AU8987598A - Meningococcus vaccine comprising the valence of bz83 strain - Google Patents

Description

Meningococcal Vaccine Containing the BZ83 Strain Valency The subject of the present invention is a new 5 pharmaceutical composition intended for the treatment or prevention of meningococcal infections which contains the TbpB (Tbp2) subunit of the human transferrin receptor (HTR) of a Neisseria meningitidis strain of the ET-5 complex, group BZ83. 10 Generally speaking, meningitis is either of viral origin or of bacterial origin. The bacteria mainly responsible are N. meningitidis and Haemophilus influen zae, which are involved, respectively, in approximately 40 and 50% of cases of bacterial meningitis. 15 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 2500 to 3000 per annum. The species N. meningitidis is subdivided into 20 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 25 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 30 be overcome by conjugating these polysaccharides to a carrier protein. In contrast, the polysaccharide of N. menin gitidis group B displays little or no immunogenicity in man, irrespective of whether or not it is in conjugated 35 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 polysaccharide based vaccine.

-2 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. 5 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 10 iron-transport proteins such as transferrin and lacto ferrin, since the amount of iron in free form is negli gible in man (of the order of 1018 M), in any case insufficient to permit bacterial growth. Thus, N. meningitidis possesses a receptor for 15 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 human transferrin receptor of N. 20 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 25 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 30 for the purposes of the present patent application, referred to as the human transferrin receptor (HTR), and the polypeptides constituting it, as the subunits. In the text which follows, the subunits of high molecular weight and of lower molecular weight are referred to, 35 respectively, as TbpA (Tbpl) and TbpB (Tbp2). Since the pioneering work of Schryvers et al., it has been discovered that the N. meningitidis species is divided essentially into 2 types of strain which differ in the constitution of their respective HTRs -3 (see WO 93/6861 and WO 93/7172). A first type of strain, termed type M982 (or IM2169), possesses a TbpB which reacts with an antiserum directed against the HTR of the M982 strain but which does not react with an 5 antiserum directed against the HTR of the B16B6 (or IM2394) strain; whereas the other type, termed type B16B6, possesses a TbpB which reacts with an antiserum directed against the HTR of the B16B6 strain but which does not react with an antiserum directed against the 10 HTR of the M982 strain. An antigenic diversity therefore exists at the level of the subunit of lower molecular weight. This diversity is however limited since it is reduced to 2 main types, contrary to what is suggested by Griffiths et al., FEMS Microbiol. Lett. 15 (1990) 69:31. Type M982 strains are for example the M982 strain for which the sequence of the tbpB gene is described in patent application EPA 586 266 and in the EMBL data bank under the reference number Z15130; the 20 strains 6940, M978 and S3032 have [sic] the 5' sequences of the tbpB genes are described in WO 95/33049; the strains BZ83 and 8680 for which the sequences of the tbpB genes are described in WO 95/33049 and WO 97/13860, respectively (these sequences 25 can also be found in the EMBL data bank under the respective reference numbers Z50732 and Y09977); or alternatively the strains 32/94 and 8710 whose sequences can be found in the EMBL data bank under the respective accession numbers Y09617 and Y09618. These 30 strains, as well as others, are available from Dr Caugant, Centre de R6ference des M6ningocoques collaborating with the World Health Organization, National Institute of Public Health, Oslo, Norway (see doctoral thesis -presented before Claude Bernard 35 University - Lyon I by Bachra Rokbi, presented on 10 Oct. 1995, entitled "Etude de la variability antig6nique et mol6culaire du r6cepteur de la transferrine humaine de N. meningitidis" [Study of the antigenic and molecular variability of the human -4 transferrin receptor of N. meningitidis]). The charac teristics of the strains cited are presented in Table IV below. The DNA sequence coding for the TbpB of the B16B6 strain is described in EPA 586 266. 5 The study of the DNA fragments coding for various TbpBs has revealed a difference in the size of these fragments depending on the type from which they were derived. This has made it possible to make changes to the definitions of types M982 and B16B6, originally 10 provided in WO 93/6861. It is now appropriate to define the strain types according to the size of the TbpB gene: about 2.1 to 2.3 kb for TbpB of type M982 and 1.8 kb for TbpB of type B16B6. The protein TbpB, rather than Tbpl, has a number 15 of characteristics which make it a potential vaccinal candidate: a ubiquitous expression, accessibility at the surface of the microorganism, the capacity to induce bactericidal antibodies and a limited variability since, as has just been stated, two major groups have so far 20 been identified. By virtue of this discovery, pharmaceutical