TECHNICAL FIELD This invention is in the field of virology, more particularly in the field of influenza A virus. It provides proteins, nucleic acids and antibodies which are useful for the detection, diagnosis, prevention and/or treatment of infection by influenza A virus.

BACKGROUND ART Novel influenza viruses continuously emerge in the human population. Three times during the twentieth century, an avian influenza virus subtype crossed the species barrier, starting a pandemic and establishing itself for decades in man. As the 1997 H5N1 event in Hong Kong indicated, the occurrence of another pandemic in the near future cannot be excluded. Sufficient vaccine may not be available to ameliorate the consequences of such an event because of time shortages. Surveillance for new influenza A virus subtypes may reduce this risk [Offringa et al.

(2000) J Virol Methods 88 (1) : 15-24].

Fifteen haemagglutinin subtypes (H1 to H15) of influenza A virus have been reported. The present invention is based on the discovery of a sixteenth subtype in gulls in Sweden and the Netherlands.

DISCLOSURE OF THE INVENTION The invention provides a protein comprising any one of amino acid sequences SEQ ID 1,2,3 or 4. These are the haemagglutinin (HA) proteins from four isolates of a new influenza A virus subtype, designated H16 herein.

The invention also provides a protein comprising the HA1 subunit or the HA2 subunit of the full-length haemagglutinins (HAO) set forth as SEQ IDs 1 to 4. The HA1 subunit of SEQ IDs 1-3 is SEQ ID 5; the HA2 subunit is SEQ ID 6. The HA1 subunit of SEQ ID 4 is SEQ ID 7; the HA2 subunit is SEQ ID 8.

The invention also provides a protein comprising both the HA1 and HA2 subunits, covalently bonded via a disulphide bridge. Preferred pairs of HA1/HA2 are SEQ ID 5/6 and SEQ ID 7/8.

This protein is preferably in the form of a trimer.

Of the fifteen previous haemagglutinin subtypes, the H16 HA protein sequence is most closely related to that of H13, showing about 80% homology over the complete sequence (75% for HA1, 87% for HA2). Within the H16 subtype, the two isolates represented by SEQ IDs 2 and 4 show 89% sequence identity (88% for HA1, 92% for HA2). The invention provides a protein comprising an amino acid sequence which has at least 80% sequence identity to one or more of SEQ IDs 1, 2,3 & 4. It also provides a protein comprising an amino acid sequence which has at

least 75% sequence identity (preferably at least 88%) to SEQ ID 5 and/or 7. It also provides a protein comprising an amino acid sequence which has at least 87% sequence identity (preferably at least 92%) to SEQ ID 6 and/or 8.

The degree of sequence identity to any particular SEQ ID is preferably greater than the minimum values specified in the previous paragraph e. g. at least 85%, more preferably at least 90%, and most preferably at least 95% (e. g. 296%, 297%, >98%, or 299% sequence identity). These include mutants and allelic variants of the SEQ IDs. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

The invention further provides a protein comprising a fragment of one of amino acid sequences SEQ IDs 1 to 8. The fragment should comprise at least n consecutive amino acids from the sequences, wherein n is 7 or more (e. g. 8,10,12,14,16,18,20,30,40,50,75,100 or more).

Preferably the fragment comprises one or more epitope (s) from the sequence. Preferred fragments are SEQ IDs 13 to 35, which are common to all of SEQ IDs 1,2,3 and 4. A preferred fragment of SEQ IDs 1 and 3 is SEQ ID 2. Preferred fragments of SEQ IDs 1 to 4 lacks the 32 C-terminal amino acids, which are the transmembrane and cytoplasmic domains.

The proteins of the invention can, of course, be prepared by various means (e. g. native expression, recombinant expression, purification from viral culture or allantoic fluid, chemical synthesis etc.) and in various forms (e. g. native, fusions etc.). They are preferably prepared in substantially pure form (ie. substantially free from other influenza or host cell proteins). It is preferred that the proteins of the invention are glycosylated and/or fatty acid acetylated.

It is preferred that the proteins of the invention are not immunologically cross-reactive with the haemagglutinin proteins from influenza A virus subtypes HI to HI 5. It is also preferred that the proteins retain immunological cross-reactivity with subtype H16 (e. g. with one or more of SEQ IDs 1 to 8). Preferred proteins of the invention are thus able to distinguish the H16 subtype from the previous subtypes e. g. in a standard haemagglutination inhibition assay.

According to a further aspect, the invention provides antibodies which bind to the proteins of the invention. These may be polyclonal or monoclonal and may be produced by any suitable means.

The antibodies are preferably specific for the H16 subtype i. e. they do not bind to the haemagglutinin proteins from influenza A virus subtypes HI to H15. These antibodies are thus able to distinguish the H16 subtype from the previous subtypes. Antibodies of the invention may also be able to distinguish isolates within the H16 subtype.

According to a further aspect, the invention provides nucleic acid comprising any one of nucleotide sequences SEQ IDs 9 to 12.

The invention also provides nucleic acid comprising a nucleotide sequence which has at least 80% sequence identity to any one of SEQ IDs 9 to 12. Preferably, the degree of sequence identity is at least 85%, more preferably at least 90%, and most preferably at least 95% (e. g.

296%, #97%, >98%, or 299% sequence identity).

The invention also provides nucleic acid which can hybridise to any one of SEQ IDs 9 to 12, preferably under"high stringency"conditions (e. g. 65°C in a O. lxSSC, 0.5% SDS solution).

