MXPA00006900A - Neisseria meningitidis - Google Patents

Abstract

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

Description

- - ANTIGENS OF NEISSERIA MENINGITIDIS FIELD OF THE INVENTION This invention relates to antigens from the bacteria Neisse ia me ni ng i t i di s.

BACKGROUND OF THE INVENTION Neisseria meningi tidi s is a gram-negative, non-mobile, diplococcal human pathogen. Colonizes the pharynx, causes meningitis and, occasionally, septicemia in the absence of meningitis. It is closely related to N. gonor rhoeae, although a feature that clearly differentiates meningococci from gonococci is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.

N. meningitidis causes both epidemic and endemic diseases. In the United States, the rate of attack is 0.6-1 per 100,000 people per year, and it may be much greater during outbreaks of the disease (see Lieberman et al., 1996). Safety and Immunity of Serogroups A / C Neisseria meningi tidi s 01 igosacchar ide- Pro tei n Get together 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, the rates of endemic diseases are much higher and during situations or proportions of epidemic incidence they can reach 500 cases per 100,000 people 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. meningi tidi s is the leading cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).

Based on the capsular polysaccharide of the organism, 12 serogroups of N. meningi tidi s have been identified. Group A is the pathogen - which is most commonly involved in the 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 W134 and Y are responsible for the rest of the cases in the United States and in developed countries. The meningococcal vaccine in the present is in use and is a tetravalent polysaccharide vaccine, composed of serogroups A, C, Y, and W135. Although effective in adolescents and adults, it induces a poor immune response and short duration of protection, and can not be used in infants [eg Morbidity and Mortality eekly report, Vol. 46, No. RR-5 (1997) ] This is because polysaccharides are antigens independent of T cells and induce a very weak immune response that can not be amplified by repeated immunization. Following the success of the H vaccine. i n fl? In Zae, conjugate vaccines against serogroups A and C have been developed and are in the final stage of the clinical trial (Zollinger WD "New and - Vaccine Vaccines Against Meningococcus 1 Disease" in: New Generation Vaccines, s? pra, pp. 469-488; Lieberman et al (1996) supra; Constantino et al (1992) Development and phase I clinical testing of a conjugate vaccine against eningococcus A and C. Vaccine 10: 691-698).

However, meningococcus B continues to be a problem. This serotype is currently responsible for approximately 50% of the total meningitis in the United States, Europe, and South America. The polysaccharide can not be used because the menB capsular polysaccharide is a polymer of a N-acetyl neuraminic acid linked to a (2-8) which is also present in the tissue of mammals. This results in a tolerance to the antigen; indeed, if an immune response is generated, it would be anti-self and therefore undesirable. In order to avoid the induction of auto-inity and to induce a protective immune response, the capsular polysaccharide, for example, has been chemically modified by substituting the N-acetyl groups with the N-p rop onion groups. 1 or, leaving the specific antigenicity unaltered (Romero &Outschoorn (1994) Current status of Meningococcal group B vaccine candidates: capsular or non-capsular? Cl in My crobi ol R ev 7 (4): 559 - 575) .

Alternative methods to menB vaccines have used complex mixtures of outer membrane proteins (OMPs), which contain either OMPs alone, or OMPs enriched in porins, or deleted from class 4, it is believed that OMPs induce antibodies that block bactericidal activity. This method produces vaccines that are not well characterized. They are able to protect against the homologous strain, but they are not effective in the long run when there are many antigenic variants of the outer membrane proteins. To overcome the antigenic variability, have been constructed multivalent vaccines containing up to nine different porins (eg Poolman JT (1992) Development of a meningococcal vaccine In f e c t A ge n t s Di s 4:... 13-28). The additional proteins that are going to be used in the outer membrane vaccines have been - the opa and opc proteins, but none of these approaches or methods has been able to overcome the antigenic variability (for example Ala'Aldeen &Borriello (1996 Proteins 1 and 2 that bind to the meningococcal transferrin are both exposed surfaces and generate bactericidal antibodies capable of killing the homologous and heterologous strains Vac ci ne 14 (l): 49-53).

A certain amount of sequence data is available for the meningococcal and gonococcal genes and proteins (e.g. EP-A-0467714, WO 96/29412), but is not complete in any way. The provision of additional sequences could provide an opportunity to identify proteins exposed on the surface or secreted that are considered to be targets for the immune system and that are not variable at all. For example, some of the proteins identified could be components of effective vaccines against meningococci B, some could be components of vaccines against all serotypes of meningococci, and others could be components of vaccines against all pathogenic Neisseria.

DESCRIPTION OF THE INVENTION The invention provides proteins comprising the amino acid sequences of N. meningitidis described in the examples.

Sequences comprising homologous proteins (ie having sequence identity) are also provided to the amino acid sequences of N. meningitidis described in the examples. Depending on the particular sequence, the degree of identity of the sequence is preferably greater than 50% (for example 60%, 70%, 80%, 90%, 95%, 99% or more). These homologous proteins include mutants and allelic variants of the sequences described in the examples. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. The identity between the proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using a search-like separation with parameters of open penalty of separation = 12 and penalty. of extension of separation = 1.

The invention further provides proteins comprising fragments of the amino acid sequences of N. meningi tidi s described in the examples. The fragments should comprise at least n consecutive amino acids of the sequences and, depending on the particular sequence, n is 7 or greater (for example, 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope of the sequence.

The proteins of the invention can, of course, be prepared by various means (e.g., recombinant expression, purification of the cell culture, chemical synthesis, etc.) and in various forms (e.g., fusions, native, etc.). They are preferably prepared in a substantially pure form (ie substantially free of other - host cell proteins or N. meningitidis) According to a further aspect, the invention provides antibodies that bind to these proteins. These can be polyclonal or monoclonal and can be produced by any suitable method.

According to a further aspect, the invention provides nucleic acids comprising the nucleotide sequences of N. meningitidis described in the examples. In addition, the invention provides nucleic acids comprising homologous sequences (i.e., having sequence identity) to the nucleotide sequences of N. meningi tidi s described in the examples.

In addition, the invention provides nucleic acids that can hybridize to the N. meningitidis nucleic acid described in the examples, preferably under conditions of "high stringency" (for example 65 ° C in an O.lx SSC solution, 0.5% SDS) .

- - Nucleic acids comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides of the sequences of N. meni n gi ti di sy, depending on the particular sequence n is 10 or greater (for example 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).

According to a further aspect, the invention provides nucleic acids encoding the proteins and protein fragments of the invention.

It should also be appreciated that the invention provides nucleic acids comprising sequences complementary to those described above (for example for antisense or soda generation purposes).

The nucleic acids according to the invention can, of course, be prepared in many ways (for example by chemical synthesis, from genomic or AD? C libraries, from the organism itself etc.) and can take various forms (for example from a single strand, - double-strand, vectors, probes, etc.).

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

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

According to a further aspect, the invention provides compositions comprising proteins, antibodies, and / or nucleic acids according to the invention. These compositions may be suitable as vaccines, for example, or as diagnostic reagents, or as immunogenic compositions.

The invention also provides nucleic acids, proteins, or antibodies according to the invention for use as medicaments - - (for example as vaccines) or as diagnostic reagents. It also provides for the use of the nucleic acid, the protein, or the antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to the Neisseria bacterium; (ii) a diagnostic reagent to detect the presence of the Neisseria bacteria or the antibodies generated against the Neisseria bacteria; and / or (iii) a reagent that can generate antibodies against the Neisseria bacteria. Said Neisseria bacterium can be any species or strain (such as N. gonorrhoeae) but preferably N. meningitidis, especially 1-a strain A, strain B or strain C.

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

According to additional aspects, the - -invention provides several processes.

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

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

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

A process for detecting proteins of the invention is also provided, comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of complexes of the body-an í geno; and (b) detecting said comp far.

Unlike the sequences described in PCT / I B98 / 01665, the sequences described in the present application are believed to have no significant N-homologs. gon o r rh o e a e. Accordingly, the sequences of the present invention also find use in the preparation of reagents to distinguish between N. -me n i n gi t i di s and N. gon orrh or ea e.

A summary of the standard techniques and procedures that can be employed to perform the invention (e.g. to use the described sequences for vaccine or diagnostic purposes) are presented below. This summary is not a limitation of the invention but, on the contrary, gives examples that can be used, but are not required General The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill in the art. Such techniques are fully explained in the literature for example Sambrook Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D.? Glover ed., 1985); 01 i gonu cl eotide Synt-hesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames &S.J. Higgins eds, 1984); Transcription and Translation (B. D. Hames &S.J. Higgins eds., 1984); Animal Cell Culture (R. Freshney, 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J.H.

- -Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemi cal Methods in Cell and Molecular Biology. { Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D.M. Weir and C. C. Blackwell eds 1986).

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

All publications, patents, and patent applications cited herein are fully incorporated by reference. In particular, the contents of British patent applications 9800760.2, 9819015.0 and 9822143.5, are incorporated herein.

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

The term "comprising" means "including" as well as "consisting" for example a composition "comprising" X may consist exclusively of X or may include some material additional to X, such as X + Y.

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

An "origin of replication" is a polypeptide sequence that initiates and regulates the replication of polynucleotides, such as an expression vector. The origin of the replication behaves as an autonomous unit of the replication of the polynucleotide within a cell, capable of replication under its own control. An origin of replication may be necessary for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of appropriate proteins within the cell. The origin examples are the sequences that are replicated autonomously, 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 that - differs but has the sequence identity with the native or described sequence. Depending on the particular sequence, the degree of sequence identity between the native sequence or sequence described and the mutant sequence is preferably greater than 50% (eg 60%, 70%, 80%, 90%, 95%, 99 % or more, calculated using the Smi th-Wat erman algorithm as described above). As used herein, an "allelic variant" of a nucleic acid molecule, or region, for which the nucleic acid sequence is provided herein is a nucleic acid molecule, or region, which occurs essentially in the same place in the genome of a different or second isolate, and which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. An allelic variant coding region typically encodes a protein that has activity similar to that of the protein encoded by the gene to which it is to be compared. An allelic variant may also comprise an alteration in the 5 'or - 3' untranslated regions of the gene, such as in the regulatory control regions (for example see US Patent 5,753,235).

S i s t e s of expression The nucleotide sequences of Neisseria can be expressed in a variety of different expression systems; for example those used with the cells of mammals, baculoviruses, plants, bacteria and 1 evacuations. i. Mammal systems Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding the mammalian RNA polymerase and initiating transcription in the (3 ') direction of a coding sequence (eg, the structural gene) in the mRNA. A promoter will have a transcription initiation region, which is usually positioned at the 5 'end of the coding sequence, and a TATA box, usually located at 25-30 pairs of bars (bp) in the 5' direction of the transcription initiation site. The TATA box is thought to be targeted to RNA polymerase II to initiate RNA synthesis at the correct site. A mammalian promoter will also contain a promoter element in the 5 'direction, usually located within 100 to 200 bp in the 5' direction of the TATA box. A promoter element in the 5 'direction determines the rate at which transcription is initiated and can act in any orientation [Sambrook et al. (1989) "Expression of Cloned Genes in Mammal and Cells." In Molecular Cloning: A Laboratory Manual, 2nd ed.].

Mammalian mammalian genes are often highly expressed and have a fairly wide host range; therefore, the sequences encoding the mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, the LTR promoter of mouse mammary tumor virus, the adenovirus major late promoter (Ad MLP). And the promoter of the herpes simplex virus. In addition, sequences derived from non-viral genes, such as the murine me t a 1 or murine t one iin gene, also provide useful promoter sequences. The expression can be either constitutive or regulated (inducible), depending on the promoter it can be induced with glucocorticoids in the cells that respond to the hormone.

The presence of an enhancing element (enhancer), combined with the promoter elements described above, will usually increase d-e expression levels. An enhancer is a DNA regulatory sequence that can stimulate transcription up to 1000-fold when binding to homologous or heterologous promoters, with synthesis starting at the site of normal RNA initiation. The enhancers are also active when placed in the 5 'or 3' direction of the transcription initiation site, in any normal or released orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236: 1231; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. The enhancer elements derived from viruses can be particularly useful, because they usually have a host range more than one.

Examples include the early gene enhancer [Dijke et al (1985) EMBO J. 4: 761] and the meto rdo s / p romo t s derived from the long terminal repeat (LTR) of the Sarcoma Rous virus [Gorman et al. (1982b) Proc. Nati Acad. Sci. 79: 6111] and of the human omegal ovi ru s [Bosh-art et al. (1985) Cell 41: 521]. Additionally, some enhancers are adjustable and become active only in the presence of an inducer, such as a hormone or a metal ion [S a s sone - Co r s i and Borelli (1986) Trends Genet. 2: 215; Maniatis et al. (1987) Science 236: 1237].

A DNA molecule can be expressed in vitro in cells of mammals. A promoter sequence can be directly linked to 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 can be excised from the protein by incubation with cyanogen bromide.

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

Usually, the transcription termination and polyadenylation sequence 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 'terminal of the mature mRNA is formed by cleavage and polyadenylation post-site-specific cryptsallation [Birnstiel et al. (1985) Cell 41: 349; Proudfoot and Whitelaw (1988) "Termina t ion 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 that can be translated into the polypeptide encoded by the DNA. Examples of transcription / polyadenylation terminator signals include those derived from SV40 [Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloning: A Lab ora t ory Ma n ua l] Usually, the components described above, comprise a promoter, a polyadenylation signal, and the transcription termination sequence that are put together in expression constructs. Enhancers, introns with acceptor and donor sites of a cleavage or a functional splice, and leader sequences can also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an e xpert element (eg, plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require tumor factors to replicate. For example, plasmids containing the papovavirus replication systems, such as SV40 [Gluzman (1981) C ell 23: 1 1 5] opo 1 i ornavi ru s, replicated with an extremely high copy number in the presence of the antigen Appropriate viral T Additional examples of mammalian replicons include those derived from bovine pap i 1 ornavi ru and Epstein-Barr virus. Additionally, the replicon can 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 vectors that carry mammalian bacteria include pMT2 [Kaufman et al. (1989) Mol. Cel l. Bi ol. 5: 946] and pHEBO [Shimizu et al. (1986) Mo l. Ce l l. Bi or l. 6: 1074].

The transformation procedure used depends on the host being transformed. Methods for the 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 polynucleides in liposomes, and directed microinjection of DNA in the nucleus.

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 cells ( CHO), HeLa cells, baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepa t ocellular carcinoma cells (e.g. Hep "G2), and a number of other cell lines. ii. Baculovirus systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques that are known in the art. In general, the components of the expression system include a transfer vector, usually a bacterial plasmid that contains both a fragment of the baculovirus genome and a suitable restriction site for the insertion of the gene or heterologous genes to be expressed; a wild-type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for homologous reconnaissance of the heterologous gene in the baculovirus genome); and appropriate insect host cells and growth medium.

After inserting the DNA sequence encoding the protein into the transfer vector, the wild-type viral vector and genome are transfected into u-n to insect host cell where the vector and viral genome allow recombination. The packaged recombinant virus is expressed and the recombinant plaques are first identified and purified. The materials and methods for the insect expression systems of 1 to 1 rus / cé 1 u 1 s of insect are commercially available in packages of, among others, Invitrogen, San Diego CA ("MaxBac" equipment). These techniques are generally known to those skilled in the art and are fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter "Summers and Smith").

Before inserting the DNA sequence encoding the protein in the baculovirus genome, the components described above, comprising a promoter, the leader (if desired), the coding sequence of interest, and the transcription termination sequence, they are usually assembled in a translocation construct of the intermediary (transfer vector). This construct may contain a single gene - and be operably linked to the regulatory elements; multiple genes, each of which contains its own set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. The translocation constructs of intermediates are sometimes maintained in a replicon, such as an ex-chromosomal element (eg, plasmids) capable of being stable 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.

At present, the most commonly used transfer vector for the introduction of foreign genes into AcNPV is pAc373. Many other vectors, known to those skilled 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 32 base pair BamHI cloning site downstream from the ATT; see Luckow and Summers, Virology ( 1989) 17:31.

The plasmid usually also contains the polyadenylation signal of the polyhedrin (Miller et al (1988) Ann. Rev. Microbiol., 42: 177) and a prokaryotic ampicillin resistance gene (amp) and the origin of replication for the 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 transcription downstream (from 5 'to 3') of a coding sequence (eg, the structural gene) in the mRNA. A promoter will have a transcription initiation region which is usually placed near 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 can also have a second domain called an enhancer, which, if present, is usually distant from the structural gene. The expression can be either regulated or constitutive.

The structural genes, abundantly transcribed in recent times in a cycle of viral infection, provide particularly useful promoter sequences. Examples include sequences derived from the genes encoding the viral polyhedron protein Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression", in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); Publ. EPO Nos. 127 839 and 155 476; and the gene encoding the plO protein, Vlak et al., (1988), J. Gen Virol. 69: 165 The DNA encoding the appropriate signal sequences can be derived from genes for secreted baculovirus or insect proteins, such as the baculovirus polyhedrin gene (Carbonell et al (1988) Gene, 73: 409). Alternatively, since signals for post-translational modifications of mammalian cells (such as peptide cleavage signal, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and Signals required for nuclear secretion and accumulation also appear to be conserved between invertebrate cells and vertebrate cells, origin leaders that do not belong to insects, such as those derived from genes encoding human -al to-interferon, Maeda et al., '(1985), Nature 315: 592; the human gastrin releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8: 3129; Human IL-2, Smith et al., (1985) Proc. Nafl Acad. Sci. USA, 82: 8404; Mouse IL-3 (Miyajima et al., (1987) Gene 58: 213; and glucerebroses from human, Martin et al. (1988) DNA, 7:99, can also be used for secretion in insects. A recombinant polypeptide or polyprotein - it can be expressed in t ra m e rmen t e, if expressed with the appropriate regulatory sequences, can be secreted. A good intracellular expression of unfused, foreign proteins usually requires heterologous genes that ideally have a short leader sequence that contains suitable translation initiation signals that precede an ATG start signal. If desired, the methionine at the N-terminus can be excised from the mature protein by incubation in vitro with cyanogen bromide. Alternatively, recombinant proteins that are not naturally secreted can be secreted from insect cells by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that It provides the secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids that direct the protein pathway of the protein to the endoplasmic reticulum.

After the insertion of the DNA sequence and / or the gene encoding the precursor of the protein expression product, an insect cell host is taken form with the heterologous DNA of the transfer vector and the wild-type baculovirus genomic DNA - usually by co-transfection. The transcription termination sequence and the promoter of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing the heterologous DNA at 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. Bíol. (1983) 3: 2156; and Luckow and Summers (1989)). For example, the insertion can be within a gene such as the polyhedrin gene, by double homologous cross-recombination; the insert can also be in a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioassays 4:91. The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5 'and 3' by the specific polyhedrin sequences and is placed in the 3 'direction of the polyhedrin promoter.

The baculovirus expression vector formed in a novel manner is subsequently packaged in an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, most of the viruses produced after co - testation are still wild type viruses. Therefore, a method is necessary to identify the recombinant viruses. An advantage of the expression system is a visual screen that allows to distinguish the recombinant viruses. The polyhedrin protein, which is produced by the native virus, occurs at very high levels in the nucleus of infected cells at later times after viral infection. The accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, which gives them an appearance of brightness that is easily visualized under the light of the microscope. Cells infected with recombinant viruses lack the occlusion bodies. To distinguish recombinant viruses from wild type viruses, the transfection supernatant is placed in a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plates are selected under the light of the microscope for the presence (indicative of the wild type virus) or absence (indicative of the recombinant virus) of the 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).

The expression vectors of the recombinant baculovirus have been developed for infection in several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegyptii, Autographa californica, Bombyx mori, Drosophila melanogas ter, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell-et al., (1985) J. Virol 56: 153; Wright (1986) Nature 321: 118; Smith et al., (1983) Mol Cell. Biol. 3: 2156, and see in general, Fraser et al. (1989) In Vitro Cell, Dev. Biol. 25: 225).

Cells and culture medium of cells are commercially available for direct expression and fusion of heterologous polypeptides in a baculovirus / expression system; Cell culture technology is generally known to those skilled in the art. See, for example Summers and Smith s upra The modified insect cells can be grown in a suitable nutrient medium, which allows stable maintenance of the plasmids present in the modified insect host. When the gene of the expression product is under inducible control, the host can be grown at high density, and the expression can be induced. Alternatively, when the expression is constitutive, the product will be continuously expressed in the medium and the nutrient medium can be circulated continuously, removing the product of interest and increasing the nutrients eliminated. The product can be purified by techniques such as chromatography, for example HPLC, affinity chromatography, ion exchange chromatography, etc .; electrophoresis, density gradient centrifugation, solvent extraction, or the like. As appropriate, the product can be further purified, as required, to substantially remove any insect protein that is also secreted into the medium or resulting from the lysis of insect cells, to provide a product that is less substantially free of host debris, eg, proteins, lipids and polysaccharides.

To obtain the expression of the protein, the recombinant host cells derived from the transformants are incubated under conditions that allow the expression of the sequence encoding the recombinant protein. These conditions will vary, depending on the selected host cell. However, conditions are easily achievable for those with ordinary skill in the art, based on what is known in the art. iii Plant Systems There may be several plant cell cultures and plant expression systems, genetic, known in the art. Cell gene expression systems of exemplary plants include those described in the patents, such as: US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of gene expression in plant cell cultures have been described by Zenk, Phytochemi stry 30: 3861-3863 (1991). Descriptions of the peptides of the signal from the plant proteins can be found in addition in the references described above in Vaulcombe et al., Mol. Gen. Genet. 209: 33-40 (1987); Chandler et al., Plant Molecular Biology 3: 407-418 (1984); Rogers, J. Biol. Chem. 260: 3731-3738 (1985); Rothstein et al., Gene 55: 353-356 (1987); Whittier et al., Nucleic Acids Research 15: -2515-2535 (1987); Wirsel et al., Molecular Microbiology 3: 3-14 (1989); Yu et al., Gene 122: 247-253 (1992). A description of the regulation of gene expression of plants by phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R.L. Jones and J. MacMillin, Gibbe re 11 ins: in: Advanced Plant Physiology. Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, PP. 21-52. - 4 - References describing other metabolically regulated genes are: Sheen, Pl a n t Cel l, 2: 1027-1038 (1990); Maas et al., EMBO J. 9: 3447-3452 (1990); Benkel and Hickey, Pr o c. Na t i. A ca d. S ci. 84: 1337-1339 (1 87).

Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising the genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with accompanying sequences downstream and downstream from the expression cassette suitable for expression in a plant host. The accompanying sequences will be of viral or plasmid origin and will provide necessary features to the vector to allow the vectors to move the DNA from an original cloning host, such as a bacterium, to the desired plant host. The basic bacterial vector / plant construct will preferably provide a wide range of prokaryotic origin of replication hosts; - a prokaryotic selectable marker; and, for Agrobacterium transformations, T-DNA sequences for the Agrobacterium-mediated transfer to the plant chromosomes. Where the heterologous gene is not readily detectable, the construct will preferably also have a suitable selectable marker gene to determine whether a plant cell has been transformed. A general review of suitable markers, for example for members of the grass family, is found in Wilmink and Dons, 1993, Pl a n t Mol. B i or l. R ep t r, 11 (2): 165-185.

The appropriate sequences to allow the integration of the heterologous sequence in the genome of the plant are also recommended. These could include transposon sequences and the like for homologous recombination as well as Ti sequences that allow random insertion of a heterologous expression cassette into a plant genome. Suitable selectable prokaryotic markers include resistance to antibiotics such as ampicillin or racitic. 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 present invention can be included in an expression cassette for the expression of the proteins of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the sequence encoding the heterologous protein the following elements, a promoter region, untranslated sequences in the plant - 5 ', the initiation codon which depends on whether or not the structural genes are equipped with some, and a transcription and transcription termination sequence. The unique restriction enzyme sites at the 5 'and 3' ends of the cassette will allow easy insertion into a preexisting vector.

A heterologous coding sequence can be for any protein that is related to the present invention. The sequence encoding the protein of interest will encode a signal peptide that allows processing and translocating the protein, as appropriate, and will usually lack any sequence that can 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 that is expressed and translocated during germination, employing the signal peptide that is provided for the tumor, someone can also provide the 1 s of the protein of interest. In this way, the proteins of interest will be translocated from the cells in which they are expressed and can be harvested efficiently. Typically the secretion in the seeds is through the aleurone or escutellar epithelial layer towards the endosperm of the seed. While the protein is not required to be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

- - Since the last expression of the desired gene product will be in a eukaryotic cell, it is desirable to determine whether any portion of the cloned gene contains sequences that will be processed outwardly as introns by the host's splicosome machinery. If so, the site-directed mutagenesis of the "intron" region can be conducted to avoid the loss of a portion of the genetic message as a false intron code, Reed and Maniatis, Ce l 41: 95-105, 1985.

The vector can be directly or directly in the cells of the plants using micropipettes for mechanical transfer of the recombinant DNA. Cr.ossway, Mo l. Gen Gen et, 202: 179-185, 1985. The genetic material can also be transferred to plant cells using polyethylene glycol, Krens, et al., Na ture, 296, 72-74, 1982. Another method of introducing the nucleic acid segments is the high-speed ballistic penetration by small particles with the nucleic acid either within the matrix of small beads of particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, 1991, Planta, 185: 330-336? they teach the bombardment of particles from the endosperm of barley to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid surface bodies, Fraley, et al., Proc. Nati Acad. Sci. USA 79, 1859-1863, 1982.

The vector can also be introduced into plant cells by electroporation. (Fromm et al., Proc. Nati, Acad. Sci. USA 82: 5824, 1985). In this technique, the protoplasts of the plants are electroporated in the presence of plasmids containing the gene construct. The high-strength electric field impulses permeate the reversible biomembranes that allow the introduction of the plasmids. The protoplasts of the plants in the soil reform the cell wall, divide, and form calluses in the plants.

All plants from which the protoplasts can be isolated and cultured to give the complete regenerated plants can be transformed by the present invention so that the total plants are recovered and contain the transferred gene. It is known that virtually all plants can be regenerated from cultured cells or tissues, which include but are not limited to the main species of sugarcane, sugar beet, cotton, fruits and other trees, legumes and vegetables. Some suitable plants include, for example, species of the genera F agaria, Lotus, Medi ca go, Onobrychis, Trifolium, Trigonel la, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanu s, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Maj ora na, Ci chorium, Helianth s, Lactuca, Bromus, Aspar agu s, Antirrhinum, Hereroca 11 is, Nemesia, - 4 Pelar gonium, Panicu , Penni setum, Ran unc lu s, Senecio, Salpi gl os si s, Cucumi s, Browaal ia, Glycine, Lolium, Zea, Triticum, Sor hum, and Da tura.

Means for regeneration vary from species to species of plants, but generally a suspension of the transformed protoplasts containing copies of the heterologous gene is provided first. The callus tissues are formed and can induce shoots from the calluses and subsequently the root can be generated. Alternatively, the formation of the embryo can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain several amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for species such as corn and alfalfa. The shoots and roots will normally develop simultaneously. Efficient regeneration will depend on the medium, the genotype, and the history of the crop. If these variables are controlled, then the regeneration is totally reproducible and repeatable.

In some plant cell culture systems, the desired protein of the invention can be excreted or alternatively, the protein can be extracted from the total plant. Where the desired protein of the invention is secreted into the medium, it can be harvested. Alternatively, embryos and seeds with semi-embryo or other plant tissues can be mechanically disrupted to release any protein secreted between cells - and tissues. The mixture can be suspended in a buffer solution to obtain the soluble proteins. The conventional purification and isolation methods of the proteins will then be used to purify the recombinant proteins. The parameters of time, temperature, pH, oxygen, and volumes will be adjusted through routine methods to optimize the expression and recovery of the heterologous protein. iv. Bacterial Systems Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding the bacterial RNA polymerase and initiating transcription in the direction (3 ') of the coding sequence (eg structural gene) to the mRNA. A promoter will have a transcription initiation region that will usually be placed proximate 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 can also have a second domain called an operator, which can be overlapped to an adjacent RNA polymerase binding site and which initiates RNA synthesis. The operator allows regulated negative transcription (inducible), as a protein repressor of the gene that can bind to the operator and therefore inhibit the transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator, in addition, positive regulation can be achieved by the binding sequence of the activating protein of the gene, which, if present is usually close to (5) ') to the RNA polymerase binding sequence. An example of a gene activating protein is the catabolite activating protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18: 113]. Regulated expression can therefore either positive or negative thereby improving or reducing transcription.

The enzymes of the sequence-encoding metabolic pathways provide particularly useful promoter sequences. Examples include promoter sequences derived from enzymes that metabolize sugars, such as galactose, lactose. { lac) [Chang et al. (1977) Nature 198: 1056], and maltose.

- Additional examples include the promoter sequences derived from the enzymes bios int ethicas such as tryptophan (trp) [Goeddel et al. , (1980) Nuc. Acids Res. 8: 4057; Yelverton et al. , (1981) Nucí. Acids Res. 9: 131; North American Patent No. 4,738,921; EP-A-0036776 and E P-A-0121775]. The promoter system g-laotamasa (bla) [Weissmann (1981) "The cloning of interferon and other mistakes." In Inferieron 3 (ed I. Gresser)].

The bacteriophage lambda PL promoter systems [Shimatake et al. (1981) Nature 292: 128] and T5 [US Patent 4,689,406] also provide useful promoter sequences.

In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, the transcriptional activation sequences of a bacterial or bacteriophage promoter can be linked to the sequences of the operon of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [US Patent No. 4,551,433]. For example, the tac promoter is a trp-lac hybrid promoter comprised of either the tpp promoter or the lac operon sequences that are regulated by the lac repressor [Amann et al. (1983) Gene 25: 161; de Boer et al. (1983) Proc. Nati Acad. Sci. 80:21]. In addition, a bacterial promoter can include naturally occurring promoters that are not of bacterial origin that have the ability to bind to the bacterial RNA polymerase and initiate transcription. A promoter that occurs naturally of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage of the po-ime ras / promoter system of T7 RNA is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189: 113; Tabor et al. (1985) Proc. Nati Acad. Sci. 82: 1074]. In addition, a hybrid promoter may also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an 'initiation codon (ATG) and a sequence of 3-9 nucleotides in length located at nucleotides 3-11 e 5 'direction of the initiation codon [Shine et al. (1975) Nature 254: 34]. The SD sequence is thought to promote the binding of mRNA to the ribosome by pairing the bases between the SD and 3 'sequence and E. coli 16S rRNA [Steitz et al. (1979) "Genetic Signals and Nucleotide Sequences in Messenger RNA-" In Biological Regulation and Development: Gene Exprés si on (ed R.F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome binding sites [Sambrook et al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular Cloning: A Laboratory Manual].

A DNA molecule can be expressed i n t ra ce 1 u 1 a rme n t e. A promoter sequence can be directly linked to 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, the methionine at the N-terminus can be cleaved from the protein by incubation in vitro with cyanogen bromide or by incubation in vivo with a N-terminal peptidase of bacterial methionine (EPO-). A-0 219 237).

The 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, that is fused to the 5 'end of the heterologous coding sequences. After expression, this construct will provide a fusion of the two amino acid sequences. For example, the gene of the bacteriophage lambda cell can be linked to the 5 'terminal of a foreign gene and can be expressed in the bacterium. 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) Nat? Re 305: 810]. The fusion proteins can also make consequences for the lacZ genes [Jia et al. (1987) Gene 60: 191], trpE [Alien et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135: 11], and Chey [EP-A-0 324 647]. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g., the ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, the foreign native protein can be isolated [Miller et al. (1989) Bio / Technology 7: 698].

Alternatively, the foreign proteins can also be secreted from the cells by creating chimeric DNA molecules that encode a fusion protein comprised of a fragment of the signal peptide sequence that provides secretion of the foreign protein in the bacterium [ U.S. Patent No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of the hydrophobic amino acids that direct the secretion of the protein from the cell. The protein is either secreted in the growth medium (gram-positive bacteria) or in 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 peptide fragment of the signal and the gene.

The DNA encoding the appropriate signal sequences can be derived from the genes for the secreted bacterial proteins, such as the outer membrane protein gene from E. coli (ompA) (Masui et al. (1983), in : Experimental Manipulation of Gene - Expression; Ghrayeb et al. (1984) EMBO J. 3: 2431] and the signal sequence of the alkaline phosphatase of E. coli (phoA) [Oka et al. (1985) Proc. Acad. Sci. 82: 1212] As a further example, the alpha-amylase en signal sequence of several Bacillus strains can be used to secrete the heterologous proteins of B. subtilis [Palva et al. (1982)]. Proc. Nati, Acad. Sci. USA 79: 5582; EP-A-0 244 042].

Usually, the transcription termination sequences recognized by the bacterium are regulatory regions located 3 'up to the translation stop codon, and thus with the promoter flanking the coding sequence. These sequences direct the transcription of an mRNA that can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of approximately 50 nucleotides capable of forming the basic circuit structures that aid in the transcription of the determination. Examples include transcription termination sequences derived from genes with strong promoters, such as t rp in E. c ol i as well as other bi genes i n t e t i eos.

Usually, the components described above, which comprise a promoter, a signal sequence (if desired), a coding sequence of interest, and a transcription termination sequence, are readily placed in the expression constructs. Expression constructs are often maintained in a replicon, such as an element or non-synergists (eg, psemids) capable of being stable in a host, such as a bacterium. 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 can be either a high or low copy number plasmid. A high copy number plasmid will generally have a range of copy number from about 5 to - about 200, and usually from 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 20 plasmids. Either a vector with high or low copy number can be selected, depending on the effect of the vector and the foreign protein in the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. The integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. The integrations appear to result from recombinations between the homologous DNA in the vector and the bacterial chromosome. For example, integration vectors constructed with DNA from several strains of Bacillus are integrated into the Bacillus chromosome (EP-A-0 127 328). The integrating vectors can also be comprised of bacteriophages or transposon sequences.

- - Usually, the integrating and extrachromosomal expression constructs may contain selectable markers that allow the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and can include genes that render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) An n u. R e v. My cr obi o l. 32: 4 6 9]. Selectable markers may also include genes b i os i n t e t i eos, such as those in the b i or s in t e t i ca s pathways of histidine, tryptophan and leucine.

Alternatively, some of the components described above can be readily used in the transformation vectors. Transformation vectors are usually comprised of a selectable marker that either remains in a replicon or develops into an integrating vector, as described above.

- The vectors of expression and transformation, whether they are extirpated extérieurs extérieurs or integrating vectors, have been developed for the transformation into a many bacteria. For example, expression vectors have been developed for, among others, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Nati Acad Sci. USA 79: 5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292: 128; Amann et al. (1985) Gene 40: 183; Studier et al. (1986) J.

Mol Biol. 189: 113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol 54: 655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ Microbiol. 54: 655], Streptomyces lividans [North American Patent No. 4,745,056].

Methods for the introduction of exogenous DNA into bacterial hosts are well known in the art, and usually include either the transformation of the bacterium - treated with CaCl 2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. The transformation procedures usually vary with the bacterial species that are to be transformed. See for example [Masson et al. (1989) FEMS Microbiol. Lett. 60: 213; Palva et al. (1982) Proc. Nati Acad Sci. USA 79: 5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Nati Acad Sci. -85: 856; Wang et al. (1990) J. Bacteriol. 172: 949, Campy1 oba ct e r], [Cohen et al, (1973) Proc. Nati Acad. Sci. 69: 2110; Dower et al. (1988) Nucleic Acids Res. '15: 6127; Kushner (1978) "An improved method for transformation of Escherichia coli with ColE 1 - from ri ved plasmids In Genetic Engineering: Proceedi ngs of the International S ysumium on Genetic Engineering (eds HW Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53: 159; Taketo (1988) Biochim Biophys., Acta 949: 318; Escherichia], [Chassy et al. (1987) FEMS.

Microbiol Lett. 44: 113 Lactobacillus]; Fiedler et al. (1988) Anal. Biochem 170: 38, - -Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. 'Lett. 66: 203, S t aphylococcus], [Barany et al. (1980) J. Bacteriol. 144: 698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation, in: S treptococcal 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. 4 th Evr. Cong. Biotechnology 1: 412, Streptococcus]. v. Expression in Yeasts Yeast expression systems are well known to someone with ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding to the RNA polymerase of the yeast and initiating transcription in the (3 ') direction of a coding sequence. (for example structural genes) in mRNA. A promoter will have a transcription initiation region that is usually positioned proximal to the 5 'end of the coding sequence. This -transcription initiation region usually includes an RNA polymerase binding site (the "TATA Box") and a transcription initiation site. A yeast promoter can also have a second domain called an activating sequence in the 5 'direction (UAS), which, if present, is usually distant from the structural gene. The UAS allows regulated (inducible) expression. Constitutive expression is carried out in the absence of UAS. Regulated expression can be either positive or negative, thus improving • or reducing transcription.

Yeast is a fermentation organism with an active metabolic pathway, therefore the sequences encoding the enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-fat or isomerase, g-1-aldehydo-3-phosphate-dehydrogenase (GAP or GAPDH), Hexokinase, f ooks f oof a ct, 3 - f og 1 i cer ato mutase, and pyruvate kinase (Py K) (E PO-A- 0 329 203). The yeast gene PH05, which encodes acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Pr o c. Na t i. A ca d. S ci. USA 8 0: 1].

In addition, synthetic promoters that do not occur in nature also function as yeast promoters. For example, the UAS sequences of a yeast promoter can be linked to the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the activation region of GAP-transcription (U.S. Patent Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters consisting of the regulatory sequences of either the ADH2, GAL4, GAL IO, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP -A-0 164 556). In addition, a yeast promoter can include promoters that occur naturally from non-yeast sources and that have the ability to bind the yeast RNA polymerase and initiate transcription. Examples of such promoters include, among others, [Cohen et al. (1980) Proc. Nati Acad. Sci. USA 77: 1078; Henikoff et al. (1981) Nature 283: 835; Hollenberg et al. (1981) Curr. Microbiol. Immunol. 96: 119; Hollenberg et al. (1979) "The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae," in: Plasmids of Medical, Envi ronme ta 1 and Commercial Importance (eds. K.N. Timmis and A. Puhler); I r cera- Pu i ga 1 on et al. (1980) Gene 11: 163; Panthier et al. (1980) Curr Genet. 2: 109;].

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

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused at the 5 'end of the heterologous coding sequences. After 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 to the 5 'of a foreign gene and expressed in the yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See for example EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for the processing of the 7-enzyme (eg, processing protease). • specific to ubiquitin) to cleave the ubiquitin from the foreign protein. Through this method, therefore, the native foreign protein can be isolated (for example WO 88/024066).

Alternatively, the foreign proteins can also be secreted from the cell in the growth medium by creating the chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides secretion in the yeast of a foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be excised either in vi or in vi t ro. The leader sequence fragment usually encodes a signal peptide comprised of the hydrophobic amino acids that direct the secretion of the protein from the cell.

The DNA encoding the appropriate signal sequences can be derived from the genes - for the secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873, JPO 62,096,086) and the factor A gene (U.S. Patent No. 4,588,684). Alternatively, the leaders of the non-yeast origins, such as the leader interferon, exist in such a way that they are also provided in the secretion in the yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha factor gene, which contains both a "pre" signal sequence and a "pro" region: The types of alpha factor fragments that can be employ the leader of the full length pre-pro alpha factor (approximately 83 amino acid residues) as well as the truncated alpha factor leaders (usually about 25 to about 50 amino acid residues) (US Patent Nos. 4,546,083 and 4,870,008; A-0 324 274). Additional leaders who employ a fragment of the alpha factor leader that provides secretion include alpha hybrid factor leaders made with a presequence of a first yeast, but also a pro-region of a second yeast alpha factor (e.g. , see WO 89/02463).

Usually, the transcription termination sequences recognized by the yeasts are the regulatory regions located 3 'up to the translation stop codon, and thus with the promoter flanking the coding sequence. These sequences direct the transcription of an mRNA that can be translated into the polypeptide encoded by the DNA. Examples of the transcription terminator sequence and other termination sequences recognized by the yeasts, such as those encoding the enzymes g 1 i co 1 i t i c a s.

Usually, the components described above, which comprise a promoter, a leader (if desired), a coding sequence of interest, and the -7-transcription termination sequence, and are put together in the expression constructs. Expression constructs are often maintained in a replicon, such as an ex-chromosomal element (e.g., plasmids) capable of being stable in a host, such as a yeast or bacterium. The replicon can have two replication systems, thus allowing it to be maintained, for example, in the yeast for expression in a prokaryotic host for cloning and amplification. Examples of such vectors transporting b a c t er i - 1 evadu r a s include Yep24 [Botstein et al. (1979) Gene 8: 17-24], pCl / 1 [Brake et al. (1984) Proc. Nati Acad. Sci USA 81: 4642-4646], and YRpl7 [Stinchcomb et al. (1982) J. Mol. Biol. 158: 151]. In addition, a replicon can be either a high or low copy number plasmid. A plasmid with high copy number will generally have a range of copy number from about 5 to about 200, and usually from about 10 to about 150. A host containing a plasmid with high copy number will preferably have at least about 10 copies. , and more preferably at least about 20. Enter a vector with a high or low copy number can be selected, depending on the effect of the vector and the foreign protein in the host. See, for example, Brake et al. , supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integration vector. The integration vectors usually contain at least one sequence homologous to a chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. The integrations seem to result from the recombination between the homologous DNA and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101: 228-245. An integration vector can be directed to a specific place in the yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression constructs can integrate, possibly affecting the levels of the recombinant protein produced [Riñe et al. (1983) Proc. Nati Acad. Sci. USA 80: 6150]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the complete vector, or two segments homologous to the adjacent segments in the chromosome and flanking the expression construct in the vector , which results in the stable integration of only the expression construct.

