MANASP - Development of novel management strategies for invasive aspergillosis

EC contribution: : € 2.914.800
Duration: : 36 months
Starting date: : 01/01/2007
Funding scheme: : Specific Targeted Research Project
Keywords: : immunotherapy, vaccine, antigens, T-cells, dendritic cells, A. fumigatus, PCR diagnostics, monoclonal antibodies, GMP-conditions, aspergillosis, transplantation
Contract/Grant agreement number: : LSHM-CT-2006-037899
Project web-site: : http://www.manasp.org

Background:

The overall objective of the MANASP project was to develop new treatment strategies for Invasive Aspergillosis (IA) - the major infectious complication of treating haematological malignancies with intensive chemotherapy or haematopoietic stem cell transplantation (HSCT).

Ineffective host immune responses facilitate fungal invasion of the pulmonary system and other vital organs leading to death. In order to redress this immunological imbalance we propose to develop new immunotherapeutic strategies which will augment current antifungal treatments and reduce morbidity and mortality.

Facilitated by recent publication of the A. fumigatus (the principal pathogen of the genus) genome sequence we will exploit new knowledge and techniques in genomics and post-genomics. We will identify the immunologically important fungal molecules to design immunotherapies based on vaccines using fungus-primed innate immune cells, monoclonal antibodies or adoptive transfer of specific T lymphocyte clones.

We will also generate new nucleic acid based diagnostic tests to inform when and how immunotherapy can be optimally applied.

The outcomes will have strong commercial applications which will be delivered by three leading European SMEs within the consortium.

Problem:

Over the past decade, invasive aspergillosis (IA) has emerged as the most serious life-threatening infectious complication of intensive remission-induction chemotherapy and allogeneic HSCT in patients with a variety of haematological malignancies. Aspergillus fumigatus is the most commonly isolated species from cases of IA and is the focus of our research although we believe the intended outcomes will act as a paradigm for management of IA due to less common species such as A flavus and the emerging A terreus. Despite the limited improvements that have been made with preventative strategies and the development of new antifungal drugs, IA has an incidence of 10 - 30% and is still associated with high mortality rates as high as 90% in some surveys. T lymphocytes provide a critical secondary defense against this and other fungal pathogens. Therefore, Aspergillus-specific T-cell immunity, transferred through the infusion of ex vivo-generated, donor-derived, Aspergillus-specific T-cells might be beneficial for recipients of allogeneic HSCT.
There are only raw data regarding immunotherapy of patients with invasive fungal infection, and minimal data relating to IA. This might be due, at least in part, to the complex antigenic properties of A. fumigatus, which have been less well characterized compared to viruses such as CMV or EBV. Only a minority of the hundreds of (glyco)proteins of A. fumigatus reported in the literature have been characterized at either a molecular and/or biochemical level.
The diagnosis of IA is a particular challenge. Conventional microbiological methods fail to detect most cases so that current diagnostic categories are based on a combination of clinical, imaging, and histopathological (often post-mortem) criteria. There have been some advances with non-culture techniques based on PCR based amplification of fungal DNA or detection of Aspergillus antigens in blood samples. However these have not been validated as tools for identifying patients at risk or for monitoring responses to treatment by determination of fungal loads.

Aim:

The aims of The overall MANASP goal of our STREP is to develop more effective management strategies for IA with the following aimsincluded:

dDevelopment of immunotherapeutic strategies for Invasive AspergillosisIA.;
dThis will be achieved by identifying the T-cell clones that react with specific Aspergillus targets and evaluating their efficacy for prevention and treatment of IA. Success will be measured by characterization of the generated T-cells, and demonstration of their functional properties against Aspergillus.
Development of improved diagnostic tests for IA with commercial potential. This will be achieved by identifying appropriate nucleic acid targets, extraction and amplification protocols and demonstrating their sensitivity and specificity in clinical samples.;
vValidation of a dendritic cell (DC)- based vaccine immunotherapy strategy in animal models to generate protective immunity against Aspergillus.;
Uuse of genomic and proteomic techniques to identify new Aspergillus targets that interact with the host's immune system.

Expected and obtained results:

The results of the MANASP include:

identification of different pattern recognition receptors in response to A. fumigatus and their role in activating DCs;
identification of PAMPs of A. fumigatus useful for immunotherapy strategies;

Identification of murine DC subsets capable of inducing antifungal Th responses
Adoptive transfer of manipulated DC into mice suffering from IA

Characterization of Aspergillus-specific T-cell response in healthy individuals

Identification of a protective T-cell response in patients surviving IA

Development of a clinical protocol under current cGMP-conditions for treatment of patients with IA

Modulation of Aspergillus-specific immune response by transduced T-cells

Preparation of a bank of monoclonal antibodies against A. fumigatus

Selection of the most efficient antibodies by in vitro and in vivo analysis

Development of an assay to detect Aspergillus DNA with high specificity
Development of an assay to detect Aspergillus RNA with high sensitivity
Clinical evaluation to confirm sensitivity in detecting Aspergillus infection using standardised definitions produced by International consensus and comparison to different well established assays

Commercialization of the assay into an affordable and rapid diagnostic test

Potential applications:

Surveillance of patients being treated for haematological malignancies by chemotherapy or HSCT using DNA/RNA based molecular diagnostics. This will generate greater ascertainment of possible and probable cases of IA and earlier therapeutic intervention leading to improved survival rates. Incorporation of these diagnostic tests into internationally accepted EORTC/MSG criteria for clinical application in categorising cases of IA to facilitate research trials of new antifungal agents or other novel therapies Development of immunotherapeutic strategies based on demonstrated efficacy against Aspergillus in vitro and in animal models. This will either be through adoptive transfer of donor T-cells, or administration of monoclonal antibodies targeted against immunologically relevant Aspergillus antigens/epitopes. wider application of this technology will enable the treatment of other groups of patients (outside the Haematological Malignancy field).

Coordinator:

Prof. Hermann Einsele
University of Wuerzburg
Medizinische Klinik II
Klinikstrasse 6 - 8
97070 Wuerzburg, Germany
Tel. +49 931 20170010
Fax +49 931 20136409
E-mail: einsele_h@klinik.uni-wuerzburg.de

Photos:

Conidiophores of A. Fumigatus

Germination of a conidium in a macrophage of an immunocompromised host

Macrophage - conidia interaction

European Commission