Biomedical subjects

C Andréoni

Publications and source records attributed to C Andréoni.

10 recordsLinked to original sources

PubMed1999

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus.

This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky's disease virus) DNA vaccine. Aujeszky's disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-gamma. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-gamma). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-gamma had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky's disease.

Adjuvants, Immunologic↗

PubMed1997

Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding.

The cholera toxin B (CTB) subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotentiating properties, where coupling has been obtained either by genetic fusion or chemical conjugation. For genetically fused immunogens both N- and C-terminal fusions have been used. Only shorter extensions have previously been evaluated and in some reports these fusions have impaired the biological functions of CTB, such as the ability to form pentamers and to adhere to its cell receptor, the GM1 ganglioside. Here we report the first systematic study where the same fusion partner has been used for either C-terminal, N-terminal or dual fusions to CTB. The serum albumin binding region (BB, approximately 25 kDa) from streptococcal protein G, which is known to fold independently of N- or C-terminal fusions, was selected as fusion partner. The three fusion proteins CTB-BB, BB-CTB and BB-CTB-BB were expressed in Escherichia coli, where they were efficiently secreted to the periplasmic space, and could be purified by affinity chromatography on human serum albumin (HSA) columns. The CTB fusion proteins were compared for their ability to form pentamers, by gel electrophoresis and size-exclusion chromatography, and it was concluded that all three fusion proteins were able to pentamerize. Interestingly, the C-terminal fusion to CTB showed most efficient pentamerization, while the dual fusion was much less efficient. Purified pentamer fractions from all three fusions where found to react to a monoclonal antibody described to react only to pentameric forms of CTB. In addition, the purified pentamer fractions were analyzed in an enzyme-linked immunosorbent assay (ELISA) for their ability to bind GM1, and it was found that the C-terminal fusion (CTB-BB) showed significant GM1-binding, but that also the N-terminal and dual CTB fusion proteins bound GM1, although less efficiently. The implications of the results for the design and use of CTB fusion proteins as subunit vaccines are discussed.

Animals↗

PubMed1997

Challenge of BALB/c mice with respiratory syncytial virus does not enhance the Th2 pathway induced after immunization with a recombinant G fusion protein, BBG2NA, in aluminum hydroxide.

The polypeptide of aa 130-230 of the G protein (G2Na) of respiratory syncytial virus (RSV) was fused to BB, the albumin-binding region of streptococcal G protein, producing BBG2Na, which induced protective immune responses in rodent models. Evaluation of the immune response in mice immunized with BBG2Na in the adjuvant alhydrogel revealed high amounts of interleukin (IL)-5 and some IL-4 in splenocytes restimulated in vitro. This is compatible with a Th2 response. The activation of the Th2 pathway in such mice was further supported by the detection of IL-5 and G2Na-specific IgE in vivo. Of interest, in contrast to immunization with formalin-inactivated RSV, immunization of mice with BBG2Na followed by intranasal RSV challenge did not lead to increased production of IL-5- or G2Na-specific IgE. However, IgG1- and IgG2a-specific antibodies were boosted. These results demonstrate that the Th2 pathway is not enhanced by RSV challenge in BBG2Na-immunized mice.

Adjuvants, Immunologic↗

PubMed1997

Flow cytometric quantification of surface-displayed recombinant receptors on staphylococci.

Surface display of recombinant proteins on bacteria and phages has become an important tool in bioscience. To evaluate the various host systems, a great need exists for quantitative methods to determine the densities of displayed proteins and peptides on the bacteria and phage surfaces. Here we describe how a method previously applied for quantification of surface proteins on mammalian cells has been adapted for quantification of chimeric receptors surface-displayed on bacteria; in this study, the bacteria being recombinant staphylococci. The presented method takes advantage of fluorescence-activated cell sorting (FACS) technology and a new type of nonfluorescent plastic beads, similar in size (2 microns in diameter) to bacterial cells, and thus suitable for generation of calibration curves from which the number of chimeric receptors can be obtained. The method was used to estimate the number of antigenic sites on two types of recombinant staphylococci, both carrying heterologous chimeric receptors, and it was found that the recombinant Staphylococcus carnosus cells carried approximately 10(4) surface-displayed antigenic sites, while recombinant Staphylococcus xylosus exposed approximately 3 x 10(3) sites per cell. The use of the deviced method for different applications is discussed.

Animals↗

PubMed1996

Surface display on staphylococci: a comparative study.

Two different host-vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence-activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.

Flow Cytometry↗

PubMed1995

Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus.

Protein engineering has been employed to investigate the effect of specific amino acid changes on the targeting of heterologous proteins to the outer cell surface of the Gram-positive bacterium Staphylococcus xylosus. Three different variants, corresponding to a 101 amino acid region of the major glycoprotein (G protein) of human respiratory syncytial virus (RSV), were generated in which multiple hydrophobic phenylalanine residues were either substituted or deleted. The different G protein fragments were expressed as one part of recombinant receptors designed for surface display on S. xylosus cells. The engineered variants of the RSV G protein hybrid receptors were, in contrast to a non-engineered fragment, efficiently targeted to the outer cell surface of recombinant S. xylosus cells as determined by different methods, including fluorescence-activated cell sorting. In addition, immunization of mice with live recombinant S. xylosus demonstrated that surface exposure was required to generate receptor-specific antibodies. The present strategy of hydrophobic engineering should be of general interest in surface-display applications and for secretion of proteins otherwise difficult to translocate through host cell membranes.

