CZ20004395A3 - Antigenic polypeptides Neisseria meningitidis, corresponding polynucleotides and protective antibodies - Google Patents

Abstract

The solution is provided by BASB030 N.meningitidis polypeptides polynucleotides encoding BASB030 polypeptides and methods for producing such polypeptides by recombinant techniques. It also provides antibodies and their diagnostic, prophylactic and therapeutic use.

Description

Technical field

The present invention relates to polynucleotides (referred to herein as BASB030 polynucleotides, polypeptides encoded by said polynucleotides (referred to herein as BASB030 or BASB030 polypeptides)), recombinant materials and methods for their preparation. In another aspect, the present invention relates to methods of using such polypeptides and polynucleotides, including In another aspect, the invention relates to diagnostic tests for the detection of infection by certain pathogens.

BACKGROUND OF THE INVENTION

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium often isolated from the human upper airways. It occasionally causes invasive bacterial diseases such as bacteremia and meningitis. The incidence of meningococcal disease shows geographical and seasonal fluctuations (Schwartz,

B., Moore, P. S., Broome, C.V .; Clin. Microbiol. Roar. 2 (Supplements), S18-S24 (1989)). The most common diseases in temperate countries are caused by serogroup B strains and the incidence ranges from 1-10 / 100000 / year in the general population and sometimes reaches higher levels (Kaczmarski, E: B., (1997), Commun.

Dis. Rep. Roar. 7: R55-9, 1995; Scholten, R. J. P. M., Bijlmer, H. A., Poolman, J. T., et al., Clin. Infect. Dis. 16: 237-246 (1993); Cruz, C., pavez, G., Aguilar, E. et al., Epidemiol Infest. 105: 119-126 (1990).

There are epidemics caused by serogroup A meningococci, particularly in central Africa, which sometimes reach an incidence of up to 1000 / 100,000 / year (Schwartz, B., Moore, P.S., Broome, C.V .;

Clin. Microbiol. Roar. 2 (Supplements), S18-S24 (1989)). Almost all cases of systemic meningococcal disease are caused by serogroups A, B, C, W-135 and Y meningococci and a tetravalent A, C, W-135, Y polysaccharide vaccine is available (Armand, J., Armijon, F., mynard, MC. (Lafaix, C., J. Biol. Stand. 10: 335-39, 1982).

Polysaccharide vaccines are currently improved by their chemical conjugation to protein carriers (Lieberman, J. M., Chiu, S. S., Wong, V. K., et al., JAMA 275: 1499-1503,

1996).

The serogroup B vaccine is not currently available because B capsular polysaccharide has been found to be non-immunogenic, probably because it has structural similarity to host antigens (Wyle, FA, Artenstein, MS, Brandt, ML et al., J. Infect. Dis., 126: 514-522 (1972);

Finne, J.M. , Leinonen, M., Makela, P.M. Lancet II: 355-357 (1983).

Many years ago, attempts have been made to develop meningococcal outer membrane vaccines (de Moraes, JC, Perkins, B., Camargo, MC, et al., Lancet 340: 1074-1078, 1992; Bjune, G., Hoiby, EA, Gronnesby , JK et al., 338: 1093-1096 (1991). Such vaccines showed efficacies of 57% to 85% in older children (> 4 years) and adolescents.

Many external membrane components such as PorA, PorB, Rpm, Ope, Opa, FrpB are present in these vaccines and the contribution of these components to the observed protection has yet to be clarified.

· · ♦ ♦ ♦ ♦ ♦ ♦ ♦ * · · * * * * * * * * * * * * *

Other outer membrane components of bacteria have been defined using animal or human antibodies as potentially useful for inducing protective immunity such as TbpB and NspA (Martin, D., Cadieux, N., Hamel, J., Brodeux, BR, J. Exp. Med., 185: 1173-1183, 1997; Lissolo, L., Maitre Wilmotte, C., Dumas, P. et al., Inf. Immun. 63: 884-890,

1995). Mechanisms of protective immunity include antibody-mediated bactericidal activity and opsonophagocytosis.

The animal model of bacteremia was used to combine all of the antibody-mediated mechanisms (Saukkonen, K., Leinonen, M., Abdillahi, H., Poolman, J.T., Vaccine 7: 325328, 1989). It is generally accepted that complement-mediated bactericidal mechanisms are essential for immunity against meningococcal disease (Ross, S. C. Rosenthal, P. J., Berberic, H. M., Densen, P., J. Infect. Dis. 155: 1266-1275, 1987).

Neisseria meningitidis infections have increased dramatically in recent decades. This is attributed to the emergence of strains with multiple antibiotic resistance and an increasing population of individuals with weakened immune systems, it is not uncommon now to isolate strains of Neisseria meningitidis that are resistant to some or all of the standard antibiotics. This phenomenon creates the need for new antimicrobial agents, vaccines, drug discovery methods and diagnostic tests for the organism.

SUMMARY OF THE INVENTION

The present invention relates to BASB030, in particular BASB030 polypeptides and BASB030 polynucleotides, recombinant materials and methods for their preparation. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment and prevention of microbial diseases, inter alia. In another aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting expression or activity of BASB030 polynucleotides or polypeptides.

Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art upon consideration of the following description and other parts of the present invention.

The invention relates to BASB030 polypeptides and polynucleotides as described in more detail below. More specifically, the invention relates to polypeptides and polynucleotides of Neisseria meningitidis having amino acid homology to the PilQ outer membrane protein of Neisseria gonorrhoeae. In particular, the invention relates to BASB030 having the nucleotide and amino acid sequences set forth in SEQ ID NO: 1,3,5 and SEQ ID NO: 2,4,6, respectively. It will be appreciated that the sequences listed in the Sequence Listing are merely exemplary embodiments of the present invention, as those skilled in the art will recognize that such sequences may be used in polynucleotides in general, including ribopolynucleotides.

Polypeptides

In one aspect, the invention provides Neisseria meningitidis polypeptides referred to as BASB030 and BASB030 polypeptides, as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the polypeptides.

• ·

The present invention further provides:

(a) an isolated polypeptide comprising an amino acid sequence having at least 85% identity, preferably at least 90% identity, even more preferably at least 95% identity, preferably at least 97-99% or complete identity, with the sequence set forth in SEQ ID NO: 2,4 , 6;

(b) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence having at least 85% identity, preferably at least 90% identity, even more preferably at least 95% identity, preferably at least 97-99% or complete identity, with the sequence set forth in SEQ ID NO: 1.3 .5 over the entire length of SEQ ID NO: 1,3,5, respectively; or (c) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide having at least 85% identity, preferably at least 90% identity, even more preferably at least 95% identity, preferably at least 97-99% or complete identity, with the sequence shown in SEQ ID NO: 2,4,6.

The BASB030 polypeptides set forth in SEQ ID NO: 2,4,6 are BASB030 polypeptides from Neisseria meningitidis strains ATCC 13090 and H44 / 76.

The invention also provides an immunogenic fragment of a BASB030 polypeptide, i.e., an uninterrupted portion of a BASB030 polypeptide having the same or substantially the same immunogenic activity as a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,4,6. That is, a fragment (bound to a carrier, if necessary) can elicit an immune response that is directed against a BASB030 polypeptide. Such an immunogenic fragment may comprise, for example, a BASB030 polypeptide without an N-terminal leader sequence, and / or a transmembrane domain and / or a C-terminal anchor domain. In a preferred aspect, the immunogenic BASB030 fragment of the present invention comprises substantially the entire extracellular domain of a polypeptide having at least 85% identity, preferably at least 90% identity, even more preferably at least 95% identity, preferably at least 97-99% or complete identity. SEQ ID NO: 2,4,6 over the full length of SEQ ID NO: 2.

A fragment is a polypeptide having an amino acid sequence that is exactly the same as part, but not the entire, amino acid sequence of the present invention. For BASB030 polypeptides, the fragment may be free or may be part of a larger polypeptide in which it forms part or a region, preferably one continuous region in one larger polypeptide.

Preferred fragments are, for example, truncated polypeptides comprising a portion of the amino acid sequence of SEQ ID NO: 2,4,6 or variants thereof, such as a continuous series of residues comprising the amino- and / or carboxy-terminal amino acid sequence. Also preferred are degradation forms of the polypeptides of the present invention produced in host cells. Also preferred are fragments characterized by structural or functional properties, such as those comprising alpha-helix and alpha-helix-forming regions, betalist and beta-sheet-forming regions, bends and bend-forming regions, loops and loop-forming regions, hydrophilic regions, hydrophobic regions, alpha-amphipathic regions, beta-amphipathic regions, flexible regions, surface forming regions, and regions with high antigenic index.

Further preferred fragments are isolated polypeptides comprising an amino acid sequence having at least 15, 20,

30, 40, 50 or 100 continuous amino acids from the amino acid sequence of SEQ ID NO: 2,4,6, or isolated polypeptides comprising the amino acid sequence truncated by

at least 15, 20, 30, 40, 50 or 60 contiguous amino acids of SEQ ID NO: 2,4,6.

Fragments of the polypeptides of the present invention can be used to prepare the corresponding full-length polypeptides using peptide synthesis; therefore, these fragments can be used as intermediates for preparing the complete polypeptides of the present invention.

Especially preferred are variants with substitutions, deletions, additions, or combinations of these changes at several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids.

The polypeptides or immunogenic fragments of the present invention may be in the form of a mature protein or may be part of a larger protein, such as a precursor protein or a fusion protein. It is often advantageous to add additional amino acid sequences that are secretory or leader sequences, purification facilitating sequences, such as sequences made up of many histidine residues, or sequences for stabilization during recombinant production. Further, it is also possible to add an exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule.

In one aspect, the invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention or a fragment thereof and various portions of constant regions of heavy or light chains of immunoglobulins of different subclasses (IgG, IgM, IgA, IgE). The immunoglobulin preferably used is a constant portion of a human IgG heavy chain, particularly an IgG1, in which the fusion is carried out in the hinge region. In a particular embodiment, it may be

The Fc portion is removed by simply inserting a cleavage sequence that can be cleaved by blood clotting factor Xa.

Further, the invention relates to methods for preparing these fusion proteins by genetic engineering and their use for drug discovery, diagnosis and therapy. Another aspect of the invention relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology are disclosed in International Patent Applications Nos. WO 94/29458 and WO 94/22914.

Proteins may be chemically conjugated or expressed as recombinant fusion proteins, allowing the production of higher amounts in expression systems as compared to unfused proteins. The fusion partner may aid in the delivery of epitopes for T-helper lymphocytes (immunological fusion partner), preferably epitopes for T-helper lymphocytes recognized in humans, or may assist expression of a protein (expression enhancer) to achieve a higher yield than using natural recombinant protein. Preferably, the fusion partner is both an immunological fusion partner and an expression enhancer.

Fusion partners include protein D from Haemophilus influenzae and a non-structural influenza virus protein, NSI (hemagglutinin). Another fusion partner is a protein known as LytA. Preferably, the C-terminal portion of the molecule is used. LytA is derived from Streptococcus pneumoniae, which synthesizes Nacetyl-L-alanine amidase, LytA amidase (encoded by the lytA gene (Gene, 43 (1986), pp. 265-272)), an autolysin that specifically degrades some bonds in the peptidoglycan skeleton . The C-terminal domain of the LytA protein is responsible for affinity for choline or certain choline analogs such as DEAE. This property was used to develop E. coli C-LytA 4 4

4 4444444 4

4 4 4 4

44 4 44 4 expression plasmids which are useful for the expression of fusion proteins. Purification of hybrid proteins containing a C-LytA fragment at the amino terminus has been previously described (Biotechnology: 10 (1992), pp. 795-798). A repeating portion of the LytA molecule located at the C terminus of nucleotide 178, for example residues 188-305, may be used.

The present invention also encompasses variants of the above polypeptides, i.e., polypeptides that differ from reference polypeptides by conservative amino acid substitutions in which the residue is substituted by another residue with similar characteristics. Typically, such substitutions are made between Ala, Val, Leu and Ile; Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln; and between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr.

The polypeptides of the present invention can be prepared by any suitable method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides prepared by a combination of these methods. Methods for preparing polypeptides are well known in the art.

most preferably, the polypeptide of the present invention is derived from Neisseria meningitidis, however, it can also be obtained from another organism of the same taxonomic genus. The polypeptide of the present invention may be obtained, for example, from an organism of the same genus or order.

Polynucleotides · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The present invention provides polynucleotides encoding BASB030 polypeptides, particularly polynucleotides that encode a polypeptide designated BASB030.

In a preferred embodiment of the invention, the polynucleotides comprise a region encoding BASB030 polypeptides comprising the sequence shown in SEQ ID NO: 1,3,5, which comprises the entire gene or a variant thereof.

BASB030 polynucleotides set forth in SEQ ID NO: 1,3,5 are BASB030 polynucleotides from Neisseria meningitidis strains ATCC 13090 and H44 / 76.

In another aspect, the invention provides isolated nucleic acid molecules encoding and / or expressing BASB030 polypeptides and polynucleotides, particularly Neisseria meningitidis BASB030 polypeptides and polynucleotides, including, for example, unprocessed RNA, ribozyme NA, mRNA, cDNA, genomic DNA, and Ba Z-DNA. Other embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides and variants thereof, and compositions comprising these polypeptides and polynucleotides.

Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene that encodes a BASB030 polypeptide having a deduced amino acid sequence of SEQ ID NO: 2,4,6 and polynucleotides closely related to these polynucleotides and variants thereof.

Another preferred embodiment of the invention is a BASB030 polypeptide from Neisseria meningitidis comprising or consisting of the amino acid sequence of SEQ ID NO: 2,4,6, or variants thereof.

111 111 11 1 1111 11 1 1 119 119

191,999 91 9,999

Using the information provided herein, such as the polynucleotide sequence set forth in SEQ ID NO: 1,3,5, a polynucleotide encoding a BASB030 polypeptide can be obtained using standard cloning and screening methods, such as methods for cloning and sequencing chromosomal DNA fragments from bacteria in using Neisseria meningitidis cells as starting material to obtain a full length clone. For example, to obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence set forth in SEQ ID NO: 1,3,5, a Neisseria meningitidis chromosomal DNA clone library in E. coli or other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer oligomer. or longer, derived from a partial sequence. Clones carrying a DNA sequence identical to the probe can then be detected using stringent hybridization conditions. By sequencing the individual clones identified by hybridization with sequencing primers designed according to the original polypeptide or polynucleotide sequence, it is then possible to extend the polynucleotide sequence in both directions to obtain the entire gene sequence. Preferably, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described in Maniatis, T., Fritsch, EF, and Sambrook et al., MOLECULAR CLONING, AND LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see, in particular, Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct sequencing of genomic DNA can also be performed to obtain the complete gene sequence. In the present invention, each polynucleotide shown in SEQ ID NO: 1,3,5 has been discovered in a DNA library derived from Neisseria meningitidis.

• »· · · · · ···· ·

In addition, each DNA sequence shown in SEQ ID NO: 1,3,5 contains an open reading frame encoding a protein having approximately the same number of amino acid residues as SEQ ID NO: 2,4,6 with a derived molecular weight that can be calculated using weight amino acid residues in a manner known in the art.

The polynucleotide of SEQ ID NO: 1, between the start codon at nucleotide # 1 and the stop codon starting at nucleotide # 2308 of SEQ ID NO: 1, encodes the polypeptide of SEQ ID NO: 2.

The polynucleotide of SEQ ID NO: 3, between the start codon at nucleotide # 1 and the stop codon starting at nucleotide # 2308 of SEQ ID NO: 3, encodes the polypeptide of SEQ ID NO: 4.

The polynucleotide of SEQ ID NO: 5, between the start codon at nucleotide # 1 and the stop codon starting at nucleotide # 2308 of SEQ ID NO: 5, encodes the polypeptide of SEQ ID NO: 6.

In another aspect, the present invention further provides an isolated polynucleotide comprising or consisting of:

(a) a polynucleotide sequence having at least 85% identity, preferably at least 90% identity, even more preferably at least 95% identity, preferably at least 97-99% or complete identity, with the sequence set forth in SEQ ID NO: 1,3,5 in full length SEQ ID NO: 1,3,5, respectively; or (b) a polynucleotide sequence encoding a polypeptide having at least 85% identity, preferably at least 90% identity, even more preferably at least 95% identity, preferably at least 97-99% or complete identity, with the amino acid sequence set forth in SEQ ID NO: 2, 4.6 over the entire length of SEQ ID NO: 2,4,6, respectively.