Usually, the integrating constructs and the c xt ra c y com m ons may contain selectable markers that allow the selection of yeast strains that have been transformed.

Selectable markers can include bi genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the resistance gene G418, which confers resistance in the yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker can also provide the yeast with the ability to grow in the presence of toxic compounds, such as metals. For example, the presence of CUP1 allows the yeast to grow in the presence of copper ions [Butt et al. (1987) My crobiol Rev. 51: 351].

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

Expression and transformation vectors, either replicons or t ra c romo s or s or integration vectors, have been developed for transformation in many yeasts. For example, expression vectors have been developed for, among others, the following yeasts Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6: 142], Candida maltose [Kunze, et al. (1985) J. Basic Microbiol. 25: 141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132: 3459; Roggenkamp et al. (1986) Mol. Gen.

Genet 202: 302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158: 1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154: 131; Van den Berg et al. (1990) Bio / Technology 8: 135], Pichia gui 1 ler imondi i [Kunze et al. (1985) J. Basic Microbiol. 25: 141], Pichia pastoris [Cregg, et al. ' (1985) Mol. Cell. Biol. 5: 3316; U.S. Patent Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Nati Acad Sci. USA 75: 1929; Ito et al. (1983) J. Bacteriol. 153: 163], - S chi zosaccharomyces pombe [Beach and Nurse (1981) Nature 300: 106], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet 10: 380411 Gaillardin, et al. (1985) Curr Genet. 10:49].

Methods for the introduction of exogenous DNA into yeast hosts are well known in the art, and usually include either the transformation of spheroplasts such as intact yeast cells treated with alkaline -calcations. The transformation procedures usually vary with the yeast species that are to be transformed.

See for example [Kurtz et al. (1986) Mol. Cell. Biol. 6: 142; Kunze et al. (1985) J.

Basic Microbiol. 25: 141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132: 3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202: 302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158: 1165; De Louvencourt et al. (1983) J. Bacteriol. 154: 1165; Van den Berg et al. (1990) Bio / Technology 8: 135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5: 3376; Kunze et al. (1985) J. Basic Microbiol. 25: 141; US Patents NOS. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Nati Acad Sci. USA 75: 1929; Ito et al. (1983) J. Bacteriol. 153: 163 Saccharomyces]; [Beach and Nurse (1981) Nature 300: 1 6; Schi zosaccharo yces]; [Davidow et al. (1985) Curr. Genet 10:39; Gaillardin et al. (1985) Curr. Genet 10:49; Yarrowia].

An t i c u erp o s As used herein, the term "antibody" refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An "antibody combining site" is the three-dimensional link space with an internal surface shape and distribution charge complementary to the characteristics of an epitope of an antigen, which allows a binding of the antibody to the antigen. The "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 chromatography, preferably affinity, immunoassays, and proteins that can be used in a specific manner.

Neisseria.

Antibodies to the proteins of the invention, both polyclonal and monoclonal, can 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 serum due to the volume of serum obtainable and the availability of the labeled anti-goat and anti-rabbit antibodies. The immunization is generally carried out by mixing or emulsifying the protein in saline solution, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg / injection is typically sufficient. The immunization is generally amplified 2 to 6 weeks later with one or more injections of the protein in saline, preferably using incomplete Freund's adjuvant. An alternative way to generate antibodies is by in vitro immunization using methods known in the art, for which purposes of this invention they are considered equivalent to immunization in vivo. The polyclonal antiserum is obtained by bleeding the immunized animal in a glass or plastic container, incubating the blood at 25 ° C for one hour, followed by incubation at 4 ° C for 2-18. The serum is recovered by centrifugation (for example 1000 g for 10 minutes). Approximately 20-50 ml per bleed can be obtained from rabbits.

The monoclonal antibodies will be prepared using the standard method of Kohier & Milstein [Na t u re (1975) 56: 495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, instead of bleeding the animal to extract the serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into unit cells. If desired, the spleen cells can be separated (after removal of non-specific adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. The B cells that express the membrane binding immunoglobulin specific to the antibody bound to the plate, and do not rinse with the rest of the suspension. The resulting B cells, or all of the dissociated cells of the spleen, are then induced to fuse with the myeloma cells to form hybridomas and then cultured in a selective medium (e.g., hypoxanthine, ami n op te ri na, thymidine medium). , "HAT"). The resulting hybridomas are "plated by limiting dilution, and then assayed for the production of antibodies that specifically bind to the immunizing antigen (and which do not bind to the unrelated antigens.) The hybridomas that secrete MAb are then cultured. be in vi tro (for example in tissue culture bottles or in hollow fiber reactors), or in vi (as ascitos in mice).

If desired, antibodies (either polyclonal or monoclonal) can be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive-8-atoms (particularly 3"" 2"P and 125 I), dense electron reagents, enzymes, and ligands that have 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) or a blue pigment, when labeled with a spectrophotometer. "specific" refer 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 thereto. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couplings known in the art. It should be understood that the previous description does not attempt to categorize the different labels in different classes, as the same label can serve in different modes. For example, 125I can serve as a radioactive label or as a dense electron reagent. HRP can serve as an enzyme or as an antigen for a MAb. In addition, someone can combine several labels to give the desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, someone can label an 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 with ordinary skill in the art, and are considered as equivalents within the scope of the present invention.

Comp o s i c i ons Fa rma c e u t i ca s The pharmaceutical compositions may comprise either polypeptides, antibodies, or nucleic acids 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 condition or condition, or to exhibit a detectable therapeutic or preventive effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects may also include reduction in physical symptoms, such as a decrease in body temperature. The precise, effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the therapies or combinations of therapeutic methods 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 doctors.

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 whom it is to be administered.

A pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the 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 can be administered without undue toxicity. Suitable carriers can be large macromolecules, which are metabolized slowly, such as proteins, polysaccharides, polyacid acids, po 1 ig 1 or 1 acids, polymeric amino acids, amino acid copolymers, and virus particles. inactive Such carriers are well known to those with ordinary skill in the art.

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

The pharmaceutically acceptable carriers in the therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as suspensions or liquid injectable solutions; Solid forms suitable for solution or suspension in liquid vehicles can also be prepared before injection. Liposomes are included within the definition of an 8-pharmaceutically acceptable carrier Me m o th e s 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 are what can be treated.

The direct exposure of the compositions will generally be complemented by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the tissue interstitial space. The compositions can also be administered in a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transdermal applications (e.g., see WO 98/20734), needles, and gene guns or hypo-sprays. The dosing treatment may be a unit dose schedule or a dose schedule 11 ip 1 e. 9 -. 9 - Va c a a s The vaccines according to the invention can be either prophylactic (ie to prevent infection) or therapeutic (ie to treat the infection after the condition).

Such vaccines comprise immunizing agents, immunogens, polypeptides, proteins or nucleic acids, usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not by itself induce the production of antibodies harmful to the individuals receiving the composition. Suitable carriers are typically large macromolecules, which are slowly metabolized, such as proteins, polysaccharides, polyacid acids, polyhydric acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as droplets). oil or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers can function as immune imaging agents ("adjuvants"). In addition, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid of pathogens of diphtheria, tetanus, cholera, H. pylori, etc.

Preferred adjuvants for improving the effectiveness of the composition include, but are not limited to: (1) aluminum (alum) salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc .; (2) formulations of oil-in-water emulsions (with or without other specific immunostimulatory agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 ™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell &Newman, Plenum Press 1995), which contains 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing several amounts of * MTP-PE (see below), although not required) formulated in size-icronic particles using a such as Microfluidizer model HOY (Microfluidics, Newton, MA), (b) SAF, containing 10% squalene, 0.4% Tween 80, 5% polymer L121 blocked with pluronic and thr-MDP (see below) either my crofluidized in a submicron emulsion or generated in a vortex to generate an emulsion of larger particle size, and (c) the Ribi ™ Adjuvant System (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% of Tween 80, and one or more components of the bacterial cell walls of the group consisting of the monophosphoryl lipid A (MPL), dimhalate trehalose (TDM), and the cell wall skeleton (CWS), preferably MPL + CWS (Detox ™); (3) saponin adjuvants, such as Stimulon ™ (Cambridge Bioscience, Worcester, MA) can be used or particles generated thereof such as ISCOM (immune complexes imu 1 an t is); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (I FA); (5) cytokines, such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons - (for example interferon gamma), the macrophage colony stimulating factor (M-CSF), the tumor necrosis factor (TNF), etc .; and (6) other substances that act as immune agents are effective to improve the effectiveness of the composition. Alu and MF59 ™ are more preferred.

As mentioned above, the terms muramyl include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl normuramyl-L-alanyl-D-isoglutamine (nor -MDP), N-aceti lmu rami 1 -L-alanyl-D-isoglut a.minyl-L-alanine-2- (1'-2'-dipalmitoyl-s? -glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP) -PE), etc.

Immunogenic compositions (e.g., the immunogenic antigen / po Ipeptide / nucleic acid, pharmaceutically acceptable carriers, and adjuvants) will typically contain diluents, such as water, saline, glycerol, ethanol, etc. additionally, auxiliary substances, such as emulsifying agents - or humectants, pH buffer substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectable solutions either liquid or as suspensions; Suitable solid forms for the solution, or suspension in liquid vehicles before injection can also be prepared. The preparation can also be emulsified or encapsulated in liposomes for an improved adjuvant effect, as discussed above under the pharmaceutically acceptable carriers.

The immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the aforementioned components, as necessary. By "immunologically effective amount", it is desired to indicate 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 on the physical condition and health of the individual to be treated, the taxonomic group of the individual to be treated (eg, non-human primate, primate, etc.), the ability of the individual's immune system to synthesize the antibodies, the degree of protection desired, the formulation of the vaccine, the doctor's assessment regarding the treatment of the medical situation, and other relevant factors. It is expected that the quantity will fall in a relatively broad range that can be determined through rut analysis.

The immunogenic compositions are conventionally administered parenterally, for example, by injection, either subcutaneously, intramuscularly, or t ransderma 1 / t rans tan t (eg WO 98 / 2073'4). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. The treatment of the dosage can be a single or simple dose schedule, or a multiple dose schedule. The vaccine can be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination can be used [eg Robinson & Torres (1997) Seminar s in Immunolgy 9: 271-283; Donnelly et al. (1997) Annu Rev Immunol 15: 611-648; as will be seen later here].

Vehicles for the Supply of Genes The vehicles for gene therapy for the delivery of constructs including a coding sequence of a therapeutic method of the invention, which are to be delivered to a mammal for expression in the mammal, which can be administered either locally or through camen you These constructs can use viral or non-viral vectors in in vivo or ex vivo modalities. The expression of such a coding sequence can be induced using endogenous or heterologous promoters. The expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the nucleic acid sequences contemplated. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenovirus, adenoviral viral (AAV), viral herpes, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, ortho-virus, papovavirus, for amixo vi rus, parvovirus, p i corn a vi r u s, 'poxvirus, or the viral vector of togavirus. See generally, Jolly (1994) Cancer Gene Therapy 1: 51-64; Kimura (1994) Human Gene Therapy 5: 845-852; Connelly (1995) Human Gene Therapy 6: 185-193; and Kaplitt (1994) Nature Genetics 6: 148-153.

Retroviral vectors are well known in the art and it is contemplated that any retroviral gene therapy vector can be employed in the invention, including retroviruses of type B, C and D, xenotropic retroviruses (e.g., NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Vi rol., 53: 160) polytropic retroviruses for example MCF and MCF-MLV (see Kelly (1983) J. Vi., 45: 291), foam virus and lentivirus. See RNA Tumor Víruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the gene therapy vector can be derived from different retroviruses. For example, the LTR retrovector can 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 a synthetic origin from the second strand of a Virus of Leucosis of Birds.

These recombinant retroviral vectors can be used to generate particles of retroviral vectors capable of transduction by introducing them into appropriately packaged cell lines (see - North American Patent No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into the DNA of the host cell by incorporating a chimeric integrase enzyme into the retroviral particle (see WO 96/37626). It is preferable that the recombinant viral vector be a replication of the defective recombinant virus.

The packaging cell lines suitable for use with the retrovirus vectors described above are well known in the art, are readily prepared (see WO 95/30763 and WO 92/05266), and can be used to create producer cell lines (also so-called vector cell lines or "VCL" for the production of recombinant vector particles Preferably, the packaging cell lines are made from human progenitor cells (e.g. HT1080 cells) or mink progenitor cell lines, which they eliminate inactivation in human serum.

- - The preferred retroviruses for the construction of the vectors for retroviral gene therapy include the Virus of Leucosis of the Birds, the Bovine Leukemia Virus, the Murine Leukemia Virus, the Virus that Induces the Focus of the Mink Cells, the Virus of Sarcoma Mu rino, the Virus of Re t í cu 1 o endo te 1 ios and the Sarcoma Rous Virus. Particularly Murine Leukemia Viruses are the preferred ones and include Virus 4070A and 1504A (Hartley and Rowe (1976) J Vi rol 19: 19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR- 245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey and Rauscher Sarcoma Virus (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses can be obtained from collections or reservoirs such as the American Type Culture Collection ("ATCC") in Rockville, Maryland or isolated from known sources using commonly available techniques.

Retroviral gene therapy vectors, known in exemplary manner, which can be used in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO 89/02468; WO 89/05349, WO 89/09271, WO 90/02806, WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, WO 91/02825, WO 95/07994, US 5,219,740, US 4,405,712, US 4,861,719, US 4,980,289, US 4,777,127, US 5,591,624. See also Vile (1993) Cancer Res 53: 3860-3864; Vile (1993) Cancer Res 53: 962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33: 493-503; Baba (1993) J Neurosurg 79: 729-735; Mann (1983) Cell 33: 153; Cane (1984) Proc Nati Acad Sci 81: 6349; and Miller (1990) Human Gene Therapy 1.

The vectors of human adenoviral gene therapy are also known in the art and can be used in this invention. See, for example, Berkner (1988) Biotechnology 6: 616 and Rosenfeld (1991) Science 252: 431, and WO 93/07283, WO 93/06223, and WO 93/07282. Adenoviral gene therapy vectors - known, in exemplary fashion, that can be used in this invention, include those described in the above-referenced documents and in WO 94/12649 WO 93/03769 WO 93/19191, WO 94/28938, WO 95/11984, WO 95/00655, WO 95/27071, WO 95/29993, OR 95/34671, WO 96/05320, WO 94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95/14102, WO 95/24297, WO 95/02697, WO 94/28152, "WO 94/24299, WO 95/09241, WO 95125807, WO 95/05835, WO 94/18922 and WO 95/09654. Alternatively, administration of the DNA linked to the killed adenovirus as described in Curiel (1992) Hum. Ge n e Th e r. 3: 147-154 can also be used. The gene delivery vehicles of the invention also include adenovirus-associated virus (AAV) vectors. Preferred examples of such vectors for use in this invention are the AAV-2 based vectors described in Srivastava, WO 93/09239. The most preferred AAV vectors comprise the two inverted terminal AAV repeats in which the native D sequences are modified by the substitution of nucleotides, - - such that at least 5 native nucleotides and - up to 18 native nucleotides, more preferably at least 10 nucleotides native and up to 18 native nucleotides, more preferably 10 native nucleotides are retained and the remaining nucleotides of sequence D are deleted or replaced with non-native nucleotides. The native D sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie, there is a sequence at each end) that is not involved in the HP formation. The non-native replacement nucleotide may be any nucleotide other than that nucleotide found in the native D sequence in the same position. other exemplary AAV e ects, employable are pWP-19, pWN-1, both of which are described in Nahreini (1993) Gen e 124: 257-262. Another example of such a AAV vector is psub201 (see Samulski (1987) J. Vi r.l 61: 3096). Another exemplary AAV vector is the Double D ITR vector. Construction of the Double D ITR vector is described in U.S. Patent No. 5,478,745. Still other vectors are those described in the North American Patent of Carter 4,797,368 and the North American Patent of Muzyczka 5,139,941, the North American Patent of Chartejee 5,474,935, and of Kotin WO 94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and the albumin promoter and directs expression predominantly to the liver. Its structure and construction is described in Su (1996) Huma n Gen e Th e rapy 7: 463-470. In addition, the vectors of AAV gene therapy are described in US Patents Nos. 5,354,678, 5,173,414, 5,139,941, and 5,252,479.

The gene therapy vectors of the invention also include the herpes vectors. The preferred and preferred examples are herpes simplex virus vectors that contain a sequence encoding a thymidine kinase polypeptide such as those described in US Patent No. 5,288,641 and European Patent 0176170 (Roizman). Additional exemplary simplex herpes virus vectors include HFEM / ICP6-LacZ described in WO 95/04139 (Wistar Institute), the pHSVlac described in Geller (1988) S ci ence 241: 1667-1669 and in WO 90 / 09441 and WO 92/07945, the HSV Us3 :: pgC-lacZ described in Fink (1992) Hum an Gen e Th e rapy 3: 11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Br ea f ield), and those deposited with the ATCC with access numbers ATCC VR-977 and ATCC VR-260 Also contemplated are gene therapy vectors of the alpha virus that can be employed in this invention. The preferred alpha virus vectors are the vectors of Sindbis viruses. Togaviruses, Semliki virus Forest (ATCC VR-67; ATCC VR-1247), viruses Middleberg (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Patent Nos. 5,091, 5,217,879, and WO 92/10578. More particularly, the vectors of the alpha virus described in the patent application - North American No. 08 / 405,627, filed on March 15, 1995, WO 94/21792, WO 92/10578, WO 95/07994, and in the Patents North American Nos. 5,091,309 and 5,217,879 may also be employed. Such alpha viruses can be obtained from depositaries or from collections such as the ATCC in Rockville, Maryland or can be isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are those that are used (see USSN 08/679640).

DNA vector systems such as eukaryotic layer expression systems are also useful for the expression of nucleic acids of the invention. See WO 95/07994 for a detailed description of eukaryotic layer expression systems. Preferably, the eukaryotic layer expression systems of the invention are derived from the alphavirus vectors and more preferably from the viral vectors S i ndb i s.

- - Other viral vectors suitable for use in the present invention include those derived from polioviruses, 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 pox virus from canaries or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Nati Acad Sci 86: 317; Flexner (1989) Ann NY Acad Sci 569: 86, Flexner (1990) Vaccine 8:17; in U.S. Patent Nos. 4,603,112 and 4,769,330 and in WO 89/01973; the SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277: 108 and Madzak (1992) J Gen Virol 73: 1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made using reverse genetic techniques as described in U.S. Patent No. 5,166,057 and Enami (1990) Proc Nati Acad Sci 87: 3802-3805; Enami & Palese (1991) J Virol 65: 2711-2713 and Luytjes (1989) Cell 59: 110, - - (see also McMichael (1983) NEJ Me d 309: 13, and Yap (1978) Na ture 273: 238 and Na ture (1979) 277: 108); the human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. I saw role 66: 2731; the measles viruses, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; the Aura virus, for example ATCC VR-368; the Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; the Cabassou virus, for example ATCC VR-922; the Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; the Getah virus, for example ATCC VR-369 and ATCC VR-1243; the Kyzylagach virus, for example ATCC VR-927; the virus Mayaro, for example ATCC VR-66; the virus 'Mucapibo, for example ATCC VR-580 and ATCC VR-1244; the Ndumu virus, for example ATCC VR-371; the Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; the Túnate virus, for example ATCC VR-925; the Triniti virus, for example ATCC VR-469; the Una virus, for example ATCC VR-374; the Whataroa virus, for example ATCC VR-926; the Y-62-33 virus, for example ATCC VR-375; the O'Nyong virus, the encephalitis virus -Eastern, 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 coronaviruses, for example ATCC VR-740 and those described in Hamre (1966) Pr o c S o c Exp B i o l Me d 121: 190.

The delivery of the compositions in this invention into cells is not limited to the aforementioned viral vectors. Other methods and means of delivery may be employed such as, for example, nucleic acid expression vectors, the bound polymeric DNA bound or unbound to the killed adenovirus, alone, see for example US Patent Application No. of Series 08 / 366,787, filed on December 30, 1994 and Curiel (1992) Hum Ge ne Th 3: 147-154, the DNA bound to the ligand, see for example Wu (1989) J Bi ol Ch em 264: 16985 -16987, eukaryotic cell vehicle cells, see for example, US Patent Application Serial No. 08 / 240,030, filed May 9, 1994, and US Patent Application Serial No. 08 / 404,796. , the deposition of hydrogel materials fo t opol imer ized, manual transfer gene guns, as described in US Pat. No. 5,149,655, ionizing radiation as described in the patent American Patent No. 5,206,152 and in WO -92 / 11033, the neutralization of nucleic charges or the fusion with cell membranes. Additional methods are described in Philip (19'94) Mo l Ce l l Bi ol 14: 2411-2418 and in Woffendin (1994) Pr o c Na ti A ca d S ci 91: 1581- 1585.

The particle-mediated gene transfer can be employed, see for example US Patent Application Serial No. 60 / 023,867. Briefly, the sequence can be inserted into conventional vectors containing the conventional control sequences for high levels of expression, and then incubated with synthetic gene transfer molecules such as the binding cations with the polymeric DNA such as polylysine , protamine, and albumin, - -linked to the ligands of cell targets such as ace 1 oorosomuco ide, as described in Wu & Wu (1987) J. Bi ol. Ch em. 262: 4429-4432, insulin as described in Hucked (1990) Bi o ch em Ph a rma co l 40: 253-263, galactose as described in Plank (1992) Bi oc on ga ga Ch em 3: 533-539, lactose or transferrin.

You can also use naked DNA.

Exemplary naked DNA introduction methods are described in WO 90/11092 and in U.S. Patent No. 5,580,859. Efficiency can be improved using biodegradable latex beads. The latex beads coated with DNA are transported efficiently in the cells after the initiation of endocytosis by beads. The method can be further improved by treating beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release the DNA to the cytoplasm.

Liposomes that can act as -lli-gene delivery vehicles are described in US Patent No. 5,422,120, WO 95/13796, WO 94/23697, WO 91/14445 and EP-524,968. As described in USSN. 60 / 023,867, in the non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into the conventional vectors containing the conventional control sequences for high levels of expression, and then incubated with the synthetic gene transfer molecules. such as the binding cations of polymeric DNA such as polylysine, protamine, and albumin, bound to the target ligands of cells such as asia 1 oo ro s omu coi, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate the DNA comprising the gene under the control of a variety of tissue-specific ubiquitous or tissue-active promoters. In addition, the non-viral supply suitable for use includes mechanical delivery systems such as the method described in Woffendin e t a l (1994) Pro c. Na t i. A ca d. S ci.

- - USA 91 (24): 11581-11585. In addition, the coding sequence and the product of the expression thereof can be supplied through the deposition of hydrogel materials, topolimerized. Other conventional methods for the delivery of genes that can be used for the delivery of the coding sequence include, for example, the use of hand-held gene transfer particle guns, as described in US Pat. No. 5,149,655; the use of ionizing radiation for the activation of the transferred gene as described in U.S. Patent No. 5,206,152 and in WO 92/11033.

The delivery vehicles of exemplary liposome and po lotype genes are those described in U.S. Patent Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO 94/23697; and WO 91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Bi o ch em Bi oph ys A c t a 600: 1; Bayer (1979) Bi o ch em Bi ophys A c t a 550: 464; Ryvnay (1987) Meth Enzymol 149: 119; Wang (1987) Proc Nati Acad Sci 84: 7851; Plant (1989) Anal Biochem 176: 420.

A polynucleotide composition may comprise therapeutically effective amounts of a gene therapy vehicle as 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 whom it is to be administered.

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

The direct delivery of the compositions will generally be completed by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered in a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (e.g., see WO 98/20734), needles, and "gene guns or hypo-sprays. it can be a single-dose program or a multiple-dose program.

Methods for the ex vivo delivery and reimplantation of transformed cells in a subject are known in the art and are described for example in WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoietic cells, lymphoid cells, macrophages, dendritic cells, or tumor cells.

Generally, the delivery of nucleic acids in both ex vivo and in vitro applications can be complemented by the following procedures, for example, with dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, fusion of protoplasts, electroporation, and encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into nuclei, as is well known in the art.

Pharmaceutical compositions of polynucleotides and polypeptides In addition to the pharmaceutically acceptable carriers and the salts described above, the following additional agents can be used with the polynucleotide and / or polypeptide compositions.

A Polypeptides An example is polypeptides that include, without limitation: asiole orosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments, ferritin, interleukins; interferons; granulocytes, the macrophage colony stimulating factor (GM-CSF), the granulocyte colony stimulating factor (G-CSF), the macrophage colony stimulating factor (M-CSF), the stem cell factor and the erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, the proteins of other invasive organisms, such as the 17-amino acid peptide of the protein c r r cumspo ro z o i t of the plasmodium falciparum known as RII.

B. Hormones, Vitamins, e t c.

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

C. Polyalqui logs, polysaccharides, e t c.

Also, the polyalkylene glycols with the desired polynucleotides / polypeptides can be included. In a preferred embodiment, the pol i to i i engl i col is polyethylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE- of xt ran. Also, chitosan and poly (lactide-co-glycolide).

D. Lipids and Liposomes The desired polynucleotide / polypeptide can also be encapsulated in lipids or packaged in liposomes before being delivered to the subject or to cells derived therefrom.

Lipid encapsulation is generally complemented by using liposomes that are capable of a stable bond or of entrapment and retention of the nucleic acid. The ratio of the condensed polynucleotide to the lipid preparation may vary but will generally be about 1: 1 (mg of DNA: micromoles of lipids), or more of lipid. For a review of the use of liposomes as carriers for the delivery of nucleic acids, see Hug Y Sleight (1991) Biochim. Biophys. Minutes 1097: 1-17; Straubinger (1983) Meth. Enzymol. 101: 512-527.

Liposome preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate the intracellular delivery of the plasmid DNA (Felgner (1987) Proc. Nati, Acad. Sci. USA 84: 7413-7416); mRNA (Malone (1989) Proc. Nati, 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 [l-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 s upra). Other commercially available liposomes include transfectace (DDAB / DOPE) and DOTAP / DOPE (Boehringer). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, for example Szoka (1978) Pro c. Na t i. A ca d S ci. USA 75: 4194-4198; WO 90/11092 for a description of the synthesis r of the liposomes of DOTAP (1, 2-bis (oleoyloxy) -3- (t rime t i 1 amon i o) propane).

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), dioleoi 1 fos fa t id 1 ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate proportions. Methods for making the liposomes using these materials are well known in the art.

The liposomes can comprise multilamellar vesicles (MLV), small unilamellar vesicles (SUV), or large unilamellar vesicles (LUV). The different nucleic acid ornamented complexes are prepared using methods known in the art. See for example, Straubinger (1983) Meth. Immunol. 101: 512-527; Szoka (1978) Proc. Nati Acad. Sci. USA 75: 4194-4198; Papahadj opoulos (1975) Biochim. Biophys. Acta 394: 483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acta 443: 694; Ostro (1977) Biochem. Biophys. Res. Commun. 76: 836; Fraley (1979) Proc. Nati Acad. Sci. USA 76: 3348); Enoch & Strittmatter (1979) Proc. Nati Acad. Sci. USA 76: 145; Fraley (1989) J. Biol. Chem. (1980) 255: 10431; Szoka & Papahadj opoulos (1978) Proc. Nati Acad. Sci. USA 75: 145; and Schaefer-Ridder (1982) Science 215: 166.

E. Lipoproteins In addition, lipoproteins are included with the polynucleotides / polypeptides to be delivered. Examples of lipoproteins to be used include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments or fusions of these proteins can also be used. Also, modifications of lipoproteins that occur naturally can be used, such as acetylated LDL. These 1 proteins can be a target for the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if the lipoproteins are included with the polynucleotide to be delivered, no other target ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a portion of protein. Portions of protein are known as apopro teins. In the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; Cl, CU, CIII.

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

The amino acids of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54: 699; Law (1986) Adv. Exp. Med. Biol. 151: 162; Chen (1986) J Biol Chem 216: 12918; Kane (1980) Proc Nati Acad. Sci USA 77: 2465; and Utermann (1984) Hum Genet 65: 232.

Lipoproteins contain a variety of lipids including triglycerides, cholesterol (free and esters), and phospholipids. The composition of lipids varies in lipoproteins that occur naturally. 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 is chosen to assist in the conformation of the apoprotein for the binding activity of the receptor. The composition of the lipids can also be chosen to facilitate hydrophobic interaction and association with the binding molecules of the polynucleotide.

Naturally occurring lipoproteins can be isolated from the serum by means of an ultration, for example. Such methods are described in Meth.

Enzymol. (supra); Pitas (1980) J. Biochem. 255: 5454-5460 and Mahey (1979) J Clin. Invest 64: 743-750. The lipoproteins can also be produced in vitro or by recombinant methods by the expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15: 403 and Raddíng (1958) Biochim Biophys Acta 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Technologies, Inc., Stoughton, Massachusetts, USA. The additional description of lipoproteins can be found in Zuckermann et al. PC / US 97/14465.

F. Political Agents Polycatalytic agents can be included, with or without lipoproteins, in a composition with the desired amount that is to be delivered.

Polymeric agents typically exhibit a net positive charge at a relevant physiological pH and are capable of neutralizing the electrical charge of the 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 polypeptides useful as polycationic agents: polylysine, po 1 i a rg in i n a, po 1 i o rn i t i na, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, chromosomal proteins that are not histone, coat proteins of DNA viruses, such as (X174, transcriptional factors also contain binding domains) to DNA and therefore may be useful as nucleic acid condensation 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 to DNA sequences.

Organic polycation agents include: spermine, spermidine, and purtrescin.

The dimensions of the physical properties of a polya tional agent can be extrapolated from the above list, to construct other polypeptide agents or to produce synthetic polycatalytic agents. . Synthetic polycathionic agents that are useful include, for example, DEAE-dexthan, polybrene, Lipofectin ™, and the 1 ipo fe ctAMINE ™ are monomers that form poly cationic complexes when combined with polynucleotides / polypeptide .

Immunodiagnostic assays The Neisseria antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Nei antibodies can be used to detect antigen levels). Immunoassays based on well-defined recombinant antigens can be developed to replace invasive diagnostic methods. Antibodies to Neisseria proteins within biological samples, including for example, blood or serum samples, can be detected. The design of immunoassays is subject to a large number of variations, and a variety of these are known in the art. The protocols for immunoassay can be based, for example, on competition, or on direct reaction, or on sandwich assays. The protocols can also, for example, use support solids, or they can be by immunoprecipitation. Most trials involve the use of labeled antibodies or polypeptides; labels can be, for example, fluorescent, chemiluminescent, radioactive, or dyed molecules. The assays that amplify probe signals are also known; examples of which are assays that use biotin and avidin, and immunoassays mediated and labeled with enzymes, such as ELISA assays.

Suitable equipment for immunodiagnostics and containing the appropriate labeled reagents are constructed by packing the appropriate materials, including the compositions of the invention, in suitable containers, together with reagents and remaining materials (eg, suitable buffers, saline solutions, etc.) required for the conduct of the test, as well as an adequate set of test instructions.

Hibri di za tio n of the Acci e s Nu c e s Hybridization refers to the association of two nucleic acid sequences to one another via a hydrogen bond. 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 each other under conditions that favor hydrogen bonding. Factors that affect this link include: the type and volume of the solvent; the reaction temperature; the time of hybridization; the agitation; agents that block non-specific binding of the sequence from the liquid phase to the solid support (reagent from Denhardt or BLOTTO); the concentration of the sequences; the use of compounds to increase the proportion of association of the sequences (dextran sulfate or polyethylene glycol); and the severity of the washing conditions after the hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 a 9. 57 The 'severity' refers to the conditions in a hybridization reaction that favors the association of very similar sequences on sequences that are different.

For example, the combination of temperature and salt concentration should be chosen to be appropriate at 120 to 200 ° C below the calculated average temperature of the hybrid under study. The conditions of temperature and saline conditions can often be determined empirically in preliminary experiments preliminary experiments in which samples of the genomic DNA immobilized in the filters are hybridized to the sequence of interest and then washed under conditions of different severity. See Sambrook et al. on page 9.50.

The variables to be considered when performing, for example, a Southern spotting are (1) the complexity of the DNA to be run and (2) the homology between the probe and the sequences that are detected. The total amount of the fragments to be studied can vary by a magnitude of 10, from 0.1 to 1 μg for a plasmid or digestion phage at 10 ~ 9 to 10"8 g for a single copy gene in a genome highly complex eukaryotic For polynucleotides of lesser complexity, the staining is substantially shorter, the time and the exposure times are shorter, a smaller amount of starting polynucleotides, and a lower specific activity of the probes that are going to be used, for example, a yeast gene of a single copy can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, generating staining for two hours, and hybridization for 4-8 hours with a probe of 108 cpm / μg For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, spotting overnight, and hybridization overnight in the presence of 10% dextran sulfate using a probe greater than 108 cpm / μg, resulting in an exposure time of approx. 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 the probe is not 100% homologous to the fragment. Other variables commonly found include the length and total G + C content of the hybridization sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all these factors can be approximated by a single equation: - - Tm = 81 + 16.6 (log? 0Ci) + 0.4 [% (G + C)] - 0.6 (% formamide) - 600 / «- 1.5 ( % unpaired)]. where Ci is the concentration of salt (monovalent ions) and n and s the length of the hybrid in the base pairs (slightly modified from Meinkoth &Wahl (1984) An al., Bi or ch., 138: 267-284).

In the design of a hybridization experiment, some factors that affect the hybridization of the nucleic acid can be conveniently altered. The temperature of the hydration and the washings and the salt concentration during the washings are the simplest to adjust. As the temperature of the hybridization increases (ie the severity), it becomes less frequent for the hybrid to occur between the strands that are non-homologous, and as a result, the funds decrease. If the radio-labeled probe is not completely homologous to the immobilized fragment (as is often the case in the gene family and in the int-species hybridization experiments), the hybridization temperature should be reduced, and the Fund should be increased. The temperature of the washings affects the intensity of the hybridization band and the degree of the bottom in a similar manner. The severity of the washings was reduced with the decrease in salt concentrations.

In general, suitable hybridization temperatures in the presence of 50% formamide are at 42 ° C for a probe with a 95% to 100% homology to the target fragment, at 37 ° C for a 90% to 95% homology %, and 32 ° C for a homology of 85% to 90%. For lower homologies, the formamide content should decrease and the temperature should be adjusted appropriately, using the above equation. If the homology between the probe and the target fragment is not known, the simplest approach is to start both with the hybridization probe and with the washing conditions that are not severe. If the nonspecific bands or the high background are observed after an additional period, the filter can be washed at a higher severity and can be re-exposed. If the time required for exposure makes this method impractical, hybridization should be tested and the severities should be tested in hybridization and / or washing in parallel.

In s ayos with Son da de Aci do Nu cl i co Methods such as PCR, tests with DNA probes, branched, or staining or transfer techniques using nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. It is considered that a probe "hybridizes" with a sequence of the invention if it can form a double-stranded complex, which is sufficiently stable to be detected.

The nucleic acid probes will hybridize to the Neisseria nucleotide sequences of the invention (including sense and antisense strands). Although many different nucleotide sequences will encode the amino acid sequence, the sequence of - 1 - Native Neisseria is preferred because it is the current sequence present in the cells. The mRNA represents a coding sequence and therefore a probe should be complementary to the coding sequence; the single-stranded cDNA is complementary to the mRNA, and therefore a cDNA probe should be complementary to the non-coding sequence.

The sequence of the probe need not be identical to the Neisseria sequence (or its complement) - some variation in sequence and length can lead to an increased assay sensitivity if the nucleic acid probe can form a double strand with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the double form. The additional sequence of Neisseria can also be useful as a label to detect the double form. For example, a non-complementary nucleotide sequence can be attached to the 5 'end of the probe, with the remainder of the probe sequence being complementary to a Neisseria sequence. Alternatively, longer or non-complementary base sequences can be spaced within the probe, provided that the probe sequence has sufficient complement to that of a Neisseria sequence to hybridize with the probe. same and therefore form a double strand that can be det ected.

The sequence and exact length of the probe will depend on the conditions of hybridization, such as temperature, saline condition and the like. For example, diagnostic applications, which depend on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be more Short that. Short primers usually require colder temperatures to form sufficiently stable hybrid complexes with the standard. -''o jetivos, original, as a pattern or template. After a minimum amount I nucleic acid targets are generated by the polymerase, can be detected by 1"I more traditional methods, such as spotting or Southern blots When using the spotting or trnsference method Southern, the labeled probe will hybridize to the Neisseria sequence (or its complement).

Also, the mRNA or cDNA can be detected by traditional staining techniques described in Sambrook et al [s upra], the mRNA, or cDNA generated from the mRNA using a polymerase enzyme, can be purified and separated using electrophoresis in gel. The nucleic acids in the gel are then transferred to 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 duplicates containing the labeled probe are detected. Typically, the probe is labeled with a radioactive portion.

- BRIEF DESCRIPTION OF THE DRAWINGS - Figures 1-7 show the biochemical data and the analysis of the sequence belonging to Examples 1, 2, 3, 7, 13, 16 and 19, respectively, with ORFs 40, 38, 44, 52, 114, 41 and 124. Ml and M2 are molecular weight markers. The arrows indicate the position of the main recombinant product or, in the Western blots, the position of the main immuno-reactive band of -N. meningi t idi s. TP indicates the total protein extract of N. meningitidis; OMV indicates the preparation of the vesicle of the outer membrane of N. meningi tidi s. The results of bacterial tests: a diamond () shows preimmune data; a triangle (A) sample, GST control data; a circle (•) shows the data with the recombinant protein of N. meningitidis. The computer analysis shows a graph of hydrophilicity (upper), a graph of antigenic index (medium), and an AMPHI analysis (lower). The AMPHI program has been used to predict the epitopes of T cells [Gao et al. (1989) J. Immunol. 143: 3007; Roberts et al. (1996) AIDS Res Hum Retrovir _12_: 593; Quakyi et al. (1992) Scand J Immunol suppl. 11: 9) and is available in the Protean package from DNASTAR, Inc. (1228 South Park Street, Madison, Wisconsin 53715 USA).

EXAMPLES The examples describe the nucleotide sequences that have been identified in N. meningitidis, together with their putative translation products. All nucleic acid sequences are complete, that is, they encode less than the full-length native type proteins. It is believed in the present that none of the sequences of AD? described here have significant counterparts in N. gonorrhea e.

The examples are generally of the following format: • a nucleotide sequence that has been identified in N. meningitidis (strain B) - - • the putative translation product of this sequence • a computer analysis of the translation product based on the comparisons of the database • a corresponding gene and the sequence of the protein identified in N. meningitidis (strain A) • a description of the characteristics of the proteins indicating that they could be suitable antigens • the highlights of the biochemical analysis (expression, purification, ELISA, FACS, etc.).

The examples typically include details of the sequence homology between species and strains. Proteins that are similar in sequence are generally similar in structure and function, and homology often indicates an evolutionary or common origin. The comparison with the sequences of proteins of known function is widely used as a guide for the assignment of the function of the putative protein to a new sequence and has proved particularly useful in the analyzes of the complete genome.

Sequence comparisons are performed in NCBI (t tp: / www. Ncb i.nlm.n ih.gov) using the algorithms BLAST, BLAST2, BLASTn, BLASTp, BLASTn, BLASTx, & tBLASTx [for example, see also Altschul et al. (1997) Gapped BLAST and PSI-BLAST; a new generation of protein database search programs. Nu cl ei c A ci ds R es ea ch 25: 2289-3402]. Investigations were performed against the following databases: non-redundant sequences GenBan k + EMBL + DDB J + PDB and the sequences of the CDS translations of GenBank + PDB + SwissProt + SPupdate + PIR non-redundant.

The points within the nucleotide sequences (eg, position 288 in Example 12) represent nucleotides that have been arbitrarily introduced to maintain a reading structure. Of the same ma was, the underlined nucleotides doubly were eliminated. Lowercase letters (for example-position 589 in Example 12) represent ambiguities that arose during the alignment of the independent sequencing reactions (some of the nucleotide sequences in the examples are derived from the combination of the results of two or more experiments).

