AU2004201096B2 - Neisseria genomic sequences and methods of their use - Google Patents

Description

AUSTRALIA

Patents Act 1990 CHIRON CORPORATION, THE INSTITUTE FOR GENOMIC RESEARCH COMPLETE SPECIFICATION STANDARD PATENT Invention Title.

Neisseria genomic sequences and methods of their use The following statement is a full description of this invention including the best method of performing it known to us:- NEISSERIA GENOMIC SEQUENCES AND METHODS OF THEIR USE This application is a divisional application of Patent Application No. 12022/00, the contents of which are incorporated herein by reference.

This invention relates to methods of obtaining antigens and immunogens, the antigens and immunogens so obtained, and nucleic acids from the bacterial species: Neisseria meningitidis. In particular, it relates to genomic sequences from the bacterium; more particularly its serogroup.

BACKGROUND

Neisseria meningitidis is a non-motile, gram negative diplococcus human pathogen. It colonizes the pharynx, causing meningitidis and, occasionally, septicaemia in the absence of meningitidis. It is closely related to N. gonorrhoea, 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. (1996) Safety and Immunogenicity of a Serogroups A/C Neisseria meningitidis Oligosaccharide-Protein Conjugate Vaccine in Young Children. JAMA 275(19):1499-1503; Schuchat et al (1997) Bacterial Meningitis in the United States in 1995. N. Engl J Med 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. meningitidis is the major cause of bacterial meningitis at all ages in the United States (Schucaht et al (1997) supra).

Based on the organism's capsular polysaccharide, 12 serogrourps of N.

meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for m:\specifications\090000\93000\502278clmhxg.doc WO 00/22430 PCT/US99/23573 -2the 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 Morbidity and Mortality weekly report, Vol. 46, No. RR-5 (1997)). This is because polysaccharides are Tcell independent antigens that induce a weak immune response that cannot be boosted by repeated immunization. Following the success of the vaccination against H. influenzae, conjugate vaccines against serogroups A and C have been developed and are at the final stage of clinical testing (Zollinger WD "New and Improved Vaccines Against Meningococcal Disease". In: New Generation Vaccines, 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 (menA) and C (menC) (Vaccine 10:691-698)).

Meningococcus B (MenB) remains a problem, 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 Poolman JT (1992) Development of a meningococcal vaccine. Infect. Agents Dis. 4:13-28).

WO 00/22430 PCT/US99/23573 -3- 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 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. Vaccine 14(1):49-53).

A certain amount of sequence data is available for meningococcal and gonococcal genes and proteins EP-A-0467714, WO96/29412), but this is by no means complete.

The provision of further sequences could provide an opportunity to identify secreted or surface-exposed proteins that are presumed targets for the immune system and which are not antigenically variable or at least are more antigenically conserved than other and more variable regions. Thus, those antigenic sequences that are more highly conserved are preferred sequences. Those sequences specific to Neisseria meningitidis or Neisseria gonorrhoeae that are more highly conserved are further preferred sequences. 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 identification of sequences from the bacterium will also facilitate the production of biological probes, particularly organism-specific probes.

It is thus an object of the invention is to provide Neisserial DNA sequences which encode proteins predicted and/or shown to be antigenic or immunogenic, can be used as probes or amplification primers, and can be analyzed by bioinformatics.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates the products of protein expression and purification of the predicted ORF 919 as cloned and expressed in E. coli.

Fig. 2 illustrates the products of protein expression and purification of the predicted ORF 279 as cloned and expressed in E. coli.

Fig. 3 illustrates the products of protein expression and purification of the predicted ORF 576-1 as cloned and expressed in E. coli.

Fig. 4 illustrates the products of protein expression and purification of the predicted ORF 519-1 as cloned and expressed in E. coli.

WO 00/22430 PCT/US99/23573 -4- Fig. 5 illustrates the products of protein expression and purification of the predicted ORF 121-1 as cloned and expressed in E. coli.

Fig. 6 illustrates the products of protein expression and purification of the predicted ORF 128-1 as cloned and expressed in E. coli.

Fig. 7 illustrates the products of protein expression and purification of the predicted ORF 206 as cloned and expressed in E. coli.

Fig. 8 illustrates the products of protein expression and purification of the predicted ORF 287 as cloned and expressed in E. coli.

Fig. 9 illustrates the products of protein expression and purification of the predicted ORF 406 as cloned and expressed in E. coli.

Fig. 10 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 919 as cloned and expressed in E. coli.

Fig. 11 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 279 as cloned and expressed in E. coli.

Fig. 12 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 576-1 as cloned and expressed in E. coli.

Fig. 13 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 519-1 as cloned and expressed in E. coli.

Fig. 14 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 121-1 as cloned and expressed in E. coli.

Fig. 15 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 128-1 as cloned and expressed in E. coli.

Fig. 16 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 206 as cloned and expressed in E. coli.

Fig. 17 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 287 as cloned and expressed in E. coli.

Fig. 18 illustrates the hydrophilicity plot, antigenic index and AMPHI regions of the products of protein expression the predicted ORF 406 as cloned and expressed in E. coli.

WO 00/22430 PCT/US99/23573 THE INVENTION The invention is based on the 961 nucleotide sequences from the genome of N. meningitidis shown as SEQ ID NOs: 1-961 of Appendix C, and the full length genome of N. meningitidis shown as SEQ ID NO. 1068 in Appendix D. The 961 sequences in Appendix C represent substantially the whole genome of serotype B of N. meningitidis There is partial overlap between some of the 961 contiguous sequences ("contigs") shown in the sequences in Appendix C, which overlap was used to construct the single full length sequence shown in SEQ ID NO. 1068 in Appendix D, using the TIGR Assembler [G.S.

Sutton et al., TIGR Assembler: A New Tool for Assembling Large Shotgun Sequencing Projects, Genome Science and Technology, 1:9-19 (1995)]. Some of the nucleotides in the contigs had been previously released. (See ftp: 11ftp.tigr.org/pub/data/n_meningitidis on the world-wide web or The coordinates of the 2508 released sequences in the present contigs are presented in Appendix A. These data include the contig number (or as presented in the first column; the name of the sequence as found on WWW is in the second column; with the coordinates of the contigs in the third and fourth columns, respectively.

The sequences of certain MenB ORFs presented in Appendix B feature in International Patent Application filed by Chiron SpA on October 9, 1998 (PCT/IB98/01665) and January 14, 1999 (PCT/IB99/00103) respectively.

In a first aspect, the invention provides nucleic acid including one or more of the N. meningitidis nucleotide sequences shown in SEQ ID NOs:1-961 and 1068 in Appendices C and E. It also provides nucleic acid comprising sequences having sequence identity to the nucleotide sequence disclosed herein. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% 60%, 70%, 80%, 90%, 95%, 99% or more). These sequences include, for instance, mutants and allelic variants. The degree of sequence identity cited herein is determined across the length of the sequence determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following parameters: gap open penalty 12, gap extension penalty 1.

The invention also provides nucleic acid including a fragment of one or more of the nucleotide sequences set out herein. The fragment should comprise at least n consecutive nucleotides from the sequences and, depending on the particular sequence, n is 10 or more 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 100 or more). Preferably, the fragment is unique to the genome of N. meningitidis, that is to say it is not present in the genome of another organism. More preferably, the fragment is unique to the genome of strain B of N. meningitidis. The invention also provides nucleic acid that hybridizes to those provided herein. Conditions for hybridizing are disclosed herein.

In one embodiment, the invention provides an isolated or recombinant nucleic acid comprising a fragment of 100 or more nucleotides from position 1 to position 19530 or position 25265 to position 50955 of nucleotide sequence SEQ ID The invention also provides nucleic acid including sequences complementary to those described above for antisense, for probes, or for amplification primers).

Nucleic acid according to the invention can, of course, be prepared in many ways by chemical synthesis, from DNA libraries, from the organism itself, etc.) and can take various forms single-stranded, double-stranded, vectors, probes, primers, etc.). The term "nucleic acid" includes DNA and RNA, and also their analogs, such as those containing modified backbones, and also peptide nucleic acid (PNA) etc.

It will be appreciated that, as SEQ ID NOs:1-961 represent the substantially complete genome of the organism, with partial overlap, references to SEQ ID NOs: l- 961 include within their scope references to the complete genomic sequence, e.g., where two SEQ ID NOs overlap, the invention encompasses the single sequence which is formed by assembling the two overlapping sequences. Thus, for instance, a nucleotide sequence which bridges two SEQ ID NOs but is not present in its entirety in either SEQ ID NO is still within the scope of the invention. Additionally, such a sequence will be present in its entirety in the single full length sequence of SEQ ID NO:1068.

The invention also provides vectors including nucleotide sequences of the invention expression vectors, sequencing vectors, cloning vectors, etc.) and host cells transformed with such vectors.

According to a further aspect, the invention provides a protein including an amino acid sequence encoded within a N. meningitidis nucleotide sequence set out herein. It also provides proteins comprising sequences having sequence identity to those proteins. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% 60%, 70%, 80%, 90%, 95%, 99% or more).

Sequence identity is determined as above disclosed. These homologous proteins include mutants and allelic variants, encoded within the N. meningitidis nucleotide sequence set out herein.

In one embodiment, the invention provides an isolated or recombinant protein comprising an amino acid sequence encoded within position 1 to position 19530 or position 25265 to position 50955 of the N. meningitidis nucleotide sequence SEQ ID In another embodiment, the invention provides an isolated or recombinant protein comprising an amino acid sequence having greater than 70% sequence identity to an amino acid sequence encoded within position I to position 19530 or position 25265 to position 50955 of the N. meningitidis nucleotide sequence SEQ ID The invention further provides proteins including fragments of an amino acid sequence encoded within a N. meningitidis nucleotide sequence set out in the sequence listing. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope from the sequence.

In one embodiment, the invention provides an isolated or recombinant protein comprising a fragment of at least 10 amino acids from an amino acid sequence encoded within position 1 to position 19530 or position 25265 to position 50955 of the N.

meningitidis nucleotide sequence SEQ ID The proteins of the invention can, of course, be prepared by various means recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms native, fusions etc.). They are preferably prepared in substantially isolated form substantially free from other N. meningitidis host cell proteins).

Various tests can be used to assess the in vivo immunogenicity of the proteins of the invention. For example, the proteins can be expressed recombinantly or chemically synthesized 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; the protein is an immunogen. This method can also be used to identify immunodominant proteins.

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

In a further aspect, the invention provides a computer, a computer memory, a computer storage medium floppy disk, fixed disk, CD-ROM, etc.), and/or a computer database containing the nucleotide sequence of nucleic acid according to the invention. Preferably, it contains one or more of the N. meningitidis nucleotide sequences set out herein.

This may be used in the analysis of the N. meningitidis nucleotide sequences set out herein. For instance, it may be used in a search to identify open-reading frames (ORFs) or coding sequences within the sequences.

In a further aspect, the invention provides a method for identifying an amino acid sequence, comprising the step of searching for putative open reading frames or protein-coding sequences within a N. meningitidis nucleotide sequence set out herein.

Similarly, the invention provides the use of a N. meningitidis nucleotide sequence set out herein in a search for putative open reading frames or protein-coding sequences.

Open-reading frame or protein-coding sequence analysis is generally performed on a computer using standard bioinformatic techniques. Typical algorithms or program used in the analysis include ORFFINDER (NCBI), GENMARK [Borodovsky Mclninch (1993) WO 00/22430 PCT/US99/23573 -8- Computers Chem 17:122-133], and GLIMMER [Salzberg et al. (1998) Nucl Acids Res 26:544-548].

A search for an open reading frame or protein-coding sequence may comprise the steps of searching a N. meningitidis nucleotide sequence set out herein for an initiation codon and searching the upstream sequence for an in-frame termination codon. The intervening codons represent a putative protein-coding sequence. Typically, all six possible reading frames of a sequence will be searched.

An amino acid sequence identified in this way can be expressed using any suitable system to give a protein. This protein can be used to raise antibodies which recognize epitopes within the identified amino acid sequence. These antibodies can be used to screen N. meningitidis to detect the presence of a protein comprising the identified amino acid sequence.

Furthermore, once an ORF or protein-coding sequence is identified, the sequence can be compared with sequence databases. Sequence analysis tools can be found at NCBI (http://www.ncbi.nlm.nih.gov) the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx, tBLASTx [see also Altschul et al. (1997) Gapped BLAST and PSI- BLAST: new generation of protein database search programs. Nucleic Acids Research 25:2289-3402]. Suitable databases for comparison include the nonredundant GenBank, EMBL, DDBJ and PDB sequences, and the nonredundant GenBank CDS translations, PDB, SwissProt, Spupdate and PIR sequences. This comparison may give an indication of the function of a protein.

Hydrophobic domains in an amino acid sequence can be predicted using algorithms such as those based on the statistical studies of Esposti et al. [Critical evaluation of the hydropathy of membrane proteins (1990) Eur JBiochem 190:207-219]. Hydrophobic domains represent potential transmembrane regions or hydrophobic leader sequences, which suggest that the proteins may be secreted or be surface-located. These properties are typically representative of good immunogens.

Similarly, transmembrane domains or leader sequences can be predicted using the PSORT algorithm (http://www.psort.nibb.ac.jp), and functional domains can be predicted using the MOTIFS program (GCG Wisconsin PROSITE).

WO 00/22430 PCT[US99/23573 -9- The invention also provides nucleic acid including an open reading frame or proteincoding sequence present in a N. meningitidis nucleotide sequence set out herein.

Furthermore, the invention provides a protein including the amino acid sequence encoded by this open reading frame or protein-coding sequence.

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 mneans known to those skilled in the art.

The antibodies of the invention canbe used in a variety of ways, for confirmation that a protein is expressed, or to confirm where a protein is expressed. Labeled antibody fluorescent labeling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein, for instance.

According to a further aspect, the invenition provides compositions including protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as vaccines, as immunogenic compositions, or as diagnostic reagents.

The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments 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. Said Neisserial bacteria may be any species or strain (such as N. gonorrhoeae) but are preferably N. meningitidis, especially strain A, strain B or strain C.

In still yet another aspect, the present invention provides for compositions including proteins, nucleic acid molecules, or antibodies. More preferable aspects of the present invention are drawn to immunogenic compositions of proteins. Further preferable aspects of the present invention contemplate pharmaceutical immunogenic compositions of proteins or vaccines and the use thereof in the manufacture of a medicament for the treatment or prevention of infection due to Neisserial bacteria, preferably infection of MenB.

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.

WO 00/22430 PCTIUS99/23573 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 which may further include chemical synthesis of proteins and/or chemical synthesis (at least in part) of nucleotides.

A process for detecting polynucleotides of the invention is provided, comprising the steps of: contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and detecting said duplexes.

A process for detecting proteins of the invention is provided, comprising the steps of: contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and detecting said complexes.

Another aspect of the present invention provides for a process for detecting antibodies that selectably bind to antigens or polypeptides or proteins specific to any species or strain of Neisserial bacteria and preferably to strains ofN. gonorrhoeae but more preferably to strains ofN. meningitidis, especially strain A, strain B or strain C, more preferably MenB, where the process comprises the steps of: contacting antigen or polypeptide or protein according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and detecting said complexes.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Methodology Summary of standard procedures and techniques.

General This invention provides Neisseria meningitidis MenB nucleotide sequences, amino acid sequences encoded therein. With these disclosed sequences, nucleic acid probe assays and expression cassettes and vectors can be produced. The proteins can also be chemically synthesized. The expression vectors can be transformed into host cells to produce proteins.

The purified or isolated polypeptides can be used to produce antibodies to detect MenB proteins. Also, the host cells or extracts can be utilized for biological assays to isolate WO 00/22430 PCT/US99/23573 -11agonists or antagonists. In addition, with these sequences one can search to identify open reading frames and identify amino acid sequences. The proteins may also be used in immunogenic compositions and as vaccine components.

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 Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D.N Glover ed. 1985); Oligonucleotide Synthesis Gait ed, 1984); Nucleic Acid Hybridization Hames S.J. Higgins eds. 1984); Transcription and Translation Hames S.J. Higgins eds. 1984); Animal Cell Culture Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 155; Gene Transfer Vectorsfor MaInmalian Cells Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, and Handbook of Experimental Immunology, Volumes I-IV 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.

Expression systems The Neisseria MenB nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, plant cells, baculoviruses, 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 transcription of a coding sequence structural gene) into mRNA. A WO 00/22430 PCT/US99/23573 -12promoter will have a transcription initiating region, which is usually placed proximal to the end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box.

An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation (Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian Cells." In Molecular Cloning: A Laboratory Manual, 2nd ed.).

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible). Depending on the promoter selected, many promotes may be inducible using known substrates, such as the use of the mouse mammary tumor virus (MMTV) promoter with the glucocorticoid responsive element (GRE) that is induced by glucocorticoid in hormone-responsive transformed cells (see for example, U.S. Patent 5,783,681).

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter (Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer (Dijkema et al (1985) EMBO J. 4:761) and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777) and from human cytomegalovirus (Boshart et al. (1985) Cell 41:521). Additionally, some enhancers are regulatable and WO 00/22430 PCT/US99/23573 -13become active only in the presence of an inducer, such as a hormone or metal ion (Sassone- Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237).

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

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

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation (Bimstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) "Termination and 3' end processing of eukaryotic RNA. In Transcription and splicing (ed. B.D. Hames and D.M.

Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105). These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA.

Examples of transcription terminator/polyadenylation signals include those derived from SV40 (Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloning: A Laboratory Manual).

Usually, the above-described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.

Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element plasmids) capable of WO 00/22430 PCT/US99/23573 -14stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 (Gluzman (1981) Cell 23:175) or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 (Kaufman et al. (1989) Mol. Cell. Biol. 9:946) and pHEBO (Shimizu et al. (1986) Mol.

Cell. Biol. 6:1074).

The transformation procedure used depends upon the host to be transformed.

Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells Hep G2), and a number of other cell lines.

ii. Plant Cellular Expression Systems There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. 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 WO 00/22430 PCT/US99/23573 Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R.L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology,.

Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBOJ. 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 ofplasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

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

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only WO 00/22430 PCT/US99/23573 -16one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5' untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

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

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of WO 00/22430 PCT/US99/23573 -17introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into,the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence ofplasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, 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 germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

WO 00/22430 PCT/US99/23573 18- 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.

iii. Baculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego 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. 1555 (1987) (hereinafter "Summers and Smith").

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an i WO 00/22430 PCT/US99/23573 -19intermediate 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 plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

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

The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al.

(1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

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

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression," in: The Molecular Biology ofBaculoviruses (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 baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al.

WO 00/22430 PCT/US99/23573 (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 (alpha) ainterferon, 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'lAcad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

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

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

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus usually by cotransfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers 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 WO 00/22430 PCT/US99/23573 -21 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 thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 gim in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. Current Protocols in Microbiology Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (PCT Pub. No. WO 89/046699; Carbonell et al., (1985)J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol.

3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

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

WO 00/22430 PCT/US99/23573 -22- 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, HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, 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.

iv. Bacterial Systems Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream transcription of a coding sequence structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal to the WO 00/22430 PCT/US99/23573 -23- RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli coli) (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1977) Nature 198:1056), and maltose.

Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl.

Acids Res. 9:731; U.S. Patent 4,738,921; EPO Publ. Nos. 036 776 and 121 775). The betalactamase (bla) promoter system (Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. I. Gresser)), bacteriophage lambda PL (Shimatake et al. (1981) Nature 292:128) and T5 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 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 polymerase and initiate transcription. A naturally occurring promoter of nonbacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al.

(1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO Publ. No. 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 WO 00/22430 PCT/US99/23573 -24binding site is called the Shine-Dalgaro (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' end ofE. coli 16S rRNA (Steitz et al. (1979) "Genetic signals and nucleotide sequences in messenger RNA." In Biological Regulation and Development: Gene Expression (ed. R.F.

Goldberger)). To express eukaryotic genes and prokaryotic genes with weak ribosomebinding site, it is often necessary to optimize the distance between the SD sequence and the ATG of the eukaryotic gene (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 or in vitro incubation with a bacterial methionine Nterminal peptidase (EPO Publ. No. 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 (EPO Publ. No. 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 ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated (Miller et al. (1989) Bio/Technology 7:698).

WO 00/22430 PCT/US99/23573 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 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 phosphatase signal sequence (phoA) (Oka et al. (1985) Proc.

Natl. Acad. Sci: 82:7212). As an additional example, the signal sequence of the alphaamylase 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; EPO Publ. No. 244 042).

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

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will WO 00/22430 PCT/US99/23573 -26generally 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 (EPO Publ. No. 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.

Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

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

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis (Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. 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; EPO Publ. Nos. 036 776, 136 829 and 136 907), Streptococcus cremoris (Powell et al. (1988) WO 00/22430 PCT/US99/23573 -27- Appl. Environ. Microbiol. 54:655); Streptococcus lividans (Powell et al. (1988) Appl.

Environ. Microbiol. 54:655), Streptomyces lividans 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 CaCI 2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. (See use of Bacillus: Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541; use of Campylobacter: Miller et al. (1988) Proc. Natl.

Acad. Sci. 85:856; and Wang et al. (1990) J. Bacteriol. 172:949; use of Escherichia coli: Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res.

16:6127; Kushner (1978) "An improved method for transformation of Escherichia coli with 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; use of Lactobacillus: Chassy et al. (1987) FEMS Microbiol. Lett. 44:173; use of Pseudomonas: Fiedler et al.

(1988) Anal. Biochem 170:38; use of Staphylococcus: Augustin et al. (1990) FEMS Microbiol. Lett. 66:203; use of Streptococcus: Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun.

32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc.

4th Evr. Cong. Biotechnology 1:412.

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 transcription of acoding sequence structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the "TATA Box") and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), WO 00/22430 PCT/US99/23573 -28which, 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) (EPO Publ. No. 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO Publ. No. 329 203). The yeast PH05 gene, encoding acid phosphatase, also provides useful promoter sequences (Myanohara et al. (1983) Proc. Natl.

Acad. Sci. USA 80:1).

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatorysequence linked to the GAP transcription activation region Patent Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556).

Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, (Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; 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 ofMedical, Environmental and Commercial Importance (eds. K.N. Timmis and A. Puhler); Mercerau- Puigalonet 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 Nterminus of the recombinant protein will always be a methionine, which is encoded by the WO 00/22430 PCT/US99/23573 -29- ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, plant, 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 EPO Publ. No. 196056. 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 ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated W088/024066).

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

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EPO Publ. No. 012 873; JPO Publ. No.

62:096,086) and the A-factor gene Patent 4,588,684). Alternatively, leaders of nonyeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EPO Publ. No. 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro" region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about to about 50 amino acid residues) Patent Nos. 4,546,083 and 4,870,008; EPO Publ.

I

WO 00/22430 PCT/US99/23573 No. 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 alpha factor. (See PCT Publ. No. WO 89/02463.) Usually, transcription termination sequences recognized by yeast are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 (Botstein et al.

(1979) Gene 8:17-24), pCl/1 (Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646), and YRpl7 (Stinchcomb et al. (1982) J. Mol. Biol. 158:157). In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome (Orr- Weaver et al. (1983) Methods in Enzymol. 101:228-245). An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for WO 00/22430 PCT/US99/23573 31 inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced (Rine et al. (1983) Proc.

Natl. Acad. Sci. USA 80:6750). The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.

Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUPI 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 and methods of introducing exogenous DNA into yeast hosts have been developed for, inter alia, the following yeasts: Candida albicans (Kurtz, et al. (1986) Mol.

Cell. Biol. 6:142); Candida maltosa (Kunze, et al. (1985) J. Basic Microbiol. 25:141); Hansenula polymorpha (Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302); Kluyveromycesfragilis (Das, et al. (1984) J. Bacteriol.

158:1165); Kluyveromyces lactis (De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135); Pichia guillerimondii (Kunze et al. (1985) J.

Basic Microbiol. 25:141); Pichia pastoris (Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S.

Patent Nos. 4,837,148 and 4,929,555); Saccharomyces cerevisiae (Hinnen et al. (1978) Proc.

Natl. Acad. Sci. USA 75:1929; Ito et al. (1983)J. Bacteriol. 153:163); Schizosaccharomyces WO 00/22430 PCTIUS99/23573 -32pombe (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 [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. Genei. 202:302; Hansenula]; [Das et al. (1984) J.

Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol.

5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Patent Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al.

(1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Definitions A composition containing X is "substantially free of' Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term "heterologous" refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene.

Another example is where a Neisserial sequence is heterologous to a mouse host cell.

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 WO 00/22430 PCT/US99/23573 -33reproduced 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 a DNA, RNA or amino acid sequence differing from but having homology with the native or disclosed sequence. Depending on the particular sequence, the degree of homology between the native or disclosed sequence and the mutant sequence is preferably greater than 50% 60%, 70%, 80%, 90%, 95%, 99% or more) which is calculated 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 at essentially 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.

(see, for example, U.S. Patent 5,753,235).

Antibodies As used herein, the term "antibody" refers to a polypeptide or group ofpolypeptides 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, humanized 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 Neisseria MenB proteins.

Antibodies elicited against the proteins of the present invention bind to antigenic polypeptides or proteins or protein fragments that are present and specifically associated with strains of Neisseria meningitidis MenB. In some instances, these antigens may be associated with specific strains, such as those antigens specific for the MenB strains. The antibodies of WO 00/22430 PCT/US99/23573 -34the invention may be immobilized to a matrix and utilized in an immunoassay or on an affinity chromatography column, to enable the detection and/or separation ofpolypeptides, proteins or protein fragments or cells comprising such polypeptides, proteins or protein fragments. Alternatively, such polypeptides, proteins or protein fragments may be immobilized so as to detect antibodies bindably specific thereto.

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 g/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 for one hour, followed by incubating at 4 0 C for 2-18 hours. The serum is recovered by centrifugation 1,000g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler Milstein (Nature (1975) 256:495-96), or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells that express 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 hypoxanthine, aminopterin, thymidine medium, WO 00/22430 PCTIUS99/23573 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 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 32 P and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. "Specific binding partner" refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art.

It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, 1251 may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 125I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

Antigens, immunogens, polypeptides, proteins or protein fragments of the present invention elicit formation of specific binding partner antibodies. These antigens, immunogens, polypeptides, proteins or protein fragments of the present invention comprise immunogenic compositions of the present invention. Such immunogenic compositions may further comprise or include adjuvants, carriers, or other compositions that promote or enhance or stabilize the antigens, polypeptides, proteins or protein fragments of the present WO 00/22430 PCTIUS99/23573 -36invention. Such adjuvants and carriers will be readily apparent to those of ordinary skill in the art.

Pharmaceutical Compositions Pharmaceutical compositions can include 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, when given to a patient that is febrile. 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 judgment of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.

The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

WO 00/22430 PCT/US99/23573 -37- Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

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

Delivery Methods Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal and transcutaneous applications, 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 to prevent infection) or therapeutic to treat disease after infection).

Such vaccines comprise immunizing antigen(s) or immunogen(s), immunogenic polypeptide, protein(s) or protein fragments, or nucleic acids ribonucleic acid or deoxyribonucleic acid), usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to WO 00/22430 PCT/US99/23573 -38the 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 immunogen or antigen 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: aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; 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 MF59 (PCT Publ. No. WO 90/14837), containing 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), SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr- MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and Ribi T M adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL CWS (DetoxTM); saponin adjuvants, such as Stimulon T (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); cytokines, such as interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin a pertussis toxin or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129; see, WO 93/13302 and WO 92/19265; and other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59 are preferred.

WO 00/22430 PCT/US99/23573 -39- As mentioned above, muramyl peptides include, but are not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-Disoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

The vaccine compositions comprising immunogenic compositions which may include the antigen, 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. Alternatively, vaccine compositions comprising immunogenic compositions may comprise an antigen, polypeptide, protein, protein fragment or nucleic acid in a pharmaceutically acceptable carrier.

More specifically, vaccines comprising immunogenic compositions comprise an immunologically effective amount of the 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 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.

Typically, the vaccine compositions or 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.

The immunogenic compositions are conventionally administered parenterally, by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal and transcutaneous applications. Dosage treatment may be a single dose schedule WO 00/22430 PCT/US99/23573 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 Robinson Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648).

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

Expression of the coding sequence in vivo can be either constitutive or regulated.

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

Retroviral vectors are well known in the art, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J.

Virol. 53:160) polytropic retroviruses MCF and MCF-MLV (see Kelly (1983) J. Virol.

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

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

WO 00/22430 PCT/US99/23573 -41 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 lines or "VCLs") for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells 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 Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190).

Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection ("ATCC") 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) JNeurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

WO 00/22430 PCT/US99/23573 -42- Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and W093/07283, W093/06223, and W093/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in W094/12649, W093/03769, W093/19191, WO94/28938, W095/11984, 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 there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.

Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in US Patent 5,478,745.

Still other vectors are those disclosed in Carter US Patent 4,797,368 and Muzyczka US Patent 5,139,941, Chartejee US Patent 5,474,935, and Kotin W094/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470.

WO 00/22430 PCT/US99/23573 -43- 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 comprising sequences 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 WO95/04139 (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 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in US patents 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Serial No. 08/405,627, filed March 15, 1995,WO94/21792, WO92/10578, WO95/07994, US 5,091,309 and US 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see USSN 08/679640).

DNA vector systems such as eukarytic layered expression systems are also useful for expressing the nucleic acids of the invention. SeeW095/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox WO 00/22430 PCT/US99/23573 -44virus 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 NYAcad Sci 569:86, Flexner (1990) Vaccine 8:17; in US 4,603,112 and US 4,769,330 and W089/01973; 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 NatlAcad Sci 87:3802-3805; Enami Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJMed 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol.

66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP- 0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see US Serial No. 08/366,787, filed December 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) JBiol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see US Serial No.08/240,030, filed May 9, 1994, and US Serial No. 08/404,796, deposition of WO 00/22430 PCT/US99/23573 45 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 AcadSci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see US Serial No.

60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu Wu (1987) J Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Naked DNA may also be employed to transform a host cell. 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 efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S. 5,422,120, W095/13796, W094/23697, W091/14445 and EP-524,968. As described in USSN.

60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) 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 WO 00/22430 PCT/US99/23573 -46photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. 5,206,152 and WO92/11033 Exemplary liposome and polycationic gene delivery vehicles are those described in US 5,422,120 and 4,762,915; inWO 95/13796; WO94/23697; and W091/14445; in EP- 0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprise a 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 directly to the subject; delivered ex vivo, to cells derived from the subject; or 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, transdermally or transcutaneously, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a tumor or lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule. See WO98/20734.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in 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.

WO 00/22430 PCT/US99/23573 -47- Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

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

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

B. Hormones, Vitamins, Etc.

Other groups that can be included in a pharmaceutical composition include, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included in a pharmaceutical compositions with the desired polynucleotides and/or polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccarides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide) may be included in a pharmaceutical composition.

WO 00/22430 PCT/US99/23573 -48- D. Lipids, and Liposomes The desired polynucleotide or 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 or polypeptide. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl.

Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem.

265:10189-10192), in functional form.

Cationic liposomes are readily available. For example, N(1-2,3-dioleyloxy)propyl)-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, 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, 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.

WO 00/22430 PCT/US99/23573 -49- The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See Straubinger (1983) Meth.

Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem.

Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.

E. Lipoproteins In addition, lipoproteins can be included with the polynucleotide or polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery ofpolynucleotides 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, Al, All, AIV; CI, CII, CIII.

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

The amino acid sequences 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 )JBiol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.

WO 00/22430 PCT/US99/23573 Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phopholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem.

255:5454-5460 and Mahey (1979)J Clin. Invest 64:743-750.

Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443.

Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Massachusetts, USA.

Further description of lipoproteins can be found in Zuckermann et al., PCT. Appln.

No. US97/14465.

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

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications.

Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples of useful polypeptides include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as OX 174, transcriptional WO 00/22430 PCT/US99/23573 -51factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

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

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

G. Synthetic Polycationic Agents Synthetic polycationic agents which are useful in pharmaceutical compositions include, for example, DEAE-dextran, polybrene. LipofectinTM, and lipofectAMINETM are monomers that form polycationic complexes when combined with polynucleotides or polypeptides.

Immunodiagnostic Assays Neisseria MenB antigens, or antigenic fragments thereof, of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisseria MenB 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 Neisseria MenB proteins or fragments thereof within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the WO 00/22430 PCT/US99/23573 -52invention, 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 Hybridization "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 0

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 the complexity of the DNA being blotted and the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 tg 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 gg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 WO 00/22430 PCT/US99/23573 -53cpm/gg. For a single-copy mammalian gene a conservative approach would start with 10 Ag of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 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 probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation: Tm= 81 16.6(logioCi) 0.6(%formamide) 600/n where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth Wahl (1984) Anal. Biochem. 138:267-284).

In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42 0 C for a probe with is 95% to 100% homologous to the target fragment, 37 0 C for to 95% homology, and 32 0 C for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this WO 00/22430 PCT/US99/23573 -54approach 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.

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 sufficient complementarity with the a Neisserial sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least WO 00/22430 PCT/US99/23573 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. 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 backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. 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 see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384- 386).

One example of a nucleotide hybridization assay is described by Urdea et al. in international patent application W092/02526 (see also U.S. Patent 5,124,246).

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in: Mullis et al. (Meth.

Enzymol. (1987) 155: 335-350); US patent 4,683,195; and US patent 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 polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labeled 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 polymerase WO 00/22430 PCT/US99/23573 -56enzyme, 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 labeled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labeled with a radioactive moiety.

EXAMPLES

The invention is based on the 961 nucleotide sequences from the genome of N. meningitidis set out in Appendix C, SEQ ID NOs:1-961, which together represent substantially the complete genome of serotype B of N. meningitidis, as well as the full length genome sequence shown in Appendix D, SEQ ID NO 1068.

It will be self-evident to the skilled person how this sequence information can be utilized according to the invention, as above described.

The standard techniques and procedures which may be employed in order to perform the invention to utilize the disclosed sequences to predict polypeptides useful for vaccination or diagnostic purposes) were summarized above. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

These sequences are derived from contigs shown in Appendix C (SEQ ID NOs 1-961) and from the full length genome sequence shown in Appendix D (SEQ ID NO 1068), which were prepared during the sequencing of the genome ofN. meningitidis (strain The full length sequence was assembled using the TIGR Assembler as described by G.S. Sutton et al., TIGR Assembler: A New Tool for Assembling Large Shotgun Sequencing Projects, Genome Science and Technology, 1:9-19 (1995) [see also R. D. Fleischmann, et al., Science 269, 496- 512 (1995); C. M. Fraser, et al., Science 270, 397-403 (1995); C. J. Bult, et al., Science 273, 1058-73 (1996); C. M. Fraser, et. al, Nature 390, 580-586 (1997); Tomb, et. al., Nature 388, 539-547 (1997); H. P. Klenk, et al., Nature 390, 364-70 (1997); C. M. Fraser, et al., Science 281, 375-88 (1998); M. J. Gardner, et al., Science 282, 1126-1132 (1998); K. E.

Nelson, et al., Nature 399, 323-9 (1999)]. Then, using the above-described methods, putative translation products of the sequences were determined. Computer analysis of the translation products were determined based on database comparisons. Corresponding gene and protein sequences, if any, were identified in Neisseria meningitidis (Strain A) and Neisseria WO 00/22430 PCT/US99/23573 -57gonorrhoeae. Then the proteins were expressed, purified, and characterized to assess their antigenicity and immunogenicity.

In particular, the following methods were used to express, purify, and biochemically characterize the proteins of the invention.

