HK1014991B - Proteinase k resistant surface protein of neisseria meningitidis - Google Patents

Description

proteinase K degradation resistant neisseria meningitidis surface proteins

Field of the invention

The present invention relates to a highly conserved, immunologically accessible antigen present on the surface of neisseria meningitidis. This unique antigen can provide the basis for new immunotherapeutic, prophylactic and diagnostic agents useful in the treatment, protection and diagnosis of neisseria meningitidis disease. More precisely, the invention relates to: a proteinase K resistant surface protein of neisseria meningitidis having an apparent molecular weight of 22 kDa: the corresponding nucleotide sequence and the derived amino acid sequence (SEQ ID NO: 1 to SEQ ID NO: 26); recombinant DNA methods for producing such a 22kDa surface protein of Neisseria meningitidis; antibodies that bind to a 22kDa surface protein of Neisseria meningitidis, and methods and compositions for the diagnosis, treatment and prevention of Neisseria meningitidis disease.

Background

Worldwide, neisseria meningitidis is a leading cause of death and morbidity. Neisseria meningitidis can cause both endemic and infectious diseases, mainly meningitis and meningococcemia [ Gold, evolution of Neisseria meningitidis disease 69 p., Vedros N.A., CRC Press (1987), Schwartz et al,review of clinical microbiology2, p.s118 (1989). Indeed, in the united states, this organism is second only to haemophilus influenzae type b, the most common cause of bacterial meningitis, accounting for approximately 20% of all cases, and there is ample information that the primary defense mechanism against neisseria meningitidis is the viability of bactericidal sera. Moreover, protection against bacterial invasion is associated with anti-meningitis antibodies present in serum (Goldschneider et al,journal for experimental medicinePages 129, 1307 (1969); the Goldschneider et al,journal for experimental medicinePages 129, 1327 (1969).

Neisseria meningitidis can be divided into groups of serotypes, depending on the capsular antigen. Currently, 12 serotype groups are known, however, of these the serotype groups A, B, C, Y and W-135 are most common. Within the group of serotypes, the proteins and lipopolysaccharides in the capsule can be subdivided into various serotypes, subtypes and immunotypes [ Frasch et al,overview of infectious diseasesPage 7, 504 (1985).

The capsular polysaccharide vaccines currently available are not effective against all neisseria meningitidis isolates and are also not effective in inducing the production of protective antibodies in children [ Frasch,clinical microorganism Review of science2, p.s134 (1989); reingold et alLancetPage 114 (1985); zollinger in Woodrow and Levine,new generation vaccinePage 325, marceledekker inc.n.y (1990). Currently, capsular polysaccharides from serotype groups A, C, Y and W-135 are used to produce vaccines against this bacterium. These polysaccharide vaccines are effective in a short period of time. However, vaccinated individuals are unable to develop immunological memory, so they must be re-immunized within three years to maintain a level of defense.

Further, these polysaccharide vaccines do not induce sufficient levels of bactericidal antibodies to achieve the desired protective effect in children under the age of two, the main victim of which is the child. Serotype group B is one of the major causes of meningitis disease in developed countries. However, there is currently no effective vaccine against this bacterium. In fact, the polysaccharide of serotype group B is not a good immunogen, it induces only a very low IgM response, and IgM has a poor specificity and thus is also poorly protective [ Gotschlich et al,journal for experimental medicinePage 129, 1349 (1969); the Stevakis et al,infectious disease magazine149, 387 (1984); zollinger et al, in the same manner,journal of clinical research63, page 836 (1979). Furthermore, the presence of very similar and cross-reactive structures in glycoproteins from human brain tissue of newborns may frustrate attempts to increase the immunogenicity of polysaccharides from serogroup B.

In order to obtain a more effective vaccine, other surface antigens of neisseria meningitidis are being investigated, such as lipopolysaccharides, hair (pili) proteins and some proteins located on the outer membrane. There are also reports of certain immunoreactions and bactericidal antibodies against the aforementioned surface protein antigens present in the sera of immunized volunteers and convalescent patients [ Manderell and Zollinger,immunization against infectious diseasesP 57, 1590 (1989); the process of Poolman et al,infection and immunityPage 40, 398 (1983); the Rosenquist et al, for example,journal of clinical microorganismsPage 26, 1543 (1988); the results of the Wedge and Froholm,infection and immunityPp 51, 571 (1986); the combination of Wedge and Michaelsen,journal of clinical microbiologyPage 25, 1349 (1987).

Further, in the case of a liquid crystal display,direct anti-outer membrane proteins, such as monoclonal antibodies class 1, 2/3 and class 5, have also been reported to have bactericidal effects and to protect animals from infection in experiments [ Brodeur et al,infection and immunity50, page 510 (1985); the processes of Frasch, et al,clinical medical researchPage 9, 101 (1986); the Saukkonen et al, in the name of,microbiology of pathogeny3,261 (1987); the Saukkonen et al, in the name of,vaccine7,325 (1989).

Antigens prepared based on outer membrane proteins of neisseria meningitidis have shown a role as common immunogens in both animals and humans, some of which have been clinically tested [ Bjune et al,hand (W.E.) Surgical knife1093 (1991); costa, and the like, in the art,HIPH yearbookPage 14, 215 (1991); the processes of Frasch, et al,medicine and nutritionPages 43, 177 (1982); the processes of Frasch, et al,european clinic Journal of microbiology4,533 (1985); frasch et al in Robbins,bacterial plague Seedling(s)Page 262 Praeger Publications, n.y. (1987); the processes of Frasch, et al,infectious diseases MagazinePage 158, 710 (1988); the Moreno et al,infection and immunity47, page 527 (1985); rosenqvist et al,journal of clinical microbiologyPage 26, 1543 (1988); a Sierra and the like,NIPH yearbookPage 14, 195 (1991); the results of the Wedge and Froholm,feeling of Dyeing and immunizationPage 51, 571 (1986); the combination of Wedge and Michaelsen,clinical microbiology MagazinePage 25, 1349 (1987); zollinger et al, in the same manner,journal of clinical research63, 836 (1979); zollinger et al, in the same manner,NIPH yearbookPage 14, 211 (1991). However, the outer membrane proteins present a great deal of antigenic variability within the strain, which limits their use in vaccines [ Frasch,clinical microbiologyReview p.S134 (1989). Indeed, bactericidal antibodies prepared from antigens extracted from bacteria of one serotype are strictly only effective against that serotype or cells of related serotypes [ Zollinger in Woodrow and Levine,new one Vaccine substitutePage 325 Marcel Dekker inc. n.y. (1990) ] -furthermore, the protective effect produced by these vaccines in children remains to be clarified. Highly conserved neisseria meningitidis outer membrane proteins, such as class 4 [ Munkley et al,microbial pathogens11, 447 pages (1991) ]eggThe production of bactericidal antibodies is not induced by the proteins of the group consisting of the proteins of the genus albumin and of the lipoproteins (also called H.8), and is therefore not an ideal immune candidate. To improve these vaccines, a highly conserved protein on the surface of all strains of Neisseria meningitidis is required and can induce the production of bactericidal antibodies in order to develop a broad spectrum vaccine.

Current laboratory methods for detecting neisseria meningitidis include: after preparation of the smear, a gram stain, latex agglutination or co-agglutination is carried out, the bacteria are cultured and isolated on enriched and selective media [ Morello et al in Balows,clinical microbiology manualP 258, the American Association of microbiology, Washington (1991). Carbohydrate degradation tests are commonly used to demonstrate the identification of isolates of Neisseria meningitidis. Most of the above experimental procedures are time consuming and require trained personnel. Commercially available agglutination or coaggregation kits contain multivalent sera that are directed against capsular antigens expressed by the most prevalent group of serotypes and are therefore useful for rapid identification of neisseria meningitidis. However, these multivalent sera are generally non-specific and cross-react with other bacteria and, as such, must be used in conjunction with methods such as gram staining and bacterial culture. Therefore, there is a need for an effective alternative to improve the speed and reliability of diagnosis.

DISCLOSURE OF THE INVENTION

The present invention solves the aforementioned problems by providing a highly conserved antigen that is located on the surface of N.meningitidis and is immunologically accessible. The invention also provides recombinant DNA molecules encoding the aforementioned antigens, unicellular hosts transformed with such DNA molecules, and methods of producing substantially pure recombinant antigens. The invention also provides antibodies specific against the aforementioned neisseria meningitidis antigens. The antigens and antibodies of the invention provide a unique method and pharmaceutical composition basis for the detection, prevention and treatment of neisseria meningitidis diseases.

Preferred antigens are the 22kDa surface protein of Neisseria meningitidis, including fragments, analogues and derivatives thereof. Preferred antibodies are also the monoclonal antibodies Me-1 and Me-7 which are specific against the 22kDa surface protein of Neisseria meningitidis. These antibodies are highly soluble in Neisseria meningitidis and passively protect mice from experimental infection.

The invention further provides a method which can be used to isolate novel neisseria meningitidis surface antigens and antibodies specific for the same.

Brief Description of Drawings

FIG. 1 shows the nucleotide sequence and deduced amino acid sequence of the 22kDa surface protein of strain 608B of Neisseria meningitidis (SEQ ID NO: 1; SEQ ID NO: 2). Amino acid residues are conventionally three-letter representations of the open reading frame from a 143 base start codon to a 667 base stop codon. The box indicates the generally recognized ribosome binding site, and the generally recognized-10 promoter sequence is shown underlined. A19 amino acid peptide is also underlined.

FIG. 2 shows the results of an alpha-chymotrypsin and trypsin digestion of the outer membrane of strain 608B Neisseria meningitidis (B: 2 a: P1.2), followed by staining with 14% SDS-PAGE and Coomassie blue. The following molecular weight standards are in lane 1: phosphorylase b (97,400); bovine serum albumin (66, 200); ovalbumin (45,000); carbonic anhydrase (31,000); soybean trypsin inhibitor (21,500); and lysozyme (14,400). Lane 2 shows an undigested outer membrane preparation control. Lane 3 shows the α -chymotrypsin digested preparation (2mg enzyme/mg protein); lane 4 shows the trypsinized preparation.

FIG. 3a shows the results of proteinase K digestion of an outer membrane preparation of strain 608B Neisseria meningitidis (B: 2 a: P1.2), followed by staining with 14% SDS-PAGE and then with Coomassie blue. Lanes 1, 3, 5, 7 show the undigested controls. Lanes 2, 4, 6, 8 show the outer membrane preparations after digestion with proteinase K (2IU/mg protein). Lanes 1 to 4 show the treatment of the preparations at pH 7.2. Lanes 5 to 8 are preparations treated at pH 9.0. Lanes 1, 2, 5 and 6 show preparations treated without SDS. Lanes 3, 4, 7, 8 show preparations treated with SDS. Molecular weight standards are shown on the left (in kilodaltons).

FIG. 3b is a graph showing the electrophoretic migration of the recombinant 22kDa protein after affinity purification, after staining with 14% SDS-PAGE and then with Coomassie blue. Lane 1 contains the following molecular weight standards: phosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400). In lane 2 is 5 μ g of the affinity purified recombinant 22kDa protein control. Lane 3 shows an electrophoretogram of 5. mu.g of the affinity-purified recombinant 22kDa protein after being heated at 100 ℃ for 5 minutes, and lane 4 shows an electrophoretogram of 5. mu.g of the affinity-purified recombinant 22kDa protein after being heated at 100 ℃ for 10 minutes. In lane 5 is an electrophoretogram of 5. mu.g of affinity purified recombinant 22kDa protein after treatment with heat at 100 ℃ for 15 minutes.

FIG. 4 shows staining with Coomassie Brilliant blue after 14% SDS-PAGE. And their corresponding Western blots, which show the reactivity of monoclonal antibodies specific against the 22kDa surface protein of Neisseria meningitidis. Outer membrane preparations (A) prepared from the Neisseria meningitidis 608B strain (B: 2 a: P1.2) were untreated; (B) treated with proteinase K (2IU/mg protein). Lane 1 is the characteristic migration pattern of the molecular weight standards and outer membrane preparations after electrophoresis on 14% SDS-PAGE gels. Lane 2 shows Me-2; lane 3 shows Me-3; lane 4 shows Me-5; lane 5 shows Me-7; lane 6 is an irrelevant monoclonal antibody as a control. Molecular weight standards are phosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400). The immunoblotting results for Me-2, Me-3, Me-5, Me-6 and Me-7 shown in FIG. 4 were consistent with those for Me-1.

FIG. 5 shows the binding capacity of monoclonal antibodies to untreated bacterial cells. The results for the representative monoclonal antibodies Me-5 and Me-7 are shown in counts/min on the vertical axis. The different bacterial strains used in this test are indicated on the horizontal axis. A haemophilus influenzae porin-specific monoclonal antibody was used as a negative control. Background below 500 counts/min was recorded and subtracted from the binding data.

FIG. 6 shows the results of enrichment of the culture supernatant of E.coli strain BL21 (DE3) and purification of the recombinant 22kDa surface protein from Neisseria meningitidis, electrophoresed on a 14% SDS-PAGE gel, stained and subjected to the corresponding Western blot. FIG. 6(A) is the results of Coomassie blue and silver staining after 14% SDS-PAGE, lane 1 containing the following molecular weight standards, phosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400). Lane 2 is an outer membrane protein preparation from an extract of Neisseria meningitidis 608B strain (serotype B: 2 a: P1.2). Lane 3 shows the culture supernatant of concentrated E.coli BL21 (DE3) (10 mg). Lane 4 shows the affinity purified Neisseria meningitidis recombinant 22kDa surface protein (1 mg). FIG. 6(B) is an electrophoretogram of affinity purified recombinant 22kDa surface protein from Neisseria meningitidis after digestion with alpha-chymotrypsin, trypsin and proteinase K, on a 14% SDS-PAGE gel and staining with Coomassie blue. Lane 1 includes the following molecular weight standards phosphorylase b (97,400), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400). Lanes 2 to 5 are purified Neisseria meningitidis recombinant 22kDa surface protein (2 mg). Lanes 7 to 10 are bovine serum albumin (2 mg). In lanes 2 and 7 is undigested protein ("NT"). Lanes 3 and 8 are alpha-chymotrypsin ("C") treated proteins (2mg enzyme/mg protein). Lanes 4 and 9 are trypsin ("T") treated protein (2mg enzyme/mg protein). Lanes 5 and 10 are proteinase K ("K") treated protein (2IU/mg protein). FIG. 6(C) is an image obtained after Western blotting of another identical gel using the monoclonal antibody Me-5.

