WO2009133463A1

WO2009133463A1 - Soluble influenza vaccine

Info

Publication number: WO2009133463A1
Application number: PCT/IB2009/005633
Authority: WO
Inventors: Kjell Ove Hakansson; Allan Randrup Thomsen; Jan Pravsgaard Christensen
Original assignee: Københavns Universitet
Current assignee: Københavns Universitet
Priority date: 2008-04-30
Filing date: 2009-04-30
Publication date: 2009-11-05
Anticipated expiration: 2010-10-30

Abstract

The invention provides fusion proteins comprising: (i) a first portion comprising an influenza A M2e polypeptide, and (ii) a second portion comprising a rotaviral NSP4 polypeptide. The fusion proteins mimics the native tetrameric structure of native M2 protein. Additionally it is highly soluble and as disclosed herein can be produced and isolated in large quantities extremely quickly and efficiently. Also provided are related materials and methods, including use of the fusion proteins as vaccines.

Description

Soluble influenza vaccine

Technical field

The present invention relates generally to improved methods and materials for use in preparing soluble influenza vaccines.

Background art

Influenza A virus is an enveloped, negative sense single stranded RNA virus with the ability to infect a wide variety of mammalian and avian species. Influenza A virus escapes the immune response of the hosts by a high mutation rate (genetic drift), reassortment of the segmented genome in cells infected by more than one strain (genetic shift) and by crossing the species barriers (reviewed in e.g. [1 ,2]). Vaccines against influenza A must be frequently updated in response to the genetic instability and the production of adequate, up-to-date vaccines is compromised by the relatively long development and production time.

Matrix protein 2 (M2) is an integral membrane protein which occurs as a homotetramer, and has a small external domain (M2e) of around 23 amino acid residues (after the terminal Met is removed). Natural M2-protein is present in a few copies in the virus particle, but in abundance on virus infected cells. Although M2e is almost non- immunogenic its sequence is highly conserved [6,7].

US published application 20040223976 relates to M2 peptide conjugates.

In one strategy for developing a universal Influenza A vaccine, the M2e peptide has been linked to a virus-like particle (VLP) based on the Hepatitis B Virus core (HBc) [18, 21]. It has been reported that in this context, M2e was highly immunogenic, and that the M2e- HBc vaccine induces antibodies that protected mice against influenza-induced death and morbidity. This recombinant construct (M2e linked to a Hepatitis B core protein) has apparently been used in trials a vaccine candidate ("ACAM-FLU-A™) and is asserted to provide protection against ¹A' strains of flu, without side effects [6,11-18] (see also US 7361352 and www.acambis.co.uk.). lmmunoprotection depends on destruction of virus infected cells, rather than neutralization of virus particles [15], and is therefore weaker than what is obtained with immunization with inactive viruses. Chemically synthesized M2e peptides have been conjugated to keyhole limpet hemocyanin and Neisseria meningitidis outer membrane protein complex 2004 [12]. Sera raised against these constructs offered protections in mice against heterologous strains with Asp21Gly, although there was a lack of cross- reactivity with H5N1 M2e (ProiOLeu, Ile11Thr, Glu14Gly).

Synthetic branched polypeptide with four copies of M2e induced an antibody response in mice and resulted in a reduced viral replication in these animals after challenge with live virus [24,25].

M2e constructs with the transmembrane region deleted and fused to glutathione S- transferase were also, after immunization, reported to enhance viral clearance in mice challenged with live virus [14]. Glutathione S-transferase fusion proteins with multiple copies of M2e sequence similarly protected mice upon administration of lethal doses of influenza virus. However, since antibodies raised against human M2e glutathione S- transferase fusion proteins display no or weak affinity for the avian variants, a bivalent vaccine composition with two M2e sequences might be necessary for a universal cover [7]. As pointed out by de Filette et a/., direct comparison of the result of all these studies is not meaningful as different protocols and viral doses have been used [11].

WO02074795 relates to chimeric protein comprising an antigen and an oligomerisation domain, and complexes thereof, in addition to their use for the manufacture of a vaccine. This lists several different possible domains, of which the preferred type is a leucine zipper.

De Filette et al. J Biol Chem. 2008 Feb 5 reported an M2e based construct whereby M2e was fused to a sequence variant of the leucine zipper domain from the yeast transcription factor GCN4, forming all parallel four-chain coiled-coils in order to try and obtain a tetrameric structure with the conformation of the native M2 ectodomain. It is asserted that the fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies. Nevertheless, it will be appreciated that the identification of further particular oligomerisation domains, particularly those which can lead to production efficiencies of the recombinant vaccine, would provide a contribution to the art.

Disclosure of the invention

The present inventors provide herein a fusion protein between the M2 extracellular region and rotavirus NSP4. The fusion protein mimics the native tetrameric structure of native M2. Additionally it is highly soluble and as disclosed herein can be produced and isolated in large quantities extremely quickly and efficiently. In preferred embodiments the fusion comprises two engineered histidines making Ni-NTA (Nickel-nitrilotriacetic acid) chromatography rapid and efficient.

As with previous M2e vaccines, since the fusion of the present invention is one of the most conserved parts of the influenza genome, and it may be expected that it would provide protection against most present and future influenza strains.

WO0127335 relates to the use of rotaviral enterotoxin NSP4 genotypes as an adjuvant with different antigens - the cytosolic region between amino acid residues 131 to 140 of the sequence was considered to be particularly relevant to enterotoxicity and adjuvant activity. NSP4 is a glycoprotein, and the use of the glycosylated part (near the N- terminus, which locates in the ER) in lectin affinity chromatography is discussed. However the use of NSP4 in the context of the present invention is not discussed.

Thus in one aspect the present invention describes:

A fusion protein comprising:

(i) a first portion comprising an influenza A M2e polypeptide, and

(ii) a second portion comprising a rotaviral NSP4 polypeptide.

As described herein the fusion protein may be used to mimic the tertiary and quaternary structure of the native M2 ectodomain, but wherein the hydrophobic transmembrane region is replaced by a water soluble coiled-coil.

Optionally the fusion also contains: (iii) a third portion comprising one or more (preferably at least 2, 3, or 4) His residues.

This type of truncated-tag has been shown in the Examples herein to facilitate expression and hence recovery of fusions according to the present invention compared to a longer tag, such as is more commonly used in the art. Additionally, it is believed that the shorter tag may be more physiologically acceptable than a longer tag.

The invention further provides nucleic acids, vectors and host cells suitable for production of the fusion protein and oligomeric protein complexes (e.g. homotetramer) formed therefrom.

