CA2317815A1 - Neisseria meningitidis antigens - Google Patents

Abstract

The invention provides proteins from Neisseria meningitidis (strains A and B), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data. The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.

Description

NEISSERIA MENINGITIDIS ANTIGENS
This invention relates to antigens from the bacterium Neisseria meningitides.
BACKGROUND
Neisseria meningitides is a non-motile, gram negative diplococcus human pathogen. It colonises the pharynx, causing meningitis and, occasionally, septicaemia in the absence of meningitis. It is closely related to N.gonorrhoeae, although one feature that clearly differentiates meningococcus from gonococcus is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.
N.meningitidis causes both endemic and epidemic disease. In the United States the attack rate is 0.6-1 per 100,000 persons per year, and it can be much greater during outbreaks (see Lieberman et al: ( 1 ~ 96) Safety and Immunogenicity of a Serogroups A/C Neisseria meningitides Oligosaccharide-Protein Conjugate Vaccine in Young Children. JAMA 275(19):1499-1503;
Schuchat et al (1997) Bacterial Meningitis in the United States in 1995. NEngl JMed 337(14):970-976). In developing countries, endemic disease rates are much higher and during epidemics incidence rates can reach 500 cases per 100,000 persons per year. Mortality is extremely high, at 10-20% in the United States, and much higher in developing countries.
Following the introduction of the conjugate vaccine against Haemophilus influenzae, N. meningitides is the major cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).
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 W 135 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.
Although efficacious in adolescents and adults, it induces a poor immune response and short duration of protection, and cannot be used in infants [eg. Morbidity and Mortality weekly report, Vo1.46, No.
RR-5 (1997)]. This is because polysaccharides are T-cell independent antigens that induce a weak immune response that cannot be boosted by repeated immunization. Following the success of the vaccination against H. influe»zae, conjugate vaccines against serogroups A and C have been developed and are at the final stage of clinical testing (Zollinger WD '~Tew and Improved Vaccines Against Meningococcal Disease" in: New Generation Yaccines, supra, pp. 469-488; Lieberman et al ( 1996) supra; Costantino et al ( 1992) Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C. Yaccine 10:691-698).
Meningococcus B remains a emblem, however. This serotype currently is responsible for approximately 50% of total meningitis in the United States, Europe, and South America. The polysaccharide approach cannot be used because the menB capsular polysaccharide is a polymer of a(2-8)-linked N acetyl neuraminic acid that is also present in mammalian tissue. This results in tolerance to the antigen; indeed, if an immune response were elicited, it would be anti-self, and therefore undesirable. In order to avoid induction of autoimmunity and to induce a protective immune response, the capsular polysaccharide has, for instance, been chemically modified substituting the N acetyl groups with N propionyl groups, leaving the specific antigenicity unaltered (Romero & Outschoom ( 1994) Current status of Meningococcal group B
vaccine candidates: capsular or non-capsular? Clin Microbiol Rev 7(4):559-575).
Alternative approaches to menB vaccines have used complex mixtures of outer membrane proteins (OMPs), containing either the OMPs alone, or OMPs enriched in porins, or deleted of the class 4 OMPs that are believed to induce antibodies that block bactericidal activity.
This approach produces vaccines that are not well characterized. They are able to protect against the homologous strain, but are not effective at large where there are many antigenic variants of the outer membrane proteins. To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed (eg. Poohnan JT (1992) Development of a meningococcal vaccine.
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 (eg. Ala'Aldeen & Borriello (1996) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Yaccine 14(1):49-53).
A certain amount of sequence data is available for meningococcal and gonococcal genes and proteins (eg. EP-A-0467714, W096/29412), but this is by no means complete. The provision of further sequences could provide an opportunity to identify secreted or surface-exposed proteins that WO 99/36544 PCT/IB99~0103 are presumed targets for the immune system and which are not antigenically variable. For instance, some of the identified proteins could be components of efficacious vaccines against meningococcus B, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against all pathogenic Neisseriae.
THE INVENTION
The invention provides proteins comprising the N.meningitidis amino acid sequences disclosed in the examples.
It also provides proteins comprising sequences homologous (ie. having sequence identity) to the N.meningitidis amino acid sequences disclosed in the examples. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (eg.
60%, 70~/0, 80%, 90%, 95%, 99% or more). These homologous proteins include mutants and allelic variants of the sequences disclosed in the examples. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between the proteins is preferably determined by the Smith-Waternian homology search algorithm as implemented in the MPSRCH
program (Oxford Molecular), using an affuie gap search with parameters gap open penalty=12 and gap extension penalty=1.
The invention further provides proteins comprising fragments of the N.
meningitides amino acid sequences disclosed in the examples. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope from the sequence.
The proteins of the invention can, of course, be prepared by various means {eg. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (eg. native, fusions etc.). They are preferably prepared in substantially pure form (ie.
substantially free from other N. meningitides or host cell proteins) According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means.

-ø_ According to a further aspect, the invention provides nucleic acid comprising the N. meningitides nucleotide sequences disclosed in the examples. In addition, the invention provides nucleic acid comprising sequences homologous (ie. having sequence identity) to the N.
meningitides nucleotide sequences disclosed in the examples.
Furthermore, the invention provides nucleic acid which can hybridise to the N.
meningitides nucleic acid disclosed in the examples, preferably under "high stringency" conditions (eg. 65°C in a O.IxSSC, 0.5% SDS solution).
Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the N. meningitides sequences and, depending on the particular sequence, n is 10 or more (eg 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.
It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).
Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc. ) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).
In addition, the tenor "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 (eg. 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 (eg. as vaccines) 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. meningitides, especially strain A, strain B or strain C.
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.
Unlike the sequences disclosed in PCT/IB98/01665, the sequences disclosed in the present application are believed not to have any significant homologs in N.gonorrhoeae. Accordingly, the sequences of the present invention also find use in the preparation of reagents for distinguishing between N.meningitidis and N.gonorrhoeae A summary of standard techniques and procedures which may be employed in order to perform the invention (eg. 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 eg. 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 8c 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 Pur~cation:
Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes 1 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.
In particular, the contents of UK patent applications 9800760.2, 9819015.0 and 9822143.5 are incorporated herein.
De nitio»s 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" eg. a composition "comprising"
X may consist exclusively of X or may include something additional to X, such as X+y.
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 S 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 Neisserial sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.
An "origin of replication" is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.
A "mutant" sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence, identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an "allelic variant" of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5' or 3' untranslated regions of the gene, such as in regulatory control regions (eg. see US patent 5,753,235).

-g Fxwression systems The Neisserial 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 (eg. 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 I00 to 200 by 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
I 5 Laboratory Manual, 2nd ed J.
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 marine metallotheionein gene, also provide useful promoter sequences.
Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
The presence of an enhancer element (enhancer); combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the nornlal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orien-tation, 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.

_g 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. AcaaL 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 colon. 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 firm 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 mamnnalzan cells.
Usually, tl°anscription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop colon 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 terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloning: A Laboratory 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 (eg. 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 traps-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981} Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T
antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO
[Shimizu et al.
(1986) Mol. Cell. Biol. 6:1074].
The transformation procedure used depends upon the host to be transformed.
Methods for introduction of heterologous polynuchtides 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 (eg.
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 wo ~r~sa4 pcT~s~rooio3 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 CA ("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. 1 SSS ( 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 (eg.
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 polymerise and initiating the downstream {5' to 3') transcription of a coding sequence (eg. 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
polymerise 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.
Structiual 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, Vlak et al., (1988), J. Gen.
Virol. 69:765.
DNA encoding suitable signal sequences can be derived from genes for secreted insect or IS 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 a-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'1 Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebmsidase, 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 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-transfornned with the heterologous DNA of the transfer vectorand 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-Skb 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 1 S pln 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 plagued 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 alias Aedes aegypti , Autographa cal:fornica, 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 baculovinas/expression system; cell culture technology is generally known to those skilled in the art. See, eg. 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, eg. HPLC, afl'mity 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, eg. proteins, lipids and polysaccharides.
In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence.
These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.
iii. Plant Systems There are many plant cell culture and whole plant genetic expression systems known in the art.
Exemplary plant cellular genetic expression systems include those described in patents, such as:

US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, 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 RL. 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 bacteriaUplant 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. Reptr, 11 (2):165-185.
Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the proteins) 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 colon 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 proteins) 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 electtoporation.
(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, Solamrm, Petunia, 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 genrninate 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 _lg_ 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 polymerise and initiating the downstream (3') transcription of a coding sequence (eg. 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 polymerise 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 polymerise 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 polymerise 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; US
patent 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and TS [US
patent 4,689,406]
promoter systems also provide useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters.
For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [US patent 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) 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 polymerise and initiate tt~anscription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerise 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. Acid Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851 ).
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 165 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 (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) BiolTechnology 7:698].
Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [US patent 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene:
DNA encoding suitable signal sequences can be derived from genes for secreted.bacterial proteins, such as 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 WO 99/36544 PCT/IB99/00i03 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 tn~nslated into the polypeptide encoded by the DNA. Transcription terniination sequences frequently include DNA sequences of about SO 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 (eg. 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 numuer 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.

wo ~r~ssaa -22- Pc~rnB~rooio3 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. (I988) Appl. Ermiron. Microbiol.
54:655]; Streptococcus lividans [Powell et al. (1988) App1 Environ. Microbiol. 54:655], Streptomyces lividans [CJS patent 4,745,056].
Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl2 or other agents, such as divalent canons and DMSO. DNA can also be introduced into bacterial cells by electroporanon.
Transformation procedures usually vary with the bacterial species to be transformed. See eg.
[Masson et al. (1989) FEll~f 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 dl. (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 ColEl-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. Fen-etti and R. Curtiss III); Perry et al.
(198I) Infect. Immun.
32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al.
(1987) Proc. 4th Evr. Cong. Biotechnology 1:412, StreptococcusJ.
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 (eg. 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 polymera.se 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-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphoiructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203).
The yeast PHOS gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. ( 1983) Proc. Natl. Acacl Sci. USA 80:1 J.
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 (LTS Patent Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR
PHOS genes, WO 99136544 -24- PGT/iB99/00103 combined with the transcriptions! activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occuwing 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; Henikoffet 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 pmtein 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 eg. EP-A-0 196 056. Another example is a ubiquitin fusion pmtein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg.
ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. W088/024066).
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion pmtein comprised of a leader sequence fi~agment 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 (LTS
patent 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).
A preferred class, of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro" region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usu lly about 25 to about 50 amino acid residues) (L1S Patents 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast aIphafactor. (eg. see WO
89/02463.) Usually, transcription termination sequences recognized by yeast are regulatory regions located 3' to the translation stop colon, 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 (eg. glasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing itto 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 YRp 17 [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 S to about 200, and WO 99/36544 -2~ PCT/IB99/00103 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 erect of the vector and the foreign protein on the host. See eg. 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 v~tor 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 [Rive 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.
Sel~table markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRPl, and ALG7, and the 6418 resistance gene, which confer resistance in yeast cells to tunicamycin and 6418, 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 CUPl 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 maltose [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 [Des, et al. (1984) J. Bacteriol.
158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al.
(1990) BiolTechnology 8:135], Pichia guillerimondii [Kunze et al. (1985) J.
Basic Microbiol.
25:141], Pichia pastoris (Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; US
Patent Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl.
Aced. 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 eg. [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]; [Des et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J.
Bacteriol. 154:1165; Van den Berg et al. (1990) BiolTecknology 8:135;
Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol.
25:141; US Patent Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Aced.
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].
A t' dies 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 Neisseriai 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 pg/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 (eg. 1,OOOg 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 removal 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 (eg.
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 (eg. 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 ~ZSI), 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 {T'MB) to a blue pigment, quantifiable with a spectrophotometer. "Sp~ific 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 Iabel may serve in several different modes. For example, ~Z~I may serve as a radioactive label or as an electron-dense reag~t. I-iRP 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 ~~I, 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 instant 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 SO mg/kg or 0.05 mg/kg to about 10 mglkg 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.

WO 99/36544 -31- Pf.:'T/IB99/00103 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 (eg. see W098/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Vaccines Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
Such vaccines comprise immunising antigen(s), immunogen(s), polypeptiide(s), proteins) 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 (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.
Powell & Nevvlnan, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (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, MA), (b) SAF, containing 10~/° Squalane, 0.4%
Tween 80, 5% pluronic-blocked polymer L 121, and thr-MDP (see below) either microfluidizsd into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi"~' adjuvant system (R.AS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),, preferably MPL + CWS
(Detoxl'"'); (3) saponin adjuvants, suchas Stimulon"''' (Cambridge Bioscience, Worcester, MA) 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 (eg.
IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor ('INF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition: Alum and MF59TM 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-t,-alanyl-v-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (M'TP-PE), etc.
The immunogenic compositions (eg. the immunising antigen/immunogen/polypepdde/protein/
1 S 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 (eg. 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, eg.
by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg.
W098/20734).
Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdenmal applications. Dosage treatrnent 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 [eg. Robinson &
Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].
Gene Deliverv 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-I93; and Kaplitt (1994) Nature Genetics 6:148-I53.
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 Xl, NZB X2 and NZB9-1 (see ONeill (1985) J. Yirol.
53:160}~polytropic retroviruses eg. MCF and MCF-MLV (see Kelly (1983) J. ~rol. 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 S from a Rous Sarcoma Virus; a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see US patent 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA
by incorporation of a chimeric integrase enzyme into the retroviral particle (see W096/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.
Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see W095/30763 and W092/05266), and can be used to create producer cell lines (also termed vector cell Iines or "VCLs'~ for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (eg. 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 Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscber (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") in Rockville, Maryland or isolated from known sources using commonly available techniques.
Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, W089/02468;
W089/05349, W089/09271, W090/02806, W090/07936, W094/03622, W093/25698, W093/25234, W093/11230, W093/10218, W091/02805, W091/02825, W095/07994, US
5,219,740, US 4,405,712, US 4,861,719, US 4,980,289, US 4,777,127, US
5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J
Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349;
and Miller (1990) Human Gene Therapy 1.
Human adenoviral gene then3py vectors are also known in the art and employable in this invention.
See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and .
W093/07283, W093/06223, and W093/07282. Exemplary known adenoviral gene then;py vectors employable in this invention include those described in the above referenced documents and in W094/12649, W093/03769, W093/19191, W094/28938, W095/1I984; W095/00655, W095/27071, W095/29993, W095/34671, W096/05320, W094/08026, W094/11506, W093/06223, W094/24299, W095/14102, W095/24297, W095/02697, W094/28152, W094/24299, W095/09241, W095/25807, W095/05835, W094/18922 and W095/09654.
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, W093/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262.
Another example of such an AAV vector is psub201 (see Samulski (1987) J. Yirol. 61:3096). Another exemplary AAV
vector is the Double-D ITR v~tor. Construction of the Double-D ITR vector is disclosed in US
Patent 5,478,745. Still other vectors are those disclosed in Carter US Patent 4,797,368 and Muzyczka US Patent 5,139,941, Chartejee US Patent 5,474,935, and Kotin W094/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver.
Its structure and construction are disclosed in Su (I996) Human Gene Therapy 7:463-470.
Additional AAV gene therapy vectors are described in US 5,354,678, US
5,173,414, US 5,139,941, and US 5,252,479.
The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in US 5,288,641 and EP0176170 (Roizman).
Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in W090/09441 and W092/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH I OS and GAL4 described is EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.
Also contemplated are alpha virus gene therapy vectors that can be employed in this invention.
Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC
VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC
VR-373;
ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250;
ATCC
VR-1249; ATCC VR-532), and those described in US patents 5,091,309, 5,217;879, and W092/10578. More particularly, those alpha virus vectors described in US
Serial No. 08/405,627, filed March 15, 1995,W094/21792, W092l10578, W095/07994, US 5,091,309 and US
5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see USSN
08/679640).
DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See W095/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol.
Standardization 1:115; rhinovilus, for example ATCC VR-I 110 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, FIexner ( 1990) Vaccine 8:17; in US
4,603,112 and US
4,769,330 and W089/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533;
influenza virus, for example ATCC VR 797 and recombinant influenza viruses made employing reverse genetics techniques as described in US 5,166,057 and in Enami (1990) Proc Notl Acad Sci 87:3802-3805;
Enami & Palese (1991) J Yirol 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 e~:ample 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; ONyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC
VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre ( 1966) Proc Soc Exp Biol Med 121:190.
Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see US Serial No. 08/366,787, filed December 30,1994 and Curiel (1992}
Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see US Serial No.08/240,030, filed May 9, _38_ 1994, and US Serial No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in US Patent 5,149,655, ionizing radiation as described in US5,206,152 and in W092/11033, 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 US Serial No.
60/023,867.
Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding rations 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 US 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efl'lciently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
Liposomes that can act as gene delivery vehicles are described in US
5,422,120; W095/13796, W094/23697, W091/14445 and EP-524,968. As described in USSN. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding rations 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 wo ~r~ssaa rcr~a~rooioa hand-held gene transfer particle gun, as described in US 5,149,655; use of ionizing radiation for activating transferred gene, as described.in US 5,206,152 and W092/11033 Exemplary liposome and polycationic gene delivery vehicles are those described in US 5,422,120 and 4,762,915; in WO 95/13796; W094/23697; and W091/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; S2oka (1980) Biachem Biophys Acta 600:1; Bayer (1979) Biachem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang ( 1987) Proc Natl Acad Sci 84:7851; Plant ( 1989) Anal Biochem I
76:420.
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 (eg. see W098/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg. W093/14778. 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.
Polvnucleotide and polvp~ptide 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 fi~agments; 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.Polval~Cylenes, Polysacchari~,e~. e(c.
Also, polyalkylene glycol can be included with the desired poiynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a prefewed 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.
~5 Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. l0l :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, NY. (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, eg. Szoka (1978) Proc. Natl. Acad Sci. USA 75:4194-4198; W090/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others.
These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SIJVs), or large unilamellar vesicles (LIJVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See eg. Straubinger (1983) Meth. Immunol.
101:512-527; Szoka (1978) Proc. Natl. Acac~ Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell I7:77); 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.Lipoyroteins In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered.
Examples of lipoproteins to be utilized include: chylomicrons, HDL, ff)L, 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, and E, over time these lipoproteins lose A
and acquire C and E apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.
The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) 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 Utemu~m (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) JClin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30:
443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Massachusetts, USA. Further description of lipoproteins can be found in Zuckermann et al. PCT/US97/14465.
F.Polvcationic Agents Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.
Polycatianic 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, polyonvthine, and protamine. Other examples include histories, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA
viruses, such as (X 174, 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-l, 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 ar to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example, DEAE-dextrau, polybrene.
LipofectinTM, and IipofectAMINE''M are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.
Immunodia~2nostic Assavs Neisserial antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisserial 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 Neisserial 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 invalve 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 inununoassays, 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 Hvbridisation "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 fragments) to be studied can vary a magnitude of 10, from 0.1 to 1 ~g 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 pg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/~g. For a single-copy mammalian gene a conservative approach would start with 10 ~g of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10$ cpm/~g, 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 pmbe 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(log,°Ci) + 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 8c 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 (ie.
stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar mannei. 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 temperatwe 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 Neisserial nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native Neisserial 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.

wo ~r~ssaa pcrn899rooio3 ~7 The probe sequence need not be identical to the Neisserial 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 Neisserial 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 Neisserial sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has su~cient complementarity with the a Neisserial sequence in order to hybridize therewith arid 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 sui~ciently 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. Natl.
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 eg.
backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half life, alter RNA arty, increase nuclease resistance etc. [eg. 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 [eg. see Corey (I997) 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. Enrymol.
(1987) 155: 335-350]; US patents 4,683,195 and 4,683,202. Two "primer"
nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired Neisserial 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 polymerise, 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 Neisserial 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 polymerise 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 pmbe and then washed to remove ariy unhybridized probe. Next, the duplexes containing the Labeled probe are detected.
Typically, the probe is labelled with a radioactive moiety.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1-7 show biochemical data and sequence analysis pertaining to Examples 1, 2, 3, 7, 13, 16 and 19, respectively, with ORFs 40, 38, 44, 52, 114, 41 and 124.. M1 and M2 are molecular weight markers. Arrows indicate the position of the main recombinant product or, in Western blots, the position of the main N. meningitides immunoreactive band. TP indicates N.
meningitides total protein extract; OMV indicates N.meningitidis outer membrane vesicle preparation. In bactericidal assay results: a diamond (1) shows preimmune data; a triangle ( ~ ) shows GST
control data; a circle (~) shows data with recombinant N.meningitidis protein. Computer analyses show a hydrophilicity plot (upper), an antigenic index plot (middle), and an AMPHI
analysis (lower). The AMPHI program has been used to predict T-cell epitopes [Gao et al. (1989) J.
Immunol. 143:3007;
Roberts et a1 ( 1996) AIDS Res Hum Retrovir 12:593; Quakyi et al. ( 1992) Scand J Immunol suppl.l 1:9) and is available in the Protean package of DNASTAR, Inc. (1228 South Park Street, Madison, Wisconsin 53715 USA).

