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

Executive Summary:
The over-arching aim of FLUTCORE was to develop a universal vaccine to provide protection against the variants of the major strains of influenza A virus (IAV) that account for the major outbreaks of seasonal epidemic influenza and also provide protection against IAV strains that might cause pandemic influenza including novel recombinants derived from animal and human hosts.
Influenza is a major global healthcare problem causing up to half a million deaths per year and up to 5 million cases of severe disease with significant impact on health, work productivity and quality of life. Most cases are seasonal, caused by epidemics in the wet seasons in each hemisphere. The influenza virus (IAV) can be classified into two main groups, each of which contains several known sub-types and strains. This diversity can be attributed in part to genetically encoded differences in the highly variable globular head region of haemagglutinin (HA) and neuraminidase (NA) proteins that are expressed on the outer surface of the virus. These proteins also form the major targets of immune responses that protect immune individuals from re-infection and control new infections.
Current vaccines for IAV are based on killed viral strains that have caused recent seasonal outbreaks in the opposite hemisphere. These viruses are grown in eggs, harvested, killed and purified. Administration elicits immune responses to immundominant HA and NA epitopes. The selection of the correct strain is based on epidemiological surveillance of prevalent strains to match the vaccine to the seasonal IAV and often results in the use of the “wrong” strain as the seasonal vaccine resulting in failure to protect.
FLUTCORE set out with the goal of developing a universal IAV vaccine capable of eliciting immune responses that will protect recipients against all strains of IAV. For this purpose, it was critical to identify conserved regions of important IAV proteins that could be targeted by protective immune responses. Commonly, well conserved sequences are poor immunogens so it was essential that FLUTCORE should find a means of presenting these well conserved IAV antigens to the immune system in such a way that they elicit vigorous and protective immune responses. Virus-like particles are formed of proteins, often derived from viruses, that mimic infectious virions but lack the genetic machinery to replicate. Exposure of VLP to the immune system elicits many of the same responses induced by viruses but without the risk of leading to infection. They can be genetically and biochemically manipulated to express specific “target” proteins – in this case IAV antigens – and manufactured to high degrees of purity. VLP vaccines are increasingly being recognised as a preferred vaccine platform and replacing existing killed or attenuated virus vaccines.
We have identified conserved epitopes in the long alpha helix of HA and in the Matrix protein and genetically engineered these antigens into the major insertion sites of hepatitis B tandem core molecules. We have expressed these proteins in bacteria to form virus-like particles. We have used these VLP to immunise mice and selected the most immunogenic constructs to express in yeast, a preferred expression system for the manufacture of human vaccines. In parallel we have developed engineering processes to ferment yeast to express VLP efficiently and then developed purification processes capable of purifying the VLP for human use. The resulting VLP have been evaluated in mice for their immunogenicity and their ability to protect vaccinated mice against infection with lethal doses of pathogenic strains representing Group 1 and Group 2 IAV.

Beyond the funding period of FLUTCORE the partners intend to take their lead vaccine through to a Phase I clinical trial in human subjects using a trial design that will explore both safety and proof of mechanism.

Project Context and Objectives:
Project context

Influenza is a major global healthcare problem.
Influenza virus infection is a major global healthcare problem causing respiratory distress, myalgia, arthralgia and fever that may last for 10 days to two weeks, impacting work productivity and daily function. In a minority of cases it may cause a much more severe illness culminating in hospitalization and death, particularly in the young, the elderly and the immunocompromised. It is estimate that there are 3-5 million cases of severe flu and 250,000 to 500,000 deaths each year.
Influenza A virus (IAV) variants are subtyped on the basis of heterogeneity of the antigenic viral surface proteins haemagglutinin (HA) and neuraminidase (NA). These proteins are the major targets for natural host immunity but due to antigenic drift (the continual accumulation of amino acid changes), immunity to one strain of IAV is unlikely to protect against another strain. Seasonal IAV epidemics occur during the winter months in the northern and southern hemispheres but can occur year-round in tropical regions. Animal species including birds and swine can be infected with IAV and reassortment between animal and human strains can lead to the creation of new strains of IAV not previously encountered by man and to which man has no natural immunity. These strains can lead to rapidly spreading pandemics which may be associated with very high mortality rates such as the pandemic in 1919 that caused more deaths than the whole of the 1914-18 World War.