FIG. 7 shows the effect of a protein-A purified monoclonal antibody against the 22kDa surface protein of Neisseria meningitidis on the 608B strain of Neisseria meningitidis (B: 2 a: P1.2), i.e.its bactericidal activity. The vertical axis of the graph shows the percent survival of neisseria meningitidis after exposure to different concentrations of monoclonal antibody (monoclonal antibody concentrations are shown on the horizontal axis).

FIG. 8 shows the nucleotide sequence and deduced amino acid sequence of the 22kDa surface protein of strain MCH88 from Neisseria meningitidis (SEQ ID NO: 3, SEQ ID NO: 4). Amino acid residues are indicated by conventional three-letter symbols. The open reading frame is from a start codon of 116 bases to a stop codon of 643 bases.

FIG. 9 shows the nucleic acid sequence and deduced amino acid sequence of the 22kDa surface protein of strain Z4063 of Neisseria meningitidis (SEQ ID NO: 5; SEQ ID NO: 6). Amino acid residues are indicated by conventional three-letter symbols. The open reading frame shows a start codon of 208 bases to a stop codon of 732 bases.

FIG. 10 shows the nucleic acid sequence and deduced amino acid sequence of the 22kDa surface protein of strain b2 of Neisseria gonorrhoeae (SEQ ID NO: 7, SEQ ID NO: 8). Amino acid residues are indicated by conventional three-letter symbols. The open reading frame is from the start codon at base 241 to the stop codon at base 765.

FIG. 11 shows a consensus sequence of DNA sequences between four strains of Neisseria, and also reveals substitutions or insertions of specific nucleotides in several strains.

FIG. 12 shows the consensus sequence of the protein sequence between four strains of Neisseria, and reveals at the same time the insertion or substitution of specific amino acids in each strain.

FIG. 13 shows the time course of the immune response in rats to the recombinant 22kDa protein, as reflected by the corresponding ELISA (enzyme linked immunosorbent assay) titers. Rat-injected outer membrane preparations were derived from E.coli strain JM 109 harboring plasmid pN2202 or a control plasmid pWRS 30. The development of specific humoral immunity was also analyzed by ELISA, which uses an outer membrane preparation from the 608B strain of Neisseria meningitidis (B: 2 a: P1.2) as the coating antigen.

FIG. 14 shows the time course of the immune response to the recombinant 22kDa protein in cynomolgus monkeys expressed as the corresponding ELISA titres. The two monkeys were immunized with 100. mu.g (K28) and 200. mu.g (I276) of affinity purified 22kDa protein per injection. Control monkeys (K65) were immunized with 150. mu.g of unrelated recombinant protein following the same procedure. The development of specific humoral immunity was also analyzed by ELISA, which uses an outer membrane preparation from the 608B strain of Neisseria meningitidis (B: 2 a: P1.2) as the coating antigen.

FIG. 15 shows the synthetic peptide stretch of the invention and its relative position in a full-length 22kDa protein, the 22kDa protein being that of Neisseria meningitidis strain 608B (B: 2 a: P1.2).

FIG. 16 is a map of plasmid pNP2204 containing the gene encoding the Neisseria meningitidis 22kDa surface protein: 22 kDa; ampicillin resistance gene: amp^iR(ii) a The amino resistance coding region, ColE1, origin of replication; CI857, phage λ CI857 temperature sensitive repressor gene: lambda PL, phage lambda transcription promoter, T1 transcription terminator, the direction of transcription being indicated by the arrow. The restriction sites used for the 22kDa gene inserted on the p629 plasmid were Bgl II and BamHI.

Detailed Description

In our study of the superstructure of the outer membrane of Neisseria meningitidis, we have found a novel low molecular weight 22kDa protein with unique properties. Furthermore, the membrane proteins are highly resistant to treatment with proteolytic enzymes such as proteinase K, a serine protease from Candida albicans (mold). This is quite surprising since proteinase K resistant proteins are rare in nature due to the strong hydrolytic power of proteinase K, the broad pH optimum range and the low peptide bond specificity of the enzyme [ Barrett, A.J.and N.D.Rawlings, Proc. Biochemical Association ](1991)19: 707-715) there are only a few reports describing the resistance of some proteins of prokaryotic origin to proteinase K. Proteinase K resistant proteins are found only in species within leptospira (Nicholson, v.m.and j.f.prescott,microbiology of veterinarian(1993)36: 123-,immunology of infection(1991)59: 1037-1042 ] spiroplama mirum [ Basian F.O. et al,journal of clinical microbiology(1987)25: 2430-2431 [ Onodera, T, etc. ], and viruses and prions,microbial immunology(1993)37: 311 to 316; prusiner, S.B., etc.,united states of America Journal of national academy of sciences(1993)90: 2793 + 2797 ]. Here we describe the use of this protein as a means to improve the prevention, treatment and diagnosis of N.meningitidis infections.

Thus, in one aspect of the invention we provide a highly conserved, immunologically accessible surface protein of N.meningitidis, as well as fragments, analogues and derivatives thereof. As used herein, "Neisseria meningitidis surface protein" refers to any Neisseria meningitidis surface protein encoded by a naturally occurring Neisseria meningitidis gene. According to the invention, the Neisseria meningitidis protein may be of natural origin or may be obtained through the use of molecular biology techniques, with the aim of producing a recombinant protein, or fragment thereof.

As used herein, "highly conserved" means that the genes encoding such surface proteins of Neisseria meningitidis and their protein products are present in more than 50% of known Neisseria meningitidis strains. Preferably, the gene and its protein are present in more than 99% of known strains of Neisseria meningitidis. Examples 2 and 4 provide a method by which one skilled in the art can detect surface proteins of a number of different Neisseria meningitidis bacteria in determining whether they are "highly conserved".

As used herein, "immunologically accessible" means that the surface proteins of N.meningitidis are located on the surface of the bacterial body and that antibodies are accessible to and interact with it. Example 2 provides a method by which one skilled in the art can detect a number of different neisseria meningitidis surface proteins to determine whether they are "immunologically accessible". The immunological accessibility may be determined by other methods including agglutination assays, ELISA assays, RIA, immunoblot assays, dot-enzyme assays, surface accessibility assays, electron microscopy assays, or combinations thereof.

"fragments" of a surface protein of Neisseria meningitidis as used herein include polypeptides having at least one peptide epitope, or analogs and derivatives thereof. Such peptides may be obtained through the use of molecular biology or may be synthesized using conventional liquid or solid phase peptide synthesis techniques.

"analogs" of a Neisseria meningitidis surface protein, as used herein, include proteins, or fragments thereof, in which one or more amino acid residues in the naturally occurring sequence are replaced with another amino acid residue, provided that the overall function of the protein and its protective characteristics are preserved. These analogs can be produced synthetically or by recombinant DNA techniques. Such as the production of an analogue by mutation of a naturally occurring surface protein of Neisseria meningitidis, are well known within the art.

For example, as depicted in FIG. 10, one such analog is selected from the recombinant proteins encoded for production by the gene encoding the 22kDa protein of strain b2 of Neisseria gonorrhoeae. Another analog is obtained by isolating the corresponding gene from Neisseria lactococcus.

As used herein, a "derivative" of a Neisseria meningitidis surface protein refers to a protein or protein fragment that has been covalently modified, for example, with dinitrophenol, in order to render it immunogenic in humans. The derivatives of the present invention also include derivatives of the amino acid analogs of the present invention.

It will be appreciated that by way of example of the present invention, a person skilled in the art may determine, by appropriate experimentation, whether a particular fragment, analogue or derivative is useful in the diagnosis, defence or treatment of N.meningitidis disease.

The invention also includes multimeric forms of neisseria meningitidis surface proteins, fragments, analogues and derivatives. These multimeric forms include, for example, one or more polypeptides crosslinked together by a crosslinking agent such as avidin/biotin, glutaraldehyde or dimethyloctadiimide (subyimide). Such multimeric forms also include polypeptides comprising 2 or more neisseria meningitidis sequences in tandem or in reverse proximity, such polypeptides being produced from polycistronic mRNA produced by recombinant DNA techniques.

The invention provides substantially pure neisseria meningitidis surface proteins. The term "substantially pure" means that the surface protein according to the invention is free of other proteins of neisseria meningitidis origin. Preparation of substantially pure neisseria meningitidis surface proteins can be accomplished by a variety of conventional methods, such as those described in examples 3 and 11.

In another aspect, the invention provides, inter alia, a 22kDa surface protein of Neisseria meningitidis, or a fragment, analogue or derivative thereof, having the amino acid sequence shown in FIG. 1(SEQ ID NO: 2).

In another aspect, the invention provides, inter alia, a 22kDa surface protein of Neisseria meningitidis, and fragment analogues or derivatives thereof, having the amino acid sequences shown in FIG. 8(SEQ ID NO: 4) and FIG. 9(SEQ ID NO: 5). This fragment may be selected from the peptides listed in FIG. 15(SEQ ID NO: 9 to SEQ ID NO: 26).

In another aspect, the invention provides a 22kDa surface protein of Neisseria gonorrhoeae or a fragment, analogue or derivative thereof having the amino acid sequence shown in FIG. 10(SEQ ID NO: 8) from the above, it will be clear that any discussion of a 22kDa protein of Neisseria meningitidis also includes 22kDa proteins isolated from or produced by genes isolated from other species of Neisseria such as Neisseria gonorrhoeae or Neisseria lactosyli.

According to the invention, the 22kDa surface protein of Neisseria meningitidis can be further characterized by the following features.

(1) The molecular weight was estimated to be about 22kDa on SDS-PAGE gel;

(2) its mobility on SDS-PAGE gel is not affected by treatment with reducing agent;

(3) the isoelectric Point (PI) ranges from pI8 to pI 10;

(4) it is highly resistant to degradation by proteolytic enzymes such as alpha-chymotrypsin, trypsin and proteinase K;

(5) periodate oxidation does not eliminate specific binding of antibodies directed against the 22kDa protein on the surface of Neisseria meningitidis;

(6) it is a highly conserved antigen;

(7) on the surface of untreated neisseria meningitidis, the antibody may be contacted with the protein;

(8) it can induce the generation of bactericidal antibodies;

(9) it can induce the production of antibodies which can resist experimental infection;

(10) when injected into an animal host, it induces the development of an immune response that is protective against infection by Neisseria meningitidis.

The present invention also provides for the first time a DNA sequence (SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7) encoding a Neisseria meningitidis 22kDa surface protein.

Preferred DNA sequences of the invention are selected from:

(a) the DNA sequence of FIG. 1(SEQ ID NO: 1);

(b) the DNA sequence of FIG. 8(SEQ ID NO: 3);

(c) the DNA sequence of FIG. 9(SEQ ID NO: 5);

(d) the DNA sequence of FIG. 10(SEQ ID NO: 7);

(e) analogs or derivatives of the aforementioned DNA sequences;

(f) a DNA sequence which is degenerate with the aforementioned DNA sequence; and

(g) a fragment of any of the foregoing DNA sequences; wherein said sequence encodes a product which exhibits the immunological activity of a Neisseria meningitidis 22kDa surface protein.

Such fragments are preferred peptides, as depicted in FIG. 15(SEQ ID NO: 9, SEQ ID NO: 26).

Preferably, the present invention provides for the first time a DNA sequence (SEQ ID NO: 1) encoding a Neisseria meningitidis 22kDa surface protein. More preferred DNA sequences of the invention are selected from:

(a) the DNA sequence of FIG. 1(SEQ ID NO: 1);

(b) analogs or derivatives of the aforementioned DNA sequences;

(c) a DNA sequence which is degenerate with any of the above sequences; and

(d) a fragment of any one of the preceding DNA sequences, wherein said sequence encodes a product which displays the immunological activity of a neisseria meningitidis 22kDa surface protein.

An analogue or derivative of the gene encoding the Neisseria meningitidis 22kDa surface protein will hybridise to the gene encoding the 22kDa protein using the conditions described in example 4.

It is an object of the present invention that a fragment, analogue or derivative of a Neisseria meningitidis 22kDa surface protein has the "immunological activity" of the 22kDa surface protein if it is capable of eliciting the development of an immune response upon injection into an animal host, and if the immune response is also capable of combating Neisseria meningitidis infection. One skilled in the art can determine whether a particular DNA sequence encodes a product that has the immunological activity of the Neisseria meningitidis 22kDa protein by the method set forth in example 6.

The method for separating the surface protein of the neisseria meningitidis comprises the following steps:

a) isolating-a neisseria meningitidis culture;

b) isolating the outer membrane fraction from the bacterial culture; and is

c) Isolating said antigen from the outer membrane fraction.

It is particularly noted that the aforementioned step (c) may comprise further steps: the protein extract of the outer membrane of Neisseria meningitidis is treated with proteinase K and the protein is separated into fractions by conventional separation techniques such as ion exchange and gel chromatography, electrophoresis and the like.

Another and preferred method is to produce the Neisseria meningitidis surface protein of the present invention by molecular biology techniques, as described in particular in example 3, which are particularly useful for preparing substantially pure recombinant Neisseria meningitidis 22kDa surface protein.

Thus, in another aspect the invention provides a method of producing a recombinant Neisseria meningitidis 22kDa surface protein, and also fragments, analogues and derivatives thereof, comprising the steps of (1) culturing a unicellular host bacterium transformed with a recombinant DNA molecule comprising a coding sequence for a protein of interest or a fragment, analogue or derivative thereof, and comprising one or more expression control sequences associated with the coding sequence, and (2) collecting the substantially pure protein, fragment, analogue or derivative.

In order for a transfected gene to be highly expressed in a host, the gene must be operatively linked to transfection and translation expression control sequences that are functional in the chosen expression host, as is well known in the art. Preferably, the expression control sequences and the gene of interest are contained in an expression vector comprising a bacterial selection marker and an origin of replication, and if the expression host is a eukaryotic cell, the expression vector also comprises an expression marker effective in the expression host.

