Unit: Antiviral immunity, Biotherapy and Vaccines 

Director: GOUGEON Marie-Lise

Our unit is involved in the study of host-pathogen interactions, and we are particularly interested in the characterization of adaptive immune response in humans, in the context of non persistent or chronic viral infections (measle virus, HIV, HBV, HCB), of cancer (melanoma) and autoimmune diseases (multiple sclerosis, primary biliary cirrhosis (PBC), stiff-man-syndrome (SPS)…). We focus on several points: cellular and molecular characterization of the induction and persistence of immunological memory induced by viruses or vaccines; 2- Analysis of the diversity of TCR and BCR repertoire in physiological and pathological situations; 3- Analysis of viral strategies developed by HIV to escape the imuune response.

In addition, we have set up in 2005 a laboratory devoted to the evaluation of immunological responses of patients enrolled in clinical trials (immunotherapy, chemotherapy, vaccinotherapy) or healthy volunteers enrolled in vaccine trials, to evaluate the immunogenicity of new vaccine candidates developed at Pasteur Institute.

Characterization of immunological memory induced by viral infection or by a vaccine (Anne Bristeau-Leprince, Fabrice Lemaître, François Huetz).

Immunological factors involved in the induction and maintenance of antigen-specific memory T cells are not well known, and their characterization is essential for the development of new vaccines which should induce long-term protective memory against pathogens. In a murine model, allowing the analysis of central memory CD8 TCM and effector memory TEM spécific for the male peptide Smcy3 in the context H2-D, we showed that two thirds of TCM et TEM clones originate from a common precursor. In addition TCM and TEM populations behaved differently in vivo: TCM clones were stable in the absence of re-stimulation, but they strongly responded to an antigenic challenge and differentiated into both TCM et TEM, although a fraction of TCM generatesdTEM. In contrast, TEM did not persist in the absence of antigen, and they were unable to mount a secondary response, male cells being eliminated by a primary response from the host. These data provide new insights into the drastically different behaviors of TCM and TEM CD8 subsets in vivo and show that vaccinologists should be careful not to generate the maximum quantity of memory T cells, but rather the right quality of memory cells. We also study T cell memory induced by measles virus and measles vaccine. Measles specific memory is long-term persisting and we ask the question of the factors involved. We have developed a number of molecular tools and technological approaches to study these factors, including peptide-HLA class I and -class II tetramers, multiparametric flow cytometry analysis, TCR repertoire analysis with Immunoscope technology.

Influence of terminal deoxynucleotidyl transferase (TdT) on Valpha and Vbeta public repertoires (Nicolas Fazilleau, Fabrice Lemaitre, Jean Kanellopoulos)

T cell repertoires observed in response to immunodominant and subdominant peptides include private, i.e., specific for each individual, as well as public, i.e., common to all mice or humans of the same MHC haplotype, Valpha-Jalpha and Vbeta-Dbeta-Jbeta rearrangements. To measure the impact of N-region diversity on public repertoires, we have characterized the alpha beta TCRs specific for several CD4 or CD8 epitopes of wild-type mice and of mice deficient in the enzyme TdT. We find that V, (D), J usage identified in public repertoires is strikingly conserved in TdT(o/o) mice, even for the CDR3 loops which are shorter than those found in TdT(+/+) animals. Moreover, the 10- to 20-fold decrease in alpha beta T cell diversity in TdT(o/o) mice did not prevent T cells from undergoing affinity maturation during secondary responses. A comparison of the CDR3beta in published public and private repertoires indicates significantly reduced N-region diversity in public CDR3beta. Our findings suggest that public repertoires are produced more efficiently than private ones by the recombination machinery. Alternatively, selection may be biased in favor of public repertoires in the context of the interactions between TCR and MHC peptide complexes and we hypothesize that MHC alpha helices are involved in the selection of public repertoires.

Analysis of TCR and BCR repertoire diversity and clonotypic detection in vivo (Annick Lim, Brigitte Lemercier, Xavier Wertz, François Huetz)

Since its conception (1992), Immunoscope has been improved and adapted to the clinical follow-up of T cells in immunotherapy trials and in pathological situations. The improvements include quantitative analyses of T cell repertoire diversity, clonotypic Immunoscope analyses on peptide-specific T cells sorted on the basis of peptide-HLA class I and class II tetramers, and the design of protocoles adapted to a prospective and retrospective clinical follow-up. The recent development of Immunoscope for B cells extends its application to clinical and vaccinal situations. For example, Immunoscope approach revealed, in children with X-linked severe combined immune deficiency (SCID-X1) involved in a gene-therapy trial to correct the γc gene deficiency, the clonal expansion of mature T cells associated with LMO-2.We designed clonotypic probes that allowed to follow up in vivo these clonal populations and analyze the impact of chemotherapy on these expansions. Immunoscope approach also allowed to show that, in the context of an adoptive therapy in melanoma patients, the infusion of Melan-A/Mart1-specific autologous T cell clones leads to the recruitment of new tumor-specific clonotypes, and this was associated with tumor regression. At the B cell level, we are currently analyzing the impact of anti-IgE treatment on the restoration of B cell diversity in patients with atopic eczema.