Chromosomal DNA Preparation N. meningitidis strain 2996 was grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% Sucrose, 50 mM Tris-HC1, mM EDTA, adjusted to pH After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml lysis solution (50 mM NaCI, 1% Na-Sarkosyl, 50 Ig/ml Proteinase K), and the suspension was incubated at 37 0 C for 2 hours. Two phenol extractions (equilibrated to pH 8) and one ChC1 3 /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 4 ml buffer (10 mM Tris-HC1, ImM EDTA, pH The DNA concentration was measured by reading the OD at 260 nm.

Oligonucleotide design Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF, using the meningococcus B sequence when available, or the gonococcus/meningococcus A sequence, adapted to the codon preference usage of meningococcus. Any predicted signal peptides were omitted, by deducing the amplification primer sequence immediately downstream from the predicted leader sequence.

For most ORFs, the 5' primers included two restriction enzyme recognition sites (BamHI-NdeI, BamHI-Nhel, or EcoRI-NheI, depending on the gene's 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 pET21b+ (using either NdeI-XhoI or NheI-XhoI).

primer tail: CGCGGATCCCATATG (BamHI-NdeI) CGCGGATCCGCTAGC (BamHI-Nhel) WO 00/22430 PCT/US99/23573 -58- CCGGAATTCTAGCTAGC (EcoRI-NheI) 3'-end primer tail: CCCGCTCGAG (XhoI) For some ORFs, two different amplifications were performed to clone each ORF in the two expression systems. Two different 5' primers were used for each ORF; the same 3' Xhol primer was used as before: primer tail: GGAATTCCATATGGCCATGG (NdeI) primer tail: CGGGATCC (BamHI) Other ORFs were cloned in the pTRC expression vector and expressed as an amino-terminus His-tag fusion. The predicted signal peptide may be included in the final product. NheI-BamHI restriction sites were incorporated using primers: primer tail: GATCAGCTAGCCATATG (NheI) 3'-end primer tail: CGGGATCC (BamHI) As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridizeed to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the whole primer, and was determined for each primer using the formulae: Tm 4 2 (tail excluded) Tm= 64.9 0.41 GC) 600/N (whole primer) The average melting temperature of the selected oligos were 65-70 0 C for the whole oligo and 50-55°C for the hybridising region alone.

Oligos were synthesized by a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2 ml NH 4 -OH, and deprotected by 5 hours incubation at 56 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 100l.1 or Iml of water. OD 260 was determined using a Perkin Elmer Lambda Bio spectophotometer and the concentration was determined and adjusted to 2-10 pmol/l.

Table 1 shows the forward and reverse primers used for each amplification. In certain cases, it might be noted that the sequence of the primer does not exactly match the sequence in the ORF. When initial amplifications are performed, the complete 5' and/or 3' sequence WO 00/22430 PCT/US99/23573 -59may not be known for some meningococcal ORFs, although the corresponding sequences may have been identified in gonoccus. For amplification, the gonococcal sequences could thus be used as the basis for primer design, altered to take account of codon preference. In particular, the following codons may be changed: ATA-ATT; TCGT-CT; CAG4CAA; AAG->AAA; GAG-)GAA; CGA and CGG-CGC; GGG->GGC.

Amplification The standard PCR protocol was as follows: 50-200 ng of genomic DNA were used as a template in the presence of 20-40 gM of each oligo, 400-800 iM dNTPs solution, lx PCR buffer (including 1.5 mM MgCl2), 2.5 units Taql DNA polymerase (using Perkin-Elmer AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase, or Tahara Shuzo Taq polymerase).

In some cases, PCR was optimsed by the addition of 10l.t DMSO or 50 il 2M betaine.

After a hot start (adding the polymerase during a preliminary 3 minute incubation of the whole mix at 95 0 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 0

C.

The standard cycles were as follows: Denaturation Hybridisation Elongation First 5 cycles 30 seconds 30 seconds 30-60 seconds 0 C 50-55 0 C 72 0

C

Last 30 cycles 30 seconds 30 seconds 30-60 seconds 0 C 65-70 0 C 72 0

C

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 fragment 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 WO 00/22430 PCT/US99/23573 60 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 30pl or 50Jl of either water or 10mM Tris, pH Digestion of PCR fragments The purified DNA corresponding to the amplified fragment was split into 2 aliquots and double-digested with: NdeI/XhoI or NheI/XhoI for cloning into pET-21b+ and further expression of the protein as a C-terminus His-tag fusion BamHI/XhoI or EcoRIIXhoI for cloning into pGEX-KG and further expression of the protein as a GST N-terminus fusion.

For ORF 76, Nhel/BamHI for cloning into pTRC-HisA vector and further expression of the protein as N-terminus His-tag fusion.

Each purified DNA fragment was incubated (37 0 C for 3 hours to overnight) with units of each restriction enzyme (New England Biolabs) in a either 30 or 40 p 1 l 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) pl of either water or 10mM Tris-HC1, pH 8.5. The final DNA concentration was determined by 1% agarose gel electrophoresis in the presence of titrated molecular weight marker.

Digestion of the cloning vectors (pET22B, pGEX-KG and pTRC-His A) [Lg plasmid was double-digested with 50 units of each restriction enzyme in 200 pl reaction volume in the presence of appropriate buffer by overnight incubation at 37 0 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 p.l of 10 mM Tris-HC1, pH 8.5. The DNA concentration was evaluated by measuring OD 26 0 of the sample, and adjusted to 50 p.g/pl. 1 4l of plasmid was used for each cloning procedure.

WO 00/22430 PCT/US99/23573 -61 Cloning The fragments corresponding to each ORF, previously digested and purified, were ligated in both pET22b and pGEX-KG. In a final volume of 20 ftl, a molar ratio of 3:1 fragment/vector was ligated using 0.5 .1I of NEB T4 DNA ligase (400 units/pl), 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 Boheringer "Rapid Ligation Kit", following the manufacturer's instructions.

In order to introduce the recombinant plasmid in a suitable strain, 100 fl E. coli competent cells were incubated with the ligase reaction solution for 40 minutes on ice, then at 37 0 C for 3 minutes, then, after adding 800 pl LB broth, again at 37 0 C for 20 minutes. The cells were then centrifuged at maximum speed in an Eppendorf microfuge and resuspended in approximately 200 pl of the supernatant. The suspension was then plated on LB ampicillin (100 mg/ml).

The screening of the recombinant clones was performed by growing randomly-chosen colonies overnight at 37 OC in either 2 ml (pGEX or pTC clones) or (pET clones) LB broth 100 p.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 30 .1l. 5 tl of each individual miniprep (approximately Ig) were digested with either NdeIIXhoI or BamHI/XhoI 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.

Cloning Certain ORFs may be cloned into the pGEX-HIS vector using EcoRI-PstI, EcoRI-SalI, or SalI-PstI cloning sites. After cloning, the recombinant plasmids may be introduced in the E. coli host W3110.

Expression Each ORF cloned into the expression vector may then be transformed into the strain suitable for expression of the recombinant protein product. 1 p.1 of each construct was used to WO 00/22430 PCT/US99/23573 -62transform 30 il of E.coli BL21 (pGEX vector), E.coli TOP 10 (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 gg/ml), incubated at 37 0 C overnight, then diluted 1:30 in 20 ml of LB+Amp (100 g/ml) in 100 ml flasks, making sure that the OD 6 oo ranged between 0.1 and 0.15. The flasks were incubated at 30 0 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 addiction of ImM IPTG, whereas in the case ofpGEX system the final concentration of IPTG was 0.2 mM. After 3 hours incubation at 30 0 C, the final concentration of the sample was checked by OD. In order to check expression, Iml 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.

GST-fusion proteins large-scale purification.

A single colony was grown overnight at 37 0 C on LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid colture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600 ml of fresh medium and allowed to grow at the optimal temperature (20-37 0 C) to OD 5 50 0.8-1. Protein expression was induced with 0.2mM IPTG followed by three hours incubation. The culture was centrifuged at 8000 rpm at 4 0 C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml cold PBS. 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. The supematant was collected and mixed with 150pl 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 4C. The resin was washed twice with 10 ml cold PBS for 10 minutes, resuspended in Iml cold PBS, and loaded on a disposable column. The resin was washed twice with 2ml cold PBS until the flow-through reached OD 2 80 of 0.02-0.06.

The GST-fusion protein was eluted by addition of 700.l cold Glutathione elution buffer 10mM reduced glutathione, 50mM Tris-HCI) and fractions collected until the OD 28 0 was 0.1.

WO 00/22430 PCT/US99/23573 -63- 21pl 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, kDa) or Amersham Rainbow Marker (220, 66, 46, 30, 21.5, 14.3 kDa) as standards. As the MW of GST is 26kDa, this value must be added to the MW of each GST-fusion protein.

His-fusion soluble proteins large-scale purification.

A single colony was grown overnight at 37 0 C on a LB Amp agar plate. The bacteria were inoculated into 20ml of LB+Amp liquid culture and incubated overnight in a water bath shaker. Bacteria were diluted 1:30 into 600ml fresh medium and allowed to grow at the optimal temperature (20-37 0 C) to OD 550 0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000 rpm at 4C, the supernatant was discarded and the bacterial pellet was resuspended in 7.5ml cold 10mM imidazole buffer (300 mM NaCI, 50 mM phosphate buffer, mM imidazole, pH The cells were disrupted by sonication on ice for 30 sec at using a Branson sonifier B-15, frozen and thawed two times and centrifuged again. The supernatant was collected and mixed with 150[il Ni2+-resin (Pharmacia) (previously washed with 10mM imidazole buffer) and incubated at room temperature with gentle agitation for minutes. The sample was centrifuged at 700g for 5 minutes at 4 0 C. The resin was washed twice with 10 ml cold 10mM imidazole buffer for 10 minutes, resuspended in Iml cold imidazole buffer and loaded on a disposable column. The resin was washed at 4°C with 2ml cold 10mM imidazole buffer until the flow-through reached the O.D 2 8 o of 0.02- 0.06. The resin was washed with 2ml cold 20mM imidazole buffer (300 mM NaCI, 50 mM phosphate buffer, 20 mM imidazole, pH 8) until the flow-through reached the O.D 28 0 of 0.02- 0.06. The His-fusion protein was eluted by addition of 700pl cold 250mM imidazole buffer (300 mM NaCI, 50 mM phosphate buffer, 250 mM imidazole, pH 8) and fractions collected until the O.D 280 was 0.1. 21l of each fraction were loaded on a 12% SDS gel.

His-fusion insoluble proteins large-scale purification.

A single colony was grown overnight at 37 °C on a LB Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600ml fresh medium and let to grow at the WO 00/22430 PCT/US99/23573 -64optimal temperature (37 0 C) to O.D 550 0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000rpm at 4 0 C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml buffer B (urea 8M, 10mM Tris-HC1, 100mM phosphate buffer, pH 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 stored at -20°C, while the pellets were resuspended in 2 ml guanidine buffer (6M guanidine hydrochloride, 100mM phosphate buffer, 10 mM Tris-HCl, pH 7.5) and treated in a homogenizer for 10 cycles. The product was centrifuged at 13000 rpm for 40 minutes. The supernatant was mixed with 150.l Ni2+-resin (Pharmacia) (previously washed with buffer B) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for minutes at 4 0 C. The resin was washed twice with 10 ml buffer B for 10 minutes, resuspended in lml buffer B, and loaded on a disposable column. The resin was washed at room temperature with 2ml buffer B until the flow-through reached the OD 28 0 of 0.02-0.06.

The resin was washed with 2ml buffer C (urea 8M, 10mM Tris-HC1, 100mM phosphate buffer, pH 6.3) until the flow-through reached the O.D 280 of 0.02-0.06. The His-fusion protein was eluted by addition of 700tl elution buffer (urea 8M, 10mM Tris-HC1, 100mM phosphate buffer, pH 4.5) and fractions collected until the OD 28 0 was 0.1. 211pl of each fraction were loaded on a 12% SDS gel.

His-fusion proteins renaturation glycerol was added to the denatured proteins. The proteins were then diluted to using dialysis buffer I (10% glycerol, 0.5M arginine, 50mM phosphate buffer, reduced glutathione, 0.5mM oxidised glutathione, 2M urea, pH 8.8) and dialysed against the same buffer at 4 0 C for 12-14 hours. The protein was further dialysed against dialysis buffer II (10% glycerol, 0.5M arginine, 50mM phosphate buffer, 5mM reduced glutathione, oxidised glutathione, pH 8.8) for 12-14 hours at 4 0 C. Protein concentration was evaluated using the formula: Protein (mg/ml) (1.55 x OD 28 0 (0.76 x OD 2 6 o) I WO 00/22430 PCT/US99/23573 Mice immunisations of each purified protein were used to immunise mice intraperitoneally. In the case of some ORFs, Balb-C 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 other ORFs, CD1 mice could be immunised using the same protocol. For other ORFs, CD1 mice could be immunised using Freund's adjuvant, and the same immunisation protocol was used, except that the immune response was measured on day 42, rather than 56. Similarly, for still other ORFs, CDI mice could be immunised with Freund's adjuvant, but the immune response was measured on day 49.

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 7ml of Mueller-Hinton Broth (Difco) containing 0.25% Glucose. Bacterial growth was monitored every 30 minutes by following OD 62 0 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 10000 rpm. 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 0 C with stirring. 100.1 bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4 0 C. The wells were then washed three times with PBT washing buffer Tween-20 in PBS). 200 gl of saturation buffer Polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37 0 C. Wells were washed three times with PBT. 200 .l of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN 3 in PBS) were added to each well and the plates incubated for 90 minutes at 37 0 C. Wells were washed three times with PBT. 100 pl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37 0

C.

Wells were washed three times with PBT buffer. 100 pl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 il of H 2 0) were added to each well and the plates were left at room temperature for 20 minutes. 100 pl H 2

SO

4 was added to each WO 00/22430 PCT/US99/23573 -66well and OD 4 90 was followed. The ELISA was considered positive when OD490 was times the respective pre-immune sera.

FACScan bacteria Binding Assay procedure.

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37 0 C. Bacterial colonies were collected from the agar plates using a sterile dracon 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

OD

62 0 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 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer BSA, 0.4% NaN 3 and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD 6 20 of 0.07. 100u1 bacterial cells were added to each well of a Costar 96 well plate. 1001 of diluted (1:200) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4 0 C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200pl/well of blocking buffer in each well. 100p1 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 0 C. Cells were spun down by centrifugation at 4000rpm for 5 minutes and washed by addition of 200.tl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200pl/well 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 Treshold:92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gains 7.1; FL-2 PMT: 539. Compensation values: 0.

OMV preparations Bacteria were grown overnight on 5 GC plates, harvested with a loop and resuspended in 10 ml 20mM Tris-HC1. Heat inactivation was performed at 56 0 C for 30 minutes and the bacteria disrupted by sonication for 10' on ice 50% duty cycle, 50% output Unbroken cells were removed by centrifugation at 5000g for 10 minutes and the total cell envelope fraction recovered by centrifugation at 50000g at 4 0 C for 75 minutes. To extract cytoplasmic membrane proteins from the crude outer membranes, the whole fraction was resuspended in WO 00/22430 PCT/US99/23573 -67- 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 50000g for 75 minutes to pellet the outer membranes. The outer membranes were resuspended in 10mM Tris-HC1, pH8 and the protein concentration measured by the Bio-Rad Protein assay, using BSA as a standard.

Whole Extracts preparation Bacteria were grown overnight on a GC plate, harvested with a loop and resuspended in Iml of 20mM Tris-HC1. Heat inactivation was performed at 56 0 C for 30' minutes.

Western blotting Purified proteins (500ng/lane), outer membrane vesicles (5 pbg) and total cell extracts tg) derived from 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 0 C, in transferring buffer (0.3 Tris base, 1.44 glycine, 20% methanol). The membrane was saturated by overnight incubation at 4 0 C in saturation buffer (10% skimmed milk, 0.1% Triton XI00 in PBS). The membrane was washed twice with washing buffer skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37 0 C with 1:200 mice sera diluted in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labeled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

Bactericidal assay MC58 strain was grown overnight at 37 0 C on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7ml Mueller-Hinton broth. The suspension was incubated at 37 0 C on a nutator and let to grow until OD 620 was in between 0.5-0.8. The culture was aliquoted into sterile 1.5ml 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 OD 6 2 0 of 0.5, diluted 1:20000 in Gey's buffer and stored at 25 0

C.

WO 00/22430 PCT/US99/23573 -68- 504l of Gey's buffer/l% BSA was added to each well of a 96-well tissue culture plate. 254l of diluted (1:100) mice sera (dilution buffer: Gey's buffer/0.2% BSA) were added to each well and the plate incubated at 4 0 C. 25p. of the previously described bacterial suspension were added to each well. 251l of either heat-inactivated (56 0 C waterbath for minutes) or normal baby rabbit complement were added to each well. Immediately after the addition of the baby rabbit complement, 22tl of each sample/well were plated on Mueller- Hinton agar plates (time The 96-well plate was incubated for 1 hour at 37 0 C with rotation and then 22ul of each sample/well were plated on Mueller-Hinton agar plates (time After overnight incubation the colonies corresponding to time 0 and time Ih were counted.

The following DNA and amino acid sequences are identified by titles of the following form: m, or a] or pep], where means a sequence from N. gonorrhoeae, "m" means a sequence from N. meningitidis B, and means a sequence from N. meningitidis A; means the number of the sequence; "seq" means a DNA sequence, and "pep" means an amino acid sequence. For example, "g001.seq" refers to an N. gonorrohoeae DNA sequence, number 1. The presence of the suffix or to these sequences indicates an additional sequence found for the same ORF. Further, open reading frames are identified as ORF where means the number of the ORF, corresponding to the number of the sequence which encodes the ORF, and the ORF designations may be suffixed with or indicating that the ORF corresponds to a N. gonorrhoeae sequence or a N. meningitidis A sequence, respectively. Computer analysis was performed for the comparisons that follow between and peptide sequences; and therein the "pep" suffix is implied where not expressly stated.

EXAMPLE 1 The following ORFs were predicted from the contig sequences and/or the full length sequence using the methods herein described.

Localization of the ORFs ORF: contig: 279 gnm4.seq The following partial DNA sequence was identified in N. meningitidis <SEQ ID 962>: WO 00/22430 PCT/US99/23573 69 m279.seq 1 ATAACGCGGA TTTGCGGCTG CTTGATTTCA ACGGTTTTCA GGGCTTCGGC 51 AAGTTTGTCG GCGGCGGGTT TCATCAGGCT GCAATGGGAA GGTACGGACA 101 CGGGCAGCGG CAGGGCGCGT TTGGCACCGG CTTCTTTGGC GGCAGCCATG 151 GCGCGTCCGA CGGCGGCGGC GTTGCCTGCA ATCACGATTT GTCCGGGTGA 201 GTTGAAGTTG ACGGCTTCGA CCACTTCGCT TTGGGCGGCT TCGGCACAAA 251 TGGCTTTAAC CTGCTCATCT TCCAAGCCGA GAATCGCCGC CATTGCGCCC 301 ACGCCTTGCG GTACGGCGGA CTGCATCAGT TCGGCGCGCA GGCGCACGAG 351 TTTGACCGCG TCGGCAAAkAT TCAATGCGCC GGCGGCAACG AGTGCGGTGT 401 ATTCGCCGAG GCTGTGTCCG GCAACGGCGG CAGGCGTTTT GCCGCCCGCT 451 TCTAAATAG This corresponds to the amino acid sequence <SEQ ID 963; ORF 279>: m279.pep 1 ITRICCCLIS TVFRASASLS AAGFIRLQWE GTDTGSGRAR LAPASLAAAM 51 ARPTAAALPA ITICPGELKL TASTTSLWAA SAQMALTCSS SKPRIAAIAP 101 TPCGTADCIS SARR.RTSLTA SAKFNAPAAT SAVYSPRLCP ATAAGVLPPA 151 SK* The following partial DNA sequence was identified in N.gonorrhoeae <SEQ ID) 964>: g279. seq 1 atgacgcgga tttgcggctg cttgatttca acggttttga gtgtttcggc 51 aagtttgtcg gcggcgggtt tcatcaggct gcaatgggaa ggaacggata 101 ccggcagcgg cagggcgcgt ttggctccgg cttctttggc ggcagccatg 151 gtgcgtccga cggcggcggc gttgcctgca atcacgactt gtccgggcga 201 gttgaagttg acggcttcga ccacttcgcc ctgtgcggat tcggcacaaa 251 tctgcctgac ctgttcatct tccaaaccca aaatggccgc cattgcgcct 301 acgccttgcg gtacggcgga ctgcatcagt tcggcgcgca ggcggacgag 351 tttgacggca tcggcaaaat ccaatgcttc ggcggcgaca agcgcggtgt 401 attcgccgag gctgtgtccg gcaacggcgg caggcgtttt gccgcccact 451 tccaaatag This corresponds to the amino acid sequence <SEQ ID 965; ORE 279.ng>: 927 9.pep 1 MTRICGCLIS TVLSVSASLS AAGFIPLQWE GTDTGSGRAR LAPASLAAAN 51 VRPTAAALPA ITTCPGELKL TASTTSPCAD SAQICLTCSS SKPIGnAAIAP 101 TPCGTADCIS SARRRTSLTA SAKSNASAAT SAVYSPRLCP ATAAGVLPPT 151 SK* ORE 279 shows 89.5% identity over a 152 aa overlap with a predicted ORF (ORE 279.ng) from N. gonorrhoeae: 20 30 40 50 m2 79 .pep ITRI CGCLISTVFRASASLSAAGFIRLQWEGTDTGSGRARLAPASLAAAMARPTAAALPA g279 MTRICGCLISTVLSVSASLSAAGFIRLOWEGTDTGSGRARLAPASLAAAMVRPTAAALPA 10 20 30 40 50 80 90 100 110 120 m279 .pep ITICPGELKLTASTTSLWAASAQMALTCSSSKPRIAAIAPTPCGTADCISSARRRTSLTA 9279 ITTCPGELKLTASTTSPCAflAQICLTCSSSKPKMAAIAPTPCGTACISSARRTSLTA 80 90 100 110 120 130 140 150 m279.pep SAXFNAPAATSAVYSPRLCPATAAGVLPPASKX 1 1 1 g279 SAKSNASAATSAVYSPRLCPATAAGVLPPTSKX 130 140 150 WO 00/22430 PCT/US99/23573 70 The following partial DNA sequence was identified in N meningitidis <SEQ ID 966>: a279. seq 1 ATGACNCNGA TTTGCGGCTG CTTGATTTCA ACGGTTTNNA GGGCTTCGGC 51 GAGTTTGTCG GCGGCGGGTT TCATGAGGCT GCAATGGGAA GGTACNGACA 101 CNGGCAGCGG CAGGGCGCGT TTGGCGCCGG CTTCTTTGGC GGCAAGCATA 151 GCGCGCTCGA CGGCGGCGGC ATTGCCTGCA ATCACGACTT GTCCGGGCGA 201 GTTGAAGTTG ACGGCTTCAA CCACTTCATC CTGTGCGCAT TCGGCGCAAA 251 TTTGTTTTAC CTGTTCATCT TCCAAGCCGA GAATCGCCGC CATTGCGCCC 301 ACGCCTTGCG GTACGGCGGA CTGCATCAGT TCGGCGCGCA NGCGCACGAG 351 TTTGACCGCG TCGGCAAAAT CCAATGCGCC GGCGGCAACN AGTGCGGTGT 401 ATTCGCCGAN GCTGTGTCCG GCAACGGCGG CAGGCGTTTT GCCGCCCGCT 451 TCCGAATAG This corresponds to the amino acid sequence <SEQ ID 967; ORE 279.a>: a279.pep 1 MTXICGCLIS TVXRASASLS AAGFMRLQWE GTDTGSGRAR LAPASLAASI 51 ARSTAAALPA ITTCPGELKL TASTTSSCAD SAQICF1'CSS SKPRIAAIAP 101 TP-CGTADCIS SARXRTSLTA SAKSNAPAAT SAVYSPXLCP ATAAGVLPPA 151 SE* m279/a279 OR~s 279 and 279.a showed a 88.2% identity in 152 aa overlap 20 30 40 50 m279 .pep ITRICGCLISTVFASASLSAAGFIRLQWEGTDTGSGRARLAASLAAAMARPTAAALPA 1 1 1 1 1 1 1 a2 79 MTXICGCLI STVXRASASLSAAGFMRLQWEGTDTGSGPARLAPASLAAS IARSTAAALPA 20 30 40 50 80 90 100 110 120 m27 9. pep ITICPGELKLTASTTSLWAASAQMALTCSSSKPRIAAIAPTPCGTADCISS3ARRRTSLTA a27 9 ITTCPGELKLTASTTSSCADSAOICE'rCSSSKPRIAAIAPTPCGTADCISSARXRTSLTA 80 90 100 110 120 130 140 150 m279 .pep SAX<FNAPAATSAVYSPRLCPATAAGVLPPASKX a27 9 SAKSNAPAATSAVYSPXLCPATAAGVLPPASEX 130 140 519 and 519-1 gnm7.seq The following partial DNA sequence was identified in N. meningitidis <SEQ ID 968>: rn5l9.seq (partial) I .TCCGTTATCG GGCGTATGGA GTTGGACAAA ACGTTTGAAG AACGCGACGA 51 AATCAACAGT ACTGTTGTTG CGGCTTTGGA CGAGGCGGCC GGGgCTTgGG 101 GTGTGAAGGT TTTGCGTTAT GAGATTAAAG ACTTGGTTCC GCCGCAAGAA 151 ATCCTTCGCT CAATGCAGGC GCAAATTACT GCCGAACGCG AAAAACGCGC 201 CCGTATCGCC GAATCCGAA.G GTCGTAAAAT CGAACAAAtC AACCTTGCCA 251 GTGGTCAGCG CGAAGCCGAA ATCCAACAAT CCGAAGGCGA GGCTCAGGCT 301 GCGGTCAATG CGTCAAATGC CGAGAAAATC GCCCGCATCA ACCGCGCCAA 351 AGGTGAAGCG GAATCCTTGC GCCTTGTTGC CGAAGCCAAT GCCGAAGCCA 401 TCCGTCAAAT TGCCGCCGCC CTTCAAACCC AAGGCGGTGC GGATGCGGTC 451 AATCTGAAGA TTGCGGAACA ATACGTCGCT GCGTTCAACA ATCTTGCCAA 501 AGAAAGCAAT ACGCTGATTA TGCCCGCCAA TGTTGCCGAC ATCGGCAGCC 551 TGATTTCTGC CGGTATGAAA ATTATCGACA GCAGCAAAAC CGCCAAaTAA WO 00/22430 WO 0022430PCT/US99/23573 71 This corresponds to the amino acid sequence <SEQ ID 969; ORE 519>: m519.pep (partial) 1 .SVIGRM4ELDK TFEERDEINS TVVAALDEAA GAWGVKVLRY EIKDLVPPOE 51 ILRSMQAQIT AEREKRARIA ESEGRKIEQI NLASGQREAE IOOSEGEAQA 101 AVNASNAEKI ARINRAKGEA ESLRLVAEAN AEAIRQIAAA LQTQGGADAV 151 NLKIAEQYVA AFNNLAKESN TLIMPANVAD IGSLISAGMK IIDSSKTAK* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 970>: 9519. aeq 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 atggaatttt atcctttgtc ggcgtttcca atcgaccgcg acccagccag gcatcatcta agcaactaca cgttatcggg tcaacagtac gtgaaagtcc ccttcgcgca gtattgccga ggtcagcgtg ggtcaatgcg gcgaagcgga cgtcaaattg tctgaagatt aagacaatac aattttcggc tcattatctt gtcatccccc tcgcgccctg tcgcctaccg gtctgcatca tttccaagta ttatggcaat cgtatggagt cgtcgtctcc tccgttacga a tgcaggcac atccgaaggc aagccgaaat tccaatgccg atccctgcgc ccgccgccct gcgggacaat gcggattaag ggcatgaaaa gttggcagcc agcaggaagt acggccggtt ccattcgctg cgcgcgataa accgatccca tacccagctt tggacaaaac gccctcgatg aatcaaggat aaattaccgc cgtaaaatcg ccaacaatcc agaaaatcgc Cttgttgccg tcaaacccaa acgttaccgc cccgccaagg attttcgcca gtcgccgttt ccacgttgtc tgaatatttt aaagaaatcc tacgcaattg aact cgcct c gcccaaacga gtttgaagaa aagccgccgg ttggttccgc cgaacgcgaa aacaaatcaa gaaggcgagg ccgcatcaac aagccaatgc agcggggcgg gttcaaaaat ttgccgaaat gaagcaaaaa tcggcttcaa gaaaggctcg gat tccc t tt ctttagacg: actgttgacg atacggttcg cgctgcgttc cgcgacgaaa ggcttggggt cgcaagaaa: aaacgcgccc ccttgccagt ctcaggctgc cgcgccaaag cgaagccaac atgcggtcaa cc tgccaaag.

cgggaaccct cggccaaata 951 a This corresponds to the amino acid sequence <SEQ ID 971; ORE 51 9.ng>: 19.pep 1 MEFFIILLAA VAVFGFKSFV 51 IDP.VAYRHSL KEIPLDVPSO 101 SNYIMAITQL AQTTLRSVIG 151 VKVLRYEIKD LVPPQEILRA 201 GQREAEIQQS EGEAQAAVNA 251 RQIAAALQTQ SGADAVNLKI 301 NFRRHEKFSP EAKTAK* VI PQQEVHVV

VCITRDNTQL

RMELDKTFEE

MQA I TAERE

SNAEKIARIN

AGQYVTAFKN

ERLGRFHRAL TAGLNILIPF TVDGIIYFQV TDPKLASYGS RDE INSTVVS ALDEAAGAWG KRARIAESEG RKIEQINLAS RAKGEAESLR LVAEANAEAN LAKEDNTRI K PAKVAEIGNP ORE 519 shows 87.5% identity over a 200 aa overlap with a predicted ORE ('ORE 519.ng) from N. gonorrhoeae: mS 19/g 519 m519 .pep 20 SVIGRMELDKTFEERflEINSTVVAALDEAA II II II 11111111II11 :111 I

YFQVTDPKLASYGSSNYIMAITQLAOTTLRSVIGRMELDKTFEERDEINSTVVSALDEAA

100 110 120 130 140 g519 s0 60 70 80 19. .pep GAWGVKVLRYEIKDLVPPQEILRSMQAQITAEREKRARIAESEGRKIEQINLASGQREAE g519 GAWGVKVLRYEIKDLVPPQEILRAMQAOITAEREKRARIAESEGRKIEOINLASGOREAE 150 160 170 180 190 200 100 110 120 130 140 150 rn~l9.pep IQQSEGEAQAAVNASNAEKIARINRAKGEAESLRLVAEANAEAIRQIAAALQTQGGADAV WO 00/22430 PCTIUS99/23573 72 19 I QQSEGEAQAAVNASNAEKIARINRAKGEAESLRLVAEANAEANRQIAAALQTQSGADAV 210 220 230 240 250 260 160 10 180 190 200 r1519 .pep NLKIAEQYVAAFNNLAKESNTLIMPANVADIGSL-ISAGMKIIDSSKTAK 9519 NLKI AGQYVTAFfUNLAKEDNTRI KPAKVAE IGNPNFRRHEKFS PEAKTAK 270 280 290 300 310 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 972>: a519. seq 1 ATGGAATTTT TCATTATCTT GCTGGCAGCC GTCGTTGTTT TCGGCTTCAA 51 ATCCTTTGTT GTCATCCCAC AGCAGGAAGT CCACGTTGTC GAAAGGCTCG 101 GGCGTTTCCA TCGCGCCCTG ACGGCCGGTT TGAATATTTT GATTCCCTTT 151 ATCGACCGCG TCGCCTACCG CCATTCGCTG AAAGAAATCC CTTTAGACGT 201 ACCCAGCCAG GTCTGCATCA CGCGCGACAA TACGCAGCTG ACTGTTGACG 251 GTATCATCTA TTTCCAAGTA ACCGACCCCA AACTCGCCTC ATACGGTTCG 301 AGCAACTACA TTATGGCGAT TACCCAGCTT GCCCAAACGA CGGTGCGTTC 351 CGTTATCGGG CGTATGGAAT TGGACAAAAC GTTTGAAGAA CGCGACGAAA 401 TCAACAGCAC CGTCGTCTCC GCCCTCGATG AAGCCGCCGG AGCTTGGGGT 451 GTGAAGGTTT TGCGTTATGA GATTAAAGAC TTGGTTCCGC CGCAAGAAAT 501 CCTTCGCTCA ATGCAGGCGC AAATTACTGC TGAACGCGAA AAACGCGCCC 551 GTATCGCCGA ATCCGAAGGT CGTAAAATCG AACAAATCAA CCTTGCCAGT 601 GGTCAGCGCG AAGCCGAAAT CCAACAATCC GAAGGCGAGG CTCAGGCTGC 651 GGTCAATGCG TCAAATGCCG AGAAAATCGC CCGCATCAAC CGCGCCAAAG 701 GTGAAGCGGA ATCCTTGCGC CTTGTTGCCG AAGCCAATGC CGAAGCCATC 751 CGTCAAATTG CCGCCGCCCT TCAAACCCAA GGCGGTGCGG ATGCGGTCAA 801 TCTGAAGATT GCGGAACAAT ACGTCGCCGC GTTCAACAAT CTTGCCAAAG 851 AAAGCAATAC GCTGATTATG CCCGCCAATG TTGCCGACAT CGGCAGCCTG 901 ATTTCTGCCG GTATGAAAAT TATCGACAGC AGCAAAACCG CCAAATAA This corresponds to the amino acid sequence <SEQ ID 973; ORE 519.a>: a519 .pep 1 MEFFIILLAA VVFGFKSFV VIPQQEVHVV ERLGRFHRAL TAGLNILIPF 51 IDRVAYRHSL KEIPLDVPSQ VCITRDNTQL TVDGIIYFQV TDPKLASYGS 101 SNYIMAITQL AQTTLRSVIG RMELDKTFEE RDEINSTVVS ALDEAAGAWG 151 VKVLRYEIKD LVPPQEILRS MQAQITAERE KRARIAESEG RKIEQINLAS 201 GQREAEIQQS EGEAQAAVNA SNAEKIARIN RAKGEAESLR LVAEANAEAI 251 RQIAAALQTQ GGADAVNLKI AEQYVAAFNN LAKESNTLIM PANVADIGSL 301 ISAGMAIIDS SKTAK* M519/a519 ORrs 519 and 519.a showed a 99.5% identity in 199 aa overlap 10 20 m519.pep SVIGRMELDKTFEERDEINSTVVAALDEAA a51 9 YFQVTDPKLASYGSSNYIMAITQLAQTTLRSVIGPRNELDKTFEERDEINSTVVSALDEAA 100 110 120 130 140 50 60 70 80 m519 .pep GAWGVKVLRYEIKDLVPPQEILRSMQAQITAEREKRARIAESEGRKIEQINLASGQREAE a51 9 GAWGVKVLRYEIKDLVPPQEILRSMQAQITAEREKRARIAESEGRKIEQINLASGQREAE 150 160 170 180 190 200 100 110 120 130 140 150 r~l 9. pep IQQSEGEAQAAVNASNAEKIARINRKGEAESLRLVAEANAEAIRQIALQTQGGADAV a51 9 IQQSEGEAQAAVNASNAEKIARINRAKGEAESLRLVAEANAEAIRQIAAALQTQGGADAV 1210 220 230 240 250 260 WO 00/22430 PCTIUS99/23573 -73- 160 170 180 190 200 9. pep NLKIAEQYVAAFNNLAKESNTLIMPANVADIGSLISAGMKIIDSSKTAKX a51 9 NLKIAEQYVAAFNNLAKESNTLIMPAI4VADIGSLISAGMKIIDSSKTAKX 270 280 290 300 .310 Further work revealed the following DNA sequence identified in N. meningitidis <SEQ ID 974>: m519-1. se 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901

ATGGAATTTT

ATCCTTTGTT

GGCGTTTCCA

ATCGACCGCG

ACCCACCAG

GCATCATCTA

AGCAACTACA

CGTTATCGGG

TCAACAGTAC

GTGAAGGTTT

CCTTCGCTOA

GTATCGCCGA

GGTCAGCGCG

GGTCAATGCG

GTGAAGCGGA

CGTCAAATTG

TOTGAAGATT

AAAGCAATAC

ATTTCTGCCG

TCATTAT OTT

GTCATCCCAC

TOG CGCCCTG

TCGCCTACCG

GTCTGCATCA

TTTCCAAGTA

TTATGGCGAT

CGTATGGA6T

TGTTGTTGCG

TGCGT TAT GA

ATGOAGGCGC

ATCCGAAGGT

AAGCCGAAAT

TCAAATGCCG

ATCCTTGCGC

CCGCCGCCCT

GCGGAACAAT

GCTGATTATG

GTATGAAAAT

GTTGGTAGCC

AACAGGAAGT

ACGGcCGGTT

CCATTCGCTG

CGCGCGACAA

ACCGACCCCA

TACCCAGCTT

TGGACAAAAC

GCTTTGGACG

GATTAAAGAC

AAATTACTGC

CGTP.AAATCG

CCAACAATCC

AGAAAATCGC

CTTGTTGCCG

TCAAACCAA

ACGTCGCTGC

CCCGCCAATG

TAT OGACAGC

GTCGCCGTTT

CCACGTTGTC

TGAATATTTT

AAAGAAATCC

TACGCAGCTG

AACTCGCCTC

GCCCAAAOGA

GTTTGAAGAA

AGGCGGCCGG

TTGGTTCOG C

CGAACGCGAA

AACAAATCAA

GAAGGCGAGG

CCGCATCAAC

AAGCCAATGC

GGCGGTGCGG

GTTCAACAAT

TTGCCGACAT

AGCAAAACCG

TCGGTTTCAA

GAAAGGCTGG

GATTCCCTTT

CTTTAGACGT

ACTGTTGACG

ATACGGTTCG

CGCTGCGTTC

CGCGACGAAA

GGCTTGGGGT

CGCAAGAAAT

AAACGCGCCC

CCTTGCCAGT

CTCAGGCTGC

CGCGCCAAAG

CGAAGCCATC

ATGCGGTCAA

OTT GCCAAAG

CGGCAGCCTG

CCAAATAA

This corresponds to the amnino acid sequence <SEQ ID 975; ORF 519- 1>: m519-1.

