AU720789B2 - Neisseria meningitidis TBP2 subunit - Google Patents

Description

WO 97/13860 PCT/FR96/01580 NEISSERIA MENINGITIDIS TBP2 SUBUNIT The present invention relates to a new variant of the Tbp2 subunit of the transferrin receptor of Neisseria meningitidis, to its use as a medicament, as well as to the DNA fragment coding for this variant.

Generally speaking, meningitis is either of viral origin or of bacterial origin. The bacteria mainly responsible are N. meningitidis and Haemophilus influenzae, which are involved, respectively, in approximately 40 and 50% of cases of bacterial meningitis.

Approximately 600 to 800 cases per annum of N.

meningitidis meningitis are recorded in France. In the United States, the number of cases amounts to approximately 2,500 to 3,000 per annum.

The species N. meningitidis is subdivided into serogroups according to the nature of the capsular polysaccharides. Although a dozen serogroups exist, of cases of meningitis are attributable to 3 serogroups: A, B and C.

Effective vaccines based on capsular polysaccharides exist to prevent meningitis caused by N.

meningitidis serogroups A and C. These polysaccharides as such display little or no immunogenicity in children under 2 years of age and do not induce immune memory.

However, these drawbacks may be overcome by conjugating these polysaccharides to a carrier protein.

In contrast, the polysaccharide of N. meningitidis group B displays little or no immunogenicity in man, irrespective of whether or not it is in conjugated form. Thus, it is seen to be highly desirable to seek a vaccine against meningitis induced by N. meningitidis in WO 97/13860 2 PCT/FR96/01580 particular of serogroup B other than a polysaccharidebased vaccine.

To this end, different proteins of the outer membrane of N. meningitidis have already been proposed.

In this connection, special attention has been directed towards the membrane receptor for human transferrin.

Generally speaking, the large majority of bacteria need iron for their growth, and they have developed specific systems for acquiring this metal. As regards N. meningitidis in particular, which is a strict pathogen of man, the iron may be taken only from human iron-transport proteins such as transferrin and lactoferrin, since the amount of iron in free form is negligible in man (of the order of 10 1 in any case insufficient to permit bacterial growth.

Thus, N. meningitidis possesses a receptor for human transferrin and a receptor for human lactoferrin which enable it to bind these iron-chelating proteins and thereafter to take up the iron needed for its growth.

The transferrin receptor of N. meningitidis strain B16B6 has been purified by Schryvers et al.

(WO 90/12591) from a membrane extract. This protein as purified is seen to consist essentially of 2 types of polypeptide: a polypeptide with a high apparent molecular weight of 100 kD and a polypeptide with a lower apparent molecular weight of approximately 70 kD, as visualized after polyacrylamide gel electrophoresis in the presence of SDS.

The product of the purification carried out, in particular, by Schryvers is, by arbitrary definition and for the purposes of the present patent application, WO 97/13860 3 PCT/FR96/01580 referred to as the human transferrin receptor (HTR), and the polypeptides constituting it, as the subunits. In the text which follows, the subunits of high molecular weight and of lower molecular weight are referred to, respectively, as Tbpl and Tbp2.

Since the pioneering work of Schryvers et al., it has been discovered that there are in fact, at least 2 types of strain which differ in the constitution of their respective transferrin receptors. This was demonstrated by studying outer membrane extracts of several tens of strains of N. meningitidis of miscellaneous origins. These membrane extracts were first subjected to polyacrylamide gel electrophoresis in the presence of SDS, and then electrotransferred onto nitrocellulose membranes (Western blotting). These nitrocellulose membranes were incubated: a) in the presence of a rabbit antiserum directed against the transferrin receptor purified from N. meningitidis strain B16B6, also referred to as IM2394; b) in the presence of a rabbit antiserum directed against the transferrin receptor purified from N. meningitidis strain M982, also referred to as IM2169; or c) in the presence of human transferrin conjugated to peroxidase.

As regards a) and the recognition of the transferrin receptor subunits is visualized by adding an anti-rabbit immunoglobulin antibody coupled to peroxidase, and then by adding the substrate for this enzyme.

Tables I and II below show the profile of some representative strains as appears on 7.5% polyacrylamide WO 97/13860 4 PCT/FR96/01580 gel after electrophoresis in the presence of SDS; the bands are characterized by their apparent molecular weights expressed in kilodaltons (kD): NB: In brackets, the serogroup, serotype, subtype and immunotype are shown in order.

13 860-5 5 PCT/FR96/01580 Strains Table 11 2169 1000 1604 132 1001 876 1951 2449 867 9: P1.9 nd) (B :nd) (C:15:P1.16) (A:4:P1.9) (B:19:P1.6) (A:nd) (B:nd) (B:2b:P1.2) Detection with anti-2394 receptor 96 98 98 98 98 96 94 94 93 antiserum Detection with 96 98 98 98 98 96 94 94 93 anti-2169 receptor atsrm87 85 83 81 79 88 87 85 8 Detection with peroxidase 87 85 83 81 79 88 87 85 trans ferrin N.B. In brackets, the serogroup, serotype, subtype and immunotype are shown in order.

WO 97/13860 6 PCT/FR96/01580 The results entered on the first 2 lines of the tables show that there are 2 types of strain: The first type (Table I) corresponds to strains which possess a receptor both of whose subunits are recognized, under the experimental conditions used, in Western blotting, by the anti-IM2394 receptor antiserum, while only the high molecular weight subunit is recognized by the anti-IM2169 receptor antiserum.

The second type (Table II) corresponds to strains which possess a receptor both of whose subunits are recognized, under the experimental conditions used, in Western blotting, by the anti-IM2169 receptor antiserum, while only the high molecular weight subunit is recognized by the anti-IM2394 receptor antiserum.

Consequently, an antigenic diversity exists in respect of the lower molecular weight subunit. This diversity is, however, restricted since it resolves into 2 major types, contrary to the suggestion made by Griffiths et al., FEMS Microbiol. Lett. (1990) 69: 31.

On the basis of these observations, application WO 93/6861 distinguishes strains of the M982 (IM2169) type or of the B16B6 (IM2394) type.

Besides the strains mentioned in Table II, M982 type strains are, for example, the strains S3032 (12, P 1.12.16), 6940 (19, P M978 P 1.1, 2223 nd), 1610 nd), C708 4, P M981 4), also referred to as 891, and 2996 2b, P The Applicant received, as a free gift, the strains S3032, M978 and M981 from Dr. J. Poolman (RIVM, Bilthoven, Holland), and the strain C708 from Dr. Achtman (Max Planck Institute, Berlin, Germany).

WO 97/13860 7 PCT/FR96/01580 The strain IM2154 (serogroup C) is mentioned by way of example as being of the B16B6 type.

On the basis of the above findings, the assumption was possible that a vaccine which is effective against all N. meningitidis infections could be adequately composed of the high molecular weight subunit, irrespective of the strain of origin of the receptor, since this subunit is recognized by both types of antiserum. However, it seems that this cannot be the case, inasmuch as the high molecular weight subunit appears to be incapable of inducing the production of neutralizing type antibodies. Only the smaller of the 2 receptor subunits (Tbp2) appears to be capable of performing this function.

Indeed, the protein Tbp2, rather than Tbpl, has a number of characteristics which make it a potential vaccinal candidate: an ubiquitous expression, accessibility at the surface of the microorganism, the capacity to induce neutralizing antibodies and a limited variability since, as has just been stated, two major groups have so far been identified.

By virtue of this discovery, pharmaceutical compositions have already been proposed which contain: the HTR of at least one strain of the B16B6 type and the HTR of at least one strain of the M982 type (WO 93/6861); or (ii) the Tbp2 of at least one strain of the B16B6 type or the Tbp2 of at least one strain of the M982 type (WO 93/7172) To obtain large quantities of proteins for development on the industrial scale, the DNA fragments coding i.a. for various Tbp2s were cloned. The amino WO 97/13860 8 PCT/FR96/01580 acid sequences of the Tbp2 subunits of the strains M982 and B16B6 have been disclosed in patent application EPA 586,266 (published on 9th March 1994), as well as the corresponding DNA fragments. These sequences are repeated in SEQ ID NO 1 to 4 of the present application.

The study of the Tbp2 subunits, irrespective of the strain of origin, has made it possible to demonstrate three main structural domains, at least one of which is associated with special properties. By definition, the domains of M982 Tbp2 and B16B6 Tbp2 have been fixed as shown in the table below, by indicating the position of the amino acids, limits of the different domains included, and by reference to the numbering appearing in SEQ ID NO 1 and 3.

M982 Tbp2 B16B6 Tbp2 N-terminal domain or first domain 1-345 1-325 Hinge domain or second domain 346-543 326-442 C-terminal domain or third domain 544-691 443-579 This definition applies similarly to all M982 or B16B6 type Tbp2s, after alignment of an M982 or B16B6 type sequence with the reference sequence, to maximum homology. Thus, by way of example and by reference to Figure 1, the position is shown of the domains of the Tbp2 subunit of the strain 8680 as follows: first domain (1-334), second domain (335-530) and third domain (531- S677).

WO 97/13860 9 PCT/FR96/01580 It is now known that the N-terminal domain or first domain contains the transferrin binding site in its entirety and is hence very probably exposed to the outside. Consequently, the sole N-terminal domain constitutes a component of choice for i.a. vaccinal purposes.

Furthermore, the comparative study of the M982 and B16B6 Tbp2 sequences aligned to maximum homology has demonstrated four hypervariable portions localized within the hinge region (also called hinge domain, as appears in the table above) of M982 Tbp2, which portions are absent from the B16B6 Tbp2. These hypervariable portions exist in all the M982 type Tbp2s (Figure 2).

The B16B6 type strains lack them. In the text which follows, "M982 type hinge region" is understood to mean a hinge region which has these four hypervariable portions.

The study of the sequences has made it possible to change the definitions of the M982 and B16B6 types, originally provided in WO 93/6861. There is now agreement to define the types of strain on the basis of the presence or of the absence of the hypervariable regions and, consequently, on the basis of the size of the tbp2 gene: about 2.1 kb for the M982 type tbp2 and 1.8 kb for the B16B6 type tbp2.

By comparison of the sequences of various M982 type Tbp2s, it is also known that the variability of the hinge region is greater than that of the other two domains. Since the hinge region did not appear to be essential for the human transferrin binding function, it has been postulated that the role of this region is, at least in part, to induce a "screen"-type variability in WO 97/13860 10 PCT/FR96/01580 order to avoid recognition of a strain by the immune system of an individual who had subsequently been in contact with another strain.

On this assumption, the hinge region of the M982 type strains constitutes a major problem in the preparation of a vaccine. Indeed, if this region is immunodominant, during an immunization, the antibodies induced will be predominantly directed against this region and consequently the immune response will be specific for the Tbp2 protein of the homologous strain.

Taking into account the observations and assumptions mentioned above, it has already been proposed to use, i.a. for vaccinal purposes, not entire M982 type Tbp2s anymore, but Tbp2s at least deleted of their four hypervariable portions or, alternatively, deleted of the second and third domains.

A region essential for the induction of a broad spectrum immunity being probably the first domain, a pharmaceutical composition of choice appeared as being capable of being, for example, at least composed: of a polypeptide corresponding to an M982 type Tbp2 at least partially deleted of the second or third domain, e.g. of a polypeptide corresponding to the first domain of an M982 type Tbp2; and (ii) of a B16B6 type Tbp2 or of a polypeptide corresponding to a B16B6 type Tbp2, at least partially deleted of the second or third domain, e.g. of a polypeptide corresponding to the first domain of a B16B6 type Tbp2 (PCT application, filing No. PCT/FR95/000701; reference is made to WO 97/13860 11 PCT/FR96/01580 the initial definitions of the types of strain).

Subsequently, the study of the immunodominance of the hinge region of the M982 type Tbp2s was continued and completed by analysing, in relation to a large number of M982 type strains, the cross-reactivity of the sera obtained against: the entire M982 Tbp2 protein (100% Tbp2); (ii) the truncated M982 Tbp2 protein containing only the first 350 N-terminal amino acids (50% Tbp2); and (iii) the M982 Tbp2 protein deleted of the four hypervariable portions (A 362-379; A 418- 444; A 465-481; and A 500-520) of the hinge region (80% Tbp2).

To do this, the entire, truncated and deleted Tbp2 proteins were first produced in E. coli by the recombinant route. As regards the entire M982 Tbp2, its production is described in EPA 586,266 (published on the 9th March 1994). Those of the other two M982 Tbp2s are reported in Examples 4 and 6 of the present application.

The preparation of the hyperimmune sera as well as the analysis of these sera, prior to the study of immunodominance, is the subject of Example 8 of the present application. The study of cross-reactivity is detailed further in Example 9.

Fifty-four strains classified in the M982 type either by analysis of the outer membrane proteins by SDS-Page electrophoresis followed by immunorevealing, or on the basis of the size of the PCR-amplified tpb2 gene, were studied by dot blotting (analysis on whole microorganisms) for their cross-reactivity (see Example WO 97/13860 12 PCT/FR96/01580 9) These strains were obtained free of charge from Dr D.A. Caugant. They were isolated from various regions of the world and at designed times. Consequently, this collection should be representative. 14 strains are not recognized by .the anti-100% M982 Tbp2 serum, whereas only 4 escape recognition by the anti-80% M982 Tbp2 serum and 1 by the anti-50% M982 Tbp2 serum.

These results confirm the initial hypothesis according to which a Tbp2 protein, deleted of the hypervariable portions, induces a higher crossreactivity than that observed with an entire protein.

The cross-reactivity is again enhanced in the case of a Tbp2 protein which has conserved only the transferrinbinding domain since only one strain out of 54 is not recognized. The immunogenicity of a Tbp2 is influenced by the presence of the hinge region which induces antibodies specific for the homologous strain. By eliminating at least the four hypervariable portions, the immune response against epitopes situated outside the hinge region appears to be promoted.

Only 4 strains out of the 54 strains of the M982 type did not react with the anti-80% Tbp2 serum. This result may be explained especially by the fact that at least one of these strains is very different from the M982 reference strain. Consequently, it appears necessary to add at least a third valency to the pharmaceutical compositions already known.

Accordingly, the invention relates to a protein, in a substantially purified form, which is the subunit of lower molecular weight (Tbp2) of the human transferrin receptor (HTR) of an N. meningitidis strain, the strain being characterized in that it is not WO 97/13860 13 PCT/FR96/01580 recognized in dot blot by an antiserum obtained against a polypeptide corresponding to the Tbp2 of the N.

meningitidis M982 strain (M982 Tbp2) deleted of the hypervariable portions of its hinge region (M982 Tbp2 A 362-379; A 418-444; A 465-481; A 500-520), and the Tbp2 subunit being characterized in that it is encoded by a DNA fragment of about 2.1 kb.

In other words, since the Tbp2 subunit according to the invention is characterized in that it is encoded by a DNA fragment of about 2.1 kb, the N.

meningitidis strain from which it is derived is itself characterized in that its genome comprises an open reading frame (ORF) coding for the said Tbp2 subunit of about 2.1 kb.

Since the protein according to the invention is encoded by a DNA fragment of about 2.1 kb, it is quite obvious, taking into account what has been said above, that this protein has an M982 type hinge region.

The reactivity between an anti-80% Tbp2 serum and an anti-50% Tbp2 serum may also be explained by the fact that in the construct which served for the production of an anti-80% Tbp2 serum, some variable sequences remain whereas in the 50% proteins, all these sequences are eliminated. Indeed, only one strain among the 54 M982 type strains did not react with the Tbp2 serum. It is the strain 8680 B:15:P1.3, isolated in Chile during the 1987 epidemic and belonging to the electrophoretic group. This strain comes from Dr D.A.

Caugant's collection, WHO Collaborating Centre for Reference and Research on Meningococci, National Institute of Public Health, Oslo, Norway. The Tbp2 14 protein derived from this strain therefore constitutes most particularly a vaccinal candidate of choice.

Accordingly, a Tbp2 subunit according to the invention is derived advantageously from an N.

meningitidis strain which is not recognized in dot blot by an antiserum obtained against an M982 Tbp2 fragment extending from the amino acid at position 1 to the amino acid at position 350, that is to say against a polypeptide corresponding to the Tbp2 of the N.

meningitidis M982 strain substantially deleted of the second and third domains.

A Tbp2 subunit according to the invention is derived especially from an N. meningitidis strain whose Tbp2 subunit is recognized in Western blotting of membrane fractions by an antiserum obtained against the HTR of the M982 strain (anti-M982 HTR antiserum) and by an antiserum obtained against the HTR of the B1616 [sic] strain (anti-Bl6B6 HTR antiserum). Such antisera have been described in patent application WO 93/6861.

20 "Tbp2 subunit which is derived from an N.

meningitidis strain" is obviously understood to mean a derivative taken in its most general sense; that is to say not limited to a physical process, but the result of an intellectual process. Thus, this expression covers i.a. a Tbp2 produced by the recombinant route e.g. in E.

coli.

A Tbp2 subunit according to the invention comprises an amino acid sequence whose degree of homology (of identity) with the amino acid sequence of the M982 Tbp2, as shown in SEQ ID No. 2 is 60 to A advantageously about 65%. From another point of view, a Tbp2 subunit according to the invention comprises an 15 amino acid sequence whose degree of homology with the amino acid sequence of the 8680 Tbp2, as shown in SEQ ID No.6 from the amino acid at position 1 to the amino acid at position 677 is 80 to 100%, advantageously 90 to 100%, preferably 95 to 100%. According to a specific embodiment, it comprises an amino acid sequence substantially as shown in SEQ ID No. 5, starting with the amino acid residue at position 1 and ending with the amino acid residue at position 677.

A Tbp2 subunit according to the invention may be in a form dissociated from the high molecular weight subunit (Tbpl) of the N. meningitidis strain from which the Tbp2 is derived or alternatively in association with this Tbpl, thus forming a Tbpl-Tbp2 complex considered 15 to be the human transferrin receptor. Whether it is in a dissociated form or in the form of a Tbpl-Tbp2 complex, *the Tbp2 subunit according to the invention should be substantially purified, that is to say separated from the medium in which it exists naturally. This may be, 20 inter alia, a preparation especially free of H.pylori Scytoplasmic and periplasmic proteins.

The invention also relates to an isolated

DNA

fragment coding for a protein according to the invention, as well as for a precursor of this protein; the latter comprising a signal peptide homologous or heterologous to the protein according to the invention.

