AU706090B2

AU706090B2 - Tbp2 fragments of the transferrine receptor of neisseria meningitidis

Info

Publication number: AU706090B2
Application number: AU26757/95A
Authority: AU
Inventors: Eric Jacobs; Michele Legrain; Ling Lissolo; Veronique Mazarin; Marie-Jose Bernadette Jacqueline Millet
Original assignee: Transgene SA; Pasteur Merieux Serum et Vaccines SA
Current assignee: Transgene SA; Sanofi Pasteur SA
Priority date: 1994-05-31
Filing date: 1995-05-30
Publication date: 1999-06-10
Anticipated expiration: 2015-05-30
Also published as: WO1995033049A2; NO960332L; WO1995033049A3; FI960428A; HUT75992A; FR2720408B1; FI960428A0; NO960332D0; AU2675795A; JPH09501059A; HU220116B; EP0720653A1; CA2167936A1; FR2720408A1

Description

WO 95/33049 PCT/FR95/00701 Tbp2 FRAGMENTS OF THE TRANSFERRIN RECEPTOR OF NEISSERIA MENINGITIDIS The present invention relates to polypeptides derived from the Tbp2 subunit of the transferrin receptor of Neisseria meningitidis, to their use in a therapeutic capacity, in particular for vaccination, as well as to the DNA fragments coding for these polypeptides.

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 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, 90 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 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.

WO 95/33049 2 PCT/FR95/00701 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 REPLACEMENT SHEET (RULE 26) man (of the order of 10- 18 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, referred to as the transferrin receptor, and the polypeptides constituting it, as 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.

Furthermore, 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 membrane extracts of several tens of strains of N. meningitidis of miscellaneous origins. These membrane extracts were first A I WO 95/33049 3 PCT/FR95/00701 subjected to polyacrylamide gel electrophoresis in the presence of SDS, and then electrotransferred onto nitrocellulose membranes. 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 gel after electrophoresis in the presence of SDS; the bands are characterized by their apparent molecular weights expressed in kilodaltons (kD): Strains Table I 2394(B;2a;Pl.2:L2,3) 2234 nd) 2228 nd) 2154 nd) 550 (C;2a:) 2170 (B;2a:Pl.l:L3) 2448 nd) 179 (C;2a:P1.2) Detection with 93 93 99 anti-2394 receptor antiserum 68 69 69 Detection with anti-2169 93 93 99 receptor antiserum Detection with peroxidase- 68 69 69 transferrin NB: In brackets, the serogroup, serotype, subtype and immunotype are shown in order.

rv Strains Table 11 2169 1000 1604 132 1001 876 1951 2449 867 (B:9:Pl.9) (B:nd) (B:nd) (C:15:P1.16) (A:4:Pl.9) (E:19:Pl.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 antiserum 87 85 83 81 79 88 87 85 Detection with peroxidase- 87 85 83 81 79 88 87 85 trans ferrin NB: In brackets, the serogroup, serotype, subtype and immunotype are shown in order.

WO 95/33049 5 PCT/FR95/00701 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, 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, 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.

In conformity with this, reference will be made in the text which follows to IM2169 type or IM2394 type strains.

Besides the strains mentioned in Table II, IM2169 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 the free gift of 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).

The strain IM2154 (serogroup C) is mentioned by way of example as being of the IM2394 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 01e WO 95/33049 6 PCT/FR95/00701 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 two receptor subunits (Tbp2) appears to be capable of performing this function.

The amino acids sequences of the Tbp2 subunits of the strains IM2169 and IM2394 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.

In SEQ ID NO 5 to 10, the sequences of the Tbp2 subunits of the IM2169 type strains, namely the strains M978, 6940 and S3032, are presented.

It is shown, furthermore, that the sequence of the IM2154 (IM2394 type) Tbp2 subunit differs by two amino acids from the sequence of the IM2394 Tbp2 subunit, at positions 306 and 510.

It has now been found that a Tbp2 subunit, irrespective of the strain of origin, possesses in structural terms three main domains, at least one of which is associated with special properties. By definition, the domains of IM2169 Tbp2 and IM2394 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.

Tbp2 Im2169 Tbp2 IM2394 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 IM2169 WO 95/33049 7 PCT/FR95/00701 or IM2394 type Tbp2's, after alignment of an IM2169 or IM2394 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 M978 as follows: first domain (1- 346), second domain (347-557) and third domain (558-705).

Furthermore, it was also found that the N-terminal domain or first domain and/or the hinge domain or second domain could be necessary and sufficient for the purpose of inducing a vaccinal effect in humans; accordingly, it would not be essential to use a Tbp2 in a complete form. It was found in particular that the first domain contained the transferrin binding site virtually in its entirety, was hence very probably exposed to the outside and consequently constituted a component of choice for vaccinal purposes.

Lastly, it was found that certain regions of the second domain of the IM2169 type Tbp2's were quite generally variable and immunodominant. Two approaches are hence possible with a view to a vaccine; either it is considered that the immunodominant epitopes may mask other epitopes of vaccinal interest, and they are consequently deleted, or use is made of this variability in order to retain only these regions in a vaccine.

Accordingly, the invention provides a polypeptide having an amino acid sequence which is derived from that of a Tbp2 subunit of the transferrin receptor of an IM2169 or IM2394 type N. meningitidis strain whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394; in particular by total or partial deletion of at least one domain of the said IM2169 or IM2394 type Tbp2 subunit, provided the first and second domains are not totally deleted simultaneously.

"Sequence which is derived from another sequence" is obviously understood to mean a sequence originating from this other sequence by the use of a mental process.

More especially, a polypeptide according to the WOo 95/33049 8 PCT/FR95/00701 invention possesses an amino acid sequence which is derived from an IM2169 or IM2394 type Tbp2 subunit: in particular by total or partial deletion of at least one domain of the said Tbp2 subunit, selected from the second and third 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 0 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; Al1, 02, A3; 1, 2, 03; 1, 02, 03; 1, A2, 03; 01, 2, 03; 01, 02, 03; 01, A2, 03; Al1, 2, 03; Al1, 02, 03.

Interest also attaches to a polypeptide according to the invention derived from an IM2169 type Tbp2 subunit by partial deletion of the second domain, which contains in their entirety or virtual entirety the first and third domains; that is to say the combination 1, 02, WO 95/33049 9 PCT/FR95/00701 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 the region homologous to that of IM2169 Tbp2 extending from the amino acid at position 1 to the amino acid approximately at position When a polypeptide according to the invention is derived, in particular, by partial deletion of the second domain of an IM2169 type Tbp2 subunit, this partial deletion advantageously affects one or more regions of the second domain which is/are the homologue(s) of the regions of the IM2169 sequence extending: from the amino acid at position 362 to the amino acid at position 379; (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 simultaneously affects the four regions to (iv) described above.

When a polypeptide according to the invention is derived, in particular, by complete deletion of the third domain and virtually complete deletion of the second domain of an IM2169 type Tbp2 subunit and contains the whole of the first domain, or is derived, in addition, by deletion of the N-terminal portion of the first domain, the virtually complete deletion of the second domain extends over the region which: in the case of a polypeptide derived from an IM2169 type Tbp2 subunit, is the homologue of the region of the second domain of the IM2169 Tbp2 subunit extending from the amino acid in one of the positions 346 to 361 to the amino acid at position 543; WO 95/33049 10 PCT/FR95/00701 -in the case of a polypeptide derived from an IM2394 type Tbp2 subunit, is the homologue of the region of the second domain of the IM2394 Tbp2 subunit extending from the amino acid in one of the positions 326 to 341 to the amino acid at position 442.

When a polypeptide according to the invention is derived, in particular, by partial deletion of the first domain of an IM2169 or IM2394 type Tbp2 subunit, this partial deletion advantageously affects all or part of the region: which is the homologue of the region of the first domain of the said IM2169 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid at position 281; or (ii) which is the homologue of the region of the first domain of the said IM2394 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid at position 266.

By way of example of the foregoing, a deletion of interest affecting the region: which is the homologue of the region of the first domain of the said IM2169 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid approximately at position 40; or (ii) which is the homologue of the region of the first domain of the said IM2394 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid approximately at position may be mentioned.

The IM2169 or IM2394 type sequence from which that of a polypeptide according to the invention is derived displays a degree of homology with the respective reference sequence, IM2169 or IM2394, advantageously of at least 70-75 preferably of at least 80 and, as a more special preference, of at least 90 7 7*1-' A(T wO 95/33049 11 PCT/FR95/00701 According to a most especially preferred embodiment, a polypeptide according to the invention possesses a sequence derived from that of the IM2169 or IM2394 Tbp2 subunit.

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 Figures 1 to 4 and 8 to 10. 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 described 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, 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 ID SEQ NO 1, from the amino acid at position 1 to the amino acid at position 345; (ii) the sequence as shown in ID SEQ NO 3, from the amino acid at position 1 to the amino acid at position 325 or 442; (iii) the sequence as shown in ID SEQ NO 1, from the amino acid at position 1 to the amino acid at position 691 or 543, from which the regions 362-379, 418-444, 465-481 and 500- 520 have been deleted; (iv) the sequence as shown in ID SEQ NO 1, from the amino acid at position 346 to the amino acid at position 543, WO 95/33049 12 PCT/FR95/00701 may be mentioned.

Polypeptides corresponding to the definition given in the paragraph above are illustrated as follows: A polypeptide according to the invention whose sequence is substantially as shown in ID SEQ NO 1, 5, 7, 9, 36 or 38, from the amino acid at position 1 to the amino acid at position 350, 351, 354, 358, 322 or 346, respectively; (ii) A polypeptide according to the invention whose sequence is substantially as shown in ID SEQ NO 3, from the amino acid at position 1 to the amino acid at position 330; (iii) A polypeptide according to the invention whose sequence is substantially as shown in: ID SEQ NO 1, from the amino acid at position 1 to the amino acid at position 691, from which the regions 362-379, 418-444, 465-481 and 500-520 have been deleted; ID SEQ NO 5, from the amino acid at position 1 to the amino acid at position 705, from which the regions 365-382, 421-453, 474-495 and 514-534 have been deleted; ID SEQ NO 7, from the amino acid at position 1 to the amino acid at position 693, from which the regions 366-383, 422-448, 469-485 and 504-524 have been deleted; ID SEQ NO 9, from the amino acid at position 1 to the amino acid at position 699, from which the regions 372-389, 428-454, 475-491 and 510-529 have been deleted; ID SEQ NO 36, from the amino acid at position 1 to the amino acid at position 699, from which the regions 339-356, 395- 421, 443-458 and 477-497 have been deleted; or ID SEQ NO 38, from the amino acid at position 1 to the amino acid at position 699, from which the regions 363-380, 429- WO 95/33049 13 PCT/FR95/00701 445, 467-482 and 501-521 have been deleted; and A polypeptide according to the invention whose sequence is substantially as shown in: (iv) ID SEQ NO 1, from the amino acid at position 346 to the amino acid at position 543, ID SEQ NO 5, from the amino acid at position 347 to the amino acid at position 557, ID SEQ NO 7, from the amino acid at position 350 to the amino acid at position 557, ID SEQ NO 9, from the amino acid at position 354 to the amino acid at position 551, ID SEQ NO 36, from the amino acid at position 323 to the amino acid at position 521, or ID SEQ NO 38, from the amino acid at position 345 to the amino acid at position 544.

Particular polypeptides corresponding to the definitions in points to (iv) are described in the examples which follow.

A polypeptide according to the invention possesses an amino acid sequence which comprises at least advantageously at least 20, preferably at least and, as an absolute preference, at least 100, amino acids.

Obviously, a polypeptide according to the invention can also comprise, additionally, an amino acid sequence which does not display homology with the sequences of the Tbp2 subunits of strains IM2169 and IM2394; which sequences are shown in ID SEQ NO1 and 3 from the amino acid at position 1 to the amino acid at the C-terminal position.

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

WO 95/33049 14 PCT/FR95/00701 For example, an additional sequence can 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 ID SEQ NO 1 to 4. Furthermore, it may be pointed out that a suitable heterologous signal sequence can be a signal sequence of a gene coding for a lipoprotein.

The subject of the invention is also: an isolated DNA fragment coding for 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 a suitable 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.

"Isolated DNA fragment" means that a DNA fragment according to the invention is not integrated in a DNA fragment coding for a complete Tbp2 subunit.

In the expression cassette, the DNA fragment according to the invention can 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 a person skilled in the art. His general competence will enable him to choose a particular signal region or promoter which will be suited to the host cell in which he envisages expression.

For the purposes of the method according to the invention, the host cell can be a mammalian cell, a WQO 95/33049 15 PCT/FR95/00701 bacterium or a yeast, the latter two being preferred.

Here too, the choice of a particular line is within the capacity of a person skilled in the art.

The invention also relates to a monoclonal antibody: capable of recognizing an epitope present in the first domain of an IM2169 or IM2394 type Tbp2 subunit; the said epitope having a sequence homologous to that present in the first domain of the Tbp2 subunit of the strain IM2394 and selected from YKGTW (SEQ ID NO 32), EFEVDFSDKTIKGTL (ID SEQ NO 33), EGGFYGPKGEEL (ID SEQ NO 34) and AVFGAK (ID SEQ NO 35); and optionally, (ii) incapable of recognizing the epitope present in the third domain of the said IM2169 or IM2394 type Tbp2 subunit, whose sequence is homologous to that of the epitope of the first domain which is recognized.

In order to illustrate the above point it may be pointed out by way of example that the sequences of the third domain of the IM2394 Tbp2 subunit which are homologous, two by two, to those of the first domain occur at positions 443-447, 472-485, 537-548 and 568-573, respectively.

Preferably, a monoclonal according to the invention is: capable of recognizing the region present in the first domain of an IM2169 or IM2394 type Tbp2 subunit whose sequence is homologous to the sequence EGGFYGPKGEEL present in the first domain of the Tbp2 subunit of the strain IM2394; and optionally, (ii) incapable of recognizing the epitope present in the third domain of the said IM2169 or IM2394 type Tbp2 subunit, an epitope equivalent to the one which is recognized, whose sequence is homologous to the sequence SGGFYGKNAIEM present in the third domain of the Tbp2 subunit of the WO 95/33049 16 PCT/FR95/00701 strain IM2394.

A preferred monoclonal is: capable of recognizing the epitope GFYGPK present in the first domain of a Tbp2 subunit of the strain IM2394; and (ii) incapable of recognizing the equivalent epitope present in the third domain of the said IM2394 Tbp2 subunit.

In effect, such a monoclonal has been recognized as bactericidal, and consequently it is possible to envisage using it as active principle in a pharmaceutical composition in passive immunotherapy to combat an N.

meningitidis infection.

Lastly, the invention also relates to a pharmaceutical composition comprising as active principle at least one polypeptide according to the invention.

A pharmaceutical composition according to the invention is, in particular, 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 advantageously comprises as active principle at least two polypeptides according to the invention; that is to say at least one first polypeptide whose sequence is derived from that of an IM2169 type Tbp2 subunit, and at least one second polypeptide whose sequence is derived from that of an IM2394 type Tbp2 subunit. Alternatively, a composition according to the invention can also contain at least one polypeptide whose sequence is derived from that of an IM2169 type Tbp2 subunit and at least one IM2394 type Tbp2 subunit.

As regards the IM2394 type polypeptide, a component of the pharmaceutical composition, it is greatly preferable for this polypeptide to contain all or part of the sequence which is homologous to that of the first domain of the IM2394 Tbp2 subunit from which it is derived. The portion of the sequence which should preferably be maintained is the homologue of the region of the WO 95/33049 17 PCT/FR95/00701 IM2394 Tbp2 subunit extending from the amino acid at position 267 to the amino acid at position 325. The sequence of such a polypeptide can be derived from that of an IM2394 type Tbp2 subunit, in particular by total or partial deletion of the region of the second or third domain of the IM2394 type Tbp2 subunit.

Thus, for the purposes of a pharmaceutical composition containing two types of component (IM2394 type and IM2169 type), the following IM2394 type polypeptides are more especially preferred: 1, 2, 03; 1, 2, A3; 1, 02, A3; 1, A2, A3 01, 2, 03; 01, 2, A3; 01, 02, A3; 01, A2, A3.

As regards the IM2169 type polypeptide, a component of the pharmaceutical composition, two preferred approaches are possible: Either to combine with the IM2394 type polypeptide a polypeptide which contains all or part of the sequence which is homologous to that of the first domain of the IM2169 Tbp2 subunit from which it is derived. In this case, the portion of the sequence which should preferably be maintained is the homologue of the region of the IM2169 Tbp2 subunit extending from the amino acid at position 282 to the amino acid at position 345. The sequence of such a polypeptide may be derived from that of an IM2169 type Tbp2 subunit, in particular by total or partial deletion of the region of the second or third domain of the IM2169 type Tbp2 subunit.

Thus, according to this alternative and for the purpose of a pharmaceutical composition containing two types of component (IM2394 type and IM2169 type), the following IM2169 type polypeptides are more especially preferred: 1, 2, 03; 1, 2, A3; 1, 02, A3; 1, A2, A3 01, 2, 03; 01, 2, A3; 01, 02, A3; 01, A2, A3.

1, 02, 3; 01, 02, 3.

WO 95/33049 18 PCT/FR95/00701 As regards the last two possibilities 02, 3; 01, 02, the partial deletion of the second domain may very advantageously affect one or more regions of the second domain which is/are the homologue(s) of the regions of the IM2169 sequence extending: from the amino acid at position 362 to the amino acid at position 379; (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.

Or to combine with the IM2394 type polypeptide a polypeptide whose sequence is derived by partial deletion of the second domain and by total or virtually total deletion of the first or third domain of the IM2169 type Tbp2 subunit, and contains the second domain in its entirety (Al, 2, A3). In this alternative, the pharmaceutical composition containing two types of component (IM2394 type and IM2169 type) may advantageously contain several IM2169 type polypeptides (Al, 2, A3); for example two or more of the polypeptides selected from (Al, 2, A3) IM2169, M978, 6940 and S3032.

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, especially 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 1 Lu

_N

WO 95/33049 19 PCT/FR95/00701 individual being treated or the mode of administration.

In order to establish the subject of the present invention, it may be pointed out that the N. meningitidis strains IM2394 and IM2169 are available to the public from the Collection Nationale de Culture des Microorganismes [National Collection of Microorganism Cultures] (CNCM), Pasteur Institute, 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 and by reference to Figures 1 to Figures 1 to 3, 8 and 9 present, respectively, the alignments of the M978, 6940, S3032, BZ83 and BZ163 Tbp2 sequences with the IM2169 Tbp2 sequence, to maximum homology. The respective degrees of homology are 78.9, 81.2, 79.6, 71.3 and 81.8 Figure 4 presents the alignments to maximum homology of the sequences of the hinge domains (second domain) of IM2169 6940 2223 C708 M978 1610 867 S3032 and 891 Tbp2. The numbering of IM2169, as it appears in ID SEQ NO 2, is given in italics. The sequences which may be deleted according to a preferred embodiment appear in bold type.

indicates the consensus sequence.

Figures 5 to 7 illustrate the construction of the plasmids pTG5782, pTG5755 and pTG5783, respectively.

Figure 10 presents the alignments to maximum homology of the sequences of the hinge domains (second domain) of IM2169 2223 708 M528 6940 M978 1610 S3032 867 BZ83 and BZ163 (11) Tbp2. The numbering of IM2169, as it appears in ID SEQ NO 2, is given in italics. The sequences which may be deleted according to a preferred embodiment appear in bold type. indicates the consensus sequence.

EXAMPLE 1 Polypeptide T/2169 02, A3; 1-350) including the sequence as shown in ID SEQ NO 1 (IM2169), from the amino acid at position 1 to the amino acid at position 350 WO 95/33049 20 PCT/FR95/00701 1A 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, to generate the plasmid pTG4704.

From plasmid pTG3721, a fragment containing the sequence coding for the secretion signal of RIpB and from the beginning of the sequence coding for mature Tbp2 up to the internal HaeII site is amplified by PCR using the primers OTG4915 and OTG4651.

OTG4915 AAACCCGGATCCGTTGCCAGCGCTGCCGT HaellI OTG4651 BspHI TTTTTTCATG AGA TAT CTG GCA ACA TTG 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 AGT TTC The PCR fragment is then digested with BspHI and HaeII and inserted simultaneously with the HaeII-HindIII fragment of pTG4704, which contains the 3' portion of the region coding for Tbp2, into the plasmid pTG3704 described in Application EPA 586,266, digested with NcoI and HindIII, to generate the plasmid pTG5768.

From plasmid pTG3721, a fragment containing the sequence coding for the N-terminal portion of Tbp2 is amplified by PCR using the primers OTG4928 and OTG5011.

WO 95/33049 21 PCT/FR95/00701 SphI OTG4928 GTG TTT TTG TTG AGT GCA TGC CTG GGT GGC Val Phe Leu Leu Ser Ala Cys Leu Gly Gly _cleavage of the signal peptide OTG5011 TGCGCAAGCTTACAGTTTGTCTTTGGTTTTCGCGCTGCCG HindIII 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 thereby generated.

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

1B Production and purification of T/2169 (1-350) An E. coli strain (Xac-I) is transformed with pTG5782. The transformants are cultured at 37 0 C in M9 medium 0.5 succinate 50 gg/ml arginine 100 Ag/ml ampicillin. In the exponential phase, 0.2 of arabinose (inducer) is added. After one hour of induction, cells are removed and extracts are prepared. Western blot analysis followed by visualization with hTF-peroxidase enables a preponderant band to be detected, 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 WO 95/33049 22 PCT/FR95/00701 inducing bactericidal antibodies, and should consequently be useful for vaccinal purposes.

EXAMPLE 2: Polypeptide T/2394 02, A3; 1-340) including the sequence as shown in ID SEQ NO 2 (IM2394), from the amino acid at position 1 to the amino acid at position 340.

2A 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 containing the region coding for the C-terminal portion of the hTf binding domain is amplified by PCR using the primers OTG4873 and OTG4877.

This fragment is then digested with MiuI and HindIII.

OTG4873 AAAAAGCATGCATAAAAACTACGCGTTACACCATTCAAGC MIul OTG4877 :TATATAAGCTTACGTTGCAGGCCCTGCCGCGTTTTCCCC HindIII Plasmid pTG4710 is digested with MluI and HindIII. The MluI-HindIII fragment containing the 3' portion of the sequence coding for Tbp2 is replaced by the PCR fragment coding for the C-terminal portion of the hTf binding domain. The plasmid pTG5707 is thereby generated. A BamHI-MluI fragment containing the araB promoter and the beginning of the sequence coding for Tbp2 is then replaced, in plasmid pTG5707, by a BamHI- MluI fragment of pTG4764 described in Application EPA 586,266, which contains the araB promoter, and the sequence coding for the RlpB secretion signal fused to the sequence coding for the N-terminal domain of Tbp2.

The plasmid pTG5755 is thereby generated. This vector contains the araB promoter, and the sequence coding for the secretion signal of RlpB fused to the sequence coding for the N-terminal domain of Tbp2 (1-340).

WO 95/33049 23 PCT/FR95/00701 2B Production and purification of T/2394 (1-340) T/2394 (1-340) is produced and purified as described in Example 1B.

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 should consequently be useful for vaccinal purposes.

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

3A 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 N. meningitidis strain IM2169 is amplified by PCR (polymerase chain reaction) using specific primers complementary to the 5' and 3' regions (AS' and A3', respectively), on 10 ng of genomic DNA extracted from a culture of bacteria of the strain IM2169.

5' CCCGAATTCTGCCGTCTGAAGCCTTATTC 3' A3' 5' CCCGAATTCTGCTATGGTGCTGCCTGTG 3' A DNA fragment is thereby obtained and, after digestion with EcoRI, it comprises 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. Implementation of the deletions.

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

In brief, the phage form of the recombinant phagemid pSK/2169tbp2 is obtained after rescue with the helper phage VCS M13 according to the technique described by Stratagene, the supplier of the parent vector, and WO 95/33049 24 PCT/FR95/00701 used to infect the bacterial strain CJ236. The outcome of the dut and ung mutations carried by strain CJ236 is the synthesis of DNA molecules which have incorporated the nucleotide precursor dUTP.

The phages are harvested and the single-stranded DNA is extracted with a phenol/chloroform mixture. This DNA is hybridized under standard conditions with the following oligonucleotides: 2169d1 5' CGCATCCAAAACCGTACCTGTGCTGCCTGA 3' 2169d2 5' TTTATCACTTTCCGGGGGCAGGAGCGGAAT 3' 2169d3 5' GTTGGAACAGCAGACAGCGGTTTGCGCCCC 3' 2169d4 5' GAACATACTTTGTTCGTTTTTGCGCGTCAA 3' The hybridization reaction is continued for 30 min at a temperature decreasing from 70°C to The complementary second strand is then completed by total synthesis in the presence of the four deoxynucleotides, T4 DNA polymerase and T4 DNA ligase, according to standard conditions.

The strain E. coli SURE (Stratagene) is transformed with the DNA thereby obtained. In this strain, the molecules carrying dUTP, that is to say unmutated molecules, are destroyed.

The phages obtained are analysed by the standard techniques of rapid preparation of plasmid DNA and of 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.

3B Construction of the expression vector pTG5783 Plasmid pTG5768 described above is digested with HpaI and XcmI. An XcmI-XcmI fragment of pTG5768 and the HpaI-XcmI fragment of plasmid pSK/2169ed#7 are inserted simultaneously into this vector to generate the plasmid pTG5783. This vector contains the araB promoter, and the sequence coding for the secretion signal of RipB fused to WO 95/33049 25 PCT/FR95/00701 the modified tpb2 sequence (deletions dl to d4).

