Community Research and Development Information Service - CORDIS

FP7

OPTIMALVAC Report Summary

Project ID: 223126
Funded under: FP7-HEALTH
Country: Germany

Final Report Summary - OPTIMALVAC (Initiative on Optimizing Malaria Vaccine Lab Assays Evaluation)

Executive Summary:
A broad-range of candidate malaria vaccines derived from diverse technologies have resulted from the multiple approaches being taken by different groups in developing malaria vaccines. Individual groups have developed assays within the context of vaccine discovery, with identification of processes for measurement of parasite growth and virulence to test specific vaccine antigens. In-house assays are strain, stage and even process specific and the ability to compare results between different candidates is further limited by diverse methodologies and assay components such as differing parasites, cells and reagents. Lack of an enabling environment for comparability of research results generated in different laboratories could lead unfortunately to scepticism of assay results, which in turn generates uncertainty about the efficacy of the vaccines and rationale(s) of vaccine development pathways.

To compare the relative merits of different candidate vaccines and approaches in a credible and informed manner, efforts must be made to harmonise key assays that can be utilised a) in the evaluation of malaria vaccines, and b) throughout the vaccine development process. Consistent, reproducible, and comparable intra- and inter- laboratory performance and increased accuracy and precision of assay data will strengthen the quality of the data on vaccine performance, generate greater confidence in candidate vaccine potential, and support the process of rational vaccine selection.

The OPTIMALVAC project officially started on 01 April 2009 and carried on for three years, ending in March 2012. The project involved twelve partner institutions and was funded by the European Commission's Framework Program 7 (FP7) as a Coordination and Support Action, project number: 223126, with EC funding of €1,000,000 with additional complementary contributions from the PATH Malaria Vaccine Initiative and the Centers for Disease Control and Prevention.

The overall goal of this three year project was to develop harmonised assays to facilitate comparison of results and improve decision making on vaccine construct development, product characterisation, down-selection of vaccine candidates and/or vaccine formulations, and enhancement of clinical development plans. OPTIMALVAC covered the following priority actions for each respective group:
• Humoral assays: To optimise and harmonise assay protocols for antibody recognition of parasite proteins and to develop reference standards
• Functional assays: To optimise and harmonise antibody-dependent cellular parasite inhibition (ADCI) assays and establish assay performance criteria
• Cell mediated immunity assays: To harmonise ELISPOT and Intracellular Cytokine Staining (ICS) protocols, and to liaise with HIV and TB initiatives on harmonisation

OPTIMALVAC was a success in bringing for the first time the main actors in malaria vaccine development to agree to participate in the harmonisation of very complex immunoassays, including assays that have lacked reproducibility between laboratories for the last decade. OPTIMALVAC partners have developed and exchanged reference reagents, guidance documents for ADCI, ICS and ELISPOT, SOPs for IFA and software tools which have been made available at the www.malariaresearch.eu, www.optimalvac.eu and www.euvaccine.eu websites. Consent form templates have been developed to accommodate regulatory and ethical considerations of the OPTIMALVAC project. OPTIMALVAC has facilitated links between HIV, TB, cancer and malaria assay harmonisation communities in much more robust ways than was the case previously. The establishment of service/reference centres for the above mentioned assays is envisaged. Malaria Vaccine assay harmonisation can now be viewed as a jointly planned activity at the level of funding organisations.

Project Context and Objectives:
In the past, the lack of harmonisation of malaria vaccine assay conduct coupled with the lack of an enabling environment for comparing research results lead to scepticism about the comparability of assay results. Assay harmonisation was and is a central priority to enable rational malaria vaccine design and for decision-making in vaccine development.

The overall goal of this three year project was to develop harmonised assays to facilitate comparison of results and improve decision making on vaccine construct development, product characterisation, down selection of candidates and/or formulation, and clinical development plans.

This project covered the following priority actions for each respective group:
Humoral assays: To optimise and harmonise assay protocols for antibody recognition of parasite proteins and to develop reference standards
Functional assays: To optimise and harmonise cell dependent parasite inhibition assays and establish assay performance criteria
Cell mediated immunity assays: To harmonise ELISPOT and ICS protocols, to liaise with HIV and TB initiatives on harmonisation

In addition, support was given towards the following activities:
• To establish repositories for protocols and SOPs
• To establish repositories for standards and reagents for specific assays
• To establish common data management and statistical analysis plans
• To establish assay hands-on training and transfer trouble-shooting capacity
• To contribute to the establishment of future reference centres for selected assays
• To include essential Regulatory and Ethical Considerations
• To ensure comprehensive Coordination and Management

Objective 1. To apply a uniform strategic approach to the optimisation, harmonisation and qualification of key assays
This approach towards harmonisation of assays is aimed at facilitating comparison of assay results from Phase I to Phase II trials. Full validation of assays which is usually undertaken in preparation of a Phase III pivotal trial is beyond the scope of this proposal as the validation process is both vaccine and antigen dependent.