The nucleotide sequences were scanned or explored in all six reading structures to predict the presence of hydrophobic domains using an algorithm based on the statistical studies of Esposti et al. [Critical evaluation of the hydropathy of membrane proteins (1990) E u r J B i o ch em 190: 207-219]. These domains represent potential transmembrane regions or hydrophobic leader sequences.

Open reading structures were predicted from fragmented nucleotide sequences using the ORFFINDER program (NCBI).

- The underlined amino acid sequences indicate the possible transmembrane domains or leader sequence in the ORFs, as predicted by the PSORT algorithm (http://www.psort.nibb.ac.jp). Functional domains were also predicted using the MOTIFS program (G C G Wisconsin &PROSITE).

Several tests were used to assess the immunogenicity of the proteins identified in the examples. For example, proteins can be expressed recombinantly and used to select sera from patients by immunostaining. A positive reaction between the protein and the patient's serum indicates that the patient has previously generated an immune response to the protein in question, ie the protein is an immunogen. This method can also be used to identify immunodominant proteins.

The recombinant protein can also be conveniently used to prepare antibodies for example in the mouse. These can be used to direct confirmation that a protein is located on the cell surface. The labeled antibody (for example, fluorescent labeling for FACS) can be incubated with intact bacteria and the presence of labels on the bacterial surface confirms the location of the protein.

In particular, the following methods (A) to (S) is used to express, purify and characterize biochemically the proteins of the invention: A) Preparation of Chromosomal DNA Strain 2996 of N. Men in gi ti di s was grown to an exponential phase in 100 ml of GC medium, harvested by centrifugation and resuspended in 5 ml of buffer (20% Sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH 8 ). After 10 minutes of incubation on ice, the bacteria were used by the addition of 10 ml of a lysis solution (50 mM NaCl, 1% Na-Sarcosil, 50 μg / ml Proteinase K), and the suspension was incubated at 37 ° C for 2 hours. Two extractions were carried out with phenol (balanced at pH 8) and one extraction with Ch C 13 / i s or i 1 to 1 coh ol (24: 1) The DNA was precipitated by the addition of 0.3 M sodium acetate and 2 volumes of ethanol, and was collected by centrifugation. The pellets were washed once with 70% ethanol and redissolved in 1 ml of buffer (10 M Tris-HCl, 1 mM EDTA, pH 8). The concentration of DNA was measured - by reading OD at 260 nm.

B) Design of the oligonucleotides The synthetic oligonucleotide primers were designed based on the coding sequence of each ORF, using (a) the meningococcal B sequence when available, or (b) the gonococcal / meningococcal A sequence, adapted to the codon of choice of use of meningococcus as needed. Any predicted signal peptide was omitted by deducing the sequence of the amplification primer at the 5 'end immediately in the 3' direction from the predicted leader sequence.

The 5 'primers include two restriction enzyme recognition sites (Ba HI-Ndel, BamHl-Nhel, or EcoRI-Nhel, which depend on the restriction pattern of the gene itself); the 3 'primers include an 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 BamHl-Xhol or EcoRI -Xh oí), and pET21b + ( using either Ndel-Xnol or Nhel-Xhol). tail of end primer 5 CGCGGATCCCATATG (BamHI-Ndel) CGCGGATCCGCTAGC (BamHI-Nhel) CCGGAATTCTAGCTAGC (EcoRI-Nhel) tail of primer of end 3 CCCGCTCGAG (Xhol) As well as they contain the recognition sequences of the restriction enzyme, the primers include the nucleotides that hybridize to the sequence to be amplified. The number of nucleotides hybridized zan t is dependent on the melting temperature of the complete primer and was determined for each primer using the formulas: Tm = 4 (G + C) + 2 (A + T) (glue excluded) Lm 64.9 + 0.41 GC) - 600 / N (complete primer) The average melting temperature of the selected oligos was 65-70 ° C for the complete oligo and 50-55 ° C for the hybridization region alone.

Table I shows the forward and reverse primers used for each amplification. The oligos were synthesized by a DNA / RNA Synthesizer Perkin Elmer 394, eluted from the columns in 2 ml of NH 4 OH, and deprotected by 5 hours of incubation at 56 ° C. The oligos were precipitated by the addition of 0.3 M Na-acetate and 2 volumes of ethanol. The samples were then centrifuged and the pellets resuspended in either 100 μl or 1 ml of water. The - D260 was determined using a Perkin Elmer Lambda Bio spectrophotometer and the concentration was determined and adjusted to 2-10 pmol / μl.

C) Amplification The standard PCR protocol was as follows: 50-200 ng of genomic DNA was used as a standard in the presence of 20-40 μM of each oligo, 400-800 μM of solution dNTPs, lx PCR buffer (which include -1.5 mM MgCl2), 2.5 units of the DNA polymerase Ta ql (using the Per-Elmer AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase, or the Tahara Shuzo Taq polymerase).

In some cases, PCR was optimized by the addition of 10 μl of DMSO or 50 μl of 2M betaine.

After a warm start (adding the polymerase during a 3-minute preliminary incubation of the complete mixture at 95 ° C), each sample underwent a double stage amplification: the first five cycles were performed using the temperature of hybridization of one of the oligos excluding the tails of the restriction enzymes, followed by 30 cycles performed according to the hybridization temperature of the full-length oligos. The cycles were followed by a 10-minute extension stage at 72 ° C.

The standard cycles were as follows The lengthening time varied according to the length of the ORF that. it's going to be amplified.

Amplifications were performed using either the Perkin Elmer GeneAmp 9600 or 2400 PCR System. To verify the results, 1/10 of the amplification volume was loaded on a 1-1.5% agarose gel and - the size of each fragment amplified was compared to a molecular weight marker of the DNA.

The amplified DNA was either directly loaded onto a 1% agarose gel or first precipitated with ethanol and resuspended in a suitable volume to be loaded on a 1% agarose gel. The DNA fragment corresponding to the right band that was eluted and purified from gel, using the Qiagen Gel Extraction Kit, following the manufacturer's instructions. The final volume of the DNA fragment was 30 μl or 50 μl in either water or in 10 mM Tris, pH 8.5.

D) Digestion of PCR fragments The purified DNA corresponding to the amplified fragments was divided into 2 aliquots and digested twice with: - Nd e l / Xh or l or Nh e l / Xh or l for the cloning in pET-21b + and the additional - - expression of the protein as a His-tag fusion in the C-terminal BamHI / XhoI or EcoRI / Xhol for cloning into pGEX-KG and the express additional ion of the protein as a GST fusion at the C-terminus - EcoRI / PstI, EcoRI / SalI, SalI / PstI for cloning into pGex-His and additional expression of the protein as a His-tag fusion in the C-terminal Each fragment of purified AD'N was incubated (37 ° C for 3 hours overnight) with 20 units of each restriction enzyme (New England Biolabs) in a final volume of either 30 or 40 μl in the presence of the buffer appropriate. 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 μl of either water or 10 mM Tris-HCl, pH -8.5. The final concentration of DNA was determined by electrophoresis on a 1% agarose gel in the presence of the titrated molecular weight marker.

E) Digestion of the cloning vectors (pET22B, pGEX-KG, pTRC-His, A and pGex-His) μg of the plasmid was double-digested with 50 units of each restriction enzyme in 200 μl of the reaction volume in the presence of the appropriate buffer by incubation overnight 37 ° C. After loading the complete digestion on a 1% agarose gel, the band corresponding to the digested vector was purified from the gel using the Extraction of Qiagen QIAquick Gel and DNA was eluted in 50 μl of 10 mM Tris-HCl pH 8.5. The DNA concentration was evaluated by measuring the OD260 of the sample, and adjusted to 50 μg / μl. 1 μl of the plasmid was used for each cloning procedure.

The pGEX-His vector is a modified pGEX-2T-vector that carries a region encoding six histidine residues in the 5 'direction to the thrombin cleavage site and containing the multiple cloning site of the pTRC99 vector (Pharmacia ).

F) Cloning The fragments corresponding to each ORF, previously digested and purified, were ligated with both pET22B and pGEX-KG. In a final volume of 20 μl, a 3: 1 fragment molar fraction was ligated using 0.5 μl of NEB T4 DNA ligase (400 units / μl), in the presence of the buffer supplied by the manufacturer. The reaction was incubated at room temperature for 3 hours.

In some experiments, the ligation was performed using the "Rapid Ligation Kit" from Boehringer, following the manufacturer's instructions.

To introduce the recombinant plasmid into a suitable strain, 100 μl of competent DH5 cells of E were incubated. c or i with the ligase reaction solution for 40 minutes on ice, then at 37 ° C for 3 minutes, subsequently, after adding 800 μl of the LB broth, again at 37 ° C for 20 minutes. The cells were centrifuged at a maximum speed in an Eppendorf microcentrifuge and resuspended in approximately 200 μl of the supernatant. The suspension was plated on ampicillin LB (100 mg / ml).

The selection of the recombinant clones was performed by the growth of 5 colonies randomly chosen overnight at 37 ° C in either 2 ml DE (clones pGEX or pTC) or 5 ml (clones pET) broth LB + 100 μg / ml of ampicillin. The cells were pelleted and the DNA was extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions, at a volume of 30 μl. 5 μl of each individual miniprep (approximately 1 g) were digested with either Nd / Xh ol or Ba mHl / Xh oly were loaded on the complete digestion on a 1-1.5% agarose gel (depending on the size of the insert expected ), in parallel with the -molecular weight marker (1Kb DNA Ladder, GIBCO). The selection of positive clones was made based on the correct insert size.

G) Expression Each ORF cloned in the expression vector was transformed into the suitable strain for the expression of the product of the recombinant protein. 1 μl of each construct was used to transform 30 μl of BL21 of E. c or l i (vector pGEX), TOP 10 of E. c or l i (vector pTRC) or BL21-DE3 of E. col i (pET vector), as described above. In the case of the vector pGEX-His, the same strain of E. col i (W3110) was used for cloning and initial expression. The unique recombinant colonies were inoculated in 2 ml of LB + Amp (100 μg / ml), incubated at 37 ° C overnight, then diluted 1:30 in 20 ml of LB + Amp (100 μg / ml) in 100 ml flasks, making sure that the ODgQO value varies between 0.1 and 0.15. The flasks were incubated at 30 ° C in rotating water bath agitators until the OD indicated adequate exponential growth - for the induction of expression (0.4-0.8 OD for the pET and pTRC vectors; 0.8-1 OD for the vectors pGEX and pGEX-His). For the pET vectors, pTRC and pGEX-His, the expression of the protein was induced by the addition of 1 M of IPTG, while in the case of the pGEX system the final concentration of the IPTG was 0.2 mM. After 3 hours of incubation at 30 ° C, the final concentration of the sample was verified by OD. To verify the expression, 1 ml of each sample was removed, centrifuged in a microcentrifuge, the pellets were resuspended in PBS, and analyzed by 12% SDS-PAGE with Coomassie Blue staining. The entire sample was centrifuged at 6000 g and the pellets were resuspended in PBS for future use.

H) Large scale purification of GST fusion proteins The single colony was grown overnight at 37 ° C on an LB + Amp agar plate. Bacteria were inoculated in 20 ml - of liquid culture of LB + Amp in a water bath shaker and grown overnight. Bacteria were diluted 1:30 in 600 ml of fresh edium and allowed to grow at the optimum temperature (20-37 ° C) to OD550 0.8-1. Protein expression was induced with 0.2 mM IPTG followed by three hours of incubation. The culture was centrifuged at 8000 rpm at 4 ° C. The supernatant was discharged and the bacterial pellets were suspended in 7.5 ml of cold PBS. The cells were broken by sonication on ice during seconds at 40W using a sound system Branson B-15, se. they were frozen and rinsed twice and centrifuged again. The supernatant was collected and mixed with 150 μl of Glu t a t i ona-S epharo s 4B (Pharmacia) resin (previously washed with PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4 ° C. The resin was washed twice with 10 ml of cold PBS for 10 minutes, resuspended in 1 ml of cold PBS, and loaded onto a disposable column. The resin was washed twice with 2 ml of cold PBS until a total flow of OD28O of 0.02-0.06 was reached. The GST fusion protein was eluted by the addition of 700 μl cold Glutathione buffer (10 mM reduced glutathione, 50 mM Tris-HCl) and the fractions were collected until the OD28O was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel using either the standard broad molecular weight range of Biorad SDS-PAGE (Ml) (200, 116.25, 97.4, 66.2, 45, 31, 21.5, 14.4 , 6.5 kDa) or the Marker -Amersham Rainbow (M2) (220, 66, 46, 30, 21. '5, 14.3 kDa) as patterns. According to the molecular weight of GST. was 26 kDa, this value must be added to the molecular weight of each GST fusion protein.

) Analysis of the solubility of the His fusion To analyze the solubility of the His fusion expression products, the pellets of 3 ml cultures were resuspended in the Ml buffer [500 μl of PBS pH 7.2]. 25 μl of lysozyme (10 mg / ml) were added and the bacteria incubated - for 15 minutes at 4 ° C. The pellets were sonicated for 30 seconds at 40W using a Branson B-15 sonicator, frozen and washed twice and then dried again in pellets and the supernatant by a centrifugation step. The supernatant was collected and the pellets were resuspended in M2 buffer [8 M urea, 0.5 M NaCl, 20 mM imidazole and 0.1 M NaH2 P0] and incubated for 3 to 4 hours at 4 ° C. After centrifugation, the supernatant was collected and the pellets were resuspended in M3 buffer [guanidinium-6M HCl, 0.5M NaCl, 20mM imidazole and 0.1M? AI_2P? 4] overnight at 4 ° C. Supernatants from all stages were analyzed by SDS-PAGE.

J) Large scale purification of His fusion A single colony was grown overnight at 37 ° C on an LB + Amp agar plate. The bacteria were inoculated in 20 ml of the liquid LB + Amp culture and incubated overnight in a water bath shaker.

- The bacteria were diluted 1:30 in a fresh 600 ml medium and allowed to grow to the optimum temperature (20-37 ° C) to OD550 0.6-0.8. Expression of the protein was induced by the addition of 1 mM of IPTG and the culture was further incubated for three hours. The culture was centrifuged at 8000 rpm at 4 ° C, the supernatant discharged and the bacterial pellets resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM -imidazole, pH 8) for soluble proteins or (ii) buffer B (8M urea, 10 mM TrisHCl, 100 mM phosphate buffer, pH 8.8) for insoluble proteins.

The cells were disrupted by sonication on ice for 30 seconds at 40W using a Branson B-15 sonicator, frozen and rinsed twice and centrifuged again.

For the insoluble proteins, the supernatant was stored at -20 ° C, while the pellets were resuspended in 2 ml of the - shock absorber C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5 ) and were treated in a homogenizer for 10 cycles. The product was centrifuged at 13,000 rpm for 40 minutes.

The supernatants were collected and mixed with 150 μl of Ni2 + -resin (Pharmacia) (they were previously washed with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4 ° C. The resin was washed twice with 10 ml of buffer A or B for 10 minutes, resuspended in 1 ml of buffer A or B and loaded onto a disposable column. The resin was washed either at (i) 4 ° C with 2 ml of cold buffer A or (ii) at room temperature with 2 ml of buffer B, until the total flow reached OD28O of 0.02-0.06.

The resin was washed with either (i) 2 ml of the 20 mM imidazole buffer, cold (-300 M NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8) or (ii) buffer D (8 M urea, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3) until the total flow reached O.D28O of 0.02-0.06. The His fusion protein was eluted by the addition of 700 μl of either (i) buffer A in cold elution (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8) or (ii) B elution buffer (8M urea, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5) and the fractions were collected until the -D280 was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel.

K) Renaturing of His fusion proteins % glycerol was added to the denatured proteins. The proteins were then diluted to 20 μg / ml using the dialysis buffer I (10% glycerol, 0.5 M arginine, 50 mM phosphate buffer, 5 mM reduced glutathipine, 0.5 mM oxidized glutathione, 2 M urea, pH 8.8) and dialyzed against the same shock absorber at 4 ° C for 12- - -14 hours. The protein was further dialyzed against the dialysis buffer II (10% glycerol, 0.5M arginine, 50mM phosphate buffer, 5mM reduced glutathione, 0.5mM oxidized glutathione, 2M urea, pH 8.8) for 12-14 hours at 4 hours. ° C. The concentration of the protein was evaluated using the following formula: Protein (mg / ml) = (1.55 x OD280) - (0.76 x OD260) L) Large scale purification of His fusion 500 ml of bacterial cultures were induced and the fusion proteins were obtained soluble in buffer Ml, M2 or M3 using the procedure described above.

The crude extract of the bacteria was loaded onto a Ni-NTA superflow column (Qiagen) equilibrated with Ml, M2 or M3 buffer depending on the solubilization buffer of the fusion proteins. The non-agglutinated material was eluted by washing the column with the same buffer. The specific protein was eluted with the corresponding buffer-containing 500 mM imidazole and dialyzed against the corresponding buffer without imidazole. After each run, the columns were sanitized by washing with at least two volumes of the 0.5 M sodium hydroxide column and re-equilibrated before the next use.

M) Immunizations of mice μg of each purified protein was used to immunize in rater i tionally to the mice. In the case of ORF 44, CD1 mice were immunized with Al (OH) 3 as an adjuvant on days 1, 21 and 42, and the immunological response was verified or monitored by taking samples on day 56. For the ORF 40, CD1 mice were immunized using Freund's adjuvant, instead of Al (OH) 3, and the same immunization protocol was used, except that the immune response was mediated at day 42, instead of day 56. Similarly, for ORF 38, CD1 mice were immunized with Freund's adjuvant, but the immune response was measured on day 49.

N) ELISA assay (serum analysis) The non-encapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37 ° C. Bacterial colonies were harvested from the agar plates using a sterile dracon brush and inoculated in 7 ml of the Mué 1 Broth ler-Hin t on (Difco) containing 0.25% Glucose. Bacterial growth was monitored or monitored every 30 minutes following the ODg20-bacteria. They grew until the value of 0.3-0 was reached. '4. The culture was centrifuged for 10 minutes at 10,000 rpm. The supernatant was discharged and the bacteria washed once with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then overnight at 4 ° C with shaking. 100 μl of the bacterial cells were added to each well of a 96-well Greiner plate and incubated overnight at 4 ° C. The wells were - then washed three times with the • PBT wash buffer (0.1% Tween-20 in PBS). 200 μl of the saturation buffer (Polyvinylpyridinium 10 to 2.7% in water) was added to each well and the plates were incubated for 2 hours at 37 ° C. The wells were washed three times with PBT. 200 μl of diluted serum (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN3 in PBS) were added to each well and the plates were incubated for 90 minutes at 37 ° C. The wells were washed three times with PBT. 100 μl of rabbit anti-mouse serum conjugated with HRP (Dako), diluted 1: 2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37 ° C. The wells were washed three times with PBT buffer. 100 μl of the substrate buffer for HRP (25 ml of citrate buffer pH 5, 10 mg of O-phenidine and 10 μl of H20) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl of H2SO4 were added to each well and the OD 90 was continued. The ELISA assay was considered positive when the OD490 was 2.5 times the respective pre-immune serum. 0) Bacterial Binding Assay Procedure FACScan The non-encapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37 ° C. Bacterial colonies were harvested from the agar plates using a draconic brush sterile and inoculated into 4 tubes each containing 8 ml of the Mueller-Hinton Broth (Difco) containing 0.25% glucose. The bacterial growth was monitored every 30 minutes following the ODg20- The bacteria were allowed 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 discharged and the pellets were resuspended in a blocked buffer (BSA 1%, NaN3 0. 4%) and centrifuged for 5 minutes at 4000 rpm. The cells were resuspended in the blocked buffer and reached ODg20 of 0.07. 100 μl of bacterial cells were added to each well of a 96-well Costar plate. 100 μl of the diluted serum (1: 200) (in buffer and blocked) was added to each well and the plates were incubated for 2 hours at 4 ° C. The cells were centrifuged for 5 minutes at 4000 rpm. The supernatant was aspirated and the cells were washed by the addition of 200 μl / well of the blocking buffer in each well. 100 μl of the goat anti-mouse F (ab) 2 conjugate R- P i coeri t ri na, diluted 1: 100, was added to each well and the plates were incubated for 1 hour at 4 ° C. The cells were separated by centrifugation at 4000 rpm for 5 minutes and washed with the addition of 200 μl / well of blocking buffer. The supernatant was aspirated and the cells resuspended in 200 μl / well of PBS, 0.25% formaldehyde. The samples were transferred to FACScan tubes and read. The condition for adjusting the FACScan was: FLI on, FL2 and FL3 off, FSC-H threshold: 92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gain 7.1; FL-2 PMT: 539; compensation values: 0.

- - OMV Preparations The bacteria were grown overnight in 5 GC plates, harvested with a hook and resuspended in 10 ml of Tris-HCl. The heat inactivation was carried out at 56 ° C for 30 minutes and the bacteria were broken by sonication for 10 minutes on ice (heavy cycle 50%, exit 50%). The unruptured cells were removed by centrifugation at 5000 g for 10 minutes and the total cell shell fraction was recovered by centrifugation at 50000 g at 4 ° C for 75 minutes. To extract proteins from the cytoplasmic membrane of the crude outer membranes, the entire fraction was resuspended in 2% sarcosil (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000 g for 10 minutes to remove the aggregates, and the supernatant was ulcerated and subsequently flushed at 50000 g for 75 minutes to obtain pellets from the outer membranes. The outer membranes were resuspended in 10-mM Tris-HCl, pH 8 and the concentration of the protein was measured by the BioRad Protein assay, using BSA as a standard.

Q) Preparation of the Complete Excerpts The bacteria were grown overnight on a GC plate, harvested with a hook and resuspended in 1 ml of 200 mM Tris-HCl. The inactivation by heat was carried out at 56 ° C for 30 minutes.

A) Staining or Western transfer The purified proteins (500 ng / lane), the vesicles of the outer membrane (5 μg) and total cell extracts (25 μg) derived from the MenB 2996 strain were loaded on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was carried out for 2 hours at 150mA at 4 ° C, in transfer buffer (0.3% Tris base, 1.44% glycine, 20% methanol). The membrane was saturated overnight by incubation at 4 ° C in saturation buffer - (10% skimmed milk, 0.1% Triton XlOO in PBS). The membrane was washed twice with wash buffer (3% skimmed milk, 0.1% Triton XlOO in PBS) and incubated for 2 hours at 37 ° C with mouse serum diluted 1: 200 in wash buffer. The membrane was washed twice and incubated for 90 minutes with a 1: 2000 dilution of anti-mouse Ig labeled with horseradish peroxidase. The membrane was washed twice with 0.1% Triton XlOO in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by the addition of water.

S) Bactericidal test The MC58 strains were grown overnight at 37 ° C on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7 ml of the Mué 11 er-Hi n t on broth. The suspension was incubated at 37 ° C in a nutator and allowed to grow until ODg20 was 0.5-0.8. The culture was divided into aliquots in 1.5 ml sterile Eppendorf tubes and centrifuged for 20 minutes at maximum speed in a microcentrifuge. The pellets were washed once in a > Gey buffer (Gibco) and resuspended in the same buffer to an OD620 of 0.5, diluted 1: 20000 in the Gey buffer and stored at 25 ° C. 50 μl of the Gey buffer / 1% BSA was added to each well of a 96-well tissue culture plate. 25 μl of the mouse serum (1: 100 in the - Gey shock absorber / 0.2% BSA, to each well and the plate was incubated at 4 ° C. 25 μl of the bacterial suspension previously described was added to each well. 25 μl of either the normal or heat-inactivated baby rabbit complement (water bath 56 ° C for 30 minutes), were added to each well. Immediately after the addition of the baby rabbit complement, 22 μl of each sample / well was plated on Mué 11 er-Hin t on agar plates. (time 0). The 96 well plates were incubated for 1 hour at 37 ° C with rotation and then 22 μl of each sample / well were placed on Mué 11 er-Hin t on agar plates (time 1). After an overnight incubation, the colonies corresponding to time and time 1 were counted.

Table II gives a summary of the results of cloning, expression and purification.

Example 1 The following partial DNA sequence was identified in N. m in i n gi t i di s (SEQ ID NO: 1): 1 .ACACTGTTGT TGGCAACGGT TCAGGCAAGT GCTAACCAAT GAAGAC5CAAG 51 AAGAAGATTT MMrTTftGRC CCCGTACAAC GCftCTGlTGC CGGCTSATA 101 GTCAATTCCG KGAAAGAI-GG CACGGGAGAA. AAAGifAAAAG TftG? AC? AP * 151 TTCAGATTGG GCAGTATA? TCAACGAGAA AGGftGTACTA ACAGCÍAGAG 201 AAATCACCyT CAAAGCCGGC GACAACCTGA AAATCAAACA AAACGÍ? CACA 251 AACTTCACCT ACTCGCTG? A AAAAGACC C GAAS? TCTGA CCAGTtJTTGG 301 AACTGAAAAA TTAGCGTTTA GCGCAAACGG CAATAAAGTC AACATcíACftA 351 GCGJWACCAA AGGCTTG? AT TTTGCGAAAG AAACGGCTGG sACGAACGgC 401 GACACCACGG TTCATCTGAA CGGTATTGGT TCGACTTTGA CCGAT CGCT 451 GCTGAATACC GGAGCGAGCA CAAACGTAAC CAACGACAAC GTTAD5GATG 501 ACGAGAAAAA ACGTGCGGCA AGCGTTAAAG ACGTATTAAA CGCTGGCTGG 551 AACATTAAAG GCGTTAAACC CGGTACAACA GCTCCGATA ACGTT6ATTT 601 CGTCCGCACT TACGACACAG TCGAGTTCTT GAGCGCAGAT ACGAAAACAA 651 CGACTGTTAA TCTGGAAAGC AAAGACAACG GCAAGAAAAC CSAAGtTAAA 701 ATCGGTGCGA AGACTTCTGT TATTAAAGAA AAAGAC ... This corresponds the amino acid sequence (SEQ ID NO. 2; ORF40) 1 .TLLFATVQAS AHOEEOEEDL YL0PV0RTVA VL1VHSDKEG TGEKEKVEES 61 SDWAVYFUEK GVLTAREITJÍ KAGDNLKIKQ NGTN? TYSL KD TDLTSVG l_l TEKLSGSANG NKVTJGTSD K GLNFAKETAG TNGDTTVH N G? GSTLTDTL LNTGATTKV NDVTDDEKK RAASVfTDVLN AGWNIKGVKP G TASDNVDF 2P1 VRT? DTVEFL SADTKTTTW VESKDNGKK EVKXGAKTSV IKEKD ...

- - Additional work revealed the complete DNA sequence (SEQ ID NO: 3): 1 ATGAACAAAA TATACCGCAT CATTTG_3VAT ACTSCCCTCA ATGCCTGGGT 51 CGTCGTATCC QAGCTCACAC GCAACCACAC CA? ACGCGCC TCCGCAACCG 101 TGAAGACCGC CGTAGTGGCG ACACTGTTGT TTGCAACGGT GCAGGCAAGT 151 GCTAACAATG AAGAGCAAGA AGAAGATTTA TATTTAGACC CCGTACAACG 201 CACTGTTGCC GTGrríSATAG TCAATTCCGA TAAAGAAGGC ACGGGAGAAA 251 AAGAAAAAGT AGAAGAAAAT CAC3.TTGGG CASTATATT CAACGAGAAA 301 GGAGTACTAA CAGCCAGAGA AATCACCCTC AAAGCCGGCG ACAACCTGAA 351 AATCAAACAA AACGGCACAA ACTTCACCTA CT SCTGAAA AAAGACCTCA 401 CASftTCTGAC CA6TGTTSGA? CTGAAAAAT TATCG TTAG CGCAAACGGC 451 AATAAAGTCA ACATCRCAAG CGACACCAAA GGCTTGAATT TTGCGAAAGA 501 AACGGCTGGC ACGAACGGCG ACACCACGGr GC? GCTGAA GGTATTGGTT 551 CGACTT GAC CGATACGCTG CTGAATACCG GAjGCGACCAC AAACGTAACC 601 AACGACAACG TTACCGATGA CGAGAAAAAA CGTGCGGCAA GCGTTAAAGA 651 CGTATTAAAC GCTGGCTGGA ACATTAAAGG CGTTAAACCC GGTACAACAG 701 CTTC 5A.TAA GTTGAT TC GTCCGCACTT ACGACACAGT CGAGTTCTTG 751 AGCGCAGATA CGAAAACAAC GACTGTTAAT GTGGñA? GCA AAGACAACGG 6 ?? CAAGAAAACC GAAGTTAAAA TCGG GCGAA GACTTCTGTT ATTAAAG? AA 851 AASACGGTAA GTTGGTTACT GGTAAAGACA AAGGCGAGAA TGGTTCTTCT 901 ACAGACGAAG GCGAAGGCTT AGTGACTGCB AAAGAAGTGA TTGATGCAGT 951 AAACAAGGCT GGTTGGñGAA .TGAAAACAAC AACCGCTAAT GGTCfiAACAG 1001 GTCAAGCTGA CAftSTTTGAA ACCGTTACAT CAGGCACAAA TßTA? CCTTT 1051 GCTAGTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCA? GGCAA 1101 CATCACTGTT ATGTATGATG TAAATGTCGG CGATGCCCTA AAOGTCAATC 1151 AGCTGCAAAA CAGCG < ? TTGG AATTTGGArp CCAAAGCGGT TGCAGGTTCT 1201 TCGGGCAAAG TCATCAGCGG CAfcTGTTTCG CCGAGCAAGG GAAAGATGGA 1251 TGAAACCGTC AACATTAATG CCGGCAACAA CATCGAGATT ACCCGCAACG 1301 GAAAAATAT CGACATCGCC ACWCGATGA CCCCGCAGTT TTCCAGCGTT 1351 TCGCTCCGCG CGGGGGCGGA TGCGCCCACT TTGAGCGTGG ATGGGGACGC 1401 ATTGftATGTC GGCAGCAAGA AGGACAACAA ACCCSTCCGC ATACCMÍGG 1451 TCGCCCCGGG CGTTAAAGAG GGGGATGTTA CAAACGTCGC ACAACTfAAA 1501 GGCGTQGCGC AAAACTTGAA CAACCGCATC GACAATGTGG ACGGCAACGC 1551 GCGTGCGGGC ATCGCCCAAG CGitreGCAAc 1601 ATGTGCCCGG CAAGAGTATG ATGGCGATCG GCGGCGGCAC GTATCGCGGC 1651 GAAGCCGGTT ACGCCA CSG CTACTCCAGT ATGTCCG? GC GCGGAAATTG 1731 GATTATCAAA ßdCACGGCTT CCfißCAATTC GGGCGGCCAT TTCGGTfiCTT 1751 CCGCAGCTGT CGGTTATCAG TGSTAA This corresponds to the amino acid sequence (SEQ ID NO: 4; ORF40-1): 1 MNKIYR1IWN SALNflWWVS EtTRNHTKRA SATVKTAVLA GÍÜGATVQAS 51 ANNEBQEEDL AND DPVORTVA VLXVBSDKEG TGEKEKVEEN SEWAV? FMEK 101 GVLTAREIT KAdWOLKIKO NGTNFTYSL KDtTDLTSVG TEKLSGSANG 151 NKVMITSDT CLNE KETAG TNGDGTVHLW GIGSGTDT NTGATTNVT 201 NDHVTDOEKK FAASVKDVP * AGV0.1KGV P G GASDMVDF VRTYDTVEFL 251 SADTKTTTVN VESKDNGKKT BV IßAKTSV tXEKDSKLVT GKDKGENGSS 301 TDEGEGLVTA KEVIDAVHKA. GWBKKtTTAK GQTÍJOAD GE TVTSGTHVTF 351 ASGKGTTATV SKODQGNITV H? DVNVGE? T WWQLQHSGI. NLDSKAV? GS 401 SGKVtSGXVS FSKGKMDETV NIKAGNNIEI GKKID1A TSKTPQFSSV 451 TR SI-GAGADAPT LSVDGDALMV GSKKDNKPVR ITNVAPGVKE CDVT VAO K 501 GVAQNI? NRI DNVDGNARAG XAOAIATAGL VQAYLPGKSM MAIGGGTYRG 551 EAG? AIGYSS ISDGOTW? IK GTASGttSRGÍ. flGASASVSYQ Additional work identified the corresponding gene in strain N. of N. meningitidis (SEQ ID NO: 5): 1 ATGAACAAAA TATACCGCAT CATTTGGAAT AGTtJCCCTCA ATßCCTGNGT 51 aSCCG ?? TCC GAGCTCACAC GCAACCACAC CAAACGCGCC TCCGCAACCG 101 TGAAGACCGC s »tAt. t < SGCG ACACTGTTGT TT CAACGGT TCAGGCGAAT 151 GCTACCGATG AAGATGAAGA AGAAGAGTTA GAATCCGTAC AACGCTCTGT 201 CGTAGGGftGC ATSCAAGCCA GTATGGAAGG CAGCGGCGAA TTGGAAACGA 251 ATCATTATC AATGACTAAC G? CAGCA? GG AATTTCTAGA CCCATACATA 301 G JW3TTACCC TCAAAGCCGG CGACAACCGG AAAATCAAAC AAAACACCAA 351 TGAAAACACC AATGCCAGTA GCTTCACCTA CTCGCTGAAA AAAGACCTCA 401 CAGGCCTGAT CAATGTTGAN ACTGAAAAAT TATCGTTTGG CG & AACGGC 451 AAGAAA6TCA ACATCATAAG CGACACCAAA GGCTTGAATT TCGCGAAAGA 501 AACGGCTGGG ACGAACGGCG ACACCAGCT TCATCTGAAC GGTATCGGTT 551 CGACTTTGAC CGATACGCTT GCGGGTTCTT CTGCTTCTCA CGTK3ATGCG 601 GGTAACCNAA GTACACATTA CACTCGTGCA GCAAGTATTA AGGATGTGTT 651 GAATGCGG6T TGGAAT? TTA AGGGTGTTAA AÍWNGGCTCA ACAACTGGTC 701 AATCAGAAAA TGTCGATTTC GTCCGCACTT ACGACACAGT CGAGTTCTTG 151 AGCGCftGATA CGHAAACAAC GACNGTGAAT GTGGAftAGCA AAGACAACGG 801 CAAGAGAACC GAAGTTAAAA TCGGTGCGAA GACTTCTGTT ATTAAAGAAA 851 AAGACGGTAA GTTGGTTACT GGTAAAGGCA AAGGCGA6AA TGGTTCTTCT 901 ACAGACGAAG GCGAAGGCTT AGTGACTGCA AAftGAAGTGft TTGftTGCAiST 951 AAACAAGGCT GGTTGGAGAA TGAAAACAAC AACCGCTAAT GGTCAAACAG 1001 GGCAAGCTGA CAAGT TGAA ACCGTTACAT CAGGCACAAA TGTAACCTTT 1051 GCTACTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCAAGGCAA 1101 CATCACTGTT ATGTATGATG TAAATGTCGO CGftTGCCCTA AACGTCAATC 1151 AGCTGCAAAA CAGCGGTTGG AATT7G ATT C AAAGCGGT TGC? GGTTCT 1201 TC6GGCAAA / S TCATCAGCGG CAATGTGTCG CCGftGCAAGG GAAAGATGGB 1251 TGAAACCGTC AACATTAATG CCSGCAACAA CATCGAGATT AGCCGCAACG 1301 GTAAAAATAT CGACATCGCC A TTCGATGG CGCCGCAfiTT TTCCAGCGTT 1353 TCGCTCGGCG CGGGGGCAGA TGCGCCCACT TTAAGCGTGG ATGACGAGGG i 401 CGCSTTGAAT GTCGGCAGCA AGGATGCCAA CAAACCCGTC CGCATTACCA - 1452 ATGTCGCCCC GGGCGTTAAA GANGGGGATG TTACAAACGT CNCACAACTT 1501 AAAGGCGTGG CGCAAAACTT GAACAACCGC ATCGACAATG TGGACGGCAA 1551 CGCGCGTGCK GGCATCGCCC AAGCGATTGC AACCGCAGGT CTGGTTCAGG 1601 CGTATCTGCC CGGCAAGAGT ATSATGGCGA TCGGCGGCGG CACTTATCGC 1651 GGCGAAGCCG GTTACGCCAT CGGCTACTCC AGTATTTCCG ACGGCGGAAA 1701 TTGGATTATC AAAGGCACGG CTTCCGGCAA TTCGCGCGGC CATTTCGGTG 1751 CTTCCGCATC TsTCGGTTAT CAGGGGTAA This encodes a protein having the amino acid sequence (SEQ ID NO: 6; ORF4 Oa): 1 MNKIYRIIW * SALNAXVAVS £ STSMBTKR__ SWVKTAV1? TLl.FATVQj! _j 31 ATDEDEEEEL ESVQRSWGS XQASMEGSGE feTISLSM N DSKEVD ?? I 1Ó1 WTLKAGDNI »KGKQNTÍJEHT SASSFTYSLK K <GLIÍ? VX TEKLSFGANG 151 K VNIISDTK GLNFAKETAG TNGOTTVHLN GÍGSTLTOGL AGSSASHVDA 201 GNXSTHYTRA A5IKD NAG tíNlKGVXXG? TTGQsmv? R VRTYDTVfcFL 251 SADTXTTTVN VESKDNGKRT EVKIGMTSV IJOSKDGKIiVT G G GEHQ S 301 TDEGEGLVTA KEVIDAVKKA GHKJMKTTTAN GQTGQADKFE TVTSGTNVTF 351 ASGICGTTATV SKDDQGfclTV MYDVNVGW NVKQLQ SGW N DSKAVJ &S 401 SGKVISGSV5 PSKGKHDETV K1HAGNIEI SfcHGKNIDIA SMA3E ^ FSS 451 SLGAGADAPT SVDDSQftN VGSKDANKPV KtTNVAPGVK XGDVTOVJ? QL 501 KGVAQN NNR IDNVDG &ARA GIAQAIATAG VGAYLPGKS MMAIGGGTYR 551 GEAGYAIG? S SISDGGfcWXI KGASGHS G HfGASRSVGY The sequence of the 'partial B strain originally identified (ORF40) shows a 65.7% identity in an overlap of 254aa with ORF40a: ID 20 30 orf 0. ep TÜFATVOASAMOE_e? EEDI.YU) PVQRTVA I Htl I I I |: |;: | r: | I: | : II. : l orf 0a SA NAXVAVSELTWíH? KIWSATVinAVIATSiE? TVOSNATDEDEEEEI ^ 20 30 40 50 € 0 40 50 60 70 80 orf 40. ep VLIWSDlKECTCBeKEKVEB. -SW? VYENE ^ GT i .... ii.it i.; :: |; : : I I I I I I I I 1 :: orf 0a VGSÍQASMeGSSEIJETISLSMTNDSKEFVDPrrv rTLWtoD »lJCTKQ t * ENT AS 70 80 90 100 110 120 I 150 ISO no 180 190 200 orf40.p «p STLTDTLI_» GAT? -IVTNDNVTDI > S! Aa »ASVK ^ III lili :: t |:: f I:: I I H: I M M 1 I IM! IH lü 1: 1 I lli orf 40 * STLTDTIA (KSAS-HVDftsrtXST-HYTWlftSI? A? VT? AG ^ 190 200 210 220 230 240 .210 220 230 240 orf 0, pep BTYOTVEFLSADTKT TTO? VESK__Nraap_EVKlGUUÍTSVIKEKD M II II I ll ll i I I I MIIMI: MIIIII i 11 III I i orf40í TYDTVEFLSAOTXTTTVHVESKWtaWTEVKIGftKTSVIKEKDGKLVTÍ »250 260 270 280 Z90 30C The sequence of the complete B strain (ORF40-1) and ORF40a shows 83.7% identity in the 601 aa superposition: 20 30 40 50 60 orf 40-1. pep MNKIYRI IWNSALNAW SE ^ .TRNKT RA5A- Ip • AVI? TIJFATVQASA 3EEQEE_ L orf40a MNKIYRIIWNSA NAXVAVSELTIWHTKRASATVKTAVIATLLF? TVQAWATDEDeEEEL 10 20 30 40 50 60 70 80 90 100 110 119 orf40-1. pep V DPV RTVAVLIVNS0KEt-TGEKEKVEEN-SD AVV-NEKGVL_, AREIT KAGßN_KIK: I I I: I I:::: 1 I: I I I::::: I:::: i 1 I I I I 1 I I I arf40i -ESV0RSV-VGSI0ASMEGSGE ETlS1.3l_TH0SJtEFVDPYIV VTLKAGDNLKI 70 80 90 100 110 120 130 140 150 160 170 otf 0-1. ep QN G __._ TYSlR DtTDLTSVGT «SF5A_Í íKVNITSDTKGLNFAKETAlSTNG II: -: i! I / 11 fj J I I: I _ I I - "» 11: III I Ml 11 I I I 1111 (I I I orf 0 * CtTrN2OTT »ASSFTVSl.KKDLTGLlNVXTE LSnWU4GKXVNlJ5_TXGLNFAKeTAst G 120 130 140 150 160 170 180 190 200 210 220 230 300 310 320 330 540 350 orf 40-1. ep KPKGfttGSSTDEGEßLVTRKEVIDAVNXA ^^ I UIIIIIMIII orf 40a KGKGENGSSTDEGEGtVTAKEVIDftVNK? ^ RiWíTTTA ^^ 300 310 320 330 340 35D 360 370 3B0 390 400 410 orf 40- 1. pep S «KGTTATVSKI.DQGNr-WYDVNVG0AIl¡pmQUW5Gim ^ UU Ill tlIlll J l | l II II II Ixf 40a SOTGTT? TVSKDDQGNiatanrDVNVGr »I ^^ 360 370 360 390 400 410 -420 430 440 450 460 470 Orí 40 -I. p ^ SKOKMD? rrWi? GNNIEITRNCTCKIDIATSip 11 or 11 m ¡minim ¡11: ppnp 11 m M 11 M i • - 1111 orf i 0a SKGKHDETVNIK-U ^ IEISÍUN ^ IDIATSKA ^ SSV ^ 420 430 440 450 460 470 480 4 & 0 500 510 520 S30 orf 40-1 -pep GSKKDfpCPVRITNVaPGV EGDVTKVA01JGVA (WI? ImiDIWDGNAIlAGI 111 piiiiiipini in ¡11 11 r 11 ?? p. I ..111 pp 11 p 11/11 p 11 orf 40a GSKOAHKPVR? _W? _GVKXGD \ r ™ vXQLKW ^ 480 490 500 510 520 530 54D 550 560 570 580 590 orf 40-1.pep VOAYIJ «_- & ftIGGGTYRGI? CTAIGYSSISDGGíWlIKGT ^ 111 inpp mm p N u ni p H (fi n ¡i i iiiiiiN a i mu 11 orf 40a O? _apGKSWMAICWTYIlGEAGyAIGYSSlSD-aWIIKCTASaNSBGH GM 540 550 560 570 500 590 orf 0-1. Pep wx 1 I srf40a wx The computer analysis of these amino acid sequences gave the following results: -Homology with the Hsf protein encoded by the place of type b surface fibrils of H. influenzae (access number U41852) The ORF40 and the Hsf protein show the 54% aa identity in overlapping 251 aa: Orf i 0 1 TLLFATVQñSANOEEOEED YLDPV? RTVAV IVNSDXXXXXXXXXXXXNSOWAVYpffiK 60 r ?, FATVOft + A E ++ JE LDFV RT VL + SD NS + W + YF + K fisf 4 I TLLFATVOANATDEDEE tE, PVVRTAPVLSFHSDKEGTGEKEVTE - NSNWGIYÍTWK 95 Orf40 61 GVLTAREXTXKñGDNLKI ON GTNÍ YSLKKDLrDI, TSVGTEKLSFSANGNKVN 114 GVL A IT KAGDN K? KON ++ FTYSLKK_UDLTSV TEK SF ANG + KV + Hsf 96 GVLKAGAITt_KAGONLKIK? , DESTt »SSFTYSLKECOLTDLTSVAtEKIJSFGMlGDKVD 155 Orf40 115 rTSßTKGLNFAKETAGTNGDTTVHLBG IGSTLTPTL NTSAXXXXXXXXXXXXEKKBAAS 1 4 1TSD GL AK G + VHLNG + STL 0 + »TG EK? A + ÜYÍ 156 rTSDANGLKLAK TGNGWVHL GLnSTLPPAVTMTGVLSSSSFTPNDV-EKTRAAT 209 Orf 40 175 VKDVl_NAGWNIX < ? VKPGTTASDNVDFWTY_ ^ 234 VKDVJLRAGWNIKC K ++ VO V Y + VEF ++ D T V + + K + NGK TEW Kaf 210 V DVLNAcaw? GAKTAGG VESVDLVSAYN VEFrTGOIWTLD LTAKEl? GKTTEVKF 269? Rf40 235 GA? CRSVIKE O 245 KTSVGKEKD Hsf 270 TPKTSVIKEKD 280 The ORF40a also shows the homology to Hsf: gil 1666683 (U41852) product of the hsf gene [Haemophilus influenzae] Length = 2353 Record = 153 (67.7 bits), Expected = 1.5e-116, Sum P (ll) = 1. 5e-116 Identities = 33/36 (91%), Positive = 34/36 (94%) Interrogating: 16 VAVSlU-TRNHTKlUlSATVKT VXATLS_ ATV? AT AT 51 V VSELTR «TKRASATV + TAV1AT I» FATV0ARAT Subject: 17 VWSELTRTHTKRASATVETAVIATLLFATVQANAT 52 Record = 161 (71.2 bits), Expected = 1.5e-116, Sum P (ll) = 1. 5e-116 Identities = 32/38 (84%), Positive = 36/38 (94%) - - Interrogating: 101 VTLKPXSDNLKl QWTHEKTlIASS TYSl SKOLTs lHV 138 + TLKAGDI_LíaKQ! IT + E + TMASSFTYSLKKDLT L + V. Subject: 103 ITLKAGINLi QNTDESTNASSFTYSLKKDLTDLTSV 140 Record = 110 (48.7 bits), Expected = 1.5e-116, Sum P (ll) 1. 5e-116 Identities = 21/29 (72%), Positive = 25/29 (86%) Interrogative: 13 B VTEK SFGANGKKVNIISDTKGUSIE7ÍKET 166 V ++ K &5 G KG KVNI 5DT SI? FAK ++ subject: 143S VSDK S GTSGHKV ?. ITSD KG1? FW-DS 1467 Record = 85 (37.6 bits), Expected = 1.5e-116, Sum P (ll) 1. 5e-116 Identities = 18/32 (56%), Positive = 20/32 (62%) Interrogative:? 9 T GipTViiliíGIGSTiaT T AGSSASHVDAGN 200 T D + HLNGI STLTDTL S A + GN Subject: 14 eg rGDDAKIHL GIAST TDTLI * NSGATTNLGGN 1500 Record = 92 (40.7 bits), Expected = 1.5e-116, Sum P (ll) 1. 5e-116 Identities = 16/19 (84%), Positive = 19/19 (100%) Interrogation: 206 RñA_.IKDVL AGíífíIKGVK 224 RAAS +? O.V? JÍAGWN ++ GVK subject: 1509 RAASVKDVLHAGWNVRGVK 1527 Record = 90 (39.8 bits), Expected = 1.5e-116, Sum P (li; 1.5e-116 Identities = 17/28 (60%), Positive = 20/28 (71%) Question: 226 STTGQSENVDFVRTYDTVEFLSAD TTTT 253 S Q EN + QF TYDTV + F45 p TT Subject: 1530 SANNQVENIDFVATYDTVDFVSGDKDTT 2557 Based on homology with Hsf, it was predicted that this protein from N. meningitidis, - and its epitopes, could be useful antigens for vaccines or diagnostics.

