact and MTT assay showed >80 % cell viability after 72 h while unmodified and surface modified NPs achieved 17 % and 48 % mucin binding respectively. In conclusion, surface modified PLGA NPs have been shown to be safe for potential oral protein-based vaccine delivery.
Topics: Humans; Drug Delivery Systems; Antigens; Alginates; Ovalbumin; Vaccines; Nanoparticles; Drug Carriers; Particle Size
PubMed: 36599187
DOI: 10.1016/j.colsurfb.2022.113121 -
Nucleic Acids Research Jul 1982The "ovalbumin Y" gene, one of three which constitute the ovalbumin gene family in chicken has been completely sequenced. The exact location of exons can be derived from...
The "ovalbumin Y" gene, one of three which constitute the ovalbumin gene family in chicken has been completely sequenced. The exact location of exons can be derived from the comparison with the ovalbumin gene sequence and from the map previously established by electron microscopy analysis. During evolution of the Y gene, selective pressure has operated to retain a sequence coding for an ovalbumin-like protein. The location of splice junctions, the length of protein coding exons and the reading phase are as in the ovalbumin gene. The overall homology between the Y and ovalbumin protein coding sequences is 72.6% (resulting in a 58% homology for the amino acid sequences). A significantly high number of base changes within coding sequences are present in clusters, which appear in several cases to be correlated with the occurrence of direct repeats. The 3' untranslated sequences of the Y and ovalbumin mRNAs have diverged much more, and the Y sequence contains a peculiar U(T) rich region. Corresponding introns of the ovalbumin and Y genes differ extensively both in sequence and in length. They share however characteristic biases in their base distribution.
Topics: Animals; Base Sequence; Biological Evolution; Chickens; DNA, Single-Stranded; Genes; Microscopy, Electron; Ovalbumin
PubMed: 7122240
DOI: 10.1093/nar/10.14.4363 -
Journal of Immunology Research 2018Therapeutic vaccines that arouse the cytotoxic T cell immune response to reject infected cells have been investigated extensively for treating disease. Due to the large...
Therapeutic vaccines that arouse the cytotoxic T cell immune response to reject infected cells have been investigated extensively for treating disease. Due to the large amounts of resident antigen-presenting cells (APCs) and T cells in lymph nodes, great efforts have been made to explore the strategy of targeting lymph nodes directly with nanovaccines to activate T cells. However, these nanovaccines still have several problems, such as a low loading efficiency and compromised activity of antigens and adjuvants derived from traditional complicated preparation. There are also safety concerns about materials synthesized without FDA approval. Herein, we construct an assembled nanoparticle composed of an antigen (ovalbumin, OVA) and adjuvant (CpG) to ensure its safety and high loading efficiency. The activity of both components was well preserved due to the mild self-assembly process. The small size, narrow distribution, negative charge, and good stability of the nanoparticle endow these nanovaccines with superior capacity for lymph node targeting. Correspondingly, the accumulation at lymph nodes can be improved by 10-fold. Subsequently, due to the sufficient APC internalization and maturation in lymph nodes, ~60% of T cells are stimulated to proliferate and over 70% of target cells are specifically killed. Based on the effective and quick cellular immune response, the assembled nanoparticles exhibit great potential as therapeutic vaccines.
Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Female; Lymph Nodes; Lymphocyte Activation; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocytes, Cytotoxic; Vaccines
PubMed: 30018987
DOI: 10.1155/2018/3714960 -
Journal of Biochemistry May 1976Hen's oviduct polysomes are present in the precipitates prepared from the oviduct homogenates by low-speed centrifugation, in constrast other eukaryotic polysomes. The...
Hen's oviduct polysomes are present in the precipitates prepared from the oviduct homogenates by low-speed centrifugation, in constrast other eukaryotic polysomes. The polysomes possessed synthesizing activity for ovalbumin. The amount of the released form of ovalbumin (soluble ovalbumin) synthesized in cell-free system A or B, consisting of the cell sap and the total ribosomal fraction or the polysomes, respectively, was about a half of that bound to the polysomes (nascent ovalbumin). The amount of soluble ovalbumin synthesized in cell-free system C, consisting of the pH 5 fraction and the polysomes, was only about 5% of that of nascnet ovalbumin. These results indicate that factors required to release nascent ovalbumin from polysomes are present in the pH 5 supernatant fraction. The soluble and nascent ovalbumins, which were purified by chromatography on a CM-cellulose column and by the use of antiovalbumin antiserum, respectively, seemed to be elongation products the initiations of which were supposed to occur in the oviducts before preparation of the cell-free system. The initiated chains in vitro were found to exist as nascent peptides bound to polysomes. Thus, the cell-free systems prepared in the present study lacked the ability to complete initiated peptide chains. The soluble ovalbumin synthesized in the cell-free systems was indentical with ovalbumin A1 containing two residues of phosphates, which was crystallized from hen's egg-white and was different soluble ovalbumin devoid of the prosthetic group (ovalbumin A3) prepared in the oviduct minces. This result suggests that an enzyme necessary for incorporation of the phosphate is present in the cell sap.
Topics: Adenosine Triphosphate; Animals; Cell-Free System; Chickens; Chromatography, Affinity; Dithiothreitol; Female; Kinetics; Leucine; Ovalbumin; Oviducts; Polyribosomes; Proline; Protein Biosynthesis
PubMed: 956139
DOI: 10.1093/oxfordjournals.jbchem.a131154 -
Biochemistry May 2021Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations....
Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive center loop that lies adjacent to the OVA 323-339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins, to study how changes in loop size and protein stability influence the processing and presentation of the OVA 323-339 epitope. We observed that the OVA R339T loop insertion increases the stability and protease resistance, resulting in the reduced presentation of the OVA 323-339 epitope . These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools.
Topics: Animals; Binding Sites; Chickens; Epitopes; Kinetics; Ovalbumin; Peptide Fragments; Serpins; Thermodynamics
PubMed: 33956428
DOI: 10.1021/acs.biochem.1c00095