Vaccination anti-tumorale dans les hémopathies malignes (étude de deux antigènes tumoraux candidats)

De nos jours, malgré l'efficacité des thérapeutiques classiques dans les proliférations malignes hématopoïétiques, il est difficile d'obtenir des rémissions cliniques durables. En effet, de par la persistance d'une maladie résiduelle, un certain nombre de patients rechutent. L'avancée des connaissances concernant le rôle du système immunitaire dans le contrôle des tumeurs a donné un nouvel élan à la vaccination anti-tumorale. L'objectif de ce travail a été d'évaluer deux antigènes tumoraux dans une perspective de vaccination. Dans une première partie, nous avons évalué l'immunogénicité de l'AID, antigène tumoral candidat dans le lymphome folliculaire. Les résultats obtenus in vitro chez l'homme, lors de l'induction de lymphocytes T cytotoxiques, et in vivo chez la souris, dans un modèle d'immunisation anti-AID, ont montré la faible immunogenicité de cette protéine. En parallèle, nous avons développé un modèle animal de lymphome systémique à partir de la lignée cellulaire A20. Le suivi de l'évolution des masses intra-abdominales chez des souris, inclues dans un protocole de vaccination, a été effectué par tomographie par émission de positon (TEP) avec une machine dédiée au petit animal. Nous avons ainsi pu démontrer l'intérêt de la TEP dans l'évaluation de l'efficacité d'une stratégie de vaccination anti-tumorale. Enfin dans une dernière partie, nous avons démontré l'efficacité du vaccin plasmidique p.DOM-peptide, appliqué à l'antigène tumoral candidat dans les leucémies aiguës, WT1. Nous avons également mis en évidence pour la première fois l'immunogénicité et la présentation par les cellules tumorales leucémiques de l'épitope WT1.37.To date, despite efficacy of classical treatments in haematopoïetic cancers, durable clinical remissions ar difficult to obtain. Indeed, because of the existence of a residual disease, some patients relapse. The improvement of knowledge about the role of the immune system in tumour control has given a boost to anti-tumour vaccination. The objective of this work was to assess two tumour antigens as veccination targets. Firstly we assessed immunogenicity of AID, a tumour antigen candidate in follicular lymphoma. Human cytotoxic T lymphocyte inductions in vitro, and anti-AID immunisations in mice, showed the low immunogenicity of this protein. Secondly, we developed an animal model of systemic lymphoma using the lymphoma cell line, A20. Intra-abdominal disease in mice was visualised by positon emission tomography (PET) with a machine designed for small animals. Thus, we demonstrated that PET can be used to assess the efficacy of an anti-tumour vaccination strategy. Finally, we demonstrated efficacy of the p.DOM-peptide DNA vaccine, applied to the leukaemia tumour antigen, WT1. We also highlighted for the first time the immunogenicity and the presentation by leukaemic cells of the WT1.37 epitope.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Protection patterns in duck and chicken after homo- or hetero-subtypic reinfections with H5 and H7 low pathogenicity avian influenza viruses: a comparative study.

Avian influenza viruses are circulating continuously in ducks, inducing a mostly asymptomatic infection, while chickens are accidental hosts highly susceptible to respiratory disease. This discrepancy might be due to a different host response to the virus between these two bird species and in particular to a different susceptibility to reinfection. In an attempt to address this question, we analyzed, in ducks and in chickens, the viral load in infected tissues and the humoral immune response after experimental primary and secondary challenge infections with either homologous or heterologous low pathogenicity avian influenza viruses (LPAIV). Following homologous reinfection, ducks were only partially protected against viral shedding in the lower intestine in conjunction with a moderate antibody response, whereas chickens were totally protected against viral shedding in the upper respiratory airways and developed a stronger antibody response. On the contrary, heterologous reinfection was not followed by a reduced viral excretion in the upper airways of chickens, while ducks were still partially protected from intestinal excretion of the virus, with no correlation to the antibody response. Our comparative study provides a comprehensive demonstration of the variation of viral tropism and control of the host humoral response to LPAIV between two different bird species with different degrees of susceptibility to avian influenza

Viral excretion in lower digestive tract of ducks (A) or in upper airways of chickens (B) of LPAIV H7N2.