The ORF40-1 (61 kDa) was cloned with the pET and pGex vectors and expressed in E. c or l i, as described above. The products of the expression and purification of the protein were analyzed by SDS-PAGE. Figure IA shows the results in the affinity purifications of the His fusion protein, and Figure IB shows the results of the expression of the GST fusion in E. c or l i. The purified His fusion protein was used to immunize the mice, whose serum was used for the FACS analysis.

(Figure 1C), a bactericidal test (Figure ID), and ELISA (positive result). These experiments confirm that ORF40 is a protein exposed on the surface and that it is a useful immunogen.

Figure 1E shows the hydrophilicity charts, antigenic index, and the AMPHI regions for ORF40-1.

- - E jmplo 2 The following partial DNA sequence was identified in N. meningitidis (SEQ ID NO: 7): 1 ATGTTACGTt TGACTGCtTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC 51 GTGTTCGCCG CAAAATTCCG ACTCTGCCCC AAAGCCAAA GaACAGGCGG 101 TTTCCGCCGC ACAA..CCGP? GgCGCGTCCG TTACCGTCAA AACCGCGCGC 152 GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT 201 GGGTATG C 5ACACCTT6A GCAAACTGGG CGTGAftAACC GGTTTGTCCG 251 TCGATAAAAA CCGCCTGCCG TATTTAGAG6 AATATTTCAA AACGACAAAA 301 CCTGCCGGCA CTTTGTTOSA GCCGGATTAC GAAACGCTCA ACGCTTACAA 351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC CgCCAAGGCG TTTGACAAAT 401 TGAAcGAAAT CGCGCCGhCC ATCGpowTGA CCGCCGATAC CGCCAACCTC 4SI AAAGAAACTG CCAA? GAGGC ATCGACGCTG GCGCAAATCT TC ..

This corresponds to the amino acid sequence (SEQ ID NO: 8; ORF38): 1 MLRLTALAVC TA &GAC5P QKSDSASOAfc EQJWSAAQTE GASVTVKTAK 51 GDVQIPQIÍPE RIAVYDtGML DTLSFXGVKT GLSVDKW.LP YLEEYFKTTK. 101 PAGTLFEPDY £ TLNAY_0? QL IXICSRAAKA FD NEIAPT IXXTADTANL 151 KESAKEASTL AQIF ..

Additional work revealed the complete nucleotide sequence (SEQ ID NO: 9): - 1 ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG TTTGGGCGC 51 GTGTTCGCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG 101 TTTCCGCCGC ACAAACCGAñ GGCGCGTCCG TTACCGTCAA AACCGCGCGC 151 GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT 201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG 251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTT AA AACGACAAAA 301 CCTGCCGGCA CTTTGTTCGJ. GCCGTOTAC GAAACGCTCft ACGCTTACAA 351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC CGCCAAGGCG TTTGACAAAT 401 TGAACGAAAT CGCGCCGACC ATCGAAOTGA CCGCCGATAC CGCCAACCTC 451 AAAGAAAGTG CCAAAGAGCG CATCGACGCG CTGGCGCftAA TCTTCGGCAA 501 A W3GOGGAA GCCGACAAGC TG2VAGGCGGA AATCGACGCG TCT7TTGAAG 551 CCGCGAAAAC TGCCGCACAA GGTAAGGGCA AAGGTTTGGT GATTTTGGTC 6C1 AACGGCGGCA AGATGTCGGC TTTCGGCCCG TCTTCACGCT TGGGCGGCTG 651 GCTGCACAAA GACATCGGCG TTCCCGCTGT CGATGAATCA AT AAAGAAG 701 GCAGCCACGG TCAGCCTATC AGCTTCGAAT ACCTGAAAGA GAAAAATCCC 751 GACTGGCTGT TTGTCCTTGA CCGAAGCGCG GCCATC5GCG AAGAGGGTCA 801 GGCGGCGAAA GACGTGTTGG ATAATCCGCT GGTTGCCGAA ACAACCGCT 851 GGAAAAAAGG ACAGGTCGTG TACCTCGTTC CTGAAACTTA TTTGGCAGCC 901 GGTGGCGCGC AñGAGCTGCT GAATGCAAGC AAACAGGTTG CCGACGCTTT 9SI TAACGCGGCA AAATAA This corresponds to the amino acid sequence (SEQ ID NO: 10; ORF38-1): 1 HLRLTALAVC TA1ALGACSP QNSDSAPQAK EQAVSAAQTE GAS TVKTAI. 51 GDVQ2PQNPE RIAVYDLGML DTLSKLGVKT GLSVDKNRLP YLEEYFKTTK 101 PAGTLFEPDY ETUJAYKPOL IIIGSRAA TO FDKLNEIAPT IEMTADTANL 151 KESAKERIDA IAQIFG QAE A0KLKAEIDA SFEAAfCTAAQ GKGKGLVILV 201 NGGKMSAFGP SSR1GGWLHK DIGVPAVDES IKEGSHGQPI = FEY KE NP 251 D LFVLD SA AIGEEGQAAK DVLDNPLVAE TTAWKKGQVV YLVPETYLAA 301 GGAQELLSA5 KQVADAFNAA K * Computer analysis of this amino acid sequence revealed a lipid binding site of putative prokaryotic membrane lipoprotein (underlined).

- - The additional work identified the corresponding gene in strain A of N. meningi tidi s [SEC. ID? O: 11]: January 1 ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC 5511 GTGTTCGCCG CñAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG 101 TTTCCGCCGC ACAATCCGAA GGCGTGTCCG TTACCGTCAA AACGGCGCGC 151 GGCGATGTTC AAATACCGCA AAACCCCSAA CGTATCGCCG TTTACGATTT 201 GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG 251 TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA 301 CCTGCCGGAA CTTTGTTCGA GCCGGfcTTAC GAAACGCTCA ACGCTTACAA 351 ACCGCAGCTC ATCATCATCG GCAGCCGCGC AGCCAAAGCG TTTGACAAAT 401 TGAACGAAAT CGCGCCGACC ATCGAAATGA CCGCCGATAC CGCCAACCTC 451 AAAGAAAGTG CCAAAGAGCG TATCGACGCG CTGGCGCAAA TCTTCGGCAA 501 AAAGGCGGAA GCCGACAAGC TGAAGGCGGA AATCGACGCG TCTTTTGAAG 551 CCGCGAAAAC TGCCGCGCAA GGCAAAGGC? AGGGTTTGGT GATTTTGGTC 601 AACGGCGGCA AGATGTCCGC TTCCGCCCG TCTTCACGAC TGGGCGGCTG 651 GCTGCACAAA GACATCGGCG TTCCCGCTGT TGACGAAGCC ATCAAAGAAG 701 GCAGCCACGG TCASCCTATC AGCTTTGAAT ACCTGAAAGA GAAAAATCCC 7 75511 GACTGGCTGT TTGTCCTTGA CCGCAGCGCG GCCATCGGCG AAGAGGGTCA 8 80011 GGCGGCGAAA GACGTGTTGA ACAATCCGCT GGTTGCCGAA ACAACCGCTT 851 GGAAAAAAGG ACAAGTCGTT TACCTTGTTC CTGAAACTTA TTTGGCftGCC 901 GGTGGCGCGC AAGAGCTACT GAATGCAAGC AAACAGGTTG CCGACGCTTT 951 TAACGCGGCA AAATAA This encodes a protein having the amino acid sequence [SEQ. ID? O: 12; O R F 38 a]: 1 M RI.TAAVC TAIAI ^ SACSP QHSDSAPQAK e SS SS GVSVTVKTAR 51 GDVQIPQ? PE IAVYDGML DTLSIOGVKT GLSVQK? R P YLEEYFKTTK 101 PAGTlíEPDY ETLKAYKPQL IIIGSRAA TO FCKL? EIAPT lEMTADTA? L 151 XESAXEfclfcA LAQIFGKKJIE ADK AE1HA SGEAAKTAAQ GKGKGLVILV 201? GGKMSAFGP SSRLGGWHK DIGVPAVDEA IKEGSHGQPI SF? YIJ? SK? P 251 D? F FVLDRSA AIGEEGQftAK DV tWPLVAE TTAWKKGQW YLVPeTYLAA 301 GGAQELLÍÍAS KQVAPAF? AA K * The sequence of the B strain originally identified (ORF38) shows a 95.2% identity in an overlap of 165aa with ORF38a: - - 20 30 40 50 60 orf d - P «p MI_BLTAL? VCTAlJU ^ acSiX? WSDSAP0AKEORVSAAQTEGASVTV TARGDV0IPgHPE Ull I I I II III II I II III Hf lilili III llil II: III I I II II lili MI 11 III orf38a MI_RtT IAVsi l acaroHSPS PQAKEQ VSA 1C 30 40 50 60 20 70 80 90 100 110 120 orf38.pep RIAVYDLGKLDTLSKLGV T5LSVD N? UPYLEEYFKTTKPAGTLFEPDYETLN? YKIO IIIIMIIIIII! IIIIII 1 MI 1 IIII) II 1 II IMHIIIH [MIIIIIMII itl I orf 38a RIA -.? 'DI MLDTl ^ KLGVKTGLSTOKNRLPYLEEYFKTTKPAGTl.FePDYETLNAYKPQ 70 80 90 100 110 120 130 140 150 160 orf 38 -PEP lIIGSRAAKAFDKLHEIAPtiXXTAOTANL ESAKE-ASTI OIF my me iim 11 um iifiiipniii m M 11 orr38a I IlGSRAAKAFDKlJIEIA EwrADtAKLKESAKER? DMAQI FGIQ? ^ 130 140 150 160 170 180 orf 38a S?? UUCT? AQGKGKGLVI VMGGKMSJ-FGPSSRLSGWri? KOlGVPAVDEAIKEGSKGQPl 190 200 210 220 230 240 The sequence of the complete B strain (ORF38-1) and ORF38a shows the 98.4% identity in the 321 aa overlap: ? ORF3 a.pep HI.R-.TALAUCTAlALGACSPQKSDSAPOAKECAVSAA0SEGVSVTVKTARGDV0IP NPE ???? ?? m ?? m pm m p u ???? ??????:? Ii: i ????? pt? i? i ????? x f 38- 1 MIJÍLRA_? VCTAI? LGACSPC »? 5nSAP0AKE0AVSAAQTEGASVTVKTARGDV0I PQN pE orf 36a • pep KIAVYDLG« iyri Kl? \ pCTGLSVDKimLPYl__EYFípTTtPAGTLFEP0YETL? .AYKPQ_. u 111 n m 11 M i M i m 11 m 11 J i N m i m 11111 111111 m 111! O? F3B-l RIAVTDWSMLDT SKlJWKTGLeVDKNRLPYLEEYFK T PAGTLFePDYETLNAYKPC.1, or F38A .pep IIIGSRAAKAFDK NEIAPTIEMTADTAMLKESAKERIDAlAQIFGKKAEADi or 11 KAEinA ii) J f! N m i p m J m 11 m M m i m m 1111 N N 111111.- orf38-i 11 i-ftroK IIÍGSrOVAÍ NEIAWIEMTAOTANLKESAReRIDftlAOIPGKOAE ^ orf 3 B? . pep SFeAAKTAAOGKGKGLVILVNGGÍtt AFCPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI my ^ i IIIII I i niiiiiiii mu iniin one niimim i > . or f 38 -i S FEAA? CTAAQGKGKGLVII.VNGGKMSAFGPSSRl? GWI? KDrGVPAV £ KSIKEGSHGQPl orfSBa.pep GGAQELLNA = KCVADAPKftAK mmimim iiiiip orf3B-l GGAQELLNASKQVADAFNAAK The computer analysis of these sequences revealed the following: - -Homology with a lipoprotein (lipo) of C • jejuni (access number X82427) The ORF38 and lipo show 38% identity aa in the overlap 96 aa: Orf 8: 40 EGASVTVKTARGDVQIPQNPERIAVYOI? MLDT ^ 9B EG S VK + G + + P + NP ++ + OLG + LDT L + ++ V J_P + FK I_lpo: 51 EGD5FLVK_SLGENKTF1WP5KV \ aLDLGILOTFI? fa? D 110 Orf 38: 99 TKPAGTLFEPD-Ff-_MAYKe? LII? GSRftAKAFOlL 134 G + + 0 + E + HA KP 1211 B * K + PKL Lipo: 111 KPSVGGVOQVDFEAINALKPDLIIISßROSXFraKL 146 Based on this analysis, it was predicted that this N. meningi tidi s protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

The ORF38-1 (32 kDa) was cloned with the pET and pGex vectors and expressed in E. coli, as described above. The products of the expression and purification of the protein were analyzed by SDS-PAGE. Figure 2A shows the results of the affinity purification of the His fusion protein, and Figure 2B shows the results of the expression of the GST fusion in E. coli. The purified His fusion protein was used to immunize the mice, whose - - serum was used for Western blot or spot analysis (Figure 2C) and FACS analysis (Figure 2D). These experiments confirm that ORF38-1 is a protein exposed on the surface and that it is a useful immunogen.

Figure 2E shows the hydrophilicity charts, antigenic index, and the AMPHI regions for ORF38-1.

E j emp lo 3 The following DNA sequence from N. meningi tidis was identified - [SEC. ID NO: 13]: 1 ATGAAACTTC TGACCACCGC AATCCTGTCT TCCGCAATCG CGCTCAGCAG : TATGGCTGCC GCCGCTGGCA CGGACAACCC CACTGTTGCA AAAAAAACCG 101 TCAGCTACGT CTGCCAGCAA GG AAAAAG TCAAAGTAAC CTACGGCTTC 151 AACAAACAGG GTCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCAAACG 201 CGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT 251 ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCGGCGTGAT GGATG6CAAA 301 TCCTACCGCA AACAGCCCAT TATGATTACC 6CACCTGACA ACCAAATCGT 351 CTTCAAAGAC TGTTCCCCAC GTTAA This corresponds to the amino acid sequence [SEC. ID NO: 14; ORF44]: 1 MKLLTTAILS SAIA55MAA AAGTDNPTVA KKTVSYVCQQ 5KKVKVTYGF 51 NKQGLTTYAS AVIHGKRVQM FtfNLDKSPHV ETFYG EGGY VLGTGVKDG 101 SYRKOPIMIT APDUOIVFKD CSPR «- - Computer analysis of this amino acid sequence predicted the leader peptide shown underlined.

Additional work identified the corresponding gene in strain A of N. meningitidis [SEC. ID? O: 15]: 1 ATGAAACTTC TGACCACCGC AATCCTGTCT CCGCAATCG CGCTCAGCAG 51 TATGGCTGCT GCTGCCGGCA CGRACAA CC CACCGTTGCC AAAAAAACCG 101 TCAGCTACGT CTGCCAGCAA GGTAAAAAAG TCAAAGTAAC CTACGGCTTT 151 AACAAACAGG GCCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCA CG 201 TGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT 251 ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCSGCGTGAT GGATGGCAAA. 301 TCCTATCGCA AACAGCCTAT TATGATTACC GCACCT6ACA ACCAAATCGT 351 CTTCAAAGAC TGTTCCCCAC GTTAA This encodes a protein having the amino acid sequence [SEQ. ID? O: 16; I 0RF44a]: 1 MK LTTAILS SAIALSSMAA AAGTH? PTVA KKTVSYVCQQ GKKVKVTYGF 51 KKQGLTrYAS AVIKGia.VC3K PV? LDKSDSV ETFYGKEGGY V GTGVHDGK 101 SYRKQPIMIT AP _ ?? QIVFK__ CS PR * The sequence of strain B (ORF44) shows 99.2% identity in an overlap of 124aa with ORF44a: - - 20 30 40 50 60 orf44.pep HBa_pAI-SSAIAL5SMflA G-DWPtVAKKrv5YVCQQGKy ^^ i m 1111 í 11 n H i m M 11 ¡.- n 11111] i m 11111 p 11 N 1111111 m i orf 4 a. H. LTTAr SSA__P 5SMAAAA? FfrNH_TVaKCTVSYVCCXre ^ 10 20 30 40 50 60 70 80 90 100 110 120 orf 44.pep A IireK VQHP NLDKSDíroE FYGK £ GGYVI? ^ G \ ra $ x? S p MI I! H N! I I I I I ll I I I! I I I I I L I I I I I I I I I I I I I I I I I orpha 44a AVINGKRVCJMPVNLDKSONVETFYGKEGsYVI-GTsVMDGKSYRKOPIMITAPDNQIVFKD 70 80 90 100 110 120 orf 44. ep CSPRX mil orf a CSPRX The computer analysis gave the following results: Homology with the LecA adhesin from Eikenella cor r odens (access number D78153) The ORF44 and the LecA protein show 45% identity aa in the 91 aa superposition: Orf 44 33 TVSYVCQ_GK_V VTYGETJKX; LTTYASAVrNGKRVQMPVK__DKSÍWV_T_YGKEGGYV ^ 92 + V + YVCCQG ++ - I- V and FN G + T A + N + +++ P NL SDNV + T * _Y _ LecA 135 SVAYVCQQGRRIJW YRFtJSAGVP SAE RVHHFiNIJUJPYMLSñSDHVDTVF-SANGYRL 193 Orí 44 93 GTGVMDG SYRKQPIMITAPDNDIVFKDCSP 123 T KD + YR Q I +++ AP + Q +++ KDCSP LecA 194 TTNAMDSANYRSQDIIVSAPNGQMLYKDCSP 224 Based on homology with adhesin, it was predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

The ORF44-1 (11.2 kDa) was cloned into the pET and pGex vectors and expressed in E. coli, - as described above. The products of the expression and purification of the protein were analyzed by SDS-PAGE. Figure 3A shows the results of the affinity purification of the His fusion protein, and Figure 3B shows the results of the expression of the GST fusion in E. c or l i. The purified His fusion protein was used to immunize the mice, whose serum was used for ELISA, which gave positive results, and for a bactericidal assay (Figure 3C). These experiments confirm that ORF44-1 is a protein exposed on the surface and that it is a useful immunogen.

Figure 3D shows the graphs of h i dr of il iity, antigenic index, and the AMPHI regions for ORF44-1.

E j empl o 4 The following partial DNA sequence was identified in N. men in gi ti di s (SEQ ID NO: 17.): - - 1 -GGCACCGft T TCAAAACCAC C TTTCCGGA GCCSACATAC AGGCAGGGGT 51 GGGTGAAAAA (CSAGCCG A GCGAAAAT TATCCTAAAA GGCATCGTTA 101 ACCGCATCCA AACGGAAGAA AAGCTGGAA CCAÍCTCGAC CGTATGGCAA 151 AAGCAGGCCG GAAGCGGCAG CACGGTTGAA ACGCTGAAGC TACCGAGCT 201 TGAAGGGCCG? GCAC GCCGA AGCTGACCGC TCCCGGCGGC TATATCGCCG 251 ACATCCCCAA AGGCAACCTC AAAACCGAAA TCGAA? AGCT GGCCAAACAG 301 CCCGAATATG CCTATCTGAA ACAGCTTCAG ACGGTCAAG € 351 GAACCAAGTA ACGTGAACTG CAGCTCGCTT AOSACWATG GGACTATAAA CAGGAAGGCC 401 TAACCGGAGC CGGAGCCGCA ATTANCGCAC TGGCCGTTAC CGTGGTCACC 451 TCAGGCGCAG GAACCGGAGC CGTATTGGGA TTAANACGNG GGCCGCCGC 501 CGCAACCGAT GCAGCATTT.

This corresponds to the amino acid sequence [SEC. ID NO: 18; ORF49]: 1 .. GTEFTTLSG ADIQAGWGEK ARADAKI11K GIVWR1QTEE Kl.ES »STVtfQ 51 KOAGSGSTVE TÍCLPSÍEGP ALPKTAPGG YIADIFKGN KTEIEKLAKC 101 PEYAYLKQC TVKDVtWNQV QlAYDKWDY ?. QEGLTGAGAA IXAAVTWT 151 SGAGTGAVG LXRVAAAATD AAf ..

Additional work revealed the complete nucleotide sequence [SEC. ID NO: 19]: 1 ATGCAACTGC TGGCAGCCG? AdGCATTCAC CAACACCAAT TGAATGTTCA 51 GAAAAGTACC CGTTTCATCG GCATCAAAGT GGGTAAAAGC AATTACAGCA 101 AAAACGAGCT GAAC5AAACC AAACTGCCCG TACGCGTTAT CGCCCAAACA 151 GCCAAAACCC GTTCCGGCTG GGATACCGTA CTCGAAGGCA CCGAATTCAA 201 AACCACCCTT TCCGGAGCCG ACATACAGGC AGGGGTGGGT GAAAAAGCCC 251 GAGCCGATSC GAAAATTATC CTAAAAGGCA TCGTTAACCG CATCCAAACC 301 GAAGAAAAGC TGGAATCCAA CTCGACCGTA TGGCAAAAGC AGGCCGGAAG 351 CGGCAGCACG GTTGAAACGC TGAAGCTACC GAGCTTTGAA GGGCCGGCAC 401 ^ GCCTAAGCT GACCGCTCCC GGCGGCTATA TCGCCGACAT CCCCAAAGGC 451 AACCTCAAAA CCGAAATCGA AAAGCTGGCC AAACAGCCCG AATATGCCTA 501 TCTGAAACAG CTTCAGACGG TCAAGGACGT GAACTGGAAC CAAGTACAGC 551 TCGCTTACGA CAAATGGGAC TATAAACAGG AAGGCCTAAC CGGAGCCGGA 601 GCCGCAATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC 651 CGGAGCCSTA tTGGGATTAA ACGGTGCGGC CGCCGCCGCA ACCGATGCAG - - 701 CATTTGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT CAACAACAAA 751 GGCAATATCG GTAACACCCT GAAAGAGTG GGCAGAAGCA GCACGGTGAA 801 AAATCTGATG GTTGCCGTCG CTACCGCAGG CGTAGCCGAC AAAATCGGTG 851 CTTCGGCACT GAACAATGTC AGCGATAAGC AGTGGATCAA CAACCTGACC 901 GTCAACCTGG CCAATGCGGG CAGTGCC6CA CTGATTAATA CCGCTGTCAA 951 CGGCGGCAGC CTGAAAGACA ATCTGGAAGC GAATATCCTT GCGGCTTTGG 1001 TGAATACTGC GCATGGAGAG GCAGCAAGTA AAArCAAACA GTTGGATCAG 1051 CACTACATTG CCCATAAGAT TGCCCATGCC ATAGCGGGCT GTGCGGCAGC 1101 GGCGGCGAAT AAGGGCAAGT GTCAAGATGG TG GATCGGT GCGGCGGTCG 1151 GTGAAATCCT TGGCGAAACC CTACTGGACG GCAGAGACCC TGGCAGCCTG 1201 AATGTGAAGG ACAGGGCAAA AATCATTGCT AAGGCGAAGC TGGCAGCAGG 1251 GGCGGTTGCG GCGTTGAGTA AGGGGGATGT GAGTACGGCG GCGAATGCGG 1301 CTGCTGTGGC GGTAGAGAAT AATTCTTTAA ATGATATACA GGATCGTTTG 1351 TTGAGTGGAA ATTATGCTTT ATGTATGAGT GCAGGAGGAG CAGAAAGCTT 1401 TTGTGAGTCT TATCGACCAC TGGGCTTGCC ACACTTTGTA AGTGTTTCAG 1451 GAGAAATGAA ATTACCTAAT AAATTCGGGA ATCGTATGGT TAATGGAA & A 1501 TTAATTATTA ACACTAGAAA TGGCAftTGTA TATTTCTCTG TAGGTAAAAT 1551 ATGGAGTACT GTAAAATCAA CAAAATCAAA TATAAGTGGG GTATCTGTCG 16C1 GTTGGGTTTT AAATGTTTCC CCTAATGATT ATTTAAAAGA AdCATCTATG íßai AATGATTTCA GAAATAGTAA TCAAAATAAA GCCTATGCAG AAATGATTTC 1701 CCAGACTTTG GTAGGTGAGA GTGTTGGTGG TAGTCTTTGT CTGACAAGAG 1751 CCTGCTTTTC GGTAAGTTCA ACAATATCTA AATCTAAATC .TCCTTTTAAA 1801 GATTCAAAAA TTATTGGGGA AATCGGTTTG GGAAGTGGTG TTGCTGCAGG 1851 AGTAGAAAAA ACAATATACA TAGGTAACAT AAAAGATATT GATAAATTTA 1901 TTAGTGCAAA CATAAAAAAA TAG This corresponds to the amino acid sequence [SEC-ID NO: 20; ORF49-1]: 1 MQtlAAEGIH QHQLNVQKST RFÍGIKVGKS NYSKNELNET KLPVRVIAgT 51 AKTRSGWDTV LEGGEGKTTJ. SGAOIQAGVG EKABADAKII L CIV RIQT 101 EEKLESNSTV WQKOAGSGST VET L ^ SFE GPALPXVTA- * GGYXADIVKG 151 NLKTEIEKI? KQPEYAYKQ -QTVKDVMW. QVQ AYDKWD YKQBGLTGAG 201 AAIIALAVTV VT36AGTGAV LGlKGftAAAA TDAA? ASIAS QASVSFINH 251 GNJGNTUE G..SSTVHIH VAVATAGVAD KIGASALSBV SOKQBINNLT 101 VHIANAGSAI- LIHTAVSGGS KOKLERKI AALWHTAKGE AASKIKQ DO 351 HY? AHKIAHA IAGCAAAAAH KGKCQGAIG AAVGEI1GET LLDGRDPGSI, 401 NVKDKAKIIA KAKXAAGAVA ALSGDVSTA? KAAAVAVEN NSLNDIQDRL 451 LSGNYALCKS AGGAESFCSS YRPLGLPHGV SVSGEMKLFN KfGNRMVNGK 501 LIINTSKGNV YFSVGKIWST VKSTKSNISG VSVGWVWJVS PNDYLKEASM 551 NDFRNSNQNK AYAEMISQTL VGESVGGSLC LTRACÍTSVSS TIS SKSPFK 601 DSKIIGEIGL GSGVAAGVEK TIYIGNIKDI DKF? SANIKK The computer analysis predicted a transmembrane domain and also indicates that the ORF49 does not have significant amino acid homology with the known proteins. However, a corresponding ORF of strain N. of N. meningitidis was identified: The ORF49 shows 86.1% identity in an overlap of 173aa with an ORF (ORF49a) of strain N. of N. meningitidis: 20 30 orf 49. pep GTEFCTtLSGADlQAGWGEKASU-t? KIXLE ni ii M i: mi p ii mi: i go 1111 arf 49¿t SK? EI-? ETKLFV VVAOXAATRSWDTVIÍGTEFKTTIAGADIOAsvXEKARVDAKIILK .0 50 60 70 90 9C 40 50 60 70 60 90 oxf 49.pep GI Wr-EKlí.SMSt WjKQJ ^ eGST E-iKLPSFEGP? ISKi.'IAPGsYI? DIPKGM ipmim 11 m M 1111 M i ni: p ip M i im iim ii piiin orf 9 * GIVKRIQSEE ^ LET? STV OKQAGRCSTIETLKLPSFeSPTPPKLSAPGGYIVDlPKG? L 100 110 120 130 140 150 100 110 120 130 14C 150 orf 4 S. pßp KTE1EKLM.0 EYM LKQI ^ VKQVÍWMQVQLAYiaCWPYKOEGL-G G- ^ I AlAVTVV lll Ml l: I M 111 l 1 I I :: I :: I MUIII I l: 1 I? II MI MI MI MI 49a K EIEKLSKOPeYAYLKOLQVAKWI? WWQVQLAYDRWDYKQEGLTEAGAAlIALAVTVVT 160 170 180 190 200 210 160 170 of 49. pep SGAGTGAVLGLXRVAAAATDAAF lll: i 111 i II orf 49a SGAGTGAVl_3L? GA_y_WTDAAFAS_ASaASVSFIOT «CGDVG TlJ ^ l? RSSTVK? _. VVA 220 230 240 250 260 270 The ORF49-1 and the ORF49a show 83.2% identity in the overlap 457 aa: - - orf 49a. pep XQI ^ AEEGIHHELDVQKSRRFIGlKVGXSNYSKNEI? ETia.P \ ^ VVAQ > ÜVA.TRSGKDTV III I I [I I: I: I • I M I I III III I I I I I I I I I I I I I; I II I I I I. ' orf 49- 1 KWAAEGIHOHQI? VOKSTRFIGIKVGlKNYSKIffil ^ ETKLPVRVIAQTAK RSG «_tv orf 49a- pep LEGTEpCTTLAGAD? CAGVXEKARVüLSI-JCGl ^ II I I f if III: I I III II! I: I II I II MII MI I: I II I i f IM 1 IIII lll orf 49-1 _? GTEFCTTI £ GMIC »GVGE_? RA_AKIIIJre ^ orf 49a - pep IETWCLPSFE_PTFPKL» PGGYIVDrPRGNLK__XIEKI_SKQPEYAYU0LOVAKNINWH: III 1 III 11 • ' 1: IM: IIIIIMIM f 1 MI I 1 I ll! : I III I I J I II I I:: I:: 1 I I orf 49-1 V_ _J PSFEGPAL_TCLTAPGGYIADIPK < _NIJKTEIEia_W ^ orf 49a. pep QVQlAYDRMDYKQeG_TEAGA ^ XMAVTVVTS ( "GTGAVI ^ IHI IIMimillll III lili Hll I MI !! i lili I) Ml i m m i I i i orí49-¿aVQIAY0KWDYKQEGLTG" 5A \ IIAI * VTVVTS ^^ orf 49a .pep QASVS IlWKGDVGKTIJCEI RSSTV NLVVAAATAGV [> KlG »_iALXNVSDKOWI NLT M M M IM 1 h: I M I I! I MI I M I M: 11: I I M I 11 I I I I I I I I I I I I I I 1 or f 49- 1 QASVSraNNKGNIGNTUEI? RSSTVKNl_4VAVATAI_VADra or f 49 & . pep VNI? NAGW_LX_rr AVNGGSU? DX? -EAHIl? J ^^ i M 111 M mu? m ti ?? p mi u muim um 11 u u u i uu i orf 49-1 VN_ANAGSAA IOTAWGGSIJDOT £ 3WI1JUIL _ ^^ orf 49a. pep GWJMDWAAA? ÃAA £ VAVKNHQ_ OTEGREÍT) NEMT / ^^ :: II i :: U III I Il: ||: |: I: I :::; :: I orf 49- 1 ALSKGDVSTAAN? AAVAVEN SLNDI0DRLLSGNYALCKSAGGAE5FCES YRPLGLPHFV orf 49a. pep KRLAAS1AICTDISRSTECRTIRKQÍU_IDSRSLHSSWEÁGL1GKDDEWYKLFSKSYTQAD orf 49- 1 SVSGEMKLPNKFGNRMVNGKLI INTR GNVYFSVGKIWS VKSTKSNISGVSVGWVLKVS The full length ORF49a nucleotide sequence [SEC. ID NO: 21] is: - - 1 KTGCAACTGC TGGCAGAAGA AGGCATCCAC AAGCACGAGT TGGATGTCCA 51 AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGTNAGAGC AATTACAGTA 101 AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT CGCCCAAANT 152 GCAGCCACCC GTTCAGGCTG GGATACCGTG CTCGAAGGTA CCGAATTCAA 201 AACCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC GAAAAAGCCC 251 GTGTCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG TATCCAGTCG 301 GAAGAAAAAT TAGAAAC AA CTCAACCGTA TGGCAGAAAC AGGCCGGACG 351 CGGCAG ACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA AGCCCTACTC 401 CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACAT TOCGAAAGGC 451 AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG AGTATGCCTA 501 TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT CAGGTGCAGC 551 TTGCTTACGA CAGATGGGAC TACAAACAGG AGGGCTTAAC CGAAGCAGGT 601 GCGGCGATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC 651 CGKAGCCGTA TTGGGATTAA ACGGTGCGNC CGCCGCCGCA ACCGATGCAG 701 CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCG TCAT CAACAACAAA 751 GGCGATGTCG GCAAAACCCT GAAAG? GCTG GGCAGAAGCA GCACGGTGAA 601 AAATCTGGTG GTTGCCGCCG CTACCGCAGG CGTAGCCGAC AAAATCGGCG 851 CTTCGGCACT GAHCAATGTC AGCGftTAAGC AGTGGATCAA CAACCTGACC 901 GTCAACCTAG CCAATGCGGG CAGTGCCGCA CTGATTAATA CCGCTGTCAA 951 CGGCGGCAGC CTGAAAGACA RTCTGGAAGC GAATATCCTT GCGGCTTTGG 1001 TCAATACCGC GCATGGAGAA GCAGCCAGTA AAATCAAACA GTTGGATCAG 1051 CACTACATAG TCCACAAGAT TGCCCATGCC ATAGCCGGCT GTGCGGCAGC 1101 GGCGGCGAAT AAG GCAAGT STCAGGATGG TGCGATAGGT GCGGCTGTGC 1151 GCGAGATAGT CGGGGAGGCT TTGACAAACG GCAAAAATCC TGACACTTTG 12D1 ACAGCTAAAG AACGCGAACA GATTTTGGCA TACAGCAAAC TGGTTGCCGG 1251 ACGGTAAGC GGTGTGGTCG GCG6CGATGT AAATGCGGCG GCGAATGCGG 1301 CTGAGGTAGC GGTGAAAAAT AATCAGCTTA GCGACHAAGA GGGTAGAGAA 1351 TTTGATAACG AAATGACTGC ATGCGCCAAA CAGAATANTC CTCAACTGTG 1401 CAGAAAAAAT ACTGTAAAAA AGTATCAAAA TGTTGCTGAT AAAAGACTTG 1451 CTGCTTCGAT TGCAATATGT ACGGATATAT CCCGTAGTAC TGAATGTAGA 1 01 ACAATCAGAA AACAACATTT GATCGATAGT AGAAGCCTTC ATT ATCTTG 1551 GGAAGCAGGT CTAATTGGTA AAGATGATGÁ ATGGTATAAA TTATTCAGCA 1601 AAT TTACAC CCAAGCAGAT TTGGCTTTAC AG CTTATCA TTTGAATACT 1 S1 GCTGCTAAAT CTTGGCTTCñ ATCGGGCAAT ACAAAGCCTT TATCCGAATG 2701 GAGGTCCGAC CAAGfGTTATA AC? TAG? TC AGGASTTM.T C AGATTCft 1751 TTCCAATACC AAGAGGGTTT TAAAACAAA ATACACCTAT TACTAATGTC 1801 AAATACCCGG AAGGCATCAG TTTCGATACA AACCTAMAAA GACATCTGGC 1B51 AAATGCTGAT GGTTTTAGTC AAGAACAGGG CATTAAAGGA GCCCATAACC 1901 GCACCAATNT TATGGCAGAA CTAAATTCAC GAGGAGGANG NGTAAAATCT 1951 GAAACCCAHA CTGATATTGA AGGCATTACC CGAATTAAAT ATGAGATTCC 2001 TACACTAGAC AGGACAOGTA AACCTGATGG TGGATTTAAG GAAATTTCAA 2051 GTATAAAAAC TGTTTATAAT CCTAAAAART TTTtWGAXGA TAAAATACTT 2101 CAAATGGCTC AANATGCTGN TTCACAAGGA TATTCAAAAG CCTCTAAAAT 2151 TGCTCAAAAT GAAAGAACTA AATCAATATC GGAAAGAAAA AATGTCATTC 2201 AATTCTCñGA AAC TTTGAC GGAATCAAAT TTASANNNTA TNTWGATGTA 2251 AATACAGGAA GAATTACAAA CATTCACCCA GAATAATTTA A This encodes a protein having the amino acid sequence [SEQ. ID NO: 22]: - 1 XQLLAEEGIH KHELDVQK5R SFIGIKVGXS NYSKNELNET KLPVRWAQX 52 AATRSGWDTV LEGTEFRTTJ- AGADIQAGVX EKARVUAKII LKGGVNRIQS 101 EEKLETNSTV WOKCAGRGST IETLKLPSFE SPTPPKLSAP GGYIVDIPKG 151 H KTEIEKLS QPEYAYKQ LQVAKNIKWN QVQIAYORWD YKQEGLTEAG 201 AAIIALAVTV VTSGAGTGAV LGLNGAXAAA TDAAFASLAS QASVSF1NNK 251 GDVGTLKEL GRSSTVKNLV VAAATAGVAD ICCG &SAXOTV SDKQIflNKLT 301 VNLANAGSAA LZNTAV GGS LKDXLEAKIL AALVNTAHGE AASKIKQLDC 351 HYIVH IAHA IAGCAAAAAK KGKCQDGAIG AAVGEIVGEA T GKNPDT 401 TAKEKEQI TO YSKLVAGTVS GWGGDVNAA ANAAEVAVKS NQLSWCEGRE 451 FDNEMTACAK QKXPOCRKN TVKKYQHVAD KRLAA5IAIC TOISRSTECR 501 TIRKQHUOS RS HSSWEAG IGKDDEIÍYK LFSKSYTOA0 LALQSYfíLNT 551 AAKSWLQSGN TKPSEHMSD QGYTLISGVN PRFIPIPRGF VKgNTPITNV 601 KYPEGISÍDT «XRHIANAD GFSQEQGIKG AHNRTKXMAE I? SRGGXVKS 651 ETJCTDIEG1T RIKYEIPTD RTG PDGGFK EISS1KTVW PKXFXDDKIL 701 QMAQXAXSQG YSKASKIAQN ERTKSISERK KVIQFSETFD GIKFRXYXDV 751 TGRGTNIHP E * Based on the presence of a putative transmembrane domain, these N proteins are predicted. Glyphs and their epitopes could be useful antigens for vaccines or diagnostics.