Nucleic acid comprising a fragment of SEQ IDs 9 to 12 is also provided. The nucleic acid should comprise at least n consecutive nucleotides from any one of SEQ IDs 9 to 12, wherein n is 10 or more (e. g. 12,14,15,18,20,25,30,35,40,50,75,100,200,300 or more).

The invention also provides nucleic acid encoding a protein of the invention.

It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (e. g. for antisense or probing purposes).

Nucleic acid according to the invention can, of course, be prepared in many ways (e. g. by chemical synthesis, from genomic or cDNA libraries, from the virus itself etc.) and can take various forms (e. g. single stranded, double stranded, vectors, probes etc.).

In addition, the term"nucleic acid"includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (e. g. expression vectors) and host cells transformed with such vectors.

According to a further aspect, the invention provides an influenza A virus having a full-length haemagglutinin amino acid sequence which has at least 80% sequence identity to one of SEQ IDs 1 to 4. Preferably, the degree of sequence identity is at least 85%, more preferably at least 90%, and most preferably at least 95% (e. g. 296%, 297%, 298%, or >99% sequence identity).

The virus may be an inactivated virus (e. g. by treatment with formaldehyde or propiolactone), an attenuated virus (e. g. by serial passage in embryonated eggs, by chemical mutation, or by passage at low temperatures), or may be a'split'virus (e. g. by treatment with detergent to solubilise the lipid bilayer).

The invention also provides compositions (preferably immunogenic compositions) comprising protein and/or nucleic acid and/or antibody and/or virus according to the invention. These compositions are suitable for diagnostic, immunisation and vaccination purposes.

Influenza vaccines are approved for use in humans and are widely available [e. g. see Saito & Tashiro (2000) Pediatr Int 42 (2): 219-25]. Current vaccines comprise an inactivated virus, a split virus, or purified surface glycoproteins. Success with DNA vaccines has also been reported [e. g.

Ross et al. (2000) Nature Immunology 1: 127-131]. The materials of the present invention can be used in these vaccines to give specificity for the H 16 subtype.

Thus, the invention provides an influenza A virus vaccine, comprising an immunologically effective amount of protein, nucleic acid (e. g. in the form of an expression vector) and/or virus according the invention. Vaccines of the invention may be prophylactic or therapeutic.

Vaccines of the invention may include an adjuvant. A preferred adjuvant is MF59 [see: W090/14837 ; Chapter 10 in Vaccine design : the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995; Martin (1997) Biologicals 25: 209-213], containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 formulated into a submicron oil-in-water emulsion. This adjuvant is used in the FluadTM vaccine together with HA protein [see: De Donato et al. (1999) Vaccine 17: 3094-3101 ; Minutello et al. (1999) Vaccine 17: 99-104].

The vaccine may or may not include a preservative (e. g. thiomersal). Preservative-free vaccines (e. g. Begrivac) are preferred for individuals sensitive to preservative ingredients.

Other possible components include: sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride hexahydrate, calcium chloride dihydrate, sodium citrate, and citric acid.

The protein or nucleic acid present in the vaccine may be a fusion of HA (or a fragment thereof e. g. lacking the transmembrane and cytoplasmic domains) with complement protein C3d [Dempsey et al. (1996) Science 271: 348-350], in particular with 3 tandem repeats of C3d [Ross et al. (2000) Nature Immunology 1: 127-131]. The HA and C3d sequences may be joined by a glycine-rich linker sequence, such as (Gly-Gly-Gly-Gly-Ser) 2.

The invention also provides nucleic acid and/or protein and/or antibody according to the invention for use as medicaments (e. g. vaccines). It also provides the use of nucleic acid and/or protein and/or antibody according to the invention in the manufacture of a medicament for treating or preventing infection due to influenza A virus.

The invention also provides a method of treating a patient, comprising administering to the patient a prophylactically or therapeutically effective amount of nucleic acid and/or protein and/or antibody according to the invention.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided, comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.

A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.

A process for detecting H16 subtype influenza A virus is provided, wherein protein and/or nucleic acid and/or antibody according to the invention is contacted with a biological sample (e. g. a sample from a patient). The process may be a standard haemagglutination inhibition assay. The process is preferably able to distinguish the H16 subtype from subtypes H1 to H15. It may also be able to distinguish isolates within the H16 subtype.

A process for producing virus of the invention is provided, wherein virus is grown in eggs (e. g. embryonated hen eggs) and is subsequently harvested from the allantoic fluid. This is the standard procedure for growing flu virus. After purification, inactivation, and standardisation for HA content, the virus may be formulated as a vaccine.

The virus may also be grown in cell culture. Where cell culture is used, it is preferred to use animal cells which can grow in suspension in protein-free or serum-free media (see W097/37000). Virus yield may be increased by culturing between 30-36°C, preferably-33°C (see W097/37001).

A summary of standard techniques and procedures which may be employed in order to perform the invention (e. g. to utilise the HA proteins for vaccination) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

General The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature e. g. Sambrook Molecular Clorairig ; A Laboratory Manual, Second Edition (1989) ; DNA Cloning, Volumes I and ii (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Ariimal Cell Culture (R. I. Freshney ed. 1986) ; Irnrmobilized Cells and Enzymes (IRL Press, 1986); B.

Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155 Gene Transfer Vectors for Manlrnalian Cells (J, H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Irnrrmnochemical Methods in Cell and Molecular

Biology (Academic Press, London); Scopes, (1987) Protein Purification : Principles and Practice, Second Edition (Springer-Verlag, N. Y.), and Handbook of Experimental lmmtbnology, Volumes l-IV (Weir & Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification.

Definitions The term"comprising"means"including"as well as"consisting"e. g. a composition"comprising"X may consist exclusively of X or may include something additional to X, such as X+Y, A composition containing X is"substantially free of"Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term"heterologous"refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where an influenza sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.

