we are now approaching the end of the third year of the FLUCOP project, our efforts to standardise and harmonise assays utilized for measuring immune responses in humans against seasonal influenza vaccines are bearing more and more fruits now.
In this newsletter we will give you an update on the recent progress we have achieved in the development of assays to detect neuraminidase (NA)-specific antibody responses for evaluating influenza vaccines immunogenicity and in the development of new technologies that eventually could be applied to population based evaluations of influenza vaccines.
As pointed out previously, interactions between the project partners work very well and demonstrate how productive such collaborations in the context of public-private partnerships can be.
Working on this project it is stunning to see how fruitful collaborative work between industry and academic partners can be.
Rapid progress is being made on developing and standardizing assays to evaluate NA-specific antibody responses in influenza vaccinees.
The contribution of anti-NA responses to the immunogenicity of influenza vaccines has recently gained increased attention amongst manufacturers, researchers and regulators. Objective of this work is to advance the understanding and application of NA-Antibody titration assays as tools for evaluating influenza vaccine performance.
Since the beginning of the FLUCOP project, the progress has been considerable. After a thorough investigation and inventory of specific NA-antibody titration assays, followed by the comparison of the available protocols within the different partner laboratory, the work focused on two assays: the Enzyme-Linked-Lectin-Assay (ELLA) and the Enzyme-linked Immunosorbent Assay (ELISA).
Subsequently, a comprehensive compilation of all ELLA variable parameters has been established. For each parameter, further analyses were performed to determine whether this parameter should be fixed or included in the upcoming pilot study. The data generated during this preliminary work has enabled the work package to define a common protocol and narrow the list of variable parameters to focus on along our first pilot study.
In parallel, the development and optimization of an ELISA targeting specifically NA-antibody is progressing and mainly conducted at the Paul-Ehrlich-Institute (PEI). In the first year, PEI has successfully established the materials and protocol needed for NA-expression. During the second year, a basic approach suitable for the solubilisation of the expressed NA-protein required for ELISA application has been developed. Additionally, preliminary work has shown the possibility to titrate NA-antibody with a commercially available recombinant NA protein.
The leadership of this part of the FLUCOP work plan is joint responsibility of Dr. Hanna Sediri and Dr. Ralf Wagner from PEI and Dr. Damien Friel from GSK. The expertise of the industrial partner in assay validation is valuable to develop, optimize, standardize and validate the ELLA and ELISA assays.
Other involved partners are:
Public Health England, Universitetet i Bergen, University of Siena, National Institute for Biological Standards and Control (NIBSC), Sanofi Pasteur, Abbott Biologicals BV, Seqirus.
Novel methodology to detect ADCC responses to influenza has uncovered an additional, potentially cross protective role for the hemagglutinin stalk region.
Erasmus MC previously reported on the development of an antibody-dependent cellular cytotoxicity (ADCC) assay to measure alternative, immunologically relevant, activity of antibodies. These antibodies are areas of intense current interest in influenza research, as they have been recognized as a potential correlate of protection and are predominantly directed to conserved regions of the hemagglutinin glycoprotein. The assay developed at Erasmus MC is based on a continuous NK cell line that expressed the Fcg receptor (CD16) and recombinant hemagglutinins as source of viral antigen. Activation of NK cells by serum is subsequently assessed by CD107a expression measured by flow cytometry. In an initial publication on this assay (de Vries RD, Vaccine 2017, see full references further below), de Vries et al showed that both after infection and vaccination influenza A(H1N1)pdm09 virus-specific ADCC mediating antibody responses were induced. Trivalent vaccination led to the additional induction of influenza A(H3N2) and B virus-specific ADCC mediating antibodies. In a follow-up publication (de Vries RD, Vaccine 2017), the group at Erasmus MC now showed that primary infection of humans with either influenza B virus lineage induces hemagglutinin-specific ADCC mediating antibodies. In addition, they showed that only the stalk-specific antibodies mediated ADCC, and displayed substantial cross-reactivity with IBV of both lineages. In a paper recently published in the Journal of Infectious Diseases (de Vries RD, J Infec Dis. 2017) the authors speculate that potentially, ADCC-mediating antibodies directed to the HA stalk of influenza B virus contribute to cross-protective immunity to IBV of both lineages.
Proceedings from the 1st Assay validation workshop held on June 7-8, 2016 site in Wavre, Belgium, and 2nd Assay validation workshop held on April 5th 2017 in Siena, Italy.
