Final Report Summary - FIGHTMAL (Correlating protection against malaria with serum profiles against Plasmodium falciparum antigen repertoires)
The FIGHTMAL consortium includes a small number of academic and industrial partners with cutting edge expertise in protein microarrays, immunoassay development, immunology, protein expression, epidemiology and working towards the objective of translating the genome sequence information of Plasmodium falciparum into a useful tool to unravel correlates of protection against human malaria. The objective is to develop a microarray immunoassay covering the entire repertoire of surface secreted P. falciparum proteins to compare the antigen-antibody recognition profiles of protected and non-protected persons in malaria-exposed communities, thus facilitating the identification of the antigens that function as targets of protective immunity. In terms of research activities, task distribution, mobility plan and management, the project was structured with a view to facilitate the exchange of skills, knowledge and resources not only between industry and academia, but also between European and disease endemic countries, as the partnership includes two Uganda-based research centres.
Work package 1: Identification of putative surface and secreted P.Falciparum proteins
A wide range of bioinformatics tools were utilized to identify possible P. falciparum antibody targets to be arrayed. An extensive bioinformatics analysis was performed by the team at the University of Perugia (UNIPG) on the parasite proteome searching for 3 different protein sets to be spotted onto 3 different chips: i) vaccine candidates, ii) putative immune targets, iii) variant surface antigens. This initially resulted in: CHIP 1: a selection of 25 genes; CHIP 2: a total of 252 unique entries were selected; CHIP 3: an initial list of 74 candidates was created, representing the (PfEMP1s) erythrocyte membrane protein 1 family. A dedicated database was developed to store and retrieve all the bioinformatics and experimental information generated by the project.
Work package 2: Expression and conformation analysis of recombinant parasite proteins
A substantial research effort (both on small and large scale) was undertaken by UNIPG in collaboration with Microtest Matrices (MtM) and Protein Therapeutics (PX) to identify optimal protocols and effective strategies for high-throughput cloning, protein expression and purification, with a view to identify parasite proteins for inclusion within the putative immune targets chip. Despite the intensive efforts of the participating laboratories involved in these activities, every phase of the production of putative candidate antigens selected via the bioinformatics analysis proved very challenging, narrowing down the expected overall performance (63%, 283 soluble-expressed clones out of the starting 448). Protein expression represented the major obstacle, in spite of the consortium expertise. The project managed to achieve a satisfying success rate, thanks to the development of an enriched medium and the optimization of both the expression and purification protocols. This is an unprecedented, outstanding and innovative result for the purification process of the P. falciparum proteins never achieved so far.
Work package 3: Microarray production and immunoassay development
In parallel, microarray production and immunoassay development activities were carried out by MtM in collaboration with UNIPG to produce three different protein chips containing i) vaccine candidates, ii) putative immune targets and iii) variant surface antigens. In order to achieve standardized protocols for characterizing serum reactivity samples, initial studies were carried out by using the 4 most common and commercially available vaccine candidates to investigate the antigen-antibody kinetics, identify the optimal incubation time and dilution for the serum samples, evaluate the analytical precision for the reproducibility of the assay and assess the feasibility for detecting not only total human IgG but also IgG subclasses. A panel of both European negative and African positive sera was then tested, showing that the proteins did not cross react with negative sera, but only with positive African sera with different profiles, as expected. Analytical tests indicated a good reproducibility of the assay immunoassay, as for the feasibility of detecting in microarray one specific IgG subclass, this proved possible, with a potential to detect two different IgG subclasses, given adequate performance conditions.
Work package 4: Assay development and validation
An assay development and validation activity was performed by MtM in collaboration with UNIPG and the London School of Hygiene and Tropical Medicine (LSHTM) to compare the performance of the developed microarray immunoassay to ELISA, using serum samples from subjects with known reactivity to malaria antigens. To this end stored sera collected during previous studies in The Gambia, Tanzania and Uganda by LSHTM were used for testing against new antigens (produced as part of WP 1 and WP2) by both ELISA and microarray immunoassay (WP3) to assess whether the data generated agreed in terms of antibody prevalence and concentration in both systems. Three antigens for analysis by ELISA and initial experiments have been performed to establish an optimized ELISA protocols.