An"origin of replication"is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

A"mutant"sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (e. g. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an"allelic variant"of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5'or 3' untranslated regions of the gene, such as in regulatory control regions (e. g. see US patent 5,753,235).

Expression systems The influenza nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast. i. Mammalian Systems Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e. g. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5'end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the

correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989)"Expression of Cloned Genes in Mammalian Cells," In Molecular Cloning : A Laboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000- fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site.

Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236 : 1237 ; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4 : 761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) PNAS USA 79: 6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41 : 521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet.

2 : 215; Maniatis et al. (1987) Science 236: 1237].

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3'to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3'terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polya- denylation [Birnstiel et al. (1985) Cell 41 : 349; Proudfoot and Whitelaw (1988)"Termination and 3'end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14 : 105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989)"Expression of cloned genes in cultured mammalian cells."In Molecular Cloning : A Laboratory Matiuall.

Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhances, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e, g. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23 : 175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al.

(1989) Mol. Cell. Biol. 9: 946] and pHEBO [Shimizu et al. (1986) Mol. Cell, Biol. 6: 1074].

The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the ATCC, including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e. g. Hep G2), and a number of other cell lines. ii. Baculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art.

Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form e. g. from Invitrogen, San Diego CA ("MaxBac"kit). These techniques are generally known to those skilled in the art and fully described in Summers & Smith, Texas Agricultural Experi1nent Station Bulletin No. 1555 (1987) (hereinafter"Summers & Smith").

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (e. g. plasmids)

capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17 : 31.

The plasmid usually also contains a polyhedrin polyadenylation signal (Miller (1988) Ann. Rev. Microbiol. 42: 177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5'to 3') transcription of a coding sequence (e. g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5'end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986)"The Regulation of Baculovirus Gene Expression,"in The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EP-127839 & EP-155476; and the gene encoding the plO protein, Vlak et al. (1988), J. Gen. Virol. 69: 765.

DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73 : 409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human D-mterferon, Maeda et al., (1985), Nature 315 : 592 ; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8: 3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82: 8404; mouse IL-3, (Miyajima et al., (1987) Gene 58 : 273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7: 99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus--usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5kb section of the baculovirus genome. Methods for introducing heterologous DNA into the

desired site in the baculovirus virus are known in the art. (See Summers & Smith supra ; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3 : 2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4 : 91, The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5'and 3'by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 Om in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. Current Protocols in Microbiology Vol. 2 (Ausubel et al. eds) at 16, 8 (Supp. 10,1990); Summers & Smith, supra ; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia : Aedes aegypti, Autographa californica, Bombyx mori, Drosophila rnelanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56 : 153; Wright (1986) Nature 321 : 718 ; Smith et al., (1983) Mol, Cell. Biol. 3 : 2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25 : 225).

Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art.

See, e. g. Summers & Smith supra.

The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid (s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, e. g. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis ; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, e. g. proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.

iii. Plant Systems There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytocheiiiistry 30 : 3861-3863 (1991), Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al,, Mol, Gen. Genet. 209 : 33-40 (1987); Chandler et al., Plant Molecular Biology 3: 407-418 (1984); Rogers, J. Biol. Cherra, 260: 3731-3738 (1985); Rothstein et al., Gene 55 : 353-356 (1987); Whittier et al,, Nucleic Acids Research 15: 2515-2535 (1987); Wirsel et al,, Molecular Microbiology 3 : 3-14 (1989); Yu et al., Gene 122: 247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins : in : Advanced Plant Physiology,. Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2 : 1027-1038 (1990); Maas et al., EMBO J. 9: 3447-3452 (1990); Benkel & Hickey, PNAS USA 84 : 1337-1339 (1987) Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, e. g. for the members of the grass family, is found in Wilmink & Dons, 1993, Plant Mol, Biol. Reptr, 11 (2): 165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein (s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5'untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5'and 3'ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein (s) of interest will be translocated

from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the"intron"region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41 : 95-105,1985.

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202 : 179-185,1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296,72-74,1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327,70-73,1987 and Knudsen and Muller, 1991, Planta, 185 : 330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., PNAS USA, 79,1859-1863,1982.

The vector may also be introduced into the plant cells by electroporation. (Fromm et al., PNAS USA 82 : 5824,1985).

In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct.

Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linus, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphafaus, Sinapis, Atropa, Capsicurra, Datura, <BR> <BR> <BR> Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca,<BR> <BR> <BR> <BR> <BR> Bromus, Asparagus, Antirrhirium, Hererocallis, Nemesia, Pelargonium, Pariicurn, Pennisetuni, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension.

These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable, In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution

to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein. iv. Bacterial Systems Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e. g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5'end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.

In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in E. coli [Raibaud et al. (1984) Annu, Rev. Genet. 18 : 173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198 : 1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8 : 4057; Yelverton et al. (1981) Nucl, Acids Res.

9: 731; US patent 4, 738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981)"The cloning of interferon and other mistakes."In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al, (1981) Nature 292 : 128] and T5 [US patent 4, 689,406] promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of a bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [US patent 4, 551, 433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25 : 167; de Boer et al. (1983) PNAS USA 80 : 21].

Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189 : 113; Tabor et al, (1985) PNAS USA 82 : 1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EP-A-0267851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al, (1975) Nature 254 : 34]. The SD sequence is thought to promote binding of mRNA to the ribosome by base-pairing between the SD sequence and the 3'end of 16S rRNA [Steitz et al. (1979)"Genetic signals

and nucleotide sequences in messenger RNA."In Biological Regulation and Development : Gene Expression (ed. R. F.

Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al.

(1989)"Expression of cloned genes in Escherichia coli."In Molecular Cloning : A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5'end of heterologous coding sequences.

Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5'terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309 : 810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60 : 197], trpE [Allen et al. (1987) J. Biotechnol. 5: 93; Makoff et al. (1989) J.

Gen. Microbiol. 135 : 11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e. g. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) BiolTechnology 7: 698].

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [US patent 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as E. coli outer membrane protein gene (pompa) [Masui et al. (1983) in : Experimental Manipulation of Gene Expression ; Ghrayeb et al. (1984) EMBO J 3 : 2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) PNAS USA 82 : 7212]. As a further example, signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) PNAS USA 79: 5582; EP-A-0244042].

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3'to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e. g. plasmids) capable of stable maintenance in

a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from-5 to-200, and usually-10 to-150. A host containing a high copy number plasmid will preferably contain at least-10, and more preferably at least-20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector.

Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0127328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Ajarau. Rev. Microbiol, 32 : 469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) PNAS USA 79: 5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292 : 128; Amann et al. (1985) Gene 40 : 183; Studier et al. (1986) J. Mol. Biol. 189 : 113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ, Microbiol. 54 : 655]; Streptococcus lividans [Powell et al. (1988) Appl.

Environ. Microbiol. 54 : 655], Streptomyces lividans [US patent 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with Caca2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See e, g. [Masson et al. (1989) FEMS Microbiol. Lett. 60: 273; Palva et al. (1982) PNAS USA 79 : 5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) PNAS USA 85 : 856; Wang et al. (1990) J. Bacteriol. 172 : 949, Campylobacter], [Cohen et al. (1973) PNAS USA 69: 2110; Dower et al.

(1988) Nucleic Acids Res. 16 : 6127; Kushner (1978)"An improved method for transformation of Escherichia coli with ColEl-derived plasmids. In Genetic Engineering : Proceedirvgs of the Iriternational Symposimn on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al, (1970) J. Mol. Biol. 53 : 159; Taketo (1988) Biochi} Biophys. Acta 949 : 318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44 : 173 Lactobacillus]; [Fiedler et al, (1988) Anal. Biocllem 170 : 38, Pseudomonas] ; [Augustin et al. (1990) FEMS Microbiol. Lett. 66: 203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144 : 698; Harlander (1987)"Transformation of Streptococcus lactis by electroporation, in : Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III) ; Perry et al. (1981) Infect.

/mum.. 32: 1295; Powell et al. (1988) Appl. Environ. Microbiol. 54 : 655; Somkuti et al, (1987) Proc. 4th Evr. Cong.

Biotechnology 1 : 412, Streptococcus].

General guidance on expression in E. coli and its optimisation can be found in Baneyx (1999) Curr. Opin. Biotech.

10 : 411-421 and Hannig & Makrides (1998) TIBTECH 16: 54-60. v. Yeast Expression Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e. g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5'end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the"TATA Box") and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0284044), glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, enolase, glucokinase, and pyruvate kinase (PyK) (EPO-A-0329203). The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara etal. (1983) PNAS USA 80 : 11.

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (US Patent Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) PNAS USA 77 : 1078; Henikoff et al. (1981) Nature 283 : 835; Hollenberg et al. (1981) Curr. Topics Microbiol. Ittimutiol, 96 : 119; Hollenberg et al. (1979)"The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,"in : Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11 : 163; Panthier et al. (1980) Curr. Genet. 2: 109 ;].

A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5'end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5'terminus of a foreign gene and expressed in yeast. The DNA sequence

at the junction of the two amino acid sequences may or may not encode a cleavable site. See e. g. EP-A-0 196 056.

Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e. g. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (e. g. W088/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (US patent 4,588,684). Alternatively, leaders of non- yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a"pre"signal sequence, and a"pro"region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 aa residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (US Patents 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (e. g. see WO 89/02463.) Usually, transcription termination sequences recognized by yeast are regulatory regions located 3'to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e. g. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast- bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8 : 17-24], psi/1 [Brake et al. (1984) PNAS USA 81 : 4642-4646], and YRpl7 [Stinchcomb et al. (1982) J. Mol. Biol. 158 : 157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from-5 to ~200, and usually-10 to-150. A host containing a high copy number plasmid will preferably have at least-10, and more preferably at least-20. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See e. g. Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector.

Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al.

(1983) Methods in Enzymol. 101 : 228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) PNAS USA 80 : 6750]. The chromosomal sequences included in the vector can occur either as a single segment in the

vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRPI, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51 : 351].

Alternatively, some of the above described components can be put together into transformation vectors.

Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6 : 142], Candida maltosa [Kunze, et al.

(1985) J. Basic Microbiol. 25 : 141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132 : 3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202 : 302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol.

158 : 1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154 : 737; Van den Berg et al. (1990) BiolTechnology 8 : 135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25 : 141], Pichia pastoris [Cregg, et al, (1985) Mol. Cell. Biol, 5 : 3376; US Patent Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) PNAS USA 75 : 1929; Ito et al. (1983) J. Bacteriol. 153 : 163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300 : 706], and Yarrowia lipolytica [Davidow, et al, (1985) Curr. Genet, 10 : 380471 Gaillardin, etal. (1985) Curr. Genet. 10 : 49].

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See e. g. [Kurtz et al. (1986) Mol. Cell. Biol. 6: 142; Kunze et al. (1985) J. Basic Microbiol. 25 : 141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132 : 3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202: 302; Hansenula]; [Das et al, (1984) J. Bacteriol. 158 : 1165; De Louvencourt et al. (1983) J.