1 MEFFIILLVA VAVFGFKSFV 51 IDRVAYRH-SL KEIPLDVPSQ 101 SNYIMAITOL AQTTLRSVIG 151 VKVLRYEIKD LVPPQEILRkS 201 GQREAEIQQS EGEAQAAVNA 251 RQIAAALQTQ GGADAVNLKI 301 ISAGMKIIDS SKTAK* VI PQEVHVV VCIT RDNTQL RM'E LDKT FEE

MQAQITAERE

SNAEKIARIN

AEQ.YVAAFNN

ERLGRFHRAL

TVDGI IYFQV RDE INSTVVA

KRARIAESEG

RAKGEAESLR

LAKESNTLIM

TAGLNILIPF

T DPKLASYG S

ALDEAAGAWG

RKIEQINLAS

LVAEANAEAI

PANVADIGSL

The following DNA sequence was identified in N. gonorrhoeae <SEQ ID 976>: g519-1 seq 1 ATGGAATTTT TOATTATOTT GTTGGOAGOO GTOGOOGTTT TOGGCTTOAA 51 ATOOTTTGTO GTOATOOOOO AGOAGGAAGT OOAOGTTGTO GAAAGGOTOG 101 GGOGTTTOOA TOGOGOOOTG AOGGOCGGTT TGAATATTTT GATTOOOTTT 151 ATOGAOOGOG TOGOOTAOOG OOATTOGOTG AAAGAAATOO OTTTAGAOGT 201 ACOCAGOCAG GTOTGOATOA OGCGOGATAA TAOGOAATTG AOTGTTGAOG 251 GOATOATOTA TTTOOAAGTA AOOGATOOOA AAOTOGOOTO ATAOGGTTOG 301 AGOAAOTAOA TTATGGCAAT TACCCAGCTT GCCCAAAOGA CGCTGCGTTC CGTTATOGGG OGTATGGAGT TOAAOAGTAO OGTOGTOTOO GTGAAAGTOO TOOGTTAOGA CCTTCGCGCA ATGCAGGCAC GTATTGOOGA ATOOGAAGGC GGTOAGOGTG AAGOOGAAAT GGTOAATGOG TOOAATGOOG GOGAAGCGGA ATCOOTGOGO CGTOAAATTG OOGOOGOOOT TOTGAAGATT GOGGAACAAT TGGAOAAAAC GTTTGAAGAA CGCGACGAA GOOOTOGATG AAGOOGOOGG GGOTTGGGGT AATOAAGGAT TTGGTTCCGC OGOAAGAAAT AAATTACCGC OGAAOGCGAA AAAOGOGCO CGTAAAATOG AACAAATOAA OOTTGOOAGT OOAAOAATOO GAAGGOGAGG OTOAGGOTGO AGAAAATCGO OOGOATOAAC OGCGCOAAAG OTTGTTGOOG AAGOOAATGO OGAAGOOATO TOAAAOOOAA GGOGGGGOGG ATGOGGTOAA AOGTAGOOGC GTTOAAOAAT OTTGCCAA;AG WO 00/22430 PCTIUS99/23573 74 851 AAAGCAATAC GCTGATTATG CCCGCCAATG TTGCCGACAT CGGCAGCCTG 901 ATTTCTGCCG GCATGAAAAT TATCGACAGC AGCAAAACCG CCAAATAA This corresponds to the amino acid sequence <SEQ ID 977; ORF 519-l.ng>: g519-1.pep 1 MEFFIILLAA VAVFGFKSFV VIPQQEVHVV ERLGRFHRAL TAGLNILIPF 51 IDRVAYRHSL KEIPLDVPSQ VCITRDNTQL TVDGIIYFQV TDPKLASYGS 101 SNYIMAI1TQL AQTTLRSVIG RMELDKTFEE RDEINSTVVS ALDEAAGAWG 151 VKVLRYEIKD LVPPQEILRA MQAQITAERE KRARIAESEG RKIEQINLAS 201 GQREAEIQQS EGEAQAAVNA SNAEKIARIN RAKGEAESLR LVAEANAEAI 251 RQIAAALQTQ GGADAVNLKI AEQYVANN LAKESNTLIM PANVADIGSL 301 ISAGMKIIDS SKTAK* m519-1/g519-1 ORFs 519-1 and 519-1.ng showed a 99.0% identity in 315 aa overlap 20 30 40 50) g519-1 .pep MEFFIILLAAVAVFGFKSFVVIPQQEVHVVERLGRFHRALTAGLNILIPFIDRVAYRSL 111i1111II11111II 1111 zn519-1 MEFFIILLVAVAVFGFKSFVVIPQQEVHVVERLGRFHFALTAGLNILIPIFIDRVAYRHSL 20 30 40 50 80 90 100 110 120 g519-1 .pep KEIPLDVPSQVCITRDNTQLTVDGIIYFQVTDPKLASYGSSNYIMAITQLAQTTLRSVIG m519-1 KEIPLDVPSQVCITRDNTQLTVDGIIYFQVTDPBCLASYGSSNYIMAITQLAQTTLRSVIG 80 90 100 110 120 130 140 150 160 170 180 g519-1 .pep RMELDKTFEERDEINSTVVSALDEAAGAWGVKVLRYEIKDLVPPQEILRAN0QAQITAERE m51 9-1 RMELDKTFEERDEINSTVVAALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITAERE 130 140 150 160 170D 180 190 200 210 220 230 240 g 519-1. pep KRARIAESEGRKIEQINLASGQREAEIQQSEGEAQAAVNASNAEKIARINRAKGEAESLR m5S19-1 KRARIIAESEGRKIEQINLASGQREAEIQQSEGEAQAAVNASNAEKIARINRAKGEAESLR 190 200 210 220 230 240 250 260 270 280 290 300 g519-1 .pep LVAEANAEAIRQIAAALQTOGGADAVNLKIAEQYVAAFNNLAKESNTLIMPANVADIGSL m519-1 LVAEANAEAIP.0IAAALQTQGGADAVNLKIAEQYVAAFNNLAKESNTLIMPANVADIGSL 250 260 270 280 290 300 310 g519-1.pep ISAGMKIIDSSKTAKX I II t1111111111 I 1, mn519-1 ISAGMKIIDSSKTAKX 310 The following DNA sequence was identified in N. meningitidis <SEQ ID 9'78>: a519-1.seq 1 ATGGAATTTT TCATTATCTT GCTGGCAGCC GTCGTTGTTT TCGGCTTGAA 51 ATCCTTTGTT GTCATCCCAC AGCAGGAAGT C CACGTTGTC GAAAGGCTCG 101 GGCGTTTCCA TCGCGCCCTG ACGGCCGGTT TGAATATTTT GATTCCCTTT 151 ATCGACCGCG TCGCCTACCG CCATTCGCTG AAAGAAA"CC CTTTAGACGT 201 ACCCAGCCAG GTCTGCATCA CGCGCGACAA TACGCAGCTG ACTGTTGTLCG 251 GTATCATCTA TTTCCAAGTA ACCGACCCCA AACTCGCCTC ATACGGTTCG 301 AGCAACTACA TTATGGCGAT TACCCAGCTT GCCCAAACGA CGCTGCGTTC WO 00/22430 PCTIUS9923573 75 351 CGTTATCGGG CGTATGGAAT TGGACAAAAC GTTTGAAGAA CGCGACGAAA 401 TCAACAGCAC CGTCGTCTCC GCCCTCGATG AAGCCGCCGG AGCTTGGGGT 451 GTGAAGGTTT TGCGTTATGA GATTAAAGAC TTGGTTCCGC CGCAAGAAAT 501 CCTTCGCTCA ATGCAGGCGC AAATTACTGC TGAACGCGAA AAACGCGCCC 551 GTATCGCCGA ATCCGAAGGT CGTAAAATCG AACAAATCAA CCTTGCCAGT 601 GGTCAGCGCG AAGCCGAAAT CCAACAATCC GAAGGCGAGG CTCAGGCTGC 651 GGTCAATGCG TCAAATGCCG AGAAAATCGC CCGCA~TCAAC CGCGCCAAAG 701 GTGAAGCGGA ATCCTTGCGC CTTGTTGCCG AAGCCAATGC CGAAGCCATC 751 CGTCAAATTG CCGCCGCCCT TCAAACCCAA GGCGGTGCGG ATGCGGTCAA 801 TCTGAAGATT GCGGAACAAT ACGTCGCCGC GTTCAACAAT CTTGCCAAAG 851 AAAGCAATAC GCTGATTATG CCCGCCAATG TTGCCGACAT CGGCAGCCTG 901 ATTTCTGCCG GTATGAAAAT TATCGACAGC AGCP.AAACCG CCAAATAA This corresponds to the amino acid sequence <SEQ ID 979; ORF 519-1 a519-1.pep.

1 MEFFIILLAA VVVF'GFKSFV VIPQQEVHVV EP.LGRFHRAL TAGLNILIPF 51 IDRVAYP.HSL KEIPLDVPSQ VCITR0NTQL TVDGIIYFQV TDPKLASYGS 101 SNYIMAITQL AQTTLRSVIG RS4ELDKTFEE RDEINSTVVS ALDEAAGAWG 151 VKVLRYEIKD LVPPQEILRS MQAQITAERE KRARIAESEG RKIEQINLAS 201 GQREAEIQQS EGEAQAAVNA SNAEKIARIN RAKGEAESLR LVAEANAEAI 251 RQIAAALOTQ GGADAVNLKI AEQYVAAFNN LAKESNTLIM PANVADIGSL 301 ISAGMKIIDS SKTAK* m519-1/a519-1 ORFs 519-1 and 519-l.a showed a 99.0% identity in 315 aa overlap 20 30 40 so a519-1.pep MEFFIILLAAVVVFGFKSFVVIPQQEVHVVERLGRFHRALTAGLNILIPFIDRVAYRHSL m519-1 MEFFIILLVAVAVFGFKSFVVIPQQEVHVVERLGRFHRALTAGLNILIPFIDRVAYRHSL 20 30 40 s0 80 90 100 110 120 a519-1 .pep KEIPLDVPSQVCITRDNTQLTVDGIIYFQVTDPKLASYGSSNYIKAITQLAQTTLRSVIG 1111 1111 1111 1111li 111 1111 11 m519-1 KEIPLDVPSQVCITRDNTQLTVDGIIYFQVTDPKLASYGSSNYIMAITQLAQTTLRSVIG 80 90 100 110 120 130 140 150 160 170 180 a519-1.pep RMELDKTFEERDEINSTVVSALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITAERE m51 9-1 RMELDKTFEERDEINSTVVAALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITAERE 130 140 150 160 170 180 190 200 210 220 230 240 a519-1.pep KRARIAESEGRKIEQINLASGQREAEIQQSEGEAQAAVNASNAEKIARINRAKGEAESLR m519-1 KRARIAESEGRKIEQINLASGQREAEIQQSEGEAQAAVNASNAEKIARIN4RAKGEAESLR 190 200 210 220 230 240 250 260 270 280 290 300 a519-1 .pep LVAEANAEAIRQIAAALQTQGGADAVNLKIAEQYVAAFNNLAKESNTLIMPANVADIGSL 19-1 LVAEANAEAIRQIAAALOTQGGADAVNLKIAEQYVAAFNNLAKESNTLIIMPANVADIGSL 250 260 270 280 290 300 310 a519-1.pep ISAGMKIIDSSKTAKX m519-1 ISAGMKIIDSSKTAKX 310 WO 00/22430 PCTIUS99/23573 76 576 and 576-1 gnm22.seq The following partial DNA sequence was identified in N. meningitidis <SEQ ID 980>: m576.seq.. (partial) 1 .ATGCAGCAGG CAAGCTATGC GATGGGCGTG GACATOGGAC GCTCCCTGAA 51 GCAAATGAAG GAACAGGGCG CGGAAATCGA TTTGAAAGTC TTTACCGAAG 101 CCATGCAGGC AGTGTATGAC GGCAAAGAAA TCAAAATGAC CGAAGAGCAG 151 GCTCAGGAAG TCATGATGAA ATTCCTTCAG GAACAACAGG CTAAAGCCGT 201 AGAAAAACAC AAGGCGGACG CGAAGGCCAA TAAAGAAAAA GGCGAAGCCT 251 TTCTGAAAGA AAATGCCGCC AAAGACGGCG TGAAGACCAC TGCTTCCGGC 301 CTGCAATACA AAATCACCAA ACAGGGCGAA GGCAAACAGC CGACCAAAGA 351 CGACATCGTT ACCGTGGAAT ACGAAGGCCG CCTGATTGAC GGTACGGTAT 401 TCGACAGCAG CAAAGCCAAC GGCGGCCCGG TCACCTTCCC TTTGAGCCAA 451 GTGATTCCGG GTTGGACCGA AGgCGTACAG CTTCTGAAAG AAGGCGG.CGA 501 AGCCACGTTC TACATCCCGT CCAACCTTGC CTACCGCGAA CAGGGTGCGG 551 GCGACAAAAT CGGTCCGAAC GCCACTTTGG TATTTGATGT GAAACTGGTC 601 AAAATCGGCG CACCCGAAAA CGCGCCCGCC AAGCAGCCGG CTCAAGITCGA 651 CATCAA-AAA GTAAATTAA This corresponds to the amino acid sequence <SEQ ID 981; ORF 576>: m576.pep.. (partial) 1 MQQASYAMGV DIGRSLKQMK EQGAEIDLKV FTEANQAVYD GKEIKMTEEQ 51 AQEVMMKFLQ EQQAI(AVEKH KADAKANKEK GEAFLKENAA KDGVK'TTASG 101 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVFDSSKAN GGPVTFPLSQ 151 VIPGWTEGVQ LLKEGGEATF YIPSNLAYRE QGAGDKIGPN ATLVFDVKLV 201 KIGAPENAPA KQPAQVDIKK VN* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 982>: g576.seq.. (partial) 1 .atgggcgtgg acatcggacg ctccctgaaa caaatgaagg aacagggcgc ggaaatcgat gcaaagaaat ttcctgcagg gaaggccaac aagacggcgt cagggtgaag cgaaggccgc gcggcccggc ggcgtacggc caaccttgcc ccactttggt gcgcccgcca ttgaaagtct caaaatgacc agcagcaggc aaagaaaaag gaagaccact gcaaacagcc ctgattgacg caccttccct ttctgaaaga taccgcgaac atttgacgtg agcagccgga ttaccgatgc gaagagcagg taaagccgta gcgaagcctt gcttccggtc gacaaaagac gtaccqtatt ttgagccaag aggcggcgaa agggtgcggg aaactggtca tcaagtcgac catgcaggca cccaggaagt gaaaaacaca cctgaaggaa tgcagtacaa gacatcgtta cgacagcagc tgattccggg gccacgttct cgaaaaaatc aaatcggcgc atcaaaaaag gtgtatgacg gatgatgaaa aggcggatgc aatgccgccg aatcaccaaa ccgtggaata aaagccaacg ttggaccgaa acatcccgtc ggtccgaacg acccgaaaac taaattaa This corresponds to the amino acid sequence <SEQ ID 983; ORE 576.ng>: g576.pep..(partial) 1 MGVDIGRSLK QMKEQGAEID LKVFTDAMQA VYDGKEIKMT EEQAQEVMMK 51 FLQEQQAKAV EKHKADAKAN KEKGEAFLKE NAAEDGVKTT ASGLQYKITK 101 QGEGKQPTKO DIVTVEYEGR LIDGTVFDSS KANGGPATFP LSQVIPGWTE 151 GVRLLKEGGE ATFYIPSNLA YREQGAGEKI GPNATLVFDV KLVKIGAPEN 201 APAKQPDQVD IKKVN* Computer analysis of this amino acid sequence gave the following results: Homology with a predicted ORF from N. gonorrhoeae m576/g576 97.2% identity in 215 aa overlap WO 00/22430 PCTIUS99/23573 77 20 30 40 50 m57 6. pep MQQASYAI1GVDIGRSLKQMKEQGAEIDLKVFTEAM4QAVYDGKEIKDMTEEQAQEVMMKFLQ g576 MGVDIGRSLKQMKEQGAEIDLKVFTDAM'QAVYDGKEIKMTEEQAQEVMMKFLQ 20 30 40 80 90 100 110 120 m57 6. pep EQQAKAVEKHKADAKANKEKGEA FLKENAAKDGVKTTASGLQYKITKQGEGKQPTKDDIV 1111 1111 111 11111111li ii I111 g57 6 EQQAKAVEKHKADAKANKEKGEAFLKENAAEDGVKTTASGLQYKITKQGEG(QPTKDDIV 70 80 90 100 110 130 140 150 160 170 180 m57 6. pep TVEYEGRLIDGTVFDSSKANGGPVTFPLSQVIPGWTEGVQLLKEGGEATFYIPSNLAYRE g57 6 TVEYEGRLIDGTVFDSSKANGGPATFPLSQVIPGWTEGVRLL(EGGEATFYIPSNLAYRE 120 130 140 150 160 170 190 200 210 220 m57 6. pep QGAGDKIGPNATLVFDVKLVKIGAPENAPAKQPAQVDIKKVNX g57 6 QGAGEKIGPNATLVFDVKLVKIGAPENAPAKQPDQVDIKKVNX 180 190 200 210 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 984>: a576.seq 1 ATGAACACCA TTTTCAAAAT CAGCGCACTG ACCCTTTCCG CCGCTTTGGC 51 ACTTTCCGCC TGCGGCAAAA AAGAAGCCGC CCCCGCATCT GCATCCGAAC 101 CTGCCGCCGC TTCTTCCGCG CAGGGCGACA CCTCTTCGAT CGGCAGCACG 151 ATGCAGCAGG CAAGCTATGC GATGGGCGTG GACATCGGAC GCTCCCTGAA 201 GCAAATGAAG GAACAGGGCG CGGAAATCGA TTTGAAAGTC TTTACCGAAG 251 CCATGCAGGC AGTGTATGAC GGCAAAGAAA TCAAAATGAC CGAAGAGCAG 301 GCTCAGGAAG TCATGATGAA ATTCCTTCAG GAACAACAGG CTAAAGCCGT 351 AGAAAAACAC AAGGCGGACG CGAAGGCCAA TAAAGAAAAA GGCGAAGCCT 401 TTCTGAAAGA AAATGCCGCC AAAGACGGCG TGAAGACCAC TGCTTCCGGC 451 CTGCAATACA AAATCACCAA ACAGGGCGAA GGCAAACAGC CGACCAAAGA 501 CGACATCGTT ACCGTGGAAT ACGAAGGCCG CCTGATTGAC GGTACGGTAT 551 TCGACAGCAG CAAAGCCAAC GGCGGCCCGG TCACCTTCCC TTTGAGCCA 601 GTGATTCTGG GTTGGACCGA AGGCGTACAG CTTCTGAAAG AAGGCGGCGA 651 PLGCCACGTTC TACATCCCGT CCAACCTTGC CTACCGCGAA CAGGGTGCGG 701 GCGACAAAAT CGGCCCGAAC GCCACTTTGG TATTTGATGT GAAACTGGTrC 751 AAAATCGGCG CACCCGAAAA CGCGCCCGCC AAGCAGCCGG CTCAAGTCGA 801 CATCAAAAAA GTAAATTAA This corresponds to the amnino acid sequence <SEQ ID, 985; ORF 576.a>: a576.pep 1 MNTIFKISAL TLSAALALSA CGKKEAAPAS ASEPAAASSA QGDTSSIGST 51 MQQASYAM'GV DIGRSLKQMK EQCAEIDLKV FTEAMQAVYO GKEIKM~TEEQ 101 AQEVMMKFLQ EQQAKAVEKH KADAKANKEK GEAFL!(ENAA KDGV1<TTASG 151 LQYKITKQGE GKQPTKDOIV TVEYEGRLID GTVFDSSKAN GGPVTFPLSQ 201 VILGWTEGVQ LLKEGGEATF YIPSNLAYRE QGAGDKIGPN ATLVFDVKLV 251 KIGAPENAPA KQPAQVDIKK VN* m576/a576 ORFs 576 and 576.a showed a 99.5% identity in 222 aa overlap 20 m576.pep MQQASYAMGVDIGRSLKQMKEQGAEI DLKV a57 6 CGKKEAAPASASEPAAASSAQGDTSSIGSTMQQASYAMGVDIGRSLKQMKEQGAEIDLKV 40 50 60 70 WO 00/22430 PCTIUS99/23573 78 50 60 70 80 m57 6. pep FTEAMQAVYDGKEIKMTEEQAQEVMMKF'LQEQQAKAVEKHKADAKANKEKGEAFLKENAA a576 FTEAMQAVYDGKE IKMTEEQAQEVMM'KFLQEQQAKAVEKHKADAKANKEKGEAFLKENAA 90 100 110 120 130 140 100 110 120 130 140 150 m57 6.pep KDGVKTTASGLQYKITKQGEGK(QPTK00IVTVEYEGRLIDGTVFDSSKANGGPVTFPLSQ a57 6 KDGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLIDGTVFDSSKANGGPVTFPLSQ 150 160 170 180 190 200 160 170 180 190 200 210 m57 6.pep VIPGWTEGVQLLKEGGEATFYIPSNLAYREQGAGDKIGPNATLVFDVKLVKIGAPENAPA 1II111111111F1111111111li 1111Fli a576 VILGWTEGVQLLKEGGEATFYIPSNLAYREQGAGDKIGPNATLVFDVKLVKIGAPENAPA 210 220 230 240 250 260 220 rn516.pep KQPAQVOIKKVNX a576 KQPAQVDIKKVNX 270 Further work revealed the following DNA sequence identified in N. meningitidis <SEQ ID 986>: m576-..seq I ATGAACACCA TTTTCAAAAT CAGCGCACTG ACCCTTTCCG CCGCTTTGGC 51 ACTTTCCGCC TGCGGCAAAA AAGAAGCCGC CCCCGCATCT GCATCCGAAC 101 CTGCCGCCGC TTCTTCCGCG CAGGGCGACA CCTCTTCGAT CGGCAGCACG 151 ATGCAGCAGG CAAGCTATGC GATGGGCGTG GACATCGGAC GCTCCCTGAA 201 GCAAATGAAG GAACAGGGCG CGGAAATCGA TTTGAAAGTC TTTACCGAAG 251 CCATGCAGGC AGTGTATGAC GGCAAAGAAA TCAAAATGAC CGAAGAGCAkG 301 GCTCAGGAAG TCATGATGAA ATTCCTTCAG GAACAACAGG CTAAAGCCGT 351 AGAAAAACAC AAGGCGGACG CGAAGGCCAA TAAAGAAAAA GGCGAAGCCT 401 TTCTGAAAGA AAATGCCGCC AAAGACGGCG TGAAGACCAC TGCTTCCGG3C 451 CTGCAATACA AAATCACCAA ACAGGGCGAA GGCAAACAGC GGACCAAAGA 501 CGACATCGTT ACCGTGGAAT ACGAAGGCCG CCTGATTGAC GGTACGGTAT 551 TCGACAGCAG CAAAGCCAAC GGCGGCCCGG TCACCTTCCC TTTGAGCCAA 601 GTGATTCCGG GTTGGACCGA AGGCGTACAG CTTCTGAAAG AAGGCGGCGA 651 AGCCACGTTC TACATCCCGT CCAACCTTGC CTACCGCGAA CAGGGTGCGG 701 GCGACAAAAT CGGTCCGAAC GCCACTTTGG TATTTGATGT GAAACTGGTC 751 AAAATCGGCG CACCCGAAAA CGCGCCCGCC PAGCAGCCGG CTCAAGTCGA 801 CATCAAAAAA GTAAATTAA This corresponds to the amino acid sequence <SEQ ID 987; ORF 576-1>: m576-1 .pep 1 MNTIFKISAL TLSAALALSA CGKKEAAPAS ASEPAAASSA QGDTSSIGST 51 MQQASYANGV DIGRSLKQMK EQGAEIDLKV FTEAMQAVYD GKEIKNTEEQ 101 AQEVMMKFLQ EQQAKAVEKH KAJDAKANKEK GEAFLKENAA KDGVKTTASG 151 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVFDSSKAN GGPVTFPLSQ 201 VIPGWTEGVQ LLKEGGEATF YIPSNLAYRE QGAGOKIGPN ATLVFDVFKLV 251 KIGAPENAPA KQPAQVDIKK VNk The following DNA sequence was identified in N. gonorrhoeae <SEQ ID 988>: g576- seq 1 ATGAACACCA TTTTCAAAAT CAGCGCACTG ACCCTTTCCG CCGCTTTC;GC 51 ACTTTCCGCC TGCGGCAAAA AAGAAGCCGC CCCCGCATCT GCATCCGAAC 101 CTGCCGCCGC TTCTGCCGCG CAGGGCGACA CCTCTTCAAT CGGCAGCACG 151 ATGCAGCAGG CAAGCTATGC AATGGGCGTG GACATCGGAC GCTCCCTGAA WO 00/22430 PCTIUS99/23573 79

ACAAATGAAG

CCATGCAGGC

GCCCAGGAAG

AGAAAAACAC

TCCTGAAGGA

CTGCAGTACA

CGACATCGTT

TCGACAGCAG

GTGATTCCCG

AGCCACGTTC

GCGAAAAAAT

AAAATCGGCG

CATCAAAAAA

GAACAGGGCG

AGTGTATGAC

TGATGATGAA

AAGGCGGATG

AAATGCCGCC

AAATCACCAA

ACCGTGGAAT

CAAAGCCAAC

GTTGGACCGA

TACATCCCGT

CGGTCCGAAC

CACCCGAAAA

GTAAATTAP.

CGGAAATCGA TTTGAAAGTC TTTACCGAT3 GGCAAAGAAA TCAAAATGAC CGAAGAGCAG ATTCCTGCAG GAGCAGCAGG CTAAAGCCGT CGAAGGCCAA CAAAGAAAAA GGCGAAGCCT AAAGACGGCG TGAAGACCAC TGCTTCCGGT ACAGGGTGAA GGCAAACAGC CGACAAAAGA ACGAAGGCCG CCTGATTGAC GGTACCGTAT GGCGGCCCGG CCACCTTCCC TTTGAGCCAA AGGCGTACGG CTTCTGAAAG AAGGCGGCGA CCAACCTTGC CTACCGCGAA CAGGGTGCGG GCCACTTTGG TATTTGACGT GAAACTGGTC CGCGCCCGCC AAGCAGCCGG ATCAAGTCGA This corresponds to the amino acid sequence <SEQ ID 989; ORF 576-1 .ng>: g576- pep 1 MNTIFKISAL TLSAALALSA CGI KEAAPAS ASEPAAASAA QGDTSSIGST 51 MQQASYANGV DIGRSLKQMK EQGAEIDLKV FTDAMQAVYD GKEIKNTEEQ 101 AQEVMMKFLQ EQQAKAVEKH KADAKANKEK GEAFLKENAA KDGVKTTASG 151 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVFOSSKAN GGPATFPLSQ 201 VIPGWTEGVR LLKEGGEATF YIPSNLAYRE QGAGEKIGPN ATLVFDVKIV 251 KIGAPENAPA KQPDQVDIKK VNg576-1/=576-1 ORE's 576-1 and 576-Ing showed a 97.8% identity in 272 aa overlap g576-1 .pep m576-1 g576-1 .pep m576-1 g57 6-1. pep m57 6-1 g576- pep m576-1 g576-1 .pep mn576-1 20 30 40 50 MNTI FKI SALTLSAALALSACGKKEAAPASASEPAAASAAQGDTSS IGSTMQQASYAM~GV MNT IFEKI SALTLSAALALSACGKKEAAPASASE PAAASSAQGOTS SIGSTMQQASYAM'GV 20 30 40 50 80 90 100 110) 120 DIGRSLKQMKEQGAEI DLKVFTDAMQAVYDGKEIKMTEEQAQEVME4KFLQEQQAKAVEKH DIGRSLKQMKEQGAEI DLKVFTEAMQAVYDGKEIKMTEEQAQEVNNKFLQEQQAKAVEKH 80 90 100 110 120 130 140 150 160 170 180 KADAKANKEKGEAFLKENAAKDGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLI 0 KADAKANKEKGEAFLKENAAKDGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLI D 130 140 150 160 170 180 190 200 210 220 230 240 GTVFDSSKANGGPATFPLSQVIPGWTEGVRLLKEGGEATFYI PSNLAYREQGAGEKIGPN GTVFDSSKANGGPVTFPLSQVI PGWTEGVQLLKEGGEATFYI PSNLAYREQGAGDKIGPN 190 200 210 220 230 240 250 260 270

