# Patent

## Title
Antigenic meningococcal peptides

## Abstract

WO99/36544 discloses a large number of proteins from Neisseria Meningitidis. The present invention relates to fragments of those proteins which comprise at least one antigenic determinant. Homologous sequences and proteins comprising these fragments are also disclosed.

## Background
All documents cited herein are incorporated by reference in their entirety. In particular, the contents of international patent application WO99/36544 are fully incorporated herein.
FIELD OF THE INVENTION
This invention relates to antigenic peptide sequences from the bacterium Neisseria meningitidis. 
BACKGROUND
Neisseria meningitidis is a non-motile, Gram-negative diplococcus that is pathogenic in humans.
Based on the organism's capsular polysaccharide, 12 serogroups of N. meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the rest of the cases in the United States and developed countries.
The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Meningococcus B remains a problem, however. The polysaccharide approach cannot be used because the menb capsular polysaccharide is a polymer of α(2-8)-linked N-acetyl neuraminic acid that is also present in mammalian tissue. One approach to a menB vaccine uses mixtures of outer membrane proteins (OMPs) To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed [e.g. Poolman J T (1992) Infect. Agents Dis. 4:13-28]. Additional proteins to be used in outer membrane vaccines have been the opa and opc proteins, but none of these approaches have been able to overcome the antigenic variability [e.g. Ala'Aldeen & Borriello (1996) Vaccine 14(1):49-53].
THE INVENTION
The invention provides fragments of the proteins disclosed in international patent application WO99/36544, wherein the fragments comprise at least one antigenic determinant.
Thus, if the length of any particular protein sequence disclosed in WO99/36544 is x amino acids (see Table II), the present invention provides fragments of at most x−1 amino acids of that protein. The fragment may be shorter than this (e.g. x−2, x−3, x−4, . . . ), and is preferably 100 amino acids or less (e.g. 90 amino acids, 80 amino acids etc.). The fragment may be as short as 3 amino acids, but is preferably longer (e.g. up to 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, or 100 amino acids).
Preferred fragments comprise the meningococcal peptide sequences disclosed in Table I, or sub-sequences thereof. The fragments may be longer than those given in Table I e.g. where a fragment in Table I runs from amino acid residue p to residue q of a protein, the invention also relates to fragments from residue (p−1), (p−2), or (p−3) to residue (q+1), (q+2), or (q+3).
The invention also provides polypeptides that are homologous (i.e. have sequence identity) to these fragments. Depending on the particular fragment, the degree of sequence identity is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more). These homologous polypeptides include mutants and allelic variants of the fragments. Identity between the two sequences 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 also provides proteins comprising one or more of the above-defined fragments.
The invention is subject to the proviso that it does not include within its scope proteins comprising any of the 45 protein sequences disclosed in WO99/36544 (i.e. the even SEQ IDs: 2, 4, 6, 8, 10, . . . , 86, 88, 90 of WO99/36544).
The proteins of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (e.g. native, C-terminal and/or N-terminal fusions etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other Neisserial or host cell proteins). Short proteins are preferably produced using chemical peptide synthesis.
According to a further aspect, the invention provides antibodies which recognise the fragments of the invention, with the proviso that the invention does not include within its scope antibodies which recognise one of 45 complete protein sequences in WO99/36544. The antibodies may be polyclonal or, preferably, monoclonal, and may be produced by any suitable means.
The invention also provides proteins comprising peptide sequences recognised by these antibodies. These peptide sequences will, of course, include fragments of the meningococcal proteins in WO99/36544, but will also include peptides that mimic the antigenic structure of the meningococcal peptides when bound to immunoglobulin.
According to a further aspect, the invention provides nucleic acid encoding the fragments and proteins of the invention, with the proviso that the invention does not include within its scope nucleic acid encoding one of the 45 protein sequences in WO99/36544.
In addition, the invention provides nucleic acid comprising sequences homologous (i.e. having sequence identity) to these sequences. Furthermore, the invention provides nucleic acid which can hybridise to these sequences, preferably under “high stringency” conditions (e.g. 65° C. in a 0.1×SSC, 0.5% SDS solution).
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 organism 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 compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions.
The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (e.g. as vaccines or as immunogenic compositions) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria. Said Neisserial bacteria may be any species or strain (such as N. gonorrhoeae) but are preferably N. meningitidis, especially strain A or strain B.
The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, 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 polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting said duplexes.
A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
A summary of standard techniques and procedures which may be employed in order to perform the invention (e.g. to utilise the disclosed sequences for vaccination or diagnostic purposes) 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 Cloning; 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); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized 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 Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical 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 Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).
Standard abbreviations for nucleotides and amino acids are used in this specification.
All publications, patents, and patent applications cited herein are incorporated in full by reference.


DEFINITIONS
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 “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.
The term “antigenic determinant” includes B-cell epitopes and T-cell epitopes.
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 a meningococcal 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.
Expression Systems
The meningococcal 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 metallothionein 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) Proc. Natl. Acad. Sci. 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 polyadenylation [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 terminator/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual]. 
Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, 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 replication 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 American Type Culture Collection (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 from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and 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 the polyhedrin polyadenylation signal (Miller et al. (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); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Viak 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 α-interferon, 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 2-5 kb 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 and 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 μm 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 and 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 melanogaster, 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 and 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: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 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. Chem. 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 and Hickey, Proc. Natl. Acad. Sci. 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, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Repir, 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., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.
The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. 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, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Pelunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, 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 Escherichia coli (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; U.S. Pat. No. 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 [U.S. Pat. No. 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 one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 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) Proc. Natl. Acad. Sci. 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) Proc Natl. Acad. Sci. 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 the pairing of bases between the SD sequence and the 3′ and of E. coli 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) Bio/Technology 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 [U.S. Pat. No. 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 the E. coli outer membrane protein gene (ompA) [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) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the 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) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].
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 about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 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-0 127 328). 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) Annu. 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) Proc. Natl. Acad. Sci. 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 [U.S. Pat. No. 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 CaCl2 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) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids”. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 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. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus]. 
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), enolase, glucokinase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EP-A-0329203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].
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 (U.S. Pat. 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 PHO5 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) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 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. WO88/024066).
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 yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 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 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 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], pCl/1 [Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [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 about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter 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) Proc. Natl. Acad. Sci. 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, TRP1, 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) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. 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, et al. (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. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. 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]. 
Antibodies
As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.
Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying meningococcal proteins.
Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours. The serum is recovered by centrifugation (e.g. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.
Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g. hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (e.g. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).
If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, 125I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 125I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the invention.
Pharmaceutical Compositions
Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed 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 DNA constructs in the individual to which it is administered.
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 WO98/20734), 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 may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection).
Such vaccines 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) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) 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) MF59TM adjuvant (Chiron Corporation, Emeryville, Calif.; WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% TWEEN 80 surfactant (Uniqema, New Castle, Del.), and 0.5% SPAN 85 surfactant (Uniqema) (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% TWEEN 80 surfactant (Uniqema), 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI adjuvant system (RAS), (Corixa Corporation, Seattle, Wash.) 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 DETOX adjuvant (MPL+CWS) (Corixa Corporation); (3) saponin adjuvants, such as STIMULON adjuvant (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59TM adjuvant (Chiron Corporation) 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-MLP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-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 subcutaneously, intramuscularly, or transdermally/transcutaneously (e.g. WO98/20734). 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 Immunology 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 Kaplit (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 U.S. Pat. No. 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 WO96/37626). 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 and WO92/05266), 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 and Rowe (1976) J Virol 19: 19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. 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 American Type Culture Collection (“ATCC”) at 10801 University Boulevard, Manassas, Va. 20110-2209 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, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 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) Proc Natl Acad Sci 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 WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. 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, WO93/09239. 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 (i.e. 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 U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. 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 U.S. Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S. Pat. No. 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 U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ 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 U.S. Pat. Nos. 5,091,309, 5,217,879, and W092/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, W094/21792, W092/10578, W095/07994, U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC at 10801 University Boulevard, Manassas, Va. 20110-2209 or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).
DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 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) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; 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 U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Natl Acad Sci 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 U.S. Ser. No. 08/366,787, filed Dec. 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 U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, 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) Proc Natl Acad Sci 91:1581-1585.
Particle mediated gene transfer may be employed, for example see U.S. Ser. 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 WO 90/11092 and U.S. Pat. No. 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 U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. Ser. No. 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) Proc. Natl. Acad. Sci. 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 U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033
Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 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 Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal 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) in 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 WO98/20734), 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 in 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.
Polynucleotide and Polypeptide Pharmaceutical Compositions
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 polyethylene 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) Biochim. 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) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.
Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 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, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphosphatidyl 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 in the art.
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) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 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, AII, AIV; CI, CII, CIII.
A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C & E, over time these lipoproteins lose A and acquire C & E apoproteins. VLDL comprises A, B, C & E apoproteins, LDL comprises apoprotein B; HDL comprises apoproteins A, C, & E.
The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci 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 Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 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 Technologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in Zuckermann et al. WO98/06437.
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-1, 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. LipofectinTM, and lipofectAMINETM are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.
Immunodiagnostic Assays
Meningogoccal antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-meningococcal antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to meningococcal proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.
Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.
Nucleic Acid Hybridisation
“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° C. below the 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 1 μg for a plasmid or phage digest to 10−9 to 10−8 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 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/μg, 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(log10Ci)+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 & Wahl (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 (i.e. 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 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. 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 Assays
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 meningococcal nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native meningococcal 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 meningococcal 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 meningococcal 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 a meningococcal sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a meningococcal 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. [Proc. Nat. Acad. Sci. 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]; U.S. Pat. Nos. 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 meningococcal 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 meningococcal 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.
Examples of Preferred Fragments
The protein sequences disclosed in WO99/36544 have been subjected to computer analysis to predict antigenic peptide fragments within the full-length proteins. Three algorithms have been used in this analysis:

    
    
        AMPHI This program has been used to predict T-cell epitopes [Gao et al. (1989) J. Immunol. 143:3007; Roberts et al. (1996) AIDS Res Hum Retrovir 12:593; Quakyi et al. (1992) Scand J Immunol suppl. 11:9] and is available in the Protean package of DNASTAR, Inc. (1228 South Park Street, Madison, Wis. 53715 USA).
        ANTIGENIC INDEX as disclosed by Jameson & Wolf (1988) The antigenic index: a novel algorithm for predicting antigenic determinants. CABIOS 4:181:186.
        HYDROPHILICITY as disclosed by Hopp & Woods (1981) Prediction of protein antigenic determinants from amino acid sequences. PNAS USA 78:3824-3828
    
    


Table I indicates preferred fragments of the proteins disclosed in WO99/36544. The three algorithms often identify the same fragments (e.g. ORF38-1—the fragments from residue 37-42 and 143-146 are both identified twice). Such multiply-identified fragments are particularly preferred. The algorithms often identify overlapping fragments (e.g. ORF40-1—AMPHI identifies residues 161-165, and Hydrophilicity identified residues 163-175). The invention explicitly includes fragments resulting from a combination of these overlapping fragments (e.g. the fragment from residue 161 to residue 175 in the case of ORF40-1). Fragments separated by a single amino acid are also often identified (e.g. ORF40-1 Antigenic Index 423-426 and 428-438). The invention also includes fragments spanning the two extremes of such “adjacent” fragments (e.g. 423-438 for ORF40-1).