compositions have already been proposed which contain: (i) the HTR of at least one strain of the B16B6 type and the HTR of at least one 25 strain of the M982 type (WO 93/6861); or (ii) the TbpB of at least one strain of the B16B6 type or the TbpB of at least one strain of the M982 type (WO 93/7172). The strains responsible for meningitis cases 30 worldwide (isolated cases or epidemic) are collected, studied and classified based on various criteria. A first method which is universally used consists in classifying the meningococci into serogroups, serotypes or subserotypes on the basis of antigenic differences 35 at the level of the capsular polysaccharides, of the class 2/3 outer membrane protein and of the class 1 outer membrane protein, respectively. This classi fication is carried out with the aid of monoclonal antibodies. For example, the M982 strain is thus -5 classified as being of serogroup B, serotype 9, subserotype P1.9 (B:9:Pl.9). Another classification method which is used internationally is based on the electrophoretic 5 mobility of certain metabolic enzymes. It is MLEE (Multi-Locus Electrophoresis Enzyme) whose use is in particular described in Olyhoek et al., in "The Pathogenic Neisseriae", (1985), ed. Schoolnick G.K. (American Society for Microbiology) : 530; Caugant et 10 al., J. Gen. Microbiol. (1986) 132: 641; Selander et al., Appl. Env. Microbiol. (1986) 51: 873; Caugant et al., Genetics (1981) 98:464; Caugant et al., PNAS (1986) 83: 4931. Generally speaking, the following 15 enzymes are analysed: malic enzyme (MAE), glucose-6 15 phosphate dehydrogenase (G6P), peptidase (PEP), isocitrate dehydrogenase (IDH), nicotinamide adenine dinucleotide (NAD) phosphate-dependent glutamate dehydrogenases (GD1 and GD2), alcohol dehydrogenase (ADH), fumarase (FUM), alkaline phosphatase (ALK), 20 indophenol oxydases (IP1 and IP2), adenylate kinase (ADK) and dehydrogenase (UDH). The electromorphs (or alloenzymes) of each enzyme correspond to alleles of the same genetic locus. A special combination of the alleles of these 15 enzymes constitutes an 25 electrophoretic type (ET) . These types can be grouped into a family or complex (cluster). For example, the ET-5 complex consists of 22 electrophoretic types which are very closely linked (maximum genetic distance is 0.16). In the table which follows, the allelic profile 30 of the strains of the ET-5 electrophoretic type is indicated (allele number per locus for each of the 15 enzymes). The enzymes (G6P and UDH) for which the allele(s) are likely to vary within the ET-5 complex are indicated in bold. 35 0N ~ r N N C: -1 -7 Various related electrophoretic complexes or types may exist within lineages. To date, 10 lineages (I to X), each representing a set of at least five electrophoretic types which are related to each other, 5 have been defined as described for example in Caugant et al., J. Infect. Dis. (1990) 162: 867. For example, lineage III groups together ET types 24, 24.1 - 4 and 25. During the past 25 years, the strains 10 responsible for a large number of meningitis cases worldwide (e.g. United States (Oregon, State of Washington), Norway, Cuba, Chile, Brazil, the Netherlands, New Zealand) have been found to belong to the ET-5 complex or to the III lineage. It therefore 15 very rapidly became desirable to provide a vaccine containing an antigen, for example a TbpB obtained from a strain of this complex. In fact, this is what is already done in the various vaccine compositions already known since they use a TbpB obtained from the 20 8680 strain (see WO 97/13860). This strain indeed belongs to the ET-5 complex. However, it has now been discovered that clinical isolates belonging to the ET-5 complex exhibited a certain variability at the level of the 25 tbpB gene. The study was carried out on 31 isolates responsible for recent meningitis epidemics worldwide. The tbpB genes were analysed (a) on the one hand for their profiles of restriction by the enzymes AvaIl, HincII, VspI and XhoI after amplification of the gene 30 from the genomic DNA and with the aid of the primers P1 (SEQ ID NO: 9; Pl corresponds to positions 115 to 113 of the sequence of the M982 tbpB gene, as found in the EMBL library, under accession number Z15130) and P2 (antisense; SEQ ID' NO: 10; P2 corresponds to positions 35 2264 to 2244 of the sequence of the M982 tbpB gene, as found in the EMBL library, under accession number Z15130); and (b) on the other hand for the size of the PCR (polymerase chain reaction) fragment, derived from the tbpB gene which may be amplified from the genomic -8 DNA with the aid of the primers P3 (SEQ ID NO: 11) and P4 (antisense; SEQ ID NO: 12). The restriction profiles of the tbpB genes of strains M982, BZ83 and 8680 are presented in Table II 5 below. It should be noted that the M982 strain does not belong to the ET-5 complex. However, it is studied because it serves as a reference for all the strains possessing a tbpB gene of 2.1 to 2.3 kb. Table II 10 AvaII VspI XhoI HincII M982 NC 1311 nt NC 1299 nt BZ83 1184 nt NC NC 1155 nt 477 nt 385 nt 415 nt 276 nt 257 nt 8680 1507 nt 1298 nt 1551 nt 1191 nt 445 nt 470 nt 483 nt 527 nt NC: not cut. These profiles were called by letters in Table III 15 below: Table III AvaII VspI XhoI HincII M982 N G N I BZ83 A N N B 8680 C D E F 20 The result's for all the strains are presented in Table IV below: 0 4-Jq 0 4 a H 0

