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Genome scale analysis of hte immune response against pathogenic micro-organisms; leading to diagnostic and vaccine candidates and development of an integrated micro array platform for clinical use (MICROBEARRAY)

Final Report Summary - MICROBEARRAY (Genome scale analysis of hte immune response against pathogenic micro-organisms; leading to diagnostic and vaccine candidates ... for clinical use)

The genome sequences of microbial organisms responsible for diseases of world-wide medical importance have been sequenced or will be available in the very near future. Combinatorial cloning technologies for producing large numbers of proteins have been developed and high-throughput assays such as protein micro arrays have been clinically validated for detecting the presence in the serum of antibodies directed against microbial antigens. These scientific and technical achievements offer the opportunity of investigating the natural immune response against the whole proteome of a variety of micro-organisms. A powerful combination of genomic information, molecular tools and immunological assays are available to identify the antigens that, either alone or in combination, function as targets of protective immunity or could be used as markers for serodiagnosis. We proposed to identify in micro-organisms of great medical relevance such as M. pneumoniae, C. pneumoniae, L. pneumophila, coronavirus spp and P. falciparum, a large collection of surface and secreted proteins as well as putative endotoxins. This repertoire of proteins was to be produced as recombinant molecules or as sets of overlapping synthetic peptides and printed on array slides. The serum reactivity of groups of individuals with proven history of exposure to the selected micro-organisms were to be analysed against the arrayed microbial proteins to identify diagnostic markers and correlates of protection. This project will further develop SMEs and research partners proprietary technology for high throughput protein expression, software tools, surface peptides synthesis, protein and peptide surface capture, and array reader instrumentation to create an integrated platform of great commercial and research value.

It was anticipated that the proposed experimental approaches and technological developments will generate valuable intellectual property and unravel how the humoral immune response interacts with the microbial proteomes thus filling the gap between genomic data and development of both novel vaccines and diagnostic tools.

The project has yielded a variety of results that are suitable for publishing (either in their entirety now or as part of further studies in the future). We have analysed the antibody reactivity profiles to a group of recombinant malaria antigens in around 200 children showing different levels of clinical immunity to P. falciparum infection. The analysis was carried out using an antibody capture micro-array immunoassay that incorporated as a substrate 18 recombinant arrayed antigen chosen amongst the most promising available blood stage vaccine candidates. Unexpectedly we found a great heterogeneity in individual antibody reactivity to malaria antigens with individuals tending to recognise distinct combinations of antigens. Such diversity likely reflects the complexity of the parasite proteome and the combined genetic diversity of the human and parasite populations and provides an intriguing explanation to the difficulties that have been encountered in vaccine development. Serum reactivity profile clustering and comparisons carried out with two independent statistical approaches revealed that reactivity to certain combination of antigens AMA1 and two MSP2 variants (3D7 and Fc27) is significantly associated with protective immunity. When we looked for association between protection against malaria and serum reactivity to any of the 18 individual antigens we failed to find any correlation. These findings demonstrate that immunity to malaria is associated with antibody recognition of multiple rather than individual antigens, and that reactivity to AMA1 and the two MSP2 variants has some association with the development of clinical immunity in children. The simultaneous assessment of hundreds of distinct antigen-antibody reactions should dramatically facilitate the identification of the parasites antigens that, in combinations, function as targets of the protective immune response and hence facilitate both the development and the evaluation of anti-malarial vaccines.

This work has implications that go beyond the field of malaria to date more than 320 prokaryotic genomes have been completed or are close to completion, including numerous pathogenic organisms. Serum profiling against microbial antigen repertoire could also be applied to identify correlates of protection for many of these other micro-organisms. These results of this work have been accepted (subject to final editing) as an article in a highly reputable scientific journal.

Currently, the market for micro array is focused around large scale, expensive units which cost upwards of GBP 50 000 to GBP 600 000. Few companies have the financial or logistical capability to enter into this market. The cost of the instrumentation also limits the growth of the total market as micro array technology would be far too expensive for public health, private research organisations and those businesses wanting to enter into the diagnostic sales or service provision market. The work of this co-operative project will be instrumental in developing protein micro array for clinical application near to commercial reality. This proposal brings together a group of SMEs with different and complimentary skills into an academic network of excellence to carry out work which will benefit each company far more than if they worked alone. The output of the proposed activity will create several different commercial opportunities for the particular SMEs involved but also address the wider competitiveness of the European sector for micro array technology through production of an integrated platform for low cost, flexible micro array assays.