Analysis of viral strategies to escape immune attack (Peggy Masdehors-Taoui, Pauline Gardès, Delphine Marsac, Isabelle Liberman)

MHC class I-specific inhibitory receptors (iNKRs) are expressed by a subset of memory CD8+T cells. Similar to NK cells, these receptors might exert on CD8 T cells an important negative control that participates to the prevention of autologous damage but may also contribute to viral escape. We have shown that chronic HIV-infection induces an up-regulation of iNKRs on the surface of CD8 T cells. This positive regulation is driven by in vivo viral replication, and it is associated with inhibition of effector functions (proliferation, perforin expression, cytokine synthesis) in HIV-specific CD8 T cells. Studies are ongoing on the impact of antiretroviral therapies on these receptors. These observations suggest that the negative control exerted by these receptors may contribute to the alterations of CTL functions and to viral escape.

T cell dynamics and antiviral immunity in HIV+ patients receiving IL-2 (Béatrice Poirier-Baudoin, Peggy Masdehors-Taoui, Valérie Seffer, Pierre-Henri Commere, in collaboration with Pr. Yves Levy, Hôpital Henri Mondor, Créteil)

Combined antiretroviral therapy inhibiting HIV reverse transcriptase and protease induces a clinical benefit in a large fraction of chronically HIV-infected patients, while reducing the plasmatic viral load to undetectable levels and restoring the level of CD4 T cells. However, because of severe adverse effects, including metabolic complications, new therapeutic strategies are required to spare antiretroviral regimens. For example, antiretroviral therapy has been associated with IL-2 immunotherapy to restore qualitatively and quantitatively the pool of CD4 T lymphocytes. With the aim to better understand the mechanisms whereby IL-2 induces a significant increase in CD4 T cells in HIV+ patients, we have developed several methodological approaches to detect ex-vivo recent thymic emigrants (TREC), to identify lymphocytes submitted either to homeostatic proliferation, or to programmed cell death by apoptosis. In parallel, HIV-specific immunity has been analyzed after stimulation with viral antigens and multiparametric analysis by FACS. In the context of three clinical trials ((ANRS 048, ANRS 079, Silcaat), we have shown that T cell dynamics is modified under the impact of IL-2, inducing increased survival and homeostatic proliferation and, in the long-term, IL-2 therapy could preserve effector T cells specific for HIV. We are currently involved in two ongoing clinical trials, evaluating the impact of IL-2 on CD4 T cell restoration in the context of antiretroviral therapeutic interruption (ANRS 118), and the influence of IL-2 therapy on the delay in treating naïve patients with antiretrovirals (ANRS 119).

Phase II Vaccinal trial SC599 against Shigellosis (Béatrice Poirier-Baudoin, Valérie Seffer, Lucy Henno).

The annual number of episodes of Shigella infection is 163.2 million, the majority being observed in developing countries and 70% of cases are children. The annual number of deaths is 1.1 million, 61% being children below 5 years of age. A vaccine is urgently needed. The vaccine trial SC599, double-blinded vs placebo, evaluates the tolerance and compares immunogenicity of two doses of a vaccine composed of attenuated genetically modified Shigella dysenteriae sérotype 1 (SD1). 111 healthy volunteers have been enrolled by two centers (Centre Vaccinal Pasteur-Cochin in Paris and the St George Vaccine Centre in London). The vaccine immune response of the volunteers is evaluated both in our laboratory and the laboratory of David Lewis in London. Two immunogenicity criteria are evaluated: 1- the frequency of B lymphocytes producing IgM, IgG, IgA specific for SD1 LPS is determined with the Elispot technique, developed in our laboratory ; 2- the humoral response specific for SD1 and B-subunit, the antibody titers being determined by Elisa. The primary criteria is the number of B cells forming anti-SD1 IgA spots. This trial started in 2005 and should end before the end of 2006.

Proteomic analysis of ash allergens (Pascal Poncet, in collaboration with Gabriel Peltre, ESPCI, et Jean-Michel Wal, INRA)

In Europe, sensitization to ash pollen is underestimated because of a lack of precise pollinisation calendar, lack of standardized extract for diagnosis and wide cross reactivities with other pollens. Ash is member of the Oleaceae family (olive, privet, forsythia…). In order to identify the panel of ash pollen allergens and to decipher the IgE response, 62 sera from patients diagnosed for ash, olive or grass allergies were screened by IgE immunoblot of an aquaeous extract of ash pollen separated by SDS-PAGE. Six sera were selected for 2D IgE immunoblot analysis followed by mass spectrometry for protein identification. We have shown that 92% of allergic patients to ash had IgE reactivity against Fra e, the major ash pollen allergen, and 46% react with profilin. The overlapped patterns determined 5 zones of IgE reactivity based on pI, defining the allergome of ash pollen. Protein identification by mass spectrometry and MALDI-TOF analysis revealed the existence of isoforms of Fra e 1, and 10 proteins did not exhibit IgE reactivity. This work contributes to a better delineation of ash pollen allergens and pattern of sensitization. The investigation of diagnostic and therapeutic relevance of these identified allergens may help to predict the evolution of symptoms and to refine an "a-la-carte" treatment of an allergy that results for 70% in asthma.

Keywords: Immune memory, TCR and BCR repertoire diversity, Immunoscope, T cells specific for viruses and tumors, AIDS, clinical trials and immunotherapy

Activity Reports 2005 - Institut Pasteur

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