.-

H 0U U a) : ~ I N jC N N (j N N N Ln Ln Lf 4 4 r40~ >1 0 a)~ a) z z z~ 7- - o o~ u ~ 0~~o~ z0 CM rr4 *- *- -~ 0 0 0)~~~~U mH44flfr)fln Of F44 E-4 mj wl co z r-c 0mmmmCz00)O o0) 0 o0 k.0 to tl 110 I'D n I'

OJ

IX 4 1 E4U L 4-4 4-4n 0 Q) Q-) 0 .HH 4-) ) 4-4 0.. 4 oVc 0) a 4-) wH m a ()m 4 m o000 oc Mz T )O )0)C)M0 ' - 11 The asterisk means the strain M982 which is not part of the ET-5 complex. (a) The size of the fragment indicated is that of the fragment amplified from the genomic DNA with the 5 aid of the primers P3 and P4, calculated to within one base when the complete sequence of the gene was available (strains M892, BZ83 and 8680) or determined after migration on a 3% agarose gel by comparison with the PCR fragment generated for the strains M892, BZ83 10 and 8680. To carry out this study to a successful conclusion, the genomic DNA was first extracted from strains cultured on Mueller-Hinton agar plates (MHA, Difco). The DNA was extracted using a rapid guanidium isothiocyanate method (Pitcher et al., Letters in 15 Applied Microbiology (1989) 8: 151). The PCR reactions were carried out in a volume of 100 pm containing 200 pM (each) of dCTP, dGTP, dATP and dTTP (Pharmacia LKB); 0.2 pM of each of the primers and 2.5 U of Taq polymerase (Appligene). The amplifications took place 20 in a DNA thermocycler (Biometra, Trio-thermobloc) programmed as follows: initial denaturing at 95*C for 5 min and then 25 cycles; each comprising a consecutive denaturation (30 sec, 95*C), annealing (30 sec, 58 0 C) and extension of the DNA chain (1 min, 72"C). The size 25 of the amplified fragments is determined after electrophoresis of the amplification product on a 3% agarose gel. (b) Restriction profiles of the genes amplified by PCR from the genomic DNA, with the aid of the primers 30 P1 and P2; the letters A to G and N refer to the restriction profiles indicated in Table III (H corresponds to the restriction profile: 1230, 320, 270, 210 and 80 nt; J corresponds to the restriction profile: 1350, 570'and 330 nt). To carry out this study 35 to a successful conclusion, the DNA of the strains was prepared as described above in (a) and then amplified under the following conditions: 25 cycles; each comprising a denaturation of the DNA (1 min, 94*C), annealing (2 min, 58 0 C), and extension (3 min, 72 0

C).

- 12 Each of the amplified fragments was purified on a Qiaquick column (Qiagen) and digested with the four enzymes in four separate reactions and following the manufacturer's instructions (New England Biolabs). The 5 restriction products were separated by electrophoresis on a 2% agarose gel. It has thus been shown that the variability mentioned above is reduced to 3 major groups (see Table 10 IV) . The most important group contained 17 strains, including the BZ83 strain (percentage representation 54.8%). It was followed by a group of 10 strains characterized by a PCR fragment having a single size (844 nt) identical to that of the PCR fragment obtained 15 with the M982 strain. Although the M982 strain is itself not part of this group, it is however designated in the text which follows under the name of the M982 group because of this size identity (it will be noted that the terms M982 group and M982 type do not 20 designate the same thing) . Among these 10 strains, the restriction profiles of the tbpB genes exhibit some heterogeneity: none of the genes is cut with AvaII or with XhoI; as regards VspI, either it has no restriction site, or it offers a G type restriction 25 profile; and the restriction profiles obtained with HincII are even more heterogeneous. In terms of representation, next was a group of 3 strains among which was the 8680 strain. Although the 8710 strain is characterized by a PCR fragment which has a smaller 30 size than those which characterize the strains of the M982 group, the restriction profile of its tbpB gene indeed shows that this strain is related to the M982 group. To confirm the predictive value of the 35 classification based on the PCR fragment type, the tbpB genes of the strains 32/94, 8726 and 8710 (cited in Table IV above) were cloned, sequenced and aligned with the sequences of the tbpB genes of the prototype strains BZ83, M982 and 8680 using the Infobiogen - 13 multiple Clustal alignment programme (Dessen et al., Bisance: "a French programme for accessing the biomolecular sequences databases") (1990) Cabios 6: 355). The results of the comparisons are given in Table 5 V below, in terms of percentage homology (identity) and indeed confirm the relevance of the classification by PCR analysis. Table V 10 32/94 8680 8710 8726 BZ83 M982 32/94 100 74 77 75 99 78 8680 - 100 79 90 74 75 8710 - - 100 80 77 86 8726 - - - 100 76 74 BZ83 - - - - 100 79 M982 - - - - - 100 The work relating to the analysis of the tbpB gene reported above has been the subject of a publication (Rokbi et al., Clin. Diag. Lab. Immunol. 15 (Sept. 1997) 4 (5) : 522. Since then, it has been discovered that the strains not belonging to the ET-5 complex had tbpB genes whose restriction enzyme profile was identical to that of the tbpB gene of the BZ83 strain. Some of these 20 strains belong to the III lineage; one is for example the 90/94 strain (Dr Caugant collection). Knowing that the selection of the isolates tested represents epidemics of varied territorial origin and given the large representation among them of 25 the group illustrated by the BZ83 strain, it is not dubious that the strains of this group are predominant in recent epidemics. It appears desirable to include in a vaccine a TbpB obtained from a strain of the group illustrated by the BZ83 strain, called hereinafter BZ83 30 group. Accordingly, this subject of the invention is a pharmaceutical composition comprising (i) the subunit - 14 of lower molecular weight (TbpB) of the human transferrin receptor (HTR) of a Neisseria meningitidis strain possessing a DNA sequence coding for TbpB, (a) which contains 2 AvaII restriction sites, 3 HincII 5 restriction sites, no VspI restriction site and no VspI and XhoI restriction site or, preferably and, (b) from which it is possible to generate by PCR (polymerase chain reaction) with the aid of the primers P3 of formula 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of 10 formula 5'-GAAGACGAGTCGGAAACAAAGGGATG-3', a poly nucleotide of 765 to 775 nucleotides, preferably of 772 nucleotides (strain of the BZ83 group); or (ii) a fragment of the said TbpB. "TbpB subunit of a strain, or which is derived, 15 comes or is obtained from an N. meningitidis strain" is obviously understood to mean a derivative taken in its most general sense; that is to say not limited to a physical process, but the result of an intellectual process. Thus, this expression covers i.a. a TbpB 20 produced by the recombinant route e.g. in E. coli. The N. meningitidis strain from which TbpB, which is useful for the purposes of the present invention, is obtained is preferably a strain belonging to the ET-5 complex or to the III lineage. For example, 25 a strain of the BZ83 group which is part of the ET-5 complex is the 32/94 strain. A strain of the BZ83 group which is part of the III lineage is the 90/94 strain. The sequences of the tbpB genes of these strains are given in the sequence identifier below. 30 According to an advantageous embodiment, the TbpB entering into the composition according to the invention is obtained from an N. meningitidis strain possessing a DNA sequence coding for TbpB which exhibits an AvaII 'restriction profile consisting of 3 35 fragments of 1184, 477 and 415 nt and an HincII restriction profile consisting of 4 fragments of 1155, 385, 276 and 257 nt. Preferably, this TbpB has the amino acid sequence as shown in SEQ ID NO: 2 (BZ83 strain).

