Surface display of heterologous proteins on bacterial cells is an important objective for many applications in microbiology and molecular biology. In gram-negative bacteria, different types of surface proteins have been exploited for this purpose. These include the following: (i) outer membrane proteins LamB (4), PhoE (1), and OmpA (35); (ii) lipoproteins TraT (19) and the peptidoglycan-associated lipoprotein (10); (iii) the fimbria protein fimbrillin (20, 21); and (iv) the flagellar protein flagellin (32). The different surface display systems have been used extensively to express heterologous antigenic determinants on the bacterial cells for the purpose of developing live bacterial vaccine vehicles. Enteric bacteria, such as Escherichia coli and Salmonella typhimurium, have been studied in this context, and the cell surface presentation has been considered advantageous to induce an antibody response to the exposed antigens with live cells for immunization (11,28,41).The expression of functional single-chain antibodies on the surface of E. coli cells (7, 10) has opened the discussion of whether this strategy would be an alternative to the rapidly developing phage technology for the selection of peptides or recombinant antibody fragments from large libraries (31). An interesting application for bacterial cells exposing heterologous proteins might be the development of whole-cell adsorbents by the surface expression of suitable protein ligands. Immobilized recombinant bacteria cannot be considered for separation processes in pharmaceutical industries but might because of their low cost constitute competitive adsorbents for certain separations (3, 11). The use of enzyme-coated bacteria as novel biocatalysts has also been envisioned, because enzymes with retained activity have been surface displayed on E. coli cells (8, 9).Investigations with gram-positive bacteria for cell surface display of heterologous proteins have recently been initiated. Expression systems for the mouth commensal bacterium Streptococcus gordinii (37) and the nonpathogenic bacterium Staphylococcus xylosus (18) have been developed on the basis of the fibrillar M6 protein from Streptococcus pyogenes and protein A from Staphylococcus aureus, respectively. These cell surface display systems have been used for the surface expression of several antigenic determinants, and immunization with live recombinant bacteria induces both local and systemic antibody responses to the hybrid receptors (33, 37), suggesting that gram-positive bacteria might constitute potential live bacterial vaccine delivery systems. The surface receptors of gram-positive bacteria seem to be more permissive for the insertion of extended sequences of foreign proteins (6) than the different gram-negative systems, in which both translocation through the cytoplasmic membrane and correct integration into the outer membrane are required for proper surface exposure of the heterologous polypeptide. Considering the development of whole-cell adsorbents or bacterial biocatalysts by surface ...

SUMMARYDendritic cells (DCs) are bone marrow-derived antigen-presenting cells that have an exquisite capacity to interact with T cells and modulate their responses. Little is known about porcine DCs despite the fact that they represent an important target in strategies that are aimed at modulating resistance to infection in pigs and may be of major importance in transplantation biology. We generated immature monocyte-derived porcine dendritic cells (MoDCs) directly from adherent peripheral blood cells treated with porcine granulocyte±macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells were observed via electron microscopy and their phenotype was characterized using monoclonal antibodies. The functionality of the porcine MoDCs was demonstrated showing that the cells were capable of different specialized functions relevant to antigen capture and were potent stimulators in a primary allo-mixed leucocyte reaction. Treatment of the MoDCs with porcine cell line-derived necrotic factors resulted in the phenotypic and functional maturation of MoDCs. We con®rmed also that monocyte-derived DCs were differentially regulated by cytokines, showing that transforming growth factor-b1 (TGF-b1) is able to redirect monocytic precursors into the differentiation pathway of Langerhans' cells presenting typical Birbeck granules. Interestingly, and in contrast to the human and murine model, we showed that the monocyte-derived porcine Langerhans'-type cells (MoLCs) were much more potent activators of allogeneic T cells than MoDCs obtained without TGF-b1.

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.

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Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37 061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties. OmpA [20, 21] have been demonstrated to mediate expression of gram-negative bacteria, present at about 10 5 copies/cell. It is believed to occur in a monomeric form in its native state. A of peptides on the surface of live enterobacterial vectors. OmpC, the outer-membrane protein complex of Neisseria meningitidis, typical feature of OmpA is that it can be modified by heat: the mobility of Escherichia coli OmpA on SDS/PAGE decreases has been shown to be effective in humans as a conjugate vaccine with Haemophilus influenzae, pneumococcal and meningococcal when it is heated in the presence of SDS [1]. OmpA is highly conserved among gram-negative bacteria and is thought to con-capsular polysaccharides [22Ϫ25]. Other clinically useful carrier proteins have been derived from bacteria: diphteria and tetanus sist of two domains. The N terminus, including amino acid residues 1Ϫ170, forms a membrane-spanning domain and crosses toxoids are both successfully used in conjugated H. influenzae vaccines to transform the capsular polysaccharide, which is a Tthe outer membrane eight times in antiparallel β-strands, leading to a typical amphiphilic β-barrel [2Ϫ4]. The C-terminal moiety independent antigen, into a T-dependent antigen [26]. KeywordsIn this paper we describe the expression and production in of the protein is thought to be periplasmic [5]. The protein seems to be multifunctional. In addition to non-physiological functions, E. coli and purification of Klebsiella pneumoniae OmpA [27].Using various analytical criteria, the purity and structural integsuch as serving as a receptor for phages and colicins [6], it serves as a mediator in F-factor-dependent conjugation [7]. It is rity of the protein were evaluated. We demonstrate the presence of at least one T-cell epitope on the molecule and its potential also required for the structural integrity of the outer membrane and the generation of normal cell shape [8]. The capacity to use as a carrier protein for conjugated peptides. The immunological carrier properties of the recombin...