EP1129195A2 - Nucleic acids and polypeptides specific of the neisseria genus pathogenic strains - Google Patents

Abstract

The invention concerns nucleic acids coding for polypeptides specific of the Neisseria genus pathogenic strains, the corresponding polypeptides, and their diagnostic and therapeutic applications.

Description

 Nucleic acids and polypeptides specific for pathogenic strains of the genus Neisseria.

The present invention relates to nucleic acids encoding polypeptides specific for pathogenic strains of the genus Neisseria, in particular useful for preventing or treating infection by Neisseria meningitidis. Generally speaking, meningitis is either of viral origin or of bacterial origin. The main responsible bacteria are: Haemophilus influenzae type b, Neisseria meningitidis and Streptococcus pneumoniae. The Neisseria meningitidis species is subdivided into serogroups according to the nature of the capsular polysaccharides. Although there are a dozen serogroups, 90% of meningitis cases are caused by three serogroups: A, B and C.

There are effective vaccines based on capsular polysaccharides to prevent meningitis with Neisseria meningitidis serogroups A and C. These polysaccharides as such are only slightly or not immunogenic in children under two years of age and do not induce immune memory. However, these drawbacks can be overcome by conjugating these polysaccharides to a carrier protein.

On the other hand, the polysaccharide of Neisseria meningitidis serogroup B is not or only slightly immunogenic in humans, whether it is in conjugated form or not. It therefore appears highly desirable to seek a vaccine against meningitis caused by Neisseria meningitidis, in particular serogroup B, other than a polysaccharide-based vaccine.

To this end, various proteins of the outer membrane of N. meningitidis have already been proposed, such as the membrane receptor for human transferrin (WO 90/12591 and WO93 / 06861).

Neisseria meningitidis is genetically very close to Neissena gonorrhoeae and Neisseria lactamica. N. gonorrhoeae is mainly responsible for localized infections in the urogenital tract. It colonizes the genital mucosa, then crosses the epithelium, then invades the subepitheium where it multiplies and is responsible for a strong inflammatory reaction. In contrast, N. lactamica is considered a non-pathogenic species.

Sequences present in N. gonorrhoeae and N. meningitidis, absent from N. lactamica were disclosed in the patent application WO 98/02 547, but this earlier patent application does not locate and identify the coding sequences.

The authors of the present invention have now managed to identify some of these genes by searching the meningococcal genome for open reading frames specific for pathogenic strains of the genus Neisseria, by implementing the following strategy:

Some of the sequences disclosed in patent application WO98 / 02547 (designated in said previous application SEQ ID No. 66, 67, 69, 70, 72 to 96, 98, and 99), have been positioned on the genome sequence of the N. meningitidis strain of serogroup B (ATCC 13090), available from the Pathoseq® bank of Incyte Pharmaceuticals as well as on the genome sequence of the Neisseria meningitidis Z2491 strain (Sanger Center). This made it possible to identify, in the genome of N. meningitidis which has 2.3 mega bases, 19 contigs representing 220,000 base pairs.

The authors of the present invention then analyzed, for each of the 19 contigs, the presence of open reading frames (ORF), containing at least 100 amino acids (and by definition bounded by a codon of initiation and a stop codon), using the Gene Jockey II sequence processor software (Biosoft). This analysis made it possible to select around 400 candidate ORFs.

The sequences of each of these ORFs were then

(R) analyzed using Codon Use software (Conrad Halling), which takes into account the frequency of use of codons in N. meningitidis. Only ORFs whose sequences have a maximum frequency of use of these codons have been selected. At the end of this analysis, 197 candidate ORFs were selected.

The ORFs selected by this double analysis were subjected to a search for homologies on all the banks

(S) available using the BLASTX program from access to the Incosete Pharmaceuticals Pathoseq® bank. This search for homologies made it possible to exclude ORFs coding a priori for cytoplasmic proteins or periplasmics, especially proteins of metabolism. The ORFs were also subjected to an analysis of possible protein motifs, to

(R) using the DNA Star Protean program (Lasergene software).

The authors of the present invention then investigated whether the ORFs selected at the end of the previous step (118 in number) were actually absent from N. lactamica, as provided for in the application of the prior art WO 98/02547 .

To this end, PCR amplification was used. This amplification proved inoperative for 78 of the 118 ORFs tested. Only ORFs for which the amplification in N. lactamica was negative (sequences called "lactamica ^" ") were retained. In order to verify that these negative results were not" false negatives ", the lactamica sequences ^" retained been subject to dot blot control. At the end of this stage, only 23 ORFs were confirmed N. meningitidis / N. lactamica ^' . Finally, these 23 ORFs were repositioned as a whole on the genome of N. meningitidis ATCC13090. This made it possible to demonstrate that three ORFs previously eliminated on the basis of their putative protein function, appeared localized in the vicinity or even surrounded by some of the 23 ORFs N. meningitidis / N. lactamica ^' . These three ORFs (SEQ ID No. 29, 35 and 37) were reintroduced into the study, and it turned out that they too were N. meningitidis / N. lactamica ^' .

The authors of the present invention then sought to know whether the ORFs identified from the genome of the N. meningitidis strain of serogroup B ATCC 13090 were also present in the genomes of N. meningitidis Z2491 (Sanger Center) of serogroup A and of N. gonorrhoeae FA1090 (Advanced Center of Genome Technology, Oklahoma University). Then they compared the sequences derived from these different genomes, by multiple alignment (Clustal, Infobiogen). This made it possible to redefine for some of the ORFs the most probable position of the initiation and end of translation codons. The sequences of the open reading frames originating from the ATCC13090 strain are given in SEQ ID N ° 1 - 51 (odd numbers) and the amino acid sequences which are deduced therefrom, in SEQ ID N ° 2 - 52 (numbers peers). The present invention therefore relates to a nucleic acid in isolated form coding for a polypeptide, or an antigenic fragment thereof, excluding the nucleic acids disclosed in SEQ ID Nos. 70, 73, 74, 77, 80, 81, 87, 88, 89, 94, 95 and 98 of application WO 98/02547 (sequences annexed to this description and numbered SEQ ID No. 70A, 73A, 74A, 77A, 80A, 81 A, 87A, 88A, 89A, 94A, 95A, 98A to distinguish them from the sequences of the invention); said polypeptide having an amino acid sequence which is identical or homologous to a sequence chosen from those of group II; group II consisting of the sequences shown in SEQ ID No. 2 - 52 (even numbers) and the sequence SEQ ID No. 53.

Preferably, said nucleic acid may have a nucleotide sequence chosen from those of group I, group I consisting of the sequences shown in SEQ ID No. 1-51 (odd numbers).

The term "nucleic acid" includes and also means ORF, gene, polynucleotide, DNA and RNA. The term "nucleic acid in isolated form" means a nucleic acid separated from the biological environment in which it is found under natural conditions. For example, a DNA molecule exists under natural conditions when it is integrated into a genome or when it is part of a gene bank. In this case, it cannot be in isolated form. On the other hand, the same molecule separated from the genome by cloning (for example following an amplification by PCR) must be considered as being in isolated form. Typically, a DNA molecule in isolated form does not contain the coding regions which are contiguous to it in 5 'and 3' in the genome from which it originates. The nucleic acids in isolated form can be integrated into vectors (for example plasmids or viral or bacterial vectors) without thereby departing from their characteristic of being separated from their natural environment.

The authors of the present invention have more particularly found that the ORFs which, when they came from the ATCC 13090 strain, were characterized by the sequences as shown in SEQ ID No. 19, 27, 39, 45, 47 and 49 were specific for Neisseria meningitidis insofar as it was not possible to identify identical or homologous sequences in the genome of N. gonorrhoeae. They also found that the ORF characterized by the strain sequence as shown in SEQ ID No. 39 was specific for serogroup B Neisseria meningitidis.

A subject of the invention is also a polypeptide in isolated form or a fragment thereof; said polypeptide having an amino acid sequence identical or homologous to a sequence chosen from those of group II.

The amino acids framed in the sequence SEQ ID No. 8 correspond to the signal sequence, the amino acid in bold represents the first amino acid of the mature form. The amino acid sequence of the mature protein form is shown in SEQ ID No. 53.

In the context of the present invention, the terms of

"polypeptide" and "protein" are equivalent and interchangeable with each other. They designate any chain of amino acids, whatever its length and its post-translational modifications (for example, phosphorylation or glycosylation).