A wide variety of expression host/vector combinations may be used to express the DNA sequences of the present invention. Useful eukaryotic host expression vectors include, for example, those derived from SV₄₀Expression regulatory sequences for bovine papilloma virus, adenovirus and cytomegalovirus. Useful bacterial host expression vectors include known bacterial plasmids such as E.coli-derived plasmids ColE1, pCR1, pBR322, pNB9 and derivatives thereof, broad host plasmids such as RP4, phage DNA such as numerous derivatives of bacteriophage lambda such as NM989, and other DNA phages such as M₁₃And filamentous single-stranded DNA phages. Useful yeast cell expression vectors include 2 μ plasmid and its derivatives. An effective insect cell expression vector includes pVL 941.

In addition, a number of expression control sequences are available for use in these vectors to express the DNA sequences of the present invention. Such effective expression control sequences include those of the structural gene to which the aforementioned expression vector is ligated. Examples of effective expression control sequences include, for example, SV₄₀Or the early and late promoters of adenovirus, the Lac system, the trp system, the TAC and TRC systems, the master operon and promoter regions of phage lambda, the control regions of fd coat protein, the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, such as Pho5, the promoters of the yeast alpha-binding system and other known sequences which control the expression of genes in prokaryotic and eukaryotic cells or which control the expression of genes in prokaryotic and eukaryotic viruses, and combinations thereof. The expression control sequence for the 22kDa surface protein of Neisseria meningitidis is very efficient for expressing this protein in E.coli (example 3).

Transformation of host cells with the aforementioned vectors constitutes a further aspect of the invention. Many unicellular hosts are effective in expressing the DNA sequences of the invention. These include well-known eukaryotic and prokaryotic hosts such as some strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COS1, COS7, BSC1, BSC40 and BMT10, and tissue-cultured human and plant cells. Preferred hosts include bacteria such as E.coli and Bacillus subtilis, and mammalian cells in tissue culture.

Of course, it should be understood that not all vectors and expression control sequences are equally good for expressing the DNA sequences of the present invention, nor are all host systems equally good for the same expression system. However, a person skilled in the art can select between these vectors, expression control sequences and hosts within the scope of the present invention by appropriate experimentation. For example, in selecting a vector, the host must also be considered, since the vector must replicate in the host. The copy number of the vector, the ability to control copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, are also contemplated.

Many factors also need to be considered in selecting expression control sequences. These factors include, for example, the relative strength of the sequence, its control, and its compatibility with the DNA sequences of the invention, especially with regard to its potential secondary structure, the selection of a unicellular host with regard to its compatibility with the chosen vector, the toxicity of the products encoded by the DNA sequences of the invention, their secretory properties, their ability to fold the protein correctly, their fermentation or culture requirements, and the products encoded by the DNA sequences of the invention should be readily purified from the host.

In these references, one skilled in the art can select different vector/expression control sequence/host combinations to express the DNA sequences of the present invention by fermentation or large-scale animal culture.

The polypeptides encoded by the DNA sequences of the present invention may be isolated by fermentation or cell culture and purified by a variety of conventional methods. Those skilled in the art can select the most suitable separation and purification techniques within the scope of the present invention.

The neisseria meningitidis surface proteins of the present invention will be useful for the prevention, treatment and diagnosis of diseases caused by neisseria meningitidis infection.

The neisseria meningitidis surface proteins of the present invention will be useful for the prevention, treatment and diagnosis of diseases caused by neisseria gonorrhoeae or neisseria lactosami infection.

According to the invention, the surface proteins of Neisseria meningitidis are particularly suitable for raising antibodies against Neisseria meningitidis diseases and for generating protective responses against diseases.

According to the invention, the surface proteins of Neisseria meningitidis are particularly suitable for raising antibodies against Neisseria gonorrhoeae disease or Neisseria lactis disease and for generating protective responses against diseases.

In particular, we propose a Neisseria meningitidis 22kDa surface protein, or a fragment, analogue or derivative thereof, for use as an immunogen and as a vaccine, the protein having the amino acid sequence shown in FIG. 1(SEQ ID NO: 2).

In particular, we propose a Neisseria meningitidis 22kDa surface protein, or a fragment, analogue or derivative thereof, for use as an immunogen and as a vaccine, having the amino acid sequence shown in FIG. 1(SEQ ID NO: 2), FIG. 8(SEQ ID NO: 4), FIG. 9(SEQ ID NO: 6) or FIG. 10(SEQ ID NO: 8).

Standard immunization techniques can be applied to the surface protein of this Neisseria meningitidis to make it useful as an immunogen and vaccine. In particular, any suitable host may be injected with a pharmacologically effective dose of Neisseria meningitidis 22kDa surface protein to produce monoclonal or multivalent antibodies against Neisseria meningitidis, or to elicit the development of a protective immune response against Neisseria meningitidis disease. Suitable particles of neisseria meningitidis surface proteins may be formed before the host is injected, thus we provide a pharmaceutical composition comprising one or more neisseria meningitidis surface antigens or fragments thereof. Preferably, the antigen is a Neisseria meningitidis 22kDa surface protein or a fragment, analogue or derivative thereof. One or more pharmaceutically acceptable excipients are additionally added. As used herein, "pharmacologically effective dose" refers to a dose of one or more Neisseria meningitidis surface antigens or fragments thereof that induces a sufficiently high titer of anti-Neisseria meningitidis antibodies that can be used to treat or prevent Neisseria meningitidis infection.

The neisseria meningitidis surface proteins of the present invention may also provide a basis for diagnostic testing of neisseria meningitidis infection. Several diagnostic methods are possible. For example, the invention provides a method for detecting neisseria meningitidis antigens in a biological sample, the sample containing or suspected of containing neisseria meningitidis antigens, the method comprising:

a) separating a biological sample from a patient;

b) incubating the biological sample with an anti-Neisseria meningitidis 22kDa surface protein antibody or fragment thereof to form a mixture, and

c) detecting the specifically bound antibody or fragment in the mixture to indicate the presence of the neisseria meningitidis antigen.

Preferred antibodies for use in the aforementioned diagnostic methods are Me-1 and Me-7.

In another aspect, the invention provides a method for detecting antibodies specific for an antigen of Neisseria meningitidis in a biological sample, which sample contains or is suspected of containing said antibodies, which method comprises:

a) separating a biological sample from a patient;

b) incubating the biological sample with a neisseria meningitidis surface protein or fragment thereof of the present invention to form a mixture; and are

c) Detecting in the mixture the specifically bound antigen or fragment thereof to indicate the presence of antibodies specific against Neisseria meningitidis. One skilled in the art will recognize that this diagnostic test may take several forms, including immunoassays such as enzyme-linked immunosorbent assays (ELISAs), injection immunoassays or latex agglutination assays, to determine whether antibodies specific for the protein are present in the organism.

The DNA sequences of the invention may also be used to design probes and detect the presence of pathogenic Neisseria in a biological sample containing or suspected of containing Neisseria. The detection method of the invention comprises the following steps:

a) separating a biological sample from a patient;

b) incubating a biological sample with a probe comprising a DNA sequence of the invention to form a mixture; and are

c) Detecting the specifically bound DNA probe in the mixture to indicate the presence of Neisseria.

Preferred DNA probes contain the base sequences shown in the following figures: FIG. 1(SEQ ID NO: 1), FIG. 8(SEQ ID NO: 3), FIG. 9(SEQ ID NO: 5), or FIG. 10(SEQ ID NO: 7), or is the consensus sequence of FIG. 11(SEQ ID NO: 9).

More preferably, the DNA probe should have the 525 base pair sequence of FIG. 1(SEQ ID NO: 1).

As a method for diagnosing Neisseria meningitidis infection, the DNA probes of the present invention may also be used to detect Neisseria meningitidis circular nucleic acid in a sample, for example, using polymerase chain reaction probes which may be synthesized by conventional techniques and may be immobilized on a solid support, or the probes may be labeled with a detectable label.

A preferred DNA probe for this application is an oligonucleotide comprising a stretch of oligonucleotide complementary to at least 6 contiguous nucleotides of a gene of the Neisseria meningitidis 22kDa surface protein, the sequence of the gene being as shown in FIG. 1(SEQ ID NO: 1), FIG. 8(SEQ ID NO: 3), FIG. 9(SEQ ID NO: 5), FIG. 10(SEQ ID NO: 7), or FIG. 11(SEQ ID NO: 9) for the consensus sequence.

A more preferred DNA probe for this application is an oligonucleotide containing a stretch of oligonucleotide complementary to at least 6 consecutive nucleotides of the gene for the 22kDa surface protein of Neisseria meningitidis, the gene sequence being shown in FIG. 1(SEQ ID NO: 1).

Another diagnostic method for detecting neisseria meningitidis in a patient comprises the steps of:

a) labeling an antibody or fragment thereof of the invention with a detectable label;

b) administering the labeled antibody or labeled fragment to a patient, and

c) detecting the specifically bound labeled antibody or fragment in the patient to reveal the presence of Neisseria meningitidis.

For the purification of any anti-Neisseria meningitidis surface protein antibody, affinity chromatography may be used, which requires immobilised Neisseria meningitidis surface proteins as affinity media.

Thus, according to another aspect of the invention we provide a 22kDa surface protein of Neisseria meningitidis covalently bound to an insoluble support, or a part or analogue thereof, the amino acid sequence of which protein comprises the sequence of FIG. 1(SEQ ID NO: 2), FIG. 8(SEQ ID NO: 4), FIG. 9(SEQ ID NO: 6) or FIG. 10(SEQ ID NO: 8).

According to a preferred aspect of the invention we provide a 22kDa surface protein of Neisseria meningitidis covalently bound to an insoluble support, or a part or analogue thereof, the amino acid sequence of which protein comprises the sequence of figure 1(SEQ ID NO: 2).

Another feature of the invention is the use of the Neisseria meningitidis surface proteins of the invention as immunogens to generate specific antibodies, thereby allowing the diagnosis, or in particular treatment, of infection by Neisseria meningitidis. Suitable antibodies can be determined by suitable screening methods, such as measuring the ability of a particular antibody to passively protect against N.meningitidis infection in a test model. One example of an animal model, a mouse model, is described in the examples. The antibody may be a whole antibody or an antigen-binding part thereof and may generally belong to any kind of immunoglobulin, and the antibody or fragment may be of animal origin, in particular of mammalian origin, further in particular of murine, rat or human origin. The antibody may be a natural antibody or a fragment thereof, and if necessary, a recombinant antibody or a fragment thereof. The term recombinant antibody or antibody fragment means an antibody or antibody fragment produced using molecular biology techniques. The antibody or antibody fragment may be polyclonal or, preferably, of monoclonal origin. It may be specific for a number of epitopes associated with a surface protein of Neisseria meningitidis but preferably it is specific for one epitope, preferably the antibody or fragment thereof is specific for one or more epitopes associated with a 22kDa surface protein of Neisseria meningitidis. The Me-1 and Me-7 monoclonal antibodies described herein are also preferred.

Examples

In order that the invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example 1 describes the treatment of a preparation of outer membrane protein of Neisseria meningitidis with proteolytic enzymes and subsequent identification of the 22kDa surface protein of Neisseria meningitidis.

Example 2 describes the preparation of a monoclonal antibody specific for the Neisseria meningitidis 22kDa surface protein.

Example 3 describes the preparation of recombinant 22kDa surface protein from Neisseria meningitidis.

Example 4 describes the use of DNA probes to identify organisms expressing the Neisseria meningitidis 22kDa surface protein.

Example 5 describes the protection of mice from infection by Neisseria meningitidis with monoclonal antibodies against the 22kDa surface protein of Neisseria meningitidis.

Example 6 describes the use of a purified recombinant 22kDa surface protein to induce a protective response against Neisseria meningitidis infection.

Example 7 describes the identification of the sequence of the 22kDa protein and the gene sequences encoding proteins from other strains of Neisseria meningitidis (MCH88 and E4063), and also identifies a strain of Neisseria gonorrhoeae.

Example 8 describes the immune response of rabbits and monkeys to this 22kDa Neisseria meningitidis surface protein.

Example 9 describes a method for mapping different immunological epitopes of the 22kDa Neisseria meningitidis surface protein

Example 10 describes the large scale production of the 22kDa surface protein by heat induction of an expression vector.

Example 11 describes the purification of this 22kDa surface protein when it is produced by recombinant techniques.

Example 12 describes the use of a 22kDa surface protein as a vaccine for humans.

Example 1 identification of a proteolytic enzyme-treated outer membrane preparation of Neisseria meningitidis followed by a Neisseria meningitidis 22kDa surface protein that is resistant to enzymatic hydrolysis.

Several antigen preparations prepared from whole cells, extracted with lithium chloride, or sodium lauryl sarcosine were used to study the superstructure of the outer membrane of N.meningitidis. The outer membrane of gram-negative bacteria serves as the interface between the interior of the cell and the environment, and contains the vast majority of antigens that are freely exposed to the host's immune defenses. The main goal of this study was to identify a novel antigen that induces a protective response against N.meningitidis. One method used by the inventors to identify such antigens is to partially degrade the antigen preparation described above with proteolytic enzymes. The antigenic determinants produced by this enzymatic treatment can then be identified by analysis of the immunity and protection of these treated antigen preparations. Surprisingly, after separation of the outer membrane extract of the Neisseria meningitidis lithium chloride by electrophoresis, we observed a low molecular weight band that was not destroyed by proteolytic enzyme treatment, stained with Coomassie Brilliant blue R-250. Coomassie Brilliant blue stains proteins or polypeptides and has little or no affinity for other major components of the outer membrane, polysaccharides or lipids. The fact that the low molecular weight antigen can be stained with Coomassie Brilliant blue suggests that at least part of the antigen is composed of a peptide that is not digested by proteases or protected from enzymatic degradation by other outer membrane structures. Further, as shown below, even when over-treated, very powerful proteinase K cannot degrade this low molecular weight antigen.

