title: A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.
abstract: The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.
authors: Laboratório de Doenças Infecciosas, CIISA, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
authors: Afonso P
authors: AP
authors: Basto
authors: João
authors: J
authors: Piedade
authors: Ruben
authors: R
authors: Ramalho
authors: Susana
authors: S
authors: Alves
authors: Helena
authors: H
authors: Soares
authors: Pierre
authors: P
authors: Cornelis
authors: Carlos
authors: C
authors: Martins
authors: Alexandre
authors: A
authors: Leitão
mesh_terms: D000358:African Swine Fever Virus / Q000235:genetics; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D003713:Dendritic Cells / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000956:Antigens, Viral; D001426:Bacterial Proteins; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C060507:outer membrane lipoprotein I, Bacteria