NO304188B1 - Nucleotide sequence encoding an outer membrane protein from Neisseria meningitis - Google Patents

Description

The present invention lies in the area of genetic manipulation and biotechnology. More particularly, the invention is characterized by a nucleotide sequence which codes for a protein P64k from Neisseria meningitidis where the P64k protein has the amino acid sequence indicated in SEQ ID NO:1. The nucleotide sequence codes for a protein that belongs to the outer membrane of said bacterium. This gene is cloned and expressed in the host organism Escherichia coli. The peculiarities of the protein formed from this gene as well as its capacity to induce immunologically active antibodies (bactericidal antibody) in its natural host organism allow its use in vaccine preparations against pathogenic strains of this microorganism.

The gram-negative bacterium N. meningitidis is responsible for one in three cases of bacterial meningitis in the world. It was first described by Anton Weichselbaum in 1887 (I. DeVoe, 1982, Microbiol. Revs. 46: 162-190), and humans are its only natural host until today.

In the first half of this century, certain essential aspects were found in connection with the metabolism and the serological differentiation of this microorganism. The first successful attempts to obtain vaccine preparations were based on its capsular polysaccharide (E. Kabat et al., 1945, J. Exp. Med. 80: 299-307). According to the chemical composition of this capsular polysaccharide, the bacterium N. meningitidis is serogrouped into A, B, C, 29-E, H, ■

I, K, L, W-135, X, Y or Z, and the major percentage of disorders are caused by A, C, Y, W-135 and B. Non-enveloped strains are not associated with the invasive disease.

By using different purification methods for these polysaccharides (E. Gotschlich et al., 1969, J. Exp. Med.

129: 1349-1365) the first four polysaccharides (PS) proved to be good immunogens and induce bactericidal antibodies in humans (E. Gotschlich et al., 1969, J. Exp.

With. 129: 1367-1384). The presence of this type of antibody has previously been correlated with non-susceptibility to the infection (I. Goldschneider et al., 1969, J. Exp. Med. 129: 1307-1326). Today, mono-, bi- or tetravalent vaccines have been well studied for serotypes A, C and W-135 (F. Ambrosch et al., 1983, Bulletin of the WHO 61: 317-323; I. Vodopija et al., 1983 , Infect. Immunol. 42: 599-604; M. Cadoz et al., 1985, Vaccine 3: 340-342; H. Peltola et al., 1985, Pediatrics 76: 91-96).

These vaccines have been prescribed for use in humans in various countries (Centers for Disease Control, 1985, Morbid. Mortal. Weekly Report 34: 255-259) and some of them are commercially available from various companies and manufacturers (Connaught Laboratories, USA; Smith Kline-RIT, Belgium; Institute merieux, France; Behringwerke Aktien-gesellschaft, Germany; Istituto Sieroterapico e Vaccino genea Toscano "Sclavo", Italy; Swiss Serum and Vaccine Institute, Berne, Switzerland; among others).

However, the usual vaccines against N. meningitidis serogroup C do not induce sufficient levels of bactericidal antibody in children under 2 years of age who are the main victims of this disease. It has been shown that the titer of specific antibody against N. meningitidis in children under four years of age, after three years of vaccination, is similar in vaccinated and in non-vaccinated children (H. Kayhty et al., 1980, J. of Infect. Dis. 142: 861-868). In addition, no memory response was found against N. meningitidis after 8 years of vaccination in young adults (N. Rautonen et al., 1986, J. of Immunol. 137: 2670-2675).

The polysaccharide corresponding to N. meningitidis serogroup B is weakly immunogenic (E. Gotschlich et al., 1969, J. Exp. Med. 129: 1349-1365) and induces a small response of IgM with little specificity (W. Zollinger et al., 1979 , J. Clin. Invest. 63: 836-848). There are various theories regarding this problem, such as cross-reactivity between B-polysaccharide and fetal brain structures, antigenic structures modified in solution, and sensitivity to neuroaminidases (C. Moreno et al., 1985, Infect. Immun. 47: 527-533). Recently, a chemical modification of PS B was achieved which induced a response in the host organism (H. Jennings et al., 1988, US patent 4,727,136; F. Ashton et al., 1989, Microb. Pathogen. 6: 455-458 ), but the safety of this vaccine in humans has not been demonstrated.

Due to the lack of an effective vaccine against N. meningitidis B, and because the risk of endemic infection is small and mainly limited to children, a routine immunization with polysaccharides is not recommended (C. Frasch, 1989, Clin. Microbiol. Revs. 2: S134-S138) except in the case of an epidemic.

Since after the Second World War, the disease was caused in most cases by N. meningitidis B, where vaccines against serogroup B gained special importance.