E j emp lo 5 The next sequence of AD? partial was identified in N. m e n i n gi t i di s [SEC. ID? O: 23]: 1 CGGATCGTTG TAGGTTTGCG GATTTCTTGC GCCGTAGTCA CCGTAGTCCC 51 AAGTATAACC CAAGGCT7TG TCTTCGCCTT TCATTCCGAT AAGGGATATG 1C1 ACGCTTTGGT CGGTATAGCC GTCTTGGGAA CCTTTGTCCA CCCAACGCAT 151 ATCTGCCTGC GGATTCTCAT TGCCGCTTCT GGCTGCTGA TTTTTCTGCC 201 TTCGCGTTTT TCAACT CGC GCT GAGGGC TTCGGCATAT TTGTCGGCCA 251 ACGCCATTTC TTTCGGATGC AGCT6CCTAT TGTTCCAATC TACATTCGCA 301 CCCACCACAG CACCACCACT ACCACCAGTT GCATAG - - This corresponds to the amino acid sequence [SEC. ID NO: 24; ORF50]: 1 .. IRWGLRISC AWRVPSIT QGFVFAFHSD KGYDALVGXA VLGTFVHPTH 51 ICLRIL1AAS HLLIFLPSRF STSRLRASAY LSANAISFSC SCLLFQSTFA 101 PTTAPPLPPV A- The computer analysis predicts two transmembrane domains and also indicates that the ORF50 does not have significant homology of the amino acids with the known proteins.

Based on the presence of a putative transmembrane domain, it is predicted that this N. meningi tidi s protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

E emp lo 6 The next sequence of AD? partial was identified in N. meningitidis [SEC. ID? O: 25]: 1 AAGTTTGACT TTACCTGGTT TATTCCGGCG GTAATCAAAT ACCGCCGGTT 51 GTTTTTTGA? GTAGGGGTGG TGTCGGTSGT srrGCftßCTG TTTGCGCTGA 102. TTACGCCTCT GTTTTTTCCAA GTGGTGATGG ACAAGGTGCT GGTACATCGG 151 GGATTCTCTA CTTTGGATGT GGTGTCGGTG GCTTTGTTGG TGGTGTCGCT 201 GTTTGAGATT GTGTTGGGCG GTTTGCGGAC GTAtCTGTTT GCACATACGA 251 CTTCACGTAT TGATGTGGAA TTGGGCGCGC GTTTGTTCCG GCWCTGCTT 301 TCCCTGCCTG TATCCTATGT CGAGCACAGA CGAGT3GGTG ATACGGTGGC 351 TCGGGGTGCGG GAATTGGAGC AÍ3ATTCGCAA TTTCTTGACC GGXCAGGCGC 40 L tGAcrtoSGT ¡S TGGATTGG GCGTTTTCGT TTRTCTTTCT GGCGGTGATG 451 T6GTATTACA GCTCCACTCT GACTTGGGTG GTATTSGCTT CGÍTG // 1451 1501 ATTTGCGC 1551 CAACCGGACG GTGCTGATTA TCGCCCACCG TCTGTCCACT GTÍAAAACGG 1601 CACACCGGAT CATTGCCATG GATAAAGGCA GGATTGTGGA &SCGGGAACA 1651 CAGCAGGAAT TGCTGGCGAA CG..AACGGA TATTACOGCT AT TGTATGA 1701 TTTACAGAAC GGGTAG This corresponds to the amino acid sequence [SEC. ID NO: 26; ORF39]: 1 ..? TDFTWFIPA VTKYRÍ.LFFE VLWSWLQL FALITPI? TF. WHD V1.VHR 51. GFSTLDWSV ALLWSLÍ? I VLGGLRTYLF AHTTSRIDVE LGARLFRHI.L 101 SLPLSYFEHR HVGDTVARVR ELEQIRHeLT GQALTSVLBL AFSFIFLftVM 151 tfYYSSTLTWV VLASL // 501 ICANRT VLIIAHRLST VKTAHRIlAK D GRTVBAGT 551 GQEU? KXNG YYRYLYOLN G * Additional work revealed the complete sequence of nucleotides [SEC. ID NO: 27]: - 1 ATGTCTATCG TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT 51 CGCCCATTAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT 101 TTGTftJCTTC CQCACAGAGC GATTTAAATG AAACGCAATG GCTGTTAGCC 151 GCCAAATCTT TGGGATTGAA GGCAAA6GTA GTCCGCCAGC CTATTAAACG 201 TTTGGCTATG GCGACTTTAC CCGCAT? GGT ATGGTGTGAT GACGGCAACC 251 ATTTCATTTT GGCCAAAACA GACGGTGAGG GTGAGCATGC CCAATTTTTG 301 ATACAGGATT TGGTTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT 351 TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG 401 TATTGGGCAG TTTGGCAAAG TttGA rrrA CC GGTGTAT TCCGGCGGT ?. 451 ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGT CGGTGGTGTT 501 GCAGCTGTTT GCGCTGATTA CGCCT TGTT TTTCCAAGTG GTGATGGACA 551 AGGTGCTGGT ACATCGGGGA TTCTCTACTT TGGATGTGGT GTCGGTGGCT 601 TTGTTGG GGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA 651 TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT 701 TGTTCCGGCA TCTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA 751 GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT T01 CTTGACCGGT CAGGCGCTGA CT CGGTGTT GGATTTGGCG TTTTCGTTTA S51 TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTGGTA 901 TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT 951 ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACAACCAGT 1001 CGTTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATGGCG 1051 GTGGRGC GC AGATGACGCA GCGTTGSGAC AATCAGTTGG CGGCTTATGT 1101 GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG 1151 TGCAGCTGAT TCAGAAGCTG GT6ACGGTGG CGACGTTGTG GATTGGCGCA 1201 CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA 1251 TATGCTCTCG GGACAGGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT 1301 GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT GGGGGATATT 1351 CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGGCTT TGCCCGATAT 1401 CCGGGGGGAG ATTACGTTCG AACATGTCGA 7TTCCGCTAT AAGGCGGACG 1451 GCAGGCTGAT TTTGCftGGAT TTGAACCTGC GGATTCGGGC GGGGGAAGTG 1501 CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT 1551 GGTGCAGCGT CTGTATGTAC CGGAGCAGGG ACGGGTGTTG GTGGACGGCA 1601 ACGATTTGGC TTTGGCCGCT CCTGCCTGGC TGCGGCGGCA GGTCGGCGTG 1651 GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC 1701 GCTGACGGAT ACGGGTATGC CGCTGGAACG CATTATCGAA GCAGCCAAAC 1751 TGGCGGGCGC ACACGAGTTT AT ATGGJW3C TGCCGGAftGG CtACGGCACC 1801 GTGGTGGGCG AACAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT 1851 TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG 1901 AAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC 1951 ATGCAGGCCA GGGGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT 2001 GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA 2051 TTGTGGAAGC GGGAACACAG CAGGRATTGC TGGCGAAGCC GAACGGATAT 2101 TACCGCTATC TGTATGATTT ACAGAACGGG TAG This corresponds to the amino acid sequence [SEC. ID NO: 28; ORF39-1]: - 1 MSIVSAFtPA LSALIIIAKY HGIAAHPAOI QHEFCTSAQS DLNETOWUA 51 AKSLGLKWV VftQPIKRUW ATLPAVWCD DGNHFXIAKT DGEGEHAQFL 101 IQD VTNKSA VLSFAEFSKR SGKLILVAS RASVLGSÍAK? JFTWFIPAV 151 IKY RLFFEV ALITPLFFQV VMDKVLVHRG FST PW5VA 201 ILWSLFEIV LGGLRTYLÍA "HTTSRIDVEL GARXJFRH1LS LPLSYFEHRR 251 GDTVARVRE LEOIRKFLTG OA TSVLD A FSFIGLAVMW YYSSTLTWVV 301 L? S PAYAGW SAFISPILRT RI? IDRFARNR DNQSFLVBSI TAVGTVKAMA 351 EPCMQRHC "01AAYVASG FRVTKIAWG QQGVQ I3K1. VTVATIAÍIGA 401 RVIE5KXTV GQLIAFKKLS GQVAAPVIR AQUíQDFQQV G SVARLGDI 451 LNAPTENASS HLALPDIRGE GTEEHVDRY KftDGRLIIiQD LNLRIRAGEV 501 LGIVGRSGSG KSTLTKVQR LYVPEQGRVL. VDGNDLAI? A PAHJ ^ RRQVGV 551 VLOBNVLLNR SIRDHIA TD TGMPLERI1E AAKLAGAHEP IMELPEGYGT 601 WGEQGAG S GGQRQRIAIA RALITNPRIL IFDEATSALD YESERAIMQN 651 MQAJCA? RTV LIIAHRSTV KTAHRIIAMD KGRIVEACTQ QEUAKPNGY 701 YRYLYBLQNG Computer analysis of this amino acid sequence gave the following results: Homology with a predicted QRF of N. meningitidis (strain A) The ORF39 shows 100% identity in a 165aa overlap with an ORF (ORF39a) of strain N. of N. meningitidis: - - 20 30 orf 39. pep KFDFnriPAVIKYRR-.FFEVlWSWLQL my! tmiiuu i u? m ?? i orf 39a AVLS FAE FSNR YSGKLI VA = RASVLGSLAKFPFTWFI PA I KYRRLFFEVL WSWLQL 110 120 130 140 150 160 40 50 SO 70 80 90 orf 39. pep FALITPLFFQWMPKV LVHRGFST ^ PWSVALLWSLFEIVLGGLRTYLFAH-TSRIDVE III II III III II IIIII UUI lll m Ul UI Ul U IIIUU II I III MI 11 or orf FALITPLFFQiWMDKVLVHRGFSTf.PWSVALLVVSI 39a 'E_IVLGGLRTYLFAHTT3RIDVE 1"I0 180 190 200 210 220 100 110 120 130 140 150 orf 39. pep! ^ ARLFRHIJ ^ LPLSYEEHRRVGPTVARVRELeQrRNrLTGCAI_r5VLD AFSFIFLAVW 11 II IIIUII II IIIUI II II III II II II II II II II III III II II IUI II II orf 39a I ^ RLFRH SLPI ^ YF? HRRVGDf A VREl-5QI NF TGQA: SVLD? AFSRIFIA H 230 240 250 260 270 JlT uuummiu oíf 9a WYY = STLTWVVLA3LgAYAg «SAFlSPSLRTRL-.DKFARHAD« OSFLVESIT? VGTV AM 290 300 310 320 330 340 The ORF39-1 and the ORF39a show the 99.4 identity in an overlap 710 aa: - - orf39-.pep MSIVSAPLPALSA IIIJUIYHGIAAHPADIQKErrtSAQSDI_lPrQl # L * A e_GI.KAKV mu i MMi IM M m iiMimimi IIIMIMI? MI mm m m u f orf 39a MSIVSAPLPA1_SAIJ: IIAHYHG1AA_.PADI «E_CT-? 0SD1_I_T0W_IAAKSLGI.KAKV orf 39-1. oep VRQPIIOlIJWATLPALVWCODGNflFI AKTDGEGEHAOFLIQDLVtWKSAVLSFAEFSNR m m 11 m p 11 ?? 11 p u 11111 m i ?? m: m u. • i m 111 u m p i orf 39a VRQÍIKIU? lttTLPA_-V »CÜIGHHFIIAKTEK3GGEHAQYl, lQDLT_ «KSAVLSrAEFSNR orf39-l -pep YSGiai_.VASRASVIßS_AK-_FTB-TPAVI and? UU.FEEVLVVSVV? FA! -itPLFFQV i ?? ?? i my I? MI M 11 II? M IMIM iiMiiiiMiiiim? u m u p? YS 39a orf? KLII.VASRASVLGSLAKF_F? irFTPAVIKYRR_FFBVLVVSVV -I.FALITP_FFCV orf 39-1.pep VKDKVLVHRGF5TUl SVAIiWSLrciVIy_ £? ^ i ty FABTre imiuriu? m mppum III a my iiiiu iiiiumm m orf39a MDKVLViraGFSr.?lWS Al? VVSl ^ IVLGGI? TYLFAH orf 39- 1. p «p LPLSYFEB. { ? VG_rrW * VRELEQIHNFLTG0 ^ TSVLDIAF3FIFIAVMHYYSSTLT «VV itiiiuiiimuí MMiMipimuiiuiimiMiumuii m ??? or f 39a LPLS YFEa_UlVG_rtVARVREI £? I «UíF_-TCOALtSVXDLAFSrrFIAVMWYYSSTLT V- 'orf 39-1. pep IA_LPAYAFW = aFlSPll? TRl_HD __ ^ ia} AON0Sp.VESITAVGTVKAM? VEPOM QRWD il mi IMIUMUI? Mimimimmimí mili i m i M M or f 39a IASLPA? AFWSAFJSPI I ^ TRI? DK Aia? AI »<; .ST-VesiTAVGTV AMAVEPC? MC «WD arf 39- 1. pep NQl ^ YVASGYkVfíOAVVGQQ Ql? ^ C ^^ 11 I lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll IC ^ CLVTVATU.IGJUlI, VIES_a.rVG0LI ___ TlMI.S © rf39-l. p «? GQVAAPVISlLAQlWODFCX? TCISV? RI? Dil ^^ ipipiiiiíiiMiiiiiiiiiiiiiiiiiiipiiiiiiiiHiiiiiiiiiinti orf39A GOVAA? VlRLA0L »^ IllüVPTEHASSHIAI.PDIRGEITFEHVDFRY ODFXWGrsVARIJ orf 9-1.pep KADGRLIlQDUI? J ^ RAGEVLGIVGRSGSGKSTLTK milMlUU lllll I IIUIIIIMIIHUpif lilili or rf 39a KADGM-Dl ^ lI? L_ lRAGEVl? IVGRSGSGKSTI, TK_VOKl.?VPAQGidensesVLV?GND_-ALAñ orf 39- 1. pep P? WI_MC ^ GVVLQ_rHV _J_RSI.u3NIALTDTCH ^^ M p MM mmmm I MMIIM my IIM lllll lll IM fll MI l IMM orf 39a PAWLRROV? VVXQENVLUÍRSIKDNIALTDTGMPLERI IEAAKLAGAHEFIWELPEGYGT orf39-l -pep WGEQGAGI ^ GGQ Q IAI R? L? TO I iroEAtSft? Yfc ^ RAIMQH ^^ II MM go IM II II MIIIMIIMIMIIM! I I I II I I III I M Ul I I I I orf39a WGEOGAGLSGGOR0RlAIARALrTt.PRXLIFOEATSAlDYESE? AIM0 MOAICANRTV orf 39- 1. pep LIIAHRLSTVKTAHRI IAHDKGRIVEAGTQQELIAKPNGYYRYLYDLQNGX IIMII IIHIMIIM I I I I I M II II M II IU IM I I I IM I l My M or £ 39a LIIAHR_S-VKTAHÍÍIIAMDKGRIV AGTQ0ELLAKPNGYYRY_.YDi. { 3NGX The full length 0RF39a nucleotide sequence [SEQ. ID NO: 29] is: - - 1 ATGTCTATCS TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT 51 CGCCCATTAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT 101 TTT5TACTTC CGCACAGAGC GATGTAAAGG AAACG AATG GCTGTTAGCC 151 GCCAAATCTT TGGGATTGAA GGCAAA5GTA GTCCOCCAGC CTATTAAACG 201 TTTGGCTATG GCGACTTTAC CCGCATTGGT ATGGTGTGAT GACGGCAACC 251 ATTTTATTTT GGCTAAAACA GACGGTGGGG GTGftGCATGC CCAATATCTA 301 ATACAGGATT TAACTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT 351 TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG _oi TATTGGGCAG TTTGGCAAAG TTT6ACTTTA CCTGGTTTAT TCCGGCGGTA 451 ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGG CGGTGGTGTT 501 GCAGCTGTTT GCGCTGATTA CGCCTCTGTT TTTCCAAGTG GTGATGGACA 551 AGGTGCTGGT ACATCGGGGA TTCTCTACTT TGGATGTGGT GTCGGTGGCT 501 TTGTTGGTGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA 651 TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT 701 TGTTCCGGCA CTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA 751 GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT 801 CTTGACCGGT CAGGCGCTGA CTTCGGTGTT GGATTTGGCG TTTCGTTTA 851 TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTt? 3TA 901 TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT 952 ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACA? O- AGT 1001 CGTTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATßGCG 1051 GTGGAGCCGC AGATGACGCA GCGTTGGGAC AATCAGTTGG CGGCTTATGT 1101 GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG 1151 TGCAGCTGAT TCAGAAGCTG GTGACGGTGG CGACGTTGTG GATTGGCGCA 1201 CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA 1251 TATGCTCTCG GGACAGGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT 1301 GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT GGGGGATATT 1351 CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGG TT TGCCCGATAT 14. 01 CCGGGGGGAG ATTACGTTCG AACATG CGA TTTCCGC GAT AAGKCGGACG 1451 GCAGGCTGAT TTTGCAGGAT TTGAACCTGC GGATTCGGGC GGGGGAAGTG 1501 CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT 1551 GGTGCAGCGT CTGTATGTAC CGGCGCAQGG ACGGGTSTTG GTGGACGGCA 1601 ACGATTTGGC TTTGGCCGCT CCTGCTTGGC TGCGGCGGCA GGTCGGCGTG 1651 GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC 17Q1 GCTGACGGAT ACGGGTATGC CGCTGGAACG CATATCGAA GCAGCCAAAC 1751 TGGCGGG GC ACACGAGTTT ATTATGGAGC TGCCGGAAGG CTACGGCACC 1801 GTGGTGGGCG A? CAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT 1851 TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG 1901 PAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC 1551 ATGCAGGCCA TTTGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT 2001 GTCCACTGT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA 2051 TTGTGGAAGC GGGAACACAG CAGGAATTGC TGGCGAAGCC GARCGGATAT 2101 TACCGCTATC TGTATGATTT ACAGAACGGG TAG This encodes a protein having the amino acid sequence [SEQ. ID NO: 30]: - 1 M5IVSAFLPA 5ALIILAHY HGIAANPADI QHEPCTSAQS DLNETO LLA 51 AKSLGLKAKV VR &? PIKftlAM ATLPAfcVWCD DGHH IIAKT DGGGSHAQ L 101 IOD TTKKSA VI ^ FAEFSKK YSG ILVAS RASVLGSLAK FDP1WFIPAV 151 IKYRRLBTEV WSWQLF ALIGPUTQV VMDKVVHRG FSTLPWSVA 201 LLWSLEEIV I G RTY FA HTTSRI0VEL? GARLFRRLLS PLSYFEHRR 251 VGDTVARVRE LEO? RNFLTG QALTSVLPLA FSFIFIAVMW YYSSTLTHVV 301 LASLPAYAFW SAFISPILRT RLNDKFARKñ DNQSFLVE5I 351 TAVGTVKAMA VB) MTC? IWD KQ1AAYVASG RVT? OAVVG QQGVQUQ L VTVATLWIGA 401 RLVIESKLTV GOLIAFNMLS GQVAAPVIRL AQUIQDFQQV GISVARLGDI 451 LNAPTE ASS H ALPDI GE ITFEHVDFRY KADGRLTLQD LNLRISAGEV 501 LGIVGRSGSG KSTLTKLVQR X.YVPAQGRVL VDGNDXALAA PAWLFU.QVGV 551 VLQENVLLNR SIRDNIALTD TGMPERIIE AAKLAGAHEF IMELPEGYGT 601 WGEQGAGLS GGQRQRIAIA RA1ITMPRIL IFDEATSALD YESEAAIMQN 651 MQAICA RTV LIIAHR ^ STV KTAHRIIAMD KGRIVEAGTQ OELLAKPNGY 701 YRYL DLQNG ORF39a is homologous to a form of cytolysin of A. pl eur op neumoniae: sp | P26760 | RTlB_ACTPL RTX-I DETERMINING B OF TOXIN (PROTEIN) ATP LINK OF SECRETION OF TOXIN RTX-1) (APX-IB) (CYTOLYSIN IB) (CLY-IB). > gi 197137] pir MD43599 cytolysin IB - Actinobacillus pleuropneumoniae (serotype 9) > gi [38944 (X61112) Clyl-B protein [Actinobacillus pleuropneumoniae] Length = 707 Record = 931 bits (2379), Expected = 0.0 Identities = 472/690 (68%), Positive = 540/690 (77%), Spaces - 3/690 (0%) intßrroflßntßi 20 YHGlAANSAIlIQHE? CTSAOSt? .E QWXXXXXXXXXXXXVVRCPrKR? UWiTX.PALV C 79 YH IA NP + -M-H + F + L + T H V ++ I RLA LPALVK ßu.βto? 20 YH GAWPEELKH FDI ^ GKG-LDLTAHLIAAKSLE KAKJV AIDRIAFIA PALV R 78 Question: 80 DDWHFIIAKTKKMElttOYLIODLTTN SAV SFAEFSNRYSGKLIlVASRIASVX ^ SLA 139 + DG HFI- K D E + YLI Di T + -.1 A £ F + Y Gj i VASSAS + H-6 LA Suaeto »79 EtWKBFI TKIDN-'EA? IFDI ^ THWPRlLEOAEFESLYQGKLILVASRASTveKLA 136 Question: 140 K? O? ^ IPAV? -tylU? T? XXXXjaa xxxxxxxiTF ^^ l9c KfTF «ÍFIPAVIlRR + ITPLFfOWMDKVLVHRGF Subject, 137 KFOWWFIPAVIKYBKIFlETUVSIFI ^ IFAL ^ PLYFsVVMtí VLVHSGFSTlNV'ITV 196 Question: 200 XXXXXXXFEIVl? GI-RTY FAHr? _ »I0VEI * MlFRHI?? LPL3Y_EHRR \ DTVARVR 259 - - FEIVL GLRTY + FAH * TSRllWELGARLFRKLL + LPtSYFE + RRVGDTVARVR Bujeta: 197 AIAIVV -TEIVLMG_RTYIFAHST_RIDVE_ßARl__UJI_lALPISYFE_-RRVGDTVMVR 256 Question: 60 EIiEQIRNFLTGOALRSVLDIAFSFI-TAVM «YYSStLr-WVV] ASLPAVAFHSAFlSPIl.? - 319 EL + QIRHFLTGQALTSVLOL FSFIF AVMHYYS LT V + L SLP AND WS FISPILR Subject: 257 EL00IRHp.T ^ ALTSVLDLI__3FirFAV14ífYYSPKLTLVILG _._ J > _YMGWSIFISPII_R 316 Question: 320 TRLNDKFAR ADNOSFL VXSITAVGTVKAMAVEPOKrQRWDNOlAAYVASGFRVTKLAW 79 RÍ. ++ FAR ACNOSFLVES + TA + T + KA + AV PCMT WD QI_A + YV ++ GFRVT LA + Subject: 317 RRLDEKFARGAIWQSFl.VESV AIKTIKAUlVTI 3ÍÍ N WnK0IJlS_Ví? GfRVTriATI 376 Interrogating: 3 SO K30GV0LlQK VTVATI-WIGARLVIESKLTVGQI, I AF «K SGOVAAPVIRl OLMQDF (_i- 439 CCQGVQ IOK + V V TL + GA LVI ++ GQL1AFNHLSGQV APVIRLAQLWQDF SO Subject: J77 GQCKVQFICKVVHVGGLBLGAHLVISGDLSIGQLIAFNKLSGOV? APVIRIAQLWQDFOQ 436 I terrogant i 440 VGISVARLGDSI ^ A__EHASSH] _ .PDIRGEITre_n ^ D-TlYKADsR_ILQßLNIJlIRAGE 4 9 VGISV RLGD + LM + PTE + LALP + I + G + ITF - - FRYK D + IL D + NL 1 * GE Subject: 437 VGXSVTRl? - WI_ (? S_-TSYOGKlALPEIKGDXTFRHIRFRYKPIW \ PVII r? VNLSIQCGE 96 Question: 500 VU3IVGRSGS ?? STLTKLVQR¿YVPA0GRVI, VD ^ 5 & ? V-K? IVGRSGSGKSTL-KL + QR Y + P G + VL + CG + OLAIA P WtRRfVdVVLO + HVLLN Subject: 4g IGl to «GSGtóTLltarC» FYIPENGQV_íIIX-HDLA__JJ_WWUWQVGW OiWVLL? Í S 56 Question: 560 RSIRIWlALtstGMPliRIlEAAK__W »HEFSliEl, PEGYGTvVGE» aGtSGGORQRIAI 619 RSIRDNIAL D Q4P + E + I + AAKIAGAHEFX THE EGY T + VGEQGAGLSGGQRQRIAI Subject i 5J, 7 RSIRn_IñIADPGMPMEKTVHAAKIAG? Ffi: FISELREG_HTIVGEQGAGLSGGQRORIAl 616 Interrogan-be; 620 ARALITNPRI IFDEATSALOYESERAIKQKHQftlCMlRTVl,! lAHRLSlVKT AJiR? IAM 67 p ARAL + NF + ILIFDEATSALDYESE IM + NM IC RTV + IIAHRLSTV A RII M Card: 61 Al_lLVT.NreiIlFraATSALDYESEB? IKRNMHQlCKGRTVIIIAHRI_STV NAI) RirVM 676 Question: 680 DKGRIVEAGTQQELIAKPMGYYRYLY0LON 709 + KG + rVE G + ÜL1A PNG and YL + LQ + Subject: 677 EKGQIVEOGKHKELLADPNGLYKYIJJOLQS 706 Homology with the protein prototype of ATP of leukotoxin secretion HlyB of 1 Ha emoph i lu s to etinomyeetemeomí tan tans (access n umme X53955 The ORF39 and the HlyB protein show the 71% and 60% amino acid identity are overlapped 167 and 55 in the N- and C-terminal regions, respectively: - Or £ 39 1 K_OFtWFIPAVXKYR_ttX »aaXXXía? TXXXXITPlFFQN ^^ 60 KFDFTWFIPAV3KYR + ITPLFFOWHDKVL? HRGF HlyB 137 KFDFT« FIPAVIKYRKIFIETLlVSIFLQIFALITPlFFOVTO? i, VBRGFSTLNVZTV 196 Orf39 121 ELS0IR "GTLS? ALT5Vl __? L? FSFIFLAVHWYySSTL WVVlASLIC QIRNFLTGQALTS + 167 + LDI, FSFIF AVMWYYS LT Wt SL HlyB C 257 // 303 ELDQIRHFLTGOW-TSILtrLLFSFIFFAVMWYYSPKLTLWLGSLPC Orf39 2CANRTVLIIAHRLSTVXTAHRIIÁKS 16S) KGRIVEAGT (IC ^ EIJJUIX_.sYYRYLYDl_ ?. 22C KRTVLIJAKRLSTVK A RII MDKG I + EG QELL + GY YL + LO HlyB 651! CCKRTVLIIAIiRI_! TVKHADRIIVMDRSEIIE <? GKHQE_lJ_) BKGLYSYIiH? _C 705 Based on this analysis, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 7 The next sequence of AD? partial was identified in N. meningitidis [SEC. ID? O: 31]: 1 ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT 51 CTACGCCTGC CAACCGCAAT CCGAAGCCGC ACTGCAAGTC AAGGCTGAAA 101 ACAGCCTGAC CGCTATGCGC TTAGCCG CG CCGACAAACA GGCAGAGATT 151 GACGGGTTGA ACGCCCAAAk sGACGCDSAA ATCAGA ...

This corresponds to the amino acid sequence [SEC. ID? O: 32; ORF52]: 1 M YLIKTAIj. AVAAAGIYAC QFQSEAAVQV KAE? SLTAMR lAVADKQAEI 51 DGL? AQXDAE IR ..

- - Additional work revealed the complete nucleotide sequence [SEC. ID NO: 33]: 1 ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT 51 CTACGCCTGC CAACCGCAAT CCGAAGCCSC AGTGCAftGTC AAGGCTGAAA 101 ACAGCCTGAC CGCTATGCGC TTAGCCGTCG CCGACAAACA GGCAGAGATT 151 GACGGGTTGA ACGCCCAAAT CGACGCCGAA ATCAGACAAC GCGAAGCCGA 2 DI AGAATTGAAA GACTACCGAT GGATACACGG CGACGCGGAA GTGCCGGAGC 251 TGGAAAAATG A This corresponds to the amino acid sequence [SEC. ID NO: 34; ORF52-1]: 1 MKY IRTALL AVAAAGIYAC QPQSEAAVQV KAEHS TAMB. LAVADKQNEI 51 DGLNAQIDAE IRQRBAEELK DYRW1HGDAE VPELEK * Computer analysis of this amino acid sequence predicts a lipid binding site of the prokaryotic membrane lipoprotein (underlined).

ORF52-1 (7 kDa) was cloned into the pGex vectors and expressed in E. coli, as described above. The products of the expression and purification of the protein were analyzed by SDS-PAGE. Figure 4A shows the results of the affinity purification of the GST fusion. Figure 4B shows the hydrophilicity, antigenic index, and AMPHI regions for ORF52-1.

- - Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

E emp lo 8 The next sequence of AD? partial was identified in N. meningi tidi s [SEC. ID? O: 35]: 1 ATGGTTATCG GAATATTACT CGCATCAAGC AAGCATGCTC TTG? CATTAC 51 TCTATTGTTA AATCCCGTCT TCCATGCATC CAGTTGCGTA TCGCGTTsGG 101 CAATACGGAA TAAAAtCTGC TGTTCTGCTT TGGCTAAATT TGCCAAATTG 151 TTTATTGTTT CTTTAGGaGC AGCTTGCTTA GCCGCCTTCG CTTTCGACAA 201 CGCCCCCACA GGCGCTTCCC AAGCgTTGCC TACCGTTACC GCACCCGTGG 251 CGATTCCCGC GCCCGCTTCS < * CAGCCTGA This corresponds to the amino acid sequence [SEC. ID? O: 36; ORF56]: I MVIGILLASS KHAI? MTLLL PVFHASSCV SRXAIR? KIC C5ALAKFA L 51 FIVSLGAACL AAFAFD? APT GASQALPTVT APVAIPAPAS AA * Additional work revealed the complete nucleotide sequence [SEC. ID? O: 37]: 1 ATGGCTTGTA AGG rr A GGTTTTTCCG TTAATGGTTA TCGGARTAT7 51 ACTTGCATCA AGCAAGCCTG CCCTTTCCT TACTCTATTC TTAñATCCCG 101 TCTTCCATGC ATCCAG? GC GTATCGCGTT GGGCAATACG GAATAAAATC 151 TGCTGTTCTG CTTGGGCTAA ATTTGCCAAA TTGTTTATTG TTTCTTTAGG 201 AGCAGCTTGC TTAGCCGCCT TCGCTTTCGA CAACGCCCCC ACAGGCGCTT 251 CCCAAGCGTT GCCTACCGTT ACCGCACCCG TGGCGATTCC CGCGCCCGCT 301 TCGGCAGCCT GA - - This corresponds to the sequence of amino acids [SEC. ID NO: 38; ORF56-1]: 1 MACTG KVFP LMVXGILLAS SKPAPFLTU, 1NPVFHASSC VSR AIRHKI 51 CCSALAKFAK LFTVSLGAAC LAAFAFD AF TGASQALPTV TAPVAIPAPA 101 SAA " Computer analysis of this amino acid sequence predicts a leader peptide (underlined) and suggests that ORF56 could be a membrane or protein pe r ip 1 s um.

Based on this analysis, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

E j empl o 9 The next sequence of AD? partial was identified in N. meningitidis [SEC. ID? O: 39]: 1 ATGTTCAGTA TTTTAAATGT GTTTCTTCAT TGTATTCTGG CTTGTGTAGT 51 CTCTGGTGAG ACGCCTACTA TATTTGGTAT CTTGCTCTT TTTTACTTAT 1D1 TGTATCTTTC TTATCTTGCT GTTTTTAAGA TTTCTTTTC TTTTTTCTTA 151 GACAGAGTTT CACTCCG5TC TCCCAGGCTG GAGTGCAAAT GGCATGACCC 201 TTTGGCTCAC TGGCTCACGG CCACTTCTGC TATTCTGCCG CCTCAGCCTC 251 CAGGG.-.

- - This corresponds to the sequence of amino acids [SEC. ID NO: 40; ORF63]: 1 MFSILNVFLH CILACWSGE TPTIFGILAL FYLLYLSYLA VFKlFFSFfL 51 DRVSLRSPRJL. ECKWHDPÍAH «LTATSAILP PQPPG ...

Computer analysis of this amino acid sequence predicts a region of transmembrane Based on this analysis, it is predicted that this N. meningi tidi s protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

E j emp lo 10 The following partial DNA sequence was identified in N. meningitidis [SEC. ID? O: 41]: ..GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTT6AAAT ACCCGTTGTT 51 GCTTTGGATT GCGGATATGT TGCTGTACCG GTTGTTGGGC GGCGCGGAAA 101 tC AAtaCGG CCGTTGCCCT C5TGCCGCC5A TGACGGATTG GCAGCATTTT 151 TTGCCGGCGA TGGGAACGGT GTCGGCTTGG G GGCGGTGA TTTGGGCATA 201 CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA This corresponds to the amino acid sequence [SEC. ID? O: 42; ORF69]: - - 1., VRTWLVFWLQ F.LKYPLLLWI AOMLLYRLLG GAEIECGR P VPPMTDWQHF 51 LPAMGTVSAW VAVIHAYLMI ESEKNGKY * Computer analysis of this amino acid sequence predicts a transmembrane region.

A corresponding ORF of strain N. of N. meningitidis was also identified.

Homo loggia with a predicted ORF of _ /. me ingiti is (strain A) The ORF69 shows 96.2% identity in an overlap of 78aa with an ORF (ORF69a) of strain N. of N. meningitidis: 70 79 orf69.p «p VAVIWAYLHIESEKKGRYX UUIIIUUUUUU orf 69a VAVIWAYLMIESEKHGRYX 70 The nucleotide sequence ORF69a [SEQ ID? O: 43] is: 1 GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTCTT 51 GCTTTGTATT GCGGATATGC TGCTGTACCG GTTGTTGGGC GGCGCGGAAA 101 TCGAATGCGG CCGTTGCCCT GTACCGCCGA TGACGGATTG GCAGCATTTT 151 TTGCCGACGA TGGGAACGGT GGCGGsTTGG GT43GCGGTGA TTTGGGCATA 201 CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA - - This encodes a protein having the amino acid sequence [SEQ. ID NO: 44]: 1 VRTWLVFWLQ RLKYPLLLCI A WLLYRL G GAEIECGRCP VPPHTDWQHF 51 LPTHGTVAAW VAVItfAYLWI £ SEKNGRY * Based on this analysis, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 11 The following DNA sequence was identified in N. meningitidis [SEC. ID? O: 45]: 1 ATGTTTCAAA ATTTTGATTT GGGCGTGTTC CTGCTTGCCG TCCTCCCCGT 51 GCTGCCCTCC ATTACCGTCT CGCACGTGGC GCGCGGCTAT ACGGCGCGCT 101 ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC 151 CTGCCCCATA TCGATTTGGT CGGCACAATC ATCgTACCGC TGCTTACTT 201 GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT 251 CGCGCAACTT CCGCAACCCG cGCCTTGCCT GGCGTTGCGT TGCCGCGTCC 301 GGCCCGCTGT CGAATCTA5C GATGGCTGTw CTGTGGGGCG TGGTGGGGGT 351 GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG 401 CAAACTACGG TATTCTGATC AATGCGATTC TGTTCGCGCT CAACATCATC 451 CCCATCCTGC C TGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC 501 GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA 551 TCCTACTGCT GATGCTGACC sGGGTTTTGG GTGCGTTTAT GCACCGATT 601 STGCGGraTGc GTGATTGCrT TTGTGCAGAT GTwCGTCTGA CTGGCTTTCA 651 GACGGCATAA This corresponds to the amino acid sequence [SEC. ID? O: 46; O R F 77]: - - i MF? NFDLGVF LLAVLPVLPS ITVSHVARGY TARYWGDNTA EOYGRLTLNP 51 PHIDLVGTI I PLLTLMFT PFLFGK? PI PIDSR FRNP RIAWRCVAAS 1Q1 GPLSNLAMAV LWGWLVLTP YVGGAYQMPL AQMA1TYGILI NAII-FALN I 151 PILPWDGGIF IDTFLSAKYS QAFFTKIEPYG T IILLLMLT XVLGAFIAPI 201 XRXRDCXCA VRLTGFQTA * Additional work revealed the complete nucleotide sequence [SEC. ID NO: 47]: 1 ATGTTTCAAA ATTTTGATTT GGGCGTGTTT CTGCTTGCCG TCCTGCCCGT 51 GCTGCTCTCC ATTACCGTCA GGGAGGTGGC GCGCGGCTAT ACGGCGCGCT 101 ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC 151 CTGCCCCATA TCGATTTGGT CGGCACAATC ATCGTACCGC TGCTTACTTT 201 GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT 251 O? CGCAACTT CCGCAACCCG CGCCTT6CCT GGCGTTGCGT TGCCCCGTCC 301 GGCCCGCTGT CGAATCTAGC GATGGCTGTT CTGTGGGGCG TGGTTTTGGT 351 GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG 401 CAAACTACGG TATTCTGATC AATGCGATTC TG? TC5CGCT CAACATCATC 451 CCCATCCTGC CTTGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC 501 GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA 551 TCCTACTGCT GAT6CTGACC GGGGTTTTGG GTGCGTTTAT TGCACCGATT 601 GTGCGGCTGG TGATTGCGTT TCTGCAiSATG TTCGTCTGA This corresponds to the amino acid sequence [SEC. ID NO: 48; ORF77-1]: 1 WFQWFDLGVF LtAV PVLLS ITVR £ VARGY TARYWGDMTA EQYGRLTL P 51 LPHIDLVGT1 IVPLLTLMFT PFLFGWA PI PIOSRHFRHP RlAWRCVAAS 101 GgLSKIxfiHAV I? FGWLVLTP YVGGAYQMFL AQMAHVGILI flAlLFALNII 151 PILPWPGGIF IDTFLSA YS QAFRKIEPYG TffllLLLMLT GVLGAFIAPI 201 VRLVIAFVQM FV * Computer analysis of this amino acid sequence reveals a putative leader sequence and several transmembrane domains.

