EP1943269A2 - Neisseria-meningitidis-antigene - Google Patents
Neisseria-meningitidis-antigeneInfo
- Publication number
- EP1943269A2 EP1943269A2 EP06806276A EP06806276A EP1943269A2 EP 1943269 A2 EP1943269 A2 EP 1943269A2 EP 06806276 A EP06806276 A EP 06806276A EP 06806276 A EP06806276 A EP 06806276A EP 1943269 A2 EP1943269 A2 EP 1943269A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- peptide
- antibody
- antigen
- nucleic acid
- meningitidis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to a peptide consisting of one antigen of N. meningitidis of any of the SEQ ID NOS: 1 to 184 or a functional active fragment or derivative thereof, optionally further consisting of additional amino acid residue(s); a nucleic acid coding for the same; a pharmaceutical composition, especially a vaccine, containing a polypeptide comprising or consisting of said peptide or said nucleic acid; a nucleic acid coding for said polypeptide or a nucleic acid complementary to the same; an antibody or functional active fragment thereof; a hybridoma cell line which produces said antibody; a method for producing said antibody; a pharmaceutical composition comprising said antibody; the use of said peptide or said nucleic acid for the manufacture of a medicament for the immunization of a subject; the use of said antibody or functional fragment thereof for the manufacture of a medicament for the treatment of an infection; a method of diagnosing a N. meningitidis infection; a method for identifying a lig
- Neisseria meningitidis is a leading cause of purulent meningitis and septicemia world-wide and affects predominantly infants between 6 months and 2 years of age as well as teenagers. Meningococcal meningitis and sepsis are devastating diseases that can kill children and young adults within hours despite the availability of effective antibiotics.
- the disease is caused by Neisseria meningitidis, or meningococcus, an aerobic gram- negative diplococcus, that is closely related to Neisseria gonorrhea, and to several nonpathogenic Neisseria species, such as N. lactamica.
- the organism has both an inner (cytoplasmic) and outer membrane, separated by a cell wall.
- the outer membrane contains several protein structures, which enable the bacteria to interact with the host cells, as well as other functions.
- the outer membrane is surrounded by a polysaccharide capsule that is necessary for pathogenicity because it helps the bacteria to resist phagocytosis and complement-mediated lysis.
- the outer membrane proteins and the capsular polysaccharide comprise the main surface antigens of the organism.
- Meningococci are classified using serological methods based on the structure of the polysaccharide capsule. Thirteen antigenically and chemically distinct polysaccharide capsules have been described. Some strains, often those found to cause asymptomatic nasopharyngeal carriage, are not groupable, and do not have a capsule. Almost all invasive disease is caused by one of five serogroups: A, B, C, Y, and W-135. The relative importance of each serogroup depends on geographic location, as well as other factors, such as age. The serogroup B meningococcus is responsible for the majority of cases of meningococcal disease in temperate countries.
- Meningococcal disease may manifest itself as either a systemic infection of the bloodstream (meningococcaemia) or an inflammation of the meninges of the brain and spinal cord (meningitis). Meningitis is the most common presentation of invasive meningococcal disease and results from hematogenous dissemination of the organism. Meningeal infection is similar to other forms of acute purulent meningitis, with sudden onset of fever, headache, and stiff neck, often accompanied by other symptoms, such as nausea, vomiting, photophobia (eye sensitivity to light), and altered mental status. Meningococci can be isolated from the blood in up to 75 % of persons with meningitis.
- Meningococcal sepsis occurs without meningitis in 5 % - 20 % of invasive meningococcal infections. Less common presentations of meningococcal disease include pneumonia (5 % - 15 % of cases), arthritis (2 %), otitis media (1 %), and epiglottitis ( ⁇ 1 %).
- the case fatality rate of invasive meningococcal disease is 9 % - 12 %, even with appropriate antibiotic therapy.
- the fatality rate of meningococcemia is up to 40 %. Up to 20 % of survivors have permanent sequelae, such as hearing loss, neurologic damage, or loss of a limb.
- Meningococcal disease occurs worldwide in both endemic and epidemic form. The reported annual incidence of meningococcal disease varies from 0.5 to 10 per 100,000 persons. However, during epidemics the incidence can rise above 400 per 100,000. The case fatality rate ranges from 5 % to 15 %, and up to 25 % of survivors are left with neurological sequelae. Recently, 2,600 and 5,600 cases were reported annually in the United States and Europe, respectively. Humans are the only natural reservoir of meningococcus. Up to 10 % of adolescents and adults are asymptomatic transient carriers of N. meningitidis, most of which are not pathogenic (i.e., strains that are not groupable). Primary transmission mode is by respiratory droplet spread or by direct contact. The communicability of N.
- meningitidis is generally limited. In studies of households in which a case of meningococcal disease has occurred, only 3 % - 4 % of households had secondary cases. Most households had only one secondary case. Estimates of the risk of secondary transmission are generally 2-4 per
- Serum bactericidal activity is considered to be the major protective immune mechanism and the most important parameter to be analysed when testing antigens to be used in vaccines.
- Meningococcal infection kills 50 % of untreated patients and 5 % - 15 % of patients who receive currently available treatments, according to the National Center for Genome Resources in the US. Since the vast majority of strains are susceptibly to Penicillin, it is the primary choice of treatment.
- a vaccine can contain a whole variety of different antigens.
- antigens are whole-killed or attenuated organisms, subfractions of these organisms/tissues, proteins, or, in their most simple form, peptides.
- Antigens can also be recognized by the immune system in form of glycosylated proteins or peptides and may also be or contain polysaccharides or lipids.
- Short peptides can be used since for example cytotoxic T-cells (CTL) recognize antigens in form of short usually 8 - 11 amino acids long peptides in conjunction with major histocompatibility complex (MHC).
- CTL cytotoxic T-cells
- MHC major histocompatibility complex
- B-cells can recognize linear epitopes as short as 4 - 5 amino acids, as well as three-dimensional structures (conformational epitopes).
- adjuvants need to trigger immune cascades that involve all cells of the immune system.
- adjuvants are acting, but are not restricted in their mode of action, on so-called antigen presenting cells (APCs). These cells usually first encounter the antigen(s) followed by presentation of processed or unmodified antigen to immune effector cells. Intermediate cell types may also be involved. Only effector cells with the appropriate specificity are activated in a productive immune response.
- the adjuvant may also locally retain antigens and co-injected other factors.
- the adjuvant may act as a chemoattractant for other immune cells or may act locally and/or systemically as a stimulating agent for the immune system.
- capsular polysaccharide as the basis of a vaccine for prevention of MenB diseases has been problematic.
- the MenB capsular polysaccharide is identical to a widely distributed human carbohydrate [a(238)7V- acetyl neuraminic acid or polysialic acid], which, being a self-antigen, is a poor immunogen in humans.
- use of this polysaccharide in a vaccine may elicit autoantibodies.
- An alternative approach to vaccine development is based on surface- exposed proteins contained in outer membrane vesicles (OMVs). These vaccines have been shown both to elicit serum bactericidal antibody responses and to protect against developing meningococcal disease.