3C Preparation and purification of D4/2169.

D4/2169 is produced and purified according to Example IB.

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

EXAMPLES 4 to 8: Polypeptides 4) C/2223, 5) C/M981, 6) C/1610, 7) C/M978 and 8) C/C708 corresponding to the second domain (hinge region) of Tbp2's of various strains.

The DNA fragments coding for the Tbp2's of N.

meningitidis strains 2223, M981, 1610, M978 and C708 were cloned by PCR amplification as described in Example 3A, using the same two primers. Similarly, these fragments were inserted at the EcoRI or EcoRI/BamHI sites of the phagemid pBluescriptSK(-). Sequencing of the region coding for the second domain was performed, and the amino acid sequence deduced as each one of it [sic] is seen in Figure 4.

On the basis of each of the nucleotide sequences, primers specific to each of the two domains are created by introducing suitable cleavage sites for the purpose of a future cloning in frame with [lacuna] r1pB signal sequence, under the control of the araB promoter. These primers are used in PCR to amplify the region coding for the second domain of each of the Tbp2's. These regions are cloned as described above into a plasmid containing the rlpB signal sequence, under the control of the araB promoter.

Expression of the peptides is carried out as described in Example 1B.

EXAMPLE 9: Vaccine composition (T/2169-T/2394) intended for preventing N. meningitidis infections Sterile solutions of T/2169 and T/2394 as purified in Examples 1B and 2B are thawed. In order to prepare one litre of vaccine containing 100 Ag/ml of each of WO 95/33049 26 PCT/FR95/00701 the active principles, the following solutions are mixed under sterile conditions: Solution of T/2394 [lacuna] 1 mg/ml in buffer C (500 mM phosphate buffer, pH 8, 0.05 Sarkosyl) 100 ml Solution of T/2169 containing 1 mg/ml in buffer C 100 ml Buffered physiological solution (PBS) pH 6.0 300 ml Aluminium hydroxide containing 10 mg Al+++/ml 50 ml 1 merthiolate in PBS 10 ml PBS qs 1,000 ml EXAMPLE 10: Vaccine composition (D4/2169-Tbp2/2394) intended for preventing N. meningitidis infections A sterile solution of D4/2169 as purified in Example 3C is thawed. A sterile solution of Tbp2/2394 as prepared and purified in Example 3 of EPA 586,266 is treated similarly. In order to prepare one litre of vaccine containing 100 Ag/ml of each of the active principles, the following solutions were mixed under sterile conditions: Solution of Tbp2/2394 containing 1 mg/ml in buffer C 100 ml Solution of D4/2169 1 mg/ml in buffer C 100 ml Buffered physiological solution (PBS) pH 6.0 300 ml Aluminium hydroxide containing 10 mg Al+++/ml 50 ml -1 merthiolate in PBS 10 ml PBS qs 1,000 ml EXAMPLE 11: Obtaining an antibody capable of recognizing the epitope GFYGPKGE of the first domain of IM2394 Tbp2.

11A Immunization of mice and production of hybridomas MRL/Lpr-Lpr mice, known to produce more IgG2a, IgG2b and IgG3 than Balb/C mice Immunol. Methods (1991) 144: 165), receive a first intraperitoneal injec- NO 95/33049 27 PCT/FR95/00701 tion of 50 fg of the IM2394 membrane fraction in the presence of Freund's complete adjuvant. The membrane fraction which is used is prepared as follows: The strain IM2394 stored in lyophilized form is taken up and cultured on Mueller-Hinton agar overnight at 37 0 C in an atmosphere containing 20 of CO 2 The layer is taken up and is used to inoculate an Erlenmeyer containing Mueller-Hinton broth to which 30 IM EDDA (ethylenediaminedi(ortho-hydroxyacetic acid) [sic] Sigma) has been added. After 5 hours of incubation at 37 0 C with rotary stirring, the culture is centrifuged.

The pellet is taken up with Tris-HCl buffer, pH 8, and the suspension is lysed in an ultrasound apparatus operating at high pressure (Rannie, model 8.30H). The suspension obtained is centrifuged at low speed to remove cell debris and the membranes are collected by ultracentrifugation (140,000 xg, 75 min, 4 0 The membrane fraction is finally taken up in 50 mM Tris-HCl buffer, pH 8, and its protein concentration determined.

This first injection is followed by two booster injections 21 and 49 days later. The booster doses contain 25 Ag of the Tbp2 protein as purified in Example 3 of EPA 586,266, in the form of an emulsion in Freund's incomplete adjuvant.

56 days later, the mouse which has developed the highest antibody titre (monitoring of immune sera by ELISA) is selected for the production of specific monoclonal antibodies. This mouse receives a final booster injection (78 days after the initial injection), inoculating 25 Ag of the Tbp2 protein as purified in Example 3 of EPA 586,266, both intravenously and intraperitoneally.

3 days later, the animal's spleen is removed and the spleen cells are fused with P3 x 63 Ag 8653 mouse myeloma cells in a ratio of one myeloma cell to 4 spleen cells.

The fusion protocol used is derived from that described initially by G. K8hler and C. Milstein, Nature (1975) 256: 495. After fusion, the cells are arranged in sterile microwells (Nunc) coated with a nutrient "feeder", on the basis of 100,000 cells per well in a volume of 200 .l of WO 95/33049 28 PCT/FR95/00701 selective medium [DMEM medium containing 20 of FCS and a hypoxanthine/azaserine/thymidine mixture at a concentration of 2 (Gibco. Ref 043-01060H)]. The selective medium is replaced 6 days later by a non-selective medium [DMEM medium containing 20 of FCS and a hypoxanthine/thymidine mixture at a concentration of 2 (Gibco. Ref 043-01065H)].

11B Screening of the hybridomas The culture supernatants of the hybridomas are tested by ELISA according to the following method: Into ELISA plate microwells "sensitized" overnight at +4 0 C with 100 1l of a solution containing gg/ml of RT 2394 in carbonate buffer (50 mM pH 9.6) and then saturated for 1 hour at 37°C with 200 il of a 0.1 M phosphate buffer containing 1 of bovine serum albumin (weight/volume) (PBS-BA), there are introduced 100 gl of hybridoma culture supernatant (or dilutions of immune sera prepared in PBS-BA buffer containing 0.05 of Tween (PBS-T-BA). After a further incubation for 1 h 30 min at 37°C followed by 5 washes in PBS-Tween, the wells are coated with 100 pl of a mixed solution of antibodies conjugated to alkaline phosphatase (AP) which are specific for the mouse isotypes IgG2a, IgG 2 b and IgG 3 so as to select only hybridomas secreting specific antibodies which are functional in the test of bactericidal effect.

The mixed solution of conjugated antibodies is prepared by diluting the following 3 goat immune sera, namely goat anti IgG 2 a-AP (Caltag), goat anti IgG2b-AP (Caltag) and goat anti IgG 3 -AP (Caltag), to 1/1500 in PBS-T-BA buffer.

After incubation of the solution of conjugated antibodies for 1 h 30 min at 37 0 C followed by 5 washes, the enzyme reaction is visualized with 100 Al of a solution of paranitrophenyl phosphate containing 5 mg/ml in 0.1 M diethanolamine buffer, pH 9.8. The development of the reaction is stopped after 30 min by adding 50 Al of IN sodium hydroxide, before spectrophotometric analysis at 405 nm.

Clones which are positive after this first screening are analysed for their capacity to recognize the Tbp2 subunit by Western blotting.

WO 95/33049 29 PCT/FR95/00701 To this end, the transferrin receptors of IM2394 (0.863 mg/ml) and of IM2169 (0.782 mg/ml) as prepared in Examples 1 and 2 of W093/6861 are diluted to 1/10 in 1M Tris buffer, pH 6.8, and then denatured by adding 10 of a 25 solution of SDS in TE buffer (100 mM Tris-HCl, 10 mM EDTA), pH 8.0, and 5 of f-mercaptoethanol. After treatment for 15 min at 56 0 C, a 110 pl aliquot containing the denatured IM2394 or IM2169 transferrin receptor is applied to a 7.5 polyacrylamide gel. After migration (1 hour at 200 volts in a Biorad cell), the proteins are electrotransferred onto a nitrocellulose membrane (100 volts for 50 min). The membrane is saturated overnight at room temperature in 20 mM Tris buffer, 137 mM NaCl, pH 7.6 (TBS) containing 5 of skimmed milk powder, then mounted on a miniblotter. The antibodies which are tested are adjusted to a concentration of 25 gg/ml in TBS buffer containing 1 of milk powder before being applied in the proportion of pl per channel.

After 45 min of incubation, followed by rinses in TBS buffer/i milk, 50 Al of a rabbit anti-mouse IgG.A.M immune serum (Zymed) conjugated to alkaline phosphatase and diluted beforehand 1000 times in TBS buffer/1 milk are applied to each channel.

After a further incubation for 45 min, followed by rinses, the enzyme reaction is visualized using a chromogenic substrate (BCIP/NBT (Sigma Fast The reaction is stopped after 15 min by dipping into distilled water. Positive clones are characterized by their capacity to disclose a band corresponding to a protein of approximately 69 kD (Tbp2 subunit) after electrotransfer of the IM2394 transferrin receptor onto a nitrocellulose membrane.

At the end of this second screening by Western blotting, the clones are analysed for their capacity to produce an immunoglobulin which reacts with the peptide sequence GFYGPKGE in an ELISA system; the methodology is identical to that described above, except for the sensitization of the plates, which is carried out by adding

I

WO 95/33049 30 PCT/FR95/00701 100 gl of a solution of peptide GFYGPKE containing 2 Ag/ml into each well.

Among the hybridomas tested, one which proves capable of reacting with the peptide is selected; it is then stabilized by successive clonings (at least 2) on the basis of 5 cells/well during the first cloning and one cell/well during subsequent clonings.

11C Production and purification of the monoclonal antibody The monoclonal antibody is produced in ascites of male nude Swiss mice.

days after injection of 500 pl of pristane intraperitoneally, the nude mice receive a second intraperitoneal injection of 7 million cells originating from the hybridoma.

The ascites fluids are withdrawn under sterile conditions and then purifed by affinity chromatography on a column of G protein. The ascites diluted to 1/5 in 0.1M phosphate buffer, pH 7.4, and filtered through a 0.22 A Millipore filter is passed through a column G protein previously equilibrated in the same phosphate buffer, at the rate of 40 ml/hour.

The antibodies bound to the column are eluted using 0.1M glycine buffer, pH 2.7. The eluted fractions are immediately neutralized using 1 M Tris buffer pH (in the proportion of 1 volume of Tris to 10 volumes of eluate).

The eluate is then dialysed overnight at +4 0 C in 0.1M phosphate buffer, pH 7.4, aliquoted and stored frozen.

The purity of the antibody is monitored by electrophoresis on 7.5 polyacrylamide gel and by permeation chromatography on Superose 12. The degree of purity is generally greater than 95 By applying the protocol described above and screening approximately 800 hybridomas, a monoclonal capable of reacting with the epitope GFYGPKGE of the first domain of IM2394 Tbp2, and incapable of reacting with the corresponding epitope located in the third WO 95/33049 31 PCT/FR95/00701 domain (that is to say GFYGKNAI), was selected in particular.

This monoclonal (referred to as 475 E 7 is an IgG 2 b of isoelectric point between 7.8 and 8.1, and possesses a bactericidal titre of 512.

This titre was determined as follows: From a solution of Mab 475 E 7 doubling dilutions are made and incubated in the presence of 50 il of a suspension of meningococci containing 1x10 4 CFU/ml and 50 il of young rabbit complement [the bacterial suspension is obtained by culturing N. meningitidis strain B16B6 at 37 0 C for 5 hours in Mueller-Hinton-Difco broth containing 30 AM EDDA (ethylenediaminedi(orthohydroxyphenylacetic acid) Sigma)].

After one hour of incubation at 37C, 25 Al of mixture are withdrawn and cultured on supplemented Mueller-Hinton agar. The agar dishes are incubated overnight at 37°C under an atmosphere containing 10 of CO 2 The colonies are counted and the bactericidal titre is expressed as the reciprocal of the final dilution in the presence of which 50 or more lysis of the bacteria is observed relative to the control.

Under these conditions, it was determined that the Mab 475 E 7 possessed a bactericidal titre of 512.

EXAMPLE 12: Demonstration of the bactericidal activity of immunoglobulins specific for the protein T/2169 (1-350) with respect to various N. meningitidis strains.

12A Production and purification of T/2169 (1-350) A strain of E. coli B is transformed with plasmid pTG5782 described in Example 1. The transformant selected is amplified to give seeding batches. From a tube of E.

coli B transformed with pTG 5782, amplification of the culture is carried out in M9 medium 0.5 succerate [sic]. Culturing is carried out in a 20-1 fermenter.

In the exponential phase, arabinose (expression inducer) is added. After one hour of incubation, the cells are harvested and ruptured in an apparatus operating at high pressure (Rannie) and the membrane fraction WO 95/33049 32 PCT/FR95/00701 is harvested by centrifugation.

Western blot analysis followed by visualization with transferrin-peroxidase enables a preponderant band to be detected whose molecular weight corresponds to that expected for this truncated form. The protein is purified by preparative SDS-PAGE from 10 acrylamide gel.

12B Production of immunoglobulins specific for T/2169 (1-350) The protein fraction thereby obtained is used to immunize rabbits. Briefly, rabbits (New Zealand White) are immunized on D/0 with 50 Ag of protein T/2169 prepared as described in 12A, in the presence of Freund's complete adjuvant, and (ii) on D/21 and D/42 with 50 Ag of protein T/2169 in the presence of Freund's incomplete adjuvant. On D/56, the rabbits are sacrificed and the serum is harvested. From this serum, the immunoglobulins are purified by affinity chromatography on a protein A-Sepharose (Pharmacia) resin [sic]. The purification is carried out according to the supplier's recommendations.

The purified IgG fraction is lyophilized, and the lyophilisate is taken up with a certain volume so that the final protein concentration of the solution is in the region of 25 mg/ml.

12C Test of bactericidal effect In parallel with the purification of T/2169, a purification is carried out by preparative SDS-PAGE of a fraction of E. coli B obtained after transformation with the plasmid pTG3704 (this vector is identical to plasmid pTG5782 but does not include any sequence of Tbp2). The protein fraction obtained by preparative SDS-PAGE is used to immunize rabbits as is described above, and the IgGs are purified from the serum harvested.

Hence two serum fractions designated T/2169 IgG and Control IgG are obtained. They are analysed for their capacity to lyse different strains of N. meningitidis in the test of bactericidal effect, as described in Example 4 of WO 93/6861 (published on 15.04.1993).

The results obtained on different isolates are WO 95/33049 33 PCT/FR95/00701 summarized in the table below, and demonstrate that the purified protein T/2169 proves capable of inducing antibodies which are bactericidal with respect to several strains of the IM2169 type group. These results of cross bactericidal effect demonstrate that T/2169 should be useful for vaccinal purposes.

Determination of the bactericidal activity of immunoglobulins specific for the protein T/2169 in comparison with control immunoglobulins with respect to six N. meningitidis strains Bactericidal titres* Strain Serogroup Control IgG T/2169 IgG Serotype/subtype 2169 B:9;P1.9 4 128 RH 873 B;8;P.1.17 4 16 RH 876 B;19,P1.6 4 64 351 B:NT;P1.7 4 256 NG G40 4 512 EG 328 B;NT;- 4 64 Bactericidal titres are expressed as the reciprocal of the dilution for which 50 lysis of the initial colonies is observed EXAMPLE 13: Demonstration of the bactericidal activity of immunoglobulins specific for the protein D4/2169 with respect to various strains of N. meningitidis.

13A Production and purification of D4/2169 D4/2169 is produced and purified according to Example 12A.

13B Production of immunoglobulins specific for D4/2169 This production is performed in a similar manner to that described in Example 12B.

13C Test of bactericidal activity WO1 95/33049 34 PCT/FR95/00701 Two immunoglobulin fractions designated D4/2169 IgG and Control IgG are obtained. They are analysed for their capacity to lyse different strains of N.

meningitidis in the test of bactericidal activity as described in Example 4 of WO 93/6861 (published on 15.04.93).

The results obtained on different isolates are summarized in the table below, and demonstrate that purified D4/2169 proves capable of inducing antibodies which are bactericidal with respect to several strains, and should consequently be useful for vaccinal purposes.

Determination of the bactericidal activity of immunoglobulins specific for the protein D4/2169 in comparison with control immunoglobulins with respect to six N. meningitidis strains Bactericidal titres* Strain Serogroup Control IgG D4/2169 IgG Serotype/subtype 2169 B:9;P1.9 4 32 RH 873 B;8;P.1.17 4 8 RH 876 B;19,P1.6 4 16 351 B:NT;P1.7 4 128 NG G40 4 64 EG 328 B;NT;- 4 16 *Bactericidal titres are expressed as the reciprocal of the dilution for which 50 lysis of the initial colonies is observed.

WO 95/33049 35 PCT/FR95/00701 SEQ ID NO Project name Sequence 1, 2 IM2169-2 IM(2169 Tbp2 complete 3, 4 IM(2394-2 IM(2394 Tbp2 complete 6 M(978 M(978 Tbp2 complete 7, 8 6940 6940 Tbp2 complete 9, 10 S3032 S3032 Tbp2 complete 11 2D IM(2169 2nd domain of 11(2169 Tbp2 12 2D 6940 2nd domain of 6940 Tbp2 13 2D 2223 2nd domain of 2223 Tbp2 14 2D C708 2nd domain of C708 Tbp2 2D M(978 2nd domain of M(978 Tbp2 16 2D 1610 2nd domain of 1610 Tbp2 17 2D 867 2nd domain of 867 Tbp2 18 2D S3032 2nd domain of S3032 Tbp2 19 2D 891 2nd domain of M(981 Tbp2 OTG 4915 OTG 4915 21 OTG 4651 OTG 4651 22 OTG 4928 OTG 4928 23 OTG 5011 OTG 5011 24 OTG 4873 OTG 4873 OTG 4877 OTG 4877 26 A 51 A 51 27 A 31 A 3' 28 2169 Dl 2169D1 29 2169 D2 2169D2 2169 D3 2169D3 31 2169 D4 2169D4 32 MABi 1ot box of the lot domain of 11M2169 Tbp2 33 MAB2 2nd box of the 1ot domain of IM(2169 Tbp2 34 1(AB3 3rd box of the lot domain of IM(2169 Tbp2 1(AB4 4th box of the lot domain of IM(2169 Tbp2 36, 37 BZ83 BZ83 Tbp2 complete 38, 39 BZ163 BZ163 Tbp2 complete 36 41 42 2D BZ83 2D BZ163 2D M528 2nd domain of BZ83 Tbp2 2nd domain of BZ163 Tbp2 2nd domain of M528 Tbp2 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.

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WO 95/33049 37 PCT/FR95/00701 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Pasteur Merieux serums et vaccins STREET: 58, avenue leclerc CITY: Lyons COUNTRY: France POSTAL CODE: 69007 NAME: Transgene STREET: 11, rue de Molsheim CITY: Strasbourg COUNTRY: France POSTAL CODE: 67000 (ii) TITLE OF INVENTION: Tbp2 fragments of N. meningitidis (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Tape COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25

(EPO)

INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2230 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: IM2169 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

sig_peptide 60..119 mat_peptide 120..2192 (ix) FEATURE: NAME/KEY: CDS LOCATION: 60..2192 (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

misc feature 120..1154 miscfeature 1155..1748 misc feature 1749..2192 miscbinding 127..1169 'Y'V o<' Wo 95/33049 38 (xi) SEQUENCE DESCRIPTION :SEQ ATTTGTTAAA .AATAAATAAA ATAATAATCC TTATCATTCT PCT/FR95/00701 ID NO: 1: TTAATTCA6AT TGGGTTAT ATG AAC AAT CCA TTG GTA AAM CAG GCT GCT ATG CTG CTC CCT GTG TTT Met Asa Asn Pro LoU Val Asa Gin Ala Ala TTG TTG ACT Lau Lou Set CTC GAT ACC Val Asp Thr is TCC GRA AA Ser Clu Lys ATG AGG TTG Met Ar; Lau CT? AAA CTG Val Lys Lau CCC AAG GAPA Pro Lys Ciu ACA GAC GCC Thr Asp Cly AAC CAT CAA~ Asn His Gin 110 CAG CCA ACA Gin Ala Thr 125 TAT AAA CAT Tyr Lys His TCA CCC CAC Ser Giy Asp CAK CTT CCT Gin Leu Pro 175 GCC TGT Ala Cys 1 CAA GCC Clu Ala CCG CA Pro Gin AAA CCC Lys Arg AAC GAG Asn Ciu CT? CC? Lau Pro s0 GAC AC Asp Ser AAC GCC Asa Cly CC? CAC Cly His CC-A CC Ala Ala 145 CAT CC? Asp Cly 160 -15 CTC GC Lou Cly CCG CC? Pro Ar; CCC CA Ala Gin 35 ACG AAM Ar; Asa 5o ACT CAT Sex Asp AAA CCC Lys Ax; GAT AT? Asp Ile AGC CC? Ser Ala 115 GAA AAT Ciu Asa 130 ACT CA Ser Clu TAT ATC Tyr Ile CCC CC= Cly Gly 5 ccc C Pro Ala 20 AAA CAC Lys Asp TCC TAT Trp Tyr TGC GAG Trp Glu CAA AA Gin Lys 85 TAT TC? Tyr Ser 100 GGC AAC Cly Asa TTC CAA Ph* Gin AAA CAT Lys Asp TC TAT Ph. Tyr 165 CT? ATC Val Ile 180 Val Lau TC CAT Phe Asp TAT CA Tyr Gin OCA TAC Ciy Tyr CCA CPA Ala Glu GGA6 TTC Cly Lou AT? CA Ile Glu TAT CC Tyr Lau 105 AAT CAAL Asn Gin 120 TAT TCC Tyr 5cr AAC AA Asn Lys CAA AA Giu Lys Pro Val Phe CT? GAT TCT Lau Asp Ser CAT CTT TCT Asp Val Ser CC? TI? CC Cly Phe Al1a CPA6 ACC GAG Ciu Sc: Clu CCC ACA AAA Pro Thr Lys AAA, GTA CA Lys Vol Ciu ACA CCA TCA Thr Pro Ser CCT AAA AAT Pro Lys Asn CC? ?CG TT? Gly Trp Phe 140 AAA AT? AAC Lys Ile Lys 155 CC? TCC CGA Pro Ser Ar; 170 TGG CAT T?? Trp His Ph.

C TCT OCA AAA Ala Ser Cly Lys TAC AAA CC? GTC Tyr Lys Gly Val 185

'~T

WO 95/33049 39 PCT/FR95/ 00701 GTA ACC Val Thr 190 GAT ACA AAA AAG GGT CA^ GAT Asp Thr Lys Lys Giy Ciii Asp 195L CC? GAA ATC CG CC? Ph. Arg Glu Ile Ile Gin Pro 200 TCA AAA AAA CAA GGC GAC AGG 205 Glu

GT

Gly

TTG

Lau

AAT

An

ACA

The 235

MAT

An

GGC

Gly

AGC

Sex

AAA

Lys

TCC

Sex 365

CTT

Val

CTC

Lou

ATT

Ile

AAA

Lys Lys Lys Gin GMA TAT TCC Glu Tyr 5cr TAT GGT 7?? Tyr Gly Phe 240 ACG GG? AAA Thr Gly Lys 255 AAT GAC AAA Asn Asp Lys 270 GGC AAC CC Gly Asn Axg GAA ACC AAA Glu Thr Lys GGC ?TC Giy Ph. Ph.

320 GAC GAT CAA Asp Asp Gin 335 CTG GAA AAT Lau Giu An 350 GCC C? GCC Cly Gly Ala TG GA? CCG Leu Asp Ala GAC AAC TTC Asp Ann Phe 400 CCC CC CTG Pro Lou Lou 415 GGT AAA AAC Gly Lys An 430 Giy Asp AMg 210 TAT AGvC GA T?? Tyr Sac Gly Ph.

215 CC2 Cly AAC AAA AAC CMA 7CC ACG CTG AAA CAT Ann Lys Asn Glu Ser ?hr Lou Lys Asp 225 230 ACC ?CC AAT ?TA GAA CC GAT TC GCC ?hr sex Ann Leu Glu Val Asp Phe Cly 245 ?TA ATA CCC AA? AAT C AGC CTA AAT Lou Ile AMg Asn Ann Ala Sac Lou An 260 265 CAT ACC ACC CAA TAC TAC AGC CT? GAT His Thx Thr Gin Tyr Tyr Sex Lou Asp 275 280 TC AMC GGC AG GCA ACG GCA ACT GAC Ph. Ann Cly Thr Ala ?hr Ala Thx Asp 290 295 CTA CAT CCC TCC CAC 7CC TCT Lou His Pro Ph. Val Sex Asp Sex Sex 305 310 CCC CCC CAG CC? GAG GAA TC CC? TC Gly Pro Gin Cly Ciu Ciu Lau Gly Ph.