- - A corresponding ORF of strain A of N. meningitidis was also identified.

H ome logy with a predicted ORF of N. meningi tidis (strain A The ORF77 shows 96.5% identity in a 173rd s ia tion with an ORF (ORF77a) of strain N. of N. meningitidis: 20 30 40 50 60 orf7 .pep M_MFDLGVF AVLPVLP5ITVSHVARGYTARYWSDirrAEQYGRLtL-shore .PLgHIPLVGti iiiniiimiiiimimiiiiHimi RGYTARYWGDtiyAEQYGRLTIJlPLPHIDLVGtJ October 20, 30? Or 80 ao? Oo no orf 120 # 77.p p IVPLLTI_MrrPFLF ARglPlDSRWFR ^? ^ PRlAHRCVAASGPI WLAMRVI.WGVVLVLTP pnnu 11 or 11 one mu mu 1 iiiiiiiiiimi 1 uni 11 mi 1 orf 77a IVPLLTI ^ TPFLFGtfARPIFIDSRMFR? PRLftWRCVAASGPLSMLft AVLWGVVLVLTP 40 50 60 70 80 90 130 140 150 160 170 180 arf 77.pßp .yGGAYWPlACy < HYGILI »^ IL ftlJfrlPIfcP DGGiriPTFLSA YSO RKrEPYG iipumiiiiiii num uiuuumuuuum imuiui? orf77a YVGGAYQMglAQMA? YXILIHArLXAL? riPIL ^ 100 110 120 130 140 150 190 200 210 220 orf 7. pep TWI ILLL-4LTKVLGAFIAPIXRyRDC3CAPVRLTGFOTA_U UU UU UU UU UU UUUU TW I IXLLMLTGVLGAXIAPIVQLVI AFVQHFVX 360 17C 180 The ORF77-1 and ORF77a show 96.8% identity in an overlap 185 aa: - - 20 30 4C 50 € 0 orf 77-1.pßp MFQHFDLGVFUAVL-VLLSíTVREVARGYTARYHGMITAEOYGRLTIJIPLPHIOLVGTI MUÍ MIUUU U UUUUU UMIU U > orf77a RGYTARYttóMrrAEÜYGRLTU_PLPHIDLVGTI ID 30 70 80 20 90 100 110 120 orf77-l .pep IVPLLTLHFTPFLFG ARPIPIDSRNFRNPRIAWRCVAASGPLSNIAMAVLWGWLVLTP or iimiii (uu M an u iiiiiutuimiii ms your niimii my orf 7 XVPLLTI? FTPFLFG ARPIPIOSRNFRKPRLAKRCVAASGPLSNIAMAVLWGVVLVLTP 40 50 60 70 80 90 130 140 150 160 170 1B0 arf77 - 1. PSSP YVGa "And MPIAQHWYGIUNAIIÍALNJIPrLtíDGGI_IDTFLSAKYSQAFR rEPYG IIIIIH UUUUIU orf77a YVGGAYCJMPLAQMAKYXILlNArLXALNIIPILPHDGGrFIDTFLSAKXSOAFR SEPYG 100 110 120 130 140 150 190 200 210 orf 77- 1. pbw TW IILLI LTGVLGAFIAPIVRLVlAFVQHFVX, \\ > iimiiiii piiiüiiimnii orf77a TW1IXLLMLTGVLGAXIAPIVQLVIAFVQMFVX 160170 1B0?? U.na nucleotide sequence ORF77a [SEC. ID NO: 49] was identified 1 .. CGCGGCTATA CAGCGGCTA CTGGGGTGAC AACACTGCCG AACAATACGG 51 CAGG TGACA CTGAACCCCC TGCCCCATAT CGATTTGGTC GGCACAATCA 101 TCGTACCGCT GCTTACTTTG ATGTTTACGC CCTTCCTG T CGGCTGGGCG 151 CGTCCGATTC CTATCGATTC GCGCAACTTC CGCAACCCGC GCCTTGCCTG 201 GCGTTGCGTT GCCGCGTCCG GCCCGCTGTC GAATCTGGCG ATGGCTGTTC 251 TGTGGGGCGT GGTTTTGGTG CTGACTCCGT ATGTCGGTGG GGCGTATCAG 301 ATGCCGTTGG CNCAAATGGC AAACTACNW ATTCTGATCA ATGCGATTCT 351 GTRCGOGCTC AACATCATCC CCATCCTGCC TTGGGACGGC 6GCATTTTCA 01 TCGACACCTT CCTGTCGGC AAATANTCGC AAGCGTTCCG CAAAATCGAA 451 CCTTATGGGA CGTGGATTAT CCMGCTGCTT ATGCTGACCG GGGTTTTGGG SQ1 TGCGTWTATT GCACCGATTG TGCAGCTGGT GATTGCGTTG GTGCAGATGT 551 TCGTCTGA This encodes a protein having the amino acid sequence [SEQ. ID NO: 50]: 1 . -RGYT &RYWGO KTAEQYGRLT HP gaiD, GTUVgL TL MFTPFLFGWA 51 RPIPIDSRHF RNPRLA BCV AA3GPLSHLA MAVLWGWI / LTPYVGGAYQ 101 MFL QMAWyy I INAILXAL HIIPILPWDG GIFIDTFLSA KXSQAFR IE 151 PYGTWIIXU. MLTGVLGAXI APIVQLV1AF VÚMFV * Based on this analysis, it is predicted that This protein from N. meningitidis, and its - - epitopes, could be useful antigens for diagnosis or diagnosis.

Example 12 The following partial DNA sequence was identified in N. men i n gi t i di s [SEC. ID ? O: 51]: 1 ATGAACCTGA TTTCACGTTA CATCATCCGT CñAATGGCGG TTA GGCGGT 51 TTftCGCGCTC CTTGCCTTCC TCGCTTTGTft CAGCTTTTTT GAAATCCTGT 101 AC AAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGCTG 151 GGCTACACCG CCCTCAAAAT GCCCGCCCGC CCCTACGAAC TGATTCCCCT 201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA 2S1 GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG 301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCOTCGCGCT 351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAS 401 CCGCCGCCAT CAACGGCAAA ATCAGCACCG 6CAATACCGG CCTTTGGCTG 451 AAAGAAAAAA ACAGCGTGAT CAAÍ "GTGCGC GAAATGTTGC CCGACCAT ..

This corresponds to the amino acid sequence [SEC. ID? O: 52; ORF112]: 1 M? L? SRYIIB. MAVMAVYAL? AFLALYSFF E? LYETGSLG KGSYG? WEML 51 GYTALKMPAR AYELIPLAVL IGGI.VSLSQL AAGSELTVIK ASG ST KLL 101 LILSQFGFIF AIATVALGEM VAP? LSQKAE? IKAAAIKGK ISTG? TGLW 151 KEK? SVI? VR EMLPDH ...

The additional work revealed the partial nucleotide sequence [SEC. ID? O: 53]: - - 1 ATGAACCTGA TTTCAC5TTA CATCATCCGT CAAATGGCGG TTATGGCGGT 51 TTACGCGCTC CTT6CCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT 101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCñTATG GGAAATGCTG 151 gGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC TGATTCCCCT 201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA 251 GCGAACTGAC CGTCATCAAA GCCftGCGGCA TGAGCACCAA AAAGCTGCTG 301 TTGATTCTGT CGCAGTTCGG TTTATTTTT GCTATTGCCA CCGTCGCGCT 351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG 401 CCGCCGCCAT CAACGGCAAA ATCAGCACCG GCAATACCGG CCTTTGGCTG 451 AñAGAAAAAA ACAGCrTAT CAATGTGCGC GAAATCTTGC CCGACCATAC 501 GCTTTTGGGC ATCAAAATTT GGGCGCGCAA CGATAAAAAC GAATTGGCAG 551 AGGCAGTGGA AGCCGATTCC GCCGTT GGA ACAGCGACGG CAGTTGGCAG 601 TTGAAAAACA TCCGCCGCAG CACGCTT5CC GAAGACAAAG TCGAGGTCTC 651 TATTGCGGCT GAAGAAAACT GGCCGATTTC CGTCAAACGC AACCTGATGG 701 ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA ACTGACCACC 751 TACATCCGCC ACCTCCAAAA CAACAGCCAA AACACCCGAA TCTACGCCAT BOC CGCATGGTGG CGCAAATTGG TTGACCCCGC CGCACCCTGG GTGATGGCGC 851 TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC 901 TTAAAACTCT TCGGCGGCAT CTGTsTCGGA TTGCTGTTCC ACCTTGCCGG 951 ACGGCTCTTT GGSTTTACCA GCCAACTCGG- This corresponds to the amino acid sequence [SEC. ID NO: 54; 0RF112-1]: 1 HNLI5RYIIR CWAVMAVYAL LAGS LYSFF EILYETGNLG KGSYGGWEML 51 GYTALMPAR AYELrPLAVL IGGLV5LS0L AAGSELTVGK ASGMSTKKLL 101 LILS FGFIF AIATVALGEW VAPTLSQKAE NIKAAAXNGK ISTG TGLWL 151 EKKSXISVR EMLPDHTLLG IKIWARNDKN E1AEAVEADS AVLNSDGSW0 201 LKNIRRSTLG EDKVEVS? ÑA SEHV.PISV E- tflttDVLLVXP DQMSV5ELTT 251 YIRHLQNNSC STRIYAIAWW RKLVYPAAASf VMALVAFAFT PCTTRHGNMG 301 LKLFGGICXG LLFHIAGRLF GFTSC.L.

Computer analysis of this amino acid sequence predicts several transmembrane domains.

A corresponding ORF of strain N. of N. meningitidis was also identified.

- -Homology with a predicted ORF of N. meningitidis (strain A) ORF112 shows 96.4% identity in an overlap of 166aa with an ORF (0RFll2a) of strain N. of N. meningitidis: ? 70 80 90 100 110 120 orf 112. pep AY LrWAVLIGGLVS ___ QI_A & SE_TVIKASGí-S l TKKLLLILSQFGFIFAIATVA GE III:.? II 1 I II I l ... t IIKIIIIÜlil orf l llll lllll 112a AyELKPLAVLrGGLV = XSOLAAGSELXVlKASGMSTKKLLLILSOroFIFAIATVALGEW 7C 80 90 100 110 120 130 140 1S0 160 OX f 112. ep VAÍ ^ LSOKAE? IKAAAl? GKISTs? TGL L EKWSVIKVREMLPOH uiu 11 m MI MU uuu 111 muu uu a uiu orf 112a VAPTLSOKAE? LKAAAI? GKlSTG? TGLWLKEKKISIIWVReMLPDKTLLGIKIWAR-ÍDK? 130 140 150 160 170 180 or f 112a ELAEAV ^ ADSAVL? SDGStfQLK? IRRSTl? EDKVEVSIAAEEXWPlSVra? UroVLLVKP 1 »0 200 210 220 230 240 A "partial" nucleotide sequence of ORF112a [SEQ. ID? O: 55] was identified: - - 1 ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT 51 TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CftGCTTrTTT GAAATCCTGT 101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGHTG 151 GGNTACACCG CCCTCAAAAT GNCCGCCCGC GCCTACGAAC TGATGCCCCT 201 CGCCGTCCTT ATCGGCGGAC TGGTCTCTNT CAGCCAGCTT GCCGCCGGCA 251 6CGAACTGAN CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG 301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT 351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG 401 CCGCGGCCAT CAAOSGCAAA ATCAGTACCG GCAATACCGG CCTTTGGCTG 451 AAAGAAAAAA ACAGCATTAT CAATGTGCGC GAAATGTTGC CCGACCATAC 501 CCTGCTCGGC ATTAAAATCT GGGCCCGCAA CGATAAAAAC GAACTGGCAG 551 AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG CAGTTGGCAG 601 TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG TCGAGGTCTC 653 TATTGCGGCT GAAGAAAAT GGCCGATTTC CGTCAAACGC AACCTGATGG 701 ACGTATTGCT CGTCAAACCC GACCAAATGT CC6TCGGCGA ACTGACCACC 751 TACATCCGCC ACCTCCAAA NACAGCCAA AACACCCGAA TCTACGCCAT ßoi CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG GTGATGGCGC 651 TCGTCGCCTG TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC 901 TAAAANTCT TCGGCGGCAT CTGTCTCGGA TTGCTGTTCC ACCTTGCCGG 951 NCGGCTCTTC HGGTTTACCA GCCAACTCTñ CGGCATCCCG CCCTTCCTCG 1001 NCGGCGCACT ACCTACCATA GCCTTCGCCT TGCTCGCCGT TTGGCTGATA 1051 CGCAAACAGG AAAAACGCTA A This encodes a protein having the amino acid sequence [SEQ. ID NO: 56]: 1 HKLISRYIIR QMAVMftVYAL LftFIALYSFT EILYETGHLG KG5YGIWSMX 51 GYTA KHXftR AY_.LW_.IAVL IGGI.VSX__QL AAGSELXVIK ASGMSTKKLL 101 LILSQFGFIF AIATVALGEW VAPLSQKAE NIKAAAING ISTGNTGLWL 151 KEKNSIINVR EMLPDHTLLG IKItfAlWDKN E AEAVEADS AVLNSDG5H? Q 01 LKNIRRSTLG EDKVEVSIAA EEXtfPISVKR NLMOVLLVKJ? DQMSVGELTT 251 YIRHLQXXSQ RTRIYAIAW RKLVYPAAAW V ALVAFAFT PQTTRHGKMG 301 KXFGGICLG LLFHLAGRLF XFTSQLYGIP PFLXGALP I AFALLAVWLI 351 RKQEKR * The 0RF112a and the 0RF112-1 show 96.3% identity in an overlap 326 aa: - orfll2 * .pßp MNLISRYIlRQHAVM? VYAU? FLALYSFí? ILYETG IiGKGSYGIHEtKGrrALKMXAR uiiuiiiiiiui i ?? iiiiiiii iiiiiin mmuiuuii imiii or orf 112-1 M? ^ ISRYlIRQMAV? íAVYALLAFLALYSFBEILYETGNLGKGSYGIírEMLGYTALKMPAR orf 112a. pSp AYEIlíPLAVLlGGLVSXSOlAAGSELXVI A-GMSTKKLLLILSQFGRIFAIATVALGE UII? ÍI mu m uuu UMUIII iiuup 11 m om m orm 112-1 AYELIPLAVLIGGLVSI ^ ÜLAAGSELTVIKASGMS? KKLLLILSOFGFIFAIATVALGEW orf 112a. pep VAPrLSQBWSNIKAAAINGKISTGNTGL LKEKNSrim EMLPDHTLLGIKIWARND K u m u i m m u u i ?? u u m u u u u u m m 11 u orf 112-1 VA-ti_SQKAENlKAAAINGKISTGNTGLW_KEKNSXSNVREMLPDHTLLGIKItfAR DKN orf 112a. pep EI_MAVEJU5SAVI ^ S0GSWOLK rRRSTI_GEll! fEVS? AA ££ XWP? SVKRNU-DVLLVKP muiuiim mui u iinummuí u i n ummi u m or f 112-1 EI_AEAVE? 0SAVl ^ S? X3SWQUWIRRSTl ^^ t > KVErVSlAñEENWPISVKKNlíroVLl, VKP orf 112a .pep DQHSVGELTTYIRHWXXSí »rrRlYñIA m < KL \ r ¥ -FÍWAWVMALVAFAFTPQTTRHGNMG lumiuuu m m m mu u m i iiiuuu joined uu m or f 112-1 DQMSVGEITTYSRfíL ON S0STR1 YAJAWHRKLVYPAAAWVMALVAFAFTPOGTKHGHMG orf 112a .pep LíQCFGGICLGLL-HLAGRLFXFTSQLYGIPPFLXGALP IAFALLAVWLIRKOEKRX M IIIII m 11 orfll2-l LKLFGGICXGLLFHIAGRLFGFTSOL Based on this analysis, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

E xemployment 13 The next sequence of AD? partial was identified in N. meningitidis [SEC. ID? O: 57]: - - 1 -- . 1 -GCAGTAGCCG AAACTGCCAA CAGCCAGGGC AAAGGTAAAC AGGCAGGCAG 51 TTCGGTTTCT GTTTCACTGA -AACTTCAGG CGACCTTTGC GGCAAACTCA 101 AAACCACCCT TAAAACTTTG GTCTGCTCTT TGGTTTCCCT GAGTATGGTA 151 TTGCCTGCCC ATGCCCAAAT TACCACCGAC AAATCAGCAC CTAAAAACCA 201 GCAGGTCGTT ATCCTTAAAA CCAACACTGG TGCCCCCTTG GTGAATATCC 251 AAACTCCGAA TGGACGCGGA TTGAGCCACA ACCGCTA.TA CGCATTTGAT 301 GTTGACAACA AAGSGGCAGT GTTAAACAAC GACCGTAACA ATAATCCGTT 351 TGTGGTCWA GGCAGTGCGC AATTGATTTT GAACGAGGA CGCGGTACGG iQl CTAGCAAACT CAACGGCATC GTACCGTAG GC6GTCAAAA GGCCGACGTG 451 ATTATTGCCA ACCCCAACGG CATTACCGTT AATGGCGGCG GCTTTAAAAA 501 TGTCGGTCGG GGCATCTTAA CTACCGGTGC GCCCCAAATC GGCAAAGACG 551 GTGCACTGAC AGGATTTGAT GTG £ GTCAAG GCACATTG? A CCGTAdrAGC 601 AGCAGGTTGG AATGATAAAG GCGGAGCmrm yTACACCGGG GTACTTGCTC 651 GTGCAGTTGC TTTGCAGGGG AAATTwnmGG GTAAA.AACT GGCGGTTTCT 701 ACCGGTCCTC AGAAAGTAGA TTACGCCAGC GGCGAAATCA GTGCAGGTAC 751 GGCW3CGGGT ACGAAACCGA CTATTGCCCT TGATACTGCC GCACTGGGCG SOI GTATGTACGC CGACAGCATC ACACTGATTG CCAATGAAAA AGGCGTAGGC 851 GTCTAA This' corresponds to the amino acid sequence [SEC. ID NO: 58; ORF114]: 1 ..AVAETANSQG KGKQAGS = VS VSLKTSGDLC GKLKTTLKTL VCSLVgLSMV 51 LPAHAQITTD KSAPKNQQW ILKTNTGAPL VNI0TPNGRG LSSWRXYAFD 101 v? Í? KGAVLN? Í DBJSNNPFWK GSAQLXLNEV RGTASKUíG? VTVGGQKADV 151 IIANPNGITV HGGGFKNVGR GJLTTGAP0I GKDGALTGFD WKAHWTVXA 201 AGWNDKGGAX YTGVLARAVA LQGKXXGKXL AVSTGPQKVD YA5GEISAGT 251 AAGTKPT1AL DTAALGGMYA DSITLIANEK GVGV * Additional work revealed the complete nucleotide sequence [SEC. ID NO: 59]: - 1 ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT 51 GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG 101 GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA 151 CTCAAAACCA CCCTTAAAAC TTTGGTCTGC TCTTTGGTTT CCCTGAGTAT 202 GGTATTGCCT GCCCATGCCC AAATTACCAC CGACAAATCA GCACCTAAAA 251 ACCAGCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT 301 ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT 351 TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC 401 CGTTTGTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT 451 ACGGCTAGCA AACTCAACGG CATCGTTACC GTA.GGCGGTC AAAAGGCC & fc 501 CGTGATTATT GCCAACCCCA ACGGCATTAC CGTTAATGGC GGCGGCTTTA 551 AAAATGTCGG TCGGGGCATC TTAACTACCG GTGCGCCCCA AATCGGCAAA € 01 GA.CGGt < SC_VC. TX3CTOTG03V CAASGCACAT GACCGTAGG 651 AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG 702 CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AGGGTAAAAA CCTGGCGGTT 751 TC ACCGGTC CTCAGMAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG 801 TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG 851 GCGGTATGTA CGCCGACAGC ATCACACTGA TTGCCAATGA AAAAGGCGTA 901 GGCGTCAAAA ATGCCGGCAC ACTCGAAGCG GCCAAGCAAT TGATTGTGAC 951 TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG 1001 GCACCGAAGC TTCACCGACT TATCTCTCCA TCGAAACCAC CGAAAAAGGA 1051 GCGGCAGGCA CATTTATCTC CAATGGT6GT CGGATCGAGA GCAAAGGCTT 1101 ATTGGTTATT GAGACGGGAG AAGATATCAG CTTGCGTAAC GGAGCCGTGG 1151 TGCAGAATAA CGGCAG CGC CCAGCTACCA CGGTATTAAA TGCTGGTCAT 1201 AATTTGGTGA TTGAGAGCAA AACTAATGTG AACAATGCCA AAGGCCCGGC - - * B51 CGCTTTGGCT GGGTGTGTTA GCGGATTGGT ACAAGGAAAA TGTAAAGACG 4901 GGGCAATTGG CGCAGCAGTT GGGGAAATCG TAGCCGACTC CATGCTTGGC 4951 GGCAGAAACC CTGCTACACT CAGCGATGCG GAAAAGCATA AGGTTATCAG 5001 TTACTCGAAG ATTATTGCCG GCAGCGTGGC GGCACTCAAC GGCGGCGATG 5051 TGAATACTGC GGCGAATGCG GCTGAGGTG5 CGGTAGTGAA TAATGCTTTG 5101 AATTTTGACA GTACCCCTAC CAATGCGAAA AAGCATCAAC CGCAGAAGCC 5151 CGACAAAACC GCACTGGAAA AAATTATCCA AGGTATTATG CCTGCACATG 5201 CAGCAGGTGC GATGACTAAT CCGCAGGATA AGGATGCTGC CATTTGGATA 5251 AGCAATATCC GTAATGGCAT CACASGCCCG ATTGTGATTA CCAGCTATGG 5301 GGTTTATGCT GCA5GTTGGA CAGCTCCGCT GATCGGTACA GCGGGTAAAT 5351 TAGCTATCAG CACCTGCATG GCTAATCCTT CTGGTTGTAC TGTCATGGTC 5401 ACTCAGGCTG CCGAAGCGGG CGCGGGAATC GCCACGGGTG CGGTAACGGT 5451 AGGCAACGCT TGGGAAGCGC CTGTGGGGGC GTTGTCGAAA GCGAAGGCG5 5501 CCAAGCAGGC TATACCAACC CAGACAGTTA AAGAACTTGA TGGCTTACTA 5551 CAAGAATCAA AAAATATAGG TGCTGTAAAT ACACGAATTA ATATAGCGAA 5601 TAGTACTACT CGATATACAC CAATGAGACA AACGGGACAA CCGGTATCTG 5651 CTGGCTTTGA GCATGT CTT GAGGGGCACT TCCATAGGCC TATTGCGAAT 5701 AACCGTTCAG TTTTTACCAT CTCCCCAAAT GAATTGAAGG TTATACTTCA 5751 AAGTAATAAA GTAGTTTCTT CTCCCGTATC GATGACTCCT GATGGCCAAT SB01 ATATGCGGAC TGTCGATGTA GGAAAAGTTA TTGGTACTAC TTCTATTAAA 5851 GAAGGTGGAC AACCCACAAC TACAATTAAA GTATTTACAG ATAAGTCAGG 5901 AAATTTGATT ACTACATACC CAGTAAAAGG AAACTAA This corresponds to the amino acid sequence [SEC. ID NO: 60; ORF114-1]: - - 1 MHKGLHRI1F SKKHSTMVAV AETANSQGKG KOAGSSVSVS LKTSGDLCGK 51 KTTLKT1.VC SLVS SW7LP AHAQITTDKS ABKNQQW? KTÍJTGAPLVN 101 IQTPNGRGI HNRYTQF0VD NKGAVLNI? DR NNFFWKGS AQLILNEVRG 151 TASKL GIVT VGGQKADVII ANPNGGTVNG GGFKNVGRGI LTTGAPQIGK 201 DGALTGFDVR QGTLTVGAAG WNDKGGA0YT GVLARAVALQ GKLOGKHLAV 251 STGPQKVDYA SGEISAGTAA GTKPTIALDT AALGGHYADS ITLIAHEKGV 3Q1 GVKNAGTLEA AKQLIVTSSG RIBNSGRIAT TADGTEASPT AND SIETTEKG 351 AAGTFISNGG RIESKGLLVI ETGEDISLRN GAWQttHGSR PATTVLNAGH 401 NLVIES TNV NNAKGPATLS ADGRTVI EA SIQTGTTVYS SSKGNAELGN 451 WrRlTaftD VLSNGTISSS AVID &KDTAB I? AGKP S E ASTVTSDIRL 501 NGGSZKGG Q LALLADDKIT AKTTKLK PG NLYVHTGKDL NLNVDKDLSA 551 ASIHLKSDNA AHXTCT5KTL TA5KDHGVEA GSL? ÍVTNTNL RTNSGNLHIO 601 AAKGNIQLRN TKLHAAKALE TTALQGNIVS DGLHAVSADG HVSLLANGNA S51 DFTGHNTLTA KADVUAGSVG BGRLKADNTN ITSSSsDITL VAGNGIQLGP 701 GKQBNSINGK msif8NGGN ADLKKLHVHA KSGAHIHSB RA 5IENTKL 751 ESTH TfíLKA OHERVTLSQV DAYAHRHLSI TGSQIWQNDK LPSANKLVAN 801 GVIALHARYS Q1ADNTTLRA GA1 LTAGTA LVKRGNINWS TVSTKTLEDN 851 AELKP1? GRL NGEAGSG LT IEPANRT3AH TDLSÜCTGGK LLLSAKGGHA 901 GAPSAQVSSL EAKGNIRLVT GETDLRGSKI TAGKNLWAT TKGKLH2EAV 351 NtfSFSNYFPT OAAELNQS KELEOOIAQL KKSSPKSKLI FTLQEERDRL 1001 AFYIQAINKE VKGKKPKGKE AND QAKSAQN IDLISAOGIE 2SGSDITASK 1051 KLNLHAAGVL PKAADSEAAA ILIDGITDQY EIGKPTYKSB YDKAALNKPS 1101 RLTGRTGVSI RAAARLDDAR IIIGASE? KA PSGSIDIKAH SO? VLEAGí? Ti 1151 DAYTFLKTKG KSGKIIRKTK FTSTRDHLIM PAPVELTA G ITLOAGGNIE 1201 ANTTRFNAPA GKVTLVAGEE LQLLAESGIH KBELDVQKSR RFIGIKVGKS L251 NYSKNELNET KLPVRWAQT AATRSGWDTV LEGTEFKTTL AGADIOAGVG L301 EKARADAKII LKGIVRIQS EEKLETNSTV OKQAGRGST IETLKLPSFE L351 SPTPPKLTAP GGYGVDIPKG NLKTEIEKLA KQPEYAYKQ LQVAKNVffiW 1401 QVQLAYDKWD YKQEGLTRAG AA? VTirVTA LTYGYGATAA GGVAASGSST 1451 AAAAGTAATT TAAATTVSTA TAMQTAALAS LYSQAAVSII NNKGDVGKAL 1501 KDLGTSOTVK 01VTSALTAS AI? QMGAD1A QLSKTOTE FSSTGNQT2 .551 NLGGRLATNL SNAGJSAGIN TAVNGGSLKD NLGNAALGAL VNSFQGEAAS .601 KIKTTFSDDY VAKQFAHALA GCVSGLVQGK CKDGAIGAAV GEIVADSHLG .651 GR PATLSDA EKHKVISYSK 1IAGSVAALN GGDVNTAANA AEVAWWíAL .701 NFDSTPT AK KHQPQKPDKT ALEKIIQGIM FAHAAGAMTN PQDKDAAIWI .751 SNIR GITGP IVITSYGVYA AGWTAPUGT AGKLA1STCM AHPSGCTVMV .BO TOAAEAGAGI ATGAVTVGNA WEAPVGALSK AKAAKQA1PT QTVKELDGLL BS. ESKNIGAVN TRINIAMSTT RYTPMRQTGQ PVSAGFEHVL EGHFHRPIA .5.01 BRSVFTISPH ELKVILQSNK WSSPVS TP DGOYHRTVDV GKVIGTTSI 951 EGGQPTTTIK VFTDKSGMLI TTYPVKGN * Computer analysis of this amino acid sequence predicts a transmembrane region and also gives the following results: - Homo loggia with a predicted ORF of N. meningitidis (strain A) The 0RF114 shows 91.9% identity in an overlay of 284aa with an ORF (ORFll4a) from strain N. of N. meningitidis: 20 30 .0 orf 114.pßp AVAETAKSQOKGKQftGSSVSVSr TSGDLCGKLKTTLKTLVC ?? luuiuuuu muí m uunuuum orf 114a MHKGLHRIIFSKKHSTMVAVAETAMSQGKGK QAGSSVSVSLKTSGOLCGKLKrTLK LVC 10 20 30 40 50 60 50 60 70 80 90 100 or f 114. pep SLVSI ^ MVLPAHAOITTDKSAPK? QsVVILKTIíTGAPLV? IQTP? GRsLSH? RXYAFDVD I I I II I! II III llllli llllíl IIUUUU MMIIUI m uu orf 114 SLVSLS_0__OKKQITTDKSAProiXQVVIIjppW? AWLV? IQTP_IGRGLSH? RYTQF0VD 70 80 90 100 110 120 110 120 130 140 150 160 m uum A? P? GITV G 180 170 180 190 200 210 220 orf 114.pßp GGFK .GRGILTTGAPQIGKDG? LTGFDVVKAHWrvXAA5 »mDKCP3AXYTsVIJj? IVAl? ! I U III lili I I | II I I I II lll I I I:: II II f II I III lili 11 lll I 11 orf 114a GG_TOíVGRGILTIG_APQIGKDGALTGFDVRQsí'LTVGAAG »KDKGGA0YTGVLARAVAL. 190 200 210 220 230 2 D 230 240 250 26D 270 280 orf 114. pßp GKXXGKXIAVStGPOKVDYASGErSAGTAAGTKPTIALDITU GMSMYADSITLIAHEKGV U I I I I I I I I I I I III IU IM III I I I M M I I I I M I M I M UU orflHe GKIOGK? WVSTGPafCTOYASGeiSAGffAAGTJOTIAI ^ 250 260 270 280 290 300 or f 11 a G VK? AGTLEAAKQL rVTSSGRIE? SGR 1 ATTADGTEAS PTYLXI ETTEKGAXGTFI SKGG 310 320 330 340 350 360 The full-length nucleotide sequence 0RF114a [SEQ. ID? O: 61] is: - - 1 ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT 51 GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG 101 GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA 151 CTCAAAACCA CCCTTAAAAC CTTGGTCTGC TCTTTGGTTT CCCTGAGTAT 201 GGNATTNCHN NNCNNTl-CCC AAATTACCAC CGACAAATCA GCACCTAAAA 251 ACCANCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT 301 ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT 351 TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC 401 CGTTTCTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT 451 ACGGCTAGCA AACTCAACGG CATCGTTACC GTAGGCGGTC AAAAGGCCGA 501 CGTGATTATT GCCAACCCCA ACGGCATTAC CGTTAATGGC GGCGGCTTTA 551 AAAATGTCGG TCGGGGCATC TTAACTATCG GTGCGCCCCA AATCGGCAAA 601 GACGGTGCAC TGACAGGATT TGATGTGCGT CAAGGCACAT TGACCGTAGG 651 AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG 701 CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AGGGTAAAAA CCTGGCGGTT 751 TCTACCGGTC CTCAGAAAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG BOl TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG 851 GCGGTATGTA CGCCGACAGC ATCACACTGA TTGCCAMTGA AAAAGGCGTA 901 GGOGTCAAAA ATGCCGGCAC ACTCGPAGCG GCCAAGCAAT TGATTGTGAC 951 TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG 1001 GCACCGAAGC TTCACCGACT TATCTNNCNA TCGAAACCAC CGAAAAAGGA 1051 GCNNCAGGCA CATTTATCTC CAATGGTGGT CGGATCGAGA GCAAAGGCTT no: ATTGGTTATT GAGACGGGAG AAGATATCAN CTTGCGTAAC GGAGCCGTG6 1151 TGCAGAATAA CGGCAGTCGC CCAGCTACCA CGGTATTAAA TGCTGGTCAT 1201 AATTTGGTGA TTGAGAGTAA AACTAATGTG AACAATGCCA AAGGCTCGNC - - 1251 TAATCTGTCG GCCGGCGGTC GTACTACGAT CAATGATGCT ACTATTCAAG 1301 CGGGCAGTTC CGTGTACAGC TCCACCAAAG GCGATACTGA KTTGGGTGAA 13 yes AATACCCGTA TTAT? ICCTGA AAACGTAACC GTATTAI: TA ACGGGAGTAT 01 TGGCAGTGC GCTGTAATTG AGGCTAAAGA C? CTGCACAC ATTGAATCGG 1451 GCAAACCGCT TTC7TTAGAA ACCTCGACCG TTGCCTCCAA CATCCGTTTG 1501 AACAACGGTA ACATTAAAGG CGGAAAGCAG CTTGCTTTAC TGGCAGaCGA 1551 TAftCATTACT GCCM ACTft CCAATCTGAA TACTCCCCGC AATC GTATG TTCATACAGG 1601 TA? AGATCTG AATTTGAATG TTCATA ?? GA TTTGTCTGCC 1651 GCCAGCATCC ATTTGAAATC GGATAACGCT GCCCATATTA CCGGCACCAG 1701 TAAAACCCTC ACTGCCTCAA AAGACATGGG TGTGGAGGCA GGCTTGCTGA 1751 AtGTTACCAA TACCARTCCG CGTACCAACT CGGGTIATCT GCACATTCAG 1801 GCAGCCAAAG GCñAT? TTCA GCTTCGCAAT ACCAAGCTGA ACGCAGCCAA 1851 GGCTCTCGAA ACCACCGCAT TGCAGGGCAA TATCGTTTCA GACGGCCTTC 1901 ATGCTGTTTC TGCAGACGGT CATGTATCCT TATTGGCCAA CGGTAATGCC 1951 GACTTTACCG GTCACAATAC CCTGACAGCC AAGGCCGATG TCHATGCAGG 2001 ATCGCTTGGT AAAGGCCGTC TGAAAGCAGA CAATACCAAT ATCACTTCAT Z0S1 CTTCAGGAGA TATTACGTTG GTTGCCGN N NCGGTATTCA GCTTGGTGAC 2101 GGAAAACAAC GCAATTCAAT CAACGGAAAft CACATCAGCA TCAAAAACAA 2151 CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC AAAAGCGGGG 2201 CATTGAACAT TCATCCCGAC CGGGCRTTGA 6CA? ASAARA TACNAAGCTG 2251 GAGTCTACCC ATAATACGCA TCTTAATGCA CAACACGAGC GGGTAACGCT 2301 CAACCAAGTA GATGCCTACG CACACCGTCA TCIAAGCATT JU4CGGCAGCC 2351 ACATTTGGCA AAACOWCAftA CTGCCTTCTG CCAACAAGCT GGTGGCTAAC 2401 GGTGTATTGG CAHTCftATGC GCGCTATTCC CAAATTCCCG ACAACACCAC 2451 GCTGAGACCG GGTGCAATCA ACCTTACTGC CGGTACCGCC CTAGTCAAGC 2501 GCSGCAACAT CAATTGGAGT ACCGTTTCGA CCAAGACTTT GGAAGATAAT 2551 GCCGAATTAA AACCATTG5C CGGACGGCTG? ATATTGAAG CAGCTAGCGG 2601 CACATTAACC "TCGftACCTG CCftftCCGCAT CAGTGCGCAT ACCGACCTGñ 2651 GCATCAAAAC AGGCGGAAAA TTGCTGTTGT CTGCAAARGG AGGAAATGCA 2701 GGTGCGCOTA GTGCTCA? GT TTCCTCATTG GAAGCAAAAG GCAATATCCG 2751 TCTGGTTACA GGAGWA? CAG ATTTAAGAGG TTCTAAAATT AC? GCCGGTA 2801 AAAACTTGGT TGTCGCCACC ACCAAAGGCA AGTK »ATA«. CGJ? 5CCCTA 2051 AACAACTCAT TCAGCAATTA TTTTCHTACA CAAAAAGNGN NWGNNCTCAA 2901 CCAAAAATCC AAAGAATTGG AAC? GCAGAT TGCGCAGTTG AAAAAñAGCT 2951 CGCNTAAAAG CA? GCTGATT CCAACCCTGC AAG? AG? ACG C? ACCGTCTC 3001 GCTTTCTATA TTCAAGCCAT CftAO &OGAA < ? WAA &se_? AAMkACCCAA 3051 ACGCAAAGAA TACCTGCAAG CCAAGCTTTC TGCAC-UV? AT ATrGACTTGA 3101 TTTCCGCACA ASGCATCGRA ATCAGCGGTT CCGATATTAC CGCTTsCAAA 3151 AAACTG3_ICC TTCACdCCGC AGGCGTATTG CCAAAGGCftG CAGATTCAGA 3201 GGCGGCTGCT ATTCTGAITG ACGCrCATAAC CGACCAATAT GAARTEGßCA 3251 AGCCCACCTA CAAGAGTCAC TACßACAAAG CTGCTCTßAA CAAGCCTTCft 3301 CGTTTGACCG CACGTACOGG GGTAAGTATT CATGCAGCTG CC5CACTCGA 3351 TOATGCACGT ATTATTATCG GTGCATCCGA AATCftAAGCT CCCTCAGGCA 3401 GCATAGACAT CAAAGCCCAT AGTGATATTG TACTGGAGGC TGGACAAAAC 3451 GATGCCTATA CCTTCTTABA AACCAAAGGT AAAAGCGGCA HAATNATCAG 3501 AAAAACHAAG TTTACCAGCA CCN6CGAHCA CCTGATTATG CCAGCCCCNG 3551 TCGAGCTGAC CGCCAACGGT ATCACGCTTC AGGCAGGCGG CAACATCGAA 3601 GCTAATACCA CCCGCTTCAA TGCCCCTGCA GGTAAAGTTA CCCTGGTTGC 3651 GGGTGAAHAG NTGCAACTGC TGGCAGAAGA AGGCATCCAC AASCACGftGT 3701 TG6ATGTCCA AA? AAGCCGC CGCTTTATCG GCATCAASGT AGGTNAGAGC 3751 AATTACAGTA AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT 3801 CGCCCAAANT GCAGCCACCC G TCAGGCTG «ATACCGTG CTCGAAGGTA 3851 CCGAATTCAñ AA CCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC 3901 GAAAAAGCCC GTGTCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG 3951 TATCCAGTCG GAAGAAAAA TAGAAACCAA CTCAACCGTA TGGCAGAAAC 001 AGGCCGGACG CGGCAGCACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA "051 AGCCCTACTC CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACñT 4101 TCCßAAAGGC AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG i5i AGTATGCCGA TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT 4201 CAGGTGCAGC TTGCTTACGA CAGATGGGAC TACAAACASG AGGGCTTAftC 4Z51 CGAAÍ5CAGGT GCGGO? ATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG 4301 GC5CAGGAAC CGGAGCCGTA TTGGGATTAA ACGGTGCCaiC CGCCGCCGCA 4351 ACCGATGCAG CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT 4401 CAACAACAAA GGCGATGTCG GCAAAACCCT GAAASAGCTG GGCAGAAGCA 4451 GCACGOTGAA AAATCtGGrtG GTTßCCSCCG C? ACCGCAGG C_TAGCCGA_ 4501 AAAA CGGCG TTCGGCACT GANCAATGTC AGCGATAASC AGTGGATCAA 4551 CAACCTGACC 6TCAACCTAG CCAATGNCGG GCAGTGCCGC ACTGAttaa - - This encodes a protein that has the amino acid sequence [SEC. ID NO: 62]: 1 MHKGLHRI 1? SKKK5TMVAV ASTANSQGKG KQAG5SVSV5 LK SGDLCGÍ; 51 LKT LKTLVC SLVSLSMXXX XXXQITTDKS APKNXQWIL KTNTGAPLVN 101 IQTPNGRGLS HNRYTOFDVD NKGAVLNNDR NNNPFLVKGS AOLILNEVRG 151 TASKLNGIVT GGQKADVII ANPNGITVNG G FKVGRGI LTIGAPOIGK 201 DGALTGFDVR QGTLTVGAAG WNDKGGADYT GVIARAVALO GKLOGKNLAV 251 STGPQKVDYA SGEISAGTAA GT PTIALDT AALGGMYAOS IT IAXEKGV 301 GVKMAGLEA AKQLGVSSG RIENSGRIAT TADGTEASPT YLXIETTEKG 351 AXG? SNGG KIESKGLLVI ETGEDIXLRS GAWQNHGSR PATTVLNAG_3 401 NLVIESKTNV NNAXGSXNLS AGGRTTINJDA TI0AGS5VY5 STKGDTXLGE 451 NTR1IAEUVT VLS GS1GSA AVLEAKDTAH 1ESGKPLSLE TSTVASNIRL 5ci HMGNIKGGKC LALLADDNIT AKTTNLNTPG NLYVHTGKDL NLNVDKDLSA 551 ASIHLKSDNA AHITGTSKTL TA5K0MGVEA GLLNVTNTKL RTNSGNLHJQ 601 AAKGHTQLRK TKL AAKALE TTALQGMIVS DGLHAVSADG HVSLLANGNA 651 DFTGRKTLTA KftDVXAGSVG KGWJKABN H ITSSSL VAXXGIQLGD 701 GKQRNSIN5K HISIKNHGGN ADLKNLNVHA KSGALNXHSD RALSIENTKL 751 ESTBHTH NA QHERVTLSQV DA? AHRHLS1 XGSQSWQHDf- LPSANKLVftH BOV GV AXNARYS QIAD TTLRA GAIHLTAGTA LVKRGNINWS TVSTKTLEDN 851 AELKPLAGRL NIEASSSTLT IE.2ANRISAH TDLSIKTGGK LLLSAKGGN A 901 GAXSAQVSSL EAKGNIBLVT GXÍDLRGSKI TAGKN WAT TKGKLRIEAV 951 KNSFSRYFXT QKXXXLNQKS KELEQQIAQL KKSSXKSKLI PTLQEERDRL 1001 AFYIQAISKE VKGKKPKGKE YL0AKLSAQN IDLISAQGIE ISGSDITASK 1051 LNLHAAGVL PKAADSEAAA ILÍDGITDQY EIGKPTYKSH YDKAALNKPS 1101 RLTGRTGVSI HAAAALDDAR IIÍGASEIKA PSGSIDIKAH SDIVLEAGQN 1151 DAYTFLXTKG KSGXXIRKTK FTSTXXHLIM PAPVELTANG ITLQAGGNIE 1201 ANTTRFNAPA GKVTLVAGEX XQ LAEEGIH KHELDVO SR RFIGIKVGXS 1251 NYSKNEI? ET KLPVRWAQX AATRSGMDTV LEGTEFKTTL AGADIQAGVX 1301 LK EKARVDAKII I? lIQS EEfíLETNSTV WQKQAGRGST IETLKLPSFE 1351 NL TEIEKLS SPTPPKLSAP GGYIVDJPKG KPPEYAYLKQ LQVAKN1NW QVQLAYDRWD 1401 YKQEGLTEAG AAIIALAVTV VTSGAGTGAV LGLNGAXAAA H51 TOAA ASLAS OASVSFIWNK GDVGKTLICeL GRSSTVKNLV VAAATAGVAD 1501 KIGASALXNV SDK? JWlNNLT VNLANXGQCR TD * ORF114-1 and ORF114a show 9.9% identity in an overlap 1564: - - orf 114a. ep M ^ GL! »____ KKBSTMVAVAETANSQGia_KQ_GSSVS ^^ mu mu i ?? ura um iiuunuuuu II ui iii IMI or orf-WAVAETANS MNKGLHRIIFSI_atS 114-1 (XpGKQAGSSV ^ or fll4 to .pep SLVSLSMCX__XXC_TTD S '? 3' 0WIL! ^^ umiiuii IMIIII uiiiun ni i i ipiiiui orf114-1 mu SLVSLSMVLPAHACITTDKSAPIOKK? WriJCTNTGAPLWIQTPNGRGLSHNRYTQFDVD orf11 a.pep HKGAVL NORNNNPFLVKGSAQLILNEVRGTASKL .GIVTVGGQKADVIIANPNGITVNG II 1 IUIMI I i u MJMMMI lll mm ?? i M i mm I II II orf 114-1 NKGAVIjmDRNimPFVVKGSAOLII _VKsTASKI_JGIVTVGGQKAOVIIñl.PNGITWG orf I4A -PEP GGíTWVGKGILTIGAIOlGKWa TGFDVRQGTLTVGAA (WNDKGGADYTGVIARAVALD my III My tumm iiuu IIUM IIMI MIIU ?? ium orf 114 mu -1 GGFKtm ^ GILTTGAPQIGKlWALTGFDVRQGTLTVGAAGWíDKGGADYTGVSARAVAlQ orf 114 pbw?. ???. ^ GKL KNL VSTGPQCTDYASGEI-AGTAAGTKrriAlDrAALGanrADSITLIAXEKGV uiuumimuuiu mmuui luumuumu uu M my orf 114-1 GKN__ñV aa ^ ^ STGro VDYASGEISAGTAAGTK- IALDTAAI__GHYADSITLIANEKGV or f 114 pbw GVKNAC-TLEAAKQLIVTSSGRIEHSGRiATTADGTEASPrYLXIETrEKGAXsTFISNGG jmuuuuuu 11 umi lumpm ?? i a i umu umui orf 114 - 1 GVKNAGTLEAA aLTVTSSGBIENSGRIATTADGTEASPTYLSIETIE GAAGTFISNGG orfll4a .pßp RlESKGL, LVTETGEDIXLBN < 3AWWNGSRPATTVL-JAGHNLVIESKTNVNNAKGSXNLe M III IU IM Mf i I II II II II M MiMii MI Me or f 11-1 RlESKGLLVI ^ GEDISI NGAVVQNl.G3RPA_ VLKAGHNLVlESKT ™ NAKGPATLS orf 114a ep?. AGWTTTNTTNGSSVYSSTKGDTXl? EKTRIIAElfip ^ LSNGSisSAAVIEAKDTAP |. U r I r M: II: U: II U II:: II: I I U. 'U II II U: I: I: I I I: I 1 III I or f 114-1 ADGRTVIKEASIQTGTTVYSSSKGNAE-GNKTRITGADVTVLSNGTISSSAVIDAKDTAK orfll4a .pep IESGKPLSLETSTVASt.IRl? NGNIK (»Q__AL_AQCNITAKTTKI_rrPGNLYVKTGKX3] _ M M I U U I: U U: u u u u u l l ll 11 IU U I U U I U U R I 4 1 I_AGKP_S_? AST-TSDIRUaSGSSKCGrouU »lAIH ^? T ^^ orf 114a. ep NLBVDKDLSAASIHLKSDNAAHITGTS TLTAS OHGVEAGLLNVTHTNLRTNSGNLHIC u p M m u m i n m u m i m i ii ii m 11 n u i u 11 u i m 11 -. - - orfll «-l N_? VDKDLSAASIHLKSDNAAHITGTSKTLTASt03« -VeAGSI.-VTNTHlRTN_GNLHI0 orflUa-pep AAKGNlQI? irTKLNAAKALETTA_? GHIV5_. { aJiAVSADGiiVSLrA GNADFTCHNTLTA I I I IIIII IIHIIimf ll l UIIIIIHinlMI IMIII IHMMrllll.i orflH-1 AAKGNIQLRNTKI? AAKALET AL3GNIVSDG1JÍAVSADGHVSLIA GNA1.FTGHHTLTA orf 114. PSSP ADVXAGSVCXGRLKAONTHITSSSGDITLVAXXGIOLGDGKÍJRNSINGKHISIKNNGGN I-1) 11 I I I II IMHl IMMMIM II M I I It II MI I I I II I I I M M J_ADV GRSP orflH-1 < .KtyiIJ? -DNTNITSSSGDITI.VAGHGIQLGDGKQR?! SINGKHrSIKNNG_N orflKß. PSSP -_DU8I VHAKSG IKSDRAI ^ ___ ^ I_N-TCIXSTH II II I II II III 11 11 f II I II II II I IM 11 M MIIIHIIIIM II f |? i I 111 IIM orflH-1 ADlJCN_NVHA SGALÍ.l_S_RALSIE -.___ JSST8 Thij HERVTI ?? ? QVDAYAHRHLSl orflUa.pep XGSOIWQNI »XPSAípaVANGVlAX ARysCJAi» WTIJUM_AINLTAGTALVKRGNI-IWS MI IHIMIII MI1MMM IM 1111111111 IIM 11 II II III II IIIHM orfll4-l TGSOIW? ^ DKLPSAHiaVAKGVIAWARysOIADirrTLRAGAIHLTAGTALVKRGNIN S arf 114a. ßp TVSTKTI _: _ NAEI_ÍPIAGRIJ.I_AGSGTI.TIEPAMRISAHTDLSIK__GK1_ SAKGGNA I nuil! i IIM H i npi MIHIMI II II II II MI I UIHHHI II orf 11 -: TVST_ ri_EDtó_: LKPLAGRl_IIEAGSGTLTX_PANRISAH DI ^ IKTGG ___ I ^ AKGGNA orf 114a. ep GAXSA0VSSLeAKGMlRLVTGXTDJ-RG__ MMMIIII ITAGKNLVVATTKGKJ.SIEAVm.SF5NYFXT II MI I lili IIII I II 11 11 II II 11 II MII MII 11 II i I il II orfH4-l-AXGNXRLVTS_TDt GAPSAQVSSI. £ GSXITAaXl.LWA7rKGKLN? EAV_MSF5MyFFT M Mllll ll lll II II II Htl I II I MU l! HHII HH II HI o.fll-í-1 QKAAEIJ3K = E_? QQIAQXJKSSPKSKLIPTLQ__E -? _ A? FYlOAlNKE VKGKKPKGKE orf 114a .pep YI? 3AKLSAQNIDLISAC ^ I £ ISGSC? TA3KKlM ?? AAGVLy_aUiDSEAAAI_.II) GlTD0 ? MY i ?? mu MI MI IIII IUHMII MIIIII MIMIIIHMM II? III? ? T I orfll4-l ^ YI AKX ACNIOLISAQGIEISGSDITASKKUJLHAASVXPKAAOSBAAAILIDGITDQY rfll * '- p * p ^ eiSKOTYKSK DBaUU PSKI.TG & TGVSI_U "AA ^ lilili I MIIMIIIMII IIMIMIIIMIIIim orf 11 -1 ElcaíP TCSHYDKAAlJWPSRLTGRtGV = leu AALDDAfilIlGAfSEIKAPSGSiDlKAi?. orfllla.pep SDIVI__AGQSDAYT_ ^ TKGKSGX.p: R TBTtCTXXm ^ iiiiMiimiiiM MIIII imipp n uí uní MUÍ ni ?? i Oril 4-l SDIVL__AGgN AYTFL KGKSGKZZ-UCrKFTS R HLZMP PVELTAHGITLOAGGHI £ orfllía.pßp Am, TRF APAsKVTLVAGE3X0U EEGIHKHEUWQKSIUlF15IKVGXS >!????; IYSK E N: t MIMI IflIMMH I II II I I il II I, II I l II II 1 I I I I II II I 1 I I 1 I I ull orflH-1 ANTrRFNAPAGKV LVAGEELOLIAEeGiaiCHELDVjKíSBRFIGIKVGKSNYSKKELNET orfllía.pep KI > VRVVA0XAATRSGWpTVIJ > ETEFKTTIJ «» DI) AGV5_EKARVDAKriLKGIVNRI0S M III II I MI lllll IHHIiMIMipillllMI 1111:? 111 I MI orfilí-i KLPVSVVAQTAATRSGWDTVLEGTEFKTTl GADIQAGVGEKARAimKIILKGIVHRIQS orfllía.pep Eeki TNSTVWQKOAGRGSTlETLKLPSFESP-'PPKLSA-'GGYXVDIPKGKLKTeiEKLS I 1 H) I f) HMMI II IMMIIIIIII) I f IIHI. ' I I I M I I! 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- -Homology with putative N secreted protein. men i n gi t i di s (access number AF030941) The 0RF114 and the pspA protein show 36% identity aa in the 302aa overlap: Orf 114: 1 AVAETA? SQGKGKQAGSSVSVSL KTSGDXXXXXXXXXXXXXXXXXXXXXXXPAHA? 56 AVAE + GK 0 + SV + S PA A pspA: 19 AVAEHVHRDGKSMQDSEAASVKVTGAASVSSABAfiFGFRMAJ-FSVMtALGVAAFS PAPAS 78 Orf 114: 57 -ITTDKSAPKMQO ^ W! TIJ THTGAPLV? ICTWGRGLSin.RXYAFDVI »IKGAVL ?? DR ?? - 11. I OKSAPK? QQ VIL + T G P V? IQTP + + G + S? R FDVD KG + L ?? R + t- pspA: 79 GIIADKSAPIOí .OAVII OTñ? GLPOV IQ PSSQGVSV? RFKQFDVDEKiGVIL ?? SRS T 138 Orf 114: 115 «PFVVKGSAQL? Lt? EV-RGTASKl? ^ IVTVGGOKAI? VllA? PMGITV? GG 163 MP + + G A ++ I +? + -» - 5 LKG + VGG ++ A + V ++ A? P + GI V? GG papA: 139 ^ OLGGMIOG? PHIARGEARVIV? QIDSS? PSLIJÍGYIEVGGKRAEVVVAÍIPSGIRV? GG 198 Orf 114; 164 GFK? VG GI TT APQIGKI and rGFD KAHWTV ^^ 223 G? LT + G P + + G LTGFDV + G O A YT + L + RA * pspA: 199 GLIWAASVTLT5GV_VL-? Ira? LTGFrrVSSGKVVIGGKGL-DTSDADYTRILSRAAEI? A 256 Orf 114; 224 KXXGKJCIAVSTGPQKVDYASGEISAGTAAGTK PT? ALsTAALGGMYADSITLIA? E 279 GK + V + G K + D + + A + PT + A + DTA LGGMYAD ITLI + * pspñ: 257 GVWGKDV VVSGK? KI? FDGSIAKTASAPSSSDSVTPTVAIDTATLGGMYADK? Ti.ISTD 316 Orf 114; 280 KG 281 G pspA: 317? G 318 The ORFll4a is also homologous to pspA: gil 2623258 (AF030941) putative secreted protein [? eisseria meningitidis] Length = 2273 Record = 261 bits (659), Expected = 3e-68 Identities = 203/663 (30%), Positive = 314/663 (46%), Spaces = 76/663 (11%) - - Question: 1 W_ G_HRIiraKKHSTMVAVAETA S0GKÍ_ Í) A5SSVSVSI? TSG3XXXXXXXXX 55 MKK + -M-IF + KK S M + AVAE + GK Q + SV * + S Subject: i Mma? C ?? vtFNi «Rsa *? V? Ijjvfpax3C3M0DKyw 60 Interrogate: 56 xxxxxxKXffiKxxx xxxorrrwcsArarao viLXT ^ 115 I DKSAPKH Q VI + TGP VNI_TP + + G + S HR + SI FSVMI? L¿ ^ AArSPAPAS6IIA_KSAPKNQQ * VILCTANG P0VNI0TFSSQGVSVNRF 120 Subject: Question: 115 OFDVDKKGAVLNN8N NPp.VKGSflßlIlHEV-i < < ? TASKlHGIVTVG_? G3 QFDVD 1CG + UJN R + H HP 1 + C A ++ I + * f * + S £ KG + VGG Subject: 121 QFDVDEKOTIUINSRSNTC OI S ^ IQOTPHIJUIGEARVIVNOT 1BC Interrogator: 16 - QKADVIXWPtWITVNSGGF ?. { Wß «GXLT» _AMIGKK? AM ^ 223 ++ A + V-W-ANP-K.I VNG3G H LT G V + + G LTGFDV G + + G G D Subject: 19] KRAEVWAHPSGIRVNSSGLINAASVTLTSsm ^ -OTGNLTGFIWSSGfarVIGGJffi 238 Interrogation: ZZ1 < M-J «TGVXÍUUi? C3Gr QGKH_AVSTSPaKV0YASGEI-iAGTAAfi'r? PtíALP 279 ADYT + L + RA + + SK ++ V + G K + D + * A + PT + AtD Subject: 239 TSDADYTRlLSRAAEIl «GVS» GKip KVV-SGKHKtDroGS_A! P? WAPSSSPSVTPTVArD 29B Interrogator: 280 TAAI? »RYADSIT IAXEKGVGVKllAGrLEAftK-QLrVTSSGRIEÍIS RIATTADGRIAS 338 TA LGGHYAD ITLI ++ G ++ "G * M. - ++ - & + - * NS < _ I + P. + Subject: 299 TATI¿a_K? AtraTLISTDKCAVIRMB__RIFA_, TGGVTLSADdK SNSSSI- DAA 351 Interrogator: 339 Prp.XIETTEKGAX [rrFISW3GRIESKGll, V_eTGEDIXLR_IGñ? VQMNsSRPATrV l.A 396 + + T + »G l. V ** * _ + G + GS * + Subject: 3S2 eiTIS? Q VD KRQG? RSGKGSVIJWSDGIiajpAGL? GSAGliDIBDT 39B Interrogating: 399 GHNLVIESKTNVNNAKGS XN SAGGRTriND? TIOAGSSVlfSSTja.ip'X CfEHTRI ib * 5 * S ++ HH < 3 * ++ £ ++ SD + A V S + D G + Subject: .00 G --- »- KSSIJ ^ T ^ ™ TDGTIIAGKD SUJAKSL-« 3DGI_. AARDV-sVSlJ8DDFAGKRDIE 453 Interrogate: 455 IAE V VLSNGS? GSAAVlEAKDTAHIESGKPI? LETSTVAS IRUWIGNIKGGKQLAU 51 4 * T - »3» + + I + ft DI * * * t »t S B G L-r Subject: 454 AGRTLtFStQGRI-K_? RIIQAGDTVSLTAAOIONTVSGK10SGSRTGL «GKNGITNRGLT 513 question. 515 ADDNJT AKTrNIJíTPGNLYVHTGKDLKLNVDKDLSAASlHLKBONAASpTsTSKT 569 + IT AK + N T G + Y G + - * - D L4 AA Subject. 5l4 f.SNGITLLCT £ AKSDNAGT-GRrY GSRVAV? ADTLUJREETVNGETKAA V 562 question. 570 ^ TASKDM_n / EAGXX > O (XXXXXXXXSGNUJI0AA --- KGNICI? NTKL- AAKALETTAL? _ 625. A + + + A SG + LHI + A + Q NT L + A + E ++ Subject: 563 f AARERLDIGAREIENREAAÜSSSGDLHlGSALNGSROVGGANTSLHNRSA- 1ESS 619 Question: 626 (NI 628 subject: 62c (SKI 622 Record = 37.5 bits (85), Expected = 0.53 Identities = 87/432 (30%), Positives 159/432 (36%), Spaces = 62/432 (14%) - - Question i 239 ^ < aafarai ^ BTGSWr HASGE _._ l ?? T? * CTKTO ^ 290 LQG LQC5KN + + G t + G I A A K A i- + S T + Subject. 1023 lQfi _ * _? 5 »íiraAAGSDrrt» - TßSIG * eHAIi_K ASHHiesRSETRSNONE 1072 question. 299 < -U * G, IOÜlGPr_BAAK0LIVTSSGRI - EKSGRIATrADsTí »SWYI_XIErrEKßAltG - ?, F 355 V + M G + A L -W3 + + I W E T + G T ß subject. 1073 QGSVRNl5RV-AGIYLTCR0HßSV_LJ »GNM_V_.TAS ELTNOSEDGQTV U2G Iptßrrogantßi 35ß ISNSGRIESKGI? VXE GÍDIXLRN-AVVQlM-? SRPAT VIJÍAGJOlLVIESK T «OS ++ fifi I S + I • * •? + * * + T-» G Hl. + t. Subject. 1121 I __? ßGDIRSDTrGISRH (^ IFI> SONYVIRraQNEVGSTIRTR < rI «, SLNAKsDrBIRAA 1179 question mark .09 MVNHAKGSXHLSAGGRTTrBOATIOAÍ SS VYSSTKtrDTXLGENtRIIAeNVT 4 SO V + + GL * AG D + • -» -_- + Y + G + T_ f ßujetot 118C EVaSKQ6MJCI »AAS RDIKV £ AffKAHTErED? IIcrrGRSCKWIKOKMTRHIICNQN-G 123. question. * ßl L_HCSSGS AVlBAXIT.7? I__ < 3CPl.StCTSTV? SN ^ 520 + G ++ +1 + G + + T + S HH + K + + A + t. 1235 WS! _TI? JKE__WSGRDITV_CMIIAra LS .-- An3N ^ 1292 question mark »52 To TTNL? .rPG-IJLYVETsKß« I_? VDKOLSARSIR KSDl J? HITGTSKTLTA 572 EKSGlMGSGGlG rAGSKK3) QTt SErtfSim: S VGSI_SGOT115AGKH i!! 1352 Question mark. 373 SK-DMGV £ AGXXXX_XXXXXXX_GN_H-_A * and ICI? NTKLNAAKALETTAI2_ g2. + 1 > + G + - + G. * | CG ++ * NT + A A ** G ß subject. 1353 P_G0 I $ «paSIDAAQimYSO £ SKCVYEQEGVrVArSVTV ^^ nj.2 Question mark. S27 NIVSDG1AAVSA 630 Bu ject. 1413 KSKSSRVUAMAA 1424 Amino acids 1-1423 of 0RF114-1 were cloned into the pGex vector and expressed in É. coli, as described above. The GST fusion expression was visible using SDS-PAGE, and Figure 5 shows the hydrophilicity plots, antigenic index, and the AMPHI regions for 0RF114-1.

