FLUCOP Newsletter February 2019

Welcome to the fifth FLUCOP newsletter

Dear all,

In this newsletter we will give you an update on the recent progress we have achieved in the standardisation of assays that in the future could play an important role for the analysis of clinical trial testing novel influenza vaccines, and concerning the understanding of assays that provide important supportive information for the evaluation of the immunogenicity of influenza vaccines.

The next FLUCOP Annual Meeting will take place in April 2019, another great opportunity for the partners to strengthen their interactions and networking.

Kindest regards,

Your FLUCOP Coordination team

Progress towards standardisation of the haemagglutination inhibition and virus neutralisation assays: large collaborative studies planned to start early 2019

This part of the FLUCOP work programme is fully dedicated to the standardisation of the two main serologic assays used in the evaluation of influenza vaccines: the haemagglutination inhibition assay (HAI) and the virus neutralisation assay (VN).

After collection and analysis of HI and VN protocols completed during year 1, key parameters and reagents that can have a major impact on assay variability were identified and evaluated in a set of pilot studies designed using a Design of Experiment (DOE) approach. Studies were conducted with four strains of the seasonal vaccine during year 2 and 3 in 6 participating labs; data were collected in a large database and analysed using both a classical statistical analysis and a proprietary algorithm of data analysis developed by Quinten.

Results of the first HI pilot study allowed defining optimal experimental conditions to be included in a consensus protocol that was then used in a second set of confirmatory experiments, in comparison to lab-specific protocols and reagents. Altogether, data showed that the use of common protocols and reagents associated with the use of standards can significantly reduce the inter-lab variability of the HI assay. These results were shared and discussed at the annual FLUCOP meeting (April 2018); a manuscript for publication is under preparation so that these findings can be disseminated to the wider influenza research community.

For VN assay, two distinct formats of the assay (short and long form) were selected for evaluation using the same approach than for HI assay. Consensus protocols were developed for both formats and pilot studies, one for each of the two assay formats, were designed. The pilot study for the short form VN assay has been completed, and the study for the long form assay is on-going.

In the upcoming year, collaborative studies are planned to start in at least 10 laboratories, to confirm the findings from the pilot studies, first for HI and then for VN. In support of these HI and VN collaborative studies, a clinical trial was conducted at the University of Ghent to collect large volume of sera from 30 subjects vaccinated with the 2017-2018 quadrivalent vaccine.  The use of a common set of samples for both studies will allow comparison of HAI and VN results. Finally, prior to collaborative studies, a video of HI and VN consensus protocols will be prepared and distributed to all participating laboratories as training material. 

Advancing the understanding and application of cell-mediated immunity

Another focus of FLUCOP is to advance the understanding and application of cell-mediated immunity (CMI) assays as tools for evaluating influenza vaccine performance. This particular part of the FLUCOP work plan consists of three major tasks, namely (1) the collection of biological materials to conduct the surveys, (2) the creation of a standardized protocol for the separation, freezing, storage and thawing of peripheral blood mononuclear cells (PBMC) and (3) the standardisation and evaluation of assays to measure influenza-specific cell-mediated immunity.

Significant progress has been made since the start of the FLUCOP project. The first task, namely the creation of a biobank with PBMC, has been initiated in 2015. Blood samples have been collected from subjects vaccinated during influenza seasons 2015/2016 and 2016/2017. During the clinical studies, conducted mainly for this purpose, PBMC were isolated before and 1 weeks after vaccine administration. Once the cellular immune responses against the haemagglutinin of the vaccine strains were determined, the samples were used for the third task. In addition to the blood samples, also buffy coats have been collected from healthy blood donors. These have been provided to us by the Flemish division of the Red Cross.

The second task, namely the creation of a standardized protocol for PBMC processing and cryopreservation, was completed by the annual meeting in Siena, April 2017 where the results have been presented. A series of test protocols were designed that allowed to investigate the impact of critical parameters that were selected based on a literature study and on the quality of cell preparation and preservation. Different laboratories processed blood samples according these test protocols and their own in-house protocol. The integrity and functional qualities of the PBMC thus obtained were analysed by two laboratories using enzyme-linked immunospot assays (ELISPOT) (in one lab) and intracellular cytokine staining (ICS) is a very useful and widely used flow cytometry based assay which detects the production and ICS (in the other lab) technologies. Statistical analyses where then applied to examine whether certain protocol variables had an impact on PBMC qualities. Based on these data a standardized operating procedure has been drafted on how to separate, freeze, store and thaw PBMC.

In the past year further progress was made in the first part of the third task, namely the creation of standardized protocols for flu-specific ELISpot and ICS assays. This process was initiated by two pilot studies aiming to examine PBMC analysis and data analysis performed by the different work package partners. In the first pilot study, focusing on PBMC analysis, a set of PBMC samples was distributed to the participating partners in order to execute ELISPOT and/or ICS assay(s) according to their in-house protocol(s) and a predefined immune read-out(s). Antigens for “in vitro” stimulation of PBMC were provided by the coordinating partner and consisted of recombinant HA proteins of A/California/2009pdm and B/Phuket. In Pilot Study 2 the focus was on data analysis. An ELISpot plate was prepared by University of Oxford and distributed to the other participating partners to perform a read-out using their equipment and protocol. An ICS dataset of 10 paired samples has also been sent for data analysis. After statistical analysis, the results were reviewed together with all applied protocols. This survey has enabled us to reach a consensus on a standard protocol for ELISpot and ICS.