WO 99/36544 ~9- PCT/IB99/00103 EXAMPLES
The examples describe nucleic acid sequences which have been identified in N.meningitidis, along with their putative translation products. Not all of the nucleic acid sequences are complete ie. they encode less than the full-length wild-type protein. It is believed at present that none of the DNA
sequences described herein have significant homologs in N.gonorrhoeae.
The examples are generally in the following format:
~ a nucleotide sequence which has been identified in N. meningitides (strain B) ~ the putative translation product of this sequence ~ a computer analysis of the translation product based on database comparisons ~ a corresponding gene and protein sequence identified in N. meningitides (strain A) ~ a description of the characteristics of the proteins which indicates that they might be suitably antigenic ~ results of biochemical analysis (expression, purification, ELISA, FACS etc.
) The examples typically include details of sequence homology >xtween species and strains. Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin. Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein fimction to a new sequence and has proved particularly useful in whole-genome analyses.
Sequence comparisons were performed at NCBI (http://www.ncbi.nlm.nih.gov) using the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx, & tBLASTx [eg. see also Altschul et al. (199?) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:2289-3402]. Searches were performed against the following databases: non-redundant GenBank+EMBL+DDBJ+pDB sequences and non-redundant GenBank CDS translations+pDB+SwissProt+Spupdate+PIR sequences.
Dots within nucleotide sequences (eg. position 288 in Example 12) represent nucleotides which have been arbitrarily introduced in order to maintain a reading frame. In the same way, double-underlined nucleotides were removed. Lower case letters (eg. position 589 in Example 12) represent ambiguities which arose during alignment of independent sequencing reactions (some of the nucleotide sequences in the examples are derived from combining the results of two or more experiments).
Nucleotide sequences were scanned in all six reading frames to predict the presence of hydrophobic domains using an algorithm based on the statistical studies of Esposti et al.
[Critical evaluation of the hydropathy of membrane proteins (1990) Eur J Biochem 190:207-219]. These domains represent potential transmembrane regions or hydrophobic leader sequences.
Open reading frames were predicted from fragmented nucleotide sequences using the program ORFFINDER (NCBI).
Underlined amino acid sequences indicate possible transmembrane domains or leader sequences in the ORFs, as predicted by the PSORT algorithm (http://www.psort.nibb.ac;jp). Functional domains were also predicted using the MOTIFS program (GCG Wisconsin &
PROSITE).
Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot. A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie. the protein is an immunogen. This method can also be used to identify immunodominant proteins.
The recombinant protein can also be conveniently used to prepare antibodies eg. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (eg. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.
In particular, the following methods (A) to (S) were used to express, purify and biochemically characterise the proteins of the invention:
A) Chromosomal DNA preparation N. meningitidis strain 2996 was grown to exponential phase in 100m1 of GC
medium, harvested by centrifugation, and resuspended in Sml buffer (20% Sucrose, SOmM Tris-HCI, SOmM EDTA, pH8).
After 10 minutes incubation on ice, the bacteria were lysed by adding l Oml lysis solution (SOmM
NaCI, 1% Na-Sarkosyl, SOp,g/ml Proteinase K), and the suspension was incubated at 37°C for 2 wo ~r~ssaa -51- pcrns~rooio3 hours. Two phenol extractions (equilibrated to pH 8) and one ChCl3/isoamylalcohol (24:1 ) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes ethanol, and was collected by centrifugation. The pellet was washed once with 70%
ethanol and redissolved in 4ml buffer (IOmM Tris-HCI, ImM EDTA, pH 8). The DNA
concentration was measured by reading the OD at 260 nm.
B) Oligonucleotide design Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF, using (a) the meningococcus B sequence when available, or (b) the gonococcus/meningococcus A
sequence, adapted to the codon preference usage of meningococcus as necessary.
Any predicted signal peptides were omitted, by deducing the 5'-end amplification primer sequence immediately downstream from the predicted leader sequence.
The 5' primers included two restriction enzyme recognition sites (BamHi-NdeI, BamHI Nhel, or EcoRI-NheI, depending on the gene's own restriction pattern); the 3' primers included a XhoI
restriction site. This procedure was established in order to direct the cloning of each amplification product (corresponding to each ORF) into two different expression systems:
pGEX-KG (using either BamHI Xhol or EcoRI XhoI), and pET21 b+ (using either NdeI XhoI or Nhel XhoI).
5'-end primer tail: CGCGGATCCCATATG (BamHI-NdeI ) CGCGGATCCGCTAGC (BamHI-NheI) CCGGAATTCTAGCTAGC (EcoRI-NheI) 3'-end primer tail: CCCGCTCGAG (XhoI) As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridised to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the whole primer, and was deternlined for each primer using the formulae:
T,~ = 4 (G+C~ 2 (A+'17 (tail excluded) T,~ 64.9 + 0.41 (% GC) - 600/N (whole primer) The average melting temperature of the selected oligos were 65-?0°C for the whole oligo and 50-55°C for the hybridising region alone.

WO 99!36544 PCTIIB99I00103 Table I shows the forward and reverse primers used for each amplification.
Oligos were synthesized by a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2m1 NIi40H, and deprotected by 5 hours incubation at 56°C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were then centrifuged and the pellets resuspended in either 100p1 or lml of water. ODD was determined using a Perlcin Eliner Lambda Bio spectrophotometer and the concentration was determined and adjusted to 2-l Opmol/~,1.
C) Amplification The standard PCR protocol was as follows: 50-200ng of genomic DNA were used as a template in the presence of 20-40~,M of each oligo, 400-800pM dNTPs solution, I x PCR
buffer (including I.SmM MgCI~, 2.5 units Taql DNA polymerise (using Perkin-Elmer AmpliTaQ, GIBCO
Platinum, Pwo DNA polymerise, or Tahara Shuzo Taq polymerise).
In some cases, PCR was optimised by the addition of 10.1 DMSO or 50~12M
betaine.
After a hot start (adding the polymerise during a preliminary 3 minute incubation of the whole mix at 9S°C), each sample underwent a double-step amplification: the first 5 cycles were performed using as the hybridization temperature the one of the oligos excluding the restriction enzymes tail, followed by 30 cycles performed according to the hybridization temperature of the whole length oligos. The cycles were followed by a final 10 minute extension step at 72°C.
The standard cycles were as follows:
DenaturationHybridisationElongation 30 seconds 30 seconds 30-60 seconds First 5 cycles 30 seconds 30 seconds 30-60 seconds Last 30 cycles The elongation time varied according to the length of the ORF to be amplified.

The amplifications were performed using either a 9600 or a 2400 Perkin Elmer GeneAmp PCR
System. To check the results, 1/10 of the amplification volume was loaded onto a 1-1.5% agarose gel and the size of each amplified fiagment compared with a DNA molecular weight marker.
The amplified DNA was either loaded directly on a 1 % agarose gel or first precipitated with ethanol and resuspended in a suitable volume to be loaded on a 1% agarose gel. The DNA
fragment corresponding to the right size band was then eluted and purified from gel, using the Qiagen Gel Extraction Kit, following the instructions of the manufacturer. The final volume of the DNA
fragment was 30,1 or SOEI.I of either water or l OmM Tris, pH 8.5.
D) Digestion of PCR fragments The purified DNA corresponding to the amplified fragment was split into 2 aliquots and double-digested with:
- NdeIlXhoI or lVlreIlXhoI for cloning into pET-21 b+ and further expression of the protein as a C-terminus His-tag fusion - BamHllXhol or EcoRllXhol for cloning into pGEX-KG and further expression of the protein as N-terminus GST fusion.
- EcoRllPstl, EcoRllSall, SalllPstl for cloning into pGex-His and further expression of the protein as N-terminus His-tag fusion Each purified DNA fragment was incubated (37°C for 3 hours to overnight) with 20 units of each restriction enzyme (New England Biolabs ) in a either 30 or 401 final volume in the presence of the appropriate buffer. The digestion product was then purified using the QIAquick PCR
purification kit, following the manufacturer's instructions, and eluted in a final volume of 30 or 50,1 of either water or 1 OmM Tris-HCI, pH 8.5. The final DNA concentration was determined by 1 % agarose gel electrophoresis in the presence of titrated molecular weight marker.
E) Digestion of the cloning vectors (pET22B, pGEX-KG, pTRC-His A, and pGea-His) 10~g plasmid was double-digested with 50 units of each restriction enzyme in 200p,1 reaction volume in the presence of appropriate buffer by overnight incubation at 37°C. After loading the whole digestion on a 1 % agarose gel, the band corresponding to the digested vector was purified from the gel using the Qiagen QIAquick Gel Extraction Kit and the DNA was eluted in 50,1 of 1 QmM Tris-HCI, pH 8.5. The DNA concentration was evaluated by measuring OD2~
of the sample, and adjusted to SOwg/~.I. 1 ~1 of plasmid was used for each cloning procedure.
The vector pGEX-His is a modified pGEX-2T vector carrying a region encoding six histidine residues upstream to the thrombin cleavage site and containing the multiple cloning site of the vector pTRC99 (Phannacia).
1~ Cloning The fragments corresponding to each OItF, previously digested and purified, were ligated in both pET22b and pGEX-KG. In a final volume of 20.1, a molar ratio of 3:1 fragment/vector was ligated using 0.51 of NEB T4 DNA ligase (400 units/p,l), in the presence of the buffer supplied by the manufacturer.
The reaction was incubated at room temperature for 3 hours. In some experiments, ligation was performed using the Boehringer "Rapid Ligation Kit", following the manufacturer's instructions.
In order to introduce the recombinant plasmid in a suitable strain, 100p1 E.
coli DHS competent cells were incubated with the ligase reaction solution for 40 minutes on ice, then at 37°C for 3 minutes, then, after adding 800p1 LB broth, again at 37°C for 20 minutes. The cells were then centrifuged at maximum speed in an Eppendorf microfuge and resuspended in approximately 2()ON,1 of the supernatant. The suspension was then plated on LB ampicillin ( 1 OOmg/ml ).
The screening of the recombinant clones was performed by growing 5 randomly-chosen colonies overnight at 37°C in either 2ml (pGEX or pTC clones) or Sml (pET
clones) LB broth + 1 OO~g/ml ampicillin. The cells were then pelletted and the DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions, to a final volume of 30p.1. 5~,1 of each individual miniprep (approximately 1 g ) were digested with either NdeUXhoI or BamHIlXhoI and the whole digestion loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1Kb DNA Ladder, GIBCO). The screening of the positive clones was made on the base of the correct insert size.

G) Expression Each OItF cloned- into the expression vector was transformed into the strain suitable for expression of the recombinant protein product. 1 pl of each construct was used to transform 30.1 of E. coli BL21 (pGEX vector), E coli TOP I 0 (pTRC vector) or E.coli BL21-DE3 (pET
vector), as described above. In the case of the pGEX-His vector, the same E. coli strain (W3110) was used for initial cloning and expression. Single recombinant colonies were inoculated into 2ml LB+Amp (100~g/ml), incubated at 37°C overnight, then diluted 1:30 in 20m1 of LB+Amp (100~,g/ml) in 100m1 flasks, making sure that the OD,~ ranged between 0.1 and 0.15. The flasks were incubated at 30°C into gyratory water bath shakers until OD indicated exponential growth suitable for induction of expression (0.4-0.8 OD for pET and pTRC vectors; 0.8-1 OD for pGEX and pGEX-His vectors). For the pET, pTRC and pGEX-His vectors, the protein expression was induced by addition of 1 mM IPTG, whereas in the case of pGEX system the final concentration of IPTG was 0.2mM. After 3 hours incubation at 30°C, the final concentration of the sample was checked by OD. In order to check expression, 1 ml of each sample was removed, centrifuged in a microfuge, the pellet resuspended in PBS, and analysed by 12% SDS-PAGE with Coomassie Blue staining.
The whole sample was centrifuged at 6000g and the pellet resuspended in PBS
for further use.
H) GST-fusion proteins large-scale purification.
A single colony was grown overnight.at 37°C on LB+Amp age Plate. The bacteria were inoculated into 20m1 of LB+Amp liquid culture in a water bath shaker and grown overnight.
Bacteria were diluted 1:30 into 600m1 of fresh medium and allowed to grow at the optimal temperature (20-37°C) to ODs 0.8-1. Protein expression was induced with 0.2mM IPTG followed by three hours incubation. The culture was centrifuged at 8000rpm at 4°C. The supernatant was discarded and the bacterial pellet was resuspended in 7.Sml cold PBS. The cells were disrupted by sonication on ice for 30 sec at 40W using a Branson sonifier B-15, frozen and thawed twice and centrifuged again.
The supernatant was collected and mixed with 1501 Glutatione-Sepharose 4B
resin (Pharmacia) (previously washed with PBS) and incubated at room temperature for 30 minutes.
The sample was centrifuged at 700g for 5 minutes at 4°C. The resin was washed twice with l Oml cold PBS for 10 minutes, resuspended in lml cold PBS, and loaded on a disposable column. The resin was washed twice with 2ml cold PBS until the flow-thmugh reached OD2~ of 0.02-0.06. The GST-fusion protein was eluted by addition of 700,1 cold Glutathione elution buffer (IOmM
reduced glutathione, SOmM Tris-HCl) and fractions collated until the ODD was 0.1. 21 Nl of each fraction were loaded on a 12% SDS gel using either Biorad SDS-PAGE Molecular weight standard broad range (Ml) (200, 116.25, 97.4, 66:2, 45, 31, 21.5, 14.4, 6.5 kDa) or Amersham Rainbow Marker (M2) (220, 66, 46, 30, 21.5, 14.3 kDa) as standards. As the MW of GST is 26kDa, this value must S be added to the MW of each GST-fusion protein.
1) His-fusion solubility analysis To analyse the solubility of the His-fusion expression products, pellets of 3ml cultures were resuspended in buffer M1 [5001 PBS pH 7.2J. 25,1 lysozyme (lOmg/ml) was added and the bacteria were incubated for 15 min at 4°C. The pellets were sonicated for 30 sec at 40W using a Branson sonifier B-15, frozen and thawed ,twice and then separated again into pellet and supernatant by a centrifugation step. The supernatant was collected and the pellet was resuspended in buffer M2 [8M urea, O.SM NaCI, 20mM imidazole and 0.1 M NaH2 PO,J and incubated for 3 to 4 hours at 4°C: After centrifugation, the supernatant was collected and the pellet was resuspended in buffer M3 [6M guanidinium-HCI, O.SM NaCI, 20mM imidazole and O.IM NaHZPO4J
overnight at 4°C. The supernatants from all steps were analysed by SDS-PAGE.
.T) His-fusion large-scale purification.
A single colony was grown overnight at 37°C on a LB + Amp age. plate.
The bacteria were inoculated into 20m1 of LB+Arnp liquid culture and incubated overnight in a water bath shaker.
Bacteria were diluted 1:30 into 600m1 fresh medium and allowed to grow at the optimal temperature (20-37°C) to ODs 0.6-0.8. Protein expression was induced by addition of 1mM IPTG
and the culture further incubated for three hours. The culture was centrifuged at 8000rpm at 4°C, the supernatant was discarded and the bacterial pellet was resuspended in 7.Sm1 of either (i) cold buffer A (300mM NaCI, SOmM phosphate buffer, l OxnM imidazole, pH 8) for soluble proteins or (ii) buffer B (urea 8M, l OmM Tris-HCI, 100mM phosphate buffer, pH 8.8) for insoluble proteins.
The cells were disrupted by sonication on ice for 30 sec at 40W using a Branson sonifier B-15, frozen and thawed two times and centrifuged again.

For insoluble proteins, the supernatant was stored at -20°C, while the pellets were resuspended in 2ml buffer C (6M guanidine hydrochloride, 100mM phosphate buffer, IOmM Tris-HCI, pH 7.5) and treated in a homogenizes for 10 cycles. The product was centrifuged at 13000rpm for 40 minutes.
Supernatants were collected and mixed with 1501,x.1 Ni2+-resin (Pharmacia) (previously washed with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700g for 5 minutes at 4°C. The resin was washed twice with 1 Oml buffer A or B for 10 minutes, resuspended in 1 ml buffer A or B and loaded on a disposable column. The resin was washed at either (i) 4°C with 2m1 cold buffer A or (ii) room temperature with 2ml buffer B, until the flow-through reached OD2~ of 0.02-0.06.
The resin was washed with either (i) 2ml cold 20mM imidazole buffer (300mM
NaCI, SOmM
phosphate buffer, 20mM imidazole, pH 8) or (ii) buffer D (urea 8M, IOmM Tris-HCI, 100mM
phosphate buffer, pH 6.3) until the flow-through reached the O.D2~ of 0.02-0.06. The His-fusion protein was eluted by addition of 7001 of either (i) cold elution buffer A
(300mM NaCI, 50mM
phosphate buffer, 250mM imidazole, pH 8) or (ii) elution buffer B (urea 8M, lOmM Tris-HCI, 100mM phosphate buffer, pH 4.5) and fractions collected until the O.D2~ was 0.1. 21 ~l of each fraction were loaded on a 12% SDS gel.
1~ His-fusion proteins renaturation 10% glycerol was added to the denatured proteins. The proteins were then diluted to 20~,g/ml using dialysis buffer I (10% glycerol, O.SM arginine, SOmM phosphate buffer, SmM
reduced glutathione, O.SmM oxidised glutathione, 2M urea, pH 8.8) and dialysed against the same buffer at 4°C for 12-14 hours. The protein was further dialysed against dialysis buffer II (10%
glycerol, O.SM arginine, SOmM phosphate buffer, SmM reduced glutathione, O:SmM oxidised glutathione, pH

8.8) for 12-14 hours at 4°C. Protein concentration was evaluated using the formula:
Protein (mg/ml) _ (1.55 x ODZ~) - (0.76 x ODi~) L) His-fusion large-scale purification 500m1 of bacterial cultures were induced and the fusion proteins were obtained soluble in buffer M1, M2 or M3 using the procedure described above. The clvde extract of the bacteria was loaded _Sg_ onto a Ni-NTA superflow column (Qiagen) equilibrated with buffer M1, M2 or M3 depending on the solubilization buffer of the fusion proteins. Unbound material was eluted by washing the column with the same buffer. The specific protein was eluted with the corresponding buffer containing 500mM imidazole and dialysed against the corresponding buffer without imidazole.
After each run the columns were sanitized by washing vrri~ at least two column volumes of 0.5 M
sodium hydroxide and reequilibrated before the next use.
M) Mice immunisations 20pg of each purified protein were used to immunise mice intraperitoneally. In the case of ORF 44, CD1 mice were immunised with Al(OH)3 as adjuvant on days 1, 21 and 42, and immune response was monitored in samples taken on day 56. For ORF 40, CD1 mice were immunised using Freund's adjuvant, rather than Al(OH)3, and the same immunisation protocol was used, except that the immune response was measured on day 42, rather than 56. Similarly, for ORF
38, CD 1 mice were immunised with Freund's adjuvant, but the immune response was measured on day 49.
l~ ELISA assay (sera analysis) The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37°C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 7m1 of Mueller-Hinton Broth (Difco) containing 0.25% Glucose.
Bacterial growth was monitored every 30 minutes by following ODD. The bacteria were let to grow until the OD
reached the value of 0.3-0.4. The culture was centrifuged for 10 minutes at 10000rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in PBS
containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then overnight at 4°C with stirring. 100p.1 bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4°C. The wells were then washed three times with PBT washing buffer (0.1 % Tween-20 in PBS). 200p1 of saturation buffer (2.7%
Polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37°C. Wells were washed three times with PBT. 200E,i,1 of diluted sera (Dilution buffer: 1 % BSA, 0.1 % Tween-20, 0.1 % NaN3 in PBS) were added to each well and the plates incubated for 90 minutes at 37°C. Wells were washed three times with PBT. 100p1 of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2(?00 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37°C. Wells were washed three times with PBT buffer. 104p1 of substrate buffer for HRP (25m1 of citrate buffer pHS, 1 Omg of O-phenildiamine and l Opl of HZO) were added to each well and the plates were left at room temperature for 20 minutes. I OOE.iI HZS04 was added to each well and OD~o was followed. The ELISA was considered positive when OD,~o was 2.5 times the respective pre-immune sera.
O) FACScan bacteria Bonding Assay procedure.
The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37°C. Bacterial colonies were collected from the agar plates using a sterile drawn swab and inoculated into 4 tubes containing 8ml each Mueller-Hinton Broth (Difco) containing 0.25%
glucose. Bacterial growth was monitored every 30 minutes by following OD6~o.
The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1%
BSA, 0.4% NaN3) and centrifuged for S minutes at 4000rpm. Cells were resuspended in blocking buffer to reach OD6xo of 0.07. 100E.i1 bacterial cells were added to each well of a Costar 96 well plate. 100N,1 of diluted (1:200) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4°C. Cells were centrifuged for 5 minutes at 4000rpm, the supernatant aspirated and cells washed by addition of 200pUwell of blocking buffer in each well. 1001 of R-Phicoerytrin conjugated F(ab)2 goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4°C. Cells were spun down by centrifugation at 4000rpm for 5 minutes and washed by addition of 200~,Uwell of blocking buffer. The supernatant was aspirated and cells resuspended in 200p,Uwel1 of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL1 on, FL2 and FL3 off; FSC-H
threshold:92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gains 7.1; FL-2 PMT:
539;
compensation values: 0.
P) OMV preparations Bacteria were grown overnight on 5 GC plates, harvested with a loop and resuspended in 10 ml 20mM
Tris-HCI. Heat inactivation was performed at 56°C for 30 minutes and the bacteria disrupted by sonication for 10 minutes on ice (50% duty cycle, 50% output). Unbroken cells were removed by centrifugation at SOOOg for 10 minutes and the total cell envelope fraction recovered by centrifugation at SOOOOg at 4°C for 75 minutes. To extract cytoplasmic membrane proteins from the crude outer membranes, the whole fraction was resuspended in 2% sarkosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000g for 10 minutes to remove aggregates, and the supernatant further ultracentrifuged at SOOOOg for 75 minutes to pellet the outer membranes. The outer membranes were resuspended in lOmM Tris-HCI, pH8 and the protein concentration measured by the Bio-Rad Protein assay, using BSA as a standard.
Q) Whole Extracts preparation Bacteria were grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20mM Tris-HCI. Heat inactivation was performed at 56°C for 30 minutes.
R) Western blotting Purified proteins (SOOng/lane), outer membrane vesicles (SUg) and total cell extracts (25ug) derived finm MenB strain 2996 were loaded on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150mA at 4°C, in transferring buffer (0.3 Tris base, 1.44 % glycine, 20% methanol). The membrane was saturated by overnight incubation at 4°C in saturation buffer (10% skimmed milk, 0.1% Triton X1()a in PBS). The membrane was washed twice with washing buffer (3% skimmed mills, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37°C with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with O.I% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
S) Bactericidal assay MC58 strain was grown ovennight at 37°C on chocolate agar plates. S-7 colonies were collected and used to inoculate 7ml Mueller-Hinton broth. The suspension was incubated at 37°C on a nutator and let to grow until OD~o was 0.5-0.8. The culture was aliquoted into sterile l.Sml Eppendorf tubes and centrifuged for 20 minutes at maximum speed in a microfuge. The pellet was washed once in Gey's buffer (Gibco) and resuspended in the same buffer to an OD6ZO of 0.5, diluted 1:20000 in Gey's buffer and stored at 25°C.