- 15 Alternatively, it is possible to use fragments obtained from a TbpB of an adhoc strain. Fragments useful for vaccinal purposes can be defined according to the method proposed in WO 95/33049. 5 WO 95/33049 teaches that the study of the TbpB subunits, irrespective of the strain of origin, makes it possible to demonstrate three main structural domains, at least one of which is associated with special 10 properties. By definition, the domains of M982 TbpB and B16B6 TbpB have been fixed as shown in Table VI 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 4 and 8. 15 Table VI M982 TbpB B16B6 TbpB N-terminal domain or first domain 1-345 1-325 Hinge domain or second domain 346-543 326-442 C-terminal domain or third domain 544-691 443-579 This definition applies similarly to all M982 or B16B6 type TbpBs, after alignment of an M982 or B16B6 type sequence with the reference sequence, to maximum 20 homology. Thus, by way of example, the position is shown of the domains of the TbpB subunit of the strain 8680 is indicated as follows: first domain (1-334), second domain (335-530) and third domain (531-677). Knowing that the N-terminal domain or first 25 domain contains the transferrin binding site in its entirety and is hence very probably exposed to the outside, the sole N-terminal domain consequently - 16 constitutes a component of choice for i.a. vaccinal purposes. Furthermore, the comparative study of the M982 and B16B6 TbpB sequences aligned to maximum homology has 5 demonstrated four hypervariable portions localized within the hinge region (also called hinge domain, as appears in the table above) of M982 TbpB, which portions are absent from the B16B6 TbpB. These hypervariable portions exist in all the M982 type TbpBs. The B16B6 10 type strains lack them. In the text which follows, "M982 type hinge region" is understood to mean a hinge region which has these four hypervariable portions. By comparison of the sequences of various M982 type TbpBs, it is also known that the variability of the 15 hinge region is greater than that of the other two domains. Since the hinge region did not appear to be essential for the human transferrin binding function, it has been postulated that the role of this region is, at least in part, to induce a "screen"-type variability in 20 order to avoid recognition of a strain by the immune system of an individual who had subsequently been in contact with another strain. On this assumption, the hinge region of the M982 type strains constitutes a major problem in the 25 preparation of a vaccine. Indeed, if this region is immunodominant, during an immunization, the antibodies induced will be predominantly directed against this region and consequently the immune response will be specific for the TbpB protein of the homologous strain. 30 Taking into account the observations and assumptions mentioned above, it has already been proposed to use, i.a. for vaccinal purposes, not entire M982 type TbpBs anymore, but TbpBs at least deleted of their four hypervariable portions or, alternatively, 35 deleted of the second and third domains.

- 17 A composition according to the invention may also contain a TbpB obtained from a strain of the M982 group or of the group illustrated by 8680 (8680 group). Thus, a composition according to the invention 5 may comprise in addition (i) a first additional TbpB obtained from an N. meningitidis strain which is part of the ET-5 complex and which has a DNA sequence coding for TbpB (a) which does not contain AvaII and XhoI restriction sites or, preferably and, (b) from which it 10 is possible to generate by PCR with the aid of the primers P3 of formula 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5'-GAAGACGAGTCGGAAACAAAGGGATG-3', a polynucleotide of 840 to 850 nucleotides, preferably of 844 nucleotides (strain of the M982 group); or (ii) a 15 fragment of the said first additional TbpB. According to an advantageous embodiment, the first additional TbpB is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which contains 1 VspI restriction site and 20 3 HincII restriction sites. Preferably, this TbpB is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which exhibits a VspI restriction profile consisting of 2 fragments of 1311 and 769 nt and an HincII restriction profile consisting 25 of 4 fragments of 1229, 319, 281 and 259 nt. A pharmaceutical composition according to the invention may also comprise in addition (i) a second additional TbpB obtained from an N. meningitidis strain which is part of the ET-5 complex and which has a DNA 30 sequence coding for TbpB (a) which contains 1 AvaII restriction site, 2 VspI restriction sites, 1 XhoI restriction site and 2 HincII restriction sites or, preferably and, (b) from which it is possible to generate by PCR with the aid of the primers P3 of 35 formula 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5' -GAAGACGAGTCGGAAACAAAGGGATG-3', a polynucleotide of 800 to 810 nucleotides, preferably of 805 nucleotides (strain of the 8680 group); or (ii) a fragment of the said second additional TbpB.