- OMVs outer membrane vesicles
- the object has been solved by a peptide consisting of one antigen of N. meningitidis of any of the SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96
- the fragment is characterized by being derived from the antigen as defined above by one or more deletions.
- the deletion(s) may be C-terminally, N-terminally and/or internally.
- the fragment is obtained by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3,
- the functional active fragment of the invention is characterized by having a biological activity similar to that displayed by the complete protein, including the ability to exhibit reactivity with Men B specific antibodies, to mediate seroconversion and/or to show bactericidal activity.
- the fragment of an antigen is functional active in the context of the present invention, if the activity of the fragment amounts to at least 10 %, preferably at least 25 %, more preferably at least 50 %, even more preferably at least 70 %, still more preferably at least 80 %, especially at least 90 %, particularly at least 95 %, most preferably at least 99 % of the activity of the antigen without sequence alteration.
- These fragments may be designed or obtained in any desired length, including as small as about 5 to 8 amino acids in length.
- SEQ ID NOS: 1 to 184 are characterized in tables 3 to 5 of the present specification disclosing the underlying amino acid sequences and their immunologic properties.
- the peptides of SEQ ID NOS: 1 to 184 have been shown to exhibit reactivity with Men B specific antibodies, such as with human sera of convalescing Men B and optionally Men C patients or their family members (see Example 1 and 3).
- the antigenic peptide is also able to mediate seroconversion.
- Seroconversion in the context of the present invention refers to the development of detectable specific antibodies to the antigenic peptide in the serum as a result of infection or immunization. Examples of such antigenic peptides exhibiting strong seroconversion are those consisting of the SEQ ID NO: 1 to 55. Examples of antigenic peptides exhibiting weak seroconversion are those consisting of the SEQ ID NO: 56 to 94. Seroconversion may be assessed by calculating the fold increase of antibody levels in convalescent samples relative to that in acute sera (e.g. > 2 fold). Methods for determining antibody levels and seroconversion are known to the person skilled in the art and described below (see e.g. Example 3).
- the antigenic peptides show bactericidal activity.
- Bactericidal activity in the context with the present application refers to the ability of a peptide of inducing immunofunctional antibodies against N. meningitidis serogroup B. Bactericidal activity can be tested with epitope-induced hyperimmune mouse sera. Such a test for determining bactericidal activity is e.g. described in Example 4.
- the antigen of N. meningitidis can be any of the antigens as defined above, particularly as defined in any of the SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,
- the functional active fragment is as defined above.
- the additional amino acid residue(s) of (i) and/or (ii) may be any amino acid, which may be either an L-and/or a D-amino acid, naturally occurring and otherwise.
- the amino acid is any naturally occurring amino acid such as alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine.
- the amino acid may also be a modified or unusual amino acid.
- those are 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4- aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'- diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycinem N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproloine, 4-hydroxyproloine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, 6-N-Methyllysine, N-methylvaline, norvaline, norleucine or orni
- the amino acid may be subject to modifications such as posttranslational modifications. Examples of modifications include acetylation, amidation, blocking, formylation, ⁇ -carboxy glutamic acid hydroxylation, glycosilation, methylation, phosphorylation and sulfatation. If more than one additional or heterologous amino acid residue is present in the peptide, the amino acid residues may be the same or different from one another. In one embodiment the peptide of the invention further encompasses at least one amino acid residue heterologous to the antigen.
- heterologous amino acid or “amino acid heterologous to the antigen” refers to any amino acid which is different from that amino acid located adjacent to theantigen in any naturally occurring protein of N.
- the protein of the invention encompassing at least one heterologous amino acid refers to a protein which is different from any naturally occurring protein of N. meningitidis or fragment thereof, particularly which is different from that of N. meningitidis B, especially strain MC58.
- the full length genome of N. meningitidis B strain MC58 and the encoded proteins are disclosed e.g. in WO 00/022430.
- the peptide of the invention comprising additional amino acid residue(s) as defined above is characterized in that it comprises at least 2, preferably at least 3 antigens as defined above.
- the antigenic peptide may be flanked by the amino acid residue(s) as defined in (i) and/or (ii) C-terminally, ⁇ -terminally or C- and ⁇ -terminally.
- the peptide as defined above comprises at least one amino acid sequence of the SEQ ID NO: 185 to 194, 196 to 199, 202 to 205, 207, 209 to 211, 213, 215, 217 to 225, 227 to 234 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 196, 197, 198, 199, 202, 203, 204, 205, 207, 209, 210, 211, 213, 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 227, 228, 229, 230, 231, 232, 233 and/or 234.
- the functional active fragment may be further characterized by structural features. Accordingly, in one preferred embodiment of the invention the functional active fragments consists of at least 60 %, preferably at least 70 %, more preferably at least 80 %, still more preferably at least 90 %, even more preferably at least 95 %, most preferably 99 % of the antigenic peptide of any of the SEQ ID NOS: 1 to 184, more preferably of the antigenic peptide of any of the SEQ ID NOS: 1 to 94 or 110 to 184, still more preferably of the antigenic peptide of any of the SEQ ID NOS: 1 to 94 and most preferably of the antigenic peptide of any of the SEQ ID NOS: 1 to 55.
- the functional active fragment as defined above may be derived from the peptide by one or more amino acid deletions. The deletions may be C-terminally, N-terminally and/or internally.
- Another preferred embodiment of the invention relates to a peptide as defined above in the previous embodiments, wherein the antigen is a functional active variant of an antigen of any of the SEQ ID NOS: 1 to 184 and wherein the variant has at least 50 % sequence identity to the antigen of any of the SEQ ID NOS: 1 to 184, preferably of the SEQ ID NOS: 1 to 94 or 110 to 184, more preferably of the SEQ ID NOS: 1 to 55.
- the functional active variant has a sequence identity of at least 60 %, preferably at least 70 %, more preferably at least 80 %, still more preferably at least 90 %, even more preferably at least 95 %, most preferably 99 % to the antigen of any of the SEQ ID NOS: 1 to 184, preferably of the SEQ ID NOS: 1 to 94 or 110 to 184, more preferably ofthe SEQ ID NOS: l to 55.
- the percentage of sequence identity can be determined e.g. by sequence alignment. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms have been described e.g. in Smith and Waterman, Adv. Appl. Math. 2: 482, 1981 or Pearson and Lipman, Proc. Natl. Acad. Sci.US. A. 85: 2444, 1988.
- NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. MoI. Biol. 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Variants of an antigen of any of the sequences of SEQ ID NOS: 1 to 184 are typically characterized using the NCBI Blast 2.0, gapped blastp set to default parameters.
- NCBI National Center for Biotechnology Information
- the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
- the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set t default parameters (open gap 9, extension gap 1 penalties).
- the functional active variant of an antigen is obtain by sequence alterations in the antigen, wherein the antigen with the sequence alterations retains a function of the unaltered antigen, such as it specifically binds a Men B specific antibody that binds an unaltered form of the antigen.
- sequence alterations can include, but are not limited to, conservative substitutions, deletions, mutations and insertions.