A DNA fragment coding for a protein according to the invention is described in SEQ ID NO 5 from the nucleic acid at position 30 to the nucleic acid at position 2061. A DNA fragment coding for a precursor of a protein i according to the invention is described in SEQ ID NO WO 97/13860 16 PCT/FR96/01580 from the nucleic acid at position 1 to the nucleic acid at position 2061.

A DNA fragment according to the invention may also comprise a nucleic acid sequence other than that shown in SEQ ID NO 5 provided that it can hybridize to the DNA fragment shown in SEQ ID NO 5, under conditions of high stringency.

Hybridization methods as well as the use of conditions of high stringency are within the capability of persons skilled in the art. For example, hybridization procedures are described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley Sons Inc., 1994; Silhavy et al., Experiments with Gene Fusions, Cold Spring Harbor Lab., 1984: Davis et al., A Manual for Genetic Engineering: Advanced Bacterial Genetics, CSH Lab., 1980. For the general procedure to be followed, important parameters are indicated which should be taken into account in order to optimize the hybridization conditions occur [sic] in the formula which makes it possible to calculate a critical value, the melting temperature above which two complementary strands separate (Casey Davidson, Nucl.

Acid Res. (1977) 4: 1539). This formula is as follows: Tm 81.5 0.5 X G+C) 1.6 log (positive ion concentration) 0.6 X formamide). Under conditions of high stringency, the hybridization temperature is about 20-25 0 C below the melting temperature as calculated. For example, conditions of high stringency are obtained by carrying out the prehybridization and hybridization incubations for 4 16 h, at about 55 0 C, in 6 x SSC solution (1 M NaC1, 0.1 M sodium Scitrate, pH WO 97/13860 17 PCT/FR96/01580 The invention also relates to a polypeptide derived especially by deletion of a Tbp2 subunit according to the invention, and (ii) an isolated DNA fragment coding for such a polypeptide.

This is more particularly a polypeptide having an amino acid sequence which is derived from that of a Tbp2 subunit according to the invention whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the M982 reference strain, especially by total or partial deletion of at least one domain of the said Tbp2 subunit according to the invention, provided that the first and second domains are not simultaneously and totally deleted.

"Sequence which is derived from another sequence" is obviously understood to mean a sequence originating from this other sequence by an intellectual process.

From another point of view, it is a polypeptide capable of binding to human transferrin and derived-from a Tbp2 subunit according to the invention, especially by deletion of one or more amino acids localized on the side of the C-terminal end or in the region of the first forty amino acids of the Tbp2 subunit. The C-terminal end is defined as being the Tbp2 part corresponding to the second and third domains.

More especially, a polypeptide according to the invention possesses an amino acid sequence which is derived from a Tbp2 subunit according to the invention: in particular by total or partial deletion of at least one domain of the said Tbp2 7s. subunit, selected from the second and third WO 97/13860 18 PCT/FR96/01580 domains; preferably by total or partial deletion of the third domain or of the second and third domains; (ii) in particular by total deletion of the first and third domains, or (iii)in particular by complete deletion of the third domain and by partial deletion of the first domain, optionally by partial deletion of the second domain.

Advantageously, a polypeptide according to the invention has a partial, virtually total or total deletion of the third domain, preferably a total deletion.

In this case, the first and also the second domain may be maintained in their entirety, partially or totally deleted, independently of one another.

The following combinations are possible (given that the first, second and third domains in their entirety are represented by 1, 2 and 3, respectively, and that O and A mean partially and totally deleted, respectively): 1, 2, A3; 1, 02, A3; 1, A2, A3; 01, 2, A3; 01, 02, A3; 01, A2, A3; Al, 2, A3; Al, 02, A3; 1, 2, 03; 1, 02, 03; 1, A2, 03; 01, 2, 03; 01, 02, 03; 01, A2, 03; Al, 2, 03; Al, 02, 03; Interest also attaches in particular to a polypeptide derived from a Tbp2 subunit according to the invention by partial deletion of the second domain, WO 97/13860 19 PCT/FR96/01580 which contains in their entirety or virtual entirety the first and third domains; that is to say the combination i, 02, 3. ("Domain maintained in its virtual entirety" is understood here and in the text which follows to mean a domain modified at a very small number of positions, not more than 5 approximately.) A polypeptide according to the invention can also correspond to the combination 01, 02, 3, the partial deletion of the first domain advantageously affecting all or part of the region homologous to that of M982 Tbp2 extending from the amino acid at position 1 to the amino acid approximately at position Advantageously, when a polypeptide is derived especially by partial deletion of the second domain of a Tbp2 subunit according to the invention, this partial deletion substantially affects one or more regions of the second domain which are the homologues of the regions of the M982 sequence extending: from the amino acid at position 388 to the amino acid at position 396; (ii) from the amino acid at position 418 to the amino acid at position 476; and (iii)from the amino acid at position 499 to the amino acid at position 521.

Alternatively, a partial deletion of the second domain substantially affects one or more hypervariable portions. These portions are, after alignment to maximum homology, the homologues of the portions of the M982 sequence extending: from the amino acid at position 362 to the amino acid at position 379; WO 97/13860 20 PCT/FR96/01580 (ii) from the amino acid at position 418 to the amino acid at position 444; (iii)from the amino acid at position 465 to the amino acid at position 481; and (iv) from the amino acid at position 500 to the amino acid at position 520.

Preferably, the partial deletion substantially affects the four portions to (iv) described above.

When a polypeptide according to the invention is derived, especially by complete deletion of the third domain and a virtually complete deletion of the second domain of a Tbp2 subunit according to the invention and comprises the entire first domain, or is derived, in addition, by deletion of the N-terminal part of the first domain, the virtually complete deletion of the second domain extends over the region which is the homologue of the region of the second domain of the M982 Tbp2 subunit extending from the amino acid at one of the positions 346 to 361 to the amino acid at position 543.

When a polypeptide is derived especially by partial deletion of the first domain of a Tbp2 subunit according to the invention, this partial deletion advantageously affects all or part of the region which is the homologue of the region of the first domain of the M982 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid at position 281.

By way of example of the above, there may be mentioned a deletion of interest affecting all or part of the region which is the homologue of the region of the first domain of the said M982 type Tbp2 subunit extending from the amino acid at position 1 to the amino 7Xacid approximately at position WO 97/13860 21 PCT/PR96/01580 The degree of homology may be readily calculated by aligning the sequences so as to obtain the maximum degree of homology; to this end, it may be necessary to introduce vacant positions artificially, as illustrated in Figure i. Once optimum alignment is achieved, the degree of homology is established by reckoning up all the positions in which the amino acids of the two sequences turn out to be identical, relative to the total number of positions.

It would be tedious to describe homologous sequences other than in a generic manner, on account of the excessively large number of combinations. A person skilled in the art knows, however, the general rules which enable one amino acid to be replaced by another without abolishing the biological or immunological function of a protein.

By way of a preferred example, there may be mentioned a polypeptide according to the invention whose sequence possesses at least 70-75%, advantageously at least 80%, preferably at least 90%, and, as an absolute preference, 100%, homology with: the sequence as shown in SEQ ID NO 5, from the amino acid at position 1 to the amino acid at one of the positions 334 to 351, advantageously at position at position [sic] 334; (ii) the sequence as shown in SEQ ID NO 5, from the amino acid at position 1 to the amino acid at position 677, deleted of the 377- 385, 407-465 and 488-508 regions; (iii)the sequence as shown in SEQ ID NO 5, from the amino -acid at position 1 to the amino WO 97/13860 22 PCT/FR96/01580 acid at position 677, deleted of the 351- 368, 407-433, 454-470 and 489-508 portions, or with (iv) the sequence as shown in SEQ ID NO 5, from the amino acid at position 335 to the amino acid at position 530.

A polypeptide according to the invention possesses an amino acid sequence which comprises at least 10, advantageously at least 20, preferably at least 50, most preferably at least 100 amino acids.

Quite obviously, a polypeptide according to the invention may also comprise, additionally, an amino acid sequence which does not exhibit homology with the sequences of the Tbp2 subunits according to the invention.

Generally speaking, an additional sequence can be that of any other polypeptide excluding Tbp2.

For example, an additional sequence may be that of a signal peptide localized at the N-terminal position of a polypeptide according to the invention. Examples of a signal sequence are shown in SEQ ID NO 1 to 4.

Furthermore, it may be pointed out that an appropriate heterologous signal sequence may be a signal sequence found in the precursor of a lipoprotein.

A Tbp2 subunit according to the invention may be directly purified from a native strain or, advantageously, may be obtained by the recombinant route in a heterologous or homologous expression system. The polypeptides according to the invention are preferably recombinant products. In order to use an expression by the recombinant route, the DNA fragment coding for the NX Tbp2 of the strain 8680-was cloned and sequenced.

WO 97/13860 23 PCT/FR96/01580 The subject of the invention is also: an isolated DNA fragment coding for a Tbp2 subunit or a polypeptide according to the invention; (ii) an expression cassette which comprises at least one DNA fragment according to the invention, placed under the control of elements capable of providing for its expression in an appropriate host cell, and (iii)a method of production of a polypeptide according to the invention, according to which a host cell containing an expression cassette according to the invention is cultured.

In the expression cassette, the DNA fragment according to the invention may be combined or otherwise with a DNA block coding for a signal peptide which is heterologous or otherwise to the polypeptide encoded by the said DNA fragment, depending on whether or not secretion of the polypeptide is sought. Preferably, this secretion will be sought.

Elements such as a DNA block coding for a heterologous signal peptide (signal region) or a promoter already exist in quite large numbers, and are known to persons skilled in the art. Their general competence will enable them to choose a particular signal region or promoter which will be suited to the host cell inwhich the expression is envisaged.

For the purposes of the method according to the invention, the host cell may be a mammalian cell, a bacterium or a yeast, the latter two being preferred.

WO 97/13860 24 PCT/FR96/01580 Here too, the choice of a particular line is within the capacity of persons skilled in the art.

Lastly, the invention also relates to a pharmaceutical composition comprising, as active principle, a Tbp2 subunit or a polypeptide according to the invention.

A pharmaceutical composition according to the invention is especially useful for inducing an immune response in humans against N. meningitidis, inter alia a vaccinal effect so as to protect humans against N.

meningitidis infections, in prevention or in therapy.

A composition according to the invention may comprise in addition: an additional Tbp2 subunit which is derived from an N. meningitidis strain whose Tbp2 subunit is recognized in Western blotting of membrane fractions by an anti-M982 HTR antiserum and is not recognized by an anti-B16B6 HTR antiserum, or (ii) a polypeptide capable of binding human transferrin and derived from a Tbp2 subunit of an N. meningitidis strain whose Tbp2 is recognized in Western blotting of membrane fractions by an anti-M982 HTR antiserum and is not recognized by an anti-Bl6B6 HTR antiserum, especially by deletion of one or more amino acids localized on the side of the C-terminal end of the Tbp2 subunit, or (iii) an additional Tbp2 subunit which is derived from an N. meningitidis strain whose Tbp2 subunit is not recognized in Western blotting of membrane WO 97/13860 25 PCT/FR96/01580 fractions by an anti-M982 HTR antiserum and is recognized by an anti-B16B6 HTR antiserum, or (iv) a polypeptide capable of binding human transferrin and derived from a Tbp2 subunit of N. meningitidis whose Tbp2 subunit is not recognized in Western blotting of membrane fractions by an anti-M982 HTR antiserum and is recognized by an anti-B16B6 HTR antiserum, especially by deletion of one or more amino acids localized on the side of the C-terminal end of the Tbp2 subunit.

A composition according to the invention advantageously comprises at least one, preferably two additional elements chosen from the four listed above.

The additional Tbp2 subunit of the M982 type advantageously comprises an amino acid sequence whose degree of homology with the amino acid sequence of the M982 Tbp2, as shown in SEQ ID No. 1, is 90 to 100%. It is preferably the M982 Tbp2 subunit.

The additional Tbp2 subunit of the B16B6 type advantageously comprises an amino acid sequence whose degree of homology with the amino acid sequence of the B16B6 Tbp2, as shown in SEQ ID No. 3, is 90 to 100%. It is preferably the B16B6 Tbp2 subunit.

Preferably, the additional polypeptide (ii) is derived from a Tbp2 subunit of an N. meningitidis strain whose Tbp2 is recognized in Western blotting of membrane fractions by an anti-M982 HTR antiserum and is not recognized by an anti-B16B6 HTR antiserum, especially by partial deletion of the second domain, inter alia by substantial deletion of the hypervariable regions of the second domain of the -Tbp2 subunit or by substantial WO 97/13860 26 PCT/FR96/01580 deletion of the second or third domain of the Tbp2 subunit.

Advantageously, this polypeptide is derived from a Tbp2 subunit which comprises an amino acid sequence whose degree of homology with the amino acid sequence of the M982 Tbp2, as shown in SEQ ID No. 1, is 90 to 100%.

Preferably, it is a polypeptide which is derived from the M982 Tbp2.

Preferably, the additional polypeptide (iv) is derived from a Tbp2 subunit of an N. meningitidis strain whose Tbp2 is not recognized in Western blotting of membrane fractions by an anti-M982 HTR antiserum and is recognized by an anti-B16B6 HTR antiserum, especially by substantial deletion of the second or third domain of the Tbp2 subunit, preferably of the second and third domains.

Advantageously, this polypeptide is derived from a Tbp2 subunit which comprises an amino acid sequence whose degree of homology with the amino acid sequence of the M982 Tbp2, as shown in SEQ ID No. 3, is 95 to 100%.

Preferably, it is a polypeptide which is derived from the B16B6 Tbp2.

According to a preferred embodiment, a composition according to the invention comprises: a polypeptide which is derived from a Tbp2 subunit according to the invention, especially by partial deletion of the second domain of the Tbp2 subunit, inter alia by substantial deletion of the hypervariable portions of the second domain of the Tbp2 subunit; (ii) a polypeptide which is derived from a Tbp2 Ssubunit of an N. meningitidis strain whose Tbp2 WO 97/13860 27 PCT/FR96/01580 is recognized in Western blotting of membrane fractions by an anti-M982 HTR antiserum and is not recognized by an anti-B16B6 HTR antiserum, especially by partial deletion of the second domain, inter alia by substantial deletion of the hypervariable portions of the second domain of the Tbp2 subunit, and (iii) a Tbp2 subunit which is derived from an N.

meningitidis strain whose Tbp2 subunit is not recognized in Western blotting of membrane fractions by an anti-M982 HTR antiserum and is recognized by an anti-B16B6 HTR antiserum.

A pharmaceutical composition according to the invention may be manufactured in a conventional manner.

In particular, the polypeptide(s) according to the invention is/are combined with an adjuvant, a diluent or a vehicle which is acceptable from a pharmaceutical standpoint. A composition according to the invention may be administered by any conventional route in use in the vaccine field, in particular subcutaneously, intramuscularly or intravenously, for example in the form of an injectable suspension. The administration can take place in a single dose or a dose repeated one or several times after a certain time interval. The appropriate dosage varies in accordance with various parameters, for example the individual being treated or the mode of administration.

In order to determine the subject of the present invention, it may be pointed out that the N.

meningitidis strains IMP2394 (M982) and IM2169 (B16B6) are available to the public from the Collection Nationale de Culture des Microorganismes (CNCM) Institut 28 Pasteur, 25 rue du Dr Roux 75015 Paris under the respective registration numbers LNP N 1511 and LNP N 1520.

The invention is described in greater detail in the examples below Pnd by reference to Figures 1 to Figure 1A and 1B presents, respectively, the alignments of the M982 and 8680 Tbp2 sequences to maximum homology according to the Kanehisa programme (Bisance CITI-2) The degree of homology is Figure 2A and 2B presents the alignments to maximum homology of the sequences of the hinge domains (second domain) of M982 6940 2223 C708 M978 1610 867 S3032 and 981 Tbp2. The numbering of the M982 Tbp2 sequence, as it appears in 15 SEQ ID NO 1, is given in italics. The sequences of the hypervariable portions which may be deleted according to a preferred embodiment appear in bold type.

(C)

indicates the consensus sequence.

Figures 3 to 5 illustrate, respectively, the 20 construction of the plasmids pTG5782, pTG5755 and pTG5783.

EXAMPLE 1: Purification of the HTR of the

N.

meningitidis 8680 strain .5 1A Culture A frozen sample of the N. meningitidis 8680 strain is taken up in about 1 ml of Muller Hinton broth (BMH, Difco). The bacterial suspension is then spread on the solid Muller Hinton medium containing cooked blood After 24 h of incubation at 37 0 C under an Satmosphere containing 10% CO 2 the bacterial layer is recovered in order to inoculate 150 ml of BMH pH 7.2, WO 97/13860 29 PCT/FR96/01580 distributed in 3 Erlenmeyer flasks of 250 ml. The incubation is continued for 3 h at 37 0 C, with stirring.

Each of the 3 cultures thus prepared makes it possible to inoculate 400 ml of BMH pH 7.2 supplemented with 30 |m of Ethylenediamine-Di(O-Hydroxyphenylacetic acid (EDDA, Sigma), which is an iron-chelating agent in free form.

After 16 h of culture at 37 0 C, with stirring, the cultures are checked for their purity by observing under a microscope after Gram staining. The suspension is centrifuged, the pellet containing the microorganisms is weighed and stored at -20 0

C.

1B Purification The method of purification is essentially that described by Schryvers et al (supra).

The bacterial pellet obtained in 1A is thawed and then resuspended in 200 ml of 50 mM Tris HC1 buffer, pH 8.0 (buffer The suspension is centrifuged for min at 15,000 xg at 40C. The pellet is recovered and then resuspended in buffer A at the final concentration of 150 g/l. Fractions of 150 ml are treated for 8 min at 800 bar in a cell lyser working at high pressure (Rannie, model 8.30H). The cell lysate thus obtained is centrifuged for 15 min at 4 0 C at 15,000 x g. The supernatant is recovered and then centrifuged for 75 min at 4°C at 200,000 x g. After removal of the supernatant, the pellet is taken up in buffer A and after assaying the proteins according to Lowry, the concentration of the suspension is adjusted to 5 mg/ml.

1.75 mg of biotinylated human transferrin are added to 1.4 ml of the membrane suspension according to IA the process described by Schryvers. The final WO 97/13860 30 PCT/FR96/01580 concentration of the membrane fraction is 4 mg/ml. The mixture is incubated for 1 hour at 37 0 C and then centrifuged at 100,000 xg for 75 min at 40C. The membrane pellet is taken up in buffer A containing 0.1 M NaCi and incubated for 60 min at room temperature.

