We are now two years into the FLUCOP project, two years towards standardisation and development of assays for assessment of influenza vaccine correlates of protection. Working on this project it is stunning to see how fruitful collaborative work between industry and academic partners can be. After an initial phase of logistical planning and organization, laboratory work is now well underway in all the Work Packages (WP). In this newsletter we are pleased to present to you an overview of the first standardisation pilot studies that have been conducted and a description of the role of some of the key-partners involved. Furthermore, you can read about new technologies developed under FLUCOP, targeting population based evaluations of influenza vaccines and correlates of protection.
Please enjoy reading our newsletter and don’t forget to have your calendar ready when browsing under ‘upcoming events’ for interesting meetings & events.
The FLUCOP project has several main objectives, which are also reflected in its work package (WP) structure. In this newsletter we would like to share progress made on standardisation of the haemagglutination inhibition assay (HAI, WP1) and on cell-mediated immunity (CMI) assays as tools for evaluating influenza vaccine performance (WP2).
Standardisation of Haemagglutination inhibition assay:
The first steps have successfully been taken on the path towards a standardised HAI assay, namely: protocols from collaborating laboratories were shared and analysed to identify sources of variability, the parameters most likely to have an impact on the assay performance were identified and an initial consensus protocol was distilled for further evaluation. In order to facilitate the conduct of collaborative studies, a centralized serum sample management facility was established for the whole Consortium, to provide all participating partners with identical samples and reagents. Given that decisions on how to improve and standardise assays should not only be based on theoretical considerations, but empirically tested in the laboratory, a pilot study was designed to assess parameters judged to be potentially capable of generating variability in the results obtained by different laboratories. This DoE (design of experiment)-based study was designed in a collaborative effort between laboratory scientists, statisticians and data management experts and was carried out by the participating laboratories using similar serum samples. The data that were generated in this study are being analysed by the statistical and data management teams at the time of writing. We will keep you updated about the final results via the FLUCOP webpage, newsletter and publications. To gain more insight now, you can get to know some key-partners involved in this study under ‘Inside FLUCOP’, or better yet say ‘hi’ and discuss with them during an Influenza meeting.
Besides a leading role of NIBSC – as described below under ‘Inside FLUCOP’ – Dr Catherine Caillet from Sanofi Pasteur acts as a co-leader of in WP1. As an industrial partner her expertise in process validation processes and interaction with regulatory agencies is key to develop standardised and validated HAI and VN assays. Other involved partners are:
Department of Health, Universitetet i Bergen, Fondazione IRCCSCG Ca’ Granda Ospedale Maggiore Policlinico, University of Siena, Quinten, Biomedical Primate Research Centre, GlaxoSmithKline, CRUCELL Holland BV, Abbott Biologicals BV
Cell-mediated immunity (CMI) assays:
Besides classic serological assays, such as HAI, FLUCOP also strives to advance the understanding and application of cell-mediated immunity (CMI) assays as tools for evaluating influenza vaccine performance. This work is undertaken in WP2 and consists of three major tasks, namely (1) the collection of biological materials to conduct WP2 studies, (2) to create a standardized protocol for the separation, freezing and shipment of peripheral blood mononuclear cells (PBMC) and (3) to standardize and evaluate assays to measure influenza-specific cell-mediated immunity.
The first task, namely the creation of a biobank with PBMC, was initiated in 2015 and is now largely accomplished. PBMC have been collected pre- and post-vaccination, from blood samples from subjects vaccinated during flu seasons 2015/2016 and 2016/2017, as well as from blood donors (buffy coats from which the most influenza-responsive ones will be selected). In total c. 2000 vials have been cryopreserved from the blood samples collected. The influenza-specific responses of these cells will be measured using the ICS technique (intracellular cytokine staining).
In the past 12 months the main focus of WP2 has been on the standardisation of PBMC isolation and freezing. Similar to the preparation of the pilot study in WP1, Standard Operation Procedures (SOP) from all participating partners have been collected and from these a selected number of critical parameters identified. Test protocols were designed to investigate the impact of these critical parameters on the quality of cell preparation and preservation. Three participating laboratories have processed blood samples according to these proposed test protocols and compared them to their own in-house protocols, with which they were most familiar. Analyses of the integrity and functional qualities of the cells using ELISPOT and ICS allowed the Consortium to discern the impact of the critical parameters initially identified. Results will be presented during the upcoming annual meeting in Siena, April 2017.
The main upcoming event with regard to the quality of CMI assessment is a workshop on assay validation, which will take place immediately after the annual meeting in April 2017. Representatives of Ghent University, GSK and Crucell will jointly give a presentation on the validation of ELISPOT and ICS assays.
