Community Research and Development Information Service - CORDIS

Periodic Report Summary 2 - FLUNIVAC (InFLUenza virus UNIVersal VACcine development program)

Project Context and Objectives:
Effectively protecting the human population from seasonal and pandemic influenza has proven to be a challenge, since influenza viruses continue to escape from and evade immunity. Current influenza vaccines fail to provide long-lasting and broad protection against multiple strains of influenza. For the development of a universal influenza vaccine, we have to “do better than Nature”, since even natural influenza virus infections induce broad protective immunity to a limited extent.
FLUNIVAC aims to develop a candidate influenza vaccine based on recombinant Modified Vaccinia virus Ankara (rMVA) encoding both B-cell and T-cell response-inducing proteins, ready to commence Phase I clinical trials. Our working hypothesis is that in order to develop a universal influenza vaccine that provides longer-lasting and broader protection against multiple strains of influenza virus both broad-reactive antibody (B-cell) and T-cell responses need to be induced. Our approach is to target Influenza A virus proteins that i) are established vaccine antigens or have extensively been explored as potential vaccine antigens (haemagglutinin (HA), neuramidase (NA), nucleoprotein (NP), matrix-1 (M1), matrix-2 (M2)), and ii) hitherto have largely been overlooked as potential antigens, but which are relatively conserved and which would broaden the immune response (non-structural protein 1 (NS1) and the polymerase proteins (PB1, PB2, PA)). We will genetically design and optimize the targeted proteins as well as the MVA as vector in such a way that the antigens are optimally expressed and presented to appropriate cells of the immune system by antigen presenting cells of the host.

Therefore, FLUNIVAC has the following objectives for the 48 month duration of the project:
1. To generate rMVA that express the individual targeted influenza A proteins:
a. To generate rMVA expressing modified surface proteins HA, NA, and M2e individually for the induction of a broad virus neutralizing antibody response directed to the conserved regions of these proteins
b. To generate rMVA expressing internal proteins NP, M1, NS1, and the polymerase proteins PB1, PB2 and PA individually for the induction of broadly protective T-cell responses
c. To optimize the immune response to the abovementioned proteins by genetically modifying the genes of the targeted proteins (“modification by design”)
2. To determine the capacity of the generated individual rMVAs to induce the desired immune response in vitro and in mice
3. To evaluate selected combinations of rMVAs resulting from objective 2 for the induction of broad protective immunity in vivo
4. To assess the longevity of protective immunity induced with the rMVAs by comparison with adjuvanted vaccine preparations (‘gold-standard’) in vivo
5. To prepare for Phase I clinical trial
These objectives are complemented by the following two supportive objectives:
6. To optimize the MVA vector in terms of protein-expression kinetics and antigen delivery
7. To explore viable MVA production platforms as alternative to chicken embryo fibroblast (CEF) cells

The “modification by design” of the proteins on one hand, and of the MVA as vector on the other hand, should induce an immune response that provides better and longer-lasting protection against influenza A viruses of various subtypes than the natural variant of these proteins. Moreover, the combination of rMVA expressing optimized conserved targets for both B and T cell responses has strong potential to induce long-lasting robust cross-protective immunity. This will be tested by in vitro and in vivo assessment of the generated rMVAs in a selective procedure, as well as by comparison to adjuvanted vaccine preparations (‘gold-standard’) to assess the longevity of the immune response. In addition, we will explore the use of an avian cell line for the production of our MVA-based universal influenza vaccine candidate to improve production consistency and may help to ensure robust supplies of an affordable vaccine.

Project Results:
In the second project period, FLUNIVAC focused on the generation and testing of recombinant MVAs (rMVAs) expressing wild type and genetically modified surface and internal influenza virus proteins in vitro and in vivo. Several rMVAs were produced and tested to confirm the identity, expression and functional integrity of the influenza viral proteins under investigation. The set of rMVAs expressing (modified) NP has been tested extensively in vitro. The modifications of NP had the desired effect on antigen presentation and subsequent T cell activation. Upon further in vivo testing, it was shown that a rMVA expressing NP alone was sufficient to induce protective immunity against a lethal challenge infection. However, the modifications did not further improve the induction of protective immunity and another mouse strain was considered to demonstrate the added value of the modification in vivo. MVA expressing wild type and modified HA were constructed and tested. However, two out of three modified HA proteins were not recognized by antibodies specific for the stalk region of HA and therefore these two constructs were deemed not suitable for further testing in vivo. As a back-up strategy two new rMVA were designed and produced. In addition, rMVA were produced that drive the expression of a subtype 1 neuraminidase gene. MVAs with a chimeric A56-M2e fusion protein have been produced and fully characterized. Two different constructs were produced, and several modifications were tested. The first MVA A56-M2e constructs have been tested in vivo with very promising results that justify further evaluating of these in constructs. Vaccination of mice with the MVA A56-M2e construct (without cysteine) afforded full protection against a lethal challenge with influenza virus, which correlated with the induction of M2e specific antibodies and CD4+ T cells. The antibody mediated protection proved to be dependent on FcγR signaling.