• ** ** 9 9

9 9 9 9999999 ·

9 9 9 9 9

9,999 99 · ·· ·

Polynucleotides encoding polypeptides of the present invention, including homologues and orthologs from strains other than Neisseria meningitidis, may be obtained by a process comprising the step of screening a suitable library under stringent hybridization conditions (e.g., using temperatures ranging from 45 to 65 ° C and SDS concentration from A suitable labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO: 1,3,5 or a fragment thereof; and isolating the full length gene and / or genomic clones containing said polynucleotide sequence.

The invention provides a polynucleotide sequence identical over its entire length to the coding sequence (open reading frame) of SEQ ID NO: 1,3,5. the invention also provides a coding sequence for the mature polypeptide or fragment thereof itself, as well as a coding sequence for the mature polypeptide or fragment thereof in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre- or pro- or prepro-protein sequence. . The polynucleotide of the present invention may also comprise at least one non-coding sequence, including, for example, at least one non-coding 3 'or 5' sequence, such as a transcribed but untranslated sequence, termination signals (such as rho-dependent and rhoindependent termination signals), binding sites for ribosomes, Kozak sequences, mRNA stabilizing sequences, introns and polyaenylation signals. The polynucleotide sequence may also comprise additional coding sequences encoding additional amino acids. For example, marker sequences may be encoded to facilitate purification of the fused polypeptide. In some embodiments, the marker sequence is a hexa-histidine peptide as supplied in a pQE vector (Qiagen, Inc) and described in Genz et al., Proc. Nati. Acad. Sci. USA 86: 821-824 (1989), or HA peptide tail (Wilson et al., Cell 37: 767

• ·· • • • ·· • • • • Φ · ···· ·· • • • · ♦ • • φ « • · • · · «· ··· • · «·

(1984), where both of these peptides can be used to purify the fusion polypeptide sequences fused to them. The polynucleotides of the present invention also include, for example, polynucleotides comprising a structural gene and sequences associated with it that direct gene expression.

The nucleotide sequence encoding the BASB030 polypeptide of SEQ ID NO: 2,4,6 may be identical to the sequence encoding the polypeptide comprised in nucleotides 1 to 2307 of SEQ ID NO: 1, or the sequence encoding the polypeptide comprised at nucleotides 1 to 2307 of SEQ ID NO: 3, or with a polypeptide coding sequence comprised at nucleotides 1 to 2307 of SEQ ID NO: 5, respectively. Alternatively, it may be a sequence resulting from the degeneracy of the genetic code that also encodes the polypeptide of SEQ ID NO: 2,4,6.

The term polynucleotide encoding a polypeptide as used herein refers to polynucleotides that comprise a sequence encoding a polypeptide of the present invention, in particular a bacterial polypeptide, and in particular a Neisseria meningitidis BASB030 polypeptide having the amino acid sequence of SEQ ID NO: 2,4,6. The term also encompasses polynucleotides that comprise one uninterrupted region or an interrupted region encoding a polypeptide (for example, polynucleotides interrupted by an integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or interrupted due to RNA editing or genomic DNA reorganization). which may also contain coding and / or non-coding sequences.

The invention further relates to variants of the polynucleotides described herein encoding variant polypeptides having derived amino acid sequences of SEQ ID NO: 2,4,6. Fragments of the polynucleotides of the present invention can be used, for example, to synthesize the complete polynucleotides of the present invention.

Particularly preferred embodiments of the invention are polynucleotides encoding BASB030 variants having the BASB030 amino acid sequences of the polypeptide of SEQ ID NO: 2,4,6, in which there were several, 5 to 10, 1 to 5, 1 to 3, 2, 1 or none of the amino acid residues substituted, modified, deleted and / or added, or which have been modified by a combination of these changes. Particularly preferred are those strong substitutions, additions, and deletions that do not alter the properties and activities of the BASB030 polypeptide.

Another preferred embodiment of the invention are polynucleotides that are at least 85% identical over their entire length to a polynucleotide encoding a BASB030 polypeptide having the amino acid sequence set forth in SEQ ID NO: 2,4,6 and polynucleotides that are complementary to such polynucleotides. In this regard, polynucleotides identical at least 90% over their entire length, and more preferably at least 95% identical, are particularly preferred. More preferably, the polynucleotides are at least 97% identical, more preferably 98% and most preferably at least 99%.

Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of SEQ ID NO: 1,3,5.

In some embodiments of the invention, polynucleotides are provided that hybridize, particularly under stringent conditions, to BASB030 polynucleotide sequences, such as the polynucleotides set forth in SEQ ID NO: 1,3,5.

• · ·····

The invention further relates to polynucleotides that hybridize to the polynucleotide sequences disclosed herein. In particular, the invention relates herein to polynucleotides that hybridize under stringent conditions to the polynucleotides of the present invention. The terms stringent conditions and stringent hybridization conditions mean that hybridization only occurs when there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is incubation overnight at 42 ° C in a solution containing: 50% formamide, 5x SSC (150 mM NaCl, 15 mM sodium tripotate), 50 mM sodium phosphate (pH 7.6), 5x Denhart's solution, 10% dextran sulfate and 20 μς / ιηΐ denatured, smeared salmon sperm DNA where incubation is followed by washing the hybridization membrane in 0.1 x SSC at approximately 65 ° C. Hybridization and wash conditions are well known and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spirng Harbor, NY, (1989), particularly in Chapter 11. Hybridization in solution can be performed in using the polynucleotide sequences of the present invention.

The invention also provides polynucleotides consisting of or comprising a polynucleotide sequence obtained by examining a suitable library comprising the complete gene for the polynucleotide sequence set forth in SEQ ID NO: 1,3,5 under stringent hybridization conditions using a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO: 1,3,5 or a fragment thereof; and a method for isolating said polynucleotide sequence. Fragments useful for obtaining such polynucleotides are, for example, probes and primers described herein.

as disclosed herein for the polynucleotide assays of the present invention, the polynucleotides of the present invention can be used as hybridization probes for RNA, cDNA and genomic DNA to isolate complete cDNA and genomic clones encoding BASB030 and to isolate cDNA and genomic clones of other genes having high identity, particularly high sequence identity, to the BASB030 gene. Such probes will usually contain at least 15 nucleotide residues or base pairs. Preferably, such probes will contain at least 30 nucleotide residues or base pairs and may contain at least 50 nucleotide residues or base pairs. Particularly preferred probes comprise at least 20 and less than 30 nucleotide residues or base pairs.

The coding region of the BASB030 gene can be isolated using the DNA sequence set forth in SEQ ID NO: 1,3,5 for oligonucleotide probe synthesis, a labeled oligonucleotide having a sequence complementary to the gene sequence of the present invention is then used to screen a cDNA library, genomic DNA or mRNA to determine to which member of the library the probe hybridizes.

There are several methods in the art for obtaining full-length or shorter DNAs, such as methods based on rapid cDNA end-amplification (RACE) (see, for example, Frohman et al., PNAS USA 85: 8998-9002, 1988). New modifications to this technique, such as Marathon ^™ technology (Clontech laboratories Inc), have greatly simplified the search for longer cDNAs. In Marathon ^™ technology, cDNA is prepared from mRNA extracted from selected tissue and the 'adapter' sequence is ligated to each end. Nucleic acid amplification (PCR) is then performed to amplify the missing 5 'end of the DNA using a combination of gene-specific oligonucleotide primers and PCR. For the adapter. The PCR is then repeated using nested primers, i.e. primers designed for a thermal reaction with the amplified product (usually an adapter specific primer that is thermally bound 3 'to the adapter sequence and a gene specific primer that is thermally bound further 5 'to the selected gene sequence). The product of this reaction is then analyzed by DNA sequencing and complete DNA is prepared by binding the product directly to existing DNA to form the complete sequence, or by performing separate PCR for the complete sequence using new sequence information to design the 5 'primers.

The polynucleotides and polypeptides of the present invention can be used, for example, as research reagents and materials for the development of drugs and diagnostic agents for diseases, particularly human diseases, as discussed below in connection with polynucleotide assays.

The polynucleotides of the present invention, which are oligonucleotides derived from SEQ ID NOs: 1-6, can be used in the methods disclosed herein, preferably in PCR, to determine whether the polynucleotides of the present invention are transcribed in bacteria in infected tissue. It is known that such sequences are also useful for diagnosing the stage of infection and the type of pathogen.

The invention also provides polynucleotides that encode polypeptides that are mature proteins, plus additional amino or carboxy-terminal amino acids, or amino acids within the mature polypeptide (for example, when the mature form is made up of more than one polypeptide chain). Such sequences may be of importance for processing the protein from the precursor to the mature form, may allow protein transport, shorten or increase the half-life of the protein, or facilitate protein manipulation in assays or production. In general, in vivo these additional amino acids may be removed from the mature protein. cellular enzymes.

For each polynucleotide of the present invention, a polynucleotide complementary to said polynucleotide is provided. It is preferred that these complementary polynucleotides be completely complementary to the respective polynucleotide.

A precursor protein having the mature form of a polypeptide fused to one or more of the sequences may be an inactive form of the polypeptide. When the prose sequences are deleted, such inactive precursors are usually activated, some or all such prose sequences may be removed prior to activation. Generally, such precursors are referred to as preproteins.

in addition to the standard A, G, C T / U designations for nucleotides, the designation Ν may also be used in the description of the polynucleotides of the present invention. N indicates that any of the four DNA or RNA nucleotides may be present at said position of the DNA or RNA sequence, except that it is preferred that N is not a nucleic acid that, when combined with adjacent nucleotide positions, reads in the correct position. reading frame, could create a premature termination codon in such reading frame.

In summary, a polynucleotide of the present invention may encode a mature protein, a mature protein with a leader sequence (which may be referred to as a preprotein), a mature protein precursor containing one or more non-preprotein leader sequences, or a preprotein that is

2b is a preprotein precursor comprising a leader sequence and one or more prosequences, which are usually removed during processing to produce active and mature forms of the polypeptide.

In one aspect, the invention provides the use of the polynucleotide of the present invention for therapeutic or prophylactic purposes, particularly for genetic immunization.

The use of the polynucleotide of the present invention in genetic immunization will advantageously employ a suitable method of administration, such as direct injection of plasmid DNA into muscles (Wolff et al., Hum. Mol. Genet. (1992), 1: 363; Manthorpe et al.

Hum. Gene Ther. (1983), 4: 419), administration of DNA complexed with specific protein carriers (Wu et al., J. Biol. Chem. (1989), 264: 16985), coagulation of DNA with calcium phosphate ((Benvenisty and Reshef, PNAS USA (1986) 83: 9551), DNA encapsulation in various forms of liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment tang et al., Nature (1992) 356: 152; Eisenbraun et al. , DNA Cell Biology (1993), 12: 791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA (1984) 81:

5849).

Vectors, host cells, expression systems

The invention also relates to vectors comprising the polynucleotide or polynucleotides of the present invention, host cells that are genetically engineered vectors, and production of the polypeptides of the present invention by recombinant techniques. Cell-free translation systems can also be used for the production of such proteins

using RNAs derived from the DNA constructs of the present invention.

The recombinant polypeptides of the present invention can be prepared by methods well known to those skilled in the art from genetically engineered host cells containing expression systems. Accordingly, in another aspect, the invention relates to expression systems comprising the polynucleotide or polynucleotides of the present invention, host cells that are genetically engineered by such expression systems, and production of the polypeptides of the present invention by recombinant techniques.

For recombinant production of the polypeptides of the present invention, the host cells may be genetically engineered to incorporate the expression systems or portions thereof or polynucleotides of the present invention. Insertion of the polynucleotide into the host cell may be accomplished by methods described in standard laboratory manuals such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986), and in Sambrook et al., MOLECULAR CLONING, and LABORATORY MANUAL, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) which include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic transfection and infection.

Examples of suitable hosts include bacterial cells such as streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, Moraxella catarrhalis, Haemophilus influenzae, and Neisseria meningitidis; fungi such as yeast, Kluveromyces, Saccharomyces, basidiomyces, candida albicans and Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells such as gymnosperm and angiosperm cells.

Various expression systems can be used to produce the polypeptides of the present invention. Such vectors include, but are not limited to, chromosomal, episomal, and viral vectors, for example, vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, from viruses such as baculoviruses, papa viruses, SV40, vaccinia viruses. adenoviruses, poultry pox viruses, pseudorabies viruses, picornaviruses, retroviruses and alphaviruses and vectors derived from combinations thereof, such as vectors prepared from plasmid and bacteriophage genetic elements such as cosmids and phagemids. The expression system may comprise control regions that regulate the intended expression. In general, any system or vector suitable for maintaining, propagating or expressing polynucleotides and / or expressing a polypeptide in a host can be used for expression in the present invention. The appropriate DNA sequence may be inserted into the expression system by any of the conventional techniques, such as those described in Sambrook et al., MOLECULAR CLONING, and LABORATORY MANUAL (supra).

In recombinant expression systems in eukaryotic organisms, the expressed polypeptide may contain appropriate secretion signals to secrete the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment. The signals may be endogenous to the polypeptide or may be heterologous signals.

• ·

2ο

The polypeptides of the present invention can be obtained and purified from recombinant cell cultures by well known techniques such as ammonium sulfate precipitation or ethanol, acid extraction, anionic or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectectin chromatography. . Preferably, metal ion affinity chromatography (IMAC) is used for purification. Well known protein folding techniques can be used to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation, or purification.

The expression system may also be a recombinant live microorganism such as a virus or bacterium, the selected gene may be inserted into the genome of the live recombinant virus or bacterium. Inoculation and in vivo infection with this vector will result in in vivo expression of the antigens and induction of an immune response. Viruses and bacteria useful for this purpose include, for example: poxviruses (e.g. vaccinia virus, poultry pox virus, canary pox virus), alphaviruses (Sindbis virus, Semliki forest virus, Venezuelan equine encephalitis virus), adenoviruses, adeno-associated viruses, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella-zoster virus, etc.), Listeria,

Salmonella, Shigella, Neisseria, BCG. These viruses and bacteria may be virulent or may be attenuated in various ways to obtain live vaccines. Such live vaccines also form part of the present invention.

Diagnostic, prognostic, serotyping and mutation tests

The present invention also relates to the use of BASB030 polynucleotides and polypeptides of the present invention as diagnostic agents. Detection of BASB030 polynucleotides and / or polypeptides in eukaryotes, particularly in mammals, particularly humans, is a diagnostic method for diagnosing a disease, determining the extent of a disease or the response of an infectious organism to treatment. Eukaryotes, particularly mammals, especially humans, especially individuals infected with, or suspected of having, an organism containing a BASB030 gene or protein, may be screened at the nucleic acid or amino acid level using a variety of known techniques, as well as using the methods disclosed herein.

Polypeptides and polynucleotides for prognostic, diagnostic or other analysis may be obtained from presumably infected and / or infected samples from an individual.

Polynucleotides from any such source, particularly DNA or RNA, can be used for detection directly or can be amplified enzymatically using PCR or any other amplification technique prior to analysis. RNA, especially mRNA, cDNA and genomic DNA can be used in the same manner. Using amplification, characterization of the species and strain of an infectious or present organism in an individual can be performed by analyzing the genotype of the selected polynucleotide of the organism. Deletions and insertions can be detected by changing the size of the amplified product compared to the genotype of a reference sequence obtained from a related organism, preferably from another species of the same genus or from another strain of the same species. Point mutations can be identified by hybridizing the amplified DNA to labeled BASB030 polynucleotide sequences. Perfectly or significantly corresponding sequences can be distinguished from incomplete or more significantly mismatched duplexes by digesting DNAse or RNAse, for DNA and RNA respectively, or by detecting differences in melting points or renaturation kinetics.