Current vaccines
Current approaches to vaccination rely on the use of killed influenza virus to prevent disease. Immunity is strain specific and so vaccines for seasonal IAV epidemics in the Northern Hemisphere are based on prevalent strains causing influenza in the preceding wet season in the Southern Hemisphere. IAV is grown in eggs and then killed to form the seasonal vaccine. The main vaccine targets are the globular head domain of HA and NA. Because immunity is strain specific, vaccines have to be developed and administered afresh each year.

Potential for a universal vaccine
For many years investigators have sought to design a universal influenza vaccine that might induce protective immunity against many strains of influenza, obviating the need to re-immunise the at-risk population each year.
The requirements for such a universal, or broad vaccine include the induction of protective immune responses that target proteins expressed by the virus that are conserved between at least the major strains that are responsible for seasonal influenza. These proteins should be of biological importance in the virus’s life cycle (so that mutation will lead to a loss of fitness or pathogenicity) and, if not readily immunogenic, they must have the potential to be immunogenic if presented in the correct immunological context (through antigen presentation or as a result of the addition of an adjuvant).
The FLUTCORE consortium set out to develop a candidate universal influenza vaccine capable of inducing protective immune responses that will prevent or limit seasonal influenza in the vast majority of individuals irrespective of the prevalent seasonal strain; and provide protection against pandemic influenza caused by reassortant strains, obviating the need to re-design and manufacture influenza vaccines each year.

The FLUTCORE Partners
The group of partners assembled to develop and deliver the FLUTCORE project included the lead and coordinating partner iQur Limted, a London based SME focussed on the use of the Hepatitis B tandem core VLP technology as a platform for vaccine design.
University of Leeds invented the tandem core vaccine platform and provided expertise in the development of analytical methods for the analysis and study of VLP vaccines. The Biochemical Engineering department at UCL, London has experience and expertise in the manufacture of yeast based vaccines and provided in vaccine production.
The Latvijas Biomedicinas Petijumu un Studiju Centrs (LMBC) based in Riga, Latvia, has critical expertise in the manufacture of VLP in yeast and experience in production of tandem core based VLP.
Centre de Recherche Public de la Santé in Luxembourg (renamed Luxembourg Institute for Health (LIH) has expertise in immunological and molecular studies of influenza and brought expertise in the identification of relevant IAV antigens to include in the vaccine.
3P Biopharmaceuticals is a GMP manufacturer of VLP based vaccines based in Pamplona, Spain. 3P aided with the design, development and delivery of a GMP manufacturing process for the FLUTCORE vaccine.
The Institut Catala de la Salut is a Phase I clinical trials unit based in Barcelona capable of helping design and deliver a Phase I clinical trial of a vaccine for influenza.
UCL European Research and Innovation Office provided support with coordination of the FLUTCORE project.



Project Objectives

Design of a universal influenza Virus-like particle vaccine based on tandem core technology
The over-arching aim of FLUTCORE was to develop a universal vaccine to provide protection against the variants of the major strains of influenza A virus (IAV) that account for the major outbreaks of seasonal epidemic influenza and also provide protection against IAV strains that might cause pandemic influenza including novel recombinants derived from animal and human hosts.
The first stage in this process was the selection of a vaccine platform. The hepatitis B tandem core virus-lie particle platform was selected on the basis that the resulting particles are immunogenic, capable of incorporating large and complex inserts, well tolerated by humans and safer than live attenuated viruses.
The second step was the selection of well conserved IAV antigens that could be used to elicit immune responses with the potential to control a wide range of IAV strains.

Characterization of VLPs
It was essential to develop methods that could be used to characterise the VLP vaccine in order to determine its characteristics for the development of manufacturing processes and to facilitate studies of composition and stability.