- the functional active variant exhibits reactivity with human sera of convalescing Men B patients, more preferably mediates seroconversion and most preferably shows bactericidial activity. These characteristics of the functional active variant can be assessed e.g. as detailed in Examples 1 , 3 and 4.
- a variant specifically binds a Men B specific antibody, exhibits reactivity with human sera of convalescing Men B patients, mediates seroconversion or shows bactericidial activity, if the activity of the variant amounts to at least 10 %, preferably at least 25 %, more preferably at least 50 %, even more preferably at least 70 %, still more preferably at least 80 %, especially at least 90 %, particulary at least 95 %, most preferably at least 99 % of the activity of the antigen without sequence alterations.
- allelic variant includes naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants.
- an allelic variant is an alternate form of a (poly)peptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does essentially not alter the biological function of the polypeptide.
- biological function is meant a function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells.
- the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
- the biological function is distinct from the antigenic function.
- a polypeptide can have more than one biological function.
- allelic variation is the rule.
- any bacterial species e.g. N. meningitidis
- a polypeptide that fulfils the same biological function in different strains can have an amino acid sequence that is not identical in each of the strains.
- Such an allelic variation is equally reflected at the polynucleotide level.
- allelic variation is very common within the meningitides species.
- allelic diversity of the two transferring-binding protein B gene isotypes among a collection of N. meningitidis strains representative of serogroup B has been studied by Rokbi B and co workers (Rokbi B et al., 2000, Infection and Immunity 68: 4938-4947).
- the functional active variant or fragment derived from the antigen by amino acid exchanges, deletions or insertions may also conserve, or more preferably improve, the activity (reactivity, seroconversion and/or bactericidal activity as defined above).
- these peptides may also cover epitopes, which trigger the same or preferably an improved T cell response. These epitope are referred to as "heteroclitic". They have a similar or preferably greater affinity to MHC/HLA molecules, and the ability to stimulate the T cell receptors (TCR) directed to the original epitope in a similar or preferably stronger manner. Heteroclitic epitopes can be obtained by rational design i. e.
- Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc..
- one conservative substitution is included in the peptide.
- two conservative substitutions or less are included in the peptide.
- three conservative substitutions or less are included in the peptide.
- conservative amino acid substitutions include, but are not limited to, those listed below:
- the peptide as defined above may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity (as defined above for fragments and variants) as the modified peptides, and optionally having other desirable properties.
- carboxylic acid groups of the protein whether C-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form an ester, or converted to an amide.
- Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be converted to an amide. Hydroxyl groups of the peptide side chains may be converted to alkoxy or to an ester using well recognized techniques.
- Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with alkyl, alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
- halogen atoms such as fluorine, chlorine, bromine or iodine
- alkyl, alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids amides of such carboxylic acids.
- Thiols can be protected with any one of a number of well recognized protecting groups, such as acetamide groups.
- Peptides of this invention may be in combination with outer surface proteins or other proteins or antigens of other proteins.
- the antigen may be in the form of a fusion protein.
- the antigen of the invention may be optionally fused to a selected polypeptide or protein derived from other microorganisms.
- an antigen or polypeptide of this invention may be fused at its N-terminus or C-terminus to a polypeptide from another pathogen or to more than one polypeptide in sequence.
- Polypeptides which may be useful for this purpose include polypeptides identified by the prior art.
- the peptide of the invention is fused to an epitope tag which provides an epitope to which an anti-tag substance can selectively bind.
- the epitope tag is generally placed at the amino- or carboxyl-terminus of the peptide but may be incorporated as an internal insertion or substitution as the biological activity permits.
- the presence of such epitope-tagged forms of a peptide can be detected using a substance such as an antibody against the tagged peptide.
- provision of the epitope tag enables the peptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
- Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his), poly-histidine-glycine (poly-his-gly) tags, the HA tag polypeptide, the c-myc tag, the Strep tag and the FLAG tag.
- Fusions also may include the peptides or antigens of this invention fused or coupled to moieties other than amino acids, including lipids and carbohydrates.
- antigens of this invention may be employed in combination with other vaccinal agents described by the prior art, as well as with other species of vaccinal agents derived from other microorganisms. Such proteins are useful in the prevention, treatment and diagnosis of diseases caused by a wide spectrum of Neisseriae isolates.
- fusion proteins are constructed for use in the methods and compositions of this invention. These fusion proteins or multimeric proteins may be produced recombinantly, or may be synthesized chemically.
- the peptides of the invention may be prepared by any of a number of conventional techniques. Desired peptides may be chemically synthesized.
- An alternative approach involves generating the fragments of known peptides by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes, expressing the digested DNA and isolating the desired fragment.
- Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired peptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed as the 5' and 3' primers in the PCR.
- cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)), PCR mutagenesis, or other known techniques can be performed on the cloned DNA to produce the peptide of the invention.
- Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA e.g. obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
- the DNA may be triple- stranded, double- stranded or single-stranded.
- Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
- Nucleic acid molecule as used herein also refers to, among other, single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded, or triple-stranded, or a mixture of single- and double-stranded regions.
- nucleic acid molecule as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the nucleic acid may be a fragment of a nucleic acid occurring naturally in N. meningitidis, particularly in N. meningitidis B, especially in N. meningitidis B strain MC58.
- the nucleic acid also includes sequences that are a result of the degeneration of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all nucleotide sequences are included in the invention which result in the peptide as defined above.
- nucleic acid may contain one or more modified bases.
- Such nucleic acids may also contain modifications e.g. in the ribose-phosphate backbone to increase stability and half life of such molecules in physiological environments.
- DNAs or RNAs with backbones modified for stability or for other reasons are "nucleic acid molecule" as that feature is intended herein.
- DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are nucleic acid molecule within the context of the present invention. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
- nucleic acid molecule as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of nucleic acid molecule, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
- nucleotide substitutions can be made which do not affect the polypeptide encoded by the nucleic acid, and thus any nucleic acid molecule which encodes an antigen or fragment or functional active variant thereof as defined above is encompassed by the present invention.
- any of the nucleic acid molecules encoding an antigen of the invention or fragment or functional active variant thereof can be functionally linked, using standard techniques such as standard cloning techniques, to any desired regulatory sequences, whether a N. meningitidis regulatory sequence or a heterologous regulatory sequence, heterologous leader sequence, heterologous marker sequence or a heterologous coding sequence to create a fusion protein.
- the nucleic acid of the invention may be originally formed in vitro or in a cell in culture, in general, by the manipulation of nucleic acids by endonucleases and/or exonucleases and/or polymerases and/or ligases and/or recombinases or other methods known to the skilled practitioner to produce the nucleic acids.
- the nucleic acid is located in a vector.
- a vector may additionally include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication, one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art such as regulatory elements directing transcription, translation and/or secretion of the encoded protein.
- the vector may be used to transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell.
- the vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like. Numerous types of appropriate expression vectors are known in the art for protein expression, by standard molecular biology techniques.
- Such vectors are selected from among conventional vector types including insects, e.g., baculovirus expression, or yeast, fungal, bacterial or viral expression systems.