After solubilization, a certain volume of N Lauroyl Sarkosine and 500 mM EDTA is added to this suspension so that the final Sarkosyl and EDTA concentrations are 0.5% and 5 mM respectively. After an incubation of 15 min at 37 0 C, with stirring, 1 ml of streptavidin-agarose resin (Pierce), previously washed with buffer A, is added. The suspension is incubated for min at room temperature and then centrifuged at i000 x g for 10 min. The resin is then conditioned in a column and the eluate is eliminated.

The resin is washed with 3 column volumes of mM Tris-HCl buffer pH 8.0 containing I [sic] M NaC1, mM EDTA 0.5% Sarkosyl (buffer B) and then with one column volume of buffer B containing 750 mM guanidine HC1. The transferrin receptor is then eluted with buffer B containing 2 M guanidine HC1. The eluate is collected in fractions whose volumes correspond to IV, in tubes containing IV of 50 mM Tris HC1 pH 8.0, I [sic] M NaC1.

The optical density at 280 nm of the eluate is measured at the outlet of the column with the aid of a UV detector.

The fractions corresponding to the elution peak are recovered, dialysed against 10 mM phosphate buffer, pH 8.0 containing 0.05% Sarkosyl and lyophilized. The lyophilized product is taken up in water at a concentration 10 times higher. The solution is dialysed RA a second time against- 50 mM phosphate buffer pH WO 97/13860 31 PCT/FR96/01580 containing 0.05% Sarkosyl (buffer C) and then the solution is filtered on a membrane of porosity 0.22 rm.

The protein content is determined and adjusted to 1 mg/ml by addition of buffer C, under aseptic conditions. This preparation is thawed at EXAMPLE 2: Purification of the Tbp2 subunit of the N.

meningitidis 8680 strain by hydrophobic chromatography.

The culture of the N. meningitidis 8640 strain, as well as the purification steps up to the preparation of the membrane suspension are carried out under conditions identical to those described in Example 1.

To one volume of the membrane suspension, there is added an identical volume of 50 mM Tris-HCl pH containing 2 M NaCl, 20 mM EDTA, 1% Sarkosyl. The mixture is incubated for 15 min at 37°C, with gentle stirring. One volume of this suspension is then brought into contact with an identical volume of Sepharose 4B resin coupled to human transferrin. This affinity resin was coupled by grafting human transferrin (Sigma, St Louis USA) to Sepharose 4B-CNBr (Pharmacia) according to the recommendations of the manufacturer. The density of the ligand and [sic] of 5 mg transferrin/ml of resin.

The contact is made in a bath for 1 h at room temperature, with gentle rotary stirring. The resin is then conditioned in a column, the direct eluate is eliminated.

The resin is washed with 3 column volumes of mM Tris-HCl buffer pH 8.0 containing 1 M NaCl, 10 mM EDTA 0.5% Sarkosyl (buffer B) and then with one column volume of buffer B containing 750 mM guanidine HC1. The transferrin receptor is then eluted with the 50 mM Tris- WO 97/13860 32 PCT/FR96/01580 HC1 buffer pH 8.0, 1 M NaCi 10 mM EDTA 0.05% Sarkosyl and 2 M guanidine HC1. The optical density at 280 nm of the eluate is measured at the outlet of the column with the aid of a UV detector. The fractions corresponding to the elution peak are combined and the protein is precipitated by addition of three volumes of cooled ethanol.

After incubating overnight at the protein is recovered by centrifugation for one hour at 10,000 x g. The precipitate is taken up with a certain volume of 10 mM phosphate buffer pH 7.0 containing 0.5 M NaCl, 5 M guanidine HC1 (buffer D) so that the final protein concentration is about 1 mg/ml. The solution is brought into contact with the phenyl-Sepharose resin (Pharmacia) previously equilibrated with the same buffer. The incubation is carried out in a bath with rotary stirring for 2 hours at room temperature. The gel is then conditioned in a column.

Under these conditions, the high molecular weight subunit (Tbpl) is recovered in the direct eluate, whereas the lower molecular weight subunit (Tbp2) is bound to the resin. The column is rinsed with three volumes of buffer D and then with 5 volumes of 10 mM phosphate buffer pH 7.0. Tbp2 is eluted with the 10 mM phosphate buffer pH 7.0 containing 0.5% Sarkosyl. The excess Sarkosyl contained in the Tbp2-elution buffer is eliminated by ethanol precipitation, the protein is then taken up in 50 mM phosphate buffer pH 8.0 containing 0.05% Sarkosyl (buffer C).

The solution is then filtered on a membrane of porosity 0.22 pm. The protein content is determined and WO 97/13860 33 PCT/FR96/01580 adjusted to 1 mg/ml by addition of buffer C, under aseptic conditions. This preparation is thawed at -700C.

EXAMPLE 3: Cloning of the DNA Fragment coding for 8680 Tbp2 DNA is extracted by a conventional phenol chloroform method. For a quantity of microorganisms corresponding to 1 to 2 g of wet weight, the pellet is taken up in 25 ml of lysis solution (50 mM glucose, mM EDTA, 25 mM Tris pH 8.0) supplemented with 1 ml of proteinase K (Sigma) at 10 mg/ml. The mixture is incubated for 10 min at room temperature and then 0.5 ml of 20% sarkosyl is added. This is followed by incubation for 10 min at 4 0 C. After passing the suspension through an 18G needle, 1 ml of RNAse A (Sigma) at 2 mg/ml is added and then the suspension is incubated at 37 0 C for min. At the end of this stage, the DNA is extracted by addition of 0.5 volume of phenol. The extraction is achieved by stirring for 10 min, and 0.5 volume of chloroform-isoamyl alcohol (24:1) is then added. After centrifugation of the mixture at 5000 revolutions/min, for 15 min, the supernatant is collected and is again treated with phenol/chloroform. The phenol/chloroform extraction step is repeated until the aqueous phase becomes clear.

At the end of the extraction, the DNA is precipitated with 2 volumes of absolute ethanol in the presence of 0.3 M sodium acetate pH 7 and then rinsed with 70% ethanol. The DNA is taken up in 1 ml of distilled water and then assayed in a spectrophotometer at 260 nm and at 280 nm. One OD 260 nm unit corresponds to 50 ng of DNA/Il. The DNA preparations are considered WO 97/13860 34 PCT/FR96/01580 to be satisfactory and utilizable if the OD 260/OD 280 ratio is between 1.8 and 2.

From 10 ng of genomic DNA, the tbp2 gene is amplified by PCR with the aid of the following primers: 5' Interl Bam: 5'-CCACGGATCCTGCCGTCTGAAGCCTTATTC-3' 3' Met 2 Eco: 5'-CGCGGATCCTGCTATGGTG-3' The reaction medium is as follows: 10 ng of genomic DNA; 0.5 pM of 5' and 3' primers; 200 pM of dNTPs (Boehringer); 10 .1 of 10x PCR buffer (Biotaq) and 2.5 U of Taq polymerase (Biotaq). The reaction volume is adjusted to 100 il with distilled water. The amplification conditions in a Trioblock apparatus (Biolabs) are the following: denaturation: 940C, 1 min; annealing: 580C, 2 min; extension: 720C, 3 min; number of cycles: The DNA fragment amplified by PCR is subjected to electrophoresis on 1% preparative agarose gel ("Electrophoresis grade", BRL) is [sic] prepared in TAE buffer (0.04 M Tris acetate, 0.002 M EDTA pH The amplified band corresponding to the tbp2 gene is cut out. The DNA contained in the agarose is purified with the aid of a Geneclean kit (Bio 101). The DNA is then digested with EcoRI and BamHI for 2 h at 37°C. The digested DNA is then repurified by phenol/chloroform extraction, precipitated with 2 volumes of ethanol and then taken up in 20 .l of Tris-EDTA (TE) buffer (10 mM Tris-HCL, 1 mM EDTA pH The vector pBSK (Stratagene) is digested with EcoRI and BamHI and then purified as described above for the EcoRI/BamHI insert.

The EcoRI/BamHI insert and pBSK are ligated in 1A the presence of T4 ligase (Boehringer) at 160C overnight WO 97/13860 35 PCT/FR96/01580 under the following conditions: vector 100 ng; insert 200-300 ng; 10X T4 ligase buffer (Boehringer) 1 p1; ligase 5U; water qs 10 pl.

E. coli XL1-Blue (recAl, endAl, gyrA96, thi-1, hsdR17, supE44, relAl, lac, proAB, lacqZ6M15, Tn1O (tetr)) is precultured in 10 ml of SB medium (Difco) in the presence of tetracycline at 10 pg/ml overnight at 37 0 C. 500 lI of preculture are used to inoculate 1 1 of SB medium. When the culture reaches the exponential phase (OD 600 nm the microorganisms are washed three times in 10% glycerol in water, reducing the wash volumes from 500 ml to 120 ml. The supernatant is eliminated after centrifugation at 2500 x g for 15 min at 4°C. After the last wash, the bacteria are taken up in 3 ml of 10% glycerol, 1 mM Hepes pH 7.5. The microorganisms are aliquoted in 100 pi in tubes placed in liquid nitrogen. The electrocompetent bacteria are stored for one month at -70 0

C.

Five p of the ligation reaction are used to transform 100 p4 of E.coli XL-1 bacteria by electroporation in cuvettes 2 mm thick (Eurogentec) under a voltage of 2500 volts. After electroporation, the microorganisms are incubated for 1 hour at 37 0 C and then 1/10 and 9/10 of the volume of the preparation are spread on plates of LB medium (Difco) plus ampicillin (100 jg/ml) supplemented with X-gal (25 4g/ml) and IPTG jg/ml) allowing a white/blue selection of the recombinant clones.

The recombinant clones (white) are analysed after mini-preparation of plasmid according to a conventional method (Maniatis et al., 1989) by double EcoR1 [sic] BamHI digestion. The clones selected are WO 97/13860 36 PCT/FR96/01580 those which have integrated a 2.1 kb fragment. The direction of integration of the insert is determined after digestion with HincII. The plasmid DNA of the clones of interest is prepared in a large quantity by a maxi-preparation using a method of purification on a tip-250 column (Kit Quiagen).

The column-purified plasmid DNA (Quiagen) is sequenced using the Sequenase kit (USB, version This technique is derived from the sequencing method described by Sanger (Sanger et al., 1977). Three to five ug of DNA are used per sequence reaction. The sequence reactions are loaded onto a 6% acrylamide gel in the presence of 8 M urea. The nucleotide sequence revealed and the deduced amino acid sequence are those shown in SEQ ID No. EXAMPLE 4: Polypeptide T/2169 02, A3; 1-350) including the sequence as shown in SEQ ID NO 1 (IM2169), from the amino acid at position 1 to the amino acid at position 350 4A Preparation of the DNA fragment coding for T/2169 (1-350): Construction of the vector pTG 5782.

From the plasmid pTG3721 described in application EPA 586,266, a HindIII restriction site is introduced, by site-directed mutagenesis, downstream of the sequence coding for Tbp2, in order to generate the plasmid pTG4704.

From the plasmid pTG3721, a fragment comprising the sequence coding for the signal for secretion of RlpB and the beginning of the sequence coding for mature Tbp2 WO 9 7/13860 37 PCT/FR96/01580 up to the internal HaeII site is amplified by PCR with the aid of the primers OTG4915 and 0TG4651.

OTG4 915 AAACCCGGATCCGTTGCqAG-CGCTGCCGT HaeII OTG4651 EspHI TTTTTCATGAGA TAT CTG GCA ACA TrG TTG TTA TCT CTG Met Arg Tyr Leu Ala Thr Leu Leu Leu Ser Leu GCG GTG TTA ATC ACC GCC GGG TGC CTG GGT GGC Ala Val Leu Ile Thr Ala Gly Cys Leu Gly -cleavage of the signal peptide GGC GGC ACT TTC The PCR fragment is then digested with BspHI and HaeII and inserted simultaneously with the Hae II-HindIII fragment of pTG4704, which comprises the 3' part of the region coding for Tbp2, into the plasmid pTG3704 described in application EPA 586,-266, digested with NcoI and Hind!II, in order to generate the plasmid pTG5768.

From the plasmid pTG3721, a fragment comprising the sequence coding for the N-terminal part of Tbp2 is amplified by PCR with the aid of the primers OTG4928 and 11.

SphI OTG4928 GTG TTT TTG TTG AGT GCA TOC CTG GGT GCC Val Phe Leu Leu Ser Ala Cys Leu Gly Gly _Cleavage of the signal peptide OTG 5011 TGCGCAAGCTTACAG TTT GTCTTTGGT TTT CGCGCTGCCG HindIII WO 97/13860 38 PCT/FR96/01580 This PCR fragment is digested with SphI and HindIII and then cloned into the plasmid pTG4710 described in application EPA 586,266; the plasmid pTG5740 is thus generated.

The HaeII-HindIII fragment of pTG5740 comprises the 3' part of the sequence coding for the human transferrin binding domain (hTf) of the region coding for the first domain) is inserted into the plasmid pTG3704 digested with BamHI and HindIII, simultaneously with the BamHI-HaeII fragment of pTG5768 comprising the araB promoter, the signal sequence rlpB and the beginning of the coding sequence of Tbp2; the plasmid pTG5782 is thus generated. This vector comprises the araB promoter, the sequence coding for the signal for secretion of RlpB fused to the sequence coding for the N-terminal domain of Tbp2 (1-350).

4B Production and purification of T/2169 (1-350) An E. coli strain is transformed by pTG5782. The transformants are cultured at 37 0 C in M9 medium succinate 0.5% arginine 50g/ml ampicillin 100 pg/ml. In the exponential phase, 0.2% of arabinose (inducer) is added. After one hour of induction, the cells are collected and extracts are prepared. A Western Blot analysis followed by revealing with hTF-peroxidase makes it possible to detect a predominant band whose MW corresponds to that expected for this truncated form of Tbp2.

In a test as described in Example 4 of W093/6861 (published: 15.04.93), purified T/2169 proves capable of inducing bactericidal antibodies, and consequently should be useful for vaccinal purposes.

.n a A WO 97/13860 39 PCT/FR96/01580 EXAMPLE 5: Polypeptide T/2394 02, A3; 1-340) including the sequence as shown in SEQ ID NO 2 (IM2394), from the amino acid at position 1 to the amino acid at position 340.

Preparation of the DNA fragment coding for T/2394 (1-340): Construction of the vector pTG 5755 From the plasmid pTG4710 described in application EPA 586,266, a fragment comprising the region coding for the C-terminal part of the hTf binding domain is amplified by PCR with the aid of the primers OTG4873 and OTG4877. This fragment is then digested with MluI and HindIII.

OTG4873 AAAAAGCATGCATAAAAACTACGCGTTACACCATTCAAGC Ml u OTG4877 :TATATAAGCTTACGTTGCAGGCCCTGCCGCGTTTTCCCC HindIII The plasmid pTG4710 is digested with MluI and HindIII. The MluI-HindIII fragment comprising the 3' part of the sequence coding for Tbp2 is replaced by the PCR fragment coding for the C-terminal part of the hTf binding domain. The plasmid pTG5707 is thus generated. A BamHI-MluI fragment comprising an araB promoter and the beginning of the sequence coding for Tbp2 is then replaced, in the plasmid pTG5707, by a BamHI-MluI fragment of pTG4764 described in application

EPA

586,266, which contains the araB promoter, the sequence coding for the signal for secretion RIpB fused to the sequence coding for the N-terminal domain of Tbp2. The plasmid pTG5755 is thus generated. This vector comprises S the araB promoter, and the sequence coding for the WO 97/13860 40 PCT/FR96/01580 signal for secretion of R1pB fused to the sequence coding for the N-terminal domain of Tbp2 (1-340).

Production and purification of T/2394 (1-340) T/2394 (1-340) is produced and purified as described in Example 4B.

In a test as described in Example 4 of WO93/6861 (published: 15.04.93), purified T/2394 proves capable of inducing bactericidal antibodies, and consequently should be useful for vaccinal purposes.

EXAMPLE 6: Polypeptide D4/2169 02, 3) whose sequence is identical to that shown in SEQ ID NO 1, from the amino acid at position 1 to the amino acid at position 691, deleted of the regions 362-379, 418-444, 465-481 and 500-520.

Polypeptide D4/2169 02, 3) whose sequence is identical to that shown in SEQ ID NO 1, from the amino acid at position 1 to the amino acid at position 691, deleted of the regions 362-379, 418-444, 465-481 and 500-520.

6A Preparation of the DNA fragment coding for D4/2169 1.1. Cloning of the DNA fragment.

The DNA fragment coding for the Tbp2 subunit of the N. meningitidis IM2169 strain is amplified by PCR (Polymerase chain reaction) with the aid of specific primers complementary to the 5' and 3' regions, (A5' and A3', respectively), on 10 ng of genomic DNA extracted from a culture of bacteria of the strain IM2169.

WO 97/13860 41 PCT/FR96/01580 5' CCCGAATTCTGCCGTCTGAAGCCTTATTC 3' A3' 5' CCCGAATTCTGCTATGGTGCTGCCTGTG 3' A DNA fragment is thus obtained and after digestion with EcoRI, it has 2150 nt. This EcoRI fragment is then ligated to the dephosphorylated EcoRI ends of the phagemid pBluescriptSK(-) (Stratagene) to give the recombinant phagemid pSK/2169tbp2.

1.2. Carrying out the deletions.

The clone pSK/2169tbp2 containing the tbp2 sequences of the strain M982 is deleted by -the Kunkel technique, PNAS (1985) 82: 448.

In short, the phage form of the recombinant phagemid pSK/2169tbp2 is obtained after rescuing by the "helper" phage VCS M13 according to the technique described by Stratagene, supplier of the parent vector, and used to infect the bacterial strain CJ236. The dut and ung mutations carried by the CJ236 strain have, as consequence, the synthesis of DNA molecules having incorporated the nucleotide precursor dUTP.

The phages are recovered and the single-stranded DNA is extracted with a phenol/chloroform mixture.

This DNA is hybridized under conventional conditions, to the following oligonucleotides: 2169d1 5' CGCATCCAAAACCGTACCTGTGCTGCCTGA 3' 2169d2 5' TTTATCACTTTCCGGGGGCAGGAGCGGAAT 3' 2169d3 5' GTTGGAACAGCAGACAGCGGTTTGCGCCCC 3' 2169d4 5' GAACATACTTTGTTCGTTTTTGCGCGTCAA 3' WO 97/13860 42 PCT/FR96/01580 The hybridization reaction is continued for min in decreasing temperature from 70 0 C to 300C.

The second complementary strand is then completed by total synthesis in the presence of the four deoxynucleotides, T4 DNA polymerase and T4 DNA ligase, under conventional conditions.