By "antigenic fragments of the polypeptides specific for pathogenic strains of the genus Neisseria" is meant the polypeptides derived from the polypeptides of the invention as defined above, by deletions of parts of said polypeptides without destruction of the antigenicity (for example, without loss significant of the antigenic activity) of said polypeptides. The specific antigenicity can be determined using various methods known to those skilled in the art, as explained below.

These fragments are preferably at least 12 amino acids long, more preferably at least 20 amino acids long, preferably 50 amino acids long, more preferably 75 amino acids long, preferably 100 amino acids long.

These fragments can be used to reveal epitopes which can be masked in parent polypeptides. They are also advantageous for inducing a protective immune response dependent on T lymphocytes. Deletions can indeed make it possible to eliminate immunodominant regions of high variability between different strains.

Such fragments can be obtained using standard techniques known to those skilled in the art (for example, Ausubel et al, Current Protocols in Molecular Biology, John Wiley & sons Inc, 1994), for example by PCR, RT-PCR, restriction enzyme treatment of cloned DNA molecules, or by the method of Kunkel et al (Proc. Natl. Acad. Sci. USA (1985) 82: 448).

By "homologous amino acid sequence" is meant a sequence which differs from one of the group II sequences by substitution, deletion, and / or insertion of one or more amino acids, at positions such that these modifications do not destroy not the specific antigenicity of the polypeptide in question. Said substitutions are preferably conservative substitutions, that is to say substitutions of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (such as asparagine, glutamine, serine, threonine, and tyrosine), amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid); amino acids with apolar side chains (such as glycine, alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine, tryptophan, and cysteine).

Advantageously, a homologous amino acid sequence has a degree of homology (ie, identity) of at least 75% with one of the sequences of group II; preferably this degree of homology is at least 80%, very preferably, at least 90%. The homologous amino acid sequences include in particular the sequences which are substantially identical to one of the Group II sequences. By "substantially identical sequence" is meant a sequence whose degree of homology (Le., Of identity) with one of the sequences of group II is at least 90%, advantageously at least 95%, preferably d '' at least 97%, most preferably at least 99%. In addition, it may differ from the reference sequence only by a majority of conservative substitutions.

The degree of homology (also called degree of identity) is usually determined using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison , Wl 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e. identity). To this end, it may be necessary to artificially introduce spaces ("gaps") in the sequence. Once the optimal alignment has been achieved, the degree of homology (i.e. identity) is established by recording all the positions for which the amino acids of the two sequences compared are identical, relative to the total number of positions.

By "homologous nucleotide sequences" is meant sequences which differ from the sequences of group I by substitution of a nucleotide or more nucleotides, or by deletion and / or insertion of one or more codons, at positions such that these sequences code (i) always for polypeptides having the sequences of group II, by effect of the degeneration of the genetic code; or (ii) for polypeptides having homologous sequences as defined above.

Advantageously, a homologous nucleotide sequence has a degree of homology of at least 60% with one of the sequences of group I; preferably this degree of homology is at least 80%, most preferably at least 90%.

Typically, a homologous nucleotide sequence hybridizes specifically to sequences complementary to the sequences of the group I under stringent conditions. The temperature at which the hybridization test is performed is an important factor influencing stringency. Conventionally, this temperature, called hybridization temperature (Th), is chosen from 5 to 40 ° C, preferably from 20 to 25 ° C below the temperature at which 50% of the paired strands separate (Tm) . In general, it is considered that conditions of high stringency are met when Th is less than Tm by 5 to 25 ° C approximately, for example by 5 to 10 ° C or more often by 20 to 25 ° C approximately. A moderate stringency is established when Th is less than Tm from 30 to 40 ° C. For sequences with more than 30 bases, the temperature

Tm is defined by the relationship: Tm = 81.5 + 0.41 (% G + C) + 16.6 Log (cation concentration) - 0.63 (% formamide) - (600 / number of bases). Thus, the ionic strength has a major impact on the value of Tm. The temperature Tm increases by 16.6 ° C each time the concentration of monovalent cation increases by a factor 10. The addition of formamide in the buffer hybridization, on the other hand, lowers the value of Tm. (For a complete reference see Sambrook et al, Molecular Cloning, A laboratory manual, Cold Spring Harbor laboratory Press, 1989, pages 9.54-9.62).

Conventionally, the hybridization experiments are carried out at a temperature of 60 to 68 ° C, for example at 65 ° C. At this temperature, stringent hybridization conditions can for example be implemented in 6xSSC, advantageously in 2xSSC or 1xSSC, preferably in 0.5xSSC, 0.3xSSC or 0.1xSSC (in the absence of formamide). A 1xSSC solution contains 0.15 M NaCI and 0.015 sodium citrate.

This is why, in other words, the subject of the invention is a polynucleotide in isolated form, which is capable of hybridizing under stringent conditions with a DNA molecule having one of the nucleotide sequences as shown in the SEQ ID N ° 1 - 51 (odd numbers) or their complement.

A particular class of homologous sequences is made up of those found in nature by virtue of the phenomenon extremely common allelic variation. A bacterial species, for example, N. meningitidis or N. gonorrhoeae, consists of a wide variety of strains which differ from each other by minor variations, called allelic variations. Thus, a polypeptide which is present in different strains and which obviously fulfills in each of them, the same biological function, may have an amino acid sequence which is not identical from one strain to another. In other words, the sequences resulting from the allelic variation are purely equivalent or alternative sequences to those of group II. The class of allelic variant sequences of one of the sequences of group II consists of the sequences of the polypeptide as it is found in a pathogenic species of the genus Neisseria (for example N. meningitidis or of N. gonorrhoeae) other than the strain of N. meningitidis ATCC 13090. The biological function which is associated with the allelic variant sequences is the same as that which is associated with the reference sequence. The differences (substitution, deletion or addition of one or more amino acids) that they present between themselves (including the reference sequence) do not alter the biological function of the polypeptide. By "biological function" is meant the function exerted by the polypeptide in the cells which produce it naturally. The allelic variation is also expressed at the level of the coding sequences. A polynucleotide encoding a polypeptide having a sequence which is an allelic variant of one of the group I sequences can be easily cloned by amplification of the genomic DNA of the strains of pathogenic species of the genus Neisseria, for example by PCR (reaction in polymerase chain), using synthetic oligonucleotide primers capable of hybridizing at the 5 'and 3' ends of the coding region. The sequences of such primers can easily be established by a person skilled in the art from the nucleotide sequences given in SEQ ID Nos. 1 - 51 (odd numbers). Primers generally have 10 to 40 nucleotides, preferably 15 to 25 nucleotides.

This is why, in other words, the subject of the invention is a DNA molecule in isolated form which can be amplified and / or cloned by PCR. from the genome of a pathogenic Neisseria strain using a pair of 5 'and 3' PCR primers; the sequences of these primers being established from one of the nucleotide sequences as shown in SEQ ID Nos. 1 - 51 (odd numbers). An example is given for each pair of primers in Example 1.1 below.

A more particular subject of the present invention is the allelic variants having the nucleotide sequences SEQ ID No. 54 to 76 (even numbers) and the products coded by these nucleotide sequences, having the amino acid sequences SEQ ID No. 55 to 77 ( odd numbers). The polypeptides of the invention can be fused to other polypeptides, for example by translation of a hybrid gene. Vectors for the expression of fusion polypeptides are commercially available, such as the vectors pMal-c2 or pMal-p2 from New England Biolabs, in which the protein to which the polypeptides of the invention can be fused is a protein of binding to maltose, or the glutathione-S-transferase system from Pharmacia, or the His-Tag system from Novagen. Such systems are in particular useful for purification of the polypeptides of the invention. The polypeptides of the invention can be fused to polypeptides exhibiting adjuvant activity, such as, for example, the B subunit of cholera toxin or of the heat-sensitive toxin of E. coli.

The nucleic acids of the present invention can be used (i) in a process for the production of polypeptides encoded by said nucleic acids in a recombinant host system, (ii) for the construction of vaccine vectors such as poxviruses, intended for use in methods and compositions for preventing and / or treating infection with pathogenic strains of Neisseria, in particular by Neisseria meningitidis, (iii) as a vaccine agent in naked form or in combination with a vehicle promoting transfer to cells target and, (iv) in the construction of attenuated Neisseria strains which can overexpress a nucleic acid of the invention or express it in a non-toxic, mutated form.

