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  Director : LECLERC Claude (cleclerc@pasteur.fr)


  abstract

 

The activity of our laboratory is focused on the understanding of the mechanisms that control the presentation of antigens to T lymphocytes by dendritic cells and on the development of new strategies of vaccination. We have recently developed several strategies of activation of CTL responses as well as fully synthetic glycopeptide carrying saccharidic epitopes to induce anti-tumoral immune responses.



  report

cale

A. The adenylate cyclase toxin (CyaA) of Bordetella pertussis: a new vector targeting dendritic cells

1. Identification of the receptor of the adenylate cyclase (M. El-Azami El-Idrissi in collaboration with D. Ladant, IP and P. Sebo, Prague)

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor which can invade eukaryotic cells. We previously demonstrated that CyaA uses the integrin CD11b/CD18 as a cell receptor, but the CyaA domain that interacts with CD11b is still undetermined. Using CyaA mutants and CyaA fragments, we showed that the 373-1706 fragment inhibits CyaA binding to CD11b. Furthermore, the inhibitory effect of the 373-1706 fragment on anti-CD11b monoclonal antibody binding to CD11b demonstrated that this CyaA fragment is acting through its specific interaction with CD11b and therefore contains the CyaA/CD11b interaction domain.

These observations were strengthened by a direct binding assay, using anti-CyaA monoclonal antibodies.These results will help us to design a highly efficient vector capable of targeting a wide range of antigens to CD11b+ antigen presenting cells, leading to a more efficient immune responses.

2. Induction of antiviral and antitumoral CTL responses by CyaA (C. Fayolle and G. Dadaglio)

The ability of CyaA to induce specific CTL responses against human epitopes was addressed. Several recombinant CyaA bearing HLA-A2-restricted melanoma epitopes were constructed and tested in HLA-A2 transgenic mice (collaboration with F. Lemonnier, IP). Strong melanoma-specific CTL responses were induced in immunized mice indicating that CyaA could be a good vector to deliver human epitopes. Importantly, these responses can be detected 5 months after immunization indicating that recombinant CyaA induce specific memory immunity. Simultaneous injection of recombinant CyaA carrying different melanoma epitopes induced CTL against these epitopes, demonstrating the capacity of CyaA to induce multispecific CTL responses in vivo against different antigens expressed by tumoral cells.

We have also explored the capacity of the CyaA vector to deliver several different CD8+ T-cell epitopes inserted into sites previously identified to stimulate CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from LCMV, the V3 region of HIV-gp120 and ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed upon delivery by CyaA and presented in vitro to specific T cell hybridoma. Immunization of mice with CyaA toxoids carrying the polyepitope triggered specific CTL responses for each of the three epitopes, as well as protection against a lethal LCMV challenge. Moreover, mice primed against CyaA or a recombinant CyaA were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying another foreign CD8+ CTL epitope.

These results highlighted the potency of the adenylate cyclase vector to induce protective ctl responses with multiple specificity and/or broad MHC restriction.

We explored an alternative approach in which the epitopes are chemically linked to CyaA. We showed that a CD8+ T cell epitope from ovalbumin chemically coupled to CyaA is presented to CD8+ T cells by a mechanism requiring proteasome processing, the transporters associated with antigen presentation (TAP1/2 molecules) and neosynthesis of MHC class I molecules. In addition, high CTL responses were induced in mice immunized with this protein. The expected advantage of this novel strategy is its versatility as we can easily couple any defined epitope to CyaA.

B. Mechanisms of delivery of exogenous viral pseudo-particles into the MHC Class I pathway (G. Morón in collaboration with I.Casal and P. Rueda)

Chimeric porcine parvovirus virus-like particles (PPV-VLPs), prepared by self-assembly of the VP2 capside protein of this virus and carrying heterologous epitopes at its N terminus is an efficient antigen delivery system and elicit strong CD4+ and CD8+ T cells responses specific for the foreign epitopes in the absence of adjuvant. As usually exogenous antigens can not enter into the MHC Class I pathway, PPV-VLPs constitute a very interesting antigen carrier to trigger CTL response. We have demonstrated that only dendritic cells (DC) are able to present PPV-VLPs to specific CD8+ hybridoma. Besides, DCs purified from mice injected with PPV-VLPs-OVA induce a CTL response after injection to naïve mice. MHC Class I presentation of PPV-VLPs requires intracellular processing, following a non classical pathway involving macropinocytosis, vacuolar acidification and lysosomal proteases, but also processing by the proteasome complex and translocation of epitopes from the cytosol to the endoplasmic reticulum using TAP molecules. The correct processing of the epitopes carried by PPV-VLPs is dependent of the amino-acidic sequences flanking the epitope.

C. Analysis of CTL and Th responses induced by dendritic cell subpopulations (G. Dadaglio and G. Schlecht)

It is now generally accepted that fully mature DC are the only professional antigen presenting cells able to induce primary T cell-mediated immune response. Two distinct DC subpopulations have been identified in mice and distinguished on the basis of their differential CD8 expression.