Other outer membrane components of a N. meningitidis include phospholipids, lipopolysaccharides (LPS or endotoxins), piliproteins, and others. Different immunotypes of LPS have been described for N. meningitidis (W. Zollinger and R. Mandrell, 1977, Infect. Immun. 18: 424-433; CM. Tsai et al., 1983, J. Bacteriol. 155: 498-504 ) and immunogenicity using non-toxic derivatives were attempted (H. Jennings et al., 1984, Infect. Immun. 43: 407-412), but their variability (H. Schneider et al., 1984, Infect. Immun. 45: 544-549) and pyrogenicity (when conjugated with lipid A) are currently limiting factors.

The pills, structures that are necessary to attach cells to the nasopharyngeal mucous membrane (D. Stephens et al., 1983, The J. Infect. Dis. 148: 369-376) have antigenic differences i. different strains (J. Greenblatt et al., 1988, Infect. Immun. 56: 2356-2362) with some common epitopes (D. Stephens et al., 1988, The J. Infect. Dis. 158: 332-342). At present, some doubts exist regarding the effectiveness of a vaccine based on these structures. However, some of these vaccine types have been obtained without known results in connection with their use in humans (C. Brinton, 1988, US patent 4,769,240).

Recently, attention has focused on other proteins in the outer membrane of this bacterium. There are many immunological types of these protein complexes.

The strains of N. meningitidis are divided into serotypes according to the presence of specific epitopes in the main protein P1/P2 and into subtypes according to other epitopes in protein Pl (C. Frasch et al., 1985, Rev. Infect. Dis. 7 : 504-510).

There are several published articles and patent applications regarding vaccines based on mixtures of these proteins, with previous selective removal of endotoxins using biocompatible detergents. The immunogenicity of these mixtures in animals and humans has been demonstrated (W. Zollinger et al., 1979, J. Clin. Invest. 63: 836-848; C. Frasch and M. Peppler, 1982, Infect. Immun. 37: 271 -280; E. Beuvery et al., 1983, Infect. Immun. 40: 369-380 E. Rosenqvist et al., 1983, NIPH Annals 6: 139-149; L. Wang and C. Frasch, 1984, Infect. Immun. 46: 408-414; C. Moreno et al., 1985, Infect. Immun. 47: 527-533; E. Wedege and L. Froholm, 1986, Infect. Immun. 51: 571-578; C. Frasch et al., 1988, The J. Infect. Dis. 158: 710-718; M. Lifely. and Z. Wang, 1988, Infect. Immun. 56: 3221-3227; J. Poolman et al., 1988, In J. Poolman et al (Eds), Gonococci and Meningococci, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 159-165; E. Rosenqvist et al., 1988, J. Clin. Microbiol. 26: 1543-1548). including results in massive field trials such as Capetown, South Africa in 1981 (C. Frasch, 1985, Eur. J. Clin. Microbiol. 4: 533-536); Iquique, Chile, 1987 (W. Zollinger, 1988, Proceedings of the Sixth International Pathogenic Neisseria Conference. Callaway Gardens Conference Center) and Cuba 1986 and 1988 (G. Sierra, 1988, Proceedings of the Sixth International Pathogenic Neisseria Conference. Callaway Gardens Conference Centre). However, with the exception of the last case, the bacterial antibodies induced by these preparations were limited to the same serotype strains or related ones.

One of these vaccines is referred to in US patent 4,601,903 which is limited to one of the Neisseria types that cause meningitis (serotype 2) with high frequency, but other serotypes have also been isolated with high frequency from patients, such as serotype 4 ( Cuba from 1981 to 1983, H. Abdillahi et al., 1988, Eur. J. Clin. Microbiol. Infect. Dis. 7: 293-296; Finland from 1976-1987, H. Kayhty et al., 1989, Scand. J. Infect. Dis. 21: 527-535); 8 (Australia from 1971 to 1980, F. Ashton et al., 1984, Can. J. Med. Biol. 30: 1289-1291) and 15 (Norway from 1982 to 1984; L. Froholm et al., 1985 Proceedings of the Fourth International Symposium on Pathogenic Neisseria. American Society for Microbiology; Chile from 1985 to 1987, S. Ruiz et al., 1988, Proceedings of the Sixth International Pathogenic Neisseria Conference. Callaway Gardens Conference Center) as well as strains of undefined serotype ( F. Ashton et al., 1980, Can. J. Microbiol. 26: 1480-1488; Australia from 1971 to 1980, F. Ashton et al., 1984, Can. J. Med. Biol. 30: 1289-1291; Finland from 1976 to 1987, H. Kayhty et al., 1989, Sean. J. Infect. Dis. 21: 527-535).

The Cuban vaccine obtained in 1988 at the Centro Nacional de Biopreparados (European Patent Application No. 301,992) has proven to be highly effective. It is based on an antigenic complex of high molecular weight. It has a wide range of cross-reactivity with other strains and forms and maintains bactericidal antibody in the immunized host organism.

However, the methods used to obtain this type of vaccine begin with the propagation in an appropriate culture of a microorganism that is highly pathogenic, with the associated biological risk of handling the bacterium directly. Furthermore, this type of lipopolysaccharide preparation contains a contaminant which, although it can increase the product's effectiveness, at the same time shows unwanted secondary effects due to its strong pyrogenicity. In addition, its variation in small antigenic components that form parts of the preparation cannot be controlled in the different portions, which makes it difficult to follow important parameters related to reactogenicity and immunogenicity.