- Other appropriate expression vectors of which numerous types are known in the art, can also be used for this purpose. Methods for obtaining such expression vectors are well-known (see, e.g. Sambrook et al, Molecular Cloning. A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory, New York (1989)).
- the vector is a viral vector.
- Viral vectors include, but are not limited to, retroviral and adenoviral vectors.
- Suitable host cells or cell lines for transfection by this method include bacterial cells.
- E. coli the various strains of E. coli are well-known as host cells in the field of biotechnology.
- Various strains of B. subtilis, Pseudomonas, Streptomyces, and other bacilli and the like may also be employed in this method.
- Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the peptides of the present invention.
- Other fungal cells or insect cells such as Spodoptera frugipedera (Sf9) cells may also be employed as expression systems.
- mammalian cells such as human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-I cell line or murine 3T3 cells derived from Swiss, BALB/c or NIH mice may be used.
- CHO Chinese hamster ovary cells
- NIH mice NIH mice
- Still other suitable host cells, as well as methods for transfection, culture, amplification, screening, production, and purification are known in the art.
- a peptide of the invention may be produced by expressing a nucleic acid of the invention in a suitable host cell.
- the host cells can be transfected, e.g. by conventional means such as electroporation with at least one expression vector containing a nucleic acid of the invention under the control of a transcriptional regulatory sequence.
- the transfected or transformed host cell is then cultured under conditions that allow expression of the protein.
- the expressed protein is recovered, isolated, and optionally purified from the cell (or from the culture medium, if expressed extracellularly) by appropriate means known to one of skill in the art.
- the proteins are isolated in soluble form following cell lysis, or extracted using known techniques, e.g. in guanidine chloride.
- the peptides or fragments of the invention are produced as a fusion protein.
- fusion proteins are those described above.
- the molecules comprising the peptides and antigens of this invention may be further purified using any of a variety of conventional methods including, but not limited to: liquid chromatography such as normal or reversed phase, using HPLC, FPLC and the like; affinity chromatography (such as with inorganic ligands or monoclonal antibodies); size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; and the like.
- affinity chromatography such as with inorganic ligands or monoclonal antibodies
- size exclusion chromatography such as with inorganic ligands or monoclonal antibodies
- size exclusion chromatography such as with inorganic ligands or monoclonal antibodies
- size exclusion chromatography such as with inorgan
- Such purification provides the antigen in a form substantially free from other proteinaceous and non-proteinaceous materials of the microorganism.
- Another subject of the invention is a pharmaceutical composition, especially a vaccine, comprising at least one peptide according to the invention; and/or at least one polypeptide comprising a peptide according to the invention.
- a peptide or polypeptide of the invention may be used for methods for immunizing or treating humans and/or animals with the disease caused by infection with N. meningitidis, particularly serogroup B. Therefore, the (poly)peptide may be used within a pharmaceutical composition.
- the pharmaceutical composition of the present invention may further encompass pharmaceutically acceptable carriers and/or excipients.
- the pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the (poly)peptides herein disclosed.
- parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
- pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
- physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
- solid compositions e. g. powder, pill, tablet, or capsule forms
- conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
- compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
- non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
- the pharmaceutical composition further comprises an immunostimulatory substance such as an adjuvant.
- the adjuvant can be selected based on the method of administration and may include mineral oil-based adjuvants such as Freund's complete and incomplete adjuvant, Montanide incomplete Seppic adjuvant such as ISA, oil in water emulsion adjuvants such as the Ribi adjuvant system, syntax adjuvant formulation containingmuramyl dipeptide, or aluminum salt adjuvants.
- the adjuvant is a mineral oil-based adjuvant, most preferably ISA206 (SEPPIC, Paris, France).
- the immunostimulatory substance is selected from the group comprising polycationic polymers, especially polycationic peptides such as polyarginine, immunostimulatory deoxynucleotides (ODNs), peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, neuroactive compounds, especially human growth hormone, alumn, adjuvants or combinations thereof.
- polycationic polymers especially polycationic peptides such as polyarginine, immunostimulatory deoxynucleotides (ODNs), peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, neuroactive compounds, especially human growth hormone, alumn, adjuvants or combinations thereof.
- the combination is either a polycationic polymer and immunostimulatory deoxynucleotides or of a peptide containing at least two LysLeuLys motifs and immunostimulatory deoxynucleotides.
- the immunostimulatory substance is at least one immunostimulatory nucleic acid.
- Immunostimulatory nucleic acids are e.g. neutral or artificial CpG containing nucleic acids, short stretches of nucleic acids derived from non-vertebrates or in form of short oligonucleotides (ODNs) containing non- methylated cytosine-guanine dinucleotides (CpG) in a defined base context (e.g. as described in WO 96/02555).
- ODNs long oligonucleotides
- CpG non- methylated cytosine-guanine dinucleotides
- nucleic acids based on inosine and cytidine as e.g.
- deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues may preferably be used as immunostimulatory nucleic acids in the present invention.
- mixtures of different immunostimulatory nucleic acids are used in the present invention.
- the aforementioned polycationic compounds may be combined with any of the immunostimulatory nucleic acids as aforementioned.
- such combinations are according to the ones described in WO 01/93905, WO 02/32451, WO 01/54720, WO 01/93903, WO 02/13857 and WO 02/095027 and the Australian patent application A 1924/2001.
- such vaccine composition may comprise a neuroactive compound.
- the neuroactive compound is human growth factor, e.g. described in WO 01/24822.
- the neuroactive compound is combined with any of the polycationic compounds and/or immunostimulatory nucleic acids as defined above.
- the composition may be used e.g. for immunization or treatment of a subject.
- the pharmaceutical composition encompasses at least one peptide of the invention; however, it may also contain a cocktail (i.e., a simple mixture) containing different peptides (including fragments and variants) or polypeptides of the invention, optionally mixed with different antigenic proteins or peptides of other pathogens. Such mixtures of these peptides, polypeptides, proteins or fragments or variants thereof are useful e.g. in the generation of desired antibodies to a wide spectrum of Neisseriae isolates.
- the (poly)peptide(s) of the present invention may also be used in the form of a pharmaceutically acceptable salt. Suitable acids and bases which are capable of forming salts with the peptides of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases.
- Still another subject of the invention is a pharmaceutical composition containing a nucleic acid selected from the group consisting of:
- nucleic acid sequences may further be used as components of a pharmaceutical composition.
- the composition may be used for immunizing or treating humans and/or animals with the disease caused by infection with N. meningitidis, particularly serogroup B.
- nucleic acid sequences of this invention may further be used in compositions directed to actively induce a protective immune response in a subject to the pathogen.
- These components of the present invention are useful in methods for inducing a protective immune response in humans and/or animals against infection with N. meningitidis, particularly serogroup B.
- nucleic acid delivery compositions and methods are useful, which are known to those of skill in the art.
- the nucleic acid of (a), (b) or (c) may be employed in the methods of this invention or in the compositions described herein as DNA sequences, either administered as naked DNA, or associated with a pharmaceutically acceptable carrier and provide for in vivo expression of the antigen, peptide or polypeptide.