Based on these results that include homology with the putative secreted protein of N. meningitidis and in the presence of a transmembrane domain, it is predicted that this N. meni ngi tidi s protein, and its epitopes, could be useful antigens for - Vaccinations or diagnoses.

E j a lo 14 The following partial DNA sequence was identified in N. meningi tidi s [SEC. GONE . 63]: 1 .. CGCTTCATTC ATGATGAAGC AGGCGGCAGC AACATCGGCG GCGGCAAAAT 51 GATTGTTGCA GCCGGGCAGG ATATCAATGT ACGCGGCAnA AGCCTTATTT 101 CTGATAAGGG CATTG TTTA AAAGCAGGAC ACGACATCGA TATTTCTAC 151 GCCCATAATC GCTATACCGG CAATGAATAC CACGAGAGCA wAAAwTCAGG 201 CGTCAGGGG ACTGGCGGAT TGGGCTTTAC TATCGGTAAC CGGAAAACTA 251 CCGATGftCAC TGATCGTACC AAATTGTsC ATACAGGCAG CATTATAG_5_C 301 AGCCTßAaTG GAGACA CGT TACAGTTGCA GGAAACCGCT ACCGACAAAC 351 CGGCAGTACC GTCTCCAGCC CCGAGGGGCS CAATACCGTC ACAGCCAAAw 401 GCATAGATGT AGAGTTCGCA AACAACCGGT ATGCCACTGA CTACGCCCAT 451 ACCCAgGGAA CAAAAAGGCC TTACCßTCGC CC_CAATGTC CCGGTTGTCC 501 AAGCTGCACA AAACTTCATA CAAGCAGCCC AAAAT TGGG CAAAAGTAAA 551 AATAAACGCG TTAATGCCRT GGCTGCAGCC AATGCTGC T GGCAGAGTTA 602 TCAAGCAACC CAACAAATGC AACAATTT5C TCCAAGCAGC AGTGCGGGAC 651 AAGGTCAAAA CTACAftTCAA AGCCCCAGTA TCAGTGTGTC CATTAC-TAC 701 GGCGAACAGA AAAGTCGTAA CGAGCAAAAA AGACATTACA CCGAAgCGGC 751 AgCAACTCAA ATTATCGGCA AAGGGCAAAC CACACTTGCG GCAACAGGAA 801 GTGGGGAGCA GTCCAATATC AATATTACAG GTTCCGATGT CATCGGCCAT 651 6CAGGTACTC C. CTCATTGC CGACAACCAT ATCAGACTCC AATCTGCCA-. 901 ACAG3ACGGC AGCGAGCAAA GCAAAAACAA AAGCAGTGGT TGGAATGCAG 951 GCGTACGT? .n CAAAATAGGC AACGGCATCA GGTTTdGAAT TACCGCCGGA 1001 GGAAATATCG GTAAAGGTAA AGAGCAAGGG GGAAGTACTA CCCACCGCCA 1051 CACCCATGTC GGCAGCACAA CCGGCAAAAC TACCATCCGA AGCCGCGGGg 1101 GATACCACCC TCAAAGGTGT GCAGCTCATC GGCAAAGGCA TACAGGCAGA 1151 7ACGC6CAAC CTGCAGATAG AAAGTGTTCA AGATAC GAA ACCTATCAGA 1201 GCAAACAGCA AAACGGCAA7 GTCCAAGTT ^ ACTGTCGGTT ACGGATTCAG 1251 TdCAAGCGGC AGTTACCGCC AAAGCAAAGT CAAAGCAGAC CATGCCTCCG 1301 TAACCGGGCA AAgCGGTATT TATGCCGGAG AAGACGGCTA TCAAATyAAA 1351 GTyAGAGACA ACACAGACCT and? AGGGCGGT ATCATCACGT CTAGCCAAAG 1. 01 C6CAGAAGAT AAGGGCAAAA ACCTTTTTCA GACGGCCACC CTTACTGCCA 1451 GCGACATTCA AAACCACAGC CGCTACGAAG GCAGAAGCTT CGGCATAGGC 1501 GGCAGTTTCG ACCTGAACGG CGGCTGGGAC GGCACGsTTA CCGACAAACA 1551 AGGCAGGCCT ACCGACAGGA TAAGCCCGGC AGCCGGCTAC GGCAGCGACG 1601 GAGACAGCAA AAACAGCACC ACCCGCAGCG GCGTCAACAC CCACAACATA 1651 CACATCACCG AC6AAGCGGG ACAACTTGCC GGAACAGGCA GGACTGCAAA by AGAAACCGAA GCGCG? ATC? ACACCGGCAT CGACACCGAA ACTGCGGATC 1751 AACACTCAGG CCATCTGAAA AACAGCTTC- AC ...

- This corresponds to the sequence of \ amino acids [SEC. ID NO: 64; 0RF116]: 1 ..RFIHDEAVGS K1GGGKH1VA AGQDINVRGX SL? SDKGIVL KAGHD1DIST 51 AHNRYTGNEY HESJU.SGVMG TGGLG TIÍ2Í RKTTDDTDRT KIVHTGSIIG 101 SLNGDTVTVA GNRY3.QTGST VSSPßGRNTV TA XIDVEFA NNRYATDYAH 151 TQEQKGLTVA LNVPWQAAQ HFIQ? AQNVG KSKWKRVNAM AAANAAWQ5Y 201 QATQQMQQG? FSSSAGQGQN YKQSPSISVS IX? GEQKSBK EQKRHYTEAA 251 ASQIIGKGQT T AATGSGEC. SNIN? TGSDV IGHAGTXLIA PNHIRLQSAK 301 QDGSEOSK K SSGWNAGVRX K? GNGIRFGI TAGGNIGKGK EQGGSTTHRH 351 THVGSTTGKT TSRSGGDTTL KGVQLIGKGI QADTRNLHIE SVQDTETYOS 401 KQQNGNVQVT VG? GFSASGS YRQSKVKAOH ASVTGQSGIY AGEDGYQIKV 451 RDKTDLKGGI ITSSQSAEDK GKNLFQTATL TASDIQNHSR YEGRSFGIGG 501 SFDLHGGWDG TVTDKQGRPT DRSSPAAGYG SDGDSKNSTT RSGVNTHMIH 551 ITDBAGQLAR TGRTAKETEA RIY 6I0TET ADQHSGHLKR SFD ..,alysis of this amino acid sequence gave the following results: Homology with the putative secreted pspA protein of N. meningitidis (accession number AF030941) The ORF116 and the pspA protein show 38% aa identity in the 502aa overlap: Orf 116: 6 EAVGS? IGGGKMTVAA? QDIMVRGXSLISDKGIVLKAGHDIDIS'rftHH 'p'G? EYHESX.X 65 + AV + G ++ I + + G + O? V G ++ I + D + L A ++ I + A R £ ++ PspA: 1 235 OAVSGTLDG EIILVS < 3R0ITVtGS? IlAD? I_TILSAK? RVLB »AETR5RSAEMHKKEK 1294 Orf 116: fiS XXXXXX3ÜXXXXXX? RIO. { XXXXXRTKTVKTGSIjGS? K3DTVTVAGHRYROTGSTvesPE 125 ++ K + HT S ++ GSL? G + T4 AG AND 0TGST4SSP + P *? A: 1295 SGLMGSCGIGFTAtMKKDTQTHRSETVSHTESVVGSL? Girri ^ SAQisiYTCp'GSTlSS ^ 1354? R_U6: 12G GRNTV AKXIDVE AKNRYATDYARTQEOKGLTVALHVPIXXX XXXXXXXXXXXG S 182 C +++ 1 + A HRY + EQKG + TVA4 + VP GKS PspA. 1355 < pVGISSGKIS! DAAÍ ^ RYSQESKOVYEQKGVTVAISVPVVNTVMGAVüAVKAVQTVGKS 1414 Orf 116: 183 KNIOlVXXXXXXXXXWQSY0ATQ. { »Q0FA - PSSSAGOG (__ IYHQSPSISVSIX_GE_KSRN 2.0 KK RV * • + A P + AGQG? SVS + YGECK + 4 PapA: 1415 KKSRVNAHAAANALRKGVDSGVALYNAARNPK AAC? QG ISVSVTYGEQKNTS 1466 Orf 116: 241 E0KRHYTEAAASOIIGKGDTTIAATGSGE0SI-1NXTGSDVIGHAG XLIADNHIRL0SAK 300 E + T4 +1 G 5+ A + G G S 1 ITGSOV G GT L A + N ++++ A + PspA: 1467 ESRlKGTWCffiGKITGGGKVSLTASGAGKDSR? TITGSDVYGGKG R KAENAVOIEAAR 152? Orfllfi: 301 OIX-SEQS-? KSSGWASVRXKIGNGÍRFGITAXXICXXXXXXXXSTTHRip'HVGS.T'Gi ^ 360 Q E + S + HKS + G + NAGV I Gl l. TA T 4R + H GS * T P < PA: 1527 OTHQERSENKSAGFNAGVAlAINKGISrcrrAGANYGKGYGNGDETAYRilSHlGSKDSQT 1586 Orfll €: 361 TIRSGGrrrCLKGVOLIGKGIQADTMJLHIESVOCTETYOSKQOHGínrQfVTVG? GFSASGS 420 I SGGCT MIG OL GKG + 4-LRIES + QOT ++ KQ + N QVTVGYGFS GS PspA: 1587 AIESGGD VIKGGQL G GVGVTAESLHIESLODTAVFKSKOENVSACVTVGYGFSVGG = 1646 Orfllß: 42Z YR. { ._pVJGlDHASrVTGOSGIYAGEDGYQIKVRDÍ.TDLKGGIitSSOSAEDKGKNLFQTATL 48C Y + SK .D + ASV QSGI + AG DGY + I + V _ G + S DK KNL + T * * PspA: 1647 YNRSKSSSDYASWEQSGIEAGGDGYR1RVNGKTGLVGAAWSD ADKSKNLLKTSEI 1703 Orf 116: 481 TA_-DIQ_.HSR? EGRSFGIGGSF 502. IQNH + 4 G4 G _ PspA. 1704 HKDICWHASAAASALGI-SGGF 1725 Based on the homology with pspA, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

E j p lo 15 The following partial DNA sequence was identified in N. meningitidis [SEC. ID NO: 65]: 1 ..ACGACCGGCA GCCTCGGCGG CATACTGGCC GGCGGCGGCA CTTCCCTTGC 51 CGCACCGTAT TTGGACAAAG CGGCGGAAAA CCTCGGTCCG GCGGGCAAAG 101 CGGCGGTCAA CGCACTGGGC GGTGCGGCCA TCGGCTATGC AACTGGTGGT 151 AGTGGTGGTG CTGTGGTGGG TGCGAATGTA GATTGGAACA ATAGGCAGCT 201 GCATCCGAAA GAAATGGCGT TGGCCGACAA ATATGCCGAA GCCCTCAAGC 251 GCGAAGTTGA AAAACGCGAA GCAGAAAAA TCAGCAGCCA AGAAGCGGCA 301 ATGAGAATCC GCAGGCAGAT ATGCGTTGGG TGGACAAAGG TTCCCAAGAC 351 GGCTATACCG ACCAAAGCGT CAfATCCCTT ATCGGAATGA This corresponds to the amino acid sequence [SEC. ID NO: 66; 0RF118]: 1 .. TTGSLGG3- LA GGGTSLAAPY LPKAASH GP AGKAAVNALG GA SGYATGG 51 SGGAWGRKV DWWSRQLHPK EMA ADKYAE 3MXREVEKRE GRK? SSQEAA 101 MRIRRQICVG T VPKTAIP TKASYP SE * Computer analysis of this sequence revealed two putative transmembrane domains.

Based on this analysis, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

E n g it 16 The next sequence of AD? partial was identified in N. meningi tidi s [SEC. ID? O: 67]: 651 AATCAAACAG TTGGATCAGC ACTACATTAC CCACAAGATT GCCCaTSCCA 701 TAGCGGGCTG TGCGGcTGCG GCGGCGAATA AGGGCAAGTG TCAGGATGGT 751 GCGATAgfGTG CGGCTGTGGG CGAGATAGTC GGGGAgGCTT TGACAAACGG ßoi CAAAAA CCT GACACTTTGA CASCTAAAgA ACGCGaACAG ATTTTGGCAT TS1 ACAGCAAACT GGTTGCCGGT ACGGTAAGCG GTGTGGTTCGG CGGCGATGTA 901 AATGCGGCGG CGAATGCGGC TGAGGTAGCG GTGAAAAATA ATCAGCTTAG_951_CGACAAAtGA This corresponds to the amino acid sequence [SEC. ID NO: 68; ORF41]: 1 ..QCR KSSQFY RRKLLCKYIY RFPXYCPXAC VAEOTPYACY LXQLQVTKDV 51 NWNQVXLAYD KWPYKQEGLJ GAG AAl I ALA VTWTAGAGA GAALGLtKSA 101 AAATDAAFAS LASOASVSI-1 NNKGNIGNTI, KELGRSSTVK NLMVAVATAG 151 VADKIGASAL N »V5DKQ» IK NLTVNLANAG SAALINTAVN GGSLKDNLEA 201 NILAALVSTA KGEAASKIKQ LDQHYITKK? AfiAIAGCAAA AANKGKCQDG 251 AIGAAVGE? V GEAÍ.THGKSP DTLTAKEREQ 2XAYSKLVAG TV5GWGGDV 301 NAAAHAAEVA VKNNQLSDK * Additional work revealed the complete nucleotide sequence [SEC. ID NO: 69]: 1 ATGCAAGTAA ATATTCAGAT TCCCTATATA CTGCCCAGAT GCGTGCGTGC 51 TGAAGACACC CCCTACGCTT GCTATTTGAA ACAGCTCCAA GTCACCAAAG 101 ACGTCAACTG GAACCAGGTA CA? CTGGCGT ACGACAAATG GGACTATAAA 151 CAGGAAGGCT TAACCGGAGC CGGAGCAGCG ATTATTGCGC TGGCTGTTAC 201 CG GGTTACT GCGGGCGCGG GAGCCGGAGC CGCACTGGGC TTAAACGGCG 251 CGGCCGCAGC GCAACCGAT GCCGCATTCG CCTCGCTGGC CAGCCAGGCT 301 TCCGTATCGC TCATCAACAA CAAAGGCAAT ATCGGTAACA CCCTGAAAGA 351 GCTGGGCAGA AGCAGCACGG TGAAAAATCT GATGGTTGCC GTCGCTACCG 401 CAGGCGTAGC C5? CAAAATC GGTGCTTCGG CACTGAACAA TGTCAGCGAT 451 AAGCAGTGGA TCAACAACC7 GACCCTCAAC CTGGCCAATG CGGGCAGTGC 501 CGCACTGATT AATACCGCTG TCAACGGCGG CAGCCTGAAA GACAATCTGG 551 AAGCGAATAT CCTTGCGGCT TTGGTGAATA CTGCGCATGG AGAAGCAGCC 601 AGTAAAATCA AACAGTTGGA TCAGCACTAC ATTACCCACA AGATTGCCCA 651 TGCCATAGCG GGCTGTGCGG CTGCGGCGGC GAATAAGGGC AAGTGTCAGG 701 ATGGTGCGAT AGGTGCGGCT 6TGGGGGAGA TAGTCGGGGA GGCTTTGACA 7S1 AACGGCAAAA ATCCTGACAC TTTGACAGCT AAAGAACGCG AACAGATTTT BOG GGCATACAGC AAACTGGTTG CCSGTACGGT AAGCGGTGTG GTCGGCGGCG 851 ATGTAAATGC GGCGGCGAAT GCGGCTG? GG TAGCGGTGAA AAATAATCAG 9Q1 CTTAGCGACA AAGAGGGTAG AGAAT TGAT AACGfiAATGA. CTGCATGCGC 951 CAAACAGAAT AATCCTCAAC TGTGCAGAAA AAATACTGTA AAAAAGTATC 1001 AAAATGTTGC TGATAAAAGA CTTGCTGCTT CGATTGCAAT ATGTACGGAT 1QS1 ATATCCCGGA GTACTGAATG TASAACAA.TC AGAAAACAAC ATTTGATCGA 1101 TAGTAGAAGC CTTCATTCAT CTTGGGAAGC AGGTCTAATT GGTAAAGATG 1151 ATGAATGGTA TAAATTATTC AGCAAATCTT ACACCCAAGC AGATTTGGCT 1201 TTACAGTCTT ATCATTTGAA TACTGCTGCT AAATCTTGGC TTCAATCGGG 125? CAATACAAAG CCTTTATCCG AATGGATGTC CGACCAAGGT TATACACTTA 1301 TTTCAGGAGT TAATCCTAGA TTCATTCCAA TACCAAGAGG GTTTGTAAAA 1351 CAAA &TACAC CTATTAC AA TGTCAAATAC CCGGAAGGCA TCAGTTTCGA 1401 TACAAACCTA AAAAGACATC TGGCAAATGC TGATGGTTTT AGTCAAAAAC 1451 AGGGCAGGAA AGGAGCCCAT AACCGCACCA ATTTTATGGC AGAACTAAAT 1501 TCACGAGGAG GACGCGTAAA ATCTGAAACC CAAACTGATA TT6AAGGCAT 1551 TACCCGAATT AAATATGAGA TTCCTACACT AGACAGGACA GGTAAACCTG 1601 ATGGTGGATT TAAGGAAATT TCAAGTATAA AAACTGTTTA TAATCCTAAA 1651 AAA.THWCTG ATGATAJAAT ACTTCAAATG GCTCAAAATG CTGCTTCACA 1701 AGGATATTCA AAAGCCTCTA AAATTGCTCA AAATGAAAGA ACTAAATCAA 1751 TATCGGAAAG AAAAAATGTC ATTCAATTCT CAGAAACCTT TGACGGAATC 1801 AAATGGAG? T CATATTTTGA TGTAAATACA GGAAGAATTA CAAACATTCA 1851 CCCAGAATAA This corresponds to the amino acid sequence [SEC. ID NO: 70; ORF41-1]: 1 MQVNIQIPYI LPRCVRAEOT PYACYLKOLQ VTKDVN N V 0LAYDKWDYK SI QEGLTGAGAA IXAIAVTWT AGAGAGAA G NGAAAAATD AAFASLA50A 101 SVSLINNKGN IGNTLKELGR SSTVKKLMVA VATAGVADKI GASALNNVSD LSI KQWINNLTVU LANAGSAALI W A? 3GGS; DHLEANIY? LVNTAHGEAA 201 SKIKQLDQHY ITHKIAHAIA GCAAAAANKG KCQDGAIGAA VGEIVGEALT 251 NGKNPDTLTA KEREQIIAYS KLVAGTVSGV VGGDVNAAAK AAEVAVKN Q 301 LSDKEGREFO? Eu ACA_.g NPfLCRKNTV KKYCS3VMK ?. LAASIAICTD 351 ISRSTECRTI RKQHLIDSR3 LHSSWEAGLI GKDDEWYKLF SKSYTQADLA 401 LQSYHLMTAA KSWLQSGNT PLSEWMSOQG YTLISGVNPR FIPIPRGFVK 451 QNTPITNVKY PEGISFDTH1, KRHLAHADGF SQKQGIKGAH RTNFKAELN 501 SRGGRVKSET QTEIEGITRI KYEIPTLDRT GKPDGGFKEI SSIKTVYNPK 551 KFSDDKILQK AQNAASQGY5 KASKIAONER TKSISERK V IQFSETFDGI 501 KFRSYFDVNT GRITNIHPE * Computer analysis of this amino acid sequence predicts a transmembrane domain and homology with an ORF of N. meningi tidi s (strain A) was also found.