<p>Birds were either primo-infected at six weeks of age (H7 groups) or re-infected at six weeks of age with H7N2 virus, three weeks after H5N3 first inoculation (H5H7 groups). Viral titrations were measured 2 days (d2, for chickens) in oropharyngeal swabs, 3 days (d3, for ducks) in cloacal swabs, or 8 days (d8, for both birds species in their respective swabs) and were expressed as viral RNA copies per sample and compared between primo-infected (H7 groups, black bars) and re-infected birds (H5H7 groups, white bars). All the swabs were eluted in 1.5 ml PBS and were analysed using strictly the same protocol for RNA extraction and RT-PCR. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p

Antibody response to LPAIV H7N2 in ducks (top) or in chickens (bottom).

<p>Antibody titers were measured by ELISA (left) or HI method (right) from serum collected at the indicated time in days (d) after primary infection (H7 groups, black bars) or heterologous secondary infection (H5H7 groups, white bars) and were expressed as the reciprocal of the dilution used for measurement as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105189#s2" target="_blank">Materials and Methods</a> section. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p

Antibody response to the H5N3 virus in ducks (top) or in chickens (bottom).

<p>Antibody titers were measured by ELISA (left) or HI method (right) from serum collected at the indicated time in days (d) after primary infection (H5 groups, black bars) or secondary infection (H5H5 groups, white bars) and were expressed as the reciprocal of the dilution of serum used for measurement as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105189#s2" target="_blank">Materials and Methods</a> section. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p

Viral excretion in upper airways (left) or in lower digestive tract (right) of LPAIV H5N3 in ducks (top) or in chickens (bottom).

<p>Birds were either primo-infected at six weeks of age (H5 groups) or re-infected at six weeks of age with the same virus as that used three weeks before (H5H5 groups). Viral titrations were measured at 2 days (d2, for chickens), 3 days (d3, for ducks) or 8 days p.i. (d8, for both bird species) and were expressed as viral RNA copies per sample and compared between primo-infected (H5 groups, black bars) and re-infected birds (H5H5 groups, white bars). All the swabs were eluted in 1.5 ml PBS and were analysed using strictly the same protocol for RNA extraction and RT-PCR. Significant differences between groups are indicated with asterisks (*, P<0.05; **: P<0.01; ***: P<0.001).</p

Viral excretion in upper airways (left) or in lower digestive tract (middle) and antibody response (right) to LPAIV H5N3 in ducks (top) or in chickens (bottom).

<p>Viral load in oropharyngeal swabs (A, D) and cloacal swabs (B, E) was expressed as viral RNA copies per sample and compared between control (naïves) and challenged birds two days (for chickens, corresponding to the peak of infection in lung) or three days (for ducks, corresponding to the peak of infection in cloacum) after inoculation. All the swabs were eluted in 1.5 ml PBS and were analysed using strictly the same protocol for RNA extraction and RT-PCR. Antibody titration (C, F) was expressed as inverse dilution of serum used for measurement by ELISA (vertical scores) or haemagglutination inhibition method (HI, draught-board).</p

[F-18]-Fluoro-2-deoxy-D-glucose positron emission tomography as a tool for early detection of immunotherapy response in a murine B cell lymphoma model.

[F-18]-fluoro-2-deoxy-D: -glucose positron emission tomography (FDG-PET) is a non-invasive imaging technique which has recently been validated for the assessment of therapy response in patients with aggressive non-Hodgkin's lymphoma. Our objective was to determine its value for the evaluation of immunotherapy efficacy in immunocompetent Balb/c mice injected with the A20 syngeneic B lymphoma cell line. The high level of in vitro FDG uptake by A20 cells validated the model for further imaging studies. When injected intravenously, the tumour developed as nodular lesions mostly in liver and spleen, thus mimicking the natural course of an aggressive human lymphoma. FDG-PET provided three-dimensionnal images of tumour extension including non-palpable lesions, in good correlation with ex vivo macroscopic examination. When mice were pre-immunized with an A20 cell lysate in adjuvant before tumour challenge, their significantly longer survival, compared to control mice, were associated with a lower incidence of lymphoma visualized by PET at different time points. Estimation of tumour growth and metabolism using the calculated tumour volumes and maximum standardized uptake values, respectively, also demonstrated delayed lymphoma development and lower activity in the vaccinated mice. Thus, FDG-PET is a sensitive tool relevant for early detection and follow-up of internal tumours, allowing discrimination between treated and non-treated small animal cohorts without invasive intervention

DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I–binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2–restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT137–45–specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT137–45, have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients. <br/