TABLE I







1769 fragments of the proteins disclosed in WO99/36544










Fragment#





(SEQ ID NO)
WO99/36544 ORF
Algorithm
Amino Acids













1.
38-1
AMPHI
 6-14


2.
38-1
AMPHI
57-59


3.
38-1
AMPHI
67-76


4.
38-1
AMPHI
 92-100


5.
38-1
AMPHI
127-137


6.
38-1
AMPHI
149-166


7.
38-1
AMPHI
210-215


8.
38-1
AMPHI
231-236


9.
38-1
AMPHI
270-272


10.
38-1
AMPHI
303-320


11.
38-1
Antigenic Index
16-34


12.
38-1
Antigenic Index
37-42


13.
38-1
Antigenic Index
46-64


14.
38-1
Antigenic Index
72-91


15.
38-1
Antigenic Index
 94-112


16.
38-1
Antigenic Index
114-117


17.
38-1
Antigenic Index
124-136


18.
38-1
Antigenic Index
143-146


19.
38-1
Antigenic Index
148-160


20.
38-1
Antigenic Index
167-195


21.
38-1
Antigenic Index
201-216


22.
38-1
Antigenic Index
218-240


23.
38-1
Antigenic Index
244-252


24.
38-1
Antigenic Index
257-278


25.
38-1
Antigenic Index
282-290


26.
38-1
Antigenic Index
308-314


27.
38-1
Hydrophilicity
21-34


28.
38-1
Hydrophilicity
37-42


29.
38-1
Hydrophilicity
47-55


30.
38-1
Hydrophilicity
57-61


31.
38-1
Hydrophilicity
72-74


32.
38-1
Hydrophilicity
76-78


33.
38-1
Hydrophilicity
82-91


34.
38-1
Hydrophilicity
 94-101


35.
38-1
Hydrophilicity
108-112


36.
38-1
Hydrophilicity
126-136


37.
38-1
Hydrophilicity
143-146


38.
38-1
Hydrophilicity
148-160


39.
38-1
Hydrophilicity
167-195


40.
38-1
Hydrophilicity
221-223


41.
38-1
Hydrophilicity
226-236


42.
38-1
Hydrophilicity
244-250


43.
38-1
Hydrophilicity
257-274


44.
38-1
Hydrophilicity
282-286


45.
38-1
Hydrophilicity
311-314


46.
38a
AMPHI
 6-14


47.
38a
AMPHI
57-59


48.
38a
AMPHI
67-76


49.
38a
AMPHI
 92-100


50.
38a
AMPHI
127-137


51.
38a
AMPHI
149-166


52.
38a
AMPHI
210-215


53.
38a
AMPHI
223-225


54.
38a
AMPHI
231-236


55.
38a
AMPHI
270-272


56.
38a
AMPHI
303-320


57.
38a
Antigenic Index
16-34


58.
38a
Antigenic Index
37-42


59.
38a
Antigenic Index
46-64


60.
38a
Antigenic Index
72-91


61.
38a
Antigenic Index
 94-112


62.
38a
Antigenic Index
114-117


63.
38a
Antigenic Index
124-136


64.
38a
Antigenic Index
143-146


65.
38a
Antigenic Index
148-160


66.
38a
Antigenic Index
165-195


67.
38a
Antigenic Index
201-216


68.
38a
Antigenic Index
218-240


69.
38a
Antigenic Index
244-252


70.
38a
Antigenic Index
257-278


71.
38a
Antigenic Index
282-290


72.
38a
Antigenic Index
308-314


73.
38a
Hydrophilicity
21-34


74.
38a
Hydrophilicity
37-42


75.
38a
Hydrophilicity
47-55


76.
38a
Hydrophilicity
57-61


77.
38a
Hydrophilicity
72-74


78.
38a
Hydrophilicity
76-78


79.
38a
Hydrophilicity
82-91


80.
38a
Hydrophilicity
 94-101


81.
38a
Hydrophilicity
108-112


82.
38a
Hydrophilicity
126-136


83.
38a
Hydrophilicity
143-146


84.
38a
Hydrophilicity
148-160


85.
38a
Hydrophilicity
165-195


86.
38a
Hydrophilicity
221-223


87.
38a
Hydrophilicity
226-236


88.
38a
Hydrophilicity
244-250


89.
38a
Hydrophilicity
257-273


90.
38a
Hydrophilicity
282-286


91.
38a
Hydrophilicity
311-314


92.
39-1
AMPHI
 6-13


93.
39-1
AMPHI
21-24


94.
39-1
AMPHI
37-40


95.
39-1
AMPHI
60-75


96.
39-1
AMPHI
118-122


97.
39-1
AMPHI
134-139


98.
39-1
AMPHI
165-183


99.
39-1
AMPHI
192-195


100.
39-1
AMPHI
233-241


101.
39-1
AMPHI
247-267


102.
39-1
AMPHI
273-275


103.
39-1
AMPHI
299-308


104.
39-1
AMPHI
310-319


105.
39-1
AMPHI
322-330


106.
39-1
AMPHI
338-347


107.
39-1
AMPHI
358-364


108.
39-1
AMPHI
366-368


109.
39-1
AMPHI
376-378


110.
39-1
AMPHI
385-392


111.
39-1
AMPHI
413-416


112.
39-1
AMPHI
421-424


113.
39-1
AMPHI
429-438


114.
39-1
AMPHI
445-454


115.
39-1
AMPHI
456-458


116.
39-1
AMPHI
498-500


117.
39-1
AMPHI
512-519


118.
39-1
AMPHI
576-587


119.
39-1
AMPHI
589-600


120.
39-1
AMPHI
650-652


121.
39-1
AMPHI
670-674


122.
39-1
Antigenic Index
26-32


123.
39-1
Antigenic Index
35-45


124.
39-1
Antigenic Index
54-69


125.
39-1
Antigenic Index
79-84


126.
39-1
Antigenic Index
88-96


127.
39-1
Antigenic Index
105-110


128.
39-1
Antigenic Index
117-124


129.
39-1
Antigenic Index
152-154


130.
39-1
Antigenic Index
190-192


131.
39-1
Antigenic Index
222-231


132.
39-1
Antigenic Index
246-265


133.
39-1
Antigenic Index
292-295


134.
39-1
Antigenic Index
318-335


135.
39-1
Antigenic Index
353-362


136.
39-1
Antigenic Index
370-372


137.
39-1
Antigenic Index
402-404


138.
39-1
Antigenic Index
406-408


139.
39-1
Antigenic Index
419-421


140.
39-1
Antigenic Index
446-449


141.
39-1
Antigenic Index
453-460


142.
39-1
Antigenic Index
465-469


143.
39-1
Antigenic Index
476-487


144.
39-1
Antigenic Index
491-499


145.
39-1
Antigenic Index
505-514


146.
39-1
Antigenic Index
522-536


147.
39-1
Antigenic Index
557-567


148.
39-1
Antigenic Index
569-575


149.
39-1
Antigenic Index
577-580


150.
39-1
Antigenic Index
593-599


151.
39-1
Antigenic Index
603-619


152.
39-1
Antigenic Index
626-628


153.
39-1
Antigenic Index
634-637


154.
39-1
Antigenic Index
639-647


155.
39-1
Antigenic Index
655-658


156.
39-1
Antigenic Index
672-674


157.
39-1
Antigenic Index
677-686


158.
39-1
Antigenic Index
688-691


159.
39-1
Antigenic Index
693-699


160.
39-1
Antigenic Index
707-710


161.
39-1
Hydrophilicity
28-32


162.
39-1
Hydrophilicity
38-44


163.
39-1
Hydrophilicity
54-69


164.
39-1
Hydrophilicity
80-83


165.
39-1
Hydrophilicity
89-96


166.
39-1
Hydrophilicity
117-119


167.
39-1
Hydrophilicity
121-123


168.
39-1
Hydrophilicity
152-154


169.
39-1
Hydrophilicity
224-231


170.
39-1
Hydrophilicity
247-265


171.
39-1
Hydrophilicity
318-332


172.
39-1
Hydrophilicity
357-361


173.
39-1
Hydrophilicity
402-404


174.
39-1
Hydrophilicity
406-408


175.
39-1
Hydrophilicity
446-449


176.
39-1
Hydrophilicity
454-459


177.
39-1
Hydrophilicity
465-469


178.
39-1
Hydrophilicity
476-487


179.
39-1
Hydrophilicity
491-499


180.
39-1
Hydrophilicity
506-514


181.
39-1
Hydrophilicity
525-535


182.
39-1
Hydrophilicity
560-567


183.
39-1
Hydrophilicity
573-575


184.
39-1
Hydrophilicity
577-580


185.
39-1
Hydrophilicity
594-596


186.
39-1
Hydrophilicity
605-607


187.
39-1
Hydrophilicity
611-619


188.
39-1
Hydrophilicity
634-637


189.
39-1
Hydrophilicity
639-647


190.
39-1
Hydrophilicity
672-674


191.
39-1
Hydrophilicity
677-686


192.
39-1
Hydrophilicity
688-690


193.
39-1
Hydrophilicity
693-695


194.
39a
AMPHI
 6-13


195.
39a
AMPHI
21-24


196.
39a
AMPHI
37-40


197.
39a
AMPHI
60-75


198.
39a
AMPHI
118-122


199.
39a
AMPHI
134-139


200.
39a
AMPHI
165-183


201.
39a
AMPHI
192-195


202.
39a
AMPHI
233-241


203.
39a
AMPHI
247-267


204.
39a
AMPHI
273-275


205.
39a
AMPHI
299-308


206.
39a
AMPHI
310-319


207.
39a
AMPHI
322-330


208.
39a
AMPHI
338-347


209.
39a
AMPHI
358-364


210.
39a
AMPHI
366-368


211.
39a
AMPHI
376-378


212.
39a
AMPHI
385-392


213.
39a
AMPHI
413-416


214.
39a
AMPHI
421-424


215.
39a
AMPHI
429-438


216.
39a
AMPHI
445-454


217.
39a
AMPHI
456-458


218.
39a
AMPHI
498-500


219.
39a
AMPHI
512-520


220.
39a
AMPHI
576-587


221.
39a
AMPHI
589-600


222.
39a
AMPHI
650-652


223.
39a
AMPHI
670-674


224.
39a
Antigenic Index
26-32


225.
39a
Antigenic Index
35-45


226.
39a
Antigenic Index
54-69


227.
39a
Antigenic Index
79-84


228.
39a
Antigenic Index
89-96


229.
39a
Antigenic Index
103-110


230.
39a
Antigenic Index
117-124


231.
39a
Antigenic Index
152-154


232.
39a
Antigenic Index
190-192


233.
39a
Antigenic Index
222-231


234.
39a
Antigenic Index
246-265


235.
39a
Antigenic Index
292-295


236.
39a
Antigenic Index
318-335


237.
39a
Antigenic Index
353-362


238.
39a
Antigenic Index
370-372


239.
39a
Antigenic Index
402-404


240.
39a
Antigenic Index
406-408


241.
39a
Antigenic Index
419-421


242.
39a
Antigenic Index
446-449


243.
39a
Antigenic Index
453-460


244.
39a
Antigenic Index
465-469


245.
39a
Antigenic Index
476-487


246.
39a
Antigenic Index
491-499


247.
39a
Antigenic Index
505-514


248.
39a
Antigenic Index
529-535


249.
39a
Antigenic Index
557-567


250.
39a
Antigenic Index
569-575


251.
39a
Antigenic Index
577-580


252.
39a
Antigenic Index
593-599


253.
39a
Antigenic Index
603-619


254.
39a
Antigenic Index
626-628


255.
39a
Antigenic Index
634-637


256.
39a
Antigenic Index
639-647


257.
39a
Antigenic Index
655-658


258.
39a
Antigenic Index
672-674


259.
39a
Antigenic Index
677-686


260.
39a
Antigenic Index
688-691


261.
39a
Antigenic Index
693-699


262.
39a
Antigenic Index
707-710


263.
39a
Hydrophilicity
28-32


264.
39a
Hydrophilicity
38-44


265.
39a
Hydrophilicity
54-69


266.
39a
Hydrophilicity
80-83


267.
39a
Hydrophilicity
89-95


268.
39a
Hydrophilicity
105-108


269.
39a
Hydrophilicity
117-119


270.
39a
Hydrophilicity
121-123


271.
39a
Hydrophilicity
152-154


272.