We analyzed the CTL responses triggered either by CD8+ or CD8- DC subsets. We showed that both DC subsets are able to induce CTL activity although the CD8- DC seems more efficient. We did not observe a strict polarization of Th cells according to the DC subset used for immunization, since in both cases we observed the production of both Th1 and Th2 cytokines by CD4+ T cells although CD8- DC induce a higher amount of Th2 cytokines than CD8+ DC. We also showed that the route of injection influences the polarization of the induced T cell responses, since CD8+ DC injected subcutaneously lost their ability to induce Th2 cytokines. Taken together, these data indicate that both CD8+ or CD8- DC subsets could be efficient in vaccination protocols against infections or tumors.

D. Analysis of CD4+ T cell immune responses induced against a antigen delivered by BCG (R. Lo-Man and X. Jiao in collaboration with the Unit of Génétique Mycobactérienne)

T cell immunity to intracellular bacteria results from the interaction with the host phagocytic APC involved in MHC presentation of bacterial Ag to T cells. In order to better document the cellular events taking place during a mycobacterial infection, we characterized the APC involved in vivo in Ag presentation to T cells in the early phase of infection. Using in situ detection and ex vivo characterization of cells that have been infected by rBCG.MalE, we showed that in the spleen, about 1-2 % of macrophages as well as DCs are infected. However, in infected mice, immunogenic peptides-MHC II complexes were detected only on DC, but not on macrophages, using an ex vivo T cell read-out assay. Together with the display of mycobacteria-derived peptides/ MHC II complexes, costimulatory molecules such as B7 are quickly upregulated in vivo only on DC. Interestingly, CD8+ and CD8- spleen DC are equally potent APC in vivo, but the production of IL-12 was mainly associated with the CD8+ subset. All these events occur transiently during a 48-hour period following infection. As the infection develops, there is a massive production and recruitment of DC in the spleen of infected mice. Strikingly, BCG bacilli survive without growing in DC during the first two weeks of infection as their number does not increase in this leukocyte subset. In conclusion, we documented that DC are infected in vivo by mycobacteria and represent the major leukocyte subset involved in the triggering of the immune response to mycobacteria in vivo. In this process, DC activities are only transient and are limited to the early phase of infection, despite the fact that DC remain infected for a much longer period of time. Survival of mycobacteria within DC may also represent a reservoir for infection

E. Role of CD8+ T-cells in anti-mycobacterial immunity (L. Majlessi and M. Rojas)

The evidence of the important role of the CD8+ T-cell subset in the acquired anti-mycobacterial immunity prompted us (i) to investigate the mechanisms of the processing and presentation of mycobacterial antigens in major histocompatibility complex class I pathway and (ii) to evaluate new vaccinal vectors - elaborated in our laboratory- able to generate efficacious Th1-CD4+ as well as T CD8+ T-cell responses in controlling infection with Mycobacterium tuberculosis.

F. Elaboration of a fully synthetic immunogen bearing a carbohydrate tumor marker for immunotherapy (R. Lo-Man and E. Deriaud in collaboration with S. Bay and S. Vichier-Guerre from Unité de Chimie Organique)

Over the last few years, anti-cancer immunotherapy has emerged as a new exciting area for controlling tumors. Carbohydrate antigens are potential targets for such immune intervention. We developed multiple antigenic glycopeptides (MAG) based on a lysin core extended with peptidic arms displaying a carbohydrate tumor antigen (Tn antigen = GalNAc-O-Ser/Thr expressed on carcinoma cells). Monomeric Tn, a cluster of 3 Tn (Tn3) and 6 Tn (Tn6) motifs were introduced in a MAG construct. The resulting dendrimeric MAGs were able to induce anti-Tn IgG antibodies in a T cell dependent manner that recognized murine as well as human tumor cell lines that express Tn. In mice, prophylactic vaccination using these MAG provided a 80- to 90% protection rate of mice against TA3/Ha tumor (expressing Tn) challenge, but was inefficient against the CT26 tumor (that do not express Tn). When used in active specific immunotherapy, the MAG based on the Tn3 cluster showed a strong capacity to promote the survival of tumor-bearing mice. Together, these results demonstrate that the MAG represents a safe and highly efficient system to induce anti-carbohydrate antibodies and is a potent alternative strategy to the traditional carbohydrate-protein conjugates which are developed for vaccine and therapeutic purposes. We are now developing MAG potentially active in humans.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

PIRES Servanne (spires@pasteur.fr)

DADAGLIO Gilles, IP (Researcher, gdadag@pasteur.fr)

LECLERC Claude, IP (Head of Unit, cleclerc@pasteur.fr)

LO-MAN Richard, IP (Researcher, rloman@pasteur.fr)

MAJLESSI Laleh, IP (Researcher, lmajless@pasteur.fr)

BOISGERAULT Florence, Postdoc

EL-AZAMI EL-IDRISSI Mohammed, Postdoc

MORON Gabriel, Postdoc

MOTIEIAN NAJAR Hossain, Postdoc

SCHLECHT Géraldine, PhD student

SUN Cheng-Ming, PhD student

DERIAUD Edith (Assistant-ingeneer, ederiaud@pasteur.fr)

FAYOLLE Catherine (Ingeneer, cfayolle@pasteur.fr)

ROJAS Marie (Technician, mrojas@pasteur.fr)


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