ATLVFDVKLVKIGAPENAPAKQPDQVDIKKVNX

11111111111111111111111 III 111111

ATLVFDVKLVKIGAPENAPAKQPAQVDIKKVNX

250 260 270 The following DNA sequence was identified in N. meningitidis <SEQ ID 990>: a576- seq 1 ATGAACACCA TTTTCAAAAT CAGCGCACTG ACCCTTTCCG CCGCTTTGGC 51 ACTTTCCGCC TGCGGCAAAA AAGAAGCCGC CCCCGCATCT GCATCCGAAC 101 CTGCCGCCGC TTCTTCCGCG CAGGGCGACA CCTCTTCGAT CGGCAGCACG WO 00/22430 PCTlUS99/2 80 151 ATGCAGCAGG CAAGCTATGC GATGGGCGTG GACATCGGAC GCTCCCTGAA 201 GCAAATGAAG GAACAGGGCG CGGAAATCGA TTTGAAAGTC TTTACCGAAG 251 CCATGCAGGC AGTGTATGAC GGCAAAGAAA TCAAAATGAC CGAAGAGCAG 301 GCTCAGGAAG TCATGATGAA ATTCCTTCAG GAACAACAGG CTAAAGCCGT 351 AGAAAAACAC AAGGCGGACG CGAAGGCCAA TAAAGAAAAA GGCGAAGCCT 401 TTCTGAAAGA AAATGCCGdC AAAGACGGCG TGAAGACCAC TGCtTCCGGC 451 CTGCAATACA AAATCACCAA ACAGGGCGAA GGCAAACAGC CGACCAAAGA 501 CGACATCGTT ACCGTGGAAT ACGAAGGCCG CCTGATTGAC GGTACGGTAT 551 TCGACAGCAG CAAAGCCAAC GGCGGCCCGG TCACCTTCCC TTTGAGCCAA 601 GTGATTCTGG GTTGGACCGA AGGCGTACAG CTTCTGAAAG AAGGCGGCGA 651 AGCCACGTTC TACATCCCGT CCAACCTTGC CTACCGCGAA CAGGGTGCGG 701 GCGACAAAAT CGGCCCGAAC GCCACTTTGG TATTTGATGT GAAACTGGTC 751 AAP.ATCGGCG CACCCGAAAA CGCGCCCGCC AAGCAGCCGG CTCAAGTCGA 801 CATCAAAAA GTAAATTAA This corresponds to the amino acid sequence <SEQ ID 991; ORE 576-1.a>: a576-1 .pep 1 MNTIFKISAL TLSAALALSA CGKKEAAPAS ASEPAAASSA QGDTSSIGST 51 MQQASYANGV DIGRSLKQM( EQGAEIOLKV FTEAM'QAVYD GKEIKMTEE;Q 101 AQEVMMKFLQ EQQAKAVEKH KADAKANKEIK GEAFLKENAA KDGVKTTASG 151 LQYKITKQGE GKQPTKDDIV TVEYEGRLID GTVB'OSSKAN GGPVTFLSQ 201 VILGWTEGVQ LLKEGGEATF YIPSNLAYRE QGAGDKIGPN ATLVFDVKILV 251 KIGAPENAPA KQPAQVDIK( VNa576-1/m576-1 ORFs 576-1 and 576-l.a 99.6% identity in 272 aa overlap 20 30 40 50 a57 6-1. pep MNTIFKISALTLSAALASACGKKEAAPASASEPAAASSAGDTSSIGSTMQQASYAMGV m576-1 MNTIFKISALTLSAALALSACGKKEAAPASASEPAA.ASSAQGDTSSIGSTMQQASYAMGV 20 30 40 50 80 90 100 110 120 a576-1 .pep DIGRSLKQMKEQGAEIDLKVFTEAI4QAVYDGKEIKMTEEQAQEVrMiKFLQEQQAKAVEKH mn57 6-1 DIGRSLKQMKEQGAE IDLKVFTEANQAVYDGKE IKMTEEQAQEVMMKFLQEQQAKAVEKH 80 90 100 110) 120 130 140 150 160 170 180 a 576-1 .pep KADAKANKEKGEAFLKENAAKDGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLID m57 6-1 KADAKANKEKGEAFLKENAAKDGVKTTASGLQYKITKQGEGKQPTKDDIVTVEYEGRLID 130 140 150 160 170 180 190 200 210 220 230 240 a576- pep GTVFDSSKANGGPVTFPLSQVILGWTEGVQLLKEGGEATFYIPSNLAYREQGAGDKIGPN m57 6-1 GTVFDSSKANGGPVTFPLSQVIPGWTEGVQLLKEGGEATFYIPSNLAYREQGAGDKIGPN 190 200 210 220 230 240 250 260 270 a576-1 .pep ATLVFDVKLVKIGAPENAPAKQPAQVDIKKVNX m57 6-1 ATLVFDVKLVKIGAPENAPAKQPAQVDIKKVNX 250 260 270 919 and 919-2 gnm43.seq ~3573 WO 00/22430 PCTIUS99/23573 The following partial DNA sequence was identified in N.meningitidis <SEQ ID 992>: M919. Seq 1 ATGAAAAAAT ACCTATTCCG CGCCGCCCTG TACGGCATCG CCGCCGCCAT 51 CCTCGCCGCC TGCCAAAGCA AGAGCATCCA AACCTTTCCG CAACCCGACA 101 CATCCGTCAT CAACGGCCCG GACCGGCCGG TCGGCATCCC CGACCCCGCC 151 GGAACGACGG TCGGCGGCGG CGGGGCCGTC TATACCGTTG TACCGCACCT 201 GTCCCTGCCC CACTGGGCGG CGCAGGATTT CGCCAAAAGC CTCCAATCCT 251 TCCGCCTCGG CTGCGCCAAT TTGAAAAACC GCCAAGGCTG GCAGGATGTG 301 TGCGCCCAAG CCTTTCAAAC CCCCGTCCAT TCCTTTCAGG CAAAACAGTT 351 TTTTGAACGC TATTTCACGC CGTGGCAGGT TGCAGGCAAC GGAAGCCTTG 401 CCGGTACGGT TACCGGCTAT TACGAACCGG TGCTGAAGGG CGACGACAGG 451 CGGACGGCAC AAGCCCGCTT CCCGATTTAC GGTATTCCCG ACGATTTTAT 501 CTCCGTCCCC CTGCCTGCCG GTTTGCGGAG CGGAAAAGCC CTTGTCCGCA 551 TCAGGCAGAC GGGAAAAAAC AGCGGCACAA TCGACAATAC CGGCGGCACA 601 CATACCCCG ACC'rCTCCcG ATTCCCCATC ACCGCGCGCA CAACAGCAAT 651 CAAAGGCAGG TTTGAAGGAA GCCGCTTCCT CCCCTACCAC ACGCGCAACC 701 AAATCAACGG CGGCGCGCTT GACGGCAAAG CCCCGATACT CGGTTACGCC 751 GAAGACCCTG TCGAACTTTT TTTTATGCAC ATCCAAGGCT CGGGCCGTCT 801 GAAAACCCCG TCCGGCAAAT ACATCCGCAT CGGCTATGCC GACAAAAACG 851 AACATCCyTA CGTTTCCATC GGACGCTATA TGGCGGATAA GGGCTACCTC 901 AAACTCGGAC AAACCTCCAT GCAGGGCATT AAGTCTTATA TGCGGCAAAA 951 TCCGCAACGC CTCGCCGAAG TTTTGGGTCA AAACCCCACC TATATCTTTT 1001 TCCGCGAGCT TGCCGGAAGC AGCAATGACG GCCCTGTCGG CGCACTGGGC 1051 ACGCCGCTGA TGGGGGAATA TGCCGGCGCA GTCGACCGGC ACTACATTAC 1101 CTTGGGTGCG CCCTTATTTG TCGCCACCGC CCATCCGGTT ACCCGCAAAG 1151 CCCTCAACCG CCTGATTATG GCGCAGGATA CCGGCAGCGC GATTAAAGGC 1201 GCGGTGCGCG TGGATTATTT TTGGGGATAC GGCGACGAAG CCGGCGAAC'T 1251 TGCCGGCAAA CAGAAAACCA CGGGATATGT CTGGCAGCTC CTACCCAACG 1301 GTATGAAGCC CGAATACCGc CCGTAA This corresponds to the amino acid sequence <SEQ ID 993; ORF 919>: m919.Pep 1 MKKYLFRAAL YGIAAAIIAA CQSKSIOTFP QPDTSVINGP DRPVGIPDPA 51 GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN L1KNRQGWQDV 101 CAQAFQTPVH SFQAKQFFER YFTPWOVAGN GSLAGTVTGY YEPVLKGDIDR 151 RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGQN SGTIDNTGGT 201 HTADLSRFPI TARTTAIKGR FEGSRFLPY4 TRNQINGGAL DGKAPILGYA 251 EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DIK4EHPYVSI GRYMAflKGYL 301 KLGQTSMQGI KSYMRQNPQR LAEVLGQNPS YIF8'RELAGS SNDGPVGALG 351 TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG 401 AVRVDYI'WGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P* The following partial DNA sequence was identified in N.meningitidis <SEQ ID 994>: m919-2. seq 1 ATGAAAAAAT ACCTATTCCG CGCCGCCCTG TACGGCATCG CCGCCGCCAT 51 CCTCGCCGCC TGCCAAAGCA AGAGCATCCA AACCTTTCCG CAACCCGACA 101 CATCCGTCAT CAACGGCCCG GACCGGCCGG TCGGCATCCC CGACCCCGCC 151 GGAACGACGG TCGGCGGCGG CGGGGCCGTC TATACCGTTG TACCGCACCT 201 GTCCCTGCCC CACTGGGCGG CGCAGGATTT CGCCAAAAGC CTGCAATCCT 251 TCCGCCTCGG CTGCGCCAAT TTGAAAAACC GCCAAGGCTG GCAGGATGTG 301 TGCGCCCAAG CCTTTCAAAC CCCCGTCCAT TCCTTTCAGG CAAAACAGTT 351 TTTTGAACGC TATTTCACGC CGTGGCAGGT TGCAGGCAAC GGAAGCCTTG 401 CCGGTACGGT TACCGGCTAT TACGAACCGG TGCTGAAGGG CGACGACAGG 451 CGGACGGCAC AAGCCCGCTT CCCGATTTAC GGTATTCCCG ACGATTTTAT 501 CTCCGTCCCC CTGCCTGCCG GTTTGCGGAG CGGAAAAGCC CTTGTCCGCA 551 TCAGGCAGAC GGGAAAAAAC AGCGGCACAA TCGACAATAC CGGCGGCA.CA WO 00/22430 PCT/U599/23573 82 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201

CATACGGCCG

CAAAGGCAGG

AAATCAACGG

GAAGACCCTG

GAAAACCCCG

AACATCCCTA

AAACTCGGAC

TCCGCAACGC

TCCGC GAG CT

ACGCCGCTGA

CTTGGGTGCG

CCC'rCAACCG

GCGGTGCGCG

ACCTCTCCCG

TTTGAAGGAA

CGGCGCGCTT

TCGAACTTTT

TCCGGCAAAT

CGTTTCCATC

AAACCTCCAT

CTCGCCGAAG

TGCCGGAAGC

TGGGGGAATA

CCCTTATTTG

CCTGATTATG

TGGATTATTT

ATTCCCCATC ACCGCGCGCA CAACAGCAAT GCCGCTTCCT CCCCTACCAC ACGCGCAACC GACGGCAAAG CCCCGATACT CGGTTACGCC TTTTATGCAC ATCCAAGGCT CGGGCCGTCT ACATCCGCAT CGGCTATGCC GACAAAAACG GGACGCTATA TGGCGGATAA GGGCTACCTC GCAGGGCATT AAGTCTTATA TGCGGCAAAA TTTTGGGTCA AAACCCCAGC TATATCTTTT AGCAATGACG GCCCTGTCGG CGCACTGGGC TGCCGGCGCA GTCGACCGGC ACTACATTAC TCGCCACCGC CCATCCGGTT ACCCGCAAAG GCGCAGGATA CCGGCAGCGC GATTAAAGGC TTGGGGATAC GGCGACGAAG CCGGCGAACT 1251 TGCCGGCAAA CAGAAAACCA CGGGATATGT CTGGCAGCTC CTACCCAACG 1301 GTATGAAGCC CGAATACCGC CCGTAA This corresponds to the amino acid sequence <zSEQ I) 995; ORF 919-2>: m919-2.pep MKKYLFRAAL YGIAAAILAA CQSKSIQTFP QPDTSVINGF GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN CAQAFQTPVH SFQAKQFFER YFTPWQVAGN GSLAGTVTGY RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN HTAOLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI KLGQTSMQGI KSYMRQNPQR LAEVLGQNPS YIFFRELAGS TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMI(PEYR DRPVGI PDIIA

LKNRQGWQDV

YEPVLKGODR

SGT I NTGGT

DGKAPILGYA

GRYMADKGYL

SNDGPVGALG

AQDTGSAIKG

The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 996>: g919. seq 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 ATGAAAAAAC ACCTGCTCCG CctcgCCGCC

CATCCGTCAT

GGAACGACGG

GTCCATGCCC

TCCGCCTCGG

TGCGCCCAAG

TTTTGAACGC

Caggtacgt

CGGACGGAAC

CTCCGTCCCG

TCAGGCAGac

CATACCGCCG

caaaGGCAGG AAAtcaacGG GAagaccCcG GAAAACCCcg AACAtccgTa AAoctcgggc

TCCGCAACGC

TCCGCGAGCT

ACGCCACTGA

CTTGGGCGCG

CCCTCAACCG

GCGGTGCGCG

TGCCGGCAAA

GCATGAAGCC

TGCCAAAgca

CAACGGCCCG

TTGCCGGCGG

CACTGGGCGG

CTGCGCCAAT

CCTTTCAAAC

TATTTCACGC

TACCGGCTAT

GGGCCCGCTT

CTGCCTGCCG

g9GGAAAAAC

ACCTCTCCCG

TTTGAaggAA CGGCgcgcTT tcgaacttTT tccggcaaat tgtttccatc agACCTCGAT

CTCGCCGAAG

TGCCGGAAGC

TGGGGGAATA

CCCTTATTTG

CCTGATTATG

TGGATTATTT

CTCCGCCCTG

gGAGCATCCA

GACCGGCCGG

CGGGGCCGTC

CGCaggATTT

TTGAAAAACC

CCCCGTGCAT

cgtGGCaggt

TACGAACCGG

CCCGATTTAC

GTTTGCGGGG

AGCGGCACGA

ATTCCCCATC

GCCGCTTCCT

GACGGCAAag

TTTCATGCAC

acatCCGCAt ggACGctaTA GCAGGgcatc

TTTTGGGTCA

GGCAATGAGG

TACGGcatCG CCGCCgccAT AACCTTTCCG CAACCCGACA CCGGCATCCC CGACCCCGCC TATACCGTTG TGCCGCACCT TGCCAAAAGC CTGCAATCCT GCCAAGGCTG GCAGGATGTG TCCTTTCAGG CAAAGcGgTT tgcaggcaAC GGAAGcCTTG TGCTGAAGGG CGACGGCAGG GGTATTCCCG ACGATTTTAT CGGAAAAAAC CTTGTCCGCA .TCGACAATGC CGGCGGCACG ACCGCGCGCA CAACGGcaa t CCCTTACCAC ACGCGCAACC cccCCATCCT CggttacgcC AtccaaggCT CGGGCCGCCT cggaTacgcc gacAAAAACG TGGCGGACAA AGGCTACCTC aaagcCTATA TGCGGCAAAA AAACCCCAGC TATATCTTTT GCCCCGTCGG CGCACTGGGC CGCCGGCGCA ATCGACCGGC ACTACATTAC TCGCCACCGC CCATCCGGTT ACCCGCAAAG GCGCAGGATA CAGGCAGCGC GATCAAAGGC TTGGGGTTAC GGCGACGAAG CCGGCGAACT CAGAAAACCA CGGGATACGT CTGGCAGCTC CTGCCCAACG CGAATACCGC CCGTGA WO 00/22430 PCT/US99/23573 83 This corresponds to the amino acid sequence <SEQ ID 997; ORF 919.ng>: g919.pep 1 MKKGHLLRSAL YGIAAAILAA CQSRSIQTFP QPDTSVINGP DRPAGIPDPA 51 GTTVAGGGAV YTWVPHIJSMP HWAAQDFAKS LOSFRLGCAN L1UNRQGWQDV 101 CAQAFQTPVH SFQAXRFFER YFTPWOVAGN GSLAGTVTGY YEPVLKGDGR 151 RTERARFPIY GIPDDFISVP LPAGLRGGKN LVRIRQTGQN SGTIDNAGGT 201 HTADLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA 251 EDPVELFFMH IQGSGRLKTP SGKYIRIGYA D1UNEHPYVSI GRYMADKGYL 301 KLGQTSMOGI KAYMRQNPOR LAEVLGQNPS YIFFRELAGS GNEGPVGALG.

351 TPLMGEYAGA IDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG 401 AVRVDYFWGY GDEAGELAGI( QKTTGYVWQL LPNGMKPEYR P- ORE 919 shows 95.9 identity over a 441 aa overlap with a predicted ORE (ORE 919.ng) from N. gonorrhoeae: rn919/g919 20 30 40 50 m919 .pep MIKYLRAALYGIAAAI LAACQSKS IQTPPQPDTSVINGPDRPVGI PDPAGTTVGGGGAV 1 I:I: I I I I I 1 1 I I I I 11: 11111111 11111 :111111 I I I I I IIIII-IIIII g919 r.KKHLLRSALYGI AAAI LAACQSRS IQTFPQPDTSVI NGPDRPAG IPDPAGTTVAGGGAV 20 30 40 50 m919 .pep g919 m919 .pep g919 80 90 100 110 120 YTVVPHLSt4PHWAAQDFAKSLQS FRLGCANLKNRQGWQDVCAQAFQTPVHSFQAKRFFER 80 90 100 110 120 130 140 150 160 170 180 YFTPWQVAGNGSLAGTVTGYYEPVLKGDDRRTAQARFPIYGI PDDFI SVPLPAGLRSGKA YFT PWQVAGNGSLAGTVTGYYEPVLKGDGRRTERARFPIYGI PDDFI SVPLPAGLRGGN 130 140 150 160 170 180 190 200 210 220 230 240 m919.pep LVRIRQTGIGSGTIONTGGTHTADLSRFPITARTTAI KGRFEGSRFLPYH[TRNQINGGAL g919 LVRIRQTGKNSGTIDNAGGTHTADLSRFPITARTTAIKGRFEGSRFLPYHTRNQINGGAL 190 200 210 220 230 240 250 260 270 280 290 300 rn919 .pep DGKAPILGYAEDPVELFP'MHIQGSGRLKTPSGKYIRIGYADKNEHPYVSGRYMADKGYL g9 19 DGKAPI LGYAEDPVELFFMHIQGSGRLKTPSGKYIRIGYADKEHPYVSIGRYMADKGYL 250 260 270 280 290D 300 310 320 330 340 350 360 m919 .pep KLGQTSMQGI KSYMRONPQRLAEVLGONPSYI FFRELAGSSNDGPVGALi:TPLMGEYAGA 11111111111 I:111 1111111 11111111 I I I:11111111111111111 g919 KLGQTSMQGIKAYMRQNPQRLAEVLGONPSYIFFRELAGSGNEGPVGALGTPLMGEYAGA 310 320 330 340 350 360 370 380 390 400 410 420 m919 .pep VDRHYI TLGAPLFVATAHPVTRKALNRLIMAQDTGSAI KGAVRVDYFWGYGDEAGELAGK 991 9 I DRHYITLGAPLFVATAHPVTRKALNRLIMAQDTGSAI KGAVRVDYFWGYGDEAGELAGK 370 380 390 400 410 420 WO 00/22430 PCTIUS99/23573 84 430 440 m919.pep QKTTGYVWQLLPNGMKPEYRPX g919 QKTTGYVWQLLPNGMKPEYRPX 430 440 The following partial DNA sequence was identified in NMmeningitidis <SEQ ID 998>: a919.seq 1 ATGAAAAAAT ACCTATTCCG CGCCGCCCTG TGCGGCATCG CCGCCGCCAT 51 CCTCGCCGCC TGCCAAAGCA AGAGCATCCA AACCTTTCCG CAACCCGACA 101 CATCCGTCAT CAACGGCCCG GACCGGCCGG TCGGCATCCC CGACCCCGCC 151 GGAACGACGG TCGGCGGCGG CGGGGCCGTT TATACCGTTG TGCCGCACCT 201 GTCCCTGCCC CACTGGGCGG CGCAGGATTT CGCCAAAAGC CTGCAATCCT 251 TCCGCCTCGG CTGCGCCAAT TTGAAAAACC GCCAAGGCTG GCAGGATGTrG 301 TGCGCCCAAG CCTTTCAAAC CCCCGTCCAT TCCGTTCAGG CAAAACAGTT 351 TTTTGAACGC TATTTCACGC CGTGGCAGGT TGCAGGCAAC GGAAGCCTTrG 401 CCGGTACGGT TACCGGCTAT TACGAGCCGG TGCTGAAGGG CGACGACAGG 451 CGGACGGCAC AAGCCCGCTT CCCGATTTAC GGTATTCCCG ACGATTTTAT 501 CTCCGTCCCC CTGCCTGCCG GTTTGCGGAG CGGAAAAGCC CTTGTCCGCA 551 TCAGGCAGAC GGGAAAAAAC AGCGGCACAA TCGACAATAC CGGCGGCACA 601 CATACCGCCG ACCTCTCCCA ATTCCCCATC ACTGCGCGCA CAACGGCAAT 651 CAAAGGCAGG TTTGAAGGAA GCCGCTTCCT CCCCTACCAC ACGCGCAACC 701 AAATCAACGG CGGCGCGCTT GACGGCAAAG CCCCGATACT CGGTTACGCC 751 GAAGACCCCG TCGAACTTTT TTTTATGCAC ATCCAAGGCT CGGGCCGTCT 801 GAAAACCCCG TCCGGCAAAT ACATCCGCAT CGGCTATGCC GACAAAAACG 851 AACATCCCTA CGTTTCCATC GGACGCTATA TGGCGGACAA AGGCTACCTC 901 AAGCTCGGGC AGACCTCGAT GCAGGGCATC AAAGCCTATA TGCAGCAAAA 951 CCCGCAACGC CTCGCCGAAG TTTTGGGGCA AAACCCCAGC TATATCTTTT 1001 TCCGAGAGCT TACCGGAAGC AGCAATGACG GCCCTGTCGG CGCACTGGGC 1051 ACGCCGCTGA TGGGCGAGTA CGCCGGCGCA GTCGACCGGC ACTACATTAC 1101 CTTGGGCGCG CCCTTATTTG TCGCCACCGC CCATCCGGTT ACCCGCAAAG 1151 CCCTCAACCG CCTGATTATG GCGCAGGATA CCGGCAGCGC GATTAAAGGC 1201 GCGGTGCGCG TGGATTATTT TTGGGGATAC GGCGACGAAG CCGGCGAACT 1251 TGCCGGCAAA CAGAAAACCA CGGGATATGT CTGGCAGCTT CTGCCCAACG 1301 GTATGAAGCC CGAATACCGC CCGTAA This corresponds to the amino acid sequence <SEQ ID 999; ORF 919.a>: a919.pep 1 MI<KYLFRAAL CGIAAAILAA CQSKSIQTF'P QPDTSVINGP DRPVGIPDPA 51 GTTVGGGGAV YTVVPHLSLP HWAAQOE'AKS LQSFRLGCAN LKNRQGWQDV 101 CAQAFQTPVH SVQAKQFFER YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR 151 RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT 201 HTADLSOFPI TAR.TTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA 251 EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMADKGYL 301 KLGQTSMQGI KAYMQQNPQR LAEVLGQNPS YIFFRELTGS SNDGPVGALG 351 TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSA1KG 401 AVRVOYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P* m919/a919 ORFs 919 and 919.a showed a 98.6% identity in 441 aa overlap 20 30 40 50 m919 .pep MKKYLFP.AALYGIAAAILAACQSI(SIQTFPQPDTSVINGPDRPVGIPDIPAGTTVGGGGAV a919 MKKYLFRAALCGIAAAILAACQSKSIQTFPQPDTSVINGPDRPVGIPDPAGTTVGGGGAV 20 30 40 50 80 90 100 110 120 m919 .pp YTVVPH-LSLPHWAAQDFAISLQSFRLGCANLKNRQGWQDVCAQAFQTPVHSFQAKQFFER 601111 I 111 111 II 1111 I I I III a 919 YTVVPHLSLPHWAAQDFAKSLQS FRLGCANLKNRQGWQDVCAQAFQTPVHSVQAKFFR WO 00/22430 WO 0022430PCTIUS99/23573 85 80 90 100 110 120 130 140 150 160 170 180 m919.pep YFTPWQVAGNGSLAGTVTGYYEPVLKGDDRRTAQARFPIYGIPDDFISVPLPAGLRSGKA 51111 ii 11111Iii I11111 Ilil 11111 a91 9 YFTPWQVAGNGSLAGTVTGYYEPVLKGDDRRTAQARFPIYGI PDDFI SVP2LPAGLRSGKA 130 140 150 160 170 180 190 200 210 220 230 240 m919 .pep LVRIRQTGKNSGTIDNTGGTHTADLSRFPITARTTAIKGRFEGSRFLPYHTRNQINGGAL a919 LVRIRQTGKNSGTIDNTGGTHTADLSQFPITARTTAIKGRFEGSRFLPYHTrRNQINGGAL 190 200 210 220 230 240 250 260 270 280 290 300 m919 .pep DGKAPILGYAEDPVELFFMHIQGSGRLKTPSGKYIRIGYA0KNEHPYVSIGRYMADKGYL a91 9 DGKAPILGYAEDPVELFFMHIQGSGRLKTPSGKYIRIGYADKNEHPYVSIGRYMADKGYL 250 260 270 280 290 300 310 320 330 340 350 360 zn919.pep KLGQTSrQGIKSYMRQNPQRLAEVLGQNPSYIFFRELAGSSNDGPVGALGTPLMGEYAGA a919 KLGQTSMQGIKAYMQQNPQRLAEVLGQNPSYIFFRELTGSSNDGPVGALGTPLMGEYAGA 310 320 330 340 350 360 370 380 390 400 410 420 m919 .pep VDRHYITLGAPLFVATAHPVTRKALNR~LIMAQDTGSAIKGAVRVDYFWGYGDEAGELAGK a91 9 VDRHYITLGAPLFVATAHPVTRKALNRLIMAQDTGSAIKGAVRVDYFWGYGDEAGELAGK 370 380 390 400 410 420 430 440 m9 19 .pep QKTTGYVWQLLPNGMKEYRPX 1111 1111111 a91 9 QKTTGYVWQLLPNGMKPEYRPX 430 440 121 and 121-1 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1000>: m121. seq 1 ATGGAAACAC AGCTTTACAT CGGCATCATG TCGGGAACCA GCATGGACGG 51 GGCGGATGCC GTACTGATAC GGATGGACGG CGGCAAATGG CTGGGCGCGG 101 AAGGGCACGC CTTTACCCCC TACCCCGGCA GGTTACGCCG CCAATTGCI'G 151 GATTTGCAGG ACACAGGCGC AGACGAACTG CACCGCAGCA GGA1'TTTGTC 201 GCAAGAACTC AGCCGCCTAT ATGCGCAAAC CGCCGCCGAA CTGCTGTGCA 251 GTCAAAACCT CGCACCGTCC GACATTACCG CCCTCGGCTG CCACGGGCAA 301 ACCGTCCGAC ACGCGCCGGA ACACGGTTAC AGCATACAGC TTGCCGATTT 351 GCCGCTGCTG GCGxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx 401 xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx 451 xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx 501 xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx 551 xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx 601 xxxxxxCAGC TTCCTTACGA CAAAAACGGT GCAAAGTCGG CACAAGGCAA 651 CATATTGCCG CAACTGCTCG ACAGGCTGCT CGCCCACCCG TATTTCGCAC 701 AACGCCACCC TAAAAGCACG GGGCGCGAAC TGTTTGCCAT AAATTGGCTC 751 GAAACCTACC TTGACGGCGG CGAAAACCGA TACGACGTAT TGCGGACGCT 801 TTCCCGT2'TT ACCGCGCAAA CCGTTTGCGA CGCCGTCTCA CACGCAGCG WO 00/22430 WO 0022430PCTIUS99/23573 86 851 CAGATGCCCG 901 TTAATGGCGG 951 CACCGCCGAC 1001 CGTGGTTGGC 1051 GCAACCGGCG 1101 A TCAAATGTAC ATTTGCGACG ATTTGGCAGA ATGTTTCGGC CTGAACCTCG ATCCGCAATG GGCGTGTTGG ATTAATCGCA CATCCAAACC GTGTATTCTG

GCGGCATCCG

ACACGCGTTT

GGTGGAAGCC

TTCCCGGTAG

AnCGCGGGAT CAA1'CCTGTT

CCCTGCACAG

GCCGnATTTG

TCCGCACAAA

ATTATTATTG

This corresponds to the amino acid sequence <SEQ ID 1001; ORF 121>: m12l. pep 1U 1 METQLYIGIM 51 DLQDTGADEL 101 TVRHAPEHGY 151 xxxxxxxxxx 201 xxQLPYDKNG 251 ETYLDGGENR 301 LMADLAECFG 351 ATGASKPCIL SGTSMDGADA VLIRMDGGKW LGAEGHAFTP HRSRILSQEL SRLYAQTAAE LLCSQNLAPS SIQLADLPLL Axxxxxxxxx xxxxx xxxxxxxxxx xxxxxxxxxx xxxxxxxxxx AKSAQGNILP QLLDRLLALHP YFAQRHPKST YDVLRTLSRF TAQTVCDAVS HAAADARQMY TRVSLHSTAD LNLDPQWVEA AXFAWLAACW

XAGYYY*

YPGRLRRQLL

DITALGCHGQ

xxxxxxxxxx xxxxxxxxxx

GRELFAINWL

ICDGGIRNP'V

INRIPGSPHF

The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 1002>: g121. seq I ATGGAAACAc AGCTTTACAT CGGCATTATG TCGGGAACCA GTATGGACGG 51 GGCGGATGCC GTGCTGGTAC GGATGGACGG 101 AAGGGCACGC CTTTACCCCC T1ACCCTGACC 151 GATTTGCAGG ACACAGGCAC 201 GCAAGAACTC AGCCGCCTGT 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051

GTCAAAACCT

ACCGTCCGAC

GCCGCTGCTG

GCCGCGACCT

CACGAAGCCC

CGGCGGGATT

GCTTCGACAC

cacTGccagc catatTGCcg AACCCca ccc gaaacctAcc ttcccgattc

CAGATGCCCG

TTAATGGCGG

CACCGCCGAA

cgtggttggC

GCGACCGGCG

CGCTCCGTGC

ACGCGCCGGA

GCGGAACTGa

TGCTGCCGGC

TGTTCCGCGA

GCCAACATCA

AGGGCCGGGC

TGCCTTACGA

cAACTGCTCG aaAAAGCACG ttgacggcgg accgcgcaaA

TCAAATGTAC

ATTTGGCAGA

CTGAACCTCG

GGCGTGTTGG

CATCCAAACC

AGACGAACTG

ACGCGCAAAC

GACATTACCG

ACACGGTtac cgcggatttT GGacaAGGTG

TGACAGGGAA

GCGTACTCCC

AATATGCTGA

CAAAAacggt gcaggctGCT GGgcGCGaac cgaaaaccga ccg'rttggga

ATTTGCGGCG

ATGTTTCGGC

ATCCTCAATG

ATTAACCGCA

GTGTATTCTG

CGGCAAATGG

GGTTGCGCCG

CACCGCAGCA

CGCCGCCGAA

CCCTCGGCTG

AGCATACAC

TACCGTCggc

CGCCGCTCGT

ACACGCGTGG

CCCCGGCGCA

TGGAcgcgtg gcAAAGgcgg CGCCcaccCG TgtttgcccT tacgacgtat cgccgtctca

GCGGCATCCG

ACACGCGTTT

GGTGGAGGCG

TTCCCGGTAG

GGCGCGGGAT

CTGGGCGCGG

CAAATTGCTG

GGATGTTGTC

CTGCTGTGCA

CCACGGGCAA

TTGCCGATTT

gacttcCGCA

CCCCGCCTTT

TACTGAACAT

CCCGCCTTCG

gacgcaggca cacAAGGCPA

TATTTCTCAC

AAa ttggct c t gcggacgc~t

CACGCAGCGG

CAATCCTGTT

CCCTGCACAG

gccgCATTtg

TCCGCACAAA

ATTATTATTG

1101 A This corresponds to the amino acid sequence <SEQ ID 1003; ORF 121.ng>: gl21 .pep

METQLYIGIM

DLQDTGTDEL

TVRRAPEHGY

HEAL FRDDRE

HWQLPYDKNG

ETYLDGGENR

LMADLAECFG

ATGASKPCIL

SGTSMDGADA VLVRMDGGKW HRSRMLSQEL SRLYAQTAAE S IQLADLPLL AELTRI FTVG TP.VVLNIGGI ANISVLPPGA AKAAQGNILP QLLGRLLAHP YDVLRTLSRF TAQTVWDAVS TRVSLHSTAE LNILDPQWVEA

GAGYYY*

LGAEGHAFT P LLC SQN LAPC

DFRSRDLAAG

PAFGFDTGPG

YFSQPHPKST

HAAADARQMY

AAFAWLAACW

YPDRLRRKLL

DITALGCHGQ

GQGAPLVPAF

NMLMDAWTQA

GRELFALNWL

ICGGGIRNPV

INRIPGSPHKI

ORF 121 shows 73.5% identity over a 366 aa overlap with a predicted ORE (ORF121.ng) from N. gonorrhoeae: m12l/gl2l WO 00/22430 PCT1US99/23573 87 20 30 40 50 m121 .pep METQLYIGIMSGTSMDGADAVLIRNDGGKWLGAEGHAFTPYPGRLRRQLLDLQDTGADEL g12 1 METQLYIGIMSGTSMDGADAVLVRMDGGKWLGAEGHAFTPYPDRLRRKLLDLQDTGTDEL 10 20 30 40 50 80 90 100 110 120 ml21 pep HRSRILSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHGYSIQLAOLPLL g121 HRSRMLSQELSRLYAQTAAELLCSQNLAPCDITALGCHGQTVRHAPEHGYSIQLADLPLL 70 80 90 100 110 120 130 140 150 160 170 180 m12l pep AXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX g12 1 AELTRI FTVGDFRSRDLAAGGQGAPLVPAFHEALFRDDRETRVVLNIGGI:ANISVLPPGA 130 140 150 160 170 180 190 200 210 220 230 240 m12 1.pep XXXXXXXXXXXXXXXXXXXXXXQLPYDKNGAKSAQGNILPQLLDRLLAHPYFAQRHPKST g1 21 PAFGFDTGPGNMLMDAWTQAHWQLPYDKNGAKAAQGNILPQLLGRLLAHPYFSQPHPKST 190 200 210 220 230 240 250 260 270 280 290 300 m121.pep GRELFAINWLETYLDGGENRYDVLRTLSRFTAQTVCDAVSHAAAOARQMY'ICDGGIRNPV g12 1 GRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVWDAVSHAAAOARQMYICGGGIRNPV 250 260 270 280 290 300 310 320 330 340 350 360 m121 pep LMADLECFGTRVSLHSTADLNLDPQWVAAXFAWLAACWINRIPGSPHICATGASKPCIL g121 LMADLAECFGTRVSLHSTAELNLDPQWVEAAAB'AWLAACWINRIPGSPHKATGASKPCIL 310 320 330 340 350 360 rnl2l.pep XAGYYYX HIM11 g121 GAGYYYX The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1004>: a121. seq 1 ATGGAAACAC AGCTTTACAT CGGCATCATG TCGGGAACCA GCATGGACGG 51 GGCGGATGCC GTACTGATAC GGATGGACGG CGGCAAATGG CTGGGCGCGG 101 AAGGGCACGC CTTTACCCCC TACCCCGGCA GGTTACGCCG CAAATTGCTG 151 GATTTGCAGG ACACAGGCGC GGACGAACTG CACCGCAGCA GGATGTTGTC 201 GCAAGAACTC AGCCGCCTGT ACGCGCAAAC CGCCGCCGAA CTGCTGTGCA 251 GTCAAAACCT CGCGCCGTCC GACATTACCG CCCTCGGCTG CCACGGGCA.A 301 ACCGTCAGAC ACGCGCCGGA ACACAGTTAC AGCGTACAGC TTGCCGATTT 351 GCCGCTGCTG GCGGAACGGA CTCAGATTTT TACCGTCGGC GACTTCCGCA 401 GCCGCGACCT TGCGGCCGGC GGACAAGGCG CGCCGCTCGT CCCCGCCTTT 451 CACGAAGCCC TGTTCCGCGA CGACAGGGAA ACACGCGCGG TACTGAACAT 501 CGGCGGGATT GCCAACATCA GCGTACTCCC CCCCGACGCA CCCGCCTTCG 551 GCTTCGACAC AGGACCGGGC AATATGCTGA TGGACGCGTG GATGCAGGCA 601 CACTGGCAGC TTCCTTACGA CAAAAACGGT GCAAAGGCGG CACAAGGCAA 651 CATATTGCCG CAACTGCTCG ACAGGCTGCT CGCCCACCCG TATTTCGCAC 701 AACCCCACCC TAAAAGCACG GGGCGCGAAC TGTTTGCCCT AAATTGGCTC 751 GAAACCTACC TTGACGGCGG CGAAAACCGA TACGACGTAT TGCGGACGCT 801 TTCCCGATTC ACCGCGCAAA CCGTTTTCGA CGCCGTCTCA CACGCAGCGG 851 CAGATGCCCG TCAAATGTAC ATTTGCGGCG GCGGCATCCG CAATCCTGTT 901 TTAATGGCGG ATTTGGCAGA ATGTTTCGGC ACACGCGTTT CCCTGCACAG 951 CACCGCCGAA CTGAACCTCG ATCCGCAATG GGTAGAAGCC GCCGCGTTCG 1001 CATGGATGGC GGCGTGTTGG GTCAACCGCA TTCCCGGTAG TCCGCACAAA 1051 GCAACCGGCG CATCCAAACC GTGTATTCTG GGCGCGGGAT ATTATTATTG 1101 A WO 00/22430 PCTIUS99/23573 -88- This corresponds to the amino acid sequence <SEQ ID 1005;) ORF 121.a>: al21. pep 1 METOLYIGIM SGTSMOGADA VLIRMDGGKW LGAEGHAFTP YPGRLRRKLL 51 DLQDTGADEL HRSRMLSQEL SRLYAQTAAE LLCSQNLAPS DITALGCHGQ 101 TVRHAPEHSY SVQLADLPLL AERTQIFTVG DFRSRDLAAG GQGAPLVPAF 151 HEALFRDDRE TRAVLNIGGI ANISVLPPDA PAWGFDTGPG NMLMDAWMQA 201 IWQLPYDKNG AKAAQGNILP QLLDRLLAHP YFAQPHPKST GRELFALNWL 251 ETYLDGGENR YDVLRTLSRF TAQTVFDAVS HAAADARQMY ICGGGIRNPV 301 LMADLAECFG TRVSLHSTAE LNLDPQWVEA AAWAWMAACW VNRIPGSPHK 351 ATGASKPCIL GAGYYY* m121/a121 ORFs 121 and 121.a 74.0% identity in 366 aa overlap 10 20 30 40 50 m12 1.pep METQLYIGIMSGTSMDGADAVLIRMDGGKWLGAEGHAFTPYPGRLRRQLLDLQDTGADEL a12 1 METQLYIGIMSGTSMDGADAVLIRMDGGKWLGAEGEAFTPYPGRLRRKLLDLQDTGADEL 20 30 40 50 80 90 100 110 120 m121 .pep HRSRILSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHGYSIQLADLPLL a12 1 HRSRMLSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHSYSVQLADLPLL 70 80 90 100 110 120 130 140 150 160 170 180 m121 .pep AXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX a121 AERTQIFTVGDFRSRDLAAGGQGAPLVPAFHEALFRDDRETRAVLNIGGINISVLPPDA 130 140 150 160 170 180 190 200 210 220 230 240 m121 .pep XXXXXXXXXXXXXXXXXXXXXXQLPYDKNGAKSAQGNILPQLLDRLLAHPYFAQlHPKST 111 111 11111 11 IIII a12 1 PAFGFDTGPGNMLMDAWMQAHWQLPYDKNGAKAAQGNILPQLLDRLLAHP'YFAQPHPKST 190 200 210 220 230 240 250 260 270 280 290 300 m121 .pep GRELFAINWLETYLDGGENRYDVLRTLSRE7TAQTVCDAVSHAAADARQMYICDGGIRNPV a1 21 GRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVFDAVSHAAADARQMYICGGGIRNPV 250 260 270 280 290 300 310 320 330 340 350 360 rnl2l .pep LMADLAECFGTRVSLHSTADLNLDPQWVEAAXFAWLAACWINRIPGSPHKATGASKPCIL a121 LMAOLAECFGTRVSLHSTAELNLDPQWVEAAAFAWMAACWVNRIPGSPHK<ATGASKPCIL 310 320 330 340 350 360 ml21.pep XAGYYYX