Contributing partner: Ghent University, CRUCELL Holland BV , The Chancellor, Masters and Scholars of University of Oxford, University of Bergen
Part of the WP2 team at the FLUCOP annual meeting 2016
New technologies for population based evaluations of influenza vaccines
The goal of WP4 is to develop and test new, potentially useful technologies that will aid in defining biomarkers for correlates of protection against influenza, induced either by vaccination or infection. With the advent of novel generation vaccines using different production and antigen presentation platforms, new correlates will have to be established and implemented for the evaluation of efficacy and effectiveness. Under WP4 novel methods and assays are developed that could supplement existing technologies in future influenza vaccination studies. At the Erasmus Medical Center a protein microarray technology was developed. This method allows the detection of serum antibodies to a large panel of influenza viruses simultaneously with only small volumes of serum samples. This method is particularly useful for assessing the history of previous exposures with influenza viruses and for determining pre-existing immunity prior to vaccination, which is one of the most important factors that influence vaccine efficacy. Thus, results obtained with the protein micro array should advance our understanding of the performance of influenza vaccines and the impact of pre-existing immunity.
Also at the Erasmus Medical Center, an assay was developed to measure an alternative, immunologically relevant, activity of antibodies, i.e. antibody dependent cellular cytoxicity (ADCC). In addition to antibodies with virus neutralizing capacity, also antibodies that mediate ADDC have been recognized as a correlate of protection. Furthermore, because a proportion of these antibodies are also directed to conserved regions of the hemagglutinin molecule, they may also afford some degree of cross-protective immunity. For the development of the ADCC assay the investigators took advantage of the use of a NK cell line that expresses the Fcγ receptor (CD16) and recombinant hemagglutinins as source of viral antigen. Activation of the NK cells was assessed by CD107a expression and used as a surrogate for ADCC activity. It was shown that both after infection and vaccination influenza A(H1N1)pdm09 virus-specific ADCC mediating antibody responses were induced. Upon vaccination with a trivalent vaccine also the induction of ADCC antibodies to the influenza B and A(H3N2) component was demonstrated. This FLUCOP funded work was published recently (de Vries et al. Vaccine 2017 35(2):238-247). At the Artemis One Health Research Foundation, methods were developed for metadata analysis regarding influenza vaccine effectiveness. The methods and algorithms were applied to the comparison of the vaccine efficacy and effectiveness of quadrivalent and trivalent influenza vaccines which indicated an age-dependent benefit of the use the QIV over TIV. This study also sets the benchmark for future influenza vaccines. In the future these novel approaches to study vaccine efficacy can be used to define novel correlates of protection of new alternative vaccine preparations.
Inside FLUCOP: Two to standardise them all / Partner profile – NIBSC
The major focus of the FLUCOP project lies on standardisation of immunoassays to advance R&D in the field of influenza vaccination. Who would then be a better suited specialist to work on tasks of standardisation than the Influenza Resource Centre (IRC) of the National Institute for Biological Standards and Control (NIBSC), particularly, with NIBSC being the leading laboratory for international standardisation of biological medicines in the world? NIBSC, which is part of the Medicines and Healthcare products Regulatory Agency in the UK, is involved in two work packages of FLUCOP (WP1, WP3). Their larger effort is concentrated on WP1, for which NIBSC’s Dr Othmar Engelhardt is a work package co-leader, and to which Dr Joanna Waldock contributes as a postdoctoral scientists.
More than 95% of World Health Organisation (WHO) International Standards are supplied by NIBSC. In addition, NIBSC also produces various other types of standards. In the influenza area, NIBSC is one of four WHO Essential Regulatory Laboratories (ERLs). One of their main tasks is to produce reagents for the standardisation of potency testing of inactivated influenza vaccines. Due to the ever-changing nature of influenza viruses, this is comparable to a Sisyphean task: As the WHO reviews influenza epidemiology and evolution to issue recommendations on the components of seasonal influenza vaccines, new reagents have to be generated and calibrated every time a component of the vaccine changes.
In addition to its activities as an ERL, NIBSC has a long standing interest in the standardisation of influenza serology. Through collaborative studies, some organised by NIBSC, it is known that the traditional influenza serology assays (haemagglutination inhibition [HAI], virus neutralisation [VN] and single radial haemolysis [SRH]) are highly variable. This makes comparison of data generated in different laboratories challenging. As a consequence, the strength of HAI as a widely accepted correlate of protection has been questioned. NIBSC generated the first WHO International Standards for antibody to influenza; these were shown to reduce inter -laboratory variability in HAI and VN. Our strong background in serology standardisation meant that the NIBSC team was excited about working with a network of European serology and industrial laboratories within FLUCOP to advance standardisation. Since the start of the FLUCOP project, NIBSC has been very active in particular on work related to the harmonisation of the HAI assay, and is also making important contributions to the standardisation of the VN assay, as well as to developing methodologies to measure responses against Neuraminidase.