The strain E. coli SURE (Stratagene) is transformed with the DNA thus obtained. In this strain, the molecules carrying dUTP, that is to say not mutated, are destroyed.

The phages obtained are analysed by the conventional techniques of rapid preparation of plasmid DNA and digestion with the appropriate restriction enzymes. The presence of the desired mutation is then verified by nucleotide sequencing.

The clone pSK2169#7, carrying the four mutations A 1203-1256, A 1371-1451, A 1512-1562 and A 1617- 1679, is selected.

6B Construction of the expression vector pTG5783 The plasmid pTG5768 described above is digested with HpaI and XcmI. An XcmI-XcmI fragment of pTG5768 and the HpaI-XcmI fragment of the plasmid pSK/2169ed#7 are simultaneously inserted into this vector in order to generate the plasmid pTG5783. This vector comprises the araB promoter, and the sequence coding for the signal for secretion of RlpB fused to the modified tbp2 sequence (deletions dl to d4).

6C Preparation and purification of D4/2169.

D4/2169 is produced and purified according to Example 4B.

In a test as described in Example 4 of W093/6861 (published: 15.04.93),- purified D4/2169 proved capable WO 97/13860 43 PCT/FR96/01580 of inducing bactericidal antibodies and consequently should be useful for vaccinal purposes.

EXAMPLE 7: Determination of the level of expression of the HTRs of the N. meningitidis strains The level of expression of the HTR protein by N.

meningitidis is determined by measuring the capacity of a defined number of microorganisms to bind hTf coupled to peroxidase.

A preculture of 18 hours at 37 0 C on MHA serves to inoculate an Erlenmeyer flask containing 50 ml of MHB supplemented with 30 iM EDDA. The optical density is measured at 600 nm at the beginning of the culture and after 5 hours of culture; the difference in optical density between these two times is called growth. The expression of the HTR is measured after 5 hours of culture as follows: The bacterial density of the culture is calculated from the absorbance of the suspension measured at 600 nm knowing that one absorbance unit corresponds to 3x10 9 CFU/ml.

One ml of a bacterial culture is centrifuged for 15 min at 2500 g and then the bacterial pellet is taken up in a volume of 50 mM Tris-HCl pH 8.0 so as to obtain a suspension of 1x10 8 CFU/ml. In the same buffer, a 2-fold serial dilution is carried out using this suspension (1/2 to 1/2048). Fifty il of each of these samples are deposited in each of the wells of a "dotblot" apparatus (Biorad) operating under vacuum and equipped with a 0.45 lm nitrocellulose membrane (Schleicher and Schull) previously impregnated with a buffer. After depositing the samples, the membrane is incubated for 1 h at room temperature in blocking buffer WO 97/13860 44 PCT/FR96/01580 mM Tris-HCl pH 8.0, 9 g/l NaCl, 10 g/l skimmed milk). The membrane is then incubated for 1 h at 370C, with stirring, with a solution of hTf-peroxidase at 1 g/ml in blocking buffer. After three washes in blocking buffer, the binding of hTf-peroxidase is revealed by the addition of a colorimetric peroxidase substrate 4-chloro-l-naphthol (4-Cl-N) at 0.01% in 1% ethanol.

After 20 min of incubation at room temperature, the titre for expression of HTR is determined; it corresponds to the reciprocal of the last dilution for which a purple colour is still visible. The reading is performed either visually or using a densiometer (Sebia- Preference apparatus).

EXAMPLE 8: Preparation and titration of the antirecombinant Tbp2 (M982 Tbp2 100%, M982 Tbp2 and M982 Tbp2 anti-M982 HTR and anti-Bl6B6 HTR hyperimmune sera.

8A Preparation of the immunogens A culture of the E. coli strain described in Example 4B above or in Example 6B above is lysed by passing through a lysis apparatus (Rannie) and then centrifuged. The insoluble material is loaded onto a acrylamide gel 5 mm thick (10 mg of insoluble proteins per gel) and then subjected to electrophoresis. The Tbp2 protein as well as the 80 and 50% forms are visualized by Coomassie Blue staining and then extracted from the gel. In parallel, a negative control corresponding to the extraction of an E. coli protein of the same size as the Tbp2 protein is prepared.

The. M982 and B16B6 HTRs are, for their part, purified as described in Examples 1 and 2 of WO 93/6861.

WO 97/13860 45 PCT/FR96/01580 8B Preparation of the antisera New Zealand albino rabbits receive subcutaneously, multisite, 50 gg of one of the products described above in the present example, in the presence of complete Freund's adjuvant. 21 days and 42 days after the first injection, the rabbits again receive 50 Ig of the same product but this time in the presence of incomplete Freund's adjuvant. 15 days after the last injection, the animal serum is collected and then decomplementized for 30 min at 560C and sterilized by filtration on a membrane of porosity 0.22 Im (Millipore).

When the immunogen used is an HTR, the filtrate is subsequently depleted by contact with the initial strain (M982 or B16B6) which, to do this, was cultured beforehand in the presence of iron in free form (under these conditions, the synthesis of the transferrin receptor is repressed). The details regarding the contacting are as follows: 10 ml of the filtrate are added to 1010 cfu (colony forming units) of a culture of the initial strain. The adsorption is continued overnight at 4°C, with stirring. The bacteria are then removed by centrifugation for 15 min at 2500 x g. The supernatant is recovered and then again subjected to 3 successive operations of adsorption as previously described. The adsorbed sera are filtered on a membrane of porosity 0.22 gm (Millipore) and stored at In the case of the sera directed against recombinant Tbp2 proteins, the adsorption is performed against the E. coli strain which served to produce the recombinant proteins, cultured for 5h in LB medium (Difco) supplemented with ampicillin at 100 Ag/ml.

WO 97/13860 46 PCT/FR96/01580 8C Titration of the bactericidal activity of the sera The capacity of the anti-HTR or anti-Tbp2 sera to induce lysis of the various N. meningitidis strains is evaluated by a test of bactericidal activity. This technique consists in bringing into contact two volumes of 2-fold dilutions (1/4 to 1/2048) of the adsorbed and decomplementized serum to be titrated, one volume of young rabbit complement diluted 1/1.5 and one volume of bacterial suspension containing 70 microorganisms derived from a 5h culture at 37°C in MHB broth pH 7.2, AM EDDA. The reagents are mixed with each other in cups and incubated for 30 min at 370C, with stirring.

The reaction of bactericidal activity of the serum towards the microorganisms is stopped by the addition of 1 ml of agar consisting of MH broth pH 7.2, 20% G supplement (Pasteur Diagnostic) and 15% noble agar (Difco). The cups are then incubated overnight at 370C in the presence of 10% CO 2 The reading of the results consists in counting the colonies formed from the nonlysed bacteria. The titre is defined as the reciprocal of the last dilution of the serum inducing the lysis of of the bacteria initially introduced.

8D Characterization of the anti-recombinant M982 Tbp2 sera The specificity of four hyperimmune sera directed respectively against a protein Tbp2-100% (serum 577), Tbp2-80% (580), Tbp2-50% (583) or alternatively against an Escherichia coli non-sense protein (control serum 582) is analysed either by a "dot-blot" method on whole microorganisms, or by the "Western-blot" method on outer membrane proteins.

WO 97/13860 47 PCT/FR96/01580 1.1. Analysis on whole microorganisms The preparation of the microorganisms, their deposition on nitrocellulose membrane are identical to that described in Example 9. M982 and B16B6 are cultured in the absence or in the presence of chelating agents.

After the step of saturating with blocking buffer, the membrane is incubated with one of the various hyperimmune sera to be analysed (diluted in blocking buffer: dilutions ranging from 1/500 to 1/10000) for 1 h at 37 0 C. The membrane is then washed twice with blocking buffer, with vigorous stirring, and then, in order to reveal the first serum, it is again incubated with a goat anti-rabbit IgG serum coupled to peroxidase (Zymed) diluted 1/1000 in blocking buffer. To increase the sensitivity of the detection of the signal, the peroxidase substrate used is a chemiluminescent substrate (Kit ECL, Amersham) used according to the recommendations of the manufacturer.

The results which are obtained indicate that the anti-E.coli protein serum (serum 582) is a good negative control in the sense or [sic] it makes it possible to detect neither the M982 strain nor the B16B6 strain irrespective of the culture conditions. The other three sera recognize only the M982 strain and at a higher level when it is cultured in the presence of EDDA, which corresponds to a recognition of the HTR. Indeed, the M982 strain expresses the HTR at a basal level when it is cultured in the WO 97/13860 48 PCT/FR96/01580 absence of chelating agent and this level increases in the presence of chelating agent.

The results indicate that the anti-Tbp2 100% (577), anti-Tbp2 80% (580) and anti-Tbp2 (583) sera are specific for the HTR of the M982 strain.

1.2. Analysis on outer membrane proteins The M982 strain is cultured in a medium depleted of iron. Five ml of exponential phase culture (about 1.108 microorganisms/ml) are centrifuged at 1500 g for 15 min. The pellet is taken up in 1.5 ml of 125 mM Tris-HC1, pH The suspension is lysed by sonication (Branson- Sonifer 450 apparatus, probe 3 mm in diameter) for 2 min. The lysis is continued by adding Triton X100 to 1% final and incubating for min in ice. The suspension is centrifuged at 8000 g in order to remove the non-lysed bacteria and then the supernatant is centrifuged for min at 10000 g in order to obtain the outer membranes in the form of a pellet. The pellet is taken up in 125 mM Tris-HCl, pH 8.0 and then assayed for proteins by a micromethod (Kit Micro-BCA, Pierce) used according to the recommendations of the manufacturer.

Electrophoresis is carried out on polyacrylamide gel in the presence of SDS according to the Laemmli method (Laemli. 1970).

The separating gel contains 12.5% acrylamide and the stacking gel contains 5% thereof. The samples to be analysed are diluted one half in Ssample buffer (0.125 M Tris buffer at pH 6.8; WO 97/13860 49 PCT/FR96/01580 of glycerol, 2.5% 2-mercaptoethanol, 0.001% Bromophenol blue) and hydrolysed at 100 0 C. Fifty Ag of proteins are deposited for a big gel and 1 Ag for the Phast gels (Pharmacia, crosslinking The migration is performed at a constant voltage 50 V for 15x12 cm gels (Biorad vertical apparatus) and 250 V on Phast gels.

After a step of fixing for 30 min in a solution of 10% acetic acid, 25% isopropanol, the gels are stained with Coomassie blue (0.25% blue; 45% methanol; 10% acetic acid). The gels are then gradually destained in successive baths of 20% methanol, 10% acetic acid.

The proteins are transferred onto nitrocellulose membrane (Schleicher Schull; 0.45 im) according to the Towbin method (Towbin et al., 1979). The transfer is made at constant amperage (1 mA/cm 2 for 90 min for a big gel and 1 hour for a Phast gel. The efficiency of the transfer is revealed by staining with Ponceau red (Kodak). After transfer, the nitrocellulose is washed in blocking buffer (0.5 M Tris-HC1 pH 1% skimmed milk; 150 mM NaCl) for 1 hour at room temperature.

The incubation with the antiserum diluted in blocking buffer takes place for one hour at 370C. The first serum is revealed by an antiserum coupled to peroxidase (goat antirabbit, Zymed) for one hour at 37 0 C. The peroxidase substrate is 4-chloro-l-naphthol at 1% (purple colour).

WO 97/13860 50 PCT/FR96/01580 The results obtained indicate that the Tbp2 protein from the M982 strain is specifically recognized by the anti-Tbp2 100%), anti-Tbp2 and anti-Tbp2 50% antisera. No other N.

meningitidis protein is recognized by these sera. The anti-E. coli protein serum does not recognize the Tbp2 of the N. meningitidis M982 strain.

8E Titration of the anti-recombinant M982 Tbp2 sera In order to be able to compare the anti-Tbp2 100, 80 and 50% hyperimmune sera adsorbed on E. coli, it is necessary to know their titre in relation to the homologous strain. Accordingly, a "dot-blot" titration on the M982 microorganisms cultured in a medium depleted of iron is carried out using the procedure described in 9.D.2.

An identical concentration of microorganisms (Ixl0 8 microorganisms/ml) is deposited on nitrocellulose filters which are then revealed with 2-fold dilutions of the various sera to be titrated. The titres are defined as the reciprocal of the last dilution for which a positive signal is observed. With colorimetric revealing, the titres obtained are the following: 512 for the anti-Tbp2 100% serum (serum 577); 256 for the anti-Tbp2 80% serum (serum 580); 256 for the anti-Tbp2 serum (serum 583).

This indicates that the three anti-Tbp2 100, and 50% hyperimmune sera have within a 2-fold range the same titre in relation to the M982 strain. Because of this, for the analysis of their cross-reactivity towards various M982 type strains, they may be used at the same concentration.

WO 97/13860 51 PCT/FR96/01580 EXAMPLE 9: Analysis of the cross-reactivity of the anti-recombinant M982 Tbp2 sera The determination of the level of crossreactivity towards the M982 type strains is carried out by the dot blot method as described in Example 8.

After culturing in MHB medium 30 mM EDDA for hours, 2-fold dilutions of the various bacterial suspensions are deposited on 5 different nitrocellulose filters: a first filter is revealed with human transferrin coupled to peroxidase (HTG) [sic] and makes it possible to define a titre for binding HTF; the second is revealed with an anti-E. coli protein serum (serum used at 1/2000; negative control); the third is revealed with a serum directed against an entire recombinant M982 Tbp2 protein (100%); the fourth is revealed with a serum directed against a recombinant M982 Tbp2 protein deleted of the hypervariable portions of the "hinge" region and the fifth is revealed with a serum directed against a recombinant M982 Tbp2 protein [lacuna] corresponds to the N-terminal region. The titres are defined as the reciprocal of the last dilution for which a colour is visible.

In order to be able to quantify the crossreactivity of each of the sera with each of the strains, M& it is necessary to define a value which takes into WO 97/13860 52 PCT/FR96/01580 account the variability at the level of the expression of the HTR among the strains analysed.

Accordingly, an index corresponding to the serum titre expressed in terms of the titre for expression of transferrin is calculated. This index is the value selected for the analysis because it takes into account the variable level of expression of the HTR from one strain to another under our culture conditions. However, this index cannot account for a possible difference in the affinity of the HTRs.

For information, a summary of the values of the indices obtained for the 54 strains is presented in the table below.

Number of strains recognized Serum Anti-Tbp2 Anti-Tbp2 Anti-Tbp2 recognition 100% 80% index serum 577 serum 580 serum 583 0 14 4 1 0.016 0 1 1 0.031 6 3 1 0.062 10 8 0.125 5 10 7 0.25 5 9 12 5 8 9 1 7 4 2 2 4 8 4 0 3 2 8 0 0 3 WO 97/13860 53 PCT/FR96/01580 EXAMPLE 10: Study of cross-bactericidal activity Preparation of an anti-N. meningitidis 8680 HTR antiserum A preparation of 8680 HTR as obtained in Example 1 is used as the immunogen in order to prepare an antiserum according to the procedure described in Example 8B.

Culture of the strains The N. meningitidis 92/04, M871, 504/91, 8680, 8726, BZ83, 44, 52 and 8710 strains (strains obtained from the meningocci reference laboratory: Dr. Caugant, WHO-collaborating center for reference and research on meningococci, NIPH, Oslo, Norway, and which form part of the ET5 electrophoretic group Mohamed-Rokbi, thesis presented on 10 October 1995, before the Claude Bernard University, Lyon, France for the award of the Doctorate degree) as well as the M982 and B16B6 strains are cultured as described in Example 1A.

Test of bactericidal activity The bactericidal activity of the antiserum prepared in 10A is tested against the strains cultured in 10B according to the procedure described in Example 8C.

The results are presented in the table below: WO 97/13860 54 PCT/FR96/01580 Strain Serotype/subtype Bactericidal titre Rabbit 1 Rabbit 2 B16B6 B:2a:P1.2 <4 nd M982 B:9:P1.9 <4 <4 92/94 B:15:P1.7, 9, 16 128 32 M871 B:15:P1.7, 16 256 32 504/91 512 128 8680 B:15:P1.3 1024 256 8726 B:4:P1.3 1024 256 BZ83 B:15:- 32 64 44 nd 64 <4 52 nd 16 16 8710 B:15:P1.3 256 64 The bactericidal titres obtained against the strains of the ET5 electrophoretic group demonstrate a cross-bactericidal activity. Those obtained against the B16B6 and M982 strains, on the other hand, indicate an absence of cross-bactericidal activity.

These results tend to support the proposition made by the present application, namely the possible addition of a Tbp2 protein or of an HTR which is derived from an 8680 type strain, to a vaccinal composition containing a Tbp2 or an HTR which is derived from an M982 type strain and (ii) a Tbp2 or an HTR which is derived from a B16B6 type strain, without undermining the results already observed as regards recognition of the 8680 strain in Western blotting, by anti-M982

HTR

and anti-B16B6 HTR antisera.

WO 97/13860 -5 55 PCT/FR96/01580 SEQ ID NO Project name 1, 2 IM2169-2 3, 4 IM2394-2 6 8680 7 2D IM2169 8 2D6940 9 2D2223 2D 708 11 2D M978 12 2D 1610 13 2D867 14 2D S3032.