Also provided herein are processes for producing the nucleic acids, vectors, host cells, fusion proteins, oligomeric protein complexes, and vaccines based thereon.

Also provided herein are influenza vaccines and uses thereof.

The fusion proteins and recombinant oligomeric protein complexes may be crystallised and used in structural studies and these provided further aspects of the invention.

Some preferred embodiments and further aspects of the present invention will now be discussed in more detail:

First portion sequence

Thus the first portion of the fusion protein comprises an influenza A M2e polypeptide which is the N-terminal portion of the highly conserved membrane protein M2.

M2 is one of the smallest known membrane proteins consisting of only 97 amino acids. It is a tetrameric [8,9] ion channel specific for passive transport of protons [10]. The first 24 amino acids form the extracellular domain, M2e, located on the outside of the membrane of the virus and of infected cells. This is followed by an α-helical transmembrane region and a relative small cytoplasmic domain.

Preferably this portion has the sequence of a characterised strain. In the Examples below the H1 N1 M2e (M2 ectodomain) was used, which represents a good consensus of strains sequenced to date. This has the following sequence (SEQ ID No 1) M S L L T E V E T P I R N E W G C R C N D S S D (HlNl consensus) 1 2 3 4 5 6 7 8 9 101112131415161718192021222324

As noted below, the terminal Met may preferably be absent at position 1 , and any disclosure of a sequence herein in which the Met is shown to be present will be understood to likewise disclose the same sequence in which the Met is absent.

Numerous other strains have been characterised to date and there is a very high level of conservation. Indeed heterologous protection has been demonstrated between strains ([6,11,13,18]).

If desired, fewer than 24 amino acids of the sequence may be used -for example 20, 21 22, or 23 amino acids. Preferably at least 20 amino acids are used.

However preferably longer sequences from the M2 protein may be also used e.g.30, 40 or more amino acids i.e. extending beyond the ectodomain into the helical domain.

An example full length M2 sequence is:

mslltevetp irnewgcrcn dssdglwaa siigilhlil wildrlffkc vyrpfkhglk rgpstegvpe smreeyrkeg qnavdaddgh fvsiele

In the Examples below, a Pro which may be part of the transmembrane domain (underlined above) was included. Thus any disclosure of an M2e sequence herein will be understood to likewise disclose the same sequence in which the Pro is present.

Preferred M2e sequences of the invention will preferably have equal to or no more than 1,2,3,4,5,6,7,8 or 10 substitutions, deletions, or additions in the consensus sequence SEQ ID No 1, most preferably no more than 1, 2 or 3 substitutions deletions, or additions in the consensus sequence.

Typically the M2e portion sequence will share at least 75%, 80%, 90%, 95%, 96%, 97% or 98% identity with the consensus sequence. The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, the comparison is done by comparing sequence information using a computer program. An exemplary, preferred computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, 'GAP' (Devereux et al., 1984, Nucl. Acids Res. 12: 387). The preferred default parameters for the ^'GAP^' program includes: (1) The GCG implementation of a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; or other comparable comparison matrices; (2) a penalty of 30 for each gap and an additional penalty of 1 for each symbol in each gap for amino acid sequences, or penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps.

In one embodiment an artificial derivative based on the consensus sequences may be prepared by the skilled person in the light of the present disclosure. Such derivatives may be prepared, for instance, by site directed or random mutagenesis, or by direct synthesis. Preferably a variant nucleic acid (for example) is generated either directly or indirectly (e.g. via one or more amplification or replication steps) from an original nucleic acid having all or part of the sequence shown herein. Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation. Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the fusion protein. In all aspects of the present invention the N-terminal methionine of the first portion may be absent, and indeed this post-translational removal is expected in to occur in the recombinant processes of the invention.

Preferably, however, the sequence, will reflect only changes made in naturally occurring strains.

The following represents a consensus sequence showing the variable residues characterised in existing human strains and H5N1:

MS(QF)(QP)TEVETP(TM)R(NVKVS)(EXG)W(EVG)C(RXK)C(SVN)(DXG)SS(DVN) (invariant residues are in bold)

Preferably any substitution in respect of the consensus is selected from one in any of these 10 positions (3, 4, 11 , 13, 14, 16, 18, 20, 21 , 24).

Preferably any substitution in respect of the consensus is selected from the change shown in these 10 positions. Preferably positions 1 to 10 are as in the consensus sequence.

Non-limiting examples including substitutions at the defined positions are as follows. These have up to four substitutions, including positions 11 , 13, 16 and 20:

H5nl (above) MSLLTEVETP IRNEWGCRCN DSSD x swine flu' MSLLTEVETP TR£3EWECRC£3 DSSD h5nl (alternate) MSLLTEVETP TRSEWECRCS3 DSSD h5nl (alternate) MSLLTEVETP TRNEWECRCN DSSD

The following represents a consensus sequence showing the variable residues characterised in existing human strains:

MS(LVF)(LVP)TEVETP(TVI)R(NVKVS)(EVG)W(EVG)C(RVK)CN(DVG)SS(DVN) (invariant residues are in bold) Preferably any substitution in respect of the consensus is selected from one in any of these 9 positions (3, 4, 11, 13, 14, 16, 18, 21, 24).

Preferably any substitution in respect of the consensus is selected from the change shown in these 9 positions. Preferably positions 1 to 10 are as in the consensus sequence.

Thus, for example, the corresponding 24 amino acids from H5N1 may be used.

MSLLTEVETP TRNEWECRCS DSSD

Or any human M2e sequence meeting the consensus below (from Fiers et al (2004)

M2eOF WMAN INFLUENZA A STRAINS

S L L T E V E.. T PJ R₁ NJ₁₁ W G C R C N D S S D

E G

% 100 ICO 100 1rø 15U ItM tOO IM 1W 98 86 IHJ 1OO 1WJ 76 WO rø TOO 1<» TS 100 100 1(W

It will be appreciated that the production strategy of the invention, in addition to use with any of the panel of sequences described above, may likewise be applied to further influenza strains of interest - for example newly characterised M2e sequences from newly emergent strains which may arise in epidemics or pandemics may be cloned and produced as described herein.

Second portion sequence

The second portion of the fusion protein comprises a rotaviral nonstructural protein (NSP4) polypeptide, and in particular a soluble tetrameric coil sequence therefrom.