In vivo studies have been performed to compare the immunogenicity of rMVA with that of protein with the adjuvant Matrix-M1, which was considered the “gold standard”. To test the antibody and B cell responses in more detail the technology to immortalize antigen-specific mouse B-cells was further improved and will be used using spleen cells obtained from mice immunized with the respective HA constructs.

To optimize the MVA as vector to further improve its immunogenicity, five new promoter sequences were designed to optimize the expression of recombinant genes. To test the efficiency and functionality we cloned the influenza virus NP gene under the control of these different promoter candidates as a model target antigen and produced rMVAs. After extensive in vitro characterization, these MVA were tested in mice which demonstrated that with the use of a synthetic new promotor the immunogenicity of MVA can be even further improved.

During the second period, work on a MVA production platform based on a continuous cell line as alternative to primary chicken embryo fibroblasts, continued to focus on the development of a purification process for rMVAs to meet the regulatory requirements that a vaccine derived from a cell line must fulfill.

Overall, good progress has been made towards the overall project objectives of FLUNIVAC. Several interesting leads and products are in the pipeline, which justify further testing and assessing their potential to induce broad protective immunity in the third period. The collaboration between the partners within WP and across WP is excellent and fruitful. The FLUNIVAC consortium investigators continued to publish in peer-reviewed scientific journals, and acted as speakers at national and international conferences, symposia, and seminars to inform the scientific community, but also policy makers and industry. Furthermore, the FLUNIVAC project website has been regularly updated ( The FLUNIVAC newsletter has been disseminated to the growing list of FLUNIVAC stakeholders, annually.

Potential Impact:
Respiratory infections are amongst the top five leading causes of death worldwide. In case of influenza, currently used vaccines have relatively narrow immunogenicity. FLUNIVAC is expected to result in a universal influenza vaccine candidate that provides broad-protective and long-lasting immunity to different subtypes of influenza A viruses, as proven in pre-clinical animal studies. The proposed approach will significantly contribute to addressing one of today’s largest unmet medical needs: an universal influenza vaccine that provides longer-lasting and broader protection against ideally all manifestations of influenza in humans (seasonal, avian and pandemic). If successful, this approach will pave the way for eliminating the need for yearly updates of current seasonal influenza vaccines, and will significantly contribute to pandemic preparedness. By using MVA as vector and combining surface and internal proteins as targets in one vaccine to induce B-cell as well as T-cell responses, broad and long-lasting immunogenicity as well as virtually unlimited production capacity for large vaccination campaigns can be realized. Furthermore, we envisage mass vaccination campaigns to be reduced to one vaccination campaign every decade instead of the current rate of at least one vaccination campaign per year. If the production of the MVA-based vaccine is successful on the avian cell line, this enables fast and robust production of the vaccine against a reasonable price per vaccine. The reduced number of large scale vaccination campaigns and improved production of the vaccine will significantly contribute to the cost-effectiveness of our influenza vaccination. Finally, it will contribute to pandemic preparedness, which is currently hampered since the use of vaccines in case of a pandemic is limited by the time required for its development and deployment. It will contribute in two ways: i) this vaccine is expected to provide protection to ideally all manifestations of influenza A viruses, including pandemic influenza, and ii) the vaccine can be produced faster using the avian cell line that circumvents the limitations of currently used CEF cells for the production of MVA-based vaccines. In addition, FLUNIVAC aims to engage research intensive SMEs into the development of a new universal influenza vaccine, thereby enhancing Europe’s research network in the development of novel vaccines with a real potential to contribute significantly to human health.

The approach taken by the FLUNIVAC consortium and the state-of-the-art technologies that we use are unique in many ways, and can contribute significantly to the development of novel vaccines beyond the scope of this project. The key concept of the FLUNIVAC proposal is to induce robust and broadly protective immune responses by modifying the antigens and the MVA as vector in such a way that these are optimally expressed and presented to cells of both arms of the immune system. The novel knowledge generated on the specific induction of the immune response upon selective modifications of the antigens will open up new possibilities in future vaccine development, which extends far beyond the field of influenza. Furthermore, the usefulness of the novel FLUNIVAC technologies and insights developed by this consortium can be readily expected to extend to other candidate vaccines beyond influenza vaccine development. For example, the approach to use a modified MVA protein for enhanced presentation of recombinant influenza antigen is highly innovative and still awaits proof-of-principle. When successful, it is likely to also improve the MVA delivery of various other important antibody target polypeptides. In addition, the know-how to be developed as a result of the optimization of the MVA vector is likely to have high generic impact on the next generation poxivirus vector vaccines to be developed against various infectious diseases and cancer.

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