The differences in the polynucleotide sequence may also be:

Z3 detected by alterations in electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This can be done with or without denaturing agents. Differences in polynucleotides can also be detected by direct DNA or RNA sequencing. See, for example, Myers et al., Science 230: 1242 (1985). Sequence changes at specific sites can also be detected by nuclease protection assays, such as RNAs, VI and S1 protection assays, or by chemical cleavage. See, for example, Cotton et al., Proc. Nati. Acad. Sci. USA 85: 4397-4401 (1985).

In another embodiment, a set of oligonucleotide probes comprising a BASB030 nucleotide sequence or fragments thereof may be prepared to perform efficient screening, for example, of genetic mutations, serotype determination, taxonomic classification or identification. Methods for preparing kits are well known and can be used to solve many problems in molecular genetics, such as gene expression, genetic binding, and genetic variability (see, for example, Chee et al., Science 274: 610 (1996)).

Thus, in a further aspect, the present invention relates to a diagnostic kit comprising:

(a) a polynucleotide of the present invention, preferably a nucleotide sequence of SEQ ID NO: 1,3,5, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably a polypeptide of SEQ ID NO: 2,4,6, or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably a polypeptide of SEQ ID NO: 2,4,6.

• ·· fl • 9 »fl fl ♦ · • • • • fl • • • f f f f

It will be appreciated that in any such kit, (a), (b), (c), or (d) may form the essential component. Such kits will be useful in the diagnosis of disease or susceptibility to disease, inter alia.

the invention also relates to the use of the polynucleotides of the present invention as diagnostic agents. Detection of the mutated form of the polynucleotide of the present invention, preferably SEQ ID NO: 1,3,5, which is associated with a disease or pathogenic potential, will provide a diagnostic means that will assist or determine the diagnosis of the disease, prognosis of the disease course, susceptibility to disease resulting from overexpression, overexpression, or overexpression of a polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such a polynucleotide can be detected at the polynucleotide level by various techniques, such as those described herein.

Cells from organisms carrying mutations or polymorphisms (allelic variations) in the polynucleotide and / or polypeptide of the present invention can also be detected at the polynucleotide or polypeptide level by various techniques, for example, for serotyping. For example, RT-PCR can be used to detect mutations in RNA. Particularly preferred is the use of RT-PCR together with an automatic detection system such as Gene Scan. RNA, cDNA or genomic DNA can be used for the same purpose. For example, PCR primers complementary to a polynucleotide encoding a BASB030 polypeptide can be used to identify and analyze mutations.

The invention further provides primers having 1, 2, 3 or 4 nucleotides deleted from the 5 'and / or 3' ends. These primers may

2 / být · · · · # # be used, for example, to amplify BASB030 DNA and / or RNA isolated from a sample from an individual, such as a body sample.

The primers can be used to amplify polynucleotides isolated from an infected individual, and the polynucleotide can then be processed by various techniques to evaluate the polynucleotide sequence. In this way, mutations in the polynucleotide sequence can be detected and can be used to diagnose and / or determine the prognosis of an infection or its stage or serotype, and / or to classify an infectious agent.

The invention further provides a method for diagnosing a disease, preferably a bacterial infection, more specifically an infection caused by Neisseria meningitidis, comprising determining an overexpression of a polynucleotide having the sequence of SEQ ID NO: 1,3,5 in a sample from an individual. Increased or decreased expression of BASB030 polynucleotides can be measured by any method for quantifying polynucleotides, such as amplification,

PCR, RT-PCR, RNAs protection, Northern hybridization, spectrometry and other hybridization methods.

Further, the diagnostic assays of the present invention for detecting overexpression of a BASB030 polypeptide compared to normal tissue control samples can be used, for example, to detect the presence of infection. Assay techniques that can be used to determine levels of BASB030 polypeptide in a sample from an individual, such as a body sample, are well known in the art. Such assay methods include radioimmunoassays, competitive binding assays, western hybridization, antibody sandwich assays, antibody detection and ELISA assays.

The polynucleotides of the present invention may be used as components of polynucleotide assemblies, preferably in a polynucleotide set

9 · * · * · «« 9 · ♦ ·· * 1 • 9,999 · 99 ^ZO »······

999 · 9 · ♦ · · 99 9 high density assemblies or grids. These high density kits are particularly suitable for diagnostic or prognostic purposes. For example, a set of stains, wherein each stain contains a different gene further comprising a polynucleotide or polynucleotides of the present invention, may be used for probing, for example, by hybridizing or amplifying nucleic acids, using a probe obtained from a sample from an individual to determine the presence of a particular polynucleotide sequences or related sequences in an individual. Such presence may indicate the presence of a pathogen, particularly Neisseria meningitidis, and may be used to diagnose and / or prognosis the disease or the course of the disease. Grids containing many variants of the polynucleotide sequence of SEQ ID NO: 1,3,5 are preferred. Also preferred are lattices comprising many variants of the polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 2,4,6.

Antibodies

The polypeptides and polynucleotides of the present invention or variants thereof, or cells expressing these polypeptides and polynucleotides can be used to produce antibodies specific for such polypeptides and polynucleotides.

In some embodiments, the invention provides antibodies against BASB030 polypeptides and polynucleotides.

Antibodies prepared against the polypeptides and polynucleotides of the present invention can be obtained by administering to the animals, preferably non-humans, the polypeptides and / or polynucleotides of the present invention, or fragments thereof carrying epitopes, analogs or cells expressing these substances, using conventional protocols. To prepare • · • t ♦

Any of the techniques known in the art that provide antibodies produced by a continuous cell culture can be used in monoclonal antibodies. Examples are various techniques as described, for example, in Kohler, G. and Milstein, C., Nature, 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al. , pp. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, by Alan R. Liss, Inc. (1985).

Techniques for producing single chain antibodies (US Patent No. 4946778) can be adapted to produce single chain antibodies to the polypeptides or polynucleotides of the present invention. Also, transgenic mice or other organisms or animals, for example mammals, may be used to express humanized antibodies immunospecific for the polypeptides or polynucleotides of the present invention.

Alternatively, phage display technology may be used to select genes for antibodies with polypeptide binding activities of the present invention, either from a PCR repertoire of amplified lymphocyte v-genes from anti-BASB030 confirmed humans, or from naïve libraries (McCafferty et al., (1990) , Nature, 348: 552-554; Marks et al., (1992),

Biotechnology 10: 779-783). The affinity of these antibodies can also be improved, for example, by combining chains (Clackson et al., 1991, Nature 352: 628).

The above-described antibodies can be used to isolate or identify clones expressing the polypeptides or polynucleotides of the present invention to purify the polypeptides or polynucleotides, for example, by affinity chromatography.

·· «··

Antibodies against BASB030 polypeptide or BASB030 polynucleotides can be used to treat infections, particularly bacterial infections.

Polypeptide variants include antigenically, epitope or immunologically equivalent variants and form one aspect of the present invention.

Preferably, the antibody or variant thereof is modified to be less immunogenic in an individual. For example, if the subject is a human, the antibody is preferably humanized in which the complementarity determining region or regions of the hybridoma-derived antibody is transferred to a human monoclonal antibody, as described, for example, in Jones et al., (1986) Nature 321: 522-. 525 or in Tempest et al., (1991) Biotechnology 9: 266-273.

Antagonists and agonists - tests and molecules

The polypeptides and polynucleotides of the present invention can also be used to assess the binding of small molecules and ligands in, for example, cells, cell-free formulations, chemical libraries, and natural mixtures. These substrates may be natural substrates and ligands, or may be structural or functional mimetics. See, for example, Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

The screening methods can simply measure the binding of the test compound to a polypeptide or polynucleotide, or to cell membranes carrying the polypeptide or polynucleotide, or to a fusion protein containing the polypeptide, using a labeling reagent directly or indirectly associated with the test compound. Alternatively, the screening method may include competition with * * ·

Znač φ φ φ · eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným eným) Further, these screening methods can test whether the test compound results in the generation of a signal generated by activation or inhibition of polypeptides or polynucleotides, using detection systems suitable for cells containing the polypeptide or polynucleotide. Activation inhibitors are typically tested in the presence of a known agonist, and the effect on agonist activation in the presence of a test compound is monitored. Constitutively active polypeptides and / or constitutively expressed polypeptides and polynucleotides can be used in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, to test which compound causes inhibition of activation of the polypeptides or polynucleotides. Further, the assay methods may comprise the steps of mixing the test compound with a solution comprising the polypeptide or polynucleotide of the present invention to form a mixture, measuring the activity of BASB030 polypeptides and / or polynucleotides in the mixture and comparing the activity of BASB030 polypeptides and / or polynucleotides in the mixture with the standard. Fusion proteins, such as those prepared from the Fc portion of BASB030 polypeptides as described herein, can also be used for high efficiency screening assays to identify antagonists of the polypeptides of the present invention, as well as phylogenetically and / or functionally related polypeptides (see D. Bennet et al. , J. Mol Recognition 8: 52-58 (1995) and K. Johanson et al., J. Biol. Chem. 270 (16): 9459-9471 (1995)).

Polynucleotides, polypeptides and antibodies that bind to and / or interact with a polypeptide of the present invention can also be used in screening methods to detect the effect of added compounds on mRNA and / or polypeptide production in cells. For example, an ELISA assay can be designed to measure secreted or cellular concentrations of polypeptides using monoclonal or polyclonal antibodies and standard methods. This procedure can be used to detect agents that can inhibit or increase production of polypeptides (also referred to as antagonists or agonists, respectively) from appropriately engineered cells or tissues.

The invention also provides a method for screening compounds for identifying those compounds that potentiate (agonists) or block (antagonists) the effect of BASB030 polypeptides or polynucleotides, especially those compounds that are bacteriostatic and / or bactericidal. The screening method may include high-efficiency techniques. For example, for screening for agonists or antagonists, a synthetic reaction mixture, a cell compartment such as a membrane, cell envelope or cell wall, or a preparation of any of said components comprising a BASB030 polypeptide and a labeled substrate or ligand of such polypeptides is incubated in the absence or presence. test molecules and agonist or antagonist action on BASB030 polypeptide are reflected in reduced labeled ligand binding or reduced product production from such a substrate. Molecules that bind spontaneously, ie without inducing effects on the BASB030 polypeptide, are likely antagonists. Molecules that bind well and that increase the rate of product formation from the substrate, increase signal transduction, or increase chemical channel activity are antagonists. Detection of the rate or amount of product production from the signal transduction substrate or chemical channel activity may be enhanced using a reporter system. Reporter systems that may be used for this purpose include, for example, colorimetric systems, converting labeled substrate to product, reporter genes responsive to activity change

999 * 9 9 9

9

9 99

BASB030 polypeptide or polynucleotides and binding assays known in the art.

Another example of a BASB030 agonist assay is a competitive assay that combines BASB030 and potential agonists with BASB030 binding molecules, recombinant BASB030 binding molecules, natural substrates or ligands, or mimics of natural substrates or ligands, under conditions suitable for the competitive inhibition assay. BASB030 may be labeled, for example, with a radioactive or colorimetric compound, so that the number of BASB030 molecules bound to a binding molecule or converted to a product, which is necessary to evaluate the potency of potential antagonists, can be accurately determined.

Potential antagonists include, but are not limited to, small organic molecules, peptides, polypeptides, and antibodies that bind to the polypeptides and / or polynucleotides of the present invention and thereby inhibit or eliminate their activity or expression. The potential antagonist may be small organic molecules, peptides, polypeptides such as closely related proteins or antibodies that bind to the same site as the binding molecule without inducing BASB030 induced activities, preventing the action or expression of BASB030 polypeptides and / or polynucleotides by eliminating binding of BASB030 polypeptides and / or polynucleotides.

Potential antagonists include small molecules that bind to and occupy binding sites on the polypeptide, thereby preventing binding to cellular binding molecules, thus preventing normal biological activity. Examples of such small molecules are, for example, small organic molecules, peptides or peptide-like molecules. Other potential antagonists include • · ·· ·· * · · · · · • · · · · · · _{«Λ Λ} · ·· # ♦» »« ♦ «# ······· ·» · ♦ ♦ Antisense molecules (for a description of these molecules, see Okano, J. Neurochem. 56: 560 (1991): OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988)). Preferred potential antagonists include BASB030 related compounds and BASB030 variants.

In another aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention or a fragment thereof, and various portions of constant regions of heavy or light chains of immunoglobulins of different subclasses (IgG, IgM, IgA, IgE). The immunoglobulin preferably used is a constant portion of a human IgG heavy chain, particularly an IgG1, in which the fusion is carried out in the hinge region. In a particular embodiment, the Fc portion can be removed by simply inserting a cleavage sequence that can be cleaved by blood clotting factor Xa. Further, the invention relates to methods for preparing these fusion proteins by genetic engineering and their use for drug discovery, diagnosis and therapy. Another aspect of the invention relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology are disclosed in International Patent Applications Nos. WO 94/29458 and WO 94/22914.

Each of the polynucleotide sequences of the present invention can be used for screening and development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for screening for antibacterial drugs. Additional polynucleotide sequences encoding the amino-terminal regions of the encoded protein or Shine-Delgarno or other sequences facilitating translation of the respective mRNA can be used to construct antisense sequences to control expression of the selected coding sequence.

♦ «·

The invention also provides the use of a polypeptide, polynucleotide, agonist or antagonist of the present invention to achieve interference with the initial physical interaction between the pathogen or pathogens and a eukarotic organism, usually a human, responsible for the infection. In particular, the molecules of the present invention can be used to prevent the adhesion of bacteria, particularly gram-positive and / or gram-negative bacteria, to eukaryotic, preferably mammalian, intercellular matter proteins on established instruments or to extracellular cell matter proteins in wounds; for blocking bacterial adhesion between extracellular cell mass proteins in a eukaryotic organism and bacterial BASB030 proteins mediating tissue damage and / or for blocking the normal progression of infection initiated other than by implantation of established devices or by surgery.

In yet another aspect, the invention provides BASB030 agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists.

The antagonists and agonists of the present invention can be used, for example, to prevent, inhibit and / or treat diseases.

In another aspect, the invention relates to mimotopes of the polypeptide of the present invention. A mimotope is a peptide sequence that is sufficiently similar to a native peptide (in sequence or structurally) that can be recognized by antibodies that recognize the native peptide; or may induce the generation of antibodies that recognize a natural peptide when attached to a suitable carrier.

Φ φφ · · φ · φ

ΦΦΦΦ φ φ

ΦΦ · · · Μ

ΦΦΦ Φ Φ

Φ Φ Φ Φ

Peptide mimotopes may be designed for a particular purpose by addition, deletion, or substitution of selected amino acids. Thus, the peptides can be modified to facilitate conjugation to a protein carrier. It may be desirable for some chemical conjugation methods to contain a terminal cysteine. Further, it may be desirable for peptides conjugated to a protein carrier to contain a hydrophobic end distal to the conjugated end of the peptides so that the free unconjugated end of the peptides remains associated with the surface of the protein carrier. Thus, the peptide is presented in a conformation that closely mimics the conformation of the peptides in the context of the whole natural molecule, for example, the peptides may be altered to contain an N-terminal cysteine and a C-terminal hydrophobic amidated terminal. Alternatively, the addition or substitution of the D-stereoisomeric form of one or more amino acids may be made to create the desired derivative, for example, to enhance the stability of the peptides.

Alternatively, peptide mimotopes can be identified using antibodies that can bind to the polypeptides of the present invention using techniques such as phage display techniques (EP 0 552 268 B1). This technique generates a large number of peptide sequences that mimic the structure of the native peptides and are therefore capable of binding to antibodies against the native peptide, but may not have significant sequence homology to the native polypeptide themselves.

Vaccines

Another aspect of the present invention relates to a method for inducing an immunological response in an individual, particularly a mammal, preferably a human, comprising immunizing the individual with BASB030 polynucleotide and / or polypeptide, or a fragment or variant thereof, to induce antibody and / or T formation. > · · · · · · ·

Lymphocyte responses to protect an individual from infection, particularly from bacterial infection, in particular from Neisseria meningitidis infection. The invention also provides methods in which such an immunological response slows bacterial replication. Yet another aspect of the present invention relates to a method for inducing an immunological response in an individual, comprising administering to said individual a nucleic acid vector, sequence, or ribozyme to direct expression of a BASB030 polynucleotide and / or polypeptide or fragment or variant thereof, for expressing a BASB030 polynucleotide and / or of a polypeptide or fragment or variant thereof in vivo, to induce an immunological response, such as an antibody and / or T cell immune response, including, for example, induction of cytokine-producing T cells or cytotoxic T cells, to defend said individual, preferably human, before the disease, regardless of whether the disease is already present in the individual or not. One example of gene delivery is its acceleration in the form of a coating on particles into cells. Such nucleic acid vectors may comprise RNA,

DNA, ribozyme, modified nucleic acid, DNA / RNA hybrid, DNA-protein complex or RNA-protein complex.