- So-called “naked DNA” may be used to express the antigen, peptide or polypeptide of the invention in vivo in a patient. (See, e.g., J. Cohen, Science, 259:1691-1692, which describes similar uses of "naked DNA”).
- naked DNA associated with regulatory sequences may be administered therapeutically or as part of the vaccine composition e.g., by injection.
- nucleic acid encoding the antigens or peptides or polypeptides of the invention or a nucleic acid complementary thereto may be used within a pharmaceutical composition, e.g. in order to express the antigens or peptides or polypeptides of the invention in vivo, e.g., to induce antibodies.
- a preferred embodiment of the invention relates to a pharmaceutical composition, wherein the nucleic acid according to (a), (b) or (c) as defined above is comprised in a vector and/or a cell.
- Vectors and cells suitable in the context of the present invention are described above. Vectors are particularly employed for a DNA vaccine.
- An appropriate vector for delivery may be readily selected by one of skill in the art.
- Exemplary vectors for in vivo gene delivery are readily available from a variety of academic and commercial sources, and include, e.g., adeno-associated virus (International patent application No. PCT/US91/03440), adenovirus vectors (M. Kay et al, Proc. Natl. Acad. Sci. USA, 91 :2353 (1994); S.
- viral vectors e.g., various poxviruses, vaccinia, etc.
- Recombinant viral vectors such as retroviruses or adenoviruses, are preferred for integrating the exogenous DNA into the chromosome of the cell.
- Another subject of the invention relates to an antibody or functional active fragment thereof which binds specifically to the antigen of the invention.
- the present invention includes, for example, monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library.
- the antibody is a monoclonal, polyclonal, chimeric or humanized antibody or functional active fragment thereof.
- the functional active fragment comprises a Fab fragment.
- Antibodies generated against the antigens, fragments or variants thereof of the present invention can be obtained by direct injection of the antigens, fragments or variants thereof into an animal or by administering the antigens, fragments or variants thereof to an animal, preferably a non-human. The antibody so obtained will then bind the antigens, fragments or variants. Such antibodies can then be used to isolate reactive antigens, fragments or variants thereof from tissue expressing those.
- any technique known in the art which provides antibodies produced by continuous cell line cultures, e.g. a hybridoma cell line, can be used.
- Still another subject of the invention relates to a hybridoma cell line which produces the antibody of the invention.
- Hybridoma cell lines expressing desirable monoclonal antibodies are generated by well- known conventional techniques.
- the hybridoma cell can be generated by fusing a normal- activated, antibody-producing B cell with a myeloma cell.
- the hybridoma cell is able to produce an antibody specifically binding to the antigen of the invention.
- desirable high titer antibodies are generated by applying known recombinant techniques to the monoclonal or polyclonal antibodies developed to these antigens (see, e.g., PCT Patent Application No. PCT/GB85/00392; British Patent Application Publication No. GB2188638A; Amit et al., Science, 233:747-753 (1986); Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-100'33 (1989); PCT Patent Application No. WO90/07861; Riechmann et al., Nature, 332:323-327 (1988); Huse et al., Science, 246:1275-1281 (1988)).
- the present invention also provides a method for producing an antibody according to the invention, characterized by the following steps:
- step (a) administering an effective amount of the peptide according to the invention to an animal; and (b) isolating the antibody produced by the animal in response to the administration of step (a) from the animal.
- Another subject of the invention relates to a method for producing an antibody according to the invention, characterized by the following steps: (a) contacting a B cell with an effective amount of the peptide according to the invention;
- step (b) fusing the B cell of step (a) with a myeloma cell to obtain a hybridoma cell; and (c) isolating the antibody produced by the cultivated hybridoma cell.
- the antibody may be produced by initiating an immune response in a non-human animal by administrating a peptide or polypeptide of the invention to an animal, removing an antibody containing body fluid from said animal, and producing the antibody by subjecting said antibody containing body fluid to further purification steps.
- the antibody may be produced by initiating an immune response in a non- human animal by administrating an antigen, fragment or variant thereof, as defined in the present invention, to said animal, removing the spleen or spleen cells from said animal and/or producing hybridoma cells of said spleen or spleen cells, selecting and cloning hybridoma cells specific for said antigen, fragment or variant thereof and producing the antibody by cultivation of said cloned hybridoma cells.
- the antibody produced according to a method of the invention is additionally purified. Methods of purification are known to the skilled artisan.
- the antibody may be used in methods for treating an infection.
- a pharmaceutical composition especially a vaccine, comprising the antibody of the invention.
- the pharmaceutical composition may encompass further components as detailed above.
- the composition may further encompass substances increasing their capacity to stimulate T cells. These include T helper cell epitopes, lipids or liposomes or preferred modifications as described in WO01/78767.
- Another way to increase the T cell stimulating capacity of epitopes is their formulation with immune stimulating substances for instance cytokines or chemokines like interleukin-2, -7, -12, -18, class I and II interferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligand and others.
- IFN interleukin-2, -7, -12, -18, class I and II interferons
- the peptide, the polypeptide or the nucleic acid of the invention is used for the manufacture of a medicament for the immunization of a subject, preferably against N. meningitidis, more preferably against N. meningitidis serogroup B.
- the peptides, polypeptides or the nucleic acids of the invention are generally useful for inducing an immune response in a subject.
- the vaccine used for immunization may be administered to a subject susceptible to infection by N. meningitidis, preferably mammals, and still more preferably humans, in any conventional manner, including oral, intranasal, intramuscular, intra-lymph node, intradermal, intraperitoneal, subcutaneous, and combinations thereof, but most preferably through intramuscular injection.
- the volume of the dose for intramuscular administration is preferably up to about 5 ml, still more preferably between 1 ml and 3 ml, and most preferably about 2 ml.
- the volume of the dose when intramuscular injection is the selected administration route is preferably up to about 5 ml, still more preferably between 1 ml and 3 ml, and most preferably about 2 ml.
- the amount of protein comprising the antigen in each dose should be enough to confer effective immunity against and decrease the risk of developing clinical signs resulting from N. meningitidis infection to a subject receiving a vaccination therewith.
- the unit dose of protein should be up to about 5 ⁇ g protein/kg body weight, more preferably between about 0.2 to 3 ⁇ g, still more preferably between about 0.3 to 1.5 ⁇ g, more preferably between about 0.4 to 0.8 ⁇ g, and still more preferably about 0.6 ⁇ g.
- Alternative preferred unit doses of protein could be up to about 6 ⁇ g protein/kg body weight, more preferably between about 0.05 to 5 ⁇ g, still more preferably between about 0.1 to 4 ⁇ g.
- the dose is preferably administered 1 to 3 times, e.g. with an interval of 1 to 3 weeks.
- Preferred amounts of protein per dose are from approximately 1 ⁇ g to approximately 1 mg, more preferably from approximately 5 ⁇ g to approximately 500 ⁇ g, still more preferably from approximately 10 ⁇ g to approximately 250 ⁇ g and most preferably from approximately 25 ⁇ g to approximately 100 ⁇ g.
- the antibody of the invention or functional fragment thereof is used for the manufacture of a medicament for the treatment of an infection, preferably a N. meningitidis infection, more preferably a N. meningitidis serogroup B infection.