The proposed Framework of Action will guide the overall purpose and scope of activities of the action in a coherent, step-wise fashion :

• Optimisation: identification of key variables and improvement of assay components to minimise variability
• Harmonisation: identification of key assay parameters, development of common protocols, establishment of common reagents and controls as well as harmonised and detailed record-keeping
• Qualification: initiation and design of intralaboratory and interlaboratory comparison studies of a harmonised assay using standard reagents and controls to further define the performance characteristics of the assay
• Technology transfer: and capacity building among interested parties

Objective 2. To develop, share, disseminate and implement standard operating procedures (SOP) for the conduct of key assays up to GLP levels
The development of SOPs for key assays clearly elaborated in an unambiguous way (GLP-like) that are transferable between operators in different sites is a cornerstone of harmonising any methodology. Although appearing deceptively routine and simple to some, a coordinated effort is necessary to assess different methods utilised by different groups in systematic steps, evaluate similarities and key differences, and the impact on the read-outs, identify sources of heterogeneity, improve assay efficacy, agree on minimal sets of acceptance criteria for SOPs, and conduct harmonisation exercises with reference preparations in order to select the optimal SOP or SOPs, that will then be implemented.

Objective 3. To develop, share and make available reference preparations and common reagents (sera, antigens, antibodies, cells) for key assays
The quality of the assay components such as purity of the reagents, handling of the samples, parasite cultures or effector cells, as well as differences in methodologies can greatly influence the results of the assay in ways that have nothing to do with the true biological effect meant to be measured by the assay. In the case of peptide and protein antigens and reagents, the heterogeneity of biological fluids and their shift over time and storage conditions, will always be an issue in harmonisation efforts. In the optimisation process therefore, this inherent heterogeneity could be either identified or characterised for a specific assay, and harmonisation efforts set at a realistic, achievable level, which would constitute a marked improvement over the current lack of common procedures for most immunoassays used in malaria vaccine development.
A prerequisite to the success of this action is the development and availability of standards and reference materials, the conditions of their production and storage, as well as the definition and the acceptance of quality control procedures. Expertise in the development, review and maintenance of written and measurement standards will be available through partner 8 (WHO) and via contacts with NIBSC.

Objective 4. To develop, establish and make available a core set of resources and skills essential for achieving the goals of the collaborative action
• Portfolio of key assay protocols
• Portfolio of standard reagents which are components of key assays such as sera, IgG, antigens, antibodies, cells
• Assay hands-on training and transfer of trouble-shooting capacity
• Sample Handling Protocols: collection, storage, transport and utilisation methods
• Systematic database to record and maintain primary data generated
• Standard statistical approaches and tools for appropriate analysis of data
• Regulatory and Ethical considerations

In addition to the availability of key assay protocols, standards and reference reagents, the implementation of harmonised methodology will require the development of capacity within the collaborative action to respond to questions and difficulties regarding the conduct of key assays. The capacity for training and trouble-shooting in the conduct of assays and the facilitation of technology and skills transfer between the partners will be an essential element of the effort and will be integrated into each of the relevant Work Packages. Another critical component to support this effort will be the development of a database of the primary data and statistical analytical methods used in the process of development, and harmonisation of key assays.

Regulatory and ethical requirements will also be considered in the conduct of activities within the action, for example, clinical material used for work by the partners for this effort must meet the standard ethical guidelines of ICH.

Objective 5. To develop and establish a web based tool for communication and dissemination of activities and tracking progress and outcomes
Considering the challenges due to diversity of antigens, approaches and uncertainties on relevant immune responses, it is important that mechanisms be developed for sharing and managing the relevant information and knowledge between partners, whether it is methodology, materials or results, in a systematic, reliable, open and transparent manner. Progress within the overall work programme will occur in a stepwise fashion, and different groups may be at different phases in the pathway to a validated assay. It is critical to be able to document and track current activity, achievements, milestones and outputs from the action partners. A web-based tool will be developed to do this for partners with the aim that as it is further refined, it will be used for dissemination and sharing of outcomes with all interested parties.
The achievement of the objectives of this action should result in harmonised protocols, common reference preparations and reagents and improved capacities for the conduct of assays resulting in high quality scientific data that can support clinical development rationale of malaria vaccines.

Project Results:
C. Main Science & Technology results
a) Recognition of parasite proteins by antibodies
Work Package Leader: University of Edinburgh, Dr David Cavanagh
The aim of this WP was to produce a harmonised IFA method to evaluate and measure antibody reactivity against native parasite proteins. These assays use sera from malaria-exposed humans, sera from malaria vaccine immunised volunteers or antibodies raised in immunised animals. The aim was to develop, establish and disseminate a harmonised assay measuring antibody recognition of parasite proteins that is easily reproducible between laboratories, and of use for clinical trials.