Outer membrane preparations were obtained from different strains of Neisseria meningitidis by extraction with lithium chloride as described by the inventors earlier [ Brodeur et al,infection and immunityPage 50, 510 (1985). The protein content of these preparations was determined using the Lowry method, which was applied to the membrane fraction [ Lowry et al,journal of biochemistry193, 265 page (1951). Outer membrane preparations from neisseria meningitidis strain 608B were treated with alpha-chymotrypsin derived from bovine pancreas (e.c.3.4.21.1) (Sigma) or trypsin-like enzyme I derived from bovine pancreas (e.c.3.4.21.4) (Sigma) for 24 hours at 37 ℃ with constant shaking. The enzyme concentration was 2mg enzyme per mg protein treated. The same outer membrane preparations were treated with different concentrations (0.5 to 24mg/mg protein) of proteinase K from Candida albicans Linebuii (Sigma or Boehringer Mannheim Laval. Canacla) (E.C.3.4.21.14). Different experimental conditions were used for the degradation of proteins by proteinase K. The samples were incubated at 37 ℃ or 56 ℃ for 1, 2 or 24 or 48 hours with or without shaking. The pH of the sample mixture was adjusted to 7.2 or 9.0. Is also atSome samples were added with 1% Sodium Dodecyl Sulfate (SDS) (by volume). Immediately after treatment, the samples were separated by SDS-PAGE gel electrophoresis at a gel concentration of 14% (w/v) using a MiniProtean electrophoresis apparatus(Bio-Rad, Mississauga, Ontario, Canada) System electrophoresis was performed according to the manufacturer's instructions. The proteins were heated in the presence of SDS and 2-mercaptoethanol at 100 ℃ for 5 minutes, separated on a 14% SDS gel and stained with Coomassie Brilliant blue R-250.

FIG. 2 shows migration images from 14% SDS-PAGE gel electrophoresis of outer membrane preparations from Neisseria meningitidis 608B strain (B: 2 a: P1.2) after treatment with alpha-chymotrypsin and trypsin for 24 hours at 37 ℃. The high molecular weight proteins and several major outer membrane proteins were seen to be strongly hydrolytically digested in the treated samples (FIG. 2, lanes 3 and 4) compared to the untreated control (FIG. 2, lane 2). In contrast, a protein band with a molecular weight of 22kDa was not affected by either proteolytic enzyme even when treated for 24 hours.

This particular protein was further studied with proteinase K using more robust hydrolysis conditions (FIG. 3), which is one of the most powerful proteolytic enzymes due to its low peptide bond specificity and broad pH optimum. Surprisingly, the 22kDa protein was able to withstand 2 international units of proteinase K for 24 hours at 56 ℃ (FIG. 3, lane 2). Such treatments are used in our laboratory to prepare lipopolysaccharides or DNA that are almost protein-free. In fact, only small peptides are visible after this intense proteolytic treatment of the outer membrane preparation. Further, treatment for a long period of time, up to 48 hours, or treatment with high enzyme amounts (up to 24 international units) did not change the amount of the 22kDa protein. The reaction mixture was raised to pH 9.0 or 1.0% SDS, a strong protein denaturant, was added and SDS-PAGE electrophoresis showed that the amount of the 22kDa protein and its migration were not affected (FIG. 3, lanes 4, 6 and 8). Combining these two denaturing conditions typically results in complete degradation of the protein in the outer membrane preparation to leave behind amino acid residues. Low molecular weight polypeptides are often observed upon digestion and are considered fragments of sensitive proteins that are not completely digested upon enzymatic treatment. These fragments are most likely protected from further degradation by carbohydrates and lipids in the outer membrane. The bands with molecular weights of 28kDa and 34kDa in the treated sample are residual enzyme and a contaminating protein present in all the enzyme preparations tested, respectively.

Interestingly, studies on this 22kDa protein against protease showed that another protein band with an apparent molecular weight of 18kDa appeared to be resistant to enzymatic degradation as well (FIG. 3 a). After the affinity-purified recombinant 22kDa protein was subjected to SDS-PAGE, the migration pattern was analyzed to obtain clues about the 18kDa protein (FIG. 3 b). Before loading, the 18kDa band was only apparent when the affinity purified recombinant 22kDa protein was heated for a further period in sample buffer containing detergent SDS. N-terminal amino acid analysis by Edman degradation (example 3) showed that the 18kDa protein has amino acid residues (E-G-A-S-G-F-Y-V-Q) corresponding to amino acids 1 to 9(SEQ ID NO: 1). These results indicate that the 18 and 22kDa two-protein bands seen on SDS-PAGE are in fact derived from the same protein. This final result also shows that the leader sequence is cleaved from the mature 18kDa protein. Further studies will be conducted to determine molecular modifications to account for this change in apparent molecular weight and to estimate its effect on the antigenicity and protection of the protein.

In conclusion, the discovery of this Neisseria meningitidis outer membrane protein, which has very rare properties to resist proteolytic digestion, has led to further studies on its molecular and immunological properties. The recombinant 22kDa surface protein purified in example 3 and produced by E.coli also has strong resistance to degradation by proteinase K. We are now trying to understand what mechanism leads to this unusual resistance to proteolytic enzymes of the 22kDa surface protein of N.meningitidis.

EXAMPLE 2 Generation of monoclonal antibodies specific against the surface protein of 22kDa Neisseria meningitidis

Monoclonal antibodies as described hereinObtained from three independent fusion experiments. Male Balb/C mice were deimmunized with outer membrane preparations of Neisseria meningitidis strains 604A, 608B and 224/C belonging to serotype group A, B, C (Chares River laboratory, St-Constant, Quebec, Canada). The outer membrane preparation was prepared by extraction with lithium chloride as described by the previous inventors [ Brodeur et al,infection and immunityPage 50, 510 (1985). The proteins contained in these preparations were determined by the Lowry method, which is applied to the membrane fraction [ Lowry et al,biological organisms Chemical journal193, 265(1951), multiple groups of mice were injected intraperitoneally and subcutaneously with 10mg of different combinations of the above outer membrane preparations at 3 week intervals. Adjuvants used for immunization are Freund's complete or incomplete adjuvant (Gibco laboratories, Grand Island, N.Y.) or QuilA (Cedarlane laboratories, Hornby, on., Canada), depending on the mouse population. Three days prior to the fusion procedure, immunized mice received the last intravenous injection with 10mg of the outer membrane preparation described above. Fusion protocols for generating hybridoma cell lines capable of secreting the desired monoclonal antibodies have been previously proposed by the inventors [ Hamel et al,journal of medical microbiologyPage 25, 2434 (1987). The class, subclass and light chain type of monoclonal antibodies Me-1, Me-2, Me-3, Me-5, Me-6 and Me-7 were determined by ELISA previously reported [ Martin et al,clinical microbiologyPage 28, 1720 (1990) and listed in table 1.

The specificity of the monoclonal antibodies was tested by Western immunoblotting, according to the protocol described by the previous inventors [ Martin et al,european journal of immunologyPage 18, 601 (1988) and modified as follows. Outer membrane preparations obtained from different strains of Neisseria meningitidis were electrophoretically separated on a 14% SDS-PAGE gel. Proteins were transferred from the gel onto nitrocellulose membranes using a semidry apparatus (Bio-Rad). Each gel (6X 10 cm) was exposed to a current of 60mA for 10min, and the electroblotting buffer contained 25mM Tris-HCl, 192mM glycine and 20% methanol, pH 8.3. Western blot experiments clearly showed that the monoclonal antibodies Me-1, Me-2, Me-3, Me-5, Me-6 and Me-7 recognized specific epitopes of the Neisseria meningitidis 22kDa protein (FIG. 4A). SDS-PAGE gels and corresponding Western blot fractionsThe analysis also shows that the molecular weight of the protein does not change with the strain. However, the amount of the protein in the outer membrane preparation varies from species to species and is independent of the serotype group of the strain. Furthermore, these monoclonal antibodies still recognized the respective epitopes on the 22kDa surface protein of Neisseria meningitidis after treatment of the outer membrane preparation with 2IU proteinase K/mg protein (treatment described in example 1, supra) (FIG. 4B). Interestingly, after enzymatic digestion, epitopes remained unaffected, confirming that they were both accessible to antibodies and not destroyed by enzymatic treatment in the membrane preparation. The latter result suggests that the mechanism of the observed phenomenon of resistance to proteinase K is probably not related to the proximity of the surface structure repressor to the protein, or that the mechanism is not related to the protection of the membrane from proteins deeply embedded therein. The results of the immunoblotting of Me-1 were in agreement with those of the other 5 monoclonal antibodies, which are not shown in FIG. 4.

A series of experiments were also performed to characterize the Neisseria meningitidis 22kDa surface protein and distinguish it from other known Neisseria meningitidis surface proteins. No change in apparent molecular weight of the Neisseria meningitidis 22kDa surface protein was observed on SDS-PAGE gels with or without mercaptoethanol, heating the outer membrane preparation in electrophoresis sample buffer at 100 ℃ for 5 minutes, or at 37 ℃ and 56 ℃ for 30 minutes. This shows that the migration of the 22kDa surface protein present on the outer membrane is not affected by heat or 2-mercaptoethanol.

Sodium periodate oxidation was used to determine whether monoclonal antibodies reacted with carbohydrate epitopes on outer membrane preparations of neisseria meningitidis. This method is in accordance with the previous inventors [ Martin et al,infection and immunity60, 2718. page 2725 (1992). Treatment of the outer membrane preparation with 100mM sodium periodate for 1 hour at room temperature did not alter the reactivity of the monoclonal antibody with the 22kDa surface protein of Neisseria meningitidis. This treatment generally eliminates binding of the antibody to its specific carbohydrate.

Monoclonal antibody 2-1-CA2 (supplied by wo pa. bhattahaharjee, waltreed arm Institute of Research, washington, colombian) is a lipoprotein-specific antibody (also called h.8) which is a surface antigen in all neisseria pathogens. The reactivity of this monoclonal antibody with outer membrane preparations was compared to the reactivity of the Me-5 monoclonal antibody. The lipid-specific monoclonal antibody reacts with a protein band with a molecular weight of 30kDa, while the Me-5 monoclonal antibody reacts with a protein band with a molecular weight of 22 kDa. This result clearly shows that the Neisseria meningitidis 22kDa surface protein is unrelated to lipoprotein, while lipoprotein is another highly conserved outer membrane protein.

To demonstrate that the 22kDa protein is exposed on the surface of untreated Neisseria meningitidis cells, a radioimmunoassay was used, which was carried out as described by the previous inventors [ Proulx et al,infection and immunityPage 59, 963 (1991). The method used 86 and 18 hours of bacterial culture. The 6 monoclonal antibodies were reacted with 9 strains of N.meningitidis (the serotype groups of each strain are shown in parentheses in FIG. 5). 2 Neisseria gonorrhoeae strains ("NG") 2 Moraxella catarrhalis strains ("NC") and 2 Neisseria lactosylii strains ("NL"). Radioimmunoassay demonstrated that the epitopes recognized by monoclonal antibodies were surface exposed to untreated Neisseria meningitidis isolates of multiple different serotypes and serogroups and were also accessible to proteolytic enzymes (FIG. 5). The monoclonal antibody binds strongly to its target epitope located on the surface of all neisseria meningitidis bacteria tested. The binding values recorded (between 3000 and 35000 CPM) varied from strain to strain and were influenced by the physiological state of the bacteria. A haemophilus influenzae porin-specific monoclonal antibody was used as a positive control for each strain. Counts below 500CPM were recorded and then subtracted from each binding value. For the Neisseria meningitidis strains tested in this method, the results shown in FIG. 5 for the monoclonal antibodies Me-5 and Me-7 represent the results obtained with the monoclonal antibodies Me-1, Me-2, Me-3 and Me-6, and for the strains of the other bacteria tested, the binding activity shown for Me-7 represents the activity of other monoclonal antibodies recognizing the same strainBinding activity of the body.

The antigen conservation of the epitope recognized by the monoclonal antibody was also estimated. Dot-enzyme immunization is used to rapidly screen monoclonal antibodies against a large number of bacterial strains. This method was performed as described by the inventor (Lusier et al,journal of immunoassayPage 10, 373 (1989). 71 strains of Neisseria meningitidis were used in this study. The sample comprises 19 isolates of serotype group a, 23 isolates of serotype group B, 13 isolates of serotype group C, 1 isolate of serotype group 29E, 6 isolates of serotype groups W-135, 1 isolate of serotype group X, 2 isolates of serotype group Y, 2 isolates of serotype group Z, and 4 isolates ("NS") of bacteria that are not yet classified into serotype groups. These isolates were obtained from the following units: the caribbean epidemic center, spain port, terinida; eastern atlapus children hospital, ottawa, canada; department of Saskachewan Health, Regina, Canada; laboratoriare de Sant Publique Du Ouba, Montreal, Canada; max-plain institute fur Molekulare Genetik, Berlin, FRG; montreal children hospital, montreal, canada; the Victoria general Hospital, Halifax, Canada; as well as our own collection of strains. The following species were also tested: 16 Neisseria gonorrhoeae, 4 Neisseria gray, 5 Neisseria lactofermentum, 1 Neisseria flavivis, 1 Neisseria lilacinus, 3 Neisseria myxosa, 4 Neisseria profunda/Neisseria sicca, 5 Neisseria profunda, 1 Neisseria sicca, 1 Neisseria microflavia and 5 Moraxella catarrhalis, 1 Alcaligenes faecalis (ATCC8750), 1 Citrobacter freundii (ATCC 2080), 1 Edwardsiella tarda (ATCC 15947), 1 Enterobacter cloacae (ATCC 23355), 1 Enterobacter aerogenes (ATCC 13048), 1 Escherichia coli, 1 Flavobacterium odoratum, one of Haemophilus influenzae type b (Eagan strain), 1 Klebsiella pneumoniae (ATCC 13883), 1 Proteus reyi (ATCC 25932), 1 Proteus vulgaris (ATCC 13315), 1 Pseudomonas aeruginosa (ATCC 9027), 1 Salmonella typhimurium (ATCC 14028) and 1 Pasteurella marcescens (ATCC 8100))1 Shigella flexneri (ATCC 12022), 1 Shigella sojae (ATCC 9290). These bacteria are from the following units: collection by the American Standard culture Collection or Ottawa disease control laboratory center, Canada. The reactivity of the monoclonal antibodies with the most relevant strains of Neisseria is listed in Table 1. Me-7 this monoclonal antibody recognizes all 71 epitopes which detect its specificity on N.meningitidis strains. The monoclonal antibodies, as well as Me-2, Me-3, Me-5 and Me-6, also reacted with some other specific species belonging to the genus Neisseria, indicating that their specific epitopes are also expressed in some closely related species in the Neisseria family. The monoclonal antibody Me-1 reacts with an isolate from Neisseria meningitidis, in addition to being slightly reactive with Neisseria lactosamina. Me-1 was then tested further with additional 177 samples of N.meningitidis isolates, and as a result it correctly identified more than 99% of the total N.meningitidis strains tested. In addition to the neisserial strains listed in table 1, the monoclonal antibody is not reactive with any of the other species mentioned above.