2D891 16 OTG 4915 17 OTG 4651 18 OTG 4928 19 OTG 5011 OTG 4873 21 OTG 4877 22

A'

23 A 3' 24 2169 D1 2169 D2 26 2169 D3 27 2169 D4 28 Inter 1 Barn 29 Met 2 Eco Sequence complete IM2169 Tbp2 complete IM2394 Tbp2 complete 8680 Tbp2 2nd domain of IM2169 Tbp2 2nd domain of 6940 Tbp2 2nd domain of 2223 Tbp2 2nd domain of 708 Tbp2 2nd domain of M978 Tbp2 2nd domain of 1610 Tbp2 2nd domain of 2D867 Tbp2 2nd domain of S3032 Tbp2 2nd domain of M981 Tbp2 OTG 4915 OTG 4651 OTG 4928 OTG 5011 OTG 4873 OTG.4877 A A 3' 2169 D1 2169 D2 2169 D3 2169 D4 Inter 1 Barn Met 2 Eco Doculmcntl 211477/)14/I Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

oo a a •go• WO 97/13860 56 PCT/FR96/01580 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Pasteur Merieux serums et vaccins STREET: 58, avenue leclerc CITY: Lyons COUNTRY: France POSTAL CODE: 69007 (ii) TITLE OF INVENTION: N. meningitidis Tbp2 fragments (iii) NUMBER OF SEQUENCES: 29 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Tape COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE

CHARACTERISTICS:

LENGTH: 2230 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: IM2169 (ix) FEATURE: S(A) NAME/KEY: sig_peptide WO 97/13860 57 PCT/FR96/01580 LOCATION: 60..119 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

matpeptide 120..2192

CDS

60..2192 miscfeature 120..1154 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

misc_feature 1155..1748 miscfeature 1749..2192 misc_binding 237..1169 WO 97/13860 58 PCT/FR96/01580 (xi) SEQUENCE DESCRIPTION :SEQ ID NO: 1: ATTTCGrAAA AATAAATAAA ATAATAATCC TTATCATCT TTAFLTGAAT TGTTTAT ATG AAC AAT CCA TTG GTA AAT CAG GCT GCT ATG GTG CTG CCT CTG TTT- Met Ann Asn Pro Lou Val Asn GIn Ala Ala M4et Val Leu Pro Val Phe AGT GCC Ser Ala ACC GAA Thr Glu AAA CCG Lys Pro TTG AAA Leu Lys CCC AAC Leu Asn GAA CTT Glu Lou so GGC GAC Gly Asp CAA AAC Cln Asn ACA GGT Thr Gly CAT GCA His Ala TCT CTG GGC GGC Cys Leu Gly Gly GCC CCG CGT CCC Ala Pro Arg Pro 20 CAA GCC CAA AAA Gln Ala Gln Lys CGC AGC AAT TGG Arg Arg Asn Trp GAG ACT CAT TGG Glu Ser Asp Trp CCT AAA CGG CA.A Pro Lys Arg Gin AGC GAT ATT TAT Ser Asp Ile Tyr 100 GGC AGC GCT GGC Gly Ser Ala Gly 115 CAC CAA AAT TTC His Glu Asn.Phe 130 GCG ACT GAA AAA Ala Ser Glu Lys 145 CCC GC Cly Gly GCG CCA Ala Pro

TTC

Phe

TAT

Tyr GAC CMA GGC GGA Asp Cln Gly Cly TAT CCC GGG GCA, Tyr Pro Gly Ala 55 GAG C ACC GCA Clu Ala Thr Gly 70 AAA TCG CTT ATT Lys Ser Val Ile as TCT TCC CCC TAT Ser Ser Pro Tyr AAC GGT GTA MAT Ann Cly Val Asn 120 CPA TAT GTT TAT Gin Tyr Val Tyr 135 CAT TTC ACT MAC Asp Phe Ser Asti GAT CT Asp Lou CAA CAT Gin Asp TAC GGT Tyr Cly CMA GAA Glu Clu TTG CCC Leu Pro CM A Ciu Lys CTC ACA Leu Thr 105 CAA CCT Gin Pro TCC CGT Ser Cly AAA A

-S

GAT TCT Asp Ser OTT TCT Val Ser TTT CC Phe Ala ACC GAG Ser Clu 66 ACA AMA Thr Lys GTA CMA Val Clu CCA TCA Pro Ser MAA MAT Lys An TGG TT Trp Phe 140 ATT MAG Lys Lys Ile Lys 155 150 TCA CCC CAC CAT GCT TAT ATC TTC TAT CAC GGT GAA AAA CCT TCC CCA Ser Cly Asp Asp Gly Tyr Ile Phe Tyr His Gly Glu Lys Pro Ser Arg WO 97/13860 ZAA CTT CCT OCT TCT Gin Leu Pro Ala 5cr 175 GTA ACC GAT ACA AAA Val Thr Asp Thr Lys 190 TCA AAA AAA CAA GOC Ser Lys Lys Gin Gly 205 GAA GAA TAT TCC AAC Giu Glu Tyr Ser Asn 225 GOT TAT GOT TTT ACC Gly Tyr Gly Phe Thr 240 TTG ACG GOT AAA TTA Leu Thr Gly Lys Leu 255 AAT AAT GAC AAA CAT Asn Asn Asp Lys His 270 ACA GOC AAC CCC TTC Thtr Gly Asn Arg Phe 285 ;,AT GAA ACC AAA CTA Asn Clu Thr Lys Leu 305 GGC GGC TTT TTC GCC Gly Gly Phe Phe Gly 320 AGC CAC CAT CAA A Ser Asp Asp Gin Lys 335 AAA CTO GAA AAT GC Lys Leu Oiu Asn Gly 350 TCG CCC GOT OCO OCA Ser Gly Gly Ala Ala 365 59- 165 OGA AAA OTT ATC TAC Oly Lys Val Ile Tyr 180 AAG GOT CAA OAT Trr Lys Gly Gin Asp Phe 195 GAC AGO TAT AOC OGA Asp Arg Tyr Ser Gly 210 AMA AAC OAA TCC ACG Lys Asn Oiu Ser Thr 230 TCG AAT TTA GAA GTG Ser Asn Leu Giu Val 245 ATA CCC AAT PAT OCG Ile Arg Asn Asn Ala 260 ACC ACC CPA TAC TAC Thr Thr Gin Tyr Tyr 275 AMC GOC ACG OCA ACO Asn Giy Thr Ala Thr 290 CAT CCC TTT OTT TCC His Pro Phe Val Ser .310 CCG CAC GOT GAG GAA Pro Gin Gly Olu Giu 32S5 OTT GCC OTT GTC GGC Val Ala Val Val Gly 340 C GCO GCT TCA GOC Ala Ala Ala Ser Oly 355 GGC ACO TCG TCT OAA Oly Thr Ser Scr Oiu 370 CAA T70 ACA CTA PAC Oiu Leu Thr Leu Asn 390 AA 007 Lys Gly COT GMA Arg Giu 200 TIT TCT Phe Ser 215 CTG AAA Leu Lys OAT TTC Asp Phe AGC CTA Ser Leu AGC CTT Ser Leu 280 OCA ACT Ala Thr 295 GAC TCG PCT/FR96/01580 170 TOO CAT TTT Trp His Phe ATC CAG CCT Ile Gln Pro OAT GGC AGC Asp Oly Ser 220 OAT CAC GAG Asp His Giu 235 PAT MOG AAA Asn Lys Lys 250 MAT PAT ACT Asn Asn Thr OCA CPA ATA Ala Gin Ile AA AAA GAG Lys Lys Giu 300 TCT TT0 AGC 683 731 779 827 875 923 971 1019 1067 1115 1163 1211 12S9 1307 Asp Ser Ser Ser Leu Ser 315 770 GOT TTAC CCC TTT TTC Leu Gly Phe Arg Phe Leu 330 AGC 0CC AMA ACC AMA CAC 5cr Ala Lys Thr Lys Asp 345 AGC ACA OCT GCO OCA GCA Ser Thr Cly Ala Ala Ala 360 AAC ACT MAG CTO ACC ACG Asn Ser Lys Leu Thr Thr 375 380 GTT TTO CAT VJal Leu Asp OCO OTT Ala Val 385 GAC MOG AMA Asp Lys Lys ATC MAA PAT Ile Lys Asn 395 WO 97/13860 -Cc AC AAC TTC AGC Leo Asp Asn Phe Ser 400 ATT CCG CTC CTG CCC Ile Pro Leu Leu Pro 415 AA G AAA AAC GGC Lys Gly Lys Asn Gly 430 CCG GMA ACT GAT A Pro Giu Ser Asp Lys 445 GCG CAA ACC GCT TCA Ala Gin Thr Ala Ser 465 ACC TAT GMA GTC GMA Thr Tyr Giu Val Giu 480 OGA ATG TTG ACG CC Gly Met Leu Thr Arg 495 MAC ACT ACT CAA GCT Asn Ser Ser Gin Ala 510 TTZ CTC CAM GCC GAG Phe Leu Gin Oly Ciu 525 A.A GTC GTT TAT CG Asn Val Val Tyr Arg 545 ACC TOG AGC CCC MAT Ser Trp Ser Gly An 560 TTT ACT OTG MAT TTT Phe Thr Val Asn Phe 575 GMA MC AGO CAG C 60 CCC CCC CMA CTC OT T Ala Ala Gin Leu Val 405 CAT TCC GM -AGC CG Asp Ser Gbu Ser Gly 420 ACA GAA TrT ACC CC Thr Glu Phe Thr Arg 435 GAC GCC CMA GCA GOT Asp Ala Gin Ala Cly 455 ACG GCA GOT OAT ACC Thr Ala Cly Asp Thr 470 TCC TOT TCC MAC CTC Cys Cys Ser Ann Leu 485 MAC AGC MAG TCC C Ann Ser Lys Ser Ala 500 OCT MAA ACO GMA CMA Ala Lys Thr Giu Gin 515 ACC CAT GM AAA GAO Thr Asp Oiu Lys Glu 53S TCT TGG TAC -000 CAT Ser Trp Tyr Gly His 550 TCT CAT AAM GAG 0C Ser Asp Lys Glu Gly 565 GAT MAA AMA ATT ACC Asp Lys Lys Ile Thr 580 ACC TTT ACC ATT GAG Thr Phe Thr Ile Giu 595 GCG MAA ACT OCT GAG Ala Lys Thr Ala Glu 61S CGC ACG CCT MCG OCA Arg Thr Pro Lys Ala PCT/FR96/01580 GTC GAC GGC ATT ATO 1355 Val Asp Oly Ile Met 410 MAC ACT CAC GCA CAT 1403 Asn Thr Gin Ala Asp 425 AAA TTT GMA CAC ACO 14S1 Lys Phe Glu His Thr 440 ACO CAG ACG MAT GGG 1499 Thr Gin Thr Ann Gly 460 MAT GGC MAA ACA MAA 1547 Ann Gly Lys Thr Lys 4.75 MAT TAT CTG AMA TAC 1595 Ann Tyr Leu Lys Tyr 490 ATG CAG GCA GCA GGA Met Gin Ala Gly Giy OTT GMA CMA AGT ATG Val Glu Gin Scr Met 520 ATT CCA ACC GAC CMA Ile Pro Thr Asp Gin 540 ATT GCC MAC GGC ACA Ile Ala Ann Gly Thr 555 GGC MAC AGO GCO CMA Gly Ann Arg Ala Glu 570 GGC MOG TTA ACC OCT Gly Lys Leu Thr Ala 585 GCA ATO ATT CAG GGC Gly Met Ile Gin Gly 1643 1691 1739 1787 1935 1883 1931 1979 2027

CC

Ala

CMA

Glu Asn Arg Gin Ala Gln 590 MAC 0CC Tfl' GMA Asn Gly Phe Ciu 605 GAT CAA AAA MT Asp Gin Lys An GOT ACG Gly Thr 610 ACC ACC Thr Thr TCA GOT Ser Cly TAT ATC Tyr Ile TTT CAT Phe Asp ACA OAT Thr Asp WO 97/13860 61 630 PCT/FR96/01580 635 AAG GTA A]G GGC GGT TTT TAC GGG CCT AAA GCC QAA GAG TTG GGC GGA L~ys Val Lys Gly Gly 640 Phe Tyr Gly Lys Ala Glu Glu Leu Gly Gly 650 GCA ACA GCT ACA Ala Thr Ala Thr 665 TGG TTT GCC TAT Trp Phe Ala Tyr 655 CCG GGC GAT Pro Gly Asp CAA ACG GAA AAG Gln Thr Glu Lys 2075 2123 2171 2222 TCC AGC Ser Ser 670 AAA CGC Lys Arg 685 GAT GGA AAT Asp Gly Asn CAA CAG CCT Gin Gin Pro TCA GCA Ser Ala 675 AGC AGC GCG ACC G;TG GTA TTC GGT GCG 5cr Ser Ala Thr Val Val Phe Gly Ala GTG CAA TAAGCACGGT TGCCGAACAA TCAAGAATAA Val Gln 690 GGCTTCAG 2230 INFORMATION FOR SEQ ID NO: 2: Wi SEQUENCE CHARACTERISTICS: LENGTH: 711. amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 97/13860 62 WO 9/1380 2 -PCT/FR96/01580 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Leu Val1 Se r Met Val Pro Thr Asn Se r Thr Lys Leu Leu Glu Gly Pro Leu Ala Cys

I

Giu Ala Pro Gin Lys Arg Asn Glu Leu Pro Asp Ser Val Asn Gin -15 1,eu Gly Gly Pro Arg Pro 20 Ala Gin Lys Arg Asn Trp 50 Ser Asp Trp Lys Arg Gin Asp Ile Tyr 100 Ala Met Gly Ser Pro Lys Gln Gly Pro Gly Ala Thr 70 Ser Val Ser Pro Val1 Phe Tyr Gly Ala Gly Ile Tyr Leu Asp Gin Tyr Glu Leu Glu Leu Pro Leu Asp Giy Giu Pro Lys Thr Val Asp Val Phe Ser Thr Val Pro Gin Pro Lys Asn Asn His Gin Asn Gly SerAaGyAnGyVa 11011 Ala Gly Asn Gly Val 115 WO 97/13860 63 PCT/FR96/01580 125 Tyr Ser i-n Val Ser 205 Ciu Gly Leu Asn Thr 285 Gdy Se r Lys Se r 365 ValI Leu Ile Lys Ala Lys Gly Leu Thr 190 Lys Giu Tyr Thr Asn 270 Giy Giu Gly Asp Leu 350 Giy Leu Asp Pro Gly Thr His Asp Pro 175 Asp Lys Tyr Gly Gly 255 Asp Asn Thr Phe Asp 335 Giu Gi y Asp Asn Leu 415 Lys Giy Ala Asp 160 Ala Thr Gin Ser Phe 240 Lys Lys Arg Lys Phe 320 Gin Asn Ala Ala Phe 400 Leu Asn His Ala 145 Gly Ser Lys Gly An 225 Thr Leu His Phe Leu 305 Gi y Lys Gly Ala Val 385 Se r Pro Gly Glu 130 Ser Tyr Gly Lys Asp 210 L-ys Ser Ile Thr Asn 290 His Pro Val Ala Gly 370 Glu Asn Lys Gly Asn Glu Ile Lys Gly 195 Arg Asn Asn Arg Thr 275 Gly Pro Gin Ala Ala 355 Thr Leo Ala Asp Thr Phe Lys Phe Val 180 Gin Tyr Glu Leo An 260 Gln Thr Phe Gly Val 340 Ala Ser Thr Ala Ser 420 Glu Gin Asp Tyr 165 Ile Asp Ser Ser Glu 245 An Tyr Ala Val1 Gu.

32S Val Ser Ser Leo Gin 405 Glu Phe Tyr Val 135 Phe Ser iso His Gly Tyr Lys Phe Arg Giy Phe 215 Thr Leu 230 Vai Asp Ala Ser Tyr Ser Thr Ala 295 Ser Asp 310 Glu Leu dly Ser cay Ser Glu Asn 37S Asn Asp 390 Leu Val Ser Gly Thr Arg Ser Lys Lys Val 185 Ile Gly Asp Gly An 265 Asp Asp Ser Phe Lys 345 Gly Lys Lys Asp Thr 425 Phe Trp Ile 155s Ser His Gin Gly His 235 Lys Asn Gin Ly's Leu 315 Phe Lys Ala Thr Lys 395 Ile Ala His Phe 140 Lys Arg Phe Pro Ser 220 Glu Lys Thr Ile Glu 300 Ser Leu Asp Ala Thr 380 An Met Asp Thr WO 97/13860 430 Pro Glu Ser 445 Ala Gin Thr Thr Tyr Glu Gly Met Leti 495 Asn Ser Ser 510 Phe Leu Gin 525 Asn Val Val Ser Trp Ser Phe Thr Val Glu Asn Arg 590 Asn Cly Phe 605 Asp Gin Lys Lys Val Lys Trp Phe Ala Ser Ser Asp 670 Lys Arg Gin 685 64 PCT/FR96/01580 Asp Ala Val 480 Thr Gin Gly Tyr Gly 560 Asn Gin Glu Asn Gly 640 Tyr Gly Gln Lays Ser 465 Giu Arg Ala Glu Arg 545 Asn Phe Ala Gly Thr 625 Gly Pro Asn Pro 435 Asp Thr Cys Asn Ala 515 Thr Ser Ser Asp Thr 595 Ala Arg Tyr Asp Ala 675 Gln Ala Gin Ala Gly Cys Ser 485 Ser Lys 500 Lys Thr Asp Glu Trp, Tyr Asp Lys 565 Lys Lys 580 Phe Thr Lys Thr Thr Pro Gly Pro 645 Lys Gin 660 Ala Asp 470 Asri Ser Glu Lys Gly 550 Glu Ile Ile Ala Lys .630 Lys Thr 440 Thr Asn Asn Met Val 520 Ile Ile Gly Gly Gly 600 5cr Tyr Glu Lys Gin Gly Tyr Gin Glu Pro Ala Asn Lys 585 Met Gly .le Glu Al a 665 Ser Ser Ala Thr Val Val Phe Gly Ala 680 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 1808 base pairs TYPE: nucleotide STRANDEDNESS: single WO 97/13860 65 PCT/FR96/01580 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: IM2394 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

sig_peptide 1..60 mat_peptide 61..1797

CDS

1..1797 (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 61..1035 (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1036..1386 (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1387..1797 (ix) FEATURE: NAME/KEY: miscbinding LOCATION: 46..1050 (xi) SEQUENCE DESCRIPTION SEQ ID NO: 3: WO 97/138E AIC AAC AAT Met Asn Asn TTO TTG ACT Leu Leu Ser GTG OAA ACC Val Glu Thr is AAA AGC CAG Lys Ser Gin GCT TAT GGC Ala Tyr Gly C'-T AAA TAT CCA Trc GTA AAT Pro Leu Val Asn -1s GCT TOT CTG GOT Ala Cys Leu Gly O;TO CAA GAT ATG Val Gin Asp met CCT GAA AOC CAA Pro Glu Ser Gin TTT GCA GTA AAA Phe Ala Val Lys 50 AAG OAA AAG CAC -66 OCT OCT ATO Ala Ala Met -10 0CC GOC AGT Gly Gly Ser TCC AAA CCT Ser Lys Pro OAT GTA TCO Asp Val Ser CCT CGC CGG Pro Arg Arg 55 CCA TTG GOT PCT/FR96/01580 OTO TTT 48 Val Phe CAC ACC 96 Asp Ser GAT GAA 144 Asp Glu GGC OCO 192 Gly Ala =T AAT 240 Phe Asn TOO AAA 288 WO 97/13860 Pro Lys Tyr AAA CTG CAA Lys Leu Gln GAA AAA AAA 67 Lys Giu Lys AGA GGA GAA Arg Gly Glu CGC GGT AGT His Lys Pro Leu Gly 70 CCA AAT ACT TIT AGT Pro Asn Ser Phe Ser PCT/FR96/01580 Ser Met Asp Trp Lys GAG AGG CAT CAA TTG 336 Clu Arg Asp Giu Leu TCT GAA CTT ATT GAA TCA AAA TGG GAA GAT GJu Lys Lys Arg Gly Ser

GGG

Cly

GGA

cly 125

GTT

Val

TCC

Ser

TAT

Tyr

GCA

Ala

GTA

Val 205

ATG

ACT

Ser

GTT

Val Phe

GAG

Glu

ACT

Thr.