- the treatment involves administering an effective amount of an antibody of the invention to a subject, preferably a mammal, more preferably a human.
- antibodies against the antigens, fragments or variants thereof of the present invention may be employed to inhibit and/or treat infections, particularly bacterial infections and especially infections arising from N. meningitidis, especially serogroup B.
- An "effective amount" of a nucleic acid composition may be calculated as that amount capable of exhibiting an in vivo effect, e.g.
- Such amounts may be determined by one of skill in the art.
- a composition is administered parenterally, preferably intramuscularly or subcutaneously.
- it may also be formulated to be administered by any other suitable route, including orally or topically. The selection of the route of delivery and dosage of such therapeutic compositions is within the skill of the art.
- Treatment in the context of the present invention refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
- Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
- Another subject of the invention relates to a method of diagnosing a N. meningitidis infection comprising the steps of:
- the peptides or polypeptides of the invention may be used for the detection of the N. meningitidis, particularly serogroup B. Preferably such detection is for diagnosis, more preferable for the diagnosis of a disease, most preferably for the diagnosis of a N. meningitidis infection.
- the peptides or polypeptides may be used to detect the presence of a N. meningitidis-specific antibody or fragment thereof e.g. in a sample obtained from a subject.
- the sample may be e.g. a blood sample.
- the presence of a N. meningitidis-specific antigen can be detected using an antibody of the invention.
- an alternative method of diagnosing a N. meningitidis infection comprises the steps of: (a) contacting a sample obtained from a subject with the antibody according to the invention; and
- the present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of the peptides or antibodies of the present invention in cells and tissues or body fluids, including determination of normal and abnormal levels.
- diagnostic assays such as quantitative and diagnostic assays for detecting levels of the peptides or antibodies of the present invention in cells and tissues or body fluids, including determination of normal and abnormal levels.
- Assay techniques that can be used to determine levels of a peptide or an antibody, in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these, ELISAs frequently are preferred.
- An ELISA assay initially comprises preparing an antibody specific to the peptide, particularly the antigen, preferably a monoclonal antibody.
- a reporter antibody generally is prepared which binds to the monoclonal antibody.
- the reporter antibody is attached to
- the peptides or antibodies of the present invention may also be used for the purpose of or in connection with an array. More particularly, at least one of the peptides or antibodies of the present invention may be immobilized on a support.
- Said support typically comprises a variety of antigens and fragments thereof whereby the variety may be created by using one or several of the peptides or antibodies of the present invention.
- the characterizing feature of such array as well as of any array in general is the fact that at a distinct or predefined region or position on said support or a surface thereof, a distinct polypeptide is immobilized. Because of this any activity at a distinct position or region of an array can be correlated with a specific polypeptide.
- the number of different peptides or antibodies of the present invention immobilized on a support may range from as little as 10 to several 1000 different peptides or antibodies of the present invention.
- the manufacture of such arrays is known to the one skilled in the art and, for example, described in US patent 5,744,309.
- the array preferably comprises a planar, porous or non- porous solid support having at least a first surface.
- Preferred support materials are, among others, glass or cellulose. It is also within the present invention that the array is used for any of the diagnostic applications described herein.
- the nucleic acid molecules according to the present invention may be used for the generation of an array as described above.
- Another aspect of the invention relates to a method for identifying a ligand capable of binding to a peptide according to the invention comprising:
- the method may be carried out by contacting an isolated or immobilized peptide according to the invention with a candidate ligand under conditions to permit binding of the candidate ligand to the peptide, wherein the test system comprises a component capable of providing a detectable signal in response to the binding of the candidate ligand to said peptide; and detecting the presence or absence of a signal generated in response to the binding of the ligand to the peptide.
- the ligand may be a agonist or an antagonist.
- Test systems for detection binding of a ligand include e.g. binding assays with labeled ligand such as radioligands, fluorescence-labeled ligands or enzyme-labeled ligands.
- labeled ligand such as radioligands, fluorescence-labeled ligands or enzyme-labeled ligands.
- test compound can be any test compound either naturally occurring or chemically synthesized.
- Naturally occurring test compounds include in particular antibodies, preferably those showing similarity to the antibodies of the invention.
- the test compound is provided in the form of a chemical compound library.
- Chemical compound libraries include a plurality of chemical compounds and have been assembled from any of multiple sources, including chemically synthesized molecules and natural products, or have been generated by combinatorial chemistry techniques. They are especially suitable for high throughput screening. They may be comprised of chemical compounds of a particular structure or compounds of a particular creature such as a plant.
- the method for identifying a ligand may also include the following steps: (a) providing a peptide according to the invention,
- test compound inhibits or reduces the interaction activities of the peptide with the interaction partner.
- the ligands identified may be employed, for instance, to inhibit diseases arising from infection with Neisseria, especially N. meningitidis and may therefore be formulated in a pharmaceutical composition.
- the peptide according to the invention is used for the isolation and/or purification and/or identification of a ligand of the peptide, wherein the isolation and/or purification and/or identification of the ligand may be carried out as detailed above or as known to the person skilled in the art.
- an affinity device may be used.
- the affinity device may comprise as least a support material and any peptide according to the present invention, which is attached to the support material. Because of the specificity of the peptides according to the present invention for their target cells or target molecules or their interaction partners, the peptides allow a selective removal of their interaction partner(s) from any kind of sample applied to the support material provided that the conditions for binding are met.
- the sample may be a biological or medical sample, including but not limited to, fermentation broth, cell debris, cell preparation, tissue preparation, organ preparation, blood, urine, lymph liquid, liquor and the like.
- the peptide may be attached to the matrix in a covalent or non-covalent manner.
- Suitable support material is known to the one skilled in the art and can be selected from the group comprising cellulose, silicon, glass, aluminium, paramagnetic beads, starch and dextrane.
- Figure 1 shows the measurement of anti-meningococcal antibody levels and the determination of seroconversion, shows immunoblotting with human sera to detect antigenic proteins of Neisseria meningitides and determination of serum bactericidal activity (SBA) of human sera.
- SBA serum bactericidal activity
- FIG. 1 shows the generation of genomic libraries.
- Figure 3 shows the seroconversion of peptides tested with patient sera.
- Figures 4 and 5 show the detection of epitope-specific antibodies in sera of E. coli lysate immunized mice.
- Figure 6 shows the Western blot analysis for detection of MenB proteins.
- Figure 7 shows the bactericidal activity of mouse sera.
- Table 1 shows the list of serum samples obtained for antigen characterization.
- Table 2 shows the summary for the characterization of selected serum samples.
- Table 3 shows the antigens identified by bacterial surface display screens.
- Table 4 shows characteristics of MenB peptides.
- Figure 1 A shows the measurement of anti-mcningococcal antibody levels and determination of seroconversion.
- ELISA plates were coated with 10 ⁇ g/ml total protein present in bacterial lysates prepared from Neisseria meningitidis serogroup B. Standard ELISA was performed with different serum dilutions and bound IgG was detected with anti -human IgG secondary antibody. A representative experiment is shown at 10,000 X serum dilution, comparing IgG reactivities of convalescent and acute phase sera.