Another aspect of the present invention relates to immunological compositions which upon administration to an individual can induce an immunological response in an individual against a BASB030 polynucleotide and / or a polypeptide encoded therein, wherein the composition comprises a recombinant BASB030 polynucleotide and / or its encoded polypeptide and / neo DNA and / or RNA encoding and expressing an antigen of said BASB030 polynucleotide, a polypeptide encoded by it, or another polypeptide of the present invention. The immunological response may be used therapeutically or prophylactically and may be in the form of antibody immunity and / or.

Φ Φ Φ * ··· · · «

Cellular immunity, such as cellular immunity derived from CTL or CD4 + T-lymphocytes.

The BASB030 polypeptide or fragment thereof may be fused to a co-protein or chemical moiety, which may or may not produce antibodies in itself, but which may stabilize the first protein and produce a fusion or modified protein having antigenic and / or immunogenic properties, and preferably protective properties. The fusion recombinant proteins preferably further comprise an antigenic co-protein such as lipoprotein D from Haemophilus inluenzae, glutathione-S-transferase (GST) or βgalactosidase, or any other relatively large co-protein that solubilizes the protein and facilitates its production and purification. In addition, the co-protein may act as an adjuvant in the sense that it generally stimulates the immune system of the organism to which the protein is administered. The co-protein may be attached to the amino or carboxy terminus of the first protein.

The present invention provides compositions, particularly vaccine compositions, and methods comprising the polypeptides and / or polynucleotides of the present invention and immunostimulatory sequences as described, for example, in Sato, Y. et al.

Science 273: 352 (1996).

The present invention also provides methods of using the disclosed polynucleotides, or certain fragments thereof, that have been shown to encode non-variable regions of bacterial surface proteins in polynucleotide constructs used in such genetic immunization experiments in animal models of Neisseria meningitidis infection. Such experiments will be particularly useful for identifying protein epitopes capable of eliciting a prophylactic or therapeutic immune response. It is assumed that this procedure

* 9 ·· 9 99 9 99 • 9 9 9 9 9 9 • 9 9 9 9 9 9 • • · 9 9999 9 9 9 • 9 9 9 9 9 99 999 99 9 99 99

allow the subsequent preparation of monoclonal antibodies obtained from an organ of an animal resistant to infection, to develop prophylactic agents or therapeutic agents for the treatment of bacterial infection, in particular Neisseria meningitidis infection, in mammals, particularly humans.

The invention also includes a vaccine composition comprising the immunogenic recombinant polypeptide and / or polynucleotide of the present invention in combination with a suitable carrier, such as a pharmaceutically acceptable carrier. Since the polynucleotides and polypeptides may be degraded in the stomach, it is preferable to administer them parenterally, for example subcutaneously, intramuscularly, intravenously or intradermally. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostatic compounds, and ingredients that adjust the isotonicity of the composition with body fluids, preferably blood, of the subject; and non-aqueous and aqueous sterile suspensions, which may contain suspending agents or thickening agents. The compositions may be prepared in single or multi-dose containers, for example, in sealed ampoules or vials, and may be stored in a lyophilized state which requires the addition of a sterile liquid carrier immediately prior to use.

The vaccine composition of the present invention may also include an adjuvant system to enhance the immunogenicity of the composition. Preferably, the adjuvant system preferably elicits a Thi-type response.

The immune response can generally be divided into two categories, antibody and cellular immune responses (traditionally characterized by an antibody and cellular effector defense mechanism, respectively). these categories of response are referred to as a Th1 type response (cellular response) and a Th2 type response (antibody response).

The extreme Th1 response can be characterized by the production of antigen-specific, haplotype restricted cytotoxic T-lymphocytes and natural killer reactions. In mice, Thi-type responses are often characterized by the generation of antibodies of IgG2a subclass, whereas in humans, IgG1 antibodies correspond to these antibodies. Th2 immune responses are characterized by the generation of a wide variety of immunoglobulin isotypes, including IgG1, IgA and IgM (in mice).

Cytokines are believed to be the driving factor determining the type of immune response. High levels of Th1-type cytokines induce a cellular immune response to a given antigen, while high levels of Th2-type cytokines induce a humoral immune response to a given antigen.

The distinction between Th1 and Th2 type immune responses is not absolute. In fact, the individual develops immune responses that are predominantly Th1 or predominantly Th2. however, it is often desirable to evaluate cytokine families as described on murine CD4 + T cell clones in Mossman and Coffman (Mosmann, TR and Coffman, RL, (1989), Thi and Th2 cells: different patterns of lymphokine secretion lead to different functional properties (Annual Review of Immunology, 7, pp. 145-173). Traditionally, Thi-type responses have been associated with the production of IFN-γ and IL-2 cytokines by T lymphocytes. other cytokines often directly associated with the induction of a Thi-type immune response are not produced by T-lymphocytes, such as IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.

• · ·

It is known that some vaccine adjuvants are particularly suitable for stimulating either a Th1 or Th2 type response. Traditionally, the best indicator of the balance between a Th1: Th2 immune response after vaccination or infection is a direct measurement of the production of Th1 or Th2 cytokines by T lymphocytes in vitro after antigen restimulation, and / or measurement of the IgG1: IgG2 ratio in the antigen specific antibody response.

Thus, a Thi-type adjuvant is one that preferentially stimulates an isolated T-cell population to produce high levels of Thi-type cytokines upon antigen restimulation in vitro, and which induces both CD8 + cytotoxic T-lymphocytes and an antigen specific immunoglobulin response associated with Th1 isotypes -type.

Adjuvants that preferentially stimulate a Thi-type response are described in International Patent Application No. WO 94/00153 and WO 95/17209.

One such adjuvant is 3-de-O-acylated monophosphoryl lipid A (3D-MPL). This is known from GB 2220211 (Ribi). Chemically, it is a mixture of 3-de-O-acylated monophosphoryl lipid As 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form is 3-de-O-acylated monophosphoryl lipid A is described in European Patent Application 0 689 454 B1 (SmithKline Beecham Biologicals SA).

Preferably, the 3D-MPL particles are small enough to be sterile filtered through a 0.22 micron membrane (European Patent Application 0 689 454). 3D-MPL may be present in an amount of from 10 gg to 100 gg, preferably 25-50 gg per dose, wherein the antigen dose is usually in the range of 2-50 gg per dose.

• ·

Another preferred adjuvant is QS21, an HPLC purified non-toxic fraction obtained from the bark of Quillaja Saponaria Molina. This adjuvant may optionally be mixed with 3-de-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with a carrier.

A method for producing QS21 is described in US Patent No. 5,505,740.

Non-reactogenic adjuvant compositions comprising QS21 have been previously described (WO 96/33739). such formulations comprising QS21 and cholesterol have been shown to be effective adjuvants to stimulate a Th1 response when prepared together with an antigen.

Other adjuvants that preferentially stimulate a Th1-type response are immunomodulatory oligonucleotides, for example, unmethylated CpG sequences as described in WO 96/02555.

Combination of various Th1-stimulating adjuvants, as mentioned above, is also possible for the preparation of an adjuvant, which is the preferred stimulator of the Th1 response. For example, QS21 can be prepared together with 3D-MPL. The ratio of QS21: 3D-MPL is usually from 1:10 to 10: 1, preferably from 1: 5 to 5: 1 and often 1: 1. The preferred ratio for optimal synergy is 2.5: 1 to 1: 1 3D-MPL.-QS21.

Preferably, a carrier is also present in the vaccine composition of the present invention. The carrier can be an oil-in-water emulsion or an aluminum salt such as aluminum phosphate or aluminum hydroxide.

A preferred oil-in-water emulsion comprises a metabolisable oil such as squalene, α-tocopherol and Tween 80. In a particularly preferred embodiment of the vaccine compositions of the present invention

In the invention, antigens are combined with QS21 and 3D-MPL in such an emulsion. Further, the oil-in-water emulsion may comprise sleep 85 and / or lecithin and / or tricaprylin.

Usually, when administered to humans, QS21 and 3D-MPL are present in the vaccine in an amount of from 1 pg to 200 pg, preferably 10-100 pg, preferably 10-50 pg per dose. The oil-in-water emulsion typically contains 2-10% squalene, 2-10% α-tocopherol, and 0.3-3% Tween 80. Preferably, the squalene: α-tocopherol ratio is equal to or less than 1, thereby achieving greater emulsion stability. Sleep 85 may also be present at a concentration of 1%. In some cases, it is preferred that the vaccine composition of the present invention further comprises a stabilizing agent.

Non-toxic oil-in-water emulsions preferably comprise a non-toxic oil, such as squalene, an emulsifying agent, such as Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, a phosphate buffered saline solution.

A particularly effective adjuvant formulation comprising QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

The present invention also provides a polyvalent vaccine composition comprising the vaccine composition of the present invention in combination with other antigens, particularly antigens useful for the treatment of tumors, autoimmune diseases and related conditions. Such a polyvalent vaccine composition may comprise a Th1 response inducing adjuvant as described herein.

It will be appreciated that although the invention is described with respect to certain BASB030 polypeptides and polynucleotides, it includes fragments of natural polypeptides and polynucleotides and

• · • ·

similar polypeptides and polynucleotides with additions, deletions, or substitutions that do not significantly affect the immunogenic properties of the recombinant polypeptides and polynucleotides.

The antigen may also be administered in the form of whole bacteria (live or dead) or in the form of subcellular fractions, including Neisseria meningitidis.

Compositions, kits and routes of administration

In another aspect, the invention provides compositions comprising a BASB030 polynucleotide and / or a BASB030 polypeptide for administration to cells or multicellular organisms.

The invention also relates to compositions comprising the polynucleotides and / or polypeptides of the present invention or agonists or antagonists thereof. The polypeptides and polynucleotides of the present invention may be used in combination with a non-sterile or sterile carrier or carriers suitable for use in cells, tissues or organisms, such as pharmaceutical carriers suitable for administration to an individual. Such compositions comprise, for example, an additive or therapeutically effective amount of the polypeptide and / or polynucleotide of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The composition should be appropriate to the mode of administration. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the above compositions of the present invention.

• * · · · · · · · · · · · · · · · ·

The polypeptides, polynucleotides and other compounds of the present invention can be used alone or together with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered by any effective and convenient route, such as local, oral, rectal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal.

In therapeutic or prophylactic uses, the active agent may be administered to an individual in the form of an injectable preparation, for example, a sterile aqueous dispersion, preferably isotonic.

In another aspect, the present invention relates to pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and / or polynucleotide, such as a soluble form of the polypeptide and / or polynucleotide of the present invention, agonist or antagonist peptides or small molecule compounds in combination with a pharmaceutically acceptable carrier or excipient. . Such carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, or combinations thereof. The invention further relates to pharmaceutical packages and baptisms comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the present invention. The polypeptides, polynucleotides and other compounds of the present invention can be used alone or together with other compounds, such as therapeutic compounds.

The composition will be formulated for a particular mode of administration, such as systemic or oral administration. Preferred forms of systemic administration include injection, preferably intravenous injection. Other forms of injection may also be used, such as subcutaneous, intramuscular, or intraperitoneal injection. Alternative routes of systemic administration are transmucosal and transdermal administration using penetration enhancers such as bile salts or fusidic acids or other detergent agents. Further, when the polypeptide or other compound of the present invention can be prepared in an enteral or encapsulated composition, oral administration is also possible. The administration of these compounds may also be topical, for example in the form of poultices, pastes, gels, solutions, powders and the like.

For administration to mammals, particularly humans, it is contemplated that the daily dose of active agent will range from 0.01 mg / kg to 10 mg / kg, preferably about 1 mg / kg. The physician will always determine the dose best suited for the individual and this will depend on the age, weight and response of the individual. The above dosages are examples for average cases. Of course, there may be instances where higher or lower doses will be required, and such doses are within the scope of the present invention.

The dose depends on the choice of the peptides, the route of administration, the nature of the composition, the disease and the condition of the individual and the judgment of the physician. Suitable dosages are in the range of 0.1-100 Mg / kg individual weight.

The vaccine composition is preferably in injectable form. Conventional adjuvants may be used to enhance the immune response.

A suitable dose of antigen for vaccination is 0.5-5 Mg / kg, and such a dose is preferably administered 1-3 at an interval of 1-3 weeks. At the indicated dose, no adverse toxic effects were observed when the compounds of the present invention were administered, allowing them to be administered to individuals.

A wide range of possible dosages is contemplated due to the availability of many compounds and different efficacy with different routes of administration. For example, it is expected that higher doses will be required for oral administration than for intravenous injection. Dosage adjustments can be made using conventional dosage optimization methods as known in the art.

Sequence database, media sequences and algorithms

Polynucleotide and polypeptide sequences form a valuable source of information that determines their 2- and 3-dimensional structures, as well as identifying other sequences with similar homology. these procedures are best accomplished by storing sequences in a computer medium and then using the stored data in known macromolecular structure programs or to search sequence databases using known tools such as the GCG program.

The invention also provides methods for analyzing the nature of sequences or chains, particularly genetic sequences or encoded protein sequences. Preferred sequence analysis methods include, for example, sequence homology analysis methods such as identity and similarity analysis, DNA, RNA and protein structure analysis, sequence matching, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid search, analysis codon utilization, nucleic acid bases removal, and chromatographic peak analysis for sequencing.

♦ 4 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

A computer method is provided to identify homologies. The method comprises the steps of: providing a first polynucleotide sequence comprising the polynucleotide sequence of the present invention in a computer medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.

A computerized method is also provided to identify homologies. The method comprises the steps of: providing a first polypeptide sequence comprising the sequence of polypeptides of the present invention in a computer medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.

All publications and references, including patents and patent applications cited in this application, are hereby incorporated by reference in their entirety. Any patent application against which this application claims priority is also incorporated by reference in its entirety.

The term identity, as used herein, refers to a relationship between two or more polypeptide sequences or between two or more polynucleotide sequences that is determined by sequence comparison. The term identity also refers to the degree of sequence similarity between polypeptide or polynucleotide sequences, which is determined by the number of matches between the chains of such sequences. Identity can be calculated by known methods, including those described in (Computational

9 • 9 9 • 9 * • · • 9 9 · 9 • 9 9 9 9 9 9 9 9 9 .9 9 9 • 9 9 9 9 • · 9 · 9 • 9 9 • 9 9

Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Computer Science and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). The methods for determining identity are designed to give the greatest match between the sequences tested. In addition, methods for determining identity are codified in publicly available computer programs. Computer programs for determining identity between two sequences are, for example, the GAP program in the PCG program (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)); BLASTP, BLASTN (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410, (1990)); and FASTA (Pearson and

Lipman, Why. Nati. Acad. Sci. USA 85: 2444-2448 (1988)).

The BLAST family of programs is available from NCIB and other sources (BLAST Manual, Alschul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, SF et al., J. Molec. Biol. 215: 403-410, ( 1990)). The well-known Smith Waterman algorithm can also be used to determine identity.

The parameters for polypeptide sequence comparison are as follows:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970);

Comparison matrix: BLOSSUM62 from Henikoff and Henikoff, Proc. Nati. Acad. Sci. USA 89: 10915-10919 (1992);

Gap penalty: 8

Penalty penalty:

The program usable with these parameters is available as a gap program from Genetics Computer Group, Madison, WI. The above parameters are error parameters for peptide comparisons (along with zero terminal gap penalty).

The polynucleotide comparison parameters are as follows:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970);

Match matrix: matches = ++ 10, wrong pairs = 0

Gap penalty: 50

Penalty penalty:

Available as: gap program from Genetics Computer Group, Madison, WI. The above parameters are error parameters for nucleic acid comparison.

Preferred values for identity for the polynucleotides and polypeptide are set forth under (1) and (2) below.