325 AAA GCC GTT CTC CCC ACC C AAA Lys Val Ala Val Vai Cly Sex Ala Lys 340 345 CCC C GCG C? ?CA CCC AGC ACA, CC? Cly Ala Ala Ala Sex Cly Sex Thx Cly 355 360 GCA CCC ACG TCC ?CT GAhA MC ACT AAC Ala Cly Thx Sex Sex Ciu Ann Sex Lys 370 375 CTr GAA TC ACA CA AAC GAC AAG AMh Val Glu Lou Thx Lau Asn Asp Lys Lys 385 390 ACC MAT GCC CCC CMA CTC C TT CTC GAC Sex Ann Ala Ala Gin Lou Val Val Asp 405 CCC AAC GA? TCC GCM ACC CCC AMC ACT Pro Lys Asp Sex Clu Sex Cly Ann Thr 420 425 CCC GGA ACA CMA ACC CCC AMA 7?? Gly Cly Thr Clu Phe Thx Ag Lys Phe 435 440 G AT GGC AC Asp Gly Sex 220 GA? CAC GAG Asp His Glu 235 MAT AM AMA Asn Lys Lys 250 MAT MAT AC? Ann Ann Thr CCA CMA ATA Ala Gin Ile AMA AMA GAG Lys Lys Glu 300 TCT TTC AC Ser Lou Sc 315 CGC ?TT TC Mxg Ph. Lau 330 ACC AMA CAC Thx Lys Asp CC GCA GCA Ala Ala Ala CC A=C ACC Lou ?hr ?hx 360 AC AAA MT Ile Lys Ann 395 CCC ATT AC Cly Ile Met 410 CAG CCA GAT Gin Ala Asp CMA CAC ACG Ciu His Thx 779 827 875 923 971 1019 1067 1115 1163 1211 1259 1307 1355 1403 1451 WO 95/33049 CCG GMP ACT GAT AAA AA Pro Giu Ser 445 GCG CAA' ACC Ala Gin Thr ACC TAT GAP' Thr Tyr Glu GGA ATG TTG Gly Met Lau 495 AAC AG? AGT Asn Ser Ser 510 TTC CTC CPA Phe Leu Gin 525 AAC GTC GT? PAn Val Val AGC TGG AGC Trp Ser TTT ACT GG Phe Thr Val 575 GAP' AAC AGG Glu Asn Ar; 590 AAC GGC TTT Asn Gly Phe 605 GAT CAP' AAA Asp Gin Lys AAG GA AAG Lys Val Lys TGG TTT GCC Trp Ph. Ala 655 TCC AGC GAT 5cr Asp 670 Asp Lys Lys 450 GC TCP' AAT Ala Scr Asn 465 GTC GAP' GTC Val. Glu Val 480 ACG CGC AP' Thr Ar; Lys CAP' GC GAT Gin Ala Asp GGC GAG CG? Gly Glu Ar; 530 TAT CGG GGC Tyr Ar; Gly 545 GCC AAT C Gly Asn Ala 560 AP'T TTT CC Asn Phe Ala CAC GCG CAA' Gin Ala Gin GAP' GGT ACG Giu Gly Thr 610 AP'? ACC ACC Asn Thr Thr 625 GGC GOT TTT Gly Gly Phe 640 TAT CCG GGC Tyr Pro Gly GGA AAT TCA Gly Amn Ser 40 PCT/PR-95/00701 GA G CC CAA' GCA GO;T ACO. CAG ACG PAT GGG 1499 Asp Ala Gin Ala Gly Thr Gin Thr PAn GlY 455 460 ACG GCP' GOT GAT ACC AAT GGC AAP' ACA AAA 1541 Thr Ala Gly Asp Thr Asn Gly Lys Thr Lys 470 475 TGC TG? TCC AAC CTC AP'? TAT CTG AAA TAG 1595 Cys Cys Ser Asn Leu Asn Tyr Leu Lys Tyr 485 490 AP'C AC APO TCC GCO AG CAC GCA GCA GGA 1643 Asn Ser Lys Ser Al1a Met Gin Ala Gly Cly 500 505 OCT AAA ACG GAP' CAA GTT GAA CAP' AG? ATG 1691 Ala Lys Thr Glu Gin Val Glu Gin 5cr Met 515 520 ACC OAT GAP' AAA GAG AT? CCA ACC GAC CAA 1739 Thr Asp Giu Lys Giu Ile Pro Thr Asp Gin 535 540 TCT TGG TACG CG CAT AT? GCC-AAC GCC ACA 1787 Ser Tzp Tyr Gly His Il. Ala Asn Oly Thr 550 555 TCT GA? AAA' GAG GGC GGC PAA AGG GCC GAP' 1835 Ser Asp Lys Clu Gly Cly Amn Ar; Ala Ciu 565 570 GA? AAA' AAP' AT? ACC GGC AAG TA ACC GCT 1883 Asp Lys Lys Ile Thr Gly Lys Lou Thr Ala 580 585 ACC rrr ACC AT? GAG GGA ATG AT? CAG GCG 1931 Thr Ph. Thr Ile Giu Gly Hot Ile Gin Gly 595 600 GCG AAA ACT? CC GAG TCP' OCT TT GAT GTC 1979 Ala Lys ?hr Ala Giu Ser Giy Ph. Asp Lau 615 620 COGc AcG. CC? AP. OCA Tic ATC ACA GA? GCC 2027 Axrg Thr Pro Lys Ala Tyr Ile Thr Asp Ala 630 635 TAC GGG CCT AAA GCC GAP GAG TG GGC GGA 2075 Tyr Gly Pro Lys Ala Giu Glu Leu Gly Giy 645 650 GAT AP' CAP' ACG GAP' AAG GCP' ACA GCT ACA 2123 Asp Lys Gin Thr Giu Lys Ala ?br Ala ?hr 660 665 GCA AGC AGC 0CC ACC GTO GTA TTC G? GCG 2171 Ala 5cr Sex 675 Ala Thr Val Val Ph. Giy Ala 680 AAP Cac Lys Ar; 685 CAP' CAG CC? Gin Gin Pro CAP' TAAGCP'CGGT TGCCGAACAA TCAP'GAATAA Gin 2222 WO 95/33049 41

GGCTTCAG

INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 711 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: PCT/FR95/00701 2230 Met Asn Asn Pro Leu Val Ann Gin Ala Ala Met Val Leu Pro Val Phe Lou Lou Val Asp Ser Glu Met Arg Val Lys Pro Lys Thr Asp Ann His 110 Gin Ala 125 Tyr Lys Ser Giy Gin Lou Val Thr 190 Ser Lys 205 Ser Thr Lys Lou Lau Glu Giy Gin Thr His Asp Pro 175 Asp Lys Ala Glu Pro Lys An Lou Asp Asn Gly Ala Asp 160 Ala Thr Gln Cys 1 Ala Gin Arg Glu Pro Ser Gly His Ala 145 Gly 5cr Lys Gly An 225 -15 Lou Pro Ala Ar; 50 Ser Lys Asp Ser Glu 130 Se: Tyr Gly Lys Asp 210 Giy Ar; Gin 35 Asn Asp Arq Ile Ala 115 Asn Glu Ile Lys Gly 195 Arg Gly Pro 20 Lys Trp Trp Gln Tyr 100 Giy Ph.

Lys Phe Val 180 Gln Tyr Giy 5 Ala Asp Tyr Glu Lys 85 Ser Asn Gin Asp Tyr 165 Ile Asp Ser Gly Pro Gin Pro Ala 70 Ser Ser Gly Tyr Ph.

150 His Tyr Phe Gly -10 Ser Lys Gly Gly 55 Thr Val Pro Vai Val 135 Ser Gly Lys Arg Phe 215 Pho Tyr diy Ala Gly Ile Tyr An 120 Tyr Asn Glu Giy Glu 200 Ser Asp Gin Tyr Glu Lou Glu Leu 105 Gin Ser Lys Lys Val 185 Ile Gly Lou 10 Asp Cly Glu Pro Lys Thr Pro Gly Lys Pro 170 Trp Ile Asp Asp Val Phe 5cr Thr Val Pro Lys Trp Ile 155 Se: His Gin Gly Ser Ala Glu Lys Glu Ser Asn Phe 140 Lys Ar; Phe Pro Ser 220 Glu Giu Tyr Ser Lys Ann Giu Ser Thr Lou Lys Asp Asp His Glu 230 235 WO 95/33049 Gly Tyr Gly Phe 240 42 PCT/FR95/00701 Thr Sex Asn Lau Glu 245 Val Asp The Gly Asn Lys Lys 250 Ala Sac Lou sn Asn Asn Thr Lou Th: An Asn 270 Th: Gly 285 An Giu Gly Gly Ser Asp Lye Lou 350 Sa Gly 365 Val Lou Leu Asp 11 Pro Lys Gly 430 Pro Glu 445 Ala Gin Thr Tyx Gly Met Asn Sex 510 Phe Lou 525 Asn Val Gly Lys 255 Asp Lye Msn Arg Thr Lys Ph. Phe 320 Asp Gln 335 Glu Msn Gly Ala Asp Ala Asn Pb.

400 Lau Leu 415 Lys Asn Sex Asp Thr Ala Giu Val 480 Lou Thr 495 Ser Gin Gin Gly Val Tyr Lou lie His Tb: Phe Msn 290 Lou His 305 Gly Pro Lye Val Gly Ala Ala Gly 370 Val Glu 385 Ser Asn Pro Lys Gly Gly Lye Lys 450 Sac Mna 465 Glu Val Atr Lys Ala Asp Glu Arq 530 Arq Gly 545 Axq Thr 275 Gly Pro Gin Ala Ala 355 Thr Lou Ala Asp Thr 435 Asp Thr Cys Mn Ala 515 Thr SaE Asa An 260 Gin Tyr Th Ala Ph. Val Gly Glu 325 Val Val 340 Ala Sex Se: Ser Th Lou Ala Gn 405 So: Glu 420 Glu Ph.

Ala Gin.

Ala Gly.

Cys Se: 485 Ser Lys 500 Lye Thr Asp Giu Trp Tyr Tyr Thr Ser 310 Giu Gly Gly Glu Mn 390 Lau Ser Thr Ala Asp 470 Asen 5cr Glu Lys Gly 550 Sac Ala 295 Asp Lou Ser Sex Mn 375 Asp Val Gly Arg Gly 455 Th Lou Ala Gin Glu 535 His Lou 280 Thr Ser Gly Ala Thr 360 Ser Lye Val Mn Lys 440 Thr Mn Msn net Val 520 Ile Ile 265 Asp Asp Sex Phe Lye 345 Gly Lys Lye Asp Thr 425 Ph.

Gin Gly Tyr Gin 505 Glu Pro Ala Ala Lys So: Arg 330 Thz Ala Lou 110 Giy 410 Gin Glu Thr Lye Lou 490 Ala Gin Th Mn Gln Lye Lou 315 Phe Lys Ala Thr Lys 395 110 Ala His Asn Thr 475 Lye Gly Ser Asp Gly 555 Ile Glu 300 Sex Leu

ASP

Ala Thr 380 An Met Asp Thr Gly 460 Lye Tyr Giy Met Gin 540 Thr Sex Tzp So: Gly 560 Asn Ala Sex Asp Lye Glu Gly Gly Mn 565 Ar Ala Glu 570 WO 95/33049 43 PCT/FR9S/00701 Ph. Thr Val Asn Ph. Ala Asp Lys Lys Ile Thi Gly Lys Lou Thr Ala 575 580 585 Glu AMn Azg Gin Ala Gin Thr the Thr Ile Giu Gly Met Ile GiA Gly 590 595 600 Asn Gly Ph. Giu Gly Thz Ala Lys Thr Ala Giu Ser Gly Phe Asp Liu 605 610 615 620 Asp Gin Lys Asn Thr Thr Arg Thz Pro Lys Al1a Tyr Ile Thr Asp Ala 625 630 635 Lys Val Lys Gly Gly Ph. Tyr Gly Pro Lys Ala Giu Glu Lau Gly Gly 640 645 650 Trp Ph. Ala Tyr Pro Gly Asp Lys Gin Thr Glu Lys Ala Thr Ala Thr 655 660 665 Sex Ser-Asp Giy Asn Sex Ala Ser Ser Ala Thr Val Val Phe Gly Ala 670 675 680 Lys Azg Gin Gin Pro Val Gin 685 690 INFORMATION FOR SEQ ID NO: 3: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1808 base pairs TYPE: nucleic acid STRAZNDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: N. moningitidis STRAIN: 13(2394 (ix) FEATURE: NAME/KEY: sigpeptide LOCATION: 1. (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 61. .1797 (iX) FEATURE: NAME/KEY: CDS LOCATION: 1797 (ix) FEATURE: KNE/KEY: misc feature LOCATION: 61.d.035 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1036.7.1386 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1387.7.1797 (ix) FEATURE: NAME/KEY: misc binding LOCATION: 46. .1050 WO 95/33049 44 PCT/FR95/00701 (Xi) SEQMENCE DESCRIPTION SEQ ID NO: 3: ATG AAC AAT CCA 170 OTA AAT CMr OCT CT ATG Met Aen Amu Pro Lau Val Asn Gin Ala Ala Hot -15 -10 GTG CTG CC? CTG TTT Val LOU Pro Val Ph.

770 AGT OCT Lou Ser Ala GJA ACC GTG Glu Thr Val AGC CAG CCT Ser Gin Pro TAT CCC TIT Tyr Gly Phis AAA TAT AAG Lys Tyr Lys CTG CAA AGA.

La.u Gin Arg s0 AAA AMP CGC Lys Lys Ar; CAA ACT COT Gin Scr Arq 110 TAT CT? TAC Tyr Val Tyr CT? TTT COP Lou Ph. Gly AAG GAG CTG Lys Glu Lou 160 OTT ACT GAT Val Thr Amp 175 GCA CGA GGA Ala Gly Cly 190 TTG COT APT Lau Ar; Amn TGT CTG OT Cys Lou Gly 1 CAA CAT ATO Gin Asp met GAA ACC CA Giu Ser Gin 35 GCA OTA AAA Ala Val Lys GAP AhG CAC Giu Lys His GGA GMA CCA Gly 01u Pro OT AGT TCT Gly Set c GTA OTT OT VJal Val Gly 115 CT AAT AAA Lou Asn Lys 130 CC? GhC OCA Pro Asp Gly 145 CCA TCG A Pro Sex Glu GGC GGC CCC ACT TYC GAT TTO GAC AGC Gly Cly Gly Sac Ph. Asp Lau Asp Bar 5 CAC TCC AAA CCT AAC TAT GAG CAT CPA is Sex Lys Pro Lys Tyr Giu Asp 011k 20 CAG GAT OTh TCG GPA AAC ACC CCC 0CC Gin Asp Val Sc Gli AsA Ser Gly Ala CTA CC? CCC COG AAT OCA CAT TTT APT Lou Pro Ar; Az; Asua Ala His Ph* Asn 55 AAA CCA T70 CC? TCA ATG GAT TOO AAA Lys Pro Lou Gly 3.c Hot Asp Trp Lys 70 AAT ACT TT? ACT GAG AGO GAT GAA 170 Asn Set Ph. Sex Giu Arg Asp Giu Lou 85 GAA CTT AT? GAP ?CA AAA TOG CPA GAT Clu Lou Ile Glu Se: Lys TUp Glu, Asp 100 105 TAT ACA APT TC ACT TAT OTC COT TCG Tyr Thz Ann Ph. Thr Tyr Val A; 120 AAT AAT AT? GAT AT? AAM AAT AAT ATA Asn mu Ile Asp Ile Lys Asa Aen Ile 135 140 TAT CT? TAC TAT MAA GGG MAA GPA CC? Tyr Lau Tyr Tyr Lys Oly Lys Giu. Pro 150 155 AAG ATA ACT TAT MAA GOT AC? TOO OAT Lye Ile The Tyr Lys Gly Thr Trp Asp 165 170 MAA CAA AG Tr7 CPA COP. 170 COT AGT Lye Gin Azq Ph. Glu Gly Lou Cly Set 190 185 CG 0GCC 170 TCT GCA TA CPA CPA CC Gly Ala Lou Sex Ala Lou Glu Ciu Gly 200 GCA TCA TCC GOT CAT AC= GAT TT7 CC? Ala Ser Sex Cly is Thr Asp Ph. Gly 215 220 48 96 144 192 240 288 336 384 432 480 528 576 624 672 720 P.?C GAP Met Giu AAA TCO Lys Sac 195 CCA GAG Ala Giu 210 WO 95/33049 45 ATG ACT ACT GAG Tr? GAG 7T MAT TT? TC? GA? -M ACA PTA Met Thr Sex Glu Ph. Giu VJal Asp Ph. Asp Lys Thr Ile 225 230:- PCT/FR95/00701 ACA CTT TAT COT AAC AAC COT Th: Lou Tyr Ar; Asn Asn Ar; 240 CAA ATA AAA ACT ACO COT TAC Gin Ile Lys Thr Thr Ar; Tyr 255 COT TTC AAA GGT AAG GCO TO Arq Ph. Lys Gly Lys Ala Lou 270 215 ACT CAT CCC TTI AT? TCC GAC set His Pro Pbs Ile So: Asp 235 290 000 CC AA CCGC CG GAA6 CT? Gly Pro Lys Gly Giu Ciii Lau 305 AAA GTT OVA 6CGC TO TI? =OT Lys Val Ala Ala Val Ph* Cly 320 GMAA AC CC OCA G=C CC? =XA Glu Ann Ala Ala Gly Pro Ala 335 ATT ACC CCC GAG GAG TrT A Ile Tb: Gly Glu Giu Ph. Lys 350 355 GTO AAA AMO CTG CTOG TT GAC Val Lys Lys Lou Lau Val Asp 365 370 GAG GGC AAT PAG CO OCA TT7 Glu Gly Pen Lys Ala Ala Ph.

385 MCG GCA AVG GTG TOT TGT TCC Lys Ala. Thz Val Cys Cym Set 400 CTG TCA Ah GAA AAT AAA GAC Lou Sac Lys Clii Asn Lys Asp 415 CCA GTA TCC GAT GTO CC OVA pro Val Sex Asp Val Ala Ala AT? ACT CAA Ile Thr Gin 245 ACC ATT CAA Tb: Ile Gin 260 CC GCA GAT Ala Ala Asp TCC GAC ACT Sex Asp Sex PAT AAT ACT GAA Ann ABU Se: Olu 250 GCA ACT CTT CAC Ala Th: Lou Hise 265 AAA OCT GCA ACA Lye Gly Ale Tb: 260 TTC CPAA CCCc OVA Lou C.Lu Cly Gly 295 AAG CC Lye Oly 235 MAC A Ann LYe 174C AAC Cly An AAT Cv" Ann Cly TT? TAC Ph. Ty: 300

CC

Ala

C

Ala

ACG

Th: 340

A

Lys

OGA

Cly

CAG

Gin~

AAC

An

CAT

Asp 420 A=6 Arg GOT MAA TTC TIC AGC AAC GAC AAC Gly Lye Pho Lou Set Ann Asp Asa 310 315 AG CAC MAA GA MCG AG OAT G=C Lys Oln Lye Asp Lys Lye Asp Gly 325 330 CAA ACC GTG ATA GA? OCA TAC COT Ciii Tb: Val Ile Asp Ala Tyr Ax; 345 GAG CA^APTA GXC AG? Tr? GrCATA Ciii Cia Ile Asp So: Ph. Cly Asp 360 G70 GAwG CT? TCA CTG CTC CCC TCT Val Clii Lou Sac Lou Lou Pro Set 375 380 CAC rGO AT? GAG CAA AAC CCC GTG His Giu Ile Giu Gin Asa Ciy Val 390 395 TIC GAT TAC XG AGT TT? CCC AAC Lou Asp Tyr Hot So: Ph. Gly Lys 405 410 ATG TIC CTOC AA GO? OTC CGC ACT Met Ph. Lou Gin Cly Val Ar; Tb: 425 AVG GhG C? AC CCC MAA TAT CC Thx Glu Ala Ann Ala Lye Tyr Arg 440 AMC GCC ACA AGC TGO AGC GGC GMA Ann Oly Th: Sex Trp Cly Giu 455 460 816 864 912 960 1003 1056 1104 1152 1200 1248 1296 1344 1392 1.440 1488 430 435 OCT ACT TOG TAC G"A TAT CC Gly Th: Trp Tyr Gly Tyr Ile Ala 445 450 GCC TCC APT CAG Ala Sac Ann Gin GAA, GOT =O AAT Ciii Cly Gly ASA 465 AGO OVA GAG TTT Arq Ala Ciii Ph.

470 GAC OTO GAT TTT Asp Vai Asp Ph.

415 WO 95/33049 46 PCT/FR95/00701 TCC ACT. AAA AM ATC A= GG ACA CTG AC C AM CAt CT A C Sac Thr Lys Lys lie Sox Gly Thr Lau Thr Ala Lys Asp Arg Thx Set 480 485 490 CCT GCG TTT ACT ATT ACT GC ATG ATI AAG CAt AAC CCT TTT TCA GGT Pro Ala Ph* Thr Ile Ths Ala Meot Il Lys Asp Mn Gly Ph* Sex Gly 495 500 505 GTG GCG AAA ACC GGT GAA AAC GGC TTT CCG CTG CAT CCG CAA AAT ACC Val Ala Lys Thr Gly Glu Asn Gly Pbs Ala Lau Asp Pro Gin Ann Th 510 515 GGA AAT TCC CAt TAT ACO CAT ATT GAA GCC ACT GTA TCC GCC CT TTC Cly Asn Ser His Tyr Thr His II Glu Ala Th: Val Sac oly Gly Ph.

525 530 535 540 TAC GGC AAA AAC CCC ATC GAG ATG GCC GGA TCG TTC TCA TFr C= GCA Tyr Gy Lye Asn Ala le Ciu Met Gly Gly Sex Phe Set Ph. Pro Gly 545 550 AAT GC CCA GAG GGA AAA CAA GM AM GCA 7CC 070 GTA TTC CT CC An Ala Pro Giu Gly Lys Gin Glu Lys Ala Se: Val Val Ph* Gly Ala 560 565 570 AAA CCC CAA tG CTT GTG CA TAACCACG=C T Lye Ar; Gin Gin Lou Val CIi 575 1536 1584 1632 1680 1723 1776 1806 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 599 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Asn Asn Pro Lou Val Asn Gin Ala Ala Net Val Lou -15 -10 Lau Lou Set Ala Cys Lau Gly Gly Gly Giy Set Ph. Asp 1 5 Val Glu Thr Val Gin Asp Met His Set Lys Pro Lys Tyr 20 Lys Sex Gin Pro Giu Set Gin Gin Asp Val Set Giu Ann 35 Ala Tyr Giy Phe Ala Val Lys Lou Pro At; Ar; Asn Ala 50 55 Pro Lye Tyr Lys Giu Lys His Lys Pro Lou Gly Set Met 70 Lys Lou Gin Ar; Gly Giu Pro An Set Phe Se: Glu A:; 85 Glu Lys Lye Ar; Gly Set Set Giu Lou Ile Glu Sex Lys 100 105 Pro Val Phe Lou Asp 'Sr Glu Asp Glu Sex Gly Ala His Ph* Asn Asp Trp Lye Asp Giu Lou Trp Giu ASp WO 95/33049 -47 PCT/FR9S/00701 Gly Gin Set Arg Val 110 Gly Tyr Val Tyr Lou 125 Val Lau Ph. Gly Pro 145 Set Lys Giu Lau Pro 160 Tyr Val Thr Asp Ala 175 Ala Ala Gly Gly Asp 190 Val Lou Ar; Asn Gin 205 Met Thr Sex Glu Ph.

225 ThZ Lou Tyr Arg Msn 240 Val Asn 130 Asp Ser met Lys Ala 210 Glu ksn Gly Tyr Thi 115 Lys Asn As2 Gly Tyr Lou Glu Lys Ile 165 Glu Lys Gin 180 Se Gly Ala 195 Glu Ala Set Val Asp Ph.

Ax; Ile Thr Asn Ph. Thr 120 Ile Asp Ile 135 Tyr Tyr Lys 150 Thr Tyr Lys Arg Ph. Glu Lou Sex Ala 200 Ser Giy His 215 Se Asp Lys 230 Gln Mn Mn Tyl Lys G1) Giy Gly 185 Lou Thr rh: set r Val Arg Mn Asn Lys Glu 155 Thr Trp 170 Lou Gly Giu Glu Asp Ph.

lie Lye 235 Giu An I Sor 140 Pro Asp Se: Gly Gly 220 lys UYr 245 250 Gin Ii Lys 255 Thr Thr Arg Tyr Ilo Gin Ala Th: Lou His Gly Asn 265 Ar; Ph. Lys Gly 270 Lys Ala Lou Ala Ala Asp Lys 275 So: His 285 Gly Pro Lye Val Glu Msn 11 Th: 350 Val Lys 365 Giu Gly Lye Ala Lou Sor Pro Lye Ala Ala 335 Gly Lys Msn Thr Lye 415 Phe Ile Set Asp Sox Gly Ala 320 Ala Giu Lou Lye Val 400 Glu Glu 305 Val Gly Glu Lou Ala 385 Cys Msn 290 Glu Ph.

Pro Ph.

Val 370 Ala Cys Lye Lou Ala Gly Ala Ala Th: 340 Lys Lye 355 Asp Gly Phe Gin Set Asn Asp Asp 420 Asp Gly Lys 325 Glu Glu Val His Lau 405 mot Se: Lye 310 Gin Th: Gin Glu Glu 390 Asp Lou 295 Ph.

Lye Val Ii.