IM

a121 GAGYYYX Further work revealed the DNA sequence identified in N. meningitidis <SEQ ID 1006>: m121-1. seq 1 ATGGAAACAC AGCTTTACAT CGGCATCATG TCGGGAACCA GCATGGACGG 51 GGCGGATGCC GTACTGATAC GGATGGACGG CGGCAAATGG CTGGGCGCGG 101 AAGGGCACGC CTTTACCCCC TACCCCGGCA GGTTACGCCG CCAATTGCTG 151 GATTTGCAGG ACACAGGCGC AGACGAACTG CACCGCAGCA GGATTTTGTC 201 GCAAGAACTC AGCCGCCTAT ATGCGCAAAC CGCCGCCGAA CTGCTGTGCA WO 00/22430 PCTIUS99/23573 89 251 GTCAAAACCT CGCACCGTCC GACATTACCG CCCTCGGCTG CCACGGGCAA 301 ACCGTCCGAC ACGCGCCGGA ACACGGTTAC AGCATACAGC TTGCCGATTT 351 GCCGCTGCTG GCGGAACGGA CGCGGATTTT TACCGTCGCC GACTTCCGCA 401 GCCGCGACCT TGCGGCCGGC GGACAAGGCG CGCCACTCGT CCCCGCCTTT 451 CACGAAGCCC TGTTCCGCGA CAACAGGGAA ACACGCGCGG TACTGAACAT 501 CGGCGGGATT GCCAACATCA GCGTACTCCC CCCCGACGCA CCCGCCTTCG 551 GCTTCGACAC AGGGCCGGGC AATATGC'GA TGGACGCGTG GACGCAGGCA 601 CACTGGCAGC TTCCTTACGA CAAAAACGGT GCAAAGGCGG CACAAGGCAA 651 CATATTGCCG CAACTGCTCG ACAGGCTGCT CGCCCACCCG TATTTCGCAC 701 AACCCCACCC TAAAAGCACG GGGCGCGAAC TGTTTGCCCT AAATTGGCTC 751 GAAACCTACC TTGACGGCGG CGAAAACCGA TACGACGTAT TGCGGACGCT 801 TTCCCGTTTT ACCGCGCAAA CCGTTTGCGA CGCCGTCTCA CACGCAGCGG 851 CAGATGCCCG TCAAATGTAC ATTTGCGGCG GCGGCATCCG CAATCCTGTT 901 TTAATGGCGG ATTTGGCAGA ATGTTTCGGC ACACGCGTTT CCCTGCACAG 951 CACCGCCGAC CTGAACCTCG ATCCGCAATG GGTGGAAGCC GCCGNATTTG 1001 CGTGGTTGGC GGCGTGTTGG ATTAATCGCA TTCCCGGTAG TCCGCACAA 1051 GCAACCGGCG CATCCAAACC GTGTATTCTG ANCGCGGGAT ATTATTATIG 1101 A This corresponds to the amino acid sequence <SEQ ID 1007; ORE 121-1>: m121-1 .pep 1 METQLYIGIM SGTSMDGADA VLIRNDGGKW LGAEGHAFTP YPGRLRRQLL 51 DLQDTGADEL HRSRILSQEL SRLYAQTAAE LLCSQNLAPS DITALGCHGQ 101 TVRHAPEHGY SIQLADLPLL AERTRIFTVG DFRSRDLAAG GQGAPLVPAF 151 HEALFRDNRE TRAVLNIGGI ANISVLPPDA PAFGFDTGPG NMLMDAWTQA 201 HWQLPYDKNG AKAAQGNILP QLLORLLAHP YFAQPHPKST GRELFALNWL 251 ETYLDGGENR YDVLP.TLSRF TAQTVCDAVS HAAADARQMY ICGGGIP.WPV 301 LMADLAECFG TRVSLHSTAD LNLDPQWVEA AXFAWLAACW INRIPGSPHK 351 ATGASKPCIL XAGYYY* m121-1/gl21 ORFs 121-1 and 122-1.ng showed a 95.6% identity in 366 a overlap 20 30 40 50 m121- pep METQLYIGIMSGTSMDGADAVLIRMDGGKWLGAEGRiAFTPYPGRLRRQLLDLQ0TGADEL gi 21 METQLYIGIMSGTSMDGADAVLVRMDGGKWLGAEGRiAFTPYPDRLRRKLIDLQDTGTDEL 20 30 40 50 70 80 90 100 110 120 rnl21-1.pep I4RSRILSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHGYSIQLADLPLL g12 1 HRSRMLSQELSRLYAQTAAELLCSQNLAPCDITALGCHGQTVRXAPEHGYSIQLADLPLL 80 90 100 110 120 130 140 150 160 170 180 rnl2l-1 .pep AERTRIFTVGDFRSRDLAAGGQGAPLVPAFHEALFRDNRETRAVLNIGGIEANISVLPPDA g121 AELTR IFTVGDFRSRDLAAGGOGAPLV PAFHEALFR00RETRVVLN IGG IAN ISVLP PGA 130 140 150 160 170 180 190 200 210 220 231) 240 m121-1 .pep PAFGFDTGPGNMLMDAWTQAHWQLPYDKNGAKAAQGNILPQLLDRLLAHPYFAQPHPKST g12l PAFGFDTGPGNMLM1DAWTQAHWQLPYDKNGAKAAQGNILPQLLGRLLA1IPYFSQPHPKST 190 200 210 220 230 240 250 260 270 280 290 300 m121-1 .pep GREL8'ALNWLETYLDGGENRYDVLRTLSRFTAQTVCDAVSHAAADARQM1YICGGGIRNPV liI 111111111 111111111 111111111 IIIIIII 11111111 I g12 1 GRELFALNWLETYLDGGENRYDVLRTLSRBFrAQTVWDAVSHAAADARQMYICGGGIRNPV 250 260 270 280 290 300 WO 00/22430 PCTIUS99/23573 ml21-l. pep gl2l 310 320 330 340 350 360 LMADLAECFGTRVSLHSTADLNLDPQWVEAAXFAWLAACWINRI PGS PHKATGASKPCI L LMADLAECFGTRVSLHSTAELNLDPQWVEAAAFAWLAACWINRI PGS PHKATGASKPCIL 310 320 330 340 350 360.

m121-1.pep XAGYYYX HIM11 g121 GAGYYYX The following partial DNA sequence was identified in N. men ingitidis <SEQ ID 1008>: al21-1 seq 1.

51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 A'TGGAAACAC AGCTTTACAT

GGCGGATGCC

PAAGGGCACGC

GATTTGCAGG

GCAAGAACTC

GTCAAAACCT

ACCGTCAGAC

GCCGCTGCTG

GCCGCGACCT

CACGAAGCCC

CGGCGGGATT

GCTTCGACAC

CACTGGCAGC

CATATTG CCG

AACCCCACCC

GAAACCTACC

TTCCCGATTC

CAGATGCCCG

TTAATGGCGG

CACCGCCGAA

CATGGATGC

GCAACCGGCG

GTACTGATAC

CTTTACCCCC

ACACAGGCGC

AGCCGCCTGT

CGCGCCGTCC

ACGCGCCGGA

GCGGAACGGA

TGCGGCCGGC

TGTTCCGCGA

GCCAACATCA

AGGACCGGGC

TTCCTTACGA

CAACTGCTCG

TAAAAGCACG

TTGACGGCGG

ACCGCGCAAA

TCAAATGTAC

ATTTGGCAGA

CTGAACCTCG

GGCGTGTTGG

CATCCAAACC

CGGCATCATG

GGATGGACGG

TACCCCGGCA

GGACGAACTG

ACGCGCAAAC

GACATTACCG

ACACAGTTAC

CTCAGATTTT

GGACAAGGCG

CGACAGGGAA

GCGTACT CCC

AATATGCTGA

CAAAAACGGT

ACAGGCTGCT

GGGCGCGAAC

CGAAAACCGA

CCGTTTTCGA

ATTTGCGGCG

ATGTTTCGGC

ATCCGCAATG

GTCAACCGCA

GTGTATTCTG

TCGGGAACCA

CGGCAAATGG

GGTTACCCC

CACCGCAGCA

CGCCGCCGAA

CCCTCGGCTG

AGCGTACAC

TACCGTCGGC

CGCCGCTCGT

ACACGCGCGG

CCCCGACGCA

TGGACGCGTG

GCAAAGGCGG

CGCCCACCCG

TGTTTGCCCT

TACGACGTAT

CGCCGTCTCA

GCGGCATCCG

ACACGCGTTT

GGTAGAAGCC

TTCCCGGTAG

GGCCCGAT

GCATGGACG(;

CTGGGCGCGG

CAAATTGCTG

GGATGTTGTC

CTGCTGTGCA

CCACGGGCAA

TTGCCGATTT

GACTTCCGCA

CCCCGCCTTTr TACT GAACAT

CCCGCCTTCG

GATGCAGGCA

CACAAGGCAA

TATTTCGCAC

AAATTGGCTC

TGCGGACGCT

CACGCAGCGG

CAATCCTGTT

CCCTGCACAG

GCCGCGTTCG

TCCGCACAAA

ATTATTATTG

Thi s corresponds to the amino acid sequence <SEQ ID 1009; ORF 121 a>: a121-1 .pep 1 METQLYIGIM SGTSMDGADA VLIRMDGGKW LGAEGHAFTP YPGRLRRKLL 51 DLQDTGADEL HRSPJ4LSQEL SRLYAQTAAE LLCSQNLAPS DITALGCHGQ 101 TVRI-APEHSY SVQLADLPLL AERTQIFTVG DFRSRDLAAG GQGAPLVPAI 151 HEALFRDDRE TRAVLNIGGI ANISVLPPDA FAFGFDTGPG NMLMDAWMQI 201 HWQLPYDKNG AKAAQGNILP QLLDRLLAHP YFAQPI4PKST GRELFALNWI 251 ETYLDGGENR YDVLRTLSRF TAQTVFDAVS HAAADARQMY ICGGGIRNPV 301 LMADLAECFG TRVSLHSTAE LNLDPQWVEA AAFAWMAACW VNRIPGSPH? 351 ATGASKPCIL GAGYYY* I)U m121-1/a121-1 ORFs 121-1 and 121-l.a showed a 96.4% identity in 366 aa overlap 20 30 40 .50 m121-1 .pep METQLYIGIMSGTSMDGADAVLIRMDGGKWLGAEGHAFTPYPGRLRRQLLDLQDTGADEL a 121-1 METQLYIGIMSGTSMDGADAVLIERMDGGKWLGAEGHA'TPYPGRLRPI(LLDLQDTGADEL 20 30 40 50 80 90 100 110 120 m121-1 .pep HRSRILSQELSRLYAQTAAELLCSQNLAPSDITALGCHGQTVRHAPEHGYSIQLAOLPLL 1 1 1 1 :1 I 1 1 1 1 Il IIIII I I I l II 11II 11II I I II II I I IIII I I :1 I I IIII a12 1-1 HRSRtLSQELSRLYAQTAAELLCSQNLAPSOITALGCHGQTVRHAPEHSYSVQLADLPLL 80 90 100 110 120 WO 00/22430 PCT/US99/23 573 91- 130 140 150 160 170 180 m121-1 pep AERTRIFTVG0FRSRDLAAGGQGAPLVPAFHEALF'RDNRETRAVLNIGGIANISVLPPDA a121-l AERTQIFTVGDFRSRDLAAGGQGAPLVPAFHEALFRD0RETRAVLNIGGIANISVLPPDA 130 140 150 160 170 180 190 200 210 220 230 240 m121-1 pep PAFGFDTGPGNMLMDAWTQAHWQLPYDKNGAKAAOGNILPQLLDRLLAHPYFAQPHPKST 11111 11111111111 i 111 II I li li iiI a121-1 PAFGFDTGPGNMLMDAWMQAHWQLPYDKNGAKAAQGNILPQLLDRLLAHPYFAQPHPKST 190 200 210 220 230 240 250 260 270 280 290 300 m121-1 .pep GRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVCDAVSHJAJADARQMYICGGGIRNPV a121-1 GRELFALNWLETYLDGGENRYDVLRTLSRFTAQTVFDAVSAA0AQMYICGGGIRNPV 250 260 270 280 290 300 310 320 330 340 350 360 m121-1 .pep LMADLAECFGTRVSLHSTADLNLDPQWVEAAXFAWLAACWINRIPGSPHKATGASKPCIL al21 LMADLAECFGTRVSLHSTAELNLDPQWVEAAAFAWMAACWVNRIPGSPHKATGASKPCIL 310 320 330 340 350 360 m121-1.pep XAGYYYX 'Ill'I a121 GAGYYYX 128 and 128-1 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1010O>: m128.seq (partial) 1 ATGACTGACA ACGCACTGCT CCATTTGGCC GAAGAACCCC GTTTTGATCA 51 AATCAAAACC GAAGACATCA AACCCGCCCT GCAAACCGCC ATCGCCGAAG 101 CGCGCGAACA AATCGCCGCC ATCAAAGCCC AAACGCACAC CGGCTGGGCA 151 AACACTGTCG AACCCCTGAC CGGCATCACC GAACGCGTCG GCAGGATTTG 201 GGGCGTGGTG TCGCACCTCA ACTGCGTCGC CGACACGCCC GAACTGCGCG 251 CCGTCTATAA CGAACTGATG CCCGAAATCA CCGTCTTCTT CACCGAAATC 301 GGACAAGACA TCGAGCTGTA CAACCGCTTC AAAACCATCA AAAATTCCCC 351 CGAATTCGAC ACCCTCTCCC CCGCACAAAA AACCAAACTC AACCAC 1 TACGCCAGCG AAAAACTGCG CGAAGCCAAA TACGCGTTCA GCGAAACCGA 51 wGTCAAAAAA TAyTTCCCyG TCGGCAAwGT ATTAAACGGA CTGTTCGCCC 101 AAmnTCAAAAA ACTmTACGGC ATCGGATTTA CCGAAAAAAC yGTCCCCGTC 151 TGGCACAAAG ACGTGCGCTA TTkTGAATTG CAACAAAACG GCGAAmCCAT 201 AGGCGGCGTT TATATGGATT TGTACGCACG CGAAGGCAAA CGCGGCGGCG 251 CGTGGATGAA CGACTACAAA GGCCGCCGCC GTTTTTCAGA CGGCACGCTG 301 CAAyTGCCCA CCGCCTACCT CGTCTGCAAC TTCGCCCCAC CCGTCGGCGG 351 CAGGGAAGCC CGCyTGAGCC ACGACGAAAT CCTCATCCTC TTCCACGAAA 401 CCGGACACGG GCTGCACCAC CTGCTTACCC AAGTGGACGA ACTGGGCGTA 451 TCCGGCATCA ACGGCGTAkA ATGGGACGCG GTCGAACTGC CCAGCCAGTT 501 TATGGAAAAT TTCGTTTGGG AATACAATGT CTTGGCACAA rTGTCAGCCC 551 ACGAAGAAAC CGGcgTTCCC yTGCCGAAAG AACTCTTsGA CAAAwTGCTC 601 GCCGCCAAAA ACTTCCAAsG CGGCATGTTC yTsGTCCGGC AAwTGGAGTT 651 CGCCCTCTTT GATATGATGA TTTACAGCGA AGACGACGAA GGCCGTCTCA 701 AAAACTGGCA ACAGGTTTTA GACAGCGTGC GCAAAAAAGT CGCCGTCATC 751 CAGCCGCCCG AATACAACCG CTTCGCCTTG AGCTTCGGCC ACATCTTCGC 801 AGGCGGCTAT TCCGCAGCTn ATTACAGCTA CGCGTGGGCG GALAGTATrGA WO 00/22430 PCTIUS99/23573 92 851 GCGCGGACGC ATACGCCGCC TTTGAAGAAA GCGACGATGT CGCCGCCACA 901 GGCAAACGCT TTTGGCAGGA AATCCTCGCC GTCGGGGnAT CGCGCAGCGG 951 nGCAGAATCC TTCAAAGCCT TCCGCGGCCG CGAACCGAGC ATAGACGCAC 1001 TCTTGCGCCA CAGCGGTTTC GACAACGCGG TCTGA This corresponds to the amino acid sequence <SEQ ID 10 11; ORF 128>: tn128.pep (partial) 1 MTDNALLHLG EEPRFDQIKT EDIKPALQTA IAEAREQIAA IKAQTHTGWA 51 NTVEPLTGIT ERVGRIWGVV SHLNCVADTP ELRAVYNELM PEITVFFTEI1 101 GODIELYNRF KTIKNSPEFD TLSPAQKTKL NH

YSEKQLREAK

WHKDVRYXEL

QLPTAYLVCN

SGINGVXWDA

AAKNFQXGMF

QPPEYNRFAL

GKRFWQEILA

YAFSETXVKK

QQNGEXIGGV

FAPPVGGREA

VELPSQFMEN

XVRQXEFALF

SF'GHI FAGGY

VGXSRSGAES

YFPVGXVLNG

YMDLYAREGK

RLSHDEILIL

FVWEYNVLAQ

DMMIYSEDDE

SAAXYSYAWA

FKAFRGREPS

LFAQXKKLYG IGFTEKTVPV RGGAWMNDYK GRRRFSDGTL FHETGHGLHH LLTQVDELGVI XSAHEETGVP LPKELXDOCL GRL@TWQQVL DSVRKKVAVI EVLSADAYAA FEESDDVAAT IDALLRHSGF DNAV* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 1012>: g128. seq 1 atgattgaca acgCActgct ccacttgggc gaagaaccCC GTTTTaatca 51 aatccaaacc gaagACAtca AACCCGCCGT CCAAACCGCC ATCGCCGAAG 101 CGCGCGGACA AATCGCCGCC GTCAAAGCGC AAACGCACAC CGGCTGGGCG 151 AACACCGTCG AGCGTCTGAC CGGCATCACC GAACGCGTCG GCAGGATTTG 201 GGGCGTCGTG TCCCATCTCA ACTCCGTCGT CGACACGCCC GAACTGCGCG 251 CCGTCTATAA CGAACTGATG CCTGAAATCA CCGTCTTCTT CACCGAAATC 301 GGACAAGACA TCGAACTGTA CAACCGCTTC AAAACCATCA AAAATTCCCC 351 CGAATTTGCA ACGCTTTCCC CCGCACAAAA AACCAAGCTC GATCACGACC 401 TGCGCGATTT CGTATTGAGC GGCGCGGAAC TGCCGCCCGA ACGGCAGGCA 451 GAACTGGCAA AACTGCAAAC CGAAGGCGCG CAACTTTCCG CCAAATTCTC 501 CCAAAACGTC CTAGACGCGA CCGACGCGTT CGGCATTTAC TTTGACGATG 551 CCGCACCGCT TGCCGGCATT CCCGAAGACG CGCTCGCCAT GTTTGCCGCC 601 GCCGCGCAAA GCGAAGGCAA AACAGGTTAC AAAATCGGCT TGCAGATTCC 651 GCACTACCTT GCCGTTATCC AATACGCCGG CAACCGCGAA CTGCGCGAAC 701 AAATCTACCG CGCCTACGTT ACCCGTGCCA GCGAACTTTC AAACGACGGC 751 AAATTCGACA ACACCGCCAA CATCGACCGC ACGCTCGAAA ACGCATTGAA 801 AACCGccaaa cTGCTCGGCT TTAAAAATTA 851 CCAAAATGGC GGACACGCCC GAACAGGTTT 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 GCCCGCCGCG CCAAACCCTA CTTCGCCCGC GAACACCTCG GCTACGCCGG CGAAAAACTG GAAGTCAAAA AATACTTCCC CCAAATCAAA AAACTCTACG

TCTGGCACAA

ATCGGCGGCG

CGCGTGGATG

TGCAACTGCC

GGCAAAGAAG

AacCGGCCAC

TGTCCGGCAT

TTTATGGAAA

CCACGAAGAA

TcgcCGCCAA

TTCGCCCTCT

GAAAAACTGG

TCCAACCGCC

GCcggcGGCT cAGCACCGAT

AGACGTGCGC

TTTATATGGA

AACGACtaca

CACCGCCTAC

CGCGTTTAAG

GGACTGCACC

CAAcggcgtA

ACTTCGTTTG

AccgGCGAGC

AAACTTCCAG

TCGATATGAT

CAGCAGGTTT

CGA6ATACAAC

ATTCCGCAGG

GCCTACGCCG

CGCCGAAAAA

GTCTCGCCGA

CGCGAACCCA

CGTCGGCAA.A

GCATCGGATT

TATI'TTGAAT

TTTGTACGCA

AAGGCCGCCG

CTCGTCTGCA

CCACGACGAA

ACCTGCTTAC

GAATGGGACG

GGAATACAAT

CCCTGCCGAA

CGCGGTATGT

GATTTACAGT

TAGACAGCGT

CGCTTCGCCA

CTATTACAGC

CGCCGAATTG

TAAACTTCCT

GACCTCGCCG

CCCGCAGCCG

AATACGCATT

GTTCTGGCAG

CGCCGAAAAA

TGCAACAAAA

CGCGAAGGCA

CCGCTTTGCC

ACTTCGCCCC

ATCCTCACCC

CCAAGTGGAC

CGGTCGAACT

GTATTGGCAC

AGAACTCTTC

TCCTCGTCCG

GAAAGCGACG

GCGCAAAGAA

ACAGCTTCGG

TACGCATGGG

TCGCTGGCA-A

GCACGACCTC

AAGTCAAAGC

TGGGACTTGA

CAGCGAAACC

GCCTGTTCGC'

ACCGTTCCCG

CGGCAAAACC

AACGCGGCGG

GACGgcacGC

GCCCGTCGGC

TCTTCCACGA

GAACTGGGCG

GCCCAGCCAG

AAATGTCCGC

GACAAAATGC

GCAAATGGAG

AATGCCGTCT

GTcGCCGTCA CCacatctTC CCGAAGTCCt CCTTTGAAGA AAGcGACGac gtcGCCGCCA WO 00/22430 PCTIUS99/23573 93 1901 CAGGCAAACG CTTCTGGCAA GAAAtccttg ccgtcggcgg ctCCCGCAGC 1951 gcgGCGGAAT CCTTCAAAGC CTTCCGCGGA CGCGAACCGA GCATAGACGC 2001 ACTGCTGCGC CAaagcggtT TCGACAACGC gGCttgA This corresponds to the amino acid sequence <SEQ ID 1013; ORE 128.ng>: 9128 .pep 1 MIDNALLHLG EEPRFNQIQT EDIIKPAVQTA IAEARGQIAA VKAOTH-TGWA 51 NTVERLTGIT ERVGRIWGWV SHLNSVVDTP ELRAVYNELM PEITVFFTEI 101 GQDIELYNRF KTIKNSPEFA TLSPAQKTKL DHDLRDFVLS GAELPPEROA 151 ELAKLQTEGA QLSAKFSQNV LDA TDAFGIY FDDAAPLAGI PEDALAMFAA 201 AAQSEGKTGY KIGLQIPHYL AVIQYAGNP.E LREQIYRAYV TRASELSNDG 251 KFDNTANIDR TLENALKTAK LLGFKNYAEL SLATIIADTP EQVLNFLHDL 301 ARRAKPYAEK DLAEVKAFAR EHLGLADPQP WDLSYAGEKL REAKYAFSET 351 EVKKY8'PVGK VLAGLFAOIK KLYGIGFAEK TVPVWHKDVR YFELQQNGKT 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFA DGTLQLPTAY LVCNFAPPVG 451 GKEARLSHDE ILTLFHETGH GLH]ILLTOVD ELG.VSGINGV EWDAVELPSQ 501 FMENFVWEYN VLAQMSAHEE TGEPLPKELF DKM~LAANFQ RG?4FLVROME 551 FALFDMMhIYS ESDECRLKNW QQVLDSVRKE VAVIQPPEYN REANSFGHIF 601 AGGYSAGYYS YAWAEVLSTD AYAAFEESDD VAATGKRFWQ EILAVGGSI;S 651 AAESFKAFRG REPSIDALLR QSGFDNAA* ORE 128 shows 91.7% identity over a 475 aa overlap with a predicted ORE (ORE 128.ng) from N. gonorrhoeae: m128/g128 20 30 40 50 9128 .pep MIDNALLHLGEEPRFQIQTEDIKPAVQTAIAEARGIAAVKAQTHTGWANTVER.LTGIT ml128 MTDNALLHLGEEPRFDQI KTEDI KPALQTAIAEAREQIAAI KAQTHTGWANTVEPLTGI T 20 30 40 50 s0 90 100 liC0 120 9128 .pep ERVGRIWGVVSHLNSVVDTPELRAVYNELMPEITVFFTEIGQDIELYNRFKTIKNSPEFA m12 8 ERVGRIWGVVSHLNCVADTPELRAVYNELMPEITVFFTEIGQDIELYNRFKTIKNSPEFD 80 90 100 110 120 130 140 150 160 170 180 9128 .pep TLS PAOKTKLDHDLRDF'JLSGAELPPERQAELAKLOTEGAOLSAKFSQNVLDATDAFGI

Y

m128 TLSPAQKTKLNH 130 340 350 360 9128 .pep YAGEKLREAKYAFSETEVKKYFPVGKVLAG m128 YASEICLREAKYAFSETXVKICYFPVGXVLNG 20 370 380 390 400 410 420 9128 .pep LFAQ I KKLYGIGFAEKTVPVWB1DVRYFELQNGKTIGGVYMDLYAREGIRGGAWMDYK m12 8 LFAQXKKLYGI GFTEKTVPVWIi103VRYXELQQNGEX IGGVYDLYAREG)MGGAWMNDYK 40 50 60 70 80 430 440 450 460 470 480 9128.pep GRR-RFAflGTLQLPTAYLVCNFAPPVGGKEARLSHDEILTLHETGHGLHIILLTQVDELGV WO 00/22430 PCT[US99/23573 94 m12 8 GRRRFSDGTLQLPTAYLVCNFAP PVGGREARLSHDE ILILFHETGHGLHHLLTQVDELGV 100 110 120 130 140 150 490 500 510 520 530 540 g128.pep SGINGVEWDAVELPSOFMENFVWEYNVLAOMSPJ{EETGEPLPKELFDKMI.AAKNFQRGMF m1 28 SGINGVXWDAVELPSQFMENFVEYNVLAQXSAHEETGVPLPKELXDKXIAAKcFQXGMF 160 170 180 190 200 210 550 560 570 580 590 600 9128 .pep LVROMEFALFDMThIYSESDECRLIGWQQVLDSVRKEVAVIQPPEYNRFANSFGHIFAGGY m12 8 XVRQXEFALFDMMIYSEDDEGRLKNWQOVLDSVRKKVAVIQPPEYNRFALSFGHI

FAGGY

220 230 240 250 260 270 610 620 630 640 650 660 9128.pep SAGYYSYAWAEVLSTDAYAALFEESDDVAATGKRFWQEI LAVGGSRSAAES FKAFRGREPS m1 28 SAAXYSYAWAEVLSADAYAAFEESDDVAATGKRFWQEILAVGXSRSGAESF-KAFRGREPS 280 290 300 310 320 330 670 679 9128 .pep IDALLRQSGFDNAAX 1 1 1 1 1 1 m128 IDALLRHSGFDNAVX 340 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1014>: a128.seq 1 ATGACTGACA ACGCACTGCT CCATTTGGGC GAAGAACCCC GTTTTGATCA 51 AATCAAAACC GAAGACATCA AACCCGCCCT GCAAACCGCC ATTGCCGAAG 101 CGCGCGAACA AATCGCCGCC ATCAAAGCCC AAACGCACAC CGGCTGGGCA 151 AACACTGTCG AACCCCTGAC CGGCATCACC GAACGCGTCG GCAGGATTTG 201 GGGCGTGGTG TCGCACCTCA ACTCCGTCAC CGACACGCCC GAACTGCGCG 251 CCGCCTACAA TGAATTAATG CCCGAAATTA CCGTCTTCTT CACCGAAATC 301 GGACAAGACA TCGAGCTGTA CAACCGCTTC AAAACCATCA AAAACTCCCC 351 CGAGTTCGAC ACCCTCTCCC ACGCGCAAAA AACCAAACTC AACCACGATC 401 TGCGCGATTT CGTCCTCAGC GGCGCGGAAC TGCCGCCCGA ACAGCAGGCA 451 GAATTGGCAA AACTGCAAAC CGAAGGCGCG CAACTTTCCG CCAAATTCTC 501 CCAAAACGTC CTAGACGCGA CCGACGCGTT CGGCATTTAC TTTGACGATG 551 CCGCACCGCT TGCCGGCATT CCCGAAGACG CGCTCGCCAT GTTTGCCGCT 601 GCCGCGCAAA GCGAAGGCAA AACAGGCTAC AAAATCGGTT TGCAGATTCC 651 GCACTACCTC GCCGTCATCC AATACGCCGA CAACCGCAAA CTGCGCGAAC 701 AAATCTACCd CGCCTACGTT ACCCGCGCCA GCGAGCTTTC AGACGACGGC 751 AAATTCGACA ACACCGCCAA CATCGACCGC ACGCTCGAAA ACGCCCTGCA 801 AACCGCCAAA CTGCTCGGCT TCAAAAACTA CGCCGAATTG TCGCTGGCAA 851 CCAAAATGGC GGACACCCCC GAACAAGTTT TAAACTTCCT GCACGACCTC 901 GCCCGCCGCG CCAAACCCTA CGCCGAAAzAA GACCTCGCCG AAGTCAAAGC 951 CTTCGCCCGC GAAAGCCTCG GCCTCGCCGA TTTGCAACCG TGGGACTTGG3 1001 GCTACGCCGG CGAAAAACTG CGCGAAGCCA AATACGCATT CAGCGAAACC 1051 GAAGTCAAAA AATACTTCCC CGTCGGCAAA GTATTAAACG GACTGTTCGC 1101 CCAAATCAAA AAACTCTACG GCATCGGATT TACCGAAAAA ACCGTCCCCG 1151 'CTGGCACAA AGACGTGCGC TATTTTGAAT TGCAACAAAA CGGCGAAACC 1201 ATAGGCGGCG TTTATATGGA TTTGTACGCA CGCGAAGGCA AACGCGGCGG3 1251 CGCGTGGATG AACGACTACA AAGGCCGCCG CCGTTTTTCA GACGGCACGC 1301 TGCAACTGCC CACCGCCTAC CTCGTCTGCA ACTTCACCCC GCCCGTCGGC 1351 GGCAAAGAAG CCCGCTTGAG CCATGACGAA ATCCTCACCC TCTTCCACGA 1401 AACCGGACAC GGCCTGCACC ACCTGCTTAC CCAAGTCGAC GAACTGGGCG 1451 TATCCGGCAT CAACGGCGTA GAATGGGACG CAGTCGAACT GCCCAGTCAG 1501 TTTATGGAAA ATTTCGTTTG GGAATACAAT GTCTTGGCGC AAATGTCCGIC WO 00/22430 PCT/US99/23573 95 1551 CCACGAAGAA ACCGGCGTTC CCCTGCCGAA AGAACTCTTC GACAAAATGC 1601 TCGCCGCCAA AAACTTCCAA CGCGGAATGT TCCTCGTCCG CCAAATGGAG 1651 TTCGCCCTCT TTGATATGAT GATTTACAGC GAAGACGACG AAGGCCGTCT 1701 GAAAAACTGG CAACAGGTTT TAGACAGCGT GCGCAAAGAA GTCGCCGTCG 1751 TCCGACCGCC CGAATACAAC CGCTTCGCCA ACAGCTTCGG CCACATCTTC 1801 GCAGGCGGCT ATTCCGCAGG CTATTACAGC TACGCGTGGG CGGAAGTATT 1851 GAGCGCGGAC GCATACGCCG CCTTTGAAGA AAGCGACGAT GTCGCCGCCA 1901 CAGGCAAACG CTTTTGGCAG' GAAATCCTCG CCGTCGGCGG ATCGCGCAGC 1951 GCGGCAGAAT CCTTCAAAGC CTTCCGCGGA CGCGAACCGA GCATAGACGC 2001 ACTCTTGCGC CACAGCGGCT TCGACAACGC GGCTTGA This corresponds to the amino acid sequence <SEQ ID 10 15; ORE 128.a>: a128 .pep 1 MTDNALLHLG EEPRFDQIKT EDIKPALQTA IAEAREQIAA IKAQTHTGWA 51 NTVEPLTGIT ERVGRIWGVV SI{LNSVTDTP ELRAAYNELM PEITVFETEI 101 GQDIELYNRF KTIKNSPEFD TLSHAQKTKL NHDLRDFVLS GAELPPEQQA 151 ELAKLQTEGA QLSAKFSQNV LDATDAFGIY FDDAAPLAGI PEDALANFAA 201 AAQSEGKTGY KIGLOIPHYL AVIQYADNR< LREQIYRAYV TRASELSDDG 251 KFDNTANIDR TLENALQTAK LLGFKNYAEL SLATKMADTP EQVLNFLHDL 301 ARRAKPYAEK DLAEVKAFAR ESLGLADLQP WDLGYAGEKL REAKYAFSET 351 EVKYYFPVG< VLNGLFAQIK KLYGIGFTEK TVPVVWHKDVR YFELQQNGET 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFS DGTLQLPTAY LVCNFTPPVG 451 GKEARLSHDE ILTLFHETGH- GLHHLLTQVD ELGVSGI4GV EWDAVELPSQ 501 FHENFVWEYN VLAQMSAIEE TGVPLPKELF DKMLAAKNFQ RGMFLVRQME 551 FALFDMt4IYS EDDEGRLKNW QQVLDSVRKE VAVVRPPEYN RFANSFGHIF 601 AGGYSAGYYS YAWAEVLSAD AYAAFEESDD VAATGKRFWQ EILAVGGSRS 651 AAESFKAFRG REPSIDALLR HSGFDNAA* m128/a128 OR~s 128 and 128.a showed a 66.0% identity in 677 aa overlap 10 20 30 40 50 rn128.pep MTDNALLHLGEEPRFDQIKTEDIKPALQTAIAEAP.EQIAAIKAQTHTGWANTVEPLTGIT a12 8 MTDNALLHLGEEPRFDQIKTEDIKPALQTAIAEA.EQIAAIKAQTHTGWANTVEPLTGIT 20 30 40 50 80 90 100 110 120 m128.pep ERVGRIWGVVSHLNCVADTPELRAVYNELMPEITVFFTEIGQDIELYNRFKTIKNSPEFD a128 ERVGRIWGVVSHLNSVTDTPELRAAYNELMPEITVFFTEIGQDIELYNRFKTIKNSPEFD 70 80 90 100 110 120 130 m128.pep III FF111111 a12 8 TLSHAQKTKLNHDLRDFVLSGAELPPEQQAELAKLQTEGAQLSAKFSQNVLDATDAFGIY 130 140 150 160 170 180 m 126 .pep a128 FDDAAPLAGI PEDALANFAAAAQSEGKTGYKIGLQI PHYLAVIQYADNRKLREQIYRAYV 190 200 210 220 230 240 m128.pep a128 TRASELSDDGKFDNTANIDRTLENALQTAKLLGFKNYAELSLATK4ADTPEQVLNFLHDL 250 260 270 280 290 300 140 150 m12 YASEKLP.EAKYAFSETX\?KKYFPVGX WO 00/22430 WO 0022430PCT1US99/23573 96 a 128 ARRAKPYAEKDLAEVKAFARESLGLADLQPWDLGYAGEKLREAKYAFSETEVKKYFPVGK 310 320 330 340 35CI 360 160 170 180 190 200 210 m128.pep VLNGLFAOXKKLYGIGFTEKTVPVWHKDVRYXELQQNGEXIGGVYMDLYAREGKRGGAWM a128 VLNGLFAQIKKLYGIGFTEKTVPVWHKDVRYFELQQNGETIGGVYMDLYAREGKRGGAWM4 370 380 390 400 410 420 220 230 240 250 260 270 m128 .pep NDYKGRRRFSDGTLQLPTAYLVCNFAPPVGGREARLSHDEILILFHETGHGLHHLLUQVD a12 8 NDYKGRRRFSDGTLQLPTAYLVCNFTPPVGGKEARLSHDEILTLFHETGHGLHHLLTQVD 430 440 450 460 470 480 280 290 300 310 320 330 m128 .pep ELGVSGINGVXWDAVELPSQFMENFVWEYNVLAQXSAHEETGVPLPKELXDKXLAAKNFQ a128 ELGVSGINGVEWDAVELPSQFMENFVWEYNVLAQMSAHEETGVPLPKELFDKNLAAKNFQ 490 500 510 520 530 540 340 350 360 370 380 390 m128,pep XGMFXVRQXEFALFDMM4IYSEDDEGRLKNWQQVLDSVRKKVAVIQPPEYNRFALSFGHIF a128 RGMFLVRQMEFALFOM1IYSEDDEGRLKNWQQVLDSVRKEVAVVRPPEYNRFANSFGHI F 550 560 570 580 590 600 400 410 420 430 440 450 m128 .pep AGGYSAAXYSYAWAEVLSADAYAAFEESDDVAATGKRFWQEILAVGXSRSGAESFKAFRG I 11 1 11111 1 11 1 11 111 II II :1I 1111111 a12 8 AGGYSAGYYSYAWAEVLSADAYAAFEESDDVAATGKRFWQE ILAVGGSRSAAES FKAFRG 610 620 630 640 650 660 460 470 m128.pep REPSIDALLRHSGFDNAVX a128 REPSIDALLRHSGFDNAAX 670 Further work revealed the DNA sequence identified in N. meningitidis <S EQ ID 10 16>: m128-1 .seq 1 ATGACTGACA ACGCACTGCT CCATTTGGGC GAAGAACCCC GTTTTGATCA 51 AATCAAAACC GAAGACATCA AACCCGCCCT GCAAACCGCC ATCGCCGAAG 101 CGCGCGAACA AATCGCCGCC ATCAAAGCCC AAACGCACAC CGGCTGGGCA 151 AACACTGTCG AACCCCTGAC CGGCATCACC GAACGCGTCG GGAGGATTTG 201 GG GCGTGGTG TCGCACCTCA ACTCCGTCGC CGACACGCCC GAACTGCGCG 251 CCGTCTATAA CGAACTGATG CCCGAAATCA CCGTCTTCTT CACCGAAATC 301 GGACAAGACA TCGAGCTGTA CAACCGCTTC AAAACCATCA AAAATTCCCC 351 CGAATTCGAC ACCCTCTCCC CCGCACAAAA AACCAAACTC AACCACGATC: 401 TGCGCGATTT CGTCCTCAGC GGCGCGGAAC TGCCGCCCGA ACAGCAGGCA 451 GAACTGGC?.A AACTGCAAAC CGAAGGCGCG CAACTTTCCG CCAAATTCTC 501 CCAAAACGTC CTAGACGCGA CCGACGCGTT CGGCATTTAC TTTGACGATG 551 CCGCACCGCT TGCCGGCATT CCCGAAGACG CGCTCGCCAT GTTTGCCGCC 601 GCCGCGCAAA GCGAAAGCAA AACAGGCTAC AAAATCGGCF TGCAGATTCC 651 ACACTACCTC GCCGTCATCC AATACGCCGA CAACCGCGAA CTGCGCGAAC 701 AAATCTACCG CGCCTACGTT ACCCGCGCCA GCGAACTTTC AGACGACGGC 751 AAATTCGACA ACACCGCCAA CATCGACCGC ACGCTCGCAA ACGCCCTGCA.