39a
Hydrophilicity
224-231


273.
39a
Hydrophilicity
247-265


274.
39a
Hydrophilicity
318-332


275.
39a
Hydrophilicity
357-361


276.
39a
Hydrophilicity
402-404


277.
39a
Hydrophilicity
406-408


278.
39a
Hydrophilicity
446-449


279.
39a
Hydrophilicity
454-459


280.
39a
Hydrophilicity
465-469


281.
39a
Hydrophilicity
476-487


282.
39a
Hydrophilicity
491-499


283.
39a
Hydrophilicity
506-514


284.
39a
Hydrophilicity
529-535


285.
39a
Hydrophilicity
560-567


286.
39a
Hydrophilicity
573-575


287.
39a
Hydrophilicity
577-580


288.
39a
Hydrophilicity
594-596


289.
39a
Hydrophilicity
605-607


290.
39a
Hydrophilicity
611-619


291.
39a
Hydrophilicity
634-637


292.
39a
Hydrophilicity
639-647


293.
39a
Hydrophilicity
672-674


294.
39a
Hydrophilicity
677-686


295.
39a
Hydrophilicity
688-690


296.
39a
Hydrophilicity
693-695


297.
40-1
AMPHI
 6-14


298.
40-1
AMPHI
16-19


299.
40-1
AMPHI
22-27


300.
40-1
AMPHI
30-33


301.
40-1
AMPHI
41-44


302.
40-1
AMPHI
62-68


303.
40-1
AMPHI
129-139


304.
40-1
AMPHI
161-165


305.
40-1
AMPHI
181-191


306.
40-1
AMPHI
199-202


307.
40-1
AMPHI
215-220


308.
40-1
AMPHI
237-249


309.
40-1
AMPHI
298-302


310.
40-1
AMPHI
313-318


311.
40-1
AMPHI
335-342


312.
40-1
AMPHI
376-383


313.
40-1
AMPHI
399-402


314.
40-1
AMPHI
426-428


315.
40-1
AMPHI
430-433


316.
40-1
AMPHI
435-437


317.
40-1
AMPHI
479-482


318.
40-1
AMPHI
491-511


319.
40-1
AMPHI
523-525


320.
40-1
AMPHI
560-563


321.
40-1
Antigenic Index
21-32


322.
40-1
Antigenic Index
49-61


323.
40-1
Antigenic Index
64-66


324.
40-1
Antigenic Index
74-92


325.
40-1
Antigenic Index
 98-123


326.
40-1
Antigenic Index
129-135


327.
40-1
Antigenic Index
138-176


328.
40-1
Antigenic Index
193-195


329.
40-1
Antigenic Index
199-219


330.
40-1
Antigenic Index
226-240


331.
40-1
Antigenic Index
242-245


332.
40-1
Antigenic Index
251-257


333.
40-1
Antigenic Index
261-276


334.
40-1
Antigenic Index
279-306


335.
40-1
Antigenic Index
308-346


336.
40-1
Antigenic Index
352-367


337.
40-1
Antigenic Index
375-378


338.
40-1
Antigenic Index
384-406


339.
40-1
Antigenic Index
408-420


340.
40-1
Antigenic Index
423-426


341.
40-1
Antigenic Index
428-438


342.
40-1
Antigenic Index
453-459


343.
40-1
Antigenic Index
462-481


344.
40-1
Antigenic Index
485-494


345.
40-1
Antigenic Index
506-518


346.
40-1
Antigenic Index
535-539


347.
40-1
Antigenic Index
544-552


348.
40-1
Antigenic Index
559-566


349.
40-1
Antigenic Index
571-582


350.
40-1
Hydrophilicity
21-32


351.
40-1
Hydrophilicity
51-61


352.
40-1
Hydrophilicity
64-66


353.
40-1
Hydrophilicity
75-92


354.
40-1
Hydrophilicity
100-122


355.
40-1
Hydrophilicity
129-135


356.
40-1
Hydrophilicity
140-145


357.
40-1
Hydrophilicity
149-152


358.
40-1
Hydrophilicity
157-161


359.
40-1
Hydrophilicity
163-175


360.
40-1
Hydrophilicity
199-201


361.
40-1
Hydrophilicity
203-219


362.
40-1
Hydrophilicity
227-240


363.
40-1
Hydrophilicity
251-257


364.
40-1
Hydrophilicity
261-276


365.
40-1
Hydrophilicity
279-306


366.
40-1
Hydrophilicity
308-318


367.
40-1
Hydrophilicity
320-328


368.
40-1
Hydrophilicity
334-341


369.
40-1
Hydrophilicity
354-356


370.
40-1
Hydrophilicity
359-366


371.
40-1
Hydrophilicity
392-398


372.
40-1
Hydrophilicity
400-405


373.
40-1
Hydrophilicity
410-420


374.
40-1
Hydrophilicity
429-438


375.
40-1
Hydrophilicity
463-467


376.
40-1
Hydrophilicity
471-480


377.
40-1
Hydrophilicity
487-493


378.
40-1
Hydrophilicity
506-518


379.
40-1
Hydrophilicity
547-552


380.
40-1
Hydrophilicity
575-579


381.
40a
AMPHI
 6-10


382.
40a
AMPHI
19-27


383.
40a
AMPHI
30-33


384.
40a
AMPHI
41-44


385.
40a
AMPHI
61-72


386.
40a
AMPHI
78-81


387.
40a
AMPHI
92-94


388.
40a
AMPHI
128-130


389.
40a
AMPHI
132-134


390.
40a
AMPHI
161-165


391.
40a
AMPHI
181-193


392.
40a
AMPHI
197-199


393.
40a
AMPHI
204-211


394.
40a
AMPHI
213-218


395.
40a
AMPHI
227-229


396.
40a
AMPHI
237-249


397.
40a
AMPHI
298-302


398.
40a
AMPHI
313-318


399.
40a
AMPHI
335-342


400.
40a
AMPHI
376-383


401.
40a
AMPHI
399-402


402.
40a
AMPHI
426-428


403.
40a
AMPHI
435-437


404.
40a
AMPHI
475-483


405.
40a
AMPHI
492-512


406.
40a
AMPHI
524-526


407.
40a
AMPHI
561-564


408.
40a
Antigenic Index
21-34


409.
40a
Antigenic Index
50-64


410.
40a
Antigenic Index
75-83


411.
40a
Antigenic Index
88-97


412.
40a
Antigenic Index
105-122


413.
40a
Antigenic Index
129-134


414.
40a
Antigenic Index
140-176


415.
40a
Antigenic Index
190-207


416.
40a
Antigenic Index
211-217


417.
40a
Antigenic Index
224-240


418.
40a
Antigenic Index
242-245


419.
40a
Antigenic Index
250-255


420.
40a
Antigenic Index
260-276


421.
40a
Antigenic Index
279-306


422.
40a
Antigenic Index
308-346


423.
40a
Antigenic Index
352-367


424.
40a
Antigenic Index
375-378


425.
40a
Antigenic Index
384-406


426.
40a
Antigenic Index
408-420


427.
40a
Antigenic Index
423-438


428.
40a
Antigenic Index
453-468


429.
40a
Antigenic Index
471-481


430.
40a
Antigenic Index
487-493


431.
40a
Antigenic Index
507-519


432.
40a
Antigenic Index
536-540


433.
40a
Antigenic Index
545-553


434.
40a
Antigenic Index
560-567


435.
40a
Antigenic Index
572-583


436.
40a
Hydrophilicity
21-34


437.
40a
Hydrophilicity
50-64


438.
40a
Hydrophilicity
75-83


439.
40a
Hydrophilicity
88-96


440.
40a
Hydrophilicity
105-121


441.
40a
Hydrophilicity
129-134


442.
40a
Hydrophilicity
140-145


443.
40a
Hydrophilicity
148-155


444.
40a
Hydrophilicity
157-161


445.
40a
Hydrophilicity
163-175


446.
40a
Hydrophilicity
196-202


447.
40a
Hydrophilicity
211-217


448.
40a
Hydrophilicity
225-230


449.
40a
Hydrophilicity
232-240


450.
40a
Hydrophilicity
253-255


451.
40a
Hydrophilicity
261-276


452.
40a
Hydrophilicity
279-306


453.
40a
Hydrophilicity
308-318


454.
40a
Hydrophilicity
320-328


455.
40a
Hydrophilicity
334-341


456.
40a
Hydrophilicity
354-356


457.
40a
Hydrophilicity
359-366


458.
40a
Hydrophilicity
392-398


459.
40a
Hydrophilicity
400-405


460.
40a
Hydrophilicity
410-420


461.
40a
Hydrophilicity
428-438


462.
40a
Hydrophilicity
462-468


463.
40a
Hydrophilicity
472-481


464.
40a
Hydrophilicity
489-493


465.
40a
Hydrophilicity
507-519


466.
40a
Hydrophilicity
548-553


467.
40a
Hydrophilicity
576-580


468.
41-1
AMPHI
30-36


469.
41-1
AMPHI
93-98


470.
41-1
AMPHI
111-122


471.
41-1
AMPHI
126-129


472.
41-1
AMPHI
136-143


473.
41-1
AMPHI
145-150


474.
41-1
AMPHI
156-158


475.
41-1
AMPHI
186-195


476.
41-1
AMPHI
201-208


477.
41-1
AMPHI
213-223


478.
41-1
AMPHI
236-247


479.
41-1
AMPHI
250-255


480.
41-1
AMPHI
273-282


481.
41-1
AMPHI
303-309


482.
41-1
AMPHI
311-314


483.
41-1
AMPHI
329-338


484.
41-1
AMPHI
344-362


485.
41-1
AMPHI
372-377


486.
41-1
AMPHI
385-392


487.
41-1
AMPHI
409-412


488.
41-1
AMPHI
419-426


489.
41-1
AMPHI
458-463


490.
41-1
AMPHI
470-474


491.
41-1
AMPHI
486-489


492.
41-1
AMPHI
512-518


493.
41-1
AMPHI
527-551


494.
41-1
AMPHI
564-579


495.
41-1
AMPHI
593-597


496.
41-1
Antigenic Index
13-22


497.
41-1
Antigenic Index
30-38


498.
41-1
Antigenic Index
43-55


499.
41-1
Antigenic Index
73-75


500.
41-1
Antigenic Index
87-89


501.
41-1
Antigenic Index
105-112


502.
41-1
Antigenic Index
114-124


503.
41-1
Antigenic Index
136-141


504.
41-1
Antigenic Index
147-153


505.
41-1
Antigenic Index
163-166


506.
41-1
Antigenic Index
174-184


507.
41-1
Antigenic Index
195-207


508.
41-1
Antigenic Index
226-236


509.
41-1
Antigenic Index
244-246


510.
41-1
Antigenic Index
249-265


511.
41-1
Antigenic Index
281-287


512.
41-1
Antigenic Index
294-313


513.
41-1
Antigenic Index
317-342


514.
41-1
Antigenic Index
350-375


515.
41-1
Antigenic Index
379-386


516.
41-1
Antigenic Index
390-396


517.
41-1
Antigenic Index
413-422


518.
41-1
Antigenic Index
425-430


519.
41-1
Antigenic Index
436-440


520.
41-1
Antigenic Index
446-465


521.
41-1
Antigenic Index
468-495


522.
41-1
Antigenic Index
498-518


523.
41-1
Antigenic Index
520-522


524.
41-1
Antigenic Index
525-542


525.
41-1
Antigenic Index
547-558


526.
41-1
Antigenic Index
565-590


527.
41-1
Antigenic Index
595-602


528.
41-1
Antigenic Index
608-619


529.
41-1
Hydrophilicity
14-21


530.
41-1
Hydrophilicity
30-33


531.
41-1
Hydrophilicity
45-55


532.
41-1
Hydrophilicity
87-89


533.
41-1
Hydrophilicity
106-111


534.
41-1
Hydrophilicity
114-120


535.
41-1
Hydrophilicity
122-124


536.
41-1
Hydrophilicity
136-141


537.
41-1
Hydrophilicity
148-150


538.
41-1
Hydrophilicity
177-184


539.
41-1
Hydrophilicity
195-207


540.
41-1
Hydrophilicity
226-234


541.
41-1
Hydrophilicity
249-265


542.
41-1
Hydrophilicity
285-287


543.
41-1