It was agreed at the first OPTIMALVAC consortium meeting in 2009 that, given the limited resources allocated to this WP, the development of a harmonised blood stage IFA assay would be the most useful assay. The assay measures direct reactivity of antibodies to native parasite material and IFA is more easily transferable to most laboratories in Africa.

Harmonisation was conducted in 3 phases:
1. A comprehensive review of current protocols among the participating test laboratories (UEDIN, RUNMC and BPRC) and measurement of the variability between and within laboratories in a first round of IFA assay testing.
Mouse, rabbit, and rat monoclonal and polyclonal antibodies raised against P. falciparum molecules MSP-119, and AMA-1, and a negative control antigen were supplied by UEDIN and BPRC. UEDIN tested these antibodies for endpoint titre in IFA on the same batch of slides to be sent to the participating labs. Antibodies were then sent to the NIBSC for ‘blinding’. As expected, and as planned, the results of IFA assays on these non-titrated individual samples, read by each microscopist in isolation as analysed by NIBSC highlighted a variety of assay variability issues. Identification and scoring of high titre IFA positive samples was consistent between the laboratories, but there were clear discrepancies and errors in scoring negative samples and weakly positive samples.

2. Testing by UEDIN of the sources of variability identified in phase 1 (above) to minimise inter-lab variability, and development of a harmonised IFA procedure.
Testing of each of the variant parameters in the three protocols was carried out at UEDIN and a harmonised OPTIMALVAC IFA Protocol developed (Annex 1).

Variability was identified in the following areas:
• Training/inter-operator variability – experienced microscopists perform better/more accurately than less experienced microscopists
• Specificity/internal controls – need for internal antigen controls in the form of uninfected RBCs
• Microscope type and light sources – variable sensitivity of microscopes
• Fluorophores conjugated to secondary antibodies - different fluorophores have different intensities of fluorescence
• Specificity of secondary antibodies – variability in Ig isotype and/or class specificity (specificity varies - specific for heavy chain or heavy + light chain of primary Abs)
• Quality/specific antigen content of batches of parasite slides – poly-L-lysine (PLL) coated slides proved difficult to standardise due to non-specific background reactivities seen with naïve sera/secondary antibodies alone

Harmonisation was achieved by:
• Use of heavy-chain specific anti-human IgG secondary antibodies from a single supplier conjugated to a single flurophore (FITC)
• Parasite slides prepared by UEDIN in one batch, prepared using a non-PLL method and containing uninfected RBCs as internal antigen negative controls
• Issue of more specific guidelines on interpretation of IFA reactivity for microscopists, and a further emphasis on reference to control wells to assess IFA score robustly

3. Implementation and evaluation of the final harmonised OPTIMALVAC IFA Protocol in all 3 participating laboratories, using human serum samples.
A second round of testing was carried out using 1) human serum pools from African malaria-exposed adults and 2) malaria naïve controls, 3) serum from individuals vaccinated with an experimental GLURP malaria vaccine and 4) a pool of purified IgG positive for malaria antibodies. Again coded, ‘blinded’ reagents were distributed to the 3 labs and samples titrated and tested using the harmonised OPTIMALVAC IFA protocol. Results showed a high degree of concordance, with mean IFA end-points within one dilution between laboratories (Figure 1). As a test of operator variability, a microscopist with little experience in IFA testing was also asked to read the slides at UEDIN. This exercise illustrated both the need for using experienced microscopists for this assay and also the rapid improvement of inexperienced microscopists following comprehensive hands-on training.

A workshop was organised during M36 and African scientists attended and participated in discussions relating to IFA. The workshop attendees and the OPTIMALVAC technical advisory committee enthusiasm were supportive of the developed IFA protocol and participants were keen to have access to the standard reagents used.

Figure 1. High degree of correlation between IFA endpoint titres from all three laboratories in second round testing.
Legend. IFA endpoint titres from three laboratories, tested against 8 serum sample pools from immunised, malaria exposed and naïve donors. IFA endpoint titres from each lab are shown with different symbols. X-axis indicates the serum samples (A to H) tested. Unequivocal identification of IFA-reactive positive samples was achieved by all labs, with no significant statistical differences between labs in IFA titres measured (p>0.05 in all cases, Kruskal-Wallis test with post-hoc Dunn’s multiple comparisons). Significant differences between positive samples and naive samples were observed in all cases and by all labs (p<0.05 in all cases, Kruskal-Wallis test with post-hoc Dunn’s multiple comparisons). Median IFA values between samples were seen to be significantly different from each other, (p value <0.0001, Kruskal-Wallis test).