Work package 5: Epidemiological studies in malaria endemic areas
Epidemiological studies in Uganda were carried out by LSHTM with the support of Med Biotech Laboratories (MBL) local staff and MtM personnel. The preparatory cross-sectional survey enabled the selection of the study area; case definitions were defined, and a longitudinal study was conducted in an area of intense transmission in Apac in June-November 2010. The follow-up of participants in Apac was stopped in November 2010 due to logistical challenges in providing adequate support for the field study. The resulting data, however, exceed the data of the only currently available study using a protein micro-array to determine the immune profile associated with clinical protection against malaria (Crompton et al., PNAS 2010 PMID: 20351286). A second study was conducted at three sites in Uganda: Jinja, Kanungu and Tororo, in collaboration with the NIH-funded International Centre of Excellence in Malaria Research (ICEMR) at the Infectious Diseases Research Collaboration (IDRC), based at Makerere University, Kampala, as part of a two-year longitudinal programme. The study has now entered its second year of data collection and available data was deposited in the FIGHTMAL database held at UNIPG.
Work package 6: Analysis of immune response profiles to search for correlates of protection
Antibody responses were shown to malaria antigens AMA-1, MSP-1, MSP-2, CSP and to mosquito salivary proteins and were associated with age, exposure to malaria parasites and the risk of subsequent malaria episodes. While all antibody responses against all the antigens that were used in the ELISA were associated with malaria exposure, none were independently associated with the risk of clinical malaria. This illustrates how difficult it is to disentangle malaria exposure from indicators of clinical protection and underlines the importance of the FIGHTMAL project. The longitudinal studies provided unique datasets for determining the immune correlates of protection against clinical malaria which will currently remain accessible for FIGHTMAL partners and available for addressing its aim to generate a wealth of novel immunological and epidemiological information pertaining to both the complexity and variability of the naturally-induced immune response against the malaria parasite.
In terms of project structure, FIGHTMAL’s priority is harnessing the collective expertise and resources of the participating laboratories by facilitating the exchange of knowledge and personnel. To this end, the recruitment and the secondment of researchers with different skills and levels of experience were carefully implemented not only to match the project technical requirements but mainly to offer researchers the opportunity to learn and transfer new knowledge from one laboratory to another. 21 European and non-European researchers with different degrees of experience were mobilized by the FIGHTMAL consortium during the project lifetime both internationally and transectorially. 25 staff exchanges were implemented for a total of 312 full-time working months 8 experienced researchers were newly recruited to add skills, and this has proven particularly beneficial for the industrial participants that were able to widen their research networks and increment their operating potential . This has proven particularly useful forthe African research centers as well, which have been able to build new collaborative links with the European public and private sector that will undoubtedly extend far beyond the duration of the project.
The universities involved benefitted from receiving personnel working on new research methodologies thus adding value to the existing research teams and offered in exchange new learning and working environment, for example providing training on high-tech equipment or exploiting different research links. Although the mobility programme proposed by the FIGHTMAL consortium was rather ambitious and complex, important goals were achieved in terms of gender balance and staff retention. One third of the seconded and recruited researchers were in fact women; in addition to this, most of the newly employed staff continued working at the recruiting institutions, and indeed advanced in their positions, well beyond the project end However, in spite of the clear advantages for the participating institutions and the individuals success stories, the implementation of staff mobility posed a number of challenges that the consortium addressed with great efforts. These are the transferability of social security schemes, particularly in terms of pension seniority and childcare cover, academic regulations regarding staff appointment and reintegration which, added to local employment and visa requirements, represented a significant obstacle to the transfer of knowledge and to schemes, such as this Marie Curie Action, aimed at fostering and broadening researchers’ careers.
The project public website is www.figthmal.eu. The website aims at providing a substantial overview of the project, improving internal communication between the consortium via a dedicated private section and at disseminating information concerning the activities undertaken by the project.