An exemplary NSP4 sequence is as follows (see e.g. UniProtKB Entry 092323):

MEKLTDLNYT LSVITLMNNT LHTILEDPGM AYFPYIASVL TVLFALHKAS 50

IPTMKIALKT SKCSYKWKY CIVTIFNTLL KLAGYKEQIT TKDEIEKQMD 100

RWKEMRRQL EMIDKLTTRE IEQVELLKRI YDKLTVQTTG EIDMTKEINQ 150

KNVRTLEEWE SGKNPYEPRΞ VTAAM 175 Preferably the second portion is based on Ile95-Gin137 region of NSP4 (43 amino acids).

IEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLWQ

This sequence is present in several human and simian strains although analysis of protein shows that the residue at position 137 is in some strains R rather than Q.

As noted below, the C-terminus may be substituted to introduce a third portion of the fusion protein comprising 1 or more His residues at the C-terminus and that is discussed below. These are preferably introduced by substitution at positions 135 and 137 i.e.

IEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

For simplicity 135-137 are therefore omitted from the sequences shown below:

Thus the second portion may start at NSP4 residue 95, 96, 97, 98, 99, 100, 101 and extend to Leu 134. Examples of such constructs are described below.

IEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

EKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

KQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

QM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

M DRWKEMRRQ LEMIDKLTTR ΞIEQVELLKR IYDKL

DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

RWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

Typically the NSP4 portion sequence will share at least 75%, 80%, 90%, 95%, 96%, 97% or 98% identity with the sequences shown herein up to and including Leu 134. However it will be appreciated that NSP4 sequences having even lower sequence identities are known and also encompassed by the present invention e.g. the NSP4 sequence from avian rotavirus.

Exemplary NSP4 sequences are described in WO0127335 and include group A genotypes A, B, C or D. WO0127335 further references sequences corresponding to NSP4 genes that are classified in the group A rotaviruses with the corresponding GenBank Accession Numbers http://www. ncbi. nlm. nih. gov/Genbank/GenbankSearch. html): ALA (AF144792); C-11 (AF144793); R-2 (AF144794); BAP-2 (AF144795); BAPwt (AF144796); A253 (AF144797); A131 (AF144798); A411 (AF144799); A34 (AF165219); H-1 (AF144801); FI-23 (AF144802); FI-14 (AF144803); BRV033 (AF144804); B223 (AF144805); CU-1 (AF144806); OSU (D88831); SA11 (AF0871678).

Other virus strains and groups are discussed by Ciarlet M, Liprandi F, Conner ME, Estes MK (2000) Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal and human rotaviruses. Arch Virol. 145: 371-383.

Other NSP4 sequences that are classified in the non-group A rotavirus are referenced with the corresponding GenBank Accession Numbers http://www. ncbi. nlm. nih. gov/Genbank/GenbankSearch. html):

group B IDIR (U03557); group C (X83967); group C (D88353); group C (SEQ. ID. NO: 21 , L12391)

Third portion

As noted above, preferably the third portion includes fewer than 5 His e.g. 2, 3 or 4 separated by no more than a single amino acid. This has been shown to facilitate purification of the fusion on an appropriate affinity column e.g. Ni or Co metal chelate chromatography.

It is well known to use a "6xHis" or "hexa histidine"-tag for purification purposes, but it is nevertheless surprising that the addition of fewer than 5 His residues can be be highly effective.

The third portion is preferably at one or other terminus, preferably the C terminus.

The third portion may be conveniently provided by mutation of the terminal residues of one of the first and second portions i.e. substitution of the 2 or more residues (contiguous or separated by a single amino acid) in the existing viral sequences.

For example it may be introduced by substitution at or near Thr 135 of the NSP4 sequence e.g. Thr 135 and GIn 137. However it may equally be added to the end of the second portion.

Preferred fusion proteins

In the fusion protein, generally the first, second, and third sequences are arranged contiguously in that order from N to C terminus.

The first and second sequences may be separated by a short linker sequence or residue in order to facilitate expression or folding e.g. one or more gly residues. However as described in the Examples this is not necessary in the sequences of the invention.

Thus example fusion proteins between H1N1 M2e (M2 ectodomain) and a tetramerizing coiled-coil from rotavirus NSP4 protein with introduced His residues may be as follows:

MSLLTEVETP IRNEWGCRCN DSSDPIEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH MSLIITEVETP IRNEWGCRCN DSSDP EKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH MSLLTEVETP IRNEWGCRCN DSSDP KQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH MSLLTEVETP IRNEWGCRCN DSSDP QM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH MSLLTEVETP IRNEWGCRCN DSSDP M DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH MSLLTEVETP IRNEWGCRCN DSSDP DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH MSLLTEVETP IRNEWGCRCN DSSDP RWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH I I I

M2e NSP4 2 Engineered His The different constructs reflect the fact that NSP4 moiety has a less tightly wound supercoil than M2. Therefore the different junction points permit for different orientations of the amino acids in the coiled-coil, and its heptad repeats.

Preferred sequences are the third, fourth, and seventh sequences listed above.

As noted above, the M2 portion may be extended (e.g. to include a Pro from outside the M2e region) and\or the NSP4 portion shortened, in order to enhance the structural similarity between the M2-NSP4 construct and native M2e.

As noted above, apart for the M2e and NSP4 portions, the fusion protein may comprise other polypeptide sequences, such as linker sequences or polypeptide sequences that enhance the immune response.

In another aspect of the invention there is provided a recombinant oligomeric protein complex comprising a plurality of associated fusion proteins as described above. Typically the recombinant oligomeric protein complex is a homotetramer. For example the present inventors have demonstrated that the fusion proteins of the invention have the properties of higher order oligomers, as judged by column gel filtration and ultrafiltration using 3OkD cutoff Millipore centracon filters.

Having described the fusion proteins of the present invention, some other aspects of the invention will now be discussed.

Nucleic acids, vectors and host cells

As described in the Examples hereinafter, the fusion proteins of the invention are conveniently prepared from recombinant nucleic acids which can be expressed in host cells with good yield, for example from a pET vector and can be expressed in Escherichia coli.

Thus in one aspect there is provided a nucleic acid encoding a fusion protein of the first aspect. Such nucleic acid is herein referred to "fusion nucleic acid" for brevity. This may be put together, for example, using "overlap PCR". Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid). Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed. Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin, and double or single stranded. Where used herein, the term "isolated" encompasses all of these possibilities. The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Nucleic acids may comprise, consist, or consist essentially of, any of the sequences discussed hereinafter.

Aspects of the invention further embrace isolated nucleic acid comprising a sequence which is complementary to any of those discussed hereinafter.