Summary
A protocol for Immunofluorescent Antibody testing (IFA) testing has been optimised, harmonised and tested by three OPTIMALVAC laboratories (BPRC, RUNMC and UEDIN). The level of agreement in IFA endpoint titres for all samples between the three laboratories was excellent in this exercise. The assay is adaptable to specific uses, including its use with both human antibodies and antibodies raised in immunised animals. This protocol has been uploaded onto the Malaria Reference Reagent Repository website (www.malariaresearch.eu), together with additional guidance notes for modifications in the preparations of the slides for specific purposes e.g. stage specific detection of specific antibodies, or counter-staining for reaction of antibodies to two or more specific epitopes.

b) Cell dependent parasite inhibition assays
Work Package Leader: ImmunoVacc Consulting, Dr Patrice Dubois
The aim of this WP was the optimisation and harmonisation of cell dependent parasite inhibition assays (ADCI) in terms of assay performance criteria, development and selection of a common SOP, IgG or plasma preparation and storage methods, optimal parasite culture conditions (including strain selection and speed of growth), threshold of positive response and to perform intra and inter-laboratory assay variability tests using coded samples and standards.

Two pools of human sera were collected in malaria endemic regions. The first pool was collected in Ivory Coast in the 1990’s and has been characterised with respect to its activity in ADCI. This pool has been shown to contain IgG reacting to a large number of antigens including the blood stage antigen MSP-3. The second pool was collected in Gabon by Dr. Luty. It recognises MSP-1, MSP-2, AMA-1 and GLURP and has also been shown to have ADCI activity. Ethical clearance for the use of both pools in this harmonisation exercise has been granted by the NIBSC ethical review board.

The RAM-1 monoclonal antibody has been produced on a large scale under separate funding from PATH MVI. This antibody is a human IgG1 specific for P. falciparum MSP-3 (D5). The RAM-1 monoclonal antibody has demonstrated ADCI activity in vitro in Dr Druilhe’s laboratory. This monoclonal antibody serves as a standard control antibody in harmonisation assays.

Tubes that could be used for the filling of ADCI reagents while satisfying a number of pre-set criteria were identified and filled at NIBSC and QC performed by Institut Pasteur. A first set of tubes filled with reagents was sent by NIBSC to Institut Pasteur for QC testing and did not fulfil acceptance criteria. Following QC tests, it appeared that the heat-sealed this first selection of tubes was not adequate for storage of ADCI reagents. Four alternative sources of tubes were then QC tested in August 2011. The Corning® 384 (2D Bar Coded Storage Tubes, Round, Without Plug Caps, Racked) tubes were selected. Although this activity was to be initially completed by M20 it in fact required until M31 to complete. The standard reagents were then directly distributed to the participating laboratories.

A set of guidelines and a list of acceptance criteria were agreed by all WP2 participants and consensus procedure for the conduct of ADCI was made available prior to testing of standard reagents (Annex 2). Likewise, a set of guidelines for handling of reagents and filling was issued.

The intra and inter laboratory assay variability tests were completed in M36 as planned. Data shows that significant variability was observed in different laboratories. In addition, parasite inhibition by IgG alone was found to be substantial (45-50%) in data from two participating laboratories. The cause of this inhibitory effect has not been identified yet, but is under further investigation.

Practical training activities involved training of participating laboratory staff in implementing the new consensus procedure while taking into account principles outlined in the guideline on assay performance and acceptance criteria. A workshop was organised during M36 and six (6) African scientists attended and participated in discussions relating to ADCI. The attendees to the workshop showed enthusiasm for the developed protocols and were keen to have access to the reagents supplied by NIBSC.

Summary
The group has agreed on a consensus ADCI procedure, conditions for aliquoting ADCI sera and IgG preparations into small tubes were identified and implemented, the standard reagents were distributed to participating laboratories and finally, testing was performed in these participating labs.

c) CMI assays
Work Package Leader: ImmunoVacc Consulting, Dr Patrice Dubois
The aim of WP3 was to harmonise T cell-mediated immune assays in the context of malaria vaccine development. Activities included the identification of key parameters in ELISPOT and intracellular cytokine staining (ICS) which are relevant to malaria vaccine evaluation. WP3 benefited from experience gained by other groups including the HIV and TB communities which are, or have already been involved in harmonisation of these assays. The optimisation and harmonisation of the T cell assays was done in close consultation with the HIV and TB vaccine communities. It also participated in ongoing global efforts in T cell assay harmonisation sponsored by the MVI.

Standard reagents for the detection of T cell responses for the purpose of harmonisation were identified. Tetanus Toxoid (TT) and CEF peptides (a pool of MHC class 1 binding peptides derived from the CMV, EBV and influenza viruses) have been selected for activation of T cell responses. A large supply of TT has been provided by the Serum Institute of India as a donation to the OPTIMALVAC project and the CEF peptide pools have been purchased in small aliquots from a commercial source (JPT Peptide Technologies GmbH). Both antigens were used for the activation of T cells in ELISPOT and ICS assays. A supplier of human peripheral blood mononuclear cells (PBMC) has been identified (Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland). PBMC donors were screened and the ones selected which show responses to both TT and the CEF peptide pool. This activity was funded by PATH Malaria Vaccine Initiative (PATH MVI). The CHUV informed consent form used for enrolment of donors was approved by the PATH Institutional Review Board (IRB).

