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FLUTCORE Report Summary

Project ID: 602437
Funded under: FP7-HEALTH
Country: United Kingdom

Periodic Report Summary 2 - FLUTCORE (Development of a universal influenza vaccine based on tandem core technology)

Project Context and Objectives:
Influenza vaccines in use today must be redesigned and manufactured each year in an attempt to match changes that occur in the external proteins of the virus through mutation. As a consequence, one year’s vaccine is very unlikely to protect against the next season’s prevalent virus and so those at risk o infection should be vaccinated every year. If the wrong vaccine is selected, as in 2015, little or no protection is conferred through vaccination. Furthermore, existing vaccines cannot protect against new influenza subtypes arising from recombination in animals that account for worldwide infections or pandemics. In 2009, pandemic flu caused 203,000 deaths worldwide. New approaches to influenza vaccination are required.

The FLUTCORE consortium aims to harness novel technology to produce a universal influenza vaccine to combat all known and anticipate influenza A strains. Provision of such broad coverage would be a very major advance over existing vaccines. If such a vaccine can also produce lasting protection it would obviate the need for annual vaccination.
The FLUTCORE technology uses two innovative approaches to generate universal immunity to influenza A. The first is the selection of conserved vaccine targets or antigens. Conventional vaccines induce immunity to two glycoproteins found on the surface of influenza viruses, hemagglutinin and neuraminidase. However, the target or antigenic regions of these proteins are highly variable due to genetic mutation and this accounts for failure of seasonal vaccination. FLUTCORE has identified invariant conserved regions of two influenza virus surface proteins as the vaccine target antigens.

However, these conserved regions are not normally immunogenic and so a novel approach is required to induce protective immunity to these proteins. FLUTCORE’s second innovation is the use of virus-like particles (VLP) to present conserved antigens to induce protective immunity to influenza. VLPs are particulate structures that appear to the immune system to look like a virus but they lack the genetic material that allows viruses to replicate. As a result, they can induce immune responses to the proteins carried on their surface but are inherently safe compared to conventional killed or attenuated viral vaccines. Increasingly VLP technology is being adopted as a safe and effective way of producing novel vaccines.

FLUTCORE’s vaccines use VLPs formed from hepatitis B core (HBc) proteins. HBc proteins assemble spontaneously to form particles that are immunogenic (stimulate immune responses). The VLP have “spikes” on their surface to which additional antigens can be attached. Naturally occurring HBc particles can only accommodate very small insertions at the spike but due to a modification called tandem core™, FLUTCORE’s HBc particles can accommodate larger proteins such as the conserved influenza antigens identified by the consortium.

FLUTCORE has identified conserved influenza A antigens that can be incorporated into tandem core HBc VLPs. These inserts are predicted to provide immune coverage to all known strains of influenza A and potential emergent pandemic strains.

As reported in the previous period, we have successfully identified three antigens that provide apparent universal coverage with protection against a range of influenza strains. However, attempts to transfer laboratory scale production to GMP manufacture and large-scale production have encountered difficulties, revealing problems that must be addressed before a vaccine for human use can be manufactured. Much of the work in this reporting period has focussed on understanding the reasons for failure in the transfer of the manufacturing process and in beginning to identify solutions. In the meantime, partners have added to our knowledge of the physical properties and immunology of tandem core VLP vaccines for influenza.

Project Results:
1. Antigen selection: Final selection of the components of FLUTCORE’s vaccine was achieved in Period 2. This will comprise two components, each representing the variants of a conserved region of hemagglutinin (HA-stalk) found in the two main groups of influenza subtypes (Group 1 and Group 2). The third component comprises the three variants of a conserved region in the external domain of the matrix protein (M2e) expressed as a triplet. All three antigens have been encoded in tandem core VLPs, expressed in yeast and then tested in mice. They have been shown to induce antibodies that cross react with all known influenza subtypes and, when administered in combinations, they confer protection against lethality from mouse adapted Group 1 and Group 2 influenza strains, with each component providing some cross protection against a virus representing the other (heterologous) group.