For this reason, there is a growing interest in the identification and nucleotide sequence encoding highly conserved proteins in all strains and even more in the identification of bactericidal antibody-inducing proteins common to the majority of pathogenic Neisseria in order to obtain vaccine preparations with a broad spectrum of protection.

There are various high molecular weight proteins that are present in small amounts in the outer membrane of N. meningitidis when this microorganism is grown in normal culture media, but which have a strong response in affected individuals (J. Black et al., 1986, Infect. Immun. 54: 710-713; L. Aoun et al., 1988, Ann. Inst. Pasteur/ Microbiol. 139: 203-212) and/or increase in their response under special culture conditions (J. van Putten et al., 1987, Antoine van Leeuwenhoek 53: 557-5564;

A. Schryvers and L. Morris, 1988, Molecular Microbiol. 2: 281-288 and Infect. Immune. 56: 1144-1149). Some of these proteins are highly conserved among different strains, and especially those related to the acquisition of iron by the microorganism have become interesting vaccine candidates (L. Mocca et al., 1988, Proceedings of the Sixth International Pathogenic Neisseria Conference. Callaway Gardens Conference Center; C . Frasch, 1989, Clin. Microbiol. Revs. 2: Sl 34-S138).

In addition to pure proteins obtained from the microorganism or strains of related types (eg, 37 kD protein, T. Mietzner and S. Morse, 1987, US Patent 4,681,761), several related genes have been cloned and expressed. Among these proteins are the following: protease IgAl (J. Koomey and S. Falkow, 1984, Infect. Immun 43: 101-107); protein P1 (A. Barlow et al., 1987, Infect. Immun. 55: 2734-2740 and 1989, Molec. Microbiol. 3: 131-139); protein P5a (T. Kawula et al., 1988, Infect. Immun. 56:380-386); protein P5c (T. Olyhoek and M. Achtman, 1988, Proceedings of the Sixth International Pathogenic N. Converence. Callaway Gardens Conference Center); protein P4 (K. Klugman et al., 1989, Infect. Immun. 57: 2066-2071); protein P2 (K. Murakami et al., 1989, Infect. Immun. 57: 2318-2323); and from N. gonorrhoeae, which encode proteins cross-reactive with their corresponding proteins from N. meningitidis: antigen H.8 (W. Black and J.G. Cannon, 1985, Infect. Immun. 47: 322-325); macromolecular complex (W. Tsai and C. Wilde, 1988, Proceedings of the Sixth International Pathogenic Neisseria Conference. Callaway Gardens Conference Center); 37 kDa protein repressed in the presence of iron (S. Berish et al., 1988, Proceedings of the Sixth International Pathogenic Neisseria Conference. Callaway Gardens Conference Center).

The use of these proteins as active vaccine preparations has not been reported or the bactericidal tests of antibody induced against them were negative, as in the case of mouse monoclonal antibodies against H.8 (J. Woods et al., 1987, Infect. Immun. 55: 1927 -1928).

Up until today, the protein Pl located in the outer membrane of N. meningitidis is one of the best characterized and studied antigens. This protein shows no variability within the same strain. However, there are more than 17 types of protein P1 in Neisseria that have differences in three variable regions, this being the basis for the classification of N. into different subtypes. This protein is highly immunogenic in human mice (W.D. Zollinger and R.E. Mandrell, 1983, Med. Trop. 43-143-147), and elicits protective antibody (E. Wedege and L.O. Froholm, 1986, Infect. Immun. 51: 571-578 ; K. Saukkonen et al. 1987, Microb. Pathogen, 3:261-267), which gives it a special importance in vaccine preparations.

Certain subtypes of protein P1 have been cloned in E. coli starting from genomic libraries (A.K. Barlow et al., 1989, Molec. Microb. 3:131-139) or using the PCR technique (S. Butcer et al. Vllth International Congress of Neisseria, R. C. Seid, Patent Application WO 90/06696; Brian Mc Guinness et al., 1990, J. Exp. Med. 171:1871-1882, M. C. J. Maiden et al., Vllth International Conference of Neisseria, Berlin , Sept. 9-14, 1990 and 1991, Molec. Microb. 3:727; J. Suker et al., VIIth International Conference of Neisseria, Berlin, Sept. 9-14, 1990). However, until now there is no genetic construct capable of producing this protein with high expression levels. Only low expression levels

(D.A. white et al., 1990, Molec. Microb. 4:769:776) or its expression in Bacillus subtilis fused to outer membrane protein A of E. coli (omp A) (E. Wahlstrom et al., Vllth International Congress of Neisseria, September 9-14, 1990, Berlin) has been reported.

It can be confirmed that up to today no antigen has been isolated which is common to all types and serogroups of N. meningitidis and which is capable of producing bacterial antibodies. For this reason, an antigen of this type conjugated or fused to other proteins or polysaccharides of immunological interest would be relevant as a candidate for bivalent vaccine preparations.