NIBSC has been identified as the centre for distribution of activation reagents and supplies for harmonisation of T cell assay. NIBSC will distribute reagents such as TT and the CEF peptide pool. The critical activity of this WP was the distribution of standard reagents and panels including vials of frozen PBMCs from several donors which responded to the two activation reagents provided to participants (the peptide pool CEF and TT). This activity was also supported by PATH MVI.

The OPTIMALVAC guidance documents for monitoring T cell responses using ELISPOT and ICS were aligned with existing SOPs currently in use in the HIV community as these are based on several round of harmonisation. Duke University kindly provided their SOPs for ELISPOT and ICS assays in July 2010. These were used as a basis for drafting of guidance documents issued to participating laboratories (Annex 3 and 4). These guidelines also took into account the group’s consensus on parameters to investigate in the context of malaria.

The testing of PBMCs panels using ELISPOT and/or ICS was conducted in each laboratory and the results were entered into an Excel template before being sent for compilation and generation of graphs. In addition, each laboratory sent their ELISPOT plates for central reading at NIBSC while also conducting a comparative study using their own in house protocol as well as the OPTIMALVAC recommended procedure.

For ELISPOT the results of comparative testing show that cell viability was consistent in all participating laboratories. Backgrounds were found to be homogenous across labs and were generally acceptable. When looking at responses to CEF peptide pool and TT there was agreement on whether a sample was positive. However, some variations in response levels were observed when using the OPTIMALVAC consensus procedure for samples analysed in each participating lab. Variability in response levels was somewhat higher when the reading of plates was done at the central facility. A comparison of the in house protocol with the OPTIMALVAC consensus protocol is still ongoing.

For ICS, significant variability was observed for both CD4 and CD8 T cell responses regardless of the cytokine combination for all three PBMC donors. At the last OPTIMALVAC meeting (March 2012) it was recommended that all flow cytometry raw data should be uploaded on the OPTIMALVAC FTP site so as to be able to perform central analysis and help in identifying sources of variation. This analysis is ongoing. ICS reagents and guidance documents have been made available to six (6) African institutions in malaria endemic areas that are involved in malaria vaccine clinical trials.

In conclusion, this harmonisation project has helped the malaria vaccine community to identify some of the key aspects of the assays which need to be worked on in order to improve reproducibility and consistency across labs. The better overall performance of the ELISPOT assay also highlights the importance of having a single procedure used by all labs. Although this was not possible in the case of ICS assay because of the diversity of flow cytometer model used to acquire the data, it may help in the future to work with subgroups of labs using the same flow cytometry equipment.

d) Repository of resources
Work Package Leader: University of Edinburgh, Dr David Cavanagh
The aim of this WP was to establish a well maintained and managed physical Malaria Reference Reagent Repository containing standard, well-characterised parasite clones, antibody preparations, monoclonal antibodies and purified antigens, stored under agreed conditions at several sites, for dissemination on request.

Methods:

A Malaria Reference Reagent Repository has been set up by UEDIN that presently supplies the following reagents to the global malaria community, with the following uses in malaria vaccine development assays:
• Standard, characterised Plasmodium falciparum parasite lines and clones, for which serological and genotyping data is available. (ADCI, GIA, IFA)
• Parasite antigen preparations (parasite extracts for western blotting (WB), immuno precipitation etc.)
• Parasite DNA (PCR genotyping)
• Parasite antigen slides (IFA)
• Monoclonal antibodies from mouse hybridoma lines recognising MSP-1, MSP-2, GLURP, Exp-1 and -2 (controls for immunological assays such as IFA and WB)
• Humanised mouse monoclonal antibodies against MSP119 (IgG1 and IgG3 subclasses) (controls for GIA, IFA)

A website www.malariaresearch.eu has been established for management and dissemination of these resources.
In collaboration with other partner laboratories in OPTIMALVAC and EMVDA the www.malariaresearch.eu website also hosts the virtual repository with information about reagents which may be available from other laboratories in Europe including:

1. A standard rabbit IgG preparation against AMA-1 (BPRC) – used as a control for parasite GIA assays
2. RAM-1 – standard control antibody as an ADCI control
3. MalPIAG – standard pool of human serum as an ADCI positive control
A list of reagents that have been supplied from the repository over the last 3 years is available on request.

It is anticipated that more reagents will be deposited as the repository becomes more widely advertised. Submission forms are available on the website for depositors of reagents to provide information about new reagents added to the repository collection. UEDIN would be able to provide further services according to need as funding becomes available. At present the material is provided free with the cost of transport borne by the recipient. With the end of OPTIMALVAC and an additional Wellcome Trust one-year grant, which will end in Q3 2012, funding for this repository is urgently being sought.
More detailed lists of all reagents and/or material available can be supplied on request by email to david.cavanagh@ed.ac.uk

Standard Operating Procedures (SOPs) have been developed and up-loaded onto the www.malariaresearch.eu, www.optimalvac.eu and www.euvaccine.eu websites for the following procedures:

IFA
GIA
ELISA
Culture of P. falciparum
Synchronisation of P. falciparum

Protocols used by OPTIMALVAC for other WP will be added as they are finalised over the rest of 2012.