Immunogenicity studies in mice
Vaccine selection was dependent on demonstration initially of immunogenicity and then subsequently demonstration of efficacy. To this end we developed methods of evaluating immune responses to the IAV vaccine VLP in mice including antibody production, B-cell clone phenotype, T-cell receptor repertoire usage and ultimately protection in response to lethal IAV challenge.

Regulatory advice
Prior to the initiation of a Phase I clinical trial it was necessary to design a preclinical development plan and a clinical trial protocol. This was undertaken and led to the production of a briefing booklet that was submitted to the Medicines and Healthcare Regulatory Agency (MHRA) in order to obtain scientific advice about our development plan.

GMP manufacture
In order to conduct a clinical trial it will be essential that the VLP vaccine is produced in accordance with Good Manufacturing Processes (GMP). The partners worked together with the industrial manufacturing partner to design a process that would be compatible with GMP and would yield a sufficient quantity of vaccine of an adequate quality to be used in a Phase I clinical trial in humans.

Preclinical Testing
The development pathway for influenza vaccines is well understood and includes preclinical testing in ferret, a widely accepted preclinical model of influenza. In addition, based on the Scientific Advice provided by the MHRA additional safety testing in rabbits was planned. Following review of progress and future planning at and after the 6th FLUTCORE meeting the partners realised that this work could not be completed during the period of the FLUTCORE grant and so an application for an amendment to the original work plan was submitted in March 2017 and approved in April 2017.

Clinical testing
The final step in the original FLUTCORE plan was intended to be a Phase I clinical trial of the vaccine that would explore both the safety and mechanism of action of the putative universal IAV vaccine. A full Phase I trial protocol was developed and planned. However following review of progress and future planning at and after the 6th FLUTCORE meeting the partners realised that this work could not be completed during the period of the FLUTCORE grant and so an application for an amendment to the original work plan was submitted early in 2017 and approved in April 2017.

Project Results:
Please find the content of this section, including table of figures, in the attached document, under "Description of the main S&T results/foregrounds "
Potential Impact:
The final results of the project describe the selection of three VLPs carrying IAV antigens that, when combined in a single vaccine, induce broad immunity to most, if not all strains of IAV. This report provides a template for the design and development of a lead candidate Universal Influenza A virus vaccine.
At the conclusion of the project methods of manufacture of two of these VLPs have been developed at laboratory scale. The “Up Stream Processing” (USP) of both these VLP, comprising the fermentation of transformed yeast, induction of expression of the VLPs and harvesting will be described, transferred to a manufacturer and undertaken at 10L scale in a manner in accordance with a GMP process. The same USP will be used for VLP4 production and the efficacy of this process will be confirmed after the end of the FLUTCORE grant period. The “Down Stream Processes” (DSP) involved in manufacture of VLP3, comprising lysis of yeast paste, extraction and purification of VLP will have been developed to provide a Standard Operating Procedure that can be transferred to a GMP manufacturer for the production of a vaccine subunit at scale. The other DSP for VLP2 and VLP4 will require further refinement before it can be used to manufacture VLP2 and VLP4 at scale. It is assumed that the method for manufacture of VLP4 at GMP scale will be similar to the method used for VLP2.
So far the project has had little societal or socio-economic impact. Patents relating to the design and manufacture of a universal influenza vaccine have been filed and these may have value. The manufacturing methods that have been developed for TC VLP may have application in the production of other VLP vaccines. Experience gained in the conduct of FLUTCORE has been applied to enable partners to develop other grant proposals. iQur anticipates building upon the work conducted in FLUTCORE to raise further private equity funding and industrial partnership. Antibodies developed in the conduct of the research are being marketed for research use.
However the major societal implication of the project remains the successful development of a universal influenza vaccine. Whilst this goal has not been achieved in the FLUTCORE grant period, considerable progress has been made towards the selection of a credible lead candidate and the development of a manufacturing process. It is anticipated that a Phase I clinical trial will be conducted in the next year to 18 months. Should this vaccine prove effective then the societal impact will be vast, reducing the current burden of influenza, protecting individuals against pandemic flu and possibly generating great value in excess of $1bn.