- 18 According to an advantageous embodiment, the second additional TbpB is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which exhibits an AvaII restriction profile 5 consisting of 2 fragments of 1507 and 445 nt, a VspI restriction profile consisting of 3 fragments of 1298, 470 and 266 nt, an XhoI restriction profile consisting of 2 fragments of 1551 and 483 nt and an HincII restriction profile consisting of 3 fragments of 1191, 10 527 and 316 nt. Preferably, this second additional TbpB has the amino acid sequence as shown in SEQ ID NO: 6 (strain 8680). In addition to TbpBs obtained from strains of the ET-5 complex, a composition according to the 15 invention may also contain one or more TbpBs obtained from strains of other electrophoretic complexes. In particular, it is envisaged to add (i) a TbpB of a strain of the M982 type, that is to say of a strain having a tbpB gene of about 2.1 to 2.3 kb and whose 20 degree of homology (identity) with the M982 TbpB would be higher than those observed by comparing the TbpBs of the strains of the ET-5 complex and the M982 TbpB (homology at the level of the amino acid sequence) or (ii) a TbpB of a strain of the B16B6 type, that is to 25 say of a strain having a tbpB gene of about 1.8 kb. Thus, a pharmaceutical composition according to the invention may comprise in addition (i) a TbpB having an amino acid sequence whose degree of homology with the sequence shown in SEQ ID NO: 4, from the amino 30 acid at position 1 to the amino acid at position 691, is at least 85%, advantageously at least 90% or (ii) a fragment of the said TbpB. Preferably, it will be the TbpB of the M982 strain which has the amino acid sequence as shown in SEQ ID NO: 4. 35 A pharmaceutical composition according to the invention may also comprise in addition (i) a TbpB having an amino acid sequence whose degree of homology with the sequence shown in SEQ ID NO: 8, from the amino acid at position 1 to the amino acid at position 579, - 19 is at least 95%, preferably 100% (strain of the B16B6 type); or (ii) a fragment of the said TbpB. As an alternative to the various possible additional TbpBs, it is also possible to use a fragment 5 thereof. The useful fragments may be determined as is explained in WO 95/33049. The TbpBs which are useful for the purposes of the present invention may be in a form dissociated from the subunit of high molecular weight (Tbpl) of the 10 N. meningitidis strain from which TbpB is derived, or alternatively in association with this Tbpl, thus forming a Tbpl-TbpB complex which is considered as the human transferrin receptor; in other words, they may be in a form lacking their corresponding subunits of high 15 molecular weight (Tbpls) or alternatively associated therewith, so as to form an HTR. Whether it is in a dissociated form or in the form of a Tbpl-TbpB complex, the TbpB subunit according to the invention should be substantially purified; that is to say separated from 20 the medium in which it exists naturally. Inter alia, it may be a preparation lacking in particular the cyto plasmic and periplasmic proteins of N. meningitidis. A TbpB subunit which is useful for the purposes of the present invention may be in a dissociated form 25 of the subunit of high molecular weight (Tbpl) of the N. meningitidis strain from which TbpB is derived or alternatively in association with this Tbpl, thus forming a Tbpl-TbpB complex which is considered as the human transferrin receptor. Whether it is in a 30 dissociated form or in the form of a Tbpl-TbpB complex, the TbpB subunit according to the invention should be substantially purified; that is to say separated from the medium in which it exists naturally. Inter alia, it may be a preparation lacking in particular the cyto 35 plasmic and periplasmic proteins of N. meningitidis. For the purposes of the present invention, it is possible to use one or more TbpBs purified from meningococcus strains according to methods which are already known (see inter alia WO 97/13860) or - 20 alternatively which are obtained by the recombinant route in a homologous or heterologous expression system. An appropriate expression system is within the 5 capabilities of persons skilled in the art since they have the sequences of tbpB genes. They need to construct in particular an expression cassette in which a DNA fragment coding for a mature TbpB or a fragment thereof will be placed under the control of an 10 appropriate promoter. This DNA fragment may be fused or otherwise with a DNA block coding for the homologous signal peptide or for a heterologous signal peptide, depending on whether the secretion of the polypeptide is sought or not. Preferably, this secretion will be 15 sought and a signal sequence derived from a gene coding for a lipoprotein will be used. Components such as a DNA block coding for a heterologous signal peptide (signal region) or a promoter already exist in a fairly large number and are 20 known to persons skilled in the art. Their general competence will allow them to choose a signal region or a particular promoter which will be suitable for the host cell in which the expression is envisaged. For the.purposes of the method according to the 25 invention, the host cell may be a mammalian cell, a bacterium or a yeast; the latter two being preferred. Here again, the choice of a particular line is within the capabilities of persons skilled in the art. 30 The subject of the invention is also a pharma ceutical composition which comprises a DNA molecule coding for (i) the TbpB of the human transferrin 35 receptor (HTR) of a Neisseria meningitidis strain having a DNA sequence coding for TbpB, (a) which contains 2 AvaII restriction sites, 3 HincII restriction sites, no VspI restriction site and no VspI and XhoI