In conclusion, the inventors have produced 6 monoclonal antibodies that specifically react with the 22kDa surface protein of Neisseria meningitidis. Using these monoclonal antibodies, we have revealed that their specific epitopes are 1) located on a proteinase K resistant 22kDa protein present on the outer membrane of Neisseria meningitidis, 2) conserved among isolates of Neisseria meningitidis, 3) exposed to the surface of untreated Neisseria meningitidis and accessible to antibodies, and 4) the reactivity of these monoclonal antibodies with the Neisseria meningitidis 22kDa surface protein is not affected by sodium periodate treatment, suggesting that their specific epitopes are not on carbohydrates.

Although we found that Neisseria meningitidis 22kDa protein migrates to 18kDa protein when heated under stringent conditions, we found that its migration is not affected by 2-mercaptoethanol treatment.

We also revealed that this Neisseria meningitidis 22kDa surface protein is not antigenically similar to lipoproteins, whereas lipoproteins are another low molecular weight and highly conserved protein present on the outer membrane of Neisseria meningitidis.

As will be shown in example 3, these monoclonal antibodies are also reactive with a pure, recombinant 22kDa surface protein obtained by transforming E.coli strain BL21 (DE3) with the plasmid vector pNP2202 containing the gene encoding the 22kDa surface protein of Neisseria meningitidis.

TABLE 1 reactivity of monoclonal antibodies with various isolates of Neisseria

Isolates of undifferentiated serogroups

Example 3 molecular cloning, Gene sequencing, high-throughput expression and purification of the Neisseria meningitidis 22kDa surface protein

A.Molecular cloning

A genomic library of lambda GEM-11 from Neisseria meningitidis 608B strain (B: 2 a: P1.2) was created according to the manufacturer's recommendations (Promega, McMerson, Wis.). Briefly, the genomic DNA of the 608B strain was partially digested with Sau 3AI, and a fragment of 9 to 23kb was purified on agarose gel and ligated to the BamHI site of the GEM-11 arm. The resulting recombinant phage was used to transfect E.coli LE392 strain (Promega), which was then plated on LB agar plates. The library was immunoscreened using the Neisseria meningitidis 22kDa surface protein-specific monoclonal antibody of example 2 as follows, resulting in 19 positive plaques. The plate was left at-20 ℃ for 15 minutes to harden the upper agar. The nitrocellulose membrane was gently placed on the surface of the plate at 4 ℃ for 30 minutes to adsorb the proteins produced by the recombinant virus clones. The membrane was then washed with PBS-Tween 0.02% (v/v) and the immunoblots described above (Lusier et al,journal of immunoassayPage 10, 373 (1989).After amplification and DNA purification, one virus clone, designated clone 8, containing the 13kb insert was selected for subcloning. After digestion of the clone with SacI, two fragments of 5 and 8kb were obtained. These two fragments were agarose gel purified and ligated into the SacI restriction site of the low copy number plasmid pWKS30 [ Wang and Kushner, Gene 100, p.195 (1991) ]. This recombinant plasmid was used to transform E.coli JM 109 strain (Promega), and the transformation was carried out by electroporation (Bio-Rad, Mississauga Ont., Canada) according to the manufacturer's recommendations. The resulting clones were screened with the Neisseria meningitidis 22kDa surface protein-specific monoclonal antibody of example 2. Only the bacteria transformed with the plasmid containing the 8kb insert were positive for cloning. Western blot analysis of positive clones (see example 2 for methods) showed that the protein expressed by E.coli was intact and its migration on SDS-PAGE gels was consistent with the 22kDa surface protein of Neisseria meningitidis. The insert length was further reduced by digesting the clone containing the 8kb insert with Cla I to give a 2.75kb fragment, which was ligated into the Cla I site of the plasmid pWKS 30. Western blot analysis of the resulting clones again clearly showed that the E.coli expressed protein was intact and its migration on SDS-PAGE gels was consistent with the native Neisseria meningitidis 22kDa surface protein.

Restriction analysis showed that the two clones designated pNP2202 and pNP2203 contained a 2.75kb insert and were used in the opposite orientation to proceed with the sequence analysis of the gene encoding the Neisseria meningitidis 22kDa surface protein. "double stranded nesting deletion kit" purchased from pharmacia Biotechnology company (Piscataway, NJ) was used to generate a series of nesting deletions from the two clones, the procedure was performed as provided by the manufacturer, the resulting truncated inserts were used for sequencing, and M from the pWKS30 vector was sequenced₁₃The forward primer was performed using an automated sequencer available from applied biosystems Inc. (Foster City, Calif.), model 373A, according to the manufacturer's instructions, using the kit "Taq staining deoxyterminator cycle sequencing kit".

B.Sequence divisionAnalysis of

After sequencing the insert from both directions, the nucleic acid sequence showed an open reading frame of 525 nucleotides (including stop codon), the encoded protein consisted of 174 amino acid residues with the expected molecular weight of 18,000 Dalton and pI of 9.93, and the nucleic acids and resulting amino acid sequences are shown in FIG. 1(SEQ ID NO: 1, SEQ ID NO: 2).

To verify correct expression of the cloned gene, the N-terminal amino acid sequence of the native 22kDa surface protein from the Neisseria meningitidis 608B strain was determined for comparison with the deduced amino acid sequence from the nucleic acid sequence. Outer membrane preparations from the neisseria meningitidis 608B strain were separated by electrophoresis on a 14% SDS-PAGE gel and run as previously described [ Sambrook et al,molecular cloning: experimental guidelinesCold spring harbor laboratory Press (1989) on polyvinylidene fluoride membranes (Millipore Products, Bedford MA). The 22kDa protein band was excised from the gel and Edman degraded using an automated protein sequencer model 473A from Applied biosystems. The amino acid sequence E-G-A-S-G-F-Y-V-Q-A corresponds to amino acids in the open reading frame 1-10 (SEQ ID NO: 2), indicating that the 22kDA surface protein of the NeisseriA meningitidis 608B strain has A 19 amino acid leader peptide (amino acid residues-19 to-1 in SEQ ID NO: 2).

A search of the existing database showed that the 22kDa surface protein (SEQ ID NO: 2) or the gene thereof (SEQ ID NO: 1) of this strain of Neisseria meningitidis 608B has not been previously reported.

C.High-yield expression and purification of recombinant neisseria meningitidis 22kDa surface protein

The following procedure was developed in order to maximize and purify the production of recombinant Neisseria meningitidis 22kDa expressed in E.coli. This method is based on the observation that the recombinant 22kDa surface protein produced by the E.coli BL21 (DE3) strain harboring plasmid pNP2202 is present in large amounts in the outer membrane, but is also available in the culture supernatant, which is the most abundant protein. The culture supernatant was used to purify the recombinant 22kDa protein using an affinity chromatography column (FIG. 6A).

Monoclonal antibodies Me-2, Me-3 and Me-5 (described in example 2) were immobilized on cyanogen bromide-activated Sepharose 4B (pharmacia Biotech, NJ) according to the manufacturer's instructions to produce a matrix for an affinity column.

The E.coli BL21 (DE3) strain harboring the plasmid pNP2202 cultured overnight was inoculated into LB liquid medium (Gibco Laboratories, Grand Island, N.Y.) containing 25mg/ml of ampicillin (Sigma) and cultured with shaking at 37 ℃ for 4 hours to prepare a culture supernatant. The mycelia were removed from the medium by 2 times centrifugation at 4 ℃ for 10 minutes at 10,000 g. The culture supernatant was filtered through a 0.22mm membrane (Millipore, Bedfords, Mass.), and then concentrated about one hundred fold with a 10,000Daltons per cut-off ultrafiltration membrane (Amicon Co., Bererly, Mass.). To completely dissolve the membrane particles, EmpigenBB (Calbiochem co., LaJolla, CA) was added to the concentrated culture supernatant to a final concentration of 1% (vol.) suspension at room temperature for 1 hour, followed by several liters of 10mm tris-HCl buffer containing 0.05% Empigen BB (vol.) at pH 7.3, dialysis, and centrifugation at 10,000g for 20 minutes at 4 ℃. The antigen preparation was added to the affinity matrix and left overnight at 4 ℃ with constant shaking. The gel was injected onto a column and washed repeatedly with 10mM Tris-HCl buffer containing 0.05% Empigen BB (by volume). The recombinant 22kDa protein was then eluted with 1M LiCl in 10mM Tris-HCl buffer pH 7.3. The solution containing the eluted protein was dialyzed repeatedly against several liters of 10mM Tris-HCl buffer, pH 7.3, containing 0.05% Empigen BB. During each purification step, SDS-Page gel was stained with Coomassie blue and silver (Tsaiand Frash, analytical biochemistry)Analyzing biochemistry119, page 19 (1982), to estimate the purity of the recombinant 22kDa surface protein, representative results are given in FIG. 6A. Silver staining of the gel indicated that the purification process produced very pure recombinant 22kDa protein with only very small amounts of E.coli lipopolysaccharide.

The resistance of the purified recombinant 22kDa surface protein to proteolysis was also demonstrated, and the results are given in fig. 6B. Purified recombinant 22kDa surface protein 2mg/mg protein of alpha-chymotrypsin and trypsin as described in example 1 and 2IU proteinase K per mg protein were treated at 37 ℃ for 1 hour with shaking. No reduction in protein amount was observed after any treatment. In contrast, the control protein [ in this case bovine serum albumin (BSA, Simga) ] is partially or completely degraded, depending on the enzyme chosen. Further, longer treatment does not result in any change in the protein. These latter results indicate that the transformed E.coli can express the complete recombinant 22kDa surface protein and that this recombinant protein, like the naturally occurring protein of Neisseria meningitidis, is also strongly resistant to hydrolysis by three proteolytic enzymes. Furthermore, the purified recombinant 22kDa surface protein which is not embedded in the outer membrane of E.coli remains highly resistant to the action of proteolytic enzymes.

We also demonstrated the effect of enzyme treatment on the antigenic properties of the recombinant 22kDa protein. The monoclonal antibody described in example 2 was judged to recognize the recombinant 22kDa surface protein purified by the procedure described above by ELISA and Western immunoblotting (FIG. 6C). Further, the reactivity of the monoclonal antibody Me-5 and other 22kDa protein-specific monoclonal antibodies with the purified recombinant 22kDa surface protein was not altered by enzyme treatment, demonstrating that the antigenicity of the recombinant 22kDa protein appears to be similar to that of the native protein.

Important data are shown in example 3 and can be summarized as follows:

1) the complete nucleotide and amino acid sequence of the neisseria meningitidis 22kDa surface protein was obtained (SEQ ID NO: 1; SEQ ID NO: 2) (ii) a

2) The natural protein N-terminal sequence analysis proves that the Neisseria meningitidis 22kDa gene is indeed cloned;

3) this protein has not been described previously;

4) can transform a host such as Escherichia coli and obtain high-yield expression of the recombinant Neisseria meningitidis 22kDa surface protein;

5) a recombinant protein free of other neisseria meningitidis molecules is obtained and the protein is substantially free of fractions of e.coli;

6) the purified recombinant 22kDa surface protein remains strongly resistant to hydrolysis by e.g.alpha-chymotrypsin, trypsin and proteinase K; and is

7) The antigenicity of the recombinant 22kDa protein is identical to that of the native Neisseria meningitidis 22kDa surface protein.

Example 4 molecular conservation of the Gene encoding the Neisseria meningitidis 22kDa surface protein

To demonstrate the molecular conservation of the gene encoding the Neisseria meningitidis 22kDa surface protein among various isolates within the Neisseria genus, DNA dot hybridization was performed to detect different Neisseria species and other bacterial species. First, the gene encoding the 525 base pairs of Neisseria meningitidis 22kDa surface protein was amplified by PCR, purified on agarose gel and randomly primed using a nonradioactive digoxin DNA tagging and detection system (Boehringer, Laval, Canada) according to the manufacturer's instructions.

Dot blots were performed according to the manufacturer's instructions (Boehringer Mannheim). Briefly, the bacterial strains to be tested were spotted onto a charged nylon membrane (Boehringer Mannheim), dried and then prehybridized and hybridized at 42 ℃ in a solution containing 50% formamide (Sigma) according to the colony transfer instructions in the digoxin user's guide. The prehybridization solution contained 100mg/ml denatured herring sperm DNA (Boehringer Mannheim) as a supplemental blocking agent to avoid non-specific hybridization of the DNA probe. Stringent rinsing and detection with the chemiluminescent acceptor PPD-substrate were performed according to the user's instructions for the digoxin system.

The results obtained with the monoclonal antibody Me-7 were identical to those obtained with the 525bp DNA probe for the 71 Neisseria meningitidis detected. According to this result, all the strains of Neisseria meningitidis examined contained the Neisseria meningitidis 22kDa surface protein gene and all expressed proteins, since they were all recognized by monoclonal antibodies. This demonstrated that the protein is highly conserved among neisseria meningitidis isolates (table 2).

The DNA probe also detected that the gene encoding the Neisseria meningitidis 22kDa surface protein was also present in all detected strains of Neisseria gonorrhoeae.

In contrast, the monoclonal antibody Me-7 reacted only with 2 of the 16 N.gonorrhoeae strains tested, indicating that in N.gonorrhoeae the specific epitope was either not present in some way, inaccessible or modified, or that the vast majority of N.gonorrhoeae strains did not express the protein, even though they had coding sequences in their genomes (Table 2).

Good agreement was also observed between the two assays in Neisseria lactosami, since only 1 species of Neisseria lactosami was found to be a gene but not to express a protein (Table 2). The results can also be explained by the reasons in the last paragraph.

This may indicate that, although the 22kDa protein on the surface of a strain of Neisseria gonorrhoeae is not expressed or not accessible, the gene encoding the 22kDa protein of Neisseria gonorrhoeae or Neisseria lactosyli may be used in the construction of recombinant plasmids for the production of the 22kDa surface protein or analogues thereof, all of which may be used for protection, detection or diagnosis of Neisseria infection. More specifically, such infections may be from Neisseria meningitidis, Neisseria gonorrhoeae and Neisseria lactosyli. Thus, the 22kDa surface protein or an analogue thereof may be used for the production of a vaccine to combat such infections. In addition, the 22kDa protein or an analogue thereof may also be used for the production of a kit for detecting or diagnosing such an infection.