Hydrophilicity
294-297


544.
41-1
Hydrophilicity
299-313


545.
41-1
Hydrophilicity
317-321


546.
41-1
Hydrophilicity
323-342


547.
41-1
Hydrophilicity
350-371


548.
41-1
Hydrophilicity
379-386


549.
41-1
Hydrophilicity
417-422


550.
41-1
Hydrophilicity
425-427


551.
41-1
Hydrophilicity
447-449


552.
41-1
Hydrophilicity
459-462


553.
41-1
Hydrophilicity
468-475


554.
41-1
Hydrophilicity
479-482


555.
41-1
Hydrophilicity
484-491


556.
41-1
Hydrophilicity
499-518


557.
41-1
Hydrophilicity
520-522


558.
41-1
Hydrophilicity
526-542


559.
41-1
Hydrophilicity
550-558


560.
41-1
Hydrophilicity
568-590


561.
41-1
Hydrophilicity
595-598


562.
41-1
Hydrophilicity
617-619


563.
41a
AMPHI
 6-12


564.
41a
AMPHI
32-34


565.
41a
AMPHI
69-74


566.
41a
AMPHI
86-98


567.
41a
AMPHI
111-119


568.
41a
AMPHI
121-126


569.
41a
AMPHI
132-134


570.
41a
AMPHI
155-160


571.
41a
AMPHI
162-171


572.
41a
AMPHI
177-184


573.
41a
AMPHI
189-199


574.
41a
AMPHI
212-223


575.
41a
AMPHI
226-231


576.
41a
AMPHI
249-258


577.
41a
AMPHI
287-290


578.
41a
AMPHI
305-314


579.
41a
AMPHI
320-338


580.
41a
AMPHI
348-353


581.
41a
AMPHI
361-368


582.
41a
AMPHI
385-388


583.
41a
AMPHI
395-402


584.
41a
AMPHI
434-439


585.
41a
AMPHI
446-450


586.
41a
AMPHI
462-467


587.
41a
AMPHI
470-475


588.
41a
AMPHI
488-494


589.
41a
AMPHI
503-525


590.
41a
AMPHI
540-555


591.
41a
AMPHI
569-573


592.
41a
AMPHI
578-594


593.
41a
Antigenic Index
10-13


594.
41a
Antigenic Index
19-31


595.
41a
Antigenic Index
48-50


596.
41a
Antigenic Index
63-65


597.
41a
Antigenic Index
 82-101


598.
41a
Antigenic Index
112-117


599.
41a
Antigenic Index
123-129


600.
41a
Antigenic Index
139-142


601.
41a
Antigenic Index
150-160


602.
41a
Antigenic Index
171-183


603.
41a
Antigenic Index
202-212


604.
41a
Antigenic Index
220-222


605.
41a
Antigenic Index
225-241


606.
41a
Antigenic Index
257-263


607.
41a
Antigenic Index
270-289


608.
41a
Antigenic Index
293-318


609.
41a
Antigenic Index
326-351


610.
41a
Antigenic Index
355-362


611.
41a
Antigenic Index
366-372


612.
41a
Antigenic Index
389-398


613.
41a
Antigenic Index
401-406


614.
41a
Antigenic Index
412-416


615.
41a
Antigenic Index
422-441


616.
41a
Antigenic Index
444-446


617.
41a
Antigenic Index
451-471


618.
41a
Antigenic Index
475-494


619.
41a
Antigenic Index
496-498


620.
41a
Antigenic Index
501-518


621.
41a
Antigenic Index
523-534


622.
41a
Antigenic Index
540-566


623.
41a
Antigenic Index
571-578


624.
41a
Antigenic Index
582-595


625.
41a
Hydrophilicity
21-31


626.
41a
Hydrophilicity
63-65


627.
41a
Hydrophilicity
83-96


628.
41a
Hydrophilicity
 98-100


629.
41a
Hydrophilicity
112-117


630.
41a
Hydrophilicity
124-126


631.
41a
Hydrophilicity
153-160


632.
41a
Hydrophilicity
171-183


633.
41a
Hydrophilicity
202-210


634.
41a
Hydrophilicity
220-222


635.
41a
Hydrophilicity
225-241


636.
41a
Hydrophilicity
261-263


637.
41a
Hydrophilicity
270-273


638.
41a
Hydrophilicity
275-289


639.
41a
Hydrophilicity
293-297


640.
41a
Hydrophilicity
299-318


641.
41a
Hydrophilicity
326-347


642.
41a
Hydrophilicity
355-362


643.
41a
Hydrophilicity
393-398


644.
41a
Hydrophilicity
401-403


645.
41a
Hydrophilicity
423-425


646.
41a
Hydrophilicity
435-438


647.
41a
Hydrophilicity
454-458


648.
41a
Hydrophilicity
460-471


649.
41a
Hydrophilicity
475-494


650.
41a
Hydrophilicity
496-498


651.
41a
Hydrophilicity
502-518


652.
41a
Hydrophilicity
527-534


653.
41a
Hydrophilicity
544-566


654.
41a.
Hydrophilicity
571-574


655.
41a
Hydrophilicity
593-595


656.
44-1
AMPHI
57-60


657.
44-1
AMPHI
76-79


658.
44-1
Antigenic Index
22-34


659.
44-1
Antigenic Index
38-46


660.
44-1
Antigenic Index
50-55


661.
44-1
Antigenic Index
64-70


662.
44-1
Antigenic Index
72-80


663.
44-1
Antigenic Index
83-89


664.
44-1
Antigenic Index
 96-106


665.
44-1
Antigenic Index
110-124


666.
44-1
Hydrophilicity
22-34


667.
44-1
Hydrophilicity
40-46


668.
44-1
Hydrophilicity
64-69


669.
44-1
Hydrophilicity
73-80


670.
44-1
Hydrophilicity
84-89


671.
44-1
Hydrophilicity
 97-106


672.
44.1
Hydrophilicity
120-124


673.
44a
AMPHI
57-60


674.
44a
AMPHI
76-79


675.
44a
Antigenic Index
23-34


676.
44a
Antigenic Index
38-46


677.
44a
Antigenic Index
50-55


678.
44a
Antigenic Index
64-70


679.
44a
Antigenic Index
72-80


680.
44a
Antigenic Index
83-89


681.
44a
Antigenic Index
 96-106


682.
44a
Antigenic Index
110-124


683.
44a
Hydrophilicity
28-34


684.
44a
Hydrophilicity
40-46


685.
44a
Hydrophilicity
64-69


686.
44a
Hydrophilicity
73-80


687.
44a
Hydrophilicity
84-89


688.
44a
Hydrophilicity
 97-106


689.
44a
Hydrophilicity
120-124


690.
49-1
AMPHI
16-21


691.
49-1
AMPHI
44-48


692.
49-1
AMPHI
56-61


693.
49-1
AMPHI
92-97


694.
49-1
AMPHI
118-127


695.
49-1
AMPHI
130-149


696.
49-1
AMPHI
156-178


697.
49-1
AMPHI
235-240


698.
49-1
AMPHI
253-264


699.
49-1
AMPHI
268-271


700.
49-1
AMPHI
278-285


701.
49-1
AMPHI
287-292


702.
49-1
AMPHI
298-300


703.
49-1
AMPHI
328-337


704.
49-1
AMPHI
343-350


705.
49-1
AMPHI
355-365


706.
49-1
AMPHI
378-389


707.
49-1
AMPHI
422-424


708.
49-1
AMPHI
442-450


709.
49-1
AMPHI
464-481


710.
49-1
AMPHI
486-496


711.
49-1
AMPHI
514-521


712.
49-1
AMPHI
548-551


713.
49-1
AMPHI
553-557


714.
49-1
AMPHI
562-568


715.
49-1
AMPHI
573-575


716.
49-1
AMPHI
588-590


717.
49-1
AMPHI
603-605


718.
49-1
AMPHI
614-618


719.
49-1
Antigenic Index
15-21


720.
49-1
Antigenic Index
26-43


721.
49-1
Antigenic Index
50-59


722.
49-1
Antigenic Index
61-75


723.
49-1
Antigenic Index
79-87


724.
49-1
Antigenic Index
 98-108


725.
49-1
Antigenic Index
110-120


726.
49-1
Antigenic Index
122-139


727.
49-1
Antigenic Index
147-164


728.
49-1
Antigenic Index
171-179


729.
49-1
Antigenic Index
185-197


730.
49-1
Antigenic Index
214-216


731.
49-1
Antigenic Index
229-231


732.
49-1
Antigenic Index
248-266


733.
49-1
Antigenic Index
278-283


734.
49-1
Antigenic Index
289-295


735.
49-1
Antigenic Index
316-326


736.
49-1
Antigenic Index
337-349


737.
49-1
Antigenic Index
368-378


738.
49-1
Antigenic Index
386-388


739.
49-1
Antigenic Index
390-410


740.
49-1
Antigenic Index
412-414


741.
49-1
Antigenic Index
423-429


742.
49-1
Antigenic Index
438-454


743.
49-1
Antigenic Index
462-475


744.
49-1
Antigenic Index
482-500


745.
49-1
Antigenic Index
503-509


746.
49-1
Antigenic Index
521-528


747.
49-1
Antigenic Index
540-562


748.
49-1
Antigenic Index
572-579


749.
49-1
Antigenic Index
590-606


750.
49-1
Antigenic Index
610-612


751.
49-1
Antigenic Index
617-619


752.
49-1
Antigenic Index
626-634


753.
49-1
Antigenic Index
637-640


754.
49-1
Hydrophilicity
18-21


755.
49-1
Hydrophilicity
26-29


756.
49-1
Hydrophilicity
31-43


757.
49-1
Hydrophilicity
51-57


758.
49-1
Hydrophilicity
64-68


759.
49-1
Hydrophilicity
79-87


760.
49-1
Hydrophilicity
 98-107


761.
49-1
Hydrophilicity
122-125


762.
49-1
Hydrophilicity
147-164


763.
49-1
Hydrophilicity
172-175


764.
49-1
Hydrophilicity
187-197


765.
49-1
Hydrophilicity
229-231


766.
49-1
Hydrophilicity
256-262


767.
49-1
Hydrophilicity
264-266


768.
49-1
Hydrophilicity
278-283


769.
49-1
Hydrophilicity
290-292


770.
49-1
Hydrophilicity
319-326


771.
49-1
Hydrophilicity
337-349


772.
49-1
Hydrophilicity
368-376


773.
49-1
Hydrophilicity
386-388


774.
49-1
Hydrophilicity
390-410


775.
49-1
Hydrophilicity
412-414


776.
49-1
Hydrophilicity
423-429


777.
49-1
Hydrophilicity
441-451


778.
49-1
Hydrophilicity
466-472


779.
49-1
Hydrophilicity
484-490


780.
49-1
Hydrophilicity
492-494


781.
49-1
Hydrophilicity
496-498


782.
49-1
Hydrophilicity
522-528


783.
49-1
Hydrophilicity
543-562


784.
49-1
Hydrophilicity
591-606


785.
49-1
Hydrophilicity
617-619


786.
49-1
Hydrophilicity
626-632


787.
49-1
Hydrophilicity
637-640


788.
49a
AMPHI
55-61


789.
49a
AMPHI
92-97


790.
49a
AMPHI
118-127


791.
49a
AMPHI
129-135


792.
49a
AMPHI
137-145


793.
49a
AMPHI
156-178


794.
49a
AMPHI
198-200


795.
49a
AMPHI
235-240


796.
49a
AMPHI
252-264


797.
49a
AMPHI
277-285


798.
49a
AMPHI
287-292


799.
49a
AMPHI
298-300


800.
49a
AMPHI
321-326


801.
49a
AMPHI
328-337


802.
49a
AMPHI
343-350


803.
49a
AMPHI
355-365


804.
49a
AMPHI
378-389


805.
49a
AMPHI
392-397


806.
49a
AMPHI
415-424


807.
49a
AMPHI
453-456


808.
49a
AMPHI
471-480


809.
49a
AMPHI
486-504


810.
49a
AMPHI
514-519


811.
49a
AMPHI
527-534


812.
49a
AMPHI
551-554


813.
49a
AMPHI
561-568


814.
49a
AMPHI
600-605


815.
49a
AMPHI
612-616


816.
49a
AMPHI
628-633


817.
49a
AMPHI
636-641


818.
49a
AMPHI
654-660


819.
49a
AMPHI
669-691


820.
49a
AMPHI
706-721