Lou 375 Ile Tyr Gly 280 Glu Lou Asp lie Asp 360 Se: Glu Met Ala Th: Mn Gly Gly Se Lye Asp 345 Se Lou Gin Set 4y 425 Gly Phe Tyr 300 Mn Asp Axn 315 Lye Asp Gly 330 Ala Tyr.Arg Ph. Gly Asp Lou Pro Set 380 Msn Gly Vai 395 Phe Gly Lye 410 Val Arg Thr Ph. Lau Gin Pro Val 430 Set Asp Vai Ala Ala Ar; 435 Thr Glu Ala Ala Lys Tyr Arg WO 95/33049 Gly Thr Trp Tyr Gly 445 Ala Ser Asn Gin Glu 465 Ser Thr Lys Lys Ile 480 Pro Al1a Phe Thr Ile 495 Val Ala Lys Thr Gly 510 Gly Asn Ser His Tyr 525 Tyr Gly Lys Asn Ala 545 Asn Ala Pro Giu Gly 560 48 Tyr 450 Gly Ser Thr Glu Thr 530 Ile Lys Il e Gi y Gly Ala Asn 515 His Giu Gin Ala Asn Gly The 455 Asn Arg Ala Glu 470 Thr Lau Thr Ala 485 Met Ile Lys Asp 500 Gly Ph. Ala Lau Ile Glu Ala Thr 535 Met Gly Gly 5cr 550 Giu Lys Ala 5cr 565 PCT/FR9S/00701 5cr Trp, Ser Gly Giu 460 Ph. Asp Val Asp Ph.

475 Lys Asp Arg Thr Ser 490 Asn Gly Ph. Ser Giy 505 Asp Pro Gin Asn Thr 520 Val Ser Gly Gly Ph.

540 Ph. 5cr Ph. Pro Gly 555 alVlPh. Gly Ala 570 Lys Arg Gin Gin Leu Vai Gin 575 INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2255 base pairs TYPE: nucleic acid STR.MqDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: M978 (iX) FEATURE: NAME/KEY: mat peptide LOCATION: 1. .2115 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .2115 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGT CTG GGT GGC GGC GGC ACG TTC GAT CTT GAT TC? GTC GAT ACC CAA Cys Leu Gly Gly Gly Gly Thr Ph. Asp Lou Asp Ser Val Asp Thr Giu 1 5 10 GCC CCG CC? CCC CCC CCA AAA TAT CAK GAT CTT TCT TCC CAA AAA CCG Ala Pro Ar; Pro Ala Pro Lys Tyr Gin Asp Val Ser Ser Glu Lys Pro 25 CAA CCC CAA AAA GAC CAA CCC GCA TAC CC? TT? CCA ATG CCC CYC AAG GIn Ala Gin Lys Asp Gin Gly Giy Tyr Gly Phe Ala Met Ar; Leu Lys 40 WO 95/33049 -49 PCT/FR95/O0701 CGG COG AAT TOG CAT CCO CAG GCP An? CC? AM A G OAT GAG ATA AM Arg Arg Asn Trp His Pro Giz Ala Asa Pro Lys Glu Asp Glu Ile Lys 55 CTT TCT CM PAT GAT TGG GAG GCG AA G"P. TG CCA GGC PAT CCC AM Lau Sex Clu Asn Asp Trp Clu Ala Thr Gly Lau Pro Gly Asa Pro Lys 70 75 AAC TTA CCT GAG COP CAG AM TCG OTT AT GM AMA GTA AM A GGC Asa Lou Pro Glu Arg Gin Lys Sc Val lie Glu Lys Val Lys Thr Gly 90 AGC GAC ACC PAT ATT TAT TCT TC CC TAT CTC ACG CAP. TCA AAC CAT Sex Asp Se: Asa lie Tyr Set Set Pro Tyr Lau Thr Gin Se: Asn His 100 105 110 CAP AAC GOC AGT OA MC CM CCh AM PAT GM GTA AM GAT TAT AM Gin Ann Gly Ser Ala Asn Gin Pro Lys Asa Glu Val Lys Asp Tyx Lys 115 120 125 GAG TTC AM TAT GTT TAT TCC OCT TGG TTT TAC AM CAC GCT AM CTC Glu Ph. Lys Tyr Val Tyr Sc: Oly Trp Phe Ty: Lye His Ala Lys Lou 130 135 140 GAP ATC ATA AM GM AR.C AAC TP.A ATT AAG T CA OWG 60A C A= t GAG Glu Ie Ile Lys Glu Ann Asn Lou Ile Lye Gly Ala Lys So: Gly Asp 145 150 155 160 GAC GOT TAT ATC TI" TAT CAC GT CA AM CC TCC Ch CM CTT CCC Asp Gly Tyr Ile Phe Tyr His Gly Giu Lye Pro Sacr Ar; Gin Lou Pro 165 170 175 192 240 288 336 384 432 480 523 576 624 672 720 768 816 864 912 CT? TCT COP GMA GT ACC TACAM GOC G1 Val Sac Gly Glu Val Thr Tyr Lys Gly Val 180 185 ACG AMA CAG GGP CM AM TT? AAC OAT AT? Th: Lys Gin Gly Gin LysPhe P Asn Asp Ile 195 200 CAA CCC GC AGG TAT AGC GOP TI" CCG GOT Gin Gly Asp An; Tyr Set Gly Phe Pro Gly 210 215 TCC AAT AM MT GAA GCG ACT TTA CPA WC Set Asn Lye Asn Giu Ala Thr Lou Gin Gly 225 230 ACC TCA PAT TTA AM GTO GAT TTC AM Phe Thr Set Ann Lou Lys Vl Asp Phe Ann 245 250 GAA TTC ATA CCC AM PAT AG GTT ACA PAC Ciu Lou Ile Arg An Ann Az; Vi Th: Ann 260 265 TAC ACC AC CM TAT TAC AGC CTT GAG OCT Tyr Thr Thr Gin Tyr Tyr Set Lou Giu Ala 275 280 TTC MC GC AAG GC ACG OCA ACC GA= AAA TGG CAT GTA ACC GA? Trp His Phe Val Thr Asp 190 CTT GGA ACC TCA AM AM Lou Gly Thr Set Lys Lys 205 GAT GAC GC GAP GM TAT Asp Asp Gly Giu Giu Tyr 220 AG? CA AG GT TAT OT $or Gin Glu Gly Tyr Cly 235 240 AAG AM AM TIG ACO GGT Lye Lys Lys Lou Thr Gly 255 GCT ACT OCT AAC GA? AM Ala Thx Ala Ann Asp Lys 270 CAP OTA ACA C AC CCC Gin Val Thr Gly An Ar; 285 CCT GGC ACT G GMA ACC Pro Gly Thr Cly Glu Thr 300 Ph. An Gly Lys Ala 290 Thr Ala Thr Asp Lys 295 WO 95/33049 50 PCT/FR95/00701 AAA CM CAT CCC TTT OTT TCC GAC TCG TCT TOT TTG AGC GGC GCC TTT 960 Lys Gin His Pro Ph* Val Sex AMp Ser Ser Sax Lou Ser Gay Oly Phe 305 310 315 320 TTC GC CCG MG GO GAG GM TTG GGT TTC C=O TTT TTG AGO MC GAT 1005 Ph Gly Pro Lys Gly Glu Glu Lou Gly Ph* Arg Ph. Lou Sex Asa Asp 325 330 33S CM AM GTT GCC OTT GTC 000 AGC GC AA ACC CAA GAO AAA GC GCA 1056 Gin Lys Val Ala Val Val Gly Sex Ala Lys Thr Gin Asp Lys Ala Ala 340 345 350 APT GC MT ACT GCG GCO GOT TCA 0C CCC ACA GAT CG GM. CA TCA 1104 Asn Gly An Thr Ala Ala Ala Ser Gly Gly Thr Amp Ala Ala Ala Se 355 360 365 AAC GOT 0G0 GOM 0GC ACG TCG TOT GAA MC AGT AAG CTC ACC ACO OT 1152 Asn Gly Ala Ala Gly Thx Set Ser Glu An Ser Lys Lou Thx Thr Val 310 375 380 TTG GAT 000 OTT GM TTG ACA CTA AAC G AA AM ATC AM MT CTC 1200 Leu Asp Ala Val Glu Lou Thr Lou Ann Asp Lys Lys Ile Lys Asn Lou 385 390 395 400 GAO AC TTC ACC AA GCC GC CAA CTG OT GTC GAC OGO ATT A7 ATT 1248 Asp An Phe Ser An Ala Ala Cn Lou Val Val Asp Gly Ile Hot lie 405 410 415 CCG CTC OT CCC GAG ACT TO GAA AT GG AGC AAT CAG GCA GAT AM 1296 Pro Lou Lou Pro Glu Thr Set Glu Set Gay Ser Ann Gin Ala Asp Lys 420 425 430 GT AM AM GOT AM AM GOT AM AM 0C GGA ACA GAC TTT A= TAO 1344 Gly Lys Lys Gly Lys Ann Gly Lys Ann Gly Gly Thr Asp Phe Thz Tyx 435 440 445 AAA ACA ACC TAC ACG CCG AM MC CAT GAC AM OAT ACC AM 0CC CM^ 1392 Lys Thz Thx Tyr Thr Pro Lys Ann Amp Asp Lys Amp Thr Lys Al Gln 450 455 460 ACA GT 0CG OCA GGC TOT AGC 0C GCA CA AC AT TTC GOT AM GC 1440 Thr Gay Ala Ala Gly Ser Scr Oly Ala Gin Thx Amp Lou Gly Lys Ala 465 470 475 480 GAC OTT MC GGC GOT AAG CCA GA ACA AA AC TAT GAA OTC GAA GTC 148 Asp Val Amn Gay Gly Lys Ala Glu Thx Lys Thx Tyr Glu Val Glu Val 485 490 495 TGC TOT TC AMC CTC MT TAT CTO AM TAC GGA ATG TTG ACG COT AM 1536 Cys Cys Ser An Lau Asn Tyx Le Lys Tyr Gly net Lou Thx Ax; Lys 500 505 510 AAC AGC AM TOC 000 AT0 CAG OCA GA OGA AMC AOT ACT CAA GOT GAT 1584 Ann Sex Lys Sex Ala Met Gin Ala Gly Gly Ann Sex Sax Gin Ala Amp 515 520 525 OCT AM ACG GM CA GTT GM CAA ACT ATG TTC CTC CM 0GC GAG COT 1632 Ala Lys Thr Giu Gin Val Glu Gin Sex Met Phe Lou Gin Gly Glu Arg 530 535 540 ACC GAT GM AM GAG ATT CCA AM GAC CM AMC GTC OTT TAT COG 000 1680 Thx Amp Glu Lys Glu Ile Pro Ann Amp Gin An Val Val Tyr Ax; Gly 545 550 555 560 WO 95/33049 51 PCT/FR95/00701 TCT TOG TAC GOG CAT ATT GCC A=C A=C WA A=C TCC Ser Txp Tyr Gly His Ile Ala Sex Ser Thr Ser Trp 565 570 TCC AAT CCA ACG ACT CCC AA6C AGO CC CAA T ACT Scr Cly GTC AAT AAT OCT Asn Ala 575 TTC GAT 1728 1776 Sac Asn Ala Thz Ser Gly Asa Arg Ala Ohu Thr Val Asa Ph. Asp 590 580 585 ACG AAA AAA AT? AAC Thz Lys Lys le Ann 595 ACC TTT ACC ATT GhT Thzr Phe Thz Ile Asp 610 GCA AAA ACT GCT GAC Ala Lys Thx Ala Asp 625 GGC ACC CCT AAG WCA Gly Thz Pro Lys Ala 645 TAC CCC CCT AAA CCC Tyr Gly Pro Lys Ala 660 GAT AAA CAA ACC CAN Asp Lys Gin 7hr Giu 675 GCA AGC ACC CC ACC GGC ACC TTA ACC OCT MAA AhC Gly Thz Lou Thr Ala Glu NAn 600 GGT NAG AT? G G= AAC GOT Cly Lys Ile Clii Gy an Cly 615 620 TTA CC? 777 CAT CYC CAT CAN Lou Cly Ph. Ap Lau Asp Gin 630 635 TAT ATC ACA CAT C= ANG 070 Tyr Ile 7hz Ap Ala Lys Val 650 GAN GAG TC GCC WGA TOG TTT Clii Gin Lou Cly Cly Trp Ph.

665 NAG GCA ACO 02? OCA TCC GC Lys Ala Thr Val Ala Sex Cly 680 GTO GTA TC CC? CC AAA CC CAC "AG C Giln Ciii Ala TcC WOT ACO Ser Cly Thr NOC NAT ACC ACC Set Asa Thr Th: 640 cAO. G=C GOT TTT Gin Gly Oly Ph.

CCTXT C= G CC Ala Tyr Pro Oly 670 CAT C= AhT TCA Asp Cly An ser 635 CAN CAG CCT OTG 2 2 1824 1872 1920 1968 1026 064 112 165 225 255 Ala Sex Ser Ala Thx Val Val Ph. Gly Ala Lys Arg Gin Gin Pro Val 690 695 700 CAA TANCTANATr ANCTCTCTG OTGGCG GCNCC7CGA TCTTGATTCT 2 Gin 705 GTCATACCO ANCCGC ?CCCCCCCNA AAATATCANG A7TTTCTTC CGAAAANCCG 2 CAAGCCCAAN AAGACCAACC CGGATACOCT 2 INFORM4ATION FOR SEQ ID NO: 6: Wi SEQUENCE CHARACTERISTICS: LENGTH: 705 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Cys Lau Gly Gly Gly Cly Thr Ph. Asp Lou Asp Se: Val Asp Tb: Ohu 1 5 10 1 Ala Pro Ar; Pro Ala Pro Lys Tyr Gin Asp Val 5ac Set Glu Lys Pro 25 GIn Ala Gin Lys Asp Gin Gly Gly Tyr Gly Phe Ala Met Arg Leu Lys 40 wo 95/33049 52 PCT/FR95/00701 Arg Lau Arg So Asn Trp Glu Asn Ala Asn Ala Thr Pro Giy Glu Asp Pro Gly Glu Ile Asn Pro Lys Lys so Asn Lau Pro Glu Arg Gin Lys Set Val Se Asp Set Asn Ile Tyr Sec So: Pro GIr Gl.

Glu 145 Asp Val Thr Gin Ser 225 Phe Glu Tyr Pho Lys 305 Phe Gin Asn Asn a Asn I Pho 130 Ile Gly Soc Lys Gly 210 Asn Thr Lau Thr Asn 290 Gin Gly Lys Gly I Gly i 370 Gly 115 Lys Ile Tyr Gly Gin 195 Asp Lys 5cr Thr 275 ly His Pro ksn 1 355 kla 100 Scr Tyr Lys Glu 180 Gly Arg PAsn Arg 260 Gin Lys Pro 6ys U a hr La Gin Set 135 Glu Asn Asn Lou Ile Ph* 165 Val Gin Tyr Glu Lou 245 Asn Tyr Ala Phe Gly 325 Val ily 150 Tyr Thr Lys Set Ala 230 Lys Asn Tyr Tb: Val 310 Glu Val Ala Thr His Tyr Ph* Gly 215 Thr Val Arg Ser Ala 295 Set Glu Gly Ala Se.

375 Gly Lys Asn 200 Ph.

Lou Asp Val Lou 280 Thr Asp Lau Sac So: I Set 360 So: Glu Gly 185 Asp Pro Gin Ph.

Thr 265 Glu Asp Se Gly Ala 345 ly Glu 90 Tyr Asn Phe Lys Lys 170 Val Ile Gly.

Gly Asia 250 Asia Ala Lys Se: 330 Lys Gly J Asn S Clu Lys Val Lys Thr Giy Let Gl.

Tyr Gly 155 Pro Trp Lau Asp So: 235 Lys k1a ;In Pro icr 315 .rg !hr ar a Tb: I Val Lys 140 Ala Set His Gly Asp 220 Gin Lys Thr Val Gly 300 Lou Phe Gln Asp Lys 380 Gin So: 110 Lys Asp 125 His Ala Lys Se: Ar; GIn Phe Val 190 Thr Set 205 Gly Glu Glu Gly Lys Lou Ala Asia 270 Thr Gly 1 285 Thr Gly So: Gly Lou So: I Asp Lys 350 Ala Ala I 365 Lou Thr I Asi Tyr Lys Gly Lou 175 Thr Lys Giu Tyr Thr 255 kh5 ksn ;lu 1 sn I 135 Ua I a& S 'hr His Lys Lau Asp 160 Pro Asp Lys Tyr Gly 240 1ly Lys k.rg rhr ?he 320 ksp la Vat WO 95/33049 53 PCT/FR95/00701 Lou 385 Asp Pro Gly Lys Thz 465 Asp Cys Asn Ala Thr 545 Ser Sex Thr Thr Ala 625 Gly Tyr Asp Ala Gin 705 Asp Asn Lou Lys Thr 450 Giy Val Cys Val Sex Pro 420 Giy Tyr Ala Gly Asn 500 Sex Glu Lys Gly Thr 580 lie Ile Ala Lys Lys 660 Thr Ala Glu Axn 405 Glu Lys Thr Gly Gly 485 Lou Ala Gin Glu His 565 Sex Asn Asp Asp Ala 645 Ala Giu Thr Lau 390 Ala Thx Asn Pro Sex 470 Lys Asn Met Val 550 Ile Gly Gly Gly Lau 630 Tyr Glu Lys Val Thz Ala Sex Gly Lys 455 Ser Ala Tyr Gin Glu 535 Pro Ala Asn Thr Lys 615 Gly le Glu Ala Val 695 Lou Gin Glu Lys 440 Asn Gly Glu Lou Ala 520 Gin Asn Sex Arg Lou 600 l10 Phe Thr Lou Thr 680 Phe Asn Lou Sex 425 Asn Asp Ala Thr Lys 505 Gly Sex Asp Ser A1a 585 Thr Glu Asp Asp Gly 665 Val Asp Val 410 Gly Giy Asp Gin Lys 490 Tyr Giy Met Gin Thr 570 Glu Ala Giy Lou Ala 650 Gia Ala Lys 395 Val Sex Giy Lys Thr 475 Thx Gly Asn Phe Asn 555 Ser Phe Glu Asn Asp 635 Lys Trp Sex Lys Asp Awn Thx Asp 460 Asp Tyr Met Sex Lou 540 Val Txp Thr Asn Gly 620 Gin Vai Phe Gly Ile Lys Gly Ile Gin Ala 430 Asp Ph.

445 Thr Lys Lou Gly Glu Val Lou Thr 510 Sex Gln 525 Gin Gly Vai Tyr Sex Gly Val Asn 590 Arg Gin 605 Phe Ser Sex Asn Gln Giy Ala Tyr 670 Asp Gly 685 Gin Gln Asn Met 415 Asp Thx Ala Lys Glu 495 Arg Ala Giu Arg Asn 575 Phe Glu Gly Thr Gly 655 Pro Asn Pro Lau 400 Ile Lys Tyr Gin Ala 480 Val Lys Asp Arg Gly 560 Ala Asp Ala Thr Thr 640 Phe Gly Sex Val Gly Ala Lys WO 95/33049 54- INFORMATION FOR SEQ ID NO: 7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2114 base pairs TYPE: nucleic acid STRANIDRDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoaaic) (vi) ORIGINAL SOURCE: ORGAkNISM: N. meningitidis STRAIN: 6940 (iX) FEATURE: NAME/KEY: matpeptide LOCATION: l. .2079 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .2079 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: PCT/FR9S/00701 TGT TTG COGT CC= =C CCC ACG TTC GAT GAT =C GTC CmAT ACC C"A Cys Lou 1 0CC CCG Ala Pro CAA CC Gin Ala COG AGG Ar; Ar; so GAG ACT Giu Ser CCA TTA Pro Lou GAC PAC Asp Asn AY AGC Asn Set APAT TAIT Asn Tyr 130 OCT A Ala Lys 145 Gly Gly COT CCC Axq Pro CAA AA Gin Lys APT TG Asn Trp OAT TG Asp Tzp AAA CA Lys Gin APT ATT Asn Ile Ica AGC AT Ser Ile 115 AAA OAT Lys Asp AAC A Ann Giu Gly Gly Thr 5 GAC CCA JAG Asp Pro Lys GAC CA GcC Asp Gin Gly TAT TCC OCA Tyr Sex Ala 55 GAG ACG ^CA Oiu Thr Thz '70 GPA TCC GTC Giu 5cr Val TAC ACT TCC Tyr Thr Sex JAY GGC GOT Asn Cly Gly TTC AA TAT Ph. Lys Tyr 135 ATC ATA AGA Ile le AMg 15o Ph. Asp Lou Asp Ser Val Asp 10 TAT CRA MT OTT TCT TCC PA Tyr Gin Asp Val Ser Sex GlU 25 OGA TiC =C TrT 0CC ATO AGO Gly Tyr Gly Ph* Ala Net Ax; 40 GCA AAA GPA GAC GAG CG= AA Ala Lys Giu Asp Giu Val Lys G TTo CCG ACA OPA CCC AAG Oly Lau Pro Thr Giu Pro Lys 75 ATT TCA AA GTA CAA OCA MAC Ile Set Lys Val Gin Ala Amn 90 CCC TAT CTC ACC CPA TCA AAC Pro Tyr Leu Th~r Gin Ser Asia 105 110 Thr Giu is AAA CCG Lys Pro TTG A Lou Lys CTG PAC Lou Asia AAA CTG Lys Lou s0 JAA GCC Ami Gly CAT CPA His Gin 48 96 144 192 240 28 336 384 432 480 GMA AC MTG CMAM A MC GAA =TA ACA Ala Asia Lou Pro Lys 120 GT= TAT TCC 0CC TOG Val Tyr Ser Oiy Trp 140 CPA PAC ACC TCA ATT Giu Azsiac Set 1ie 155 Ciu Val TAT MA Tyr Lys COT OCh Cly Ala WO 95/33049 55 PCT/FR95/00701 AAC GCC GAC GAC GCC TAT ATC TT TAT CAC GCW AM GCM CCT TCC CGA 528 Asa Gly Amp Asp Gly Tyr lie Phe Tyr His Gly Lys Glu Pro So: Ar; 165 170 175 CM CT? CCC OCT TCT GGA ACA OTT ACC TAT A CG OT G TOG CAT TI? 576 Gn Lou Pro Ala Se: Gly Th: Val Thr Tyr Lys Cly Val ?zp His Phe 180 185 190 CCC ACC GAT GTC AAA AM TCC CMA AAT CC GAT ATT ATC CAC CC? 624 Ala Thr Asp Val Lys Lys Sac Gin Asa Phe Arg Asp Ile 11 Gn Pro 195 200 205 TCG AAA AM CM GGC GAC AGO TAT AGC CA TT? TCG GGC GAT CAT GAT 672 Se Lys Lys Gin Gly Asp Ar; Tyr Sac Gly Phe Sr Oly Asp Asp Asp 210 215 220 GM CAA TAT TCT PAT AM AAC GM TCC ATO CTG AM GAT rOT CM GAG 720 Glu Gin Tyr Sac Aan Lys Asa Glu Sac Met Lau Lys Asp Gly Gin Glu 225 230 235 240 GGT TAT GOT Trr ACC TCG PAT TTA GM GTG GAT TTC GGC AGT AM AA 768 Gly Tyr Gly Ph. Thr Set Ann Lou Clu Val Asp Phe Gay 3a Lye Lys 245 2S0 255 TTG ACO CC? AM TTA ATA CCC APT AAT ACA CTT ACA PAC OCT CC? ACT 816 Lou Th: Gly Lys Lou Ile Arg Amu Asa Ang Vol Th: Asi Ala Pro Thr 260 265 270 AAC GAT AM TAC ACC ACC CM TAC TAC AGC CTT GAT GCC CM ATA ACA 864 Asn Asp Lye Tyr Thr Thr Gin Tyr Tyr Sac Lou Asp Ala Gin Ile Tb: 275 280 285 CCC AAC CGC TIC PAC GCT AAG GCG ATA CGG ACC CAC AM CCC GhC AC? 912 Cly Asi Ax; he An Wy Lys Ala Ile Ar; Th: Asp Lys Pro Amp Thr 290 295 300 GGA GA ACC AMA CTh CAT CCC TTi OTT TCC CAC TCG TCT TC? TIC AGC 960 Gly Gly Thr Lys Lou His Pro Phe Val Sex Asp 5cr Ser Sex Lou Sac 305 310 315 320 cCC GCC TTT TIC GGT CCG AAG GCOT GAG CM TIG GT TIC =CCTT TTG 1008 Cly Cly Ph* Phe Cly Pro Lys Gly Ciu Glu Lou Gly Phe Ar; Phe Lou 325 330 335 AGC GAC CAT AM AM OTT GCG OTT GTC GGC AGC GCG AM AC AM AC 1056 Sex Asp Amp Lys Lys Val Ala Val Vol Cly Sex Ala Lys Thr Lys Amp 340 345 350 AM ACO GMA APT GC CG GTO GCT TP GGC GCC ACA GAT CG GCA CA 1104 Lys Thr Gu Asn Gly Ala Val Ala Se: Gly Gly Thz AMp Ala Ala Aa 355 360 365 TCA AAC GQT GCG GCA 0GC ACG TCG TCT CPA AAC ACT AAC CTC AC ACG 1152 Scr Asi Gly Ala Ala Gly Thr Ser Ser Clu Asn So: Lys Lou Tx Tbhr 370 375 380 GTT TTG CAT CGC GTC GAG CTG AM TTG CCC CAT APG GM CTC CM AAC 1200 Val Leu Asp Ala Vol Glu Leu Lys Lou Cly Asp Lys Gu Val Gin Lye 385 390 395 400 CTC GAC AAC TIC AGC AAC GCC GCC CM CTG GTT CTC GAC CGC AT? ATG 1248 Leu Asp An Phe Ser Asi Ala Ala Gin Lau Val Val Asp Cly Ile Met 405 410 415 i4Ji a$~Nl~o WO 95/33049 56 PCT/FR95/00701 ATT e1G CT TT CG% C C R C GGG AAM AA CXA CCC AD 4 1296 Ile Pro Lau Lou Pro ilu Ala Ser Glu Ser Gly Asn Asn Gin Ala Asn 420 -425 430 CAA OCT ACP6 AAT GGC GGA ACA CC TTT ACC CGC AAA TTT GAC CAC ACG 1344 Gin Cly Thz Asn Giy Gly Tb: Ala Ph* Tb: Arg Lys Ph. Asp ILLs Th: 435 440 445 CCC GAA ACT GAT AAA AAA GAC G=C CAA GCA OT ACG CM G APAA GGG 1392 Pro M~u Se: Asp Lys Lys Asp Ala Gin Ala Gly Thi Gin Thr Axn Gly 450 455 460 GCG CAA ACC GCT TCA AAT ACG GA GCT GAT A=O AA? GCC AA ACA~ AMh 1440 Ala Gin Tb: Ala Sac Asa Thx Ala Gly Asp Th: Ann GCly Lys Thr Lys 465 470 475 480 ACC TAT CPA CYC GAA GTC TGC TGT TCC AA.C CTC AAM TAT CTG AAA TAC 1468 Tbr Tyr Giu Val. Giu Va~l Cys Cys Se: Asa Lou Ann Tyr Lou Lys Tyr 465 490 495 GGA ATG TTN ACG CGC AMA AMC AC AhO T= CC CCC O C GCA OCA GM& 1536 Gly Met Lau Thz Acq Lys ^An Sac Lys Sac Ala Mot Gin Ala Gly Glu 500 505 510 AGC ACT ACT CAA OCT CAT GCT AAA ACG GAM CAA GTr GM CPA ACT ATG 1584 Sac Sear Gii Ala Asp Ala Lys Th: Glii Gin Val GCiii Gn 5cr Met 515 520 525 TTC CTC CAM GCC GAG CCC AC CAT CPA AMA GAG AMT CCA ACC GAG CAA 2632 Phe Lou Giii Gly Ciii Arg Tb: Asp Ciii Lys Giu Ile Pro Set Glu Gin 530 535 MAC ATC CT TAT CCC GGG TCT TOO TAC GCA TAT ATT 0CC MAC GC AM 1660 Asn Ile Val. Tyr Arg Gly Sa: Tzp Tyr Gly Tyr Ile Ala Ann Asp Lys 545 550 555 560 AGC AChA MC TGG ACC GCC MAT OCT 7CC MAT =C ACC ACT CCC AAC AGC 1728 Tb: Sa: Trp Set Cly Asa Ala Set Ann Ala Th: Sac iy Ann Ar; 565 570 575 GC GMA 777 ACT C70 AAT 777 GCC CAT AAA AMA ATT ACT GOT ACG TTA 1776 Ala Glu Pb. Th: Vai Ann Pb. Ala Asp Lys Lys Ile Tb: Cly Tb: Lou 530 585 590 ACC OCT GAC MAC AGG CAG GAG GCA A= TTT ACC AT CAT OCT AA7 AMT 1824 Th: Ala Asp Ann Ar; Gin Ciii Ala Tb: Pb. Thr Ile Asp Oly Asn Zia 595 600 605 ADO GAC AMC CCC 271 GMA GGT ACG C AAA ACT OCT GAG TCA OCT TT? 1872 Lys Asp Ann Gliy PMe Gli Gly Th: Ala Lys Tb: Ala Glu 5cr Gly Phe 610 615 620 GA? C70 CAT CAA AGC AMT MC ACC CGC P00 OCT ADO GCA TAT P.70 ACA 1920 Asp Lou Asp Giii an Tb: Thr Arg Tb: Pro Lys Al1a Tyr Ile Thr 625 630 635 640 CAT GCC AAM G70 CArv GCC COT 77 TMC 000 C=C AM GCC GAD GAG TTN 1968 Asp Ala Lys Val. Gin Gly Cly Ph. Tyr Cly Pro Lys Ala Ciii Cii Lou 645 650 655 CCC COP. TCC 77 CC TAT CCC CCC CAT AMA CAA MCG AAM MT GCA MCA 2016 Gly Gly Trp Ph. Ala Tyr Pro Gly Asp Lys Gin Tbr Lys Asn Ala Tb: 660 665 670 WO 9S/33049 57 PCT/FR95/00701 AAT GCA TCC GGC AAT AGC AGT GCA ACT GTC CTA TTC CC? C AAA CCC 2064 Aen Ala Sox Gly Asn Ser Sex Ala Thr Val Val Ph. Gly Ala Lys Ar; 675 Goo 6igs CAA CAG CCT GTG CGA TAACGCAAGC CCAAAAACAC CAAGGCGGvAT ACGGT 2114 Gin Gin Pro Val Ax;q 690 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 693 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Cys Lou Gly Giy Gly Gly Thr Ph* Asp Lou Asp Sex Val Asp Thr Glu 1 5 10 Ala Pro Ar; Pro Asp Pro Lys Tyr Gin Asp Val Ser Ser Ciii Lys Pro 25 Gin Ala Gin Lys Asp Gin Gly Gly Tyr Giy Ph. Ala Met Arg Lou Lys 40 Arg Azrg Asn Trp Tyr Ser Ala Ala Lys Giu Asp Giu Val Lys Lou Asn 55 Giu Se: Asp Trp Giu Thr Thr Gly Lou Pro Thr Glu Pro Lys Lys Lou 70 75 Pro Lou Lys Gin Glu Set Val Ile Ser Lys Val Gin Ala Asn Asn Gly 90 Asp Asn Asn Ile Tyr Thr Se: Pro Tyr Lau Thr Gin Sex Asn His Gin 100 105 110 Asn Set S: Ile Asn Gly Gly Ala Asn Lou Pro Lys Asn Giu Val Th: 115 120 125 Asn Tyr Lye Asp Ph* Lye Tyr Val Tyr Set Gly Trp Ph. Tyr Lys 'Hfis 130 135 140 Ala Lys Asn Gu Ile Ile Az; Giu Asn Ser Str Ile Lys Giy Ala Lys 145 150 155 160 Asn Giy Asp Asp Ciy Ty Ile Phe Tyr His Giy Lys Giu Pro Ser Ar; 165 170 175 Gin Lou Pro Ala Ser Giy Thr Vai Thr Tyr Lys Gly Val Trp His Ph.

