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This work investigates the formulation and in vivoefficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.

ABSTRACTFive monoclonal antibodies (MAbs) against recombinant nucleocapsid protein (NP) of severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) were developed by hybridoma technology. Epitope mapping by Western blotting showed that these anti-SARS-CoV NP MAbs bind to distinct domains of NP. These anti-SARS-CoV NP MAbs, with their high specificity, are potentially ideal candidates for developing early and sensitive diagnostic assays for SARS-CoV.

We have developed an efficient versatile in vivodendritic cell (DC) targeting vector for delivering different classes of antigens such as proteins, peptide, glycolipids and naked DNA for vaccine applications. A single chain antibody (scFv) that recognizes DEC-205 receptor of DC was fused with a core-streptavidin domain and expressed in Escherichia coliusing the T7 expression system. The bifunctional fusion protein (bfFp) was expressed as a periplasmic soluble protein and affinity-purified in its monomeric form. The bifunctional activity against DEC-205 and biotin was characterized by ELISA and Western blot. In vivoDC targeting of a diverse group of biotinylated antigens such as viral and bacterial proteins, a cancer peptide, gangliosides and DNA of certain infectious diseases was conducted in mice. Results show that in the presence of bfFp and costimulatory anti-CD40 mAb, both humoral and cell-mediated responses were augmented in either the single antigen or multiple antigen targeting strategy. Lastly, bfFp based DC targeting of antigens in low doses may be a useful strategy for the design of monovalent or polyvalent vaccines for the masses.

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody (MAb), but also possessed streptavidin-binding activity. This experimental approach can eliminate the need for chemical biotinylation of antibodies and the risk associated of antibody denaturation and can provide a stable and reproducible reagent for rapid and efficient immunoassay of VEE when detected by horseradish peroxidase (HRP)-conjugated streptavidin.

A repertoire of mouse monoclonal antibodies (MAbs) against western equine encephalitis virus (WEE) was constructed and characterized. Anti-WEE antibodies were expressed from hybridomas and purified by protein G chromatography. Each of the antibodies was functionally assessed by indirect enzyme-linked immunosorbent assays (ELISAs), Western blotting, and immunoprecipitations. All antibodies bound to WEE antigen in ELISAs, whereas only a subgroup of antibodies was found to be active in Western blotting and immunoprecipitations. A subset of antibodies was found to cross-react with other alphaviruses, such as Sindbis virus (SIN), Venezuelan equine encephalitis (VEE), and eastern equine encephalitis (EEE). Because many of the antibodies were highly reactive to WEE antigen in one or more of the assays, these antibodies are excellent candidates for immunodetection and immunotherapy studies.

A group of methyl 2-methyl-2-[2-(4-benzoyl-5-phenyl-7-halo-2-azabicyclo[4.1.0]hept-3-ene)]acetates (10–15), and the related acetamide derivative (16), that possess a variety of C-7 substituents (Br, Cl, F, H), were designed for evaluation as analgesic-antiinflammatory agents. The effect of the C-7 substituent(s) and the nature of the acetic acid ester (R¹ = Ome) or acetamide (R¹ = NH2) moiety on analgesic activity was determined using a 4% NaCl-induced abdominal constriction assay. Compounds 10–16 inhibited writhing by 36–82%, relative to the reference drugs aspirin (58% inhibition) and celecoxib (62% inhibition). The nature of the C-7 substituents was a determinant of analgesic activity in the 7,7-dihalo group of compounds where the relative activity profile was 7-Cl2 > 7-Br2 > 7-F2 > 7-Cl,7-F, and for 7-monohalo compounds where the potency order was 7-Br > 7-Cl. Elaboration of the 7,7-dibromo methyl acetate ester (10) to the corresponding acetamide derivative (16) enhanced analgesic activity. The nature of the 7-halo substituent(s) in the 7,7-dihalo group of compounds was a determinant of antiinflammatory activity, determined using the carrageenan-induced rat paw edema assay, where the relative potency order was 7-Br2 > 7-Cl2 > 7-F2 > 7-Cl,7-F. The most potent 7,7-dibromo compound (10) inhibited inflammation by 62%, relative to the reference drug ibuprofen (44%), and 10 inhibited COX-2 (IC50 = 26.4 μM) and COX-1 (IC50 = 227 μM) for a COX-2 selectivity index of 8.6. Docking 10 in the active site of human COX-2 showed it binds in the center of the COX-2 binding site with the C-5 phenyl ring oriented toward the acetylation site (Ser⁵³⁰), and the phenyl group of the C-4 benzoyl moiety oriented in the vicinity of the COX-2 secondary binding pocket near Val⁵²³. Drug Dev. Res. 49:75–84, 2000. © 2000 Wiley-Liss, Inc.

