The adenylate cyclase toxin (CyaA) of Bordetella pertussis: a new vector targeting dendritic cells(collaboration with D. Ladant, IP and P. Sebo, Prague)
Identification of the receptor of the adenylate cyclase (P. Guermonprez, M. El-Azami El-Idrissi)
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor required for the early phases of lung colonization. It can invade eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes the production of high cAMP levels. CyaA intoxication leads to toxic effects on macrophages and neutrophils. We demonstrated that CyaA uses the integrin CD11b/CD18 as a cell receptor. Indeed, the saturable binding of CyaA to the surface of various hematopoietic cell lines correlated with the presence of this integrin on these cells. Moreover, binding of CyaA to various murine cell lines and human neutrophils was specifically blocked by anti-CD11b monoclonal antibodies. The increase of intracellular cAMP level and cell death triggered by CyaA intoxication was also specifically blocked by anti-CD11b monoclonal antibodies. In addition, CyaA binds efficiently to CHO cells transfected with CD11b/CD18 but not to non-transfected cells or to cells transfected with the CD11c/CD18 integrin and triggers intracellular cAMP increase and cell death of these cells. Thus, the cell-surface expression of CD11b, notably by neutrophils, macrophages, and dendritic and natural killer cells, supports a role for CyaA in disrupting the early, innate antibacterial immune response (collaboration with the Unit of Bordetella).
Induction of antiviral and antitumoral CTL responses by CyaA (C. Fayolle and G. Dadaglio)
We investigated the potency of recombinant CyaA carrying one to four copies of a MHC-class I and -class II restricted LCMV T cell epitope to induce T cell responses. These CyaA hybrid molecules were able to induce both CTL and CD4+ Th1 responses against this epitope either in the absence or in the presence of adjuvant. Although the insertion of the larger peptides resulted in a partial lost of the invasive capacity of recombinant CyaA, the insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser extent, of the CTL responses.
The ability of CyaA to induce specific CTL responses against human epitopes was also 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 an appropriate vector to deliver human T cell epitopes to the MHC class I pathway. Importantly, these responses remained detectable 5 months after immunization indicating that recombinant CyaA induce specific memory immunity.
We have also explored the capacity of the CyaA vector carrying 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 on 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 (collaboration with M.F. Saron, IP). 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.
B. 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 capside protein of this virus (in collaboration with INGENASA, Madrid) and carrying heterologous epitopes genetically inserted at its N terminus, can be an efficient antigen delivery system without help of adjuvant and can elicit strong CD4+ and CD8+ T cell responses specific for the foreign epitopes. As exogenous soluble proteins cannot usually enter into the MHC class I pathway, PPV-VLPs constitute a promising antigen carrier to trigger CTL response. We have determined that only dendritic cells are able to present CTL epitopes delivered by PPV-VLPs to specific CD8+ T cell hybridoma. Furthermore, PPV-VLPs are rapidly uptaken by dendritic cells, and are retained inside these cells at least 24 h after uptake. PPV-VLPs follow a non classical pathway of MHC Class I processing, involving macropinocytosis, vacuolar acidification and lysosomal proteolysis, but also processing by the proteasome complex in the cytosol of dendritic cells. The produced peptides are translocated from the cytosol to the endoplasmic reticulum using TAP molecules and bind to nascent MHC class I molecules.
C. Analysis of CTL and Th responses induced by dendritic subpopulations (G. Dadaglio and G. Schlecht)
It is now generally accepted that fully mature DC are the only professional antigen presenting cells (APC) 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 CD8a expression. Important localization and functional distinctions were described between both subsets.
We analyzed CTL responses induced in the context of CD4+ T cell responses triggered either by CD8a+ or CD8a- DC subsets. We showed that both DC subsets are able to induce CTL activity although the CD8a- DC seem more efficient. Furthermore, it appears that the quality of cytotoxic responses is independent on the induction of Th responses. 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 CD8a- DC induce a higher amount of Th2 cytokines than CD8a+ DC. Taken together, these data indicate that both CD8a+ or CD8a- DC subsets could be efficient in vaccination protocols against tumors or infections.
D. Analysis of the role of different APC in the induction of CD4+ T cell immune responses against a mycobacterial antigen tumors or infections (R. Lo-Man and X. Jiao)
T cell immunity to intracellular bacteria results from the interaction with the host phagocytic APC responsible for MHC presentation of bacterial antigen to T cells. In order to better understand 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. The role of the two phagocytic APC types, namely macrophages and dendritic cells, had not been clearly addressed during an ongoing bacterial infection. Macrophages are privileged host cells for intracellular bacteria, but DC are much more potent APC. 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, CD8a+ and CD8a- spleen DC are equally potent APC in vivo, but the production of IL-12 was mainly associated with the CD8a+ 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 makes the latter a potential reservoir for these bacteria (collaboration with the Unité de Génétique Mycobactérienne, IP).
E. Elaboration of a fully synthetic immunogen bearing a carbohydrate tumor marker for immunotherapy (R. Lo-Man and E. Deriaud)
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). A monomeric Tn, a cluster of 3 Tn (Tn3) or 6 Tn (Tn6) motifs was 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 expressing Tn. In mice, prophylactic vaccination using these MAG provided a 80- 90% protection rate against TA3/Ha tumor (expressing Tn) challenge, but was inefficient against the CT26 tumor (that does not express Tn). When used in active specific immunotherapy, the MAG based on the Tn3 cluster showed a strong capacity to increase 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 that are developed for vaccine and therapeutic purposes. We are now developing MAG potentially active in humans (collaboration with S. Bay from Unité de Chimie Organique, IP).