(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least 50, 60, 70, 80, 85, 90, 95, 97, or 100% identity to the reference sequence of SEQ ID NO: 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1, or may comprise a number of nucleotide alterations as compared to a reference sequence, wherein said alterations are selected from the group consisting of a deletion, substitution, including transition and transversion, or insertion of at least one nucleotide; The 5 'or 3' end positions of the reference nucleotide sequence or anywhere between the two ends of the reference sequence in one or more continuous groups in the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by divided 100 and then subtracting the result from said total number of nucleotides in SEQ ID NO: 1, or:

n _n Xn - (xn · y), where n _n is the number of nucleotide alterations, x _n is the total number of nucleotides in SEQ ID NO: 1, y is 0.50 for 50%, 0.60 for 60%,

0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90 =, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and · Is a multiplication symbol where any other than an integer of the result x _{n and} y is rounded down to the nearest integer before subtracting from x _n . Alterations of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may generate error, nonsense, or frame-shifting mutations in that coding sequence and may therefore alter the polypeptide encoded by the polynucleotide according to such alterations.

For example, the polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:

1, that is, it may be 100% identical, or may contain a number of nucleic acid alterations to the reference sequence, such that the percent identity is less than 100%. Such alterations are selected from the group consisting of deletions, substitutions, including transitions and transversions, or insertions of at least one nucleic acid, and said alterations may occur at the 5 'or 3' end positions of the reference nucleotide ♦ 4.

4 9 9 999 9 9

9 9999999 9 • 99 9 9 sequences or anywhere between the two ends of the reference sequence, where they are either individually between the nucleic acids of the reference sequence, or in one or more uninterrupted groups in the reference sequence. The number of nucleic acid alterations is determined by multiplying the total number of nucleic acids in SEQ ID NO: 1 by the number determining the percent identity divided by 100, and then subtracting the result from the total number of nucleic acids in SEQ ID NO: 1, or:

n _n <x _n - (x _n · y), where n _n is the number of nucleic acid alterations, x _n is the total number of nucleic acids in SEQ ID NO: 1, y is 0.70 for 70%, 0.80 for 80 %, 0.85 for 85% etc .; and · is a multiplication symbol where any non-integer result number x _{n and} y is rounded down to the nearest integer before subtracting from x _n .

(1) The polypeptide embodiments further comprise an isolated polypeptide comprising a polypeptide having at least 50, 60, 70, 80, 85, 90,

95, 97, or 100% identity to the polypeptide reference sequence of SEQ ID NO: 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may comprise a number of amino acid alterations compared to a reference sequence wherein said alterations are selected from the group comprising a deletion, a substitution, including conservative and non-conservative substitutions, or an insertion of at least one amino acid, and wherein said alterations may occur at the amino- or carboxy-terminus of the reference polypeptide sequence or anywhere between the two ends of the reference sequence, a sequence, or one or more continuous groups, in a reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the number determining the percent identity divided by 100 and then subtracting the result from the indicated total number of amino acids in SEQ ID NO: 2, or:

n _and Xa - (Xa · y), where n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90 =, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for multiplication, where any other than an integer of the result x _and ay is rounded down to the nearest integer before subtracting from x _and .

For example, the polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:

2, that is, it may be 100% identical or may contain a number of amino acid alterations with respect to the reference sequence, such that the percent identity is less than 100%. Such alterations are selected from the group consisting of deletions, substitutions, including conservative and non-conservative substitutions, or insertions of at least one amino acid, and said alterations may occur at the carboxy- or amino-terminal positions of the reference polypeptide sequence or anywhere between the two ends of the reference sequence. either individually between the amino acids of the reference sequence, or in one or more continuous groups in the reference sequence. The number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the number specifying the percent identity divided by 100, and then subtracting the 9 «· 9 9 9 9 9 Result from the indicated total number of amino acids in SEQ ID NO: 2, or:

n _and Xa - (Xa · y), where n _a is the number of nucleic acid alterations, x _a is the total number of nucleic acids in SEQ ID NO: 2, y is 0.70 for 70%, 0.80 for 80%, 0 , 85 for 85% etc .; and · is a multiplication symbol where any other than an integer of the result x _and ay is rounded down to the nearest integer before subtracting from x _a .

The term "individual used in connection with an organism" refers to a multicellular eukaryotic organism, including, for example, a metazoan, a mammal, an ovid, a bovid, a monkey, a primate, and a human.

For example, a polynucleotide or polypeptide naturally present in a living organism is not isolated, but the same polynucleotide or polypeptide separated from the materials associated with the term &quot; isolated &quot; in its natural state is isolated. Further, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation, or any other recombinant method is isolated, even if it is present in an organism that may be alive or dead.

The term polynucleotide refers to a polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, including single and double stranded regions.

ΦΦΦ

Φ Φ ·· Φ

9

99

9

9 • φ

ΦΦΦ ΦΦΦ

ΦΦ

ΦΦΦ Φ Φ

Φ Φ

Φ Φ

The term variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains significant properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of a variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide.

Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations of the polypeptide encoded by the reference sequence, as set forth below. Typical variants of a polypeptide differ from another, reference polypeptide.

Generally, the differences are limited so that the sequences of the reference polypeptide and variants are closely similar and are identical in many regions, the variant and reference polypeptide may differ in amino acid sequence in one or more substitutions, additions or deletions, or a combination of these changes. A substituted or inserted amino acid residue may or may not be encoded by the genetic code. The variant of the polynucleotide or polypeptide may be a natural variant, such as an allelic variant, or it may be a non-naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques or by direct synthesis.

The term disease refers to any disease caused or related to infection by bacteria, including, for example, upper respiratory tract infections, invasive bacterial diseases such as bacteremia and meningitis.

DETAILED DESCRIPTION OF THE INVENTION

The examples below were performed using standard techniques that are well known in the art when

• «· • ♦ · · ♦ • · 9 9 9 99 • • · * • · 9 9 9 • • Φ «· · 9999 9 9 • • * • 9 9 9 • · ♦ · ·· · 99 9 99 ··

not otherwise specified. The examples are illustrative only and do not limit the scope of the present invention.

Example 1: Determination and confirmation of the DNA sequence of the BASB030 gene from two Neisseria meningitidis strains

A. BASB030 in Neisseria meningitidis serogroup B strain ATCC 13090.

BASB030 gene of SEQ ID NO: 1 was first discovered in the Incyte PathoSeq database containing undetermined genomic DNA sequences of Neisseria meningitidis strain ATCC 13090. Translation of BASB030 polynucleotide sequence shown in SEQ ID NO: 2 showed significant similarity (81% identity at 758 amino acids) with Neisseria gonorrhoeae PilQ outer membrane protein. The BASB030 gene sequence was further confirmed experimentally. For this purpose genomic DNA was extracted from 10 ^1θ Neisseria meningitidis cells (strain ATCC13090) using QIAGEN baptism for genomic DNA extraction (Qiagen Gmbh) and 1 gg of this material was subjected to DNA amplification by polymerase chain reaction using PilQl primers (5'- GGG G GCTAGC AA TAC CAA ACT GAC AAA AAT CAT TTC C-3 ') (SEQ ID NO: 7) containing an internal Nhel site (underlined) and PI1Q2 (5'-GGG G AAGCTT AT AGC GCA GGC TGT TGC CGG C-3 (SEQ ID NO: 8) containing an internal HindIII site (underlined). This PCR product was gel purified and DNA sequencing was performed using the Big Dye Cycle

Sequencing kit (Perkin Elmer) and ABI 373A / PRISM DNA sequencer. DNA sequencing was performed on both strands with redundancy of 2 and the complete sequence was assembled using

SeqMan program DNASTAR Lasergene software. The DNA sequence obtained and the derived polypeptide sequence are shown in SEQ

ID NO: 3 and SEQ ID NO: 4, respectively.

• *

B. BASB030 v Ν. meningitidis serogroup B strain H44 / 76

The BASB030 gene sequence was also designed for another N. meningitidis serogroup B strain H44 / 76. For this purpose, genomic DNA was extracted from N. meningitidis strain H44 / 76 using the experimental conditions given in Example 1. This material (1 μς) was then subjected to DNA amplification using a polymerase chain reaction using PilQ1 and PI1Q2 specific for the BASB030 gene. A 2300 bp DNA fragment was obtained, digested with Nhl / HindIII restriction endonucleases, and inserted into the appropriate sites of the pET-24b cloning / expression vector (Novagen) using standard molecular biology techniques (Molecular Cloning, A Laboratory Manual, second edition, Ed .: Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989. The recombinant pET-24b / BASB030 was then subjected to DNA sequencing using a Big Dyes kit (Applied Biosystems) and analyzed for an ABI 373 / A DNA sequencer under conditions recommended by the manufacturer. polynucleotide and derived polypeptide sequences, which are designated herein as SEQ ID NO: 5 and SEQ ID NO: 6, respectively, were obtained using the MegALign program from the DNASTAR software to assign the polynucleotide sequences of SEQ ID NOs: 1,3 and 5 and this association is shown in Figure 1, the pairwise identity matching is summarized in Table 1, which shows that three BASB030 polynucleo the gene sequences are all similar with an identity greater than 98.0%. Using the same MegAlign program, the alignment of the polypeptide sequences of SEQ ID NOs: 2,4 and 6 was performed and is shown in Figure 2; paired identity comparisons are summarized in Table 2, which shows that the three BASB030 protein sequences are all similar with an identity greater than 95.0%.

· · · ·

9 9 9 9 9 9

9 9 9 9 9 9

9 99 9 9999 9 9 9 • Φ ΦΦφ Φ Φ

ΦΦ ΦΦ · ΦΦ Φ

Taken together, these data indicate a strong conservation of the BASB030 gene sequence between two N. meningitidis serogroup B strains.

Table 1: Paired identity comparison of BASB030 polynucleotide sequences (in%)

SEQ ID NO: 3 SEQ ID NO: 5 SEQ ID NO: 1 99.9 98.9 SEQ ID NO: 3 99, 0

Table 2: Paired identity comparison of BASB030 polypeptide sequences (in%)

SEQ ID NO: 4 SEQ ID NO: 6 SEQ ID NO: 2 97.4 96, 9 SEQ ID NO: 4 99.3

Example 2: Expression and purification of recombinant BASB030 protein in E. coli

The construction of the pET-24b / BASB030 cloning / expression vector was described in Example 1B. This vector contains a BASB030 gene isolated from strain H44 / 76 fused to a chain of 6 histidine residues, placed under the control of the strong bacteriophage T7 promoter gene 10. For expression studies, this vector was inserted into Escherichia coli strain Novablue (DE3) (Novagen) containing the T7 polymerase gene placed under the control of the isopropyl-β-Dthiogalactosidase (IPTG) -controllable lac promoter. Liquid cultures (100 ml) of Novablue (DE3) (pET-24b / BASB030) of the recombinant E. coli strain were cultured at 37 ° C with shaking until the optical density at 600 nm (OD600) reached 0.6. At this time IPTG was added at a final concentration of 1 mM and the culture was continued for an additional 4 hours. The culture was then centrifuged at 10,000 rpm and the pellet was frozen at -20 ° C for at least 10 hours. After thawing, the pellet was resuspended for 30 minutes at 25 ° C in buffer A (6M) guanidine hydrochloride, 0.1 M NaH 2 PO 1, 0.01 M Tris, pH 8.0), was passed through a needle three times and clarified by centrifugation (20,000 rpm, 15 minutes). The sample was then loaded at a rate of 1 ml / min onto a Ni packed Hitrap column, and the column was washed sequentially with 40 ml of buffer B (8 M urea, 0.1 M NaClUP 0.01 M Tris, pH 8.0), 40 ml buffer C ( 8 M urea, 0.1 M NaPO 4 0.01 M Tris, pH 6.3). The recombinant BASB030 / His6 protein was then eluted from the column with 30 ml of buffer D (8 M urea, 0.1 M NaPO 4,

0.01 M Tris, pH 6.3) containing 500 mM imidazole and 3 ml fractions were collected. As shown in Fig. 3, a substantially enriched (&gt; 90% purity by coomasie staining) eluted BASB030 / His6 protein migrating at 85 kDa (determined molecular weight) from the column. This polypeptide was reactive with a murine monoclonal antibody directed against the 5-histidine motif (see Figure 3, lane 2). In addition, the denatured, recombinant PilQ-His6 protein could be solubilized in a urea-free solution. For this purpose, denatured PilQ-His6 contained in 8M urea was thoroughly dialyzed (2 hours) against buffer R (NaCl 150 mM, 10 mM NaH 2 PO 4, arginine 0.5 M, pH 6.8) containing 6M, 4M and 2M urea respectively. The PilQ preparation remained stable after freezing and thawing. Taken together, these data suggest that the BASB030 gene can be expressed and purified in both soluble and insoluble form from (BASB030 / His6) in E. coli.

Example 3: Immunization of murine BASB030 polypeptides and determination of antibody response to recombinant BASB030 polypeptide by ELISA

Partially purified BASB030 was injected three times to BALB / c mice on days 0, 14 and 28 (5 animals / group). This natural

The BASB030 polypeptide was obtained directly from N. meningitidis strain B (from J. Tommassen). The animals were injected subcutaneously with 5 gg (first fl-fl-fl injection) and 2 gg (second and third injections) of BASB030 polypeptide prepared in SBAS2 (SB62 emulsion containing 5 µg MPL and 5 pg QS21 per dose) or after adsorption of A1PO4 (with 5 gg MPL). The negative control group was mice immunized with SBAS2 only (no BASB030 polypeptide). Mice were bled on days 28 (14 after II) and 35 (7 after III) to detect specific anti-BASB030 antibodies. Specific anti-BASB030 antibodies were measured by ELISA using a partially purified recombinant BASB030 polypeptide as a microcapsule coated protein. Analyzes were performed on a pool of sera (from 5 mice) only after II (day 28).

Recognition of BASB030 epitopes on recombinant proteins, by ELISA

Briefly, microtiter plates (Maxisorb, Nunc) were coated with a 100 μΐ solution of recombinant BASB030 at a concentration of approximately 0.5 gg / ml in PBS for 2 hours at 37 ° C. The plates were then washed three times with 300 μΐ 150 mM NaCl - 0.05% Tween 20. They were then coated with 100 μΐ PBS-0,3-casein and incubated for 30 minutes at room temperature with shaking. Plates were then washed again in the same manner before incubating with antibodies. The animal sera were serially diluted twice in PBS-0.3% casein-0.05% Tween 20 and applied to the microplates (12 dilutions from 1/100 dilution) prior to incubation at ambient temperature for 30 minutes with shaking, before the next same step. washing. Biotin-conjugated anti-mouse Ig (rabbit, Dakopatts E0413) was used at a 1/2000 dilution in PBS - 0.3% casein - 0.05% Tween 20 to detect murine anti-BASB030 antibodies. After the last washing step (as described above), the plates were incubated with a solution of streptavidin-peroxidase complex diluted 1/4000 in the same solution for 30 minutes at ambient temperature with shaking.

• 9 9 99 * 99 • 9 9 999999 9 9 9 9

9 99

The results show that BASB030 polypeptide is highly immunogenic in BALB / c mice, both in the SBAS2 emulsion and after adsorption to A1PO4 / MPL (Fig. 4). These antibodies, induced after only two injections of the native protein, recognize the recombinant BASB030 polypeptide. Giant. shows that the SBAS2 emulsion is slightly less immunogenic than the formulation

A1PO4 / MPL. Purified recombinant BASB030 polypeptide was also injected into BALB / c mice to assess its immunogenicity.