The present invention relates to a recombinant polynucleotide which

codes for a protein with a molecular weight of about 64 kilodaltons. This sequence has been found in all N. meningitidis serotypes and serogroups examined as confirmed by nucleic acid hybridization, Western blot, Dot blot and ELISA.

The recombinant polynucleotide according to the present invention enables the identification of a nucleotide sequence which codes for a highly conserved protein and which is common to the majority of pathogenic strains of Neisseria (designated P64k), and to obtain the protein recombinantly with a high degree of purity and in commercially applicable quantities, so that it can be used in diagnostic procedures and as an integral part of a vaccine preparation with a broad spectrum of protection.

At the level of genetic information (DNA and RNA), the invention provides a recombinant polynucleotide, comprising a nucleotide sequence that codes for a protein P64k of Neisseria meningitidis, which protein P64k essentially has the amino acid sequence shown in SEQ ID NO:1. In a preferred embodiment, said nucleotide sequence comprises which codes for

the protein P64k of N. meningitidis substantially the nucleotide sequence shown in SEQ ID NO:1. The recombinant polynucleotide may further comprise a nucleotide sequence of a cloning or expression vector.

The invention also provides a transformed microorganism containing a recombinant polynucleotide as defined above, preferably a transformed microorganism capable of expressing the protein P64k of N. meningitidis. In a particularly preferred embodiment of the invention, the transformed microorganism is an Escherichia coli strain such as E. coli strain HB101, transformed with an expression vector containing a nucleotide sequence encoding the protein P64k of N. meningitidis, e.g. the expression vector pM-6.

The invention also provides a recombinant proteinaceous substance comprising an amino acid sequence corresponding to the amino acid sequence of at least part of a protein P64k of N. meningitidis, which protein P64k essentially has the amino acid sequence shown in SEQ ID NO:1. Said recombinant protein-containing substance may mainly comprise proteinP64k, or be a fusion protein or a protein/polysaccharide conjugate comprising the amino acid sequence of proteinP64k of N. meningitidis.

The invention further provides a vaccine composition comprising a recombinant protein as defined above, together with a suitable carrier, diluent or adjuvant. A particular embodiment of the present invention provides a vaccine composition comprising a lipoamide dehydrogenase or acetyltransferase capable of inducing antibody capable of binding to a protein P64k of N. meningitidis, together with a suitable carrier, diluent or adjuvant.

In addition, the invention provides a monoclonal antibody provided against a recombinant proteinaceous substance as defined above, or against a lipoamide dehydrogenase or acetyl transferase, and which is capable of binding a protein P64k of N. meningitidis.

In order to produce a protein P64k of Neisseria meningitidis, or a fusion protein comprising protein P64k, which protein P64k essentially has the amino acid sequence shown in SEQ ID NO:1, the steps of transforming a microorganism with an expression vector containing a nucleotide sequence encoding said protein P64k are carried out or said fusion protein, culturing the transformed microorganism to obtain expression of said protein P64k, or said fusion protein and isolating the expression product.

One particular aspect of the present invention is the gene isolated from the N. meningitidis strain B:4:Pl.15, which was named M-6 and has as a main feature its stability in E. coli vectors. This gene produces no adverse effects on the host, and allows to obtain yields of over 25% of total protein (ratio of P64k to total protein from the host strain). On the other hand, it has been shown by Southern and Western blot hybridization (E. Southern, 1975, J. Mol. Biol. 98: 503-527 and W. Burnette, 1981, Anal. Biochem. 12: 195-203) that protein P64k is present in all of the following studied strains of N. meningitidis:

N. meningitidis A

N. meningitidis B:l

N. meningitidis B:2

N. meningitidis B:4

N. meningitidis B:5

N. meningitidis B:8

N. meningitidis B:9

N. meningitidis B:ll

N. meningitidis B:15

N. meningitidis B:4:Pl.15

N. meningitidis C

N. meningitidis B:15:Pl.16

N. meningitidis B:15:Pl.16 (H 44/76)

N. meningitidis B:NT (121/85)

N. meningitidis B:NT (71/86)

N. meningitidis B:NT (210/86)

and also in N. mucosa, N. subflava and N. gonorrhoeae.

The protein is not present in N. cinerea, N. lactamica, N. sicca and N. flavescens, but these are not of interest, because they are not pathogenic.

The protein with a molecular weight of around 64 kDA can be located by electron microscopy in the outer membrane of N. meningitidis. Accordingly, this antigen is an exposed antigen that is favorable for use in a vaccine preparation. The protein was recognized in Western blot immunoidentification experiments with sera from convalescents and individuals vaccinated with the common Cuban vaccine Va-Mengoc-BC (Centro Nacional de Biopreparados, Havana, Cuba). This aspect ensures the immunogenicity of the antigen and at the same time confirms its presence in the high molecular weight protein fraction which constitutes this vaccine, and which is responsible for the sustained immune response in the disease.

