In this newsletter we will give you an update on the progress we have recently achieved in the FLUCOP standardisation of assays and in the understanding and application of cell-mediated immunity. In addition, it will report the recent advance in the development and characterisation of assays aimed at detecting NA-specific antibody responses for evaluating influenza vaccines immunogenicity.
The most recent FLUCOP Annual Meeting took place on 25 and 26 January 2021. It was another great opportunity for the partners to strengthen their interactions and networking.
We take advantage of this seventh beginning-of-the-year newsletter to wish all of you a Happy New Year.
Progress towards standardisation of the haemagglutination inhibition and virus neutralisation assays
The haemagglutination inhibition assay (HAI), the most commonly used serologic assay in influenza vaccine evaluation and influenza seroepidemiology, has been systematically evaluated by the FLUCOP consortium for possibilities for standardisation. The results from initial pilot collaborative studies showed that the use of a common (consensus) assay protocol reduced inter-laboratory variability. Similarly, the inclusion of a standard specimen consisting of a pool of human serum samples with high HAI titres against the tested viruses led to improved agreement between laboratories, while combining a common protocol with the use of a standard did not further increase harmonisation between laboratories. This work, which was recently submitted for publication, was extended by a larger collaborative study involving ten FLUCOP laboratories, with similar results. FLUCOP has generated a training video, which we intend to make publicly available to facilitate the dissemination of the consensus HAI protocol.
Following successful pilot studies assessing consensus protocols for two forms of the virus neutralisation assay (VN) (the ‘short’ and ‘long’ form of the VN), a larger collaborative study was conducted. Despite disruption caused by the COVID-19 pandemic, the study has been completed, albeit with a slightly lower number of participating laboratories than anticipated. The data generated in the study are being analysed by the team of statisticians supporting this part of the work. Results have just been presented the annual meeting of the consortium in January 2021.
We are currently conducting an assay qualification study for the HAI, which is distributed between four laboratories.
Advancing the understanding and application of cell-mediated immunity
In the past two years significant progress has been made by the FLUCOP partners involved in this part of the workplan. Shortly after a consensus on the standardized protocols for flu-specific Enzyme-Linked Immuno Spot (ELISpot) and Intracellular Cytokine Staining (ICS) assays was reached, the partial validation of the ICS and ELISpot assays was initiated by Sanofi Pasteur and Ghent University, respectively. The results of the partial ICS validation have been captured in a validation report that has been submitted to the Innovative Medicines Initiative (IMI) Secretariat. The validation of the ELISpot assay required some additional work causing some delay. The ELISpot validation report will be finalized in 2021.
In November 2019, a training session was organized in Ghent (Belgium) to instruct and familiarize the scientists involved with these protocols. Representatives of eight laboratories attended this two-day event and actively participated in the hands-on training. Their feedback was very positive and gave confidence for a proper conduct of the final task within the FLUCOP project.
To accomplish the final milestone linked to these activities, two proficiency tests were organized. In the beginning of 2020 practical instructions, samples to be tested and reagents for both the ELISpot and the ICS proficiency test were distributed to all partners involved. The flu-specific stimulating agents were kindly provided by Sanofi Pasteur who also took care of the shipments to the laboratories. Janssen (former Crucell) organized the sample shipments. Unfortunately, the COVID-19 pandemic jeopardized our plans to execute and complete these proficiency tests by the end of 2020. A few laboratories have been able to deliver the results before the end of 2020 but most are still facing delays due to COVID-related tasks and changes of priorities. Like all other FLUCOP partners we are most grateful that a no-cost extension with another year has been granted. We hope that the remaining tasks pending can be finalized in the coming months. Based on the outcome of the proficiency tests, individual reports and a general quality report will be written. The latter will present an overall comparison of the variation of the data obtained by all participants before and after implementation of the FLUCOP standard operating procedures. This analysis will show the impact of standardizing cell-mediated immunity protocols that can be applied in the evaluation of influenza vaccine performance.