NIBSC staff directly involved in FLUCOP:
Dr Joanna Waldock: Jo completed her PhD at Imperial College London in arthropod immunology in 2010. She studied the innate immune responses of mosquitoes to viral infection, to better understand the host-pathogen interactions that define the capacity of insects to spread disease. She then continued in the field of arthropod disease vector biology and infectious disease during a post-doctoral fellowship at The Cyprus Institute. Here she helped to develop a climate-based model of mosquito habitat suitability across the globe to study climate-change impacts on disease vector distribution, additionally studying viral infection in arthropod vectors at Imperial College. Continuing in the field of virology, Jo started at NIBSC in 2015, where she works in the IRC, primarily focusing on the FLUCOP project. She has led the collection of HAI protocols and the formulation of an HAI consensus protocol, produced materials that were shared between all laboratories participating in the first HAI pilot study, and completed laboratory work for this pilot study. In addition, she is working with other partners on the harmonisation of the VN assay and is conducting experiments within WP3 that will elucidate various questions about immunoassays designed to measure antibodies directed against influenza virus neuraminidase (NA), another important vaccine component.
Dr Othmar Engelhardt: After studying food and biotechnology in his home country of Austria, Othmar went into virology through his PhD research on influenza virus. With the exception of a four-year intermission working on interferon-induced antiviral proteins and the much neglected orthomyxovirus Thogoto virus, Othmar has stayed in the field of influenza since his PhD work. He joined NIBSC in 2006 and is a principal scientist in the Division of Virology. His interests at NIBSC include, in addition to influenza serology, the generation and improvement of influenza candidate vaccine viruses, in vivo models of influenza pathogenesis and for vaccine evaluation, the standardisation and control of influenza vaccines and the development and assessment of new potency assays for inactivated influenza vaccines; more recently, he has also become involved in work towards the standardisation of serology for RSV. Within FLUCOP, he is co-leader of WP 1 with Dr Catherine Caillet from Sanofi Pasteur and a member of the FLUCOP Consortium steering committee.
Dr Joanna Waldock and Dr Othmar Engelhardt at their lab in NIBSC
FLUCOP members actively involved in workshop on “Immunoassay standardisation for universal influenza vaccines”
FLUCOP partners (like Othmar Engelhardt from NIBSC) are also active in other important areas of influenza research, like the development of the next generation of influenza vaccines. One of the biggest goals in this field is the development of so-called “universal” influenza vaccines, namely, broadly reactive influenza vaccines, which would, with one immunisation, protect against a wide variety of influenza virus types, hence abolishing the need to update the composition of the vaccine every season. The development of such vaccines calls for identifying the most appropriate immunoassays to evaluate their capacity to generate broad immune responses, and also to explore a path towards the standardisation of such assays. To address this critical topic, a workshop on “Immunoassay standardisation for universal influenza vaccines” gathering more than fifty experts from around the world was organised by the EDUFLUVAC consortium and a report summarising the outcome of the discussion was recently published on Influenza and Other Respiratory Viruses journal.
The periodic replacement of viruses contained in influenza vaccines is necessary in order for the vaccines to be effective due to the constantly evolving nature of influenza viruses, including those circulating and infecting humans.
Twice annually, WHO organizes consultations with an advisory group of experts to analyse influenza virus surveillance data generated by the WHO Global Influenza Surveillance and Response System (GISRS), and issues recommendations on the composition of the influenza vaccines for the following influenza season. These recommendations are used by national vaccine regulatory agencies and the pharmaceutical companies to develop, produce and license influenza vaccines.
Influenza Vaccines for the World – IVW 2017 is the sixth international conference and exhibition in this important series of influenza vaccine meetings. The IVW conference series focuses on ‘Influenza Vaccination Issues’. The IVW series is an international forum for world renowned experts in the field of influenza vaccines and related issues (adjuvants/delivery/vaccination strategies) to report on the latest data and trends associated with current and new influenza vaccines/technologies and their availability/delivery/implementation worldwide.
19-21 April 2017, University of Lausanne, Lausanne, Switzerland
The European Scientific Working group on Influenza (ESWI) is looking forward to organizing the sixth edition of its ESWI Influenza Conferences in Riga, Latvia, on 10 – 13 September 2017. Over the past years, the ESWI Influenza Conferences have grown into the largest European scientific conferences entirely dedicated to influenza. In keeping with its excellent scientific reputation, the upcoming sixth edition will give the floor to the most renowned influenza scientists. Additionally, the conference will have a Science Policy Interface track that is a separate, tailor-made program for public health officials and opinion leaders in healthcare work. They will be given the opportunity to participate in a series of seven sessions that cover the entire spectrum of influenza policy making.
MVADS 2017 (Modern Vaccines Adjuvants & Delivery Systems) is the 6th international conference in this series addressing current research on novel vaccines and adjuvants and delivery systems for these biologics. It will focus on updating the community on new adjuvant/delivery systems/technologies associated with developing modern vaccines strategies and vaccine research.
MVADS 2017 will be of interest to researchers/contributors from academic programs, industrial, governmental and regulatory groups.
12-14 September 2017, Porto, Portugal
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The FLUCOP project is supported by the Innovative Medicines Initiative Joint Undertaking under grant agreement 115672, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP/2007-2013) and EFPIA companies’ in kind contribution. You can contact us at: http://www.flucop.eu/contact/