821.
49a
AMPHI
735-739


822.
49a
AMPHI
744-760


823.
49a
Antigenic Index
 4-23


824.
49a
Antigenic Index
27-43


825.
49a
Antigenic Index
51-62


826.
49a
Antigenic Index
64-68


827.
49a
Antigenic Index
72-75


828.
49a
Antigenic Index
79-87


829.
49a
Antigenic Index
 98-108


830.
49a
Antigenic Index
110-120


831.
49a
Antigenic Index
124-139


832.
49a
Antigenic Index
147-164


833.
49a
Antigenic Index
176-179


834.
49a
Antigenic Index
185-197


835.
49a
Antigenic Index
214-216


836.
49a
Antigenic Index
229-231


837.
49a
Antigenic Index
248-267


838.
49a
Antigenic Index
278-283


839.
49a
Antigenic Index
289-295


840.
49a
Antigenic Index
305-308


841.
49a
Antigenic Index
316-326


842.
49a
Antigenic Index
337-349


843.
49a
Antigenic Index
368-378


844.
49a
Antigenic Index
386-388


845.
49a
Antigenic Index
391-407


846.
49a
Antigenic Index
423-429


847.
49a
Antigenic Index
436-455


848.
49a
Antigenic Index
459-484


849.
49a
Antigenic Index
492-517


850.
49a
Antigenic Index
521-528


851.
49a
Antigenic Index
532-539


852.
49a
Antigenic Index
555-564


853.
49a
Antigenic Index
567-572


854.
49a
Antigenic Index
578-582


855.
49a
Antigenic Index
588-607


856.
49a
Antigenic Index
610-612


857.
49a
Antigenic Index
617-637


858.
49a
Antigenic Index
641-660


859.
49a
Antigenic Index
662-664


860.
49a
Antigenic Index
667-684


861.
49a
Antigenic Index
689-700


862.
49a
Antigenic Index
706-732


863.
49a
Antigenic Index
737-744


864.
49a
Antigenic Index
748-761


865.
49a
Hydrophilicity
 4-23


866.
49a
Hydrophilicity
31-43


867.
49a
Hydrophilicity
51-53


868.
49a
Hydrophilicity
55-57


869.
49a
Hydrophilicity
64-68


870.
49a
Hydrophilicity
79-87


871.
49a
Hydrophilicity
 98-106


872.
49a
Hydrophilicity
114-120


873.
49a
Hydrophilicity
130-139


874.
49a
Hydrophilicity
147-164


875.
49a
Hydrophilicity
187-197


876.
49a
Hydrophilicity
229-231


877.
49a
Hydrophilicity
249-262


878.
49a
Hydrophilicity
264-266


879.
49a
Hydrophilicity
278-283


880.
49a
Hydrophilicity
290-292


881.
49a
Hydrophilicity
319-326


882.
49a
Hydrophilicity
337-349


883.
49a
Hydrophilicity
368-376


884.
49a
Hydrophilicity
386-388


885.
49a
Hydrophilicity
391-407


886.
49a
Hydrophilicity
427-429


887.
49a
Hydrophilicity
436-439


888.
49a
Hydrophilicity
441-455


889.
49a
Hydrophilicity
459-463


890.
49a
Hydrophilicity
465-484


891.
49a
Hydrophilicity
492-513


892.
49a
Hydrophilicity
521-528


893.
49a
Hydrophilicity
559-564


894.
49a
Hydrophilicity
567-569


895.
49a
Hydrophilicity
589-591


896.
49a
Hydrophilicity
601-604


897.
49a
Hydrophilicity
620-624


898.
49a
Hydrophilicity
626-637


899.
49a
Hydrophilicity
641-660


900.
49a
Hydrophilicity
662-664


901.
49a
Hydrophilicity
668-684


902.
49a
Hydrophilicity
693-700


903.
49a
Hydrophilicity
710-732


904.
49a
Hydrophilicity
737-740


905.
49a
Hydrophilicity
759-761


906.
51-1
AMPHI
15-21


907.
51-1
AMPHI
40-54


908.
51-1
AMPHI
75-86


909.
51-1
AMPHI
108-110


910.
51-1
AMPHI
112-124


911.
51-1
AMPHI
141-148


912.
51-1
AMPHI
184-189


913.
51-1
AMPHI
211-216


914.
51-1
Antigenic Index
58-65


915.
51-1
Antigenic Index
123-127


916.
51-1
Antigenic Index
132-137


917.
51-1
Antigenic Index
149-153


918.
51-1
Antigenic Index
165-177


919.
51-1
Antigenic Index
198-204


920.
51-1
Antigenic Index
222-231


921.
51-1
Hydrophilicity
60-65


922.
51-1
Hydrophilicity
123-127


923.
51-1
Hydrophilicity
132-135


924.
51-1
Hydrophilicity
165-174


925.
51-1
Hydrophilicity
200-203


926.
51-1
Hydrophilicity
222-227


927.
51a
AMPHI
15-21


928.
51a
AMPHI
40-54


929.
51a
AMPHI
75-86


930.
51a
AMPHI
108-110


931.
51a
AMPHI
112-124


932.
51a
AMPHI
141-148


933.
51a
AMPHI
184-189


934.
51a
AMPHI
211-216


935.
51a
Hydrophilicity
60-65


936.
51a
Hydrophilicity
123-127


937.
51a
Hydrophilicity
132-135


938.
51a
Hydrophilicity
165-174


939.
51a
Hydrophilicity
200-203


940.
51a
Hydrophilicity
222-227


941.
52-1
AMPHI
48-50


942.
52-1
AMPHI
64-73


943.
52-1
Antigenic Index
19-26


944.
52-1
Antigenic Index
30-35


945.
52-1
Antigenic Index
42-52


946.
52-1
Antigenic Index
57-86


947.
52-1
Hydrophilicity
22-26


948.
52-1
Hydrophilicity
30-35


949.
52-1
Hydrophilicity
42-52


950.
52-1
Hydrophilicity
57-71


951.
52-1
Hydrophilicity
78-86


952.
69-1
AMPHI
25-27


953.
69-1
AMPHI
46-66


954.
69-1
Antigenic Index
32-41


955.
69-1
Antigenic Index
43-45


956.
69-1
Antigenic Index
71-78


957.
69-1
Hydrophilicity
32-38


958.
69-1
Hydrophilicity
71-78


959.
69a
AMPHI
25-27


960.
69a
AMPHI
46-66


961.
69a
Antigenic Index
32-41


962.
69a
Antigenic Index
43-46


963.
69a
Antigenic Index
71-78


964.
69a
Hydrophilicity
32-38


965.
69a
Hydrophilicity
71-78


966.
77-1
AMPHI
12-16


967.
77-1
AMPHI
23-33


968.
77-1
AMPHI
35-42


969.
77-1
AMPHI
51-57


970.
77-1
AMPHI
67-70


971.
77-1
AMPHI
73-79


972.
77-1
AMPHI
122-124


973.
77-1
AMPHI
130-134


974.
77-1
AMPHI
165-178


975.
77-1
AMPHI
191-211


976.
77-1
Antigenic Index
22-31


977.
77-1
Antigenic Index
34-44


978.
77-1
Antigenic Index
80-94


979.
77-1
Antigenic Index
101-104


980.
77-1
Antigenic Index
155-158


981.
77-1
Antigenic Index
167-181


982.
77-1
Hydrophilicity
22-28


983.
77-1
Hydrophilicity
38-44


984.
77-1
Hydrophilicity
80-92


985.
77-1
Hydrophilicity
171-178


986.
77a
AMPHI
 8-15


987.
77a
AMPHI
24-30


988.
77a
AMPHI
40-43


989.
77a
AMPHI
46-52


990.
77a
AMPHI
95-97


991.
77a
AMPHI
103-107


992.
77a
AMPHI
114-125


993.
77a
AMPHI
144-151


994.
77a
AMPHI
154-156


995.
77a
AMPHI
166-184


996.
77a
Antigenic Index
 7-17


997.
77a
Antigenic Index
53-67


998.
77a
Antigenic Index
74-77


999.
77a
Antigenic Index
128-131


1000.
77a
Antigenic Index
140-154


1001.
77a
Hydrophilicity
11-17


1002.
77a
Hydrophilicity
53-65


1003.
77a
Hydrophilicity
141-151


1004.
81-1
AMPHI
30-40


1005.
81-1
AMPHI
54-56


1006.
81-1
AMPHI
60-63


1007.
81-1
AMPHI
76-93


1008.
81-1
AMPHI
 96-101


1009.
81-1
AMPHI
104-406


1010.
81-1
AMPHI
118-126


1011.
81-1
AMPHI
190-205


1012.
81-1
AMPHI
230-233


1013.
81-1
AMPHI
239-242


1014.
81-1
AMPHI
256-258


1015.
81-1
AMPHI
264-284


1016.
81-1
AMPHI
290-297


1017.
81-1
AMPHI
317-326


1018.
81-1
AMPHI
388-396


1019.
81-1
AMPHI
403-414


1020.
81-1
AMPHI
458-463


1021.
81-1
AMPHI
476-480


1022.
81-1
Antigenic Index
1-4


1023.
81-1
Antigenic Index
35-38


1024.
81-1
Antigenic Index
86-89


1025.
81-1
Antigenic Index
95-98


1026.
81-1
Antigenic Index
100-103


1027.
81-1
Antigenic Index
128-136


1028.
81-1
Antigenic Index
154-174


1029.
81-1
Antigenic Index
197-211


1030.
81-1
Antigenic Index
220-226


1031.
81-1
Antigenic Index
232-240


1032.
81-1
Antigenic Index
244-249


1033.
81-1
Antigenic Index
251-253


1034.
81-1
Antigenic Index
255-258


1035.
81-1
Antigenic Index
276-290


1036.
81-1
Antigenic Index
292-301


1037.
81-1
Antigenic Index
307-312


1038.
81-1
Antigenic Index
318-323


1039.
81-1
Antigenic Index
334-345


1040.
81-1
Antigenic Index
352-358


1041.
81-1
Antigenic Index
364-372


1042.
81-1
Antigenic Index
376-384


1043.
81-1
Antigenic Index
387-401


1044.
81-1
Antigenic Index
409-417


1045.
81-1
Antigenic Index
423-444


1046.
81-1
Antigenic Index
452-459


1047.
81-1
Antigenic Index
486-488


1048.
81-1
Antigenic Index
490-499


1049.
81-1
Antigenic Index
507-520


1050,
81-1
Hydrophilicity
 1-4


1051.
81-1
Hydrophilicity
35-38


1052.
81-1
Hydrophilicity
95-98


1053.
81-1
Hydrophilicity
128-136


1054.
81-1
Hydrophilicity
154-164


1055.
81-1
Hydrophilicity
166-172


1056.
81-1
Hydrophilicity
202-209


1057.
81-1
Hydrophilicity
220-226


1058.
81-1
Hydrophilicity
234-238


1059.
81-1
Hydrophilicity
245-249


1060.
81-1
Hydrophilicity
251-253


1061.
81-1
Hydrophilicity
284-287


1062.
81-1
Hydrophilicity
292-299


1063.
81-1
Hydrophilicity
307-312


1064.
81-1
Hydrophilicity
321-323


1065.
81-1
Hydrophilicity
338-345


1066.
81-1
Hydrophilicity
366-368


1067.
81-1
Hydrophilicity
378-384


1068.
81-1
Hydrophilicity
387-401


1069.
81-1
Hydrophilicity
409-415


1070.
81-1
Hydrophilicity
453-459


1071.
81-1
Hydrophilicity
493-499


1072.
81-1
Hydrophilicity
507-509


1073.
81-1
Hydrophilicity
512-518


1074.
82a
AMPHI
36-40


1075.
82a
AMPHI
 95-111


1076.
82a
AMPHI
117-132


1077.
82a
AMPHI
135-137


1078.
82a
AMPHI
160-174


1079.
82a
AMPHI
183-187


1080.
82a
Antigenic Index
2-8


1081.
82a
Antigenic Index
56-60


1082.
82a
Antigenic Index
90-97


1083.
82a
Antigenic Index
104-111


1084.
82a
Antigenic Index
114-137


1085.
82a
Antigenic Index
141-151


1086.
82a
Antigenic Index
170-175


1087.
82a
Antigenic Index
180-188


1088.
82a
Antigenic Index
194-201


1089.
82a
Antigenic Index
206-209