In one aspect of the present invention, the fusion nucleic acid described above is in the form of a recombinant and preferably replicable vector.

Fusion nucleic acids can be incorporated into a specially constructed vector or can be constituted in situ by introducing nucleic acid encoding the various portions such that the whole sequence is created in frame in the vector. Such processes for making the recombinant vectors form one aspect of the invention.

It is envisaged in the present invention that fusion proteins as defined herein, based on newly characterized viruses (e.g. from pandemics) may be prepared simply and easily by sequencing, synthesis of artificial M2e-encoding DNA, followed by PCR and ligation into frame with the NSP4 portion of the fusion (e.g. using the appropriate restriction sites such as the Ndel and BamHI sites in the pET-11 vector described herein).

"Vector" is defined to include, inter alia, any plasmid, cosmid, or phage in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic or eucaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including transcriptional and translational regulatory elements, such as transcriptional enhancer sequences, translational enhancer sequences, promoters, ribosomal entry sites, including internal ribosomal entry sites, activators, translational start and stop signals, transcription terminators, cistronic regulators, polycistronic regulators, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et a/, 1989, Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. yeast or fungal cells).

A vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

However preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial cell. By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA). Example vectors include plasmids pQE, pET or other commercial or generally accessible plasmids.

A preferred vector is the pET11-a plasmid, although where expression is desired in XL1- blue, other vectors may be preferred.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter. In one embodiment, the promoter is an inducible promoter. The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. A typical inducible promoter is the lac promoter induced by IPTG.

The present invention also provides methods comprising introduction of such a construct into a cell e.g. a microbial cell (e.g. bacterial, yeast or fungal) cell or an insect cell and/or induction of expression of the construct within the cell, by application of a suitable stimulus e.g. an effective exogenous inducer.

In a further aspect of the invention, there is disclosed a host cell containing a heterologous construct according to the present invention, especially a microbial cell such as E. coli e.g. E. co// XL1-blue cells

The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention (e.g. comprising the fusion nucleic acid) especially a microbial cell such as E. coli.

The fusion protein can be produced by culturing a microbial cell as described above e.g. by growing the cells in liquid medium with appropriate selection reagents (e.g. LB medium with ampicillin).

As shown in the Examples, the expression levels are high (10-15 mg/cell of purified antigen obtainable from 1 L of bacterial culture around 3 hours after induction, or at around 6 to 7 hours total fermentation). Additionally the purification procedure can be is much quicker than that reported for the M2e-tGCN4 in DeFilette et al. (supra) and can easily be scaled up. After sonication of the bacteria, in preferred embodiments, the purification takes less than four hours and can consist of Ni-NTA-HisTag and MonoQ- ionexchange chromatography steps. The product can be very pure. Thus in another aspect of the invention provides a process for producing a composition for use in an influenza vaccine in a human or animal, the process comprising: i) providing a microbial cell of the invention described above, ii) culturing the cell such as to product the fusion protein of the invention, iii) purifying the fusion protein therefrom.

In this and other aspects of the present invention, the fusion protein is preferably in the form of an oligomeric protein complex e.g. a homotetramer, as described above.

Optionally step i) is preceded by the steps described above i.e. preparation of the vector and\or introduction into a host cell.

Optionally step iii) may be followed by formulating the fusion protein or complex as a vaccine. Vaccines are discussed in more detail below.

Preferably step iii) comprises or consists of: iiia) contacting the product of step ii) with an affinity column capable of binding a Hexa-

His-Tag e.g a Ni-NTA column. iiib) at least one anion exchange chromatography step.

The buffer used for loading during Ni-NTA chromatography is preferably imidazole-free, with elution by an imidazole gradient.

For larger scale applications monoQ might be substituted with similar ion exchangers e.g. ResourceQ.

It will be appreciated that while these steps have been shown to produce very pure product, further preparative steps are not excluded. These may include gel filtration, PEG, ammonium sulfate or ethanol precipitation, acid extraction, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, reverse phase chromatography, preparative electrophoresis, detergent solubilization, selective precipitation, centrifugation, ultracentrifugation, density gradient centrifugation, ultrafiltration through a size exclusion filter, and so on. General techniques are further described in, for example, R. Scopes, Peptide Purification: Principles and Practice, Springer- Verlag: N. Y. (1982). Vaccines and uses

The present invention provides compositions and their use as vaccines against the influenza virus, particularly influenza A. By careful selection of the M2e portion, the vaccines described herein may in principle be used as a universal influenza vaccine.

The vaccine compositions will comprise the recombinant oligomeric protein complex comprising a plurality of associated fusion proteins as described above. Typically the recombinant oligomeric protein complex is a homotetramer, preferably in substantially pure form. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, intramuscular, intraperitoneal routes - example formulations are discussed in WO95/27056.

In preferred embodiments the compositions can be combined with an adjuvant. Example adjuvants for use in anti-viral vaccines are known in the art and may be selected without undue burden by those of ordinary skill.

In one aspect the invention provides use of a vaccine of the invention in the manufacture of a medicament for inducing an immune response in a human or animal against influenza virus.

In one aspect the invention provides a vaccine of the invention for this purpose.

The invention also provides methods of reducing the likelihood of contracting a condition associated with influenza virus, e.g. in an animal or human, comprising administering an an immunologically effective dose of a vaccine of the invention.

Thus in some aspects of the present invention, the compositions can be utilized in a vaccine strategy to induce an immune response in a human or animal. The compositions can be combined with an adjuvant and administered in an effective amount to a human or animal in order to elicit an immune response. In other embodiments, the compositions are administered without an adjuvant to a human or animal. In some embodiments the composition includes immuno-stimulatory nucleic acid(s), such as CpG sequences.

The compositions of the present invention can be administered using any technique currently utilized in the art. Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the subject; the route of administration; the desired effect; and the particular composition employed. The vaccine can be used in multi-dose vaccination formats.

In one embodiment, a dose would consist of the range from about 1 μg to about 1.0 mg total protein. In another embodiment of the present invention the range is from about 0.01 mg to 1.0 mg. However, one may prefer to adjust dosage based on the amount of protein delivered. In either embodiment, these ranges are guidelines. More precise dosages can be determined by assessing the immunogenicity of the conjugate produced so that an immunologically effective dose is delivered. An immunologically effective dose is one that stimulates the immune system of the patient to establish an immunological response. Preferably, the level of immune system stimulation will be sufficient to develop an immunological memory sufficient to provide long term protection against disease caused by infection with a particular influenza virus.

