Calreticulin, a potential cell surface receptor involved in cell penetration of anti-DNA antibodies (Nabila Seddiki-Si Ahmed)
A 50-kda protein was purified as a potential receptor, using an affinity matrix containing biotinylated F14-6 or H9-3 anti-DNA mAbs derived from autoimmune (New Zealand Black x New Zealand White)F1 mouse and membrane extracts from cells. This protein was identified as calreticulin (CRT) by microsequencing. Confocal microscopy and FACS analysis showed that CRT was present on the surface of various cells. CRT protein was recognized by a panel of anti-DNA mAbs in ELISA. The binding of F14-6 to lymphocytes and Chinese hamster ovary cells was inhibited by soluble CRT. Thus, the anti-DNA mAbs used in this study bound to CRT, suggesting that CRT may mediate their penetration into the cells and play an important role in lupus pathogenesis.
Study of catalytic antibodies associated with systemic lupus erythematosus
(Barbara Mouratou, Jean-Luc Guesdon)
The objective of this work is to define the physiological role of anti-DNA catalytic antibodies found in the serums of patients suffering from the autoimmune disease, systemic lupus erythematosus (SLE). These antibodies are able to catalyze the hydrolysis of the phosphodiester bond of nucleic acids. The strain of mice F1 NZB/NZW known to develop SLE spontaniously, has been chosen as the model for our study. A rapid screening microtiter assay (catELISA) allowing to detect efficiently the presence of catalytic antibodies in a large number of samples, is currently being developed.
Obtention of human antibodies by in vitro antigenic stimulation and combinatorial library (Farida Nato)
To produce human antibodies directed against non-vaccinating antigens, it is essential to immunise the lymphocytesin vitro before their immortalisation or to make an immunised antibody library. The lymphocytes are isolated from human tonsils, a major source of B lymphocytes (50 to 60% of the total cells) present at all the stages of differentiation. It is possible to generate a primary in vitro response by use of lymphocytes CD4+ sensitised in vivo by the tetanus toxoid because of obligatory vaccination against tetanus. The immunogen used is the T epitope of tetanus toxoïd coupled to the B epitope of the antigen of interest. This approach makes it possible to obtain the switch in vitro because the polyclonal response is an IgG response (IgG2 or IgG3). It is possible to reproduce the cellular cooperation between the various immunocompetent cells present in the germinal centre. However the response remains weak and few specific B lymphocytes are present, which could explain the absence of hybridomas by immortalisation by fusion or by transformation with the Epstein Barr virus. Thus, we will use genetic engineering to produce antibody fragments. The aim is to produce specific immune libraries for antigens of interest.
Production of antibody fragments in plants (Farida Nato et Pierre Lafaye)
This work is a collaboration between Dr. Parastoo Ehsani (Pasteur Institute of Iran), Dr Aimé Nato (Laboratoire de Morphogénèse Végétale Expérimentale - University Paris XI) and our laboratory. Antibody fragment production in plant cells may provide low cost reagents for immunodiagnosis and immunotherapy.The two murine scFv genes against human IL4 and IL6 cytokines were cloned in pGEJAE1 and mobilized to Agrobacterium tumefaciens. 251 Tobacco leaf discs were cocultivated with Agrobacteria and regeneration of plants were done on selective media.The results showed that tobacco plants can produce scFvs against human IL4 and IL6. Roots secrete twice scFvs in comparison to leaves. The biochemical and immunochemical characteristics of purified tobacco produced scFvs, are the same as bacterial produced scFvs.
Peptides which bind to the LRR of Listeria monocytogenes InlB mime anti-LRR monoclonal antibodies (Iain Old et Pierre Lafaye)
This work is a collaboration with Pr Pascale Cossart (Unité des interactions bactéries cellules ). In an attempt to isolate mimotopes of monoclonal antibodies directed against the LRR (Leucine Rich Repeat) region of the InlB protein of Listeria monocytogenes, libraries of random peptides displayed on the minor coat protein (pIII) of bacteriophage M13 were screened using purified InlB. From a total of 281 clones screened by ELISA, 16 phage which bound specifically to InlB were isolated and were distributed in 5 different groups. These different phages have an exceptionally high affinity for InlB with an IC50 of between 560 pM and 1.00 nM and bound specifically to purified fragments of InlB containing the LRR region. The phage and the derived synthetic peptides competed for binding to InlB with those monoclonal antibodies (mAbs) raised against the LRR of InlB which blocked the invasion of Vero cells but did not compete for binding with anti-LRR mABs which do not block invasion. Molecular modelling of the anti-LRR mAb A13.1 suggests that a tyrosine residue present in CDR-H2 is important for the interaction with the LRR of InlB. The tyrosine residue found in each displayed InlB-specific peptide sequence may mime the tyrosine present in the antibody paratope. Accordingly, the cellular receptor of InlB, unidentified, probably presents one or several tyrosine involved in the interaction with InlB.
Design of genetically engineered immunological tools (Serge Pauillac)
The hybridoma technology described by Kholer and Milstein (1975) allows the unlimited production of specific monoclonal antibodies that are used clinically and in numerous basic or applied fields of research. The specificity and affinity of such tools can be modulated by genetic engineering by successive rounds of mutation and selection. Alternatively, these tools can be directly investigated by phage display technology by screening large libraries of antibody fragments (single chain Fv or scFv) displayed on the surface of filamentous phages.
These two strategies will be developed to produce hybrid proteins (scFv-PhoAv) comprising a potent bacterial alkaline phosphatase variant (PhoAv) linked to various scFv fragments directed to the E2 envelope protein of hepatitis C virus (in collaboration with Agata Budkowska, Laboratoire d'Epidémiologie Moleculaire des Entérovirus) or to marine toxins (e.g. saxitoxin, responsible for paralytic shellfish poisoning).
Development of rapid tests for the diagnosis of plague and cholera (Farida Nato)
This project was financed internally as part of the "Projet Transversal de Recherche" (PTR) in collaboration with Suzanne Chanteau (Peste/Tuberculose Unit at the Institute Pasteur, Madagascar), J. M. Fournier (Cholera and vibrio unit), and E Carniel (Molecular and Medical Bacteriology unit). The objective is to develop an immunochromatographic test for the rapid diagnosis of the plague and cholera which are both serious bacterial diseases that must be declared and are subject to International Health Regulations. They are caused by Y. pestis and V cholerae (serogroups O1 and O139) respectively. The F1 antigen, a capsular glycoprotein specific toY. Pestis, is excreted in large amounts and is a good candidate for the diagnosis of the plague because it is found in large quantities in bubo aspirates, at lower concentrations in the blood and urine of patients. In the case of cholera, the lipopolysaccharide is a a good candidate for diagnosis. Diagnostic methods such as PCR, dot-blot and ELISA have been described, but they are expensive and not practicable in many developing countries. Thus, we propose to develop rapid tests that can be easily carried out by health staff. The strip is soaked in a tube containing the test sample and the results appear after 10 minutes. The interpretation of the results is easy due to the use of gold coloured particles. The strips can be sent by post and scored for a few months at room temperature. The test is currently being validted in Madagascar.
Production of murine monoclonal antibodies (Farida Nato)
In collaboration with researchers at the Institut Pasteur, the laboratory use the technique developed by Köhler and Milstein (1975) to produce murine monoclonal antibodies. In addition to the value of monoclonal antibodies can for diagnosis, they are also widely used to analyse the structure-function relationship of various models studied in fundamental research. The biochemical characteristics (isotype, measurement of the afffinities, epitope) of the produced antibodies are determined in the laboratory by immunochemical methods.