Standardisation of HAI and VN assays

Previous international collaborative studies have shown that both HAI (haemagglutination inhibition) and VN (virus neutralisation) assays are highly variable between laboratories, compromising the concept of a universally applicable serological correlate of protection because results obtained in different laboratories will not necessarily be comparable. Therefore, the main aim of this work package is to improve the standardisation of HAI and VN assays.

The HAI assay requires reagents that are difficult to standardise, in particular red blood cells (RBCs) from various animal species (e.g. turkey, guinea pig, horse). Moreover, different protocols are in use in different laboratories, which may contribute to the variability of the assays.

The VN assay is even more variable than the HAI assay; a variety of protocols that differ in significant parameters (such as incubation time, method of detection, amount of input virus) are in use and differences in critical reagents (e.g. cells) may affect the results. Logistical issues make complete standardisation of certain reagents (e.g. RBCs, cells) very difficult. By contrast, harmonisation of assay protocols is a challenging, but achievable goal that could provide significant benefits. Experience from previous work has indicated that the use of antibody standards can substantially decrease inter-laboratory variability.

Therefore, this WP is working towards the standardisation of the traditional serology assays used for influenza vaccine clinical trials, i.e. the HAI and VN. Additionally, this also include a complementary study aimed at evaluating how determinations of antibody responses directed against influenza haemagglutinine obtained with the standardized HAI assays compare to those obtained using a classic Single Radial Hemolysis assay (SRH).

Although the HAI assay is most commonly used to support influenza vaccine regulatory filings, the SRH assay is still in use and considered particularly useful for evaluation of influenza B strains.

Work package co-leaders: DH-MHRA and Sanofi

Advancing the understanding and application of CMI

Although the evaluation of CMI (cell-mediated immunity) response is not considered as mandatory analysis for influenza seasonal vaccine licensing, the most recent update of the guideline “EMEA/CPMP/VEG/4717/03-Rev1” suggested that CMI analyses provide supportive information for the evaluation of vaccine immunogenicity. Protective immunity against influenza is mediated by neutralizing antibodies but CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) are known to play a role in host immunity against influenza infection. CD4+ helper T cells are instrumental for the expansion, maturation and antibody production of B lymphocytes and also for the induction and maturation of CTL. CD4+ helper T cells thus contribute to protection against infection whereas CD4+ and CD8+ cells both play a role in control of disease severity. Indeed, the numbers and qualities of influenza-specific CD4+ and CD8+ T cells have been shown to modulate the severity of the illness and to contribute to heterotypic immunity. There is evidence from clinical studies that influenza vaccines induce CMI responses in humans, particularly of the CD4+ (TIV and LAIV) and CD8+ (LAIV) types.

In view of the possible requirement of CMI assays for the evaluation of the immunogenicity of seasonal influenza vaccines, there is a need to optimize and importantly to standardise the different procedures and assays involved.

To this aim we are standardising the methods used for:

1. Peripheral Blood Mononuclear Cell (PBMC) preparation and cryopreservation;
2. CMI analysis, namely T cell-ELISPOT assay and intracellular cytokine staining-FLOW cytometry assay (ICS-FLOW)
3. Analytical data analysis and representation.

Thus, WP2 is advancing the understanding and application of CMI assays as tools for evaluating influenza vaccine performance.

Work package co-leaders: UGENT and Crucell

Assays to detect NA-specific antibody responses for evaluating influenza vaccines immunogenicity

Neuraminidase (NA) is the second major viral envelope glycoprotein of influenza viruses that plays an important functional role in the late phase of the infectious cycle. It has been reported that humoral immunity directed against NA can markedly reduce virus replication and release thereby reducing the severity and duration of illness as well as viral shedding. Cross-protection elicited by anti-NA antibodies against viruses of heterologous HA-subtypes could play a crucial protective role in humans that have previously been exposed to different influenza viruses with genetically similar N-proteins either by vaccination or natural infection. However, at present the relevance of NA-antibodies and their contribution to vaccine-induced immunity is largely unclear because the HA protein has been widely recognised as a major viral antigen, and technical development of serological assays has focused primarily on HA-specific rather than NA-specific immune responses.

The contribution of anti-NA responses to the immunogenicity of influenza vaccines has recently gained increased attention amongst manufacturers, researchers and regulators as reflected in the upcoming revised EU regulatory guidance on the quality influenza vaccines where the determination of NA component and anti-NA responses have been included. WP3 co-leader´s institutions have been instrumental in the development and preparation of this revised EMA influenza guidance documents.

In a concerted approach the experimental and regulatory expertise of WP3 members are jointly directed towards the evaluation and qualification of assays to reliably detect anti-NA antibody responses and are developing these assays into valuable tools for the evaluation of anti-NA responses elicited by influenza vaccines. Thus, WP3 will advance the understanding and application of NA-Ab titration assays as tools for evaluating influenza vaccine performance.

Work package co-leaders: PEI and GSK

Consideration of new technologies yet to be applied to population based evaluations of influenza vaccines

This work package provides an opportunity to bring forward new, potentially useful technologies that will aid in defining biomarkers for correlates of protection against influenza induced 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, some of which have been established while others still have to be defined.

WP4 is thereby developing novel methods and assays that could supplement existing technologies in future influenza vaccination studies.

Work package co-leaders: EMC and NVx

Project management, impact assessment, dissemination and ethics

WP5 is focusing on following:

• Scientific and administrative coordination of the project
• Guaranteeing the successful progress of the project within the agreed time, cost and quality limits as defined by the project contract signed with the IMI and the      Consortium Agreement signed among the participants.
• Establishing effective communication among the consortium partners.
• Impact monitoring: Impact Score Card
• Establishing and implementing effective dissemination activities
• Ethical oversight and reporting of project
• Detecting and securing exploitable IP

Work package co-leaders: SVA and Sanofi