Bispecific antibodies with specificity for tumor antigen and CD3 have been shown to redirect the cytotoxicity of T cells against relevant tumor. Our objective was to generate single-chain bispecific antibodies (bsSCA) that could retarget mouse cytotoxic T lymphocytes (CTL) to destroy human ovarian carcinoma in a xenogeneic setting. A bsSCA, 2C11 X B43.13, was constructed by genetic engineering and expressed in mammalian cells. Molecular characteristics, binding properties, and ability to retarget CTL were studied. Western blot analysis showed that the product is a 65-kDa protein. Purification of antibodies could be done by single-step affinity chromatography using protein L-agarose with an unoptimized yield of 200 mu g/L. BsSCA 2C11 X B43.13 was capable of binding to mouse CD3 and human CA125 as detected by FACS analysis of EL4 and OVCAR Nu3H2 cells, respectively. It could also bridge activated splenic T cells and human ovarian carcinoma as demonstrated by a bridge FACS assay. Redirected mouse CTL could mediate human target cell lysis in a 20-h51Cr release assay despite that they are xenogeneic. Prolonged incubation of redirected CTL and tumor targets resulted in a dramatic reduction in tumor cell number. CD28 co-stimulation enhanced redirected CTL function in both types of assays. BsSCA 2C11 X B43.13 thus can be used as a preclinical immunotherapeutic model for human ovarian cancer in a xenogeneic setting.

A group of methyl 2‐methyl‐2‐[2‐(4‐benzoyl‐5‐phenyl‐7‐halo‐2‐azabicyclo[4.1.0]hept‐3‐ene)]acetates (10–15), and the related acetamide derivative (16), that possess a variety of C‐7 substituents (Br, Cl, F, H), were designed for evaluation as analgesic‐antiinflammatory agents. The effect of the C‐7 substituent(s) and the nature of the acetic acid ester (R1= Ome) or acetamide (R1= NH2) moiety on analgesic activity was determined using a 4% NaCl‐induced abdominal constriction assay. Compounds 10–16inhibited writhing by 36–82%, relative to the reference drugs aspirin (58% inhibition) and celecoxib (62% inhibition). The nature of the C‐7 substituents was a determinant of analgesic activity in the 7,7‐dihalo group of compounds where the relative activity profile was 7‐Cl2> 7‐Br2> 7‐F2> 7‐Cl,7‐F, and for 7‐monohalo compounds where the potency order was 7‐Br > 7‐Cl. Elaboration of the 7,7‐dibromo methyl acetate ester (10) to the corresponding acetamide derivative (16) enhanced analgesic activity. The nature of the 7‐halo substituent(s) in the 7,7‐dihalo group of compounds was a determinant of antiinflammatory activity, determined using the carrageenan‐induced rat paw edema assay, where the relative potency order was 7‐Br2> 7‐Cl2> 7‐F2> 7‐Cl,7‐F. The most potent 7,7‐dibromo compound (10) inhibited inflammation by 62%, relative to the reference drug ibuprofen (44%), and 10inhibited COX‐2 (IC50= 26.4 μM) and COX‐1 (IC50= 227 μM) for a COX‐2 selectivity index of 8.6. Docking 10in the active site of human COX‐2 showed it binds in the center of the COX‐2 binding site with the C‐5 phenyl ring oriented toward the acetylation site (Ser530), and the phenyl group of the C‐4 benzoyl moiety oriented in the vicinity of the COX‐2 secondary binding pocket near Val523. Drug Dev. Res. 49:75–84, 2000. © 2000 Wiley‐Liss, Inc.

Peanut lectin (PNA) binds avidly to oligosaccharides containing the terminal sequence ß-d-Gal(1?3)a-d-GA1NAc. This disaccharide is the immunodeterminant group of the Thomsen-Friedenreich (T) antigen which is present in an exposed form on a number of human and animal adenocarcinomas and can be identified in tumor tissue sections by histochemical PNA staining techniques. We have studied the in vivo uptake of radioiodinated PNA in a murine TA3/Ha mammary adenocarcinoma model to evaluate the potential applications of radiolabeled PNA for the immunodetection of T antigen expressing carcinoma. We have found that PNA has a high in vitro binding affinity for the TA3/Ha tumor cells as well as for epiglycanin, a glycoprotein fraction shed by the TA3/Ha cells. Tissue biodistribution studies after IV 125I-PNA injection into TA3/Ha tumor-bearing mice indicated tumor: blood ratios of 7:1 and 55:1 at 24 and 48 h with corresponding tumor:muscle ratios of 33:1 and 77:1. The high tumor uptake and rapid blood clearance allowed clear scintigraphic delineation of tumor by 24 and 48 h without the necessity for background subtraction techniques. Rapid in vivo deiodination of 125I-PNA also contributed to localization of radioactivity in the stomach, salivary glands, thyroid, and kidney.

The Thomsen, Friedenreich (TF) and Tn carbohydrate antigens are expressed on the vast majority of human adenocarcinomas and are associated with aggressive behavior of certain tumors. TF and Tn antigens are also expressed on certain murine cancer cell lines including TA3-Ha, a highly lethal, transplantable mammary adenocarcinoma. TF and Tn cancer-associated carbohydrate haptens were synthesized, conjugated to protein carriers and used to demonstrate that delayed-type hypersensitivity (DTH) effector T cells can specifically recognize and respond to carbohydrate determinants on the TA3-Ha tumor-associated glycoprotein, epiglycanin. The effector cells were shown to have the helper DTH phenotype (Lytl+, Lyt2-, Thyl+) and it was demonstrated that they respond to specific carbohydrate determinants in an MHC-restricted fashion. These experiments provide the rationale for the use of synthetic tumor-associated glycoconjugates (S-TAGs) to stimulate anticancer T cell immunity. In support of this hypothesis, it was shown that preimmunization with the appropriate S-TAGs could provide a degree of protection against a subsequent tumor transplant and that antitumor effector Lytl+, Lyt2- T cells could be generated in vitro using the appropriate S-TAGs as antigens.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.