The present invention also provides (i) an expression cassette containing a polynucleotide of the invention placed under the control of elements allowing its expression, in particular under the control of a suitable promoter; (ii) an expression vector containing said expression cassette; (iii) a host cell (prokaryotic or eukaryotic) transformed with an expression cassette and / or an expression vector as defined above, as well as (iv) a method for obtaining a polypeptide coded by said polynucleotide of l the invention comprising culturing said transformed cell under conditions allowing expression of the polynucleotide of the invention, and recovering the polypeptide from cell culture.

Among the eukaryotic hosts which can be used, mention may in particular be made of yeast cells (for example, Saccharomyces cerevisiae or Pichia Pastoris), mammalian cells (for example, COS1, NIH3T3, or JEG3), arthropod cells (for example example, Spodoptera frugiperda (SF9)), and plant cells. Among the prokaryotic hosts which can be used, mention may in particular be made of E. coli.

The choice of the expression cassette depends on the host system chosen as well as on the characteristics desired for the expressed polypeptide. Generally, the expression cassettes include a promoter which is functional in the selected host system and which may be constitutive or inducible; a ribosome binding site; an initiation codon (ATG); if necessary a region coding for a signal peptide; a nucleotide sequence of the invention; a stop codon; optionally a 3 ′ terminal region (translation and / or transcription terminator). The open reading frame ("open reading frame ORF") constituted by the nucleotide sequence of the invention, alone or associated with the region coding for the signal peptide, is placed under the control of the promoter so that the translation and the transcription takes place in the host system. The promoters and the regions coding for the signal peptides are known to a person skilled in the art. Among these, there may be mentioned in particular the promoter of Salmonella typhimurium inducible by arabinosis (araB promoter) and which is functional in Gram ^" bacteria such as E. coli (US 5,028,530 and Cagnon et al., Protein Engineering (1991) ) 4 (7): 843), the promoter of the bacteriophage T7 gene coding for RNA polymerase (US 4,952,496); the signal peptide OspA and RIpB (Takase et al, J. Bact. (1987) 169: 5692) . The expressed polypeptide can be collected in substantially purified form from the cell extract or supernatant after centrifugation of the culture of recombinant cells. The recombinant polypeptide can in particular be purified by affinity purification methods using antibodies or by any other method known to those skilled in the art, such as by genetic fusion with a small binding domain.

The nucleic acids of the invention can also be useful in the field of vaccination, either by using a viral or bacterial host as a DNA delivery vehicle, or by administering the nucleic acid of interest in a free form.

Another subject of the present invention is (i) a vaccine vector containing a nucleic acid of the invention, placed under the control of elements allowing its expression; (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of said vaccine vector; (iii) a method for inducing an immune response against Neisseria in a vertebrate, in particular a mammal, preferably a human, said method comprising the administration to said vertebrate of an immunologically effective amount of said vaccine vector to cause an immune response, in in particular a protective or therapeutic response to Neisseria meningitidis; (iv) a method for preventing and / or treating infection with pathogenic strains of Neisseria, in particular by Neisseria meningitidis, which comprises the administration of a prophylactic or therapeutic amount of said vaccine vector of the invention to an individual requiring a such treatment. In combination with the polypeptides of the invention, the vaccine vector as defined above can also comprise nucleotide sequences whose expression allows the stimulation of the immune response, such as the sequences coding for cytokines.

Said vaccine vector of the invention can be administered by any conventional route in the field of vaccination, in particular by the parenteral route (for example, subcutaneous, intra-dermal, intramuscular, intravenous or intra- peritoneal). The dosage depends on many parameters known to those skilled in the art, such as the vector itself. even, the route of administration, the weight, age or sex of the animal or man to be vaccinated.

A subject of the present invention is also (i) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (ii) a method for inducing an immune response against pathogenic strains of Neisseria, in particular by Neisseria meningitidis in a vertebrate by administration to said vertebrate of an immunologically effective amount of said polynucleotide to elicit an immune response, in particular a protective immune response to pathogenic strains of Neisseria, in particular by Neisseria meningitidis; and (iii) a method of preventing and treating infection with pathogenic strains of Neisseria, in particular by Neisseria meningitidis, by administration of a therapeutic or prophylactic amount of said polynucleotide to an individual in need of such treatment. The polynucleotides of the invention (DNA or RNA) can be administered as such to a vertebrate. When a DNA molecule of the invention is used, it can be in the form of a plasmid incapable of replicating in a vertebrate cell and incapable of integrating the genome of said vertebrate. Said DNA molecule is typically placed under the control of a promoter suitable for expression in a vertebrate cell. Said polynucleotide used as a vaccine can be formulated according to various methods known to those skilled in the art. Said polynucleotide can in particular be used in a naked form, free of any vehicle promoting transfer to the target cell, such as anionic liposomes, cationic lipids, microparticles, for example gold microparticles, precipitating agents, for example calcium phosphate, or any other agent which facilitates transfection. In this case, the polynucleotide can be simply diluted in a physiologically acceptable solution, such as a sterile solution or a sterile buffer solution, in the presence or absence of a vehicle. When present, this vehicle may preferably be isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength. It can for example be a sucrose solution (for example a solution containing 20% sucrose). Alternatively, a polynucleotide of the invention can be combined with agents which facilitate transfection. It can be, inter alia, (i) associated with a chemical agent which modifies cellular permeability such as bupivacaine (WO 94/16737); (ii) encapsulated in liposomes, optionally in the presence of additional substances which facilitate transfection (WO 93/18759, WO 93/19768, WO 94/25608 and WO 95/2397, WO 93/18759 and WO 93/19768); or (iii) associated with cationic lipids or microparticles of silica, gold or tungsten.

When the polynucleotides of the invention cover microparticles, these can be injected intradermally or intraepidermally by the gene gun technique, "gene gun" (US 4,945,050, U.S. No. 5,015,580 and WO 94/24263).

The amount of DNA to be used as a vaccine depends in particular on the strength of the promoter used in the construction of the DNA, the immunogenicity of the product expressed, the individual to whom this DNA is administered, the mode of administration and the type of formulation. Generally, a therapeutically or prophylactically effective amount ranging from about 1 μg to about 1 mg, preferably from about 10 μg to about 800 μg and, preferably from about 25 μg to about 250 μg, can be administered to human adults.

The polynucleotide of the invention can be administered by any conventional route of administration, such as in particular parenterally. The choice of route of administration depends in particular on the formulation chosen. A polynucleotide formulated in association with bupivacaine is advantageously administered to the muscle. When neutral or anionic liposomes or a cationic lipid such as DOTMA (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride) or DC-Chol (3 beta- (N- (N ', N'- dimethyl aminomethane) -carbamoyl) cholesterol) are used, the formulation can be advantageously injected intravenously, intramuscularly, intradermally or subcutaneously. A polynucleotide in naked form can advantageously be administered intramuscularly, intradermally or subcutaneously. The nucleotide sequences of the invention allow the construction of specific nucleotide probes and primers useful in diagnosis. Said probes or primers are nucleic acids having sequences identical or homologous to portions of the sequences of group I or to their complementary sequences.

Preferably, said probes contain from approximately 5 to approximately 100, preferably from approximately 10 to approximately 80 nucleotides. They can contain modified bases, the sugar and phosphate residues can also be modified or substituted. The probes of the invention can be used in diagnostic tests, to capture or detect polynucleotides specific for the pathogenic strains of Neisseria. Such capture probes can be conventionally immobilized on a solid support directly or indirectly, by covalent bond or by passive adsorption. A detection probe can be labeled in particular with a radioactive isotope, an enzyme such as peroxidase or alkaline phosphatase, or enzymes capable of hydrolyzing a chromogenic, fluorogenic or luminescent substrate, or also by compounds which are themselves homogeneous, fluorogenic. or luminescent, nucleotide analogs; or biotin. A primer generally contains from about 10 to about 40 nucleotides, and can be used to initiate enzymatic polymerization of DNA in an amplification process (e.g. PCR), in an elongation process or in a method of reverse transcription. A primer of the invention can in particular be a primer as described in Example 11.1 below.