The timing of doses depends upon factors well known in the art. After the initial administration one or more booster doses may subsequently be administered to maintain antibody titers. An example of a dosing regime would be a dose on day 1 , a second dose at 1 or 2 months, a third dose at either 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months or greater than 12 months, and additional booster doses at distant times as needed.

Decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated or prevented, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The immune response so generated can be completely or partially protective against disease and debilitating symptoms caused by infection with influenza A virus. Nucleic acid-based therapeutics of the invention may be used in place of polypeptides or oligomers with conventional gene therapy vectors, such as are well known in the art (see e.g. WO0159142).

In addition to the above, the vaccines described herein may be used to protect against rotavirus infection or disease, due the presence of the NSP4 portion. The aspects described above in relation to influenza apply mutatis mutandis to against rotavirus infection.

Other aspects of the invention

The fusion proteins and recombinant oligomeric protein complexes may be crystallised and used in structural studies - including studies aimed at defining the M2e structure, and hence its potential as a drug target in combating influenza. Crystallization and structural analysis may be performed using conventional methods (see e.g. Drenth, J. Principles of Protein X-ray Crystallography. (2nd ed.) Springer Verlag, New York, 1999; McPherson, A. Crystallisation of Biological Macromolecules. Cold Spring Harbor Laboratory Press, New York, 1999). Such analysis, and methods of producing crystals, crystals, and structural data, form further aspects of the invention.

Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross- reference.

Figures

Fig. 1. The sequences of the three final clones described in this paper. The N-terminal methionine is cleaved off after translation. The amino acid sequences are identical except for the C-terminal His-tag. There is a small difference in codon usage between the clones. Fig. 2. Expression of M2e-NSP4, M2e-NSP4His₅, and M2e-NSP4His₂. The gel shows three samples from each strain. The control (-) and an IPTG induced (+) culture were resuspended in 1/10 of the original volume, sonicated and centrifuged. The first to samples (-,+) represent the entire content of control and induced cells, taken prior to centrifugation. The third sample (+sup) represents the supernatant taken after centrifugation, i.e. the soluble fraction. M2e-NSP4 and M2e-NSP4His₂ could be expressed in high yield, whereas no expression of M2e-NSP4His₅ could be detected.

Fig. 3. a) Chromatogram from Ni-NTA chromatography of M2e-NSP4His₂. b) SDS-PAGE of the soluble fraction of the sonicated cells, fractions 3, 6, 9, 13, 14 and 15 from Ni-NTA chromatography fractions (which are marked in the chromatogram) followed by the four peak fractions from the subsequent mono-Q chromatography and, for comparison, purified M2e-NSP4

Fig. 4. Stereo image of the structure of NSP4_95-137 (pdb id code 1G1 I/1G1J). Only Ca atoms, and the side chains of T135 and Q137 (NSP4 numbering) are shown.

Fig. 5. Antibody titers in M2e and M2e-NSP4 vaccinated mice. Balb/c mice were vaccinated s.c. 3 times with 10μg of the indicated constructs, 3 weeks apart. Two weeks after each vaccination animals were bleed and antibody titers determined.

Vaccinated groups were M2e peptide alone (1-mer M2e), tetramer (4-mer M2e) with and without adjuvant and unvaccinated controls (unvacc).

Antibody specificity were analysed on maxisorp ELISA plate coated with 30ng/well (100μl) M2e peptides (1-mer M2e), tetramer (4-mer M2e), and NSP4 peptides (4-mer tail).

Each dot represents one animal. No dots mean a titer 100 or less. Detection level is 200.

Fig. 6 . Protection against H7N7 infection. Balb/c mice were vaccinated s.c. 3 times with 10μg of the indicated constructs, 3 weeks apart. Six weeks after the last vaccination mice were infected intranasally with EgLon72 (H7N7) influenza virus. Weight and survival was recorded. Each line represents an individual animal. Fig. 7. Protection against H1N1 infection. Balb/c mice were vaccinated s.c. 3 times 3 weeks apart with 10μg or 50μg tetramer or left untreated. Six weeks after the last vaccination mice were infected intranasally with PR8 (H1 N1) influenza virus. Weight and survival was recorded. Control group were killed day 14 due to low weight. Each line represents an individual animal.

Examples

Material and methods

Cloning

Plasmid DNA was purified with the 'GFX plasmid DNA purification kit', while per products (when used as templates) and digested vectors were purified with the 'GFX PCR DNA and gel band purification kit' (Amersham Biosciences). The pET11-a plasmid was purchased from Novagen. Quick ligase and restriction enzymes were obtained from New England Biolabs or Fermentas. All cloning products were verified by sequencing performed by MWG-Biotech AG.

Cloning of M2e: The gene coding for the extracellular, or extraviral part of M2 ion channel was purchased from MWG-Biotech AG, using the codon corresponding to the most abundant E. coli tRNA transferases [19]. Two complementary fragments with sticky ends, mimicking Ndel and BamHI cleavage were mixed and diluted to a total concentration of 0.02 μg/ml and heated to 95° C. The solution was allowed to chill down slowly to room temperature. The DNA fragment was incorporated into a pET11-a vector linearized with Ndel and BamHI, using the quick ligase protocol. The ligated vector was heat shock transformed into E. co// XL1-blue cells, which were plated on LB-agar medium with 100 μg/ml ampicillin. Colonies were picked and grown overnight in LB-medium with ampicillin.

Cloning of NSP4₉₅.₁₃₇: A gene corresponding to region Ile95-Gln137 of nonstructural protein NSP4 from rotavirus was cloned in a similar manner. In this case, only a single colony with the NSP4 insert was found.

Cloning of M2e-NSP4: The fusion protein M2-NSP4 was constructed using overlap per with the plasmids from the M2e and NSP4₉5_-137 clones as templates. First, the M2e gene was amplified between the pET11-a Sphl site and the end of the gene using the following templates: Sphl_pet_up: ggtgcatgcaaggagatgg, primer 1: ctacacggtccatctgtttttcgatcgggtcagaagagtcgttg for 47 cycles using an annealing temperature of 57⁰C and an elongation time of 45s. The NSP4₉₅-i₃₇ gene was amplified between the start of the gene and the pET11-a Hindi 11 restriction site with the following primers: primer 2: atcgaaaaacagatggaccgt, Hindlll_pet_do: atcatcgataagctttaatgcg with the same protocol. The two products were purified using the GFX DNA purification kit and mixed and amplified with Sphl_pet_up, Hindlll_pet_do and an elongation time of 80s. The product was digested with Sphl and Hindlll and ligated into a pET11-a vector digested with the same enzymes. The ligation mixture was electrotransformed into B834.