175

GGA

Cly

CGT

Arg

AGT

GTA GTT Val Val CTr AAT Leu Ann 130 Ser

GGT

Giy 11S

AAA

Lye

GCGA

Gly

GAA

Glu

GAA

GGA CCT Gly Pro 145 CTG CCA Leu Pro 160 CAT GCT Asp Ala GGA CAT Gly Asp AAT CAG Asn Gin GAG TIT

CAC

Asp

TCG

Ser

ATG

Glu Leu Ile Glu 100 TAT ACA AAT ITC Tyr Thr Ann Phe AAT AAT ATT GAT Asn Ann Ile Asp 135 TAT Crr TAC TAT Tyr Lau Tyr Tyr 150 AAG ATA ACT TAT Lys Ile Thr Tyr 165 AAA CAA ACC rr Lye Gin Arg Phe 180 CC GCG TTG TCT Ser Lys Trp Giu Asp

'OS

ACT TAT GTC CGT TCC Thr Tyr Val Arg Ser 120 ATr AAG AAT AAT ATA Ile Lys Ann Ann Ile 140 AAA CGG AAA GAA CCT Lye Gly Lys Glu Pro 155 AA GGT ACT TGG CAT Lys Gly Thr Tip Asp 170 GAA GCA TG GG AGT Glu Gly Leu Cly Ser 185 GCA TTA CAA GAA GGC Ala Leu Ciu Ciu Gly 200 CAT ACC CAT TTT GC His Thr Asp Ph. Cly 220 AAA ACA ATA AAG CGC Lys Thr Ile Lys Gly 235 384 432 480 528 576 624 672 720 769 met Glu AAA TCG Lys Ser Cly Ala Leu Ser 195 GCA GAG GCA TCA TCC GGT Ala Giu Ala Ser-Ser Cly 210 21S GAG GT GAT T1 TCT GAT met Thr Ser Giu Phe Clu Val Asp Phe Ser Asp 225 230 CTT TAT Leu Tyr ATA AAA lie Lye 255 TTC AAA Phe Lys 270 CAT CCC His Pro CCT AAC Arg Ann 240 ACT ACC Thr Thr C AAC Cly Lya TTT ATT Phe Ile AAC CCT AT? ACT Ann Arg Ile Thr 245 CGT TAC ACC ATT Arg Tyr Thr Ile 260 GCG TTG C GCA Ala Leu Ala Aia 275 TCC CAC TCC GAC Ser Asp Ser Asp 290 CAA AAT AAT Gin Asn Asn CAA GCA ACT Gin Ala Thr CAT AAA GOT Asp Lys Cly 280 ACT TTC GAP Ser Leu Ciu 295 ACT GAA AAC AAA Ser Giu Ann Lys 250 CT? CAC GGC AAC Leu His Gly Ann 265 CCA ACA AAT GGA Ala Thr Ase Cly GGC GCA T-r TAC Cly Cly Phe Tyr 300 WO 97/13860 68 PCT/FR96/01580 3GG CCG AAA GGC GAG GAA CTT CCC CCT :;ly Pro Lys Gly Giu Giu Leu Ala Gly 305 A.AA GTT GCA C GTG TTT CGT*GCG AAG Lys Val Ala Ala Val Phe Gly Ala Lys 320 325 AAA TTC Lys Phe 310 TTG ACC Leu Ser CAG AAA GAT AAG Gin Lys Asp Lys AAC GAC AAC Msn Asp Asn 315 AAG CAT GCG Lys Asp Cly 330 GCA TAC CG'r Ala Tyr Arg TTT GGA CAT Phe Gly Asp GAA AAC GCG GCA GGG CCT GCA ACG GAA ACC GTG Glu Asn Ala Ala Gly Pro Ala Thr Ciu Thr Val ATA GAT le Asp 345 GAC ACT Asp Ser 360 ATT ACC Ile Thr 350 GTG AAA Val Lys 365 CCC GAG GAG TTT Gly Glu Glu Phe AAA GAG CAA ATA Lys Ciu Gin le AAG CTC CTG Lys Leu Leu GAC GGA G;TG Asp Gly Val GAG CTT Glu Leu 37S TCA CTG CTG Ser Leu Leu CCC TCT Pro Ser 380 CCC GTG Cly Val 395 GAG GOC AAT AAG Clu Cly Asn Lys GCA TTT CAG CAC Ala Phe Gin His ATT GAG CAA AAC Ile Giu Gin Msn AAG GCA ACG GTG TGT TGT TCC AAC Lys Ala Thr Val Cys Cys Ser Asn 400 GAT TAC ATG Asp Tyr Met ACT TTT CCC AAC Ser Phe Cly Lys 410 CTG TCA AMA CA MAT Leu Ser Lys Giu Msn 41S AMA GAC CAT Lys Asp Asp 420 ATO TrC CTG CAA Met Phe Leu Gin CTC CGC ACT Val Arg Thr 1008 1056 1104 1152 1200 1249 1296 1344 1392 1440 1488 1536 1584 1632 1680 :7CA GTA Pro Val 430 GGT ACT Cly Thr 445 ICC CAT GTG C Ser Asp Val Ala OCA AGO Ala Arg 435 ACG GAG GCA AAC CCC AMA TAT CC Thr Giu Ala Msn Ala Lys Tyr Arg 44-0 TGG TAC GCA Trp, Tyr Gly ATT CCC MAC CCC ACA ACC TOG ACC GCC Ile Ala Asn Gly Thr Scr Trp Ser Gly GCC TCC MAT CAC GAA GCT GOT MAT ACC -Ala Ser Asn Gin Giu Gly Gly Msn Arg 465 GAG TTT CAC OTG Giu Phe Asp Val CAT TTT Asp Phe 475 TCC ACT AMA Ser Thr Lys CCT C TT Pro Ala Phe 49S ATC ACT GCC Ile Ser Cly ACT ATT ACT CC Thr Ile Thr Ala ACA CTC ACO Thr Leu Thr 48S ATG ATT AAC Met Ile Lys 500 CCC TTT CC Gly Phe Ala GCA MAA GAC COT ACG TCT Ala Lys Asp Arg Thr Ser 490 GAC AAC CC? TTT TCA C Asp Asn Gly Phe Ser Cly 505 CTG CC Val Ala 510 AAA ACC GC? CMA Lys Thr Cly Oiu CTC GAT Leu Asp 520 CCC CMA MT ACC Pro Gin Msn Thr GGA MAT TCC CAC TAT ACC CAT ATT GAM CCC ACT CTA TCC CCC CCT i-rC WO 97/13860 69 G2.y Asn Ser His Tyr Thr His Ile Glu Ala Thr 525 530 S35 TAC GGC AAA AAC GCC ATC GAG; ATG GGC GGA C Tyr Gly Lys Asn Ala Ile Giu Met Gly Gly Ser 545 550 AAT GCA CCA GAG GGA AAA CAA GAA AAA GCP. WCG Asn Ala Pro Glu Gly Lys Gin Glu Lys Ala Ser 560 565 AAA CGC CAA CAG CT CTC CAA TAAGCACGGC T Lys Arg Gin Gin Leu Val Gin 575 INFORMATION FOR SEQ ID NO: 4: PCT/FR96/01580 Val Ser Gly Gly Phe 540 T1TC TCA TITr CCG GGA Phe Ser Phe Pro Gly G'TGOTA TTC GGT GCG Val Val Phe Gly Ala 570 1728 1776 1808 Wi SEQUENCE CHARACTERISTICS: LENGTH: 599 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: WO 97/13860 70 PCT/FR96/01580 Met Asn Asri Pro Leu Val Asn Gin Ala Ala Met Val Leu Pro Val Phe -15 -10 -S Leu Leu Ser Ala Cys Leu Gly Gly Gly Gly Ser Phe Asp Leu Asp Ser 1 510 Val Giu Thr Val Gin Asp Met His Ser Lys Pro Lys.Tyr Giu Asp Ciu 20 Lys Ser Gin Pro Giu Ser Gin Gin Asp Val Ser Giu .Asn Ser G ly Ala 35-40 Ala Tyr Gly Phe Ala Val Lys Leu Pro Arg Arg Asn Ala His Phe Asn 4S so 55 6O Pro Lys Tyr Lys Giu Lys His Lys Pro Lou Gly Ser Met Asp Trp Lys 70 Lys Leu Gin Arg Gly Glu Pro Ass Ser Phe Ser Giu Arg Asp Giu Leu 80o5 Giu Lys Lys Arg Giy Ser Ser Giu Leu Ile Giu 5cr Lys Trp Glu Asp 100 105 Giy Gin Ser Arg Val Val Gly Tyr Thr Ass Ph. Thr Tyr Val Arg Ser 110 115 120 Gly Tyr Val Tyr Leu Asn LysAss Asn Ile Asp Ile Lys Ass Asn Ile 125 130 135 140 Val Leu Phe Gly Pro Asp Gly Tyr Leu Tyr Tyr Lys Giy Lye Giu Pro 145 150 155 WO 97/13860 71 WO 97/3860 71 -PCT/FR96 /0 1580 ier Lys Tryr Val Ala Ala 190 Val Leu 205 Met Thi- Thr Leu Gin Ile Arg Phe 270 Ser His 285 Gly Pro Lys Val Glu Asn 11e Thr 350 Val Lys 365 Glu Gly Lys Ala Leu Ser Pro Val 430 Gly Thr 445 Glu Thr 175 Gly Arg Ser Tyr Lys 255 Lys Pro Lys Ala Ala 335 Gly Lys Asn Thr Lys 415 Ser Ti-p Leu 160 Asp Gly Asn Glu Arg 240 Thr Gly Phe Gly Ala 320 Ala Glu Leu Lys Val 400 Giu Asp Tyr Pro Ala Asp Gin Phe 225 Asn Thr Lys Ile Giu 305 Val Gly Giu Leu Ala 385 cys Asn Val1 Gly Ser Met Lys Ala 210 Giu Asri Arg Ala Se r 290 Clu Phe Pro Phe Val1 370 Ala Cys Lys Ala Tyr 450 Giu Giu Ser 195 Glu Val Arg Ty- Leu 275 Asp Leu Gly Ala Lys 3S5 Asp Phe Ser Asp Ala 435 Ile Lys Lys 1.80 Gly Ala Asp Ile Thr 260 Ala Ser Ala Ala Th- 340 Lys Gly Gin Asn Asp 420 Arg Ala Ile Thr 165 Gin Arg Ala Lou Ser Ser Phe Ser 230 Thr Gin 245 Ile Gin Ala Asp Asp Ser Gly Lys 310 Lys Gin 325 Glu Thr Glu Gin Val Giu Hi s Giu 390 Leu Asp 405 Met Phe Thr Glu Asn Gly Tyr Phe Ser Gly 215 Asp Asri Ala Lys Leu 295 Phe Lys Val Ile Leu 375 Ile Tyr Leu Ala Thr 455 Lys Glu Ala 200 His Lys Thr Gly 280 Giu Leu Asp Ile Asp 360 Ser Giu Met Gin Asn 440 Se r Thr 170 Leu Glu Asp Ile Giu 250 His Thi- Gly Asn Lys 330 Al a Phe Lou Asn Phe 410 Val Lys Se r Ti-p Gly Giu Phe Lys 235 Asn Gly Phe Asp 315 Asp Tyr Gly Pro Gly 395 Gly Arg Tyr Gly Asp Se r Gly Gly 220 01 y Lys Asn Gly Tyr 300 Asn Gly Arg Asp Ser 380 Val Lys Thr Arg Giu 460 WO 97/13860 Ser Asn Gin -er Thr Lys Lys 480 Pro Ala Phe Thr 495 Val Ala Lys Thr 510 Gly Msn Scr His 525 Tyr Gly Lys Asn Asn Ala Pro Glu 560 Lys Arg Gin Gin 575 Glu 465 Ile Ile Gly Tyr Ala 545 Gly Leu Gly Ser Thr Glu Thr 530 Ile Lys Val 72 Gly Msn Arg Gly Thr Leu 485 Ala Met Ile 500 Asn Gly Phe 515 His Ile Glu Glu Met Gly Gin Glu Lys 56S Gin Ala Glu 470 Thr Ala Lys Asp Ala Lou Ala Thz 535 Gly Ser 550 Ala 5cr PCT/FR96/01580 Asp Val Asp Phe 475 Asp Arg Thr 490 Gly Phe 5cr Gly 505 Pro Gin Msn Thr Ser Gly Gly Phe 540 Ser Phe Pro Gly 555 Val Ph. Gly Ala 570 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERI STICS: LENGTH: 2064 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 8680 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .2061 (ix) FEATURE: NAME/KEY: sig-peptide LOCATION: 1. wo 97/138' (ix) 60 73 PCT/FR96/01580

FEATURE:

NAM'E/KEY: matpeptide LOCATION: 31. .2061 (xi) SEQUENCE DESCRIPTION: SEQ ID NO ATG GTG CTG CCT OTG TTT TTG TCG AGT GCr TGT CTG GGC GGA GOC GOC Met Val Leu Pro Val Phe Leu Ser Ser Ala Cys Leu (fly Gly Gly Gly i -5 1 GGC AG? TTC GAT Crr GAT TCT GTC GAT ACC GAA GCC CG CGT CCC GCG WO 97/13860 74 PCT/ FR9 6/01580 GySer Phe Asp Leu Asp Ser Val Asp Thr Glu Ala Pro Arg Pro Ala i5 ZCA AAG TAT CAA GAT GTT TCT TCC GAA AAG CCG AAA GCC CAA AAA GAC Pro Lys Tyr Gin Asp, Val CAA GGC GGA TAC GGT TrT Gin Gly Gly Tyr Gly Phe CAG AAG GCG AAT CCT AAM Gin Lys Ala Amn Pro Lys 60 TOG GAA CAA ACCGCAT AAT Trp Giu Gin Thr Asp An AAT ATT ATT AAT GCC TTA Asn Ile le Asn Ala Leu GAT TCC AGT CAA GAA AAT Asp 5cr Ser Gin Ciu Asn 105 AAT TTC CAA TAC GTA TG Asn Phe Gin Tyr Val Trp 120 ACA ATT OAA AAA AAC GOT Thr Ile Glu Lys Ann Gly 135 140 GAC COT TAT ATT TI'? TAT Asp Gly Tyr Ile Phe Tyr 155 GTA TTG GGA CAG OTT ACO Val Leu Cly Gin Val Thr 170 GTG AAA ATA AAT CAG AAA Val Lys Ile Ann GIn Lys 185 CCC 0CC GAC CGA TAT ACT Pro Oly Asp Arg Tyr Ser 200 AAT AAA OAT AOC GAT AAG Asn Lys Asp Ser Asp Lys 215 220 ACC GAA ATA ACG CT? OAT Thr Oiu Ile Thr Val Asp 235 Ser Ser 30 CCA ATG Ala Met 45 GAA GAT Giu Asp OCT GAT Giu Lys Pro Lys Ala Gin Lys CCC TFT AAG CCC AGG MAT TOO Arg Phe Lys Arg Arg Ann Trp, GAG ATA AMA CTC TCT CA MAT Giu le Lys Leu Ser Glu Ann ATC AAA AC CCT TCC AMA CMA Gly Asp le Lye Asn Pro Ser Lys Gin Lys so as CCT GGA AAT AT OGA GGA C ACA TTG CMA Pro Gly Ann Ann Gly Cly Ala Thr Leu Gin 95 100 CAG GO;T ATA TCT MAG GTT ACG GAC TAT CAC Gin Gly le Ser Lys Val Thr Asp Tyr His 110 115 TCG COG iTrr TT TAT AAM CAG ATT AAA MAT Ser Oly Phe Phe Tyr Lye Gin le Lye Asn 125 130 TCA TCT ATA ACC OCA CCC AGA MAC OCT CCT Ser Ser Ile Thr Ala Ala Arg Ann Gly Pro 145 150 AA GCC AMA GAT CCC TCG- AGA CAM CTC CCT Lys Oly Lys-Asp Pro. Ser Arg Gin Leu Pro 160 165 TAT MAA GCC ACT TOG CAT TTC TTA ACT OAT Tyr Lys Gly Thr Trp Asp Phe Lau Thr Asp 175 180 TrT ATA CAT TTA GCC MAT ACT TCT ACO AMA Phe Ile Asp Leu Oly Ann Thr Ser Thr Lys 190 195 OCT Ti-c TCC COG GAG iTO CAT TAT ATC CTC Ala Phe Ser Gly Oiu Leu Asp Tyr Ile Val 20S 210 AAA GAC COG CAC GTA CCA MAG GCA 'XTA ACA.