A subject of the present invention is also: (i) a reagent containing a probe of the invention for the detection and / or identification of the presence of the pathogenic strains of Neisseria in a biological sample;

(ii) a method for detecting and / or identifying the presence of pathogenic strains of Neisseria in a biological sample; said method comprising the steps of a) extracting DNA or RNA from a biological sample and denature it; b) exposing said DNA or said RNA to a probe of the invention, under stringent hybridization conditions, so as to detect hybridization; and

(iii) a method for detecting and / or identifying pathogenic strains of Neisseria in a biological sample in which the DNA is extracted from a biological sample and is placed in the presence of at least one and preferably two primers of the invention and is amplified for example by PCR.

As mentioned previously, the polypeptides produced by the expression of the identified ORF sequences are useful as vaccine agents. The specific antigenicity of polypeptides homologous to group II sequence polypeptides can be assessed by testing cross-reactivity with an antiserum directed against group II sequence polypeptides. A monospecific hyperimmune antiserum can be produced against a purified group II sequence polypeptide or a fusion polypeptide, for example an expression product of the MBP, GST, or His-tag systems.

The specific antigenicity can be determined using various methods known to those skilled in the art, in particular the Western-Blot, Dot-Blot, and ELISA techniques, described below.

In the Western-Blot technique, the protein preparation to be tested is subjected to SDS-PAGE gel electrophoresis. After transfer to a nitrocellulose membrane, the material is incubated with a monospecific hyperimmune antiserum obtained after immunizing an animal with the reference material; that is, in this case, with a polypeptide having a group II amino acid sequence. This antiserum is first diluted in a dilution range ranging from approximately 1:50 to 1: 5,000, preferably from approximately 1: 100 to 1: 500. The specific antigenicity is revealed when a band corresponding to the product exhibits reactivity to one of the above dilutions.

In the ELISA test, preferably a purified protein preparation is used, although a whole cell extract can also be used. Approximately 100 μl of a preparation at approximately 10 μg / ml are distributed in the wells of a plate. The plate is incubated for two hours at 37 ° C and then overnight at 4 ° C. The plate is then washed with a saline solution of phosphate buffer (PBS) comprising 0.05% of Tween 20. The wells are saturated with 250 μl of PBS containing 1% of bovine serum albumin (BSA) to prevent non-binding specific antibody. After one hour of incubation at 37 ° C, the plate is washed with PBS / Tween buffer. The antiserum is diluted in series in PBS / Tween buffer containing 0.5% BSA. 100 μl of this dilution are added per well. The plate is incubated for 90 minutes at 37 ° C, washed and evaluated according to standard procedures. For example, when specific antibodies are produced in rabbits, a goat anti-rabbit peroxidase conjugate is added to the well. The incubation is carried out for 90 minutes at 37 ° C. and the plate is then washed. The reaction is measured by colorimetry (a reaction is positive when the optical density value is 1 if the dilution is at least 1:50, preferably at least 1: 500).

In the Dot-Blot test, a purified protein is preferably used, it being understood that it is also possible to use a whole cell extract. A protein solution at approximately 100 μg / ml is diluted twice in series in a 50 mM Tris-HCl buffer, pH: 7.5. 100 μl of each dilution are applied to a nitrocellulose membrane (BioRad device). The buffer is removed by applying vacuum to the system. The wells are washed by adding 50 mM Tris-HCl buffer (pH: 7.5) and the membrane is air dried. The membrane is then saturated in a blocking buffer (50 mM Tris-HCl (pH: 7.5) 0.15 M NaCl, 10 g / l of skimmed milk) and incubated with a dilution of antiserum ranging from approximately 1: 50 at 1: 5000, preferably at about 1: 500. The reaction is revealed according to standard procedures. For example, when specific antibodies are produced in rabbits, a goat anti-rabbit peroxidase conjugate is added to the wells. Incubation is carried out for 90 minutes at 37 ° C. The reaction is developed with the appropriate substrate and measured, for example by colorimetry, by the appearance of a colored spot (a reaction is positive when a colored spot appears in association with a dilution of at least 1:50, preferably at least 1: 500). A subject of the present invention is also (i) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (ii) a method for inducing an immune response against the pathogenic strains of Neisseria in a vertebrate, by administration to said vertebrate of an immunogenically effective amount of a polypeptide of the invention to provoke an immune response, in particular a protective immune response against pathogenic strains of Neisseria; and (iii) a method of preventing and / or treating an infection with pathogenic strains of Neisseria, by administration of a therapeutic or prophylactic amount of a polypeptide of the invention to an individual in need of such treatment.

The immunogenic compositions of the invention can be administered by any conventional route in the field of vaccination, in particular by the parenteral route (for example, subcutaneous, intra-dermal, intramuscular, intravenous or intra- peritoneal). The choice of the route of administration depends on a certain number of parameters such as the adjuvant associated with the polypeptide.

A composition of the invention contains at least one polypeptide as defined above. It may also contain at least one additional Neisseria meningitidis and / or Neisseria gonorrhoeae antigen.

The polypeptides of the invention can be formulated with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, "virus-like" particles to facilitate the transfer of the polypeptide and / or increase the immune response. The administration can be carried out by a single dose or by repeated doses if necessary at intervals which can be determined by a person skilled in the art.

For example, an initial dose may be followed by three booster doses at intervals of one or more weeks or one or more months. The appropriate dose depends on many parameters including the individual treated (adult or child), the particular vaccine antigen, the route of administration and the frequency of administration, the presence or absence or even the type of adjuvant, and the desired effect (e.g. protection and / or treatment) and can be determined by those skilled in the art. If the route of administration is parenteral, the dose is preferably less than 1 mg, preferably approximately 100 μg. The polypeptides and polynucleotides of the invention used as vaccine agents can be used sequentially, in a multistage immunization method. For example, a vertebrate can be initially sensitized with a vaccine vector of the invention, such as a poxvirus, for example by the parenteral route, and then be stimulated twice with the polypeptide encoded by the vaccine vector.

A polypeptide of the invention can also be useful as a diagnostic agent for detecting the presence of anti-Λ / e / sseπa meningitidis and / or anti-Λ / e / sser / a gonorrhoeae antibodies in a biological sample such as a blood sample.

The present invention also relates to monospecific antibodies directed against the polypeptides of the invention. By "monospecific antibody" is meant an antibody capable of reacting in a specific manner with a Neisseria polypeptide of the invention. Such antibodies can be polyclonal or monoclonal, and can be recombinant antibodies, for example chimeric (for example, constituted by a variable region of murine origin associated with a constant region of human origin), humanized and / or single chain. Said antibodies can also be in the form of immunoglobulin fragments, for example fragments F (ab) '2 or Fab. The antibodies of the invention can be of any isotype, for example IgA or IgG, the polyclonal antibodies can be of a single isotype or can contain a mixture of several isotypes.

The antibodies of the invention directed against the polypeptides of the invention can be produced and identified using standard immunological methods, for example Western-Blot analysis, a Dot-Blot assay, an ELISA test (Coligan et al , Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, NY). Said antibodies can be used in diagnostic methods for detecting the presence of a Neisseria meningitidis antigen in a sample such as in particular a biological sample (for example blood). The antibodies of the invention can also be used in affinity chromatography methods to purify a polypeptide of the invention. Finally, such antibodies can also be used in methods of passive prophylactic or therapeutic immunization. The present invention also relates to a diagnostic method for detecting the presence of pathogenic strains of Neisseria in a biological sample comprising bringing said biological sample into contact with an antibody, or a polypeptide of the invention, so that an immune complex is formed, and the detection of said complex indicative of pathogenic strains of Neisseria in the organism from which the sample originates. Those skilled in the art understand that the immune complex is formed between a component of the sample and the antibody or polypeptide of the invention, any unbound substance being able to be eliminated before detection of the complex. Thus, a polypeptide type reagent can be used for detecting the presence of anti-Λ / e / sser / a meningitidis and / or Neisseria gonorrhoeae antibodies in a sample, while an antibody of the invention can be used as a reagent for testing the presence of Neisseria meningitidis and / or Neisseria gonorrhoeae polypeptide in a sample. For use in diagnostic applications, the reagent

(for example the antibody or the polypeptide of the invention) can be in the free state or immobilized on a solid support, by direct or indirect means.

Direct means include passive adsorption or covalent bonding between the support and the reagent. By indirect means is meant that a substance which interacts with said reagent is attached to the solid support. For example, if a polypeptide type reagent is used, an antibody which binds to it can serve as an anti-reagent, it being understood that this binds to an antibody which is not involved in the recognition of antibodies in biological samples.