Recognition of BASB030 natural epitopes on cells, by whole cell ELISA

The homologous H44 / 76 MenB strain (B: 15: P1.7, 16) was used as a coated bacterium to detect specific anti-BASB030 antibodies in animal serum. Briefly, microtiter plates (Maxisorb, Nunc) were coated with 100 µl of a 1/10 dilution of H44 / 76 bacteria from a 6 hour culture in which the bacteria were killed with 400 gg / ml tetracycline. The plates were incubated at 37 ° C for at least 16 hours until the plates were completely dried. The plates were then washed three times with 300 μΐ 150 mM NaCl 0.05% Tween 20. They were then coated with 100 μΐ PBS-0,3-casein and incubated for 30 minutes at room temperature with shaking. Plates were then washed again in the same manner before incubating with antibodies. The animal sera were serially diluted twice in PBS-0.3% casein-0.05% Tween 20 and plated on microplates (12 dilutions from 1/100 dilution) before incubation at ambient temperature for 30 minutes with shaking, before the next same washing step. Biotin-conjugated anti-mouse Ig (rabbit, Dakopatts E0413) was used at a dilution of 1/2000 in PBS - 0.3% casein-0.05% Tween 20 to detect murine anti-BASB030 antibodies. After the last washing step (as described above), the plates were incubated with a solution of streptavidin complex diluted 1/4000 in the same solution for 30 minutes at ambient temperature with shaking.

As shown in Fig. 5, we conclude that there is specific recognition of BASB030 antigens on the homologous strain H44 / 76 in mice immunized with purified molecule prepared in SBAS2 emulsion as well as in A1PO4 / MPL adjuvant (5 mice / group were analyzed) . The antigenic response is higher for the SBAS2 preparation using recombinant BASB030 protein. Mice given only SBAS2 adjuvant did not show a clear positive response.

Example 4: Presence of anti-BASB030 antibodies in serum from human convalescent patients

In this assay, human sera from convalescent patients were tested by Western blotting for the presence of specific antibodies, using purified recombinant BASB030 protein as well as native BASB030 protein (from J. Toramassen, Netherlands).

Briefly, 5 µg of purified BASB030 protein (recombinant or natural) was loaded onto an SDS-PAGE gradient gel (420%, Novex, code n ° EC60252) for electrophoretic migration. Proteins were transferred to nitrocellulose membranes (0.45 µm, BioRad, code 162-0114) at 100 volts over 1 hour using a Bio-Rad Trans-blot system (code 170-3930). Then the filters were blocked with PBS-0.05% Tween 20 overnight at ambient temperature, before incubation with human serum. These sera were diluted 100-fold in PBS-0.05% Tween 20 and incubated on nitrocellulose membranes for 2 hours at ambient temperature with gentle shaking, using a mini-blotter system (Miniprotean, Biorad code 170-4017). After three repeated washes in PBS-0.05%

Tween 20 for 5 minutes, the nitrocellulose membrane was incubated at ambient temperature for 1 hour with gentle shaking with the appropriate conjugate (biotinylated anti-human IgG antibodies, sheep, Amersham, code RPN1003, or biotinylated anti-mouse Ig antibodies, rabbit,

Amersham, code RPN1001) diluted 1/500 in the same wash buffer. The membrane was then washed three times as described above and incubated for 30 minutes with shaking with streptavidin-peroxidase complex (Amersham, code 1051) diluted 1/1000 in wash buffer. After the last three washes, imaging occurred during a 10-15 minute incubation in a 50 ml solution containing 30 mg of 4-chloro-1-naphthol (Sigma), 10 ml of methanol, 40 ml of PBS and 30 μΐ H2O2. The staining was terminated by washing the membranes several times in distilled water.

The results shown in Figures 6 and 7 (Part A) show that 5-7 of 7 sera from convalescent patients recognize either native BASB030 protein of different molecular weight (Fig. 6) or recombinant BASB030 protein of molecular weight of approximately 90 kDa (Fig. 6). 7). The two convalescent sera that reacted weakly with the recombinant BASB030 polypeptide are # 261469 and # 261979 (FIG. 7), whereas for the native BASB030 polypeptide, no 262117 and 261979 showed any distinct response (FIG. 6). The sera that reacted with the highest intensity are the same for both proteins (recombinant and natural). These reactions may reflect the importance of this polypeptide as a vaccine candidate. The native BASB030 protein appears in at least three different bands, which all appear to belong to the BASB030 polypeptide, the largest being identical on both gels (approximately 90 kDa). The native BASB030 polypeptide, directly isolated from bacteria, has degradation products that have been shown as bands of 45 and 35 kDa.

Part B of both western transmissions illustrates the reaction

• 00 0 90 • 0 0 9 9 9 0 0 0 9 0 0 9 0 0 0 9 • 0 0 9909 0 0 0 9 9 0 • • · • 0 999 • 0 0 09 / 99

murine antibodies against a homologous protein from Neisseria gonorrhoeae (J. Tommassen, The Netherland). The results clearly illustrate that there is cross-reactivity between the two homologous BASB030 proteins; For the recombinant BASB030 protein, two major bands are detected, one being a 90 kDa major band that is recognized by human convalescent serum and the other having a size of approximately 70 kDa (Fig. 7). On native BASB030 protein, murine antibodies recognize not only two major bands at 90 and 45 kDa as with convalescent serum, but also a band that may be formed by degradation products (approximately 75, 70, 55, 40, 35 and 25 kDa, see Fig. 6).

Example 5: Efficacy of BASB030 vaccine: activity of anti-BASB030 antibodies

Bactericidal activity of anti-BASB030 antibodies on a homologous strain of N. meningitidis

The bactericidal activity of the animal serum (pool) was tested as described previously (1,2) with minor modifications. Briefly, N. meningitidis serogroup B (strain H44 / 76) was used to determine the bactericidal activity of animal serum. In U-well 96-well micro-plates (NUNC), 50 μΐ / well of serial 2-fold dilution is incubated with 37.5 μΐ / well of meningococcal suspension in log phase adjusted to 2.5 10 ⁴ CFU / ml and incubated for 15 minutes at 37 ° C with shaking at 210 rpm (Orbital shaker, Form Scientifics). Add 12.5 μΐ of rabbit complement from pups (Pel freeze Biologicals, US) and incubate for a further 1 hour under the same conditions. Then, 10 µl aliquots of the mixture from each well are plated on Mueller-Hinton agar plates containing 1% Isovitalex and 1% heat-inactivated horse serum, and then incubated overnight at 37 ° C with 5% CO 2. In a day

later, colonies are counted for each dilution tested and bactericidal titers are determined as the serum dilution for 50% killing, as compared to the serum-free complement control. In this way, individual colonies can be counted up to 100 CFU per spot. The titers are expressed as the dilution inducing 50% killing as calculated by regression analysis.

The results illustrated in Table 3 show that anti-BASB030 antibodies have a strong bactericidal effect on the homologous strain H44 / 76 as also observed for the anti-PorA monoclonal antibody used as a positive control. At a 1/2560 dilution the killing percentage is still very high (91%).

Links:

1. Hoogerhout, P., Donders, E.M.L.M., van Graas-van den Brink,

J. A: M. Kuipers, B., Brugge, H. F., van Unen, L. M.,

Timmermans, H.A.M, the Hove, G.J., de Jong, A.D.P.J.M,

Peeters, C.C.A.M., Wiertz, E.J.H.J, and Poolman, J.T., Infection and Immunity, Sept. 1995, vol. 63, no. 9, pp. 3473-3478.

2. Maslanka, S: E., Gheesling, L. L., Libutti, D.E., Donaldson,

Harakeh, HS, Dykes, JK, Arhin, FF, Devi, SJN, Frasch, CE, Huang, JC, Kriz-Kuzemenska, P., Lemmon, RD, Lorange, M., Peeters, CCAM, Quataert, S: , Tai, JY, Carlone, GM, and the Multilaboratory Study Group, in Clin. Diagn. Lab. Immunol. 1997, 4: 156-167.

Table 3: Bactericidal effect of anti-BASB030 antibodies

Antibodies tested Dilutions tested Killing earned (%) Anti-PorA monoclonal Ab (positive control) 1/10000 100 ALIGN! Anti-BASB030 1/40 96 polyclonal 1/80 68 antibodies 1/160 56 1/320 68 1/640 56 1/1280 96 1/2560 91 Negative control 1/40 0

Example 6: Efficacy of anti-BASB030 antibodies in a passive protection model (young rats)

Ani-BASB030 antibodies obtained from immunized mice (5 / group, two groups) were evaluated for protective efficacy in a protective model in young rats. The assay measured the rate of elimination of N. meningitidis strain B by antibodies injected 24 hours prior to infection.

Briefly, 100 μΐ of 1/10 dilution of mouse serum (with specific anti-BASB030 antibodies) is injected intraperitoneally (IP) into 7-day-old Sprague-Dawley rats 24 hours before infection with live bacteria (day -1). On day 0, rats randomized and passively immunized with mouse serum on day -1, after IP 10 mg of iron dextran at 100 μΐ, for 30 minutes official XP by administration of 10 ⁷ live bacteria (100 μΐ) of one or more N. meningitidis strains H44 / 76 (B15: P1.7,16), previously passaged on rats (twice). The N. meningitidis strain was grown in chapped TBS medium for approximately 2 hours. The bacteria are then diluted in PBS to obtain a suspension of 1.10 ⁸ CFU / ml. For this test, rats were used at 7 days of age. Groups are composed of 8 rats that are randomly selected from litters prior to IP injection of 100 μΐ test sera. Three hours after infection, 20 μΐ of blood obtained by cardiac puncture under anesthesia is diluted in PBS (1/10, 1/100, 1/1000 and 1/10000) and several dilutions are applied to Mueller

Hinton colony forming unit counting (CFU) media. Then (after 24 hours) the number of CFUs is evaluated and compared with the number of CFU / ml blood in rats passively immunized with PBS.

The control group is rats injected with PBS.

Weighted averages are calculated for each animal and the average for each group is compared to a different average.

The results obtained with anti-BASB030 antibodies (see Figure 8) illustrate that these specific antibodies have an elimination effect on N. meningitidis strain H44 / 76 as compared to the negative control group. A difference of up to 1 log 10 in favor of anti-BASB030 antibodies is observed, compared to the non-specific antibodies present in mice injected with PBS only. The protective effect observed for our anti-BASB030 antibodies is the same as that obtained using a well characterized anti-PorA monoclonal antibody (H44 / 29, anti-Pl. 16).

Legend to Fig. 3 /

Substantially pure (&gt; 80%) fractions of BASB030 protein were obtained on a 4-20% gradient polyacrylamide gel (NOVEX) under SDS-PAGE conditions in parallel to the protein molecular weight marker. Gels were either stained with Coomasie Blue R250 or analyzed by Western blotting using an anti- (His5) monoclonal antibody.

Stored materials

The deposit containing Neisseria meningitidis serogroup B strain was deposited with the American Type Culture Collection (here ATCC) 22.6.1997 and was designated as accession number 13090. The deposit was described as Neisseria meningitidis (Albrecht and Ghon) and is lyophilized, 1.5-2.9 kb insert library constructed from a N. meningitidis isolate. The deposit is described in Int. Bull. Bacteriol. Nomencl. Taxon. 8: 1-15 (1958).

The deposit of the strain of Neisseria meningitidis is referred to herein as the deposited strain or as the DNA of the deposited strain.

The deposited strain contains the complete BASB030 gene. The sequences of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded by it, are in any event conflicting with any sequence description in the present invention.

The deposited strains were deposited under the Budapest Treaty on the International Deposit of Microorganisms for Patent Procedures. The tribe will be irrevocably and without restriction or condition released to the public after the patent expires. The deposited strain is listed only for the needs of those skilled in the art and is not accessible unless it is necessary for the grant of a patent, as required by 35 U.S.C. § 112.

• ·

Sequence Listing <110> Smith Kline Beecham Biologicals SA <120> Neisseria meningitidis antigenic polypeptides, relevant polynucleotides and protective antibodies <130> BM45323 <160> 8 <170> FastSeq for Windows, version 3.0 <210> 1 <211> 2310 <212 > DNA <213> bacterial <400> 1

atgaatacca aactgacaaa aatcatttcc ggtctctttg tcgcaaccgc cgcctttcag 60 acagcatctg caggaaacat tacagacatc aaagtttcct ccctgcccaa caaacagaaa 120 atcgtcaaag tcagcttcga caaagagact gtcaacccga ccggcttcgt aacctcctca 180 ccggcccgca tcgccttgga ctttgaacaa accggcattt ccatggatca acaggtactc 240 gaatatgccg atcctctgtt gagcaaaatc agtgccgcac aaaacagcag ccgtgcgcgt 300 ctggttctga atctgaacaa accgggccaa tacaataccg aagtacgcgg gaacaaagtc 360 tggatattca ttaacgaatc ggacgatacc gtgtccgccc ccgcacgccc cgccgtaaaa 420 gccgcgcctg ccgcaccggc aaaacaacag ggctgccgca ccgtctacca agtccgcagt 480 atccgtatcc aaacccttta ccccggcaaa acaacagctg ccgcaccgtt taccgagtcc 540 gtagtatccg tatccgcacc gttcagcccg gcaaaacaac aggcggcggc atcagcaaaa 600 caacagacgg cagcaccagc aaaacaacag acggcagcac cagcaaaaca acaggcggca 660 gcaccagcaa aacaaaccaa tatcgatttc cgcaaagacg gcaaaaatgc cggcattatc 720 gaattggctg cattgggctt tgccgggcag cccgacatca gccaacagca cgaccacatc 780 atcgttacgc tgaaaaacca taccctgccg accacgctcc aacgcagttt ggatgcggca 840 gactttaaaa caccggttca aaaggttacg ' ctgaaacgcc tcaataacga cacccagctg 900 accatcacaa cagccggcaa ctgggaactc gtcaacaaat ccgccgcgcc cggatacttt 960 accttccaag tcctgccgaa aaaacaaaac ctcgagtcag gcggcgcgaa caacgcgccc 1020 aaaaccttca caggccggaa aatccccctt gacttccaag atgtcgaaat ccgcaccatc 1080 ccgcagattt tggcaaaaga atccgggatg aacattgttg ccagcgactc cgtcaacggc 1140 aaaatgaccc tctccctcaa agacgtacct tgggatcagg ctttggattt ggttatgcag 1200 gcacgcaácc tcgatatgcg ccaacaaggg aacatcgtca acaccgcgcc ccgcgacgag 1260 ctgctcgcca aagacaaagc cctctcacag gcggaaaaag acattgccga tctaggcgcg 1320

• ·

7 0 • · • • • • · · • · • «· • «· · ctgtattcac aaaacttcca attgaaatac aaaaatgtgg aagaattccg cagcatcctg 1380 cgtttggaca atgccgacac aaccggaaac cgcaatacgc ttgtcagcgg caggggcagc 1440 gtgctgatcg atcccgccac caataccctg attgttaccg atacccgcag cgtcatcgaa 1500 aaattccgca aactgattga cgaattggac gtacccgcgc aacaagtgat gattgaggcg 1550 cgtatcgtcg aagcggcaga cggcttctcg cgcgatttgg gcgtcaaatt cggcgcgaca 1520 ggcaagaaaa agctgaaaaa tgatacaagc gcattcggct ggggggtaaa ctccggcttc 1680 ggcggcgacg ataaatgggg ggccgaaacc aaaatcaacc tgccgattac cgctgccgca 1740 aacagcattt cgctggtgcg cgcgatttcc tccggtgcct tgaacctgga attgtccgca 1800 tccgaatcgc tttcaaaaac caaaacgctC gccaatccgc gcgtgctgac ccaaaaccgc 1850 aaagaggcca aaatcgaatc cggttacgaa attcctttca ccgtaacctc aatcgcgaac 1920 ggcggcagca gcacgaacac ggaactcaaa aaagccgtct tggggctgac cgttacgccg 1980 aacatcacgc ccgacggcca aatcattatg accgtcaaaa tcaacaagga ctcgcctgcg 2040 caatgtgcct ccggtaatca gacgatcctg tgtatttcga ccaaaaacct gaatacgcag 2100 gctatggttg aaaacggcgg oacattgatt gtcggcggta tttatgaaga agacaacggc 2150 aatacgctga ccaaagtccc cctgttgggc gacatccccg ttatcggcaa cctctttaaa 2220 acacgcggga aaaaaaccga ccgccgcgaa ctgctgattt tcattacccc gaggattatg 2280 ggtacggccg gcaacagcct gcgctattga 2310