1090.
82a
Antigenic Index
216-218


1091.
82a
Hydrophilicity
2-8


1092.
82a
Hydrophilicity
56-60


1093.
82a
Hydrophilicity
90-97


1094.
82a
Hydrophilicity
105-108


1095.
82a
Hydrophilicity
120-128


1096.
82a
Hydrophilicity
130-134


1097.
82a
Hydrophilicity
141-151


1098.
82a
Hydrophilicity
170-175


1099.
82a
Hydrophilicity
186-188


1100.
82a
Hydrophilicity
195-201


1101.
82a
Hydrophilicity
206-209


1102.
112-1
AMPHI
6-8


1103.
112-1
AMPHI
12-34


1104.
112-1
AMPHI
45-53


1105.
112-1
AMPHI
63-65


1106.
112-1
AMPHI
70-82


1107.
112-1
AMPHI
84-86


1108.
112-1
AMPHI
107-109


1109.
112-1
AMPHI
116-123


1110.
112-1
AMPHI
183-186


1111.
112-1
AMPHI
244-246


1112.
112-1
AMPHI
248-258


1113.
112-1
AMPHI
280-282


1114.
112-1
AMPHI
302-313


1115.
112-1
Antigenic Index
35-44


1116.
112-1
Antigenic Index
57-61


1117.
112-1
Antigenic Index
81-84


1118.
112-1
Antigenic Index
91-98


1119.
112-1
Antigenic Index
125-133


1120.
112-1
Antigenic Index
140-147


1121.
112-1
Antigenic Index
149-159


1122.
112-1
Antigenic Index
161-165


1123.
112-1
Antigenic Index
174-190


1124.
112-1
Antigenic Index
192-200


1125.
112-1
Antigenic Index
202-216


1126.
112-1
Antigenic Index
218-224


1127.
112-1
Antigenic Index
228-232


1128.
112-1
Antigenic Index
239-244


1129.
112-1
Antigenic Index
255-263


1130.
112-1
Antigenic Index
290-300


1131.
112-1
Hydrophilicity
38-40


1132.
112-1
Hydrophilicity
57-61


1133.
112-1
Hydrophilicity
92-98


1134.
112-1
Hydrophilicity
125-133


1135.
112-1
Hydrophilicity
141-143


1136.
112-1
Hydrophilicity
150-159


1137.
112-1
Hydrophilicity
161-164


1138.
112-1
Hydrophilicity
175-190


1139.
112-1
Hydrophilicity
203-216


1140.
112-1
Hydrophilicity
218-224


1141.
112-1
Hydrophilicity
228-232


1142.
112-1
Hydrophilicity
239-244


1143.
112-1
Hydrophilicity
259-261


1144.
112-1
Hydrophilicity
293-297


1145.
112a
AMPHI
6-8


1146.
112a
AMPHI
12-34


1147.
112a
AMPHI
47-54


1148.
112a
AMPHI
63-65


1149.
112a
AMPHI
69-72


1150.
112a
AMPHI
84-86


1151.
112a
AMPHI
89-91


1152.
112a
AMPHI
107-109


1153.
112a
AMPHI
116-123


1154.
112a
AMPHI
183-186


1155.
112a
AMPHI
244-246


1156.
112a
AMPHI
248-258


1157.
112a
AMPHI
280-282


1158.
112a
AMPHI
302-310


1159.
112a
AMPHI
321-336


1160.
112a
Antigenic Index
35-44


1161.
112a
Antigenic Index
57-61


1162.
112a
Antigenic Index
81-84


1163.
112a
Antigenic Index
91-98


1164.
112a
Antigenic Index
125-133


1165.
112a
Antigenic Index
140-147


1166.
112a
Antigenic Index
150-158


1167.
112a
Antigenic Index
161-164


1168.
112a
Antigenic Index
174-190


1169.
112a
Antigenic Index
194-200


1170.
112a
Antigenic Index
202-216


1171.
112a
Antigenic Index
218-220


1172.
112a
Antigenic Index
222-224


1173.
112a
Antigenic Index
228-232


1174.
112a
Antigenic Index
239-244


1175.
112a
Antigenic Index
256-263


1176.
112a
Antigenic Index
290-301


1177.
112a
Antigenic Index
351-356


1178.
112a
Hydrophilicity
38-40


1179.
112a
Hydrophilicity
57-61


1180.
112a
Hydrophilicity
93-98


1181.
112a
Hydrophilicity
125-133


1182.
112a
Hydrophilicity
141-143


1183.
112a
Hydrophilicity
150-155


1184.
112a
Hydrophilicity
161-164


1185.
112a
Hydrophilicity
175-190


1186.
112a
Hydrophilicity
203-216


1187.
112a
Hydrophilicity
218-220


1188.
112a
Hydrophilicity
222-224


1189.
112a
Hydrophilicity
228-232


1190.
112a
Hydrophilicity
239-244


1191.
112a
Hydrophilicity
259-261


1192.
112a
Hydrophilicity
293-297


1193.
112a
Hydrophilicity
351-356


1194.
114-1
AMPHI
45-54


1195.
114-1
AMPHI
154-160


1196.
114-1
AMPHI
182-190


1197.
114-1
AMPHI
224-226


1198.
114-1
AMPHI
229-233


1199.
114-1
AMPHI
285-287


1200.
114-1
AMPHI
303-310


1201.
114-1
AMPHI
321-332


1202.
114-1
AMPHI
392-398


1203.
114-1
AMPHI
413-416


1204.
114-1
AMPHI
450-452


1205.
114-1
AMPHI
477-487


1206.
114-1
AMPHI
506-509


1207.
114-1
AMPHI
525-529


1208.
114-1
AMPHI
565-567


1209.
114-1
AMPHI
614-621


1210.
114-1
AMPHI
631-635


1211.
114-1
AMPHI
770-774


1212.
114-1
AMPHI
810-813


1213.
114-1
AMPHI
847-849


1214.
114-1
AMPHI
851-853


1215.
114-1
AMPHI
875-879


1216.
114-1
AMPHI
951-956


1217.
114-1
AMPHI
975-980


1218.
114-1
AMPHI
1034-1036


1219.
114-1
AMPHI
1048-1051


1220.
114-1
AMPHI
1073-1081


1221.
114-1
AMPHI
1086-1090


1222.
114-1
AMPHI
1095-1102


1223.
114-1
AMPHI
1111-1115


1224.
114-1
AMPHI
1163-1167


1225.
114-1
AMPHI
1242-1245


1226.
114-1
AMPHI
1275-1281


1227.
114-1
AMPHI
1312-1317


1228.
114-1
AMPHI
1338-1347


1229.
114-1
AMPHI
1349-1355


1230.
114-1
AMPHI
1357-1360


1231.
114-1
AMPHI
1362-1365


1232.
114-1
AMPHI
1376-1398


1233.
114-1
AMPHI
1418-1421


1234.
114-1
AMPHI
1425-1429


1235.
114-1
AMPHI
1468-1473


1236.
114-1
AMPHI
1476-1485


1237.
114-1
AMPHI
1495-1515


1238.
114-1
AMPHI
1518-1526


1239.
114-1
AMPHI
1546-1555


1240.
114-1
AMPHI
1557-1559


1241.
114-1
AMPHI
1580-1583


1242.
114-1
AMPHI
1585-1597


1243.
114-1
AMPHI
1604-1606


1244.
114-1
AMPHI
1613-1624


1245.
114-1
AMPHI
1626-1630


1246.
114-1
AMPHI
1638-1644


1247.
114-1
AMPHI
1655-1660


1248.
114-1
AMPHI
1662-1664


1249.
114-1
AMPHI
1672-1674


1250.
114-1
AMPHI
1677-1679


1251.
114-1
AMPHI
1691-1694


1252.
114-1
AMPHI
1713-1716


1253.
114-1
AMPHI
1719-1729


1254.
114-1
AMPHI
1735-1738


1255.
114-1
AMPHI
1753-1757


1256.
114-1
AMPHI
1772-1778


1257.
114-1
AMPHI
1790-1792


1258.
114-1
AMPHI
1817-1826


1259.
114-1
AMPHI
1828-1832


1260.
114-1
AMPHI
1840-1851


1261.
114-1
AMPHI
1854-1856


1262.
114-1
AMPHI
1871-1881


1263.
114-1
AMPHI
1883-1896


1264.
114-1
AMPHI
1922-1927


1265.
114-1
AMPHI
1934-1946


1266.
114-1
AMPHI
1950-1955


1267.
114-1
AMPHI
1957-1964


1268.
114-1
Antigenic Index
1-6


1269.
114-1
Antigenic Index
10-16


1270.
114-1
Antigenic Index
23-37


1271.
114-1
Antigenic Index
41-55


1272.
114-1
Antigenic Index
75-85


1273.
114-1
Antigenic Index
91-97


1274.
114-1
Antigenic Index
102-140


1275.
114-1
Antigenic Index
147-156


1276.
114-1
Antigenic Index
161-168


1277.
114-1
Antigenic Index
172-174


1278.
114-1
Antigenic Index
181-189


1279.
114-1
Antigenic Index
196-203


1280.
114-1
Antigenic Index
208-213


1281.
114-1
Antigenic Index
220-229


1282.
114-1
Antigenic Index
242-248


1283.
114-1
Antigenic Index
251-266


1284.
114-1
Antigenic Index
268-276


1285.
114-1
Antigenic Index
295-307


1286.
114-1
Antigenic Index
309-312


1287.
114-1
Antigenic Index
318-340


1288.
114-1
Antigenic Index
345-351


1289.
114-1
Antigenic Index
357-366


1290.
114-1
Antigenic Index
371-381


1291.
114-1
Antigenic Index
385-392


1292.
114-1
Antigenic Index
404-417


1293.
114-1
Antigenic Index
419-432


1294.
114-1
Antigenic Index
440-456


1295.
114-1
Antigenic Index
464-468


1296.
114-1
Antigenic Index
473-480


1297.
114-1
Antigenic Index
482-488


1298.
114-1
Antigenic Index
496-511


1299.
114-1
Antigenic Index
515-530


1300.
114-1
Antigenic Index
535-549


1301.
114-1
Antigenic Index
555-560


1302.
114-1
Antigenic Index
564-582


1303.
114-1
Antigenic Index
588-596


1304.
114-1
Antigenic Index
602-615


1305.
114-1
Antigenic Index
617-620


1306.
114-1
Antigenic Index
622-624


1307.
114-1
Antigenic Index
628-632


1308.
114-1
Antigenic Index
637-640


1309.
114-1
Antigenic Index
647-654


1310.
114-1
Antigenic Index
660-666


1311.
114-1
Antigenic Index
668-688


1312.
114-1
Antigenic Index
696-725


1313.
114-1
Antigenic Index
730-733


1314.
114-1
Antigenic Index
738-755


1315.
114-1
Antigenic Index
760-766


1316.
114-1
Antigenic Index
779-783


1317.
114-1
Antigenic Index
786-799


1318.
114-1
Antigenic Index
807-809


1319.
114-1
Antigenic Index
811-819


1320.
114-1
Antigenic Index
831-839


1321.
114-1
Antigenic Index
845-857


1322.
114-1
Antigenic Index
860-862


1323.
114-1
Antigenic Index
864-868


1324.
114-1
Antigenic Index
872-879


1325.
114-1
Antigenic Index
883-891


1326.
114-1
Antigenic Index
893-903


1327.
114-1
Antigenic Index
908-916


1328.
114-1
Antigenic Index
919-936


1329.
114-1
Antigenic Index
941-947


1330.
114-1
Antigenic Index
950-956


1331.
114-1
Antigenic Index
959-976


1332.
114-1
Antigenic Index
979-991


1333.
114-1
Antigenic Index
 993-1000


1334.
114-1
Antigenic Index
1007-1022


1335.
114-1
Antigenic Index
1041-1053


1336.
114-1
Antigenic Index
1062-1068


1337.
114-1
Antigenic Index
1075-1108


1338.
114-1
Antigenic Index
1115-1121


1339.
114-1
Antigenic Index
1126-1145


1340.
114-1
Antigenic Index
1148-1152


1341.
114-1
Antigenic Index
1156-1178


1342.
114-1
Antigenic Index
1195-1206


1343.
114-1
Antigenic Index
1208-1212


1344.
114-1
Antigenic Index
1217-1243


1345.
114-1
Antigenic Index
1246-1263


1346.
114-1
Antigenic Index
1271-1282