Among the indirect means which can be used, mention may also be made of the receptor ligand system, a molecule such as a vitamin which can be grafted onto the polypeptide type reagent and the receptor corresponding can be immobilized on the solid phase. This is illustrated by the biotin-streptavidin system. It is also possible to add a peptide tail to the reagent by chemical engineering or genetic engineering, and to immobilize the grafted or fused product by passive adsorption or covalent bond with the peptide tail.

The present invention also relates to a process for the purification, from a biological sample, of a Neisseria polypeptide of the invention, by affinity chromatography with a monospecific antibody of the invention. Said antibody is preferably of the IgG isotype. According to an exemplary embodiment, a biological sample, preferably in a buffer solution, is applied to chromatographic material, preferably equilibrated with the buffer used to dilute the biological sample, so that the polypeptide of the invention (c (i.e. antigen) can adsorb on the material. The unbound components are washed and the antigen is then eluted with an appropriate elution buffer, such as a glycine buffer or a buffer containing a chaotropic agent, for example guanidine HCI, or a high salt concentration (for example , 3M MgCl2). The eluted fractions are collected and the presence of antigen is detected, for example by measuring the absorbance at 280 nm. A subject of the present invention is also (i) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a monospecific antibody of the invention; and (ii) a method of preventing and / or treating an infection with pathogenic strains of Neisseria, by administration of a therapeutic or prophylactic amount of a monospecific antibody of the invention to an individual in need of such treatment.

To this end, the monospecific antibody of the invention is preferably of the IgG isotype, and preferably fixes the complement. Said monospecific antibody according to the invention can be administered alone or in admixture with at least one other monospecific antibody, specific for a different Neisseria meningitidis and / or Neisseria gonorrhoeae polypeptide, according to the invention. The amount of antibody can be readily determined by those skilled in the art. For example, daily administration of approximately 100 to 1000 mg of antibody over a week or three daily doses of about 100 to 1000 mg of antibody over two or three days may be an effective dosage.

Therapeutic or prophylactic efficacy can be evaluated using standard methods known to those skilled in the art, for example by measuring the induction of an immune response or the induction of protective and / or therapeutic immunity (in mice or newborn rats), by evaluation of the bacterial load in the cerebrospinal fluid). Protection can be determined by comparing the degree of Neisseria infection to a control group. Protection is highlighted when the infection is reduced compared to the control group. Such an evaluation can be made with the polynucleotides, the vaccine vectors, the polypeptides as well as the antibodies according to the invention. The therapeutic or prophylactic efficacy of a product according to the invention (polynucleotide or polypeptide) can also be evaluated in a bactericidal test as described by Danve et al., Vaccine (1993) 11 (12): 1214, against of the original meningococcal strain of the polynucleotide or polypeptide used. In the field of meningococcal vaccines, the bactericidal test is indeed recognized as being the reference test from which one can validly predict the vaccine interest of a product. In short, a product according to the invention is administered to animals such as the rabbit in order to obtain an antiserum against this product. Then this antiserum is tested for its lysis capacity. The bactericidal titer of an antiserum represents the reverse of the dilution of this antiserum for which 50% of the meningococcal load is lysed. The antiserum is considered to be bactericidal when the titer is greater than 4 with respect to the meningococcal strain of origin of the polynucleotide or polypeptide used. In this case, it is shown that the product against which the antiserum was generated, is potentially interesting from a pharmaceutical point of view.

The following examples illustrate the invention without limiting its scope. Figure legend

The appended figure represents the vector pCAMyc-His used as a cloning vector.

Details of the ORF identification strategy:

In order to select the ORF sequences specific for pathogenic strains of the genus Neisseria, an amplification by PCR is carried out on the sequences of the 1,18 ORFs retained after analysis by the Gene Jocke software, Codon Use, and the search for homologies. Only the sequences for which the amplification in N. lactamica is negative (sequences called "lactamica ^" ") are retained. In order to verify that these negative results are not" false negatives ", the lactamica sequences ^" retained are subjected to a dot blot.

A - PCR amplification:

A.1. DNA DNA extraction:

The genomic DNAs of all the Neisseria strains used in this study were prepared according to an identical protocol. The strains of N. meningitidis, N. lactamica, N. flava, N. subflava and N. mucosa were cultivated on a box of MHA medium (Muller Hinton Agar, Difco), N. gonorrhoeae were cultivated on a box of MHA medium supplemented with 10% cooked horse blood (Biomérieux) and 1% Isovitalex (Biomérieux). The culture is carried out at 37 ° C. for 18 h under an atmosphere containing 10% CO 2 overnight at 37 ° C. The cells are then harvested, washed in PBS phosphate buffer (pH 7.2) and the DNA is extracted according to protocol D of the "Rapid Prep genomic DNA isolation kit for cells and tissue" kit (Pharmacia Biotech).

The genomic DNAs were then checked on agarose gel for their integrity and by PCR reaction for their purity.

A.2. PCR reaction to screen for ORFs absent in N. lactamica 2314: PCR amplification was carried out on the genomic DNAs of the strain N. meningitidis ATCC 13090 and N. lactamica 2314 (ATCC 23970) according to the following protocol:

The PCR reaction was carried out on a volume of 50 μl with 10 ng of genomic DNA, 250 μM of each of the dNTPs, 300 nM of each of the primers, Buffer Taq DNA polymerase 1X, and 2 u of Taq DNA Polymerase (Appligen ).

The amplification cycles are:

97 ° C 45 seconds 25 cycles

56 ° C 1 minute 25 cycles

72 ° C 2.30 minutes 25 cycles

For each of the ORFs analyzed, positive and negative controls of the PCR reaction were carried out. At this stage, only the ORFs N. meningitidis + and N. lactamica - are retained.

B - Selection of N. meningitidis N. lactamica ^' ORFs by dot blot on genomic DNA:

The absence of a PCR amplification product of an ORF with genomic DNA from N. lactamica 2314 as a template does not certify the absence of this ORF in the genome of N. lactamica 2314. Indeed , a certain variability in the region where the oligonucleotides should hybridize may be responsible for the absence of amplified product for a given ORF.

In this context, an additional verification is implemented by dot blot on genomic DNA using as a probe the genomic amplification products on the strain of N. meningitidis corresponding to each of the identified reading phases. The dot blot filters contain genomic DNA from the following strains: 2 strains of N. lactamica 8064, 2314, a strain of N. flava ATCC 30008, a strain of N. mucosa ATCC 9297, 3 strains of N. meningitidis from serogroup B ATCC13090, M982, B16B6, a strain of N. meningitidis from serogroup A Z2491, a strain of N. meningitidis from serogroup C (strain Z4182), 2 strains of N. gonorrhoeae MS11 and FA1090. This dot blot analysis validates the absence of the ORF in N. lactamica 2314 and 8064 and it is also indicative of the degree of variability of an ORF within the Neisseria strains. The dot blot technique used is as follows. About 50 ng of denatured genomic DNA 5 min at 100 ° C of the different Neisseria strains are deposited under vacuum on a Hybond N + nitrocellulose membrane (Amersham) placed between the jaws of a dot blot device (Bio-Rad). Then the DNA is fixed on the membranes for 5 min at 315nm UV radiation. The membranes are incubated in prehybridization buffer (containing denatured salmon sperm DNA). They are then hybridized with a probe corresponding to the amplification product of the ORF of interest, labeled according to a cold labeling protocol, like the 'DIG DNA labeling and detection kit' system (Boehringer Mannheim). The ORF which does not hybridize on the genomic DNA of N. lactamica

2314 and 8064 is definitively selected as a potential vaccine candidate.

Example I: Cloning

1. PCR amplification

Each of the ORFs was amplified by PCR from the genomic DNA of N. meningitidis serogroup B (strain ATCC 13090), according to a standard protocol.

Two oligonucleotides, primers on the N-terminal side and on the C-Terminal side were defined for each of the ORF sequences of the invention.

The N-Terminal primer includes an enzyme restriction site for cloning, a Kozak CCACC sequence for translation initiation (M. Kozak, J. Mol. Biol. 196: 947-950), ATG of the potential ORF and approximately 17 specific bases of the 5 'part of the ORF.