- Figure 1 B shows the immunoblotting with human sera to detect antigenic proteins of Nesseria meningitidis.
- Total bacterial lysate was prepared by a freeze-thaw method, proteins separated by SDS-PAGE and transferred to nitrocellulose membrane which was probed with human serum samples at a 5,000 X dilution and mouse anti-human IgG as a secondary antibody.
- Figure 1 C shows the determination of serum bactericidal activity (SBA) of human sera.
- SBA serum bactericidal activity
- Figure 2 shows genomic libraries. Size distribution of 387 randomly picked clones of NMB300 and of 409 randomly picked clones of NMB50. Numbers in brackets indicate the expected and the actual, average insert size.
- Figure 3 shows seroconversion of peptides tested with patient sera. Seroconversion data of 2 exemplary peptides and of MenB lysate are shown for 7 serum pairs from patients. Whereas the bacterial lysate (bottom) and peptide NMB 1523.5 (top; +++++) show seroconversion, NMB 2061.2 (middle; -) is negative in this test.
- Figures 4 and 5 show detection of epitope-specific antibodies in sera of E. coli lysate immunized mice.
- Fig. 4 Western blot analysis was performed on lysates that were prepared from E. coli clones selected in BSD screens and used for immunization of mice to generate MenB antigen epitope specific antibodies. The presence of specific antibodies was noticed by the immunodetection of the epitope-carrying platform proteins, LamB or FhuA.
- Fig. 5 Peptide ELISA was performed with synthetic peptides representing displayed antigenic epitopes.
- NMB numbers represent animal sera (.1 to .5; 5 individual mice) and NMB numbers prior to data columns represent peptides as listed in Table 3.
- LamB and FhuA sera represent negative controls, induced by empty platform protein expressing E. coli.
- Figure 6 shows Western blot analysis for detection of MenB proteins with epitope- induced mouse sera.
- Total bacterial lysate was prepared from the MC58 strain and proteins were analysed by Western blotting with 5,000 X diluted mouse sera generated with immunization of epitope carrying E. coli clones.
- Figure 7 shows the bactericidal activity of mouse sera containing MenB epitope specific antibodies. Sera that induced >20 % bactericidal activity were considered positive, and were quantified based on the percentage of reduction in bacterial growth. Sera were classified as having pronounced bactericidal activity (>40 %), intermediate (30 - 40 %) or low (20 - 30 %) activity, respectively. Importantly, the bactericidal activity was IgG concentration dependent and the assay was highly reproducible.
- Table 1 Serum samples for antigen characterization.
- Serum samples were obtained from 215 donors. Column 3 referred to as "pathogen", indicates which pathogen was identified inducing disease. Column 4 shows the number of paired sera obtained from the same donor in acute and convalescent phase. N. a., not available.
- Table 2 Summary of immune characterization of selected serum samples. Sera were analysed for serum bactericidal activity (SBA) and seroconversion (SC). SBA activity is given as highest dilution of serum with bactericidal activity.
- ELISA U specifies reactivities of sera with MenB lysate. Seroconversion is given as fold increase comparing acute and convalescent phase sera.
- SE2A, SE2B, SE2C bactericidal activity of sera against MenA, B and C, respectively, expressed as highest dilution of serum with positive reaction.
- the serum numbers labeled by a * refer to the sera that were also used for peptide ELISA as summarized in Table 3.
- Peptides derived from MenB antigens were analysed with human sera for immunogenicity and seroconversion by peptide ELISA using biotinylated peptides.
- the peptide name entails the NMB designation according to the annotation of the MC58 MenB strain. The sequences and the SEQ ID NOS are listed.
- Table 4 characteristics of MenB peptides.
- the table lists all bacterial clones which were injected into mice for the generation of antibodies, including their amino acid sequence and the peptides which were synthesized according to the identified immunogenic epitope. All bacterial clones relating to one ORF (first column) were injected simultaneously. Please note that different BA experiments are separated by a horizontal line. Data obtained with the such generated sera (clone WB - Western blot with E. coli clone, WB - Western blot with MenB grown in RPMI (1) or BHI (2), BA - bactericidal activity) therefore relate to single or multiple clones. Data obtained for peptides relate always to the individual peptide (peptide ELISA, SC - seroconversion). Table 5: Summary of the characteristics of synthetic peptides and the respective clones.
- the fragments of the SEQ ID NOS: 1 to 184 were classified into 9 groups (groups I to IX) taking their bactericidal activity data and the seroconversion data of the respective clone(s) into account.
- Example 1 Selection of serum sources for the identification and characterization of MenB antigens
- Neisseria meningitidis serogroup B MC58 strain was grown in BHI medium. Overnight cultures were diluted in BHI 1 :50 and further grown until an OD 600 nm of approximately 0.6 for logarithmic phase and up to OD 600 nm of approximately 2.5 for stationary phase cells.
- Bacteria were harvested by centrifugation, re-suspended in PBS, and the bacterial suspension was freeze-thawed (incubation at -80°C followed by exposure to room temperature) and sonicated for 2 min. Samples were centrifuged and the supernatant fraction recovered and stored in aliquotes at -80 0 C. Lysate preparations were used for coating ELISA plates (10 ⁇ g/ml), for Western blot analysis (10-20 ⁇ g total protein), as well as for the induction of hyperimmune mouse sera ( ⁇ 300 ⁇ g).
- Enzyme-linked immunosorbent assay (ELISA).
- ELISA plates (Maxisorb, Millipore) were coated with 5-10 ⁇ g/ml total protein diluted in coating buffer (0.1 M sodium carbonate pH 9.2). Three dilutions of sera (2,000 X, 10,000 X, 50,000 X) were made in PBS-BSA. Highly specific Horse Radish Peroxidase (HRP)- conjugated anti-human IgG secondary antibodies (Southern Biotech) were used according to the manufacturers' recommendations (dilution: 1,000 X). Antigen-antibody complexes were quantified by measuring the conversion of the substrate (ABTS) to colored product based on OD 405 nm readings by automatic ELISA reader (TECAN SUNRISE).
- HRP Horse Radish Peroxidase
- Bacterial cells were grown overnight and were collected by centrifugation and re- suspended in SDS-sample buffer for crude, whole cell extracts. Approximately 10 to 20 ⁇ g of total cellular protein was separated by 10% SDS-PAGE and blotted onto HybondC membrane (Amersham Pharmacia Biotech, England) using a semi-dry transfer system (Bio-Rad) and visualized by Ponceau S staining. After blocking over night in 5% milk, human sera or mouse immune sera were added at 5,000-10,00Ox dilution, and HRP- labelled goat anti-mouse IgG at a dilution of 1 :5,000 (Southern Biotech) were used for specific detection. Detection was performed using the ECL detection kit (Amersham Pharmacia Biotech, England).
- Neisseria meningitidis serogroup B strain MC58 was grown on agar plates prepared with Mueller-Hinton broth supplemented with 10% fetal bovine serum (MHB-FBS, Sigma Cat.Nr.: M9677) over night at 37°C/5% CO 2 . A single colony was picked and growth repeated again. Bacteria were harvested by scraping the agar plates, washed in PBS and diluted in HBSS-B (Hanks' balanced salt solution with 0.1% BSA) to a concentration of 800 CFU per 100 ⁇ l based on OD 600nm readings of bacterial suspensions.