Related to WP3 deliverable 3.2, a specific focus was given on a common understanding of assay qualification and validation principle. It became rapidly clear that beyond WP3 participants, all WP members could take advantage of such a discussion and a decision was made to extend the principle to all Flucop participants.
A first Assay Validation workshop was organised in Wavre, Belgium, at GSK site (June 7th and 8th 2016). This very comprehensive two-day workshop on method validation was organised by WP 3, but all the topics were prepared by experts from vaccines companies for the entire FLUCOP consortium. The objective of the first validation seminar was to raise a common awareness about the critical aspects of assay development, validation and life cycle and to align the partners on assay development and validation essentials (including statistical perspective). This was an opportunity to share manufacturers’ expertise and understanding/interpretation of current assay validation guidelines with real-life examples from the influenza field. This workshop covered the basics of assay development until validation and assay maintenance and consisted of 9 presentations (speakers from Sanofi, Abbott, Seqirus and GSK). 35 participants from 13 FLUCOP partners [4 manufacturers (Sanofi Pasteur, Abbott, Seqirus, GSK), 4 universities (Oxford, Siena, Bergen, Ghent), PEI, PHE, NIBSC, EVI, Fondazione IRCCS]. This workshop was considered as the “kick off” for further FLUCOP sessions on validation in order to appropriately spread and maintain the knowledge on this important task throughout the consortium.
A second Assay Validation Workshop was held in Siena, following the FLUCOP annual meeting, on April 5th 2017. During the workshop PEI, together with WP3 Co-Leader GSK, took over the meeting chair responsibilities by giving introductory presentations and setting the stage for other talks and discussion in order to identify critical issues of common importance, develop consensus, if possible, and final conclusions on most of the meeting topics. The second Assay validation meeting was attended by 36 participants from 22 FLUCOP institutions.
Project consortium related news
Scientific representatives and leadership
Dr. Hanna Sediri has taken over from Dr. Ralf Wagner the role of Scientific Representative for PEI and as work package leader on WP3 and member of the Steering Committee.
Prof. Marion Koopmans will replace Dr. Guus Rimmelzwaan as Scientific representative for EMC and as work package leader on WP4 and member of the Steering Committee.
External Advisory Board
Prof. Van-Tam has resigned from the External Advisory Board, the entire FLUCOP consortium gratefully acknowledges his very valuable support to the project.
The Paul-Ehrlich-Institut (PEI) is the Federal Institute for Vaccines and Biomedicines. It assesses and monitors the benefit-risk balance before, during and after the marketing authorization of biomedicines for human use and immunological medicines for veterinary use. The Paul-Ehrlich-Institut has many key duties such as:
Marketing authorisation for biomedicines, authorisation of clinical trials, manufacturer-independent official experimental batch release testing, scientific advice prior to the submission of applications for marketing authorisation or clinical trials. Additionally, PEI is a World Health Organisation (WHO) Collaborating Centre for Standardization and Evaluation of Vaccines.
The marketing authorisation and monitoring of the quality, efficacy and safety of medicinal products are administered on an international level by a European and a global network. Experts from the Paul-Ehrlich-Institut are actively involved in working groups of various international organizations such as the European Medicines Agency (EMA) and WHO.
PEI is involved in two major parts of the FLUCOP work programme
On one hand, PEI is mainly involved in work related to the development of assays to detect NA-specific antibody responses for evaluating influenza vaccines immunogenicity. PEI leads and participates to all activities related to this work.
Moreover, PEI is also involved in the standardisation of HAI & virus neutralisation (VN) assays, more specifically in the VN part, where we participate to the parallel pilot study assessing the short form and long form VN.
Beyond FLUCOP, PEI has been involved in many collaborative studies such as for the Consortium for the Standardization of Influenza Seroepidemiology (CONSISE) regarding MNT, HAI and ELLA assays. The group has now long-lasting experience with these tests.
PEI staff directly involved in FLUCOP
Dr. Hanna Sediri: Hanna has completed her PhD at the University of Marburg (Germany) in 2015. She studied mutations promoting the adaptation of avian influenza virus of subtype H9N2 to mammals and the mechanism behind these adaptations. She then started her post doc at the Paul-Ehrlich-Institut, where she mainly leads the experimental work and the organization related to assays to detect NA-specific antibody responses. She is also participating in the work on the VN short and long form harmonization.