1347.
114-1
Antigenic Index
1284-1288


1348.
114-1
Antigenic Index
1292-1295


1349.
114-1
Antigenic Index
1299-1307


1350.
114-1
Antigenic Index
1318-1328


1351.
114-1
Antigenic Index
1330-1340


1352.
114-1
Antigenic Index
1344-1359


1353.
114-1
Antigenic Index
1367-1384


1354.
114-1
Antigenic Index
1395-1399


1355.
114-1
Antigenic Index
1405-1417


1356.
114-1
Antigenic Index
1445-1449


1357.
114-1
Antigenic Index
1491-1510


1358.
114-1
Antigenic Index
1526-1529


1359.
114-1
Antigenic Index
1532-1548


1360.
114-1
Antigenic Index
1552-1556


1361.
114-1
Antigenic Index
1560-1562


1362.
114-1
Antigenic Index
1573-1583


1363.
114-1
Antigenic Index
1594-1611


1364.
114-1
Antigenic Index
1627-1635


1365.
114-1
Antigenic Index
1643-1645


1366.
114-1
Antigenic Index
1647-1665


1367.
114-1
Antigenic Index
1680-1686


1368.
114-1
Antigenic Index
1700-1722


1369.
114-1
Antigenic Index
1724-1726


1370.
114-1
Antigenic Index
1739-1746


1371.
114-1
Antigenic Index
1752-1757


1372.
114-1
Antigenic Index
1780-1783


1373.
114-1
Antigenic Index
1791-1795


1374.
114-1
Antigenic Index
1804-1808


1375.
114-1
Antigenic Index
1829-1835


1376.
114-1
Antigenic Index
1841-1859


1377.
114-1
Antigenic Index
1867-1886


1378.
114-1
Antigenic Index
1897-1903


1379.
114-1
Antigenic Index
1908-1912


1380.
114-1
Antigenic Index
1917-1922


1381.
114-1
Antigenic Index
1926-1934


1382.
114-1
Antigenic Index
1938-1945


1383.
114-1
Antigenic Index
1947-1957


1384.
114-1
Antigenic Index
1961-1968


1385.
114-1
Antigenic Index
1974-1978


1386.
114-1
Hydrophilicity
4-6


1387.
114-1
Hydrophilicity
12-15


1388.
114-1
Hydrophilicity
23-34


1389.
114-1
Hydrophilicity
43-55


1390.
114-1
Hydrophilicity
76-85


1391.
114-1
Hydrophilicity
104-110


1392.
114-1
Hydrophilicity
118-123


1393.
114-1
Hydrophilicity
127-132


1394.
114-1
Hydrophilicity
147-154


1395.
114-1
Hydrophilicity
163-167


1396.
114-1
Hydrophilicity
185-187


1397.
114-1
Hydrophilicity
197-203


1398.
114-1
Hydrophilicity
208-211


1399.
114-1
Hydrophilicity
221-227


1400.
114-1
Hydrophilicity
243-245


1401.
114-1
Hydrophilicity
253-261


1402.
114-1
Hydrophilicity
263-266


1403.
114-1
Hydrophilicity
270-272


1404.
114-1
Hydrophilicity
295-301


1405.
114-1
Hydrophilicity
309-312


1406.
114-1
Hydrophilicity
320-328


1407.
114-1
Hydrophilicity
332-337


1408.
114-1
Hydrophilicity
345-351


1409.
114-1
Hydrophilicity
360-366


1410.
114-1
Hydrophilicity
371-378


1411.
114-1
Hydrophilicity
387-392


1412.
114-1
Hydrophilicity
404-415


1413.
114-1
Hydrophilicity
419-432


1414.
114-1
Hydrophilicity
441-450


1415.
114-1
Hydrophilicity
452-456


1416.
114-1
Hydrophilicity
473-480


1417.
114-1
Hydrophilicity
482-485


1418.
114-1
Hydrophilicity
496-500


1419.
114-1
Hydrophilicity
504-509


1420.
114-1
Hydrophilicity
515-520


1421.
114-1
Hydrophilicity
536-549


1422.
114-1
Hydrophilicity
555-560


1423.
114-1
Hydrophilicity
565-568


1424.
114-1
Hydrophilicity
570-579


1425.
114-1
Hydrophilicity
589-594


1426.
114-1
Hydrophilicity
602-604


1427.
114-1
Hydrophilicity
609-615


1428.
114-1
Hydrophilicity
617-620


1429.
114-1
Hydrophilicity
660-666


1430.
114-1
Hydrophilicity
668-680


1431.
114-1
Hydrophilicity
684-686


1432.
114-1
Hydrophilicity
699-708


1433.
114-1
Hydrophilicity
715-725


1434.
114-1
Hydrophilicity
730-733


1435.
114-1
Hydrophilicity
738-744


1436.
114-1
Hydrophilicity
746-754


1437.
114-1
Hydrophilicity
760-766


1438.
114-1
Hydrophilicity
789-793


1439.
114-1
Hydrophilicity
816-818


1440.
114-1
Hydrophilicity
831-836


1441.
114-1
Hydrophilicity
845-857


1442.
114-1
Hydrophilicity
860-862


1443.
114-1
Hydrophilicity
864-866


1444.
114-1
Hydrophilicity
873-879


1445.
114-1
Hydrophilicity
883-885


1446.
114-1
Hydrophilicity
887-889


1447.
114-1
Hydrophilicity
896-899


1448.
114-1
Hydrophilicity
908-916


1449.
114-1
Hydrophilicity
919-932


1450.
114-1
Hydrophilicity
941-947


1451.
114-1
Hydrophilicity
962-975


1452.
114-1
Hydrophilicity
979-989


1453.
114-1
Hydrophilicity
 993-1000


1454.
114-1
Hydrophilicity
1007-1022


1455.
114-1
Hydrophilicity
1041-1043


1456.
114-1
Hydrophilicity
1045-1053


1457.
114-1
Hydrophilicity
1062-1068


1458.
114-1
Hydrophilicity
1075-1078


1459.
114-1
Hydrophilicity
1080-1087


1460.
114-1
Hydrophilicity
1089-1104


1461.
114-1
Hydrophilicity
1115-1121


1462.
114-1
Hydrophilicity
1126-1141


1463.
114-1
Hydrophilicity
1143-1145


1464.
114-1
Hydrophilicity
1148-1151


1465.
114-1
Hydrophilicity
1157-1178


1466.
114-1
Hydrophilicity
1197-1203


1467.
114-1
Hydrophilicity
1217-1243


1468.
114-1
Hydrophilicity
1246-1263


1469.
114-1
Hydrophilicity
1271-1273


1470.
114-1
Hydrophilicity
1275-1277


1471.
114-1
Hydrophilicity
1284-1288


1472.
114-1
Hydrophilicity
1299-1307


1473.
114-1
Hydrophilicity
1318-1326


1474.
114-1
Hydrophilicity
1334-1340


1475.
114-1
Hydrophilicity
1350-1355


1476.
114-1
Hydrophilicity
1357-1359


1477.
114-1
Hydrophilicity
1367-1384


1478.
114-1
Hydrophilicity
1407-1417


1479.
114-1
Hydrophilicity
1491-1510


1480.
114-1
Hydrophilicity
1534-1540


1481.
114-1
Hydrophilicity
1576-1583


1482.
114-1
Hydrophilicity
1595-1607


1483.
114-1
Hydrophilicity
1629-1635


1484.
114-1
Hydrophilicity
1643-1645


1485.
114-1
Hydrophilicity
1649-1665


1486.
114-1
Hydrophilicity
1682-1686


1487.
114-1
Hydrophilicity
1704-1722


1488.
114-1
Hydrophilicity
1724-1726


1489.
114-1
Hydrophilicity
1740-1746


1490.
114-1
Hydrophilicity
1804-1806


1491.
114-1
Hydrophilicity
1829-1835


1492.
114-1
Hydrophilicity
1842-1855


1493.
114-1
Hydrophilicity
1876-1879


1494.
114-1
Hydrophilicity
1898-1900


1495.
114-1
Hydrophilicity
1910-1912


1496.
114-1
Hydrophilicity
1920-1922


1497.
114-1
Hydrophilicity
1928-1930


1498.
114-1
Hydrophilicity
1938-1940


1499.
114-1
Hydrophilicity
1948-1954


1500.
114-1
Hydrophilicity
1962-1967


1501.
114a
AMPHI
45-54


1502.
114a
AMPHI
154-160


1503.
114a
AMPHI
182-190


1504.
114a
AMPHI
224-226


1505.
114a
AMPHI
229-233


1506.
114a
AMPHI
285-287


1507.
114a
AMPHI
303-310


1508.
114a
AMPHI
321-332


1509.
114a
AMPHI
348-350


1510.
114a
AMPHI
392-398


1511.
114a
AMPHI
414-416


1512.
114a
AMPHI
478-486


1513.
114a
AMPHI
506-509


1514.
114a
AMPHI
525-529


1515.
114a
AMPHI
565-567


1516.
114a
AMPHI
614-621


1517.
114a
AMPHI
631-635


1518.
114a
AMPHI
770-774


1519.
114a
AMPHI
811-813


1520.
114a
AMPHI
847-849


1521.
114a
AMPHI
851-853


1522.
114a
AMPHI
875-879


1523.
114a
AMPHI
951-959


1524.
114a
AMPHI
975-981


1525.
114a
AMPHI
1034-1036


1526.
114a
AMPHI
1048-1051


1527.
114a
AMPHI
1073-1081


1528.
114a
AMPHI
1086-1090


1529.
114a
AMPHI
1095-1102


1530.
114a
AMPHI
1111-1115


1531.
114a
AMPHI
1163-1166


1532.
114a
AMPHI
1275-1281


1533.
114a
AMPHI
1312-1317


1534.
114a
AMPHI
1338-1347


1535.
114a
AMPHI
1349-1355


1536.
114a
AMPHI
1357-1365


1537.
114a
AMPHI
1376-1398


1538.
114a
AMPHI
1418-1420


1539.
114a
AMPHI
1455-1460


1540.
114a
AMPHI
1472-1484


1541.
114a
AMPHI
1497-1505


1542.
114a
AMPHI
1507-1512


1543.
114a
Antigenic Index
1-6


1544.
114a
Antigenic Index
10-16


1545.
114a
Antigenic Index
23-37


1546.
114a
Antigenic Index
41-55


1547.
114a
Antigenic Index
75-85


1548.
114a
Antigenic Index
91-97


1549.
114a
Antigenic Index
102-137


1550.
114a
Antigenic Index
147-156


1551.
114a
Antigenic Index
161-168


1552.
114a
Antigenic Index
172-174


1553.
114a
Antigenic Index
181-189


1554.
114a
Antigenic Index
196-203


1555.
114a
Antigenic Index
208-213


1556.
114a
Antigenic Index
220-229


1557.
114a
Antigenic Index
242-248


1558.
114a
Antigenic Index
251-266


1559.
114a
Antigenic Index
268-276


1560.
114a
Antigenic Index
295-307


1561.
114a
Antigenic Index
309-312


1562.
114a
Antigenic Index
318-340


1563.
114a
Antigenic Index
345-352


1564.
114a
Antigenic Index
357-366


1565.
114a
Antigenic Index
371-381


1566.
114a
Antigenic Index
385-392


1567.
114a
Antigenic Index
404-427


1568.
114a
Antigenic Index
429-434


1569.
114a
Antigenic Index
440-456


1570.
114a
Antigenic Index
465-468


1571.
114a
Antigenic Index
473-494


1572.
114a
Antigenic Index
496-510


1573.
114a
Antigenic Index
515-530


1574.
114a
Antigenic Index
535-549


1575.
114a
Antigenic Index
555-560


1576.
114a
Antigenic Index
564-578


1577.
114a
Antigenic Index
5 88-596


1578.
114a
Antigenic Index
602-615


1579.
114a
Antigenic Index
617-620


1580.
114a
Antigenic Index
622-624


1581.
114a
Antigenic Index
628-632


1582.
114a
Antigenic Index
637-640


1583.
114a
Antigenic Index
647-654


1584.
114a
Antigenic Index
660-666


1585.
114a
Antigenic Index
668-688


1586.
114a
Antigenic Index
697-725


1587.