Another new aspect is that the protein, which is an object of the invention, forms antibodies with a broad bactericidal spectrum (different serogroups, serotypes and subtypes), a characteristic that has not previously been reported for any protein from N. meningitidis.

This protein obtained in high levels in E. coli becomes an important candidate for the improvement of immunogenicity when expressed as a fusion protein with other proteins. It can also increase expression by providing increased stability and suitability in the molecular structure during the transcription and translation process. As it belongs to Neisseria, this protein can also be fused to other proteins from Neisseria to obtain vaccine preparations against this microorganism with increased immunogenicity. These fusion proteins are also objects of the present invention.

On the other hand, it was surprisingly found that the gene M-6 obtained from a genomic library of the strain N. meningitidis B:4:P1.15 showed a high homology with sequences of lipoamide dehydrogenases and acetyl transferases from other microorganisms and higher organisms. The presence of common antigenic determinants allows the use of these other related proteins as immunogens, able to provide protection by the introduction of bactericidal antibody that recognizes the antigenic determinants common to protein P64k. Consequently, the use of these lipoamide dehydrogenases and acetyl transferases (not isolated from N. meningitidis) or derivatives thereof such as peptides, fragments from enzymatic degradation, constructs by fusion with other proteins, or conjugation with proteins, polysaccharides or lipids, or insertion into complexes such as liposomes or vesicles, etc. for vaccine purposes included in the scope of the present invention.

An important target for the present invention is the nucleotide sequence which codes for the M-6 gene (SEQ ID NO:1 in the sequence listing) whose product is the protein P64k.

This gene was derived from the genome of strain B385 isolated in Cuba (N. meningitidis B:4:P1.15), by the construction of a genomic library in phage EMBL 3.

The recombinant DNA containing gene M-6 constitutes another object of the present invention, which includes the phage lambda, the plasmid pM-3 and the expression vector pM-6 for expression in bacteria.

Specifically for the intracellular expression in E. coli, the M-6 gene was cloned under the tryptophan promoter and using its own transcription termination signal and a linker fragment between M-6 and the cloning site NcoI that adds the following nucleotide sequence at the 5'- end:

The N-terminus of the protein P64k encoded by the M-6 gene inserted into plasmid pM-6 which adds 5 amino acids to the N-terminus of the original protein corresponds to:

Another object of the present invention is the micro-organisms which originate from the transformation of E. coli strain HB 101 with the pM-6 vector, and which are characterized by the expression of high levels of protein P64k, good viability

and good stability.

The transformed clone of E. coli was designated HBM64

(fig. 2), and shows an expression level of P64k that is higher than 25% in relation to the total protein of the cell (fig. 6).

The method described allows, due to the expression levels obtained for this product, to obtain an optimal purity for the use of this protein in humans.

On the other hand, the antigen obtained from the isolated sequence was very useful in the preparation of various types of potential vaccine preparations, such as bivalent vaccines with a broad immunoprotective spectrum, e.g. protein-polysaccharide conjugates, fusion proteins, etc.. EXAMPLES: These examples are intended to illustrate the invention, but not to limit the scope of the invention.

EXAMPLE 1:

For the isolation of genomic DNA from N. meningitidis, the B:4:P1.15 cells were grown in Mueller-Hinton medium (OXOID, London). The biomass from a 100 ml culture was resuspended in 8 ml Tris (hydroxymethylaminomethane) 100 mM, EDTA (ethylenediaminetetraacetic acid) 1M, pH 8. The cells were subjected to a treatment with lysozyme (10 mg/ml), followed by 200 ul self-digested pronase (20 mg/ml) and 1.1 ml of 10% SDS. The mixture was incubated at 37°C for 1 hour, then it was treated with phenol chloroform (v/v) and the remaining phenol was removed using 2-butanol. Finally, DNA was precipitated with absolute ethanol and RNA was removed with ribonuclease A (Sigma, London).

DNA of about 60 kb was subjected to partial digestion with the enzyme Sau 3A, to obtain a population of fragments of about 15 kb. This major fraction was isolated and purified by agarose gel separation (T. Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab.: Cold Spring Harbor NY).

For the construction of the genomic library, the procedure described by Maniatis was essentially followed (T. Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab.: Cold Spring Harbor NY). Four µg of purified DNA was ligated with 8 µg of BamHI-digested EMBL-3. The ligation product was packaged and the phages were finally plated on E. coli strain C66P2.

The library was screened by immunoidentification (R. Young and R. Davis, 1983, PNAS USA 80: 1194-1198) using rabbit serum obtained against a preparation of proteins belonging to the outer membrane of the strain N. meningitidis B:4:Pl.15. The clones were analyzed by Western blot (Burnette, 1981) and expression of the P64k protein with a molecular weight of about

70 kDA was found. The resulting recombinant phage was designated 31. The Western blot was also performed using a mixture of convalescent center sera of meningococceml <

free of antibody from E. coli while obtaining the same result as that using hyperimmunized rabbit serum.