501 of Gey's buf~er/1% BSA was added to each well of a 96-well tissue culture plate. 2SN,1 of diluted mice sera (1:100 in Gey's buffer/0.2% BSA) were added to each well and the plate incubated at 4°C. 2SN,1 of the previously described bacterial suspension were added to each well.
251 of either heat-inactivated (S6°C waterbath for 30 minutes) or normal baby rabbit complement S were added to each well. Immediately after the addition of the baby rabbit complement, 221 of each sample/welI were plated on Mueller-Hinton agar plates (time 0). The 96-well plate was incubated for 1 hour at 37°C with rotation and then 22N,1 of each sample/well were plated on Mueller-Hinton agar plates (time 1). After overnight incubation the colonies corresponding to time 0 and time 1 how were counted.
Table II gives a summary of the cloning, expression and purification results.
Example 1 The following partial DNA sequence was identified in N. meningitides <SEQ ID 1 >:
1 ..ACACTGTTGT TTGCAACGGT TCAGGCAAGT GCTAACCAA~
GAAGAGCAAG

IS CGTGTTGATA

TAGAAGAAAA

ACAGCCAGAG

201 AAATCACCyT CAAAGCCGGC GACAACCTGA AAATCAAACA
AAACGGCACA

251 AACTTCACCT ACTCGCTGAA AAAAGACCTC AcAGATCTGA
CCAGTGTTGG

2O AACATcACAA

sACGAACGgC

CCGATACGCT

GTTACCGATG

CGCTGGCTGG

ACGTTGATTT

ACGAAAACAA

CGAAGTTAAA

701 ATCGGTGCGA AGACTTCTGT TATTAAAGAA AAAGAC...

This corresponds to the amino acid sequence <SEQ ID 2; ORF40>:
1 ..TLLFATVQAS ANQEEQEEDL VLIVNSDKEGTGEKEKVEEN
YLDPVQRTVA

201 VRTYDTVEFL SADTKTTTVN VESKDNGKKTEVKIGAKTSVIKEKD...

Further work revealed the complete DNA sequence <SEQ ID 3>:

GGTACAACAG

CGAGTTCTTG

AAGACAACGG

S ATTAAAGAAA

TGGTTCTTCT

TTGATGCAGT

GGTCAAACAG

TGTAACCTTT

IO ATCAAGGCAA

AACGTCAATC

TGCAGGTTCT

GAAAGATGGA

ACCCGCAACG

IS TTCCAGCGTT

ATGGGGACGC

ATTACCAATG

ACAACTTAAA

ACGGCAACGC

TTATCGCGGC

GCGGAAATTG

TTCGGTGCTT

This corresponds to the amino acid sequence <SEQ ID 4; ORF40-1>:

TLLFATVQAS

SDWAVYFNEK

TEKLSFSANG

LNTGATTNVT

VRTYDTVEFL

3O 251 SADTKTTTVN VESKDNGKKT EVKIGAKTSV IIGEIfDGKLVT
GKDKGENGSS

301 TDEGEGLVTA KEVIDAVNKA GWRMKT"fTAN GQTGQADKFE
TVTSGTNVTF

NLDSKAVAGS

TSMTPQFSSV

GDVTNVAQLK

HAIGGGTYRG

Tp*

Further work identified the corresponding gene in strain A of N.meningitidis <SEQ ID S >:

ATGCCTGNGT

TCAGGCGAAT

AACGCTCTGT

TTGGAAACGA

CCCATACATA

AAAGACCTCA

CGCAAACGGC

TCGCGAAAGA

GGTATCGGTT

SO CGTTGATGCG

AGGATGTGTT

ACAACTGGTC

CGAGTTCTTG

AAGACAACGG

SS ATTAAAGAAA

TGGTTCTTCT

TTGATGCAGT

GGTCAAACAG

TGTAACCTTT

C)O ATCAAGGCAA

AACGTCAATC

TGCAGGTTCT

GAAAGATGGA

AGCCGCAACG

GS TTCCAGCGTT

ATGACGAGGG

CGCATTACCA

This encodes a protein having amino acid sequence <SEQ ID 6; ORF40a>:

TLLFATVQAN

lO 51 ATDEDEEEEL ESVQRSWGS IQASMEGSGE LETISLSMTN
DSKEFVDPYI

TEKLSFGANG

AGSSASHVDA

VRTYDTVEFL

GKGKGENGSS

IS 301 TDEGEGLVTA KEVIDAVNKA GWRhBCTTTAN GQTGQADKFE
TVTSGTNVTF

351 ~ASGKGTTATV SKDDQGNITV MYDVNVGDAL NVNQLQNSGW
NLDSKAVAGS

TSMAPQFSSV

XGDVTNVXQL

QW*

The originally-identified partial strain B sequence (ORF40) shows 65.7%
identity over a 254aa overlap with ORF40a:

orf40.pep TLLFATVQASANQEEQEEDLYLDPVQRTVA

2S IIIIIII11:1::1::11:1 . 111:1 orf40a SALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQpiNATDEDEEEEL--ESVQRSV-3O orf40.pep VLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITXKAGDNLKIKQN------GT
I
I :
Il:il I :
I I

....
orf40a . .. .
.. :
IIIIIIIIII ..
VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIKQNTNENTNAS

orf90.pep NFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLNGIG
IIII

:
orf90a IIIIII I :I IIIIII:III:IIII IIIIIIIIIIIIIIIIIIIIIIIIIII
SFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLNGIG

orf90.pep STLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTA--SDNVDFV
I
I

III
I

: :
orf40a : :
I : . I
:
IIIIIIIIIIII! I:I: 1:11111 STLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGSTTGQSENVDFV

orf40.pep RTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKD

IIIIIIIIIIIII IIIIIIIIIIIII:IIIIIIIIIIilllll SO orf40a RTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSST

The complete strain B sequence (ORF40-1) and ORF40a show 83.7% identity in 601 as overlap:

SS orf40-1. pep MNKIYRIIWNSALNAWVWSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
IIIIIIIIIilllll I:IIIIIIIIIIIIIIIIIIIilllllllllll:1::1::11:1 orf40a MNKIYRIIWNSALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL

orf40-1. pep YLDPVQRTVAVLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITLKAGDNLKIK
. III:I I .... II: II I : . .. . I : .. :IIIIIIIIIII

-~
orf90a --ESVQRSV-VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIK

S

orf90-1. pep QN------GTNETYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNG

II :::IIIlllllll I :I IIIIII:III:IIII lllllllllllllllll orf40a QNTNENTNASSFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKG
N

L
FAKETAGTNG

1~ 180 190 200 210 220 230 orf40-1. pep DTTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTT

IIIIIIIIiillllll-I :::I: :I I : : lili:lllillllllllll orf90a I:I
DTTVHLNGIGSTLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGST

orf90-1. pep A--SDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGA

KTSVIKEKDGKLVTG
' i:llllliilllllllllll IIIIIIIII1111:1111111111111111111111 orf40a TGQSENVDFVRTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVT

G
2~ 240 250 260 270 280 290 orf40-1. pep KDKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFA

I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllll 2S orf40a KGKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVT

SGTNVTFA

orf90-1. pep SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSP
30 Ill11111 orf40a 111lilllilllllllillllllllllilllllllllll Illilllll SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKV

ISGNVSP

orf90-i.pep SKGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGD-ALNV

IIIIIIIIIIIIIIIIIII:IIIIIIIIIIII:Illllllllllfllllllll orf90a : IIII
SKGKMDETVNINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEG

ALNV

4~ 480 990 500 510 520 530 orf90-1. pep GSKKDNKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQ~IATAGL

orf40a GSKDANKPVRITNVAPGVKXGDVTNVXQLKGVAQNLNNRIDNVDGNARAGIAQAIAT

AGL

orf90-1. pep VQAYLPGKSh~IAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQ
II

Iillllllllllllllllllllllllllllllllllllllllllllllllllllllll orf40a VQAYLPGKSI4rlAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGY

Q
S~ 540 550 560 570 580 590 orf40-l.pep WX
II
orf90a WX
SS Computer analysis of these amino acid sequences gave the following results:
Homolow with Hsf protein encoded by the type b surface fibrils locus of H
influenzae (accession number U418S2) ORF40 and Hsf protein show S4% as identity in 2S 1 as overlap:
Orf90 1 TLLFATVQASANQEEQEEDLYLDPVQRTVAVLIVNSDXXXXXXXXXXXXNSDWAVYFNEK 60 C)O TLLFATVQA+A E++E LDPV RT VL +SD NS+W +YF+ K
Hsf 91 TLLFATVQANATDEDEE----LDPVVRTAPVLSFHSDKEGTGEKEVTE-NSNWGIYFDNK 95 Orf40 61 GVLTAREITXKAGDNLKIKQN------GTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVN 119 GVL A IT KAGDNLKIKQN ++FTYSLKKDLTDLTSV TEKLSF ANG+KV+
C)S Hsf 96 GVLKAGAITLKAGDNLKIKQNTDESTNASSFTYSLKKDLTDLTSVATEKLSFGANGDKVD 155 Orf40 115 ITSDTKGLNFAKETAGTNGDTTVHLNGIGSTLTDTLLNTGAXXXXXXXXXXX7~E~ 174 ITSD GL AK G+ VHLNG+ STL D + NTG EK RAA+
S Hsf 156 ITSDANGLKLAK-----TGNGNVHLNGLDSTLPDAVTNTGVLSSSSFTPNDV-EKTRAAT 209 Orf40 175 VKDVLNAGWNIKGVKPGTTASDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKI 239 VKDVLNAGWNIKG K ++VD V Y+ VEF++ D T V + +K+NGK TEVK
Hsf 210 VKDVLNAGWNIKGAKTAGGNVESVDLVSAYNNVEFITGDKNTLDWLTAKENGKTTEVKF 269 Orf90 235 GAKTSVIKEKD 245 KTSVIKEKD
Hsf 270 TPKTSVIKEKD 280 ORF40a also shows homology to Hsf gi~1666683 (U41852) hsf gene product (Haemophilus influenzae) Length = 2353 1S Score a 153 (67.7 bits), Expect = 1.5e-116, Sum P(11) = 1.5e-116 Identities = 33/36 (91%), Positives = 34/36 (99%) Query: 16 VAVSELTRNHTKRASATVKTAVLATLLFATVQANAT 51 V VSELTR HTKRASATV+TAVLATLLFATVQANAT
ZO Sbjct: 17 VWSELTRTHTKRASATVETAVLATLLFATVQANAT 52 Score = 161 (71.2 bits), Expect = 1.5e-116, Sum P(11) = 1.5e-116 Identities = 32/38 (84%), Positives = 36/38 (94%) 2S Query: 101 VTLKA'GDNLKIKQNTNENTNASSFTYSLKKDLTGLINV 138 +TLKAGDNLKIKQNT+E+TNASSFTYSLKKDLT L +V
Sbjct: 103 ITLKAGDNLKIKQNTDESTNASSFTYSLKKDLTDLTSV 190 Score = 110 (48.7 bits), Expect = 1.5e-116, Sum P(11) = 1.5e-116 30 Identities = 21/29 (72%), Positives = 25/29 (86%) Query: 138 VTEKLSFGANGKKVNIISDTKGLNFAKET 166 V++KLS G NG KVNI SDTKGLNFAK++
Sbjct: 1439 VSDKLSLGTNGNKVNITSDTKGLNFAKDS 1967 Score = B5 (37.6 bits), Expect = 1.5e-116, Sum P(il) = 1.5e-116 Identities = 18/32 (56%), Positives = 20/32 (62%) 4O Query: 169 TNGDTTVHLNGIGSTLTDTLAGSSASHVDAGN 200 T D +HLNGI STLTDTL S A+ GN
Sbjct: 1969 TGDDANIHLNGIASTLTDTLLNSGATTNLGGN 1500 Score = 92 (90.7 bits), Expect = 1.5e-116, Sum P(11) = 1.5e-116 Identities = 16/19 (89%), Positives = 19/19 (100%) Query: 206 RAASIKDVLNAGIiPTIKGVK 229 RAAS+KDVLNAG~it~1++GVK
Sbjct: 1509 RAASVKDVLNAGNNVRGVK 1527 S0 Score = 90 (39.8 bits), Expect = 1.5e-116, Sum P(11) = 1.5e-116 Identities = 17/28 (60%), Positives = 20/28 (71%) Query: 226 STTGQSENVDFVRTYDTVEFLSADTTTT 253 S Q EN+DFV TYDTV+F+S D TT
SS Sbjct: 1530 SANNQVENIDFVATYDTVDFVSGDKDTT 1557 Based on homology with Hsf, it was predicted that this protein from N.
meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
ORF40-1 (6lkDa) was cloned in pET and pGex vectors and expressed in E.coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. Figure 60 lA shows the results of affinity purification of the His-fusion protein, and Figure 1B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for FACS analysis (Figure 1 C), a bactericidal assay (Figure 1 D), and ELISA (positive result). These experiments confirm that ORF40-1 is a surface-exposed protein, and that it is a useful immunogen.
S Figure lE shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF40-1.
Example 2 The following partial DNA sequence was identified in N. meningitides <SEQ ID
7>
1 ATGTTACGTt TGACTGCtTT AGCCGTATGC ACCGCCCTCG
CTTTGGGCGC

lO GaACAGGCGG

101 TTTCCGCCGC ACAAACCGAA GgCGCGTCCG TTACCGTCAA
AACCGCGCGC

TTTACGATTT

GGTTTGTCCG

AACGACAAAA

IS ACGCTTACAA

351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC CgCCAAGGCG
TTTGACAAAT

901 TGAAcGAAAT CGCGCCGACC ATCGrmwTGA CCGCCGATAC
CGCCAACCTC

451 AAAGAAAGTG CCAArGAGGC ATCGACGCTG GCGCAAATCT
TC..

This corresponds to the amino acid sequence <SEQ ID 8; ORF38>:

151 KESAKEASTL AQIF..
Further work revealed the complete nucleotide sequence <SEQ ID 9>:
1 ATGTTACGTT TGACTGG~TT AGCCGTATGC ACCGCCCTCG
ZS CTTTGGGCGC

GAACAGGCGG

AACCGCGCGC

TTTACGATTT

GGTTTGTCCG

AACGACAAAA

ACGCTTACAA

TTTGACAAAT

CGCCAACCTC

TCTTCGGCAA

TCTTTTGAAG

GATTTTGGTC

TGGGCGGCTG

ATTAAAGAAG

GAAAAATCCC

AAGAGGGTCA

ACAACCGCTT

e51 GGAAAAAAGG ACAGGTCGTG TACCTCGTTC CTGAAACTTA
TTTGGCAGCC

CCGACGCTTT

This corresponds to the amino acid sequence <SEQ ID 10; ORF38-1>:
4S 1 MLRLTALAVC TALALGACSP QNSDSAPQf4FC EQAVSAAQTE GASVTVKTAR

301 GGAQELLNAS KQVADAFNAA K*
Computer analysis of this amino acid sequence reveals a putative prokaryotic membrane lipoprotein lipid attachment site (underlined).
Further work identified the corresponding gene in strain A of N.meningitidis <SEQ ID 11>:

GAACAGGCGG

AACGGCGCGC

TTTACGATTT

GGTTTGTCCG

lO 251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA
AACGACAAAA

ACGCTTACAA

TTTGACAAAT

CGCCAACCTC

TCTTCGGCAA

TCTTTTGAAG

GATTTTGGTC

TGGGCGGCTG

ATCAAAGAAG

GAAAAATCCC

AAGAGGGTCA

ACAACCGCTT

TTTGGCAGCC

CCGACGCTTT

2S This encodes a protein having amino acid sequence <SEQ ID 12; ORF38a>:

301 GGAQELLNAS KQVADAFNAA K*
The originally-identified partial strain B sequence (ORF38) shows 95.2%
identity over a l6Saa overlap with ORF38a:

orf38.pep MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE

Illlllllllllllllllllllllllllllillfllil:II:IIIIIIIIIIIIIIIIII
orf38a MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE

orf38.pep RIAVYDLGMLDTLSKLGVKTGLSVDKNALPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII.11111111111111111111111 orf38a RIAWDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL

4S 7o eo 90 loo ll0 120 orf38.pep IIIGSRAAKAFDKLNEIAPTIXXTADTANLKESAKE-ASTLAQIF
Iilllllilllllllllllll IIIIIIIII1111 ::11111 SO orf38a IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEADKLKAEIDA

orf38a SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAZKEGSHGQPI

SS The complete strain B sequence (ORF38-1) and ORF38a show 98.4% identity in 321 as overlap:

orf38a.pep MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE
Illllllllllllllllillllllllllllllllllil:II:IIIIIIIIIIIIIIIIII
orf38-1 MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE
S orf38a.pep RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
IIIIIIIIIIIIIIIIIIIIIIIIIilllllllllllllllllllllllllllllfllll orf38-1 RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
orf38a.pep IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEApKLKAEIDA
1~ IIIIIIIIIIIillllllllllllllllllllllllllll,lllllll:llllllllllll orf38-1 IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKQAEADKLKAEIDA
orf38a.pep SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI
Iilllllllllllllllllllllllllllllllllllllllllllilil:1111111111 IS orf38-1 SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDESIKEGSHGQPI

orf38a.pep SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLNNPLVAETTAWKKGQVVYLVPETYLAA

IIIIIIIIIIIIIilllllllllllllllllll:IIIIIIIIIIIIIIIIIIIIIIIIII
orf38-1 SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLDNPLVAETTAWKKGQVVYLVPETYLAA

orf38a.pep GGAQELLNASKQVADAFNAAK
I

llllllliilllllllllll orf38-1 GGAQELLNASKQVADAFNAAK

Computer analysis of these sequences revealed the following:
2S Homology with a linonrotein (lipo) of C.fetuni (accession number X82427) ORF38 and lipo show 38% as identity in 96 as overlap:
Orf38: 40 EGASVTVKTARGDVQIPQNPERIAVYDLGMLDTLSKLGVKTGLS-VDKNRLPYLEEYFKT 98 EG S VK + G+ + P+NP ++ + DLG+LDT L + ++ V LP + FK
Lipo: 51 EGDSFLVKDSLGENKTPKNPSKWILDLGILDTFDALKLNDKVAGVPAKNLPKYLQQFKN 110 Orf38: 99 TKPAGTLFEPDYETLNAYKPQLIIIGSRAAKAFDKL 139 G + + D+E +NA KP LIII R +K +DKL
Lipo: 111 KPSVGGVQQVDFEAINALKPDLIIISGRQSKFYDKL 196 Based on this analysis, it was predicted that this protein from N.
meningitides, and its epitopes, could 3S be useful antigens for vaccines or diagnostics.
ORF38-1 (321cDa) was cloned in pET and pGex vectors and expressed in E.coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. Figure 2A shows the results of affinity purification of the His-fusion protein, and Figure 2B shows the results of expression of the GST-fusion in E.coli. Purified His-fusion protein was used to immunise mice, whose sera were used for Western blot analysis (Figure 2C) and FACS
analysis (Figure 2D).
These experiments confirm that ORF38-1 is a surface-exposed protein, and that it is a useful immunogen.
Figure 2E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF38-1.
Example 3 4S The following N. meningitides DNA sequence was identified <SEQ ID 13>:

wo ~r~ss~ rc~rnsmooio3 g_ This corresponds to the amino acid sequence <SEQ ID 14; ORF44>:
lO 1 MKLLTTAILS SAIALSSMAA AAGTDNPTVA KKTVSYVCQQ GKKVKVTYGF
51 NKQGLTTYAS AVINGKRVQiM PVNLDKSDNV ETFYGKEGGY VLGTGVMDGK
101 SYRKQPIMIT APDNQIVFKD CSPR*
Computer analysis of this amino acid sequence predicted the leader peptide shown underlined.
Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 1 S>:

This encodes a protein having amino acid sequence <SEQ ID 16; ORF44a>:

101 SYRKQPIMIT APDNQIVFKD CSPR*
The strain B sequence (ORF44) shows 99.2% identity over a 124aa overlap with ORF44a:

orf44.pep MKLLTTAILSSAIALSSMAAAAGTDNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS
30 IIIIiIIIIIIn IIIIIIIIIII:iIIIIiIn IIIIIIIIIIIIIIIIIillllllll orf49a MKLLTTAILSSAIALSSMAAAAGTNNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS

3S orf94.pep AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD
Iilllllllllllllllllllllllllllllllllllllllllllllllllllllllill orf44a AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD

40 orf49.pep CSPRX
IIIII
orf94a CSPRX
Computer analysis gave the following results:
Homolo~~y with the LecA adhesin of Eikenella corrodens (accession number D781 S3) 4S ORF44 and LecA pmtein show 4S% as identity in 91 as overlap:
Orf99 33 ~VSYVCQQGKKVKVTYGFNKQGLTTYASAVINGKRVQMPVNLDKSDNVETFYGKEGGYVL 92 +V+YVCQQG+++ V Y FN G+ T A +N + +++P NL SDNV+T + GY L
LecA 135 SVAYVCQQGRRLNVNYRFNSAGVPTSAELRVNNRNLRLPYNLSASDNVDTVF-SANGYRL 193 S0 Orf49 93 GTGVMDGKSYRKQPIMITAPDNQIVFKDCSP 123 T MD +YR Q I+++AP+ Q+++KDCSP

LecA 194 TTNAMDSANYRSQDIIVSAPNGQMLYKDCSP 229 Based on homology with the adhesin, it was predicted that this protein from .N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
ORF44-1 ( 11.2kDa) was cloned in pET and pGex vectors and expressed in E.
coli, as described S above. The products of protein expression and purification were analyzed by SDS-PAGE. Figure 3A shows the results of affinity purification of the His-fusion protein, and Figure 3B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for ELISA, which gave positive results, and for a bactericidal assay (Figure 3C). These experiments confirm that ORF44-I is a surface-exposed protein, and that it is a useful immunogen.
Figure 3D shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF44-1.
Example 4 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
17>
1 ..GGCACCGAAT TCAAAACCAC CCTTTCCGGA GCCGACATAC
IS AGGCAGGGGT

GGCATCGTTA

CGTATGGCAA

TACCGAGCTT

TATATCGCCG

ACGTGAACTG

CAGGAAGGCC

CGTGGTCACC

TGGCCGCCGC

501 CGCAACCGAT GCAGCATTT...

2S This corresponds to the amino acid sequence <SEQ ID 18; ORF49>:
1 ..GTEFKTTLSG ADIQAGVGEK ARADAKIILK GIVNRIQTEE KLESNSTVWQ

151 SGAGTGAVLG LXRVAAAATD AAF..
30 Further work revealed the complete nucleotide sequence <SEQ ID 19>:

TGAATGTTCA

AATTACAGCA

CGCCCAAACA

CATCCAAACC

AGGCCGGAAG

GGGCCGGCAC

AATATGCCTA

CAAGTACAGC

CGGAGCCGGA

GCGCAGGAAC

ACCGATGCAG

WO ~~~ PCT/IB99/00103 CAACAACAAA

GCACGGTGAA

AAAATCGGTG

CAACCTGACC

CCGCTGTCAA

GCGGCTTTGG

GTTGGATCAG

GTGCGGCAGC

GCGGCGGTCG

TGGCAGCCTG

1201 AATGTGAAGG ACAGGGCAAA AATCATTGCT A1~GGCGAAGC
TGGCAGCAGG

GCGAATGCGG

GGATCGTTTG

CAGAAAGCTT

IS. 1901 TTGTGAGTCT TATCGACCAC TGGGCTTGCG ACACTTTGTA
AGTGTTTCAG

TAATGGAAAA

TAGGTAAAAT

GTATCTGTCG

AGCATCTATG

AAATGATTTC

CTGACAAGAG

TCCTTTTAAA

TTGCTGCAGG

GATAAATTTA

2S 1901 TTAGTGCAAA CATAAAA?~AA TAG

This corresponds to the amino acid sequence <SEQ ID 20; ORF49-1>:

51 AKTRSGWDTV LEGTEFKT"TL SGADIQ~4GVG EKARADAKII LKGIVNRIQT

501 LIINTRNGNV YFSVGKIWST VKS'!'KSNISG VSVGWVLNVS PNDYLKEASM

601 DSKIIGEIGL G$CsVI~AGVEK TIYIGNIKDI DKFISANIKK
40 Computer analysis predicts a transmembrane domain and also indicates that ORF49 has no significant amino acid homology with known proteins. A corresponding ORF from N. meningitides strain A was, however, identified:
ORF49 shows 86.1% identity over a 173aa overlap with an ORF (ORF49a) from strain A of N.
meningitides:
45 l0 20 30 orf49.pep GTEFKTTLSGADIQAGVGEKARADAKIILK
(1111111:11111111 IIII:IIIIIII
orf99a SKNELNETKLPVRWAQXAATRSGWDTVLEGTEFKTTLAGADIQAGVXEKARVDAKIILK

orf99.pep GIVNRIQTEEKLESNSTVWQKQAGSGSTVETLKLPSFEGPALPKLTAPGGYIADIPKGNL
IIIIIII:IIIII:IIIIIIIIII III:IIIIIIIII:i: III:IIilll:lllllll orf99a GIVNRIQSEEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNL

orf99.pep KTEIEKLAKQPEYAYLKQLQTVKDVNWNQVQLAYDKWDYKQEGLTGAGAAIXALAVTVVT
IIIIIiI:IIIIIIIIiIIi::I::IIIIIIIIII:IIIIIIIII Illll Ililllll -~z-orf99a KTEIEKLSKQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTVVT

S orf99.pep SGAGTGAVLGLXRVAAAATDAAF
(IIIIIIIIII : Ill orf99a SGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGRSSTVKNLWA

ORF49-1 and ORF49a show 83.2% identity in 4S7 as overlap:
lO orf49a.pep XQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNETKLPVRWAQXAATRSGWDTV
IIII IIII:I:i:llll illlllll Illlllilllllillll:ll:l orf49-1 IIIIIIII
MQLLAAEGIHQHQLNVQKSTRFIGIKVGKSNYSKNELNETKLPVRVIAQTAKTRSGWDTV

orf49a.pep LEGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQS$EKLETNSTVWQKQAGRGST
1S IIIIIIIIII:IIIIIIII IIII:IIIIIIiilllill:LII11:1111111111 orf49-1 III
LEGTEFKTTLSGADIQAGVGEKARADAKIILKGIVNRIQTEEKLESNSTVWQKQAGSGST

orf99a.pep IETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLSKQPEYAYLKQLQVAKNINWN
:IIIIIIIII

:I: III:111111:11111111111111:IIIIIIIIIIII::I::III
2O orf49-1 VETLKLPSFEGPALPKLTAPGGYIADIPKGNLKTEIEKLAKQPEYAYLKQLQTVKDVNWN

orf99a.peg QVQLAYDRWDYKQEGLTEAGAAIIALAVTVVTSGAGTGAVi,GLNGAXAAATDAAFASLAS
IIIIIII:IIIIIIIII IIIIllllllllllllllllllllllll orf99-1 IIIIIIIIIIIII
2S QVQLAYDKWDYKQEGLTGAGAAIIALAVTWTSGAGTGAVLGI,NGAAAAATDp,AFASLAS

orf49a.pep QASVSFINNKGDVGKTLKELGRSSTVKNLWAAATAGVADKIGASALXNVSDKQWINNLT
IIIIIIIIIII::I:IIilllllllllll:II:IIIIIIIIIIIIII
orf99-1 IIIIIIIIIIII
30 orf99a.pep QASVSFINNKGNIGNTLKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLT
VNLANAGSAALINTAVNGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHA

IIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIilllllllllllllllll:111111 orf49-1 VNLANAGSAALINTAVNGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYIAHKIAHA

orf99a.pep IAGCAAAAANKGKCQDGAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVS
3S IIIIIIIIIIII11111111111111:II:I :1::1 :I::I:1 orf99-I :I:1 :11:11:1:
IAGCAAAAANKGKCQDGAIGAAVGEILGETLLDGRDPGSLNVKDRAKIIAKAKLAAGAVA

orf49a.pep GWGGDVNAAANAAEVAVKNNQLSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVAD
III::illti Ili:ll:l:l : I ::::::
4O orf49-1 ALSKGDVSTAANAAAVAVENNSLNDIQDRLLSGNYALCMSAGGAESFCESYRPLGLPHFV

orf49a.pep KRLAASIAICTDISRSTECRTIRKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQAD
orf49-1 SVSGEMKLPNKFGNRMVNGKLIINTRNGNVYFSVGKIWSTVKSTKSNISGVSVGWVLNVS
4S The complete length ORF49a nucleotide sequence <SEQ ID 21> is:

TGGATGTCCA

AATTACAGTA

CGCCCAAANT

SO CCGAATTCAA

GAAAAAGCCC

TATCCAGTCG

AGGCCGGACG

AGCCCTACTC

SS TCCGAAAGGC

AGTATGCCTA

CAGGTGCAGC

CGAAGCAGGT

GCGCAGGAAC

E)O ACCGATGCAG

CAACAACAAA

GCACGGTGAA

AAAATCGGCG

CAACCTGACC

GS GCGCTGTCAA

GCGGCTTTGG

GTTGGATCAG

GTGCGGCAGC

GCGGCTGTGG

TGACACTTTG

TGGTTGCCGG

GCGAATGCGG

GGGTAGAGAA

CTCAACTGTG

AAAAGACTTG

TGAATGTAGA

lO 1501 ACAATCAGAA AACAACATTT GATCGATAGT AGAAGCCTTC
ATTCATCTTG

TTATTCAGCA

TTTGAATACT

TATCCGAATG

CCTAGATTCA

TACTAATGTC

GACATCTGGC

GCCCATAACC

NGTAAAATCT

ATGAGATTCC

GAAATTTCAA

TAAAATACTT

CCTCTAAAAT

AATGTCATTC

TNTNGATGTA

A

This encodes a protein having amino acid sequence <SEQ ID 22>:

KHELDVQKSR
RFIGIKVGXS
NYSKNELNET
KLPVRWAQX

LEGTEFKTTL
AGADIQAGVX
EKARVDAKII
LKGIVNRIQS

WQKQAGRGST
IETLKLPSFE
SPTPPKLSAP
GGYIVDIPKG

ICQPEYAYLKQ
LQVAKNINi4N
QVQLAYDRWD
YKQEGLTEAG

QASVSFINNK

SDKQ'PINNLT

LINTAVNGGS
LKDXLEANIL
AALVNTAHGE
AASKIKQLDQ

IAGCAAAAAN
KGKCQDGAIG
AAVGEIVGEA
LTNGKNPDTL

YSKLVAGTVS
GWGGDVNAA
ANAAEVAVKN
NQLSDXEGRE

QNXPQLCRKN
TVKKYQNVAD
KRLAASIAIC
TDISRSTECR

RSLHSSWEAG
LIGKDDEWYK
LFSKSYTQAD
LALQSYHLNT

TKPLSEIiMSD
QGYTLISGVN
PRFIPIPRGF
VKQNTPITNV

NLXRHLANAD
GFSQEQGIKG
AHNRTNXMAE
LNSRGGXVKS

RIKYEIPTLD
RTGKPDGGFK
EISSIKTVYN
PKXFXDDKIL

YSKASKIAQN
ERTKSISERK
NVIQFSETFD
GIKFRXYXDV

E*

Based on the presence of a putative tcansmembrane domain, it is predicted that these proteins from N. meningitides, and their epitopes, could be useful antigens for vaccines or diagnostics.
4S Example 5 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
23>
1 ..CGGATCGTTG TAGGTTTGCG GATTTCTTGC GCCGTAGTCA CCGTAGTCCC

This corresponds to the amino acid sequence <SEQ ID 24; ORF50>:
SS 1 ..RIWGLRISC AWTWPSIT QGFVFAFHSD KGYDALVGIA VLGTFNHPTH

101 PTTAPPLPPV A*

Computer analysis predicts two transmembrane domains and also indicates that ORF50 has no significant amino acid homology with known proteins.
Based on the presence of a putative transmembrane domain, it is predicted that this protein from N. meningitides, and its epitopes, could be useful antigens for vaccines or diagnostics.
S Ezxmple 6 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
2S>
1 .AAGTTTGACT TTACCTGGTT TATTCCGGCG GTAATCAAAT
ACCGCCGGTT

TTTGCGCTGA

GGTACATCGG

TGGTGTCGCT

GCACATACGA

GCATCTGCTT

ATACGGTGGC

GGTGAGGCGC

GGCGGTGATG

CGTTG.....

//

1451 .......... .......... .......... ..........
..........

1501 .ATTTGCGC

GTTAAAACGG

AGCGGGAACA

1651 CAGCAGGAAT TGCTGGCGAA CG..AACGGA TATTACCGCT
ATCTGTATGA

This corresponds to the amino acid sequence <SEQ ID 26; ORF39>:
2S 1 ..KFDFTWFIPA VIKYRRLFFE VLWSWLQL FALITPLFFQ WMDKVLVHR

151 WYYSSTLTWV VLASL..... ..... .. .......... ..........
//~
3O 501 .......... ....ICANRT VLIIAHRLST VKTAHRIIAM DKGRIVEAGT
551 QQELLANXNG YYRYLYDLQN G*
Further work revealed the complete nucleotide sequence <SEQ ID 27>:

TCATCATCCT

CAGCATGAAT

GCTGTTAGCC

CTATTAAACG

GACGGCAACC

CCAATTTTTG

TTGCCGAATT

CGCGCTTCGG

TCCGGCGGTA

CGGTGGTGTT

GTGATGGACA

551 AGGTGCTGGTACATCGGGGA TTCTCTACTT TGGATGT~GT
GTCGGTGGCT

TGCGGACGTA

GGCGCGCGTT

GCACAGACGA

TTCGCAATTT

TTTTCGTTTA

TTGGGTGGTA

CAGCAGGGGG

GATTGGCGCA

TTGCGTTTAA

S GCGCAGTTGT

GGGGGATATT

TGCCCGATAT

AAGGCGGACG

GGGGGAAGTG

lO TCACCAAATT

GTGGACGGCA

GGTCGGCGTG

ACAATATCGC

GCAGCCAAAC

IS CTACGGCACC

GGCAGCGTAT

ATTTTTGATG

TATGCAGAAC

CCCACCGTCT

2001 'GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT

GAACGGATAT

This corresponds to the amino acid sequence <SEQ ID 28; ORF39-1>:

HGIAANPADI
QHEFCTSAQS
DLNETQWLLA

ATLPALVWCD
DGNHFILAKT
DGEGEHAQFL

YSGKLILVAS
RASVLGSLAK
FDFTWFIPAV

FSTLDWSVA

LPLSYFEHRR

QALTSVLDLA
FSFIFLAVMW
YYSSTLTWW

RLNDKFARNA
DNQSFLVESI
TAVGTVKAMA

FRVTKLAWG QQGVQLIQKL
VTVATLWIGA

GQVAAPVIRL
AQLWQDFQQV
GISVARLGDI

ITFEHVDFRY
KADGRLILQD
LNLRIRAGEV

LYVPEQGRVL
VDGNDLALAA
PAWLRRQVGV

TGMPLERIIE
AAKLAGAHEF
IMELPEGYGT

RALITNPRIL
IFDEATSALD
YESERAIMQN

KTAHRIIRMD
KGRIVEAGTQ
QELLAKPNGY

Computer analysis of this amino acid sequence gave the following results:
Homolotzv with a predicted ORF from N meningitides (strain A_) 40 ORF39 shows 100% identity over a l6Saa overlap with an ORF (ORF39a) from strain A of N.
meningitides:

orf39.pep KFDFTWFIPAVIKYRRLFFEVLWSWLQL
IIIIIIIIIIillllllllllllllillll 4S orf39a AVLSFAEFSNRYSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLWSWLQL

40 50 60 70 80 g0 orf39.pep FALITPLFFQVVMDKVLVHRGFSTLDWSVALLWSLFEIVLGGLRTYLFAHTTSRIDVE
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllllilllllllllllll orf39a FALITPLFFQVVMDKVLVHRGFSTLDWSVALLWSLFEIVLGGLRTYLFAHTTSRIDVE
170 180 190 200 21~ 220 SS orf39.pep LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQ,ALTSVLDLAFSFIFLAVM
Illllllilllllllllllllfll11111llllllllllllllllillllllllllllli orf39a LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQ~1LTSVLDLAFSFIFLAVM

orf39.pep WYYSSTLTWWLASLXXXXXXXXXXXXXXXXXXXXXXXXXXXXICANRTVLIIAHRLSTV

_76_ Ilillil 111111 orf39a WYYSSTLTWWLASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAM

_ 290 300 310 320 330 390 ORF39-1 and ORF39a show 99.4% identity in 710 as overlap:

S orf39-1. pep MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV

IIIIIillllllllllllllilllllllllllllllllllllllllllllllllllllll orf39a MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV

orf39-1. pep VRQPIKRLAMATLPALVWCDDGNHFILAKTDGEGEHAQFLIQDLVTNKSAVLSFAEFSNR
IIIIIIIIIIIIIIIIIIIII

IIIIIIIIIII Illll:lllll:lllllllllllllll orf39a VRQPIKRLAMATLPALVWCDDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNR

orf39-1. pep YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLWSWLQLFALITPLFFQV
IIIIIIIIIIIIiIIIIIIIIII1111111111111Illlllilllllllll11111111 IS orf39a YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLWSWLQLFALITPLFFQV

orf39-1. pep VMDKVLVHRGFSTLDWSVALLWSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

orf39a VMDKVLVHRGFSTLDWSVALLWSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS

orf39-1. pep LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWW

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllll orf39a LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWW

2S orf39-1. pep LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEpQMTQRWD
Illillllllllllllllllllllllllllllllllllllllllllllllllllilllll orf39a LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWD

orf39-1. pep NQLAAYVASGFRVTKLAWGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS
1111111111111illlllllllllilllfllllllllllll1111n orf39a NQLAAYVASGFRVTKLAWGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS

orf39-1. pep GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY

III11111111111111111lllllllllllllllllllllllllllllllllillllll 3S orf39a GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY

orf39-1. pep KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPEQGRVLVDGNDLALAA

Illllillllllllllllllllllllllllllllllllllllll orf39a IIIIIIIIIFIIIII
KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPAQGRVLVDGNDLALAA

orf39-1. pep PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIMELPEGYGT

IIIIIIIIIIIIIilllllllllllllllllllllllllIIII11111111111111111 orf39a PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIMELPEGYGT

4S orf39-1. pep WGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV

IIIIIIIIIIIIilllllllIIIIIIIilllllllllllllllllllllllllliillll orf39a WGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV

orf39-1. pep LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX
SO Illllllllllllllllllllllllllllllllllllllllllllllllll orf39a LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX

The complete length ORF39a nucleotide sequence <SEQ
ID 29> is:

GCTCCCCGCC

CCGCCAATCC

GATTTAAATG

GGCAAAGGTA

CCGCATTGGT

GACGGTGGGG

TAAGTCTGCG

C)O 351 TTCTAACAGA TATTCGGGCA GGTTGCTTCCCGCGCTTCGG
AACTGATATT

TTTGACTTTA

TTTTGAAGTA

CGCCTCTGTT

TTCTCTACTT

C)S 601 TTGTTGGTGG TGTCGCTGTT TTGGGCGGTTTGCGGACGTA
TGAGATTGTG

CACGTATTGA

GCACAGACGA

TTCGCAATTT

TTTTCGTTTA

S TTGGGTGGTA

TCAGTCCGAT

GACAACCAGT

GGCGATGGCG

CGGCTTATGT

CAGCAGGGGG

GATTGGCGCA

TTGCGTTTAA

GCGCAGTTGT

GGGGGATATT

IS TGCCCGATAT

AAGGCGGACG

GGGGGAAGTG

TCACCAAATT

GTGGACGGCA

GGTCGGCGTG

ACAATATCGC

GCAGCCAAAC

CTACGGCACC

GGCAGCGTAT

ATTTTTGATG

TATGCAGAAC

CCCACCGTCT

AAAGGCAGGA

GAACGGATAT

30 This encodes a pmtein having amino acid sequence <SEQ ID 30>:

- IiGIAANPADI HEFCTSAQS DLNETQWLLA

DGGGEHAQYL

VLSFAEFSNR
YSGKLILVAS
RASVLGSLAK
FDFTWFIPAV

ALITPLFFQV
VMDKVLVHRG
FSTLDWSVA

LGGLRTYLFA
HTTSRIDVEL
GARLFRHLLS
LPLSYFEHRR

LEQIRNFLTG
QALTSVLDLA
FSFIFLAVMW
YYSSTLTWW

SAFISPILRT
RLNDKFARNA
DNQSFLVESI
TAVGTVKAMA

NQLAAYVASG
FRVTKLAWG
QQGVQLIQKL
VTVATLWIGA

4O GQLIAE1~MLS
GQVAAPVIRL
AQLWQDFQQV
GISVARLGDI

HLALPDIRGE
ITFEHVDFRY
KADGRLILQD
LNLRIRAGEV

KSTLTKLVQR
LWPAQGRVL
VDGNDLALAA
PAWLRRQVGV

SIRDNIALTD
TGMPLERIIE
AAKLAGAHEF
IMELPEGYGT

GGQRQRIAIA
RALITNPRIL
IFDEATSALD
YESERAIMQN

LIIAHRLSTV
KTAHRIIAMD
KGRIVEAGTQ
QELLAKPNGY

ORF39a is homologous to a cytolysin from A.pleuropneumoniae:
spIP267601RT1B ACTPL RTX-I TOXIN DETERMINANT B (TOXIN RTX-I SECRETION ATP-BINDING PROTEIN) (APX-IB) (HLY-IB) (CYTOLYSIN IB) (CLY-IB) >gi1971371pirllD93599 cytolysin IB - Actinobacillus pleuropneumoniae (serotype 9) SO >gi138994 (X61112) ClyI-B protein [Actinobacillus pleuropneumonise] Length = 707 Score = 931 bits (2379), Expect = 0.0 Identities = 472/690 (68%), Positives = 590/690 (77%), Gaps = 3/690 (0%) Query: 20 YHGIAANPADIQHEFCTSAQSDLNETQWXXXXXXXXXXXXVVRQPIKRLAMATLPALVWC 79 SS YH IA NP +++H+F + L+ T W V++ I RLA LPALVW
Sbjct: 20 YHNIAVNPEELKHKFDLEGKG-LDLTAWLLAAKSLELKAKQVKKAIDRLAFIALPALVWR 78 Query: 80 DDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNRYSGKLILVASRASVLGSLA 139 +DG HFIL K D E +YLI DL T+ +L AEF + Y GKLILVASRAS++G LA
60 Sbjct: 79 EDGKHFILTKIDN--EAKKYLIFDLETHNPRILEQAEFESLYQGKLILVASRASIVGKLA 136 Query: 140 KFDETWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 199 KFBFTWFIPAVIKYR+ ITPLFFQVVMDKVLVHRGF
Sbjct: 137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQWMDKVLVHRGFSTLNVITV 196 Query: 200 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSZ,PLSYFEHRRVGDTVARVR 259 FEIVL GLRTY+FAH+TSRIDVELGARLFRHLL+LP+SYFE+RRVGDTVARVR
Sbjct: 197 ALAIWLFEIVLNGLRTYIFAHSTSRIDVELGARLFRHLLALPISYFENRRVGDTVARVR 256 Query: 260 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWWLASLPAYAFWSAFISPILR 319 S EL+QIRNFLTGQALTSVLDL FSFIF AVMWYYS LT V+L SLP Y WS FISPILR
Sbjct: 257 ELDQIRNFLTGQALTSVLDLMFSFIFFAVMWYYSPKLTLVILGSLPFYMGWSIFISPILR 316 Query: 320 TRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWDNQLAAWASGFRVTKLAW 3?9 RL++KFAR ADNQSFLVES+TA+ T+KA+AV PQMT WD QLA+W++GFRVT LA +
lO Shjct: 317 RRLDEKFARGADNQSFLVESVTAINTIKALAVTPQMTNTWDKQLASYVSAGFRVTTLATI 376 Query: 380 GQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLSGQVAAPVIRLAQLWQDFQQ 439 GQQGVQ IQK+V V TLW+GA LVI L++GQLIAFNMLSGQV APVIRLAQLWQDFQQ
Sbjct: 377 GQQGVQFIQKVVMVITLWLGAHLVISGDLSIGQLIAFNMLSGQVIAPVIRLAQLWQDFQQ 436 Query: 990 VGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRYKADGRLILQDLNLRIRAGE 499 VGISV RLGD+LN+pTE+ LALP+I+G+ITF ++ FRYK D +IL D+NL I+ GE
Sbjct: 937 VGISVTRLGDVLNSPTESYQGKLALPEIKGDITFRNIRFRYKPDAPVILNDVNLSIQQGE 996 20 Query: 500 VLGIVGRSGSGKSTLTKLVQRLWPAQGRVLVDGNDLALAAPAWLRRQVGWLQENVLLN 559 V+GIVGRSGSGKSTLTKL+QR Y+P G+VL+DG+DLALA P WLRRQVGWLQ+NVLLN
Sbjct: 497 VIGIVGRSGSGKSTLTKLIQRFYIPENGQVLIDGHDLALADPNWLRRQVGWLQDNVLLN 556 Query: 560 RSIRDNIALTDTGMPLERIIEAAKLAGAHEFIMELPEGYGTWGEQGAGLSGGQRQRIAI 619 ZS RSIRDNIAL D GMP+E+I+ AAKLAGAHEFI EL EGY T+VGEQGAGLSGGQRQRIAI
Sbjct: 557 RSIRDNIALADPGMPMEKIVHAAKLAGAHEFISELREGYNTIVGEQGAGLSGGQRQRIAI 616 Query:'620 ARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTVLIIAHRLSTVKTAHRIiAM 679 ARAL+ NP+ILIFDEATSALDYESE IM+NM IC RTV+IIAHRLSTVK A RII M
3O Sbjct: 617 ARALVNNPKILIFDEATSALDYESEHIIMRNMHQICKGRTVIIIAHRLSTVKNADRIIVM 676 Query: 680 DKGRIVEAGTQQELLAKPNGYYRYLYDLQN 709 +KG+IVE G +ELLA PNG Y YL+ LQ+
Sbjct: 677 EKGQIVEQGKHKELLADPNGLYHYLHQLQS 706 Homolottv with the HIvB leucotoxin secretion ATP-binding protein of Haemophilus actinomvcetemcomitans taccession number XS39SS~
ORF39 and HIyB protein show 71% and 69% amino acid identity in 167 and SS
overlap at the N-and C-terminal regions, respectively:
40 Orf39 1 KFDFTWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 60 KFDFTWFIPAVIKYR+ ITPLFFQWMDKVLVHRGF
HlyB 137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQVVMDKVLVHRGFSTLNVITV 196 Orf39 61 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSLPLSYFEHRRVGDTVARVR 120 4S FEI+LGGLRTY+FAH+TSRIDVELGARLFRHLL+LP+SYFE RRVGDTVARVR
HlyB 197 ALAIWLFEIILGGLRTWFAHSTSRIDVELGARLFRHLLALPISYFEARRVGDTVARVR 256 Orf39 121 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWWLASLIC 167 EL+QIRNFLTGQALTS+LDL FSFIF AVMWYYS LT WL SL C
SO HlyB 257 ELDQIRNFLTGQALTSILDLLFSFIFFAVMWYYSPKLTLWLGSLPC 303 //
Orf39 166 ICANRTVLIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLANXNGYYRYLYDLQ 220 SS IC NRTVLIIAHRLSTVK A RII MDKG I+E G QELL + G Y YL+ LQ
HlyB 651 ICQNRTVLIIAHRLSTVKNADRIIVMDKGEIIEQGKHQELLKDEKGLYSYLHQLQ 705 Based on this analysis, it is predicted that this protein from N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
Example 7 60 The following partial DNA sequence was identified in N. meningitides <SEQ
ID 31 >

r79,~

151 GACGGGTTGA ACGCCCAAAk sGACGCCGAA ATCAGA...
This corresponds to the amino acid sequence GSEQ ID 32; ORF52>:
1 MItYLIRTALL AVAAAGIYAC QPQSEAAVQV KAENSLTAMR LAVADKQAEI
51 DGLNAQXDAE IR..
Further work revealed the complete nucleotide sequence <SEQ ID 33>:

This corresponds to the amino acid sequence <SEQ ID 34; ORF52-1>:

51 DGLNAQIDAE IRøREAEELK DYRWIHGDAE VPELEK*
Computer analysis of this amino acid sequence predicts a prokaryotic membrane lipoprotein lipid attachment site (underlined).
ORF52-1 (7kDa) was cloned in the pGex vectors and expressed in E.coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE.
Figure 4A shows the results of affinity purification of the GST-fusion. Figure 4B shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF52-1.
Based on this analysis, it is predicted that this pmtein from N. meningitides, and its epitopes, could be useful antigens for vaccines or diagnostics.
Eiample 8 The following DNA sequence was identified in N.meningitidis <SEQ ID 35>

51 TCTATTGTTA AATCCCGTCT TCCATGCATC CAGTTGCGTA TCGCGTTsGG
3O 101 CAATACGGAA TAAAAtCTGC TGTTCTGCTT TGGCTAAATT TGCCAAATTG
151 TTTATTGTTT CTTTAGGaGC AGCTTGCTTA GCCGCCTTCG CTTTCGACAA
201 CGCCCCCACA GGCGCTTCCC AAGCgTTGCC TACCGTTACC GCACCCGTGG

This corresponds to the amino acid sequence <SEQ ID 36; ORF56>:

51 FIVSLGAACL AAFAFDNAPT GASQALPTVT APVAIPAPAS AA*
Further work revealed the complete nucleotide sequence <SEQ ID 37>:

TTAAATCCCG

GAATAAAATC

TTTCTTTAGG

ACAGGCGCTT

CGCGCCCGCT

This corresponds to the amino acid sequence <SEQ ID 38; ORFS6-1>:

51 CCSALAKFAK LFIVSLGAAC LAAFAFDNAP TGASQ~LPTV TAPVAIPAPA
101 SAA*
Computer analysis of this amino acid sequence predicts a leader peptide (underlined) and suggests that OIZFS6 might be a membrane or periplasmic protein.
Based on this analysis, it is predicted that this protein from N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
1 S Example 9 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
39>

CTTGTGTAGT

TTTTACTTAT

TTTTTTCTTA

GGCATGACCC

CCTCAGCCTC

251 CAGGG...

This corresponds to the amino acid sequence <SEQ ID 40; ORF63>:
1 MFSILNVFLH CILACWSGE TPTIFGILAL FYLLYLSYLA VFKIFFSFFI.
2S 51 DRVSLRSPRL ECKWHDPLAH WLTATSAILP PQPPG...
Computer analysis of this amino acid sequence predicts a transmembrane region.
Based on this analysis, it is predicted that this protein from N.
meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
Example 10 30 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
41>
1 ..GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTGTT

This cowesponds to the amino acid sequence <SEQ ID 42; ORF69>:
1 ..VRTWLVEWLQ RLKYPLLLWI ADMLLYRLLG GAEIECGRCP VPPMTDWQHF
51 LPAMGTVSAW VAVIWAYLMI ESEKNGRY*

_81 Computer analysis of this amino acid sequence predicts a transmembrane region.
A corresponding ORF from strain A ofN.meningitidis was also identified:
Homolotav with a predicted ORF from N menin,~itidis (strain A) ORF69 shows 96.2% identity over a 78aa overlap with an ORF (ORF69a} from strain A of N.
meningitides: ' orf69.pep VRTWLVFWLQRLKYPLLLWIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPAMGTVSAW
IIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIillllll:1111:11 orf69a VRTWLVFWLQRLKYPLLLCIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPTMGTVAAW

orf69.pep VAVIWAYLMIESEIQ~IGRYX
IIIIlllllllllllllll 1S orf69a VAVIWAYLMIESEIQdGRYX
The ORF69a nucleotide sequence <SEQ ID 43> is:

This encodes a protein having amino acid sequence <SEQ ID 44>:

2S 51 LPTMGTVAAW VAVIWAYLMI ESEKNGRY*
Based on this analysis, it is predicted that this protein from N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
Example 11 30 The following DNA sequence was identified in N. meningitides <SEQ ID 45>

TCCTCCCCGT

ACGGCGCGCT

ACTGAACCCC

151 CTGCCCCATA TCGATTTGGT CGGCACAATC ATCgTACCGC

CCTATCGATT

251 CGCGCAAGTT CCGCAACCCG cGCCTTGCCT GGCGTTGCGT
TGCCGCGTCC

301 GGCCCGCTGT CGAATCTAGC GATGGCTGTw CTGTGGGGCG
TGGTTTTGGT

GCTCAAATGG

TCCTGTCGGC

ACGTGGATTA

551 TCCTACTGCT GATGCTGACC sGGGTTTTGG GTGCGTTTAT
wGCACCGATT

601 sTGCGGmTGc GTGATTGCrT TTGTGCAGAT GTwCGTCTGA
CTGGCTTTCA

45 This corresponds to the amino acid sequence <SEQ ID 46; ORF77>:

5I LPHIDLVGTI IVPLLTLMFT PFLFGWARPI pIDSRNFRNP RLAWRCVAAS

201 XRXRbCXCAD VRLTGFQTA*
Further work revealed the complete nucleotide sequence <SEQ ID 47>:

TCCTGCCCGT

ACGGCGCGCT

IO ACTGAACCCC

TGCTTACTTT

CCTATCGATT

TGCCGCGTCC

TGGTTTTGGT

IS GCTCAAATGG

CAACATCATC

TCCTGTCGGC

ACGTGGATTA

TGCACCGATT

20 This corresponds to the amino acid sequence <SEQ ID 48; ORF77-1>:

51 LPHIDLVGTI IVPLLTLMFT PFLFGWARPI PIDSRNFRNp RLAWRCVAAS
101 GPLSNLAMAV LWGWLVLTP YVGGAYQMpL AQMANYGILI NAILFALNII

2S 201 VRLVIAFVQM FV*
Computer analysis of this amino acid sequence reveals a putative leader sequence and several transmembrane domains.
A corresponding ORF from strain A of N. meningitides was also identified:
Iiomolo>zv with a predicted ORF from N meningitides (strain Al 30 ORF77 shows 96.5% identity over a 173aa overlap with an ORF (ORF77a) from strain A of N.
meningitides:

orf77.pep MFQNFDLGVFLLAVLPVLPSITVSHVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
~~~~~~~~ii~i~~~~lllllllllllllllll 3S orf77a RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI

orf77.pep IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGWLVLT
P

40 ~Illllll 1111 111111IllllllllllllllllllllllllllillilIII 1 orf77a 11 IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLT

_ P

130 140 i50 160 170 orf77.pep _YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPY

G

_ orf77a IIIIIIIIilllllll 1111111 IIIIIIIflllllllllllllll IIIIlll1111 _YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAIQCSQAFRKIEPY

G

orf77.pep TWIILLLMLTXVLGAFIAPIXRXRDCXCADVRLTGFQTAX
Ilil IIIII 1111 1111 orf77a TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX

ORF77-1 and ORF77a show 96.8% identity in 185 as overlap:

orf77-1. pep MFQNFDLGVFLLAVLPVLLSITVREVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
~ii~iiiiiiiliillllllillllllllllll S orf77a RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI

orf77-1. pep IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGWLVLTP
1~ IIIIIIIIIIIIIIIIIIIIilllllllllIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
orf77a IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGWLVLTP

ZS orf77-1. pep YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPYG
IIIIIIIIIIIIIIII 1111111 IIIIIIIillllllillllllll 11111111111 orf77a YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAKXSQAFRKIEPYG

l90 200 210 orf77-1. pep TWIILLLMLTGVLGAFIAPIVRLVIAFVQMFVX
IIII IIIIIIiII) IIIII:IIIIIIIIIII
orf77a TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX

A partial ORF77a nucleotide sequence <SEQ ID 49> was identified:
1 ..CGCGGCTATA CAGCGCGCTA CTGGGGTGAC AACACTGCCG
AACAATACGG

GGCACAATCA

GCCTTGCCTG

ATGGCTGTTC

GGCGTATCAG

ATGCGATTCT

CAAAATCGAA

GGGTTTTGGG

GTGCAGATGT

This encodes a protein having amino acid sequence <SEQ ID SO>:
4O 1 ..RGYTARYWGD NTAEQYGRLT LNPLPHIDLV GTIIVPLLTL MFTPFLFGWA

151 PYGTWIIXLL MLTGVLGAXI APIVQLVIAF VQMFV*
Based on this analysis, it is predicted that this protein from N.
meningitides, and its epitopes, could 4S be useful antigens for vaccines or diagnostics.
Eaample 12 The following partial DNA sequence was identified in N.meningitidfs <SEQ ID
S1>

451 31~F11'~GAAAAAA ACAGCGTGAT CAATGTGCGC GAAATGTTGC CCGACCAT..
This corresponds to the amino acid sequence <SEQ ID S2; ORF112>:

-51 GYTALKMPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK ASGMSTKK_LL

151 KEKNSVINVR EMLPDR...
Further work revealed further partial nucleotide sequence <SEQ ID S3>:

TTATGGCGGT

GAAATCCTGT

GGAAATGCTG

151 gGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC
TGATTCCCCT

GCCGCCGGCA

AAAGCTGCTG

CCGTCGCGCT

AACATCAAAG

401 CCGCCGCCAT CAACGGCAAA A~'CAGCACCG GCAATACCGG
CCTTTGGCTG

451 AAAGAAAAAA ACAGCrTkAT CAATGTGCGC GAAATGTTGC
CCGACCATAC

GAATTGGCAG

CAGTTGGCAG

TCGAGGTCTC

AACCTGATGG

ACTGACCACC

TCTACGCCAT

GTGATGGCGC

CAATATGGGC

901 TTAAAACTCT TCGGCGGCAT CTGTsTCGGA TTGCTGTTCC
ACCTTGCCGG

951 ACGGCTCTTT GGGTTTACCA GCCAACTCGG...

This corresponds to the amino acid sequence <SEQ ID S4; ORF112-1>:

QMAVMAVYAL
LAFLALYSFF
EILYETGNLG
KGSYGIWEML

AYELIP IGGLVSLSQL
AAGSELTVIK
ASGMSTKKLL

AIATVA VAPTLSQKAE
NIKAAAINGK
ISTGNTGLWL

EMLPDIiTLLG
IKIWARNDKN
ELAEAVEADS
AVLNSDGSWQ

EDKVEVSIAA
EENWPISVKR
NLMDVLLVKP
DQMSVGELTT

NTRIYAIAWW PQTTRHGNM
RKLVY G

301 LKLFGGICXG _ LLFHLAGRLF L...
GFTSQ

Computer analysis of this amino acid sequence predicts two transmembrane domains.
A corresponding ORF from strain A of N. meningitides was also identified:
HomoloQV with a predicted ORF from N. meningitidislstrain A,~
40 ORF 1 I 2 shows 96.4% identity over a 166aa overlap with an ORF (ORF 112a) from strain A of N.
meningitides:

orf112.pep MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMPAR
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIfllllllllllll 1111111 II
4S orf112a MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR

orf112.pep AYELIPLAVLIGGLVSLSQLAAGSELTVIKASGMSTKKLLLILSQFGFIFAIATVALGEW
SO IIII:Iillllfllll IIIIIIIII:IIIIIIIIIIIIIIIIIIillilllllllllll orf112a AYELMPLAVLIGGLVSXSQLAAGSELXVIKASGMSTKKLLLILSQFGFIFAIATVALGEW

130 , 190 150 160 orf112.pep VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSVINVREMLPDH
IIIIIII1111111111111IIIIillllllllll:llllllllll orf112a VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN

orf112a ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP

A partial ORF112a nucleotide sequence <SEQ ID 55> was identified:

TTATGGCGGT

GAAATCCTGT

GGAAATGNTG

TGATGCCCCT

IS GCCGCCGGCA

AAAGCTGCTG

CCGTCGCGCT

AACATCAAAG

CCTTTGGCTG

501 CCTGCTGGGC ATTAAAATCT GGGCCCGCAA CGATAAAA,AC
GAACTGGCAG

CAGTTGGCAG

TCGAGGTCTC

AACCTGATGG

TCTACGCCAT

GTGATGGCGC

CAATATGGGC

ACCTTGGCGG

NCGGCGCACT ACCTACCATA GCCTTCGCCT TGCTCGCCGT

This encodes a protein having amino acid sequence <SEQ ID 56>:

EILYETG

NIKAAAINGK ISTGNTGLWL

ELAEAVEADS AVLNSDGSWQ

NLMDVLLVKP DQMSVGELTT

VMALVAFAFT pQTTRHGNMG

PFLXGALPTI AFALLAVWLI

40 351 RKQExR*

ORF112a and ORF112-1 show 96.3% identity in 326 as overlap:
orf112a.pep MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
orf112-1 IIIIiII II
MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMPAR

orf112a.pep AYELMPLAVLIGGLVSXSQLAAGSELXVIKASGMSTKKLLLILSQFGFIFAIATVALGEW

IIII:IIIIIIIIIII IIIIIIIII:IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
orf112-1 AYELIPLAVLIGGLVSLSQLAAGSELTVIKASGMSTKKLLLILSQFGFIFAIATVALGEW

SO orf112a.pep VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN

Illllllllllllllllllillllllillllllll illlllllllllllllllllllll orf112-1 VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSXINVREMLPDHTLLGIKIWARNDKN

orf112a.pep ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP
55 IIIIIII1 illlllllIIIIIIIIIIIIIIIiIIIIiIII IIIIIIIIIIIIIIIII
orf112-1 ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEENWPISVKRNLMDVLLVKP
orf112a.pep DQMSVGELTTYIRHLQXXSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG
IIIIIIIillllllll IIIIIIIIIIIIilllllllllllllllllllllilllllll 6O orfll2-1 DQMSVGELTTYIRHLQNNSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG
orf112a.pep LKXFGGICLGLLFHLAGRLFXFTSQLYGIPPFLXGALPTIAFALLAVWLIRKQEKRX
II IIIII IIIIIIIIIII IIIiI

orf112-1 LKLFGGICXGLLFHLAGRLFGFTSQL
Based on this analysis, it is predicted that this protein from N.
meningitides, and its epitopes, could be useful antigens for vaccines or diagnostics.
S Example 13 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
S7>
1 ..GCAGTAGCCG AAACTGCCAA CAGCCAGGGC AAAGGTAAAC
AGGCAGGCAG

GGCAAACTCA

IO GAGTATGGTA

CTAAAAACCA

GTGAATATCC

251 AAACTCCGAA TGGACGCGGA TTGAGCCACA ACCGCTA.TA
CGCATTTGAT

ATAATCCGTT

IS CGCGGTACGG

GGCCGACGTG

GCTTTAAAAA

GGCAAAGACG

551 GTGCACTGAC AGGATTTGAT GTG~GTCAAG GCACATTGgA
CCGTAGrAGC

601 AGCAGGTTGG AATGATAAAG GCGGAGCmrm yTACACCGGG
GTACTTGCTC

651 GTGCAGTTGC TTTGCAGGGG AAATTwtt~dGG GTAAA.AACT
GGCGGTTTCT

GTGCAGGTAC

GCACTGGGCG

AGGCGTAGGC

2S This corresponds to the amino acid sequence <SEQ ID S8; ORF114>:
1 ..AVAETANSQG KGKQAGSSVS VSLKTSGDLC GKLKTTLKTL
VCSLVSLSMV

LSHNRXYAFD

VTVGGQKADV

YASGEISAGT

251 AAGTKPTIAL DTAAZ,GGMYA DSITLIANEK GVGV*

Further work revealed the complete nucleotide sequence <SEQ ID S9>:

ACAGCACCAT

TTGCGGCAAA

CCCTGAGTAT

GCACCTAAAA

CTTGGTGAAT

AACAATAATC

GGTACGCGGT

AAAAGGCCGA

GGCGGCTTTA

TGACCGTAGG

GGGGTACTTG

CCTGGCGGTT

TCAGTGCAGG

SO GCCGCACTGG

AAAAGGCGTA

TGATTGTGAC

ACTGCCGACG

CGAAAAAGGA

SS GCAAAGGCTT

GGAGCCGTGG

TGCTGGTCAT

AAGGCCCGGC

_87_ AGTATTCAGA

ATTAGGCAAT

ACGGCACCAT

S ATCGAAGCAG

TATCCGCTTA

TGGCAGACGA

AATCTGTATG

TTTGTCTGCC

IO CCGGCACCAG

GGCTCGCTGA

GCACATTCAG

ACGCAGCCAA

GACGGCCTTC

IS CGGTAATGCC

TCAATGCAGG

ATCACTTCAT

GCTTGGTGAC

TCAAAAACAA

2151 ~CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC
ZO AAAAGCGGGG

TACCAAGCTG

GGGTAACGCT

ACCGGCAGCC

GGTGGCTAAC

ZS ACAACACCAC

CTAGTCAAGC

GGAAGATAAT

CAGGTAGCGG

ACCGACCTGA

GCAATATCCG

ACAGCCGGTA

CGAAGCCGTA

CTGAACTCAA

2901 CCAAAAATCC AAA('~AATTGG AACAGCAGAT TGCGCAGTTG

CGACCGTCTC

AAAAACCCAA

ATTGACTTGA

CGCTTCCAAA

GAAATTGGCA

CAAGCCTTCA

CGGCACTCGA

CCCTCAGGCA

-AACCAAAGGT AAAAGCGGCA AAATCATCAG

CCAGCCCCCG

CAACATCGAA

CCCTGGTTGC

SO AAGCACGAGT

AGGCAAGAGC

TCCGCGTCGT

CTCGAAGGTA

AGGTGTAGGC

SS TTGTGAACCG

TGGCAGAAAC

CAGCTTCGAA

TCGTCGACAT

AAACAGCCCG

E)O CAACTGGAAC

AAGGCTTAAC

CTGACTTATG

AAGTAGTACA

CTACTACCGT

E)S TTGTATAGCC

CAAAGCGTTG

CTTCTGCCCT

CAATTGAACA

AACTATTGCC

AACTTAGGCA

AGCCGCCAGC

AGTTCGCCCA

_88_ TGTAAAGACG

CATGCTTGGC

AGGTTATCAG

S GGCGGCGATG

TAATGCTTTG

CGCAGAAGCC

CCTGCACATG

CATTTGGATA

lO CCAGCTATGG

GCGGGTAAAT

TGTCATGGTC

CGGTAACGGT

GCGAAGGCGG

IS TGGCTTACTA

ATATAGCGAA

CCGGTATCTG

TATTGCGAAT

TTATACTTCA

ZO GATGGCCAAT

TTCTATTAAA

ATAAGTCAGG

This corresponds to the amino acid sequence ~SEQ ID 60; ORF114-1>:

ZS LKTSGDLCGK

KTNTGAPLVN

AQLILNEVRG

LTTGAPQIGK

GKLQGKNLAV

ITLIANEKGV

3O . 301 GVKNAGTLEA AKQLIVTSSG RIENSGRIAT TADGTEASPT
YLSIETTEKG

PATTVLNAGH

SSKGNAELGN

ASTVTSDIRL

NLNVDKDLSA

RTNSGNLHIQ

HVSLLANGNA

VAGNGIQLGD

RALSIENTKL

TVSTKTLEDN

LLLSAKGGNA

TKGKLNIEAV

PTLQEERDRL

ISGSDITASK

YDKAALNKpS

SDIVLEAGQN

ITLQAGGNIE

RFIGIKVGKS

AGADIQAGVG

IETLKLPSFE

LQVAKNVNWN

GGVAASGSST

NNKGDVGKAL

FSSTGNQTIA

VNSFQGEAAS

GEIVADSMLG

AEVAWNNAL

PQDKDAAIWI

ANPSGCTVMV

QTVKELDGLL

EGHFHRPIAN

GKVIGTTSIK

1951 EGGQPTTTIK VFTDKSGNLI TTYPVKGN*

Computer analysis of this amino acid sequence predicts a transmembrane region and also gives the 6S following results:

Homoloev with a predicted ORF from rreenin 'tidis stain Al, ORF 114 shows 91.9% identity over a 284aa overlap with an ORF (ORF I 14a) from strain A of N.
meningitidis:

S orf114.pep AVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
Illllllllllllillilllllllllllllllllllllilll orfil4a MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC

orf114.pep SLVSLSMVLPAfiAQITTDKSAPKNQQWILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVD
1111111 IIIIIIIIIII Illllllllllllllllillllllllll IIII

orf114a SLVSLSMXX7CXXXQITTDKSAPKNXQWILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD

orf114.pep NKGAVLNNDRNNNPFVVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG
IIIIIIIIIIIIiII:IIIIIIIIIIIillllllllllllllllllllllllllllllll orf114a NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG

orf114.pep GGFKNVGRGILTTGAPQIGKDGALTGFDWKAHWTVXAAGGPNDKGGAXYTGVLARAVALQ
I
II
I
I

ZS orf114a IIIIIIIIIIII IIIIIIIIIIIIIIII : II IIIII
Ii IIII
IIIIII
GGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWtJDKGGADYTGVLARAVALQ

orf119.pep GKXXGKXLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIANEKGV

n 11 11111111 11111111 1111111111111111111111111111 orf114a GKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV

3$ orf114.pep GVX
II
orfil9a GVKNAGTLEAAKQLIVTSSGRIENSGRIATTADGTEASPTYLXIETTEKGAXGTFISNGG

The complete length ORF 114a nucleotide sequence <SEQ ID 61 > is:

ACAGCACCAT

AAACAGGCAG

TTGCGGCAAA

CCCTGAGTAT

GCACCTAAAA

CTTGGTGAAT

ATACGCAGTT

AACAATAATC

GGTACGCGGT

AAAAGGCCGA

GGCGGCTTTA

AATCGGCAAA

TGACCGTAGG

GGGGTACTTG

CCTGGCGGTT

TCAGTGCAGG

GCCGCACTGG

AAAAGGCGTA

TGATTGTGAC

ACTGCCGACG

E)0 1001 GCACCGAAGC TTCACCGACT TATCTNNCNA TCGAAACCAC
CGAAAAAGGA

GCAAAGGCTT

GGAGCCGTG~' TGCTGGTCAT

AAGGCTCGNC

ACTATTCAAG

NTTGGGTGAA

ACGGTAGTAT

ATTGAATCGG

CATCCGTTTG

TGGCAGACGA

AATCTGTATG

TTTGTCTGCC

IO CCGGCACCAG

1701 TAAAACCC,TC ACTGCCTCAA AAGACATGGG TGTGGAGGCA
GGCTTGCTGA

GCACATTCAG

ACGCAGCCAA

GACGGCCTTC

CGGTAATGCC

TCNATGCAGG

ATCACTTCAT

GCTTGGTGAC

TCAAAAACAA

AAAAGCGGGG

TACNAAGCTG

GGGTAACGCT

ANCGGCAGCC

GGTGGCTAAC

ACAACACCAC

CTAGTCAAGC

GGAAGATAAT

CAGGTAGCGG

ACCGACCTGA

AGGAAATGCA

GCAATATCCG

ACAGCCGGTA

CGAAGCCGTA

NNGNNCTCAA

CGACCGTCTC

AAAAACCCAA

ATTGACTTGA

CGCTTCCAAA

GAAATTGGCA

CAAGCCTTCA

CGGCACTCGA

CCCTCAGGCA

NAATNATCAG

CCAGCCCCNG

CAACATCGAA

3601 GCTAATACCA CCCGCTTCAA TGCCCCTGCA.GGTAAAGTTA
CCCTGGTTGC

SO AAGCACGAGT

AGGTNAGAGC

TCCGCGTCGT

CTCGAAGGTA

AGGTGTANGC

TTGTGAACCG

TGGCAGAAAC

CAGCTTCGAA

TCGTCGACAT

AAACAGCCCG

4151 AGTATGCCTA TCTGAAACAG CTCCAAGTAG CGAAAAP,CAT
CAACTGGAAT

E)O 4201 CAGGTGCAGC TTGCTTACGA CAGATGGGAC TACAAACAGG
AGGGCTTAAC

GTCACCTCAG

CGCCGCCGCA

TATCGTTCAT

GGCAGAAGCA

C)S 4451 GCACGGTGAA AAATCTGGTG GTTGCCGCCG CTACCGCAGG
CGTAGCCGAC

AGTGGATCAA

ACTGAttaa This encodes a protein having amino acid sequence <SEQ ID 62>:
1 MNKGLIiRIIF SKKHSTMVAV AETANSQGKG KQAGSSVSVS LKTSGDLCGK

KTNTGAPLVN

AQLILNEVRG

LTIGAPQIGK

S GKLQGKNLAV

ITLIAXEKGV

YLXIETTEKG

PATTVI,NAGH

STKGDTXLGE

lO TSTVASNIRL

NLNVDKDLSA

RTNSGNLHIQ

HVSLLANGNA

VAXXGIQLGD

701 GKQRNSINGK HISIKNNGGN,ADLKNLNVHA KSGALNIHSD
IS RALSIENTKL

LPSANKLVAN

TVSTKTLEDN

LLLSAKGGNA

TKGKLNIEAV

ZO PTLQEERDRL

1001 AFYIQAINKE VKGKKPKGKE YLQ,AKI,SAQN IDLISAQGIE
ISGSDITASK

YDKAALNKPS

1101 RLTGRTGVSI HAAAAI,DDAR IIIGASEIKA PSGSIDIKAH
SDIVLEAGQN

ITLQAGGNIE

ZS RFIGIKVGXS

AGADIQAGVX

IETLKLPSFE

1.351 SPTPPKLSAP GGYIVDIPKG NLKTEIEKLS KQP
EYAYLKQ LQVAKNINWN

1401 _ QVQLAYDRWD YKQEGLTEAG AAIIALAVTV VTSGAGTGAV
LGLNGAXAAA

1501 KIGASALXNV SDKQWINNLT VNLANXGQCR TD*

ORF114-1 and ORF114a show 89.8% identity in 1564 as overlap orf114a.pep MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC

1111111(illllillilllllllllllllillltilllllllllillllllllllllll orf114-1 :~T~GLHRIIFSKKHSTMVAVAETAtJSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLV

C

orf119a.pep SLVSLSMXXXXXXQITTDKSAPKNXQWILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD

1111111 IIIIIIIIIII IIIlttll111111111111111111111111111 orf114-1 SLVSLSMVLPAHAQITTDKSAPKNQQWILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD

4O orf114a.pep NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG

IIIIIIII1111111:IIIIIIIIIIIIIilllllllllllllllltlllllllllllll orf114-1 NKGAVLNNDRNNNPFWKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG

orf119a.pep GGFKNVGAGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ
4S I tllllil ll 1111111I~IIIIIIIIIIII IIIIlIIllII1111111111111 orf119-1 GGFKNVGRGILTTGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ

orf114a.pep GKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
SO orf119-1 IIII
GKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIA

NEKGV

orf114a.pep GVKNAGTLEAAKQLIVTSSGRIENSGRIATTADGTEASPTYLXIETTEKGAXGTFISNGG

orf114-1 IIIIIIII
GVKNAGTLEAAKQLIVTSSGRIENSGRIATTADGTEASPTYLSIETTEKGAAG

TFISNGG
SS

orf119a.pep RIESKGLLVIETGEDIXLRNGAWQNNGSRPATTVLNAGHNLVIESKTNVNNAKGSXNLS

iillllllilllllll II.Illlllllllllllllillltlllllllllllllll orf114-1 :II
RIESKGLLVIETGEDISLRNGAWQNNGSRPATTVLNAGHNLVIESKTNVNNAKGPATLS

6O orf119a.pep AGGRTTINDATIQAGSSVYSSTKGDTXLGENTRIIAENVTVLSNGSIGSAAVIEAKDTAH

I Ill:l::t:ll:l::llll:ll:: 11:1111 : :IIIIIII:I:I:III:IIIIII
orf114-1 ADGRTVIKEASIQTGTTVYSSSKGNAELGNNTRITGAOVTVLSNGTISSSAVIDAKDTAH

orf114a.pep IESGKPLSLETSTVASNIRLNNGNIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL
6S 1 :11111 11:111:1:1111:1:1 lltllllllllllilllliltllllllllllll orf119-1 IEAGKPLSLEASTVTSDIRLNGGSIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL

orf114a.pep NLNVDKDLSAASIHLKSDNAAHITGTSKTLTASKDMGVEAGLLNVTNTNLRTNSGNLHIQ
11111111Iltlllllllltlillllllllllllllll111 IIIillilllllllllll orf114-1 NLNVDKDLSAASIHLKSDNRAHITGTSKTLTASKDMGVEAGSLNVTNTNLRTNSGNLHIQ
orf114a.pep AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA
III(iilllllllllllllllllflllllllllllllllllllllllillllllllllll orf114-1 AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA
orf114a.pep KADVXAGSVGKGRLKADNTNITSSSGDITLVAXXGIQLGDGKQRNSINGKHISIKNNGGN

1111 IIIIIIIIIIIIIIIIIIillllllll IllllIIIIIIIIIIIIIIIIIIIII
orf119-1 KADVNAGSVGKGRLKADNTNITSSSGDITLVAGNGIQLGDGKQRNSINGKHISIKNNG

GN

orf114a.pep ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI

IIIIIIII-11111111111111111111111111111111111111111111illlllll orf114-1 ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI

IS orf119a.pep XGSQIWQNDKLPSANKLVANGVLAXNARYSQIADNTTLRAGAINIaTAGTALVKRGNINWS
:IIII1 orf114-1 1111111111111111 IIIIIIIIIIIilllllllllllllllllllllll TGSQIWQNDKLPSANKLVANGVLALNARYSQIADNTTLRAGAINLTAGTALVKRGNINWS

orf114a.pep TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA
Il llllillililllllllllllilllllllllilllllllllllll1111111111111 orf114-1 TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA

orf119a.pep GAXSAQVSSLEAKGNIRLVTGXTDLRGSKITAGKNLWATTKGKLNIEAVNNSFSNYFXT

il IIIIilllilllllllll IIIIIIIIillllllllllllll1111111111111 2S orf114-1 I
GAPSAQVSSLEAKGNIRLVTGETDLRGSKITAGKNLWATTKGKLNIEAVNNSFSNYFPT

orfil4a.pep QKXXXLNQKSKELEQQIAQLKKSSXKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKGKE

11 IIIIIIIIIIIIIIIiIII Ilillliliilllllilillllllllllllllllf orf114-1 QKAAELNQKSKELEQQIAQLKKSSPKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKG

KE

orf119a.pep YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY

11111111111111111fllllllllllllilllllllllllllllllllllllllllil orfil4-1 YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY

3S orf114a.pep EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH

IIIIIIIIIIIIIIIIIiIIIlIIIIIIIIII1111111111-Illllillllllllllll orf114-1 EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH

orf114a.pep SDIVLEAGQNDAYTFLXTKGKSGXXIRKTKFTSTXXHLIMPAPVELTANGITLQAGGNIE

1111111111111111 111111 111111111 II1111111111111111111li1 orf119-1 SDIVLEAGQNDAYTFLKTKGKSGKIIRKTKFTSTRDHLIMPAPVELTANGITLQAGGNIE

orf119a.pep ANTTRFNAPAGKVTLVAGEXXQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNET

1111111111111111 I llllllillllll11111111111111 4S orf114-1 11111111 I
ANTTRFNAPAGKVTLVAGEELQLLAEEGIHKHELDVQKSRRFIGIKVGKSNYSKNELNET

orfil4a.pep KLPVRWAQXAATRSGWDTVI.EGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQS

IIIIIIIII:IIIII111111111111111111111111 IIII:IIIII1111111111 orf114-1 KLPVRWAQTAATRSGWDTVLEGTEFKTTLAGADIQAGVGEKARADAKI

ILKGIVNRIQS

orf114a.pep EEKLETNSTVWQKQ~GRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLS

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllllll:Illllllllillllllllill:
orf119-1 EEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLTAPGGYIVDIPKGNLKTEIEKLA

SS orf119a.pep KQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTWTSGAGTGAV

111111IIII111111:Illllilill:lllllllll:lllll::: If::l orf114-1 I I: I:
KQPEYAYLKQLQVAKNVNWNQVQLAYDKWDYKQEGLTRAGAAIVTIIVTALTYGYGATAA

orf114a.pep LGLNGA------------- ~TD----_----,~p~FASLASQASVSFINNKGDVGKTL

I: :: :liil II: III 111:1 :1111 II1:~
orf114-1 GGVAASGSSTAAAAGTAATTTAAATTVSTATAMQTAALASLYSQAAVSIINNKGDV

orf114a.pep KELGRSSTVKNLWAAATAGVADKIGA----------SALXNVSDKQWINNL----TVNL

1:11 1:111::1::1 III: :::II : I : : :I I II ':II
ES orf114-1 KDLGTSDTVKQIVTSALTAGALNQMGADIAQLNSKVRTELFSSTGNQTIANLGGR

orf114a.pep ANXGQCRTDX

:I I
70 orf119-1 SNAGISAGINTAVN...

HomoloQV with ~sroA putative secreted protein of N meningitides (accession number AF0309411 ORF114 and pspA protein show 36% as identity in 302aa overlap:
Orf119: 1 AVAETANSQGKGKQ,AGSSVSVSL----KTSGDXXXXXXXXXXXXXXXXXXXXXXXPAHAQ 56 AVAE + GK Q + SV + S PA A
S pspA: 19 AVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAAFSVMLALGVAAFSPAPAS 78 Orf119: 57 -ITTDKSAPKNQQWILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVDNKGAVLNNDRNN- 119 I DKSAPKNQQ VIL+T G P VNIQTP+ +G+S NR FDVD KG +LNN R+N
pspA: 79 GIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFKQFDVDEKGVILNNSRSNT 138 Orf119: 115 ----------NPFVVKGSAQLILNEV-RGTASKLNGIVTVGGQKADVIIANPNGITVNGG 163 NP + +G A++I+N++ S LNG + VGG++A+V++ANp+GI VNGG
pspA: 139 QTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGGKRAEVWANPSGIRVNGG 198 1S Orf114: 169 GFKNVGRGILTTGAPQIGKDGALTGFDVVKAHWTVXAAGWNDKGGAXYTGVLARAVALQG

G N LT+G P + +G LTGFDV + G D A YT +L+RA +
pspA: 199 GLINAASVTLTSGVPVL-NNGNLTGFDVSSGKWIGGKGL-DTSDADYTRILSRAAEINA 256 Orf114: 229 KXXGKXLAVSTGPQKVDYASGEISAGTAAGTK----PTIALDTAALGGMYADSITLIANE 279 2O GK + V +G K+D+ +A + PT+A+pTA LGGMYAD ITLI+ +
pspA: 257 GVWGKDVKWSGKNKLDFDGSLAKTASAPSSSDSVTPTVAIDTATLGGMYADKITLISTD 316 Orfil4: 280 KG 281 G
2S pspA: 317 NG 318 ORF114a is also homologous to pspA:
gi12623258 (AF030941) putative secreted protein [Neisseria meningitides]
Length Score = 261 bits (659), Expect ~ 3e-68 30 Identities = 203/663 (30%), Positives = 319/663 (46%), Gaps = 76/663 (11%) Query: 1 MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLK-----TSGDXXXXXXXXX 55 MNK +++IF+KK S M+AVAE + GK Q + SV + +S
Sbjct: 1 MNKRCYKVIFNKKRSCMMAVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAA 60 Query: 56 XXXXXXXXXXX?CXXXXXXQITTDKSAPKNXQWILKTNTGAPLVNIQTPNGRGLSHNRYT 115 I DKSAPKN Q VIL+T G P VNIQTP+ +G+S NR+
Sbjct: 61 FSVMLALGVAAFSPAPASGIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFK 120 40 Query: 116 QFDVDNKGAVLNNDRNN-----------NPFLVKGSAQLILNEV-RGTASKLNGIVTVGG 163 QFDVD KG +LNN R+N NP L +G A++I+N++ S LNG + VGG
Sbjct: 121 QFDVDEKGVILNNSRSNTQTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGG 180 Query: 164 QKADVIIANPNGITVNGGGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWND 223 4S ++A+V++ANP+GI VNGGG N LT G P + +G LTGFDV G + +G G D
Sbjct: 181 KRAEVWANPSGIRVNGGGLINAASVTLTSGVPVL-NNGNLTGFDVSSGKWIGGKGL-D 238 Query: 229 KGGADYTGVLARAVALQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTK----PTIALD 279 ADYT +L+RA + + GK++ V +G K+D+ +A + pT+A+D
S0 Sbjct: 239 TSDADYTRILSRAAEINAGVWGKDVKWSGKNKLDFDGSLAKTASAPSSSDSVTPTVAID 298 Query: 280 TAALGGMYADSITLIAXEKGVGVKNAGTLEAAK-QLIVTSSGRIENSGRIATTADGTEAS 338 TA LGGMYAD ITLI+ + G ++N G + AA + +++ G++ NSG I +A+
Sbjct: 299 TATLGGMYADKITLISTDNGAVIRNKGRIFAATGGVTLSADGKLSNSGSI-------DAA 351 SS
Query: 339 PTYLXIETTEKGAXGTFISNGGRIESKGLLVIETGEDIXLRNGAWQNNGSRPATTVLNA 398 + +T + + G I S V++ + I + G + GS + +
Sbjct: 352 EITISAQTVD--------NRQGFIRSGKGSVLKVSDGINNQAGLI----GSAGLLDIRDT 399 C)0 Query: 399 GHNLVIESKTNVNNAKGS----XNLSAGGRTTINDATIQAGSSVYSSTKGDTXLGENTRI

G +S ++NN G+ ++S ++ ND + A V S + D G+
Sbjct: 900 G-----KSSLHINNTDGTIIAGKDVSLQAKSLDNDGILTAARDV-SVSLHDDFAGKRDIE 953 Query: 455 IAENVTVLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASNIRLNNGNIKGGKQLALL 519 E)S +T + G + t +I+A DT + + + + + + S R G L+

_94-Sbjct: 454 AGRTLTFSTQGRLKNTRIIQAGDTVSLTAAQIDNTVSGKIQSGNRTGLNGKNGITNRGLI

Query: 515 ADDNIT-----AKTTNLNTPGNLYVHTGKDLNLNVDKDLSAASIHLKSDNAAHITGTSKT

+ IT AK+ N T G +Y G + + D L+ AA

S Sbjct: 519 NSNGITLLQTEAKSDNAGT-GRIY---GSRVAVEADTLLNREETVNGETKAA-------V

Query: 570 LTASKDMGVEAGXXXXXXXXXXXXSGNLHIQAA---KGNIQLRNTKL-NAAKALETTALQ

+ A + + + A SG+LHI +A +Q NT L N + A+E++

Sbjct: 563 IAARERLDIGAREIENREAALLSSSGDLHIGSALNGSRQVQGANTSLHNRSAAIESS---Query: 626 GNI 628 ~I

Sbjct: 620 GNI 622 IS

Score = 37.5 bits (85), Expect = 0.53 Identities = 87/932 (20%), Positives = 159/432 (36%), Gaps =

(14%) Query: 239 LQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEK

2O LQG LQGKN+ + G + +G I A A K A + + S T +

Sbjct: 1023 LQGDLQGKNIFAAAGSDITN--TGSIGAENALLLK--------ASNNIESRSETRSNQNE

Query: 299 GVGVKNAGTLEAAKQLIVTSSGRI--ENSGRIATTADGTEASPTYLXIETTEKGAXG-TF

V+N G + A L +G + + I TA E T + G T

ZS Sbjct: 1073 QGSVRNIGRV-AGIYLTGRQNGSVLLDAGNNIVLTAS-----------ELTNQSEDGQTV

Query: 356 ISNGGRIESKGLLVIETGEDIXLRNGAWQNNGSRPATTVLNAGHNLVIESK---++ GG I S + I + V++ + +T+ G NL + +K

Sbjct: 1121 LNAGGDIRSDTTGISRNQNTIFDSDNWIRKEQNEVGSTIRTRG-NLSLNAKGDIRIRAA

Query: 909 NVNNAKGSXNLSAGGRTTINDATIQAGSS--------VYSSTKGDTXLGENTRIIAENVT

V + +G L+AG D ++AG + Y+ G + TR +

Sbjct: 1180 EVGSEQGRLKLAAG-----RDIKVEAGKAHTETEDALKYTGRSGGGIKQKMTRHLKNQNG

3S Query: 461 VLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASNIRLNNGNIKGGKQLALLADDNIT

+G++ +I +G + + T+ S NN +K + + A+ N

Sbjct: 1235 QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILS--AKNNIVLKAAETRSRSAEMNKK

Query: 521 AKTTNLNTPG-NLYVHTGKDLNLNVDKDLSAASIHLKSDN-------AAHITGTSKTLTA

4O K+ + + G + KD N + +S + S N H T T T+++

Sbjct: 1293 EKSGLMGSGGIGFTAGSKKDTQTNRSETVSHTESWGSLNGNTLISAGKHYTQTGSTISS

Query: 573 SK-DMGVEAGXXXXXXXXXXXXSGNLHIQAAKG-----NIQLRNTKLNAAKALETTALQG

+ D+G+ +G + + KG ++ + NT + A A++ G

4S Sbjct: 1353 PQGDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPWNTVMGAVDAVKAVQTVG

Query: 627 NIVSDGLHAVSA 638 + ++A++A

Sbjct: 1413 KSKNSRVNAMAA 1424 Amino acids1-1423 of ORF114-1 were cloned in the pGex vector and expressed in E.coli, as described above.
GST-fusion expression was visible using SDS-PAGE, and Figure 5 shows plots of hydrophilicity, antigenic index, and AMPI~iI
regions for ORF114-1.

Based on these results, including the homology with the putative secreted protein of N. meningitides SS and on the presence of a transmembrane domain, it is predicted that this protein from N. meningitides, and its epitopes, could be useful antigens for vaccines or diagnostics.
Example 14 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
63>

WO ~~~ PCT/IB99/00103 _gs_ 1 ..CGCTTCATTC ATGATGAAGC AGTCGGCAGC AACATCGGGG
GCGGCAAAAT

51 GATTGTTGCA GCCGGGCAGG ATATCAATGT ACGCGGCAnA
AGCCTTATTT

TATTTCTACT

S wAAAwTCAGG

CGGAAAACTA

251 CCGATGACAC TGATCGTACC AATATTGTsC ATACAGGCAG
CATTATAGGC

301 AGCCTGAaTG GAGACACCGT TACAGTTGCA GGAAACCGCT
ACCGACAAAC

ACAGCCAAAw CTACGcCCAT

lO 451 ACCCA~GGAA CAAAAAGGCC TTACCGTCGC CCTCAATGTC
CCGGTTGTCC

CAAAAGTAAA

GGCAGAGTTA

AGTGCGGGAC

IS CATTAC.TAC

CCGAAgCGGC

751 AgCAAGTCAA ATTATCGGCA AAGGGCAAAC CACACTTGCG
GCAACAGGAA

CATCGGCCAT

851 GCAGGTACTC C.CTCATTGC CGACAACCAT ATCAGACTCC
AATCTGCCAA

951 GCGTACGTnn CAAAATAGGC AACGGCATCA GGTTTGGAAT
TACCGCCGGA

CCCACCGCCA

AGCGGCGGG~

TACAGGCAGA

ACCTATCAGA

2S 1201 GCAAACAGCA AAACGGCAAT GTCCAAGTT~ ACTGTCGGTT
ACGGATTCAG

CATGCCTCCG

1301 TAACCGGGCA AAgCGGTATT TATGCCGGAG AAGACGGCTA
TCAAATyAAA

1351 GTyAGAGACA ACACAGACCT yAAGGGCGGT ATCATCACGT
CTACCCAAAG

CGGCATAGGC

CCGACAAACA

GGCAGCGACG

CCACAACATA

ACTGCGGATC

1751 AACACTCAGG CCATCTGAAA AACAGCTTCG AC...

This corresponds to the amino acid sequence <SEQ ID 64; ORF 11 ~>:
1 ..RFIHDEAVGS NIGGGKMivA AGQriNVRGX SLISDKGIVL KAGHDIDIST
51 AHNRYTGNEY HESXXSGVfiGGLGFTIGN RKTTDDTDRT NIVHTGSIIG
4O 101 SLNGDTVTVA GNIi,~~GST VSSPEGRNTV TAKXIDVEFA NNRYATDYAH
151 TQEQKGLT~;!A L~pWQ~Q NFIQAAQNVG KSKNKRVNAM AAANAAWQSY
201 (~,P:T~~MQQFA PSSSAGQGQN YNQSPSISVS IXYGEQKSRN EQKRHYTEAA

3u1 QDGSEQSKNK SSGWNAGVRX KIGNGIRFGI TAGGNIGKGK EQGGSTTHRH

451 RDNTDLKGGI ITSSQSAEDK GKNLFQTATL TASDIQNHSR YEGRSE'GIGG

551 ITDEAGQLAR TGRTAKETEA RIYTGIDTET ADQHSGHLKN SFD...
SO Computer analysis of this amino acid sequence gave the following results:
Homolosty with DSDA vutative secreted protein of N menin 'tidis ~a~ccession number AF0309411 ORF116 and pspA protein show 38% as identity in S02aa overlap:
Orf116: 6 EAVGSNIGGGKMIVAAGQDINVRGXSLISDKGIVLKAGHDIDISTAHNRYTGNEYHESXX 65 +AV + G ++I+ +G+DI V G ++I+D +L A ++I + A R E ++
SS PspA: 1 235 QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILSAKNNIVLKAAETRSRSAEMNKKEK

Orf116: 66 XXXXXXXXXXXXXXNRKXXXXXXRTNIVHTGSIIGSLNGDTVTVAGNRYRQTGSTVSSPE 125 ++K + HT S++GSLNG+T+ AG Y QTGST+SSP+
PspA: 1295 SGLMGSGGIGFTAGSICICDTQTNRSETVSHTESWGSLNGNTLISAGKHYTQTGSTISSPQ 1359 Orf116: 126 GRNTVTAKXTDVEFANNRYATDYAHTQEQKGLTVALNVPXXXX---XXXXXXXXXXXGKS 182 G +++ I ++ A NRY+ + EQKG+TVA++VP GKS
PspA: 1355 GDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVNTVMGAVDAVKAVQTVGKS 1914 S Orf116: 183 KNKRVXXXXXXXXXWQSYQATQQMQQFA--KN RV + + + A P +AGQG ISVS+ YGEQK+ +

PspA: 1915 KNSRVNAMAAANALNKGVDSGVALYNAARNPKKAAGQG--------ISVSVTYGEQKNTS1466 Orf116: 291 EQKRHYTEAAASQIIGKGQTTLAATGSGEQSNINITGSDVIGHAGTXLIADNHIRLQSAK300 lO E + T+ +I G G+ +L A+G+G+ S I ITGSDV G GT L
A+N +++++A+

PspA: 1967 ESRIKGTQVQEGKITGGGKVSLTASGAGKDSRITITGSDVYGGKGTRLKAENAVQIEAAR1526 Orf116: 301 QDGSEQSKNKSSGWNAGVRXKIGNGIRFGITAXXXXXXXXXXXXSTTHRHTHVGSTTGKT360 Q E+S+NKS+G+NAGV I GI FG TA T +R++H+GS +T

IS PspA: 1527 Orf116: 361 TIRSGGDTTLKGVQLIGKGIQADTRNLHIESVQDTETYQSKQQNGNVQVTVGYGFSASGS920 I SGGDT +KG QL GKG+ +LHIES+QDT ++ KQ+N + QVTVGYGFS
GS

PspA: 1587 AIESGGDTVIKGGQLKGKGVGVTAESLHIESLQDTAVFKGKQENVSAQVTVGYGFSVGGS1646 Orf116: 921 YRQSKVKADHASVTGQSGIYAGEDGYQIKVRDNTDLKGGIITSSQSAEDKGKNLFQTATL480 Y +SK +D+ASV QSGI+AG DGY+I+V T L G + S DK KNL
+T+ +

PspA: 1647 YNRSKSSSDYASVNEQSGIFAGGDGYRIRVNGKTGLVGAAWSD---ADKSKNLLKTSEI1703 2S Orf116: 481 TASDIQNHSRYEGRSFGIGGSF 502 DIQNH+ + G+ G F
PspA: 1709 WHKDIQNHASAAASALGLSGGF 1725 Based on homology with pspA, it is predicted that this protein from N.
meningitides, and its epitopes, could be useful antigens for vaccines or diagnostics.
30 Eiample 15 The following partial DNA sequence was identified in N.meni»gitidis <SEQ ID
6S>
1 ..ACGACCGGCA GCCTCGGCGG CATACTGGCC GGCGGCGGCA
CTTCCCTTGC

GCGGGCAAAG

AACTGGTGGT

ATAGGCAGCT

GCCCTCAAGC

AGAAGCGGCA

TTCCCAAGAC

40 This corresponds to the amino acid sequence <SEQ ID 66; ORF118>:
1 ..TTGSLGGILA GGGTSLAAPY LDKAAENLGP AGKAAVNALG GAAIGYATGG

101 MRIRRQICVG WTKVPKTAIP TKASYPLSE*
Computer analysis of this amino acid sequence reveals two putative transmembrane domains.
4S Based on this analysis, it is predicted that this protein from N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
Example 16 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
67>
1 ..CAATGCCGTC TGAAAAGCTC ACAATTTTAC AGACGGCATT TGTTATGCAA

51 GTACATATAC AGATTCCCTA TATACTGCCC AGrkGCGTGC
GTgGCTGAAG

101 ACACCCCCTA CGCTTGCTAT TTGrAACAGC TCCAAGTCAC
CAAAGACGTC

151 AACTGGAACC AGGTACwACT GGCGTACGAC AAATGGGACT
ATAAACAGGA

S GTTACCGTGG

251 TTACTGCGGG CGCGGGAgCC GGAGCCGCAC TGGGcTTAAA
CGGCGCGGCc AGGcTTCCGT

351 ATCGCTCATC AaCAACAAAG GCAATATCGG TAaCACCCTG
AAAGAGCTGG

401 GCAGAAGCAG CACGGTGAAA AATCTGATGG TTGCCGTCGc tACCGCAgGC

451 GTagCcgaCA AAATCGGTGC TTCGGCACTG AACAATGTCA
lO GCGATAAGCA

AGTGCCGCAC

551 TGATTAATAC CGCTGTCAAC GGCGGCAGCc tgAAAGACAA
TCTGGAAGCG

CAGCCAGTAA

GCCCaTGCCA

701 TAGCGGGCTG TGCGGcTGCG GCGGCGAATA AGGGCAAGTG
IS TCAGGATGGT

751 GCGATAgGTG CGGCTGTGGG CGAGATAGTC GGGGAgGCTT
TGACAAACGG

801 CAAAAATCCT GACACTTTGA CAGCTAAAgA ACGCGaACAG
ATTTTGGCAT

CGGCGATGTA

ATCAGCTTAG

951 CGACAAAtGA

20 This corresponds to the amino acid sequence <SEQ ID 68; ORF41>:
1 ..QCRLKSSQFY RRHLLCKYIY RFPIYCPXAC VAEDTPYACY
LXQLQVTKDV

GAALGLNGAA

NLMVAVATAG

201 NILAALVNTA HGEAASKIKQ LDQHYITFIKI AfiAIAGCAAA
AANKGKCQDG

TVSGWGGDV

301 NAAANAAEVA VKNNQLSDK*

Further work revealed the complete nucleotide sequence <SEQ ID 69>:

GTCACCAAAG

GGACTATAAA

TGGCTGTTAC

TTAAACGGCG

CAGCCAGGCT

CCCTGAAAGA

GTCGCTACCG

TGTCAGCGAT

CGGGCAGTGC

GACAATCTGG

AGAAGCAGCC

AGATTGCCCA

AAGTGTCAGG

GGCTTTGACA

AACAGATTTT

GTCGGCGGCG

AAATAATCAG

CTGCATGCGC

AAAAAGTATC

1001 AAAATGTTGC TGATAAAA('~A CTTGCTGCTT CGATTGCAAT
SO ATGTACGGAT

ATTTGATCGA

GGTAAAGATG

AGATTTGGCT

TTCAATCGGG

TATACACTTA

GTTTGTAAAA

TCAGTTTCGA

AGTCAAAAAC

AGAACTAAAT

TTGAAGGCAT

GGTAAACCTG

TAATCCTAAA

CTGCTTCACA

ACTAAATCAA

1751 TATCGGAAAG AAAAAATGTC ATTCAATTCT CAGAAACCT~' C TGACGGAATC
S

) AAATTTAGAT CATATTTTGA TGTAAATACA GGAAGAATTA

WO 99!36544 PCT/IB99/00103 This corresponds to the amino acid sequence <SEQ ID 70; ORF41-1>:

QLAYDKWDYK

AAFASLASQA

101 SVSLINNKGN IGNTLKELGR SSTVKNIdHVA VATAGVADKI
GASALNNVSD

LVNTAHGEAA

VGEIVGEALT

AAEVAVKNNQ

LAASIAICTD

SKSYTQADLA

FIPIPRGFVK

NRTNFMAELN

SSIKTVYNPK

IQFSETFDGI

601 KFRSYFDVNT GRITNIHPE*

1 S Computer analysis of this amino acid sequence predicts a transmembrane domain, and homology with an ORF from N.meningitidis (strain A) was also found.
ORF41 shows 92.8% identity over a 279aa overlap with an ORF (ORF41 a) from strain A of N.
meningitides:

orf4l.pep YRRHLLCKYIYRFPIYCPXACVAEDTPYACYLXQLQVTKDVNWNQVXLAYDKWDYKQEGL
II

Iill:l::lllll flll:llllllli orf4la YLKQLQVAKNTNWNQVQLAYDRWDYKQEGL

orf4l.pep TGAGAAIIALAVTWTAGAGAGAALGLNGAAAAATDAAFA9LASQASVSLINNKGNIGNT

11111111:111:11:111111 Illlllllllllllllll:lllll::l:l orf4la TEAGAAIIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKT

orf4l.pep LKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAV

IIIIIIIIIIIII:II:illlllllllllll IIIIIIIIIIIIIIIIIIIIIIIIIiII
orf9la LKELGRSSTVKNLWAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALIN

TAV

orf4l.pep NGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQD
IIIilll IIIIIIIIIIilillllllllllllllll:Illlllllllllilllllllll 4O orf4la NGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQD

orf9l.pep GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGWGGDVNAAANAAEV
4S lilllll11111lIIIllli~lIIIIll111lillllllllllllillllllllllllll orf4la GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGWGGDVNAAANAAEV

SO orf4l:pep AVKNNQLSDKX

IIIIIIIII
orf9la AVKNNQLSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVADKRLAASIAICTDISRS

A partial ORF4la nucleotide sequence <SEQ ID 71> is:
SS 1 ..TATCTGAAAC AGCTCCAAGT AGCGAAAAAC ATCAACTGGA ATCAGGTGCA

wo ~r~6saa Pcr~s~rooio3 ATCAACAACA

CAGCACGGTG

ACAAAATCGG

AACAACCTGA

TACCGCTGTC

TTGCGGCTTT

CAGTTGGATC

CTGTGCGGCA

GTGCGGCTGT

. CCTGACACTT

ACTGGTTGCC

CGGCGAATGC

GAGGGTAGAG

TCCTCAACTG

IS 901 TGCAG,AAAAA ATACTGTAAA AAAGTATCAA AATGTTGCTG
ATAAAAGACT

ACTGAATGTA

TCATTCATCT

AATTATTCAG

CATTTGAATA

TTTATCCGAA

ATCCTAGATT

ATTACTAATG

AAGACATCTG

GAGCCCATAA

NGNGTAAAAT

ATATGAGATT

AGGAAATTTC

GATAAAATAC

AGCCTCTAAA

AAAATGTCAT

TATNTNGATG

This encodes a protein having the partial amino acid sequence <SEQ ID 72>:

SLASQASVSF

AHGEAASKIK

VGEALTNGKN

IAICTDISRS

TQADLALQSY

PRGFVKQNTP

XMAELNSRGG

TVYNPKXFXD

4S 551 IAQNERTKSI SERKNVIQFS YXDVNTGRIT NIHPE*
ETFDGIKFRX

ORF4la and ORF41-1 show 94.8% identity in S9S as overlap:

orf4la.pep YLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAA

Illllll:l::llllllllll:lllllifll IIII
SO orf41-1 MQVNIQIPYILPRCVRAEDTPYACYLKQLQVTKDVNWNQVQLAYDKWDYKQEGLTGAGAA

orf9la.pep IIALAVTWTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGR

SS Illlllllll:lll:ll:llllli III111111111111111:11111::1:1111111 orf41-1 IIALAVTWTAGAGAGAALGLNGAAAAATDAAFASLASQASVSLINNKGNIGNTLKELGR

