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  • Periodic Report Summary 2 - EDUFLUVAC (Combinatorial immunization strategy to educate the immune system towards cross recognition and coverage against antigenic drift in seasonal influenza virus exposure)

EDUFLUVAC Report Summary

Project ID: 602640
Funded under: FP7-HEALTH
Country: Germany

Periodic Report Summary 2 - EDUFLUVAC (Combinatorial immunization strategy to educate the immune system towards cross recognition and coverage against antigenic drift in seasonal influenza virus exposure)

Project Context and Objectives:
Current influenza vaccines afford only limited protection against seasonal as well as pandemic influenza. As influenza viruses can accumulate three-four amino acids substitutions per year and frequently change antigenically to escape population immunity, vaccine composition has to be updated and vaccines need to be administered annually. As a consequence, persistent monitoring and selection of viruses as well as production and formulation of vaccines are necessary every year. A major shortcoming is therefore that the relatively long production time results in a vaccine that does not always have a sufficient antigenic match with the epidemic strain.
A significant advance in human infectious disease research would be the development of a new generation influenza vaccine that stimulates production of a robust, broadly neutralising antibody response. Thus, the development of a “universal” influenza vaccine that can provide broad coverage against different strains within a subtype or even across subtypes has become a key public health priority in both industrialised and low- and middle-income countries.
EDUFLUVAC aims at developing a novel influenza vaccine candidate encompassing a combination of multiple influenza haemagglutinin (HA) or neuraminidase (NA) antigenic variants within a single (sub)type. This vaccine concept, using the proven, modern technology of baculovirus-derived virus-like particles (VLPs), is expected to elicit a broad neutralising immunity that will confer longer-lasting and broader protection against multiple strains of influenza virus. Proof of principle of the EDUFLUVAC strategy will be demonstrated in relevant animal models for influenza vaccines. The project will take note of new influenza vaccine regulatory guidance, and will be geared towards the development of a complete Investigational Medicinal Product Dossier (IMPD) ready for transfer into current Good Manufacturing Practice (cGMP) production for early phase clinical testing. Finally, the knowledge generated will be disseminated and relevant networking will be addressed, e.g. via a workshop series.

Project Results:
To define which strains would represent an appropriate coverage within one (sub)type, five HA antigens were selected for each of the H1, H3 and B (sub)types based on sequence diversity and serological cross-reactivity; the selected strains represent major antigenic drift groups in each (sub)type. The innovation of this approach lies in the selection of antigenic variants with maximal sequence diversity (each (sub)type is addressed separately), resulting in diluted strain-specific epitopes and enhanced presentation of common epitopes to the immune system, thereby increasing the breadth of the antibody response. Concomitantly, three NA antigens from the N1 subtype and six HA antigens from Group 2 subtypes were selected by similar criteria. As reagents to the selected HAs are critical in the determination of HA content, strains for which reagents were readily available at the Department of Health - Medicines and Healthcare products Regulatory Agency (DH-MHRA) were selected.

The selected HA and NA sequences were assembled into baculovirus expression vectors using the assembly platform rePAX® technology of Redbiotec AG (RBT). The design of the expression cassettes (promoters, polyadenylation signals and order of the HA sequences) was further improved for some vectors in order to increase the stability and the expression levels of the recombinant baculovirus vectors. Successful assembly, transfer into the baculovirus expression vector system and expression of both the surface antigens and matrix protein were confirmed prior to the delivery of the constructs to the Instituto de Biologia Experimental e Tecnológica (iBET) for influenza VLP production. A platform for production and purification of the target VLPs in sufficient quantity and quality for subsequent mouse immunisation studies was assembled at iBET. An initial screening was carried out to select suitable culture conditions and optimise the infection strategy. Concomitantly, a robust downstream processing platform for VLP purification, capable of removing the majority of host and viral contaminants while ensuring high product yield and quality, was developed. Profiting from these improved processes, the VLP production for mouse immunogenicity studies was successfully completed. Further process optimisation was then undertaken to produce VLPs at a larger scale for ferret and non-human primate studies and suitable for further GMP production. As part of the EDUFLUVAC analytical development plan, several potential batch release assays have been implemented at iBET. Additional analytical techniques for in-process monitoring of influenza VLPs during the production and purification processes, and for rational upstream and downstream process optimisation were also developed.