This experiment was repeated using sera from several healthy individuals and the signal obtained was negative against the recombinant protein P64k.

EXAMPLE 2:

For subcloning in bacteria, the 17 kb insert corresponding to the phage was isolated from the library, cloned into the plasmid pUC18 after separation from the phage arms using the enzyme SalI. This resulted in the construct pM-1 (Fig. 2), which was subjected to restriction analysis (Fig. 3).

The SalI-HindIII fragment of about 6 kb was recloned into the plasmid pUC18 and the construct pM-2 was obtained (Fig. 2). To obtain a more precise location of the gene encoding protein P64k, deletions were performed with the enzymes ClaI, EcoRI and HincII. The complete fragment of the gene M-6 was finally located as an EcoRI-HindIII insert corresponding to the construct pM-3 (Fig. 2).

In all constructs, the presence of the gene was confirmed by recognition of the protein by colony immunoidentification and Western blot using hyperimmunized rabbit serum.

The sequence of the insert in pM-3 was determined by the method of Sanger (F. Sanger et al., 1977, PNAS USA 74. 5463-5467).

From the sequence obtained, the approximate molecular weight of the protein encoded by the gene was derived.

To obtain a construct for high expression of the protein P64k, the plasmid pM-3 (Fig. 2) was linearized with the enzyme EcoRI, and repeated suppressions of the gene were performed by incubating the sample with the nucleases ExoIII and Sl.

The resulting fragments were separated from the rest of the vector pUC18 by digesting with the restriction enzyme HindIII and were cloned fused to a stabilizer fragment (European Patent Application EP-A-0 416 673) using an Xba-butt adapter to conserve the XbaI site of the stabilizer gene :

The constructs where the fused fragment coincided with the reading frame were selected by immunoidentification using hyperimmune rabbit serum.

The insert sequences were established using Sanger's method (F. Sanger et al., 1977, PNAS USA 74: 5463-5467). From the sequences obtained, the approximate molecular weight of the protein encoded by this gene was deduced.

The fusion region between the proteins was located in the gene sequences. In the clone pILM-25 (Figure 4), the ATG fused to the gene predetermined by the sequence of the DNA insert isolated from the library with the fusion site.

The NcoI-XbaI fragment corresponding to the stabilization peptide coding sequence was cleaved from pILM-25 to obtain a non-fused protein expressed under the tryptophan promoter with its original terminator from N. Meningitidis B:4:Pl.15 according to pM-6- the construction (figure 1).

The pM-6 plasmid was transformed into different strains of E. coli such as W3110, JA-221, HB-101, LE-392 and MC-1061, and the expression of P64k was compared. The best results were obtained in W3110, JA-221 and HB-101. These tribes became

chosen to increase fermentation, and expression levels up to 25% of total cell protein were obtained.

EXAMPLE 3:

To confirm the correct expression of the cloned gene, the N-terminal region of the intact protein was subjected to the Edman degradation method (P. Edman, 1950, Acta Chem. Scand. 4: 283-293). This technique provides the sequence (primary structure) of this region of the molecule.

The p64k protein was desalted by gel filtration chromatography (PD-10, Pharmacia), eluted with water and monitored at 280 nm. The protein fraction was concentrated to 0.5 nM/µl. 1 µl of this solution was placed on a PVDF (polyvinylidene difluoride, Millipore) filter, previously activated with methanol.

The Edman degradation was performed using Knauer's Automatic Sequencer, Model 810, connected to an HPLC (high performance liquid chromatography) system, to detect the phenylthio-hydanthioine derivatives of the amino acids (PTH amino acids). Standard procedures for sequencing as recommended by the vendor for the equipment were followed. The separation of the PTH amino acids was carried out in a reversed phase column C-18 (5 µm), 250 mm x 2 mm (Merck), eluted with an acetonitrile (B buffer) in sodium acetate (A buffer) gradient, prepared according to the supplier with a 200 ul/min. current and at 42°C. The PTH amino acids were detected at 269 nm.

Data processing and recording was done in a Shimadzu model CR-6a automatic integrator using a program for data processing by subtraction of two consecutive chromatograms to facilitate the evaluation of the Edmandegradation cycles. Sequence identification was obtained by the chromatographic evaluation for the corresponding cycle analyzed and confirmed with the chromatogram obtained by subtraction to allow determination of 25 residues.

EXAMPLE 4:

To show that the protein P64k is recognized by the sera of individuals vaccinated with the Cuban Va-Mengoc-BC preparation (Centro Nacional de Biopreparados, Havana, Cuba.), a Western blot was performed with a mixture of 12 sera from adults (immunized with two doses of the Cuban vaccine) diluted in a solution containing skimmed milk (Oxoid, London). The experiment included: recombinant protein P64k, purified from E. coli HB-101 transformed with the pM-6 plasmid;

supernatant from ultrasonic cell destruction of untransformed E. coli HB-101;

the reaction was found with a protein A-colloidal gold conjugate.

It was shown that the protein P64k is recognized in the mass of sera.