6O orf4la.pep SSTVKNLWAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK
III1111:11:11111111111111 IIIIIIIIIIIIIIIIIIIIIIIIIIilllllll orf41-1 SSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK

()$ 160 170 180 190 200 210 orf4la.pep DXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQDGAIGAA

i IIIIIIIIIIIIIIIIIIIIIIIIIIIII:IIIIIIIILIIIIIIIIIIIIIIIIIII
orf91-1 DNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQDGAIGAA

orf9la.pep VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGWGGDVNAAANAAEVAVKNNQ

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiI
orf91-1 VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGWGGDVNAAANAAEVAVKNNQ

orf4la.pep LSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI
III IIIIIIIIilllllll IIIIIIIIIilllllllllllllllllllllllllllil orf41-1 LSDKEGREFDNEMTACAKQNNPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI

orf4la.pep RKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQADLALQSYHLNTAAKSWLQSGNTK

IlilllilllllllllllllliiliilllllllllllllIIIIIIIIIIIIIIIIIIiII
2O orf41-1 RKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQADLALQ$YHLNTAAKSWLQSGNTK

. 370 380 390 400 910 420 orf9la.pep PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLXRHLANADGF
2S IIIIIIIIIIIIIIIIIIIII 111111 Illillllllilllllllll IIIIIII

II
orf41-1 PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLKRHLANADGF

430 490 450 460 970 48p 9l a.pep SQEQGIKGAHNRTNXMAELNSRGGXVKSETXTDIEGITRIKYEIPTLDRTGKPDGGFKEI
orf II:IIIIIIIIIII IIIIII111 IIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIII

orf91-1 SQKQGIKGAHNRTNFMAELNSRGGRVKSETQTDIEGITRIKYEIPTLDRTGKPDGGFKEI

35 520 530 540 550 560 5?0 orf9la.pep SSIKTVYNPKXFXDDKILQMAQXAXSQGYSKASKIAQNERTKSISERKNVIQFSETFDGI
Illilllill I IIIIillll I Illlllll~lillllllllltlllllllIIIIIIII

orf41-1 SSIKTVYNPKKFSDDKILQMAQNAASQGYSKASKIAQNERTKSISERKNVIQFSETFDGI

orf4la.pep KFRXYXDVNTGRITNIHPEX

orf41-1 KFRSYFDVNTGRITNIHPEX

Amino acids 2S-619 of ORF41-1 were amplified as described above. Figure 6 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF41-1.
Based on this analysis, it is predicted that this protein from N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
SO Example 17 The following DNA sequence was identified in N.meningitidis <SEQ ID 73>

101 TTTTTGGGTT TTTGGsmrGC ATCATCGGCG GTTCAACCAA TGCCATGTCT
SS 151 CCCATATTGT TAATATTTTT GCTTAGCGAA ACAGAAAATA AAAATcgTAT

251 ATATGCTAAG AGACCAGTAT TGGTTATTAA ATAAGAGTGA ATACGdTTTA

351 GTTAAGGACT AAGATTAGCC CAaATTTTTT TAAAATGTTA ATTTTTATTG

401 tTTTATTGGT ATTGGCtCTG AAAATCGGGC AttCGGGTTT AAtCAAACTT

This corresponds to the amino acid sequence <SEQ ID 74; ORES 1>:

Further work revealed the complete nucleotide sequence <SEQ ID 7S>:

IO CAATACTGCA

GCATTGGCTT

ACCAAGCCTG

GTTTTTGGCA

ATCGGCAGCG

TCCAGTGTCT

CTGTCAATGG

GTTGCCAATA

CGGCGGTTCA

GCGAAACAGA

ATTAAATAAG

TTATTGGATT

TTTTTTAAAA

CGGGCATTCG

2S This corresponds to the amino acid sequence <SEQ ID 76; ORFS1-1>:

WALVALPSL

VKLLLILPVS

GFLAGIIGGS

LRDQYWLLNK

LVLALKIGHS

251 GLIKL*

Computer analysis of this amino acid sequence reveals three putative transmembrane domains. A
corresponding ORF from strain A of N. meningitides was also identified:
Homology with a predicted ORF from N.meningitidis (strain Al 3 S ORFS 1 shows 96.7% identity over a 1 SOaa overlap with an ORF (ORFS 1 a) from strain A of N.
meningitides:

orf5l.peP MAIITLYYSVNGILNVCAKAKNIQWANNK
IIIIIIIIIIIIIIIIIIIIIIIIIIIIII
40 orf5la YKLLAIGSWGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILNVCAKAKNIQWANNK

orf5l.pep NMVLFGFLXXIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCYLLAKIVQIYMLRDQY
4S 11111111 1 IIII111111111111 II1111:1111111111111111111111 orf5la NMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCYLLAKIVQIYMLRDQY

SO orf5l.pep WLLNKSEYXLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGHSGLIKL
IIIIIiII IIIIIIII(IIIIIIIIIIIIillllllllllllllllllllll:111111 orf5la WLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGYSGLIKL

ORES I-I and ORES 1 a show 99.2% identity in 2SS as overlap:
orf5la.pep MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKWALVALPSLLMSLLVLCSN

IIIIIIIiililillllllllllllllllllllllllllllliiilllllllllllllll orf51-1 MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKWALVALPSLLMSLLVLC

SN
S

orf5la.pep NKKGFWQEIVYYLKTYKLLAIGSWGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN

Illllllllllllllllllllllllllllllllllllillllllllllllllllllllll orf51-1 NKKGFWQEIVYYLKTYKLLAIGSWGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN

IO orf5la.pep VCAKAKNIQWANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCY

IIIIIIIIIIIIIIilllllllllllllllllllllilllllllllllllll:1111111 orf51-1 VCAKAIQJIQWANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCY

orf5la.pep LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL

orf51-1 LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL

orf5la.pep LVLALKIGYSGLIKLX
IIIIIIII:IIIIIII
20 orf51-1 LVLALKIGHSGLIKLX
The complete length ORES 1 a nucleotide sequence <SEQ ID 77> is:

CAATACTGCA

GCATTGGCTT

GTTTTTGGCA

ATCGGCAGCG

TCCAGTGTCT

301 TGGCTGCT.TT TACTGATGGC AATCATTACA TTGTATTATT
CTGTCAATGG

CGGCGGTTCA

GCGAAACAGA

CTTTTGGCAA

ATTAAATAAG

TTTTTTAAAA

CGGGTATTCA

This encodes a protein having amino acid sequence <SEQ ID 78>:

4O 51 LMSLLVLCSN NKKGFSiQEIV YYLKTYKLLA IGSWGSILG VKLLLILPVS

251 GLIKL*
4S Based on this analysis, it is predicted that this protein from N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
Ezample 18 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
79>

501 TATAAAATTT GTCAGG..
S This corresponds to the amino acid sequence <SEQ ID 80; ORF82>:

151 RLSLVCGIHS YAPCANFIKF VR..
Further work revealed the complete nucleotide sequence <SEQ ID 81 >:

TAGTTTTACA

GTTTTTCTAT

TGCTGTAAAT

TTTTATTGCC

ATAAATATAA

ATCCTCGATT

ATGACTCAAA

GTAATTAGAG

TAAATCCATA

GTGCCAATTT

AATCAACCTC

TGGAAACAAA

TTGAAAACAG

TTGCTTTTAT

This corresponds to the amino acid sequence <SEQ ID 82; ORF82-1>:

51 LLFLEKNIKN KLI.FLLPISI IIWMVIHISM INIKFYKFEH QIKEQNISSI

201 SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE LE*
Computer analysis of this amino acid sequence reveals a predicted leader peptide.
A corresponding ORF from strain A of N, meningitides was also identified:
Homology with a predicted ORF from N.meningit;dis (strain A) 3S ORF82 shows 97.1% identity over a 172aa overlap with an ORF (ORF82a) from strain A of N.
meningitides:

orf82.pep MRHMKIQNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
Illll :IIIIillllll:IIIIIilllllllllllllllllllllllllllllllllll 40 orf82a MRHMKNKNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
10 20 30 90 50 60.

orf82.pep KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
4S IIIIIIIIIIIIIIillllllllllilIIIIIIIIIIIIIIIIIIIIIIIIII1111111 orf82a KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA

SO orf82.pep KLKDNHRYGRVIRETPYIDWASDVKNKSIRLSLVCGIHSYAPCANFIKFVR
Illlflllllllllllllllllllllllllllllllllllllllllllll::
orf82a KLKDNHRYGRVIRETPYIDWASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY

WO 99/36544 -1 ~- PGT/IB99/00103 ORF82a and ORF82-1 show 99.2% identity in 242 as overlap:
orfB2a.pep ~KNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN

IIIIIIIIIIIIIIIIII:11111111111111111111111111111111111111111 orf82-1 MRHMKNKNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN

S

orf82a.pep KLLF'LLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA

Illllllllllllllllllllllllllllillfllllllllllllllllllillllllll orf82-1 KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA

lO orf82a.pep KLKDNHRYGRVIRETPYIDWASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
orf82-1 KLKDNHRYGRVIRETPYIDWASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY

orf82a.pep NQPQGDFIDNVIFEINDGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE

IIIIIIIIIIIIIIIIII:IIIIIIIIIIIIIIIIIIIIIIIIiIII
orf82-1 In II11IIII
NQP~DFIDNVIFEINDGNKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE

orf82a.pep LEX
III
20 orf82-1 LEx The complete length ORF82a nucleotide sequence <SEQ ID 83> is:

TAGTTTTACA

TGCTGTAAAT

TTTTATTGCC

ATAAATATAA

ATCCTCGATT

GTAATTAGAG

TAAATCCATA

GTGCCAATTT

AATCAACCTC

3S TGGAAAAAAp, TTGAAAACAG

TTGCTTTTAT

This encodes a protein having amino acid sequence <SEQ ID 84>:

201 SLYLLDKYKT FF2IENSVCI VLIILYLKFN LLLYRTYFNE LE*
Based on this analysis, it is predicted that this protein from N.
meningitides, and its epitopes, could 4S be useful antigens for vaccines or diagnostics.
Example 19 The following partial DNA sequence was identified in N.meningitidis <SEQ ID
8S>
1 ..ACCCCCAACA GCGTGACCGT CTTGCCGTCT TTCGGCGGAT
TCGGGCGTAC

SO GCGTTTTCGA

GCAGGCCGTG

CGGTCTTGAG

ACGGCTGACT

CCTTGACTTC

SS GCATAGTTTT

CTTGTCGATG

S This corresponds to the amino acid sequence <SEQ ID 86; ORFI24>:
1 ..TPNSVTVLPS FGGFGRTGAT INAAGGVGMT AFSTTLISVA EGAWELQAV

151 SQARADKRDN GNRLPVIRQQ FHEIHSRPPD ASR*
Computer analysis of this amino acid sequence predicts a transmembrane domain.
Further work revealed the complete nucleotide sequence <SEQ ID 87>:

GCGCGGTTGT

GCCGCTTGCA

IS TATTTTCCGT

ATGCCGACAG

CAGTTCCAGT

CCTTGTCGCG

GACTTGTAGC

TTCTCGACCT

This corresponds to the amino acid sequence <SEQ ID 88; ORF124-1>:

151 PDASR*
A corresponding ORF from strain A of N. meningitides was also identified:
Homoloav with a predicted ORF from N menirt; i~ ndis (strain A~
ORF 124 shows 87.5% identity over a 1 S2aa overlap with an ORF (ORF 124a) from strain A of N.
30 meningitides:

orf129.pep TPNSVTVLPSFGGFGRTGATINAAGGVGMTAFSTTLISVAEGAVVELQAVRAKAVNATAA
orf129a MTAFSTTLISVAEGALVELQAVMAKAVNTTAA

orf124.pep CIFTVLSKDIFDFLFIFRFQTADFRLYFRQSHADSVRLDFIFKSFRACQFQFARIVLSRQ
IIIIII

IIIIilllllllllllllll:IIIIIII:III1111 III: IIII
4O orf129a :11111 CIFTVLSKDIFDFLFIFRFQTADFRLFFRQSHADGVRLDFIFFSFRTRLFQFAGWLSRQ

orf124.pep QQGLRLVALHLVDDRLQLRKCRLVALMVRHSQARADKRDNGNRLPVIRQQFHEIHSRPPD

4S IIII111111:::111 111 III111111 l:llllll:llll1111111111 illl orfl24a QQGLRLVALHFLNDRLLLRKSRLVALNIVRHRQTRADKRDDGNRLPVIRQQFHEIHSRPPD

orf129.pep ASRX

SO .

orf129a VX

ORF124a and ORF124-1 show 89.5% identity in 1S2 as overlap:

orf124-1. pep MTAFSTTLISVAEGAVVELQAVRAKAVNATAACIFTVLSKDIFDFLFIFRFQTADFRLFF
IIIIIIIIIIIIIII:IIIIII IIIII:IIIIIIIIIIIIIIIII11111111111111 orf124a MTAFSTTLISVAEGALVELQ,AVMAKAVNTTAACIFTVLSKDIFDFLFIFRFQTADFRLFF
orf124-1. pep RQSHADSVRLDFIFFSFRACQFQFARIVLSRQQQGLRLVALHLVDDRLLLRKCRLVALMV
IIIIII:IIIIIIIIIII: III! :Illllillllllllf:::lllllll 1111111 orf129a RQSHADGVRLDFIFFSFRTRLFQFAGWLSRQQQGLRLVALHFLNDRLLLRKSRLVALMV
orf129-1. pep RHSQARADKRDNGNRLPVIRQQFHEIHSRPPDASRX
1~ II I:IIIIII:llllllllllllllllllll:
orf124a RHRQTRADKRDDGNRLPVIRQQFHEIHSRPPDVX
The complete length ORF 124a nucleotide sequence <SEQ ID 89a is:

GCGCGCTTGT

IS GCCGCCTGCA

TATTTTCCGT

ATGCCGACGG

CTGTTCCAGT

CCTTGTCGCG

GATGATGGCA

TTCTCGACCT

This encodes a protein having amino acid sequence <SEQ ID 90>:
2S 51 FQTADTFRLFF RQSHADGVRL DF"IFFSFRTR LFQFAGVVLS RIFDFLFIFR

151 PDV*
ORF124-1 was amplified as described above. Figure 7 shows plots of hydrophilicity, antigenic 30 index, and AMPHI regions for ORF124-1.
Based on this analysis, it is predicted that this protein from N.meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
It will be appreciated that the invention has been described by means of example only, and that 3S modifications may be made whilst remaining within the spirit and scope of the invention.

TABLE I - PCR primers ORF Primer Sequence Restriction sites 01ZF 38 ForwardCGCGGATCCCATATG-TCGCCGCAAAATTCCGA $~-NdeI

RBVerSeCCCGCTCGAG-TTTTGCCGCGTTAAAAGC COI

OIZF 40 Forward~GCGGATCCCATATG-ACCGTGAAGACCGCC B~_NdeI

RCVerSeCCCGCTCGAG-CCACTGATAACCGACAGA ~pI

OItF 41 FOrWardCGCGGATCCCATATG-TATTTGAAACAGCTCCAAG Bar~_NdeI

ReVerSeCCCGCTCGAG-TTCTGGGTGAATGTTA ~pI

~ FOrWaTdGCGGATCCCATATG-GGCACGGACAACCCC $g~_NdeI

ReVetSeCCCGCTCGAG-ACGTGGGGAACAGTCT COI

OihZF FOrWardGCGGATCCCATATG-AAAAATATTCAAGTAGTTGC B~_NdeI
SI

ReverseCCCGCTCGAG-AAGTTTGATTAAACCCG XhoI

OiIZF ForwardCGCGGATCCCATATG-TGCCAACCGCAATCCG $~II-NdeI

RevefSeCCCGCTCGAG-TTTTTCCAGCTCCGGCA XilOI

OItF 56 ForwardGCGGATCCCATATG-GTTATCGGAATATTACTCG $a~ NdeI

ReverseCCCGCTCGAG-GGCTGCAGAAGCTGG COI

ORF 69 ForwardCGCGGATCCCATATG-CGGACGTGGTTGGTTTT BamHI-NdeI

RCVerSeCCCGCTCGAG-ATATCTTCCGTTTTTTTCAC COI

ORF 82 ForwardCGCGGATCCGCTAGC-GTAAATTTATTATTTTTAGAA $~j-j~eI

R~~g CCCGCTCGAG-TTCCAACTCATTGAAGTA XilOI

ORF 114 ForwardCGCt3GATCCCATATG-AATAAAGGTTTACATCGCAT $~_~eI

ReVeISeCCCGCTCGAG-AATCGCTGCACC(3GCT COI

OItF 124 ForwardCGCC,1GATCCCATATG-ACTGCCTTTTCGACA $~j-I~eI

RCVerSeCCCGCTCC3AG-GCGTGAA(3C(3TCAGGA XilOI

wo ~r~6s4a PcrnB~rooio3 TABLE II - Cloning, expression and purification ORF PCR/cloningHis-fusion GST-fusion Purification expression expression orf 38 + + + His-fusion orf 40 + + + - ~s_fusion orf 41 + n.d. n.d.

off' + + + His-fusion ~

orf 51 + n.d. n.d.

orf 52 + n.d. + GST-fusion orf 56 + n.d. n.d.

orf 69 + n:d. -. -n:d.

orf 82 + n.d. - - n.d.

orf 114 + n.d. + GST-fusion orf 124 + n~a. ~ - o:a: -I

Claims (28)

1. A protein comprising an amino acid sequence selected from the group consisting of SEQ
IDs 2, 4, and 6

2. A protein having 80% or greater sequence identity to a protein according to claim 1.

3. A protein comprising a fragment of at least 16 consecutive amino acids of an amino acid sequence selected from the group consisting of SEQ IDs 2, 4 and 6.

4. An antibody which binds to a protein according to any preceding claim.

5. A nucleic acid molecule which encodes a protein according to any preceding claim.

6. A nucleic acid molecule according to claim 5, comprising a nucleotide sequence selected from the group consisting of SEQ IDs 1, 3, and 5.

7. A nucleic acid molecule comprising a fragment of at least 20 consecutive nucleotides from a nucleotide sequence selected from the group consisting of SEQ IDs 1, 3, and 5.

8. A nucleic acid molecule comprising a nucleotide sequence complementary to a nucleic acid molecule according to any one of claims 5 to 7

9. A nucleic acid molecule comprising a nucleotide sequence having 80% or greater sequence identity to a nucleic acid molecule according to any one of claims 5 to 8.

10. A nucleic acid molecule which can hybridise to a nucleic acid molecule according to any one of claims 5 to 9 under high stringency conditions.

11. A composition comprising a protein, a nucleic acid molecule, or an antibody according to any preceding claim.

12. A composition according to claim 11 being a vaccine composition or a diagnostic composition.

13. A composition according to claim 11 or claim 12 for use as a pharmaceutical.

14. The use of a composition according to claim 13 in the manufacture of a medicament for the treatment or prevention of infection due to Neisserial bacteria, particularly Neisseria meningitidis.

15. A protein comprising an amino acid sequence selected from the group consisting of SEQ
IDs 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

16. A protein having 50% or greater sequence identity to a protein according to claim 15.

17. A protein comprising a fragment of an amino acid sequence selected from the group consisting of SEQ IDs 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 85, 88, and 90.

18. An antibody which binds to a protein according to any one of claims 15 to 17.

19. A nucleic acid molecule which encodes a protein according to any one of claims 15 to 17.

20. A nucleic acid molecule according to claim 19, comprising a nucleotide sequence selected from the group consisting of SEQ IDs 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89.

21. A nucleic acid molecule comprising a fragment of a nucleotide sequence selected from the group consisting of SEQ IDs 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57. 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89.

22. A nucleic acid molecule comprising a nucleotide sequence complementary to a nucleic acid molecule according to any one of claims 19 to 21.

23. A nucleic acid molecule comprising a nucleotide sequences having 50% or greater sequence identity to a nucleic acid molecule according to any one of claims 19 to 22.

24. A nucleic acid molecule which can hybridise to a nucleic acid molecule according to any one of claims 19 to 23 under high stringency conditions.

25. A composition comprising a protein, a nucleic acid molecule, or an antibody according to any one of claims 15 to 24.

26. A composition according to claim 25 being a vaccine composition or a diagnostic composition.

27. A composition according to claim 25 or claim 26 for use as a pharmaceutical.

28. The use of a composition according to claim 25 in the manufacture of a medicament for the treatment or prevention of infection due to Neisserial bacteria, particularly Neisseria meningitidis.