Amino Acid Sequence↗

PubMed1995

Cell surface display of recombinant proteins on Staphylococcus carnosus.

A novel expression system for surface display of heterologous proteins on Staphylococcus carnosus cells has been developed. Taking advantage of the promoter and secretion signals, including a propeptide region, from the lipase gene of Staphylococcus hyicus and the cell wall-spanning and membrane-binding region of protein A from Staphylococcus aureus, efficient surface display of an 80-amino-acid peptide from a malaria blood stage antigen could be achieved. A serum albumin binding protein from streptococcal protein G was used both as a general reporter molecule and to increase the accessibility of the surface-displayed proteins. Immunoblotting, immunogold staining, and immunofluorescence on intact recombinant S. carnosus cells verified the presence of the propeptide, the malaria antigen, and the albumin-binding reporter protein on the bacterial surface. For the first time, fluorescence-activated cell sorting was used to analyze the presence of surface-displayed hybrid receptors on gram-positive bacteria.

Antigens, Protozoan↗

PubMed1991

Regulation and functional involvement of distinct determinants of leucocyte function-associated antigen 1 (LFA-1) in T-cell activation in vitro.

The expression of leucocyte function-associated antigen 1 (LFA-1) was studied by immunofluorescence method on human peripheral blood mononuclear cells (PBMC) stimulated by the phytohaemagglutinin lectin (PHA). Monoclonal antibodies (MoAb) Mas 191c, IOT18 (directed against the beta-chain, 95 kDa, CD18) and IOT16, SPVL7, MHM24 (identificating the alpha-chain, 180 kDa, CD11a) were used, defining the 'CD11a/CD18' antibody panel. By means of cross-linking or competitive experiments, we showed that these antibodies recognized at least four distinct and spatially distant domains on the LFA-1 molecule. Immunofluorescence analysis revealed that the up-regulation of LFA-1 expression was a late event, similar to the expression kinetics of the HLA DR and CD38 molecules, and followed the appearance of CD25 and CD71 molecules. Moreover, it was established that the LFA-1 up-regulation required mRNA and protein synthesis. Functional activity comparison of the different anti LFA-1 MoAb showed that the CD11a MoAb significantly inhibited the proliferation of lymphocytes stimulated by the phytohaemagglutinin to various extents, as the LFA-1 alpha determinant identified. By contrast, the CD18 MoAb did not influence strongly this cell process. We observed only a dim inhibitory effect with the CD18 MoAb recognizing an epitope common or very close to an LFA-1 alpha determinant. These results suggested that the LFA-1 antigen was important, at a molecular level, in the regulation of T-cell activation.

Antigens, Differentiation↗

PubMed1990

[Spontaneous fluctuation of extracellular matrix molecules on the surface of human skin fibroblasts in culture: flow cytometric analysis].

The evolution of five surface markers (CD 10, fibronectin, collagens I, III, IV) was analysed in cytofluorometry after immunolabelling of cultured human skin fibroblasts. The expression of the CD10 molecule and collagen IV remained stable. On the contrary, there was a spontaneous variation of the cell surface distribution of fibronectin and collagens I and III. This variation was periodical and followed the subculture rhythm. After every passage, the density of matrix components increased at the cell surface. The maximum of the density was achieved on confluent fibroblasts then decreased until the next passage. The fibronectin and collagens I and III expression was permanently altered on senescent fibroblasts. Modifications of the expression of extracellular matrix proteins on the fibroblasts surface were closely linked with cellular density and senescence.

Cells, Cultured↗

PubMedDate not supplied

Differential expression of the LFA-1 molecule on the human peripheral blood mononuclear cell subpopulations.

The expression of leukocyte function associated antigen 1 (LFA-1) was studied on human peripheral blood mononuclear cells (PBMNC) by a double immunofluorescence method. Monoclonal antibodies IOT18 (recognizing the beta chain, 95 kDa, CD18) and SPVL7 (recognizing the alpha chain, 180 kDa, CD11a) were used. These antibodies revealed the same heterogeneous distribution of the LFA-1 molecule among peripheral blood mononuclear cells. A bimodal profile characterized the lymphocytes, whereas the monocyte subpopulation presented a unimodal profile. NK cells, including CD16, CD8 dim, expressed the LFA-1 with a high density. B cells expressing the CD19 phenotype showed surface LFA-1 at a low level, whereas it appeared with an intermediate intensity on the T helpers/inducers (CD4 cells). Among the cytotoxic cells (CD8) and the immunoregulatory subpopulations associated with an immunosuppressive response (CD8+ CD11b+, CD8+ CD11b- and CD8+ leu8+) or with a contrasuppressive response (Vicia villosa cells), an heterogeneous LFA-1 expression was observed, discriminating the LFA-1 dim and the LFA-1 bright cells. Anti LFA-1 antibodies also allowed us to define three novel subsets among the CD8 cells: the CD8 dim LFA-1 bright, the CD8 bright LFA-1 bright, and the CD8 bright LFA-1 dim cell subpopulations. This heterogeneous distribution of LFA-1 among PBMNC may be associated with different functions of the different cell subsets analyzed in this study.

Antibodies, Monoclonal↗