EXAMPLE 5

The bactericidal test against B385 (B:4:Pl.15) was carried out according to the method described by Larrick et al. (Scand. J. Immunol. 32, 1990, 121-128) with modifications. With this aim, a mixture was made of a) a suspension of bacteria, cultured under special conditions (1-5 colony forming units/l), b) Gey's balanced salt solution, c) rabbit serum (3 to 4 weeks as a source of complement and d ) pooled sera from mice immunized against protein P64k in aluminum hydroxide gel, and inactivated at 56°C for 30 min. The immunization of mice was carried out according to an immunization schedule of 3 doses of 20 µg each. The amounts used in the above mixture were 1:2:1:1 in a total volume of 125 µl. The mixture was incubated at 37°C for 1 hour and plated on fresh Mueller Hinton Agar (Oxoid, London) supplemented with 5% calf serum (CubaVet, Habana). The count of surviving colonies was made after 18 hours of incubation of the plates in an atmosphere of 5% CO2 at 37°C.

The bactericidal titer was assessed as the maximum serum dilution necessary to give a 50% inhibition of bacterial growth with respect to the same mixture without test serum. It was found that 1:20 serum dilution still maintains its bactericidal activity. As negative controls (not bactericidal at 1:2 dilution), pooled serum from mice immunized with aluminum hydroxide gel, and pooled serum from mice immunized with Cuban Hepatitis B recombinant vaccine were used. The bactericidal effect was specific for the anti-P64k antibodies.

EXAMPLE 6:

The bactericidal test against different strains of N. meningitidis was carried out using: 1. An ammonium sulfate precipitate of the supernatant harvested from a culture of hybridoma cells under selection for monoclonal antibodies specific to P64k (anti P64k)/sample for analysis. 2. An ammonium sulfate precipitate of the supernatant harvested from hybridoma cells secreting monoclonal antibodies specific to the Pl.15 protein present in N. meningitidis strain B385 (anti Pl.15)/positive control of the system.

The maximum dilutions examined were always 1:16. The maximum dilutions examined which had a bactericidal effect according to EXAMPLE 5 are indicated:

As observed, the anti-P64k monoclonal antibody has significant bactericidal titers against different serogroups (A, B and C), serotypes (4, 14, 13, 15 and NT) and subtypes (7, 15, 16 and NT) of bacteria.

EXAMPLE 7:

Fusion protein M-14 (P64k and Pl.15)

To obtain a genetic construct for high expression containing the variable epitopes of the Pl.15 protein (Outer membrane protein from N. meningitidis B:4:P1.15) fused to the P64k protein, the gene encoding the Pl.15 protein was cloned by to use the polymerase chain reaction (PCR). The following region containing the variable immunodeterminants of Pl.15: was inserted into the Mlu I site of gene M-6 encoding P64k after being blunted with the klenow fragment from DNA polymerase I. The gene fusion sites of Pl .15 with M-6 are the following:

<*>: N does not belong to any of the fusion proteins and was formed by the genetic construction. The resulting fusion protein (M-14) was expressed in E. coli using a plasmid vector under the tryptophan promoter, to levels greater than 10% of total cell protein. The protein was recognized by bactericidal monoclonal antibody, and anti-P1.15 and P64k polyclonal antibody in Western blot. ;EXAMPLE 8: ;Polysaccharide/P64k conjugation ;The protein P64k was conjugated with the polysaccharide from Haemophilus influenzae using the reductive amination method. The Haemophlus influenzae polysaccharide (Polyribosyl ribitol phosphate, PRP) was purified by the cold phenol method described by Frasch, 1990 in: Bacterial Vaccines, 1990, Alan R. Liss, Inc., pp. 123-145. The final contamination of PRP with proteins or nucleic acids was less than 1%. This polysaccharide was degraded using the method of Parikh et al. 1974 (Methods in Enzymol. 34B: 77-102) with sodium periodate in PRP (ratio 1 : 5 w/w) dissolved in 0.1 M sodium acetate (pH 4.5). The incubation was carried out in the dark for 30 min. while stirring. The periodate excess was removed by addition of ribithiol. Very low molecular weight compounds were removed by dialysis (Medicell International Ltd. Membrane, London). The resulting oligo-saccharide had free aldehyde groups capable of reacting with primary amines (eg lysine residues in proteins). The conjugate is obtained by mixing protein and polysaccharide in a 1:1 ratio (w/w) by adding sodium cyanoborohydride and subjecting the mixture to incubation, first for 48 hours at 4°C and then at 37°C for 24 hours. The high molecular weight complex containing the resulting protein-polysaccharide conjugate in a 1:2.3 ratio can be separated from the non-reactive impurities by HPLC. EXAMPLE 9: Bivalent vaccine preparations against Hepatitis B virus and N. meningitidis. To obtain a bivalent vaccine preparation, different amounts of protein P64k and Hepatitis B surface antigen (Vacuna Recombinante contra la Hepatitis B, Heber Biotec, Havana, Cuba) were mixed. The antigens were adjuvanted with aluminum hydroxygel, at 2 mg/dose and inoculated into Balb/c mice with a body weight of 20 g in 3 doses of 0.5 ml each. Different variants were examined: 1. P64k20ug (P20); 2. HBsAg 20U9(H20) ;3.P64k 10 U9 + HBsAg 10 ug (P10:H10) ;4.P64K15 ug +HBsAg5ug (P15:H5) ;5. Placebo (A1(0H)3); 7 days after the immunization with the first doses, the second doses were administered. The third dose was given 14 days after the second dose. 7 days later, blood was drawn and serum from each immunized animal was separated. Antibody titers against P64k protein were measured in solid phase Enzyme-Linked Immunosorbent Assay (ELISA) using P64k at 5 mg/ml to coat the polystyrene plate. Antibody titers against HBsAg were determined with a commercial ELISA (Organon Teknika, Boxtel). Figure 5 shows the dynamics of the antibody response to P64k, using serum diluted 1/10,000. The response to P64k is not affected by the presence of the second antigen. Figure 6 shows the titers against HbsAg after each dose. The titers against this protein are not lowered by the presence of P64k in the preparation. High titers are obtained against both antigens in the same vaccine preparation. ;EXAMPLE 10: ;A program was created to examine the EMBL (European Molecular Biology Laboratory) database and demonstrate the homology between P64k and other proteins. As a result of the investigation, it was found that there is homology of a segment in the sequence of P64k with segments in the sequences of N. gonorrhoeae. This sequence was found to be characteristic of both N. gonorrhoeae and N. meningitidis (F.F. Correia, S. Inouye and M. Inouye, 1988, J. Biol. Chem. 263 No. 25, 12194-12198). ;Another region of high homology was found in two proteins of the pyruvate-d ehydrogenase complex from E. coli K12: a) acetyltransferase from E. coli and the P64k outer membrane protein from N. meningitidis. ;Homology exists between a segment comprising 100 amino acids repeated at the beginning of the amino acid sequence of the acetyltransferase ("Lipoyl Domain", including the "Lipoyl Binding Site" (P.E. Stephens et al., 1983, Eur. J. Biochem. 133, 481-489)) and a region located in the first 111 amino acids of P64k: ; where (<*>) indicates positions with the same amino acids and (-) indicates positions of conservative amino acid changes. b) .ipoamide dehydrogenase from E. coli and outer membrane P64k protein from N. meningitidis.

Homology exists between the lipoamide dehydrogenase from E. coli (a protein of 473 amino acids, P.E. Stephens et al., 1983, Eur. J. Biochem, 133, 481-489) and the protein P64k, specifically in a segment that represents almost the total protein, except from the region of homology to the "lipoyl domain" from acetyltransferase.

Where:

I — x—l: Adenine binding site (FAD)

I-2-1: Redox-active disulfide region

1-3-1: Active site histidine

Tribal deposits:

An E. coli HB-101 clone containing the plasmid pM-3 (a UC18 plasmid containing the 4.1 kb DNA fragment from Neisseria meningitidis, strain B:4:Pl.15, cloned between the EcoRI and HindIII restriction sites) was deposited on 30 August 1991 at the Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands, and was given deposit number CBS 485.91.

Claims (10)

1. Recombinant polynucleotide, characterized in that it comprises a nucleotide sequence which codes for a protein P64k from Neisseria meningitidis where the P64k protein has the amino acid sequence indicated in SEQ ID N0:1. 2. Recombinant polynucleotide according to claim 1, characterized in that the nucleotide sequence which codes for the P64k protein from N. meningitidis essentially comprises the nucleotide sequence shown in SEQ ID NO:1. 3. Recombinant polynucleotide according to claim 1 or 2, characterized in that it further comprises a nucleotide sequence from a cloning or expression vector. 4. Transformed microorganism, characterized in that it comprises a recombinant polynucleotide according to any one of claims 1 to 3. 5. Transformed microorganism according to claim 4, characterized in that it is able to express the protein P64k from N. meningitidis. 6. Transformed microorganism according to claim 5, characterized in that it is an Escherichia coli strain that has been transformed with an expression vector comprising a nucleotide sequence that codes for the protein P64k from N. meningitidis. 7. Recombinant proteinaceous substance, characterized in that it comprises an amino acid sequence that corresponds to the amino acid sequence of a protein P64k from N. meningitidis, where said P64k protein essentially has the amino acid sequence shown in SEQ ID N0:1. 8. Recombinant proteinaceous substance according to claim 7, characterized in that it is a fusion protein or a protein/polysaccharide conjugate comprising the amino acid sequence of protein P64k from N. meningitidis. 9. Vaccine composition, characterized in that it comprises a recombinant protein according to claim 7 or 8 together with a suitable carrier, diluent or adjuvant. 10. Monoclonal antibody, characterized in that it is reactive towards a recombinant proteinaceous substance according to claim 7 or 8 and which is able to bind a protein P64k from N. meningitidis.