114a
Antigenic Index
730-733


1588.
114a
Antigenic Index
738-755


1589.
114a
Antigenic Index
760-766


1590.
114a
Antigenic Index
779-783


1591.
114a
Antigenic Index
786-799


1592.
114a
Antigenic Index
806-809


1593.
114a
Antigenic Index
811-819


1594.
114a
Antigenic Index
831-839


1595.
114a
Antigenic Index
845-857


1596.
114a
Antigenic Index
860-862


1597.
114a
Antigenic Index
864-868


1598.
114a
Antigenic Index
872-879


1599.
114a
Antigenic Index
883-891


1600.
114a
Antigenic Index
893-902


1601.
114a
Antigenic Index
908-916


1602.
114a
Antigenic Index
923-936


1603.
114a
Antigenic Index
941-947


1604.
114a
Antigenic Index
950-956


1605.
114a
Antigenic Index
959-976


1606.
114a
Antigenic Index
979-989


1607.
114a
Antigenic Index
 993-1000


1608.
114a
Antigenic Index
1007-1022


1609.
114a
Antigenic Index
1041-1053


1610.
114a
Antigenic Index
1062-1068


1611.
114a
Antigenic Index
1075-1108


1612.
114a
Antigenic Index
1115-1121


1613.
114a
Antigenic Index
1126-1145


1614.
114a
Antigenic Index
1148-1152


1615.
114a
Antigenic Index
1157-1176


1616.
114a
Antigenic Index
1195-1206


1617.
114a
Antigenic Index
1208-1212


1618.
114a
Antigenic Index
1224-1243


1619.
114a
Antigenic Index
1247-1263


1620.
114a
Antigenic Index
1271-1282


1621.
114a
Antigenic Index
1284-1288


1622.
114a
Antigenic Index
1292-1295


1623.
114a
Antigenic Index
1299-1307


1624.
114a
Antigenic Index
1318-1328


1625.
114a
Antigenic Index
1330-1340


1626.
114a
Antigenic Index
1344-1359


1627.
114a
Antigenic Index
1367-1384


1628.
114a
Antigenic Index
1396-1399


1629.
114a
Antigenic Index
1405-1417


1630.
114a
Antigenic Index
1434-1436


1631.
114a
Antigenic Index
1449-1451


1632.
114a
Antigenic Index
1468-1487


1633.
114a
Antigenic Index
1498-1503


1634.
114a
Antigenic Index
1509-1515


1635.
114a
Antigenic Index
1525-1532


1636.
114a
Hydrophilicity
4-6


1637.
114a
Hydrophilicity
12-15


1638.
114a
Hydrophilicity
23-34


1639.
114a
Hydrophilicity
43-55


1640.
114a
Hydrophilicity
75-85


1641.
114a
Hydrophilicity
104-110


1642.
114a
Hydrophilicity
118-123


1643.
114a
Hydrophilicity
127-132


1644.
114a
Hydrophilicity
147-154


1645.
114a
Hydrophilicity
163-167


1646.
114a
Hydrophilicity
185-187


1647.
114a
Hydrophilicity
197-203


1648.
114a
Hydrophilicity
208-211


1649.
114a
Hydrophilicity
221-227


1650.
114a
Hydrophilicity
243-245


1651.
114a
Hydrophilicity
253-261


1652.
114a
Hydrophilicity
263-266


1653.
114a
Hydrophilicity
270-272


1654.
114a
Hydrophilicity
295-301


1655.
114a
Hydrophilicity
309-312


1656.
114a
Hydrophilicity
320-328


1657.
114a
Hydrophilicity
332-337


1658.
114a
Hydrophilicity
345-351


1659.
114a
Hydrophilicity
360-366


1660.
114a
Hydrophilicity
371-378


1661.
114a
Hydrophilicity
387-392


1662.
114a
Hydrophilicity
404-417


1663.
114a
Hydrophilicity
421-423


1664.
114a
Hydrophilicity
425-427


1665.
114a
Hydrophilicity
442-456


1666.
114a
Hydrophilicity
473-488


1667.
114a
Hydrophilicity
499-509


1668.
114a
Hydrophilicity
515-520


1669.
114a
Hydrophilicity
536-549


1670.
114a
Hydrophilicity
555-560


1671.
114a
Hydrophilicity
565-568


1672.
114a
Hydrophilicity
570-578


1673.
114a
Hydrophilicity
589-594


1674.
114a
Hydrophilicity
602-604


1675.
114a
Hydrophilicity
609-615


1676.
114a
Hydrophilicity
617-620


1677.
114a
Hydrophilicity
660-665


1678.
114a
Hydrophilicity
668-680


1679.
114a
Hydrophilicity
684-686


1680.
114a
Hydrophilicity
699-708


1681.
114a
Hydrophilicity
715-725


1682.
114a
Hydrophilicity
730-733


1683.
114a
Hydrophilicity
738-744


1684.
114a
Hydrophilicity
746-754


1685.
114a
Hydrophilicity
760-766


1686.
114a
Hydrophilicity
789-793


1687.
114a
Hydrophilicity
816-818


1688.
114a
Hydrophilicity
831-836


1689.
114a
Hydrophilicity
845-857


1690.
114a
Hydrophilicity
860-862


1691.
114a
Hydrophilicity
864-866


1692.
114a
Hydrophilicity
873-879


1693.
114a
Hydrophilicity
883-885


1694.
114a
Hydrophilicity
887-889


1695.
114a
Hydrophilicity
896-899


1696.
114a
Hydrophilicity
908-916


1697.
114a
Hydrophilicity
923-932


1698.
114a
Hydrophilicity
941-947


1699.
114a
Hydrophilicity
961-975


1700.
114a
Hydrophilicity
979-989


1701.
114a
Hydrophilicity
 993-1000


1702.
114a
Hydrophilicity
1007-1022


1703.
114a
Hydrophilicity
1041-1043


1704.
114a
Hydrophilicity
1045-1053


1705.
114a
Hydrophilicity
1062-1068


1706.
114a
Hydrophilicity
1075-1078


1707.
114a
Hydrophilicity
1080-1087


1708.
114a
Hydrophilicity
1089-1104


1709.
114a
Hydrophilicity
1115-1121


1710.
114a
Hydrophilicity
1126-1141


1711.
114a
Hydrophilicity
1143-1145


1712.
114a
Hydrophilicity
1148-1151


1713.
114a
Hydrophilicity
1158-1171


1714.
114a
Hydrophilicity
1197-1203


1715.
114a
Hydrophilicity
1224-1243


1716.
114a
Hydrophilicity
1251-1263


1717.
114a
Hydrophilicity
1271-1273


1718.
114a
Hydrophilicity
1275-1277


1719.
114a
Hydrophilicity
1284-1288


1720.
114a
Hydrophilicity
1299-1307


1721.
114a
Hydrophilicity
1318-1326


1722.
114a
Hydrophilicity
1334-1340


1723.
114a
Hydrophilicity
1350-1359


1724.
114a
Hydrophilicity
1367-1384


1725.
114a
Hydrophilicity
1407-1417


1726.
114a
Hydrophilicity
1449-1451


1727.
114a
Hydrophilicity
1469-1482


1728.
114a
Hydrophilicity
1484-1486


1729.
114a
Hydrophilicity
1498-1503


1730.
114a
Hydrophilicity
1510-1512


1731.
114a
Hydrophilicity
1527-1532


1732.
124-1
AMPHI
37-43


1733.
124-1
AMPHI
94-96


1734.
124-1
AMPHI
113-115


1735.
124-1
Antigenic Index
20-26


1736.
124-1
Antigenic Index
38-43


1737.
124-1
Antigenic Index
52-55


1738.
124-1
Antigenic Index
62-70


1739.
124-1
Antigenic Index
88-97


1740.
124-1
Antigenic Index
104-114


1741.
124-1
Antigenic Index
123-135


1742.
124-1
Antigenic Index
146-155


1743.
124-1
Hydrophilicity
20-26


1744.
124-1
Hydrophilicity
41-43


1745.
124-1
Hydrophilicity
52-55


1746.
124-1
Hydrophilicity
63-69


1747.
124-1
Hydrophilicity
91-94


1748.
124-1
Hydrophilicity
104-114


1749.
124-1
Hydrophilicity
123-135


1750.
124-1
Hydrophilicity
146-155


1751.
124a
AMPHI
19-21


1752.
124a
AMPHI
23-29


1753.
124a
AMPHI
37-43


1754.
124a
AMPHI
94-96


1755.
124a
Antigenic Index
38-43


1756.
124a
Antigenic Index
52-55


1757.
124a
Antigenic Index
62-70


1758.
124a
Antigenic Index
77-80


1759.
124a
Antigenic Index
90-96


1760.
124a
Antigenic Index
105-115


1761.
124a
Antigenic Index
120-135


1762.
124a
Antigenic Index
145-153


1763.
124a
Hydrophilicity
41-43


1764.
124a
Hydrophilicity
52-55


1765.
124a
Hydrophilicity
63-69


1766.
124a
Hydrophilicity
91-95


1767.
124a
Hydrophilicity
108-115


1768.
124a
Hydrophilicity
120-135


1769.
124a
Hydrophilicity
146-153





Key:


fragment#1 of the present application is amino acids 6-14 of ORF38-1 disclosed in WO99/36544, fragment#2 of the present application is amino acids 57-59 of ORF38-1 disclosed in WO99/36544 etc.






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







TABLE II







The present invention does not include within its scope proteins


comprising any of the 45 protein sequences disclosed in WO99/36544. As


stated above, if the length of any particular protein sequence disclosed in


WO99/36544 is x amino acids, the antigenic fragment of the present


invention has at most x − 1 amino acids of that protein. For each of the 45


protein sequences given in WO99/36544, the value of x is given, for


reference, in the following table:










SEQ ID NO:
x














2
245



4
591



6
592



8
164



10
321



12
321



14
124



16
124



18
173



20
640



22
761



24
111



26
571



28
710



30
710



32
62



34
86



36
92



38
103



40
85



42
78



44
78



46
219



48
212



50
185



52
166



54
326



56
356



58
284



60
1978



62
1532



64
593



66
129



68
319



70
619



72
595



74
150



76
255



78
255



80
172



82
242



84
242



86
183



88
155



90
153

## Claims

1. A purified polypeptide having a length of 100 amino acids or less comprising the amino acid sequence of SEQ ID NO:1331.

2. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable vehicle.

3. The polypeptide of claim 1, wherein the polypeptide has a length of 50 amino acids or less.

4. The polypeptide of claim 1, wherein the polypeptide has a length of 25 amino acids or less.

5. The polypeptide of claim 1, wherein the polypeptide has a length of 20 amino acids or less.

6. A composition comprising the polypeptide of claim 3 and a pharmaceutically acceptable vehicle.

7. A composition comprising the polypeptide of claim 4 and a pharmaceutically acceptable vehicle.

8. A composition comprising the polypeptide of claim 5 and a pharmaceutically acceptable vehicle.