801 AACCGCCAAA CTGCTCGGCT TCAAAAACT. CGCCGAATTG TCGCTGGCAA 851 CCAAAATGGC GGACACGCCC GAACAAGTTT TAAACTTCCT GCACGACCTC WO 00/22430 WO 0022430PCTIUS99/23573 97 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 GCCCGCCGCG CCAAACCCTA CGCCGAAAAA CTTCGCCCGC GAAAGCCTGA ACCTCGCCGA GCTACGCCAG CGAAAAACTG CGCGAAGCCA GAAGTCAAAA AATACTTCCC CGTCGGCAAA CCAAATCAAA AAACTCTACG GCATCGGATT TCTGGCACAA AGACGTGCGC TATTTTGAAT ATAGGCGGCG TTTATATGGA TTTGTACGCA CGCGTGGATG AACGACTACA AAGGCCGCCG TGCAACTGCC CACCGCCTAC GGCAGGGAAG CCCGCC'rGAG A.ACCGGACAC GGGCTGCACC TATCCGGCAT CAACGGCGTA TTTATGGAAA ATTTCGTTTG CCACGAAGAA ACCGGCGTTC

TCGCCGCCAA

TTCGCCCTCT

GAAAAACTGG

TCCAGCCGCC

GCAGGCGGCT

GAGCGCGGAC

CAGGCAAACG

GCGGCAGAAT

ACTCTTGCGC

AAACTTCCAA

TTGATATGAT

CAACAGGTTT

CGAATACAAC

ATTCCGCAGG

GCATACGCCG

CTTTTGGCAG

CCTTCAAAGC

CACAGCGGTT

CTCGTCTGCA

CCACGACGAA

ACCTGCTTAC

GAATGGGACG

GGAATACAAT

CCCTGCCGAA

CGCGGCATGT

GATTTACAGC

TAGACAGCGT

CGCTTrCGCCT

CTATTACAGC

CCTTTGAAGA

GAAATCCTCG

CTTCCGCGGC

TCGACAACGC

GACCTCGCCG AAGTCAAAGC TTTGCAACCG TGGGACTTCG AATACGCGTT CAGCGAAACC GTATTAAACG GACTGTTCGC TACCGAAAAA ACCGTCCCCG TGCAACAAAA CGGCGAAACC CGCGAAGGCA AACGCGGCGG CCGTTTTTCA GACGGCACGC ACTTCGCCCC ACCCGTCGGC ATCCTCATCC TCTTCCACGA CCAAGTGGAC GAACTGGGCG CGGTCGAACT GCCCAGCCAG GTCTTGGCAC AAATGTCAGC AGAACTCTTC GACAAAATGC TCCTCGTCCG GCAAATGGAG GAAGACGACG AAGGCCGTCT GCGCAAAAAA GTCGCCGTCA TGAGCTTCGG CCACATCTTC TACGCGTGGG CGGAAGTATT AAGCGACGAT GTCGCCGCCA CCGTCGGCGG ATCGCGCAGC CGCGAACCGA GCATAGACGC

GGTCTGA

This corresponds to the amnino acid sequence 'CSEQ ID 10 17; ORF 128- 1>: m128-1 .pep.

1 MTDNALLIILG EEPRFDQIKT EDIKPALQTA IAEAREQIAA IKAQTHTGWA 51 NTVEPLTGIT ERVGRIWGVV SHLNSVADTP ELRAVYNELM~ PEITVFFTEI 101 GQDIELYNRF KTIKNSPEFD TLSPAQKTKL NHDLRDFVLS GAELPPEQQA 151 ELAKLQTEGA QLSAKFSQNV LDATDAFGIY FDDAAPLAGI PEDALANFAA 201 AAQSESKTGY KIGLQIPHYL AVIQYADNRE LREQIYRAYV TRASELSDDG 251 TFDNTANIDR TLANALQTAK LLGFKNYAEL SLATKMADTP EQVLNFLHDL, 301 ARP.AKPYAEK DLAEVKAFAR ESLNLADLQP WDLGYASEKL REAKYAFSET 351 EVKKYFPVGK VLNGLFAQIK KLYGIGFTEK TVPVWHKDV. YFELQQI4GET 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFS DGTLQLPTAY LVCNFAPPVG 451 GREARLSHDE ILILFHETGH GLI*{LLTQVD ELGVSGINGV EWDAVELPSQ 501 FMENFVWEYN VLAQM5A4EE TGVPLPKELF DKMLAAKNFQ RGMFLVRQME 551 FALFDMMIYS EDDEGRLIQ)W QQVLDSVRKK VAVIQPPEYN RFALSFGHIF 601 AGGYSAGYYS YAWAEVLSAD AYAAFEESDD VAATGKRFWQ EILAVGGSRS 651 AAESFKAFRG REPSIDALLR HSGFDNAV* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 10 18>: g128-1.seq (partial)

ATGATTGACA

AATCAAAACC

CGCGCGGACA

AACACCGTCG

GGGCGTCGTG

CCGTCTATAA

GGACAAGACA

CGAATTTGCA

TGCGCGATTT

GAACTGGCAA

CCAAAACGTC

CCGCACCGCT

GCCGCGCAAA

GCACTACCTT

AAATCTACCG

AAATTCGACA

AACCGCCAAA

CCAAAATGGC

ACGCACTGCT

GAAGACATCA

AATCGCCGCC

AGCGTCTGAC

TCCCATCTCA

CGAACTGATG

TCGAACTGTA

ACGCTTT CCC

CGTATTGAGC

AACTGCAAAC

CTAGACGCGA

TGCCGGCATT

GCGAAGGCAA

GCCGTTATCC

CGCCTACGTT

ACACCGCCAA

CTGCTCGGCT

GGACACGCCC

CCACTTGGGC

AACCCGCCGT

GTCAAAGCGC

CGGCATCACC

ACT CCGTCGT

CCTGAAATCA

CAACCGCTTC

CCGCACAAAA

GGCGCGGAAC

CGAAGGCGCG

CCGACGCGTT

CCCGAAGACG

AACAGGTTAC

AATACGCCGG

GAAGAAC CCC

CCAAACCGCC

AAACGCACAC

GAACGCGTCG

CGACACGCCC

CCGTCTTCTT

AAAACCATCA

AACCAAGCTC

TGCCGCCCGA

CAACTTTCCG

CGGCATTTAC

CGCTCGCCAT

AAAATCGGCT

CAACCGCGAA

GTTTTAATCA

ATCGCCGAAG

CGGCTGGGCG

GCAGGATTTG

GAACTGCGCG

CACCGAAATC

AAAATTCCCC

GATCACGACC

ACGGCAGGCA

C CAAATT CT C

TTTGACGAT-

GTTTGCCGCC

TGCAGATTCC

CTGCGCGAAC

AAACGACGGC

ACGCATTGAA

TCGCTGGCAA

GCACGACCTC

ACCCGTGCCA GCGAACTTTC CATCGACCGC ACGCTCGAAA TTAAAAATTA CGCCGAATTG GAACAGGTTT TAAACTTCCT 901 GCCCGCCGCG CCAAACCCTA CGCCGAAAAA GACCTCGCCG AAGTCAAAGC WO 00122430 PCTIUS99/23573 98 951 CTTCGCCCGC GAACACCTCG GTCTCGCCGA CCCGCAGCCG TGGGACTTGA 1001 GCTACGCCGG CGAAAAACTG CGCGAAGCCA AATACGCATT CAGCGAAACC 1051 GAAGTCAAAA AATACTTCCC CGTCGGCAAA GTTCTGGCAG GCCTGTTCGC 1101 CCAAATCAAA AAACTCTACG GCATCGGATT CGCCGAAAAA ACCGTTCCCG 1151 TCTGGCACAA AGACGTGCGC TATTTTGAAT TGCAACAAAA CGGCAAAACC 1201 ATCGGCGGCG TTTATATGGA TTTGTACGCA CGCGAAGGCA AACGCGGCGG 1251 CGCGTGGATG AACGACTACA AAGGCCGCCG CCGCTTTGCC GACGGCACGC 1301 TGCAACTGCC CACCGCCTAC CTCGTCTGCA ACTTCGCCCC GCCCGTCGGC 1351 GGCAAAGAAG CGCGTTTAAG CCACGACGAA ATCCTCACCC TCTTCCACGPI 1401 AACCGGCCAC GGACTGCACC ACCTGCTTAC CCAAGTGGAC GAACTGGGCG; 1451 TGTCCGGCAT CAACGGCGTA AAA This corresponds to the amino acid sequence <SEQ ID 1019; ORE 128-1.ng>: g128-1.pep (partial) 1 MIDNALLHLG EEPRFNQIKT EDIKPAVQTA IAEARGQIAA VKAQTHTGWA 51 NTVERLTGIT ERVGRIWGVV SHLNSVVDTP ELP.AVYNELM PEITVFFTEI 101 GQDIELYNRF KTIKNSPEFA TLSPAQKTKL DHDLRDFVLS GAELPPERQA 151 ELAKLQTEGA QLSAKFSQNV LDATDAFGIY FDDAAPLAGI PEDALAr4FAA 201 AAQSEGKTGY KIGLQIPHYL AVIQYAGNRE LREQIYRAYV TRASELSNDG 251 KFONTP.NIDR TLENALKTAK LLGFKNYAEL SLATKMADTP EQVLNFLHDL 301 ARRAKPYAEK DLAEVKAFAR EHLGLAOPQP WDLSYAGEKL REAKYAFSET 351 EVKKYFPVGK VLAGLFAQIK KLYGIGFAEK TVPVWHKDVR YFELQQNGKT 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFA OGTLQLPTAY LVCNFAPPVG 451 GKEARLSHDE ILTLWHETGH GLHHLLTQVD ELGVSGINGV K ml2B-1/g128-1 ORFs 128-1 and 128-1.ng showed a 94.5% identity in 491 aa overlap 10 20 30 40 50 g128- pep MIDNALLHLGEEPRFNQIKTEDIKPAVQTAIAEARGQIAAVKAQTHTGWANTTVERLTGIT I111111) 11111: 111 1:1 11111(11111 11111 m12 8-1 MTDNALLHLGEEPRFDQIKTEDIKPALQTAIAEAREQIAAIKAQTHTGWANTVEPLTGIT 20 30 40 50 80 90 100 110 120 g128-1 .pep ERVGRIWGVVSHLNSVVDTPELRAVYNELMPEITVFETEIGOOIELYN.FKTIKNSPEFA m12 8-1 ERVGRIWGVVSHLNSVADTPELRAVYNELMPEITVFFTEIGQDIELYNRFKTIKNSPEFD 70 80 90 100 110 120 130 140 150 160 170 180 g128-1 .pep TLSPAQKTKLDHDLP.DFVLSGAELPPERQAELAKLQTEGAQLSAKFSQNVLDATDAFGIY m128-1 TLSPAQKTKLNHiDLRDFVLSGAELPPEQQAELAKLQTEGAQLSAKFSQNVLDATDAFGIY 130 140 150 160 170 180 190 200 210 220 230 240 g128-1 .pep FDDAAPLAGIPEDALAMFAAAAQSEGKTGYKIGLQIPHYLAVIQYAGNRELREQIYRAYV 11111 1111 111:III IIIIIII II I II I I I I 1111 m12 8-1 FDDAAPLAGIPEDALAMFAAAAQSESKTGYKIGLQI PHYLAVIQYADNREIREQIYRAYV 190 200 210 220 230 240 250 260 270 280 290 300 g128-1.pep TRASELSNDGKFDNTANIDRTLENALKTAKLLGFKNYAELSLATKMADTPE:QVLNFLHDL m128-1 TRASELSDDGKFDNTANIDRTLANALQTAKLGFKNYAELSLATKMrADTPEQVLNFLHDL 250 260 270 280 290 300 310 320 330 340 350 360 g 128-1 .pep ARRAKPYAEKDLAEVKAFAREHLGLADPQPWDLSYAGEKLREAKYAFSETEVKKYFPVGK WO 00/22430 PCT/US99/23573 99 m1 28-1 g128-1 .pep m128-1

ARRAKPYAEKDLAEVKAFARESLNLADLQPWDLGYASEKLREAKYAFSETEVKKYFPVGK

310 320 330 340 350 360 370 380 390 400 410 420

VLAGLFAQIKKLYGIGFAEKTVPVWHKDVRYFELQQNGKTIGGVYMDLYAREGKRGGAWM

VLNGLFAQIKKLYGIGFTEKTVPVWHKDVRYFELQQNGET

IGGVYMDLYAREGKRGGAWM

370 380 390 400 410 420 430 440 450 460 470 480 g128-1 .pep NDYKGRRRFADGTLQLPTAYLVCNFAPPVGGKEARLSHDEILTLFHETGHGLHHLLTQVD m12 8-1 NDYKGRRRFSDGTLQLPTAYLVCNFAPPVGGREARLSHDEILILFHETGHGLHHLLTQVD 430 440 450 460 470 480 490 g128-1.pep ELGVSGINGVK I 1111111: m12 8-1 ELGVSGINGVEWDAVELPSQFMENFVWEYNVLAQMSAHEETGVPLPKELFDKMLAAKNFQ 490 500 510 520 530 540 The following DNA sequence was identified in N. meningitidis <SEQ ID 1020>: a 128-1. seq 1 ATGACTGACA ACGCACTGCT CCATTTGGGC 51 AATCAAAACC GAAGACATCA AACCCGCCCT 101 CGCGCGAACA AATCGCCGCC ATCAAAGCCC

AACACTGTCG

GGGCGTGGTG

CCGCCTACAA

GGACAAGACA

CGAGTTCGAC

TGCGCGATTT

GAATTGGCAA

CCAAAACGTC

CCGCACCGCT

GCCGCGCAAA

GCACTACCTC

AAATCTACCG

AAATTCGACA

AACCCCTGAC

TCGCACCTCA

TGAATTAATG

TCGAGCTGTA

ACCCTCTCCC

CGTCCTCAGC

AACTGCAAAC

CTAGACGCGA

TGCCGGCATT

GCGAAGGCAA

GCCGT CAT CC

CGCCTACGTT

ACACCGCCAA

CGGCATCACC

ACTCCGTCAC

CCCGAAATTA

CAACCGCTTC

ACGCGCAAAA

GGCGCGGAAC

CGAAGGCGCG

CCGACGCGTT

CCCGAAGACG

AACAGGCTAC

AATACGCCGA

GAAGAACCCC GTTTTGATCA GCAAACCGCC ATTGCCGAAG AAACGCACAC CGGCTGGGCA GAACGCGTCG GCAGGATTTG CGACACGCCC GAACTGCGCG CCGTCTTCTT CACCGAAATC AAAACCATCA AAAACTCCCC AACCAAACTC AACCACGATC TGCCGCCCGA ACAGCAGGCA CAACTTTCCG CCAAATTCTC CGGCATTTAC TTTGACGATG CGCTCGCCAT GTTTGCCGCT AAAATCGGTT TGCAGATTCC CAACCGCAAA CTGCGCGAAC ACCCGCGCCA GCGAGCTTTC AGACGACGGC CATCGACCGC ACGCTCGAAA ACGCCCTGCA TCAAAAACTA CGCCGAATTG TCGCTGGCAA GAACAAGTTT TAAACTTCCT GCACGACCTC 801 AACCGCCAAA CTGCTCGGCT 851 CCAAAATGGC GGACACCCCC 901 GCCCGCCGCG CCAAACCCTA CGCCGAAAAA 951 CTTCGCCCGC GAAAGCCTCG GCCTCGCCGA 1001 GCTACGCCGG CGAAAAACTG 1051 GAAGTCAAAA AATACTTCCC 1101 CCAAATCAAA AAACTCTACG 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801

TCTGGCACAA

ATAGGCGGCG

CGCGTGGATG

TGCAACTGCC

GGCAAAGAAG

AACCGGACAC

TAT CCGG CAT

TTTATGGAAA

CCACGAAGAA

TCGCCGCCAA

TTCGCCCTCT

GAAAAACTGG

TCCGACCGCC

GCAGGCGGCT

AGACGTGCGC

TTTATATGGA

AACGACTACA

CACCGCCTAC

CCCGCTT GAG

GGCCTGCACC

CAACGGCGTA

ATTTCGTTTG

ACCGGCGTTC

AAACTTCCAA

TTGATATGAT

CAACAGGTTT

CGAATACAAC

ATTCCGCAGG

CGCGAAGCCA

CGTCGGCAAA

GCATCGGATT

TATTTTGAAT

TTTGTACGCA

AAGGCCGCCG

CTCGTCTGCA

CCATGACGAA

ACCTGCTTAC

GAATGGGACG

GGAATACAAT

CCCTGCCGAA

CGCGGAATGT

GAT TTACAGC

TAGACAGCGT

CGCTTCGCCA

CTATTACAGC

GACCTCGCCG AAGTCAAAGC TTTGCAACCG TGGGACTTGG AATACGCATT CAGCGAAACC GTATTAAACG GACTGTTCGC.

TACCGAAAAA ACCGTCCCCG TGCAACAAAA CGGCGAAACC CGCGAAGGCA AACGCGGCGG CCGTTTTTCA GACGGCACGC ACTTCACCCC GCCCGTCGGC ATCCTCACCC TCTTCCACGA CCAAGTCGAC GAACTGGGCG CAGTCGAACT GCCCAGTCAG GTCTTGGCGC AAATGTCCGC AGAACTCTTC GACAAAATGC TCCTCGTCCG CCAAATGGAG GAAGACGACG AAGGCCGTCTr GCGCAAAGAA GTCGCCGTCG ACAGCTTCGG CCACATCTTC TACGCGTGGG CGGAAGTAT'r AAGCGACGAT GTCGCCGCCA CCGTCGGCGG ATCGCGCAGC 1851 GAGCGCGGAC GCATACGCCG CCTTTGAAGA 1901 CAGGCAAACG CTTTTGGCAG GAAATCCTCG WO 00/22430 WO 0022430PCTIUS99/23573 100- 1951 GCGGCAGAAT CCTTCAAAGC CTTCCGCGGA CGCGAACCGA GCATAGACGC 2001 ACTCTTGCGC CACAGCGGCT TCCACAACGC GGCTTGA This corresponds to the amino acid sequence <SEQ ID 1021; ORF 128-1.a a128-1.pep 1 P4TDNALLHLG EEPRFDQIKT EDIKPALQTA IAEAREQIAA IKAQTHTGWA 51 NTVEPLTGIT ERVGRIWGVV SHLNSVTDTP ELRAAYNELM PEITVFFTEI 101 GQDIELYNRF KTIKNSPEFD TLSHAQKTKL NHDLRDFVLS GAELPPEQQA 151 ELAKLQTEGA QLSAKFSQNV LDATDAFGIY FDDAAPLAGI PEDALANFAA 201 AAQSEGKTGY KIGLQIPHYL AVIQYADNRK LREQIYRAYV TRASELSDDG 251 KFDNTANIDR TLENALQTAK LLGFKNYAEL SLATKMADTP EQVLNFLHOL 301 ARRAKPYAEK DLAEVKAFAR ESLGLADLQP WDLGYAGEKL REAKYAFSET 351 EVKKYFPVGK VLNGLFAQIK KLYGIGFTEK TVPVWHKDVR YFELQQ4GET 401 IGGVYMDLYA REGKRGGAWM NDYKGRRRFS 0GTLOLPTAY LVCNFTPPVG 451 GKEARLSHDE ILTLFHETGH 6LHHLLTQVD ELGVSGINGV EWDAVELPSQ 501 FHENFVWEYN VLAQMSA}IEE TGVPLPKELF DKMLAAKNFQ RGMFLVRQME 551 FALFDMMIYS EDDEGRLKNW QQVLDSVRKE VAVVRPPEYN RFANSFGHIF 601 AGGYSAGYYS YAWAEVLSAD AYAAFEESDD VAATGKRFWQ EILAVGGSRS 651 AAESFKAFRG REPSIDALLR HSGFDNAA* m128-1/0128-1 ORFs 128-1 and 128-l.a showed a 97.8% identity in 677 aa overlap 20 30 40 50 a128-1 .pep MTDNALLHLGEEPRFDQIKTEDIKPALQTAIAEAREQIAAIKAQTHTGWANTVEPLTGIT IIII1111 11FIIF11111II 11111 F11 l m12 8-1 MTDNALLHLGEEPRFDQIKTEDIKPALQTAIAEAREQIAAIKAQTHTGWANTVEPLTGIT 20 30 40 50 80 90 100 110 120 a128-1 .pep ERVGRIWGVVSHLNSVTDTPELRAAYNELE'WEITVFFTEIGQDIELYNRFKTIKNSPEFD m128-1 ERVGRIWGVVSHLNSVADTPELRAVYNELMPEITVFFTEIGQDIELYNRFKTIKNSPEFD 80 90 100 110 120 130 140 150 160 170 180 a128-1 .pep TLSHAQKTKLNHDLRDFVLSGAELPPEQQAELAKLQTEGAQLSAKFSQNVLDATDArGIY m1 28-1 TLSPAQKTKLNHDLRDFVLSGAELPPEQQAELAKLQTEGAQLSAKFSQNV'LDATDAFGIY 130 140 150 160 170 180 190 200 210 220 230 240 a128-1 .pep FDDAAPLAGIPEOALAMFAAAAQSEGKTGYKIGLQIPHYLAVIQYADNRKLREQIYRAYV m128-1 FDDAAPLAGIPEDALAMFAAAAQSESKTGYKIGLQIPHYLAVIQYADNRELREQIYRAYV 190 200 210 220. 230 240 250 260 270 280 290 300 a128-1 .pep TRASELSDDGKFDNTANIDRTLENALQTA(LLGE'KNYAELSLATKM4ADTPEQVLNFLHDL m128-1 TRASELSDDGKFDNTANIDRTLP.NALQTAKLLGFKNYAELSLATKMADTPEQVLNFLHDL 250 260 270 280 290 300 310 320 330 340 350 360 a128-1 .pep ARRAKPYAEKDLAEVKAFARESLGLADLQPWDLGYAGEKLPREAKYAFSETEVKKYFPVGK 55111FF111F FFIFI1111F111 11111111F 11 m126- 1 ARRAKPYAEKDLAEVKAFARESLNLADLQPWDLGYASEKLREAK(YAFSETEVKKYFPVG( 310 320 330 340 350 360 370 380 390 400 410 420 a128-1 .pep VLNGLFAQIKKLYGIGFTEKTVPVWHKDVRYFELQQNGETIGGVYMDLYA.EGKRGGAWM m12 8-1 VLNGLrAQIKKLYGIGFTEKTVPVWHKDVRYFELQQNGETIGGVYMDLYAREGKRGGAWM WO 00/22430 WO 0022430PCTIUS99/23573 101 370 380 390 400 410 420 430 440 450 460 470 480 a128-l pep NDYKGRRRFSDGTLQLPTAYLVCNFTPPVGGKEARLSHDEILTLFHETGHGLHHLLTQVD 111 11 I 1111:II:1II I11111111 111111111 m1 28-1 NDYKGRRRFSDGTLQLPTAYLVCNFAPPVGGREARLSHDEILILFHETGHGLHHLLTQVD 430 440 450 460 470 480 490 500 510 520 530 540 a128-1 .pep ELGVSGINGVEWDAVELPSQFMNFWEYNVLAQMSAHEETGVPLPKELFDK4LAAKNF'Q m128-1 ELGVSGINGVEWDAVELPSQFMENFVWEYNVLAQMSAHETGVPLPKELFDKMLAAKN'Q 490 500 510 520 530 540 550 560 570 580 590 600 a1.28-1 .pep RGMFLVRQt4EFALFDMlIYSEDDGRLKNWQQVLDSVRKEVAVVRPPEYNRFANSFGHIF m128- 1 RGMFLVRQMEFALFDMMIYSEDDEGRLKNWQQVLDSVRKKVAVIQPPEYNRFALS FGH IF 550 560 570 580 590 600 610 620 630 640 650 660 a128-1.pep AGGYSAGYYSYAWAEVLSADAYAAFEESDDVAATGKRFWQEILAVGGSRSAAESFKAFRG m128- 1 AGGYSAGYYSYAWAEVLSADAYAAFEESDDVAATGKRF*WQEILAVGGSRSAAESFKAFRG 610 620 630 640 650 660 670 679 a128-1 .pep REPSIDALLRHSGFDNAAX 11111111111 II II: m128-1 REPSIDALLRHSGFDNAVX 670 206 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1022>: m206 .seq 1 ATGTTTCCCC CCGACAAAAC CCTTTTCCTC TGTCTCAGCG CACTGCTCCT 51 CGCCTCATGC GGCACGACCT CCGGCAAACA CCGCCAACCG AAACCCAAAC 101 AGACAGTCCG GCAAATCCAA GCCGTCCGCA TCAGCCACAT CGACCGCACA 151 CAACGCTCGC AGGAACTCAT GCTCCACAGC CTCGGACTCA TCGGCACGCC 201 CTACAAATGG GGCGGCAGCA GCACCGCAAC CGGCTTCGAT TGCAGCGGCA 251 TGATTCAATT CGTTTACAAr AACGCCCTCA ACGTCAAGCT GCCGCGCACC 301 GCCCGCGACA TGGCGGCGGC AAGCCGsAAA ATCCCCGAcA GCCGCyTCAA 351 GGCCGGCGAC CTCGTATTCT TCAACACCGG CGGCGCACAC CGCTACTCAC 401 ACGTCGGACT CTACATCGGC AACGGCGAAT TCATCCATGC CCCCAGCAGC 451 GGCAAAACCA TCAAAACCGA AAAACTCTCC ACACCGTTTT ACGCCAAAAA 501 CTACCTCGGC GCACATACTT TTTTTACAGA ATGA This corresponds to the amino acid sequence <SEQ ID 1023; ORF 206>: m206 .pep..

1 MFPPDKTLFL CLSALLLASC GTTSGKHRQP KPKQTVRQI 0 AVRISHIDRT 51 QGSELM~LI4S LGLIGTPYKW GGSSTATGFD CSGMIQFVYK NALNVKLPRT 101 APflMAAASRK IPDSRXKAGD LVFFNTGGAH RYSHVGLYIG NGEFIHAPSS 151 GKTIKTEKLS TPFYAO4YLG AMTFFTE* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 1024>: g206. seq 1 atgttttccc ccgacaaaac ccttttcctc tgtctcggcg cactgctcct WO 00/22430 WO 0022430PCTIUS99/23573 -102cgcctcatgc agacagtccg caaggctcgc ctacaaatg tgattcaatt gcccgcgaca ggccggcgac acgtcggact ggcaaaacca ctaccttgga ggcacgacct gcaaatccaa aggaactcat ggc~gcagca ggtttacaaa tg 9 9cggcgc atcgtattct ctacatcggc tcaaaaccga gcgcatacgt ccggcaaaca 9ccgtccgca 9ctccacagc gcaccgcaac aacgccctca aagccgcaaa tcaacaccgg aacggcgaat aaaactctcc tttttacaga ccgccaaccg tcagccacat ctc99actca cggcttcgac acgicaagct atccccgaca cggcgcacac tcatccatgc acaccgtttt a tga aaacccaaac cggccgcaca tcggcacgcc tgcagcggC8a gccgcgcacc gccgcctcaa cgctactcac ccccggcagc acgccaaaaa This corresponds to the amino acid sequence <SEQ ID 1025; ORF 206.ng>: g206 .pep MFSPDKTLFL CLGALLLASC GTTSGIGIRQP KPKQTVRQIQ QGSQELMLHS LGLIGTPYKW GGSSTATGFD CSGMIQLVYK ARDMAAASRK I PDSRLKAGD IVFFNTGGAH RYSHVGLYIG AVRI SHI GRT

NA.LNVKLPRT

NGEFIRAPGS

GXTI KTEKLS TPFYAKNYLG AHTFFTE* ORF 206 shows 96.0% identity over a 177 aa overlap with a predicted ORF (ORF 206.ng) from N gonorrhoeae: m206/g2O6 20 30 40 50 m206 .pep MFPPDKTLFLCLSALLLASCGTTSGKHRQPKPKQTVRQ IQAVRI SHI DRTQGsQELMLHS g206 MFSPDKTLFLCLGALLLASCGTTSGKHRPKPKQTVRQIQAVRISH-IGRTQGSQELMLHS 20 30 40 50 70 80 90 100 110 120 m2 06 .pep LGLI GTPYKWGGSSTATGFDCSGMI QFVYKNALNVKIPRTARDMAAASRKI PDSRX KAGD g206 LGLIGTPYKWGGSSTATGFDCSG I QLVYKQRALNVKLPRTARDMAAASRKI PDSRLKAGD 80 90 100 110 120 130 140 150 160 170 m206 .pep LVFFN~rGGAHRYSH'GLYIGNGEFIHAPSSGKTIKrEKLSTPFYANYLGTFFTEX 9206 IVFFNTGGAHP.YSHVGLYIGNGEFIHAPGSGKTIKTEKLSTPFYAUNYLGAHTFFTE 130 140 150 160 170 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1026>: a206. seq 1 ATGTTTCCCC CCGACAAAAC CCTTTTCCTC TGTCTCAGCG CACTGCTCCT 51 CGCCTCATGC GGCACGACCT CCGGCAAACA CCGCCAACCG AAACCCAAAC 101 AGACAGTCCG GCAAATCCAA GCCGTCCGCA TCAGCCACAT CGACCGCACA 151 CAAGGCTCGC AGGAACTCAT GCTCCACAGC CTCGGACTCA TCGGCACGCC 201 CTACAAATGG GGCGGCAGCA GCACCGCAAC CGGCTTCGAT TGCAGCGGCA 251 TGATTCAATT CGTTTACAAA AACGCCCTCA ACGTCAAGCT GCCGCGCACC 301 GCCCGCGACA TGGCGGCGGC AAGCCGCAAA ATCCCCGACA GCCGCCTTAA 351 GGCCGGCGAC CTCGTATTCT TCAACACCGG CGGCGCACAC CGCTACTCAC 401 ACGTCGGACT CTATATCGGC AACGGCGAAT TCATCCATGC CCCCAGCAGC 451 GGCAAAACCA TCAAAACCGA AAAACTCTCC ACACCGTTTT ACGCCAAAAA 501 CTACCTCGGC GCACATACTT TCTTTACAGA ATGA This corresponds to the amino acid sequence <SEQ ID 1027; ORE 206.a>: a206. pep WO 00/22430 WO 0022430PCTIUS99/23573 103 MFPPDKTLFL CLSALLLASC GTTSGKHRQP KPKQTVRQIQ AVRISHIDRT QGSQELMLHS LGLIGTPYKW GGSSTATGFD CSGMIQFVYK NALNVKLPRTr ARDMAAASRK IPOSRLKAGD LVFFNTGGAH RYSHVGLYIG NGEFIHAPSS GKTIKTEKLS TPFYAKI4YLG AHTFFTE* m206/a2O6 ORFs 206 and 206.a showed a 99.4% identity in 177 aa overlap 20 30 40 50 mn206 .pep MFPPDKTLFLCLSALLLASCGTTSGKHRQPKP(QTVRQIQAVRISHIDRTQGSQEL4LHS a2 06 MFPPDKTLFLCLSALLLASCGTTSGKHRQPKPKQTVRQIQAVRISHIDRTQGSQELMLHS 20 30 40 50 80 90 100 110 120 m206 .pep LGLIGTPYKWGGSSTATGFDCSGMIQFVYKNALNVKLPRTARDMAAASRKI-PDSRXKAGD a2 06 LGLIGTPYKWGGSSTATGFDCSGMIQFVYKNALNVKLPRTARDMAAASRKI PDSRLKAGD 80 90 100 110 120 130 140 150 160 170 m206.pep LVFFNTGGAHRYSHVGLYIGNGFIHAPSSGKTIKTEKLSTPYAKNYLGAHTFFTEX a206 LVFFNTGGAHRYSHVGLYIGNGEFIHAPSSGKTIKTEI(LSTPFYAKNYLGAHTFFTEX 130 140 150 160 170 The following partial DNA sequence was identified in N. men ingitidis <SEQ ID 1028>: m287.seq 1 ATGTTTAAAC GCAGCGTAAT