Dissemination and Exploitation

Several dissemination activities were conducted as part of the FLUTCORE project. These included public engagement, training, conference presentations and publications.

The FLUTCORE story is a powerful tool for public engagement, mainly because most people for all ages have experienced or heard about ‘flu.’ The talk given to school children outlines how the current influenza vaccine is manufactured, what its limitation are and how the FLUTCORE project aims to advance the field and overcome the limitations of supply, annual change and pandemic response. Both school children and their parents engaged in the story and understood the importance of vaccination.

Our experiences in the FLUTCORE project have been shared through conference presentations to the wider scientific community and incorporated into training material to be used at participating academic institutions. The project has created enormous value to the bioprocessing community in better understand antigen/vaccine design and its impact on the manufacturing process.

We also aim to publish in Open Access journals/repositories our work on fermentation development, assay development, cell-disruption, VLP purification, vaccine testing and influenza immunology.

A broad overview of vaccine strategies and their application to influenza has been developed and used in the teaching of graduate students in the academic institutions associated with the consortium and hospitals working with them. These materials will be disseminated more widely in due course.

The development and provision of analytical techniques for the characterisation of candidate VLP vaccines had a major impact within the consortium. However these methods are being described and incorporated in publications and presentation about the development of universal influenza vaccines.


The FLUTOCRE project included:

(i) evaluation of a cloning/expression back-up strategy based on a full-length homo-tandem Hepatitis B core backbone,
(ii) structural and immunological studies of HA stalk peptide trimers, and
(iii) development, standardization and scale-up of purification method for vaccine candidates VLP2 and VLP3 based on heterotandem HBc backbone.

For the first activity, no publishable data have been obtained due to unsatisfactory results. It has been concluded that this cloning/expression strategy is not a viable process for generating chimeric VLPs of interest.
Structural and immunological studies of HA stalk peptides led to several potentially interesting findings. First, upon purification these peptides appeared in solution as trimers resembling structure of native HA. Secondly, due to high-level synthesis and stability these trimers are attractive as diagnostic antigens. Thirdly, stalk peptide trimers exhibited a notable immune-protective potential in mice and therefore might be considered as novel flu vaccine components. Immunologic data have been presented in a conference at the Latvian University [1] and after then published as an extended conference abstract [2].
Finally, data regarding the expression and purification of the first selected flu vaccine candidate subunit, VLP2 have been summarized in scientific publication to be submitted at the end of April 2017 [3]. It should be noted that the generation and purification of chimeric tandem HBc VLP from yeast is very challenging and there are few reports in the literature describing this. We think that the protocols developed and established in the conduct of the FLUTCORE project will be helpful in future activities to obtain VLPs of interest in yeast cells.


[1] Anna Kirsteina1, I-Na Lu2, Sophie Farinelle2, Claude Muller2, Kaspars Tars1, Andris Kazaks1. Expression, purification and immunological properties of influenza hemagglutinin peptides. 75th Scientific Conference of the University of Latvia. Riga, January 30, 2017.

[2] Anna Kirsteina1*, I-Na Lu2*, Sophie Farinelle2, Claude Muller2, Kaspars Tars1, Andris Kazaks1. Expression, purification and immunological properties of influenza hemagglutinin peptides. Environmental and Experimental Biology (2017) 15:59-60.

[3] Andris Kazaks1*, I-Na Lu2, Sophie Farinelle2, Alex Ramirez3, Vincenzo Crescente3, Benjamin Blaha4, Olotu Ogonah4, Tarit Mukhopadhyay4, Mapi Perez de Obanos5, Alejandro Krimer5, Inara Akopjana1, Anna Kirsteina1, Janis Bogans1, Velta Ose1, Tatjana Kazaka1, Nicola Stonehouse6, David Rowlands6, Claude P. Muller2, Kaspars Tars1 and William Rosenberg3. Production and purification of chimeric HBc virus-like particles carrying influenza virus LAH domain as potential vaccine candidates. In manuscript.