Lye Asp Cly His Val Ala Lys Cly Lau Thr 225 230 TIT GAG AMA AMA ACC CTC MAC GCA AMA TTA Phe Glu Lys Lye Thr Leu Asn Cly Lys Leu 240 245 WO 97/13860 75 PCT/FR96/01580 AT? AAA AAC AAC Ile Lys Asri An 250 ACC CAA TAC TAT Thr Gin Tyr Tyr 265 GGC AAG GCA ACG Cly Lys Ala Thr 280 CAT CCC TTT GTT OTA AGC MAT ANT GAG TTC Val Ser Ash Ann Glu Phe 255 CTT GAT GCG ACG CTT AGG Lou Asp Ala Thr Leu Arg 270 ACC GAC AAA CCT CGC ACT Thr Asp Lysn Pro Gly Thr 285 GAC TCG TCT TCT TTG AGC AAC CCT MAA TAC ACC Asn Ala Lys Tyr Thr 260 GGA AAC CGC TTC AAC Gly Asn Arg Phe Ash 275 GGA GAA ACC AAA CAA Gly Clu Thr Lys Gin 290 CCC GGC TT? TTC GGC His Pro Phe Val Ser Asp Ser Ser Ser Loeu Ser Gly Gly Phe Phe Gly 295 300 310 GAG GAA Ciu Glu 315 GTC GGC TTG GGT Leu Gly AGC C Val Ala Val Val Cly Ser Ala 330 GCG GCC GC TCA GAC GGC GAG Ala Ala Ala Ser Asp Gly Giu 345 CTA CAT CCC TCT CAA AAC CGT Leu Asp Gly Ser Glu Asn Gly 360 365 GAG CTG ACG CAC CCC CCC ACA Clu Leu Thr His Gly Cly Thr 375 380 AAC CCC CCC CAA CTC CTT OTC Asn Ala Ala Gin Lou Val Val 395 GAG OCT TCC CAA ACT CCC AAC Glu Ala Ser Ciu Ser Ciy Asn 410 GGA ACA GCC TTT ACC CGC AAA Cly Thr Ala Ph. Thr Arg Lys 425 AAA CAC ACC CAA GCA GCT ACG Lys Asp Thr Gin Ala Cly Thr 440 445 AAT ACC GCA CC? GAT ACC AJl

TTC

Phe

A

Lys

GTC

Val 350

AAG

Lys

GCA

Ala

GAC

Asp

MAT

Asn mr Phe 430

C

*Ala

GCC

CGC TTT TTC AGC CAC Arg Phe Leu Ser Asp 320 ACC CMA GAC AMA CCG Thr Gln Asp Lys Pro 335 CCC CAG CAT CMA ACC Arg Gln His Gin Thr 355 CTG ACC ACC GTT TTC Lou Thr Thr Val Leu 370 ATC AAA MAT CTC GAC Ile Lys Asn Lou Asp 385 GCC ATT ATC ATT CCC Giy Ile Met Ile Pro 400 CAA CCC MAT CMA OCT Gin Ala Asn Gin Gly 415 GAC CAC ACC CCC AMA GAT AAA AAA Asp Lys Lys 325 CCA MAT GCC Cly Ann Giy 340 CTG CGG CAC Val Arg Gin GAT CC CTC Asp Ala Val MAC TTC AC Asn Ph. 390 CC CCTr CC Leu Pro Ala 405 ACA MAT GC Thr Ann Gly 420 ACC GAT CMA 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 148 Asp His Thr Pro LYS Ser Asp Glu CC MAT CCC MAT Ala Asn Gly Ann 450 AAA ACA AAA ACC CCA CCC C TCA Pro Ala Ala Ser TAT GMA GTC CMA Tyr Ciu Val Glu Ash Thr Ala Gly Asp Thr Ann Cly Lys Thr Lys Thr 455 460 465 470 GTC TGC TCT TCC MAC CTC AXT TAT CTC AAA TAC CCC TTC CTC ACC CC WO 97/13860 Jai Cys Cys Ser AAA ACT cc CC? Lys Tkir Ala Gly 490 GCC CCC CAA ACG 76 Asn Leu 475 AAC ACG Asn Thr GAC GCG Ala Ala Gin Thr Asp Ala Gin 505 CAT GAA AAA GAG ATT CCA Asp Ciu Lys Ciu Ile Pro 520 TGC TAC GCC CAT ATT CC Trp Tyr Gly His Ile Ala 535 540 AAT CCA ACG ACT CCC AAC Asn Ala Thr Ser Cly Asn 555 AAA AAA ATT ACC CCC ATC Lys Lys Ile Thr Gly Met TTT ACC ATT GAG GGA ACC Phe Thr Ile Clu Cly Thr 585 AAA ACT C GAC TCA GCC Lys Thr Ala Asp Ser Gly 600 ACG CCT AAC OCA TAT ATC T7hr Pro Lys Aia Tyr Ile 615 620 GGG CCT AAA CCC GAA CMA

AC

Ser 525

AAC

Asn

AAG

Lys

TTA

Leu

AT?

Ile Trr Phe 605

ACA.

Thr Tyr Leia Lys Tyr 480 GGA AGC GGC MAC Gly Ser Gly Asn 495 AGT ATG TC TTA Ser Met Pkxe Leu 510 GAG CAA AAC GTC Giu Gin Asn Val AGC ACA AGC TCC Ser Thr Ser Trp 545 CC GAC ACC Ala Asp Phe Thr 560 ACC C GAA AAC Thr Ala Clu Asn 57S CAG GGC AAC CCT Gin Gly Asn Gly 590 CAT CTC CAT CAA Asp Leu Asp Cmn AAC CCC AAG CTC Asn Ala. Lys Val 62S PCT/FR96/ 01580 Gly Leu Leu Thr Arg 48S CCC ACC CCA ACC GCC 1536 CGly Ser Pro Thr Ala S00 CAA CCC GAG CGC ACC 1584 Gin Cly Ciu Arg Thr 515 GTT TAT CCC CCC TCT 1632 Val Tyr Arg Giy Ser 530 AGC GGC AAT CCT TCC 1680 Ser Cly Asn Ala Ser 550 CTC MAT TT? CCC GAG 1728* Val Asa Phe Cly Giu 565 AGO CAG C GCA ACC 1776 Arg Gin Ala Ala Thr 580 TTT TCC CCT ACO GCA 1824 Phe Ser Gly Thr Ala 595 ACC MXT ACC ACC CGC 1872 Ser Asn Thr Tkxr Cly 610 CAG GGC CC? TTT TAC 1920 Gin Gly Cly Phe Tyr ATG COT GCA TOG TT? GCT TAT CCC CCC CAC Gly Pro Lys Ala Glu Ciu Met Gly Gly Trp Phe Ala Tyr Pro Cly Asp 635 640 645 ACT CAC ACG CAG CCC 7CC C TCG CCC TCA CCC GCA TCA Ser Gin Thr Gin Arg Ser Ala Scr Cly Ser Oly Ala Scr 650 655 AGC GCG ACC GTC CTA TTC CCT C AMA CCC CMA CAG CTT Ser Ala Thr Val Val Phe Cly Ala Lys Arg Gin Gin Leu 665 670 675

TMA

INFORMATION FOR SEQ ID NO: 6: CCC CCC AMC Ala Ala Ann 660 CTG CAM Val Gin 1968 2016 2061 2064 SEQUENCE CHARACTERISTICS: LENGTH: 687 amnino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 97/13860 77 PCT/FR96/01580 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Val Leu Pro Val Ple Leu Ser Ser Ala Cj 1e Gly Pro Gin Gin Trp Asn Asp Asn Thr 135 Asp Val Val Pro Ser Lye Gly Lys Glu Ile Ser Phe 120 Ile Gly Leu Lys Gly 200 Ph. Asp Tyr Gin Gly Tyr Ala Asn Gin Thr, Ile Asn Ser Gin 105 Gin Tyr Glu Lys Tyr Ile Gly Gin 170 lie Asn 185 Asp Arg Leu Asp Giy Pro Asp Ala Glu Val Asn Phe 155 Val Gin Tyr Asp Val Phe Lys 60 An Leu Asn Trp Gly 140 Tyr Thr Lye Ser Ser Ser Ala 45 Glu Gly Pro Gin Ser 125 Ser Lys Val Asp Thr is Ser Giu Lye 30 Met Arg Phe Asp Giu Ile Asp Ile Lys 80 Gly Asn Asn 95 Gly Ile Ser 110 Gly Phe Phe Ser lie Thr Gly Lys-Asp 160 Lys Gly Thr 175 Ile Asp Leu 190 Phe Ser Gly Glu Pro Lye Lye 65 Ann Gly Lye Tyr Ala 145 Pro Leu Ala Lye Arg Leu Pro Gly Val Lye 130 Ala NO: 6: Gly Gly Gly Pro Arg Pro Ala Gin Lye Arg Asn Trp Ser Giu Asn Ser Lye Gin Ala Thr Lu 100 Thr Asp Tyr 115 Gin Ile Lye Arg Ann Gly Gly Ala Asp Tyr Asp Lys Gin His Asn Pro Ser Arg Gin Lou Pro 165 Tyr Phe Ala 205 Trp Gly Glu Val 225 Thr Leu 180 Ser Tyr Gly Gly Thr Thr Ile Leu Lye Asp Lye Val Thr 230 Leu Asn Lye Asp Ser Asp Lye 21S 220 Lye Asp Gly His Lye Thr Glu lie Thr Val Asp Phe Glu 235 Lye 240 245 Ile Lye An Asn 250 Thr Gin Tyr Tyr Ser Val Ser Leu Ser Asn Asn 255 Asp Ala Thr Giu Phe An Ala Lys Tyr 260 Leu Arg Gly Asn Arg Phe Thr Asn WO 97/13860 78 -PCT/FR96/01580 265 270 275 Lys Ala Thr Ala Thr Asp Lys Pro Gly Thr Gly Giu Thr Lys Gin 280 285 290 his Pro Phe Val SerAsp Ser Ser Ser Leu Ser Gly Gly Phe Phe Gly 295 300 305 310 Pro Lys Gly Glu Glu Leu Gly Ph. Arg Phe Leu Ser Asp Asp Lys Lys 315 320 325 Val Ala Val Val Gly Ser Ala Lys Th r Gin Asp Lys Pro Gly Asn Gly 330 335 340 Ala Ala Ala Ser Asp Gly Glu Val Arg GIn His Gin Thr Val Arg Gin 345 350 355 Leu Asp Gly Ser Glu Asn Gly Lys Leu Thr Thr Val Leu Asp Ala Val 360 365 370 Glu Leu Thr His Gly Gly Thr Ala Ile Lys Asn Leu Asp Asn Phe Ser 375 380 385 390 Asn Ala Ala Gin Lou Val Val Asp Gly Ile Met Ile Pro Lou Pro Ala 395 400 465 Glu Ala Ser Glu Ser Gly Asn Asn Gin Ala Asri Gin Gly Thr Asn Gly 410 415 420 Gly Thr Ala Phe Thr Arg Lys Phe Asp His Thr Pro Lys Ser Asp Glu 425 430 435 Lys Asp Thr Gin Ala Gly Thr Ala Ala Asn Gly Asn Pro Ala Ala Ser 440 445 450 Asn Thz- Ala Gly Asp Thr Asn Gly Lys Thr Lys Thr Tyr Giu Val Giu 45460 465 470 Val Cys Cys Ser Asn Leu Aen Tyr Leu Lys Tyr Gly Leu Leu Thr Arg 475 480 485 Lys Thr Ala Gly Asn Thr Val Gly Ser Gly Asn Gly Ser Pro Tkxr Ala 490 .495 500 Ala Ala Gin Thr Asp Ala Gin Ser Met Phe Leu Gin Gly Giu Arg Thr 505 510 515 Asp Giu Lys Glu Ile Pro Ser Glu Gin Asri Val Val Tyr Arg Gly Ser 520 525 530 Trp Tyr Gly His Ile Ala Asn Ser Thr Ser Trp Ser Gly Asn Ala Ser 535 540 545 550 Asn Ala Thr Ser Gly Asn Lys Ala Asp Phe Thr Val Asn Phe Gly Glu 555 560 565 Lys Lys Ile Thr Gly Met Leu Thr Ala Giu Asn Arg Gln Ala Ala Thr 570 575 580 WO 97/13860 79 Pne Thr Ile Glu Gly Thr Ile Gin Gly Asn Gly Phe Ser Gly Thr Ala 585 590 595 Lys Thr Ala Asp Ser Gly Phe Asp Leu Asp Gin Ser Asn Thr Thr Gly S00 605 610 Thr Pro Lys Ala Tyr Ile Thr Asn Ala Lys Val Gin Gly Gly Phe Tyr 615 620 625 630 Gly Pro Lys Ala Giu Glu Met Gly Giy Trp Phe Ala Tyr Pro Giy Asp 635 640 645 Ser Gin Thr Gin Arg Ser Ala Ser Gly Ser Gly Ala Ser Ala Ala An 650 655 660 Ser Ala Thr Val Val Phe Gly Ala Lys Arg Gin Gin Leu Val Gin 665 670 675 INFORMATION FOR SEQ ID NO: 7: SEQUENCE

CHARACTERISTICS:

LENGTH: 198 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: IM2169 WO 97/13860 80 PCT/FR96/01580 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Thr Lys Asp Lys Leu Glu Asn Gly Ala Ala Ala Ser Gly Ser Thr 1 5 10 is Ala Ala Ala Ser Gly Gly Ala Ala Gly Tkxr Ser Ser Glu Asn Ser 25 Leu Thr Thr Val Leu Asp Ala Val Glu Leu Thr Leu Asn Asp Lys 40 Ile Lys Asn Leu Asp Asn Phe Ser Asn Ala Ala Gin Leu Val Val 55 Gly Ile Met Ile Pro Leu Leu Pro Lys Asp Ser Giu Ser Gly Ass 70 75 Gin Ala Asp Lys Gly Lys ASn Gly Gly Thr Giu Phe Thr Arg Lys as 90 Glu His Thr Pro Giu Ser Asp Lys Lys Asp Ala Gin Ala Gly Thr 100 105 110 Thr Asn Gly Ala Gin Thr Ala Ser Ass Thr Ala Gly Asp Thr Msn Gly Lys Lys Asp Thr so Phe Gin cay 115 120 Lys Thr Lys Thr Tyr Glu Val Giu Val Cys Cys Ser 130 135 140 Leu Lys Tyr Gly met Leu Thr Arg Lys Ass Ser Lys 145 150 15 Ala Gly Giy Asn Ser Ser Gin Ala Asp Ala Lys Thr 165 .170 Gin Ser Met Phe Leu Gin Gly Glu Arg Thr Asp Glu 180 195 Thr Asp Gln Asn Val Val 195 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 198 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: Leu Ass Tyr Ala Met Gin 160 Gin Val Glu 175 Giu Ile Pro 190 WO 97/13860 81 ORGANISM: N. ieningitidis STRAIN: 6940 PCT/FR96/01580 (xi) SEQUENCE rhr Lys Asp Lys Thr 1 5 kla Ala Ala Ser Asn Leu Thr Thr Val Leu Val Gin Lys Leu Asp Gly Ile Met Ile Pro .ln Ala Asn Gin Gly Asp His Thr Pro Glu 100 rhr Asn Gly Ala Gin 115 Lys Thr Lys Thr Tyr 130 DESCRIPTION: SEQ ID NO: 8: Giu Asn Gly Ala Val Ala Ser Gly Gly Thr Asp Gly Thr Glu Leu Asn Ala Glu Ala Gly Thr 90 Lys Asp 105 Asn Thr Val Cys Lys Asn Glu Gly Leu Ser Thr Ala Asp 125 Asn Ser is Ser Lys Lys Glu Val Asp Asn An s0 Lys Phe Thr Gin Asn Gly Asn Tyr Met Gin 160 Val Glu 175 Glu Val 135 Leu Thr Leu Lys Tyr Gly Met 145 Ala Gly Giu Ser Ser ser Gin Ala Asp Ala Lys Thr Giu 165 170 Gin Ser Met Phe Leu Gin Giy Giu Arg Thr Asp Glu Lys 180 185 Ser Glu Gin Asn Ile Val 195 INFORMATION FOR SEQ-ID NO: 9: i) SEQUENCE CHARACTERISTICS: LENGTH: 198 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide Giu Ile Pro 190 WO 97/13860 -882 PCT/FR96/01580 (vi) ORIGINAL SOURCE: ORGANISM: N. meningiti dis STRAIN: 2223 (xi) SEQUENCE DESCRIPTION: Thr Lys Asp L~ys Thr Glu Asn Gly Ala 1 5 Ala Ala Ala Ser Asn Gly Ala Ala Gly 2S Leu Thr Thr Val Leu Asp Ala Val Glu 40 Val Gin Lys Leu Asp Asn Phe Ser Asn 55 Gly Ile Met Ile Pro Leu Leo Pro Glu 6S 70 Gin Ala Asn Gin Gly Thr Asn Gly Gly Asp His Thr Pro Glu Ser Asp Lys Lys 100 10S Ala Asn Gly Ala Gin Thr Ala Ser Asf 115 120 Lys Thr Lys Thr Tyr Glu Val Glu Val 130 135 SEQ ID NO: 9: Val Ala Ser Gly Gly Thr Asp 10 is Thr Ser Ser Glu Asn Ser Lys Leo Lys Leu Gly Asp Lys Gbu Ala Ala Gin Leu Val Val Asp Ala Ser Glu Ser Gly Asn Asn 75 so Thr Ala Phe Thr Arg Lys Phe 90 Asp Ala Gin Ala Gly Thr Gin 110 Thr Ala Gly Asp Thr Asn Gly 12S cys Cys Ser Asn Leu Asn Tyr 140 Leu Lys Tyr- Gly Met Leu Thr Arg Lys Asn Ser Lys Ser Ala Met Gin 145 I50 155 160 Ala Gly Giu Ser Ser Ser Gin Ala Asp Ala Lys Thr Glu Gin Val Gly 165 170 175 Gin Ser Met Phe Leo Gin Gly Glu Arg Thi- Asp Glu Lys Gu Ile Pro 180 185 190 Ser Glu Gin Asn Ile Val 195 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 198 amino acid TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear WO 97/13860 -883 PCT/FR96 /015 (i i) (vi) MOLECULE TYPE: peptide ORIGINAL SOUTRCE: ORGANISM: N. meningitidis STRAIN: C708 (xi) SEQUENCE DESCRIPTION: Thr Gin Asp Lys Pro Arg Asri Gly Ala SEQ ID NO: Ser Gly Ser Giu Leu Asri Gin Leu Glu Ser Phe Thr Gin Ala Gly Asp 125 Ser Asn 140 Lys Ser 145 Ala Gin Ser Gin Ala Asp Ala Lys Thr Glu Gin Val Gly 170 175 Gin Gly Giu Arg Thr Asp Giu Lys Glu Ile Pro 185 190 Asn Asp Gin Asn Val Val 195 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: WO 97/13860 84 PCT/FR96/01580 LENGTH: 211 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE:. peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: M978 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Thr Gin Asp Lys 1 Thr Asp Ala Ala Lys Leu Thr Lys Lys Ile Lys Val Asp Gly Ile Ser Asn Gin Ala Gly Thr Asp Phe 100 Lys Asp Thr Lys 115 Thr Asp Leu Gly 130 Thr Tyr Giu Vai 145 Gly Met Leu Thr Asn Ser Sez- Gin 150 Al a 5 Al a Thr Asn Met Asp Thr Ala Lys Glu Arg 165 AJla Ala Asn Ser Asn Val Leu Leu Asp 5 Ile Pro 70 Lys Gly Tyr Lys Gin Thr Ala Asp 135 Val Cys

ISO

Lys Asn Asp Ala Gly Gly Asp 40 Asn iLeu Lys Thr Gly 120 Val Cys Ser Lys Asn Ala 25 Ala Phe Leu Lys Thr 105 Al a Aen Se r Lys Thr 185 Thr Ala Val Se r Pro Gly 90 Tyr Ala Gly Ser 170 Gi u Alia Glu Asri Glu 75 Lys Thr Gly Gly Leu 155 Ala Thr Leu Ala Thr Asn Pro Ser Lys 140 Ala Ser Scr Ser Thr Leu Ala Gin Ser Giu Gly Lys Lys Asn 110 Ser Gly 125 Ala Giu Tyr Leu Gly Glu Asn Leu Se r Asa Asp Ala Thr Lys Gly Asn Asp Val Gly Gly Asp Gin Lys Tyr Gin Ala Gly Gly 175 Glu Gin Ser Met 190 Pro Asn Asp Gin 205 Phe Leu Gin Gly 195 Asn Val Val 210 Glu Arg Thr Asp 200 Giu Lys Giu Ile WO 97/13860 85 PCT/FR96/01580 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 200 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: 1610 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: WO 97/13860 Lys Arg 1 Thr Asp Ser Lys Lys Glu so Val Asp Asn Thr LYS Phe Thr Gin Asn Gly 130 Asn Tyr 145 Lys Ala 5 Ala Ala Thr Thr Lys Asn Ile Met Ala Asp His Thr 100 Asn Gly Thr Lys Lys Tyr -86 Glu Ser Gly GI Ser Asn Gly Al 25 Val Leu Asp Al Leu Asp Asn Pl, Ile Pro Leu Lie 70 Lys Gly Lys As Pro Giu Ser As 10 Ala Gin Thr Al 120 Thr Tyr Glu Va 135 Gly Leu Leu Tk I50 Gly Asn 10 Ala Gly Val Glu Ser ASn Pro Lys 75 Gly Gly Lys Lys Ser Asn Glu Val Arg Lys 155 PCT/FR96/O1580 Ala Ser Gly Gly is Ser Ser Glu Asn Lys Ser Miy Gly Ala Gin Leu Val Ser Giu Ser Gly s0 Lys Phe Thr Arg Ala Gin Ala Gly 110 Ala Gly Asp Thr 125 Cys Ser Asn Leu Ala Gly Asn Thr 160 Gly Giu Gly Gly Asr. Gly Ser Gin Thr A-a Ala Al.