More detailed lists of material available may be viewed in Annexes 2 & 3 of WP D4.4

Results:
A physical and virtual Malaria Reference Reagent repository has been set up and curated by UEDIN. The repository has over 380 P. falciparum strains/clones, as well as over 50 characterised hybridomas specific for parasite antigens. The website, www.malariaresearch.eu has been set up to allow dissemination of harmonised SOPs for malaria vaccine development assays, and to provided information regarding the characteristics and availability of the reagents.
Over the lifetime of the project 20 requests for reagents have been fulfilled and a variety of repository reagents have been supplied to the malaria community in Europe, Africa, USA and Australia.

Conclusions:
All the aims of this WP have been achieved. Reference reagents are available and a sub-section of these form standardised controls for assays. SOPs and guidance notes have been produced and are freely available via the website of the database. Other reagents and information are available on request.

Significant results:
1. A Malaria Reference Reagent Repository has been established.
2. A website www.malariaresearch.eu has been established for dissemination of reagents and protocols.
3. Standard Operating Procedures (SOPs) have been developed and up-loaded onto the www.malariaresearch.eu website for the following procedures: IFA, GIA, ELISA, culture of P. falciparum, synchronisation of P. falciparum.
4. Training of one African researcher, Emmanuel Kakra Dickson, from Noguchi Memorial Institute for Medical Research, University of Ghana in ELISA, GIA and IFA was completed in July 2010.
e) Data management and statistical analysis
Work Package Leader: Biomedical Primate Research Centre, Dr Ed Remarque
The aim of this WP was to develop and utilise standard and appropriate data management tools that fully support assay validation. This included harmonising data calculations, statistical analyses, reporting and storage of data for each of the assays described in WP1-3. In addition, it comprises the statistical analysis of data obtained in the participating laboratories for each of the assays described in WP1-3. The dissemination of results is effectuated through a web space, allowing the participating laboratories to upload results emanating from the assays optimisation and harmonisation effort, and download harmonised SOPs, software tools and the results from statistical analyses. The software tools were designed in such a way that the data audit trail remains transparent and facilitates statistical analyses.

Validated software tools are available for ELISA and GIA. Validated software tools and methods for statistical analysis were expanded to cover IFA and ADCI. These software tools provide harmonised data report formats, define how the data audit will be kept and provide validated software tools for calculating assay readouts and data management, methods for statistics analysis and validated software tools for harmonised statistical analyses. The software tools and SOPs are made available for partners and third parties via the OPTIMALVAC, EVI and www.malariaresearch.eu websites.

The web space for communication between partners has been set up and is accessible at www.optimalvac.eu. Project internal documents are secured and accessible by login with username and password.

A data upload area was created on the website (www.malariaresearch.eu), where data emanating from WP3 (FACS and ELISPOT) have been uploaded using harmonised reporting templates.
Data emanating from WP1 (IFA) were created by three participating labs and did not require a dedicated upload space, the data have been analysed and the results have been presented by the WP1 leader. Data emanating from the laboratory work in WP2 and 3 have or will be uploaded to the website. Harmonised reporting formats have been produced for WP3 and results will be analysed and made available on the website after all data have been submitted.

The main sources of variation have been identified allowing a better harmonisation of the IFA. The analysis from WP1 has revealed that a better agreement on IFA end-point titres can be achieved by clearly defining the criteria for scoring positive or negative. This was achieved by the provision of slides and sera that yielded negative, (faint) positive and strongly positive. The interpretation of IFA end points could be further improved by the preparation of serial dilutions of the antibody reagents.
Data from WP2 are limited and as no data are yet available from experiments where harmonised reagents have been used, no conclusions can be drawn on potential sources of variation. Data from WP3 are currently emanating and will be analysed soon. The project has been delayed due to late availability of reagents and cell samples, the data required for analysis have been produced for WP3, but more time is required for a thorough analysis of sources of variation.