Bacteriol. 154 : 1165; Van den Berg et al. (I990) Bio/Tecltnology 8 : 135 ; Kluyveromyces]; [Cregg et al. (1985) Mol.

Cell. Biol. 5 : 3376; Kunze et al. (1985) J. Basic Microbiol. 25 : 141; US Patent Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) PNAS USA 75 ; 1929; Ito et al. (1983) J. Bacteriol. 153 : 163 Saccharomyces]; [Beach and Nurse (1981) Nature 300 : 706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10 : 39; Gaillardin et al. (1985) Curr, Genet. 10 : 49; Yarrowia].

P11armaceutical Compositions Pharmaceutical compositions can comprise polypeptides and/or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the invention.

The term"therapeutically effective amount"as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend

upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance.

However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0. 05 mg/kg to about 10 mg/kg of the active components in the individual to which it is administered. A typical dose of HA in a vaccine is in the range 5-50 ug/dose (e. g. 10-30jig/dose, suchas 15p. dose). Adoseistypically0. 5ml.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term"pharmaceutically acceptable carrier"refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated, Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (e. g. see W098/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Vaccines Vaccines according to the invention comprise immunising antigen (s), immunogen (s), polypeptide (s), protein (s) or nucleic acid, usually in combination with"pharmaceutically acceptable carriers,"which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (e. g. W090/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80,5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0. 2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM) ; (2) saponin adjuvants, such as QS21 or StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e. g. WO00/07621 ; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e. g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e. g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-0- deacylated MPL (3dMPL) e. g. GB-2220221, EP-A-0689454; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e. g. EP-A-0835318, EP-A-0735898, EP-A-0761231 ; (7) oligonucleotides comprising CpG motifs [Krieg Vaccine 2000,19,618-622; Krieg Curr opin Mol Ther 2001 3 : 15-24; Roman et al., Nat. Med., 1997,3,849-854; Weiner et al., PNAS USA, 1997,94,10833-10837; Davis et al., J. Immunol., 1998,160,870-876; Chu et al., J. Exp. Med., 1997,186,1623-1631; Lipford et al., Eur. J. Immunol., 1997,27,2340-2344; Moldoveanu et al., Vaccine, 1988,16,1216-1224, Krieg et al., Nature, 1995,374,546-549; Klinman et al., PNAS USA, 1996,93, 2879-2883; Ballas et al., J. Immunol., 1996,157,1840-1845; Cowdery et al., J. Imnuraol., 1996,156,4570-4575; Halpern et al., Cell. ImmuIlol., 1996,167,72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988,79,866-873; Stacey et al., J. Immunol., 1996, 157,2116-2122; Messina et al., J. Immunol., 1991,147,1759-1764; Yi et al., J. Imrnunol., 1996,157,4918-4925; Yi et al., J. Immunol., 1996,157,5394-5402; Yi et al., J. Immunol., 1998,160,4755-4761; and Yi et al., J. Immunol., 1998,160,5898-5906; International patent applications W096/02555, W098/16247, W098/18810, W098/40100, W098/55495, W098/37919 and W098152581] i. e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (8) a polyoxyethylene ether or a polyoxyethylene ester e. g. W099/52549 ; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (e. g. WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (e. g. WO01/21152) ; (10) an immunostimulatory oligonucleotide (e. g. a CpG oligonucleotide) and a saponin e. g. WO00/62800 ; (11) an immunostimulant and a particle of metal salt e. g. WO/23105 ; (12) a saponin and an oil-in-water emulsion e. g. W099/11241 ; (13) a saponin (e. g. QS21) + 3dMPL + IL-12 (optionally + a sterol) e. g. W098/57659 ; (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition. Alum and MF59 are preferred.

As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphory loxy)-ethylamine (MTP-PE), etc The immunogenic compositions (e. g. the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e. g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The immunogenic compositions are conventionally administered parenterally, e. g. by injection, either subcutan- eously, intramuscularly, or transdermally/transcutaneously (e. g. W098/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.

Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be employed [e. g. Robinson & Torres (1997) Seminars in Imniuriology 9 : 271-283; Donnelly et al. (1997) Annu Rev Immunol 15: 617-648; see later herein].

Gene Delivery Vehicles Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1 : 51-64; Kimura (1994) Human Gene Therapy 5: 845-852; Connelly (1995) Human Gene Therapy 6: 185-193; and Kaplitt (1994) Nature Genetics 6: 148-153.

Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53: 160) polytropic retroviruses e. g. MCF and MCF-MLV (see Kelly (1983) J.

Virol. 45 : 291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see US patent 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see W096/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763, W092/05266), and can be used to create producer cell lines (also termed vector cell lines or"VCLs") for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (e. g. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley & Rowe (1976) J Virol 19 : 19-25), Abelson (ATCC VR-999), Friend (ATCC VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques.

Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, W089/02468 ; W089/05349, W089/09271, <BR> <BR> W090/02806, WO90/07936, W094/03622, W093/25698, W093/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, W095/07994, US 5,219,740, US 4,405,712, US 4,861,719, US 4,980,289, US 4,777,127, US 5,591,624. See also Vile (1993) Cancer Res 53: 3860-3864; Vile (1993) Cancer Res 53 : 962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33: 493-503; Baba (1993) J Neurosurg 79: 729-735; Mann (1983) Cell 33: 153; Cane (1984) PNAS USA 81: 6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6 : 616 and Rosenfeld (1991) Science 252: 431, and W093/07283, W093/06223, and W093/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in W094/12649, W093/03769, WO93/19191, W094/28938, <BR> <BR> <BR> WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, W096/05320, W094/08026, WO94/11506,<BR> <BR> <BR> <BR> W093/06223, W094/24299, W095/14102, W095/24297, W095/02697, W094/28152, W094/24299, W095/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hure. Gene Ther. 3 : 147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, W093/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.

Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124: 257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61 : 3096). Another

exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in US Patent 5,478,745. Still other vectors are those disclosed in Carter US Patent 4,797,368 and Muzyczka US Patent 5,139,941, Chartejee US Patent 5,474,935, and Kotin W094/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7 : 463-470. Additional AAV gene therapy vectors are described in US 5,354,678, US 5,173,414, US 5,139,941, and US 5,252,479.

The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in US 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in W095/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241: 1667-1669 and in W090/09441 and W092/07945, HSV Us3: : pgC-IacZ described in Fink (1992) Human Gene Therapy 3 : 11-19 and HSV 7134,2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in US patents 5,091,309, 5,217,879, and W092/10578, More particularly, those alpha virus vectors described in US Serial No. 08/405, 627, filed March 15,1995, WO94/21792, WO92/10578, WO95/07994, US 5,091,309 and US 5,217,879 are employable.

Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see USSN 08/679640).

DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See W095/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J, Biol. Standardization 1 : 115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) PNAS USA 86: 317; Flexner (1989) Ann NY Acad Sci 569 : 86, Flexner (1990) Vaccine 8: 17; in US 4,603,112 and US 4,769,330 and W089/01973 ; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277: 108 and Madzak (1992) J Gen Virol 73: 1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in US 5,166,057 and in Enami (1990) PNAS USA 87: 3802-3805; Enami & Palese (1991) J Virol 65: 2711-2713 and Luytjes (1989) Cell 59: 110, (see also McMichael (1983) NEJ Med 309 : 13, and Yap (1978) Nature 273 : 238 and Nature (1979) 277: 108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66: 2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example

ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926 ; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121 : 190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see US Serial No. 08/366, 787, filed December 30,1994 and Curiel (1992) Hum Gene Ther 3: 147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264: 16985-16987, eucaryotic cell delivery vehicles cells, for example see US Serial No. 08/240, 030, filed May 9,1994, and US Serial No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in US Patent 5,149,655, ionizing radiation as described in US5,206,152 and in W092111033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14 : 2411-2418 and in Woffendin (1994) PNAS USA 91 : 1581-1585.

Particle mediated gene transfer may be employed, for example see US Serial No. 60/023, 867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol, Chem. 262 : 4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40 : 253-263, galactose as described in Plank (1992) Bioconjugate Chem 3: 533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in W090/11092 and US 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in US 5,422,120, W095/13796, W094/23697, W091/14445 and EP-524,968. As described in USSN. 60/023, 867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) PNAS USA 91 (24) : 11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in US 5,149,655; use of ionizing radiation for activating transferred gene, as described in US 5,206,152 and W092/11033

Exemplary liposome and polycationic gene delivery vehicles are those described in US 5,422,120 and 4,762,915; in WO 95/13796 ; W094123697 ; and W091/14445 ; in EP-0524968; and in Stryer, Biochemistry, 236-240 (1975) W. H.

Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600: 1; Bayer (1979) Biochem Biophys Acta 550: 464; Rivnay (1987) Meth Ervzymol 149 : 119; Wang (1987) PNAS USA 84 : 7851; Plant (1989) Aval Biochem 176: 420.

A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

Delivery Methods Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) ill vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (e. g. see W098/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e. g. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and ill vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Polvvucleotide artd polypeptide pharmaceutical convnositions In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.

A.Polypeptides One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.

B. Hormones, Vitamins, etc.

Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a

preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran, Also, chitosan and poly (lactide-co-glycolide) D, Lipids, and Liposomes The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom, Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid, The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1 : 1 (mg DNA: micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochini. Biophys. Acta. 1097 : 1-17; Straubinger (1983) Meth. Enzymol.

101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) PNAS USA 84: 7413-7416); mRNA (Malone (1989) PNAS USA 86: 6077-6081); and purified transcription factors (Debs (1990) J. Biol, Chem. 265 : 10189-192), in functional form.

Cationic liposomes are readily available e. g. N [1-2, 3-dioleyloxy) propyl]-N, N, N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY, (See, also, Felgner supra) Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e. g. Szoka (1978) PNAS USA 75: 4194-4198; W090/11092 for a description of the synthesis of DOTAP (1, 2-bis (oleoyloxy)-3-(trimethylammonio) propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See e. g.

Straubinger (1983) Meth. Immunol. 101 : 512-527; Szoka (1978) PNAS USA 75 : 4194-4198; Papahadjopoulos (1975) Biochirrv. Biophys. Acta 394 : 483; Wilson (1979) Cell 17: 77); Deamer & Bangham (1976) Biochirn. Biophys. Acta 443 : 629; Ostro (1977) Biochem. Biophys. Res. Commun. 76^836 ; Fraley (1979) PNAS USA 76 : 3348); Enoch & Strittmatter (1979) PNAS USA 76: 145; Fraley (1980) J. Biol. C1lem. (1980) 255 : 10431; Szoka & Papahadjopoulos (1978) PNAS USA 75: 145; and Schaefer-Ridder (1982) Scietice 215: 166.

E.Lipoproteins In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors.

Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins.

At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, All, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, and E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.

The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Anne Rev.

Biochem 54 : 699; Law (1986) Adv. Exp Med. Biol. 151 : 162; Chen (1986) J Biol Chem 261 : 12918; Kane (1980) PNAS USA 77 : 2465; and Utermann (1984) Hum Genet 65: 232.

Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Math. Eiizymol. (sitpra) ; Pitas (1980) J. Bioche11l. 255 : 5454-5460 and Mahey (1979) J Clin. Invest 64 : 743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15: 403 and Radding (1958) Biochim Biophys Acta 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Massachusetts, USA. Further description of lipoproteins can be found in Zuckermann etal PCT/US97114465.

F. Polycationic Agents Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-l, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, and purtrescine.

The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. Lipofectinlm, and lipofectAMINE are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.

Nucleic Acid Hybridisatioti "Hybridization"refers to the association of two nucleic acid sequences to one another by hydrogen bonding.

Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al.

[supra] Volume 2, chapter 9, pages 9. 47 to 9.57.

"Stringency"refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200 DC belowthe calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment (s) to be studied can vary a magnitude of 10, from 0.1 to Ipg for a plasmid or phage digest to 10, 9 to 10'* g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 pg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/g. For a single-copy mammalian gene a conservative approach would start with 10 pg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/pg, resulting in an exposure time of-24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation: Tm= 81 + 16.6 (logloCi) + 0.4 [% (G + C)]-0.6 (% formamide)-600/n-1, 5 (% mismatch). where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & W ahl (1984) Anal, Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not

completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42CIC for a probe with is 95% to 100% homologous to the target fragment, 370C for 90% to 95% homology, and 320C for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.

Nucleic Acid Probe Assavs Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to"hybridize"with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.

The nucleic acid probes will hybridize to the influenza nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native influenza sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence ; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to the influenza sequence (or its complement)-some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional influenza sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5'end of the probe, with the remainder of the probe sequence being complementary to an influenza sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the an influenza sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc, (1981) 103 : 3185], or according to Urdea et al. [PNAS USA (1983) 80 : 7461], or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated e. g. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [e. g. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6: 12-19; Agrawal (1996) TIBTECH

14 : 376-387]; analogues such as peptide nucleic acids may also be used [e. g. see Corey (1997) TIBTECH 15: 224-229; Buchardt et al. (1993) TIBTECH 11 : 384-386].

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in : Mullis et al, [Meth. Enzymol. (1987) 155: 335-350]; US patents 4,683,195 and 4,683,202. Two"primer"nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired influenza sequence.

A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the influenza sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.

BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows a maximum likelihood phylogenetic tree for the HA-0 DNA sequences of various influenza A virus subtypes. 22084 trees were examined. Figure 2 shows an alignment of the DNA sequences of the HA genes of four influenza viruses according to the invention, and Figure 3 shows the corresponding amino acid sequences. These sequences are also given in the sequence listing: isolate 2/99 is represented by SEQ IDs 1 and 9; 3/99 is SEQ IDs 2 and 10; 4/99 is SEQ IDs 3 and 11 ; and 5/99 is SEQ IDs 4 and 12.

MODES FOR CARRYING OUT THE INVENTION Virus isolation During influenza A virus surveillance in wild animals, influenza A virus was found in cloacal swabs and dropping samples taken from black-headed gulls (Larus ridibundus) in Sweden and the Netherlands. Of the 110 samples analysed, 9 revealed the presence of the influenza A virus genome, as determined by RT-PCR analysis with primers and probes specific for the influenza A virus matrix gene. The 9 positive specimens were subsequently used for virus-isolation in 11-day old embryonated chicken eggs. The allantoic fluids from eggs inoculated with 6 out of these 9 samples contained a haemagglutinating agent (virus isolate) as determined by a haemagglutination assay using turkey erythrocytes. The allantoic fluids containing the agent were subsequently used for virus characterisation.

Serology Hyperimmune rabbit sera were generated by multiple immunisations with the HA and neuraminidase (NA) proteins purified from influenza A viruses of all known subtypes (H1 to H15). These hyperimmune sera are routinely used for determining the serotype of influenza A virus by haemagglutination inhibition (HAI) assays. Whereas 2 of the 6 virus isolates were neutralised in HAI assays using the rabbit anti-H13 serum, 4 isolates did not react with any of the rabbit sera in the panel, indicating the presence of a new subtype. In addition, a hyper- immune rabbit serum raised against one of the H16 strains ("A/Gull/Sweden/2/99") did not neutralise a panel of influenza A viruses representing subtypes Hl to H15:

Virus HA Hyper-immune rabbit sera raised against HA/NA proteins purified from prototype viruses Prototype Subtype 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 <BR> <BR> <BR> <BR> A/Duck/Alberta/35/76 1 2560 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10<BR> <BR> <BR> <BR> A/Singapore/1157 2 <10 5120 <10 <10 <10 40 20 <10 160 <10 <10 <10 40 <10 <10 <10 A/Duck/Ukraine/1/63 3 <10 <10 3840 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 A/Duck/Czechoslovakia/1/56 4 <10 <10 <10 320 <10 <10 <10 <10 <10 <10 <10 <10 40 <10 <10 <10 A/Tern/South Africa/1/72 5 <10 <10 <10 <10 1280 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 A/Shearwate/Australia/1/72 6 <10 <10 <10 <10 <10 2560 40 <10 20 <10 <10 20 <10 20 <10 20 A/Seal/Massachusetts/1/80 7 <10 <10 <10 <10 <10 <10 5120 <10 <10 <10 <10 <10 <10 <10 <10 <10 A/Turkey/Ontario/6118168 8 <10 160 <10 <10 <10 <10 <10 2560 <10 <10 <10 <10 40 <10 <10 <10<BR> <BR> <BR> <BR> A/Turkey/Wisconsin/1/66 9 <10 <10 <10 <10 <10 <10 <10 <10 5120 <10 <10 <10 <10 <10 <10 <10<BR> <BR> <BR> A/ChickenlGermanyl49 10 <10 <10 <10 <10 <10 <10 80 <10 <10 2560 <10 <10 640 <10 <10 <10 A/Duck/Memphis/546/74 1 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 1280 <10 <10 <10 <10 <10 A/Duck/Alberta/60/76 12 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 1920 <10 <10 <10 <10<BR> A/Gull/Maryland/704/77 13 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 2560 <10 <10 40<BR> <BR> <BR> AIGuIVMaryland/704/77 13 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 2560 <10 <10 40<BR> <BR> <BR> a/Gull/Gurjev/263/83 14 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 1920 <10 <10<BR> <BR> <BR> A/Duck/Australial341/83 15 <10 <10 20 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 960 <10 A/Gull/Sweden/2/99 16 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 80 <10 <10 10240

