AU3638600A - Proteinase K resistant surface protein of neisseria meningitidis - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): BIOCHEM PHARMA, INC Invention Title: PROTEINASE K RESISTANT SURFACE PROTEIN OF NEISSERIA

MENINGITIDIS

The following statement is a full description of this invention, including the best method of performing it known to me/us: PROTEINASE K RESISTANT SURFACE PROTEIN OF NEISSERIA KENINGITIDIS TECHNICAL FIELD OF THE INVENTION This invention relates to a highly conserved, immunologically accessible antigen at the surface of Neisseria meningitidis organisms. This unique antigen provides the basis for new inmmunotherapeutic, prophylactic and diagnostic agents useful in the treatment, prevention and diagnosis of Neisseria meningitidis diseases. More particularly, tiis invention relates to a proteinase K resistant Neisseria meningitidis surface protein having an apparent molecular weight of 22 kDa, the corresponding 15 nucleotide and derived amino acid sequences (SEQ ID NO:1 to SEQ ID NO:26), recombinant DNA methods for the production of the Neisseria meningitidis 22 kDa surface protein, antibodies that bind to' the Neisseria meningitidis 22 kDa surface protein and methods and compositions for the diagnosis, treatment and prevention of Neisseria meningitidis diseases.

k BACKGROUND OF THE INVENTION Neisseria meningitidis is a major cause of death and 25 morbidity throughout the world. Neisseria meningitidis causes both endemic and epidemic diseases, principally meningitis and meningococcemia [Gold, Evolution of meningococcal disease, p. 69, Vedros CRC Press (1987); Schwartz et al., Clin. Microbiol. Rev., 2, p. S118 (1989).. In fact, this organism is one of the most common causes, after Haemophilus influenzae type b, of bacterial meningitis in the United States, accounting for approximately 20% of all cases. It has been well documented that serum bactericidal activity is the major defense mechanism against Neisseria meningitidis and that protection against invasion by the bacteria correlates with the presence in the serum of anti-meningococcal antibodies [Goldschneider et al., J. Exp. Med. 129, p. 1307 (1969); Goldscbneider et al., J. Exp. Med., 129, p. 1327 (1969)].

Neisseria meningitidis are subdivided into serological groups according to the presence of capsular antigens. Currently, 12 serogroups are recognized, but serogroups A, B, C, Y, and W-135 are most commonly found.

Within serogroups, serotypes, subtypes and immunotypes can be identified on outer membrane proteins and lipopolysaccharide (Frasch et al., Rev. Infect. Dis. 7, p. 504 (1985)1.

The capsula': polysaccharide vaccines presently 15 available are not effective against all Neisseria meningitidis isolates and do not effectively induce the production of protective antibodies in young infants S[Frasch, Clin. Microbiol. Rev. 21 p. S134 (1989); Reingold et al., Lancet, p. 114 (1985); Zollinger, in Woodrow and Levine, New generation vaccines, p. 325, Marcel Dekker Inc. N.Y. (1990)]. The capsular polysaccharide of serogroups A, C, Y and W-135 are presently used in vaccines against this organism. These polysaccharide vaccines are effective in the short term, however the vaccinated subjects do not develop an immunological memory, so they must be revaccinated within a three-year period to maintain their level of resistance.

Furthermore, these polysaccharide vaccines do not induce sufficient levels of bactericidal antibodies to obtain the desired protection in children under two years of age, whoare the principal victims of this disease. No effective vaccine against serogroup B isolates is presently available even though these organisms are one of the primary causes of meningococcal diseases in developed countries. Indeed, the serogroup B polysaccharide is not a good immunogen, inducing only a poor response of IgM of low specificity which is not protective [Gotschlich et al., J. Exp. Med., p. 129, 1349 (1969); Skevakis et al., J. Infect. Dis., 149, p. 387 (1984); Zollinger et al., J. Clin. Invest., 63, p. 836 (1979)]. Furthermore, the presence of closely similar, crossreactive structures in the glycoproteins of neonatal human brain tissue [Finne et al., Lancet, p. 355 (1983)] might discourage attempts at improving the immunogenicity of serogroup B polysaccharide.

To obtain a more effective vaccine, other Neisseria meningitidis surface antigens such as lipopolysaccharide, pili proteins ard proteins present in the outer membrane are under investigation. The presence of a human immune 15 response and bactericidal antibodies against certain of these proteinaceous surface antigens in the sera of immunized volunteers and convalescent patients was S* demonstrated [Mandrell and Zollipger, Infect. Immun., 57, p. 1590 (1989); Poolman et al., Infect. Immun., 40, p. 398 (1983); Rosenquist et al., J. Clin. Microbiol., 26, p. 1543 (1988); Wedege and Froholm, Infect. Immun. 51, p. 571 (1986); Wedege and Michaelsen, J. Clin. Microbiol., p. 1349 (1987)].

Furthermore, monoclonal antibodies directed against 25 outer membrane proteins, such as class 1, 2/3 and 5, were also reported to be bactericidal and to protect against experimental infections in animals (Brodeur et al., Infec.

i Immun., 50, p. 510 (1985); Frasch et al, Clin. Invest.

Med., 9, p. 101 (1986); Saukkonen et al. Microb.

Pathogen., 3, p. 261 (1987); Saukkonen et al., Vaccine, 7, p. 325 (1989)1.

Antigen preparations based on Neisseria meningitidis outer membrane proteins have demonstrated immunogenic effects in animals and in humans and some of them have been tested in clinical trials [Bjune et al., Lancet, p. 1093 (1991); Costa et al., NIPH Annals, 14, p. 215 (1991); Frasch et al., Med. Trop., 43, p. 177 (1982); Frasch et al., Eur. J. Clin. Microbiol., 4, p. 533 (1985); Frasch et al. in Robbins, Bacterial Vaccines, p. 262, Praeger Publications, N.Y. (1987); Frasch et al, J.

Infect. Dis., 158, p. 710 (1988); Moreno et al. Infec.

Immun., 47, p. 527 (1985); Rosenqvist et al., J. Clin.

Microbiol., 26, p. 1543 (1988); Sierra et al., NIPH Annals, 14, p. 195 (1991); Wedege and Froholm, Infec.

Immun. 51, p. 571 (1986); Wedege and Michaelsen, J. Clin.

Microbiol., 25, p. 1349 (1987); Zollinger et al., J. Clin.

Invest., 63, p. 836 (1979) Zollinger et al., NIPH Annals, 14, p. 211 (1991)]. However, the existence of great interstrain antigenic variability in the outer membrane proteins can limit their use in vaccines [Frasch, Clin.

Microb., Rev. 2, p. S134 (1989)]. Indeed, most of these preparations induced bactericidal antibodies that were *restricted to the same or related serotype from which the antigen was extracted [Zollinger in Woodrow and Levine, New Generation Vaccines, p. 325, Marcel Dekker Inc. N.Y.

(1990)]. Furthermore, the protection conferred by these vaccines in young children has yet to be clearly established. The highly conserved Neisseria meningitidis outer membrane proteins such as the class 4 ([Munkley et al. Microb. Pathogen., 11, p. 447 (1991)] and the lip protein (also called H.8) (Woods et al., Infect. Imnmun., 55, p. 1927 (1987)] are not interesting vaccine candidates since they do not elicit the production of bactericidal antibodies. To improve these vaccine preparations, there is a need for highly conserved proteins that would be present at the surface of all Neisseria meningitidis strains and that would be capable of eliciting bactericidal antibodies in order to develop a broad spectrum vaccine.

The current laboratory diagnosis of Neisseria meningitidis is usually done by techniques such as Gram stain of smear preparations, latex agglutination or coagglutination, and the culture and isolation on enriched and selective media (Morello et al., in Balows, Manual of Clinical Microbiology, P. 258, American Society for Microbiology, Washington (1991)]. Carbohydrate degradation tests are usually performed to confirm the identification of Neisseria meningitidis isolates. Most of the described procedures are time-consuming processes requiring trained personnel. Commercial agglutination or coagglutination kits containing polyvalent sera directed against the capsular antigens expressed by the most prevalent serogroups are used for the rapid identification of Neisseria meningitidis. However, these polyvalent sera often nonspecifically cross-react with other bacterial species and for that reason should always be used in conjunction with Gram stain and, culture. Accordingly, :there is a need for efficient alternatives to these diagnostic assays that will improve the rapidity and S.reliability of the diagnosis.

DISCLOSURE OF TzE INVENTION The present invention solves the problems referred to above by providing a highly conserved, imunologically accessible antigen at the surface of Neisseria meningitidis organisms. Also provided are recombinant

DNA

*o *.:molecules that code for the foregoing antigen, unicellular hosts transformed with those DNA molecules, and a process for making substantially pure, recombinant antigen. Also provided are antibodies specific to the foregoing Neisseria meningitidis antigen. The antigen and antibodies of this invention provide the basis for unique methods and pharmaceutical compositions for the detection, prevention and treatment of Neisseria meningitidis diseases.

The preferred antigen is the Neisseria meningitidis 22 kDa surface protein, including fragments, analogues and derivatives thereof. The preferred antibodies are the Me- 1 and Me-7 monoclonal antibodies specific to the Neisseria meningitidis 22 kDa surface protein. These antibodies are highly bacteriolytic against Neisseria meningitidis and passively protect mice against experimental infection.

The present invention further provides methods for isolating novel Neisseria meningitidis surface antigens and antibodies specific thereto.

BRIEF DESCRIPTION OF THE DRAWINGS 15 Figure 1 depicts the nucleotide and derived amino acid sequences of the Neisseria meningitidis strain 608B 22 kDa surface protein (SEQ ID NO:1; SEQ ID NO:2) Conventional three letter symbols are used for the amino Sacid residues. The open reading frame extends from the start codon at base 143 to the stop codon at base 667. The box indicates the putative ribosome binding site whereas the putative -10 promoter sequence is underlined. A 19amino-acid signal peptide is also underlined.

:Figure 2 is a photograph of a Coomassie Blue stained ~25 14% SDS-PAGE gel displaying a-chymotrypsin and trypsin digests of Neisseria meningitidis strain 608B (B:2a:P1.2) outer membrane preparations. Lane 1 contains the following molecular weight markers: 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 contains undigested control outer membrane preparation.

Lane 3 contains a-chymotrypsin treated preparation (2mg of enzyme per mg of protein); lane 4 contains trypsin treated preparation.

Figure 3a is a photograph of a Coommasie Blue stained 14% SDS-PAGE gel displaying proteinase K digests of Neisseria meningitidis strain 608B (B:2a:P1.2) outer membrane preparations. Lanes 1, 3, 5, and 7 contain undigested control. Lanes 2, 4, 6 and 8 contain outer membrane preparations digested with proteinase K (2 IU per mg of protein). Lanes 1 to 4 contain preparations treated at pH 7.2. Lanes 5 to 8 contain preparations treated at pH 9.0. Lanes 1, 2, 5 and 6 contain preparations treated without SDS. Lanes 3, 4, 7 and 8 contain preparations treated in the presence of SDS. Molecular weight markers are indicated on the left (in kilodaltons).

e 15 Figure 3b is a photograph of a Coomassie Blue stained 14% SDS-PAGE gel displaying the migration profiles of V" affinity purified recombinant 22 kDa protein. Lane 1 contains molecular weight markers: 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 contains 5 pg of control affinity purified recombinant 22 kDa protein. Lane 3 contains 5 pg of affinity purified recombinant 22 kDa protein heated at 100°C for 5 min.

25 Lane 4 contains 5 Ug of affinity purified recombinant 22 kDa protein heated at 100°C for 10 min. Lane 5 contains 5 pg of affinity purified recombinant 22 kDa protein heated at 100°C for 15 min.

Figure 4 is a photograph of Coomassie Blue stained 14% SDS-PAGE gels and their corresponding Western immunoblots showing the reactivity of monoclonal antibodies specific to the Neisseria meningitidis 22 kDa surface protein. Outer membrane preparation from Neisseria meningitidis strain 608B (B:2a:P1.2) (A) untreated; Proteinase K treated (2 IU per mg of 7 protein). Lane 1 contains molecular weight markers and characteristic migration profile on 14% SDS-PAGE gel of outer membrane preparations. Lane 2 contains Me-2; Lane 3 contains Me-3; lane 4 contains Me-5; lane 5 contains Me-7; and lane 6 contains an unrelated control monoclonal antibody. The molecular weight markers 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 immunoblot results shown in Figure 4 for Me-2, Me-3, Me-5, Me-6 and Me-7 are consistent with the immunoblot results obtained for Me-!.

Figure 5 is a graphic depiction of the binding activity of the monoclonal antibodies to intact bacterial e 15 cells. The results for representative monoclonal antibodies Me-5 and Me-7 are presented in counts per minute on the vertical axis. The various bacterial strains used in the aqsay are shown on the horizontal axis. A Haemophilus influenzae porin-specific monoclonal antibody was used as a negative control.

Background counts below 500 CPM were recorded and were subtracted from the binding values.

Figure 6 is a photograph of stained 14% SDS-PAGE gels and their corresponding Western immunoblot demonstrating 25 the purification of the recombinant 22 kDa Neisseria meningitidis surface protein from concentrated culture supernatant of Escherichia coli strain BL21(DE3).

Figure 6(A) is a photograph of a Coomassie Blue and silver stained 14% SDS-Page gel. Lane 1 contains the following molecular weight markers: 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 contains outer membrane protein preparation extracted from Neisseria meningitidis strain 608B (serotype B:2a:pl.2)(10 mg). Lane 3 contains 8 concentrated culture supernatant of Escherichia coli BL21(DE3) (10 mg). Lane 4 contains affinity purified recombinant 22 kDa Neisseria meningitidis surface protein (1 mg). Figure 6(B) is a photograph of a Coomassie Blue stained 14% SDS-PAGE gel of a-chymotrypsin, trypsin and proteinase K digests of affinity purified recombinant 22 kDa Neisseria meningitidis surface protein. Lane 1 contains the following molecular weight markers: 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 contain purified reconbinant 22 kDa Neisseria meningitidis surface protein (2 mg). Lanes 7 to contain bovine serum albumin (2 mg). Lanes 2 and 7 15 contain undigested protein Lanes 3 and 8 contain a-chymotrypsin treated protein (2 mg of enzyme per mg of protein). Lanes 4 and 9 contain trypsin treated protein (2 mg of enzyme per mg of protein). Lanes and 10 contain proteinase K treated protein (2 IU 20 per mg of protein). Figure 6(C) is a photograph of the Western immunoblotting of a duplicate gel using monoclonal antibody Figure 7 is a graphical depiction of the bactericidal activity of protein A-purified anti-Neisseria meningitidis 22 kDa surface protein monoclonal antibodies against Neisseria meningitidis strain 608B (B:2a:P1.2). The vertical axis of the graph shows the percentage of survival of the Neisseria meningitidis bacteria after exposure to various concentrations of monoclonal antibody (shown on the horizontal axis of the graph).

Figure 8 depicts the nucleotide and derived amino acid sequences of the Neisseria meningitidis strain MCH88 22 kDa surface protein (SEQ ID NO:3; SEQ ID NO:4).

Conventional three letter symbols are used for the amino acid residues. The open reading frame extends from the start codon at base 116 to the stop codon at base 643.

Figure 9 depicts the nucleotide and derived amino acid sequences of the Neisseria meningitidis strain Z4063 22 kDa surface protein (SEQ ID NO:5; SEQ ID NO:6).

Conventional three letter symbols are used for the amino acid residues. The open reading frame extends from the start codon at base 208 to the stop codon at base 732.

Figure 10 depicts the nucleotide and derived amino acid sequences of the Neisseria gonorrhoeae strain b2, 22 kDa surface protein (SEQ ID NO:7; SEQ ID NO:8).

Conventional three letter symbols are used for the amino acid residues. The open reading frame extends from the start codon at base 241 to the stop codon at base 765.

15 Figure 11 depicts the consensus sequence established from the DNA sequences of the four strains of Neisseria and indicates the substitutions or insertion of nucleotides specific to each strain.

Figure 12 depicts the consensus sequence established 20 from the protein sequences of the four strains of Neisseria and indicates the substitutions or insertion of amino acid residues specific to each strain.

*Figure 13 represents the time course of the immune response to the recombinant 22 kDa protein in rabbits expressed as the reciprocal ELISA titer. The rabbits were injected with outer membrane preparations from E. coli :strain JM109 with plasmid pN2202 or with control plasmid The development of the specific humoral response was analysed by ELISA using outer membrane preparations obtained from Neisseria meningitidis strain 608B (B:2a:P1.2) as coating antigen.