Assays to detect Neuraminidase-specific antibody responses for evaluating influenza vaccine immunogenicity
After a harmonization exercise, the Neuraminidase (NA) enzyme-linked lectin assay (ELLA) consensus Standard Operating Procedure (SOP) was validated in an inter-laboratory study involving seven FLUCOP partners. This validation demonstrated that the ELLA assay is precise, linear, robust within defined limits and has good specificity.
The assay reproducibility was confirmed even across different laboratories and highlighted the importance of inclusion of a serum standard for the normalization of the NAI antibody titers and reduction of variability in results.
A validation report summarizing all experimental results was written as well as a SOP. During the last year, the ELLA consensus protocol was adapted to ensure analyze of other human NA subtypes such B neuraminidase.
University of Ghent
Ghent University (UGent), one of the five universities in Flanders (Belgium), was founded in 1817 by King William I of Orange. With a total of 36,000 students and 9,000 staff members, UGent is one of the major research universities in Belgium.
The Center for Vaccinology (CEVAC) is a research unit within the department of Diagnostic Sciences (Faculty of Medicine and Health Sciences), that contributes to modern vaccine development and evaluation. CEVAC was founded in 1986 by Geert Leroux-Roels, emeritus Professor of Medicine and since 2017 headed by Prof. dr. Isabel Leroux-Roels. Geert Leroux-Roels is still actively involved and remains acting as co-leader of work package 2 (WP2) and member of the Steering Committee of the FLUCOP consortium.
CEVAC conducts research on the improvement of existing and the development of new vaccines. In its 30-year history, CEVAC has participated in more than 200 clinical vaccine evaluations and has contributed to the development and evaluation of vaccines against hepatitis B, the first combined hepatitis A-B vaccine, a therapeutic vaccine against hepatitis C, vaccines against cervical cancer, herpes simplex, HIV and tuberculosis. CEVAC consists of two entities that execute different but complementary tasks and a constantly interactive manner: CEVAC Clinical Trial Unit and CEVAC Core Laboratory. The latter is involved in the FLUCOP consortium.
The CEVAC Core Laboratory is a GCLP compliant laboratory that provides central lab services to both industrial and academic research partners. These services consist mainly, but not exclusively, of immunological analyses to measure the immunogenicity of candidate vaccines that are evaluated in clinical vaccine trials conducted in CEVAC’s Clinical Unit or elsewhere. The Core Lab not only executes but also develops, qualifies and validates immunologic read-outs that meet the requirements of the sponsor. Today, CEVAC Core Lab team consists of 14 skilled and dedicated staff members. The laboratory is involved in Work Package 2 which aims at advancing the understanding and application of CMI assays as tools for evaluating influenza vaccine performance.
University of Gent staff directly involved in FLUCOP
Geert Leroux-Roels founded CEVAC in 1986 and directed the unit until September 2017 when he retired and Isabel Leroux-Roels took over all managerial responsibilities. He is still supporting CEVAC as an external consultant. He is an expert in immunology and infectious diseases with a focus on hepatitis and influenza. He has ample experience in the clinical evaluation of vaccines, and the development and validation of immune assays.
Frédéric Clement has more than 20 years of experience in the fields of immune-monitoring of vaccines. Many new and innovative pre-analytical or analytical methodologies have been developed, qualified and even validated up to Phase 3 trial compliance under the supervision of Frédéric. He now acts as Core Facility Manager at the Faculty of Medicine and Health Sciences and is still involved as analytical expert at CEVAC.
Gwenn Waerlop joined CEVAC in 2015 as project manager and is responsible for the general coordination of the laboratory since 2019 as successor of Frédéric Clement. She has more than 5 years of experience in the conduct of clinical vaccine trials according to GCLP guidelines and the overall project management of industry-sponsored and academic projects. She is the first point of contact for the FLUCOP project and interacts with the FLUCOP management and all WP2 partners.
Association between vaccine adjuvant effect and pre-seasonal immunity: Systematic review and meta-analysis of randomised immunogenicity trials comparing squalene-adjuvanted and aqueous inactivated influenza vaccines.
Walter E.P. Beyer, Abraham M. Palache, Leslie A. Reperant, Mimoun Boulfichd, Albert D.M.E. Osterhaus
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/