2. VLP Design and production: Several combinations of antigens were investigated employing different orientations and insertion sites in tandem core. Cloning and expression methods were developed and refined. Purification processes were investigated including different lysis conditions, filtration, centrifugation, precipitation, thermal treatment and size selection, ion exchange chromatography and affinity purification. A preferred process was selected and prepared for technical transfer to the manufacturing partner.

During laboratory scale production, it became apparent that our preferred HA-stalk/triple M2e VLP (VLP1) could not be produced reliably. The VLP was immunogenic and conferred protection in challenge experiments but contained un-assembled tandem core material, degradation products and was contaminated with host-cell proteins and nucleic acid. Attempts to purify this material failed and the consortium took the decision to abandon this construct on 12 April 2016. Subsequent work has focused on the design and production of 2 VLPs, each expressing a single influenza HA-stalk antigen representing Group 1 or Group 2 antigens; and a third VLP expressing the M2e triplet.

3. VLP Manufacture: Up Stream Process (USP) conditions for the production of VLPs in yeast were established. A mixed feed fermentation process with methanol induction was developed and transferred from laboratory scale to the manufacturing partner. This was then applied to two different VLPs, one with dual inserts of HA-stalk and triple M2e (VLP1), and one with a single HA-stalk insert (VLP2). The problems encountered with the manufacture of VLP1 at lab scale were exacerbated on transfer. Manufacture of VLP2 yielded very small quantities of VLPs and the process was deemed to be unsuitable for GMP. The Consortium agreed that VLP1 should be abandoned. It was decided that the manufacturing process for VLP2 should be revised to ensure that it is GMP compliant. In addition, it was agreed that two new vaccine components should be developed comprising a separate VLP expressing the triple M2e antigen sequence (VLP3) and if possible, a VLP expressing the Group 1 HA-stalk antigen (VLP4). Work in Period 3 will focus on the manufacture and purification of these VLPs.

4. Establishing stability tests: Physical and biochemical assays to evaluate product stability have been developed and applied to tandem core VLPs. These methods can be applied to the analysis of the vaccine when final production has been achieved.

5. Preparation for a Phase 1 Clinical Trial: A clinical trial protocol incorporating a dose escalation schedule that will be used for the Phase I clinical trial has been written and incorporated into a Briefing Booklet that was submitted to the MHRA for “scientific advice.” A favourable opinion was obtained from the MHRA in November 2015.

Potential Impact:
As a result of the difficulties and challenges encountered with the development and transfer of a GMP compliant production process for the manufacture of our preferred vaccine candidate the consortium has undertaken a detailed appraisal of progress to date and plans for the remaining grant period.

The consortium has recognised that without a GMP compliant vaccine product the planned clinical trial will not be possible within the time frame of the FLUTCORE grant. Consultation with all Project Partners has led to the development of an agreed plan for the final period of the grant and we will submit this proposal to the EU in the coming month.
Selection of the final candidate components of the FLUTCORE universal influenza vaccine is completed.

The method of expression and the conditions for fermentation of yeast expressing the VLP components of the candidate vaccine have been determined. The following tasks will be addressed in the final period of the FLUTCORE project:

1. The methods for purification of the VLPs will be further optimised to ensure that the final process is GMP compliant, provides an adequate yield of material of adequate purity and is ready for transfer to the manufacturing partner.
2. Research cell banks of VLP2 and VLP3 have been established. A third VLP RCB will be prepared for the vaccine component expressing Group 1 influenza HA-stalk antigen.
3. Master Cell Banks and Working Cell Banks for VLP2 and VLP3 will be established and qualified as GMP compliant.
4. Research and Development grade batches of VLP2 and VLP3 will be prepared and tested in mice to demonstrate immunogenicity and protection in lethal challenges with homologous and heterologous mouse adapted influenza strains representing Group 1 and Group 2 influenza strains.
5. If adequate progress is made, we will transfer VLP production methods to the manufacturing partner to permit them to conduct Research and Development grade manufacture of at least one VLP.
6. 3P and iQur will work in close partnership to develop GMP compliant analytical methods and in process controls.

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Amelie Guisseau, (European Project Manager)
Tel.: +44 20 76798329


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