Figure 14 represents the time course of the immune response to the recombinant 22kDa protein in Macaca fascicularis (cynomolgus) monkeys expressed as the reciprocal ELISA titer. The two monkeys were respectively immunized with 100g (K28) and 200g (1276) of affinity purified 22kDa protein per injection. The control monkey was immunized with 150g of unrelated recombinant protein following the same procedure. The development of the specific humoral response was analysed by ELISA using outer membrane preparations obtained from Neisseria meningitidis strain 608B (B:2a:P1.2) as coating antigen.

Figure 15 is a graphic representation of the synthetic peptides of the invention as well as their respective position in the full 22kDa protein of Neisseria meningitidis strain 608B (B:2a:P1.2).

Figure 16 is a map of plasmid pNP2204 containing the gene which encodes the Neisseria meningitidis 22 kDa surface protein 22kDa, Neisseria meningitidis 22 kDa 15 surface protein gene; Ampi, ampicillin-resistance coding region; ColEl, origin of replication; c1857, bacteriophage ci857 temperature-sensitive repressor gene; XPL, bacteriophage X transcription promoter; T1 transcription terminator. The direction of transcription is indicated by 20 the arrows. BglII and BamHl are the restriction sites used to insert the 22 kDa gene in the p 6 2 9 plasmid.

DETAILED DESCRIPTION OF THE INVENTION During our study of the ultrastructure of the outer membrane of Neisseria meningitidis we identified a new low molecular weight protein of 22 kilodaltons which has very S: unique properties. This outer membrane protein is highly resistant to extensive treatments with proteolytic enzymes, such as proteinase K, a-serine protease derived from the mold Tritirachium album limber. This is very surprising since proteinase K resistant proteins are very rare in nature because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme [Barrett, A.J. and N.D. Rawlings, Biochem. Soc. Transactions (1991) 19: 707-715]. Only a few reports have described proteins of prokaryotic origin that are resistant to the enzymatic degradation of proteinase K. Proteinase K resistant proteins have been found in Leptospira species (Nicholson, V.M. and J.F. Prescott, Veterinary Microbiol. (1993) 36:123-138], Mycoplasma species [Butler, G.H. et al.

Infec. Inmun. (1991) 59:1037-1042], Spiroplasma mirum [Bastian, F.O. et al. J. Clin. Microbiol. (1987) 25:2430- 2431] and in viruses and prions [Onodera, T. et al.

Microbiol. Immunol. (1993) 37:311-316; Prusiner, S.B.

et al. Proc. Nat. Acad. Sci. USA (1993) 90:2793-2797].

Herein, we describe the use of this protein as a means for the improved prevention, treatment and diagnosis of Neisseria meningitidis infections.

15 Thus according to one aspect of the invention we provide a highly conserved, immunologically accessible Neisseria meningitidis surface protein, and fragments, analogues, and derivatives thereof. As used herein, "Neisseria meningitidis surface protein" means any Neisseria meningitidis surface protein encoded by a naturally occurring Neisseria meningitidis gene. The Neisseria meningitidis protein according to the invention may be of natural origin, or may be obtained through the application of molecular biology with the object of 25 producing a recombinant protein, or fragment thereof.

As used herein, -highly conserved" means that the gene for the Neisseria meningitidis surface protein and the protein itself exist in greater than 50% of known strains of Neisseria meningitidis. Preferably, the gene and its protein exist in greater than 99% of known strains of Neisseria meningitidis. Examples 2 and 4 set forth methods by which one of skill in the art would be able to test a variety of different Neisseria meningitidis surface proteins to determine if they are "highly conserved'.

As used herein, *immunologically accessible" means that the Neisseria meningitidis surface protein is present at the surface of the organism and is accessible to antibodies. Example 2 sets forth methods by which one of skill in the art would be able to test a variety of different Neisseria meningitidis surface proteins to determine if they are "immunologically accessible Immunological accessibility may be determined by other methods, including an agglutination assay, an ELISA, a RIA, an immunoblotting assay, a dot-enzyme assay, a surface accessibility assay, electron microscopy, or a combination of these assays.

As used herein, "fragments" of the Neisseria meningitidis surface protein include polypeptides having at least one peptide epitope, or analogues and derivatives thereof. Peptides of this type may be obtained through S* the application of molecular biology or synthesized using conventional liquid or solid phase peptide synthesis techniques.

As used herein, "analogues* of the Neisseria ~meningitidis surface protein include those proteins, or ~fragments thereof, wherein one or more amino acid residues in the naturally occurring sequence is replaced by another amino acid residue, providing that the overall 25 functionality and protective properties of this protein are preserved. Such analogues may be produced synthetically, or by recombinant DNA technology, for example, by mutagenesis of a naturally occurring Neisseria meningitidis surface protein. Such procedures are well known in the art.

For example, one such analogue is selected from the recombinant protein that may be produced from the gene for the 22kDa protein from Neisseria gonorrhoeae strain b2, as depicted in Figure 10. A further analog may be obtained from the isolation of the corresponding gene from Neisseria lactamica.

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

It will be understood that by following the examples of this invention, one of skill in the art may determine without undue experimentation whether a particular fragment, analogue or derivative would be useful in the diagnosis, prevention or treatment of Neisseria meningitidis diseases.

15 This invention also includes polymeric forms of the Neisseria meningitidis surface proteins, fragments, analogues and derivatives. These polymeric forms include, for example, one or more polypeppides that have been crosslinked with crosslinkers such as avidin/biotin, gluteraldehyde or dimethylsuberimidate. Such polymeric forms also include polypeptides containing two or more tandem or inverted contiguous Neisseria meningitidis sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.

25 This invention provides substantially pure Neisseria meningitidis surface proteins. The term 'substantially pure" means that the Neisseria meningitidis surface protein according to the invention is free from other proteins of Neisseria meningitidis origin. Substantially pure Neisseria meningitidis surface protein preparations can be obtained by a variety of conventional processes, for example the procedure described in Examples 3 and 11.

In a further aspect, the -invention particularly provides a 22 kDa surface protein of Neisseria meningitidis having the amino acid sequence of Figure 1 14 (SEQ ID NO:2), or a fragment, analogue or derivative thereof.

In a further aspect, the invention particularly provides a 22 kDa surface protein of Neisseria meningitidis having the amino acid sequence of Figure 8 (SEQ ID NO:4), Figure 9 (SEQ ID NO:6) or a fragment, analogue or derivative thereof. Such a fragment may be selected from the peptides listed in Figure 15 (SEQ ID NO:9 to SEQ ID NO:26).

In a further aspect, the invention provides a 22kDa surface protein of Neisseria gonorrhoeae having the amino acid sequence of Figure 10 (SEQ ID NO:8), or a fragment, analogue or derivative thereof. As will be apparent from the above, any reference to the Neisseria meningitidis 22kDa protein also encompasses 22kDa proteins isolated from, or made from genes isolated from other species of Neisseriacae such as Neisseria gonorrhoeae or Neisseria lactamica.

S* A Neisseria meningitidis 22 kDa surface protein according to the invention may be further characterized by one or more of the following features: it has an approximate molecular weight of 22 kDa as evaluated on SDS-PAGE gel; its electrophoretic mobility on SDS-PAGE gel is 25 not modified by treatment with reducing agents; it has an isoelectric point (pI) in a range around pi 8 to pi it is highly resistant to degradation by proteolytic enzymes such as a(-chymotrypsin, trypsin and proteinase K; periodate oxidation does not abolish the specific binding of antibody directed against the Neisseria meningitidis 22 kDa surface protein; it is a highly conserved antigen; it is accessible to antibody at the surface of intact Neisseria menfinqi tidis organisms; it can induce the production of bactericidal antibodies; it can induce the production of antibodies that can protect against experimental infection; it can induce, when injected into an animal host, the development of an immunological response that can protect against Neisseria meningitidis infection.

This invention also provides, for the first time, a DNA sequence coding for the Neisseria meningitidis 22 kDa surface protein (SEQ ID NO:1, NO:3, NO:5, and NO:7). The preferred DNA sequences of this invention are selected from the group consisting of: the DNA sequence of Figure 1 (SEQ ID NO:l); the DNA sequence of Figure 8 (SEQ ID NO:3); *t S" the DNA sequence of Figure 9 (SEQ ID the DNA sequence of Figure 10 (SEQ ID NO:7); analogues or derivatives of the foregoing DNA sequences; DNA sequences degenerate to any of the foregoing DNA sequences; and fragments of any of the foregoing DNA sequences; wherein said sequences encode a product that displays the °coo 25 immunological activity of the Neisseria meningitidis 22 kDa surface protein.

Such fragments are preferably peptides as depicted in Figure 15 (SEQ ID NO:9, through SEQ ID NO:26).

Preferably, this invention also provides, for the first time, a DNA sequence coding for the Neisseria meningitidis 22 kDa surface protein (SEQ ID NO:1). More preferred DNA sequences of this invention are selected from the group consisting of: the DNA sequence of Figure 1 (SEQ ID NO:1); analogues or derivatives of the foregoing DNA sequences; DNA sequences degenerate to any of the foregoing DNA sequences; and fragments of any of the foregoing DNA sequences; wherein said sequences encode a product that displays the immunological activity of the Neisseria meningitidis 22 kDa surface protein.

Analogues and derivatives of the Neisseria meningitidis 22 kDa surface protein coding gene will hybridize to the 22 kDa surface protein-coding gene under the conditions described in Example 4.

For purposes of this invention, the fragments, analogues and derivatives of the Neisseria meningitidis 22 kDa surface protein have the inrunological activity" of the Neisseria meningitidis 22 kDa surface protein if they can induce, when injected into an animal host, the development of an immunological, response that can protect S* against Neisseria meningitidis infection. One of skill in the art may determine whether a particular DNA sequence ~encodes a product that displays the immunological activity of the Neisseria meningitidis 22 kDa surface protein by following the procedures set forth herein in Example 6.

The Neisseria meningitidis surface proteins of this 25 invention may be isolated by a method comprising the following steps: :i a) isolating a culture of Neisseria meningitidis .i bacteria, b) isolating an outer membrane portion from the culture of the bacteria; and c) isolating said antigen from the outer membrane portion.

In particular, the foregoing step may include the additional steps of treating the Neisseria meningitidis outer membrane protein extracts with proteinase K, followed by protein fractionation using conventional separation techniques such as ion exchange and gel chromatography and electrophoresis.

Alternatively and preferably, the Neisseria menhzngitidis surface proteins of this invention may be produced by the use of molecular-biology techniques, as more particularly described in Example 3 herein. The use of molecular biology techniques is particularly wellsuited for the preparation of substantially pure recombinant Neisseria meningitidis 22 kDa surface protein.

Thus according to a further aspect of the invention we provide a process for the production of recombinant Neisseria rneningitidis 22 kDa surface protein, including fragments, analogues and derivatives thereof, comprising the steps of culturing a unicellular host organism transformed with a recombinant DNA molecule including a .DNA se quence coding for said protein, fragment, analogue or derivative and one or more epression control sequences operatively linked to the DNA sequence, and recovering a substantially pure protein, fragment, analogue or derivative.

As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host. Preferably, the expression control sequences, and the gene of interest, will be contained in an expression vector that further comprises a bacterial selection marker and origin of replication. if the expression host is a eukaryotic cell, the expression vector should further comprise an expression marker useful in the expression host.

A wide variety of expression host/vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E.coli, including col El, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, the numerous derivatives of phage lambda, e.g. NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Useful expression vectors for yeast cells include the 2 mu plasmid and derivatives thereof. Useful vectors for insect cells include pVL 941.

In addition, any of a wide variety of expression control sequences may be used in these vectors to express 15 the DNA sequences of this invention. Such useful e expression control sequences include the expression control sequences associated with structural genes of the :.:foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, the promoter for 3phosphoglycerate kinase or other glycolytic enzymes, the 25 promoters of acid phosphatase, Pho5, the promoters of the yeast alpha-mating system and other sequences known to control expression of genes of prokaryotic and eukaryotic cells or their viruses, and various combinations thereof. The Neisseria meningitidis 22 kDa surface protein's expression control sequence is particularly useful in the expression of the Neisseria meningitidis 22 kDa surface protein in E.coli (Example 3).

Host cells transformed with the foregoing vectors form a further aspect of this invention. A wide variety of unicellular host cells are useful in expressing the DNA sequences of tbis invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as 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 COS 1. COS 7, BSC 1, ESC 40, and BMT 10, and human cells and plant cells in tissue culture. Preferred host organisms include bacteria such as E.coli and Bacillus subtilis and mnammalian cells in tissue culture.

It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

:in selecting an expression control sequence, avariety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the DNa sequences of this invention, particularly as regards potential secondary structures. Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the MNA sequences of this invention, their secretion characteristics, 1their-ability to fold the protein correctly, their fermentation or culture requirements, and the ease of purification from them of the products coded for by the DNA sequences of this invention.

Within these parameters, one of skill in the art may select various vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

The polypeptides encoded by the DNA sequences of this invention may be isolated from the fermentation or cell culture and purified using any of a variety of conventional methods. One of skill in the art may select the most appropriate isolation and purification techn:.ques without departing from the scope of this invention.

The Neisseria meningitidis surface proteins of this 15 invention are useful in prophylactic, therapeutic and diagnostic compositions for preventing, treating and diagnosing diseases caused by Neisseria meningitidis infection.

The Neisseria meningitidis surface proteins of this invention are useful in prophylactic, therapeutic and diagnostic compositions for preventing, treating and diagnosing diseases caused by Neisseria gonorrhoeae, or Neisseria lactamica infection.

The Neisseria meningitidis surface proteins according to this invention are particularly well-suited for the, generation of antibodies and for the development of a protective response against Neisseria meningitidis diseases.

The Neisseria meningitidis surface proteins according to this invention are particularly well-suited for the generation of antibodies and for the development of a protective response against Neisseria gonorrhoeae or Neisseria lactamica diseases.

In particular, we provide a Neisseria meningitidis 22 kDa surface protein having an amino acid sequence of 21 Figure 1 (SEQ ID NO:2) or a fragment, analogue, or derivative thereof for use as an immunogen and as a vaccine.

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

Standard immunological techniques may be employed with the Neisseria meningitidis surface proteins in order to use them as inmmnogens and as vaccines. In particular.

any suitable host may be injected with a pharmaceutically effective amount of the Neisseria meningitidis 22 kDa surface protein to generate monoclonal or polyvalent anti- Neisseria meningitidis antibodies or to induce the development of a protective immunological response against Neisseria meningitidis diseases, Prior to injection of the host, the Neisseria meningitidis surface proteins may be formulated in a suitable vehicle, and thus we provide a pharmaceutical composition comprising one or more Neisseria meningitidis surface antigens or fragments thereof. Preferably, the antigen is the Neisseria meningitidis 22 kDa surface protein or fragments, 25 analogues or derivatives thereof together with one or more pharmaceutically acceptable excipients. As used herein, -pharmaceutically effective amount" refers to an amount of "one or more Neisseria meningitidis surface antigens or fragments thereof that elicits a sufficient titer of anti- Neisseria meningitidis antibodies to treat or prevent Neisseria meningitidis infection.

The Neisseria meningitidis surface proteins of this invention may also form the basis of a diagnostic test for Neisseria meningitidis infection. Several diagnostic methods are possible. For example, this invention 22 provides a method for the detection of Neisseria meningitidis antigen in a biological sample containing or suspected of containing Neisseria meningitidis antigen comprising: a) isolating the biological sample from a patient; b) incubating an anti-Neisseria meningitidis 22 kDa surface protein antibody or fragment thereof with the biological sample to form a mixture; and c) detecting specifically bound antibody or bound fragment in the mixture which indicates the presence of Neisseria meningitidis antigen.

Preferred antibodies in the foregoing diagnostic method are Me-i and Me-7.

Alternatively, this invention provides a method for 15 the detection of antibody specific to Neisseria i meningitidis antigen in a biological sample containing or suspected of containing said antibody comprising: a) isolating the biological sample from a patient; b) incubating a Neisseria meningitidis surface protein of this invention or fragment thereof with the biological sample to form a mixture; and c) detecting specifically bound antigen or bound fragment in the mixture which indicates the presence of antibody specific to Neisseria meningitidis antigen.

25 One of skill in the art will recognize that this diagnostic test may take several forms, including an immunological test such as an enzyme-linked immunosorbent *"assay (ELISA), a radioimmunoassay or a latex agglutination assay, essentially to determine whether antibodies specific for the protein are present in an organism.

The DNA sequences of this invention may. also be used to design DNA probes for use in detecting the presence of the pathogenic Neisseria bacteria in a biological suspected of containing such bacteria. The detection method of this invention comprises the steps of: 23 a) isolating the biological sample from a patient; b) incubating a DNA probe having a DNA sequence of this invention with the biological sample to form a mixture; and c) detecting specifically bound DNA probe in the mixture which indicates the presence of Neisseria bacteria.