The primer on the C-Terminal side was defined so that the cloned ORF is in fusion in its 3 ′ part with a repetition of 8 histidines and a stop codon present in the vector behind the multiple cloning site, hence the insertion of a base "A" to keep the correct reading phase after cloning and the disappearance of the stop codon of the ORF. The primer on the C-Terminal side thus comprises an enzymatic restriction site for cloning, an "A" base, then approximately 20 bases specific for the 3 ′ part of the gene starting from the codon preceding the stop codon.

After searching for restriction sites absent in each of the ORFs using the MapDraw subroutine of DNASTAR (Lasergene Software), the restriction sites Xbal in 5 'and Bglll in 3' are used for the ORF SEQ ID n ° 19. For ORF SEQ ID No. 41, the 5 'Spel and 3' Bglll sites are used. The 5 'Xbal and 3' BamHl restriction sites are used to clone the remaining ORFs.

The PCR mixture comprises, for a final volume of 100 μl, 10-50 ng genomic DNA, the primers N-Terminal and C-Terminal at 200 nM each, the dNTPs at 250 μM each, the PCR buffer 1 X (Composition of the buffer PCR 10X: 200 mM Tris-HCI (pH8.8), 20 mM MgS04, 100 mM KCI, 100 mM (NH4) 2SO4, 1% TritonX-100, 1 mg / ml of nuclease-free beef serum albumin) and 2, 5 U of Polymerase.

The amplification is done as follows:

Step Temperature (° C) Time (min.) Number of cycles

Denaturation 97 0.45 25

Hybridization see table 1 25

Elongation 72 1 / kb DNA 25

The primers used and the PCR conditions presented in the table below, in which "allelic variant N. g" means that an allelic variant is present in Neisseria gonorrhoeae and "allelic variant N. m A" means that a variant allelic is present in Neisseria meningitidis of serogroup A.

2 - Cloning, transformation and selection of recombinants

The cloning vector used is the vector pCA / Myc-His or pM1070 of 6.357 kb (see figure), derived from the plasmid pCDNA 3.1 (Invitrogen). PCA / Myc-His comprises in particular the CMV ie1 promoter (bases 249-902), the intron A of the CMV ie1 gene (Chapman et al., 1991 Nucleic Acids Research, 19, 3979-3986), a multiple site of cloning (bases 1792-1852) with the sites Pmll, EcoRV, Notl, Xbal, BamHI, Kpnl and Hindlll, a sequence coding for a polyhistidine and a stop codon (bases 1908-1928), a termination sequence 3'bgh ( bases 1853-2197) and the ampiciilin resistance gene for the selection of recombinant clones in E. coli.

After purification (GeneClean Bio101 Kit), the PCR amplification products are digested for 2 hours at 37 ° C with the appropriate enzymes (Xbal-BamHI, Xbal-Bglll, or Spel-Bglll) in a final reaction volume of 20 μl. The digestion products are then ligated with the vector pCA / Myc-His previously digested with Xbal and BamHI according to the protocol of "Rapid DNA Ligation Kit" (Boehringer Mannheim). 15 μl of the ligation is used to transform 100 μl of competent E. coli XLI-blue cells (Novagen). The cells are incubated for 30 minutes in ice, 30 seconds at 42 ° C and 2 minutes in ice. Then add 500 μl of LB medium without antibiotic and incubate for 1 hour at 37 ° C. Then 50 and 550 μl of the culture are spread on dishes of LB medium plus ampicillin (50 μg / ml final concentration) and incubated overnight at 37 ° C.

The next day 36 colonies are cultured in 2 ml of LB plus ampicillin (50 μg / mi) and incubated overnight at 37 ° C.

The following day, the plasmid DNA is extracted according to the Qiagen mini-prep protocol (Qiagen) and the recombinants are identified by enzymatic restriction followed by electrophoresis on agarose gel. The cloning junctions are then verified by sequencing. Example II: Evaluation of the protective activity of the ORFs of the invention

A. Preparation of DNA intended for immunization experiments:

A colony isolated from a recombinant clone is used to inoculate a preculture in LB + ampicillin medium and 5 ml of this preculture represents the inoculate of a culture of 2.5 liters in LB + ampicillin medium. The purification protocol for preparing the plasmid DNA is that described in the EndoFree Giga Kit (Qiagen). The purified DNA is eluted from the purification column with a 10 mM Tris-HCl buffer 1 mM EDTA pH 8 and stored at -20 ° C. Before the injection, the purified recombinant plasmid is diluted at a rate of 100 μg / ml with water (of quality for injection) and the NaCl concentration is brought to 150 mM.

B. Production of a specific polyclonal serum:

B. 1. Hyperimmunization in an animal model:

The animal model used is the mouse or the rabbit. The route of administration of the injected DNA is the intramuscular route or the intradermal route. The recombinant plasmids to be injected are optionally applied to beads if they are injected into animals with a gun gene device (BioRad). The immunization protocol follows a schedule of two injections 3 weeks apart.

B.2. Analysis of the bactericidal activity of the antibodies induced:

Ten days after the last injection, the animals are bled and will be analyzed with the bactericidal test according to the protocol of Danve et al., Vaccine (1993) 11 (12): 1214. Briefly, the sera are incubated at different dilutions (reason 2) in the presence of rabbit complement and of meningocococci cultivated in the presence or in the absence of a chelating agent of iron. The bactericidal titer of a serum represents the reverse of the dilution of this antiserum for which 50% of the bacteria are lysed.

It is considered that the antiserum is not bactericidal when its titer is less than 4 against the homologous strain.

When the bactericidal titer corresponds to a seroconversion of a factor 4 against the homologous strain, the bactericidal activity of the antiserum is analyzed with respect to the other strains of Neisseria to measure the extent of the cross-reactivity of l antiserum of interest.

Example III Production of Purified Recombinant Proteins

Recombinant protein production

at. Preparation of transformants:

The PCR product obtained is then digested at 37 ° C for two hours with restriction enzymes in 20 μl of reaction volume. The digestion product is ligated into a similarly cleaved plasmid pET28a (Novagen) which is dephosphorylated before ligation by treatment with intestinal alkaline phosphatase from calves. The fusion gene constructed in this way allows one step affinity purification of the resulting fusion protein due to the presence of histidine residues at the N-terminus of the fusion protein which are encoded by this vector. The ligation reaction (20 μl) is carried out at 14 ° C. overnight, before transformation of 100 μl of fresh competent cells E. coli XL1-blue (Novagen). The cells are incubated on ice for two hours and then subjected to a thermal shock at 42 ° C. for 30 seconds before being put back into ice for 90 seconds. The samples are then added to 1 ml of LB broth in the absence of selection and cultured at 37 ° C for two hours. The cells are then spread on LB agar medium supplemented with kanamycin (50 μg / ml of final concentration) at a 10x dilution, and are incubated overnight at 37 ° C. The next day, 50 colonies are subcultured on secondary dishes and are incubated at 37 ° C overnight.

b. Protein production: The stored transformants (10 μl) are spread on selection boxes and grown overnight at 37 ° C. A few cells are collected from the dish used as an inoculum for an overnight starter culture (3 ml) at 37 ° C. The following day, a sample (time T = 0) is collected and centrifuged at 14,000 rpm for 3 minutes. The starter culture is then used to inoculate an LB medium containing kanamycin (100 μg / ml) at a dilution of 1:50 (starting optical density DO600 = 0.05-0.1). The cells are cultured at an OD600 of 1.0, a sample is collected for an SDS-PAGE (pre-induction sample) and the remaining culture is induced with 1 mM IPTG. Cultures are grown for four hours and samples are taken every hour. The culture is centrifuged at 600 x g for 20 minutes at 4 ° C. The supernatant is discarded and the pellets are resuspended in 50 mM Tris-HCl (pH: 8.0), 2 mM EDTA, and recentrifuged. The supernatant is discarded and the cells are stored at -70 ° C.

2. Purification of proteins

The pellets obtained from a liter of culture prepared according to Example 1.4 above are dried and resuspended in 20 ml of 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM Imidazole, cooled in ice. Lysozyme is added at a concentration of 0.1 mg / ml and the suspension is homogenized using a high speed homogenizer (Turrax) then treated with a sonicator (Branson, Sonofier 450). Benzonase (Merck) is used at a final concentration of 1 U / ml to remove the DNA. The suspension is centrifuged at 40,000 xg for 20 minutes and the supernatant is filtered through a 0.45 μm membrane. The supernatant is loaded onto an IMAC column (12 ml of resin) which has been prepared by immobilizing Ni cations according to the manufacturer's recommendations (Pharmacia). The column is washed with 10 column volumes of 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 60 mM Imidazole. Recombinant protein is eluted with six volumes 20 mM Tris-HCI (pH: 7.9), 0.5 M NaCI, 500 mM Imidazole, 0.1% Zwittergent 3-14.