restriction site or, preferably and, (b) from which it - 21 is possible to generate by PCR (polymerase chain reaction) with the aid of the primers P3 of formula 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5' -GAAGACGAGTCGGAAACAAAGGGATG-3', a polynucleotide of 5 765 to 775 nucleotides, preferably of 772 nucleotides (strain of the BZ83 group); or (ii) a fragment of the said TbpB. Preferably, the DNA molecule codes for a TbpB which is obtained from an N. meningitidis strain 10 belonging to the ET-5 complex or to the III lineage. According to an advantageous embodiment, this DNA molecule codes for a TbpB which is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which exhibits an AvaII restriction profile con 15 sisting of 3 fragments of 1184, 477 and 415 nt and an HincII restriction profile consisting of 4 fragments of 1155, 385, 276 and 257 nt. Preferably, this DNA molecule codes for a TbpB which has the amino acid sequence as shown in SEQ ID NO: 2 (strain BZ83). 20 A DNA-based composition according to the invention may comprise in addition a first DNA molecule coding for (i) a TbpB obtained from an N. meningitidis strain which is part of the ET-5 complex and which has a DNA sequence coding for TbpB (a) which does not 25 contain AvaII and XhoI restriction sites or, preferably and, (b) from which it is possible to generate by PCR with the aid of the primers P3 of formula 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5' -GAAGACGAGTCGGAAACAAAGGGATG-3', a polynucleotide of 30 840 to 850 nucleotides, preferably of 844 nucleotides (strain of the M982 group); or (ii) a fragment of the said TbpB. According to an advantageous embodiment, the first additional DNA molecule codes for a TbpB which is 35 obtained from an N. meningitidis strain having a DNA sequence coding for TbpB, which contains 1 VspI restriction site and 3 HincII restriction sites. Preferably, this DNA molecule codes for a TbpB which is obtained from the N. meningitidis strain having a DNA - 22 sequence coding for TbpB which exhibits a VspI restriction profile consisting of 2 fragments of 1311 and 769 nt and an HincII restriction profile consisting of 4 fragments of 1229, 319, 281 and 259 nt. 5 A DNA-based composition according to the invention may also comprise, in addition, a second additional DNA molecule coding for (i) a TbpB obtained from an N. meningitidis strain which is part of the ET-5 complex and which has a DNA sequence coding for 10 TbpB (a) which contains 1 AvaII restriction site, 2 VspI restriction sites, 1 XhoI restriction site and 2 HincII restriction sites or, preferably and, (b) from which it is possible to generate by PCR with the aid of the primers P3 of formula 15 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5'-GAAGACGAGTCGGAAACAAAGGGATG-3', a polynucleotide of 800 to 810 nucleotides, preferably of 805 nucleotides (strain of the 8680 group); or (ii) a fragment of the said TbpB. 20 According to an advantageous embodiment, the second additional DNA molecule codes for a TbpB which is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which exhibits an AvaII restriction profile consisting of 2 fragments of 1507 25 and 445 nt, a VspI restriction profile consisting of 3 fragments of 1298, 470 and 266 nt, an XhoI restric tion profile consisting of 2 fragments of 1551 and 483 nt and an HincII restriction profile consisting of 3 fragments of 1191, 527 and 316 nt. Preferably, this 30 DNA molecule codes for a TbpB which has the amino acid sequence as shown in SEQ ID NO: 6 (strain 8680). A DNA-based composition according to the invention may comprise, in addition, a DNA molecule coding for (i) a TbpB having an amino acid sequence 35 whose degree of homology with the sequence shown in SEQ ID NO: 4 is at least 85%, advantageously at least 90%; or (ii) a fragment of the said TbpB. Preferably, this will be a DNA molecule coding for the TbpB of the M982 - 23 strain which has the amino acid sequence as shown in SEQ ID NO: 4. A DNA-based composition according to the invention may also comprise, in addition, a DNA 5 molecule coding for (i) a TbpB which has an amino acid sequence whose degree of homology with the sequence shown in SEQ ID NO: 8 is at least 95%, preferably 100% (strain of the B16B6 type); or (ii) a fragment of the said TbpB. 10 The DNA molecule may be advantageously a plasmid which is incapable both of replicating and of becoming substantially integrated into the genome of a mammal. The coding sequence cited above is placed under the control of a promoter allowing expression in a 15 mammalian cell. This promoter may be ubiquitous or specific to a tissue. Among the ubiquitous promoters, there may be mentioned the cytomegalovirus early promoter (described in Patent US No. 4,168,062) and the Rous sarcoma virus promoter (described in Norton & 20 Coffin, Molec. Cell. Biol. (1985) 5 : 281). The desmin promoter (Li et al., Gene (1989) 78 : 244443; Li & Paulin, J. Biol. Chem. (1993) 268 : 10403) is a selective promoter, allows expression in muscle cells and also in the cells of the skin. A promoter specific 25 to muscle cells is for example the promoter of the myosin or dystrophin gene. Plasmid vectors which can be used for the purposes of the present invention are described i.a., in WO 94/21797 and Hartikka et al., Human Gene Therapy (1996) 7 : 1205. 30 In a DNA-based composition according to the present invention, the DNA molecule(s) may be formu lated or otherwise. The choice of formulation is highly varied. The DNA may be simply diluted in a physio logically acceptable solution with or without carrier. 35 When the latter is present, it may be isotonic or weakly hypertonic and may have a low ionic strength. For example, these conditions may be met by a solution of sucrose e.g. at 20%.