Cloning of M2e-NSP4His₅: The gene was purchased from Genscript Corporation (Piscataway, NJ, USA), with optimal E. coli codon usage. The gene was amplified from the pUC57 vector with the following primers: primer 3: tatacatatgagcctgctgaccgaag, primer 4: gcagccggatcc ttaatgatgatgatgcacatgc for 47 cycles with 57⁰C and 30s as annealing temperature and elongation time. The per product was digested with Ndel and BamHI, ligated and electrotransformed into E. coli BSZA.

Cloning of M2e-NSP4His₂: As the clones with the NSP4_95-137 and M2e-NSP4 genes accidentally contained a duplication of the last part of the sequence, this extra DNA needed to be excluded in order to proceed with C-terminal modifications. This was done by running a nested per with the primers sph1_pet_up and primer 5: cgggtggtccttactgtac with the M2e-NSP4 gene as template for 45 cycles with an annealing temperature of 57C and an elongation time of 30s. The per product was used as template for the final PCR with sph1_pet_up and primer 6: ttagcagccggatccctattagtgtacgtgcagtttgtcgtagatacgtttc as primers, and the elongation time increased to 40 s. The per product was cloned into the pET vector with the restriction enzymes Sph1 and BamHI, and heat-shock transformed into XL-1 blue. Plasmid DNA was purified from this strain and electrotransformed into E. coli B834.

Fermentation

Expression was tested by growing the cells in 5 ml LB medium with 100μg/ml ampicillin at either 30⁰C or 37⁰C under shaking in open test tubes until OD₆₀O reached 0.4. The cells were induced with 0.8 mM IPTG, which was added together with additional 100μg/ml ampicillin, and the cells were incubated for 3 hours and then harvested by centrifugation and stored at -20° C. The cells were resuspended in 0.5 ml of water, sonicated briefly and a 20 μl aliquot was subject to SDS-PAGE analysis. Large scale fermentation was performed in the same way in Erlenmeyer flasks (1 liter culture in each flask), which were started with 1 % of an overnight pre-culture. The cells were harvested by centrifugation at 400Og , washed with 5OmM Tris 5OmM NaCI pH 7.5 and centrifuged again. The pellet was stored at -2O⁰C.

Purification

Cells were thawed and resuspended in Tris buffer and sonicated with a Bandelin HD 3100 Sonopuls for 3x30 s at 30% effect. Cell debris were removed by centrifugation at 39 000g. Anion exchange was performed with resource-Q packed into a Tricorn 10/50 column or a pre-packed 10/100 mono-Q column and an Akta^'900 purifier (Amersham Pharmacia Biotech). Nikkei affinity chromatography was likewise run with an Akta 900 purifier and a Tricorn 10/50 column, this time filled with Ni-NTA superflow (Quiagen). Gel filtration was done with a Superdex 200 Hiload26/60 column and an FPLC system (Amersham Pharmacia Biotech). The buffer used for gel filtration and ion-exchange chromatography was 5OmM Tris pH 7.5, in the latter case combined with a NaCI gradient. The buffer used for Ni-NTA chromatography was initially 5OmM Tris 5OmM NaCI 2OmM imidazole, followed by elution with 25OmM imidazole, but was later optimized by using 25 mM Tris pH 8.0 followed by a imidazole gradient up to 250 mM. SDS-PAGE was run with NuPAGE Novex 10% Bis-Tris Midi gels (Invitrogen). M2e-NSP4 was sequenced at Carlsberg Research Center (Copenhagen, Denmark). An extinction coefficient of 0.87 ml/mg at 280 nm was used for estimating the concentration of purified protein,

Example 1 - Expression of fusion constructs

The pET plasmids of all clones were sequenced and found to correspond to the target proteins. In the case of the NSP_95-137 gene, the DNA corresponding to the C-terminal portion was doubled (with a frame shift) after the stop codon. As further genetic manipulations at the 3' terminus required a unique terminal sequence, the problem was rectified by running a per omitting the second copy of the final codons. The product from this per was then used as template in subsequent experiments. The translated amino acid sequences of the main products M2e-NSP4, M2e-NSP4His₅ and M2e-NSP4His₂ are shown in Fig. 1.

The expression of M2e-NSP4 and its His-tagged derivatives is displayed on SDS-PAGE gels in Fig. 2. M2e-NSP4 lacking a His-tag, is expressed with good yield in E. coli B834. The expression of the double mutant M2eNSP4His₂, where the last and the third last residue were changed to histidines, is also satisfactory. Although the molecular weight of the expressed protein appears to be smaller than the theoretical value of 8 kD, amino acid sequencing and the successful Ni-NTA chromatography (vide infra) indicates that these are full length version of the sequences in Fig. 1. Somewhat depending on the cell density at the time of induction, two other proteins with a molecular weight of app. 30 kDa are also expressed (Fig 3b). The presence of reducing agents does not change the gel pattern, and the possibility that any of the larger proteins was a tetramer of M2e-NSP4 was therefore ruled out. Moreover, the larger protein is not retained on the Ni-NTA resin (vide infra). The full HxHHHH-tagged version, M2e-NSP4His₅ do not express at all. Thus, the presence of 3 extra histidine residues at the C-terminus completely removes the good expression seen with M2e-NSP4His₂. This may be due to lack of stability and consequent degradation within the cytoplasm of the host cell or, as suggested by the relative slow growth rate of this strain, some toxic effects.

Example 2 - Purification of fusion constructs

M2e-NSP4 was purified in three steps by means of resource-Q anion exchange chromatography, gel filtration and mono-Q anion echange chromatography. Resource-Q, mono-Q and Q-sepharose resins all differed with respect to the fractionation of the proteins. M2e-NSP4 has a higher affinity for all three resins than the larger 32 kDa products, but only on resource-Q was it selectively loaded with the buffer used (5OmM Tris pH 7.5), with the larger protein flowing through without binding (data not shown). Resource-Q was therefore used for the initial purification step. Selected fractions were pooled and loaded on the Sephadex 200 gel filtration column. Finally, M2-NSP4 containing fractions were subject to mono-Q anion chromatography. This purification resulted in a relatively pure product, although small amounts of impurities can be detected on the gel (Fig. 3b). Purified M2e-NSP4 was subject to Edman sequencing and had the N-terminal sequence SLLTEVETPI... which is in agreement with the cloned DNA sequence, given that the N-terminal methionine is posttranslationally removed.