<210> 2 <211> 769 <212> PRT <213> bacterial

<400> 2 Met Asn Thr Lys Leu Thr Lys Ile Ile Ser Gly Leu Phe Wall Ala Thr 1 5 10 15 Dec Ala Ala Phe Gin Thr Ala Ser Ala Gly Asn Ile Thr Asp Ile Lys Wall 20 May 25 30 Ser Ser Leu For Asn Lys Gin Lys Ile Wall Lys Wall Ser Phe Asp Lys 35 40 45 Glu Ile Wall Asn For Thr Gly Phe Wall Thr Ser Ser For Ala Arg Ile 50 55 50 Ala Leu Asp Phe Glu Gin Thr Gly Ile Ser Met Asp Gin Gin Wall Leu 55 70 75 80 Glu Tyr Ala Asp For Leu Leu Ser Lys Ile Ser Ala Ala Gin Asn Ser 85 90 95 Ser Arg Ala Arg Leu Wall Leu Asn Leu Asn Lys For Gly Gin Tyr Asn 100 ALIGN! 105 110 Thr Glu Wall Arg Gly Asn Lys Wall Trp Ile Phe Ile Asn Glu Ser Asp 115 120 125 Asp Thr Wall Ser Ala For Ala Arg For Ala Wall Lys Ala Ala For Ala

• ·

/ 1

130 135 140 Ala For Ala Lys Gin Gin Gly Cys Arg Thr Wall Tyr Gin Wall Arg Ser 145 150 155 160 Ile Arg Ile Gin Thr Leu Tyr For Gly Lys Thr Thr Ala Ala Ala For 165 170 175 Phe Thr Glu Ser Wall Wall Ser Wall Ser Ala For Phe Ser For Ala Lys 180 185 190 Gin Gin Ala Ala Ala Ser Ala Lys Gin Gin Thr Ala Ala For Ala Lys 195 200 205 Gin Gin Thr Ala Ala For Ala Lys Gin Gin Ala Ala Ala For Ala Lys 210 215 220 Gin Thr Asn Ile Asp Phe Arg Lys Asp Gly Lys Asn Ala Gly Ile Ile 225 230 235 240 Glu Leu Ala Ala Leu Gly Phe Ala Gly Gin For Asp Ile Ser Gin Gin 245 250 255 His Asp His Ile Ile Wall Thr Leu Lys Asn His Thr Leu For Thr Thr 260 265 270 Leu Gin Arg Ser Leu Asp Wall Ala Asp Phe Lys Thr For Wall Gin Lys 275 280 285 Wall Thr Leu Lys Arg Leu Asn Asn Asp Thr Gin Leu Ile Ile Thr Thr 290 295 300 Ala Gly Asn Trp Glu Leu Wall Asn Lys Ser Ala Ala For Gly Tyr Phe 305 310 315 320 Thr Phe Gin Wall Leu For Lys Lys Gin Asn Leu Glu Ser Gly Gly Wall 325 330 335 Asn Asn Ala For Lys Thr Phe Thr Gly Arg Lys Ile Ser Leu Asp Phe 340 345 350 Gin Asp Wall Glu Ile Arg Thr Ile Leu Gin Ile Leu Ala Lys Glu Ser 355 360 365 Gly Met Asn Ile Wall Ala Ser Asp Ser Wall Asn Gly Lys Met Thr Leu 370 375 380 Ser Leu Lys Asp Wall For Trp Asp Gin Ala Leu Asp Leu Wall Met Gin 385 390 395 400 Ala Arg Asn Leu Asp Met Arg Gin Gin Gly Asn Ile Wall Asn Ile Ala 405 410 415 For Arg Asp Glu Leu Leu Ala Lys Asp Lys Ala Phe Leu Gin Ala Glu 420 425 430 Lys Asp Ile Ala Asp Leu Gly Ala Leu Tyr Ser Gin Asn Phe Gin Leu 435 440 445 Lys Tyr Lys Asn . Wall Glu Glu Phe Arg Ser Ile Leu Arg Leu Asp Asn 450 455 460

Ala Asp Thr Thr Gly Asn Arg Asn Thr Leu Wall Ser Gly Arg Gly Ser 465 470 475 480 Wall Leu Ile Asp For Ala Thr Asn Thr Leu Ile Wall Thr Asp Thr Arg 485 490 49S Ser Wall Ile Glu Lys Phe Arg Lys Leu Ile Asp Glu Leu Asp Wall For 500 505 510 Ala Gin Gin Wall Met Ile Glu Ala Arg Ile Wall Glu Ala Ala Asp Gly 515 520 525 Phe Ser Arg Asp Leu Gly Wall Lys Phe Gly Ala Thr Gly Lys Lys Lys 530 535 540 Leu Lys Asn Asp Thr Ser Ala Phe Gly Trp Gly Wall Asn Ser Gly Phe 545 550 555 560 Gly Gly Asp Asp Lys Trp Gly Ala Glu Thr Lys Ile Asn Leu For Ile 565 570 575 Thr Ala Ala Ala Asn Ser Ile Ser Leu Wall Arg Ala Ile Ser Ser Gly 580 585 590 Ala Leu Asn Leu Glu Leu Ser Ala Ser Glu Ser Leu Ser Lys Thr Lys 595 600 605 Thr Leu Ala Asn For Arg Wall Leu Thr Gin Asn Arg Lys Glu Ala Lys 610 615 620 Ile Glu Ser Gly Tyr Glu Ile For Phe Thr Wall Thr Ser Ile Ala Asn 625 630 635 640 Gly Gly Ser Ser Thr Asn Thr Glu Leu Lys Lys Ala Wall Leu Gly Leu 645 650 655 Thr Wall Thr For Asn Ile Thr For Asp Gly Gin Ile Ile Met Thr Wall 660 665 670 Lys Ile Asn Lys Asp Ser For Ala Gin Cys Ala Ser Gly Asn Gin Thr 675 680 685 Ile Leu Cys Ile Ser Thr Lys Asn Leu Asn Thr Gin Ala Met Wall Glu 690 695 700 Asn Gly Gly Thr Leu Ile Wall Gly Gly Ile Tyr Glu Glu Asp Asn Gly 705 710 715 720 Asn Thr Leu Thr Lys Wall For Leu Leu Gly Asp Ile For Wall Ile Gly 725 730 735 Asn Leu Phe Lys Thr Arg Gly Lys Lys Thr Asp Arg Arg Glu Leu Leu 740 745 750 Ile Phe Ile Thr For Arg Ile Met Gly Thr Ala Gly Asn Ser Leu Arg

755 760 765

Tyr

73 ________1 • · · · · · · <210> 3 <211> 2310 <212> DNA <213> bacterial <400> 3 aegaatacca aactgacaaa aatcatttcc ggtccctctg tcgcaaccgc cgcctttcag 60 acagcatctg caggaaacat tacagacatc aaagtttcct ccctgcccaa caaacagaaa 120 atcgccaaag tcagctttga caaagagatt gtcaacccga ccggcttcgt aacctcctca 180 ccggcccgca tcgccttgga ctttgaacaa accggcattt ccatggatca acaggtactc 240 gaatatgccg atcctctgtt gagcaaaatc agtgccgcac aaaacagcag ccgtgcgcgt 300 ctggttctga atctgaacaa accgggccaa tacaataccg aagtacgcgg gaacaaagtt 360 cggatatcca ttaacgaatc ggacgatacc gtgtccgccc ccgcacgccc cgccgtaaaa 420 gccgcgcctg ccgcaccggc aaaacaacag gctgccgcac cgtctaccaa gtccgcagta 480 tccgtatcca aaccctttac cccggcaaaa caacaggctg ccgcaccgtt. taccgagtcc 540 gtagtatccg Catccgcacc gttcagcccg gcaaaacaac aggcggcggc atcagcaaaa 600 caacagacgg cagcaccagc aaaacaacag acggcagcac cagcaaaaca acaggcggca 660 gcaccagcaa aacaaaccaa tatcgatttc cgcaaagacg gcaaaaatgc cggcattacc 720 gaattggctg cattgggctt tgccgggcag cccgacatca gccaacagca cgaccacatc 780 atcgttacgc tgaaaaacca taccctgccg accacgctcc aacgcagt 11 ggatgtggca 840 gactttaaaa caccggttca aaaggttacg ctgaaacgcc tcaataacga cacccagctg 900 attatcacaa cagccggcaa ctgggaactc gtcaacaaat ccgccgcgcc cggatacttt 960 accttccaag tcctgccgaa aaaacaaaac ctcgagtcag gcggcgtgaa caatgcgccc 1020 aaaaccttca caggccggaa aatctccctt gacttccaag atgtcgaaat ccgcaccatc 1080 ctgcagattt tggcaaaaga atccgggatg aacattgttg ccagcgactc cgtcaacggc 1140 aaaatgaccc Cctccctcaa agacgtacct tgggatcagg ctttggattt ggttatgcag 1200 gcacgcaacc tcgatatgcg ccaacaaggg aacatcgtca acatcgcgcc ccgcgacgag 1260 ctgcttgcca aagacaaagc cttcttacag gcggaaaaag acattgccga tctaggcgcg 1320 ctgtattcac aaaacttcca attgaaatac aaaaatgtgg aagaattccg cagcatcctg 1380 cgtttggaca atgccgacac aaccggaaac cgcaatacgc ttgtcagcgg caggggcagc 1440 gtgctgatcg atcccgccac caataccctg attgttaccg atacccgcag cgtcatcgaa 1500 aaattccgca aactgattga cgaattggac gtacccgcgc aacaagtgat gattgaggcg 1560 cgtatcgtcg aagcggcaga cggcttctcg cgcgatttgg gcgttaaatc cggcgcgaca 1620 ggcaagaaaa agctgaaaaa tgatacaagc gcattcggčt ggggggtaaa ctccggcttc 1680 ggcggcgacg ataaatgggg ggccgaaacc aaaaecaacc tgccgattac cgctgccgca 1740 aacagcattt cgctggtgcg cgcgatttcc tccggtgcct tgaatttgga ategtccgca 1800 tccgaatcgc tttcaaaaac caaaacgctt gccaatccgc gcgtgctgac ccaaaaccgc 1860 aaagaggcca aaatcgaatc cggttacgaa attcctttca ccgtaacctc aaccgcgaac 1920 ggcggcagca gcacgaacac ggaacCcaaa aaagccgtcc tggggccgac cgttacgccg 1980 aacaecacgc ccgacggcca aatcattatg accgtcaaaa tcaacaagga ctcgcctgcg 2040 caatgtgcct ccggtaatca gacgatcctg tgtatttcga ccaaaaacct gaatacgcag 2100

• ♦ · · »

4 · · · gctatggttg aaaacggcgg cacattgatt gtcggcggta ttcatgaaga agacaacggc aatacgctga ccaaagtccc cctgttgggc gacatccccg ttatcggcaa. cctctttaaa acacgcggga aaaaaaccga ccgccgcgaa ctgctgattt tcattacccc gaggattatg ggcacggccg gcaacagcct gcgctattga <210> 4 <211> 7S9 <212> PRT <2i3> bacterial

2160

2220

22Θ0

2310 <400> 4

Met Asn 1 Thr Lys Leu 5 Thr Lys Ile Ile Ser Gly Leu | 10 15 Dec Ala Ala Phe Gin Thr Ala Ser Ala Gly Asn Ile Thr Asp Ile Lys Wall 20 May 25 30 Ser Ser Leu For Asn Lys Gin Lys Ile Wall Lys Wall Ser Phe Asp Lys 35 40 45 Glu Ile Wall Asn For Thr Gly Phe Wall Thr Ser Ser For Ala Arg Ile 50 55 60 Ala Leu Asp Phe Glu Gin Thr Gly Ile Ser Met Asp Gin Gin Wall Leu 65 70 75 80 Glu Tyr Ala Asp For Leu Leu Ser Lys Ile Ser Ala Ala Gin Asn Ser 85 90 95 Ser Arg Ala Arg Leu Wall Leu Asn Leu Asn Lys For Gly Gin Tyr Asn 100 ALIGN! 105 110 Thr Glu Wall Arg Gly Asn Lys Wall Trp Ile Phe Ile Asn Glu Ser Asp press 120 125 Asp Thr Wall Ser Ala For Ala Arg For Ala Wall Lys Ala Ala For Ala 130 135 140 Ala For Ala Lys Gin Gin Ala Ala Ala For Ser Thr Lys Ser Ala Wall 145 150 155 160 Ser Wall Ser Lys For Phe Thr For Ala Lys Gin Gin Ala Ala Ala For 165 170 175 Phe Thr Glu Ser Wall Wall Ser Wall Ser Ala For Phe Ser For Ala Lys 130 185 190 Gin Gin Ala Ala Ala Ser Ala Lys Gin Gin Thr Ala Ala For Ala Lys 195 200 205 Gin Gin Thr Ala Ala For Ala Lys Gin Gin Ala Ala Ala For Ala Lys 210 215 220 Gin Thr Asn Ile Asp Phe Arg Lys Asp Gly Lys Asn Ala Gly Ile Ile

+420 225 230 235

240

F w »” · - -. B ······· / b · · · * - * * - - - - - - - - - - -

Glu Leu Ala Ala Leu Gly Phe Ala Gly Gin 250 For Asp Ile Ser Gin Gin 255 245 His Asp His Ile Ile Wall Thr Leu Lys Asn His Thr Leu For Thr Thr 260 265 270 Leu Gin Arg Ser Leu Asp Wall Ala Asp Phe Lys Thr For Wall Gin Lys 275 280 285 Wall Thr Leu Lys Arg Leu Asn Asn Asp Thr Gin Leu Ile Ile Thr Thr 290 295 300 Ala Gly Asn Trp Glu Leu Wall Asn Lys Ser Ala Ala For Gly Tyr Phe 305 310 315 320 Thr Phe Gin Wall Leu For Lys Lys Gin Asn Leu Glu Ser Gly Gly Wall 325 330 335 Asn Asn Ala For Lys Thr Phe Thr Gly Arg Lys Ile Ser Leu Asp Phe 340 345 350 Gin Asp Wall Glu Ile Arg Thr Ile Leu Gin Ile Leu Ala Lys Glu Ser 355 360 365 Gly Met Asn Ile Wall Ala Ser Asp Ser Wall Asn Gly Lys Met Thr Leu 370 375 380 Ser Leu Lys Asp Wall For Trp Asp Gin Ala Leu Asp Leu Wall Met Gin 385 390 395 400 Ala Arg Asn Leu Asp Met Arg Gin Gin Gly Asn Ile Wall Asn Ile Ala 405 410 415 For Arg Asp Glu Leu Leu Ala Lys Asp Lys Ala Phe Leu Gin Ala Glu 420 425 430 Lys Asp Ile Ala Asp Leu Gly Ala Leu Tyr Ser Gin Asn Phe Gin Leu 435 440 445 Lys Tyr Lys Asn Wall Glu Glu Phe Arg Ser Ile Leu Arg Leu Asp Asn 450 455 460 Ala Asp Thr Thr Gly Asn Arg Asn Thr Leu Wall Ser Gly Arg Gly Ser 465 470 475 480 Wall Leu Ile Asp For Ala Thr Asn Thr Leu Ile Wall Thr Asp Thr Arg 485 490 495 Ser Wall Ile Glu Lys Phe Arg Lys Leu Ile Asp Glu Leu Asp Wall For 500 505 510 Ala Gin Gin Wall Met Ile Glu Ala Arg Ile Wall Glu Ala Ala Asp Gly SIS 520 525 Phe Ser Arg Asp Leu Gly Wall Lys Phe Gly Ala Thr Gly Lys Lys Lys 530 S35 540 Leu Lys Asn Asp Thr Ser Ala Phe Gly Trp Gly Wall Asn Ser Gly Phe S45 550 555 560 Gly • Gly  Asp Asp Lys Trp Gly  Ala Glu Thr Lys Ile Asn Leu . For Ile