Preferred DNA probes have the base pair sequence of Figure 1 (SEQ ID NO:l), Figure 8 (SEQ ID NO:3), Figure 9 (SEQ ID NO:5), or Figure 10 (SEQ ID NO:7), or consensus sequence of Figure 11 (SEQ ID NO:9).

A more preferred DNA probe has the 525 base pair sequence of Figure 1 (SEQ ID NO:l) The DNA probes of this invention may also be used for 5 detecting circulating Neisseria meningitidis nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing Neisseria meningitidis infections. The probe may be synthesized using conventional techniques and may be immobilized on. a solid 20 phase, or may be labeled with a detectable label.

A preferred DNA probe for this application is an 0•"oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the Neisseria meningitidis 22 kDa surface protein gene of Figure 1 (SEQ ID NO:1). Figure 0S*C 25 8 (SEQ ID NO:3), Figure 9 (SEQ ID NO:5), Figure 10 (SEQ ID 0C* NO:7), or consensus sequence of Figure 11 (SEQ ID NO:9).

A more preferred DNA probe for this application is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the Neisseria meningitidis 22 kDa surface protein gene of Figure 1 (SEQ ID NO:l).

Another diagnostic method for the detection of Neisseria meaingitidis in a patient comprises the steps of: a) labeling an antibody of this invention or fragment thereof with a detectable label; b) administering the labeled antibody or labeled fragment to the patient; and c) detecting specifically bound labeled antibody or labeled fragment in the patient which indicates the presence of Neisseria meningitidis.

For purification of any anti-Neisseria meningitidis surface protein antibody, use may be made of affinity chromatography employing an immobilized Neisseria meningitidis surface protein as the affinity medium.

Thus according to another aspect of the invention we provide a Neisseria meningitidis 22 kDa surface protein having an amino acid sequence which includes the sequence of Figure 1 (SEQ ID NO:2), Figure 8 (SEQ ID NO:4), Figure 9 (SEQ ID NO:6), or Figure 10 (SEQ ID NO;8), or portion 15 thereof or an analogue thereof, covalently bound to an insoluble support.

Thus according to a preferred aspect of the invention we provide a Neisseria meningitidis 22 kDa surface protein having an amino acid sequence which includes the sequence 20 of Figure 1 (SEQ ID NO:2), or portion thereof or an analogue thereof, covalently bound to an insoluble support.

A further feature of the invention is the use of the Neisseria meningitidis surface proteins of this invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of Neisseria meningitidis infection. Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against Neisseria meningitidis infection in a test model. One example of an animal model is the mouse model described in the Examples herein. The antibody may be a whole antibody or an antigen-binding fragment thereof and may in general belong to any immunoglobulin class. The antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or if desired, a recombinant antibody or antibody fragment. The term recombinant antibody or antibody fragment means antibody or antibody fragment which were produced using molecular biology techniques. The antibody or antibody fragments may be of polyclonal, or preferentially, monoclonal origin. It may be specific for a number of epitopes associated with the Neisseria meningicidis surface proteins but it is preferably specific for one.

preferably, the antibody or fragments thereof will be specific for one or more epitopes associated with the Neisseria meningitidis 22 kDa surface protein. Also 15 preferred are the monoclonal antibodies Me-1 and Me-7 :described herein.

EXAMPLES

In order that this invention may be better 20 understood, the following examples are set forth. These :examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

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

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

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

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

26 Example S describes the use of an anti-Neisseria meningitidis 22 kDa surface protein monoclonal antibody to protect mice against Neisseria meningitidis infection.

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

Example 7 describes the identification of the sequence for the 22kDa protein and protein-coding gene for other strains of Neisseria m'eningitidis (MCH88, and Z4063), and one strain of Neissr-ia gonorrhoeae.

Example 8 describes the irununological response of rabbits and monkevs to the 22kDa Neisseria meningitidis surface protei.n.

Example 9 describes the procedure used to map the different immunological epitopes of the 22kDa Neisseria meningitidis surface protein.

Example 10''describes the induction by heat of an expression vector f or the large scale production of the 22 kna surface protein- Example 11 describes a purification process for the 22kDa surface protein when produced by recombinant technology.

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

EXPLE I Treatment Of Neoseria meningtidis outer membrane Preparat ions With Proteolytic Enzymes *~An~d The Subsequent Identification Of An Enzyme Resistant Neiaaeria meningitidis 22 kDa Surface Protein Several antigenic preparations derived-from whole cell, lithium chloride, or sarcosyl extracts were-used to study the ultrastructure of Neisseria meningitidis outer membrane. The outer membrane of Gram-negative bacteria acts as an interface between the environment and the interior of the cell and contains most of the antigens that are freely exposed to the host imune defense. The main goal of the study was the identification of new antigens which can induce a protective response against Neisseria meningi tidis. One approach used by the inventors to identify such antigens, was the partial disruption of the antigenic preparations mentioned above with proteolytic enzymes. The antigenic determinants generated by the enzymatic treatments could then be identified by the subsequent analysis of the immunological and protective properties of these treated antigenic preparations. To our surprise we observed after electrophoretic resolution of Neisseria meningicidis 15 lithium chloride outer membrane extracts, that one low :'.•*.molecular weight band, which was stained with Coomassie .°:*.Brilliant Blue R-250, was not destroyed by proteolytic enzyme treatments. Coomassie Blue is used to stain

I

proteins and peptides and has no or very little affinity for the polysaccharides or lipids which are also key components of the outer membrane. The fact that this low molecular weight antigen was stained by Coomassie blue suggested that at least part of it is made up of polypeptides that are not digested by proteolytic enzymes, or that are protected against the action of the enzymes by other surface structures. Moreover, as demonstrated below the very potent enzyme proteinase K did not digest this low molecular weight antigen even after extensive treatments.

Lithium chloride extraction was used to obtain the outer membrane preparations from different strains of Neisseria ieningitidis and was performed in a manner previously described by the inventors (Brodeur et al., Infect. immun., 50, p. 510 (1985)]. The protein content of these preparations were determined by the Lowry method adapted to membrane fractions [Lowry et al., J. Biol.

Chem. 193, p. 265 (1951)1. Outer membrane preparations derived from Neisserit meningitidis strain 608B (B:2a:P1.2) were treated for 24 hours at 37 0 C and continuous shaking with either a-chymotrypsin from bovine pancreas 3.4.21.1) (Sigma) or trypsin type 1 from bovine pancreas 3.4.21.4) (Sigma). The enzyme concentration was adjusted at 2 mg per mg of protein to be treated. The same outer membrane preparations were also treated with different concentrations (0.5 to 24 mg per mg of protein) of Proteinase K from Tritirachium album limber (Sigma or Boehringer Mannheim, Laval, Canada) (E.C.

3.4.21.14). In order to promote protein digestion by proteinase K, different experimental conditions were used.

15 The samples were incubated for 1 hour, 2 hours, 24 hours or 48 hours at 37 0 C or 56 0 C with or without shaking. The pH of the mixture samples was adjusted at either pH 7.2 or :'pH 9.0. One (vol/vol) of sodium dodecyl sulfate (SDS) was also added to certain samples. Immediately after 20 treatment the samples were resolved by SDS-PAGE gel electrophoresis using the MiniProteanIIl (Bio-Rad, Mississauga, Ontario, Canada) system on 14% (wt/vol) gels according to the manufacturer's instructions. Proteins were heated to 100 0 C for 5 minutes with 2-mercaptoethanol and SDS, separated on 14% SDS gels, and stained with Coomassie Brilliant Blue R-250.

:Figure 2 presents the migration profile on 14% SDS- PAGE gel of the proteins present in outer membrane preparations derived from Neisseria meningitidis strain 608B (B:2a:Pl.2) after treatment at 370C for 24 hours with a-chymotrypsin and trypsin. Extensive proteolytic digestion of the high molecular weight proteins and of several major outer membrane proteins can be observed for the treated samples (Figure 2, lanes 3 and 4) compared to the untreated control (Figure 2, lane On the 29 contrary, a protein band with an apparent molecular weight of 22 kDa was not affected even after 24 hours of contact with either proteolytic enzyme.

This unique protein was further studied using a more aggressive proteolytic treatment with Proteinase K (Figure Proteinase K is one of the most powerful proteolytic enzymes since it has a low peptide bond specificity and wide pH optimum. Surprisingly, the 22 kDa protein was resistant to digestion by 2 International Units (IU) of proteinase K for 24 hours at 56 0 C (Figure 3, lane This treatment is often used in our laboratory to produce lipopolysaccharides or DNA that are almost free of proteins. Indeed, only small polypeptides can be seen after such an aggressive proteolytic treatment of the 15 outer membrane preparation. Furthermore, longer treatments, up to 48 hours, or higher enzyme concentrations (up to 24 IU) did not alter the amount of the 22 kDa protein. The amount ,and migration on SDS-PAGE gel of the 22 kDa protein were not affected when the pH of .o 6.20 the reaction mixtures was increased to pH 9.0, or when of SDS, a strong protein denaturant was added (Figure 3, lanes 4, 6 and The combined use of these two denaturing conditions would normally result in the complete digestion of the proteins present in the outer membrane preparations, leaving only amino acid residues.

Polypeptides of low molecular weight were often observed 0 in the digests and were assumed to be fragments of sensitive proteins not effectively digested during the enzymatic treatments. These fragments were most probably protected from further degradation by the carbohydrates and lipids present in the outer membrane. The bands with apparent molecular weight of 28 kDa and 34 kDa which are present in treated samples are respectively the residual enzyme and a contaminating protein present in all enzyme preparations tested.

Interestingly, this study about the resistance of the 22kDa protein to proteases indicated that another protein band with apparent molecular weight of 18kDa seems to be also resistant to enzymatic degradation (Figure 3a). Clues about this 18kDa protein band were obtained when the migration profiles on SDS-PAGE gels of affinity purified recombinant 22kDa protein were analyzed (Figure 3b). The 18 kDa band was apparent only when the affinity purified recombinant 22kDa protein was heated for an extended period of time in sample buffer containing the detergent SDS before it was applied on the gel. N-terminal amino acid analysis using the Edman degradation (Example 3) clearly established that the amino acid residues (E-G-A-S- G-F-Y-V-Q) identified on the 18 kDa band corresponded to 15 the amino acids 1-9 (SEQ ID NO:l). These results indicate that the 18 and 22kDa bands as seen on the SDS-PAGE is in fact derived from the same protein. This last result also indicates that the leader sequence is cleaved from the mature 18 kDa protein. Further studies will be done to identify the molecular modifications explaining this shift "o in apparent molecular weight and to evaluate their impact ooo on the antigenic and protective properties of the protein.

In conclusion, the discovery of a Neisseria meaiagitidis outer membrane protein with the very rare property of being resistant to proteolytic digestion warranted further study of its molecular and immunological characteristics. The purified recombinant 22 kDa surface protein produced by Escherichia coli in Example 3 is also highly resistant to proteinase K digestion. We are presently trying to understand the mechanism which confers to the Neisseria meningitidis 22 kDa surface protein this unusual resistance to proteolytic enzymes.

EXAMPLE 2 Generation of Monoclonal Antibodies Specific for the 22 kDa Neisseria meningitidis Surface Protein The monoclonal antibodies described herein were obtained from three independent fusion experiments.

Female Balb/c mice (Charles River Laboratories, St- Constant, Quebec, Canada) were immunized with outer membrane preparations obtained from Neisseria meningitidis strains 604A, 608B and 2241C respectively serogrouped A, B and C. The lithium chloride extraction used to obtain these outer membrane preparations was performed in a manner previously described by the inventors. [Brodeur S:.:et al., Infect. Immun. 50, p. 510 (1985)]. The protein 15 content of these preparations were determined by the Lowry method adapted to membrane fractions [Lowry et al., J.

Biol. Chem. 193, p. 265 (1951)1. Groups of mice were .injected intraperitoneally or subcutaneously twice, at three-week intervals with 10 mg of different combinations 20 of the outer membrane preparations described above.

Depending on the group of mice, the adjuvants used for the immunizations were either Freund's complete or incomplete adjuvant (Gibco Laboratories, Grand Island, or QuilA (CedarLane Laboratories, Hornby, Ont., Canada).

Three days before the fusion procedure, the immunized mice received a final intravenous injection of 10 mg of one of the outer membrane preparations described above. The fusion protocol used to produce the hybridoma cell lines secreting the desired monoclonal antibody was described previously by the inventors [Hamel et al., J. Med.

Microbiol., 25, p. 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 as previously reported [Martin et al., J. Clin. Microbiol., 28, p. 1720 (1990)] and are presented in Table 1.

The specificity of the monoclonal antibodies was established using Western inmnoblotting following the method previously described by the inventors [Martin et al., Eur. J. Immunol. 18, p. 601 (1988)) with the following modifications. Outer membrane preparations obtained from different strains of Neisseria meningitidis were resolved on 14% SDS-PAGE gels. The proteins were transferred from the gels to nitrocellulose membranes using a semi-dry apparatus (Bio-Rad). A current of 60 mA per gel (6X10cm) was applied for 10 minutes in the electroblot buffer consisting of 25 mM Tris-HCl, 192 mil glycine and 20% (vol/vol) methanol, pH 8.3. The Western immunoblotting experiments clearly indicated that the monoclonal antibodies Me-l, Me-2, Me-3, Me-5, Me-6 and Me- S" 15 7 recognized their specific epitopes on the Neisseria ".".meningitidis 22 kDa protein (Figure 4A). Analysis of the S* SDS-PAGE gels and the corresponding Western immunoblots also indicated that the apparent molecular weight of this protein does not vary from one strain to another.

However, the amount of protein present in the outer 'o membrane preparations varied from one strain to another and was not related to the serogroup of the strain.

Moreover, these monoclonal antibodies still recognized their epitopes on the Neisseria meningitidis 22 kDa 25 surface protein after treatment of the outer membrane preparation with 2 IU of proteinase K per mg of protein :(treatment described in Example 1, supra) (Figure 4B).

Interestingly, the epitopes remained intact after the enzyme digestion thus confirming that even if they are accessible in the membrane preparation to the antibodies they are not destroyed by the enzyme treatment. This latter result suggested that the mechanism which explains the observed proteinase K resistance is most probably not related to surface structures blocking the access of the enzyme to the protein, or to the protection offered by the membrane to proteins which are deeply embedded. While not shown in Figure 4, the results of the immunoblots for Me-i were consistent with the results for the other five monoclonal antibodies.

A series of experiments were performed to partially characterize the Neisseria meningitidis 22 kDa surface protein and to differentiate it from the other known meningococcal surface proteins- No shift in apparent molecular weight on SDS-PAGE gel of the Neisseria meningitidis 22 kDa surface protein was noted when outer membrane preparations were heated at 100°C for 5 minutes, or at 37°C and 56°C for 30 minutes in electrophoresis sample buffer with or without 2-mercaptoethanol. This o o indicated that the migration of the 22kDa surface protein, 15 when present in the outer membrane, was not heat- or 2mercaptoethanol-modifiable.

So. Sodium periodate oxidation was used to determine if the monoclonal antibodies reacted with carbohydrate epitopes present in the outer membrane preparations 20 extracted from Neisseria meningitidis organisms. The method used to perform this experiment was previously described by the inventors. [Martin et al., Infect.

Immun., 60, pp. 2718-2725 (1992)]. Treatment of outer Smembrane preparations with 100 mM of sodium periodate for 1 hour at room temperature did not alter the reactivity of the monoclonal antibodies toward the Neisseria **go meringitidis 22 kDa surface protein. This treatment normally abolishes the binding of antibodies that are specific for carbohydrates.

Monoclonal antibody 2-1-CA2 (provided by Dr. A.

Bhattacharjee, Walter Reed Army Institute of Research, Washington, is specific for the lip protein (also called a surface antigen common to all pathogenic Neisseria species. The reactivity of this monoclonal antibody with outer membrane preparations was compared to the reactivity of monoclonal antibody Me-5. The lipspecific monoclonal antibody reacted with a protein band having an apparent molecular weight of 30 kDa, while monoclonal antibody Me-5 reacted with the protein band of 22 kDa. This result clearly indicates that there is no relationship between Neisseria meningitidis 22 kDa surface protein and the lip protein, another highly conserved outer membrane protein.