- 24 Alternatively, the DNA molecule(s) may be combined with agents which promote entry into the cell. This may be (ii) a chemical agent which modifies cellular permeability, such as bupivacaine (see for 5 example WO 94/16737) or (ii) an agent combining with the polynucleotide and acting as a vehicle facilitating the transport of the polynucleotide. The latter may be, in particular, cationic polymers e.g. polylysine or a polyamine e.g. spermine derivatives (see WO 93/18759). 10 This may also be fusogenic peptides e.g. GALA or Gramicidin S (see WO 93/19768) or alternatively peptides derived from viral fusion proteins. They may also be anionic or cationic lipids. Anionic or neutral lipids have been known for a long 15 time to be able to serve as transporting agents, for example in the form of liposomes, for a large number of compounds including polynucleotides. A detailed description of these liposomes, of their constituents and of the methods for their manufacture is for example 20 provided by Liposomes: A Practical Approach, RPC New Ed, IRL press (1990). The cationic lipids are also known and are commonly used as transporting agents for polynucleo tides. There may be mentioned, for example, LipofectinTm 25 also known by the name DOTMA (N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-bis (oleyloxy) -3- (trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (diocta decylaminoglycyl spermine) and cholesterol derivatives 30 such as DC-chol (3-beta-(N-(N',N'-dimethylaminoethane) carbamoyl)cholesterol). A description of these lipids is provided by EP 187,702, WO 90/11092, US Patent No. 5,283,185, WO 91/15501, WO 95/26356 and US Patent No. 5,527,928. The cationic lipids are preferably used with 35 a neutral lipid such as DOPE (dioleyl phosphatidyl ethanolamine) as is for example described in WO 90/11092. Microparticles of gold or tungsten may also be used as transporting agents, as described in WO 91/359, - 25 WO 93/17706 and Tang et al., Nature (1992) 356 : 152. In this particular case, the polynucleotide is precipitated on the microparticles in the presence of calcium chloride and spermidine and then the whole is 5 administered by a high-speed jet into the dermis or into the epidermis, with the aid of a needle-free apparatus such as those described in US Patent No. 4,945,050 and No. 5,015,580 and WO 94/24243. The quantity of DNA which can be used to 10 vaccinate an individual depends on a number of factors such as for example the strength of the promoter used to express the antigen, the immunogenicity of the product expressed, the state of the mammal for whom the administration is intended (e.g. the weight, age and 15 general state of health), the mode of administration and the type of formulation. It is indicated in particular that the intramuscular route of administration requires a larger quantity of DNA than the intradermal route of administration with the aid of 20 a needle-free apparatus. In general, an appropriate dose for a prophylactic or therapeutic use in an adult of the human species is about 1 pg to about 5 mg, preferably about 10 pg to about 1 mg and in a most particularly preferred manner about 25 pg to about 25 500 pg. When a DNA-based composition according to the present invention contains several DNA molecules (one coding for TbpB of a strain of the BZ83 group and the others for TbpB of a strain of another group or type), 30 these molecules may exist in a form separated from each other (e.g. a plasmid/coding sequence) or alternatively may constitute one or more units (e.g. one and the same plasmid/two coding sequences or more). A DNA-based composition according to the 35 present invention may, in addition, contain compounds other than the immunogenic agent itself, the nature of these compounds depending to a certain extent i.a. on the route of administration. Thus, as seen above, the pharmaceutical composition may include various formula- - 26 tion agents. By way of information, it is generally indicated that it is possible for a DNA molecule not to require the addition of an adjuvant. 5 The pharmaceutical compositions according to the invention are in particular useful for inducing an immune response in humans against N. meningitidis, 10 inter alia a vaccinal effect so as to protect humans against N. meningitidis infections, in prevention or in therapy. The compositions according to the present invention may be manufactured in a conventional manner. 15 In particular, they may be formulated with a pharmaceutically acceptable diluent, carrier or vehicle, e.g. water or a saline solution. In general, the diluent or the vehicle may be selected depending on the mode and route of administration and according to 20 standard pharmaceutical practices. Appropriate vehicles or diluent as well as what is essential for the production of a pharmaceutical composition are described in Remington's Pharmaceutical Sciences, a standard reference book in this field. The compositions 25 based on TbpBs may also contain an adjuvant. A composition according to the invention may be administered by any conventional route of administra tion in use in the field of vaccines. Advantageously, the systemic route is used, i.a. the parenteral route 30 which may itself be chosen from the intravenous, intra muscular, intradermal, intraepidermal and subcutaneous routes; the latter four being, however, preferred to the intravenous route. In order t'o obtain a protective or therapeutic 35 effect, the operation which consists in administering a pharmaceutical composition according to the present invention may be repeated once or several times, leaving a certain interval of time between each administration; which interval is of the order of a - 27 week or a month. Its precise determination is within the capabilities of persons skilled in the art and may vary according to various factors such as the nature of the immunogenic agent, the age of the individual and 5 the like. All the documents cited in the present application are incorporated by reference. EXAMPLE 1: Meningococcal vaccine containing four strain 10 valencies (BZ83, 8680, M982 and B16B6) 1A. Purification of the native HTRs The HTR of each of the strains is purified according to the method described for the HTR of the 15 8680 strain, in Example 1 of WO 97/13860. 1B. Purification of the native TbpBs The TbpB of each of the strains is purified according to the method described for the TbpB of the 20 8680 strain, in Example 2 of WO 97/13860. 1C. Preparation of the recombinant TbpBs For each of the TbpBs, expression plasmids are constructed in which the sequences coding for the 25 mature TbpBs are fused with the E. coli rlpB signal sequence (Takase et al., J. Bact. (1983) 169 : 50692) and are placed under the control of the araB promoter (of Salmonella typhimurium (Horwitz et al., Gene (1981) 14 309, Cagnon et al., Protein Engineering (1991) 30 4(7) : 843 and Legrain et al., Prot. Express. Purif. (1995) 6 : 570). These plasmids also contain a replication origin which is functional in E. coli, a gene for resistance to kanamycin and the cer locus (Summers & 35 Sherratt, Cell (1984) 36 : 1097). Each of these plasmids serves to transform the E. coli BL21 strain. To produce the TbpBs, each of the transformants selected is cultured in a 20-litre fermenter in a TGM16 - 28 medium (Slos et al., Prot. Express. Purif. (1994) 6 518) in the absence of ampicillin. During the exponen tial phase (optical density at 600 nm greater than 40), 0.2% arabinose (expression inducer) is added. After one 5 hour of induction, the cells are harvested and stored at -20*C. The bacterial pellets are resuspended in Tris buffer and subjected to high-pressure lysis (Rannie). This is followed by a series of washes and centri 10 fugations. The final pellet is solubilized in Tris buffer containing zwittergent Z3-14 and then ultra centrifuged. The supernatant is loaded onto a Q-Sepharose column. The direct eluate containing the TbpB is loaded onto a second Q-Sepharose column and the 15 TbpB is eluted in an NaCl gradient. In order to remove the degradation products and the endotoxins, the fraction containing TbpB is purified by gel filtration on an S-300 chromatography column. The final preparations are sterilized by filtration and frozen. 20 1D. Vaccinal compositions Either the 4 HTRs prepared as described in Example 1A, or the 4 TbpBs prepared as described in Example 1B, or the 4 TbpBs prepared as described in 25 Example 1C are used together. A vaccinal composition is obtained by mixing equimolar quantities of each of the BZ83, 8680, M982 and B16B6 TbpB preparations in order to obtain a final TbpB concentration of the order of 0.2 mg/ml.