«1 ·« * ·

565 570 57S Thr Ala Ala Ala Asn Ser Ile Ser Leu Wall Arg Ala Ile Ser Ser Gly 580 585 590 Ala Leu Asn Leu Glu Leu Ser Ala Ser Glu Ser Leu Ser Lys Thr Lys 595 600 605 Thr Leu Ala Asn For Arg Wall Leu Thr Gin Asn Arg Lys Glu Ala Lys 610 615 620 Ile Glu Ser Gly Tyr Glu Ile For Phe Thr Wall Thr Ser Ile Ala Asn 625 630 635 640 Gly Gly Ser Ser Thr Asn Thr Glu Leu Lys Lys Ala Wall Leu Gly Leu 645 650 655 Thr Wall Thr For Asn Ile Thr For Asp Gly Gin Ile Ile Met Thr Wall 660 665 670 Lys Ile Asn Lys Asp Ser For Ala Gin Cys Ala Ser Gly Asn Gin Thr 675 680 685 Ile Leu Cys Ile Ser Thr Lys Asn Leu Asn Thr Gin Ala Met Wall Glu 690 695 700 Asn Gly Gly Thr Leu Ile Wall Gly Gly Ile Tyr Glu Glu Asp Asn Gly 705 710 715 720 Asn Thr Leu Thr Lys Wall For Leu Leu Gly Asp Ile For Wall Ile Gly 725 730 735 Asn Leu Phe Lys Thr Arg Gly Lys Lys Thr Asp Arg Arg Glu Leu Leu 740 745 750 Ile Phe Ile Thr For Arg Ile Met Gly Thr Ala Gly Asn Ser Leu Arg 755 760 765

Tyr <210> 5 <211> 2310 <212> DNA <2i3> bacterial <400> 5 atgaatacca aactgacaaa aatcatttcc ggtctctttg tcgcaaccgc cgcctttcag 60 acagcatcgg caggaaacat tacagacatc aaagtttcct ccctgcccaa caaacagaaa 120 atcgCcaaag tcagctttga caaagagatt gtcaacccga ccggcttcgt aacctcctca 180 ccggcccgca tcgccttgga ctttgaacaa accggcattt ccatggatca acaggtactc 240 gaatatgccg atcctctgtt gagcaaaatc agtgccgcac aaaacagcag ccgtgcgcgt 300 ctggttctga atctgaaca accgggccaa tacaataccg aagtacgcgg gaacaaagtt 360 tggatattca ttaacgaatc ggacgatacc cgggcccccccc

77 1 • • ♦ 4 • 4 • gccgcgcctg ccgcaccggc aaaacaacag gctgccgcac cgtctaccaa gtccgcagta 480 tccgtatccg aaccctttac cccggcaaaa caacaggctg ccgcaccgtt taccgagtcc 540 gtagtatccg tatccgcacc gttcagcccg gcaaaacaac aggcggcggc atcagcaaaa 600 caacaggcgg cagcaccagc aaaacaacag gcggcagcac cagcaaaaca acaggcggca 660 gcaccagcaa aacaaaccaa tatcgatttc cgcaaagacg gcaaaaatgc cggcattatc 720 gaattggctg cattgggctt tgccgggcag cccgacatca gccaacagca cgaccacatc 780 atcgttacgc tgaaaaacca taccctgccg accacgctcc aacgcagttt ggatgtggca 340 gactttaaaa caccggttca aaaggttacg ctgaaacgcc tcaataacga cacccagctg 900 attatcacaa cagccggcaa ctgggaactc gtcaacaaat ccgccgcgcc cggatacttt 960 accttccaag tcctgccgaa aaaacaaaac ctcgagtcag gcggcgtgaa caatgcgccc 1020 aaaaccttca caggccggaa aatctccctt gacttccaag atgtcgaaat ccgcaccatc 1080 ctgcagattt tggcaaaaga atccggaatg aacattgttg ccagcgactc cgtcaacggc 1140 aaaatgaccc tctccctcaa ggatgtgcct tgggatcagg ctttggattt ggttatgcag 1200 gčgcgcaacc tcgatatgcg ccagcaaggg aatatcgtca acatcgcgcc ccgcgacgag 1260 ctgcttgcca aagacaaagc cctcttacag gcagaaaaag acattgccga tttgggtgcg 1320 ctgtattccc aaaacttcca gttgaaatac aaaaatgtgg aagaattccg cagcatcctg 1380 cgtttggaca atgccgacac gaccggaaac cgcaacacgc ttatcagcgg caggggcagc 1440 gtgctgatcg atcccgccac caacaccctg attgttaccg acacccgcag cgtcatcgaa 1500 aaattccgca aactgattga cgaattggac gtacccgcgc aacaagtgat gattgaggcg 1560 cgtatcgtcg aagcggcaga cggcttctcg cgcgatttgg gcgttaaatt cggcgcgaca 1620 ggcaagaaaa agctgaaaaa tgatacaagc gcattcggct ggggggtaaa ctccggcttc 1580 ggcggcgacg ataaatgggg ggccgaaacc aaaatcaacc tgccgattac cgctgccgca 1740 aacagcattt cgctggtgcg cgcgatttcc tccggtgcct tgaatttgga attgtccgca 1800 tccgaatcgc tttcaaaaac caaaacgctt gccaatccgc gcgtgctgac ccaaaaccgc 1860 aaagaggcca aaatcgaatc cggttacgaa attcctttca ccgtaacctc aatcgcgaac 1920 ggcggcagca gcacgaacac ggaactcaaa aaagccgtct tggggctgac cgttacgccg 1980 aacatcacgc ccgacggcca aatcattatg accgtcaaaa tcaacaagga ctcgcctgcg 2040 caatgtgcct ccggtaatca gacgatcctg tgtatttcga ccaaaaacct gaatacgcag 2100 gctatggttg aaaacggcgg cacattgatt gtcggcggta tttatgaaga agacaacggc 2160 aatacgctga ccaaagtccc cctgttgggc gacatccccg ttatoggcaa cctctttaaa 2220 acacgcggga aaaaaaccga ccgccgcgaa ctgctgattt tcattacccc gaggattatg 2280 ggtacggccg gcaacagcct gcgctattga 2310

<210> 6 <211> 769 <212> PRT <2i3> bacterial <400> 6

Met Asn Thr Lys Ile Ile Ser Gly Leu Phe Val Ala Thr

9 ·

»9,999 ♦ 9 * • 9,9 • 9 · 9,999

9 · · »

Ala Ala Phe Gin Thr Ala Ser Ala Gly Asn 25 Ile Thr Asp Ile 30 Lys Wall 20 May Ser Ser Leu For Asn Lys Gin Lys Ile Wall Lys Wall Ser Phe Asp Lys 35 40 45 Glu Ile Wall Asn For Thr Gly Phe Wall Thr Ser Ser For Ala Arg Ile 50 55 60 Ala Leu Asp Phe Glu Gin Thr Gly Ile Ser Met Asp Gin Gin Wall Leu 65 70 75 80 Glu Tyr Ala Asp For Leu Leu Ser Lys Ile Ser Ala Ala Gin Asn Ser 85 90 95 Ser Arg Ala Arg Leu Wall Leu Asn Leu Asn Lys For Gly Gin Tyr Asn 100 ALIGN! 105 110 Thr Glu Wall Arg Gly Asn Lys Wall Trp Ile Phe Ile Asn Glu Ser Asp 115 120 12S Asp Thr Wall Ser Ala For Ala Arg For Ala Wall Lys Ala Ala For Ala 130 13 5 140 Ala For Ala Lys Gin Gin Ala Ala Ala For Ser Thr Lys Ser Ala Wall 145 ISO 155 160 Ser Wall Ser Glu For Phe Thr For Ala Lys Gin Gin Ala Ala Ala For 165 170 175 Phe Thr Glu Ser Wall Wall Ser Wall Ser Ala For Phe Ser For Ala Lys 130 185 190 Gin Gin Ala Ala Ala Ser Ala Lys Gin Gin Ala Ala Ala For Ala Lys 195 200 205 Gin Gin Ala Ala Ala For Ala Lys Gin Gin Ala Ala Ala For Ala Lys 210 215 220 Gin Thr Asn Ile Asp Phe Arg Lys Asp Gly Lys Asn Ala Gly Ile Ile 225 230 235 240 Glu Leu Ala Ala Leu Gly Phe Ala Gly Gin For Asp Ile Ser Gin Gin 245 250 255 His Asp His Ile Ile Wall Thr Leu Lys Asn His Thr Leu For Thr Thr- 260 265 270 Leu Gin Arg Ser Leu Asp Wall Ala Asp Phe Lys Thr For Wall Gin Lys 275 230 285 Wall Thr Leu Lys Arg Leu Asn Asn Asp Thr Gin Leu Ile Ile Thr Thr 290 295 300 Ala Gly Asn Trp Glu Leu Wall Asn Lys Ser Ala Ala For Gly Tyr Phe 305 310 31S 320 Thr Phe Gin Wall Leu For Lys Lys Gin Asn Leu Glu Ser Gly  Gly Wall 325 330 335 Asn . Asn . Ala Prc • Lys Thr  Phe ! Thr Gly Arg Lys Ile : Ser  Leu Asp Phe

Λ “- •« 'β

340 345 350

Gin Asp Val Glu Ile Glu Ile Leu Gin Ile Leu Ala Lys Glu Ser

355 360 365

Gly Met Asn Ile Val Ala

370 375 380

Ser Leu Lys Asp For Pro Trp Asp Gin

385 390 395 400

Ala Arg Asn Leu Asp Met Gin Gin Gly Asn Ile Val Asn Ile Ala

405 410 415

Pro Arg Glu Glu Leu Leu Lys Asp Glu Leu Leu Gin Leu Glu

420 425 430

Lys Asp Ile Ala Asp Leu. Gly Ala Leu Tyr Ser Gin Asn Phe Gin Leu

435 440 445

Lys Tyr Lys Asn Val, Glu Glu Phe Arg Ser Ile Leu Arg Leu Asp Asn

450 455 460

Ala Asp Thr Gly Asn Arg Asn Thr Leu Ile Gly Arg Gly Ser

(+420) 465 470 475 480

Val Leu Ile Asp For Thr Asn Thr Leu Ile Val Thr Asp Thr Arg

485 490 495

Ser Val Ile Glu Lys Phe Arg

500 505 510

Ala Gin Gin Val Ile Glu Ala Arg Ile Glu Ala Ala Asp Gly

(+420) 515 320 525

Phe Ser Arg. Asp Leu Gly Val Lys

530 535 540

Leu Lys Asn As Th Asp Ser Ala Phe Gly Trp.Gly Val Asn Ser Gly Phe

+420 545 550 555 560

Gly Gl Asp Asp Lys Trp Gly Ala Glu Thr Lys Ile Asn Leu Pro Ile

565 570 575

Thr Ala Ala Ala Asn Ser Ile Ser Leu Val Arg Ala Ile Ser Ser Gly

580 585 S90

Ala Leu Asn Leu Ser Glu Ser Ser Ala Ser G Ser Ser Lys Thr Lys

595 600 605

Thr Leu Ala Asn For Arg Valu Le Thr Gin Asn Arg Lys Glu Ala Lys

610 615 620

Ile Glu Ser Gly Tyr Glu Ile Phe Thr Val Thr Ser Ile Ala Asn

(+420) 625 630 635 640

Gly Gly Ser Ser Thr Asn Thr Glu Leu

645 650 655

Thr Val Thr For Asn Ile Thr For Asp Gly Gin Ile Met Thr Val

660 565 670

9 · ** ** ** «« «

999 ·· »· ·

9 • 9 · 99

Lys Ile Asn Lys As Ser Gly Asn Gin Thr 575 680 685 Ile Leu Cys Ile Ser Thr Lys Asn Leu Asn Thr Gin Ala Met Wall Glu 590 695 700 Asn Gly Gly Thr Leu Ile Wall Gly Gly Ile Tyr Glu Glu Asp Asn Gly 705 710 715 720 Asn Thr Leu Thr Lys Wall For Leu Leu Gly Asp Ile For wall Ile Gly 725 730 735 Asn Leu Phe Lys Thr Arg Gly Lys Lys Thr Asp Arg Arg Glu Leu Leu 740 745 750 Ile Phe Ile Thr For Arg Ile Met Gly Thr Ala Gly Asn Ser Leu Arg 755 750 765

Tyr <210> 7 <211> 37 <212> DNA <213> Artificial sequences <220>

<223> Primer <400> 7 <RTIgt; gggggctagc </RTI> aataccaaac tgacaaaaat catttcc <210> 8 <211> 31 <212> DNA <2i3>. Artificial sequences <220>

<223> Primer <400> 8 ggggaagctt atagcgcagg cCgttgccgg c

Claims (30)

Claims • · · · · · · · φ · φ · φ · • IV Ure -litr An isolated polypeptide comprising an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6 over the full length of SEQ ID NO: 4 and SEQ ID NO: 6, respectively . The isolated polypeptide of claim 1, wherein the amino acid sequence has at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6 over the full length of SEQ ID NO: 4 and SEQ ID NO: 6, respectively. The polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6. An isolated polypeptide having the amino acid sequence of SEQ ID NO: 2. An immunogenic fragment of a polypeptide according to any one of claims 1 to 5, wherein said immunogenic fragment can elicit an immune response (if necessary upon binding to a carrier) that recognizes the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ. ID NO: 6. The polypeptide of any one of claims 1 to 6, wherein said polypeptide is part of a larger fusion protein. • 9 99 • 9 9 · • 9 9 9 9 · 9 • 9 9 9 9 9 9 · 9 · • 9 9 9 9 · · • 999 9 9 9 9 9 9 9 9 • 9 9 9 9 99 9 9 9 9 • 9 9 9 An isolated polynucleotide encoding a polypeptide of any one of claims 1 to 7. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6 over the full length of SEQ ID NO: 4 and SEQ ID NO: 6, v or a nucleotide sequence complementary to said isolated polynucleotide. An isolated polynucleotide comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 4 and SEQ ID NO: 6 over the entire length of the coding region, or a nucleotide sequence complementary to said isolated polynucleotide. An isolated polynucleotide comprising a nucleotide sequence having at least 97% identity to SEQ ID NO: 3 or 5 over the full length of SEQ ID NO: 3 or 5, respectively, or a nucleotide sequence complementary to said isolated polynucleotide. The isolated polynucleotide of any one of claims 8 to 11, wherein the identity is at least 99% to SEQ ID NO: 3 or 5. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 6. An isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 5. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 4 or SEQ ID NO: 6, which is Oh Can be obtained by examining a suitable library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO. NO: 3 or SEQ ID NO: 5 or a fragment of said sequences. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2. An isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, obtainable by screening a suitable library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment of said sequence. An expression vector or recombinant live microorganism comprising an isolated polynucleotide. according to any of claims 8-18. A host cell comprising the expression vector of claim 19 expressing an isolated polypeptide comprising an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6, or a host cell membrane comprising the expressed polypeptide. A method of producing a polypeptide comprising an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6, comprising culturing the host cell of claim 20 under conditions suitable for producing said polypeptide and recovering the polypeptide from the culture medium. 22. A method for expressing a polynucleotide according to any of claims 8-18, comprising transforming the host cell with an expression vector comprising at least one of said polynucleotides and culturing said host cell under conditions sufficient to express any of said polynucleotides. A vaccine composition comprising an effective amount of the polypeptide of any one of claims 1 to 7 and a pharmaceutically acceptable carrier. A vaccine composition comprising an effective amount of a polynucleotide according to any one of claims 8 to 18 and a pharmaceutically acceptable carrier. The vaccine composition of any one of claims 23 or 24, wherein said composition comprises at least one additional Neisseria meningitidis antigen. An antibody immunospecific for the polypeptide or immunological fragment of any one of claims 1 to 7. A method for diagnosing a Neisseria meningitidis infection comprising identifying a polypeptide of any one of claims 1-7, or an antibody that is immunospecific for said polypeptide, present in a biological sample from an animal suspected of having such an infection. «Φ Φ Φ Φ ΦΦ ♦♦ · · 9 9 9 99 999 9 • φ • 9 9 9,999 • « Use of a composition comprising an immunologically effective amount of the polypeptide of any one of claims 1-7 for the preparation of a medicament for use in eliciting an immune response in an animal. Use of a composition comprising an immunologically effective amount of the polynucleotides of any one of claims 8-18 for the preparation of a medicament for use in eliciting an immune response in an animal. A therapeutic composition useful for treating a human having a disease caused by Neisseria meningitidis comprising at least one antibody directed against the polypeptide of any one of claims 1-7 and a suitable pharmaceutical carrier. • · · / xn 1/17 'Vp # Giant. 1: Alignment of BASB030 polynucleotide sequences. < The identity to SEQ ID NO: 1 is represented by a dot and the dash shows the missing nucleotide