IAV vaccine strategies rely on a protective antibody response against the hemagglutinin (HA) and neuroaminodase (NA). Both of these surface glycoproteins are highly variable among IAVs circulating in humans and animals. Current vaccines induce only a narrow and strain-specific immunity. Because of antigenic drift and shift, they have to be reformulated for every season to match the circulating strains. It is therefore essential to improve current vaccines and to develop drift resistant strategies providing a broad and lasting protection. Such universal IAV vaccines are based on conserved domains of viral proteins. Usually these domains are less exposed to the host immune system and are less immunogenic resulting in less immune pressure-derived antigenic changes. In contrast to the conserved influenza matrix protein and nucleoprotein epitopes, which provide only weak protection in human challenge studies, the conserved stalk portion of HA is a much more potent candidate for a universal vaccine. In particular, the highly conserved long α-helix (LAH) of the stalk domain spanning amino acid (aa) 76-130 induces broadly reactive and protective Ab responses.
Before our study, the role of CD4 T cells in the LAH-specific immune responses was not well understood. The FLUTCORE project led to the identification of a mouse CD4+ T cell epitope that encompasses residues form the LAH domain that are detectable following sub-lethal infection of influenza. In response to stimulation with the identified epitope, splenocytes derived from the infected mice showed a significant polyfunctionality as detected by IL-2, TNF-α, and IFN-γ production as well as degranulation. Moreover, mice immunized with the peptide corresponding to this CD4+ T cell epitope exhibited inter-individual sharing of CD4+ T cell receptor β sequences and they survived better following challenge with lethal dose of pandemic H1N1 influenza virus. Thus, our data have demonstrated a crucial role of LAH-specific CD4+ T cells in the host immune response to IAV infection. (published manuscript: Lu et al.; Identification of CD4 T cell epitope in hemagglutinin stalk domain of pandemic H1N1 influenza virus and its antigen-driven protective TCR usage signature; Cell Mol Immunol, 2016; IF: 5.193)
Furthermore, due to an error-prone replication machinery, IAVs can easily adapt to immune pressure. Therefore, it is important to monitor the emergence of escape mutants in particular against conserved epitopes. The FLUTCORE consortium applied Next Generation Sequencing (NGS) to investigate the impact of antibody mediated immune responses against LAH on strain diversity within the targeted region for the first time. Unlike conventional techniques NGS provides a direct and complete snapshot of in vivo viral quasispecies. Focusing on the LAH domains, we showed that there was constrained evolution of viral quasispecies after vaccination and no emergence of a new major virus variant. These findings demonstrate that LAH is a safe and potent universal influenza vaccine candidate. (Submitted manuscript: Hauck et al., Next generation sequencing reveals a constrained viral quasispecies evolution under crossreactive antibody pressure)

As part of the characterisation of lead VLP vaccine candidates novel reagents were developed which can be used to identify the presence of LAH3 in immuno-detection assays. A mouse monoclonal antibody specific to LAH3 from H3N2 was developed de novo for the purpose of VLP2 characterisation and in-process and end-process control of VLP production. There is currently no commercial alternative available, but this antibody is of interest to the scientific community involved in influenza virology research as a way to subtype, tag or identify H3 strains of IAV. This reagent has been accepted into the BEI repository set up by the National Institute of Allergy and Infectious Disease as a valuable research reagent and has been made available for research purposes. It can be requested by research institutions worldwide from here:
https://www.beiresources.org/Catalog/BEIMonoclonalAntibodies/NR-50513.aspx

List of Websites:
Project website address: www.flutcore.eu

Contact details:
Professor William Rosenberg,
University College London, Division of Medicine
Royal Free Campus, Rowland Hill Street, Hampstead, London NW3 2PF
Tel: +44 (0)207 794 0500 x 36166
E-mail: w.rosenberg@ucl.ac.uk

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UNIVERSITY COLLEGE LONDON
United Kingdom

Subjects

Life Sciences
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