The elution profile is checked by measuring the absorbance of the fractions at an optical density of 280 nm. An aliquot fraction is analyzed on an SDS-PAGE gel and stained with Coomassie blue (Phast System - Pharmacia), and the fractions corresponding to the protein peak are then pooled and concentrated. To remove the elution buffer, the fraction is passed through a G24 Sephadex column (Pharmacia), equilibrated in PBS buffer (pH: 7.4). The protein solution is sterilized by filtration through a 0.45 μm membrane, and the protein concentration is determined by the BCA micromethod (Pierce). The protein solution is stored at -70 ° C.

Example IV Production of Monospecific Polyclonal Antibodies

1. Rabbit hyperimmune antiserum

New Zealand rabbits are injected both dermally and intravenously with 100 μg (in total) of the polypeptide purified in Example III, in the presence of complete Freund's adjuvant in a total volume of approximately 2 ml. . 21 and 42 days after the initial injection, the booster doses, which are identical to the initial doses, are administered in the same way, except for the fact that incomplete Freund's adjuvant is used. 15 days after the last injection, the animal's serum is collected, decomplemented, and filtered through a 0.45 μm membrane.

2. Liquid of hyperimmune ascites of mice

10-50 μg of the purified fusion polypeptide obtained in Example II are injected into 10 mice subcutaneously, in the presence of complete Freund's adjuvant, in a volume of approximately 200 μl. 7 and 14 days after the initial injection, booster doses, which are identical to the initial doses, are administered in the same way, except that incomplete Freund's adjuvant is used. 21 and 28 days after the initial injection, the mice receive 50 μg of the antigen alone intraperitoneally. On day 21, the mice are also injected intraperitoneally with sarcoma 180 / TG CM26684 cells 180 / TG (Lennette & Schmidt, Diagnostic procedures for viral, rickettsial, and chlamydial infections, (1979) 5th Ed. Washington DC, American Public Health Association ). The ascites fluids are collected 10 to 13 days after the first injection.

Example V: Purification of the polypeptides of the invention by immunoaffinity

1. Purification of specific IgG

An immune serum as prepared in Example IV is applied to a column of protein A Sepharose 4 Fast Flow (Pharmacia) balanced with 100 mM Tris-HCl (pH: 8.0). The resin is washed by applying 10 volumes of 100 mM Tris-HCl columns and 10 volumes of 10 mM Tris-HCl columns (pH: 8.0) to the column. The IgGs are eluted with a 0.1 M glycine buffer (pH: 3.0) and are collected in 5 ml fractions to which 0.25 ml of 1 M Tris-HCl (pH: 8.0) is added. The optical density of the eluate is measured at 280 nm and the fractions containing the IgG are pooled and if necessary stored at -70 ° C.

2. Preparation of the column

An appropriate amount of Sepharose 4B gel activated by CNBr (1 g of dried gel providing approximately 3.5 ml of hydrated gel, and gel capacity ranging from 5 to 10 mg of coupled IgG per ml of gel) manufactured by Pharmacia ( 17-0430-01) and suspended in 1 mM HCI buffer and washed with a buchner by adding small amounts of 1 mM HCI buffer. The total volume of the buffer is 200 ml per gram of gel. The purified IgGs are dialyzed for four hours at 20 ± 5 ° C against 5 volumes of 500 mM PBS buffer (pH: 7.5). Then they are diluted in 500 mM PBS (pH: 7.5) for a final concentration of 3 mg / ml.

The IgGs are incubated with the gel overnight at 5 ± 3 ° C, with shaking. The gel is packed in a chromatography column and washed with 2 column volumes of phosphate buffer, 500 mM (pH: 7.5) then a volume of 50 mM NaCl sodium buffer (pH: 7.5). The gel is then transferred to a tube then incubated with 100 mM ethanolamine, (pH: 7.5) for 4 hours at room temperature with stirring, then washed twice with two column volumes of PBS. The gel is then stored in 1/10 000 merthiolate PBS. The amount of IgG coupled to the gel is determined by measuring the optical density at 280 nm of the IgG solution and of the direct eluate.

3. Adsorption and elution of the antigen. An antigen solution in 50 mM Tris-HCl (pH: 8.0), 2 mM

EDTA, for example, the supernatant obtained in Example III.5 after treatment with Benzonase, centrifugation and filtration through a 0.45 μm membrane, is applied to a column balanced with 50 mM Tris-HCl (pH: 8 , 0), 2 mM EDTA at a flow rate of approximately 10 ml / hour. Then, the column is washed with 20 volumes of 50 mM Tris-HCl (pH: 8.0), 2 mM EDTA. Alternatively, the adsorption can be carried out in a "batch" which is left overnight at 5 ± 3 ° C., with stirring.

The gel is washed with 2 to 6 volumes of 10 mM PBS buffer (pH: 6.8). The antigen is eluted with a 100 mM glycine buffer (pH: 2.5). The eluate is collected in 3 ml fractions to which 150 μl of 1 mM PBS buffer (pH: 8.0) are added. The optical density is measured at 280 nm for each fraction; those containing the antigen are collected and stored at -20 ° C. Fragments of the genome of N. meningitidis Z2491 described in the patent application

WO98 / 02 547

(2) INFORMATION FOR SEQ ID NO: 70A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 243 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(l) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 70A:

GATCAGACCC ATTTTCAGCG CACCGTAAGC GCGGATTTTC TCGAATTTTT CCAAAGCTGC 60

GGCATCGTTG TTGATGTCGT CTTGCAACTC TTTGCCCGTG TAGCCCAAGT CGGCGGCATT 120

CAGGAAAACG GTCGGAATGC CCGCGTTGAT GAGCGTGGCT TTCAAACGGC CTATATTCGG 180

CACATCAATT TCATCGACCA AATTGCCGGT TGGGAACATA CTGCCTTCGC CGTCGGCTGG 240

ATC 243

(2) INFORMATION FOR SEQ ID NO: 73A:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 120 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(il) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO (iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 73A:

CGGTCAGAAA CAGGCAAGGT AATGAAAATG CCTGAGGCAC GGACTGTGCT GCGAACGAAA 60

ACTCCTTACC GAAGTCTTCT ATACCCAGGC TCAATAGCCG CTCAAGGAGA GAGCTATCAT 120

(2) INFORMATION FOR SEQ ID NO: 74A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 120 base pairs

(B) TYPE, nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 74A:

CGGTCAGAAA CAGGCAAGGT AATGAAAATG CCTGAGGCAC GGACTGTGCT GCGAACGAAA 60

ACTCCTTACC GAAGTCTTCT ATACCCAGGC TCAATAGCCG CTCAAGGAGA GAGCTATCAT 120

(2) INFORMATION FOR SEQ ID NO: 77A

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 269 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 77A:

CGGAGCATAA AATCGTTATT AAAGATAATG GTATAGGAAC GAGCTTCGAT GAAATCAATG 60

ATTTTTATTT GAGAATCGGT CGGAACAGAA GGGAAGAAAA ACAAGCCTCC CCGTGCGGAA 120

GAATTCCAAC GGGTAAAAAA GGCCTTGGTA AATTGGCATT ATTCGGGCTT GGCAACAAAA 180

TTGAAATTTC TACTATCCAG GGAAACGAAA GGGTTACTTT TACTTTGGAT TATGCAGAGA 240

TTCGAAGAAG CAAGGGTATT TATCAACCG 269

(2) INFORMATION FOR SEQ ID NO: 80A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 207 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

(iii) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 80A:

CGGGTCGCTT TATTTTGTGC AGGCATTATT TTTCATTTTT GGCTTGACAG TTTGGAAATA 60

TTGTGTATCG GGGGGGGGTA TTTGCTGACG TAAAAAACTA TAAACGCCGC GCAAAATATG 120

GCTGACTATA TTATTGACTT TGATTTTGTC CTGCGCGGTG ATGGATAAAA TCGCCAGCGA 180

TAAAGAATTT GCGAGAACCT GATGCCG 207

(2) INFORMATION FOR SEQ ID NO: 81A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 224 base pairs