The results obtained with Moraxella catarrhalis showed that 3 of the 5 strains tested reacted with the monoclonal antibody Me-7, but none reacted with the DNA probe, indicating that the gene encoding the 22kDa surface protein of Neisseria meningitidis was absent from the genome of these strains (Table 2).

Several other neisserial and other bacteria were also tested (see footnote, table 2) and found to be positive in none of the two tests. The latter result appears to indicate that the gene for the 22kDa surface protein is present only in closely related species of the Neisseria family.

TABLE 2525 base pairs reactivity of DNA probes and Me-7 monoclonal antibody with different Neisseria species

(footnotes of table 2):

the following neisseria and other bacteria were also tested in two ways and the results were negative: 1 Neisseria grayi, 1 Neisseria flavus, 1 Neisseria buffalo, 2 Neisseria myxosa, 4 Neisseria profunda/Neisseria sicca, 1 Neisseria profunda, 1 Neisseria sicca, 1 Neisseria microflavia, 1 Alcaligenes faecalis (ATCC8750), 1 Bordetella pertussis (ATCC 9340), 1 Bordetella bronchiseptica, 1 Citrobacter freundii (ATCC 2080), 1 Edwardsiella tarda (ATCC 1594), 1 Escherichia coli, 1 Flavobacterium odoratum, one of the Haemophilus influenzae types b (Eagan strain), 1 Klebsiella pneumoniae (ATCC 13883), 1 Lewy-warburghii (ATCC 25932), 1 Proteus vulgaris (ATCC 13315), 1 Pseudomonas aeruginosa (ATCC 9027), 1 Salmonella typhimurium (ATCC 14028), 1 Serratia marcescens (ATCC 8100), 1 Shigella flexneri (ATCC 12022), 1 Shigella sojae (ATCC 9290), and 1 Xanthomonas maltophilia.

In conclusion, DNA hybridization experiments clearly showed that the gene encoding the 22kDa surface protein of Neisseria meningitidis is highly conserved among pathogenic Neisseria. Furthermore, the results clearly show that DNA probes can be a useful tool for rapid and direct detection of pathogenic neisseria bacteria in clinical specimens. The probe may be even further refined to distinguish between neisseria meningitidis and neisseria gonorrhoeae.

EXAMPLE 5 bacterial lysis of monoclonal antibodies and their protective characteristics

Following the previously described method [ Brodeur et al,infection and immunity50, page 510 (1985); martin, etc. in the prior art,infection and immunity60, 2718(1992), the ability of the purified monoclonal antibody specific for the 22kDa surface protein of N.meningitidis to lyse bacteria was assessed in vitro. The purified monoclonal antibodies Me-1 and Me-7 were effective in killing Neisseria meningitidis strain 608B in the presence of guinea pig serum complement. Relatively low concentrations of each monoclonal antibody reduced viable bacteria by more than 50%. The use of higher concentrations of the purified monoclonal antibodies Me-1 and Me-7 resulted in a drastic reduction (to 99%) in the number of bacterial colony forming units. Importantly, the bactericidal activity of these monoclonal antibodies is complement dependent in that the bactericidal activity is completely lost after heat inactivation of guinea pig serum at 56 ℃ for 30 minutes. Other monoclonal antibodies did not show significant bactericidal activity against the same bacterial strain. Representative results for a combination of several experiments are given in FIG. 7, where the results for Me-7 are consistent with those for Me-1. The results for Me-2 shown are representative and consistent with those obtained for the other monoclonal antibodies Me-3, Me-5 and Me-6.

The model of infection in mice previously described by one of the inventors [ Brodeur et al,infection and immunity Epidemic disease50, page 510 (1985); brodeur et al, in general,canadian journal of microbiology32, 33 (1986) was used to measure the protection of each monoclonal antibody. Briefly, Balb/c mice were injected intraperitoneally 18 hours prior to bacterial challenge with 600ml of ascites fluid containing monoclonal antibodies. Mice were then challenged with 1ml of a suspension containing 4% mucin (Sigma), 1.6% hemoglobin (Sigma) and 1000 clonogenic units of neisseria meningitidis 608B strain. Several experimentsThe total results of (a) are shown in Table 3. It is important to note that only the monoclonal antibodies Me-1 and Me-7, which have lytic capacity, protect mice against infection by experimental Neisseria meningitidis. In fact, injection of ascites fluid containing both monoclonal antibodies prior to bacterial challenge greatly increased the survival of Balb/c mice to 70% or more, whereas the survival of the control group population receiving 600mlsp 2/0-induced ascites fluid or 600ml of ascites fluid containing unrelated monoclonal antibodies was only 9%. The results also show that injection of 400Hg protein a purified Me-7 18 hours prior to bacterial challenge can survive 80% of the mice. The experiments that were performed thereafter were used to determine the minimum antibody concentration required to protect 50% of mice from survival. For other 22kDa surface protein-specific monoclonal antibodies of Neisseria meningitidis, lower survival rates were observed: from 20% to 40%.

TABLE 3 Neisseria meningitidis 22kDa against Neisseria meningitidis 608B strain (B: 2 a: P1, 2)

Evaluation of immunoprotection Capacity of monoclonal antibodies specific to surface proteins

In conclusion, these results clearly demonstrate that antibodies specific for the Neisseria meningitidis 22kDa surface protein can effectively protect mice against challenge by experimental lethal bacteria. Protective antibodies induced by the antigen are one of the most important criteria used to determine whether to proceed with further studies of potential vaccine candidates.

Example 6 protection against bacterial challenge following immunization with purified recombinant 22kDa surface protein

The purified recombinant 22kDa surface protein was prepared according to the protocol provided in example 3 and was then used to immunize Balb/c mice to determine its protective effect against challenge with a lethal amount of Neisseria meningitidis 608B strain (B: 2 a: P1.2). In the experiments carried outIn order to ensure that no other meningitis antigens are contained in the vaccine preparation, it was decided to replace the native meningitis protein with a purified recombinant protein. The model of infected mice used in these experiments has been previously described by one of the inventors [ Brodeur et al,infection and immunity50, page 510 (1985); brodeur et al, in general,canadian journal of microbiologyPages 32, 33 (1986). Each mouse was injected three times subcutaneously with 100ml of antigen preparation containing 10 or 20. mu.g of purified recombinant 22kDa surface protein, at 3 week intervals. Quil A was used as an adjuvant in these experiments at a concentration of 25. mu.g per injection. Control mice were injected in the same manner with 10 or 20. mu.g of BSA and 20. mu.g of culture supernatant of concentrated E.coli strain BL21 (DE3) carrying plasmid pWKS30 without the insertion of the gene encoding Neisseria meningitidis protein, prepared as described in example 3. Or injecting phosphate buffer solution into the control group. Before each injection, a serum sample was taken from each mouse in order to analyze the progress of the immune response against the recombinant protein. 2 weeks after the third immunization, all mice were injected intraperitoneally with 1ml of a suspension containing 1000 clonogenic units of the strain Neisseria meningitidis 608B, which also contained 4% mucin (Sigma) and 1.6% hemoglobin (Sigma).

The results of these experiments are listed in table 4. 80% of mice immunized with purified recombinant 22kDa surface protein survived bacterial challenge, whereas the survival rate in the control group was only 0% to 42%. Importantly, mice injected with concentrated E.coli culture supernatant in the control group were not resistant to bacterial challenge. The latter results clearly indicate that the composition in the medium or the small amount of retained e.coli antigen after purification is not responsible for the observed resistance against neisseria meningitidis.

TABLE 4 protection against subsequent challenge with a meningococcal 608B strain (B: 2 a: P1.2) obtained after immunization with a purified recombinant 22kDa surface protein

Conclusion

Injection of the purified recombinant 22kDa surface protein greatly protected immunized mice from lethal infection by N.meningitidis according to the invention, antibodies are exemplified by the monoclonal antibodies Me-1 and Me-7 produced by murine hybridoma cell lines, which were deposited at 21.7.1995 in the American Type Culture Collection (ATCC) of Lokville, Mland, USA. The accession numbers are HB11959(Me-1) and HB11958 (Me-7).

Example 7 sequence analysis of other strains of Neisseria meningitidis and Neisseria gonorrhoeae

A2.75 kb ClaI digested DNA fragment containing the gene encoding the 22kDa surface protein was isolated from genomic DNA of different strains of Neisseria meningitidis and Neisseria gonorrhoeae as described in example 3.

a) MCH88 strain: the nucleic acid sequence of the MCH88 strain (clinical isolate) is shown in FIG. 8(SEQ ID NO: 3). The putative leader peptide sequence, corresponding to amino acids-19 to-1 (M-K-K-A-L-A-L-I-A-L-A-L-P-A-A-A-L-A) was deduced from experimental evidence obtained for strain 608B (example 3).

A search of the established database demonstrated that the 22kDa surface protein or gene thereof (SEQ ID NO: 3) from the strain MCH 188 of Neisseria meningitidis (SEQ ID NO: 4) has not been previously reported.

b) Z4063: z4063 strain [ Wang J.F.,infection and immunity60, page 5267 (1992) the nucleic acid sequence is shown in FIG. 9(SEQ ID NO: 5) the putative leader peptide sequence, corresponding to amino acids-19 to-1 (M-K-K-A-L-A-T-L-I-A-L-A-L-P-A-A-L-A), was deduced from experimental evidence obtained for strain 608B (example 3). Searching the established database demonstrated that the 22kDa surface protein or gene thereof (SEQ ID NO: 5) from the Neisseria meningitidis strain Z4063 (SEQ ID NO: 6) has not been previously reported.

c) Neisseria gonorrhoeae strain b 2: the nucleic acid sequence of strain Neisseria gonorrhoeae b2 (Neisseria serotype 1 national Foundation center, LCDC. Ottawa, Canada) is shown in FIG. 10(SEQ ID NO: 7). From experimental evidence obtained from strain 608 (example 3), putative leader peptide sequences were deduced, corresponding to amino acids-19 to-1 (M-K-K-A-L-A-A-L-I-A-L-A-L-P-A-A-A-L-A). Searching the established database demonstrated that the 22kDa surface protein from strain b2 of Neisseria gonorrhoeae (SEQ ID NO: 8) or its gene (SEQ ID NO: 7) has not been previously reported.

FIG. 11 shows the same sequence among the DNA sequences of all four strains that have been tested. The strain MCH88 has a codon (TCA) insertion at nucleotide 217, but in general, the four strains have striking homology.

FIG. 12 shows the homology of amino acid sequences not deduced for the four strains. In four strains, there was greater than 90% identity.

Example 8 immune response of rabbits and monkeys to the 22kDa surface protein of Neisseria meningitidis

Rabbits and monkeys were immunized with the recombinant 22kDa protein to assess antibody responses in species other than mice.

a) Rabbit

Male New Zealand rabbits were immunized with the outer membrane preparation of E.coli JM 109 strain harboring plasmid pN2202 or control plasmid pWKS30 (strains and plasmids are described in example 3). According to the method previously described by the inventors [ Brodeur et al, InfectInfection and immunity50, page 510 (1985) was extracted with lithium chloride to obtain an outer membrane preparation. The composition of the protein in the preparation was determined using the Lowry method applicable to membrane fractions [ Lowry et al,journal of biochemistry193, 265 (1951). Rabbits were injected subcutaneously and intramuscularly at several sites twice with 3 weeks of one of the outer membrane preparations described above at 150 μ g. QuilA was used as an adjuvant for these immunization experiments at a final concentration of 20% (by volume). The development of specific humoral responses was analyzed by ELISA and Western immunoblotting as described previously by the inventors [ ]Brodeur et al, in general,infection and immunity50, page 510 (1985); martin, etc. in the prior art,european journal of immunology18, 601, (1988), wherein the ELISA was coated with an antigen prepared from outer membrane extracted from Neisseria meningitidis 608B strain (B: 2 a: P1.2), alkaline phosphatase or peroxidase-labeled donkey anti-rabbit immunoglobulins were used in these methods (Jackson Immuno research laboratories, West Grove, Pa.).

Injection of the E.coli outer membrane preparation containing the 22kDa recombinant protein with QuilA adjuvant induced a strong specific humoral response in rabbits as determined by ELISA to be 1/32,000 (FIG. 13). Antibodies induced following injection of the recombinant 22kDa protein reacted with the purified recombinant 22kDa protein, but more importantly they also recognized native proteins expressed, folded and mosaicked on the outer membrane of Neisseria meningitidis. Western immunoblot experiments clearly showed that the antibodies generated after the second injection recognized the same protein band on nitrocellulose membrane as did Mab Me-2 (described in example 2), whereas Me-2 was specific against the 22kDa protein.

b) Monkey

Two cynomolgus monkeys were co-injected with 100. mu.g (K28) and 200. mu.g (I276) of affinity chromatography-purified recombinant 22kDa protein, respectively. The method for producing and purifying the protein of E.coli strain BL21DE3 is described in example 3. Alhydrogel (Cedarlane Laboratories, Hornby, on., Canada) was used as an adjuvant for these immunoassays at a final concentration of 20% (by volume). Monkeys were given two intramuscular injections at three week intervals. Control monkeys (K65) were immunized with an unrelated recombinant protein preparation following the same procedure. Serum was analyzed as described above. In these methods, alkaline phosphatase or peroxidase-labeled goat anti-human immunoglobulin (Jackson ImmunoResearch Laboratories, WestGrove, PA) is used.

The specific antibody response of K28 monkeys immunized with 100 μ g of purified protein per injection appeared faster and stronger than that of I276 monkeys injected with 200 μ g of protein (fig. 14). Antibodies specific for the native 22kDa protein were detected 21 days after the first injection in sera from immunized monkeys using Western immunoblotting, but no antibodies were present in sera from control monkeys after the second control antigen injection.

Conclusion

The data presented in examples 2 and 5 clearly show that injection of the recombinant 22kDa protein induces a protective humoral response in mice directly against Neisseria meningitidis. More importantly, the results in this example show that the immune response is not restricted to one species, and that the recombinant surface proteins can also stimulate the immune system of other species such as rabbits and monkeys.