Guidelines for performing the ADCI, ICS and ELISPOT experiments have been provided and these will form the basis for SOP’s that will be prepared after the sources of variation in the ICS and ELISPOT have been identified and adequately addressed. The latter will be placed on the www.optimalvac.eu, www.euvaccine.eu and www.malariaresearch.eu websites when available.

f) Regulatory and Ethical Considerations
Work Package Leader: European Vaccine Initiative, Dr Babatunde Imoukhuede
WP6, Regulatory and Ethical Considerations, of the OPTIMALVAC EC-project required the development of an informed consent template to be considered or modified by partners and other users outside the consortium, that could allow for utilisation of leftover samples without breaching ethical or normative guidelines.
The specific objectives of the WP were:
1. To ensure that materials utilised and conduct of activities meet with appropriate regulatory considerations for the level of development
2. To ensure that materials utilised in all activities to generate the outcomes and deliverables of the project are collected according to accepted ethical guidelines
3. The specific objective of the WP was to explore the formulation of informed consent forms in a standard way for clinical trials to enable the potential utilisation of leftover specimen for future immunoassays that are later found to be useful for gathering further information on potentially important immune responses.
A questionnaire was distributed to the project participants to assist in identifying the potential regulatory and ethical issues related to development of standardised assays through OPTIMALVAC, the partner institutions ethically acceptable procedure for dealing with collected samples for research use, the ethical and regulatory issues with transport of sera and samples from Europe to Africa and vice-versa for research purposes as well as how long samples of this nature can be stored or retained at the various institutions. The questionnaire also required that the partners provided current informed consent templates which were in use at their various sites. The templates received from partners were amended to reflect the relevant issues in the process that emerged from the questionnaire and this was incoporated into the proposed informed consent templates. The consent form templates developed were in two parts:
a. Consent form A: Written consent for blood donors
b. Consent form B: Written consent for adults or mature-minors or parents/guardians of children participating in the study
The consent forms were then reviewed by two independent reviewers, recommended by project partners and who were considered experts in the field of Ethics. The reviewers were, Dr Aisatou Toure, Institute Pasteur, Dakar, Senegal and Dr Reider Lie, National Institute for Health, USA. Their very valuable comments were taken into consideration in producing the final version of the documents (Annex 5 and 6).
These templates are expected to assist in ensuring the ability to plan for and ethically utilise leftover specimen samples for relevant analysis.

g) Management and coordination
Work Package Leader: European Vaccine Initiative, Dr Odile Leroy
The consortium management tasks in WP7 were to ensure that the project is conducted efficiently within the predetermined timing and budget, to establish management tools and methods for communication with the EC and between participants and for monitoring progress towards objectives, deliverables and milestones, to prepare regular EC progress, financial and management reports as well as a final report of the results, to organise meetings as necessary and to conduct the financial and administrative management of the project.

In 2009, the OPTIMALVAC project management team was established with Dr Odile Leroy as Coordinator who is assisted by the EVI Finance Manager and a project manager responsible for the day-to-day management and smooth running of OPTIMALVAC. A Consortium Agreement was prepared and negotiated with the project beneficiaries and was fully executed in November 2010. The Steering Committee and Technical Advisory Committee were formed.

The project kick-off meeting was held on 07 - 08 May 2009 at the WHO Headquarters, Geneva, Switzerland; the first annual meeting was held on 04 May 2010 in Berlin, Germany. The OPTIMALVAC second annual meeting was held in Heidelberg, Germany on 02 May 2011 and the final annual meeting was held on 21 March 2012 in Paris, France. These annual meetings in 2011 and 2012 were accompanied by workshops on WP1, 2 and 3 activities on 03 May 2011 and 21 – 22 March 2012. During these meetings and workshops the work achieved under OPTIMALVAC was presented and future plans were discussed.
Each face-to-face meeting was joined to a Steering Committee meeting. Two face-to-face meetings for the SC were held, 02 May 2011 in Heidelberg, Germany and 22 Mar. 2012 in Paris, France. Several SC teleconference meetings have been held in order to ensure progress and communication between participants.

One TAC telephone conference was held on 09 March 2010 and three TAC face-to-face meetings and were held on 04 May 2010 in Berlin, Germany, 03 May 2011 in Heidelberg, Germany and 22 Mar. 2012 in Paris, France.

Annual, Periodic, Final and Financial reports were submitted to the EC according to timelines in the grant agreement. Communication material like leaflets, posters, and the websites have been developped to disseminate OPTIMALVAC’s activities.

h) Global coordination
Work Package Leader: World Health Organization, Dr Vasee Moorthy
Malaria Vaccine assay harmonisation can now be viewed as a jointly planned activity at the level of the funders, with individual implementation in multiple projects (see letter published in Vaccine for further details on the global malaria vaccine assay harmonisation activity).

The outcomes of OPTIMALVAC will aid other funders in their necessary follow-on activities, and WHO, MVI, USAID and EVI are all now well integrated in their assay harmonisation activities. The EU funding through OPTIMALVAC and EMVDA has been central in developing a European platform for integration, and in linking with leading groups transatlantically.

OPTIMALVAC has facilitated links between HIV, TB, cancer and malaria assay harmonisation communities in much more robust ways than was the case previously. These links have been very helpful for the progress that has been made in T cell assay harmonisation, but have less relevance to ADCI, which has some malaria-specific elements such as parasite culture.

Links between communities are now not the limiting factor for further assay harmonisation activities; this is a major contributing factor from OPTIMALVAC.

