Homepage bandeau_genéral

  Director : LAFAYE Pierre (plafaye@pasteur.fr)



The activity of the Laboratory of Antibody Engineering is centered on studies of the function and structure of mouse and human monoclonal antibodies against microorganisms, bacterial or eukaryote toxins. The main objectives are the preparation and analysis of recombinant antibodies, in vitro antigenic stimulation, investigation of cellular receptor of autoantibodies, study of autoantibodies with catalytic activity and identification of peptides with various biological properties (e. g. enzyme inhibition) using phage display technology.

Moreover, the Laboratory establishes collaborations with Research Units of the Pasteur Institute or with other Research Institutions by preparing and characterizing mouse monoclonal antibodies of particular specificity.



Development of novel therapeutic treatments for respiratory infections using antibodies delivered at mucosal surfaces (Pierre Lafaye)

This project was financed internally as part of the "Projet Transversal de Recherche" (PTR) in collaboration with Jean-Michel Alonso and Muhammed-Kheir Taha, Neisseria unit, Hugues Bedouelle and Patrick England, Cellular Biochemistry unit and Sylvie Bay, Organic Chemistry Unit.

This project aims at developing a new therapeutic approach to fight against bacterial infections of the respiratory tract. This approach is based on the use of antibodies directed against phosphorylcholine (PC), which is a characteristic feature of many pathogenic bacteria. Our study focus on two major respiratory pathogens, Neisseria meningitidis and Streptococcus pneumoniae. Subsequently, if the results obtained are promising, this study could be extended to other pathogens expressing PC, such as Haemophilus influenzae. The three bacteria mentioned, whose natural habitat is the human upper respiratory tract, are responsible on the one hand of local infections (tonsilitis, otitis, pneumonia, etc.) and on the other hand of systemic infections such as septicemia and meningitis, after crossing the blood and hemato-encephalic barriers.

We will therefore:

- purify bacterial antigens containing PC, and synthesize chemically-defined mimes.

- produce human and murine antibodies targetting PC in its bacterial native context.

- evaluate the efficacy of the antibodies in preclinical in vitro and in vivo models.

Monoclonal antibodies against bacterial toxins (Pierre Lafaye)

Monoclonal antibodies specific for bacterial toxins may be very useful for diagnosis and therapeutics. We have developped a collaboration with the Toxins and Bacterial Pathogenesis Unit. With Michelle Mock and her team, we have obtained monoclonal antibodies against the three proteins (PA, LF and EF) responsible of the Bacillus anthracis toxicity. These antibodies have been characterized and preliminary studies show that they are able to neutralize the bacterial toxicity. The collaboration with Michel-Robert Popoff and his group started recently. Recombinant antibodies (scFvs) specific for botulinic toxins have been obtained and their characterization are in progress.

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 lymphocytes in vitro before their immortalisation and to prepare an immune 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 toxoid 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.

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 (head of the Peste/Tuberculose Unit at the Institute Pasteur, Madagascar), J. M. Fournier (head of the cholera and vibrio unit), and E Carniel (head of the 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 Yersinia pestis and Vibrio 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 internal value of F1 dipsticks is excellent (spécificity and sensitivity around 100%) . Two studies were done in Madagascar. The first concerned pilot health care centres. The agreement between the results obtained in the central plague laboratory and in the pilot centres was 90%. The second study was performed in central plague laboratory on suspect samples. The concordance between ELISA and Dipstick is 90%. Bacteriology is the reference method, but the use of dipstick increase about 59% the number of positive samples. To ensure a best epidemiological surveillance of the plague in Madagascar, the dipstick test will be used as complementory test to the bacteriology in central plague laboratory .

Concerning V. cholerae O139 et O1, a specific and sensitive dipstick test for the detection of LPS was performed and the field evaluation (in Bengladesh for O139 and in Madagascar for O1) is in progress.

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 CHO 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 and Jean-Luc Guesdon)

The objective of this work is to define the physiological role of anti-DNA catalytic antibodies found in the sera 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 spontaneously, 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, has been developed. Using our catELISA, the screening of the 1284 hybridomas obtained after the fusion of the splenocytes of ten female lupic mice has allowed the selection of some supernatants, that should contain anti-DNA catalytic antibodies.

Design of genetically engineered immunological tools (Serge Pauillac and Jean-Luc Guesdon)

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 staphylococcal enterotoxins (in collaboration with the Microbial Toxins Unit, AFSSA, Maisons-Alfort) or to marine toxins (e.g. saxitoxin, responsible for paralytic shellfish poisoning).

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.


puce Publications of the unit on Pasteur's references database


  Office staff Researchers Scientific trainees Other personnel

ESNARD Muriel – I.P. (mesnard@pasteur.fr)

DEMANGEL Caroline, I.P., actuellement en stage post-doctoral.

GUESDON Jean-Luc, I.P. (guesdon@pasteur.fr)

LEDUC Mireille, Université Paris XI Orsay. (mleduc@pasteur.fr)

LAFAYE Pierre, I.P. (plafaye@pasteur.fr)

MAZIE Jean-Claude, I.P. (jcmazie@pasteur.fr)

MOURATOU-PECORARI Barbara, I.P. (Boursière, Fondation Roux), (mouratou@pasteur.fr)

EHSANI Parastoo, Chercheur, Institut Pasteur de l'Iran.

FEKI Salma, Etudiante en thèse, Faculté des Sciences de Tunis.

LECALVEZ Florence, INSA de Lyon.

PAUILLAC Serge, Chargé de Recherche, Réseau International des Instituts Pasteur et Instituts Associés

NATO Farida, I.P., Ingénieur.

SEDDIKI-SI AHMED Nabila, I.P., Ingénieur.

DARTEVELLE Sylvie, I.P., Technicienne.

JEANNEQUIN Odile, I.P., Technicienne.

ROUYRE Sylvie, I.P., Technicienne


Page Top research Institut Pasteur homepage

If you have problems with this Web page, please write to rescom@pasteur.fr.