165 I-0 Gly Ala Gin Ser Met pkie Leu Gin Gly Glu Arg Th 180 185 Ile Pro Ser Giu Gin Asn Val Val 195 200 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 200 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide, (vi) ORIGINAL SOURCE:- ORGANISM: N. meningitidis STRAIN: 86-7 a Ala Gin 175 Lys Glu WO 97/13860 87- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: PCT/FR96/01580 13: Thr Lys Asp Lys Pro Arg Asn Gly Ala Ala Leu Ile Gly Gin Asp Thr Gin Ala Thr Lys Ile Ala His Asri Ala 130 Ala Thr Asn Met Asp Thr Gly 115 Gly Ser Val Leu Ile Lys Pro 100 Ala Lys Asn Leu Asp Pro Gly Lys Gin Thr Ala Ala Gly Ala Phe 55 Met Asn Asp Ala Ala 135 Val 40 Ser Pro Gly Glu Ser 120 Val 25 Clu Asn Giu Gly Lys 105 Gly Glu Val Ala Ser Thr Ser Ser Leu Thr Leu Ala Ala Gin Thr Ser Giu 75 Thr Ala Phe 90 Asp Thr Gin Thr Ala Gly Val Cys Cys 140 Lys Thr Ala 155 Gly Gly Glu Asn Asn Asp Leu Val Ser Gly Thr Arg Ala Gly 110 Val Thr 125 Ser Asn Asp Asn Thr Asp Gly Lys Lys Lys Val Ser Asn Asn s0 Lys Phe Thr Pro Gly. Gly Leu Asn Thr Val 160 Tyr Leu Lys Tyr Gly Leu Leu Thr Arg 145 150 Gly Ser Gly Asn Gly Ser Ser Thr Ala Ala Ala Gin Thr Ala Gin Gly 165 170 Ala Gin Ser Met Phe Leu Gin Gly Glu Arg Thr Asp Glu Lys 1SO 185 190 Pro Lys Glu Gin GIn Asp Ile Val 195 200 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 198 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOUR CE: ORGANISM: N. meningitidis 175 Giu Ile WO 97/13860 88 STRAIN: S3032 PCT/FR96/01580 (xi) Thr Lys

I

Thr Asp Ser Lys Thr Ala so Val Asp A-sn Asn Lys Thr Thr Val A-sn Gly 130 A-sn Tyr 145 Val Gly

SEQUENCE

Asp Lys Pro 5 Ala Ala Ala Leu Thr Thr Ile Lys Asn Gly Ile met Gin Pro Asp Thr Tyr Thr 100 Thr Gly Gly 115 Lys Thr Lys Leu Lys Tyr Ser Gly A-sn 165 DESCRIPTION: SEQ ID NO: 14: Ala Asn Gly A-sn Thr Ala Glu Ala Ser Gly Gly Ser Gly Vai Leu Leu Asp 55 Ile Pro 70 Gin Gly Pro Lys Thr Gin Thr Tyr 135 Gly Leu

ISO

Ser Ser Gly Asp 40 A-sn Leu

LYS

A-sn Thr 120 Giu Leu Pro A-ia 25 Ala Phe Leu A-sn Asp 105 A-ia Val Thr Thr 10 Ala Val Ser Pro Gly 90 Asp Se r Giu Arg Ala 170 Giy Glu A-sn Gin 75 Gly Lys A-sn Val Lys 155 A-la Thr Ser Leu Thr A-la A-ia A-sn Ser Thr Ala Asp Thr Thr A-ia 125 Cys Cys 140 Thr Alia Ala Gin Ser His Gln Thr Phe Lys 110 Gly Ser Gly Thr is Glu Giy Leu Gly Ile A-ia Asp A-sn A-sn Asp 175 A-sn cay Val Lys Tyr.

Gin A-la Leu Thr 160 Ala Gin Ser Ser Giu Met Gin 195 Phe Leu Gin Gly Giu Arg Thr Asp Glu A-sn Lys Ile Pro ISO 185 190 A-sn Val Val WO 97/13860 89 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 195 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: 891 PCT/FR96/01580 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: WO 97/13860 Thr Lys A~ 90 PCT/FR96/01580 Ala Ala Arg Cys ip Lys Pro Gly Asn Gly Ala Arg Leu Gin is Gly Thr Gin Ile Al a His Asn Thr Tyr 14 5 Asn Thr Thr Lys Met Asp Thr Gly Lys 130 Gly Ser Ser Vai Leu Ile Lys Pro Ala Thr Leu Ser Asn Leu Asp Pro Gly Giu 100 Gin Tyr Leu Pro Gly Asp Asn Leu Lys Ser Thr Glu Thr Thr Ala Ala Phe Leu 70 Asn Asp Ala Val Arg 150 Ala Ala Val Ser 55 Pro Gly Giu Ser Aan 135 Lys Ala Gly Gin 25 Giu Leu 40 Asn Ala Lys Asp Giu Thr Lys Asp 105 Asn Thr 120 Lou Cys Thr Ala Gin Thr Ser Lys2 Ala Ser Glu 90 Ala Ala Ser Gly Ala 170 Ser Leu Gin Glii 75 Phe Gin Gly Asn Asn 155 Gin Giu Gly Leu Ser Thr Ala Asp Leu 140 Thr Gly ksn ksp Val Gly Arg Gly Thr 125 An Gly Ala Ser Lys Val Lys Lys Thr 110 Asn Tyr Glu Gln Lys Glu Asp Asn Pile Pro Gly Leu Gly Se r 175 Leu Val Gly Gin s0 Glu~ Ser Lys Lys Gly 160 Met Pile Leu Gin Gly 180 Glu Arg Thr Asp Lys Giu Ile Pro Asn Asp Gin 190 Asn Val Val 195 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 97/13860 91 PCT/FR96/01580 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AAACCCGGAT CCGTTGCCAG CGCTGCCGT 29 INFORMATION FOR SEQ ID NO: 17: SEQUENCE

CHARACTERISTICS:

LENGTH: 85 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TTTTTTCATG AGATATCTGG CAACATTGTT GTTATCTCTG

GCGGTGTTAA

TCACCGCCGG GTGCCTGGGT GGCGGCGGCA GTTTC INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GTGTTTTTGT TGAGTGCATG CCTGGGTGGC INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleotide WO 97/13860 92 PCT/FR96/01580 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TGCGCAAGCT TACAGTTTGT CTTTGGTTTT CGCGCTGCCG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAAAAGCATG CATAAAAACT ACGCGTTACA CCATTCAAGC INFORMATION FOR SEQ ID NO: 21: SEQUENCE

CHARACTERISTICS:

LENGTH: 39 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TATATAAGCT TACGTTGCAG GCCCTGCCGC GTTTTCCCC 39 S INFORMATION FOR SEQ ID NO: 22: WO 97/13860 93 PCT/FR96/01580 SEQUENCE

CHARACTERISTICS:

LENGTH: 29 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CCCGAATTCT GCCGTCTGAA GCCTTATTC 29 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CCCGAATTCT GCTATGGTGC TGCCTGTG 28 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: CGCATCCAAA ACCGTACCTG TGCTGCCTGA 3C WO 97/13860 94 PCT/FR96/01580 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTTATCACTT TCCGGGGGCA GGAGCGGAAT INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GTTGGAACAG CAGACAGCGG TTTGCGCCCC INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) WO 97/13860 95 PCT/FR96/01580 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GAACATACTT TGTTCGTTTT TGCGCGTCAA INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CCACGGATCC TGCCGTCTGA AGCCTTATTC INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CGCGGATCCT GCTATGGTG 19

Claims (11)

1. An isolated N. meningitidis Tbp2 protein, wherein the isolated Tbp2 protein is encoded by a Tbp2 DNA having an open reading frame of about 2.1 kb and is not recognized by an antiserum obtained against a Tbp2 protein of the N. meningitidis M982 strain in which amino acid sequences 362-379, 418-444, 465-481, and 500-520 have been deleted.

2. An isolated N. meningitidis Tbp2 protein, wherein the isolated Tbp2 protein is encoded by a Tbp2 DNA having an open reading frame of about 2.1 kb and is not recognized by an antiserum obtained against a Tbp2 protein of the N. meningitidis M982 strain consisting of amino acids 1-350.

3. The Tbp2 protein according to claim 1 or 2, wherein the Tbp2 protein is S recognized by both an antiserum obtained against a human transferrin receptor of the N. meningitidis M982 strain and an antiserum obtained against a human transferrin receptor of the meningitidis B16B6 strain.

4. The Tbp2 protein according to one of claims 1 to 3 comprising an amino acid sequence which is from 60% to 70% identical to the amino acid sequence of SEQ ID NO: 2. The Tbp2 protein according to claim 4, wherein the amino acid sequence is about identical to the amino acid sequence of SEQ ID NO: 2.

6. The Tbp2 protein according to claim 5, comprising the amino acid sequence of SEQ ID NO: 6. -97-

7. The Tbp2 protein according to one of claims 1 to 6, wherein the Tbp2 protein is associated with a Tbpl protein of the N. meningitidis strain of the Tbp2 protein.

8. A Tbp2 polypeptide according to one of claims 1 to 6 which can bind to human transferrin, wherein at least one amino acid in the region of the first forty amino acids or in the region of the second or third domain has been deleted.

9. The Tbp2 polypeptide according to claim 8, wherein at least one amino acid in the second domain has been deleted. The Tbp2 polypeptide according to claim 9, wherein at least one amino acid from the regions corresponding to amino acids 388-396, 418-476, and 499-521 of the N. meningitidis M982 amino acid sequence has been deleted. be 11. The Tbp2 polypeptide according to claim 6 comprising an amino acid sequence which is from 70 to 100% identical to the amino acid sequence comprising amino acids 1-376,

386-406, 466-487 and 509-677 ofSEQ ID NO: 6. 12. The Tbp2 polypeptide according to claim 9, wherein at least one amino acid from the hypervariable region of the second domain has been deleted. be* 13. The Tbp2 polypeptide according to claim 12, wherein the polypeptide contains A the first and third domains. 14. The Tbp2 polypeptide according to claim 13 comprising an amino acid sequence which is from 70 to 100% identical to the amino acid sequence comprising amino acids 1-350, 369-406, 434-453, 471-488, and 509-677 of SEQ ID NO: 6. -98- The Tbp2 polypeptide according to claim 8, wherein at least one amino acid from the second domain and/or third domain has been deleted. 16. The Tbp2 polypeptide corresponding to claim 6 comprising the amino acid sequence of the N-terminal region of SEQ ID NO: 6, said sequence beginning with an amino acid located at position 1 to 40 and ending with an amino acid located at position 334 to 351. 17. The Tbp2 polypeptide corresponding to claim 16 wherein the amino acid sequence begins with the amino acid located at position 1 and ends with an amino acid located at position 334 to 351. 18. A pharmaceutical composition comprising either a Tbp2 protein according to one of claims 1 to 7 or a polypeptide according to one of claims 8 to 17 and a suitable carrier 19. The pharmaceutical composition according to claim 18 further comprising an Sadditional Tbp2 protein that is recognized by anti-M982 human transferrin receptor antiserum and not recognized by anti-B 16B6 human transferrin receptor antiserum. 20. The pharmaceutical composition according to claim 19 wherein the additional Tbp2 protein comprises an amino acid sequence which is from 90% to 100% identical to the amino acid sequence of SEQ ID NO: 2. 21. The pharmaceutical composition according to claim 19 wherein the additional Tbp2 protein comprises the amino acid sequence of SEQ ID NO: 2. 22. The pharmaceutical composition according to claim18 further comprising an additional Tbp2 polypeptide that can bind to human transferrin and is recognized by anti-M982 human transferrin receptor antiserum but not recognized by anti-B16B6 human transferrin -99- receptor antiserum, wherein at least one amino acid in the C-terminal region of the additional Tbp2 polypeptide has been deleted. 23. The pharmaceutical composition according to claim2 2 wherein at least one amino acid from the hypervariable region of the second domain of the additional Tbp2 polypeptide has been deleted, said deletion substantially affecting that region. 24. The pharmaceutical composition according to claim22 wherein at least one amino acid from the second domain or third domain of the additional Tbp2 polypeptide has been deleted, said deletion substantially affecting that region. The pharmaceutical composition according to any one of claims 22-24wherein the additional Tbp2 polypeptide comprises an amino acid sequence which is from 90% to 100% identical to the amino acid sequence of SEQ ID NO: 2. S* 26. The pharmaceutical composition according to claim 19 wherein the additional Tbp2 polypeptide is derived from M982 Tpb2. 27. The pharmaceutical composition according to claim 8 further comprising an additional Tbp2 protein that is recognized by anti-B16B6 human transferrin receptor antiserum and not recognized by anti-M982 human transferrin receptor antiserum. S2 28. The pharmaceutical composition according to claim 27 wherein the additional S Tbp2 protein comprises an amino acid sequence which is from 95% to 100% identical to the amino acid sequence of SEQ ID NO: 4. 29. The pharmaceutical composition according to claim 27 wherein the additional Tbp2 protein comprises the amino acid sequence of SEQ ID NO: 4. -100- The pharmaceutical composition according to claim 18 further comprising an additional Tbp2 polypeptide that can bind to human transferrin and is recognized by anti-B16B6 human transferrin receptor antiserum but not recognized by anti-M982 human transferrin receptor antiserum, wherein at least one amino acid in the C-terminal region of the additional Tbp2 polypeptide has been deleted. 31. The pharmaceutical composition according to claim 30 wherein at least one amino acid from the second domain or third domain of the additional Tbp2 polypeptide has been deleted, said deletion substantially affecting that region. 32. The pharmaceutical composition according to any one of claims 29-31 wherein the additional Tbp2 protein comprises an amino acid sequence which is from 95% to 100% identical to the amino acid sequence of SEQ ID NO: 4. 33. The pharmaceutical composition according to claim 32 wherein the additional Tbp2 protein is derived from the B16B6 Tbp2. 34. The pharmaceutical composition according to any one of claims 19-33. 35. The pharmaceutical composition according to claim3 4 comprising: a polypeptide according to any one of claims 9-14; (ii) the additional polypeptide described in claim 23 or in claims 23and 25or 2 6; and (iii) the additional Tbp2 protein described in any one of claims 27-29. -101- 36. An isolated DNA coding for a Tbp2 protein according to any one of claims 1 to 6 or for a polypeptide according to any one of claims 8 to 17 37. The Tbp2 protein according to claim 5, wherein said amino acid sequence is from to 100% identical to the amino acid sequence of SEQ ID NO: 6. 38. The Tbp2 protein according to claim 7 wherein said amino acid sequence is about 95% identical to the amino acid sequence of SEQ ID NO: 6. 39. The Tbp2 protein according to claim 11, wherein said amino acid sequence is from 80 to 100% identical to the amino acid sequence comprising amino acids 1-376, 386-406,

466-487, and 509-677 of SEQ ID NO:6. 40. The Tbp2 protein according to claim 39 therein said amino acid sequence is to 100% identical to the amino acid sequence comprising amino acids 1-376, 386-406, 466-487, and 509-677 of SEQ ID NO:6. 41. The Tbp2 protein according to claim 40 wherein said amino acid sequence comprises amino acids 1-376, 386-406, 466-487, and 509-677 of SEQ ID NO: 6. *so. 42. The Tbp2 protein according to claim 14, wherein said amino acid sequence is to 100% identical to the amino acid sequence comprising amino acids 1-350, 369-406, 434-453,

471-488, and 509-677 of SEQ ID NO: 6. 43. The Tbp2 protein according to claim 42wherein said amino acid sequence is to 100% identical to the amino acid sequence comprising amino acids 1-350, 369-406, 434-453, 471-488, and 509-677 of SEQ ID NO: 6. -102- 44. The Tbp2 protein according to claim 43 wherein said amino acid sequence comprises amino acids 1-350, 369-406, 434-453, 471-488, and 509-677 of SEQ ID NO: 6. An isolated polypeptide according to any one of claims 1-17, 37-44, substantially as hereinbefore described with reference to the figures and/or examples. 46. A pharmaceutical composition according to any one of claims 18-36, substantially as hereinbefore described with reference to the figures and/or examples. 47. An isolated DNA according to claim 36, substantially as hereinbefore described with reference to the figures and/or examples. DATED thisELEVENTH day of APRIL, 2000 Aventis Pasteur, S.A. DAVIES COLLISON CAVE PATENT ATTORNEYS FOR THE APPLICANT 9 9 9 9.99