CTGCGGGGGC

TGTCAAAACC

GAAGATGCGC

AGGCAGTCAA

GTGCGGTAAC

GATATGCCGC

CCCGGATCCG

CCGGGGAATC

GACGGAATGC

TACGGCTGCC

CTTCAGATCC

AATTTTGGAA

GCAAAATATA

ATTTCTTGGA

GATGCAGACA

TGTCGGTTTG

GGCGGTGGCG

TGCCGCCCCT

CACAGGCAGG

GATATGGCGG

AGCGGATAAT

AAAATGCCGC

AATATGCTTG

GTCTCAGCCG

AGGGGGACGA

CAAGGTGCAA

CATCCCCGCG

GGGTTGATTT

ACGTTGACCC

CGCAATGGCT

GATCGCCCGA

GTTGTTTCTG

TTCTCAAGGA

CGGTTTCGGA

CCCAAAAATG

CGGTACAGAT

CCGGAAATAT

GCAAACCAAC

TCCGTCGGCA

ATCAAGCCGG

TCAAACCCTG

GGCTAATGGC

ACTG~TAAAGG

TGTATTTTTG

TGTCAAGTCG

AAAAAGAGAC

CAGGGCGCGC

AGAAMATACA

AAGACGAGGT

AGTTCGACAC

GGAAAATCAA

CGGATATGGC

GGCGGGCAAA

AAACAATCAA

CACCTGCGAA

GTTTTGATTG

CGATTCTTGT

CAGAATTTGA

GATGGGAAGA

GAAGGGAATC

TTGCGCGATT

CCCTTTCAGC:

GCGGACACGC

AGAGGCAAAG

CAT CCGCACA

GGCAATGGCG

GGCACAAAAT

CGAATCACAC

GCAACGGATG

AAATGCGGCG

ATGCCGGCAA

GCCGCCGGTT

TGGCGGTAGC

ACGGGCCGTC

AGTGGCAATA

AAAATTAAGT

ATGATAAATT

AATCAATATA

TAGGCGTTCT

TGAAGAAGTA CAGCTAAAAT AAATAAGTAA TTACAAGAAA GTTGCCGATA GTGTGCAGAT 851 TTATCTTTTA TAAACCTAAA CCCACTTCAT 901 GCACGGTCGA GGCGGTCGCT TCCGGCCGAG ATGCCGCTGA TTCCCGTCAA 951 TCAGGCGGAT ACGCTGATTG TCGATGGGGA AGCGGTCAGC CTGACGGGGC 1001 1051 1101 1151 1201 1251 1301 1351 1401 ATTCCGGCAA TATCTTCGCG GGGGCGGAAA AATTGCCCGG ACCGGCAAAA GGCGAAATGC TACTGCATTT CCATACGGAA TTTGCCGCAA AAGTCGATTT CAGCGGCGAT GATTTGCATA ATGGAAACGG CTTTAAGGGG TCCGGAAAGT TTTACGGCCC CTATCGCCCG ACAGATGCGG CCCGAAGGGA ATTACCGGTA TCTGACTTAC

CGGATCGTAT

TTGCGGGCGC

AACGGCCGTC

CGGCAGCAAA

TGGGTrACGCA

ACTTGGACGG

GGCCGGCGAG

AAAAGGGCGG

GCCCTTCGTG

GGCCGTGTAC

CGTACCCGAC

TCTGTGGACG

AAAATTCAAA

AAAATGGCAG

GAAGTGGCGG

ATTCGGCGTG

TTCAAGGCGA

AACGGCGAAG

CAGGGGCAGG

GCATTATCGA

GCCCCATCG

CGGGGATGTT

GAAAATACAG

TTTGCCGGCA

WO 00/22430 PCTIUS99/23573 104 1451 AAAAAGAGCA GGATTGA This corresponds to the amino acid sequence <SEQ ID 1029; ORE 287>: m287 .pep 1 MFKRSVIAMA CIFALSACGG.GGGGSPDVKS ADTLSKPAAP VVSEKETEA< 51 EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT GNGGAVTADN PKNEDEVAQbl 101 DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP ANQPDMANAA DGM4QGDDPSA GGQNAGNTAA QGANQAGNNQ NFGRVDLANG VLIDGPSQNI TLTHCKGDSC DADKISNYKK DGKNDKFVGL VA0SVQMKGI ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY FAAKVDFGS( SVDGIIDSGD DLHMGTQKF< AAGSSDPIPA SNPAPANGGS SGNNFLDEEV QLKSEFEKLS NQYIIFYKPK PTSFARFRRS LTGHSGNIFA PEGNYRYLTY NGEVLHFHTE NGRPYPTRG.

AAIDGNGFKG TWTENGSGDV 451 SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEOD* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 1030>: g287 .seq 1 atgtttaaac gcagtgtgat tgcaatggct tgtatttttc ccctttcagc 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 ctgtgggggc cgtcaaaacc ctgccgaaag cgatacgcag tttcggcaga aaaaatgaag atccgcaaat CCCCCgCgtC acgaacgtgg gttgacccac aagaagcacc attaagcgat tgctgacagg cggacaaacc gagattccgC ggaagcggtc ggaattaccg tatgccctCC cacggccgtg gtCCgtaccc aaatctgtgg gcaaaaattc cggaaaatgg gaggaagtgg cggattcggc ggcggtggcg ggCCgccccc aaaagaaaga gacgcaaccg aaatacaqgc acgcgggggc caaacaggga aaaccctgcc gcaattctgt tgtaaaggcg gtcaaaatca ataaaaaaga gtaaaaaagg acctactcgt tgattCCCgt agcctgacgg gtatctgact gtgtgcaagg tacaacggcg gtccggaggc acggcattat aaagccgcca cggcggggat cgggaaaata gtgtttgccg gatcgcccga gttgttgctg tgaggaggca ccggagaagg aatggcgqtg gcaaaatgat acaaccaacc cctgcgaatg tgtgattgac attcttgtaa gaatttgaaa cgagcaacgg atggaactaa tctgcacggt caatcaggcc ggcattccgg tacggggcgg cgaaccggca aagtgctgca aggtttgccg cgacagcggc tcgatggaaa gtttccqgaa tgtcaagtcg aaaatgccgg gcgggcggtg cagccaagat cggcaacaac atgccgcaaa cgccggttct gcggtagcga ggaccgtcgc tggtgataat aattaagtga gagaattttg caaatatatc cgaggaggtc gatacgctga caatatcttc aaaaattgcc aaaggcgaaa tttccatatg caaaagtcga gatgatttgc cggctttaag ggttttacgg gcggacacgc ggaaggggt g cgccgcaagc atggcggcag ggacaacccc atgccgccga tcagattccg ttttggaagg aaaatataac ttattqgatg tgaagaaaaa tcggtttggt atcttctata gcttccggcc ttgtggatgg gcgcccgaag cggcggatcg tgcttgttgg gaaaacggcc tttcggcagc atatgggtac gggacttgga cccggccggc cagctatcgc ccgacagatg ctgaaaaggg gcaaaaaaga tcgggattga This corresponds to the amino acid sequence <SEQ ID 1031; ORE 287.ng>: g287 .pep MFKRSVIAMA CIFPLSACGG LPKEKKDEEA AGGAPQADTQ KNEDAGAQND MPQNAAESAN TNVGNSVVID GPSQNITLTH I KRYKKDEQR EN FVGLVADR EIPLIPVNQA DTLIVDGEAV YALP.VQGE PA KGEMLVGTAV KSVDGIIDSG DDLHMGTQKF EEVAGKYSYR PTDAEKGGFG GGGGSP0VKS

DATAGEGSQD

QTGNNQ FAGS

CKGDSCNGDN

VKKDGTNKYI

SLTGHSGNIF

YNGEVLHFHM

KAAI UGNGFK

VFAGKKDRD*

A DT PS K AA

MAAVSAENTG

SDSAFASNPA

LLDEEAFSKS

IFYTDKPPTR

APEGNYP.YLT

ENGRFYPFS GG

GTWTENGGGO

VVAENAGEGV

NGGAATTON P

FANGGSDFGR

E FEKLSDEEK

SARSRRSLPA

YGAEKLPGGS

RFAAKVDFGS

VSGRFYGPAG

m287/g287 OR~s 287 and 287.ng showed a 70. 1% identity in 499 aa overlap WO 00/22430 PCTIUS99/23573 105 20 30 40 49 m287.pep MFKRSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVSE------------- KETEA g2 87 MFKRSVIAMACI FPLSACGGGGGGSPDVKSADTPSKPAAPVVAENAGEGVLPKEKKDEEA 10 20 30 40 50 60 70 80 90 100 109 m287.pep KEDAPQAGSQGQGAPSAQGSQDMAAVSEENTGNGGAVTADNPKNEDEVAQNDMPQNAAGT g287 AGGAPQADTQD--ATAGEGSQDMAAVSAENTGbNGGAATTDNPKNEDAGAQNDMPQNAA-- 80 90 100 110 110 120 130 140 150 160 169 m287 .pep 0SSTPNH1TPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGGQNAGNTA g 28 7 170 180 190 200 210 220 229 zn287.pep AQGANQAGNNQAAGSSDPIPASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCKGDS g287 -ESANQTGNNQPAGSSDSAPASNPAPANGGSDFGRTNVGNSVVIDGPSQNITLTHCKGDS 120 130 140 150 160 170 230 240 250 260 270 280 289 m287 .pep CSGNNFL0F.EVQLKSEFEKLSDADKISNYKKDGKNDKFVGLVADSVQMKGINqQYIIFYKP g28'7 CNGDNLLDEEAPSKSEFEKLSDEEI<IKRYKKDEQRENFVGLVADRVKKDGTNqKYIIFYTD 180 190 200 210 220 230 290 300 310 320 330 340 349 m287 .pep KPTSFARFRRSARSP.RSLPAEMPLIPVNQADTLIVDGEAVSLTGIISGNIFAPEGNYRYLT g287 KPPT--RSARSRRSLPAEIPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLT 240 250 260 270 280 290 350 360 370 380 390 400 409 m287 .pep YGAEKLPGGSYALRVQGEPAKGEMLAGAAVYNGEVLHFHTENGRPYTRGRFAAKVD'GS g2 87 YGAEKLPGGSYALRVQGEPAKGEMLVGTAVYNGEVLHFHMENGRPYPSGGRFAAKVDFGS 300 310 320 330 340 350 410 420 430 440 450 460 469 m287.pep KSVDGI IDSGDDLHI'GTQKFKAAI GNG FKGTWTENGSGDVSGKFYGPAGEEVAGKYSYR 1111111111111 1111 11111111111 g2 87 KSVDGI IDSGDDLHMGTQKFKAAI DGNGFKGTWTENGGGDVSGR'YGPAGEEVAGK<YSYR 360 370 380 390 400 410 470 480 489 in287.pep PTDAEKGGFGVFAGKKEQDX g287 PTDAEKGGFGVFAGKKDRDX 420 430 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1032>: a287.seq 1 ATGTTTAAAC GCAGTGTGAT TGCAATGGCT TGTATTGTTG CCCTTTCAGC 51 CTGTGGGGGC GGCGGTGGCG GATCGCCCGA TGTTAAGTCG GCGGACACGC 101 TGTCAAAACC TGCCGCCCCT GTTGTITACTG AAGATGTCGG GGAAGAGGTG 151 CTGCCGAAAG AAAAGAAAGA TGAGGAGGCG GTGAGTGGTG CGCCGCAAGC 201 CGATACGCAG GACGCAACCG CCGGAAAAGG CGGTCAAGAT ATGGCGGCAG 251 TTTCGGCAGA AAATACAGGC AATGGCGGTG CGGCAACAAC GGATAATCCC WO 00/22430 PCT/US99/23573 106- 301 GAAAATAAAG ACGAGGGACC GCAAAATGAT ATGCCGCAAA ATGCCGCCGA 351 TACAGATAGT TCGACACCGA ATCACACCCC TGCACCGAAT ATGCCAACCA 401 GAGATATGGG AAACCAAGCA CCGGATGCCG GGGAATCGGC ACAACCGGCA 451 AACCAACCGG ATATGGCAAA TGCGGCGGAC GGAATGCAGG GGGACGATCC 501 GTCGGCAGGG GAAAATGCCG GCAATACGGC AGATCAAGCT GCAAATCAAG 551 CTGAAAACAA TCAAGTCGGC GGCTCTCAAA ATCCTGCCTC TTCAACCAAT 601 CCTAACGCCA CGAATGGCGG CAGCGATTTT GGAAGGATAA ATGTAGCTAA 651 TGGCATCAAG CTTGACAGCG GTTCGGAAAA TGTAACGTTG ACACATT*TA 701 AAGACAAAGT ATGCGATAGA GATTTCTTAG ATGAAGAAGC ACCACCPAA 751 TCAGAATTTG AAAAATTAAG TGATGAAGAA AAAATTAATA AATATAAAAAA 801 AGACGAGCAA CGAGAGAATT TTGTCGGTTT GGTTGCTGAC AGGGTAGAAA 851 AGAATGGAAC TAACAAATAT GTCATCATTT ATAAAGACAA GTCCGCTTCA 901 TCTTCATCTG CGCGATTCAG GCGTTCTGCA CGGTCGAGGC GGTCGCTTCC 951 GGCCGAGATG CCGCTGATTC CCGTCAATCA GGCGGATACG CTGATTGTCG 1001 ATGGGGAAGC GGTCAGCCTG ACGGGGCATT CCGGCAATAT CTTCGCGCCC 1051 GAAGGGAATT ACCGGTATCT GACTTACGGG GCGGAAAAAT TGTCCGGCGG 1101 ATCGTATGCC CTCAGTGTGC AAGGCGAACC GGCAAAAGGC GAAATGCTTG 1151 CGGGCACGGC CGTGTACAAC GGCGAAGTGC TGCATTTCCA TATGGAAAAC 1201 GGCCGTCCGT CCCCGTCCGG AGGCAGGTTT GCCGCAAAAG TCGATTTCGG 1251 CAGCAAATCT GTGGACGGCA TTATCGACAG CGGCGATGAT TTGCATATGG 1301 GTACGCAAAA ATTCAAAGCC GTTATCGATG GAAACGGCTT TAAGGGGACT 1351 TGGACGGAAA ATGGCGGCGG GGATGTTTCC GGAAGGTTTT ACGGCCCGGC 1401 CGGCGAAGAA GTGGCGGGAA AATACAGCTA TCGCCCGACA GATGCGGAAA 1451 AGGGCGGATT CGGCGTGTTT GCCGGCAAkAA AAGAGCAGGA TTGA This corresponds to the amino acid sequence <SEQ ID 1033; ORF 287.a>: a287 .pep 1 MFKRSVIAMA CIVALSACGG GGGGSPDVKS ADTLSKPAAP VVTED0VGEEV 51 LPKEKKDEEA VSGAPQADTQ DATAGKGGQD MAAVSAENTG NGGAATTDNP 101 ENKDEGPQND MPQNAADTDS STPNHTPAPN MPTRDMGNQA PDAGESAQPA 151 NQPDMANAAD GMQGDDPSAG ENAGNTADQA ANQAENNQVG GSQNPASSTN 201 PNATNGGSOF GRINVANGIK LOSGSENVTL THCKDKVCDR DFLDEEAPPK 251 SEFEKLSDEE KINKYKKDEQ RENEVGLVAD RVEKNGTNKY VIIYKDKSAS 301 SSSARFRRSA RSRRSLPAEM PLIPVNOADT LIVOGEAVSL TGHSGNIFAP 351 EGNYP.YLTYG AEKLSGGSYA LSVQGEPAKG EMLAGTAVYN GEVLHFHMEN 401 GRPSPSGGRF AAKVDFGSKS VDGIIDSGDD LHMGTQKFKA VIDGNGFKGT 451 WTENGGGDVS GRFYGPAGEE VAGKYSYRPT DAEKGGFGVF AGKKEQD k m287/a287 ORFs 287 and 287.a showed a 77.2% identity in 501 aa overlap 20 30 40 49 m2B7 .pep MFKRSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPWVSE------------ KETEA a287 MFKRSVIAI4ACIVALSACGGGGGGSPDVKSADTLSKPAAPVVTEDVGEEVLPKEKKDEEA 10 20 30 40 50 60 70 80 90 100 109 m287 .pep KEDAPQAGSQGQGAPSAQGSQDMAAVSEENTGNGGAVTADNPKNEDEVAQNDMPQNAAGT a2 87 VSGAPQADTQ--DATAGKGGQDMAVSAENTGNGGAATTDNPENKDEGPQNDMPQNAADT 80 90 100 110 110 120 130 140 150 160 169 m287 .pep DSSTPNHTPDPNMLAGNMENQATDAGESSQPANQPDMANAADGMQGDDPSAGGQNAGNTA 5 1 1 1 1 1 1 1 1 1 1 1 a28 7 DSSTPNHTPAPNMPTRDMGNQAPDAGESAQPANQPDMANAADGMQGDDPSAG-ENAGNTA 120 130 140 150 160 170 170 180 190 200 210 220 229 m287 .pep AQGANQAGNNQAAGSSDPIPASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCKGDS a2 87 DQAANQAENNQVGGSQNPASSTNPNATNGGSDFGRINVANGIKLDSGSENVTLTHCKDKV WO 00/22430 WO 0022430PCTIUS99/23573 107 180 190 200 210 220 230 240 250 260 270 280 289 m287 .pep CSGNNFLDEEVQLKSEFEKLSDAIOKISNYKKDGKNDKFVGLVADSVQMKGINQYIIFYKP 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 a2 87 CD-RDFLDEEAPPKSEFEKLS 0E8KINKYKKDEQRENFVGLVADRVEKNGT'NKYVI IYKD 240 250 260 270 280 290 290 300 310 320 330 340 in287 .pep KP--TSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRY a287 KSASSSSARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNI FAPEGNYRY 300 310 320 330 340 350 m287 .pep a287 350 360 370 380 390 400

LTYGAEKLPGGSYALRVQGEPAKGEMLAGAAVYNGEVLHFHTENGRPYPTRGRFAAKVDF

LTYGAEKLSGGSYALSVQGEPAKGEMLAGTAVYNGEVLHFHMENGRPSPSG;GRFAAKVDF

360 370 380 390 400 410 410 420 430 440 450 460 m287 .pep GSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYS a287 GSKSVDGIISGDDLH-MGTQKFKAVI 0GNGFKGTWTENGGGDVSGRFYGPAGEEVAGKYS 420 430 440 450 460 470 470 480 489 m287 .pep YRPTDAEKGGFGVFAGKKEQDX a2 87 YRPTDAEI(GGFGVFAGKKEQDX 480 490 406 The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1034>: m406 .seq 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951

ATGCAAGCAC

CGCCTGCGGG

TTGCGGTCGA

GACATGGATT

CACTATGGGC

TTGATGCACT

GATTACACCT

TTTGACAGGT

CTCGCACCCA

ATTGGCGGGA

CGACACTGCC

GCATAGACGT

ATCGACGTAT

GGCTGCTGAT ACCTATTCTT TTTTCAGTTT TTATTTTATC ACACTGACAG GTATTCCATC GCATGGCGGA GGTAAACGCT ACAAGAACTT GTGGCCGCTT CTGCCAGAGC TGCCGTTAAA TACAGGCATT ACACGGACGA AAAGTTGCATr TGTACATTGC GACCAAGGTT CAGGCAGTTT GACAGGGGGT CGCTACTCCA GATTCGTGGC GAATACATAA ACAGCCCTGC CGTCCGTACC ATCCACGTTA CGAAACCACC GCTGAAACAA CATCAGGCGG TTAACCACTT CTTTATCTAC ACTTAATGCC CCTGCACTCT ATCAGACGGT AGCGGAAGTA AAAGCAGTCT GGGCTTAAAT TGGGGGATTA TCGAAATGAA ACCTTGACGA CTAACCCGCG TTTCTTTCCC ACTTGGTACA GACCGTATTT TTCCTGCGCG TGTTTCTCCT GCCAATGCCG ATACAGATGT GTTTATTAAC TCGGAACGAT ACGCAACAGA ACCGAAATGC ACCTATACAA TGCCGAAACA CTGAAAGCCC AAACAAAACT GGAATATTTC GCAGTAGACA GAACCAATAA AAAATTGCTC ATCAAACCAA AAACCAATGC GTTTGAAGCT GCCTATAAAG AAAATTACGC ATTGTGGATG GGGCCGTATA AAGTAAGCAA AGGAATTAAA CCGACGGAAG GATTALATGGT CCATTTCTCC GATATCCGAC CATACGGCAA TCATACGGGT AACTCCGCCC CATCCGTAGA GGCTGATAAC AGTCATGAGO GGTATGGATA CAGCGATGAA GTAGTGCGAC AACATAGACA AGGACAACCT TGA WO 00/22430 PCT[US99/23573 108 This corresponds to the amino acid sequence <SEQ ID 1035; ORE 406>: m406.pep 1 MQARLLIPIL FSVFILSACG TLTGIPSHGG GKRFAVEQEL VAASARAAVK 51 DMDLQALHGR KCVALYIATMG DQGSGSLTGG RYSIDALIRG EYINSPAVRT 101 DYTYPRYETT AETTSGGLTG LTTSLSTL4A PALSRTQSDG SGSKSSLGLN 151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP ANADTDVFflJ 201 IDVFGTIRNR TEMHLYNAET LKAQTKLEYF AVDRTNKKLL IKPKTNAPEA 251 AYKENYALWM GPYKVSKGIK PTEGLMVDFS DIRPYGNHTG NSAPSVEADN 301 SHEGYGYSDE VVRQI{RQGQP* The following partial DNA sequence was identified in N. gonorrhoeae <SEQ ID 1036>: g406 .seq 1 ATGCGGGCAC GGCTGCTGAT ACCTATTCTT TTTTCAGTTT TTATTTTATC 51 CGCCTGCGGG ACACTGACAG GTATTCCATC GCATGGCGGA GGCAAACGCT 101 TCGCGGTCGA ACAAGAACTT GTGGCCGCTT CTGCCAGAGC TGCCGTTAAA 151 GACATGGATT TACAGGCATT ACACGGACGA AAAGTTGCAT TGTACATTGC 201 AACTATGGGC GACCAAGGTT CAGGCAGTTT GACAGGGGGT CGCTACTCCA 251 TTGATGCACT GATTCGCGGC GAATACATAA ACAGCCCTGC CGTCCGCACC 301 GATTACACCT ATCCGCGTTA CGAAACCACC GCTGAAACAA CATCAGGCGG 351 TTTGACGGGT TTAACCACTT CTTTATCTAC ACTTAA TGCC CCTGCACTCT 401 CGCGCACCCA ATCAGACGGT AGCGGAAGTA GGAGCAGTCT GGGCTTAAAT 451 ATTGGCGGGA TGGGGGATTA TCGAAATGAA ACCTTGACGA CCAACCCGCG 501 CGACACTGCC TTTCTTTCCC ACTTGGTGCA GACCGTATTT TTCCTGCGCG 551 GCATAGACGT TGTTTCTCCT GCCAATGCCG ATACAGATGT GTTTATTAAC 601 ATCGACGTAT TCGGAACGAT ACGCAACAGA ACCGAAATGC ACCTATACAA 651 TGCCGAAACA CTGAAAGCCC AAACAAAACT GGAATATTTC GCAGTAGACA 701 GAACCAATAA AAAATTGCTC ATCAAACCCA AAACCAATGC GTTTGAAGCT 751 GCCTATAAAG AAAATTACGC ATTGTGGATG GGGCCGTATA AAGTAAGCAA 801 AGGAATCAAA CCGACGGAAG GATTGATGGT CGATTTCTCC GATATCCAAC 851 CATACGGCAA TCATACGGGT AACTCCGCCC CATCCGTAGA GGCTGATAAC 901 ACTCATGAGG GGTATGGATA CAGCGATGAA GCAGTGCGAC AACATAGACA 951 AGGGCAACCT TGA This corresponds to the amino acid sequence <SEQ ID 1037; ORE 406.ng>: g406.pep 1 MRARLLIPIL FSVFILSACC TLTGIPSHGG GKRFAVEQEL VAASARAAVK 51 DMDLQALHGR KVALYIATMG DQGSGSLTGG RYSIDALIRG EYINSPAVRT 101 DYTYPRYETT AETTSGGLTG LTTSLSTLNA PALSRTOSDG SGSRSSLGLN 151 IGGMGDYRNE TL.TTNPRflTA FLSHLVQTVF FLRGIDVVSP ANADTDVFIN 201 IDVFGTIRNR TEMHLYNAET LKAQTKLEYF AVDRTNKKLL IKPKTNAFEA 251 AYKENYALWM GPYKVSKGIK PTEGLMVDFS DIQPYGNIITG NSAPSVEADN 301 SHEGYGYSDE AVRQHRQGQP ORE 406.ng shows 98.8% identity over a 320 aa overlap with a predicted ORE (0RE406.a) from N. gonorrhoeae.

g406/m406 20 30 40 50 g406 .pep MRARLLIPILFSVFILSACGTLTGIPSHGGGKRFAVEQELVAASARAAVKDMDLQALHGR m4 06 MQARLLI PILFSVFILSACGTLTGIPSGGGKRFAVEQELVAASARAAVKDMDLQALHGR 20 30 40 50 70 80 90 100 110 120 9406 .pep KVALYIATMGDOGSGSLTGGRYSIDALIRGEYINSPAVRTDYTYPRYETTA ETTSGGLTG WO 00/22430 PCT/US99/23573 109m4 06 KVALYIATMGDQGSGSLTGGRYSIDALIRGEYINSPAVRTDYTYPRYETTAETTSGGLTG s0 90 100. 110 120 130 140 150 160 170 180 4 06 .pep LTTSLSTLNAPAILSRTQSDGSGSRSSLGLNIGGMGDYRNETLTTNPRDTAFLSHLVQTVF m4 06 LTTSLSTLNAPALSRTQSDGSGSKSSLGLNIGGMGDYRNETLTTNPRDTAFLSHLVQTVF 130 140 150 160 170 180 [0 190 200 210 220 230 240 g4 06.pep FLRGIDVVSPANADTDVFINIDVFGTIRNRTEMHLYNAETLKAQTKLEYFAVDRTNKKLL n4 06 FLRGI DVVSPANADTDVFINI DVFGTI RNRTEMHLYNAETL.KAQTKLEYFAVDRTNKKLL 190 200 210 220 230 240 250 260 270 280 290 300 g406.pep I KPKTNAFEAAYKENYALWMGPYKVSKGIKPTEGLMVDFSDIQPYGNHTGNSAPSVEADN M4 06 I KPKTNAFEAAYKENYAIA4NGPYKVS KGI KPTEGLMVDFSDIRPYGNHTGNSAPSVEADN zo250 260 270 280 290 300 310 320 g406.pep SHEGYGYSDEAVRQHRQGQPX M406 SHEGYGYSDEVVRQHRQGOPX 310 320 The following partial DNA sequence was identified in N. meningitidis <SEQ [D 1038>: a406. seq 1 ATGCAAGCAC GGCTGCTGAT ACCTATTCTT TTTTCAGTTT TTATTTTATC 51 CGCCTGCGGG ACACTGACAG 01 TCGCGGTCGA ACAAGAACTT 51 GACATGGATT TACAGGCATT '01 AACTATGGGC GACCAAGGTT 51 TTGATGCACT GATTCGTGGC 01 GATTACACCT ATCCACGTTA 151 TTTGACAGGT TTAACCACTT 01 CGCGCACCCA ATCAGACGGT 151 ATTGGCGGGA TGGGGGATTA M1 CGACACTGCC TTTCTTTCCC 651 GCATAGACGT TGTTTCTCCT 501 ATCGACGTAT TCGGAACGAT 351 TGCCGAAACA CTGAAAGCCC 01 GAACCAATAA AAAATTGCTC 51 GCCTATAAAG AAAATTACGC 01 AGGAATTAAA CCGACAGAAG 351 CATACGGCAA TCATATGGGT )01 AGTCATGAGG GGTATGGATA )51 AGGGCAACCT TGA

GTATTCCATC

GTGGCCGCTT

ACACGGACGA

CAGGCAGTTT

GAATACATAA

CGAAACCACC

CTTTATCTAC

AGCGGAAGTA

TCGAAATGAA

ACTTGGTACA

GCCAATGCCG

ACGCAACAGA

AAACAAAACT

ATCAAACCAA

ATTGTGGATG

GATTAATGGT

AACTCTG CCC GCATGGCGGA GGTAAACGCT CTGCCAGAGC TGCCGTTAAP.

AAAGTTGCAT TGTACATTGC GACAGGGGGT CGCTACTCCA ACAGCCCTGC CGTCCGTACC GCTGAAACAA CATCAGGCGG ACTTAATGCC CCTGCACTCT AAAGCAGTCT GGGCTTAAAT ACCTTGACGA CTAACCCGCG GACCGTATTT TTCCTGCGCG ATACGGATGT GTTTATTAAC ACCGAAATGC ACCTATACAA GGAATATTTC GCAGTAGACA AAACCAATGC GTTTGAAGCT GGACCGTATA AAGTAAGCAA CGATTTCTCC GATATCCAAC CATCCGTAGA GGCTGATAAC CAGCGATGAA GCAGTGCGAC GACATAGACA This corresponds to the amino acid sequence <SEQ ID 1039; ORE 406.a>: a406 .pep 1 MQARLLIPIL FSVFILSACG TLTGIPSHGG GKRFAVEQEL VAASARAP VK DMDLQALHGR KVALYIATMG Di DYTYPRYETT AETTSGGLTG L IGGMGDYRNE TLTTNPRDTA F IDVFGTIRNR TEMHLYNAET L: AYKENYALWM GPYKVSKGIK P SHEGYGYSDE AVRRHRQGQP

QGSGSLTGG

TTSLSTLNA

LSHLVQTVF

KAQI'KLEYF

TEGLMVDFS

RYSIDALIRG

PALSRTQSDG

FLRGIDVVSP

AVDRTNKI(LL

DIQPYGNHMG

EYINSPAVRT

SGSKSSLGLN

ANADTDVFIN

IKPKTNAFEA

N SAP SVEADN WO 00/22430 PCT/US99/23573 -110m406/a406 ORFs 406 and 406.a showed a 98.8% identity in 320 aa overlap 20 30 40 50 mn406 .pep MQARLLIPILFSVFILSACGTLTGIPSHGGGKRFAVEQELVAASARAAVKDMDLQALHGR 1 111 1 1 11 1 1 1 I 1 1 111 1 1 1 1 11 1 I 1 11 11 1 111 1 11 11 11 1 11i a406 MQARLLIPILFSVFILSACGTLTGIPSHGGGKRFAVEQELVAASARAAVKDMDLQALHGR 20 30 40 50 80 90 100 110 120 mn406.pep 1VALYIATMGDQGSGSLTGGRYSIDALIPGEYINSPAVRTDYTYPRYETTAETTSGGLTG a406 KVALYIATMGDQGSGSLTGGRYSIDALIRGEYINSPAVRTDYTYPRYETTAETTSGGLTG 80 90 100 110 120 130 140. 150 160 170 180 m406 .pep LTTSLSTLNAPALSRTQSDGSGSKSSLGLNIGGMGDYRNETLTTNP.DTAFLSHLVQTVF a406 LTTSLSTLNAPALSRTQSDGSGSKSSLGLNIGGMGDYRNETLTTNPRDTAFLSHLVQTVF 130 140 150 160 170 180 190 200 210 220 230 240 m406 .pep FLRGIDVVSFANADTDVFINIDVFGTIRNRTEMHLYNAETLKAQTKLEYFAVDRTNKKLL a406 FLRGIDVVSPA1NADTDVFINIDVEGTIRNRTEMHLYNAETLKAQTKLEYFAVDRTNK(LL 190 200 210 220 230 240 250 260 270 280 290 300 m4 06. pep IKPKTNAFEAAYKENYALWMGPYKVSKGIKPTEGLMVDFS DIRPYGNHTGNSAPSVEADN a4 06 IKPKTNAFEAAYKENYALWMGPYKVSKGIKPTEGLMVDF'SDIQPYGNHMGNSAPSVEADN 250 260 270 280 290 300 310 320 m4 06.pep SHEGYGYSDEVVROHRQGQPX II i :IIII a4 06 SHEGYGYSDEAVRRHRQGQPX 310 320 The following partial DNA sequence was identified in N. meningiuidis <SEQ ID 1040>: m726. aeq 1 ATGACCATCT ATTTCAAAAA CGGCTTTTAC GACGACACAT TGGGCGCCAT 51 CCCCGAAGGC GCGGTTGCCG TCCGCGCCGA AGAATACGCC GCCCTTTTGG 101 CAGGACAGGC GCAGGGCGGG CAGATTGCCG CAGATTCCGA CGGCCGCCCC 151 GTTTTAACCC CGCCGCGCCC GTCCGATTAC CACGAATGGG ACGGCAAAAA 201 ATGGAAAATC AGCAAAGCCG CCGCCGCCGC CCGTTTCGCC AAACAAAAAA 251 CCGCCTTGGC ATTCCGCCTC GCGGAAAAGG CGGACGAACT CAAAAACAGC 301 CTCTTGGCGG GCTATCCCCA AGTGGAAATC GACAGCTTTT ACAGGCAGGA 351 AAAAGAAGCC CTCGCGCGGC AGGCGGACAA CAACGCCCCG ACCCCGATGC 401 TGGCGCAAAT CGCCGCCGCA AGGGGCGTGG AATTGGACGT TTTGATTGAA 451 AAAGTTATCG AAAAATCCGC CCGCCTGGCT GTTGCCGCCG GCGCGATTAT 501 CGGAAAGCGT CAGCAGCTCG AAGACAAATT GAACACCATC GAAACCGCGC 551 CCGGATTGGA CGCGCTGGAA AAGGAAATCG AAGAATGGAC GCTAAACATC 601 GGCTGA This corresponds to the amino acid sequence <SEQ ID 1041; OR. 726>: m726.pop 1 MTIYFKNGFY DDTLGGIPEG AVAVRAEEYA ALLAGQAQGG QIAADSDGRP 51 VLTPPRPSDY HEWDGKKWKI SKAAAAARFA KQKTALAFRL AEKADELKNS WO 00/22430 PCTIUS99/23573 101 LLAGYPQVEI DSFYRQEKEA LARQADNNAP TPMLAQIAAA RGVELDVLIE 151 KVIEKSARLA VAAGAIIGKR QQLEOKLNTI ETAPGLDALE KEIEEWTLNI 201 G* The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1042>: m907-2 .seq 1 ATGAGAAAAC CGACCGATAC CCTACCCGTT AATCTGCAAC GCCGCCGCCT 51 GTTGTGTGCC GCCGGTGCGT TGTTGCTCAG TCCTCTGGCG CACGCCGGCG 101 CGCAACGTGA GGAAACGCTT GCCGACGATG TGGCTTCCGT GATGAGGAGT 151 TCTGTCGGCA GCGTCAATCC GCCGAGGCTG GTGTTTGACA ATCCGAAAGA 201 GGGCGAGCGT TGGTTGTCTG CCATGTCGGC ACGTTTGGCA AGGTTCGTCC 251 CCGAGGAGGA GGAGCGGCGC AGGCTGCTGG TCAATATCCA GTACGAAAGC 301 AGCCGGGCCG GTTTGGATAC GCAGATTGTG TTGGGGCTGA TTGAGGTGGA 351 AAGCGCGTTC CGCCAGTATG CAATCAGCGG TGTCGGCGCG CGCGGCCTGA 401 TGCAGGTTAT GCCGTTTTGG AAAAACTACA TCGGCAAACC GGCGCACAAC 451 CTGTTCGACA TCCGCACCAA CCTGCGTTAC GGCTGTACCA 1CCTGCGCCA 501 TTACCGGAAT CTTGAAAAAG GCAACATCGT CCGCGCGCTT GCCCGCTTTA 551 ACGGCAGCTT GGGCAGCAAT AAATATCCGA ACGCCGTTTT GGGCGCGTGG 601 CGCAACCGCT GGCAGTGGCG TTGA This corresponds to the amino acid sequence <SEQ ID 1043; ORF 907-2>: m907-2 .pep 1 MRKPTDTLPV tNLQRRRLLCA AGALLLSPLA HAGAQREETL ADDVASVMRS 51 SVGSVNPPRL VFDNPKEGER WLSAM4SARLA RFVPEEEER. RLLVNIQYES 101 SRAGLDTQIV LGLIEVESAF RQYAISGVGA RGLMQVNPFW KNYIGKPAHN 151 LFDIRTNLRY GCTILRHYRN LEKGNIVRAL ARFNGSLGSN KYPNAVLGAW 201 RNRWOWR* The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1044>: m953. seq 1 ATGAAAAAAA TCATCTTCGC CGCACTCGCA GCCGCCGCCA TCAGTACTGC 51 CTCCGCCGCC ACCTACAAAG TGGACGAATA TCACGCCAAC GCCCGTTTCG 101 CCATCGACCA TTTCAACACC AGCACCAACG TCGGCGGTTT rTACGGTCTG 151 ACCGGTTCCG TCGAGTTCGA CCAAGCAAAA CGCGACGGTA AAATCGACAT 201 CACCATCCCC ATTGCCAACC TGCAAAGCGG TTCGCAACAC TTTACCGACC 251 ACCTGAAATC AGCCGACATC TTCGATGCCG CCCAATATCC GGACATCCGC 301 TTTGTTTCCA CCAAATTCAA CTTCAACGGC AAAAAACTGG TTTCCGTTGA 351 CGGCAACCTG ACCATGCACG GCAAAACCGC CCCCGTCAAA CTCAAAGCCG 401 AAAAATTCAA CTGCTACCAA AGCCCGATGG AGAAAACCGA AGTTTGTGGC 451 GGCGACTTCA GCACCACCAT CGACCGCACC AAATGGGGCA TGGACTACCT 501 CGTTAACGTT GGTATGACCA AAAGCGTCCG CATCGACATC CAAATCGAGG 551 CAGCCAAACA ATAP.