180 185 190 Ala Thr Asp Val Lys Lys Se: Gin Asn Ph* Ar; Asp Ile Ile Gin Pro 195 200 205 Ser Lys Lys Gin Gly Asp Ar; Tyr Ser Gly Ph. 5cr Gly Asp Asp Asp 210 215 220 Giu Gin Tyr Se: Asn Lys Asn Giu Set Met Lou Lys Asp Giy Gin Glu 225 230 235 240 wo 95/33049 Gly Tyr Gly Ph. Thr Sex AMn Lou 245 58 PCT/FR95/00701 Lou Asn Gly Gly 305 Gly Sex Lys Ser Val 385 Lau Ile Gin Pro Ala 465 Thr Gly Ser Phe Asn 545 Ser Thr Asp Asn 290 Giy Gly Asp Thr Asn 370 Lau Asp Pro Gly Glu 450 Gin Tyr Met Ser Lau 530 Ile Thr Gly Lys 275 Arg Thr Ph.

Asp Glu 355 Gly Asp Mn Lou Thr 435 Sex Thx Glu Lou Sex 515 Gln Val Ser Lys 260 Tyr Ph.

Lys Phe Lys 340 Asn Ala Ala Ph* Lou 420 Asr Asp Ala Val Thr 500 Gin Gly Tyr Trp Arg Gin Lys 295 Pro Lys Ala Val Thr 375 Lou Ala Ala Thr Asp 455 Thr Cys Asn Ala Thr 535 Ser Asn Asn Tyr 280 Ala Ph.

Gly Val Ala 360 Ser Lys Ala Sex Ala 440 Ala Ala Cys 5cr Lys 520 Asp Tip Ala Giu Asn 265 Tyr Ii Val Glu Val 345 Ser Sex Lau Gin Glu 425 Ph.

Gin Gly Sex Lys 505 Thr Glu Tyr Sex Val Asp 250 Ar; Val Sex Lau A.rq Thr Sex Asp 315 Glu Lou 330 Giy Sex Gly Gly Gu Asn Gly Asp 395 Lou Val 410 Sex Gly Thr Arg Ala Gly Asp Thr 475 Mn Lou 490 Ser Ala Giu Gin Lys Glu Gly Tyr 555 Asn Ala 570 Phe Gly Sex Lys 255 Thr Msn Ala Pro 270 Asp Ala Gin lie 285 Asp Lys Pro Asp 300 Ser Sex Sex Lau Gly Ph. Ar; Ph.

335 Ala Lys Thr Lys 350 Thr Asp Ala Ala 365 Sex Lys Lou Thr 380 Lys Giu Val Gin Val Asp Gly Ii 415 Mn Msn Gin Ala 430 Lys Phe Asp His 445 Thr Gin Thr An 460 Mn Giy Lys Thr Mn Tyr Lou Lys 495 Met Gln Ala Gly 510 Val Giu Gin Sex 525 Ii Pro Sex Glu 540 Ii Ala Msn Asp Thr Ser Gly Asn 575 Lys Thr Thr Thr Sex 320 Lou Asp Ala Thr Lys 400 met Asn Thr Gly Lys 480 Tyr Glu Met Gin Lys 560 Arg Wo 95/33049 59 PCT/FR9 5/00701 Ala Gu Ph. Thr Val Msn Ph. Ala Asp Lys Lys Ile Thr Gly Th: Leu 580 585 590 Thr Ala Asp Asn Arg Gin Glu Ala Thr Ph. Thr Ile Asp Gly Mn Ile 595 600 605 Lys Asp Asn Gly Phe Giu Gly Thr Ala Lys Tb: Ala Giu Gly Phe 610 615 620 Asp Leu Asp Gin Sex An Th: Thr Arg Th: Pro Lys Ala Tyr Ii Thr 625 630 635 640 Asp Ala Lye Val Gin Gly Gly Ph. Tyr Gly Pro Lys Ala Gu Giu Lou 645 650 655 Gly Gly Trp Ph. Ala Tyr Pro Gly Asp Lys Gin Thr Lye Mn Ala Tb: 660 665 670 Msn Ala S: Gly Msn Ser Sex Ala Thr Val Val Pb. Gly Ala Lye Arq 675 680 .685 Gln Gin Pro Val Arg 690 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CRLARACTERISTICS: LENGTH: 2114 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: S3032 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1..2097 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2097 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TGT TTG GGC GGA GC GGC GCC AGT TTC GAT CTT GAT TCT GTC GAT ACC 48 Cys Lou Gly Gly Gly Gly Gly Ser Phe Asp Leu Asp Sex Val Asp Thr 1 5 10 AA GCC CCO CGT CCC GCG CCA AAG TAT CA GAT OTT TCT TCC GM AAA 96 Glu Ala Pro Arg Pro Ala Pro Lys Tyr Gin Asp Val Sex Se: Glu Lys 25 CCG CM GCC CAA AAAAC AA CGC OGA TAC GGT TTT GC ATG AGG TTG 144 Pro Gin Ala Gin Lye Asp Gin Gly Gly Tyr Gly Phe Ala Mei Mg Lau 40 AAA CGG AGG AhT TOG TAT COG TOG OCA AM GM MC GAG OTT AM CTG 192 Lye Arg Ag Msn Trp Tyr Pro Sex Ala Lys Giu AMn Giu Vai Lye Lou 55 WO 95/33049 AAT GAG ACT GAT TGC GA Asa Glu Ser Asp Trp GI 7 TTA CC? GAG CGA CAC AA~ Lou Pro Glu Ar; Gin Ly GAC AGC AAT AAC AGC AA Asp Asn Amn Ser As 100 AAC CAT CAA AAC GCC AAA Asn His Gin Asn Gly Asz 115 GAP GTA ACA GAT TAC APM Glu Val Tb: Asp Tyr Lys 130 TAC AAA CAC GCC AAA CG.J Tyr Lys His Ala Lys Azq 145 150 GCA AAA MC CCC CAC CAC Ala Lys Msn Cly Asp Asp 165 TCC CGA CMA CT? CCC GC? Se: Arg Gin Lou Pro Ala 1SO CAT TTT C ACC GAT ACA His Pb. Ala Tb: Asp ?hr 195 CM CC? TCA AAA MAT CA Gin Pro Se: Lys Msn Gin 210 GAT GA.T GMA CMA TAT ?CT AsP Asp Glu Gin Tyr Se: 225 230 CAT GMA CC? TAT GOT TT His Glu Gly Tyr Gly Pb.

245 AAA AAA TTG ACG CC? AAA Lys Lys Lou Tb: Gly Lys 260 AAT ACT APT APT GAC AAA Asn Th: Asn Asn Asp Lyz 275 ACC CTT MAG GGA AAC CGC Tb: Lou Lys Gly Msa Azq 290 CCC AMA AAC GAbC CCC GMA Pro Lys Msn Asp Cly Glu 305 310 6o PCT/FR9/00701 GACC ACA GCA TTG CCA AGC AMT CCC AMA MC u Tb: Th: Gly Lou Pro So: Asn Pro Lys Ann 0 75 s0 ATCG CT? AT? GA? CMA GA GMA ACA GAT CC s Sm: Val le Asp Gin Val Clu Tb: Asp Gly 90 rAT? TAT TCT TCC CCC TAT CTC ACG CMA ?CA n Ile Tyr Se: So: Pro Tyr Lou Th: Gin Se: 105 110 ACT CCC AAC CC? OTA AMC CAA CCA AMA MC i Tb: Gly Mna Gly Val Msn Gin Pro Lys Asn 120 125 AA MTTT MAA TAT CT? TAT ?CC CCC TGG TT? Msn Phe Lye Tyr Val Tyr So: Gly Trp Pb* 135 140 GAG CT? MAC TTA C CC GMA CC? AMA AT? Giu Val Mna Lou Ala Val Glu Pro Lys Ile 155 160 CC? TAT ATC TTC TAT CAC GGT AMA GAC CCT Gly Tyr Ile Pb. Tyr Mix Gly Lys Asp Pro 170 175 TCT GGA AMA AT? ACC TAT AAA CC? GTC TGG Gly Lys Ile Tkir Tyr Lys Gly Val Trp 195 190 AAA AGC CC? CM AAA TT? CC? GMA AT? ATC Lys Ax; Cly Gin Lys Pb. Arg Giu Ile Ile 200 205 CCC GAC ACGA TA? ACC GGA ?TT TCG CC? GA? Gly Asp Arg Tyr Se: Cly Pb. Cly Asp 215 220 MT MAA M&C GMA TCC ATC CTC AAA CAT CC? Min Lye Msn Clu So: Hot Lou Lys Asp Gly 235 240 CCC TCC MRT TA CMA CG GAT ?TC CAC MAT Ala Msn Lou Ciu Val Asp Pb. Asp Msn 250 255 TTh ATA CCC MAT MAT CC MC CMA MT MAT Lou Ile Ar; Msn Asn Ala Msn Gin Asn Amn 265 270 CAC ACC ACC CM~ TAC TAC ACC CT? CAT CC His Th: Tb: Gin Tyr Tyr Se: Lou Asp Ala 280 285 TC AGC GGA AMA GCG GMA CCA ACC GAC AMA Pb. Se: Gly Lys Ala Glu Ala Tb: Asp Lys 295 300 ACC AAG GMA CAT CCC TT CT? TCC GAC TCG Tb: Lye Clu His Pro Pb. Vol So: Asp 315 320 240 298 336 384 432 480.

528 576 624 672 720 768 516 064 912 960 WO 95/33049 61 PCT/FR95/Oo701 GGT GAG GMA TTG GGT 1008 Gly Gin Giu Lou Oly 335 TCT TCT TYG AGC GGC GGC TTT TTC GGC CCG CMG So; Sex Lou Sex Gly Gly Pb. Ph* Gly pro Gin 325 330 TTC CGC TTT TYG AOC Ph* Ar; Phe Lou. Sec 340 AAA ACC AAM GAC AA Lys Tb: Lys Asp Lys 355 AMC GAT RA ASAi Asp Gin AAA OTT CC Lys Val Ala 345 OTT rwTC GOC AGC GCG Val Val Gly Set Ala 350 CCC GCA AAT GGC RAT ACT OCO GAG OCT TCA OC Pro Ala Ann Gly Asn Tb: Ala Giu Ala Set Gly 360 365 GGC ACA GAT Gly Tb: Asp 370 GCG OCA OCh Al1a Ala Ala TCG GC OT GCO& OCA GGC ACG TCG TCT RA Sex Gly Oly Ala Al1a Gly Tb: Sac Bar Giu 375 380 AMC ROT Asn Sac 305 AMG CTG ACC Lys Lau Tb: ACG CTT Tb: Val 390 TYO GAT GCG OTC GAG CTG ACG CAC GOC Lou Asp Ala Val Gin Lou ThE His Oly 395 400 GGC ACA WCA ATC Gly Tb: Ala Ile AAA RAT Lys Asn 405 CTC CAC JAC TTC AOC LOU ASP Asn the Sex 410 RAT 0CC GCC CMA CTG Asn Ala Ala G1n Lou 415 OTT OTC GA6C Val Val Asp AA.A AAT MAT Lys Ao As.

435 TAT MAA ACG Tyr Lys Tb: 450 GOC ATT Gly Ile 420 ATO ATT CCG CTC Met Ile Pro Lau .425 CTC CCT CAA RAT TCA ACA GOC Lou Pro G1. Amn S.r Tb: Gly 430 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 CMG CCC GAT Gin Pro Asp CAM GOT Gin Gly 440 RA Am G0 Lys An Giy GA ACK GCC TTT ATC Giy ThE Ala Ibe Ile 445 ACC TAC ACG CCO AA AAC GAT Tb: Tyr Tb: Pro Lys Aso Asp 455 GAO AMA Asp Lys 460 OAT ACC AMA GCC Amp Tb: Lys Ala CAA ACA OTO ACG GGC GCC Gin Thr Val Tb: Gly Gly 465 470 ACC CMA A=O OCT TM Thz Gin Tb: Ala sac 475 ACC TAT GMA 070 GMA Tb: Tyr Gin Val Giu 490 GCC AAT 000 AMA Ala AMn Gly Lys ACA A Th: Lys 485 RAT ACO OCA OT GAT Msn Tb: Ala Gly Asp 480 070 TOO TOT TOO AMC Vol Cys Cys Sur AMn 495 AAA ACT 0CC GOC ARC Lys Thx Ala Oly Aso 510 CTC AAT TAT Lou Msn Tyr OTQ AMA Lou Lys 500 TAC 000 TTG CTG ACG Tyr Gly Lou Lou Tb: 505 AC GTO GA AGC Th: Val Gly Set 515 GOC MhC AGO AGC Gly Msn Se: S.: 520 C ACC GCC Pro Thc Ala 0CC 0CC Ala Ala 525 CAA RCG GA6C Gin Thx Asp GCG CRG ACT Ala Gin Sex 530 ATG -I=CT07 Met Pb. Lau CAA 000 01n Oly 535 GAG COO ACC GAT Glu Ax; Tb: Asp 540 GMA AC AMG ATT Giu Asn Lys Ile 1534 1632 1680 1728 CCR AGC GMG CAP Pro Se: Oiu Gin 545 0CC AGO AOC ACA Ala Se: Se: Tb: AAC GTC OTT TAT CGG 000 Msn Vol Val Tyr Ar; Gly 550 AGC TOG ACC GOC RAT OCT Set Trp Sex Gly Aso Ala 565 570 TOO TAC 000 Txp Tyr Gly CAT AT! His Ile GGC 000 Gly Gly 575 TCT GAT A GAG Sex Asp Lys Giu WO 95/33049 62 PCT/FR95/00701 AAC AGG GCG GAP TZT ACT 070 AAT TTT GC GAG PAP AMP AT ACC GGC 1776 Awn Ar; Ala Glu Phe Thr Val Mn Ph'Gly Glu Lys Lys Ile Thr Gly 0 585 590 ACG TTA ACC OCT GAA AC AGO CAG GAG G= ACC Ac AT GAT GGT 1824 Thr Lou Thz Ala Glu Asn Ar; Gin Glu Ala Thr Ph. Thr Ile Asp Gly 595 600 605 AAO AT? GAG GGC AP GOT T77 TCC GWT ACG GCA AAA ACT CT GA TTA 1872 Lys Ile Glu Gly Asn Gly Phe So: Gly Thr Ala Lys Thr Ala Glu Lou 610 615 620 GGT 777 CAT CTC OAT CA AAA AT ACC AC CGC ACG CCT AAO CCA TAT 1920 Gly Phu Asp Lou Asp Gin Lys. Mn Thr Thr Ax; Th: Pro Lys Ala

T

yr 625 630 635 640 ATC ACA GAT GCC AAG OTA AAO G0 GT 77 TAC GGG CCC AAA GCC G 1968 Ile Thz Asp Ala Lys Val Lys Gly Gly Ph* Tyr Gly Pro Lys Ala Gin 645 650 655 GAG TTG G00 GGP. TOG T77 GCC TAT TOG GAC GAT AAA CAA ACG AAA AAT 2016 Gin Lou Oly Gly Trp Ph. Ala Tyr Sur Asp Asp Lys Gin Thr Lys Mn 660 665 670 GCA ACA OAT OCA CC 0CC AT OA PAT TOP GCA PC AGT-GCA ACT GTC 2064 Ala Thr Asp Ala So: Gly Mn Gly Mn 3. Ala Ser Ala T7hz Val 675 680 635 GTA TTC GOT 000 AAA CC CAP CAG CCT 070 CPA TAAACCPPGG COUTAC 2114 Val Ph. Oly Ala Lys Ar; Gln Gin Pro Val Gln 690 695 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 699 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Cys Lou Gly Gly Gly Gly Gly Sac Ph. Asp Lou Asp So: Val Asp Tht 1 5 10 Glu Ala Pro Ar; Pro Ala Pro Lys Tyr Gin Asp Vl r Sc Glu Lys 25 Pro Gin Ala Gin Lys Asp Gin Gly Oly Tyr Gly Phe Ala nt Ar; Lou 40 Lys Ar; Arg Mn Trp Tyr Pro Ser Ala Lys Glu Ann Giu Val Lys Lou 55 Asn Giu Sex Asp Trp Glu Thr Thr Gly Lau Pro Ser Asn Pro Lys Asn 70 75 s0 Lou Pro Glu Arg Gin Lys Ser Val Ile Asp Gin Val Gi Thr Asp Gly 90 WO 95/33049 Asp Ser Asn Asr Asn His Gin Asm 115 Glu Val Thx Asp 130 Tyr Lys His Ala 145 Al1a Lys Asn Gly Ser Arq Gin Lou 180 His Phe Ala Thx 195 Gin Pro Ser Lys 210 63 PCT/FR95/00701 iSex Awn Ile Tyr Sex 10! iGly Asn Thr Gly Asti 120 Tyr Lys Asn Ph. Lys 135 Lys Arq Glu Val Asn 150 Asp Asp Gly Tyr Ile 165 Pro Ala Ser Gly Lys 185 Asp Thr Lys Azq Gly 200 Asn Gin Gly Asp Arg 215 Sc: Pro Gly Val Tyr Val Lou Ala 155 Ph. Tyr 170 Ile Thr Gin Lys Tyr Sex Tyi Asn Tyr 140 Val His Tyr Ph.

Gly 220 Lou Gin 125 Sex Glu Gly Lys Arg 205 Ph.

Thz 110 Pro Giy Pro Lys Gly 190 Glu Lysn Trp Lysn Asp 175 Val Ile Gly I Sex IAwn Ph.

Ile 160 Pro Trp, :Ie Asp Asp Glu Gin Tyr Sex Asn Lys Asn Glu His Giu. Gly Tyr Ph. Ala Ser Lys Amn Thr Pro 305 Sex Ph.

Lys Gly Asn 385 Lys Thz Lou 290 Lys Se: Arg Thr Thr 370 Sex Lau Asn 275 Lys Asn Lau Phe Lys 355 Asp Lys Thr 260 Asn Gly Asp Ser Lau 340 Asp Ala Lou Asn Axq 265 Lou 250 Awn Lau Asp Msn Asp Asp 255 Asn Gly 240 AMn Awn Gly Lys Leu Ile 270 Asp Asn G1 y Gly 325 Ser Lys Al1a Thr Lys 405 Lys His Axg Ph.

295 Glu Thr 310 Giy Ph.

Awn Asp Pro Ala Ala Sex 375 Thr Val 390 Asin Lau Thr 280 Sex Lys Ph.