To verify the exposure of the 22 kDa protein at the surface of intact Neisseria meningitidis bacterial cells, a radioimmunoassay was performed as previously described by the inventors (Proulx et al., Infec. Immun.. 59, p. 963 (1991)]. Six-hour and 18-hour bacterial cultures were used for this assay. The six monoclonal antibodies were 15 reacted with 9 Neisseria meningitidis strains (the serogroup of the strain is indicated in parentheses on Figure 2 Neisseria gonorrhoeae strains 2 Moraxella catarrhalis strains and 2 Neisseria lactamica strains The radioimmunoassay confirmed 20 that the epitopes recognized by the monoclonal antibodies are exposed at the surface of intact Neisseria meningitidis isolates of different serotypes and serogroups and should also be accessible to the proteolytic enzymes (Figure The monoclonal antibodies bound strongly to their target epitopes on the surface of all Neisseria meningitidis strains tested. The recorded binding values (between 3,000 to 35,000 CPM), varied from one strain to another, and with the physiological state of the bacteria. A Haemophilus influenzae porin-specific monoclonal antibody was used as a negative control for each bacterial strain. Counts below 500 CPM were obtained and subsequently subtracted from each binding value. With respect to the Neisseria meningitidis strains tested in this assay, the results shown in Figure 5 for monoclonal antibodies Me-5 and Me-7 are representative of the results obtained with monoclonal antibodies Me-1, Me-2, Me-3 and Me-6. With respect to the other bacterial strains tested, the binding activities shown for Me-7 are representative of the binding activities obtained with other monoclonal antibodies that recognized the same bacterial strain.

The antigenic conservation of the epitopes recognized by the monoclonal antibodies was also evaluated. A dot enzyme immunoassay was used for the rapid screening of the .monoclonal antibodies against a large number of bacterial strains. This assay was performed as previously described by the inventors (Lussier et al., J. Immunoassay, 10, p.

373 (1989)]. A collection of 71 Neisseria meningitidis strains was used in this study. The sample included 19 isolates of serogroup A, 23 isolates of serogroup B, 13

S

15 isolates of serogroup C 1 isolate of serogroup 29E, 6 isolates of serogroup W-135, 1 isolate of serogroup X, 2 isolates of serogroup Y, 2 isolates of serogroup Z, and 4 isolates that were not serogrouped These isolates were obtained from the Caribbean Epidemiology Centre, Port of Spain, Trinidad; Children's Hospital of Eastern Ontario, Ottawa, Canada; Department of Saskatchewan Health, Regina, Canada; Laboratoire de Sante Publique du Quebec, Montreal, Canada; Max-Planck Institut fur Molekulare Genetik, Berlin, FRG; Montreal Children 25 Hospital, Montreal, Canada; Victoria General Hospital, Halifax, Canada; and our own strains collection. The following bacterial species were also tested: 16 Neisseria gonorrhoeae, 4 Neisseria cinerea, 5 Neisseria lactamica, 1 Neisseria flava, 1 Neisseria flavescens, 3 Neisseria mucosa, 4 Neisseria perflava/sicca, 4 Neisseria perflava, 1 Neisseria sicca, 1 Neisseria subflava and 5 Moraxella catarrhalis, 1 Alcaligenes feacalis (ATCC 8750), 1 Citrobacter freundii (ATCC 2080), 1 Edwarsiella tarda (ATCC 15947), 1 Enterobacter cloaca (ATCC 23355), 1 Enterobacter aerogenes (ATCC 13048), 1 Escherichia coli, 1 Flavobacterium odoratum, 1 Haemophilus influenzae type b (Eagan strain), 1 Klebsiella pneumoniae (ATCC 13883), 1 Proteus rettgeri (ATCC 25932), 1 Proteus vulgaris (ATCC 13315), 1 Pseudomonas aeruginosa (ATCC 9027), 1 Salmonella typhimurium (ATCC 14028), 1 Serrati marcescens (ATCC 8100), 1 Shigella flexneri (ATCC 12022), 1 Shigella sonnei (ATCC 9290). They were obtained from the American Type Culture Collection or a collection held in the Laboratory Centre for Disease Control, Ottawa, Canada. The reactivities of the monoclonal antibodies with the most relevant Neisseria strains are presented in Table 1. One monoclonal antibody, Me-7, recognized its specific epitope on 100% of the 71 Neisseria meningitidis strains tested.

This monoclonal antibody, as well as Me-2, Me-3, Me-5 and 15 Me-6 also reacted with certain strains belonging to other Neisserial species indicating that their specific epitope is also expressed by other closely related Neisseriaceae.

Except for a faint reaction with one Neisseria lactamica strain, monoclonal antibody Me-1 reacted only with 20 Neisseiria meningitidis isolates. Me-1 was further tested with another sample of 177 Neisseria meningitidis isolates and was able to correctly identify more than 99% of the total Neisseria meningitidis strains tested.

Besides the Neisseria strains presented in Table 1, the monoclonal antibodies did not react with any of the other bacterial species mentioned above.

In conclusion, six monoclonal antibodies which specifically reacted with the Neisseria meningitidis 22 kDa surface protein were generated by the inventors.

Using these monoclonal antibodies we demonstrated that their specific epitopes are 1) located on a proteinase K resistant 22 kDa protein present in the outer membrane of Neisseria meningitidis, 2) conserved among Neisseria meningitidis isolates, 3) exposed at the surface of intact Neisseria meningitidis cells and accessible to antibody, 37 and 4) the reactivity of these monoclonal antibodies with the Neisseria meningitidis 22 kDa surface protein is not modified by a treatment with sodium periodate, suggesting that their specific epitopes are not located on carbohydrates.

Although we found that the migration of the Neisseria meningitidis 22kDa protein is moved to an apparent molecular weight of about 18kDa when heated under stringent conditions, we observed that the migration is not modified by 2-mercaptoethanol treatment.

We also demonstrated that the Neisseria meningitidis 22 kDa surface protein has no antigenic similarity with the lip protein, another low molecular weight and highly conserved protein present in the outer membrane of 15 Neisseria meningitidis.

As will be presented in Example 3, these monoclonal antibodies also reacted with the purified, recombinant 22 kDa surface protein produced after transformation of Escherichia coli strain BL21 (DE3) with a plasmid vector 20 pNP2202 containing the gene coding for the Neisseria •meningitidis 22 kDa surface protein.

Table 1. ReactivitY of the monoclonal antibodies with Neisseria isolates se1egzbup of MriateUi aOnguLoitidit .m p It mr I i c 'U rr S I Srogxv s of M.±MIA m angIds..

s 2 2 3 69 0 0(71 1 Me-i w G 19 22 13 1 6 M.-2 1goca( 19 20 13 1 6 a 19CI4O 19 22 13 1 6 M-S -I.c19 22 13 1 6 M.4-6 IOC: 19 23 13 1 6 1 2 2 3 69 0 0 1 0 2 2 4 6 0 02 1 2 2 3 65 0 2 4 S 2 2 3 £9 0 2 0 1 2 2 3 24 1 6I I l iIii I I r 1 1 23 13 1 2 -isolates not serogrouped EXAMPLE 3 Molecular Cloning, Sequencing Of The Gene, High Yield Expression And purification Of The Neisseria meningitidis 22 kDa Surface Protein A. Molecular loning A LambdaGEM-11 genomic DNA library from Neisseria meningitidis strain 608B (B:2a:P1.2) was constructed according to the manufacturer's recommendations (Promega CO. Madison. WI) Briefly, the genomic DNA of the 608B 15 strain was partially digested with Sau 3AI, and fragments ranging between 9 and 23 Kb were purified on agarose gel before being ligated to the Bam HI sites of the LambdaGEM- 11 arms. The resulting recombinant phages were used to infect Escherichia coli strain LE392 (Promega) which was then plated onto LB agar plates. Nineteen positive plaques were identified after the imnmuno-screening of the library with the Neisseria meningitidis 22 kDa surface protein-specific monoclonal antibodies of Example 2 using the following protocol. The plates were incubated minutes at -20°C to harden the top agar. Nitrocellulose filters were gently applied onto the surface of the plates for 30 minutes at 4 0 C to absorb the proteins produced by the recombinant viral clones. The filters were then washed in PBS-Tween 0.02% (vol/vol) and immunoblotted as described previously [Lussier et al., J. Immunoassay, p. 373 (1989)]. After amplification and DNA purification, one viral clone, designated clone 8, which had a 13 Kb insert was selected for the subcloning experiments. After digestion of this clone with Sac I, two fragments of 5 and 8 Kb were obtained. These fragments were purified on agarose gel and ligated into the Sac I restriction site of the low copy number plasmid pWKS30 (Wang and Kushner, Gene, 100, p. 195 (1991)]. The recombinant plasmids were 1. used to transform Escherichia coli strain JM109 (Promega) by electroporation (Bio-Rad, Mississauga, Ont., Canada) following the manufacturer's recommendations, and the resulting colonies were screened with the Neisseria meningitidis 22 kDa surface protein-specific monoclonal .20 antibodies of Example 2. Positive colonies were observed only when the bacteria were transformed with the plasmid carrying the 8 Kb insert. Western blot analysis (the methodology was described in Example 2) of the positive clones showed that the protein expressed by Escherichia coli was complete and migrated on SDS-PAGE gel like the Neisseria meningitidis 22 kDa surface protein. To further reduce the size of the insert, a clone containing the 8 Kb fragment was digested with Cla I and a 2.75 Kb fragment was then ligated into the Cla I site of the plasmid. Western blot analysis of the resulting clones clearly indicated once again that the protein expressed by Escherichia coli was complete and migrated on SDS-PAGE gel like the native Neisseria meningitidis 22 kDa surface protein.

After restriction analysis, two clones, designated pNP2202 and pNP2203, were shown to carry the 2.75 Kb insert in opposite orientations and were selected to proceed with the sequencing of the gene coding for the Neisseria meningitidis 22 kDa surface protein. The "Double Stranded Nested Deletion Kit" from Pharmacia Biotech Inc. (Piscataway, NJ) was used according to the manufacturer's instructions to generate a series of nested deletions from both clones. The resulting truncated inserts were then sequenced from the M13 forward primer present on the pWKS30 vector with the "Taq Dye Deoxy Terminator Cycle Sequencing Kit" using an Applied Biosystems Inc. (Foster City. CA) automated sequencer model 373A according to the manufacturer's .o 15 recommendations.

*0 0 B. Sequence Analysis After the insert was sequenced in both directions, the nucleotide sequence reveale an open reading frame 20 consisting of 525 nucleotides (including the stop codon) encoding a protein composed of 174 amino acid residues having a predicted molecular weight of 18,000 Daltons and S. a pI of 9.93. The nucleotide and deduced amino acid sequences are presented in Figure 1 (SEQ ID NO:1; SEQ ID NO:2).

To confirm the correct expression of the cloned gene, the N-terminal amino acid sequence of the native 22 kDa surface protein derived from Neisseria meningitidis strain 608B was determined in order to compare it with the amino acid sequence deduced from the nucleotide sequencing data.

Outer membrane preparation derived from Neisseria meningitidis strain 608B was resolved by electrophoresis on a 14% SDS-PAGE gel and transferred onto a polyvinylidine difluoride membrane (Millipore Products, Bedford MA) according to a previously described method (Sambrook et al., Molecular Cloning; a laboratory manual, Cold Spring Harbor Laboratory Press (1989)]. The 22 kDa protein band was excised from the gel and then subjected to Edman degradation using the Applied Biosystems Inc.

(Foster City, CA) model 473A automated protein sequencer following the manufacturer's recommendations. The amino acid sequence E-G-A-S-G-F-Y-V-Q-A corresponded to amino acids 1-10 (SEQ ID NO:2) of the open reading frame, indicating that the Neisseria meningitidis strain 608B, 22 kDa surface protein has a 19 amino acid leader peptide (amino acid residues -19 to -1 of SEQ ID NO:2).

A search of established databases confirmed that the Neisseria meningitidis strain 608B., 22 kDa surface protein (SEQ ID NO:2) or its gene (SEQ ID NO:l) have not been 15 described previously.

C. High Yield Expression And Purification of The Recombinant Neisseria meningitidis 22 kDa Surface Protein The following process was developed in order to maximize the production and purification of the recombinant Neisseria meningitidis 22 kDa surface protein expressed in Escherichia coli. This process is based on the observation that the recombinant 22 kDa surface protein produced by Escherichia coli strain BL21(DE3) [Studier and Moffat, J. Mol. Biol., 189, p. 113 (1986)] carrying the plasmid pNP2202 can be found in large amounts in the outer membrane, but can also be obtained from the culture supernatant in which it is the most abundant protein. The culture supernatant was therefore the material used to purify the recombinant 22 kDa protein using affinity chromatography (Figure 6A).

To generate an affinity chromatography matrix, monoclonal antibodies Me-2, Me-3 and Me-5 (described in Example 2) were immobilized on CNBr-activated sepharose 4B (Pharmacia Biotech Inc., Piscataway. NJ) according to the manufacturer' s instructions.

To prepare the culture supernatant, an overnight culture of Escherichia coli strain BL21(DE3). harboring the plasmid pNP2202 was inoculated in LB broth (Gibco Laboratories, Grand Island, containing 25 mg/ml of ampicillin (Sigma) and was incubated 4 hours at 37 0 C with agitation. The bacterial cells were removed from the culture media by two centrifugations at 10,000 Xg for minutes at 4°C. The culture supernatant was filtered onto a 0.22 mm membrane (Millipore, Bedfords, MA) and then concentrated approximately 100 times using an ultrafiltration membrane (Amicon Co.. Beverly, MA) with a molecular cut off of 10,000 Daltons. To completely 15 solubilize the membrane vesicles, Empigen BB (Calbiochem Co., LaJolla, CA)) was added to the concentrated culture supernatant to a final concentration of 1% (vol/vol). The Soo suspension was incubated at room temperature for one hour, dialyzed overnight against several liters of 10 mM Tris- HC1 buffer, pH 7.3 containing 0.05% Empigen BB(vol/vol) and centrifuged at 10,OOOXg for 20 minutes at 4°C. The ~antigen preparation was added to the affinity matrix and incubated overnight at 4°C with constant agitation. The ~gel slurry was poured into a chromatography column and 25 washed extensively with 10 mM Tris-HCl buffer, pH 7.3 containing 0.05% Empigen BB (vol/vol). The recombinant 22 ~kDa protein was then eluted from the column with 1 M LiCl in 10 mM Tris-HCl buffer, pH 7.3. The solution containing the eluted protein was dialyzed extensively against several liters of 10 mM Tris-HCl buffer, pH 7.3 containing 0.05% Empigen BB. Coomassie Blue and silver stained SDS- Page gels [Tsai and Frasch, Analytical Biochem.. 119, pp. 19 (1982)] were used to evaluate the purity of the recombinant 22 kDa surface protein at each step of the purification process and representative results are presented in Figure 6A. Silver staining of the gels clearly demonstrated that the purification process generated a fairly pure recombinant 22 kDa protein with only a very small quantity of Escherichia coli lipopolysaccharide.

The resistance to proteolytic cleavage of the purified recombinant 22 kDa surface protein was also verified and the results are presented in Figure 6B.

Purified recombinant 22 kDa surface protein was treated as described in Example 1 with a-chymotrypsin and trypsin at 2 mg per mg of protein and with 2 IU of proteinase K per mg of protein for 1 hour at 37 0 C with constant shaking.

No reduction in the amount of protein was observed after any of these treatments. In comparison, partial or complete digestion depending on the enzyme selected was observed for the control protein which was in this case bovine serum albumin (BSA, Sigma). Furthermore, longer periods of treatment did not result in any modification of the protein. These latter results demonstrated that transformed Escherichia coli cells can express the complete recombinant 22 kDa surface protein and that this protein is also highly resistant to the action of these three proteolytic enzymes as was the native protein found in Neisseria meningictdis. In addition, the purified recombinant 22 kDa surface protein which is not embedded in the outer membrane of Escherichia coli is still highly resistant to the action of the proteolytic enzymes.

We also verified the effect of the enzymatic treatments on the antigenic properties of the recombinant 22 kDa protein. As determine by ELISA and Western immunoblotting, the monoclonal antibodies described in Example 2 readily recognized the recombinant 22 kDa surface protein that was purified according to the process described above (Figure 6C). Moreover, the reactivity of monoclonal antibody Me-5, as well as the reactivity of other 22 kDa protein-specific monoclonal antibodies, with the purified recombinant 22 kDa surface protein was not altered by any of the enzyme treatments, thus confirming that the antigenic properties of the recombinant 22 kDa protein seem similar to the ones described for the native protein.

Important data were presented in Example 3 and can be summarized as follows: 1) the complete nucleotide and amino acid sequences of the Neisseria meningitidis 22 kDa surface protein were obtained (SEQ ID NO:1; SEQ ID NO:2); 2) N-terminal sequencing of the.native protein confirmed that the Neisseria meningitidis 22 kDa gene was indeed cloned; *o 15 3) this protein was not described previously; 4) it is possible to transform a host such as Escherichia coli and obtain expression of the recombinant Neisseria meningitidis 22 kDa surface protein in high yield; 5) it is possible to obtain the recombinant protein free of other Neisseria meningitidis molecules and almost free of components produced by Escherichia coli; 6) the purified recombinant 22 kDa surface protein remains highly resistant to the action of proteolytic enzymes such as ac-chymotrypsin, trypsin and proteinase K; and 7) the antigenic properties of the recombinant 22 kDa protein compare to the ones described for the native Neisseria meningitidis 22 kDa surface protein.