This corresponds to the amino acid sequence <SEQ ID 1045; ORF 953>: m953 .pep 1 MKKIIFAALA AAAISTASAA TYKVDEYHAN ARFAIDHFNT STNVGGFYGL 51 TGSVEFDQAK RDGKIDITIP IANLQSGSQH FTDHLKSADI FDAAQYPDIR 101 FVSTKFNFNG KKLVSVDGNL TMHGKTAPV< LKAEKFNCYQ SPMEKTEVCG 151 GDFSTTIDRT KWGMDYLVNV GMTKSVRIDI QIEAAKQ* The following partial DNA sequence was identified in N. meningizidis <SEQ ID 1046>: orfl-l seq WO 00/22430 PCT/US99/23573 -112- ATGAAAACAA CCGACAAACG AACCGGCCGC ATCCGCTTCT TCGGCATTCT TCCCCAAGCC TACCAATACT ATCGCGACTT GGCGAAAGAT ATTGAGGTTT CAATGACAAA AGCCCCGATG 301 351 401' 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651

GTGGCGGCAT

CGGCTATAAC

ATCGTTTTAC

AAAGGCCATC

TGTCACAGAT

AATATATCGA

AGGCAATATT

ATATCATATT

CACAAAATGG

AAACATAGCC

TGGCTCACCA

ATGGGGTATT

CAGCTGGTTC

CCATTCAGTA

ACGATAATAA

CTGCCTAATA

ATCCGAGACA

GTTATCGACC

GGAAAAGGCG

ATTATATTTC

GGCAAGGCGC

GTAAACGGCG

GCACGTTCAA

GTACAGTCAT

TTTAGTGAAA

CGATAATCAG

GTTTGGATTT

GATGAAGGGG

TGGTGGGCGA

AACGTTGATT

TTATAAAATT

CTTATGGCGG

GCAGAACCTG

TCAAAATAAT

GGCGATCTGA

GCAAGTGCGT

ATCAGGTGGT

CATATGGTTT

ATGTTTATCT

GCAA.ACGGGC

GTAAAGATTG

TTCTACGAAC

TGGCACAGGA

GATTAAAAAC

GCAAGAGAAC

CAGACTGAAT

AATTGATACT

CAAGGAGATT

GGGCGTTCAT

TGGCAAACGA

GCCAAAGGGG

TTTGGATCAG

TCGGCTTGGT

TTCAACCCCG

AAACGGGCAT

CGATGATTGT

GACAACCGAA

CGCCTGCTTA

TGGGCGGGAC

TGCCGAAAAT

ACAACAAAAA

ATTGATTTTT

TCAATATATT

TTGGTGCGGA

GTGAAACGGA

CGATTAT CAT

TTGAAATGAC

TACCCTGACC

TGAAGAT GAG

ATTCTTGGCT

GGCACAGTCA

TTTACCAACA

ATGATGCCCA

AACCCCTATA

GTTCTATGAT

CACGTCAAAA

AAAATCAATG

ACGAACCGTT

CTGTTTATCA

AATGGAGAAA

TACCAGCAAC

TTACGGTCTC

ATCAGTGAAG

CCGCCTGTCC

AAAACCAAGG

CAGGCAGACG

CAGCGGCAGG

ACAAACTCTA

TCGCTTTCGT

CAACCACAAT

ATATTGCTAC

GCCTACAACG

GCTCAACCTT

ACACACCGCA

CTTAGCCATA

ACACTTATTT

AAAGGCAAGT

AGGGGAGTTG

CTGTGGTGTC

GTGAGCGTGG

AGGAAGAAAT

ATAATTATAA

ATGCCGCGTT

CAGTTATATG

GTGTTCGTAT

CCCAATAACC

CGTTGGTGGC

ACTTAGGTAG

GGAGGCTCAT

AAAGCAAAAG

TAGGAAAAAG

GAAATCTTTG

TGGGAAATAC

CCAAACATGA

CAATTGTTTA

TGCTGCAGGT

ATATTTCCTT

ATCAATCAAG

GCCTGAAAAT

ACAGTACCGT

AAAATCGGCA

CTCGATCAGC

ATAAAGGCAA

GGTACGGTGC

TTTCGGCTTT

TCCACCGTAT

CAAGACAAAG

AACCGGCAAT

GTTGGTTTGG

GTTTACCAGC

AAGCCCCGAA

TGCCTGTCGT

CGGCATCAAC

TTGCAGTCGG

GTCGGCAAAT

GCGTAACGGC

CACATAACGG

CCCGATCAAC

AGCAGGGACT

TGCATAAATT

GATGGGCGGA

TGGGGCAGGC

GCGAAAGTTC

AATACCTTTG

TGAAAAAATT

TTGGCGACAG

TGGTTAATTA

CAATGGCTTC

CTGGAGATAC

TCTTTTAACG

ACACAATTCT

ATGTTTCTTT

GGTGTCAACA

TATTGACGAA

GTGCTGGAGG

AACGAAACTT

TACTTGGAAA

AAGGCACGCT

GTGGGCGACG

AAAACAAGCC

AACTGAATGC

CGCGGCGGAC

TCAAAATACC

AATCCACCGT

AACAACAGCT

CGAGAAAGAT

CCGCCGCAGA

1701 TACCATTACA GGCAATAAAG 1751 TGGATAGCAA AAAAGAAATT 1801 ACGACCAAAA CGAACGGGCG 1851 AGACCGCACC CTGCTGCTTT CCGGCGGAAC AAATTTAAAC GGCAACATCA 1901 CGCAAACAAA CGGCAAACTG TTTTTCAGCG GCAGACCAAC 1951 TACAATCATT TAAACGACCA TTGGTCGCAA AAAGAGGGCA 2001 GGAAATCGTG TGGGACAACG ACTGGATCAA CCGCACATTT 2051 ACTTCCAAAT TAAAGGCGGA CAGGCGGTGG TTTCCCGCAA

ACCGCACGCC

TTCCTCGCGG

AAAGCGGAAA

TGTTGCCAAA

2101 GTGAAAGGCG ATTGGCATTT GAGCAATCAC GCCCAAGCAG TTTTTGGTGT CAATCTGTAC ACGTTCGGAC TGGACGGGTC 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951

CGCACCGCAT

TGACAAATTG

TTGACTAAGA

TTTAAATCTC

GCGATACACG

AGCCTCGTGG

CAACACATCG

TACAAAACGG

CATTCCGCAC

TTTTGAAAGC

CATTACACTT

GGCAATTTAA

CCACGATGCG

GCCGTTCGCG

TCGGTAGAAT

TCAGGGAACA

AATTGAAGCT

CAAAGCCACA

TGTCGAAAAA

CCGACATCAG

ACAGGGCTTG

TTATACAGTC

GCAATGCCCA

GCTTCGGGCA

CAGTCTGACG

TCAACGGTAA

AGCCGCTTTA

AAAAGACAGC

ACCTTGACAA

GCAGGGGCGC

CCGTTCGCGC

CCCGTTTCAA

TTCCGCTTTA

GGCGGAAAGT

ACCATTACCG

CGGCAATGTC

CCACACTCAA

AGCCACAACG

AGCAACATTT

ATGCTTCATT

CTTTCCGGCA

TGTCTCCCTA

CCGGACAAAT

GAATGGACGC

CGCCACCATT

AAACCGGCAG

CGTTCCCTAT

CACGCTGACG

TGTCGGAACT

TCCGAAGGCA

AAGCCTCGAA

CCGAAAACCT

TGGCGTTACC

ACGATAAAGT

GATCTTGCCG

CGGCAATCTT

CCACCCAAAA

AATCAAGCCA

TAATCTAAGC

ACGCTAAGGC

GCCGATAAGG

CAGCGGCGGC

TGCCGTCAGG

ACACTCAATT

TGCGACAGAT

TAT CCGTTAC

GTAALACGGCA

CTTCGGCTAC

CTTACACCTT

CAATTGACGG

TAATTTCACC

AACTCATCCG

GATTGCTTCA

ATCACGCTCA

AGTGCAAATG

CGGCAACCTT

CAT TAAACGG

GACCACGCCG

AAACGTAAGC

CAGTATTCCA

AAGGATACGG

CACGGAATTA

CCGCCTATCG

GCGCCGCGCC

ACCGCCAACT

AATTGAACGG

CGCAGCGACA

'GGCGGTCAAC

TAGTGGAAGG

CTGCAAAACG

CAAAGACGGC

3001 AATACCGGCA ACGAACCTGC 3051 AAAAGACAAC AAACCGCTGT 3101 AACACGTCGA TGCCGGCGCG WO 00/22430 PCT/US99/23573 113 3151 GAGTTCCGCC TGCATAATCC GGTCAAAGAA CAAGAGCTTT CCGACAAACT 3201 CGGCAAGGCA GAAGCCAAAA AACAGGCGGA AAAAGACAAC GCGCAAAGCC 3251 TTGACGCGCT GAT'rGCGGCC' GGGCGCGATG CCGTCGAAAA GACAGAAAGC 3301 GTTGCCGAAC CGGCCCGGCA GGCAGGCGGG GAAAATGTCG GCATTATGCA 3351 GGCGGAGGAA GAGAAAAAAC GGGTGCAGGC GGATAAAGAC ACCGCCTTGG 3401 CGAAACAGCG CGAAGCGGAA ACCCGGCCGG CTACCACCGC CTTCCCCCGC 3451 GCCCGCCGCG CCCGCCGGGA TTTGCCGCAA CTGCPAACCCC AACCGCAGCC 3501 CCAACCGCAG CGCGACCTGA TCAGCCGTTA TGCCAATAGC GGTTTGAGTG 3551 AATTTTCCGC CACGCTCAAC AGCGTTTTCG CCGTACAGGA CGAATTAGAC 3601 CGCGTATTTG CCGAAGACCG CCGCAACGCC GTTTGGACAA GCGGCATCCG 3651 GGACACCAAA CACTACCGTT CGCAAGATTT CCGCGCCTAC CGCCAACAAA 3701 CCGACCTGCG CCAAATCGGT ATGCAGAAAA ACCrCGCcAG CGGGCGCGTC 3751 GGCATCCTGT TTTCGCACAA CCGGACCGAA AACACCTTCG ACGACGGCAT 3801 CGGCAACTCG GCACGGCTTG CCCACGGCGC CGTTTTCGGG CAATACGGCA 3851 TCGACAGGTT CTACATCGGC ATCAGCGCGG GCGCGGGTTT TAGCAGCGGC 3901 AGCCTTTCAG ACGGCATCGG AGGCAAAATC CGCCGCCGCG TGCTGCATTA 3951 CGGCATTCAG GCACGATACC GCGCCGGTTT CGGCGGATTC GGCATCGAAC 4001 CGCACATCGG CGCAACGCGC TATTTCGTCC AAAAAGCGGA TTACCGCTAC 4051 GAAAACGTCA ATATCGCCAC CCCCGGCCTT GCATTCAACC GCTACGGCGC 4101 GGGCATTAAG GCAGATTATT CATTCAAAGC GGCGCAACAC ATTTCCATCA 4151 CGCCTTATTT GAGCCTGTCC TATACCGATG CCGCTTCGGG CAAAGTCCGA 4201 ACACGCGTCA ATACCGCCGT ATTGGCTCAG GATTTCGGCA AAACCCGCAG 4251 TGCGGAATGG GGCGTAAACG CCGAAATCAA AGGTTTCACG CTGTCCCTCC 4301 ACGCTGCCGC CGCCAAAGGC CCGCAACTGG AAGCGCAACA CAGCGCGGGC 4351 ATCAAATTAG GCTACCGCTG GTAA This corresponds to the amino acid sequence <SEQ ID 1047; ORE orfl-1>: orfl-1.pop 1 MKTTDKRTTE TH-RKAPKTGR IRFSPAYLAI CLSFGILPQA WAGHTYFGIN 51 YQYYRDFAEN KGKFAVGAKD IEVYNKKGEL VGKSMTKAPM IDFSVVSRNG 101 VAALVGDQYI VSVAHNGGYN NVDFGAEGRN PDQHRFTYKI VKRNNYKAGT 151 KGHPYGGDYH MPRLHKFVTD AEPVEMTSYM DGRKYIDQNN YPDRVRIGAG 201 RQYWRSDEDE PNNRESSYHI ASAYSWLVGG NTFAQNGSGG GTVNLGSEKI 251 KH-SPYGFLPT GGSFGDSGSP MFIYDAQKQK WLINGVLQTG NPYIGKSNGF 301 QLVRKDWFYD EIFAGDTHSV FYEPRQNGKY SFNDONNGTG KINAKHEHNS 351 LPNRLKTRTV QLFI4VSLSET AREPVYHAAG GVNSYRPRLN NGENISFIDE 401 GKGELILTSN INQGAGGLYF QGDFTVSPEN NETWGGAGVH ISEDSTVTWK 451 VNGVANDRLS KIGKGTLHVQ AKGENQGSIS VGDGTVILDQ QADDKGKKQA 501 FSEIGLVSGR GTVQLNADNQ FNPDKLYFGF RGGRLDLNGH SLSFHRIQNT 551 DEGANIVNHN QDKESTVTIT GNKDIATTGN NNSLDSKKEI AYNGWFGEKD 601 TTKTNGRLNL VYQPAAEDRT LLLSGGTNLN GNITQTNGKL FFSGRPTPHA 651 YNHLNDHWSQ KEGIPRGEIV WDNDWINRTF KAENFQIKGG QAVVSRNVAK 701 VKGDWHLSNH AQAVFGVAPH QSHTICTRSD WTGLTNCVE( TITDDKVIAS 751 LTKTDISGNV DLADHAHLNL TGLATLNGNL SANGDTRYTV SHNATQNGNL 801 SLVGNAQATF NQATLNGNTS ASGNASFNLS DHAVQNGSLT LSGNAKA4VS 851 HSALNGNVSL ADKAVFHFES SRFTGQISGG KDTALHLKDS EWTLPSGTEL 901 GNLNLDNATI TLNSAYRHDA AGAQTGSATD APRRRSRRSR RSLLSVTPPT 951 SVESRFNTLT VNGKLNGQGT FRFMSELFGY RSDKLKLAES SEGTYTLAVN 1001 NTGNEPASLE QLTVVEGKDN KPLSENLNFT LONEEVDAGA WRYQLIRXDG 1051 EFRLHNPVKE QELSDKLGKA EAKKQAEKDN AQSLIDALIAA GRDAVEKTES 1101 VAEPAIRQAGG ENVGIMQAEE EKKRVQADKD TALAKQREAE TRPATTAFPR 1151 ARRARRDLPQ LQPQPQPQPQ RDLISRYANS GLSEFSATLN SVFAVQDELD 1201 RVFAEDRRNA VWTSGIRDT( HYRSQDFRAY RQQTDLRQIG MQKNL.GSGRV 1251 GILFSHNRTE NTFDDGIGNS ARLAHGAVFG QYGIDRFYIG ISAGAGFSSG 1301 SLSDGIGGKI RRRVLHYGIQ ARYRAGFGGF GIEPHIGATR YFVQKADYRY 1351 ENVNIATPGL AFNRYRAGIK ADYSFKPAQH ISITPYLSLS YTDAASGKVR 1401 TRVNTAVLAQ DFGKTRSAEW GVNAEIKGFT LSLHAAAAKG PQLEAQHSAG 1451 IKLGYRW* WO 00/22430 PCTIUS99/23573 -114- The following partial DNA sequence was identified in N. men ingitidis <SEQ ID 1048>: orf46-2.seq 1 51 101 151 201 251 301 351 401.

451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801

TTGGGCATTT

CCTGCCGATG

GGCAGGTTCT

TTCGGCAGCA

AAAAATACAA

TTAAAGGAAA

GTCCATTCCC

CGGTAGT CCC

ACGAACACCA

CCCGCTCCCA

TGCCCAAAAT

GGCTTGCCGA

GGCGACGGAT

GGGCAATGCC

TCATCGGCGC

ATAAGCGAAG

CACCGAAAAC

TCAAAGACTA

AATGCCGCAC

CCCCATCAAA

TCACGGCACA

AAAGGGAAAT

ATACCCGTCC

CCCGCAAAAT

CATGCACACG

CGACCGTCAG

GGGGGGAACT

AGCCATCAGT

TATCGGCTAC

CCTTCGACAA

GTTGACGGAT

TCCCGCCGAC

AAGGCGCGAG

ATCCGCCTCA

CCGTTTCCAC

TCAAACGCGC

GCCGAAGCCT

GGCAGGAGAA

GCTCAAACAT

AAGATGGCGC

ATCCCTTATT

CCTCAGATTT

CATTTCGAAC

TGCCGAGCGC

TGGGCAACCT

ATTGT CCCCT CCAT G CCT CA

TTAGCCTTTA

GGCTATGACG

GGATATATAC

CTGTCCATAC TGGCAGTGTC;

GGCAAACGAT.TCTTTTATCC

CCGACGGGAA ATACCACCTA AGCGGCCATA TCGGATTGGG GATGATTCAA CAGGCGGCCA TTTCCGATCA CGGGCACGAA CATTCCGATT CTGATGAAGC CCGCATCCAT TGGGACGGAT GGCCACAGGG CGGCGGCTAT AGCTACGACA TAAAAGGCGT ACCTGACCGA CAACCGCAGC ACCGGACAAC AATGCCGGTA GTATGCTGAC GCAAGGAGTA

CACCCGATAC

TCAACGGCAC

ATTGTCGGCG

TGCTGTCATG

GCATCAACGA

AGCCCCGAGC TGGACAGATC TGCAGATATC GTTAAAAACA CAGGCGATGC CGTGCAGGGC CACGGCTTGG GTCTGCTTTC TTTGGCAGAT ATGGCGCAAC ATTGGGCAGT CCAAAACCCC AATATCTTTA TGGCAGCCAT AAAATACGGC TTGGGCGGCA TGCCGCAGCA GCCATCCGCG AAGGCATAGA AGCCGTCAGC GGGATTGGAG CTGTTCGGGG TCCTATCAAG CGGTCGCAGA TGGGCGCGAT CGCATTGCCG CCGCCGTCAG CGACAATTTT GCCGATGCGG CATACGCCAA CCTTACCATT CCCGAAATAT GTTACGGCAA AGAAAACATC ACCTCCTCAA AAAAATGTCA AACTGGCAGA CCAACGCCAC TGACGGTAAA GGGTTTCCGA ATTTTGAGAA AGCTCGATAT TCAAGAATTA TCGGGGGGCG GTGTTTGATG CGAAACCGAG ATGGGAGGTT GACAACTCGT GAGCAGGTGG ATATAAACAG TAACTTTAGC AAACTAAAAT CTGCCGATGA TACCGATAGC ATGAATGACA AAGAGAATGG CTTCACAAAT AAAGCATATA TCGTAAGAGG TGGCAGGATA CATGAATTAA ATACTAGTTG GAAAAATCCT AAGAGACCTC GTTATAGGAG

AGAAAAATGT

CAACATGCTC

AATTAATTTT

AGGCTTTTAG

CCAGTTGTGG

AAATAATRGG

AATTTAAAAA

ACTGATGTCT

TAAATAA

CCGTTCAAAC

CCGTGCCGCC

CCGAAGACAG

GCACGTGAAA

GTATACCTAA

GATAGGAAGC

TCAGGAAATA

AACTAGAGAG

GCAGATGGAA

TAGGCTTGTG

AGTACGTTGA

GTTTTTGCTG

AGTTGACTTT

TGAATGAATC

TTGGAGCAGC

GTCAAACGGC

GCGTACCGTT

TATGATACGA

GGCTAAGCCT

TTAATAAATT

AGGAACGGTA

GGAAATTAAT

TGGGAAAATT

AAATCAGTTA

AATAAATGGA

CAGAATACCT

CCTGTTCCTA

AGGTAATGTT

This corresponds to the amino acid sequence <SEQ ID 1049; ORF orf46-2>: orf46-2 .pep 1 LGISRKISLI LSILAVCLPM HAHASDLAND 51 FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ 101 VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH 151 PAPKGARDIY SYDIKGVAON IRLNLTDNRS 201 GDGFKRATRY SPELDRSGNA AEAFNGTADI 251 ISEGSNIAVN HGLGLLSTEN KMARINDLAD 301 NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG 351 KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN 401 KNVKLADQRH PKTGVPFDGK GFPNFEKHVK 451 VFDAKPRWEV DRKLNKLTTR EQVEKNVQEI 501 KLKSADEINF ADGMGKFTDS tNDKAFSRLV 551 KAYIVRGNNR VFAAEYLGRI HELKFKKVDF 601 KRPRYRSK* SFIRQVLDRQ HFEPDGKYHL QAAIKGNIGY IVRFSDHGHE WDGYEHHPAD GYDGPQGGGY TGQRLADRFH NAGSMLTQGV VKNIIGAAGE IVGAGDAVQG MAQLKDYAAA AIRDWAVQNP LGGITAHPIK LEQRYGKENI TSSTVPPSNG YDTKLDIQEL SGGGIPRAKP RNGNINSNFS QHAQLEREIN KSVKENGFTN PVVEYVEING PVPNTSWKNP TDVLNESGNV WO 00/22430 WO 002243 PCTIUS99/23573 115 Using the above-described procedures, the following oligonucleotide primers were employed in the polymerase chain reaction (PCR) assay in order to clone the ORFs as indicated: Oligonucleotides used for PCR Table I ORF Primer Sequence Restriction sites 279 Forward CGCGGATCCCATATG-TTGCCTGCAATCACGATT BamHI-NdeI <SEQ ID 1050> Reverse CCCGCTCGAG-TFVAGAAGCGGGCGGCAA <SEQ Xhol ID 1051 519 Forward CGCGGATCCCATATG-TTCAAATCCTTTGTCGTCA BamHI-Ndel <SEQ ID 1052> Reverse CCCGCTCGAG-1TrGGCGGT1TGCTGC <SEQ ID XhoI 1053> 576 Forward CGCGGATCCCATATG-GCCGCCCCCGCATCT BamH I-NdeI <SEQ ID 1054> Reverse CCCGCTCGAG-ATTrAC1TFITTGATGTCGAC XhoI <SEQ ID 1055> 919 Forward CGCGGATCCCATATG-TGCCAAAGCAAGAGCATC BamHI-NdeI <SEQ ID 1056> Reverse CCCGCTCGAG-CGGGCGGTA1TCGGG <SEQ ID XhoI 1057> 121 Forward CGCGGATCCCATATG-GMAACACAGCTTTACAT BamHI-NdeI <SEQ ID 1058> Reverse CCCGCTCGAG-ATAATMATATCCCGCGCCC <SEQ XhoI ID 1059> 128 Forward CGCGGATCCCATATG-ACTGACAACGCACT <SEQ BamHI-NdeI ID 1060> Reverse CCCGCTCGAG-GAOCGCGTTGTCGAAA <SEQ ID XhoI 1061 206 Forward CGCGGATCCCATATG-AAACACCGCCAACCGA BamH I-NdeI <SEQ ID 1062> Reverse CCCGCTCGAG-TTCTGTAAAAAAAGTATGTGC XhoI <SEQ ID 1063> 287 Forward CCGGAATTCTAGCTAGC-CTTTCAGCCTGCGGG EcoRI-NheI <SEQ ID 1064> Reverse CCCGCTCGAG-ATCCTGOTCUTTTTTGCC <SEQ ID Xhol 1065> 406 Forward CGCGGATCCCATATG-TGCGGGACACTGACAG BamHI-NdeI <SEQ ID 1066> WO 00/22430 PCT/US99/23573 -116- Reverse CCCGCTCGAG-AGGTTGTCCTTGTCTATG <SEQ Xhol ID 1067> EXAMPLE 2 Expression of ORF 919 The primer described in Table 1 for ORF 919 was used to locate and clone ORF 919.

The predicted gene 919 was cloned in pET vector and expressed in E. coli. The product of protein expression and purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of919-His fusion protein purification. Mice were immunized with the purified 919- His and sera were used for Western blot (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Symbols: Ml, molecular weight marker; PP, purified protein, TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 919 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 919 are provided in Figure 10. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, Scand JImmunol Suppl 11:9). The nucleic acid sequence of ORF 919 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 3 Expression of ORF 279 The primer described in Table 1 for ORF 279 was used to locate and clone ORF 279.

The predicted gene 279 was cloned in pGex vector and expressed in E. coli. The product of protein expression and purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 279-GST purification. Mice were immunized with the purified 279-GST and sera were used for Western blot analysis (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Symbols: Ml, molecular weight marker; TP, N.

meningitidis total protein extract; OMV, N. meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and WO 00/22430 PCT/US99/23573 -117the N. meningitidis immunoreactive band These experiments confirm that 279 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 279 are provided in Figure 11. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, ScandJ Immunol Suppl 11:9). The nucleic acid sequence of ORF 279 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 4 Expression of ORF 576 The primer described in Table 1 for ORF 576 was used to locate and clone ORF 576.

The predicted gene 576 was cloned in pGex vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 576- GST fusion protein purification. Mice were immunized with the purified 576-GST and sera were used for Western blot (panel FACS analysis (panel bactericidal assay (panel D), and ELISA assay (panel Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that ORF 576 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 576 are provided in Figure 12. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, Scand JImmunol Suppl 11:9).

The nucleic acid sequence of ORF 576 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE Expression of ORF 519 The primer described in Table 1 for ORF 519 was used to locate and clone ORF 519.

The predicted gene 519 was cloned in pET vector and expressed in E. coli. The product of WO 00/22430 PCT/US99/23573 118protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 519- His fusion protein purification. Mice were immunized with the purified 519-His and sera were used for Western blot (panel FACS analysis (panel bactericidal assay (panel D), and ELISA assay (panel Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 519 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 519 are provided in Figure 13. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, Scand Jmmunol Suppl 11:9). The nucleic acid sequence of ORF 519 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 6 Expression of ORF 121 The primer described in Table 1 for ORF 121 was used to locate and clone ORF 121.

The predicted gene 121 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 121- His fusion protein purification. Mice were immunized with the purified 121-His and sera were used for Western blot analysis (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Results show that 121 is a surface-exposed protein.

Symbols: MI, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N.

meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 121 is a surface-exposed protein and that it is a useful immunogen.

The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 121 are provided in Figure 14. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, Scand JImmunol Suppl 11:9). The nucleic acid sequence of ORF 121 and the amino acid sequence encoded thereby is provided in Example 1.

WO 00/22430 PCTIUS99/23573 -119- EXAMPLE7 Expression of ORF 128 The primer described in Table 1 for ORF 128 was used to locate and clone ORF 128.

The predicted gene 128 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 128- His purification. Mice were immunized with the purified 128-His and sera were used for Western blot analysis (panel FACS analysis (panel bactericidal assay (panel D) and ELISA assay (panel Results show that 128 is a surface-exposed protein. Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 128 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 128 are provided in Figure 15. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, ScandJlmmunol Suppl 11:9). The nucleic acid sequence of ORF 128 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 8 Expression of ORF 206 The primer described in Table 1 for ORF 206 was used to locate and clone ORF 206.

The predicted gene 206 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 206- His purification. Mice were immunized with the purified 206-His and sera were used for Western blot analysis (panel It is worthnoting that the immunoreactive band in protein extracts from meningococcus is 38 kDa instead of 17 kDa (panel To gain information on the nature of this antibody staining we expressed ORF 206 in E. coli without the His-tag and including the predicted leader peptide. Western blot analysis on total protein extracts from E.

coli expressing this native form of the 206 protein showed a recative band at a position of 38 kDa, as observed in meningococcus. We conclude that the 38 kDa band in panel B) is WO 00/22430 PCT/US99/23573 -120specific and that anti-206 antibodies, likely recognize a multimeric protein complex. In panel C is shown the FACS analysis, in panel D the bactericidal assay, and in panel E) the ELISA assay. Results show that 206 is a surface-exposed protein. Symbols: M molecular weight marker; TP, N. meningitidis total protein extract; OMV, N. meningitidis outer membrane vesicle preparation. Arrows indicate the position of the main recombinant protein product (A) and the N. meningitidis immunoreactive band These experiments confirm that 206 is a surface-exposed protein and that it is a useful immunogen. The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 519 are provided in Figure 16. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J. Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al. 1992, Scand J Immunol Suppl 11:9). The nucleic acid sequence of ORF 206 and the amino acid sequence encoded thereby is provided in Example 1.

EXAMPLE 9 Expression of ORF 287 The primer described in Table 1 for ORF 287 was used to locate and clone ORF 287.

The predicted gene 287 was cloned in pGex vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 287- GST fusion protein purification. Mice were immunized with the purified 287-GST and sera were used for FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Results show that 287 is a surface-exposed protein. Symbols: Ml, molecular weight marker. Arrow indicates the position of the main recombinant protein product These experiments confirm that 287 is a surface-exposed protein and that it is a useful immunogen.

The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 287 are provided in Figure 17. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, Scand JImmunol Suppl 11:9). The nucleic acid sequence of ORF 287 and the amino acid sequence encoded thereby is provided in Example 1.

WO 00/22430 PCT/US99/23573 121 EXAMPLE Expression of ORF 406 The primer described in Table 1 for ORF 406 was used to locate and clone ORF 406.

The predicted gene 406 was cloned in pET vector and expressed in E. coli. The product of protein purification was analyzed by SDS-PAGE. In panel A) is shown the analysis of 406- His fusion protein purification. Mice were immunized with the purified 406-His and sera were used for Western blot analysis (panel FACS analysis (panel bactericidal assay (panel and ELISA assay (panel Results show that 406 is a surface-exposed protein.

Symbols: Ml, molecular weight marker; TP, N. meningitidis total protein extract; OMV, N.

meningitidis outer membrane vescicle preparation. Arrows indicate the position of the main recombinant protein product and the N. meningitidis immunoreactive band These experiments confirm that 406 is a surface-exposed protein and that it is a useful immunogen.

The hydrophilicity plots, antigenic index, and amphipatic regions of ORF 406 are provided in Figure 18. The AMPHI program is used to predict putative T-cell epitopes (Gao et al 1989, J.

Immunol 143:3007; Roberts et al. 1996, AIDS Res Human Retroviruses 12:593; Quakyi et al.

1992, Scand JImmunol Suppl 11:9). The nucleic acid sequence of ORF 406 and the amino acid sequence encoded thereby is provided in Example 1.

The foregoing examples are intended to illustrate but not to limit the invention.

Claims (14)

1. An isolated or recombinant nucleic acid comprising N. meningitidis nucleotide sequence SEQ ID

2. An isolated or recombinant nucleic acid comprising a nucleotide sequence having greater than 70% sequence identity to nucleotide sequence SEQ ID

3. An isolated or recombinant nucleic acid comprising a fragment of 100 or more nucleotides from position 1 to position 19530 or position 25265 to position 50955 of nucleotide sequence SEQ ID

4. An isolated or recombinant nucleic acid complementary to the nucleic acid of any one of claims 1 to 3. An isolated or recombinant protein comprising an amino acid sequence encoded within position 1 to position 19530 or position 25265 to position 50955 of the N. meningitidis nucleotide sequence SEQ ID

6. An isolated or recombinant protein comprising an amino acid sequence having greater than 70% sequence identity to an amino acid sequence encoded within position 1 to position 19530 or position 25265 to position 50955 of the N. meningitidis nucleotide sequence SEQ ID

7. An isolated or recombinant protein comprising a fragment of at least 10 amino acids from an amino acid sequence encoded within position I to position 19530 or position 25265 to position 50955 of the N. meningitidis nucleotide sequence SEQ ID

8. An isolated or recombinant nucleic acid encoding a protein according to any one of claims 5-7.

9. An isolated or recombinant nucleic acid according to any one of claims 1-3, wherein the nucleic acid encodes a protein according to any one of claims 5-7. 123 CK 10. A nucleic acid probe comprising isolated or recombinant nucleic acid according to any one of claims 1-4 or 8 or 9.

11. An amplification primer comprising isolated or recombinant nucleic acid according to any one of claims 3 or 4.

12. A composition comprising isolated or recombinant nucleic acid according to any one of claims 1-4 or 8-11; and/or isolated or recombinant protein N according to any one of claims 5-7. S13. The use of a composition according to claim 12 as a medicament or as a diagnostic reagent.

14. The isolated or recombinant nucleic acid of any one of claims 1 to 4 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments and excluding, if any, comparative examples. The isolated or recombinant protein of any one of claims 5 to 7 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments and excluding, if any, comparative examples.

16. The amplification primer of claim 11 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments and excluding, if any, comparative examples.

17. The composition of claim 12 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments and excluding, if any, comparative examples.

18. The use of the composition of claim 12 substantially as hereinbefore described with particular reference to the examples and/or the preferred embodiments and excluding, if any, comparative examples.