Gin Asn 360 Gly Leu Asp, Thr Gin Tyr Tyr Sex Lou Asp Ala Gly Giu dly Lys 345 Gly Gly Asp Asan Lys His Pro 330 Val Asn Ala Ala Phe 410 Ala Pro 315 Gin Ala Th: Ala Val 395 Ser 285 Glu Ala Thx 300 Ph. Val Sex Gly Giu Giu Val Val Gly 350 Ala Glu Ala 365 Gly Thr 5cr 300 Glu Lou Thr Msn Ala Ala Asp Asp Lau 335 Ser Sex Ser His Gin 415 Lys Sex 320 Gly Ala Gly Giu Gly 400 Lau Gly Th: Al1a Ile Val Val Asp Gly 420 Ile Met Ile Pro Lou Lou Pro Gin Awn 425 Ser Thx Gly 430 WO 95/33049 Lys An An 435 64 PCT/FR95/007a1 Thr Ala P. Ile 445 Gln Pro Asp Gln Gly Lys 440 Msn Gly Gly Ty: Gin 465 ALa Lou Thr Ala Pro 545 Ala Mn Thr Lys Gly 625 Ile Glu Ala Val Lys Thr 450 Thr Val Asn Gly Asn Tyr Val Giy 515 Gin Ser 530 Se Glu Sex So: Arg Ala Leu Thr 595 lie Glu 610 Phe Asp Thr Asp Leu Gly Thr Asp.

675 Ph Gly 690 Thr Thr Lys Leu 500 Soo Ser Met Gln Th: Glu 580 Ala Giy Lau Ala Gly 660 Ala Al a Tyr Gly Th: 485 Lys Giy Ph.

Mn Se: 565 Ph.

Glu Asn Asp Lys 645 Trp Se: Lys Th Pro Lys An Asp Asp Gly 470 Lys Tyr Mn Lou Val 550 Trp Thr Msn Giy Gin 630 Val Ph.

zGl Arg 455 Th Th: Gly Se: Gin 535 Val Ser Val Arq Ph.

615 Lys Lys Ala Gln 695 Gin Tyr Lou Ser 520 Gly Tyr Gly Mn Gin 600 Se Mn Gly Tyr Gly 680 Gln Th: Glu Lau 505 Pro Glu Arg An Phe 585 Glu Gly Th Gly Se 665 Asn Pro Ala Val 490 Thr Thr Arg Gly Ala 570 Gly Ala Th: Thr Phe 650 Asp Se Val Sex 475 Glu Arg Ala Thr Ser 555 Se: Giu Thr Ala Mrg 635 Tyr Asp Ala Gln Lys 460 Msn Val Lys Ala Asp 540 Trp Asp Lys Ph Lys 620 Thr Gly Lys Se: Asp Thr Cys Thr Ala 525 Glu Tyr Lys Th 605 Th: Pro Pro Gin Se: 685 Th: Ala Cys Ala 510 Gln Asn Gly Glu Ii 590 Ile Ala Lya Lys Th 670 Ala Lys Gly So: 495 Gly Thr Lys His Gly 575 Thr Asp Glu Ala Ala 655 Lys Th Ala Asp 480 Asn Msn Asp Ile lie 560 Giy Gly Gly Lau Tyr 640 Glu Axn Val INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 198 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: poptide (vi) ORIGINAL SOURCE: ORGANISM: N. moningitidis STRAIN: 112169 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: WO 95/33049 Thr*eLys Asp Lys 1 Ala Ala Ala See Lou Thr The Val Ie. Lys Asn Lou Gly lie Met Ile Gin Ala Asp Lys Glu His The Pro 100 The Asn Gly Ala 115 Lys The Lys The 130 Lou Lys Tyr Gly 145 Ala Gly Gly Msn Gin Sex Met Phe 180 The Asp Gin Msn 195 Lou Giu Mn G1 5 Gly Gly Ala Al 65 .y Ala A 1 .a Gly T1 19 Asp Ala Val Glu 40 Mn Ph. Sex Asn 55 Lou LOu Pro Lys 70 Lys Asn Gly Gly Sex Asp Lys Lys 105 The Ala 5cr An 120 Glu Val Giu Val 135 Lou The Arg Lys 150 Ser Gin Ala Asp Gin Gly Glu Arg 185 Va].

Le la Ala 0 hr Sex u The L* Ala Asp The Asp Thr Cys Asn Ala 170 See 75 Glu Ala Ala Cys Sex 155 Lys PCT/7R95/00701 Sex Gly Secr Thr Gly See Glu Msn Ser Lys Lou Msn Asp Lys Lys Gin Leu Val Val Asp Glu See Gly Msn The Ph. The Arg Lys Ph.

Gin Ala Gly The Gin 110 Gly Asp Thr Msn Gly 125 Sex Asn Lou mn Tyr 140 Lys Ser Ala Met Gin 160 Thr Giu Gin Val Glu 175 The Asp Giu Lys Giu Ile Pro 190 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 198 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: 6940 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: The Lys Asp Lys Thr Giu Msn Gly Ala Val Ala See Gly Gly The Asp 1 5 10 Ala Ala Ala Ser Asn Giy Ala Ala Giy The Soc See Glu Asn Ser Lys 25 WO 95/33049 Lau Thr Th: Val Val Gin Lys Lou Gly Ile Met Ile Gin Al1a Asn Gin Asp His Thr Pro 100 Th: Asn Giy Ala 115 Lys Tb: Lys Th: 130 Leu Lys Tyr Gly 145 Ala Gly Giu Se: Gin Set Met Phe 180 Se: Giu Gin Msn 195 Lou Asp pro Gi y 85 Giu Gin Tyr M at set 165 Lou Ile Asp Ala Msn Phe 55 Leu Lou 70 Tb: Asn Se: Asp Th: Ala Glu Val 135 Lou Th: 150 se: Gin Gin Gly Val Val 40 Sac Pro Giy Lys Sex 120 Giu Arg Ala Glu 66 Glu LOU Msn Ala Glu Ala Giy Tb: 90 Lys ASP 105 Msn Tb: Val Cys Lys Msn Asp Ala 170 Ar; Tb: 135 Lys Ala set 75 Ala Ala Ala Cys Se: 155 Lys Asp PCT/FR95/00701 Lou Gly Asp Lys Giu Gin Lou Val Val Asp Giu set Gly Msn Asn 810 Pho Tb: Arg Lys Phe Gin Ala Gly Tb: Gin 110 Gly Asp Tb: Msn Giy 125 So: Mxn Lou Msn Tyr 140 Lys se: Ala Met Gin 160 Tb: Giu Gin Val Giu 175 Glu Lys Giu Ile Pro 190 INFORMATION FOR SEQ ID NO: 13: SEQtTENCE csARACTERISTICS: LENGTH: 198 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULZ TYPE: peptide (vi) ORIGINAL

SOURCE:

ORGANISM: N. moningitidis STRAIN: 2223 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Th: Lys Asp Lys Tb: Giu Msn Gly Ala Vai Ala So: Gly Gly Tb: Asp 1 5 10 1 Ala Ala Ala Set Msn Gly Ala.Ala Gly Tb: So: Se: Glu Msn Se: Lys 25 Lou Tb: Tb: Val Lau Asp Ala Val Glu Lou Lys Lou.Gly Asp Lys G1u 40 Val Gin Lys Lou Asp Msn Ph. Se: Msn Ala Ala G in Lou Val Val ASP 55 Gly Ile Met Ile Pro Lou Lau Pro Glu Ala se: Glu So: Gly Msn Mn 7075 s0 wo 95/33049 67 PCT/FR95/00701 Gin Ala Msn Gin Gly Thr Asn Gly Gly Th Ala Pie Thr A19 Lys Phe 95 90 AsP His Thr Pro Giu se Asp Lys Lys Asp Ala Gin Ala Gly Thr Gln 100 105 110 Ala Asn Gly Ala Gin Thr Ala Set Mn Thr Ala Gly Asp Thr Msn Gly 115 120 125 Lys Thr Lys Thr Tyr Gu Val Glu Val Cys Cys Set Amn Lau Msn Tyr 130 135 140 Leu Lys Tyr Gly Met Lou Thr Ar; Lys Mn Set Lys Sex Ala Met Gin 145 150 155 160 Ala Gly Giu Set Sex Set Gin Ala Asp Ala Lys ThIr Giu Gin Val Gly 165 170 175 Gin Ser Met Phe Leu Gin Gly Glu Ar; Thr Asp Glu Lys Giu lie Pro 180 185 190 Set Gu Gin Asn Ile Val 195 INFORMATION FOR SEQ ID NO: 14: SEQUENCE

CHARACTERISTICS:

LENGTH: 198 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL

SOURCE:

ORGANISM: N. meningitidis STRAIN: C708 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Thr Gin Asp Lys Pro Arg Asn Giy Ala Val Ala Set Gly Gly Thr Gly 1 5 10 Ala Ala Arq 5r Msn Gly Ala Ala Gly Gin Set Set Glu Msn Set Lye 20 25 Lou Thr Thr Val Lau Asp Ala Val Giu Lou Thr Lou Msn Asp Lys Lys 35 40 Ii Lys Msn Lou Asp Mn Phe 5cr Mn Ala Ala Gin Lou Val Vai Asp 50 55 Gly Ile Met Ie Pro LaU Lou Pro Glu Ala Set Giu Set Gly Lys Msn 70 75 Gin Ala Msn Gin Gly Tkr An Gly Gly Thr Ala Phe Thr Ar; Lys Phe 85 90 Msn His Thr Pro Lys Set Asp Giu Lye Asp Tht Gin Ala Gly Tb: Ala 100 105 110 Glu Msn Gly Msn Pro Ala Ala Set Msn Thr Ala Gly Asp Ala An Gly 115 120 125 WO 95/33049 68 PCT/PR9/00701 Lys Th: Lys Thr Tyr Giu Val Glu Val Cys Cys Ser Asn Lou An Tyr 130 135 140 Leu Lys Tyr Giy Met Lou Th: Arg Lys Asn Ser Lys Se: Ala Met Gin 145 150 155 160 Ala Gly Glu Set Se: Set Gin Ala Asp Ala Lys Thr Glu Gin Val Gly 165 170 175 Gin Scr Met Phe Leu Gin Gly Glu Arg Thr Asp Giu Lys Glu Ile Pro 180 185 190 Asn Asp Gin Asn Val Val 195 INFORMATION FOR SEQ ID NO: SEQURNCE CHARACTERISTICS: LENGTH: 211 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: M978 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Thr Gin Asp Lys Ala Ala Mn Giy Mn Thr Ala Ala Ala I 1 5 10 Th: Asp Ala Ala Ala Se: An Gly Ala Ala Gly Thr So: 25 Sex Lys Leu Tb: Thr Vai Leu Asp Ala Val GiU Lou Th: 40 Lys Lys Ile Lys Msn Lau AsP Msn Ph. Set An Ala Ala 4 55 Val Asp Gly Ile Met Ile Pro Leu Lou Pro Glu Th: Se: 70 75 So: Mn Gin Ala Asp Lys Gly Lys Lys Gly Lys Asn Gly 90 Gly Th: Asp Phe Thr Tyr Lys Thr: Thr Tyr Th: Pro Lys 100 105 Lys Ap Thr Lys Ala Gin ThrG ly Ala Ala Gly Sex So: 115 120 125 Thr Asp Lou Gy Lys Ala Asp Vai An Gly Giy Lys Ala 130 135 140 Thr Tyr Glu Val Giu Vai CyS Cys Set Mn Lou Mn Tyr 145 150 155 Gly Met Lou Tb: Arg Lys Mn Se: Lys Sex Ala Met Gin 165 170

I.:

atr Lau Gin Glu Lys Axn 110 Giy Glu LOu Ala Gly C Glu I Msn Lou set A.n Asp Ala Th

LYS

Gly 175 ,iy Lsn ksp, V'al Gly Gly Asp Gin Lys Tyr 160 Giy

M

WO 95/33049 69 PCT/FR95/00701 Asn Ser Sor Gin Ala Asp Ala Lys Thr Giu Gln Val Glu Gin Ser met 180 195 190 Ph* Lou Gin Gly Giu Arg Thr Asp Glu Lys Giu Ii Pro Asn Asp Gin 195 200 205 Asn Val Val 210 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 200 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: 1610 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Lys Arg Asp Lye Ala Glu Set Gly Gly Gly Asn Gly Ala Sac Gly Gly 1 5 10 Thr Asp Ala Ala Ala Ser Axn Gly Ala Ala Gly Thr Sr Sacr Gu Asn 25 Scr Lys Lou Thr Tbr Val Lou Asp Ala Val Glu Lau Lys Ser Gly Gly 40 Lys Giu Val Lye Asn Lou Asp Asn Ph. Ser Asn Ala Ala Gin Leu Val 55 Val Asp Gly Ile Met Ile Pro Lou Lou Pro Lye Asp Ser Glu Set Gly 70 75 Asn Thr Gin Ala Asp Lys Gly Lys Amn Gly Gly Thr Lys Phe Thr Atq 90 Lys Ph. Giu His Thr Pro Giu Se: Asp Lys Lys Asp Ala Gin Ala Gly 100 105 110 Thr Gin Thr Asn Giy Ala Gin Thr Ala Scr Asn Thr Ala Gly Asp Thr 115 120 125 Asn Gly Lye Thr Lye Thr Tyr Glu Val Giu Val Cys Cys 5cr Asn Lou 130 135 140 Asn Tyr Leu Lys Tyr Gly Lou Lou Thr Arg Lys Thr Ala Gly Asn Thr 145 150 155 160 Gly Giu Giy Gly Asn Gly 5cr Gin Thr Ala Ala Ala Gin Thr Ala Gln 165 170 175 Gly Ala Gin c Met Pho Lou Gin Gly Glu Arq Thr Asp Glu Lys Glu 180 185 190 Ile Pro Ser Giu Gin Asn Val Val

I

WO 95/33049 70 INFORMATION FOR SRQ ID NO: 17: Wi SEQUENCE CHARACTERISTICS: LENGTH: 200 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: 867 PCT/FR95/00701 Th: 1 Ala Lau 11e Gly Gin Asp Th: Gin Tyx 145 Gly Ala (Xi) SEQUE iAsp Lys P~ 5 kAla Sex AU Th: Val L4 i Asn Lau As Met Ile P2 Asp Lys GI es Tb: Pro Ly 100 Gly Ala G1 115 Gly Lys Th Lys Tyr Gi Gly Msn Gi 16 Ser Met Ph 180 ro Arg Amn Giy Ala In mu 'p y y s n RCE DESCRIPTION: SEQ ID NO: 17: Gly Asp Asn Lou 70 Lys Sex Th: Ty: Lou 150 Ser Leu Ala Ala Phe met Asn Asp Ala Ala 135 Lou Sex Gin Ala Gly 25 Val Glu 40 So: Msn pro Giu Gly Gly Glu Lys 105 So: Gly 120 Val Giu Tb: Arg Th: Ala Gly Giu 185 Val 200 Val Ala S.: 10 Tb: Scr Ser Lou Tb: Lou Ala Ala Gin Th: Se: Glu 75 Tb: Ala Ph.

90 Asp Tb: Gin Th: AlIa Gly Val Cys Cys 140 Lys Tb: Ala 155 Ala Ala Gin 170 Arg Tb: Asp Gly Gly Th: Asp Giu Msn Gly Lys Msn Asp Lys Lys Lau Val Val Sex Sex Gly Msn Mnn Tb: Arg Lys Ph.

Ala Gly Tbr Pro 110 Val Tb: Gly Gly 125 Se: Msn Lou Msn Asp Msn Tb: Val.

160 Tb: Ala Gin Gly 175 Giu Lys Giu Ile 190 Pro Lys Giu 195 Gin Gin Asp Ile INFORMATION FOR SEQ ID NO: 18: Ci SEQUENCE CHARACTERISTICS: LENGTH: 198 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: S3032 WO 95/33049 -71 PCT/FR95/00701 (xi) Thr: Lys Asp 1 SEQUENCE DESCRIPTION: SEQ ID NO: 18: Lys Pro Ala Asn Gly Am Thr Ala Giu Ala 5 10 Ser Gly Gly Thr Asp Ala Ala Ala Sc Gly Gly Ala Ala Ser Th: Val Asn Lys The Msn Asn 145 Val Gln Se: Lys Ala s0 Asp Asn Thr Val Gly 130 Tyr Gly Ser Glu Leu lie Gly Gln Th: Thr 115 Lys Leu Sex Met Gin 195 Thr Lys 114 Pro Tyr 100 Gly Thr Lys Gly Phe 180 Asn Thr Asn met Asp Th Giy Lys Tyr Asn 165 Lau Val Leu Iie 70 Gn Pro Thr Thr Gly 150 5er Lau Asp 55 Pro Gly Lys Gin Tyr 135 Lau Ser Asp 40 sn Lau Lys An Thr 120 Glu Lou Pro 25 Ala Ph.

Leu Asn Asp 105 Ala Val Thr Th: Val Sa: Pro Gly 90.

Asp Sex Glu Arg Ala Gly Glu Asa Gin 75 Gly Lys Msn Val Lys 155 Ala Th Lau Ala Asn Thr Asp Tb: Cys 140 Th: Ala Ser Th: Ala Ser Ala Thr Ala 125 Cys Ala Gin Se: His Gin Tb: Ph.

Lys 110 Gly Ser Gly Th: Giu As Gly Gi Lou Va Gly Ly, ile Ty.

Ala Giz Asp Ald Mn Let Asn Thr 160 Asp Ala 175 ft y 1

I

170 Gin Gly Giu Arg 185 Val Thr Asp Giu Asn Lys tie Pro 190 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 195 amino acids TYPE: amino acid TOPOLOGY: linear ii) MOLECULE TYPE: peptide vi) ORIGINAL SOURCE: ORGANISM: N. mningitidis STRAIN: 891 xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Asp Lys Pro Gly Asn Gly Ala Ag Lou Gin Ala Ala Arg Cys 5 10 Thr Lys 1 WO 95/33049 72 PCT/FR95/00701 Gly Thr Ser Asn Gly Ala Ala Gly Gln Ser Ser Glu As Ser Lys Leu 25 The Thr Val Leu Asp Ala Val Glu Leu Lys Leu Gly Asp Lys Glu Val 40 Gln Lys Leu Asp Asn Phe Ser Asn Ala Ala Gln Leu Val Val Asp Gly 55 Ile Met Ile Pro Leu Lou Pro Lys Asp Ser Glu Ser Gly Lys Asn Gin 70 75 Ala Asp Lys Gly Lys Asn Gly Glu Thr Glu Phe Thr Arg Lys Phe Glu 90 His Thr Pro Glu Sex Asp Glu Lys Asp Ala Gln Ala Gly Thr Pro Ser 100 105 110 Asn Gly Ala Gin Thr Ala Ser Asn Thr Ala GIy.Asp Thr Asn Gly Lys 115 120 125 Thr Lys Thr Tyr Glu Val Asn Leu Cys Ser Asn Leu Asn Tyr Leu Lys 130 135 140 Tyr Gly Leu Leu Thr Arg Lys Thr Ala Gly Asn Thr Gly Glu Gly Gly 145 150 155 160 Asn Ser Ser Pro Thr Ala Ala Gln Thr Ala Gin Gly Ala Gln Ser Met 165 170 175 Phe Leu Gin Gly Glu Arg Thr Asp Glu Lys Glu Ile Pro Asn Asp Gin 180 185 190 Asn Val Val 195 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AAACCCGGAT CCGTTGCCAG CGCTGCCGT 29 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 85 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: Wo 95/33049 73 PCT/FR95/00701 TT=Tr AGATATCTGG CAACATT=T GTTATCICTG GCGGGAA TCACCGCCGG GTGCCTGGT Gr&CGGCGGCA GPTTC INFORMATION FOR SEQ ID NO: 22: Wi SEQUENCE CHARACTERISTICS: CA) LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GTGTTTTTGT TGA.GTGCATG CCTGGGTGGC INFORMATION FOR SEQ ID NO: 23: Wi SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid CC) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TGCGCAAGCT TACAGTTTGT CTTTGGTTTT CGCGCTGCCG INFORMATION FOR SEQ ID NO: 24: Wi SEQUENCE3 CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid CC) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA Cgenomic) Cxi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: AAAAAGCATG CATAAAAACT ACGCGTTACA CCATTCAAGC INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genemic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TATATAAGCT TACGTTGCAG GCCCTGCCGC GTTTTCCCC WO 95/33049 -74 PCT/FR9S/00701 INFORMATION FOR SEQ ID NO: 26: Wi SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucloic acid STRKNDED1NRSS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: cccGAATTCT GCCGTCTGAA GCCTTATTC! 29 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CCCGAATTCT GCTATGGTGC TGCCTGTG 28 INFOR1MATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CGCATCCAAA ACCGTACCTG TGCTGCCTGA INFORMATION FOR SEQ ID NO: 29: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANIfEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genotmic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TTTATCACTT TCCGGGGGCA GGAGCGQAAT WO 95/33049 75 PCT/FR95/00701 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTGGAACAG CAGACAGCGG TTTGCGCCCC INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GAACATACTT TGTTCGTTTT TGCGCGTCAA INFORMATION FOR SEQ ID NO: 32: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: IM2394 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Tyr Lys Gly Thr Trp 1 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: IM2394 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Glu Phe Glu Val Asp Phe Ser Asp Lys Thr lie Lys Gly Thr Leu 1 5 10 WO 95/33049 76 PCT/FR95/00701 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: IM2394 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: Glu Gly Gly Phe Tyr Gly Pro Lys Gly Glu Glu Leu 1 5 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: N. meningitidis STRAIN: IM2394 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ala Val Phe Gly Ala Lys 1 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 2070 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: BZ83 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 1..60 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 61..2067 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2067 -77- ATG AAC AAT CCA TTG GTA AAT CAG GCT GCT ATG GTG CTG CCT GTG TTT Met Asn Asn Pro Leu Val Asn Gin Ala Ala Mot Val Leu Pro Val Phe

TTG

Leu

GTC

Val

TCC

Ser

ATG

Met

AAG

Lys

GAT

Asp

CTG

Leu

GGA

Gly

GGT

Gly 125

SAC

Asp -15

TTG

Leu

GAT

Asp

GAA

Giu

CGC

Arg

GCA

Ala

GAG

Giu

AGT

Ser

TAC

Tyr 110

GCC

Ala

GGC

Gi y

AGT

Ser

ACC

Thr

ACA

Thr

TTC

Phe

TTA

Leu

TTT

Phe

GAG

Glu 95

ACG

Thr

AAT

Asn

TAC

Tyr

GCT

Al a

GAA

Gi u

CCG

Pro

AAG

Lys

TCA

Ser

CCC

Pro 80

TCT

Ser

GAT

Asp

AAA

Lys

CTT

Leu

TGT

Cys 1

GCC

Al a

CAA

Gin

CGG

Arg

GAA

Giu

CAA

Gin

CTT

Leu

TTC

Phe

ATC

Ile

TTC

Phe 145

CTG

Leu

CCG

Pro

GCC

Al a

CGG

Arg 50

GCG

Al a

AGG

Arg

CAG

Gin

CAA

Gin

GAT

Asp 130

TAC

Tyr

GGC

Gi y

CGT

Arg

CAA

Gin 35

AAT

As n

SAT

Asp

AAT

As n

CTG

Leu

TAT

Tyr 115

TTC

Phe

AAA

Lys

GA

Gi y

CCC

Pro 20

AAA

Lys

TGG

T rp

TGG

T rp,

GAA

Giu

GGT

Gly 100

GTC

Val

CAA

Gin

GGC

Gi y GGC GGC Gly Gly 5 GCG CCA Ala Pro GAC CAA Asp Gin TAC CCA Tyr Pro GAG AAG Giu Lys 70 ATA TTG Ile Leu 85 GAG GGC Giu Gly CGC TCG Arg Ser AAA AAA Lys Lys AGC AAT Ser Asn 150 AGT TTC Ser Phe -AG TAT Lys Tyr GGC GGA Gly Gly AAA AAT Lys Asn 55 TTA GGT Leu Giy AAT ATG Asn Met GGC AAA Gly Lys GGC TAT Gly Tyr 120 ATC GCC Ile Ala 135 CCT TCC Pro Ser

SAT

Asp

CAA

Gin

TAC

Tyr

GAA

Giu

GCG

Al a

ACT

Th r

AGC

Ser 105

ATC

Ile

CTT

Leu

CAA

Gin

CTT

Leu

SAT

Asp

GGT

Gly

GAA

Giu

GGT

Giy

SAC

Asp

CGC

Arg

TAC

Tyr

TCC

Ser

GCT

Ala

GAT

Asp

GTT

Val1

TTT

Phe

SAT

Asp

AAG

Lys

GGA

Giy

GTA

Val

CGC

Arg

GGT

Gi y

CTG

Leu 155

TCT

Ser

TCT

Ser

GCA

Ala

CAT

His

CCA

Pro

ATT

Ile

GAA

Gi u

AAC

As n

CCG

Pro 140

CCG

Pro 96 144 192 240 288 336 384 432 480 528

S

000

OS

S S 0SS 0 @0 0 555 @5 5 5 55..

55 0 55 55

S.

0

S*S

00 05 00 0 S 050 5 ATG GGT AAG GTA GGT TAT AAA GGT ACT TGG SAT TAT GTA ACC GAT GCC Met Giy Lys Val 160 Gly Tyr Lys Giy Thr 165 Trp Asp Tyr Val Thr Asp Ala 170

S

SSSS..