Claims (19)

1. Pharmaceutical composition comprising (i) the subunit of lower molecular weight (TbpB) of the human transferrin receptor (HTR) of a Neisseria meningitidis strain possessing a DNA sequence coding for TbpB, (a) which contains 2 AvaII restriction sites, 3 HincII restriction sites, no VspI restriction site and no XhoI restriction site or, preferably and, (b) from whicli it is possible to generate by PCR (polymerase chain reaction) with the aid of the primers P3 of formula 5' AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5' GAAGACGAGTCGGAAACAAAGGGATG-3', a polynucleotide of 765 to 775 nucleotides, preferably of 772 nucleotides (strain of the BZ83 group); or (ii) a fragment of the said TbpB.

2. Pharmaceutical composition according to Claim 1, in which the TbpB is obtained from an N. meningitidis strain which is part of the ET-5 complex or the III lineage.

3. Pharmaceutical composition according to Claim 1 or 2, in which the TbpB is obtained from an N. meningitidis strain possessing a DNA sequence coding for TbpB which exhibits an AvaII restriction profile consisting of 3 fragments of 1184, 477 and 415 nt and an HincII restriction profile consisting of 4 fragments of 1155, 385, 276 and 257 nt (strain of the BZ83 group).

4. Pharmaceutical composition according to Claim 3, in which the TbpB has the amino acid sequence as shown in SEQ ID NO : 2 (strain BZ83).

5. Pharmaceutical composition according to one of Claims 1 to 4, which comprises in addition (i) a first additional TbpB obtained from an N. meningitidis strain which is part of the ET-5 complex and which has a DNA sequence coding for TbpB (a) which does not contain AvaII and XhoI restriction sites or, preferably and, (b) from which it is possible to generate by PCR with the aid of the primers P3 of formula -68 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5'-GAAGACGAGTCGGAAACAAAGGGATG-3', a polynucleotide of 840 to 850 nucleotides, preferably of 844 (strain of the M982 group); or (ii) a fragment of the said first additional TbpB.

6. Pharmaceutical composition according to Claim 5, in which the first additional TbpB is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which contains 1 VspI restriction site and 3 HincII restriction sites (strain of the M982 group).

7. Pharmaceutical composition according to Claim 6, in which the TbpB is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which exhibits a V.spI restriction profile consisting of 2 fragments of 1311 and 769 nt and an HincII restriction profile consisting of 4 fragments of 1229, 319, 281 and 259 nt (strain of the M982 group).

8. Pharmaceutical composition according to one of Claims 1 to 7, which comprises in addition (i) a second additional TbpB obtained from an N. meningitidis strain which is part of the ET-5 complex and which has a DNA sequence coding for TbpB (a) which contains 1 AvaII restriction site, 2 VspI restriction sites, 1 XhoI restriction site and 2 HincI! restriction sites or, preferably and, (b) from which it is possible to generate by PCR with the aid of the primers P3 of formula 5'-AAGACCAAGGCGGATACGGTTTTGC-3' and P4 of formula 5'-GAAGACGAGTCGGAAACAAAGGGATG-3', a poly nucleotide of 800 to 810 nucleotides, preferably of 805 (strain of the 8680 group); or (ii) a fragment of the said second additional TbpB.

9. Pharmaceutical composition according to Claim 8, in which the second additional TbpB is obtained from an N. meningitidis strain having a DNA sequence coding for TbpB which exhibits an AvaII restriction profile consisting of 2 fragments of 1507 and 445 nt, a VspI restriction profile consisting of 3 fragments of 1298, 470 and 266 nt, an XhoI restriction profile consisting of 2 fragments of 1551 and 483 nt and an HincII - 69 restriction profile consisting of 3 fragments of 1191, 527 and 316 nt (strain of the 8680 group).

10. Pharmaceutical composition according to Claim 9, in which the second additional TbpB has the amino acid sequence as shown in SEQ ID NO: 6 (strain 8680).

11. Pharmaceutical composition according to one of Claims 1 to 10, which comprises in addition (i) a TbpB having an amino acid sequence whose degree of homology with the sequence shown in SEQ ID NO: 4 is at least 85%, preferably at least 90% (strain of the M982 type); or (ii) a fragment of the said TbpB.

12. Pharmaceutical composition according to Claim 11, in which the TbpB has the amino acid sequence as shown in SEQ ID NO : 4 (strain M982).

13. Pharmaceutical composition according to one of Claims 1 to 12, which comprises in addition (i) a TbpB having an amino acid sequence whose degree of homology with the sequence shown in SEQ ID NO : 8 is at least 95% (strain of the B16B6 type); or (ii) a fragment of the said TbpB.

14. Pharmaceutical composition according to one of Claims 1 to 13, in which the TbpB as defined in one of Claims 1 to 4, the first additional TbpB as defined in one of Claims 5 to 7, the second additional TbpB as defined in one of Claims 8 to 10, the TbpB as defined in Claim 11 or 12, or the TbpB as defined in Claim 13 is combined with the subunit of high molecular weight (Tbpl) corresponding to it, so as to form an HTR.

15. Pharmaceutical composition which comprises a DNA molecule coding for a TbpB as defined in one of Claims 1 to 4.

16. Pharmaceutical composition according to Claim 15, which comprises in addition a first additional DNA molecule coding for a TbpB as defined in one of Claims 5 to 7.

17. Pharmaceutical composition according to Claim 15 or 16, which comprises in addition a second additional DNA molecule coding for a TbpB as defined in one of Claims 8 to 10.

18. Pharmaceutical composition according to one of Claims 15 to 17, which comprises in addition a DNA molecule coding for a TbpB as defined in Claim 11 or 12.

19. Pharmaceutical composition according to one of Claims 15 to 18, which comprises in addition a DNA molecule coding for a TbpB as defined in Claim 13.