 The second part of the third task consists of the evaluation of the quality of CMI assessment and is scheduled for the final year of the project. First a partial validation of both the ELISpot and ICS assay will be conducted by Ghent University and Sanofi Pasteur, respectively. Subsequently a training session will be organised to demonstrate and teach these validated protocols to participating partners. Finally, a proficiency test will be held to evaluate the consistency of the data generated by several partners applying these assays. 

Inside FLUCOP: Sequirus

 In July 2015, Australian pharmaceutical company CSL acquired Novartis Influenza Vaccines and merged it with its biological division, bioCSL, to create Seqirus, now the second largest influenza vaccine company in the world. With extensive research and production expertise and manufacturing plants in the US, UK and Australia, Seqirus is a transcontinental partner in pandemic preparedness and a major contributor to the prevention and control of influenza globally.

Seqirus manufacturing facility in Melbourne, Australia, has been producing egg-based flu vaccine for nearly five decades. Seqirus in Liverpool, UK, manufacturing egg-based influenza vaccine, is one of the biggest biotechnology sites in Europe and the only injectable influenza vaccine manufacturer in the UK. It also produces the only licensed adjuvanted seasonal influenza vaccine.

In addition, Seqirus has a state-of-the-art vaccine producing facility in Holly Springs, NC US, where uses a novel cell culture-based process as an alternative to traditional egg-based influenza vaccine production, giving Seqirus vital additional capacity, both for seasonal vaccine production and in the event of a pandemic.

Seqirus has a workforce of over 2,000 employees, significant manufacturing capacity, a commercial presence in twenty countries and product and geographic diversity.

Seqirus staff directly involved in FLUCOP

Dr. Giuseppe Palladino: Giuseppe obtained his MD degree in 1985 at the faculty of medicine of the Universita degli Study di Milano, in Italy. He did postdoctoral work on the mechanisms of immune response to influenza virus at the Wistar Institute in Philadelphia, then moved to Lederle Labs (later Wyeth) working on the assessment of immune responses to respiratory viruses, i.e. Influenza, RSV & PIV, in pre-clinical animal models and later in clinical trials. In 2009 he moved to Novartis Vaccines in Cambridge MA US, managing the group working on pre-clinical serology of viral vaccines. In Seqirus, he is now Head of Serology research.

Within FLUCOP, Giuseppe has represented Seqirus as steering committee member, has been earlier co-leader of the project work package on new technology and is currently co-leader another work package by coordinating the work on VN assays.


FLUCOP publications

Impact of erythrocyte species on assays for influenza serology.

Trombetta CM, Ulivieri C, Cox RJ, Remarque EJ, Centi C, Perini D, Piccini G, Rossi S, Marchi S, Montomoli E.

J Prev Med Hyg. 2018 Mar 30;59(1):E1-E7. doi: 10.15167/2421-4248/jpmh2018.59.1.870

Antibody responses to influenza A/H1N1pdm09 virus after pandemic and seasonal influenza vaccination in healthcare workers: a five-year follow-up study.

Trieu MC, Jul-Larsen Å, Sævik M, Madsen A, Nøstbakken JK, Zhou F, Skrede S, Cox RJ.

Clin Infect Dis. 2018 Jun 9. doi: 10.1093/cid/ciy487

Impact of pre-existing immunity on the induction of functional cross-reactive anti-hemagglutinin stalk antibodies following vaccination with an AS03 adjuvanted pandemic H1N1 vaccine.

Tete SM, Jul-Larsen Å, Rostami S, Lunde THF, Søland H, Krammer F, Cox RJ.

Vaccine. 2018 Apr 12;36(16):2213-2219. doi: 10.1016/j.vaccine.2018.02.022.

Immune responses after live attenuated influenza vaccination.

Mohn KG, Smith I, Sjursen H, Cox RJ.

Hum Vaccin Immunother. 2018 Mar 4;14(3):571-578. doi: 10.1080/21645515.2017.1377376.

Upcoming events

FLUCOP Meetings

• Annual consortium meeting, Siena, 3-4 April 2019 (project-internal meeting)

Other relevant events

• 1st ISIRV International Influenza Vaccine Meeting – Siena, Italy

31 March-2 April, 2019


• Influenza Vaccines for the World – Edinburgh, Scotland, UK

2-4 April, 2019


• Emerging Infectious Diseases, Toronto, Canada

10-11 April, 2019

• National Adult and Influenza Immunization Summit – Atlanta, Georgia, USA

May 14-16, 2019


• 5th International Conference on Influenza and Zoonotic Diseases – Berlin, Germany

17-18 June, 2019

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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/