EXAMPLE 4 Molecular Conservation Of The Gene Coding for the Neisseria meningitidis 22 kDa Surface Protein To verify the molecular conservation among Neisseria isolates of the gene coding for the Neisseria meningitidis 22 kDa surface protein, a DNA dot blot hybridization assay was used to test different Neisseria species and other bacterial species. First, the 525 base pair gene coding for the Neisseria meningitidis 22 kDa surface protein was amplified by PCR. purified on agarose gel and labeled by random priming with the non radioactive DIG DNA labeling .and detection system (Boehringer Mannheim, Laval, Canada) following the manufacturer's instructions.

The DNA dot blot assay was done according to the manufacturer's instructions (Boehringer Mannheim) Briefly, the bacterial strains to be tested were dotted onto a positively charge nylon membrane (Boehringer 15 Mannheim), dried and then treated as described in the DIG *System's user's guide for colony lifts. Pre- O hybridizations and hybridizations were done at 42°C with solutions containing 50% formamide (Sigma). The prehybridization solution also contained 100 mg/ml of denatured herring sperm DNA (Boehringer Mannheim) as an additional blocking agent to prevent non-specific ~hybridization of the DNA probe. The stringency washes and detection steps using the chemiluminescent lumigen

PPD

substrate were also done as described in the DIG System's user's guide.

For the 71 Neisseria meningitidis strains tested the results obtained with monoclonal antibody Me-7 and the 525 base pair MA probe were in perfect agreement. According to the results, all the Neisseria meningitidis strains tested have the Neisseria meningitidis 22 kDa surface protein gene and they express the protein since they were all recognized by the monoclonal antibody, thus confirming that this protein is highly conserved among the Neisseria meningitidis isolates (Table 2).

The DNA probe also detected the gene coding for the Neisseria meningitidis 22 kDa surface protein in all Neisseria gonorrhoeae strains tested.

On the contrary, the monoclonal antibody Me-7 reacted only with 2 out of the 16 Neisseria gonorrhoeae strains tested indicating that the specific epitope is somehow absent, inaccessible or modified in Neisseria gonorrhoeae strains, or that most of the Neisseria gonorrhoeae strains do not express the protein even if they have the coding sequence in their genome (Table 2).

A good correlation between the two detection methods was also observed for Neisseria lactamica, since only one strain of Neisseria lactamica was found to have the gene without expressing the protein (Table This result could also be explained by the same reasons presented in the last paragraph.

This may indicate that, although the 22kDa is not expressed, or not accessible on the surface of Neisseria S* gonorrhoeae strains, the 22kDa protein-coding gene of the Neisseria gonorrhoeae and Neisseria lactamica strains may be used for construction of recombinant plasmids used for the production of the 22kDa surface protein or analogs.

All such protein or analogs may be used for the prevention, detection, or diagnosis of Neisseria 25 infections. More particularly, such infections may be selected from infections from Neisseria meningitidis, Neisseria gonorrhoeae, and Neisseria lactamica. Therefore, the 22kDa surface protein or analogs, may be used for the manufacture of a vaccine against such infections.

Moreover, the 22kDa protein or analogs, may be used for the manufacture of a kit for the detection or diagnosis of such infections.

The results obtained with Moraxella catharralis strains showed that out of the 5 strains tested, 3 reacted with monoclonal antibody Me-7, but none of them reacted with the DNA probe indicating that the gene coding for the Neisseria Ineningitidi.s 22 kna surface protein is absent from the genome of these strains (Table 2).

Several other Neisserial species as well as other bacterial species (see footnote, Table 2) were tested and none of them were found to be positive by any of the two tests. This latter result seems to indicate that the gene for the 22 kna surface protein is shared only among closely related species of Neisseriacae.

Table 2. Reactivity of the 525 base pair DNA probe and monoclonlal antibody Me-7 with different Neisseria species Number of strains identified by FNelsBeeria species Monoclonal (number of strains tested)' antibody Ke-7 DNA probe Neisseria meningitidis (71) 71 71 Moraxella catharallis 3 0 Neisseria gonorrhoeate (16) 2 16 lNeiuosria actamlca 4 'The tolvwing Neiseffial species and other bacterial species were also tested with the two assays and gave negative results. 1 Neisseria cmerea. 1 Neisseria flays. I Neisseria fiavescens. 2 Neisseda mucosa. 4 Neisseria peflavsa/Cs. 1 Neisseria pedlava I N. sioca, 1 N. subflava. 1 Alfgefes feacali (ATCC 8750). 1 Botdetella pertussis (9340). 1 Bordetela brochseptiMa I Cbtvctertimufdi (ATOC 2080). 1 Edwarsella tarda (ATCC 15947), 1 Enterobacter cloaca (ATCO 23355). 1 Enterobacter aerogenes; (ATCC 13048). 1 Escherichia col, 1 Flavobacterium odoratum., 1 Haemcophilus influenzae type b (Eagan strain), 1 Klebsiella pneumoniae (ATCC 13883). 1 Proteus rettgeri (ATCC 25932). 1 Proteus vulgaris (ATC 13315), 1 pseudwwwOl8 aerighfosa (ATCC 9027). 1 Salmonella typhimuur (ATCC 14028).

1 Serrali marcescens (ATCC 8100), 1 Shigella flexneni (ATCC 12022). 1 Shigella sonnel (ATCC 9290). mid 1 Xanthomfoflas rnattoptia.

In conclusion, the DNA hybridization assay clearly indicated that the gene coding for the Neisserila 48 meninitidis 22 kDa surface protein is highly conserved among the pathogenic Neisseria. Furthermore, the results obtained clearly showed that this DNA probe could become a valuable tool for the rapid and direct detection of pathogenic Neisseria bacteria in clinical specimen. This probe could even be refined to discriminate between the Neisseria meningitidis and Neisseria gonorrhoeae.

EXAMPLE 5 Bacteriolytic And Protective Properties Of The Monoclonal Antibodies The bacteriolytic activity of the purified Neisseria meningitidis 22 kDa surface protein-specific monoclonal antibodies was evaluated in vitro according to a method S 15 described previously [Brodeur et al., Infect. Immun., p. 510 (1985); Martin et al., Infect. Immun., 60, p. 2718 (1992)]. In the presence of a. guinea pig serum complement, purified monoclonal antibodies Me-i and Me-7 efficiently killed Neisseria meningitidis strain 608B.

20 Relatively low concentrations of each of these monoclonal antibodies reduced by more than 50% the number of viable bacteria. The utilization of higher concentrations of purified monoclonal antibodies Me-i and Me-7 resulted in a sharp decrease (up to 99%) in the number of bacterial 25 colony forming units. Importantly, the bacteriolytic activity of these monoclonal antibodies is complement dependent, since heat-inactivation of the guinea pig serum **for 30 minutes at 56 0 C completely abolished the killing activity. The other monoclonal antibodies did not exhibit significant bacteriolytic activity against the same strain. The combined, representative results of several experiments are presented in Figure 7, wherein the results shown for Me-7 are representative and consistent with the results obtained for Me-1. The results shown for Me-2 are representative and consistent with the results obtained for the other monoclonal antibodies Me-3, Me-5 and Me-6.

A mouse model of infection, which was described previously by one of the inventors [Brodeur et al, Infect.

Immun., 50, p. 510 (1985); Brodeur et al., Can. J.

Microbiol., 32, p. 33 (1986)] was used to assess the protective activity of each monoclonal antibody. Briefly, Balb/c mice were injected intraperitoneally with 600 ml of ascitic fluid containing the monoclonal antibodies 18 hours before the bacterial challenge. The mice were then challenged with one ml of a suspension containing 1000 colony forming units of Neisseria meningitidis strain 608B, 4% mucin (Sigma) and 1.6% hemoglobin (Sigma). The combined results of several experiments are presented in *15 Table 3. It is important to note that only the bacteriolytic monoclonal antibodies Me-1 and Me-7 *o protected the mice against experimental Neisseria meningitidis infection. Indeed, the injection of ascitic Ifluid containing these two monoclonal antibodies before the bacterial challenge significantly increased the rate ~of survival of Balb/c mice to 70% or more compared to the 9% observed in the control groups receiving either 600 ml Sp2/0 induced ascitic fluid or 600 ml ascitic fluid containing unrelated monoclonal antibodies. Results have 25 also indicated that 80% of the mice survived the infection if they were previously injected with 400 Vg of protein A purified Me-7 18 hours before the bacterial challenge.

°Subsequent experiments are presently being done to determine the minimal antibody concentration necessary to protect 50% of the mice. Lower survival rates from 20 to were observed for the other Neisseria meningitidis 22 kDa surface protein-specific monoclonal antibodies..

Table 3. Evaluation of the immunoprotective potential of the 22 kDa surface protein-specific monoclonal antibodies against Neisseria meningitidis strain 608B (B:2a:Pl.2) Number of living mice after challenge Monoclonal of antibodies 24 h 72 h survival Me-1 29/30 23/30 76 Me-2 17/20 3/20 Me-3 5/10 2/10 11/20 8/20 Me-7 10/10 7/10 purified Me-7 13/15 12/15 Control 31/100 9/100 9 a.

a.

a In conclusion, the results clearly indicated that an antibody specific for the Neisseria meningitidis 22 kDa surface protein can efficiently protect mice against an experimental lethal challenge. The induction of protective antibodies by an antigen is one of the most important criteria to justify further research on potential vaccine candidate.

EXAMPLE 6 Trmunization With Purified Recombinant 22 kDa Surface Protein Confers protection Against Subsequent Bacterial Challenge Purified recombinant 22 kDa surface protein was prepared according to the protocol presented in Example 3, and was used to immunize Balb/c mice to determine its protective effect against challenge with a lethal dose of 51 Neisseria meningitidis 608B (B:2a:Pl.2). It was decided to use the purified recombinant protein instead of the native meningococcal protein in order to insure that there was no other meningococcal antigen in the vaccine preparation used during these experiments. The mouse model of infection used in these experiments was described previously by one of the inventors [Brodeur et al., Infec.

Inmmun., 50, p. 510 (1985); Brodeur et al., Can. J.

Microbiol., 32, p. 33 (1986)]. The mice were each injected subcutaneously three times at three-week intervals with 100 ml of the antigen preparation containing either 10 or 20 ug per mouse of the purified recombinant 22 kDa surface protein. QuilA was the adjuvant used for these experiments at a concentration of 15 25 pg per injection. Mice in the control groups were *injected following the same procedure with either 10 or 'g of BSA, 20 Vg of concentrated culture supernatant of **Escherichia coli strain BL21(DE3) carrying the plasmid without the insert gene for the meningococcal protein prepared as described in Example 3, or phosphatebuffered saline. Serum samples from each mouse were obtained before each injection in order to analyze the development of the immune response against the recombinant protein. Two weeks following the third immunization the mice in all groups were injected intraperitoneally with 1 ml of a suspension containing 1000 colony forming units of :Neisseria meningitidis strain 608B in 4% mucin (Sigma) and 1.6% hemoglobin (Sigma).

The results of these experiments are presented in Table 4. Eighty percent of the mice immunized with the purified recombinant 22 kDa surface protein survived the bacterial challenge compared to 0 to 42% in the control groups. Importantly. the mice in the control group injected with concentrated Escherichia coli culture supernatant were not protected against the bacterial 52 challenge. This latter result clearly demonstrated that the components present in the culture media and the Escherichia coli's antigens that might be present in small amounts after purification do not contribute to the observed protection against Neisseria menigitidis.

Table 4. immunization With Purified Recombinant 22 kDa Surface Protein Confers Protection Against Subsequent Bacterial Challenge with Neisseria meningitidis 608B (B:2a:P1.2) strain.

Number of living mice after challenge s of Experiment Group 24 h 48 h 72 h survival 1 10 jg of 20/20 16/20 purified 22kDa pg of BSA 17/19 8/19 42 2 20 jg of 9/10 8/10 8/10 purified 22 kDa protein pg of 7/10 5/10 2/10 concentrated E. coli supernatant ug of BSA 6/10 4/10 2/10 Phosphate 8/10 0/10 0/10 0 buffered saline

CONCLUSION

The injection of purified recombinant 22 kDa surface protein greatly protected the immunized mice against the development of a lethal infection by Neisseria meningi tidis.

Antibodies according to this invention are exemplified by marine hybridoma cell lines producing monoclonal antibodies Me-i and Me-7 deposited in the American Type Culture Collection in Rockville, Maryland, USA on July 21, 1995. The deposits were assigned accession numbers HB 11959 (Me-l)and RB 11958 (Me-7).

EXAMPLE 7 Sequence analysis of other strains of Neisseria meningitidis and of Neisseria gonorrhoeae The 2.75 kb clal digested DNA fragment containing the gene coding for the 22kDa surface protein was isolated from the genomic DNA of the different strains of Neisseria meningitidis and Neisseria gonorrhoeae as described in Example 3.

a) MCH88 strain: The nucieotide sequence of strain MCH88 15 (clinical isolate) is presented in Figure 8 (SEQ ID NO:3.).

From experimental evidence obtained from strain 608B (Example a putative leader sequence was deduced corresponding to amino acid -19 to -1 (M-K-K-A-L-A-A-L-I- A search of established databases confirmed that 22kDa surface protein from Neisseria meningitidis strain MCH 188 (SEQ ID NO:4) or its gene (SEQ ID NO:3) have not been described previously.

b) Z4063 strain: The nucleotide sequence of strain Z4063 (Wang et al. Infect. Immun., 60, p.

5 2 67 (1992)) is presented in Figure 9 (SEQ ID NO:5). From experimental evidence obtained from strain 608B (Example a putative leader sequence was deduced corresponding to amino acid 19 to -1 A search of established databases confirmed that 22kDa surface protein from Neisseria meningitidis strain Z4063 (SEQ ID NO:6) or its gene (SEQ ID NO:5) have not been described previously.

c) Neisseria gonorrhoeae strain b2: The nucleotide sequence of Neisseria gonorrhoeae strain b2 (serotype 1.

Nat.Ref. Center for Neisseria, LCDC, Ottawa, Canada) is described in Figure 10 (SEQ ID NO:7) From experimental evidence obtained from strain 608B (Example a putative leader sequence was deduced corresponding to amino acid 19 to -1 A search of established databases confirmed that 22kDa surface protein from Neisseria gonorrhoeae strain b2 (SEQ ID NO:8) or its gene (SEQ ID NO:7) have not been described previously.

Fioure 11 shows the consensus sequence established from the DNA sequence of all four strains tested. The MCH88 strain showed an insertion of one codon (TCA) at 15 nucleotide 217, but in general the four strains showed striking homology.

Figure 12 depicts the homology between the deduced .amino acid sequence obtained from the four strains. There is greater than 90% identity between all four strains.

Example 8 Immunological response of rabbits and monkeys to the 22kDa Neisseria meningitidis surface protein Rabbits and monkeys were immunized with the recombinant 22kDa protein to assess the antibody response in species other than the mouse.

a) Rabbits Male New Zealand rabbits were immunized with outer membrane preparations obtained from E. coli strain JM109 with the plasmid pN2202 or with the control plasmid (the strain and the plasmids are described in Example 3).

The lithium chloride extraction used to obtain these outer membrane preparations was performed in a manner previously described by the inventors (Brodeur et al, Infect. innun.

510 (1985)]. The protein content of these preparations were determined by the Lowry method adapted to membrane fractions (Lowry et al, J. Biol. Chem. 193, 265 (1951)]. The rabbits were injected subcutaneously and intramuscularly at several sites twice at three week intervals with 150 pg of one of the outer membrane preparations described above. QuilA, at a final concentration of 20% (vol./vol.) (CedarLane Laboratories, Hornby, Ont., Canada), was the adjuvant used for these immunizations. The development of the specific humoral 0O *6* *oo response was analyzed by ELISA using outer membrane preparations extracted from Neisseria meningitidis strain 608B (B:2a:P1.2) as coating antigen and by Western i15 immunoblotting following methods already described by the inventors [Brodeur et al., Infect. Immun. 50, 510 (1985);

S

~Martin et al, Eur. J. Immunol. 18, 601 (1988)]. Alkaline phosphatase or peroxydase-labeled Donkey anti-rabbit inmunoglobulins (Jackson ImmunoResearch Laboratories, West 20 Grove, PA) were used for these assays.