The ORF41 shows 92.8% identity in an overlap of 279aa with an ORF (ORF41a) of strain N. of N. meningitidis: - 20 30 40 50 60 69 70 ßO 90 100 110 120 129 orf 4.1. pep tGMR ii __ftvt \? rr GaGa? aaun GftAaA ^^ I I I I I I I! I I 1 1 | I: I i: I I I I I I. { I I I I I M I I I I I I: I I I 1 1: I: I orf41a T_ £ A < 3UaiAIAVTyvTS (raSTGaVLGl_NGAXA ^ 40 50 60 70 80 90 130 140 150 160 170 180 189 orf41.pep KELGRSSTV NIÍÍVAVATAGVAO IGASALNNVSPKQWTOÍ &TVNLAKAGSAALIMTAV I i I f II II f IN: II: II i III 17 IIIIII if II í IIJ II IIII! I ! I II III 1 í IN orf 41a LKELG ^ STVK LWAMTAGVAD l < and ^ AI? WSD OWINNL_ VNLAHAGSAALINtAV 100 110 120 130 1 * 0 150 250 260 270 280 290 300 309 or £ 1. pep GAIGAAVGEIV_EALTNGK ?? PDTI, TA_a_Re? _I LAYSKLVAGTVSGWGGDVNAAANAAEV 1 1 1 MJ i (i M 1 1 1 1 1 1 1 i 1 1 1 p 1 1 1 1 iiip 1 1; 1 1 1 1 f 1 1 1 p 1 1 1 p 1 1 1 1 1 1 1 1 1 orf 4 the ^ I -VVVLVLVHVWLRQVLWLRQVLWLRQVLVVVGGVVV ArtíAEV 220 230 240 250 260 270 310 32Q orf 41. pep AVKW.¡_I_SDKX p 1 1 1 1 iii orf 1a ÁVK_ Ql ^ DXEGRE- _) HEMrrA_AKOH? PQU R_NTV Kyc «VADKRIAASIAICT ISRS 280 290 300 310 320 33C The partial nucleotide sequence ORF41a [SEQ. ID NO: 71] is: 1 - . 1 - .TATCTGAAAC AGCTCCAAGT AGCGAAAAAC ATCA &CTGGA ATCAGGTGCA 51 GCTGGCTTAC GACAGATGGG ACTACAAACA GGAGCÍGCTTA ACCGAAGCAG 101 GTGCGGCGAT TATCGCACTG GCCGTTACCG TGGTCACCTC AGGCGCAGGA 151 ACCGGAGCCG TATGGGMT AAACGGTGCG NCCGC GCCG CAACCGATGC - - 201 AGCATTCGCC TCTTTGGCCA GCCAGGCTTC CGTATCGTTC ATCAACAACA 251 AAGGCGATGT CGGCAAAACC TGAAAGAGC TGGGCAGAAG ftGCACGGTG 301 AAAAATCTGG TGGTTGCCGC CGCTACCGCA GGCGTAGCCG ACAAAATCGG 351 CGCTTCGGCA CTGAtfCAATG TCAGCGATAA GCAGTGGATC AACAACCTGA 401 CCGTCAACCT AGCCAATGCG GGCAGTGCCG CACTGATTAA TACCGCTGTC 451 AACGGCGGCA GCCTSAAAGA CANTCTGGAA GCGAATATCC TTGCGGCTTT SOI GGTCAATACC GCGCATGGAG AAGCAGCCAG TAAAATCAAA CAGTTGGATC 551 AGCACTACAT AGTCCACAAG ATTGCCCATG CCATAGCGGG CTGTGCGGCA 601 GCGGCGGCGA ATAAGGGCAA GTGTCAGGAT GGTGCGATAG GTGCGGCTGT 651 GGGCGAGATA GTCGGGGAGG CTTTGACAAA CGGCAAAAAT CCTSACACTT 701 T6ACAGCTAA AGAACGCGAA CAGATTTTGG CATACAGCAA ACTGGTTGCC 151 GG ACGGTAA GCGG GTGGT CGGCGGCGA3. GTAAATGCGG CGGCGAATGC 801 GGCTGAGGTA GCGGTGAA? A ATAATCAGCT TAGCGACMAA GAGGGTAGAG 851 AATTTGATAA CGAAATGACT GCATGCGCCA AACAGAATAN TCCTCAACTG 901 TGCAGAAAAA ATAC GTAAA AAAGTATCAA AATGTTGCTG ATAAAAGACT 951 TGCTGCTTCG ATTGCAATAT GTACGGATAT ATCCCGTAGT ACTGAATGTA 1001 GAACAATCAG AAAACAACAT TTGATCGATA GTAGAAGCCT TCATTCATCT 1051 TGGGAASCAG GTCTAA5 GG TAAAGATGAT GAATGGTATA AATTATTCAG 1101 CAAATCTTAC ACCCAAGCAG ATTTGGCTTT ACAGTCTTAT CATTTGAATA 1151 CTGCTGCTAA ATCTTGGCTT CAATCGGGCA ATACAAAGCC TTTATCCGAA 1201 TGGATGTCCG ACCAAGGTTA TACACTTATT TCAGGAGTTA ATCCTAGATT 1251 CATTCCAATA CCAAGAGGGT TTGTAAAACA AAATACACCT ATTA TAATG 1301 TCAAATACCC GGAAGGCATC AGTTTCGATA CAAACCTANA AAGACATCTG 1351 GCAAATGCTG ATGGTTTTAG TCAAGAACAG GGCATTAAAG GAGCCCATAA 1401 CCGCACCAAT NTTATGGCAG AACTAAATTC ACGAGGAGGA NGHGTAAAAT 1451 CTGAAACCCA NACTGATATT GAAGGCATTA CCCGAATTAA ATATGAGATT 1501 CCTACACTAG ACAGGACAGG TAAACCTGAT GGTGGATTTA AGGAAATTTC 1551 AAGTATAAAA ACTGTTTATA ATCCTAAAAA NTTTTNNGAT GATAAAATAC 1601 TTCAAATGGC TCAANATGCT GNTTCACAAG GATATTCAAA AGCCTCTAAA 1 € 51 ATTGCTCAAA ATGAAAGAAC TAAATCAATA TCGGAAAGAA AAAATGTCAT 1701 TCAATTCTCA GAAACCTTTG ACGGAATCAA ATTTAGANNN TATKTKGATG 1751 TAAATACAGG AAGAATTACA AACATTCACC CAGAATAA This encodes a protein having the partial amino acid sequence [SEQ. ID NO: 72]: 1 YLKQLQVAKN INWNQVQLAY DR DYKQßGL TEAOUÜIAL AVTWTSGAG 51 TGAV GLUGA X? AATDAAFA SLASQASVSF INNGDVGKT LKEt-GRSSTV 101 KHLWAAATA GVADKIGA5A LXNVSDKQWI NNLTVNLANA GSAALIHTAV 151 NGGSL DX E ANI? AALVNT AKGEAASKXK QLDQHYTVHK IAHAIAGCAA 201 AAANKGK D GAI? AAVGEI GEALT GKN PDTLTAKERE QILAYSKLVA 251 GTVSGWGGD VNAAANAAEV AVKNNGLSDX EGREFDNSMT ACAKQNXF L 301 CRKNTVKJCYQ NVADKRLAAS IAICTDISRS TECRTIRKQH UDSRSLHSS 351 WEAGLIGKDD EWYKLFSKSY TQADLALQSY HLNTAAKSWL QSGTKPLSE 401 «MSDQGYTLI SGVNPRFTPI PRGFVKQWTP ITNVKYPEGI. SFDTNLXBHL 451 ANADGFS0EQ GIKCfAHNRTN XMAELNSRGG XV SETXTDI EGITRIKYET 501 PTLDRTGKPD GG? CEISSI TVYNPKXFXD DKILQMAOXA XSQGYSKASK 551 IAQNERTKSI SERKNVIGFS ETFDGIKEW- YXDVNTGRIT NIHPE * The 0RF41a and the 0RF41-1 show the 94th identity in an overlap 595 aa: 10 20 30 orf 41a. plp YI_K0LOVAKHI «WHQVQ_AYDWTO.KQEG TEAGAA lili lli: i :: ipil lll I l: f I IMIM III II orf 41-1 M (? WIQlPYIL__tCVRAEDTPYAC ^ L? CQI ^ 3VTKDV? mCWQIAYDKWDip < 0EGLTGAGA? 10 20 30 40 í > 0 60 40 50 60 70 BD 90 orf 41a .pep IIAlAVT SGAsK »VI_GI? C? __ ftAATDft ^ NIIIIIHI: II !: I í: t í 11 III 1 III l II II IIIIIHI: IIII i::«: IIIIIII orf 41- 1 IIA? VTVVTAGAGAGAALGI? GAAAAATnAArASLASOA-VSLlNNKtfNIGKTLKELGFl 1- BO? T. IDO 1JD 120 100 110 120 130 140 150 oríia e SSTVKHLWAAATAGVADKIGASALXNVSDXOTIMHLTVNLA AGSAA. GNTAVNGGSLK II I H I i: I I: I I 11 I II r I I! I I I I I II II II II I I I r I N M lll (I i orf 1-1 SSTVKNl? VAVATAGVADKrGASAI "NVSDKOWIHtrLTVip? NAGSAA_ INT? VNGGSLK 13D 140 150 160 170 180 ! (11. I II II 111 IU 11. HI it i 111. - • I 11 I 11 l tl l I III III I II i IIHIJ orf 1-1 8lfiANILAALVKTAHGEAñSKIKOl-DQHYíTHKIAHAIAGCAAAAAMKGKCODGAIGAA 190 200 210 220 230 240 220 230 240 250 260270 orf 4 pep VGEI VC5_0, T 'WPDTLTAKEpE0irAySKLVAfiTVSGVVGGOVSAAAHAAEVAVKNNC >. Innin imi miiiiii n my go miiiiiiii ppiiiip 1/1111111 orf 1- 1 VGEIVGí _ \ LTOGKNP0TLtAKEItE0I_A Stava3TVSGWVGG0VtlAAWJAAEVAVKN} l0 250,260,270 2T0 290 300 280 290 300 310??.. .??? 320330 orf 41a PSSP I IlXB fffiFDSEM AaKQNXPOI ^ UaWVlK QNV AnKRl ASIAICTBISRSTECRT lll I lllll I lll ll I 11 II II I IIIH lili II lili II 1 IIII lili IIII II I I I orf 41 -1 LSOKEGKErDNEMTAC3U < QIOT -0_3 «a nCKYQNVADKR_ * AAS 310 320 330 340 350 360 340 350 360 370 390 3S0 orf 41 *. Ep K0HWDSRSLHSSWEAGLrGK_DEOTf _SK_rr0ACIALQSYaLOT« \ KS «X? Sstí7K I 11 III III III III III III III II II II lllltll It lilili M orf i J - 1 RKQHLI DSRSLHSSWEAGLlGKDßEWYKLrSKSrrOADI LQSyHLNTAAKS LOSGNT 370 3B0 390 400 41C 420 400 410 420 430 440 450 orf. pep PLSfí? is? x ^ rn.? SCV? pscirL? t? gaFcnf.? ^ I II III I 11 II I III II III II II lili lili lllll I II II I II I l lili orf PLSEWMSDQG 41-1? TLISGVNPRFIPIPRGFTOíp 'PITNVIOrPEsiSPDO.lJFH-? NADGF 430 «0 450 460 470 IßO 460 470 480 490 500 510 orf 4 la. pSp SQEQGrKGAKNRTHXMAELRSRGGX -TSE__CTD_EGira? OT I l: II I I lili I IMIMIII Hlf I NHIIIIIUI II I lll 1 orf 41-1 SQKCX3IKGAHmTNF? "EI« SRGGRVKSETQTOIEGlpUK ^ 490 500 510 520 530 540 520 530 540 550 560 570 orf 4 la. PSSP SSIBOTra tXFXDDKt QHACKAXSQGYSKAS-CtA iiipiipi i ^ i iiiiiumipiiuiiutniíiuutuí mtiini orf 1-1 SSIKTVra _XFSDl I_ ^ tt0NAASOGYSKASKI flNERT- »I5 550,560,570 5B0 orf 590600580590 1a.p» p KFRXYXDVMTGRITH JIPEX 111 mIU I lli Jf If??? 11 orf41-a KFBSYFDVNTCRITNIHPEX 610 620 - - The amino acids 25-619 of 0RF41-1 were amplified as described above. Figure 6 shows the hydrophilicity charts, antigenic index, and the AMPHI regions for 0RF41-1.

Based on this analysis, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

E xemployment 17 The next sequence of AD? was identified in N. meningitidis- [SEC. ID? O: 73]: 1 ATGGCAATCA TTACATTGTA TTAT? CGGTC AA3SGTATTT TAAATSTATG 51 TGCAAAAGCA AAAAATATTC AAGTAGTTGC CAATAATAAG AATATGGTTC 101 TTTTTGGGTT TT GGs r GC ATCATCGGCG GTTCAACCAA TGCCATGTCT 151 CCCATATTGT TAATATTTTT GCTTAGCGAA ACAGAAAATA AAAATcgTAT 201 CGTAAAATCA AGCAATCTAT GCTATCTTG GGCGAAAñTT GTTCAAATAT 251 ATATGCTAAG AGACCAGTAT TGG ?? TTAA ATAAGAGTGA ATACGdTTTA 301 ATATTTTTAC? ^ C C ATT GTCTGTTATT GGATTGTATG TTGGAATTCG 3 l GTTAAGGACT AAGATTAGCC CAftA-TTTTTT TAAAATGTTA ATTTTTATTC 401 tTTTTATTGGT ATTGGCtCTG AAAATCGGGC AttCGGGTTT AAtCAAACTT 451 TAA This corresponds to the amino acid sequence [SEC. ID? O: 74; 0RF51]: - - 1 MAIITLYYSV NGI HVCAKA KNIOWANNK KHVLFGFLXX IIGG5TNAMS 51 PILLIFL 5E TENHÍRIVK = SNLCYLIAKI VQ1 YMLRDQY WLLNKSEYXL 101 IFL SVL5VI GLYVGIRLRT KISFNFFKHL IFIVLLVIAL KIGHSGLIKL 151 * Additional work revealed the complete nucleotide sequence [SEC. ID NO: 75]: 1 ATGCAAGAAA TAATGCAATC ?? TCGT TT GTTGCTGCCG CAATACTGCA 51 CGGAATTACA GGCATGGGAT TTCCGAGGCT CGGTACAACC GCATTGGCTT 101 TTATCATGCC ATTGTCTAAG GTTGTTGCCT TGGTGGCATT ACCAAGCCTG 151 TTAATGAGCT TGTTGGTTCT ATGCAGCAAT AACAAAAAGG GTTTTTGSGCA 201 AGAGATTGTT TATTATTTAA AAACCTA? AA ATTGCTTGCT ATCGGCAGCG 251 TCGTTGGCAG CATTTTGGGG GTGAAGTTGC TTTTGATACT TCCAGTGTCT 301 TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG 351 TATTTTAAAT GTATGTGCAA AAGCAAAAAA TATTCAAGTA GTTGCCAATA 401 ATAAGAAGAT GGT? CTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA 451 ACCAATGCCA TGTCTCCCAT ATTGTTAATA TTTTTGCTTA GCGAAACAGA 501 AAATAAAAAT CGTATCGTAA AATCAAGCAA TCTATGCTAT CTTTTGGCGA 551 AAATTGTTCA AATATATATG CTAA5AGACC AGTATTGGTT ATTAAATAAG 601 AGTGAATACG GTTTAATATT TTTAC GTCC GTATTGTCTG GATTGGATT 651- GTATGTTGGA ATTCGGTTAA GGAC AAGAT TAGCCCAAAT TTTTTTAAAA 701 TGTTAATTTT TATTGTTTTA TTGGTATTGG CTCTGAAAAT CGGGCATTCG 751 GGTTTAATCA AACTTTAA This corresponds to the amino acid sequence [SEC. ID NO: 76; "0RF51-1]: 1 MQEIMQSIVF VAAILHGIT GMGFPMLGTT ALASTMPLSK WALVALPSL 51 LM5LLVLCSN NKKGFWQEIV YY TYKLLA IGSWGSI G VKLLILPVS 101 LLLLWUÍT LYYSVW3SLN VCAKAKNIQV VAiíNKNMVLF 151 TNAHSPILLI FLLgSTEWKN RIVKSSNLCY HAKIVQGYM LRDQYWLLKK 201, EYGLIFLL "VL5VIGLYVG IRLRT I5PB FFKMLIFIVL LVLALKIGHS 251 GLIKL * Computer analysis of this amino acid sequence reveals three putative transmembrane domains. A corresponding ORF of strain A of N. men i n gi t i di s was also identified.

- - Homo loggia with a predicted ORF of N. meningi tídi's (strain A) ORF51 shows 96.7% identity in an overlap of 150aa with an ORF (0RF51a) of strain N. of N. meningitidis: 20 30 orf51.pep HAIITLYY = V? GIL? VCAKAK? IQWA ?? K iipiuiitiuiiüuniuiiiiu orf 51a And lJ? IGSVVG = IICVKLLLILPVSWLLLIA & TV? GILJ ^ CAKAKHIOVV? Ak BOK 90 100 110 120 130 40 50 60 70 80 90 orf 1. pep -_MVLFGFLXXIIGGST? AMSPIl IFLXSETE? K «RIVK_Sl.LCYL-AKIvsiYI-LRDOY iiiiiui ?????? mm ????? i ?? i? I ??? iiim ????? i ??? ?? m? p orf sia MMVLPGFIAGII ^ STWAMSPILLIFX SEI? WK? RIAKSS? LCYLLAKIVQIYMLRDQY 140 150 160 170 180 190 100 110 120 130 140 150 orf51. p p WUJ.KSEY ^ Lirij.SV gVlG YVGrRLRTKISP? rFiatLIriVLLVLALKrGKSG IKL HII? II iiHimiminiiminii iiiumimunri i.-mip orfSla WIJJ ^ SEYGI.rFLL_gVX5VlGLYVGIRLRTKISPyFrK LIFIVLLVlJUJIGYSgLIKL 200 210 220 23C 240 250 The 0RF51-1 and ORF51a show 99.2% identity in an overlap 255 aa: Orf 514 -pep MOEIMQStVFV? AAlLHGlTGMGrPt < LG-tALAFI PLS VVALVAL? Sl.LKSLLVLCS? I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I. ', I lll I 1 I! I I orf 51-1? CJEIMQSIVÍVAAAILHGITGHGFPMLGTTALAFI1.PLSKVVALVALPSLLMSLLVLCS? srf51a-p «p? KKGFWQE * V? YL rYKLLAIGSVVGSIlWKLLLILPVSW- LL« _iIITLYY_V? GIL? -iiipmuii iimiippi imiip? uni nin ni n n ni 11 m orf 51-1? KKGrWQEIVTO_KTYKL__AIGS > ? fGSlLGVKLl__ILPVS «__. LLMAlITLYYSVHGlL? srl .pep VCAXAK? IQVVAK? K MVLfX; FlAGIIGGST? AMSFIIJ-lFLLS__TE? K RlAKSSI.LCY iiipnipiinHiiiiiiiinnintiiiiipniipiiiinuiiiiip arf 51 - 1 VCAKAIWIDVVAK? K? MVLreFl? GIIGGST? AMSPIIJ.IEXLSíp'EMKKRlVKSS? LCY orfSla .pep 3JüCT Q? m £ J ^ YWL __-] KSEYGLip __- SVLSVIl? YVG_ ipilll? H II II 1 MMIMIHH IIIM? 1 II 1 f llf II 11 IM orí51-l Ll? KrVQIYMiaDOYWLLHKSEYGÍ.IFLLSVLSVTGLYVGIRLRTKISP? FFKMLIFJVL orf 51a. pep LVLSLKIGYSGLIKLX I I f I I! 11 r I 11 I I I I orf51-l LVLALKIGHSGLIEOX The full-length nucleotide sequence 0RF51a [SEQ. ID? O: 77] is - - 1 ATGCAAGAAA TAATGCAATC TATCSTTTTT GTTGCTGCCG CAATACTGCA 51 CGGAATTACA GGCATGGGAT «PTCCGATGCT CG6TACAACC GCATTGGCTT 101 TTATCATGCC ATTGTCTAAG GTTC.TTGCCT TGtíTGGCATT ACCAAGCCTG 151 TTAATGAGCT TGTTGGT CT ATGCAGCAAG AACAAAAAGG GTT T GGCA 201 AGAGATTGTT TATTATTTAA AAACCTATAA ATTGCTTGCT ATCGGCAGCG 251 TCGTTGGCAG CATT TGGGG GTGAAGTTGC TT GAtACT CCAGTGTCT 301 TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG 351 TATTTTAAAT GTRTGTSCAA AftGCAAAAAA TRTTCAAÍ3TA GTTGCCRATA 401 ATAAGAATAT GGTTCTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA 451 ACCAATGCCA T < __CTCCCAT ATTGfTTAATA TG? TGCTTA GCGAAACAGA 501 GAATAAAAAT CGTACGCAA AATCAAGCAA TCTATGCTAT CTTTTGGCAA 551 AAA GTTCA AATATATATG CTAAGAGACC AGTAT GGT ATTAAATAAG 601 AGTGAATACG GTTTAATATT TTTACTGTCC GTATTGTCTG TTAT GGATT 651 GTATGTT5GA ATTCGGTTAA GGACTAAGAT TASCCCAAAT TTTT TAAAA 01 TGTTAATTTT TATTGTTTTA TTGGTAT GG CTCTGAAAAT CGGGTATCA 751 GGTTTAATCA AACTTTAA This encodes a protein that has the amino acid sequence [SEC. ID NO: 78]: i MQEGMQSGVG VAAAILHGGT GMGIGPMLGTT AIAFIMPLS WALVALP5L 51 LMSLLVLCSW NXKGFHQErv YYLKTYKLLA IGSWGSILG VKLLLILPVS? O? WLLLLMAGIT LYYSVMGILN VCAKAKNIQV VAKNKNMVLF GFLAGI? GGS 151 THAMSPIL I FLLSETENKN RIAKSSSLCY LLA IVQIYM LRDQYWLLNK 201 SEYGLIFLLS VLSVI6LYVG IRLRTKISPt. FFKMLIFIVL LV1ALKIGYS 251 GLIK? Z Based on this analysis, it is predicted that this N. meningitidis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 18 The next sequence of AD? partial was identified in N. meningitidis [SEC. ID ? O: 79]: - - 1 ATGAGACATA TGAAAATAC ?. AAA.TTA TTA? AGTATTTA TAGTT TACA 51 TATAGCCTTG ATAGTAATTA ATATA6TGTT TGGTTATTTT GTTTTTCTAT 101 'p'GATTTTTT TíSCGTTTTTG TTTTTTGCAA ACGTCT TCT TGCTGTAAAT ßl * r R p? Mt Tf_5ftRWAW_ OOA? I? IMAC A? ATG? CT TT GATGGCC 201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA 251 AAT GATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCGCGATG 301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AAT ATGTTT ATGACTCAAA 351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTAGAG 401 AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA 451 AGATTAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT so: TATAAAATTT GTCAGG-. This corresponds to the amino acid sequence [SEC. ID NO: 80; ORF82]: 1 MRHHKIQNYL LVFIVLHIAL IVH.IVFGYF VFLF FFAFL FFANVFLAVK 51 LLFLEKKIKM LLFLLPISI IIWMVIHISM INIKFYKFEH QIKEQ SSI 101 TGVI FHD5Y NYVYDSNGYA KLKDNSRYGR GRETPYIDV VASDVK KSI 151 RLSLVCGIHS YAPCANFIKF VR ,.

Additional work revealed the complete nucleotide sequence [SEC. ID NO: 81]: 1 ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA 51 TATAGCCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT 101 TTGATTTTTT TGCGTTTTTG TTTTTTTGCAA ACGTCTTTCT TGCTGTAAAT 151 TTATTATTTT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC 201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA 251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT 301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTAGGTT ATGACTCAAA 351 TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTAGAG 401 AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA 451 AGATTAAGCT TSGTTTGTGG TATTCATTCA TATGCTCCAT GGGCCAATTT 501 TATAAAATTT GCAAAAAAAC C GTTAAAAT TTATTTGTAT AATCAACCTC 551 AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAAT5A GGAAACAAA eoi AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG 651 TGTTGGTATC GTATTAATTA TTTTATATTT AAAATTTAAT TTGCTTTTAT 701 ATAGGACTTA CT CAATGAG TTGGAATAG This corresponds to the amino acid sequence [SEC. ID NO: 82; ORF82-1]: - 1 MRHMKNKNYL__ LVFIVLHIAL IVO.IVFGYF VFLFDFEAFL FFA VTLAVN 51 LF EKNI N KL FLLPJSI HWHVXHISM INIKF? KFEH OIKEQNISSI 101 TGVIKPH SY NYVYDSNGYA K KDKfflRYGR VIRETPYIDV VASDVKNKSI 151 RLSLVCGIHS YAPCAHFIKF AXKPV IYF. WQPQGDFID »VI EIHD € N 201 SLYLLDKYKT FLIENSVCI VLIILYL Fl. LL YRTYFKE LE * Computer analysis of this amino acid sequence reveals a predicted leader peptide.

A corresponding ORF of strain N. of N. meningi tidi s was also identified.

Homo loggia with a predicted ORF of N. meníngitidis (strain A ORF82 shows 97.1% identity in an overlap of 172aa with an ORF (ORF82a) of strain N. of N. meningitidis: 20 30 40 50 60 orf 82. ep HRHMKIQMYLLVFIVLHtALlVTKIVFGyr- FLFDFFAFLFFA? VFLAVKLLFLEK I K MI M: ¡ll I III I I M; I! IIIUIM III II IIIII lllli orfB ^ a MRHHK? KMYL VFJ VLHrTLlVIWIVFGYEVFLFDrFAFLFFAítVFLAVULLFLE? W IKK 10 20 30 40 50 60 70 B0 90 100 110 120 orf 82 pbw KLLFLLPI- »IIHWVIHlSMH_IKFY FEHgIKEOHISSITGyiKPHDSYMYVYDS? GYA! Iu uuni .u? Mu ,,! U ?? muu? n \ n? nnn! iunii? Orf82a KLLrcLglSIII MVIHISHIKIKFYKFEHOIKECWISSITGVIKPHOSY YVYDSHGYA 3 80 90 100 11G 120 130 140 150 160 170 srf82.pep KLKD? HRYGRVIRETPYIDVVASDV? KSIRLSLVCGIHSYAPCA- FIKFVR i ii 11 '111 M m p 111 ii 11 ii m p p n 11 p 111111 m i i; : srf82_ KLKD? KRYGRVlRETPYIDWASDVroí SIRLSLVCGIRSYñí'CA? FIKFA KPVKIYFY 130 140 150 160 170 ISO - - The ORF82a and ORF82-1 show 99.2% identity in an overlap 242 aa: orf E2a .pep HRHMKNKIWLLVOVI ílTLIV? NIVTCYTVFLFDFFAFLFFANVFIAVNLLFI-EKNIKN i / I M i p I I I I I G I: I I N P M I I M I I I I I p M I; M i M II I M I I I I I I ori B2 -1 MRHMKNKNLVLVLVVLVLVLRQVVDWLRQVLFVFDFFAFLFFANVFIAVNLLFIJ? KNI XU orf 82a. pep KLLFLLP? sriIWMVIHISMINIKFYKFEHQIKEÍíISSITGVIKPHDSYNYVYDSNGYA III lllll Mil IIHI f 1 f IIIII Nf II 11 II fl 1 OR 1 III 1 M f NI III II II II orf82-í I__TLPISIIIWMVlHISM_NIKI? KFEHQI_aWISSIT (_VI_PH_? SYNyVYDSKGYA orf 82a. pbw KLKDNHRYGRVIR? rT-YIDVVASDVKNKSiRLSLVCGIHSYAPCAÍJFIKFAKKPVKrYFY HiiipiiiiiiMMpiiiiipiiiinpiiiipnmiipiipiiimi orfß2 -l KLKDNHRYGRVIRETPYID \ pteSIJVIWKSIl_I? LV; iHSY? PCANFIKFAKKPVKIYFY orf 82a. pep NQPQGDFIDNVTPSINDÍ__ SLYLLDKYB7rFFLI_JiSVCIVLIILYlJfTí _-__ LYRTYFNE IIIN i Nlll ININI INI I NNN INNN II III NNI I f lllli I orf? 2-1, NQPOGDFIW ^ IFEINIX? KKSLYLLDKYKTFFLIENSVCI? LirLYLK NLLLYftTYFNE orfB2a.pep LEX I M orf 82-1 LEX • The full-length ORF82a nucleotide sequence [SEC. ID NO: 83] is 1 ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA 51 TATAACCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT 101 TTGATTT TT TGCGTTTTTG TTTTTTTGCAA ACGTCTTTCT TGCTGTAAAT 151 TTATTAT7TT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC 201 GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA 251 AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT 301 ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA 351 TGGATATGCT AA CAAAAG ft.TÍUM_CM-ÍYS KI_? < 3? MwSG GSA1 _! _ 5AGI__ 401 AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA 451 AGATTAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT 501 TATAAAATTT GCAAAAAAAC CTGTTAAAAT TTATTTTTAT AATCAACCTC 551 AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAATGA TG € AAAAAAA 601 AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG 651 TGTTTGTATC GTATTAATTA T TTATATTT AAAATTTAAT TTGCTTTTAT 701 ATAGGACTTA CTTCAATGAG TTGGAATAG This encodes a protein having the amino acid sequence [SEQ. ID NO: 84]: 1 MRHMKWKNYL LVFIVLHITL IVIMIVFGYF VFXFDFFAFL FFANVFLAVH 51 LLPLEKttIKK KLLFLLPISJ IIWMVIKrSM INIKFYETEH QIKEQNJSSI 101 TGVtKPKQSY NYVYDSHGYft KLKDNHRYGR IRETPYís VA5DVKHKS1 151 RLSLVCG1HS YAPCAHFI F AKKPVKIYFY NQPQGDFIDN VTFEINPGKK 201 SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE E * - - Based on this analysis, it is predicted that this N. meningi idis protein, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 19 The next sequence of AD? partial was identified in N. meningitidis [SEC. ID? O: 85]: 1 ..ñCCCCCAACA GCGTGACCGT CTTGCCGTCT TTCGGCGGAT TCGGGCGTAC 51 CGGCGCGACC ATCAATGCAG CAGGCGGGGT CGGCATGACT GCCT7TTCGA 101. CAACCTTAAT TTCCGTAGCC GAGGGCGCGG TTGTAGAGCT GCAGGCCGTG 151 AGAGCCAAAG CCGTCAATGC AACCGCCGCT TGCATTTTTA QGGTCTTGAG 201 TAAGGACATT TTCGATTTCC TTTTTATTTT CCGTTTTCAG ACGGCTGACT 251 TCCGCCTGTA TTTTCGCCAA AGCCATGCCG ACAGCGTGCG CCTTGACTTC 301 ATATTTAAAA GCTTCCGCGC GTGCCAGTTC CAGTTCGCGC GCATAGTTTT 351 GAGCCGACñA CAGCAGGGCT TGCGCCTTGT CGCGCTCCAT CTTGTCGATG 401 ACCGCCTGCA GCTTCGCAAA TGCCGACTTG TAGCCTTGAT GCTGCGACAC 451 AGCCAAGCCC GTGCCGACAA GC CGATAAT GGCAATCGGT TGCCAGTAAT 501 TCGCCAGCAG TTTCACGAGA TTCATTCTCG ACCTCCTGAC GCTTCACGCT 551 GA This corresponds to the amino acid sequence [SEC. ID? O: 36; O R F 124]: 1 ..TP? SVTVLPS FGGFGRTGAT IMAAGGVG? ÍT? FSTTLISVA EGAWELCAV 51 RAKAV? ATAA CIFTVLSKPI FDFLFIFRFQ TADFRLYFRQ SHAD5VRLDF 101 IFKSFRACQF QFARIVLSKQ QOGLRLVALH LVDDRLQLFK CRLVALKVRH 151 SQARADKRD? G? RLPVIRQQ FHEIHSRPPD ASR * Computer analysis of this amino acid sequence predicts a transmembrane domain.

- - Additional work revealed the complete nucleotide sequence [SEC. ID NO: 87]: 1 ATGACTGCCT TTTCGACAAC CTTAATTTCC GTAGCCGAGG GCGCGGTTGT 51 AGAGCTGCAG GCCSTGAGAG CCAAAGCCGT CAATGCAACC GCCGCTTGCA 102 TTTTTTACGGT CT7GAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT 151 TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACAG 201 CGGGGCGCCT GACTTCATAT TTTTTAGCTT CCGCGCGTGC CAGTTCCAGT 251 TCGCGCGCAT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG 301 CTCCATCTTG TCGATGACCG CCTGCTGCTT CGCAAATGCC GACTTGTAGC 351 CTTGATGGTG CGACACAGCC AAGCCCGGGC CGACAAGCGC GATAATGGCA 401 ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT 451 CCTGACGCTT CACGCTGA This corresponds the sequence of amino acids [SEC. ID NO: ORF124-1]: 1 MTAFSTTLIS VAEGAWELO AVHAKAV AT AACIFTVL5K PIFPFLFIFR 51 FQTAt.FRI-FF RQSHRDSVR OFIFFSFRAC QFQFARIV5.S RQQQGLRLVA 101 LHI.VDDRLLL RKCRLVALMV RHSQARADKR DNGHRLPVIR QQFHEIHSRP 151 PDASR * A corresponding ORF of strain N. of N. meningitidis was also identified.

Homo loggia with a predicted ORF of N. meningitidis (strain A) The ORF124 shows 87.5% identity in an overlap of 152aa with an ORF (ORF124a) of strain N. of N. meningitidis: - 20 30 40 50 60 orf 124. pep TPNSV VLP SFGGPSRTGAT INAAGGVGMTAFSTTLI SVAEGAWELQAVRAKAVN ATAA I I NIIIIN i II 1'.1 III ii IIIII = II I srfl24a HTAFSTTLI SVAEGALVELQAVMAKAVN TTAA 10 20 30 70 80 90 100 110 120 orf 12 .pep Cl F? VLSKD IFDF FX FRFQTADFRLYFRQSHADSVRLDF1 FKSFRACQ FQFAH 1V SRQ I I N N II I N I 11! I I I I I M I I 11: I i I N I I: N! I I M I I I: I NI: i I I I! _ > r £ 124a CIFRIFFFLFIFRFQ ADFRLFFROSKADGVRLDFIFFSFRTRLRQFAGVVLSR ?? 40 50 60 70 80 90 130 140 150 160 170 180 orfl24.pe? QQGlALVALHLVDD LOLRKCRLVALMVRHEOARADKRDIíGNRLPVIROQFHEIHSRPPD I I I I M I I I::: J I I l l I I I I I I:: I I I M: I U I I! f i I I I I | | I N I I arfl24a QQGLRLVALKFIJ4DRLLl ^ USRLVAUfVRHRÍMIADKRDDG RLPVIRQ0FHEIHSRPPD 100 110 120 130 140 150 _ > r £ 124a VX The ORF124a and ORF124-1 show 89.5% identity in an overlap 152 aa: or¿124-1.pep HTAFSTTLlSVAEGAWeLQAVRAKAVNATAAClrrVLSKDIFDFLrifRFOTADFRLFF II II M II lll í NNiíl HII I II I:!. J 11 II IIII II II IIII i (i IIII / IM orf II 24th OTAFSTTLISV-LEGALVEWAVMAKAVMTTAACIf VLSKDIFDFLFIF FQTADFRLFF orf 124-1 pbw RQSHADSVRLDFIFFSFRACOF ^ FA I \ 'LSRQQOGX4U, VAI_HLVDORL-.LRKCRLVAL V NNIIH IIIIÍINNr \ UI: I ¡N II I ll III 11 i::: I II IM i I fl lljl? Rf! 24a ROSHADGVRLDFIFFS RRRLFOEAGWLSRQQQGLRLVALHFLNDRL-.LRKS LVALKV orf 1 -1 RHSOARAOKRDNGNRLPVIRQOFHEGKSRPPDASRX NI: IN f 1 r: i IINNI II IINUNU; orfl2_a RHRQTRADKRßDGNRLPVIRQOFHEIHSRPPDVX The full-length ORF124a nucleotide sequence [SEQ. ID NO: 89] e s 1 ATGACCGCCT TTTCGACAAC CT AATTTCC GTASCCGAGG GCGCGCTTG. 51 AGAGCTGCAA GCCGTGATGG CCAAAGCCGT CAATACAACC GCCGCCTGCA 101 TTTTTTACGGT CTTGAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT 151 TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACGG 201 CGTGCGCCTT GACTTCATAT TTTTTAGCTT CCGCACGCGC CTGTGCCAGT 2S1 TCGCGGGCGT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG 301 CTTCATTTTC TCAATGACCG CCTGCTGCTT CGCAAAAGCC GACTTGTAGC 351 CTTGATGGTG CGACACCGCC AAACCCGTGC CGACAAGCGC GATGATGGCA 401 ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT 451 CCTGACGTTT GA - - This encodes a protein that has the amino acid sequence [SEC. ID NO: 90]: l HTAFSTTL1S VAEGALVELQ VlíAKAWrr RACIFTVLSK DIFDFLFIFR 51 FQTADFRLFF RQSHADGVRL DF? FFSFRTR LFQFAGWLS RQQQGLRLVA 1C1 LHFLNDRLLL RKSR VALMV RHRQTRAPKR DDGNRLPVIR QQFHEIHSRP 151 PDV * The ORF124-1 was amplified as described above. Figure 7 shows the hydrophilicity charts, antigenic index, and the AMPHI regions for ORF124-1.

Based on this analysis, it is predicted that this N protein. Glyphs and their epitopes could be useful antigens for vaccines or diagnostics.

It will be appreciated that the invention has been described by way of example only, and that modifications can be made while remaining within the spirit and scope of the invention.

- - TABLE I PCR primers - TABLE II Cloning, expression and purification It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the manufacture of the objects to which it refers.

Having described the invention as above, property is claimed as contained in the following

Claims (28)

- - CLAIMS

1. - A protein 'characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ. ID Nos. 2, 4, and 6.

2 . - A protein characterized in that it has 80% or more of the sequence identity to a protein of quality with claim 1.

3. - A protein characterized in that it comprises a fragment of at least 16 consecutive amino acids of an amino acid sequence selected from the group consisting of SEQ. ID Nos. 2, 4, and 6.

4. An antibody characterized in that it binds to a protein according to any of the preceding claims.

5. - A nucleic acid molecule characterized in that it encodes a protein according to any of the preceding claims.

6. - A nucleic acid molecule according to claim 5, characterized in that it comprises a nucleotide sequence selected from the group consisting of SEQ. ID Nos. 1, 3, and 5.

7. - A nucleic acid molecule characterized in that it comprises a fragment of at least 20 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ. ID Nos. 1, 3, and 5.

8. - A nucleic acid molecule characterized in that it comprises a nucleotide sequence complementary to a nucleic acid molecule according to any of claims 5 to 7.

9. - A nucleic acid molecule characterized in that it comprises a nucleotide sequence having 80% or more of sequence identity to a nucleic acid molecule according to any of claims 5 to 8.

10. - A nucleic acid molecule characterized in that it hybridizes to a nucleic acid molecule according to any of claims 5 to 9 under high stringency conditions.

11. - A composition characterized in that it comprises a protein, a nucleic acid molecule, or an antibody according to any of the preceding claims.

12. - A composition according to claim 11, characterized in that it is a vaccine composition or a composition for diagnosis.

13. - A composition according to claim 11 or claim 12, for use as a drug.

- - 14. The use of a composition according to claim 13 in the manufacture of a medicament for the treatment or prevention of infection due to the Neisseria bacterium, particularly Nei sseri a men i n gi t i di s.

15. - A protein characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ. ID Nos. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 4 6, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90.

16. - A protein characterized by having 50% or more of sequence identity to a protein according to claim 15.

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

18. - An antibody characterized in that it binds to a protein according to any of claims 15 to 17.

19. - A nucleic acid molecule characterized in that it encodes a protein according to any of the rei indications 15 to 17.

20. - A nucleic acid molecule according to claim 19, characterized in that it comprises a nucleotide sequence selected from the group consisting of SEQ. ID Nos. 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 , 55, 57, 59, 61, 63, 65, 67.69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89.

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

22. - A nucleic acid molecule characterized in that it comprises a nucleotide sequence complementary to a nucleic acid molecule according to any of claims 19 to 21.

A nucleic acid molecule characterized in that it comprises a nucleotide sequence having 50% or more of sequence identity to a nucleic acid molecule according to any of claims 19 to 22.

24. - A nucleic acid molecule characterized in that it can hybridize to a nucleic acid molecule according to any of claims 19-23 - under highly stringent conditions.

25. - A composition characterized in that it comprises a protein, a nucleic acid molecule, or an antibody according to any of claims 15 to 24.

26. - A composition according to the indication 25, characterized in that it is a vaccine composition or a composition for diagnosis.

27. - A composition according to claim 25 or claim 26, for use as a drug.

28. The use of a composition according to claim 25 in the manufacture of a medicament for the treatment or prevention of infection due to the Neisseria bacterium, particularly Ne i s s eri a m e n i n g i t i di s.