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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 activation and regulation of T cell responses 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. Analysis of the interaction of the adenylate cyclase with its receptor (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. Using CyaA mutants and CyaA fragments, we have identified the region of CyaA interacting with CD11b. 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 developed a new approach in which CD8+ epitopes are chemically linked to CyaA. We showed that high CTL responses were induced in mice immunized with CyaA bearing CD8+ epitopes from ovalbumin and LCMV. In addition, we demonstrated 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 (TAP/2 molecules) and neosynthesis of MHC class I molecules. The expected advantage of this novel strategy is its versatility as we can easily couple any defined epitope to CyaA.

B. Induction of CTL responses by viral pseudo-particles and mechanism of MHC I presentation (G. Morón and F. Boisgérault in collaboration with P. Rueda, Madrid)

1. Mechanisms of delivery of exogenous viral pseudo-particles into the MHC Class I pathway (G. Morón)

Chimeric porcine parvovirus virus-like particles (PPV-VLPs), prepared by self-assembly of the VP2 capsid 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 represent a very interesting antigen carrier to trigger CTL response. Dendritic cells (DC) capture these particles very efficiently in vivo and are the only cells capable to present PPV-VLPs to specific CD8+ hybridoma. Both CD8a - and CD8a + DCs process PPV-VLPs, though they have different kinetics and T cell requirements. DC stimulation by PPV-VLPs induces acquisition of CD8a and CD205 and loss of CD4 molecules in CD8a - DCs as well as expression of co-stimulatory molecules on both DC subsets. 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.

2. Adjuvant effect of PPV-VLPs on the CD8+ T cell response to exogenous antigens (F. Boisgérault)

In order to analyze the adjuvant effect of PPV-VLPs on the CD8+ T cell response to exogenous antigens, we have chosen microspheres as a delivery system. It has been clearly established that microspheres, as well as viral pseudo-particles, can deliver antigens into the MHC I pathway. However, in the absence of costimulatory signals, beads loaded with MHC I peptides cannot induce CTL responses. We have shown that PPV-VLPs have the ability to restore CTL responses to CD8+ epitopes loaded onto beads, even in the absence of CD4+ T cells. We are currently characterizing this CD8+ T cell response and evaluating the induction of protective immunity.

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 showed that both DC subsets injected by the i.v. route 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. However the injection route can play a role in the polarization of the T cell responses, since CD8+ DC injected subcutaneously lost their ability to induce Th2 cytokines. Induction of CD8+ T cell responses by plasmacytoid DC (pDC) was also analyzed. We showed that pDC present class I restricted peptide to CD8+ T cells. However, these DC are unable to induce in vivo specific effector or memory CTL and do not induce specific tolerization. In vivo induction of CD4 T cell responses by pDC are under investigation.

D. Ontogeny of neonatal dendritic cells (CM. Sun and R. Lo-Man)

We investigated whether a developmental immaturity of the dendritic cell compartment could contribute to the high susceptibility to infections observed in newborns. We show that DCs are among the first cells to colonize the spleen, but the ontogeny of DC subsets follows distinct steps. The production of IL-12 and type I and II interferons by DCs is particularly efficient after birth. Moreover, Ag presentation by neonatal DC to T cells is also very efficient as early as day 2 and comparable to adult DCs. Therefore, despite a markedly different DC subset composition in early life as compared to adult DC compartment, neonatal DC are fully competent in their immune functions.

E. New vaccinal strategies for induction of anti-mycobacterial immunity (L. Majlessi and M. Rojas)

Generation of Th1-biased T-cell responses to mycobacterial immunogens is primordial in protection against infection with Mycobacterium tuberculosis, the ethiologic agent of tuberculosis. We evaluate the potential of the recombinant CyaA, bearing mycobacterial antigens, to induce T-cell responses and to control infection with M. tuberculosis.

Moreover, we attempt to identify the type of mycobacterial antigens able to attain MHC-I presentation pathway for generation of CD8+ T-cell responses which also play an important role in protection against this intracellular pathogen.

In collaboration with the Unit of Génétique Moléculaire Bactérienne, we investigate the impact of introduction of certain chromosomal regions of M. tuberculosis into the genome of attenuated M. bovis BCG on the immunogenicity of this vaccine by modification of host-pathogen interaction during induction of innate and acquired anti-mycobacterial immunity.

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

We have developed multiple antigenic glycopeptides (MAG) based on a lysine core extended with peptidic arms displaying a carbohydrate tumor antigen (Tn antigen). 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, therapeutic vaccination using these MAG provided a 70% survival rate 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 recently developed new MAGs potentially active in humans and we are currently testing these molecules in non-human primates.

Keywords: Vaccines, CTL, dendritic cells, cancer, T cells, immunotherapy



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  LECARPENTIER Laurette :llecarp@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

MASCARELL Laurent, Postdoc

MORON Gabriel, Postdoc

MOTIEIAN NAJAR Hossain, Postdoc

PREVILLE Xavier, Postdoc (BT PHARMA)

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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