The injection of E. coli outer membrane preparation containing the 22 kDa recombinant protein in combination

OS*

with QuilA adjuvant induced in the rabbit a strong specific humoral response of 1/32,000 as determined by ELISA (Figure 13). The antibodies induced after the injection of the recombinant 22 kDa protein reacted with the purified recombinant 22 kDa protein, but more importantly they also recognized the native protein as 56 expressed, folded and embedded in the outer membrane of Neisseria meningitidis. Western Immunoblotting experiments clearly indicated that the antibodies present after the second injection recognized on nitrocellulose membrane the same protein band as the one revealed by Mab Me-2 (described in Example which is specific for the 22 kDa protein.

b) Monkeys Two Macaca fascicularis (cynomolgus) monkeys were respectively immunized with two injections of 100 ug (K28) and 200 ug (1276) of affinity purified recombinant 22 kDa protein per injection. The methods used to produce and purify the protein from E. coli strain BL2lDe3 were 15 described in Example 3. Alhydrogel, at a final concentration of 20% (vol./vol.) (CedarLane Laboratories, Hornby, Ont., Canada), was the adjuvant used for these immunizations. The monkeys received two intramuscular injections at three weeks interval. A control monkey (K65) was immunized with an unrelated recombinant protein preparation following the same procedures. The sera were 0analyzed as described above. Alkaline phosphatase or Peroxydase-labeled Goat anti-human immunoglobulins (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for these assays.

The specific antibody response of monkey K28 which was immunized with 100g of purified protein per injection appeared faster and was stronger than the one observed for 57 monkey 1276 which was injected with 200ig of protein (Figure 14). Antibodies specific for the native 22 kDa protein as detected by Western immunoblotting were already present in the sera of the immunized monkeys twenty one days after the first injection, but were absent in the sera of the control monkey after two injections of the control antigen.

Conclusion The data presented in Examples 2 and 5 clearly showed that the injection of the recombinant 22 kDa protein can induce a protective humoral response in mice which is directed against Neisseria meninitidis strains. More importantly, the results presented in this example S* demonstrate that this immunological response is not 15 restricted to only one species, but this recombinant surface protein can also stimulate the imune system of other species such as rabbit or monkey.

Example 9 Epitope mapping of the 22kDa Neisaeria 20 meningltldis protein Neisseria meningitidis 22 kDa surface protein was epitope mapped using a method described by one of the inventors (Martin et al. Infect. Immun (1991): 59:1457- 1464]1. Identification of the linear epitopes was accomplished using 18 overlapping synthetic peptides covering the entire Neisseria meningitidis 22 kDa protein sequence derived from strain 6.08B (Figure 15) and 58 hyperimmune sera obtained after immunization with this protein. The identification of immunodominant portions on the 22 kDa protein may be helpful in the design of new efficient vaccines. Furthermore, the localization of these B-cell epitopes also provides valuable information about the structural configuration of the protein in the outer membrane of Neisseria meningitidis.

All peptides were synthesized by BioChem Immunosystems Inc. (Montreal, Canada) with the Applied Biosystems (Foster City, Calif.) automated peptide synthesizer.

Synthetic peptides were purified by reverse-phase highpressure liquid chromatography. Peptides CS-845, CS-847, CS-848, CS-851, CS-852 and CS-856 (Figure 15) were solubilized in a small volume of 6M guanidine-HCl (J.T.

15 Baker, Ontario, Canada) or dimethyl sulfoxide (J.T.

Baker). These peptides were then adjusted to 1 mg/ml with distilled water. All the other peptides were freely soluble in distilled water and were also adjusted to 1 mg/ml.

20 Peptide enzyme-linked immunosorbent assays (ELISA) were performed by coating synthetic peptides onto microtitration plates (Immulon 4, Dynatech Laboratories Inc., Chantilly, VA) at a concentration of 50 pg/ml in mM carbonate buffer, pH 9.6. After overnight incubation at room temperature, the plates were washed with phosphate-buffered saline (PBS) containing 0.05% (wt/vol) Tween 20 (Sigma Chemical Co., St.-Louis, Mo.) and blocked with PBS containing 0.5% (wt/vol) bovine serum albumin 59 (Sigma). Sera obtained from mice and monkeys immunized with affinity purified recombinant 22 kDa surface protein were diluted and lOOl per well of each dilution were added to the ELISA plates and incubated for 1 h at 37 0

C.

The plates were washed three times, and 100 ul of alkaline phosphatase-conjugated goat anti-mouse or anti-human immunoglobulins (Jackson ImmunoResearch Laboratories, West Grove, PA diluted according to the manufacturer's recommendations was added. After incubation for 1 h at 37 0 C, the plates were washed and 100 ul of diethanolamine (vol/vol), pH 9.8) containing p-nitro-phenylphosphate (Sigma) at 1 mg/ml was added. After 60 min., the reaction (X=410 nm) was read spectrophotometrically with a microplate reader.

-*j 15 Mouse and monkey antisera obtained after immunization with affinity purified recombinant 22 kDa protein (Example 8) were successfully used in combination with eighteen overlapping synthetic peptides to localize B-cell epitopes on the protein. These epitopes are clustered within three ooo 20 antigenic domains on the protein.

The first region is located between amino acid residues 51 and 86. Computer analysis using different algorithms suggested that this region has the highest probability of being immunologically important since it is hydrophilic and surface exposed. Furthermore, comparison of the four protein sequences which is presented in Figure 12 indicates that one of the major variation, which is the insertion of one amino acid residue at position 73, is also located in this region.

The antisera identified a second antigenic domain located between amino acid residues 110 and 140.

Interestingly, the sequence analysis revealed that seven out of the fourteen amino acid residues that are not conserved among the four protein sequences are clustered within this region of the protein.

A third antigenic domain located in a highly conserved portion of the protein, between amino acid residues 31 and 55, was recoanized only bv the monkeys' sera.

:..*.Example 10 Heat-inducible expression vector for the 15 large scale production of the 22 kDa surface protein The gene coding for the Neisseria meningitidis 22 kDa surface protein was inserted into the plasmid p629 (George et al. Bio/technology 5: 600-603 (1987)]. A 20 cassette of the bacteriophage X ci857 temperature sensitive repressor gene, from which the functional Pr promoter has been deleted, is carried by the plasmid p629 that uses the PL promoter to control the synthesis of the 22kDa surface protein. The inactivation of the c1857 repressor by a temperature shift from 30 0 C to temperatures above 38 0 C results in the production of the protein encoded by the plasmid. The induction of gene expression in E. coli cells by a temperature shift is advantageous 61 for large scale fermentation since it can easily be achieved with modern fermentors. Other inducible expression vectors usually require the addition of specific molecules like lactose or isopropylthio-P-Dgalactoside (IPTG) in the culture media in order to induce the expression of the desired gene.

A 540 nucleotide fragment was amplified by PCR from the Neisseria meningitidis strain 608B genomic DNA using the following two oligonucleotide primers (OCRR8: TAATAGATCTATG--AAAAGCACTTGCCAC-3' and OCRR9: 3'- These primers correspond to the nucleotide sequences found at both ends of the 22 kDa gene. To simplify the cloning of the PCR product, a Bgl II (AGATCT) restriction site was incorporated into the 15 nucleotide sequence of these primers. The PCR product was purified on agarose gel before being digested with Bgl II.

This Bgl II fragment of approximately 525 base pairs was then inserted into the Bgl II and Barn HI sites of the plasmid p629. The plasmid containing the PCR product 20 insert named pNP2204 was used to transform E. coli strain A partial map of the plasmid pNP2204 is presented in Figure 16. The resulting colonies were .screened with Neisseria meningitidis 22 kDa surfaceprotein specific monoclonal antibodies described in Example 2. Western blot analysis of the resulting clones clearly indicated that the protein synthesized by coli was complete and migrated on SDS-PAGE gel like the native Neisseria meningitidis 22 kDa surface protein. Plasmid 62 DNA was purified from the selected clone and then sequenced. The nucleotide sequence of the insert present in the plasmid perfectly matched the nucleotide sequence of the gene coding for the Neisseria meningitidis 22 kDa protein presented in Figure 1.

To study the level of synthesis of the 22 kDa surface protein, the temperature-inducible plasmid pNP2204 was used to transform the following E. coli strains: W3110, BL21, TOPP1, TOPP2 and TOPP3. The level of synthesis of the 22 kDa surface protein and the localization of the protein in the different cellular fractions were determined for each strain. Shake flask "'"cultures in LB broth (Gibco BRL, Life Technologies, Grand Island, NY) indicated that .a temperature shift from 30 0

C

15 to 39 0 C efficiently induced the expression of the gene.

Time course evaluation of the level of synthesis indicated that the protein appeared, as determined on SDS-PAGE gel, as soon as 30 min after induction and that the amount of protein increased constantly during the induction period.

Expression levels between 8 to 10 mg of 22 kDa protein per liter were determined for E. coli strains W3110 and TOPPI. For both strains, the majority of the 22 kDa p i i protein is incorporated in the bacterial outer membrane.

Example 11 Purification of the Neisseria meningitidis 22kDa protein Since the vast majority of the 22 kDa protein is found embedded in the outer membrane of E. coli strains, the purification protocol presented in this Example is different from the one already described in Example 3 where a large amount of protein was released in the culture supernatant. An overnight culture incubated at 0 C of either E coli strain W3110 or TOPPl harboring the plasmid pNP2204 was inoculated in LB broth containing vg/ml of Ampicillin (Sigma) and was grown at 30 0 C with agitation (250 rpm) until it reached a cell density of 0.6 (.=600nm) at which point the incubation temperature was shifted to 39 0 C for three to five hours to induce the 15 production of the protein. The bacterial cells were harvested by centrifugation at 8,000 xg for 15 minutes at 4 0 C and washed twice in phosphate buffered saline (PBS), o*o.

pH 7.3. The bacterial cells were ultrasonically broken (ballistic disintegration or mechanical disintegration 20 with a French press may also be used). Unbroken cells were removed by centrifugation at 5,000 xg for 5 minutes 9 and discarded. The outer membranes were separated from cytoplasmic components by centrifugation at 100,000 xg for 1 h at 100C. The membrane-containing pellets were resuspended in a small volume of PBS, pH 7.3. To solubilize the 22 kDa surface protein from the. membranes, detergents such as Empigen B8 (Calbiochem Co., LaJolla, CA) Zwittergent-3,1 4 (Calbiochem or 64 octylglucoside (Sigma) were used. The detergent was added to the membrane fraction at final concentration of 3% and the mixture was incubated for 1 h at 200C. The non soluble material was removed by centrifugation at 100,000 xg for 1 h at 100C.

The 22 kDa protein was efficiently solubilized by either three of the detergents, however P-octylglucoside had the advantage of easily removing several unwanted membrane proteins since they were not solubilized and could be separated from the supernatant by centrifuga;ion.

To remove the detergent, the 22 kDa containing supernatant was dialyzed extensively against several changes of PBS buffer.Proteinase K treatment (as in Example 1) can be used to further remove unwanted proteins 15 from the 22kDa surface protein preparation. Differential precipitation using ammonium sulfate or organic solvents, and ultrafiltration are two additional steps that can be used to remove unwanted nucleic acid and lipopolysaccharide contaminants from the proteins before gel permeation and ion-exchange chromatography can be efficiently used to obtain the purified 22 kDa protein.

*Affinity chromatography, as described in Example 3, can also be used to purify the 22 kDa protein.

Example 12 Use of 22kDa surface protein As a Human Vaccine To formulate a vaccine for human use, appropriate 22kDa surface protein antigens may be selected from the polypeptides described herein. For example, one of skill in the art could design a vaccine around the 22kDa polypeptide or fragments thereof containing an immunogenic epitope. The use of molecular biology techniques is particularly well-suited for the preparation of substantially pure recombinant antigens.

The vaccine composition may take a variety of 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 antigens of this invention may 9elicit a protective immune response when administered to a human, the compositions of this invention will be similar to those used for immunizing humans with other proteins 15 and polypeptides, e.g. tetanus and diphteria. Therefore, the compositions of this invention will preferably ~comprise a pharmaceutically acceptable adjuvant such as incomplete Freund's adjuvant, aluminum hydroxide, a muramyl peptide. a water-in-oil emulsion, a liposome, an 20 ISCOM or CTB, or a non-toxic B subunit form cholera toxin.

Most preferably, the compositions will include a water-in- 9oil emulsion or aluminum hydroxide as adjuvant.

oThe composition would be administered to the patient in any of a number of pharmaceutically acceptable forms including intramuscular, intradermal, subcutaneous or topic. Preferrably, the vaccine will be administered intramuscularly.

Generally, the dosage will consist of an initial injection,most probably with adjuvant, of about 0.01 to mg, and preferably 0.1 to 1.0 mg of 22kDa surface protein antigen per patient, followed most probably by one or more booster injections. Preferably, boosters will be administered at about 1 and 6 months after the intial injection.

A consideration relating to vaccine development is the question of mucosal immunity. The ideal mucosal vaccine will be safely taken orally or intranasally as one or a few doses and would elicit protective antibodies on the appropriate surfaces along with systemic immunity. The mucosal vaccine composition may include adjuvants, inert particulate carriers or recombinant live vectors.

:15 The anti-22kDa surface protein antibodies of this invention are useful for passive immunotherapy and immunoprophylaxis of humans infected with Neisseria meninigitidis or related bacteria such as Neisseria gonorrhoeae or Neisseria laccamica. The dosage forms and 20 regimens for such passive immunization would be similar to those of other passive immunotherapies.

An antibody according to this invention is exemplified by a hybridoma producing MAbs Me-i or Me-7 deposited in the American Type Culture Collection in Rockville, Maryland, USA on July 21, 1995, and identified as Murine Hybridoma Cell Lines, Me-1 and Me-7 respectively. These deposits were assigned accession numbers HB 11959 (Me-1) and HB 11958 (Me-7).

67 While we have described herein a number of embodiments of this invention, it is apparent that our basic embodiments may be altered to provide other embodiments that utilize the compositions and processes of this invention. Therefore, it will be appreciated that the scope of this invention includes all alternative embodiments and variations that are defined in the foregoing specification and by the claims appended thereto; and the invention is not to be limited by the specific embodiments which have been presented herein by way of example.

The entire disclosure in the complete specification of our Australian Patent Application No. 49343/96 (716225) is by this cross-reference incorporated into the present specification.

•e •coo SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Brodeur. Bernard

R

Martin. Denis Hamel. Josee Rioux. Clement (ii) TITLE OF INVENTION: PROTEINASE K RESISTANT SURFACE

PROTEIN

OF NEISSERIA

MENINGITIDIS

(iii) NUMBER OF SEQUENCES: 26 (iv) CORRESPONDENCE

ADDRESS:

ADDPSSEE: Goudreau Gage Dubuc Martineau Walker STREET: 800 Place Victoria. Suite 3400. Tour de la Eourse CITY: Montreal ST.TE: Quebec COUNTRY: Canada 2 ZIP?: H4Z 129 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

S* 30 SOFTWARE: PatentIn Release 11.0, Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/406.362 FILING DATE: 17-MAR-1995 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US (PROVIS)60/001,983 FILING DATE: 04-AUG-1995 (viii) ATTORNEY/AGENT

INFORMATION:

(A NAME: Leclerc/Dubuc/Prince, Alain/Jean/Gaetan REFERENCE/DOCKET NUMBER: BIOVAC-1 PCT (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 514-397-7400 TELEFAX: 514-397-4382 INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 830 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HypOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOuRCE: ORGANISM: NeiSSeria ineningitidis STRAIN: 608B (ix) FEATURE: NAME/ME: CDS LOCATION: 143.-667 (ix) FEATURE: NAME/KEY: sigpeptide LOCATI ON: 143- 199 (ix) F-EATURE: NAME/KEY: matpeptioe LOCATION: 2 00. 667 E-QUENCE DESCRIPTION:: SE:Q ID NO:l: 5 TZGGAAA G ACCGATAC CGcTACGTAT CTGAGTAT TGAAAATm ACGATGCAAA 6 A.AGAAAT~TAAGTATAAT ACAGCAGGAT TVITTTAACGG A P.711'17=A 12 CTACA~~.AGGAACCAA. AT A~f AAA AMA GCA CTT GCC ACA CTG ATT GCC 17 2 V. OttMet Lys Lys Ala Leu Ala Thr Leu Ile Ala -19 -15 1 CTC GCT CTC CCG GCC GCC GCA CTG GCG GAA GGC GCA TCC GGC '11T TAC 220 .Leui Ala Leu Pro Ala Ala Ala Leu Ala G1t4 Gly Ala Ser Gly Phe Tyr -5 1 GTC CAA GCC AT GCC GCA CAC GCA AAA GCC TCA AGC TCT TA GT TC7 268 VJal Gin Ala Asp Ala Ala His Ala Lys Ala Ser Ser Ser Leu Gly Ser 15 GCC AAA GGC TTC AGC CCG CGC ATC TCc GCA GGC TAC CGc ATC AAC GAC 316 Ala Lys Gly Phe Ser Pro Arg Ilie Ser Ala Gly Tyr Ar Ile Asn Asp 0. 25 30 CTC CGC TTC GCC GTC GAT TAC ACG CGC TAC AAA AAC TAT AAA GCC CCA 364 0. 45 Leu Arg Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro 45 50 0:6. TCC ACC GAT TIC AAA CTT TAC AGC ATC GGC GCG TCC GCC ATT TAC GAC 412 too Ser Thr Asp Phe Lys Leu Tyr Ser le Gly Ala Ser Ala le Tyr Asp 0 .0 50 60 65 .70 TTC GAC ACC CAA TCG CCC GTC AMA CCG TAT CTC GGC GCO CGC Tl'G AGC 460 Phe Asp Thr Gin Ser Pro Val LYS Pro TYr Leu Gly Ala Arg Leu Ser 75 80 CTC AAC CGC 0CC TCC: GTC GAC TTG GGC GGC AGC GAC AG: TC AGC CAA 508 Leu Asn Arg Ala Ser Val Asp Leu Gly Gly Ser. Asp Ser Phe Ser Gin 95 100 ACC Tcc ATC GOC CTC GGC 0Th Ti'G ACG GGC GTA AGC TAT GCC OT= ACC 556 Thr Ser Ile Gly Leu Gly Val Leu Thr-Gly Val Ser Tyr Ala Vai Tbr 105 110 115 CCG AAT GTC GAT T1' GAT GCC GGC TAC CGC TAC AAC TAC ATC GGC AAA 604 Pro Asn Val Asp Leu Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys 120 125 130 135 CTC AC ACT GTC AAA AAC GTC CGT TCC GGC GMA CTG TCC GTC GGC GMG 652 Val Asr Thr Val Lys Asn Val Arg Ser Gly Giu Leu Ser Val Gly Val 140 145 150 CGC GTC AMA TTC TGATATGCGC CTTATTCt-C AAACCGCCGA GCCTCGGCG 704 Arg Val Lys Phe 155 G'7T1 T T CTGCCACCGC AACTACACAA GCCGCC-GT TTGTACGATA ATC-CCGAAT 764 CTGCGGCTC TGCCGCCCTA Tr-"r-IYAGG 1AATCCGAA:T GTCCAACC ATCATCCACA 824 ACA 83 r f r INFoPrMA:-ION FO. SEQ ID NO:2: IcQUENCr CHARACTERISTICS: LENGT11: aminc acids "YPE: aminc acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro I -19 -15 -10 Ala Leu Ala Giu Gly Ala Ser Gly Phe Tyr Val Gin Ala Asp 1 5 His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe 20 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala 35 40 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe 55 45 Tyr Ser le Gly Ala Ser Ala le Tyr Asp Phe Asp Thr Gin 70 Val Lys Pro Tyr Lou Gly Ala Arg Lou Ser Lou Asn Arg Ala so 85 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gin Tbr Ser Ile Gly 100 105 Val Leu Thr Giy Val Ser Tyr Ala Val Thr Pro Asn Val Asp 110 115 120 Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val 130 135 Val Arg Ser Gly Giu Leu Ser Val Gly Val Arg Val Lys Phe 145 150 155 dla Ala 'la Ala Ser Pro Val Asp Lys Leu Ser Pro Ser Val Leu Gly Leu Asp 125 Lys Asn 140 INFORMATION FOR SEQ ID NO:3: SEQUENCE

CHARACTERISTICS:

LENGTH: 710 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STAIN: MCH88 (ix) FEATURE: NAME/KEY:

CDS

LOCATIONl: 116. .643

:ATURE:

NAME/KEY: sigpeptioe LOCATION: 116..'172 (ix) FEATURE: NAME/K-Y: mat.peptide LOCATION: 173. .643 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:3: GTATCT-GAG GCATGAAAA TATTACAATG CAAAAAGAAA ATTCAGTAT AATACGGCAG GATVITTTAA CGGATTCTTA ACCAT"rC TCCCTGACCA TAAAGGAATC AAGAT ATZ 118 Met 19 1 AAA AAA GCA CTT GCC GCA CTG ATT GCC CTC GCC CTC CCG GCC GCC GCA 166 Lys Lys Ala Leu Ala Ala Leu Ile Ala Leu Ala Leu Pro Ala Ala Ala -15 -10 -5 CT-.G CC GAA GOC GCA TCC GGC IT TAC GTC CAA GCC GAT GCC OCA CAC 214 Leu Ala Clu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala His 1 5 GCC AAA CCC TCA AGC TCT TTA GGT TCT GCC AAA GGC TTC AGC CCG CGC 262 Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro Arg 20 2S ATC TCC GCA GGC TAC CGC ATC AAC CAC CTC CGC TIC CCC GTC GAT TAC 310 lie Ser Ala Cly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp Tyr 35 40- ACG CGC TAC AAA AAC TAT AAA CAA CTC CCA TCC ACC GAT 7I'C AAA 358 Thr Arg Tyr Lys Asn Tyr Lys Gin Val Pro Ser Thr Asp Phe Lys Leu 50 55 TAC AGC ATC GGC GCG TCC GCC ATT TAC GAC TTC GAC ACC CAA TCC CCC 406 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gin Ser Pro 70 72 GTC AAA CC TAT CTC CGC GCG CCC TI'C AGC CMC AAC CCC CCC TCC GTC 454 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 85 AC TTr AAC GGC AGC GAC AGC TTC AGC CAA ACC TCC ACC GGC CTC GGC 502 Asp Phe Asn Gly Ser Asp Ser Phe Ser Gin Thr Ser Thr Gly Leu Gly 100 105 110 GTA TTG GCG CCC GTA AGC TAT GCC GTT ACC CCG AAT GTC CAT TlG CAT 550.

Val Leu Ala Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 115 120 125 GCC GC TAC CGC TAC AAC TAC ATC GGC AAA GTC AAC ACT GTC MA AAT 598 Ala Gly ?yr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys Asn 130 135 140 GTC CGT TCC GGC GM CTG TCC GCC CCC GTA CGC GTC AMA TTC TGATATACGC 650 Vai Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 C-TTATTZZC AAACCGCCGA GCCTT1CGGC GGT '-TTT TCCGCCGCZf-: CAACTACACA 7,C INFORATION FOR SEQ ID NO:4: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 175 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Lys Lys Ala Leu Ala Mla Leu Ile Ala Leu Ala Leu Pro Ala Ala -19 -15 -10 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gin Ala Asp Ala Ala 1 5 His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 15 20 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 35 40 Tyr Thr Arg Tyr Lys Asn Tyr Lys Gln Val Pro Ser Thr Asp Phe Lys 50 55 Leu Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Tbr Gin Ser 70 Pro Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser 80 85 Val Asp Phe Asn Gly Ser Asp Ser Phe Ser Gin Thr Ser Thr Gly Leu 100 105 Gly Val Leu Ala Cly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu 110 115 120 125 Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys 130 135 140 Asn Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 155 INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 850 base pairs TYPE: nucleic acid STRANDEDNEScS: double TOPOLOGY: linear (ii) MOLECULE TYPE: A(enmC (iii) LTYPOTHET'CA:L

NO

(iv) ANT-SENSE:

NC

(vi) ORIGIN--

SOURCE:

orGA NISN: eisser-a renincitii STRAIN: Z6 25 (ix) FEATURE: NAME/K7Y: CDS LOCA'-TION: 29E. .732 (ix) FEATURE: NA)AE/KEY: sig...peptide LOCATION: 208. .264 (ix) FEATURE: NAME/KEY: mat~peptide LOCATION: 265.-732 (xi) SEQUENCE DESCRIPTION: SEQ ID CACCCATCCG CCGCGTGATG CCGCCACCAC CATITAAAGG CAACGCGCGG GTTAACGGCT 0 TrGCCGTCGG CJAAGCAGCC GGATACCGCT ACGTATCTTG AAGTAT1TAAA AATATrACGA 120 TCCAAAAAGA AAATrTAAGT ATAATAAAGC AGAATMC=r AACGGAT1'CTr TAACAATIT 180 TCTAACTGAC CATAAAGGAA CCAAAAT ATG AAA AMA GCA CTT GCC ACA CTG 231 Met Lys Lys Ala Leu Ala Thr Lau c TCGTCC C C GCC GCA CTG GCG GMA GGC GCA. TCC GGC 279 Ile Ala Leu Ala Leu Pro Ala Ala Ala Leu Ala Giu Gly Ala Ser Gly -5 1 T T TAC GTC CAA GCC GAT GCC GCA CAC GCA'AMA GCC TCA AGC T T]TA 327 Phe Tyr Val Gin Ala Asp Ala Ala His Ala Lys Ala Ser Ser Ser Leu 10 15 GGT TCT GCC AAA GGX TTC AGC CCG CGC ATC TCC GCA GGC TAC CGC ATC 375 Gly Ser Ala Lys Gly Phe Ser Pro Arg Ile Ser Ala Gly Tyr Arg Ile 25 30 AAC GAC CTC CGC TIC GCC GTC GAT TAC ACG CGC TAC AAA AAC TAT AAA 423 Asn Asp Leu Arg Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys 45 GCC CCA TCC ACC GAT TTC AAA CTT TAC AGC ATC GGC GCG TtC GCC ATT 471 Ala Pro Ser Thr Asp Phe Lys Leu Tyr Ser Ile Gly Ala Ser Ala Ile 60 TAC GAC TIC GAC ACC CAA TCG CCC GTC AAA CZG TAT CTC GGC GCG CGc 519 Tyr Asp Phe Asp Thr Gin Ser Pro Val Lys Pro Tyr Leu Gly Ala Arg 75 80 IT AGC C= AAC CGC GCC TCC GTC GAC TIG GGC GGC AGC GAC AGC TT-C 567 Leu Ser Leu Asn Arg Ala Ser Val Asp Leu Giy Gly Ser Asp Ser Phe 90 95 100 KG-C CAA AC TCC ACC GGC CTC GGC GTA TIG GCG GGC GTA TAT GCC 615 Ser Gin Ser Thr Gly Leu Gly Val Leu Ala Gly Val Ser Tr Ala 105 1iO GTT ACC CZS ;T -TC GAT GAT GCC GGC TAC CGC TAC TC AT 663 Val T:r Prz Asn Val Asp Leu Asp Ala Gly Tyr Ara yrY r Tyr lle c125 120 GC AAA G7 C C: GTC MC GT CT TZC G: GA-. C TZ: GC: 7 Gly Lys Va Asn Thr Val Lys Asn Val Arg Ser Gly G-u Le- Ser Ala **135 140 145 GGT GT1G CCC- T C A TiC TGATA.TGCCC CTrAT tGC .AACCZ;CZA 759 Gly Val Arg Val Lys Phe 150 155 GCCGCCG G rrMTIT CTGCCACCGC AACTA ACAAk GCCGGCGCG- TGTACGATA 819 ATCCCGAATG CTGCGGCTI'C TGCCGCCCTA T 850 INFORM.ATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 174 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Lys Lys Ala Leu Ala Thr Leu lie Ala Leu Ala Leu Pro Ala Ala -19 -15 -10 Ala Leu Ala Giu Gly Ala Ser Gly Phe Tyr Val Gin Ala Asp Ala Ala 1 5 His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 20 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 35 40 Tyr Thr JArg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 50 55 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gin Ser Pro 65 70 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 80 .85 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gin Thr Ser Thr Gly Leu Gly 100 105 Val Leu Ala Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp ILeu. Asp 110 115 .120 125 Ala Gly Tyr Arg Tyr Asn Tyr le Gly Lys VJai Asn Thr Val Lys Asn 130 135 140 Val Arg Ser Giy Giu Leu Ser Ala Gly Val Arg Val Lys Phe 145 150 155 INFORMATION FOR SEQ ID NO:7: i)SEQUENCE CHARLACTERISTICS: LENGTH: 810 'base pairs TYPE: nuclic acid (Cf STRANDEDNESS: double TOPOLOGY: linear* MOLECULE TYPE: DNA (genomilc-) (iii). HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL

SOURCE:

ORGANISM: Neisseria gonorrhoeae STRAIN: b2 (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 241. .765 (ix) FEATURE: NAME/KEY: sjgpeptide LOCATION: 241. .297 (ix) FEATURE: NAME/KEY: mat-.peptide LOCATION: 298. .765 (xi) SEQuENCE DESCRIPTION: SEQ ID NO:7:' CCCCGCCTIT CGT1I CCA'Gr ~AG ACCCATCCGC CGCGTGATG-C CGCTTAGGCAACGCG CGGGTTAACG_ GATITGCCGT CGGCAAAGCA GCCGGAT'GCC 120 GCCGCGTATC TTGAGGCATT GAAAATATTA CG-AM'CAAAA AGAAAAT1=C AGTATAATAC 180 GGCAGGATTC TTTAACGGAT TAT!AACAAT T-.TCTCCT G.ACCATAAAG GA).CCAAAAT 240 ATG AAA AAA GTCA CTT GCC GCA CTG ATT GCC CTC GCA CTC CCG GCC GCC 288 Met Lys Lys Ala Lou Ala Ala Lou Ile Ala Lou Ala Lou Pro Ala Ala _i9 -i5 -10 GCA'C G GCG GMA GGC cCA Ala Leu Ala Glu Gly Ala TCC GGC TIT TAC CTc CAA GCC GAT GCC cCA Ser Gly Phe Tyr Val Gin Ala Asp Ala Ala 5

CAC

His

CGC

Arg

TAC

Tyr

TAC

GTZ:

Val

CAC

His

GTA

Val 110 AAA GCC' Lys Ala TCC c-CA Ser Ala CGC TAC Arg Tyr ATC CGC Ile Gly 65 CCG TAT Pro Tyr cC c-cc Gly C-ly GCG cGC TCA AGC Ser Ser GGC TAC cly Tyr 35 )A AAC Lys Asr 50 GCG TCC Ala Ser TT cGC Phe G1Y AcC GAC Ser Asp cTA AGC

TCT

Ser 20 ccc Arg

TAT

Tr

GTC

Val

AGC

Ser 100

TAT

TTA

Leu

ATC

Ile

AAA

Lys

AT

Ile Arg 85 TrC Phe

GCC

GGT

Gly

AAC

Asn

GCC

Ala

TAC

Ty'r 70 Leu

AGC

Ser

GTT

TCT GCC AAA Ser Ala Lys cAC CTC CGC Asp Leu Arg 40 CCA TCC ACC Pro Ser Thr 55 cAC TIC GAC Asp Phe Asp AcC CTC AAC Ser Leu Asn AAA ACC TCC Lys Thr Ser 105 ACC CCG AAT

G(C

Gly

TIC

Phe

GAT

Asp

ACC

Thr

CGC

Arg

GCC

Ala

GTC

Val rrX AGC CCG Phe Ser Pro 3CC GTC cAT kla Val Asp VIC AAA CTr Phe Lys Leu CAA TC CCC cin Ser PrcCT TCC Gr- Ala Ser Ala GGC CTC cc Gly Leu cly cAT TIc- cAT Asp Leu Asp 125 384 432 480 528 576 624 672 *9 Ala cly Val Ser Tyr Ala Val Thr Pro Asn cCC GGC TAC CGC TAC AAC TAC cTC GGC AAA GTC AAC ACT GTC AAA AAC 720 Ala Gly Tyr Arg Tyr Asn Tyr Val Gly Lys Val Asn Thr Val Lys Asn 130 135 140 GTC CcT TCC GGC GMA CTG TCC cCC cC GcT CGC GTC AMA TIC 7GATATAC-C 772 Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arg Val Lys Phe 40 145 150 155 GTTATTCCGC AMCCGCCGA GCCTrCGGC GTTrT 810 INFORMATION FOR SEQ ID NO:8: SEQUENCE

CHARACTERISTICS:

LENGTH: 174 amino acids (B)*TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Lys Lys Ala Leu Ala Ala Leu Ile Ala Leu Ala Leu Pro Ala Ala -19 -15 -10 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gin Ala Asp Ala Ala 1 5 His Ala Lys Ala ser Ser Ser Leu Gly Ser Ala Lys Gly. Phe Ser Pro 20 77 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 35 40 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 55 Tyr Ser Ile Gly Ala Ser Val Ile Tyr Asp Phe Asp Thr Gin Ser Pro 65 70 Val Lys Pro Tyr Phe Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Ala 85 His Leu Gly Gly Ser Asp Ser Phe Ser Lys Thr Ser Ala Gly Leu Gly 95 100 105 Val Leu Ala Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 110 115 120 125 Ala Gly Tyr Arg Tyr Asn Tyr Val Gly Lys Val Asn Thr Val Lys Asn 130 !35 140 Val Arg Ser Gly Glu Leu Ser Ala Gly Val Arc Val Lys Phe 145 150 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: 30 LENGTH: 16 amino acids S TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala Ala 45 1 5 10 INFORMATION FOR SEQ ID 50 SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Ala Leu Pro Ala Ala Ala Leu Ala Glu Gly Ala Ser Gly Phe 1 5 10 INFORMATION FOR SEQ ID N0:11: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Gly Ala Ser Gly Phe Tyjr Val Gin Ala Asp Ala Ala His Ala Lys 10 INFORMATION FOR SEQ ID N0:12: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids 30 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Ala Ala His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly 1 5 10 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: o(A) LENGTH: 15 amino acids 50 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Gly Ser Ala Lys Gly Phe Ser Pro Arg Ile Ser Ala Gly Tyr Arg 1 5 10 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Ser Ala Gly 'yr Arg ile Asn Asp Leu Arc Phe Ala Val Asp T S10 S. INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 3 LENGTH: 16 amino acids 30 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B 40 (xi) SEQUENCE DESCRIPTION: SEQ ID SPhe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr 1 5 10 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: 50 LENGTH: 15 amino acids S*,o TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu Tyr Ser Ile Gly Ala 1 5 10 INFORMATION FOR SEQ ID NO:17: SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Tyr Ser Ile Gly Ala Ser Ala lie Tyr .AS Phe Asp Thr Gin Ser 5. C INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids 30 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 35 (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: P: he Asp Thr Gin Ser Pro Val Lys Pro Tyr Leu Gly Ala Arg Leu 1 5 10 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL

SOURCE:

ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val Asp Leu Gly 1 5 10 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Val Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile 1 5 10 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid 25 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: 30 ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Ser Gin Thr Ser Ile Gly Leu Gly Val Leu Thr Gly Val Ser Tyr 1 5 10 INFORMATION FOR SEQ ID NO:22: 4: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein S(vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Thr Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp Ala 1 5 10 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria menincitidis STRAIN: 608B (xi) SEQUENCE DESCRITION: SEQ ID NO:23: Val Asp Leu Asp Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val 1 5 10 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids 25 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 30 (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis S STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Tyr Iie Gly Lys Val Asn Thr Val Lys Asn Val Arg Ser Gly Glu 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID Val Arg Ser Gly Glu Leu Ser Val Gly Val Arg Val Lys Phe 1 5 INFORMATION FOR SEQ ID NO:26: Wi SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid TOPOLOGY: lfinear (ii) MOLECULE TYPE: protein (vi) ORIGINAL'SOURCE: ORGANISM: Neisseria meningitidis STRAIN: 608B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Phe Ala Val Asp Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr 10 Asp Phe Lys Leu Tyr Ser I-le Gly Ala

Claims (77)

1. An isolated antigen or fragment thereof which is immunologically accessible on greater than 50% of known strains of Neisseria meningitidis.