An initial attempt to purify M2e-NSP4His₂ by Ni-NTA chromatography using batch-wise absorption, extensive washing and manual elution from Qiagen 5ml polypropylene columns resulted in very poor yield because the protein did not bind strongly enough to the resin (data not shown). Instead, 5 ml of sonicate (from 2 liter culture) were loaded through a superloop onto Ni-NTA superflow in a Tricorn 50/10 column at a speed of 0.5 ml/minute. The pH of the buffers was increased from 7.5 to 8.0, the usual 2OmM imidazole was excluded from the loading buffer and the column was washed only with a single column volume. Although M2e-NSP4His₂ starts to elute prior to the imidazole gradient, the recovery is satisfactory this time. Figs. 3a and 3b show the chromatogram and SDS-PAGE analysis of selected fractions, respectively. The yield of purified protein is 10-15 mg per litre of cell culture.

Example 3 - Discussion

Influenza A M2 ion channel is a tetrameric molecule where four transmembrane helices are lining the proton transport route through the membrane. The M2e-NSP4 construct described in this paper was designed with the purpose of retaining this quaternary structure, albeit in a soluble form suitable for high level expression in E. coli.

The present inventors surmised that using a scaffold based on the tetrameric NSP4₉5.₁₃₇ for the M2e moiety would result not only in a higher epitope density compared to a single subunit, but also a more or less native spatial distribution of the M2e-epitopes.

Detailed knowledge of the 3-dimensional structure of M2 is lacking hence there is no structure available for M2e. However the M2 gene shares an initiation site with the M1 matrix protein and the sequence of the first seven residues is the same in the two proteins. These seven residues make up two turns of an amphipathic α-helix in M1 [20], and are very likely to do so also in M2.

The structure of the transmembrane section of the M2 ion channel has been explored by X-ray crystallography and nmr spectroscopy [21, 29-31], and the NSP4_95-137 substitute used in this study is known from X-ray crystallography [22],

The fold of the NSP4 tetrameric coiled-coil is shown in Fig. 4, with the two mutated residues T-H and Q-H highlighted.

The structure of a hexahistidine tag complexed with nickel is known from X-ray chromatography; histidines 1 and 3 binds to one nickel ion and histidines 4 and 6 to another [23]. The conformation of these two nickel binding motifs is extended, which is also the case for the last three residues of NSP4_95-137, with the last and the third last side chain similarly oriented. We therefore believed that they could complex a nickel ion, and by virtue of being four in the tetramer, would retain the molecule on a Ni-NTA resin. Moreover, owing to the possibility of some conformational flexibility of the C-terminus, the last histidine on each chain may be able to complex the same nickel ions. Whatever the reason, the interaction between the double mutant M2e-NSP4His₂ and the Ni-NTA resin was sufficiently strong for purification purposes. The interaction was enhanced by increasing the pH in order to depress the degree of protonation of the histidines, and the use of competing imidazole in the loading and wash buffer was excluded.

Example 4 - Vaccination studies in mice

The following fusion protein was tested by way of non-limiting example.

MSLLTEVETP IRNEWGCRCN DSSDP QM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

Briefly, mice were vaccinated with 10 μg or 50 μg of a fusion protein of the invention formulated with an appropriate adjuvant system, as well as control compositions. Antibody titres in mice were tested using ELISA after each of 3 vaccinations with the different vaccine preparations.

The ELISA was performed as follows:

1) wells of 96-well microtitre plates (ELISA grade) were coated with antigen (peptide or tetramer, 300ng/ml) in carbonate buffer (pH9,6) with 100μl/well. Antigen-filled plates were kept at 4°C overnight.

2) wells were washed 3 times with PBS with 0.1% Tween 20

3) wells were blocked with casein (0.025g/ml) in 200μl. Incubated 1 h at 20⁰C

4) wells were washed 3 times with PBS with 0.1% Tween 20

5) serum was diluted and added to the well at different dilutions. Incubated 1 h at 20⁰C

6) wells were washed 3 times with PBS with 0.1% Tween 20

7) anti-serum antibodies (peroxides conjugated) were diluted 1:2000 (sigma cat. no A9917) and added to the wells, 100μl/well. Incubated 1 h at 20⁰C

8) wells were washed 3 times with PBS with 0.1% Tween 20

9) staining buffer with OPD (0.5g/l) and H2O2 (5μl 30% /15ml OPD) was added to the well, 100μl. Incubated 10 min in the dark at 20°C

10) 100μl 0,2M H2SO4 was added

11) plates were read at 492 nm Titer was defined as the serum dilution that gives a signal 4 times the background.

The vaccinated mice were then challenged with H1 N1 (PR8 mouse adapted) flu strain or H7N7 delivered nasally.

The results are shown in Figures 5 to 7.

As shown in Figure 5, vaccination with the M2e-NSP4 fusion of the invention plus adjuvant gave an excellent antibody response as measured by ELISA. Antibodies were produced that recognised the NSP4 peptide alone, the monomeric M2e peptide, but most especially, the (tetrameric) M2e-NSP4 construct. Without adjuvant, there was little or none of these different classes of antibodies produced. The (adjuvanted) vaccine preparation containing only (monomeric) M2e peptide produced little or no anitbody that recognised the tetrameric M2e-NSP4 peptide, though there were detectable levels of antibody against monomeric M2e peptide after the 3^rd vaccination in one of the test animals. These results demonstrate that both (tetrameric) M2e and NSP4 motifs of the M2e-NSP4 vaccine are immunogenic, and further that the monomeric M2e is only weakly immunogenic, and apparently incapable of generating detectable levels of antibody that will recognise the tetramerised form of M2e. The results also suggest that 2 vaccinations with 10 μg of M2e-NSP4 antigen given 3 weeks apart are sufficient to raise a maximal antibody titre.

As shown in Figure 6, vaccination with the M2e-NSP4 fusion of the invention protected against any lethal effects of H7N7 infection - all M2e-NSP4 vaccinated animals (3 x 10 μg antigen) survived challenge with a lethal dose of the H7N7 (mouse-adapted) virus. There was some weight loss in the protected animals, though ail recovered from the virus infection, whereas all non-vaccinated mice, and those given only the (adjuvanted) monomeric M2e vaccine (3 x 10 μg antigen) died.