The 4 non-reacting isolates (numbered 2/99,3/99,4/99 and 5/99) were further characterised in HAI assays using (a) ferret sera raised against influenza A viruses of the new (H16) subtype by nasal inoculation, (b) a hyper-immune rabbit serum raised against HA of H13 subtype A/Gull/Maryland/704/77, and (c) gull sera collected from black-headed gulls caught in the wild.

Results were as follows: Rabbitantisera Ferretantisera Gullantisera HA Duck Gull Gull Gull Gull Gull Gull Virus Subtype Memphis Maryland Maryland Sweden Sweden Sweden Sweden Hull H13 H13 H13 H16 H16 H16 A/Duck/Memphis/546/76 11 3840 <10 <10 <10 <10 <10 <10 N. T. A/Gull/Maryland/704/77 13 <10 2560 10240 2560 120 <10 <10 N. T. A/Gull/Sweden/1/99 13 <10 2560 40 640 <10 <10 <10 <10 A/Gull/Sweden/2/99 16 <10 80 <10 20 2560 320 20 N. T. A/Gull/Sweden/3/99 16 <10 160 <10407680320 <10 N. T. A/Gull/Sweden/4/99 16 <10 80 <10 <10 2560 640 80 20480 A/Gull/Sweden/5/99 16 <10 40 <10 60 3840 80 480 N. T. As can be seen, the sera clearly discriminate H13 viruses from the new H16 subtype. Rabbit sera raised against H13 influenza A virus do not neutralise influenza A viruses of the H16 subtype and vice versa. Two gull sera reacted mono-specifically with the influenza A virus of the H16

subtype. Ferret sera raised against influenza A viruses of the H16 subtype did not react with influenza A viruses of subtype H13. Moreover, these ferret sera could discriminate between the different influenza A virus isolates of the H16 subtype, suggesting these isolates are serologically diverse.

Sequence analysis and phylogeny The DNA and amino acid sequences of the HA genes of these virus isolates are shown in figures 2 and 3. The amino acid homology between viruses of the H13 and H16 subtypes is approximately 80 % for the entire HA protein (HA-0), 75 % for domain 1 of HA (HA-1), and 87 % for domain 2 of HA (HA-2).

The sequences of the HA gene segments of 4 influenza A viruses of the H16 subtype and 1 of the H13 subtype were compared with the HA gene segments from other HA subtypes. The phylogenetic tree, based on the DNA sequences of HA representing all HA subtypes is shown in figure 1. As can be seen, the genetic distance between viruses of subtype H16 and subtype H13 is similar to, or greater than the distance between viruses of other related HA subtypes (e. g. H2 and H5, H4 and H14, H7 and H15).

Comparison of the homology of H16 and H13 at the amino acid level revealed similar differences-the amino acid homology between the HA proteins of subtypes H13 and H16 is almost the same as for subtypes H2 and H5, H7 and H15 and H4 and H14. The HA1 coding sequence of the HA gene displays somewhat more homology between subtypes H13 and H16 as compared to the other related subtypes. However, if viruses of H13 and H16 subtypes were to be classified in a single group, the homology between strains in this group would be far lower than 80%, which is normal for other influenza A virus subtypes. These data demonstrate that influenza viruses A/Black-headed Gull/Sweden/2,3,4, and 5, isolated in 1999, represent a new HA subtype, distinct from H13, and referred to herein as H16.

Within the H16 subtype, SEQ IDs 1-3 and SEQ ID 4 are clearly distinct. The HA1 and HA2 domains of SEQ IDs 1-3 are 100% identical. This common HA1 domain is 88% identical to that of SEQ ID 4, and the HA2 domain is 92% identical to that of SEQ ID 4. Overall, SEQ ID 4 is 89% identical to the common sequence (SEQ ID 2) in SEQ IDs 1-3.

Experimerttal infection of black-headed gulls Experimental infection of black-headed gulls revealed that the isolated influenza A viruses are infectious. Gulls infected via oral, nasal and ocular routes start shedding virus three days after infection and continue to shed virus for approximately 8 days. There were no signs of clinical symptoms in the experimentally infected gulls.

Influenza A viruses of the H16 subtype may also infect other gull species or other bird species.

Furthermore, birds in the wild, other than black-headed gulls, may harbour influenza A viruses of subtype H 16.

Conclusion Influenza A virus HA proteins have been characterised that cannot be classified in any of the fifteen previously defined serological subtypes of influenza A virus. Serological and sequence analyses demonstrate that these viruses represent a new influenza A virus HA, designated subtype H16. The HA proteins assist in the diagnosis of influenza A virus infection of humans or animals with influenza A viruses belonging to this subtype, as well as the development of vaccines aimed to protect against infection with influenza A viruses belonging to this subtype.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.