2. The isolated antigen or fragment of claim 1 which is immunologically accessible on about 99% of known strains of Neisseria meningitidis.

3. The isolated antigen or fragment of claim 1 in which immunological accessibility is determined by using an agglutination assay, an ELISA, a RIA, an immunoblotting assay, a dot-enzyme assay, a surface accessibility assay, or a combination of these assays.

4. The isolated antigen or fragment of claim 1 which is a protein.

5. The protein of claim 4 which has a molecular weight of about 22 kilodaltons.

6. The protein of claim 4 which has a molecular weight of about 18 kilodaltons.

7. The protein of claim 5 which has the amino acid sequence selected from the sequences of: Figure 1 (SEQ ID NO Figure 8 (SEQ ID NO: Figure 9 (SEQ ID NO: and Figure 10 (SEQ ID NO:8).

8. The protein of claim 5 which has the amino acid sequence of Figure 1 (SEQ ID NO:2).

9. The protein of claim 5 in substantially pure form. The protein of claim 9, wherein said substantially pure form is obtained by the steps of: \\melb_files\home$\cincae\Keep\speci\speci 49343.96 div.doc 23/05/00 86 a) isolating a culture of Neisseria meningitidis bacteria, b) isolating an outer membrane portion from the culture of the bacteria; and c) isolating said antigen from the outer membrane portion.

11. The protein of claim 10, wherein step includes the additional step of treating the outer membrane portion with proteinase K followed by protein fractionation. 10 12. A DNA sequence encoding at least a portion of at least one antigen of the Neisseria meningitidis 22 kDa surface protein, said sequence being selected from the group consisting of: a) the DNA sequence of Figure 1 (SEQ ID NO:1); 15 b) the DNA sequence of Figure 8 (SEQ ID NO:3); the DNA sequence of Figure 9 (SEQ ID d) the DNA sequence of Figure 10 (SEQ ID NO:7); e) analogues or derivatives of the foregoing DNA sequences; 20 f) DNA sequences degenerate to any of the foregoing DNA sequences; and g) fragments of any of the foregoing DNA sequences; wherein said sequences encode a product that displays the immunological activity of the Neisseria meningitidis 22 kDa surface protein.

13. A DNA sequence encoding at least a portion of at least one antigen of the Neisseria meningitidis 22 kDa surface protein, said sequence being selected from the group consisting of: a) the DNA sequence of Figure 1 (SEQ ID NO:1); b) analogues or derivatives of the foregoing DNA sequence; \\melb_files\home$\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 87 c) DNA sequences degenerate to any of the foregoing DNA sequences; and d) fragments of any of the foregoing DNA sequences; wherein said sequences encode a product that displays the immunological activity of the Neisseria meningitidis 22 kDa surface protein.

14. A DNA sequence according to claim 12, wherein said analog is selected from the DNA of Neisseria gonorrhoeae. 10 15. A DNA sequence according to claim 12, wherein said analog is selected from the DNA of Neisseria lactamica. t

16. A DNA sequence of the formula of NO:1) from base 143 to base 667.

17. A DNA sequence of the formula of NO:1) from base 200 to base 667.

18. A DNA sequence of the formula of NO:3) from base 116 to base 643.

19. A DNA sequence of the formula of NO:3) from base 173 to base 643. A DNA sequence of the formula of from base 208 to base 732.

21. A DNA sequence of the formula of from base 265 to base 732.

22. A DNA sequence of the formula of NO:7) from base 241 to base 765. Figure 1 Figure 1 Figure 8 Figure 8 Figure 9 Figure 9 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID Figure 10 (SEQ ID \\-elbfiles\homeS\cintae\Keep\peci\speci 49343.96 div.doc 23/05/00 88

23. A DNA sequence of the formula of Figure 10 (SEQ ID NO:7) from base 298.to base 765.

24. A fragment of a DNA sequence according to claim 13, wherein said fragment is selected from the group consisting of: one of the peptides illustrated in Figure 15 (SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID MO:13, SEQ ID NO:14, SEQ ID 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, 10 SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26)

25. A fragment of a DNA sequence comprising from amino acid 31 to amino acid 55 of Figure 1 (SEQ ID NO:1).

26. A fragment of a DNA sequence comprising from amino 15 acid 51 to amino acid 86 of Figure 1 (SEQ ID NO:1).

27. A fragment of a DNA sequence comprising from amino acid 110 to amino acid 140 of Figure 1 (SEQ ID NO:1).

28. A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of the DNA sequences of claims 13, 16, or 17, and one or more expression control sequences operatively linked to the DNA sequence.

29. A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of the DNA sequences of claims 14, 15, or any one of claims 18 to 27, and one or more expression control sequences operatively linked to the DNA sequence. \\melb_files\homeS\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 89 The recombinant DNA molecule of claim 28, wherein said expression control sequence is an inducible expression vector.

31. The recombinant DNA molecule of claim 29, wherein said expression control sequence is an inducible expression vector.

32. The recombinant DNA molecule of claim 30 or 31, wherein said vector is induced by stimuli selected from: temperature, presence, of lactose, and presence of IPTG.

33. The recombinant DNA molecule of claim 32, wherein said vector is selected from: X PL, X PR, TAC, T7, T3, LC and TRP promoters. 9

34. A plasmid selected from the group consisting of: pNP2202, pNP2203, and pNP2204.

35. A unicellular host transformed with a recombinant DNA molecule of claim 28.

36. A unicellular host transformed with a recombinant DNA molecule of claim 29.

37. The unicellular host according to claim 35 or 36, wherein the host is selected from the group consisting of: strains of E.coli JM109, E.coli BL21(DE3), E.coli E.coli W3110, E.coli JM105, E.coli BL21, E.coli TOPP1, E.coli TOPP2, and E.coli TOPP3.

38. The unicellular host according to claim 35 or 36, wherein the host is selected from the group consisting of strains of E.coli JM109 and E.coli BL21(DE3). \\melbfiles\homeS\cintae\Keep\speci\speci 49343.96 div.doc 23105/00 90

39. A Neisseria meningitidis 22kDa surface protein in substantially pure form obtained by the culturing of a unicellular host according to claim 35 or 36 and isolating said protein.

40. A polypeptide coded for by a DNA sequence of any one of claims 13, 16 and 17.

41. A polypeptide coded for by a DNA sequence of any one of claims 14, 15 and 18 to 27.

42. A method for producing a DNA sequence comprising the 10 steps of culturing the unicellular host of claim 35 or *o S* 36 and isolating said DNA sequence.

43. A method for producing a polypeptide comprising the steps of culturing the unicellular host of claim 35 or o 36 and isolating said polypeptide.

44. A polypeptide in substantially pure form as obtained by the method of claim 43. A method for isolating the antigen of claim 1 comprising: a) isolating a culture of Neisseria meningitidis bacteria, b) isolating an outer membrane portion from the culture of the bacteria; and c) isolating said antigen from the outer membrane portion.

46. The method of claim 45, wherein step includes the additional steps of treating the outer membrane portion with proteinase K followed by protein fractionation. \\melb_files\home$\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 91

47. A pharmaceutical composition comprising one or more antigens or fragments thereof according to any one of claims 1 to 5, 8, 13, 16, and 17.

48. A pharmaceutical composition comprising one or more antigens or fragments thereof according to claim

49. A pharmaceutical composition comprising one or more antigens or fragments thereof according to claim 41. A pharmaceutical composition comprising one or more antigens or fragments thereof according to any one of 10 claims 6, 7, 9, 12, 14, 15, and 18 to 27. S S

51. The pharmaceutical composition of claim 49, which is a vaccine.

52. The pharmaceutical composition of claim 50, which is a vaccine. 15 53. The pharmaceutical composition of claim 49, further comprising one or more pharmaceutically acceptable excipients. *SSS

54. The pharmaceutical composition of claim 50, further comprising one or more pharmaceutically acceptable excipients. A method for preventing infection of a patient by Neisseria meningitidis comprising the administration of a pharmaceutically effective amount of the vaccine of claim 51 or 52.

56. The use of a pharmaceutically effective amount of the Neisseria meningitidis 22kDa surface protein or a fragment, analogue, or derivative thereof for the \\melb_files\homeS\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 92 prevention of Neisseria meningitidis infection in humans.

57. The use of a pharmaceutically effective amount of the Neisseria meningitidis 22kDa surface protein or a fragment, analogue, or derivative thereof for the prevention of Neisseria gonorrhoeae infection in humans.

58. The use of the Neisseria meningitidis 22 kDa surface protein or a fragment, analogue, or derivative thereof for the manufacture of a vaccine for the prevention of Neisseria meningitidis infection in humans.

59. The use of the Neisseria meningitidis 22 kDa surface protein or a fragment, analogue, or derivative thereof for the manufacture of a vaccine for the prevention of 15 Neisseria gonorrhoeae infection in humans.

60. An antibody or fragment thereof that specifically *o binds to a protein with a molecular weight of approximately 22 kilodaltons present on greater than 50% of known strains of Neisseria meningitidis. 0e

61. The antibody or fragment of claim 60 that specifically binds to about 99% of known strains of Neisseria meningitidis.

62. The antibody or fragment of claim 60 which is a monoclonal antibody or fragment thereof.

63. The monoclonal antibody or fragment of claim 62 which is of murine origin.

64. The monoclonal antibody or fragment of claim 63 which is of an IgG isotype. \\melb_files\homeS\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 93 The monoclonal antibody or fragment of claim 62 which is Me-1, Me-2, Me-3, Me-5, Me-6, or Me-7.

66. The monoclonal antibody of claim 62 which is the monoclonal antibody Me-i or Me-7.

67. A method for isolating the antibody of claim comprising: a) introducing a preparation of Neisseria meningitidis into a mammal; and b) isolating serum from the mammal containing said 99 antibody.

68. A method for isolating the monoclonal antibody of claim 62 comprising: a) introducing a preparation of Neisseria meningitidis to antibody producing cells of a r* 15 mammal; b) fusing the antibody producing cells with myeloma S: cells to form hybridoma cells; and c) isolating said monoclonal antibody from the hybridoma cells.

69. A pharmaceutical composition comprising one or more antibodies or fragments thereof according to any one of claims 60-66. The pharmaceutical composition of claim 69 which is a vaccine.

71. The pharmaceutical composition of claim 69, further comprising a pharmaceutically acceptable excipient.

72. The pharmaceutical composition of claim 69, wherein the antibody is Me-i or Me-7. \\melb.files\home\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 94

73. A method for treating a patient infected with or suspected of being infected with Neisseria meningitidis comprising the administration of a pharmaceutically effective amount of the vaccine of claim

74. A method for the detection of Neisseria meningitidis antigen in a biological sample containing or suspected of containing Neisseria meningitidis antigen comprising: 0 a) isolating the biological sample from a patient; b) incubating the antibody or fragment of claim with the biological sample to form a mixture; and c) detecting specifically bound antibody or bound o Sfragment in the mixture which indicates the presence of Neisseria meningitidis antigen. The method of claim 72 wherein the antibody is Me-1 or *Me-7.

76. A method for the detection of antibody specific to o. Neisseria meningitidis antigen in a biological sample containing or suspected of containing said antibody comprising: a) isolating the biological sample from a patient; b) incubating the antigen or fragment of claim 1 with the biological sample to form a mixture; and c) detecting specifically bound antigen or bound fragment in the mixture which indicates the presence of antibody specific to Neisseria meningitidis antigen. \\melb_files\homeS\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 95

77. The method of claim 76 wherein the antigen is the Neisseria meningitidis 22 kDa surface protein.

78. A method for the detection of pathogenic Neisseria bacteria in a biological sample containing or suspected of containing such bacteria comprising: a) isolating the biological sample from a patient; b) incubating a DNA probe having the DNA sequence of claim 13 with the biological sample to form a mixture; and 10 c) detecting specifically bound DNA probe in the mixture which indicates the presence of Neisseria S: bacteria.

79. A method for the detection of pathogenic Neisseria 15 bacteria in a biological sample containing or suspected of containing such bacteria comprising: a) isolating the biological sample from a patient; b) incubating a DNA probe having the DNA sequence of claim 12 with the biological sample to form a mixture; and c) detecting specifically bound DNA probe in the mixture which indicates the presence of Neisseria bacteria.

80. The method of claim 78 wherein the DNA probe has the 525 base pair sequence of Figure 1 (SEQ ID NO:1).

81. The method of claim 78 wherein the DNA probe has a portion of the 525 base pair sequence of Figure 1 (SEQ ID NO: 1). \\melbfiles\homeS\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 96

82. The method of claim 79 wherein the DNA probe has the full or a portion of the 528 base pair sequence of Figure 8 (SEQ ID NO:3).

83. The method of claim 79 wherein the DNA probe has the full or a portion of the 525 base pair sequence of Figure 9 (SEQ ID

84. The method of claim 79 wherein the DNA probe has a portion or the full 525 base pair sequence of Figure (SEQ ID NO:7). 10 85. The method of claim 78 wherein the DNA probe is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the Neisseria meningitidis 22 kDa surface protein of Figure 1 (SEQ ID NO:1).

86. The method of claim 79 wherein the DNA probe is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the Neisseria meningitidis 22 kDa surface protein of sequences 20 selected from: Figure 8 (SEQ ID NO:3), Figure 9 (SEQ g* ID NO:5), and Figure 10 (SEQ ID NO:7).

87. The method of claim 85 or 86 which further comprises: a) providing a set of oligomers which are primers for a polymerase chain reaction method and which flank the target region; and b) amplifying the target region via the polymerase chain reaction method.

88. A method for the detection Neisseria meningitidis in a patient comprising: \\melb.files\homeS\cintae\Keep\speci\speci 49343.96 div.doc 23/05/00 97 a) labeling the antibody or fragment of claim with a detectable label; b) administering the labeled antibody or labeled fragment to the patient; and c) detecting specifically bound labeled antibody or labeled fragment in the patient which indicates the presence of Neisseria meningitidis.

89. The use of a pharmaceutically effective amount of an antibody specific to the Neisseria meningitidis 22 kDa surface protein for the prevention of Neisseria Smeningitidis infection in humans.

90. The use of the Neisseria meningitidis 22 kDa surface protein or a fragment, analogue, or derivative thereof for the manufacture of a kit for the detection or diagnosis of Neisseria meningitidis infection in humans. oo \\melbfiles\home$\cintae\Keep\Beci\speci 49343.96 div.doc 23/05/00