Hanna became recently work package co-leader and member of the FLUCOP Consortium steering committee.
Dr. Ralf Wagner: Ralf obtained his PhD at the Institute for Virology in Marburg, where he focused his work on Influenza glycoprotein HA. After his post doc in Marburg, he became head of laboratory, enlarging his interest from influenza to rhabdoviruses and the development of attenuation for vaccine development. In 2004, Ralf became regulatory and scientific adviser in the viral vaccines section at the Paul-Ehrlich-Institut. He pursued his interest as group leader of an applied research group conducting experiment on the development and characterization of influenza vaccines. Since 2016, Ralf has become head of the viral vaccines section, with activities such as: licensure of national and European vaccines, life cycle management of vaccines and file review/qualification of vaccines (ex: Influenza) for WHO. Ralf has also published several articles demonstrating the inter-laboratory variability for HAI and VN assays and therefore the importance of standardization. Within FLUCOP, together with Dr. Hanna Sediri (PEI) and Dr. Damien Friel from GSK is co-leader of the work related to assays to detect NA-specific antibody responses, as well as a member of the FLUCOP Consortium steering committee
Influenza virus-specific antibody dependent cellular cytoxicity induced by vaccination or natural infection.
de Vries RD, Nieuwkoop NJ, Pronk M, de Bruin E, Leroux-Roels G, Huijskens EGW, van Binnendijk RS, Krammer F, Koopmans MPG, Rimmelzwaan GF.
Vaccine. 2017 Jan 5;35(2):238-247. doi: 10.1016/j.vaccine.2016.11.082. Epub 2016 Nov 30.
The present study investigated the ADCC response upon influenza virus vaccination and infection in humans using a robust ADCC assay developed by the project partners. The assay proved to offer advantages over existing methods and in the future will facilitate the assessment of ADCC mediating serum antibodies after influenza vaccination or infection and the potential value of ADCC activity as a correlate of protection.
Primary Human Influenza B Virus Infection Induces Cross-Lineage Hemagglutinin Stalk-Specific Antibodies Mediating Antibody-Dependent Cellular Cytoxicity.
J Infect Dis. 2017 Dec 27;217(1):3-11. doi: 10.1093/infdis/jix546.
In this article the authors show that, in humans, primary influenza B virus (IBV) infection induces HA-specific antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The findings indicate that potentially, ADCC-mediating antibodies directed to the HA stalk of IBV contribute to cross-protective immunity to both IBV lineages.
Conference participations and other project presentations
Development of an enzyme-linked-lectin-assay to measure influenza A virus neuraminidase specific antibodies (Hanna Sediri, PEI)
27th Annual Meeting of German Society for Virology, Marburg, Germany (22-25 March 2017)
Development of an enzyme-linked-lectin-assay to measure influenza A virus neuraminidase specific antibodies (Hanna Sediri, PEI)
Junior Scientist Zoonoses Meeting, Langen, Germany (7-9 June 2017)
The FLUCOP Program (Patricia Londono-Hayes, FLUCOP Coordinator. Sanofi Pasteur and Donata Medaglini, Managing Entity Coordinator).
Universal Influenza vaccines Workshop. What have we learnt and how can we move forward?
European Commission, Directorate General for Research & Innovation, Brussels, Belgium (14 June 2017)
Standardization and development of assays for assessment of influenza vaccines correlates of protection (Patricia Londono-Hayes, FLUCOP Coordinator. Sanofi Pasteur)
18th World Vaccine Congress, Barcelona, Spain (10-12 October 2017)
Update on progress presented to the Vaccines Working Party Meeting (Members of the FLUCOP Steering Committee)
European Medicines Agency, London, UK (17 November 2017)
• Annual meeting, Ghent, 18-20 April 2018 (project-internal meeting)
Other relevant events
• 2nd International Meeting on Respiratory Pathogens
7-9 March 2018, Singapore
• 4th International Symposium on Neglected Influenza Viruses
18-20 April 2018, Brighton, UK
• 5th International One Health Congress: free symposium on Chronic Wasting Disease
22-25 June 2018, Saskatoon, Canada
• 4th International Conference on Influenza and Zoonotic Diseases
2-3 July 2018, Vienna, Austria
• 5th European Congress in Immunology
2-5 September 2018, Amsterdam, The Netherlands
• World Vaccine Congress Europe
29-31 October 2018, Lisbon, 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/