0 -78- AAG ATG GGA CAA AAA TTT TCC GAG TTG GCT GGT TTT CCA GCG GGG GAT Lys

AGG

Arg

AGC

Ser 205

GAG

Giu

AAC

Asn

CGT

Arg

GCA

Ala go. TTT go Phe 285

GGT

GC

Vla

CG

Ala

CC

er Met Gly 175 TAT GGG Tyr Gly 190 GAG GCA Giu Ala GTG GAT Val Asp CGG ATT Arg Ile TAC GAC Tyr Asp 255 ACG GCA Thr Al a 270 GTT TCC Val Ser GAG G.AA Glu Giu GTC GGC Val Gly TCA GGC Ser Gly 335 TCT GAA Ser Glu 350 TCT GGC Ser Gly CAA CTG Gin Leu GAA AGC Glu Ser Gin Lys Phe Ser Gin Leu Aia 180

GCT

Al a

CAG

Gin

TTC

Phe

ACT

Thr 240

ATT

Ile

ACC

Th r

GAC

Asp

TTG

Leu

AGC

Ser 320

AGC

Se r

AAC

As n

GGT

Gi y

GTT

Val1

GAG

Giu 400

TTG

Leu

CAA

Gin

GCC

Al a 225

AAT

As n

CAG

Gin

GAC

Asp

TCG

Se r

GGT

Gi y 305

GCG

Al a

ACA

Th r

GGT

Gi y

AAG

-ys

GTC

Val1 385

AGC

Ser

TCT

Ser

GGT

Gi y 210

GCC

Al a

AAC

As n

GCT

Ala

AAA

Lys

TCT

Ser 290

TTC

Phe

AAA

Lys

GGT

Gi y

AAG

Lys

GAA

Giu 370

GAC

Asp

AAT

As n GCC GAG Ala Giu 195 GAG ACC Gin Thr APLG ACC Lys Thr GAA ACC Glu Thr GAC CTG Asp Leu 260 CCC AAA Pro Lys 275 TCT TTG Ser Leu CGC TTT Arg Phe ACC AAA Thr Lys GCG GCA Ala Ala 340 CTG ACC Leu Thr 355 GTC AAA Val Lys GGC ATT Gly Ile CAG GCA Gin Ala

GAA

Giu

GAT

Asp

ATG

Met

GAA

Giu 245

CAC

His

AAC

As n

AGC

Ser

TTG

Leu

GAG

Asp 325

GCA

Ala

ACG

Th r

AAT

As n

ATG

Met

GAT

Asp 405

GCG

Al a

TTC

Phe

ACC

Thr 230

AAT

As n

GGT

Gi y

GAC

Asp

GGC

Gly

AGC

Se r 310

AAA

Lys

TCG

Ser

GTT

Val

CTC

Leu

ATT

Ile 390

AA

Lys Gly Phe Pro 185 GAT GTG TTG Asp Val Leu 200 GGG CTG ACC Gly Leu Thr 215 GGC GCG CTC Gly Ala Leu AAA GCC AAA Lys Ala Lys AAC CGC TTC Asn Arg Phe 265 GAA ACC AAG Giu Thr Lys 280 GGC TTT TTC Gly Phe Phe 295 GAC GAT CAA Asp Asp Gin CTG GAA AAT Leu Giu Asn GGC GGT GCG Giy Gly Ala 345 TTG GAT GCG Leu Asp Ala 360 GAG AAC TTC Asp Asn Phe 375 CCG CTC CTG Pro Leu Leu GGT AAA AAC Gly Lys Asn

CGC

Arg

AGC

Se r

TAC

Tyr

CAA

Gin 250

AGC

Ser

GAA

Glu

GGT

Gi y

AAA

Lys

GGC

Gi y 330

GCA

Al a

GTT

Val1

AGC

Ser

CCC

Pro

GGC

Gi y 410 Ala Gly Asp

AAC

As n

GAG

Giu

CGC

Arg 235

ATT

Ile

GGC

Gi y

CAT

His

CCG

Pro

GTT

Val 315

GCG

Al a

GAT

Asp

GAG

Glu

AAT

As n

AAG

Lys 395

GGA

Giy

AAA

Lys

TTT

Phe 220

AAT

As n

AAA

Lys

AAG

Lys

CCC

Pro

AAG

Lys 300

GCC

Al a

GCG

Al a

ATG

Met

CTG

Leu

GCC

Al a 380

AAT

As n

ACA

Thr 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 GCC TTT ACC CGC AAA TTT GAA CAC ACG CCG GAA AGT GAT AAA AAA GAG Ala Phe Thr Arg Lys Phe Giu His Thr Pro Giu Ser 415 420 Asp 425 Lys Lys Asp -79- ACC CPA GCA GGT ACG GCG GAG AAT GGC AAT CCA GCC GCT TCA AAT ACG 1392 Thr Gin Ala Gly Thr Ala Giu Asn Gly Asn Pro Ala Ala Ser Asn Thr 430 435 440 GCA GGT GAT ACC PAT GGC AAA ACA AAA ACC TAT GAA GTC GAA GTC TGC 1440 Ala Gly Asp Thr Asn Gly Lys Thr Lys Thr Tyr Giu Val Giu Val Cys 445 450 455 460 TGT TCC PAC CTC PAT TAT CTG AAA TAC GGA ATG TTG ACG CGT AAA PAC 1488 Cys Ser Asn Leu Asn Tyr Leu Lys Tyr Gly Met Leu Thr Arg Lys Asn 465 470 475 AGC PAG TCC GCG ATG CAG GCA GGC GPA PAC GGT AGT CTA GCT GAC GCT 1536 Ser Lys Ser Ala Met Gin Ala Gly Giu Asn Gly Ser Leu Ala Asp Ala 480 485 490 AAA ACG GPA CPA GTT GPA CPA AGT ATG TTC CTC CPA GGC GAG CGC ACC 1584 Lys Thr Giu Gin Val Giu Gin Ser Met Phe Leu Gin Gly Giu Arg Thr 495 500 505 GAT GPA AAA GAG ATT CCA AAA GAG CPA CAA GAC ATC GTT TAT CGG GGG 1632 Asp Giu Lys Giu Ile Pro Lys Giu Gin Gin Asp Ile Val Tyr Arg Gly 510 515 520 TCT TGG TAC GGG CAT ATT GCC PAC GAC ACA AGC TGG AGC GGC PAT GCT 1680 Set Trp Tyr Gly His Ile Ala Asn Asp Thr Ser Trp Set Gly Asn Ala 525 530 535 540 *TCA GAT AGA GAG GGC GGC PAC AGG GCG GAC TTT ACC GTG PAT TTT GGT 1728 Ser Asp Arg Giu Giy Gly Asn Arg Ala Asp Phe Thr Val Asn Phe Gly C545 550 555 ACG AAA AAA ATT AAC GGA ACG TTA ACC GCT GAA PAC AGG CAG GAG GCA 1776 Thr Lys Lys Ile Asn Gly Thr Leu Thr Ala Giu Asn Arg Gin Glu Ala 560 565 570 ACC TTT ACC ATT GTG GGC GAT ATT PAG GAC AAC GGC TTT GAA GGT ACG 1824 Thr Phe Thr Ile Vai Gly Asp Ile Lys Asp Asn Giy Phe Glu Gly Thr 575 580 585 GCG AAA ACT GCT GAC TCA GGT TTT GAT CTC GAT CPA AGC AAT ACC ACC 1872 Ala Lys Thr Ala Asp Ser Giy Phe Asp Leu Asp Gin Set Asn Thr Thr 590 595 600 CGC ACG CCT PAG GCA TAT ATC ACA GAT GCC AAG, GTG AAG GGC GGT TTT 1920 Arg Thr Pro Lys Ala Tyr Ile Thr Asp Ala Lys Val Lys Gly Gly Phe 605 610 615 620 TAC GGG CCT AAA GCC GAA GAG TTG GGC GGA TGG TTT GCC TAT CCG GGC 1968 Tyr Gly Pro Lys Ala Glu Giu Leu Gly Gly Trp Phe Ala Tyr Pro Gly 625 630 635 :GAT AAA CPA ACG GPA AAG GCA ACG GTT ACA TCC GGC GAT GGA PAT TCA 2016 Asp Lys Gin Thr Giu Lys Ala Thr Val Thr Ser Gly Asp Gly Asn Set 640 645 650 GCA AGC AGT GCA ACT GTC GTA TTC GGT GCG AAA CGC CPA AAG CCT GTG 2064 Ala Ser Ser Ala Thr Val Val Phe Giy Ala Lys Arg Gin Lys Pro Val 655 660 665

CPA

Gin TAAAGTTTCG ATCTTGATTC TGTCGATACC GAAGCCCCGC GTCCCGCGCC AAATAAAA 2125 1INFORMATION(ZOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 669 amino acids TYPE: amino 'cid TOPOLOGY: linear ii) MOLECULE TYPE: protein xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: Met Leu Val Ser Met Lys Asp S.e Gly Asp As n Leu Asp Giu Arg Ala Giu Ser Tyr 110 A1a Gly Gly Met Tyr 190 Giu Val1 Arg Asn Ser Thr Th r Phe Leu Phe Glu Thr As n Tyr Lys Gly 175 Gly Ala Asp Ile Pro Leu Val Asn Gin Ala Ala Met Val Leu Pro Val Phe Al a Glu Pro Lys Ser Pro Ser Asp Lys Leu Val 160 Gin Al a Gin Phe Thr 240 Cys Al a Gin Arg Giu Gin Leu Phe Ile Phe 145 Gly Lys Leu Gin Al a 225 As n Leu Pro Al a Arg 50 Al a Arg Gin Gin Asp 130 Tyr Tyr Phe Se r Gi y 210 Ala Asn Gly Arg Gin 35 Asn Asp As n Leu Tyr 115 Phe Lys Lys Ser Al a 195 Gin Lys Glu Gly Pro 20 Lys Trp T rp Giu Gly 100 Val Gin Gly Gi y Gin 180 Giu Th r Thr Thr Gi y Ala Asp Tyr Glu Ile 85 Giu Arg Lys Ser Thr 165 Leu Giu Asp Met Giu 245 Gi y Pro Gin Pro Lys 70 Leu Gly Ser Lys Asn 150 T rp Al a Ala Phe Thr 230 Asn Ser Lys Gly Lys 55 Leu As n Gly Gly Ile 135 Pro Asp Gly Asp Gi y 215 Gi y Lys Phe Tyr Gi y Asn Gly Met Lys Tyr 120 Al a Ser Tyr Phe Val 200 Leu Ala Al a Asp Gin Tyr Giu Al a Thr Ser 105 Ile Leu Gin Val Pro 185 Leu Th r Leu Lys Leu Asp Gly Giu Gly Asp Arg Tyr Ser Al a Thr 170 Al a Arg Ser Tyr Gin 250 Asp Val Phe Asp Lys Giy Val Arg Gly Leu 155 Asp Gly As n Giu Arg 235 Ile Ser Ser Ala His Pro Ile Giu As n Pro 140 Pro Ala Asp Lys Phe 220 Asn Lys Arg Tyr Asp Ile Gin Ala Asp 255 Leu 260 His Gly Asn Arg Ser Gly Lys

WE

-81- Ala Thr Ala Thr Asp Lys Pro Lys Asn Asp Giu Thr 270 275 280 Lys Giu His Pro Phe 285 Gly Val Ala Pro Lys 365 Ala Ser Oe os.Ala Thri Ala 0* 445 Cys Ser 0@ Lys 'I Asp C Ser '1 525 Ser I Thr I Thr P Ala I Va.

Gli Va Sei Sei 350 Ser Gin Glu Phe Sin 430 Gly Ser Lys hr ;lu 10 'rp ~sp ~ys 'he ~ys i90 1 Se i Gli L Gil G1 335 Glu Gly Leu Ser Thr 415 Ala Asp Asn Ser Glu 495 Lys Tyr Arg Lys Thr 575 Thr r Asp Ser Leu Gly 305 Ser Ala 320 Set Thr Asn Gly Gly Lys Vai Val 385 Glu Ser 400 Arg Lys Gly Thr Thr Asn Leu Asn 465 Ala Met 480 Gin Val Glu Ile I Gly His Glu Gly C 545 Ile Asn C 560 Ile Val C Ala Asp I Ser 290 Phe Ser Arg Leu Phe Ser Leu Gly Ser Gly 295 Asp Phe Asp Phe Gln Gly Lys Pro Val 310 315 Asp Lys Leu Giu Asn Giy Ala Ala 325 330 Lys Thr Lys Gi Lys Glu 370 Asp Asn Phe Ala Gly 450 ryr ln lu Pro Iie 530 ;iy ;iy ;iy er Ala Ala Ala Set Gly Giy Ala Ala Asp Met Leu 355 Val Gly Gin Glu Glu 435 Lys Leu Ala Gin Lys I 515 Ala Asn 1 Thr i Asp Gly I 595 34C Thi Lys Ile Ala His 420 Asn Thr Lys Gly Ser 500 lu ksn krg Leu Ile 580 Phe 345 Thr Asn Met Asp 405 Thr Gly Lys Tyr Glu 485 Met Gin Asp Ala J Thr I 565 Lys I Asp Val Leu Ile 390 Lys Pro Asn Thr Gly 470 Asn Phe ln rhr ksp 550 kla ksp Leu

L

Leu Asp Ala I Asp 375 Pro Gly Glu Pro Tyr 455 Met Gly Leu Asp Set 535 Phe Glu Asn Asp 360 Asn Leu Lys Ser Ala 440 Glu Leu Ser Sin Ile 520 rrp rhr ksn Gly ln 600 Phe Leu Asn Asp 425 Ala Vai Thr Leu Gly 505 Val Ser Val Arg Phe 585 Ser Val Set Pro Gly 410 Lys Ser Glu Arg Ala 490 Glu Tyr Gly I Asn I Gin 570 Glu Asn Glu Asn Lys 395 Gly Lys Asn Val Lys 475 Asp Arg krg ksn Phe 555 lu ;ly 'hr Leu Ala 380 Asn Thr Asp Thr Cys 460 Asn Ala Thr Gly Ala 540 Gly Ala Thr Thr Lys 300 Ala Arg 605 Tyr Asp Ala Gin Thr Pro Gly Pro Lys Gin Set Set 655 -82- Lys Ala Tyr Ile Thr Asp Ala Lys Val Lys Giy Gly Phe 610 615 620 Lys Ala Glu Glu Leu Giy Gly Trp Phe Ala Tyr Pro Gly 625 630 635 Thr Glu Lys Ala Thr Val Thr Ser Giy Asp Giy Asn Set 640 645 650 Ala Thr Val Vai Phe Giy Ala Lys Arg Gin Lys Pro Vai 660 665 0* 00 0*0 *0 a *0 0 000 00 a 4 0000 .0 6e @0 INFOPRMATION FOR SEQ ID NO: 38: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2136 base pairs TYPE: nucieic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECUILE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meriingitidis STRAIN: BZi63 (ix) FEATURE: NAME/KEY: sigpeptide LOCATION: (ix) FEATURE: NAME/KEY: mat peptide LOCATION: 61. .2133 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .2133 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: ALTG AAC AAT CCA TTG GTA AAT CAG GCT GCT ATG GTG CTG CCT Met Asn Asn Pro Leu Val Asn Gin Ala Ala Met Val Leu Pro -20 -15 -10 TTG TTG AGT GCT TGT TTG GGC GGA GGC GGC AGT TTC GAT CTT Leu Leu 5cr Ala Cys Leu Giy Gly Giy Giy Set Phe Asp Leu 1 5 GTC GAT ACC GAA GCC CCG CGT CCC GCG CCA AAA TAT CAA GAT Val Asp Thr Glu Ala Pro Arg Pro Ala Pro Lys Tyr Gin Asp 15 20 TCC GAA AAA CCG CAA GCC CAA AAA GAC CAA GGC GGA TAC GGT Set Giu Lys Pro Gin Ala Gin Lys Asp Gin Giy Gly Tyr Gly 35 A.TG AGG TTG AAA CGG AGG AAT CGG CAT CCG CAG GCA AAA GAA Met Arg Leu Lys Arg Arg Asn Arg His Pro Gin Ala Lys Glu 50 55 GTT GAA CTA AAC CCA AAT GAT TGG GAG GAG ACA GGA TTG CCG Val Giu Leu Asn Pro Asn Asp Trp Giu Giu Thr Giy Leu Pro 70

S.

a.

000 0000 Oa *0 0 60 00 0 *00 0 0 O 0

GTG

Val

GAT

Asp

GTT

Val

TTT

Phe

GAC

Asp

AGC

Set

TTT

Phe

TCT

Ser

TCT

Set

GCG

Ala

AAA

Lys

AAG

Lys 48 96 144 240 288 -83-

CCC

Pro

ACA

Thr

AAC

Asn

GAA

Glu 125

TAT

Tyr

TCA

Ser

CAA

Gin

GAC

Asp

CAT

His 110

GTA

Val

AAA

Lys

GGC

Giy

AAC

Asn

GAT

Asp

CAA

Gin

AAA

Lys

CAT

His

GAC

TTA

Leu

GGC

Gly

AAC

Asn

GAT

Asp

GCA

Ala

GAC

Ccc Pro

AGC

Ser

GGC

Gly

TAC

Tyr

GAG

Glu 145

GGC

GAG

Glu

AAT

Asn

AGC

Set

AAA

Lys 130

AGT

Ser

TAT

CGA

Arg

ATT

ile

ACT

Thr 115

AAT

Asn

GAA

Glu

ATT

CAG

Gin

TAC

Tyr 100

AAT

Asn

TTC

Phe

AGA

Arg

TTT

CAA

Gin 85

ACT

Thr

AGC

Set

AAA

Lys

GAA

Glu

TAT

TCG

Ser

TCC

Ser

GGT

Gly

TAT

Tyr

TTC

Phe 150

CAC

GTT

Val

CCT

Pro

GCA

Ala

GTT

Val 135

AGT

Set

GGT

ATT

Ile

TAT

Tyr

AAC

Asn 120

TAT

Tyr

AAA

Lys

GAT

Asp

CTC

Leu 105

CAA

Gin

TCC

Set

ATC

Ile

GAC

AAA

Lys

ACG

Thr

CCA

Pro

GGC

Gly

AAA

Lys

CCT

GTA

Vai

CAA

Gin

AAA

Lys

TGG

Trp

TTT

Phe 155 rcc

AAA

Lys

TCA

Set

AAC

Asn

TTT

Phe 140

AAG

Lys

CGA

336 384 432 480 528 576 Asp Asp 160

AAA

Gly Tyr Ile Phe Tyr His Gly Lys Asp Pro Ser Arg 165 170 CAA CTT CCC ACT TCT GAA AAA GTT ATC TAG Gin 0 1P GTA Val *9 r c TCA C Set 205

GAA

Glu

GGT

Gly

TTG

Leu

AAC

Asn t

GGC.

Gly 285

GGA

Gly Leu Pro 175 ACC GAT Thr Asp 190 AAA GGG Lys Gly ACA ACT Thr Thr TAT GGT Tyr Gly ACG GGT Thr Gly 255 GAT AAA Asp Lys 270 AAC CGC Asn Arg GGA ACC Gly Tht Thr Set Giu Lys

ACT

Thr

CAA

Gin

TCC

Set

TTT

Phe 240

AAA

Lys

TAC

Tyr

TTC

Phe

AAA

Lys

GAA

Glu

GGC

Gly

AAT

Asn 225

ACC

Thr

TTA

Leu

ACC

Thr

AAC

Asn

CTA

Leu 305

AAG

Lys

GAC

Asp 210

AGA

Arg

TCG

Set

ATA

Ile

ACC

Thr

GGT

Gly 290

CAT

His

GGA

Giy 195

AGA

Arg

ACT

Thr

AAT

Asn

CGC

Arg

CAA

Gin 275

AAG

Lys

CCC

Pro Val 180

CAA

Gin

TAC

Tyr

GAT

Asp

TTA

Leu

AAT

Asn 260

TAC

Tyr

GCG

Ala

TTT

Phe Ile

.AAA

Lys

AGC

Set

TCC

Set

GAA

Glu 245

AAT

Asn

TAC

Tyr

ATA

Ile

GTT

Val Tyr

TTT

Phe

GGA

Gly

AAC

Asn 230

GTG

Vai

AGA

Arg

AGC

Set

GCG

Ala

TCC

Set 310 AAA GGC Lys Gly AAC GAT Asn Asp 200 TTT TCG Phe Set 215 CTT AAT Leu Asn GAT TTC Asp Phe GTT ACA Val Thr CTT GAT Leu Asp 280 ACC GAC Thr Asp 295 GAC TCG Asp Set

GTA

Val 185

ATT

TGG

Trp

CTT

CAT

His

GAA

TTT

Phe

ACC

Ile Leu Giu Tht

GGC

Gly

GAT

Asp

GGC

Gly

AAC

Asn 265

GCC

kia kAA Lys

TCT

Set GAT GAC GGC Asp Asp Gly 220

AAG

Lys

AGT

Set 250

GCT

Ala

CAA

Gin

CCC

Pro

TCT

Set

CGC

Arg 330

CAC

His 235

AAA

Lys

ACT

Thr

ATA

Ile

GAC

Asp

TTG

Leu 315

TTT

Phe

GAG

Glu

AAA

Lys

ACT

Thr

ACA

Thr

ACT

Tht 300

AGC

Set

TTG

Leu 624 672 720 768 816 864 912 960 1008 1056 GGC GGC TTT TTC GGT CCG AAG GGT GAG GAA TTG GGT TTC Gly Giy Phe Phe Gly Pro Lys Giy Giu Glu Leu Gly Phe -84- AGC GAC GAT AAA AAA GTT GCG GTT GTC GGC AGC GCG AAA ACC AAA GAC 1104 Ser Asp Asp Lys Lys Val Ala Val Val Gly Ser Ala Lys Thr Lys Asp 335 340 345 AAA ACG GAA AAT GGC GCG GTG GCT TCA GGC GGC ACA GAT GCG GCA GCA 1152 Lys Thr Giu Asn Gly Ala Vai Ala Ser Gly Gly Thr Asp Ala Ala Ala 350 355 360 TCA AAC GGT GCG GCA GGC ACG TCG TCT GAA AAC AGT AAG CTG ACC ACG 1200 Ser Asn Gly Ala Ala Gly Thr Ser Ser Glu Asn Ser Lys Leu Thr Thr 365 370 375 380 GTT TTG GAT GCG GTC GAG CTG AAA TTG GGC GAT AAG GAA GTC CAA AAG 1248 Val Leu Asp Ala Val Glu Leu Lys Leu Gly Asp Lys Glu Val Gin Lys 385 390 395 CTC GAC AAC TTC AGC AAC GCC GCC CAA CTG GTT GTC GAC GGC ATT ATG 1296 Leu Asp Asn Phe Ser Asn Ala Ala Gin Leu Val Val Asp Gly Ile Met 400 405 410 ATT CCG CTC TTG CCC GAG ACT TCC GAA AGT GGG AAC PAT CAA GCC PAT 1344 Ile Pro Leu Leu Pro Glu Thr Ser Glu Ser Gly Asn Asn Gin Ala Asn 415 420 425 CAA GGT ACA AAT GGC GGA ACA GCC TTT ACC CGC AAA TTT GAC CAC ACG 1392 Gin Gly Thr Asn Gly Gly Thr Ala Phe Thr Arg Lys Phe Asp His Thr 430 435 440 CCG GPA AGT GAT PAA AAA GAC GCC CPA GCA GGT ACG CAG ACG PAT GGG 1440 Pro Giu Ser Asp Lys Lys Asp Ala Gin Ala Gly Thr Gin Thr Asn Gly 445 450 455 460 GCG CPA ACC GCT TCA PAT ACG GCA GGT GAT ACC PAT GGC AAA ACA AAA 1488 Ala Gin Thr Ala Ser Asn Thr Ala Gly Asp Thr Asn Gly Lys Thr Lys 465 470 475 ACC TAT GAA GTC GAA GTC TGC TGT TCC PAC CTC PAT TAT CTG AAA TAC 1536 Thr Tyr Glu Val Glu Val Cys Cys Ser Asn Leu Asn Tyr Leu Lys Tyr 480 485 490 GGA ATG TTG ACG CGC AAA PAC AGC PAG TCC GCG ATG CAG GCA GGA GAA 1584 Gly Met Leu Thr Arg Lys Asn Ser Lys Ser Ala Met Gin Ala Gly Glu 495 500 505 AGC AGT AGT CPA GCT GAT GCT AAA ACG GAA CPA GTT GGA CPA AGT ATG 1632 Ser Ser Ser Gin Ala Asp Ala Lys Thr Glu Gin Vai Gly Gin Ser Met 510 515 520 TTC CTC CPA GGC GAG CGC ACC GAT GAA AAA GAG ATT CCA AGC GAG CPA 1680 Phe Leu Gin Gly Giu Arg Thr Asp Glu Lys Glu Ile Pro Ser Glu Gin 525 530 535 540 PAC ATC GTT TAT CGG GGG TCT TGG TAC GGG CAT ATT GCC AGC AGC ACA 1728 Asn Ile Val Tyr Arg Gly Ser Trp Tyr Giy His Ile Ala Ser Ser Thr 545 550 555 AGC TGG AGC GGC PAT GCT TCT GAT AAA GAG GGC GGC PAC AGG GCG GAA 1776 Ser Trp Ser Gly Asn Ala Ser Asp Lys Glu Gly Gly Asn Arg Ala Glu 560 565 570 TTT ACT GTG AAT TTT GGC GAG AAA AAA ATT ACC GGC ACG TTA ACC GCT 1824 Phe Thr Val Asn Phe Gly Giu Lys Lys Ile Thr Gly Thr Leu Thr Ala 575 580 585

GA

Gi

AA

As

GA'

As]

AAC

Ly

TG(

T rj

TCC

Set

AAA

Lys 685 (2) 0e we 0* 0 0*O 6

S

Se.

a.