A T cell assay/service centre is likely to be selected soon with some input from the OPTIMALVAC activity. It has been agreed that any reference centre for T cell assays will be available to the whole malaria vaccine community, so here a formal integration has been achieved.

ADCI is not yet advanced enough for selection of a service/reference centre, but there are laboratories that have the potential to take on this role in the future. As a result ADCI-supported antigens rest on field efficacy for proof-of-concept and remain a higher risk approach because of the lack of a challenge model, and the lack of a harmonised immunoassay.

IFA has a harmonised assay that could be progressed further towards a service/reference centre, if funds were available. Many of the existing external quality assurance, and standardisation guidelines for IFA in other contexts (e.g. clinical pathology, and clinical immunology) could be applied to develop a reference centre or qualify the assay in-house.

Multiple follow-on activities will occur, as detailed above. OPTIMALVAC has played an important role in the malaria vaccine technology roadmap area of immunoassay standardisation, and every avenue for following-on towards reference centres, or assay harmonisation will be followed by WHO.

Potential Impact:
Potential impact and dissemination activities

OPTIMALVAC has made an immdiate impact on malaria vaccine development through the creation of directly available resources such as standard reagents, guidance documents, consent forms and SOPs for malaria vaccine researchers and developers.
a) Potential Impact
Capacity building

The SOPs developed in OPTIMALVAC and the standard reagents have been made available to external collaborators in the US and India: the Infectious Disease Research Institute (IDRI), the National Institute of Health (NIH), the Seattle Biomedical Research Institute (SBRI), Walter Reed Army Institute of Research (WRAIR) and the International Centre for Genetic Engineering and Biotechnology (ICGEB). These laboratories were involved in reagent testing and in protocol harmonisation.

OPTIMALVAC contributed to several training activities, e.g. one African researcher from Ghana was trained in ELISA, GIA and IFA by UEDIN.

Within the last months, OPTIMALVAC extended its collaboration and included six (6) different African centres in malaria endemic countries. All African centres are involved in malaria vaccine clinical trials and will use the standard reagents and the harmonised ICS protocol to validate their equipment and assay performance used in vaccine clinical trials.
• Medical Research Unit Albert Schweitzer Hospital (ASH), Lambaréné, Gabon
• Centre National de Recherche et de Formation sur le Paludisme (CNRFP), Ouagadougou, Burkina Faso
• Kenya Medical Research Institute (KEMRI), Kilifi, Kenya
• Medical Research Council (MRC), The Gambia
• Malaria Research and Training Center (MRTC), Mali
• Noguchi Memorial Institute for Medical Research (NMIMR), Ghana

African scientists from these institutions were invited and attended the OPTIMALVAC Final Meeting and the WP1-3 workshop on 21 – 22 March 2012 in Paris, France: Issa Nebie Ouedraogo (CNRFP), Marguerite Massinga Loembé (ASH), Jedidah Mwacharo (KEMRI), Jainaba Njie-Jobe (MRC Gambia), Daniel Dodoo (Noguchi) and Bourema Kouriba (MRTC).

Harmonisation

The extension of the OPTIMALVAC reagent and protocol harmonisation efforts to external partners shows the increased interest of scientists in harmonisation activities. Whereas the need for harmonised assays was not obvious to researchers in the malaria community, OPTIMALVAC has contributed enormously in raising this awareness. Connections to the TB, HIV and cancer harmonisation communities are established and further joint efforts are expected to continue.
The outcomes of OPTIMALVAC will aid other funders in their necessary follow-on activities, and WHO, MVI, USAID and EVI are all now well integrated in their assay harmonisation activities.

Knowledge sharing and networking

The www.optimalvac.eu, www.euvaccine.eu and www.malariaresearch.eu websites will be important sources of information and will contribute to strengthen the vaccinologist and malaria networks by providing SOPs and protocols as well as standard reagents and parasite strains/lines to the research community.

b) Dissemination activities

The publications and other dissemination activities are listed in section 3.

Communication materials: Leaflets, posters, and websites have been developped.

Presentations:
The OPTIMALVAC activities have been presented in several conferences and meetings. Presentations have been made by WHO, or facilitated by WHO, including the role of OPTIMALVAC at the following meetings:

• American Society of Tropical Medicine & Hygiene in November 2009
• WHO MALVAC November 2010 and February 2012
• Multilateral Initiative for Malaria, Nairobi, December 2010

Harmonisation and standardisation of Immuno Assays, EVI Rendez-Vous, Heidelberg, Germany, November 2011

In addition malaria vaccine assay harmonisation has been included by WHO as a prominent item on malaria vaccine funders group meetings since 2009.

OPTIMALVAC has facilitated links between HIV, TB, cancer and malaria assay harmonisation communities. Further collaborations are initiated.

Related information

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EUROPEAN VACCINE INITIATIVE - EEIG
Germany
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