Example 922 epitope map of Neisseria meningitidis protein

The epitope of the 22kDa surface protein of N.meningitidis can be mapped using the method described by the inventors [ Martin et al,infection and immunity(1991): 59: 1457-1464 ]. The linear epitopes were successfully identified using 18 overlapping synthetic peptides covering the entire sequence of the Neisseria meningitidis 22kDa protein from strain 608B (FIG. 15) and immunised with this protein to give hyperimmune serum. The identification of an immunological determinant on the 22kDa protein contributes to the design of new effective vaccines. Further, the localization of these B-cell epitopes also provides valuable information on the structural configuration of the protein on the outer membrane of neisseria meningitidis.

All peptides were synthesized by BioChem immunology systems inc (montreal, canada) using an automated peptide synthesizer from Applied Biosystems (Foster City Calif). The synthetic peptide was purified by reverse phase high pressure liquid chromatography. The peptides CS-845, CS-847, CS-848, CS-851, CS-852 and CS-856 (FIG. 15) were dissolved in a small volume of 6M guanidine hydrochloride (J.T.Baker) or dimethyl sulfoxide (J.T.Baker). The peptides were then adjusted to 1mg/ml with distilled water. All other peptides were dissolved in distilled water and adjusted to 1 mg/ml.

Synthetic peptides were coated at a concentration of 50 μ g/ml onto microtiter plates (Immulon 4, Dynatech Laboratories Inc., Chantilly, Va.) in 50mM carbonate buffer at pH 9.6 for enzyme-linked immunosorbent assays (ELISA) of peptides. After overnight incubation at room temperature, the plates were washed with Phosphate Buffered Saline (PBS) containing 0.05% (v/v) Tween 20 and then blocked with PBS containing 0.5% (w/v) bovine serum albumin (Sigma). Mice or monkeys were immunized with the affinity purified recombinant 22kDa surface protein, then sera were taken and diluted, and 100. mu.l of the dilution was added to each well of the ELISA plate and incubated at 37 ℃ for 1 hour. Plates were washed three times and 100. mu.l of alkaline phosphatase-linked goat anti-mouse or anti-human immunoglobulin diluted as recommended by the manufacturer (Jackson ImmunoResearch Laboratories, West Grove, Pa.). After incubation for 1 hour at 37 ℃ the plates were washed and 100. mu.l diethanolamine (10% by volume pH9.8) containing 1mg/ml p-nitro-phenylphosphoric acid (Sigma) was added. After 60 minutes, the change in optical density of the reaction (λ 410nm) was read with a microtiter plate reader.

Antiserum obtained after immunization of mice and monkeys with the affinity chromatography purified recombinant 22kDa protein (example 8) was successfully used together with 18 synthetic peptides overlapping each other to localize B-cell epitopes on the protein, which were clustered in the three antigenic domains of the protein.

The first region is between amino acid residues 51 and 86. Computer analysis with different algorithms suggests that this region is most likely immunologically important because it is hydrophilic and surface exposed. Further, comparison of the four protein sequences listed in FIG. 12 shows that one of the major changes, namely the insertion of an amino acid residue at position 73, is also located in this region.

The second antigenic region identified by the antisera is located between amino acid residues 110 and 140. Interestingly, sequence analysis indicated that 7 of the 14 amino acids that were not conserved in the four protein sequences were clustered within this region of the protein.

The third antigenic domain is located within a highly conserved region of the protein, between amino acids 31 and 55, which is recognized only by monkey serum.

Example 10 Large-Scale production of the Heat-inducible expression vector for the 22kDa surface protein

The gene encoding the neisseria meningitidis 22kDa surface protein was inserted into plasmid p629 [ George et al, biology and engineering (Bio/technology) 5: 600 to 603 (1981). Plasmid p629 controls the synthesis of the 22kDa surface protein with the pL promoter and carries a break in the bacteriophage lambda cI857 temperature sensitive suppressor gene, but the functional Pr promoter of this gene has been deleted. The cI857 repressor can be inactivated by varying the temperature from 30 ℃ to above 38 ℃ with the result that the protein encoded by the plasmid is expressed. In large scale fermentations, it is advantageous to use temperature changes to induce expression of genes in E.coli, since this can be done simply with modern fermenters. Other induction plasmids typically require the addition of a specific molecule such as lactose or isopropylthio-beta-D-galactoside (IPTG) to the medium to induce expression of the gene of interest.

A540 nucleotide fragment was PCR amplified from the genomic DNA of strain 608B Neisseria meningitidis using the following primers: OCRR 8:

5'-TAATAGATCTATGAAAAAAGCACTTGCCAC-3' and OCRR 9: 3 '-CACGCGCAGTTTAAGACTTCTAGATTA-5'. These two primers correspond to the nucleotide sequences of both ends of the 22kDa gene. To clone the PCR product, a BgI II (AGATCT) site was added to the primer. The PCR product was purified on agarose gel and digested with BglII. This 525 base pair BglII fragment was then inserted into the BglII and BamHI sites of plasmid p 629. The plasmid containing the PCR product insert was designated pNP2204 and used to transform E.coli strain DH 5. alpha.F' IQ. A partial map of plasmid pNP2204 is shown in FIG. 16. The resulting plaques were screened with a monoclonal antibody specific for the Neisseria meningitidis 22kDa surface protein as described in example 2. Western blot analysis of the resulting clones clearly showed that the protein synthesized by E.coli was intact and that its migration on SDS-PAGE gels was identical to that of the native Neisseria meningitidis 22kDa surface protein. DNA was extracted from selected clones and sequenced. The nucleic acid sequence of the insert in the plasmid is identical to the sequence of the gene encoding the Neisseria meningitidis 22kDa protein shown in FIG. 1.

To investigate the synthesis level of this 22kDa surface protein, the temperature-inducible plasmid pNP2204 was used to transform the following E.coli strains W3110, JM 105, BL21, TOPP1, TOPP2 and TOPP 3. The level of synthesis of the 22kDa surface protein in each strain and its localization in different cell fractions was determined. The expression of the gene was efficiently induced by culturing in LB broth (Gibco BRL, Life technologies grade Island, NY) with shaking in a Erlenmeyer flask. Time course measurements of the level of synthesis indicated that the protein appeared 30 min after induction and that the amount of the protein increased steadily during the induction period, the results of the SDS-PAGE gels being identical to the previous results. For E.coli strains W3110 and TOPP1, an expression level of between 8 and 10mg of the 22kDa protein per liter of culture was determined. In both strains, the majority of the 22kDa protein is incorporated into the outer membrane of the bacterium.

Example 11 purification of Neisseria meningitidis 22kDa protein

Since most of the 22kDa protein was found to be embedded in the outer membrane of the E.coli strain, the purification scheme here was different from that of example 3, in which a large amount of protein was released into the culture supernatant. Escherichia coli W3110 or TOPP1 strain carrying plasmid pNP2204 was inoculated in LB broth containing 50. mu.g/ml of ampicillin (Sigma) overnight at 30 ℃, and was rotary cultured (250rpm) at 30 ℃ until the cell density was 0.6 (. lamda. gtoreq.600 nm), at which point the culture temperature was changed to 39 ℃ and the culture was carried out for 3 to 5 hours to also induce protein production. The cells were collected by centrifugation at 8000g for 15 minutes at 4 ℃ and washed twice in Phosphate Buffered Saline (PBS) at pH 7.3. The cells were disrupted by sonication (either by impact disruption or mechanical disruption using a bacterial disruptor). The unbroken cells were centrifuged at 5,000g for 5 minutes to remove and discard. The outer membrane was separated from the cytoplasm and fractionated by centrifugation at 10,000g for 1 hour at 10 ℃. The membrane-containing pellet was resuspended in a small amount of PBS pH 7.3. To dissolve the 22kDa surface protein from the membrane, detergents such as Empagem BB (Calbiochem Co., Lajolla, Calif.), Zwiffergent-3.14(Calbiochem Co., Ltd.) or beta-octyl glucoside (Sigma) are used. The final concentration of detergent added to the membrane fraction was 3%, and the mixture was incubated at 20 ℃ for 1 hour. Insoluble material was removed by centrifugation at 100,000 for one hour at 10 ℃.

The 22kDa protein is efficiently solubilized by three detergents, but the advantage of β -octyl glucoside is that it can be used to easily remove several unwanted membrane proteins, which are insoluble in it, and thus can be separated from the supernatant by centrifugation. The supernatant containing the 22kDa protein was dialyzed repeatedly against PBS buffer to remove the detergent. Proteinase K treatment (described in example 1) can be used to further remove unwanted proteins from the 22kDa surface protein preparation. Fractional precipitation with ammonium sulfate or organic solvents and ultrafiltration are additional steps that remove unwanted nucleic acids and lipopolysaccharides. After this step, gel filtration and ion exchange chromatography can be used to obtain the pure 22kDa protein. Affinity chromatography, as described in example 3, can also be used to purify this 22kDa protein.

Example 12 use of the 22kDa protein as a vaccine for humans

To prepare a vaccine for human use, an appropriate 22kDa surface protein antigen is selected from the polypeptides described herein. For example, one skilled in the art can design a vaccine for use with a 22kDa polypeptide or fragment thereof that contains immunogenic epitopes. The use of molecular biological techniques is particularly suitable for the preparation of substantially pure recombinant antigens.

The components of the vaccine may take different forms. These include, for example, solid, semi-solid and liquid dosage forms, such as powders, liquid solutions or suspensions, and liposomes. Based on our belief that the 22kDa surface protein antigen of the present invention induces a protective immune response when administered to a human, the compositions of the present invention will resemble other proteins and polypeptides such as tetanus and diphtheria which are used to immunise humans. Thus, the compositions of the present invention will preferably include a pharmacologically acceptable adjuvant such as incomplete Freund's adjuvant, aluminum hydroxide, muramyl peptide, water-oil emulsion, liposome, ISCOM or CTB, or cholera toxin in the form of a non-toxic B subunit. Most preferably the composition comprises an oil-in-water emulsion or aluminium hydroxide as an adjuvant.

The composition will be administered to the patient in one of a variety of pharmacologically acceptable forms, including intramuscular, intradermal, subcutaneous, or topical administration. Preferably, the vaccine will be administered intramuscularly.

Generally, the dosage will comprise a first injection, most with adjuvant, of about 0.01 to 10mg, more preferably 0.1 to 1.0mg, of the 22kDa surface protein antigen per patient, most likely followed by one or more further booster injections. Preferably, the booster injection will be performed 1 to 6 months after the initial injection.

One issue to consider in the development of vaccines is mucosal immunity. The ideal mucosal immune vaccine is capable of inducing the generation of systemic immunity and protective antibodies on the appropriate surface after one or more oral or intranasal doses. Mucosal vaccine compositions may include adjuvants, inert particulate carriers or recombinant live vectors.

The anti-22 kDa surface protein antibodies of the invention are useful in passive immunotherapy and in immunoprophylaxis of N.meningitidis or related bacteria such as N.gonorrhoeae or N.lactosyli which infect humans. The dosage form and system for passive immunization are similar to other passive immunotherapy vaccines.

Exemplary antibodies according to the present invention are hybridoma-produced MABs Me-1 and Me-7 antibodies, which have been deposited at 21.7.1995 in the American Type Culture Collection (ATCC) of rockville, maryland, and identified as murine hybridoma cell lines, Me-1 and Me-7, respectively. These deposits have accession numbers HB11959(Me-1) and HB11958 (Me-7).

While we have described herein a number of specific embodiments of the invention, it is apparent that other embodiments of the compositions and processes of this invention can be provided by modifying the basic embodiments. It is therefore to be understood that the scope of the invention includes all such modified embodiments and modifications as defined in the foregoing specific claims and which are defined in the appended claims; and the invention is not limited to the specific embodiments set forth herein by way of example.

Sequence listing

(1) General information:

(i) the applicant: brodeur, Bernard R

Martin，Denis

Hamel，Josee

Rioux，Clement

(ii) Title of the invention: neisseria meningitidis surface proteins resistant to proteinase K degradation

(iii) Sequence number: 26

(iv) Communication address:

(A) address: goudreau Gage Dubuc & Martineau Walker

(B) Street: 800Place Victoria, Suite 340O, Tour de la Bourse

(C) City: montreal

(D) State: kuibek

(E) The state is as follows: canada

(F) And E, postcode: H4Z 1E9

(v) A computer-readable form:

(A) media type: flexible disk

(B) A computer: IBM personal computer compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patertin Release #1.0, Version 1.25 Version

(vi) Data of the current application:

(A) application No.:

(B) application date:

(C) the category:

(vii) prior application for information:

(A) application No.: US 08/406.362

(B) Application date: 3/17/1995

(vii) Prior application for information:

(A) application No.: US (PROVIS)60001.983

(B) Application date: 8/4/1995

(viii) Attorney/firm information:

(A) name: Leclerc/Dubuc/prince

(C) Document/catalog No.: BIOVAC-1PCT

(ix) Communication information:

(A) telephone: 514-397-7400

(B) Electric transmission: 514-397-4382

(2) SEQ ID NO: 1, information:

(i) sequence characteristics:

(A) length: 830 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topology: linearity

(ii) Molecular type: DNA (genome)

(iii) The hypothesis is that: is free of

(iv) Antisense: is free of

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(ix) The properties are as follows:

(A) name/key: CDS

(B) Position: 143..667

(ix) The properties are as follows:

(A) name/key: signal peptide

(B) Position: 143..199

(ix) The properties are as follows:

(A) name/key: mature peptides

(B) Position: 200..667

(xi) Description of the sequence: SEQ ID NO: 1:

(2) SEQ ID NO: 2, information:

(i) sequence characteristics:

(A) length: 174 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 2:

(2) SEQ ID NO: 3, information:

(i) sequence characteristics:

(A) length: 710 base pairs

(B) Type (2): nucleic acids

(C) Chain: double chain

(D) Topology: linearity

(ii) Molecular type: DNA (genome)

(iii) The hypothesis is that: is free of

(iv) Antisense: is free of

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: MCH88

(ix) The properties are as follows:

(A) name/key: CDS

(B) Position: 116..643

(ix) The properties are as follows:

(A) name/key: signal peptide

(B) Position: 116..172

(ix) The properties are as follows:

(A) name/key: mature peptides

(B) Position: 173..643

(xi) Description of the sequence: SEQ ID NO: 3:

(2) SEQ ID NO: 4:

(i) sequence characteristics:

(A) length: 175 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 4:

(2) SEQ ID NO: 5, information:

(i) sequence characteristics:

(A) length: 850 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topology: linearity

(ii) Molecular type: DNA (genome)

(iii) The hypothesis is that: is free of

(iv) Antisense: is free of

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: z4063

(ix) The properties are as follows:

(A) name/key: CDS

(B) Position: 208..732

(ix) The properties are as follows:

(A) name/key: signal peptide

(B) Position: 208..264

(ix) The properties are as follows:

(A) name/key: mature peptides

(B) Position: 265..732

(xi) Description of the sequence: SEQ ID NO: 5:

(2) SEQ ID NO: 6:

(i) sequence characteristics:

(A) length: 174 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 6:

(2) SEQ ID NO: 7, information:

(i) sequence characteristics:

(A) length: 810 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topology: linearity

(ii) Molecular type: DNA (genome)

(iii) The hypothesis is that: is free of

(iv) Antisense: is free of

(vi) The source is as follows:

(A) an organism: neisseria gonorrhoeae

(B) Strain: b2

(ix) The properties are as follows:

(A) name/key: CDS

(B) Position: 241..765

(ix) The properties are as follows:

(A) name/key: signal peptide

(B) Position: 241..297

(ix) The properties are as follows:

(A) name/key: mature peptides

(B) Position: 298..765

(xi) Description of the sequence: SEQ ID NO: 7:

(2) SEQ ID NO: information of 8:

(i) sequence characteristics:

(A) length: 174 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 8:

(2) SEQ ID NO: 9, information:

(i) sequence characteristics:

(A) length: 16 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 9:

(2) SEQ ID NO: 10, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 10:

(2) SEQ ID NO: 11, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 11:

(2) SEQ ID NO: 12, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 12:

(2) SEQ ID NO: 13, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 13:

(2) SEO ID NO: 14, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 14:

(2) SEQ ID NO: 15, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 15:

(2) SEQ ID NO: 16, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 16:

(2) SEQ ID NO: 17, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 17:

(2) SEQ ID NO: 18, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 18:

(2) SEQ ID NO: 19, information of:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 19:

(2) SEQ ID NO: 20, information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 20:

(2) SEQ ID NO: information of 21:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 21:

(2) SEQ ID NO: 22 of the information:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 22:

(2) SEQ ID NO: information of 23:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 23:

(2) SEQ ID NO: information of 24:

(i) sequence characteristics:

(A) length: 15 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 24:

(2) SEQ ID NO: 25 of the information:

(i) sequence characteristics:

(A) length: 14 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 25:

(2) SEQ ID NO: information of 26:

(i) sequence characteristics:

(A) length: 25 amino acid

(B) Type (2): amino acids

(D) Topology: linearity

(ii) Molecular type: protein

(vi) The source is as follows:

(A) an organism: neisseria meningitidis

(B) Strain: 608B

(xi) Description of the sequence: SEQ ID NO: 26:

Claims (89)

1. An isolated polynucleotide which specifically hybridizes to a complementary sequence of a DNA sequence encoding a neisserial surface protein; wherein said DNA sequence comprises the consensus sequence shown in FIG. 11.

2. An isolated polynucleotide which specifically hybridizes to a complementary sequence of a DNA sequence encoding a neisserial surface protein; wherein the neisserial surface protein: including the consensus sequence shown in FIG. 12.

3. The polynucleotide of claim 1 or 2, which specifically hybridizes to a complementary sequence of a DNA sequence encoding a neisserial surface protein, wherein said DNA sequence comprises SEQ ID NO: 1, from base 200 to 664.

4. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ ID NO: 1, bases 143 to 664.

5. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ id no: 1.

6. the polynucleotide of claim 1 or 2, which specifically hybridizes to a complementary sequence of a DNA sequence encoding a neisserial surface protein, wherein said DNA sequence comprises SEQ ID NO: bases 173 to 640 of 3.

7. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ ID NO: bases 116 to 640 of 3.

8. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ id no: 3.

9. the polynucleotide of claim 1 or 2, which specifically hybridizes to a complementary sequence of a DNA sequence encoding a neisserial surface protein, wherein said DNA sequence comprises SEQ ID NO: bases 265 to 729 of 5.

10. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ ID NO: base number 208 to 729 of 5.

11. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ id no: 5.

12. the polynucleotide of claim 1 or 2, which specifically hybridizes to a complementary sequence of a DNA sequence encoding a neisserial surface protein, wherein said DNA sequence comprises SEQ ID NO: bases 298 to 762 of 7.

13. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ ID NO: bases 241 to 762 of 7.

14. The polynucleotide of claim 1 or 2, wherein the DNA sequence comprises SEQ id no: 7.

15. comprises the amino acid sequence of SEQ ID NO: 1, base 200 to 664.

16. Comprises the amino acid sequence of SEQ ID NO: 1, base 143 to 664.

17. Comprises the amino acid sequence of SEQ ID NO: 1.

18. Comprises the amino acid sequence of SEQ ID NO: 3, bases 173 to 640.

19. Comprises the amino acid sequence of SEQ ID NO: 3, bases 116 to 640.

20. Comprises the amino acid sequence of SEQ ID NO: 3.

21. Comprises the amino acid sequence of SEQ ID NO: 5, bases 265 to 729.

22. Comprises the amino acid sequence of SEQ ID NO: 5 from base 208 to 729.

23. Comprises the amino acid sequence of SEQ ID NO: 5.

24. Comprises the amino acid sequence of SEQ ID NO: 7, bases 298 to 762.

25. Comprises the amino acid sequence of SEQ ID NO: 7, bases 241 to 762.

26. Comprises the amino acid sequence of SEQ ID NO: 7.

27. A recombinant DNA molecule comprising

(i) An isolated polynucleotide as defined in any one of claims 1 to 26; and

(ii) an expression control sequence operably linked to the polynucleotide.

28. The recombinant DNA molecule of claim 27, wherein said expression control sequence comprises an inducible expression control sequence.

29. The recombinant DNA molecule of claim 28, wherein said inducible expression control sequence is inducible by a stimulus selected from the group consisting of temperature, lactose and IPTG.

30. The recombinant DNA molecule of claim 28, wherein said inducible expression control sequence is selected from the group consisting of λ PL, λ PR, TAC, T7, T3, LAC, and TRP promoters.

31. The recombinant DNA molecule of claim 27, wherein said DNA molecule is pNP 2204.

32. A unicellular host transformed with the recombinant DNA molecule of claim 27.

33. The unicellular host of claim 32, wherein the host is selected from one of the following: escherichia coli JM 109, Escherichia coli BL21 (DE3), Escherichia coli DH 5. alpha.F' IQ, Escherichia coli W3110, Escherichia coli JM 105, Escherichia coli BL21, Escherichia coli TOPP1, Escherichia coli TOPP2 and Escherichia coli TOPP 3.

34. A method for preparing a polynucleotide of any one of claims 1-26, comprising the steps of culturing a unicellular host of claim 32 and isolating said polynucleotide.

35. An isolated polypeptide encoded by the isolated polynucleotide of any one of claims 1-26, wherein the polypeptide is antigenic.

36. The isolated polypeptide of claim 35, comprising a sequence selected from the group consisting of SEQ ID NOs: 2. SEQ ID NO: 4. SEQ ID NO: 6 and SEQ ID NO: 8 in sequence (b).

37. The isolated polypeptide of claim 35, comprising a sequence selected from the group consisting of SEQ ID NOs: 2. SEQ ID NO: 4. SEQ ID NO: 6 and SEQ ID NO: 8, wherein the sequence does not contain a signal sequence.

38. The isolated polypeptide of claim 35, comprising a sequence selected from the group consisting of: SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25 and SEQ ID NO: 26.

39. the isolated polypeptide of claim 37, comprising SEQ ID NO: 2 from amino acids 31 to 55.

40. The isolated polypeptide of claim 37, comprising SEQ ID NO: 2 from amino acids 51 to 86.

41. The isolated polypeptide of claim 37, comprising SEQ ID NO: 2 from amino acids 110 to 140.

42. The isolated polypeptide of claim 37, comprising SEQ ID NO: 2 from amino acids 1 to 155.

43. The isolated polypeptide of claim 35, wherein the polypeptide is recombinant and is obtained by culturing the unicellular host of claim 32.

44. A method of isolating the polypeptide of claim 35, comprising:

a) isolating a culture of neisseria meningitidis; and

b) isolating the outer membrane fraction from the culture.

45. The method of claim 44, further comprising isolating said polypeptide from said outer membrane portion.

46. The method of claim 44, further comprising treating said outer membrane with proteinase K.

47. A pharmaceutical composition comprising a pharmaceutically effective amount of the polypeptide of claim 35 and a pharmaceutical excipient.

48. The pharmaceutical composition of claim 47, which is a vaccine.

49. A process for the preparation of a vaccine for the prevention of neisseria infection in a human which comprises admixing a pharmaceutically effective amount of a polypeptide according to claim 35 with a pharmaceutically acceptable diluent, excipient or adjuvant.

50. The method of making of claim 49, wherein said polypeptide is a Neisseria 22kDa surface protein.

51. Use of a polypeptide according to claim 35 for the manufacture of a medicament for the prevention of neisseria infection in a human.

52. The use of claim 51, wherein the polypeptide is a Neisseria 22kDa surface protein.

53. The use of claim 52, wherein the Neisseria 22kDa surface protein is Neisseria meningitidis 22kDa surface protein.

54. Use of a polypeptide according to claim 35 for the manufacture of a vaccine for the prevention of neisseria infection in humans.

55. The use of claim 54, wherein the polypeptide is a Neisseria 22kDa surface protein.

56. The use of claim 55, wherein the Neisseria 22kDa surface protein is Neisseria meningitidis 22kDa surface protein.

57. An antibody or antigen-binding fragment thereof that specifically binds to the polypeptide of claim 35.

58. The antibody of claim 57, wherein the polypeptide is a Neisseria 22kDa surface protein.

59. The antibody of claim 58, wherein the Neisseria 22kDa surface protein is Neisseria meningitidis 22kDa surface protein.

60. The antibody of claim 57 which is a monoclonal antibody.

61. The antibody of claim 60, which is murine or human.

62. The antibody of claim 61 which is of the IgG isotype.

63. The antibody of claim 60, wherein said antibody is murine and is selected from the group consisting of Me-1 and Me-7.

64. A method of isolating the antibody of claim 57 comprising

a) Introducing into a mammal (1) a neisserial preparation, (2) a neisserial outer membrane preparation, or (3) the polypeptide of claim 35; and is

b) Serum is isolated from a mammal containing the antibody.

65. The method of claim 64, wherein the Neisseria is Neisseria meningitidis.

66. A method of isolating the monoclonal antibody of claim 60 comprising

a) Introducing into a mammal (1) a neisserial preparation, (2) an outer membrane preparation of a neisserial, or (3) the polypeptide of claim 35;

b) isolating antibody-producing cells from the mammal;

c) fusing the cells with myeloma cells to produce hybridoma cells; and is

d) Isolating said monoclonal antibody from said hybridoma cells.

67. The method of claim 66, wherein the Neisseria is Neisseria meningitidis.

68. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 57.

69. The pharmaceutical composition of claim 68 which is a vaccine.

70. The pharmaceutical composition of claim 68, comprising a pharmaceutical excipient.

71. The pharmaceutical composition of claim 68, wherein said antibody is selected from the group consisting of Me-1 and Me-7.

72. A process for the preparation of a pharmaceutical composition for the prevention of neisserial infection in a human which comprises admixing a pharmaceutically effective amount of a polypeptide of claim 35 and a pharmaceutically acceptable diluent or excipient.

73. The method of making of claim 72, wherein the polypeptide is a Neisseria 22kDa surface protein.

74. Use of the antibody of claim 58 for the manufacture of a medicament for preventing Neisseria infection in humans.

75. Use of the antibody of claim 57 for the preparation of a kit for detecting Neisseria antigens in a biological sample isolated from a patient, comprising:

a) incubating the antibody of claim 57 with the biological sample; and is

b) Detecting the antibody that specifically binds to the antigen.

76. The use of claim 75, wherein said pathogen is Neisseria meningitidis.

77. The use of claim 75, wherein said antibody is selected from the group consisting of Me-1 and Me-7.

78. Use of the polypeptide of claim 35 for the preparation of a kit for the detection of antibodies specific for neisserial antigens in a biological sample isolated from a patient comprising:

a) incubating the polypeptide of claim 35 with the biological sample; and is

b) Detecting the polypeptide that specifically binds to the antibody.

79. The use of claim 78, wherein said polypeptide is a Neisseria meningitidis antigen.

80. The use of claim 79, wherein said polypeptide is a Neisseria south Americana 22kDa surface protein.

81. Use of the antibody of claim 57 for the manufacture of a kit for detecting a Neisseria pathogen in a patient, comprising:

a) labeling the antibody of claim 57 with a detectable label;

b) administering the labeled antibody to the patient; and is

c) Detecting the labeled antibody that specifically binds to the pathogen.

82. The use of claim 81, wherein the pathogen is Neisseria meningitidis.

83. Use of a polypeptide according to claim 35 for the manufacture of a kit for detecting or diagnosing neisseria meningitidis infection in a human.

Use of a DNA probe for the preparation of a kit for the detection of neisseria in a biological sample isolated from a patient comprising:

a) contacting the sample with a DNA probe that is capable of specifically hybridizing to a polynucleotide of any one of claims 15-26; wherein the DNA probe is an oligomer having a sequence complementary to at least 6 consecutive nucleotides of any one of the polynucleotides of claims 15-26; and

b) detecting hybridization of the DNA probe to the polynucleotide.

85. The use of claim 84, further comprising the step of amplifying by polymerase chain reaction the target DNA to which said DNA probe hybridizes with the use of a set of oligomers having the sequence: (i) complementary to at least 6 consecutive nucleotides in the polynucleotide of any one of claims 1-26; (ii) flanking the target DNA.

86. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6 or SEQ ID NO: 8.

87. The isolated polypeptide of claim 86, wherein the polypeptide consists of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6 or SEQ ID NO: 8, or a pharmaceutically acceptable salt thereof.

88. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, and is antigenic.

89. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, and is antigenic.