5 5 9Aee 0

S

6' 55 AAAC AGG GAG GAG GCA ACC TTT ACC ATT GAT GGT AAG u Asn Arg Gin Giu Ala Thr Phe Thr Ile Asp Gly Lys 590 595 600 CGGT TTT TCC GGT ACG GCA AAA ACT GCT GAA TTA GGT n Gly Phe Ser Gly Thr Ala Lys Thr Ala Giu Leu Gly 5 610 615 rCAA AAA AAT ACC ACC CGC ACG CCT AAG GCA TAT ATC p Gin Lys Asn Thr Thr Arg Thr Pro Lys Ala Tyr Ile 625 630 GGTG CAG GGC GGT TTT TAC GGG CCC AAA GCC GAA GAG s Val Gin Gly Gly Phe Tyr Gly Pro Lys Ala Giu Giu 640 645 TTT GCC TAT CAG GOC GAT AAA CAA ACG GAA AAT ACA )Phe Ala Tyr Gin Giy Asp Lys Gin Thr Giu Asn Thr 655 660 665 GGC AAT GGA AAT TCA GCA AGC AGT GCA ACT GTC GTA Gly Asn Giy Asn Ser Ala Ser Ser Ala Thr Val Val 670 675 680 CGC CAA AAG CCT GTG CAA TAAAGTAAAA Arg Gin Lys Pro Val Gin 690 JINFORMATIOX29OR SEQ ID NO: 39: Wi SEQUENCE 'CHARACTERISTICS: LENGTH: 692 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Asn Asn Pro Leu Vai Asn Gin Ala Ala Met Vai Leu P -15 -10 Leu Ser Ala Cys Leu Gly Gly Giy Gly Ser Phe Asp L 1 5 Asp Thr Giu Ala Pro Arg Pro Ala Pro Lys Tyr Gin A 20 25 Giu Lys Pro Gin Ala Gin Lys Asp Gin Gly Gly Tyr G 35 40 Arg Leu Lys Arg Arg Asn Arg His Pro Gin Ala Lys G 50 55 Giu Leu Asn Pro Asn Asp Trp Giu Giu Thr Gly Leu P 70 Gin Asn Leu Pro Giu Arg Gin Gin Ser Vai Ile Asp L 85 Asp Asp Gly Ser Asn Ile Tyr Thr Ser Pro Tyr Leu T 100 105

ATT

Ile

TTT

Phe

ACA

Thr

TTG

Leu 650

ACA

Thr

TTC

Phe

GAG

Gi u

GAT

Asp

GAT

Asp 635

GGC

Gi y

GTT

Vali

GGC

Gi y

CTC

Leu 620

GCC

Al a

GGA

Gi y

GCA

Al a

GCG

Ala 1872 1920 1968 2016 2064 2112 2143 Met Leu Val Ser Met Val1 Pro Thr ro eu sP ly iu ro ys hr Vai Asp Val Phe Asp Ser Val Gin Phe Ser Ser Ala Lys Lys Lys Ser -86- Asn His Gin Asn Gly Ser Thr Asn Ser Gly Ala Asn Gin Pro Lys Asn 110 115 120 Glu Val Lys Asp Tyr Lys Asn Phe Lys Tyr Val Tyr Ser Gly Trp Phe 125 130 135 140 Tyr Lys His Ala Glu Ser Glu Arg Glu Phe Ser Lys Ile Lys Phe Lys 145 150 155 Ser Gly Asp Asp Gly Tyr Ile Phe Tyr His Gly Lys Asp Pro Ser Arg 160 165 170 Gin Leu Pro Thr Ser Glu Lys Val Ile Tyr Lys Gly Val Trp His Phe 175 180 185 Val Thr Asp Thr Glu Lys Gly Gin Lys Phe Asn Asp Ile Leu Glu Thr 190 195 200 Ser Lys Gly Gin Gly Asp Arg Tyr Ser Gly Phe Ser Gly Asp Asp Gly 205 210 215 220 Glu Thr Thr Ser Asn Arg Thr Asp Ser Asn Leu Asn Asp Lys His Glu 225 230 235 Gly Tyr Gly Phe Thr Ser Asn Leu Glu Val Asp Phe Gly Ser Lys Lys 240 245 250 S" Leu Thr Gly Lys Leu Ile Arg Asn Asn Arg Val Thr Asn Ala Thr Thr 255 260 265 Asn Asp Lys Tyr Thr Thr Gin Tyr Tyr Ser Leu Asp Ala Gin Ile Thr 270 275 280 Gly Asn Arg Phe Asn Gly Lys Ala Ile Ala Thr Asp Lys Pro Asp Thr 285 290 295 300 Gly Gly Thr Lys Leu His Pro Phe Val Ser Asp Ser Ser Ser Leu Ser 305 310 315 Gly Gly Phe Phe Gly Pro Lys Gly Glu Glu Leu Gly Phe Arg Phe Leu 320 325 330 0. Ser Asp Asp Lys Lys Val Ala Val Val Gly Ser Ala Lys Thr Lys Asp 335 340 345 S Lys Thr Glu Asn Gly Ala Val Ala Ser Gly Gly Thr Asp Ala Ala Ala 350 355 360 Ser Asn Gly Ala Ala Gly Thr Ser Ser Glu Asn Ser Lys Leu Thr Thr 365 370 375 380 Val Leu Asp Ala Val Glu Leu Lys Leu Gly Asp Lys Glu Val Gin Lys 385 390 395 Leu Asp Asn Phe Ser Asn Ala Ala Gin Leu Val Val Asp Gly Ile Met 400 405 410 Ile Pro Leu Leu Pro Glu Thr Ser Glu Ser Gly Asn Asn Gin Ala Asn 415 420 425 Gin Gly Thr Asn Gly Gly Thr Ala Phe Thr Arg Lys Phe Asp His Thr 430 435 440 Pro Glu Ser Asp Lys Lys Asp Ala Gin Ala Gly Thr Gin Thr Asn Gly 0 -87- 445 450 455 460 Ala Gin Thr Ala Ser Asn Thr Ala Gly Asp Thr Asn Gly Lys Thr Lys 465 470 475 Thr Tyr Glu Val Glu Val Cys Cys Ser Asn Leu Asn Tyr Leu Lys Tyr 480 485 490 Gly Met Leu Thr Arg Lys Asn Ser Lys Set Ala Met Gin Ala Gly Giu 495 500 505 Ser Ser Set Gin Ala Asp Ala Lys Thr Giu Gin Val Gly Gin Ser Met 510 515 520 Phe Leu Gin Giy Giu Arg Thr Asp Glu Lys Giu Ile Pro Set Giu Gin 525 530 535 540 Asn Ile Val Tyr Arg Giy Ser Trp Tyr Giy His Ile Ala Set Set Thr 545 550 555 Ser Trp Set Giy Asn Ala Set Asp Lys Giu Gly Giy Asn Arg Ala Giu 560 565 570 Phe Thr Val Asn Phe Gly Giu Lys Lys Ile Thr Giy Thr Leu Thr Ala 575 580 585 Giu Asn Arg Gin Giu Ala Tht Phe Thr Ile Asp Gly Lys Ile Giu Giy 590 595 600 Asn Gly Phe Set Giy Thr Ala Lys Tht Ala Giu Leu Giy Phe Asp Leu 605 610 615 620 Asp Gin Lys Asn Tht Tht Arg Thr Pro Lys Ala Tyr Ile Thr Asp Ala 625 630 635 Lys Vai Gin Gly Giy Phe Tyr Gly Pro Lys Ala Giu Giu Leu Giy Gly 640 645 650 Trp Phe Ala Tyr Gin Gly Asp Lys Gin Thr Giu Asn Thr Thr Vai Ala 655 660 665 Ser Giy Asn Gly Asn Set Ala Ser Set Ala Thr Vai Val Phe Gly Ala *670 675 680 Lys Arg Gin Lys Pro Vai Gin 685 690 INFORMATION FOR SEQ ID, NO: 8EOENCE CHARACTERISTICS: LENGTH: 199 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Thr Lys Asp Lys Leu Glu Asn Giy Ala Ala Ala Ser Gly Ser Thr Gly 1 5 10 -88- Ala Ala Ala Ser Gly Gly Ala Ala Asp Met Pro Ser GJlu Asn Gly Lys 25 Leu Thr Thr Val Leu Asp Ala Val Glu Leu Lys Ser Gly Gly Lys Giu 40 Val Lys Asn Leu Asp Asn Phe Ser Asn Ala Ala Gin Leu Val Val Asp 55 Gly Ile Met Ile Pro Leu Leu Pro Lys Asn Ser Glu Ser Giu Ser Asn 70 75 Gin Ala Asp Lys Gly Lys Asn Gly Gly Thr Ala Phe Thr Arg Lys Phe 90 Giu His Thr Pro Giu Ser Asp Lys Lys Asp Thr Gin Ala Gly Thr Ala 100 105 110 Giu Asn Gly Asn Pro Ala Ala Ser Asn Thr Ala Gly Asp Thr Asn Gly 115 120 125 Lys Thr Lys Thr Tyr Giu Val Giu Val Cys Cys Ser Asn Leu Asn Tyr 130 135 140 Leu Lys Tyr Gly Met Leu Thr Arg Lys Asn Ser Lys Ser Ala Met Gin *145 150 155 160 Ala Gly Giu Asn Gly Ser Leu Ala Asp Ala Lys Thr Giu Gin Val Giu 165 170 175 Gin Ser Met Phe Leu Gln Gly Giu Arg Thr Asp Glu Lys Giu Ile Pro *180 185 190 *0Lys Giu Gin Gin Asp Ile Val 195 INFORMATION FOR SEQ ID"

T

NO: 41: SEQUENCE

CHARACTERISTICS:

LENGTH: 198 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: iinear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION SEQ ID NO: 41: Thr Lys Asp Lys Thr Giu Asn Gly Ala Val Ala Ser Gly Gly Thr Asp 1 5 10 Ala Ala Ala Ser Asn Gly Ala Ala Gly Thr Set Ser Glu Asn Ser Lys 25 Leu Thr Thr Val Leu Asp Ala Val Glu Leu Lys Leu Giy Asp Lys Giu 40 Vai Gin Lys Leu Asp Asn Phe Ser Asn Ala Ala Gin Leu Vai Val Asp 55 -88A- Gly Ile Met Ile Pro Leu Leu Pro Giu Thr Ser Glu 70 75 Gin Ala Asn Gin Gly Thr Asn Gly Gly Thr Ala Phe 90 Asp His Thr Pro Giu Ser Asp Lys Lys Asp Ala Gin 100 105 Thr Asn Gly Ala Gin Thr Ala Ser Asn Thr Ala Gly 115 120 Lys Thr Lys Thr Tyr Giu Val Giu Vai Cys Cys Ser 130 135 140 Leu Lys Tyr Giy Met Leu Thr Arg Lys Asn Ser Lys 145 150 155 Ala Gly Giu Ser Ser Ser Gin Ala Asp Ala Lys Thr 165 170 Gin Set Met Phe Leu Gin Giy Giu Arg Thr Asp Glu 180 185 Ser Giu Gin Asn Ile Val 195 INFORMATION FOR SEQ ID NO: 42: SEQUENCE

CHARACERISTICS:

LENGTH: 199 amino acids TYPE: amino acid

STRANDEDNESS:

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEPUENCE.DESCRIPION".- SEQ ID NO: 42: Ser Thr Ala Asp 125 Asn Set Glu Lys Gly Arg Gly 110 Thr Leu Ala Gin Glu 190 Asn Asn Lys Phe Thr Gin Asn Gly Asn Tyr Met Gin 160 Val Gly 175 Ile Pro 00 00 0@ #5 0@

S

S.

S Thr Lys 1 Thr Asp Gly Lys Lys Lys Val Asp Asn Asn Lys Phe Thr Ala Asp Asn Ala Ala 20 Leu Thr Ile Lys Gly Ile Gin Ala Ala His 100 Ala Asn Thr 5 Ala Thr Asn Met Asn Thr Gly Ala Ser Val Leu Ile 70 Gin Pro Asp Asn Asn Leu Asp 55 Pro Gly Lys Gin Gly Asn Gly Ala 25 Asp Ala 40 Asn Phe Leu Leu Thr Asn Ser Asp 105 Ala Ala Thr 10 Ala Val Ser Pro Gly 90 Glu Set Ala Gly Glu Asn Glu 75 Gly Lys Asn Ala Thr Leu Ala Ala Thr Asp Thr Ala Ser Thr Ala Ser Ala Thr Ala Ser Ser Leu Gin Glu Phe His 110 Gly Gly Glu Asn Leu Ser Thr Ala Asp Gly Asn Asp Val Gly Arg Gly Thr -88B- 115 120 125 Asn Gly Lys Thr Lys Thr Tyr Glu Val Giu Val Cys Cys Ser Asn Leu 130 135 140 Asri Tyr Leu Lys Tyr Gly Leu Leu Thr Arg Lys Thr Ala Gly Asn Thr 145 150 155 160 Gly Giu Gly Gly Asn Gly Ser Gin Thr Ala Ala Ala Gin Thr Ala Gin 165 170 175 Gly Ala Gin Ser Met Phe Leu Gin Gly Glu Arg Thr Asp Glu Lys Glu 180 185 190 Ile Pro Ser Giu Gin Asn Val 195

S#

see

S

Claims

1. A polypeptide having an amino acid sequence which is derived from that of the Tbp2 subunit of the transferrin receptor of an IM2169 or IM2394 type Neisseria meningitidis strain whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394, as shown in SEQ ID NO 1 or 3, by total or partial deletion of at least one domain of the said IM2169 or IM2394 type Tbp2 subunit, provided the first and second domains are not totally deleted simultaneously; the first, second and third domains of the sequence of the Tbp2 subunit of strain IM2169 being respectively defined from amino acid position 1 to 345, 346 to 543 and 544 to 691; and the first, second and third domains of the sequence of the Tbp2 o. subunit of strain IM2394 being respectively defined from amino acid position 1 to 325, 326 to 442 and 443 to 579. i 2. A polypeptide according to Claim 1, having an amino acid sequence which is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains :are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394 by partial deletion of the third domain of the said IM2169 or IM2394 type Tbp2 subunit.

3. A polypeptide according to Claim 1, having an amino acid sequence which is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394 by total deletion of the third domain of the said IM2169 or IM2394 type Tbp2 subunit.

4. A polypeptide according to Claim 2 or 3, having an amino acid sequence which is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394; and which contains in its entirety the second domain of the sequence from which it is derived. www P:\OPER\JLR26757-95.168 -11698 A polypeptide according to Claim 2 or 3, having an amino acid sequence which, in addition, is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394 by partial deletion of the second domain of the said IM2169 or IM2394 type Tbp2 subunit.

6. A polypeptide according to Claim 2 or 3, having an amino acid sequence which, in addition, is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394 by total deletion of the second domain of the said IM2169 or IM2394 type Tbp2 subunit.

7. A polypeptide according to Claim 4, 5 or 6, having an amino acid sequence which is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394; and which contains in its entirety the first domain of the sequence from which it is derived.

8. A polypeptide according to Claim 4, 5 or 6, having an amino acid sequence which, in addition, is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence S. of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394; by partial deletion of the first domain of the said IM2169 or IM2394 type Tbp2 subunit.

9. A polypeptide according to Claim 4 or 5, having an amino acid sequence which, in addition, is derived from that of the IM2169 or IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the respective reference strain, IM2169 or IM2394; by total deletion of the first domain of the said IM2169 or IM2394 type Tbp2 subunit. A polypeptide according to Claims 2 or 3, 4 and 7, having an amino acid sequence which is derived from WO 95/33049 91 PCT/FR95/00701 that of the IM2169 type Tbp2 subunit.

11. A polypeptide according to Claims 2 or 3, 4 and 7, having an amino acid sequence which is derived from that of the IM2394 type Tbp2 subunit.

12. A polypeptide according to Claims 2 or 3, 4 and 8, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit.

13. A polypeptide according to Claims 2 or 3, 4 and 8, having an amino acid sequence which is derived from that of the IM2394 type Tbp2 subunit.

14. A polypeptide according to Claim 12, having an amino acid sequence which, in addition, is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the reference strain IM2169, by deletion of all or part of the region which is the homologue of the region of the first domain of the said IM2169 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid at position 281. A polypeptide according to Claim 13, having an amino acid sequence which, in addition, is derived from that of the IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the reference strain IM2394, by deletion of all or part of the region which is the homologue of the region of the first domain of the said IM2394 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid at position 266.

16. A polypeptide according to Claims 2 or 3, 4 and 9, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit.

17. A polypeptide according to Claims 2 or 3, 4 and 9 having an amino acid sequence which is derived from that of the IM2394 type Tbp2 subunit.

18. A polypeptide according to Claims 2 or 3, 5 and 7, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit. WO 95/33049 92 PCT/FR95/00701

19. A polypeptide according to Claims 2 or 3, 5 and 7, having an amino acid sequence which is derived from that of the IM2394 type Tbp2 subunit. A polypeptide according to Claims 2 or 3, 5 and 8, having an amino acid sequence which is derived from that of the IM2169 type of the Tbp2 subunit.

21. A polypeptide according to Claims 2 or 3, 5 and 8, having an amino acid sequence which is derived from that of the IM2394 type Tbp2 subunit.

22. A polypeptide according to Claim 18 or 20, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the reference strain IM2169, by deletion of the region of the second domain of the said IM2169 type Tbp2 subunit which is the homologue of the region of the second domain of the IM2169 Tbp2 subunit extending from the amino acid in one of the positions 346 to 361 to the amino acid at position 543.

23. A polypeptide according to Claim 19 or 21, having an amino acid sequence which is derived from that of the IM2394 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the reference strain IM2394, by deletion of the region of the second domain of the said IM2394 type Tbp2 subunit which is the homologue of the region of the second domain of the IM2394 Tbp2 subunit extending from the amino acid in one of the positions 326 to 341 to the amino acid at position 442.

24. A polypeptide according to Claim 18 or 20, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the IM2169 Tbp2 subunit, by deletion of at least one of the regions of the second domain of the said IM2169 type Tbp2 subunit which are the homologues of the regions of the IM2169 Tbp2 subunit extending: from the amino acid at position 362 to the WO 95/33049 93 PCT/FR95/00701 amino acid at position 379; (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. A polypeptide according to Claim 24, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit, whose first, second and third domains are defined by alignment to maximum homology with the sequence of the IM2169 Tbp2 subunit, by deletion of the regions of the second domain of the said IM2169 type Tbp2 subunit which are the homologues of the said regions to (iv) of the IM2169 Tbp2 subunit.

26. A polypeptide according to Claims 20 and 24 or having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the IM2169 Tbp2 subunit, by deletion of all or part of the region which is the homologue of the region of the first domain of the said IM2169 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid at position 281.

27. A polypeptide according to Claims 3, 6 and 7, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit.

28. A polypeptide according to Claims 3, 6 and 7, having an amino acid sequence which is derived from that of the ±M2394 type Tbp2 subunit.

29. A polypeptide according to Claims 3, 6 and 8, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit. A polypeptide according to Claims 3, 6 and 8, having an amino acid sequence which is derived from that of the IRW394 type Tbp2 subunit. 94

31. A polypeptide according to Claim 1, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the reference strain IM2169; by partial deletion of the second domain of the said IM2169 type Tbp2 subunit, by deletion of at least one of the regions of the second domain of the said IM2169 type Tbp2 subunit which are the homologues of the regions of the IM2169 Tbp2 subunit extending: from the amino acid at position 362 to the amino acid at position 379, 9 (ii) from the amino acid at position 418 to the amino acid at position 444, S(iii) from the amino acid at position 465 to the amino acid at position 481, and p. (iv) from the amino acid at position 500 to the amino acid at position 520; and 20 which contains in their entirety the first and third domains of the sequence from which it is derived.

32. A polypeptide according to Claim 1, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the Tbp2 subunit of the reference strain IM2169; by partial deletion of the second domain of the said IM2169 type Tbp2 subunit, by partial deletion of the first domain and by deletion of at least one of the regions of the second domain of the said IM2169 type Tbp2 subunit which are the homologues of the regions of the IM2169 Tbp2 subunit extending: from the amino acid at position 362 to the WO 95/33049 95 PCT/FR95/00701 amino acid at position 379, (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; and which contains in its entirety the third domain of the sequence from which it is derived.

33. A polypeptide according to Claim 32, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the IM2169 Tbp2 subunit, by deletion of all or part of the region which is the homologue of the region of the first domain of the said IM2169 type Tbp2 subunit extending from the amino acid at position 1 to the amino acid position at 281.

34. A polypeptide according to one of Claims 31 to 33, having an amino acid sequence which is derived from that of the IM2169 type Tbp2 subunit whose first, second and third domains are defined by alignment to maximum homology with the sequence of the IM2169 Tbp2 subunit, as shown in ID SEQ NO 1, by deletion of the regions of the second domain of the said IM2169 type Tbp2 subunit which are the homologues of the said regions to (iv) of the IM2169 Tbp2 subunit. A polypeptide according to one of Claims 10, 12, 14, 16, 18, 20, 22, 24 to 27, 29 and 31 to 33, having an amino acid aquence which is derived from that of the IM2169 Tbp2 subunit.

36. A polypeptide according to one of Claims 11, 13, 17, 19, 21, 23, 28 and 30, having an amino acid sequence which is derived from that of the IM2394 Tbp2 subunit. WO 95/33049 96 PCT/FR95/00701

37. A polypeptide according to one of Claims 1 to 36, having a sequence which comprises at least 50 amino acids.

38. An isolated DNA fragment coding for a polypeptide according to one of Claims 1 to 37.

39. A pharmaceutical composition for inducing an immune response against N. meningitidis, comprising as active principle at least one polypeptide according to one of Claims 1 to 37.

40. A pharmaceutical composition according to Claim 39, which comprises as active principle at least one first and at least one second polypeptide according to one of Claims 1 to 37; the said first.polypeptide having a sequence which is derived from that of an IM2169 type Tbp2 subunit, and the said second polypeptide having a sequence which is derived from that of an IM2394 type Tbp2 subunit.

41. A pharmaceutical composition according to Claim in which the said at least one second polypeptide is according to one of Claims 11, 13, 15, 19, 21, 23, 28 and

42. A pharmaceutical composition according to Claim 41, in which the said at least one second polypeptide is according to one of Claims 11, 19, 23 and 28.

43. A pharmaceutical composition according to Claim 41 or 42, in which the said at least one second polypeptide has an amino acid sequence which is derived from that of the IM2394 Tbp2 subunit.

44. A pharmaceutical composition according to one of Claims 40 to 43, in which the said at least one first polypeptide is according to one of Claims 10, 12, 14, 18, 22, 27 and 29. A pharmaceutical composition according to Claim 44, in which the said at least one first polypeptide is according to one of Claims 10, 18, 22 and 27.

46. A pharmaceutical composition according to one of Claims 40 to 43, in which the said at least one first polypeptide is according to one of Claims 31 to 34.

47. A pharmaceutical composition according to one of WO 95/33049 97 PCT/FR95/00701 Claims 40 to 43, in which the said at least one one [sic] first polypeptide is according to Claim 16.

48. A pharmaceutical composition according to one of Claims 44 to 47, in which the said at least one first polypeptide has an amino acid sequence which is derived from that of the IM2169 Tbp2 subunit.

49. A pharmaceutical composition according to Claim 47, which comprises at least one third polypeptide which is according to Claim 16.

50. A monoclonal antibody:

51. capable of recognizing an epitope present in the first domain of an IM2169 or IM2394 type Tbp2 subunit; the said epitope having a sequence homologous to that present in the first domain of the Tbp2 subunit of the strain IM2394 and selected from YKGTW, EFEVDFSDKTIKGTL, EGGFYGPKGEEL and AVFGAK; and optionally, (ii) incapable of recognizing the epitope present in the third domain of the said IM2169 or IM2394 type Tbp2 subunit, whose sequence is homologous to that of the epitope of the first domain which is recognized. A monoclonal antibody according to Claim capable of recognizing the region present in the first domain of an IM2169 or IM2394 type Tbp2 subunit whose sequence is homologous to the sequence EGGFYGPKGEEL present in the first domain of the Tbp2 subunit of the strain IM2394; and optionally, (ii) incapable of recognizing the epitope present in the third domain of the said IM2169 or IM2394 type Tbp2 subunit, an 7/^ ^44 Q:\OPER\EJH\26757-95.RSP 30/3/99 -98- epitope equivalent to the one which is recognized, whose sequence is homologous to the sequence SGGFYGKNAIEM present in the third domain of the Tbp2 subunit of the strain IM2394.

52. A monoclonal antibody according to Claim 51, capable of recognizing the epitope GFYGPK present in the first domain of the Tbp2 subunit of the strain IM2394; and (ii) incapable of recognizing the equivalent epitope present in the third domain of the said IM2394 Tbp2 subunit.

53. A pharmaceutical composition for treating an N. meningitidis infection by passive immunotherapy, which comprises as active principle a monoclonal antibody according to one of Claims 50 to 52.

54. A polypeptide according to any one of claims 1 to 37 or a DNA fragment according to claim 38 or a pharmaceutical composition according to any one of claims 39 to 49 or 53 or a Smonoclonal antibody according to any one of claims 50 to 52 substantially as hereinbefore described with reference to the Figures and/or Examples. Dated this 30th day of March, 1999 PASTEUR MERIEUX Serums et Vaccins AND Transgene S.A. by their Patent Attorneys DAVIES COLLISON CAVE