The initial assessment of the influenza VLPs was performed in the mouse model. The initial studies focused on the generation of high titre reference sera to be used as standards for the Enzyme-Linked ImmunoSorbent Assay (ELISA) and for further characterisation using the virus Microneutralisation (MN) and the Haemagglutination Inhibition (HI) assays. In order to define the optimum number of vaccine components able to induce broad protection, two mouse experiments were performed: 1) pilot mouse experiments conducted with all variants of each (sub)type in order to identify the optimal vaccine dose/regimen and compare and establish kinetics and assay conditions; 2) mouse vaccination studies to analyse the breadth of protection and determine the minimum number of components required. The reference sera generated were used to fine-tune the ELISA assay and were shared with Biomedical Primate Research Centre (BPRC) and DH-MHRA to set up and perform competitive ELISA (cELISA) and MN assays respectively. The MN assay is the main decision making assay, measuring the ability of sera to neutralise both homologous and heterologous influenza viruses, and for further antigen down-selection. cELISA will provide insight into the mechanisms and demonstrate the broadening of the antibody response. Based on the immunogenicity results obtained in mice, ferrets and non-human primate (NHP) challenge studies to provide proof of concept of the EDUFLUVAC project started in July 2016 at the Wageningen Bioveterinary Research (WBVR) and BPRC respectively. Such immunogenicity and efficacy studies were preceded by the preparation and validation of the corresponding heterologous challenge models.

Besides the scientific scope of the project, the EDUFLUVAC partners are engaged in sound networking activities and are organising a workshop series with the aim to bring together the relevant and current efforts on universal influenza vaccine development and address critical topics in the field. The first workshop took place on 18-19 June 2015 at the National Institute for Biological Standards and Control (NIBSC), UK. It focused on the standardisation of immunoassays that can be used to assess broadly reactive or universal influenza vaccines and was co-organised and co-sponsored by the National Health Institute (NIH) - National Institute of Allergy and Infectious Diseases (NIAID), USA. A manuscript summarising the outcome of the workshop is in preparation.
A second workshop on experimental animal models for preclinical evaluation of universal influenza vaccines was organised on 23-24 June 2016 at BPRC, The Netherlands and was supported by Sanofi Pasteur and Viroclinics. The programme included a diversity of influenza models from well established, widely accepted models to cutting edge, newly developed animal models as well as ex-vivo approaches and human models. The third and last workshop of the EDUFLUVAC project is currently under preparation and will be organised in spring 2017.

Potential Impact:
The innovation brought by EDUFLUVAC lies in the development of a combinatorial immunisation strategy, which aims at “educating” the immune system to cross-recognise common epitopes within multiple influenza virus strains, and which is expected to confer better protection against epidemic influenza. In addition, the knowledge gathered to develop an optimised process suitable for cGMP VLP production will provide a platform for large-scale production of VLP-based influenza vaccines capable of, in the event of a pandemic, manufacturing high quantities of vaccine doses.
The aimed-for level of broad protection will reduce the burden of disease that influenza imposes across Europe and other regions on an annual basis. Importantly, the development of a vaccine that elicits broad long-lasting defence would allow considerably longer intervals between re-vaccination than at present, avoid vaccine failures due to drifted seasonal variants, and provide a high level of protection against pandemic viruses. Furthermore, it would facilitate vaccination campaigns in low and middle-income countries and thereby also confer protection against influenza in hitherto untargeted groups with limited health care programmes.
This project is aligned across the Europe 2020 growth and job creation strategy. It has created jobs in six different countries and the lead Small and Medium Enterprises (SMEs), RBT and iBET, will directly benefit from the foreground generated through this breakthrough research. The potential behind this approach gives RBT and IBET the possibility of producing a truly competitive and novel product with global applications and improving their competitiveness and attractiveness as Contract Manufacturing or Research Organisations.

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