As shown in Figure 7, vaccination with the M2e-NSP4 fusion of the invention was protective against any morbid effects of H1N1 (mouse-adapted) infection. The majority of unvaccinated mice rapidly lost weight following infection and upon reaching 60% to 70% of their healthy weight (and thereafter showing no signs of recovery) had to be euthanized in accordance with standard codes of practice for ethical treatment of laboratory animals. One animal in this treatment group appears not to have received a lethal dose of virus, as can occasionally happen with nasal administration. Some weight loss was recorded in animals receiving 3 x 10 μg antigen, but these animals quickly recovered from the potentially lethal infection. Animals vaccinated (3 x 50 μg antigen) were apparently protected against all effects of infection with a lethal dose of H1N1 virus. Single animals were lost from both vaccinated groups, but these were both before the end of day 3 and therefore were not due to a lack of protection against the flu infection.

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Claims

1 A fusion protein comprising:

(i) a first portion comprising an influenza A M2e polypeptide, and (ii) a second portion comprising a rotaviral NSP4 polypeptide.

2 A fusion protein as claimed in claim 1 further comprising: (iii) a third portion comprising between 2 and 4 His residues.

3 A fusion protein as claimed in claim 1 or claim 2 wherein the M2e polypeptide comprises:

(a) the amino acid sequence: SLLTEVETPIRNEWGCRCNDSSD, or

(b) a variant sequence having no more than 10, 5, 3, 2 or 1 substitutions deletions, or additions in the sequence of (a).

4 A fusion protein as claimed in any one of claims 1 to 3 wherein the M2e polypeptide is one found in a naturally occurring influenza A strain.

5 A fusion protein as claimed in any one of claims 1 to 4 wherein the M2e polypeptide comprises the amino acid sequence:

S(LVF)(LXP)TEVETP(TM)R(NXKNS)(EXG)W(EXG)C(RNK)C(SXN)(DXG)SS(DXN)

6 A fusion protein as claimed in any one of claims 1 to 5 wherein the M2e polypeptide comprises the amino acid sequence:

S(LXF)(LXP)TEVETP(TXI)R(NXKXS)(EXG)W(EXG)C(RXK)CN(DXG)SS(DXN)

7 A fusion protein as claimed in any one of claims 1 to 6 wherein the M2e polypeptide comprises

SLLTEVETPTRNEW(E\G)CRCSN(D\G)SSD 8 A fusion protein as claimed in any one of claims 1 to 7 wherein the M2e polypeptide comprises the amino acid sequence:

SLLTEVETPIRNEWGCRCNDSSD

9 A fusion protein as claimed in any one of claims 1 to 8 wherein the NSP4 polypeptide comprises:

(a): the amino acid sequence:

IEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLTVQ, or

(b) a sequence having at least 30 contiguous amino acids of the sequence in (a), or

(c) a sequence having no more than 3 substitutions deletions, or additions in the sequence of (a) or (b), or

(d) a sequence having at least 75% identity with the following sequence:

RWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

10 A fusion protein as claimed in any one of claims 1 to 9 wherein the NSP4 polypeptide comprises an amino acid sequence selected from:

IEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL EKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL KQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL QM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL M DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL RWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKL

11 A fusion protein as claimed in any one of claims 1 to 10 which comprises a third portion at the C terminus which includes 1 or 2 His residues. 12 A fusion protein as claimed in claim 11 wherein the third portion consists of the amino acid sequence:

HxH (where x is any amino acid)

13 A fusion protein as claimed in claim 11 or claim 12 wherein the third portion is provided by mutation of second portion.

14 A fusion protein as claimed in any one of claims 11 to 13 wherein the second and third portion comprise the amino acid sequence:

RWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

15 A fusion protein as claimed in any one of claims 1 to 14 which comprises or consists of an amino acid sequence selected from:

MSLLTEVETP IRNEWGCRCN DSSDPIEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

MSLLTEVETP IRNEWGCRCN DSSDPEKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

MSLLTEVETP IRNEWGCRCN DSSDPKQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

MSLLTEVETP IRNEWGCRCN DSSDPQM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

MSLLTEVETP IRNEWGCRCN DSSDPM DRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

MSLLTEVETP IRNEWGCRCN DSSDPDRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH

MSLLTEVETP IRNEWGCRCN DSSDPRWKEMRRQ LEMIDKLTTR EIEQVELLKR IYDKLHVH 16 A recombinant oligomeric protein complex comprising a plurality of associated fusion proteins according to any one of claim 1 to 15.

17 An oligomeric protein complex as claimed in claim 16 which is a homotetramer.

18 A nucleic acid encoding a fusion protein according to any one of claim 1 to 15.

19 A recombinant vector comprising a nucleic acid according to claim 18.

20 A recombinant vector according to claim 19 which is an expression vector.

21 A host cell containing or transformed with a nucleic acid or a recombinant vector according to any one of claims 18 to 21.

22 A host cell as claimed in claim 21 which is a microorganism which is optionally a procaryote.

23 A host cell as claimed in claim 22 which is E. coll

24 A process for preparing a soluble influenza A vaccine which process comprises culturing a host cell according to any one of claim 21 to 23.

25 A process for producing a composition for use as an influenza vaccine in a human or animal, the process comprising: i) providing a host cell according to any one of claim 21 to 23, ii) culturing the cell such as to produce a fusion protein according to any one of claims 1 to 15, iii) purifying the fusion protein therefrom.

26 A process as claimed in claim 25 wherein step iii) comprises or consists of: iiia) contacting the product of step iii) with an affinity column capable of binding a Hexa- His-Tag; iiib) at least one anion exchange chromatography step. 27 A process as claimed in any one of claims 24 to 26 wherein the fusion protein is in the form of an oligomeric protein complex which is optionally a homotetramer.

28 A process as claimed in any one of claims 24 to 27 wherein step iii) is followed by formulating the fusion protein or complex as the vaccine.

29 A vaccine composition comprising the oligomeric protein complex according to claim 16 or claim 17 or obtainable by the process of claim 28 and optionally further comprising a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or adjuvant.

30 The vaccine composition of claim 29 for use in the treatment of influenza.

31 Use of a fusion protein according to any one of claims 1 to 15, or the oligomeric protein complex according to claim 16 or claim 17, in the manufacture of a medicament for inducing an immune response in a subject against influenza A virus.

32 A methods of treating, or reducing the likelihood of contracting, a condition associated with influenza A virus, which method comprises administering an immunologically effective dose of a vaccine composition of claim 29 or claim 30 to a subject.

33 A crystal of an oligomeric protein complex according to claim 16 or claim 17.