P. Plaisant

Human monoclonal recombinant Fabs specific for HCV antigens obtained by repertoire cloning in phage display combinatorial vectors

Molecular cloning of the antibody repertoire in phage display combinatorial vectors is a powerful method enabling the dissection of the immunoresponse against a given pathogen. In this paper we describe the construction of a combinatorial library displayed on phage surface, containing the antibody repertoire of a patient with high serological response against hepatitis C virus (HCV) antigens. Following selection of the library against solid-phase-bound antigen, sixteen human antibody Fab fragments able to bind to HCV-specific antigens were generated and studied for binding characteristics. The majority of them appeared to have specificity for the HCV c33 peptide. All the clones reacting with the c33 peptide shared the same heavy-chain CDR3 sequence. This is the first report of molecular cloning in a combinatorial phage display vector of the antibody repertoire of an anti-HCV-positive patient.

A vector for the expression of recombinant monoclonal Fab fragments in bacteria

The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents. In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries. The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.

SEARCH FOR BACTERIOPHAGES SPONTANEOUSLY OCCURRING IN CULTURES OF HAEMOLYTIC INTESTINAL SPIROCHAETES OF HUMAN AND ANIMAL ORIGIN

An electron microscopic survey of the occurrence of bacteriophages which appear spontaneously in cultures of haemolytic intestinal spirochaetes of human and animal origin was made. Excluding one isometric tailed phage particle which was observed in the form of free particle in proximity to a spirochaete of the wβHIS strain HRM18, bacteriophages were never observed while examining cells of 21 weakly beta‐haemolytic human intestinal spirochaetes (wβHIS), swine Serpulina pilosicoli strain P43/6/78, and the avian strain 1380, although 50–100 cells of each spirochaetal strain were analysed. Isometric tailed bacteriophages were found associated with only three out of the 100 cells of strongly beta‐haemolytic swine Serpulina hyodysenteriae strain P18A comparatively analysed. According to our results and previous published reports, the occurrence of bacteriophages which appear spontaneously in cultures of intestinal spirochaetes is a rare event.

Electron microscopy studies of human intestinal spirochetes

The ultrastructure of twenty human intestinal spirochetes was analyzed using the electron microscope. Negatively stained cells were generally found to be loosely and irregularly waved. The isolates had cell dimensions ranging from 0.12–0.35 μm in width and from 3.9–14.2 μm in length. Twin bundles of flagella were present in the space between the cytoplasmic membrane and the outer membrane. The majority of isolates had five flagella inserted sub-terminally at each cell end. Human intestinal spirochetes divide by binary fission. They are morphologically similar to swine intestinal treponemes, both pathogenic (Treponema hyodysenteriae) and non pathogenic (Treponema innocens), and different from Treponema pallidum, Treponema phagedenis and Borrelia burgdorferi.Following treatment with sodium deoxycolate, no bundles of cytoplasmic microtubules were observed in cells obtained from cultures of human and swine intestinal spirochetes or from cells of B. burgdorferi, while these structures were present in similarly treated cells of T. pallidum and T. phagedenis.

Probing the natural antibody repertoire by combinatorial cloning of IgM and IgD isotypes in phage display vectors

It is well known that immunoglobulins with no identifiable immunogenic origin, called natural antibodies, are present in the sera of healthy individuals and their role as a defence against important pathogens has been proposed. Unfortunately, the studies are hampered by the fact that these immunoglobulins seem to have low affinity and to be polyreactive, and are commonly available in polyclonal preparations. Lately, new technologies for the production of monoclonal antibodies became available, and in particular the cloning of genes coding for antibody fragments in combinatorial phage display vectors provided a handy tool for the selection of human monoclonal antibodies. In this work, we describe the successful development of a technology for the molecular cloning of combinatorial phage display libraries containing genes coding exclusively for antibody fragment of the IgM or IgD phenotype. These libraries can be useful for molecular cloning of monoclonal antibodies of the IgM and IgD phenotype and can help elucidate the role played by natural antibodies in defence against infectious agents.