(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(1 ₁ ) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 81A:

CGGCAACGAT TTGAGCTATC GCGGTTACGA CATTCTGGAT TTGGCACAAA AATGCGAGTT 60

TGAAGAAGTC GCCCACCTGC TGATTCACGG CCATCTGCCC AACAAATTCG AGCTGGCCGC 120

TTATAAAACC AAGCTCAAAT CCATGCGCGG CCTGCCTATC CGTGTGATTA AAGTTTTGGA 180

AAGCCTGCCT GCACATACCC ATCCGATGGA CGTAATGCGT ACCG 224

(2) INFORMATION FOR SEQ ID NO: 87A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 273 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(l) TYPE OF MOLECULE: DNA (genomics)

(ni) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(x) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 87A:

AATTTCCACC TATGCCCTAC GCAGCGATTA TCCGTGGTTT ACCCAAAGGG TGATTATGGC 60

AAAAGCGCGG GGTTGAGCGA CCGCCTTTTG TTGCCGGCGT TCAAACGGGT TTTGATAGGA 120

AATGCAGGCA CGAAGCCTCG GCTGATTGTG ATGCACCTGA TGGGTTCGCA CAGTGATTTT 180

TGCACACGTT TGGATAAGGA TGCGCGGCGG TTTCAGTATC AAACTGAAAA AATATCCTGC 240 TATGTTTCCA TCAATCGCGC AAACCGATAA ATT 273

(2) INFORMATION FOR SEQ ID NO: 88A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 270 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(ni) HYPOTHETIC- NO

(iv) ANTI-SENSE. NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 88A:

AATTCTTCCG CACGGGGAGG CTTGTTTTTC TTCCCTTCTG TTCCGACCGA TTCTCAAATA 60

AAAATCATTG ATTTCATCGA AGTTCATTCC TATACCATTA TCTTTAATAA CGATTTTATG 120

CTCCGGTTTA TCGAATAACC TAACTTCCAC TTCCGTAGCA CATGCATCGT AGGCATTCGC 180

TATCAACTCG GCAATCGCAG GAACAGTGTG CGAATACAAT CTTTACACCC AAATGTTCGA 240

TTACGGTTGG CTCGAAACTC AATTTCAATT 270

(2) INFORMATION FOR SEQ ID NO: 89A:

() CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 267 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 89A:

AATTATGAAC ACACGCATCA TCGTTTCGGC TGCGTTCGTT GCGTTGGCAT TAGCAGGTTG 60

CGGCTCAATC AATAATGTAA CCGTTTCCGA CCAGAAACTT CAGGAACGTG CCGCGTTTGC 120

CTTGGGCGTC ACCAATGCCG TAAAAATCAG CAACCGCAGC AATGAAGGCA TACGCATCAA 180

CTTTACCGCA ACTGTGGGTA AGCGCGTGAC CAATGCTATG TTACCAGTGT AATCAGCACA 240

ATCGGCGTTA CCACTTCCGA TGCAATT 267

(2) INFORMATION FOR SEQ ID NO: 94A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 308 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 94A:

AATTTGTTGG GCAGATGGCC GTGAATCAGC AGGTGGGCGA CTTCTTCAAA CTCGCATTTT 60

TGTGCCAAAT CCAGAATGTC GTAACCGCGA TACGTCAAAT CGTTGCCGGT ACGCAACGGT 120

ACACAAAGCG GTATTACCGG CCGCAACGCC AGAAAGCGCA ACGGATTTTT AGGTTTGAGG 180

GTCGGGGTTT GAGTAGTTTC AGTCATGGTA TTTCTCCTTT GTGTTTTTAT GGGTTTCGGG 240

TTTTCAGACG ACCGATGCGG ATTTGTTGAA AGGCAGTCTG AAAGCGGTAA ATCATTTTTG 300

AAACAATT 30Î

(2) INFORMATION FOR SEQ ID NO: 95A: (l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 286 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 95A:

AATTCGGAGG AGCAGTACCG CCAAGCGTTG CTCGCCTATT CCGGCGGTGA TAAAACAGAC 60

GAGGGTATCC GCCTGATGCA ACAGAGCGAT TACGGCAACT TGTCCTACCA CATCCGTAAT 120

AAAAACATGC TTTTCATTTT TTCGGCAAGC AATGACGCAC AAGCTCAGCC CAACACAACT 180

GACCCTATTG CCATTTTATG AAAAAGACGC TCAAAAAGGC ATTATCACAG TTGCAGGCGT 240

AGACCGCAGT GGAGAAAAGT TCAATGGCTC CAACCATTGC GGAATT 286

(2) INFORMATION FOR SEQ ID NO: 98A:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 316 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(m) HYPOTHETIC: NO

(iv) ANTI-SENSE: NO

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 98A

AATTTGTCGG CAATCTTCCC GGGTCGCTTT ATTTTGTGCA GGCATTATTT TTCATTTTTG 60 GCTTGACAGT TTGGAGATAT TGTGTATCGG GGGGGGGTAT TTGCTGACGT AAAAAACTAT 120

AAACGCCGCA GCAAAATATG GCTGACTATA TTATTGACTT TGATTTTGTC CTGCGCGGTG 180

ATGGATAAAA TCGCCAGCGA TAAAGATTTG CGAGAACCTG ATGCCGGCCT GTTGTTGAAT 240

ATTTTCGACC TGTAATTACG ATTTGGCTTC CGCGCCGGCA CAATATGCCG CCAAGCGGCG 300

CCCACATTTT GGAAGC 316

Claims

1. Nucleic acid in isolated form, encoding a polypeptide specific for pathogenic strains of the genus Neisseria, or antigenic fragment thereof, excluding the sequences SEQ ID No. 70A, 73A, 74A, 77A, 80A, 81 A, 87A, 88A, 89A, 94A, 95A and 98A, the amino acid sequence of said specific polypeptide being identical or homologous to a sequence chosen from the sequences of group II, the group II consisting of the sequences SEQ ID No. 2 to SEQ ID No. 52 (even numbers) and the sequence SEQ ID No. 53.

2. Nucleic acid according to claim 1, the nucleotide sequence of which is identical or homologous to a sequence chosen from the sequences of group I, the group I consisting of the sequences SEQ ID No. 1 to SEQ ID No. 51 (odd numbers ).

3. Nucleic acid according to claim 1 coding for a polypeptide specific for pathogenic strains of the genus Neisseria, or antigenic fragment thereof, the amino acid sequence of said specific polypeptide being chosen from sequences SEQ ID Nos. 55 to 77 ( odd numbers).

4. Nucleic acid according to claim 3, having a nucleotide sequence chosen from sequences SEQ ID No. 54 to 7 £ (even numbers).

5. Polypeptide specific for pathogenic strains of the genus Neisseria, and antigenic fragments thereof, the amino acid sequence of said specific polypeptide being identical or homologous to a sequence chosen from the sequences of group II, consisting of the sequences SEQ ID # 2 at SEQ ID # 52 (even numbers) and the sequence SEQ ID # 53.

6. The polypeptide of claim 5 specific pathogenic strains of the genus Neisseria, and antigenic fragments thereof, the amino acid sequence of said specific polypeptide being chosen from the sequences SEQ ID NO ⁵ 55-77 (numbers peers).

7. An expression vector comprising comprising an expression cassette in which a nucleotide sequence as defined in one of claims 1 to 4 is placed under conditions allowing its expression in a host cell.

8. Host cell transformed with the expression vector according to claim 7.

9. A pharmaceutical composition comprising: a) a nucleic acid according to one of claims 1 to 4, in naked form or in combination with at least one agent which facilitates transfection; b) or a vaccine vector comprising a nucleotide sequence as defined in one of claims 1 to 4, such as in particular a virus or a bacterium; c) or a polypeptide according to one of claims 5 or 6; optionally in combination with a pharmaceutically acceptable vehicle.

¹⁰ . Monospecific antibody directed against a polypeptide according to one of claims 5 or 6.

he . Use of a nucleic acid according to one of the claims

1 to 4 or of a specific polypeptide of the pathogenic strains of Neisseήa or of antigenic fragments thereof according to one of claims 5 or 6 for the manufacture of a pharmaceutical composition intended for vaccination against Neisseήa.