- HBSS-B Hors' balanced salt solution with 0.1% BSA
- the antibodies induced by pathogens and present in serum and other body fluids constitute a molecular imprint of the in vivo expression of the corresponding proteins comprising antigens and have therefore been chosen in our approach as tools for the identification of a comprehensive set of antigens.
- Serum samples from individuals (children and adults) diseased by or exposed to Neisseria meningitidis were collected as relevant sources of antibodies for antigen identification.
- the diagnosis of meningococcal disease was based on the isolation of the pathogen from the patients' blood and serogrouping was performed according to routine clinical microbiological methods. From the majority of patients with meningitis two samples were obtained, the first in the acute phase (at the time of admission to the hospital) and the second 2 weeks later during convalescence. Single serum samples from patients were also obtained during recovery. Sera were also collected from contact persons (household members) exposed to the pathogen that diseased their family member. At the time of sampling, nasopharyngeal carriage of Neisseria meningitidis was also determined. The 290 serum samples were collected from 215 different donors and stored at -80°C until analysis (summarized in Table 1).
- Genomic DNA fragments were mechanically sheared into fragments ranging in size between 150 and 300 bp using a cup-horn sonicator (Bandelin Sonoplus UV 2200 sonicator equipped with a BB5 cup horn, 10 sec. pulses at 100 % power output) or into fragments of size between 50 and 70 bp by mild DNase I treatment (Novagen). Sonication yielded a tight fragment size distribution when breaking the DNA into fragments of the 150-300 bp size range. Fragments of 50 to 70 bp in size were obtained by mild DNase I treatment using Novagen's shotgun cleavage kit.
- a 1 :450 dilution of DNase I provided with the kit was prepared and the digestion was performed in the presence of MnCb in a 10 ⁇ l volume at 20°C for 10 min to ensure double-stranded cleavage by the enzyme. Reactions were stopped with 2 ⁇ l of "Stop-Buffer" (100 mM EDTA, 30 % glycerol, 0.5 % Orange G, 0.075 % xylene cyanol) and the fragmentation efficiency was evaluated on a 2 % TAE-agarose gel. This treatment resulted in total fragmentation of genomic DNA into near 50-70 bp fragments. Fragments were then blunt-ended twice using T4 DNA Polymerase in the presence of 100 ⁇ M each of dNTPs to ensure efficient flushing of the ends. Fragments were used immediately in ligation reactions or frozen at -20°C for subsequent use.
- "Stop-Buffer" 100 mM EDTA, 30 % glycerol, 0.5 %
- the vector pMAL4.31 was constructed on a pASK-IBA backbone with the beta-lactamase ⁇ bid) gene exchanged with the Kanamycin resistance gene. In addition the bla gene was cloned into the multiple cloning site.
- the sequence encoding mature beta-lactamase is preceded by the leader peptide sequence of ompA to allow efficient secretion across the cytoplasmic membrane.
- a sequence encoding the first 12 amino acids (spacer sequence) of mature beta-lactamase follows the ompA leader peptide sequence to avoid fusion of sequences immediately after the leader peptidase cleavage site, since e.g.
- a Smal restriction site serves for library insertion.
- the three restriction sites are inserted after the sequence encoding the 12 amino acid spacer sequence in such a way that the bla gene is transcribed in the -1 reading frame resulting in a stop codon 15 bp after the Noil site.
- a +1 bp insertion restores the bla ORF so that beta-lactamase protein is produced with a consequent gain of Ampicillin resistance.
- the vector pMAL9.1 was constructed by cloning the lamB gene into the multiple cloning site of pEHl. Subsequently, a sequence was inserted in lamB after amino acid 154, containing the restriction sites Fsel, Smal and Notl. The reading frame for this insertion was constructed in such a way that transfer of frame-selected DNA fragments excised by digestion with Fsel and Notl from plasmid pMAL4.31 yields a continuous reading frame of lamB and the respective insert.
- the vector pHIEl 1 was constructed by cloning the fliuA gene into the multiple cloning site of pEHl.
- Genomic N. meningitidis MC58 DNA fragments were ligated into the Smal site of the vector pMAL4.31.
- Recombinant DNA was electroporated into DHlOB electrocompetent E. coli cells (GIBCO BRL) and transformants plated on LB-agar supplemented with Kanamycin (50 ⁇ g/ml) and Ampicillin (50 ⁇ g/ml). Plates were incubated over night at 37 0 C and colonies collected for large scale DNA extraction. A representative plate was stored and saved for collecting colonies for colony PCR analysis and large-scale sequencing. A simple colony PCR assay was used to initially determine the rough fragment size distribution as well as insertion efficiency. From sequencing data the precise fragment size was evaluated, junction intactness at the insertion site as well as the frame selection accuracy (3n+l rule).
- Genomic DNA fragments were excised from the pMAL4.31 vector, containing the N. meningitidis MC58 library with the restriction enzymes Fsel and Notl. The entire population of fragments was then transferred into plasmids pMAL9.1 (LamB) or pHIEl l (FhuA), which have been digested with Fsel and Notl. Using these two restriction enzymes, which recognise an 8 bp GC rich sequence, the reading frame that was selected in the pMAL4.31 vector is maintained in each of the platform vectors. The plasmid library was then transformed into E. coli DH5alpha cells by electroporation.
- the column was then washed three times with 3 ml LB medium. After removal of the magnet, cells were eluted by washing with 2 ml LB medium. After washing the column with 3 ml LB medium, the 2 ml eluate was loaded a second time on the same column and the washing and elution process repeated. The loading, washing and elution process was performed a third time, resulting in a final eluate of 2 ml.
- a second and third round of screening was performed as follows.
- the cells from the final eluate were collected by centrifugation and re-suspended in 1 ml LB medium supplemented with 50 ⁇ g/ml Kanamycin.
- the culture was incubated at 37°C for 90 min and then induced with 1 mM IPTG for 30 min.
- Cells were subsequently collected, washed once with 1 ml LB medium and suspended in 10 ⁇ l LB medium. 10 to 20 ⁇ g of human, biotinylated IgGs were added again and the suspension incubated over night at 4°C with gentle shaking. All further steps were exactly the same as in the first selection round.
- Cells selected after two rounds of selection were plated onto LB-agar plates supplemented with 50 ⁇ g/ml Kanamycin and grown over night at 37°C.
- LamB or FhuA fusion proteins were detected using human serum as the primary antibody at a dilution of approximately 1 :3,000 to 1:5,000 and anti-human IgG or
- NMB 50 and NMB 300 Two genomic libraries (NMB 50 and NMB 300), which contained most, if not all, possible antigenic epitopes encoded by the MenB genome, were generated by randomly fragmenting the genomic DNA of N. meningitidis B (MC58) and cloning fragments of an approximate size of 50 and 300 bp (Fig. 2) into vector pMAL4.31.
- NMB50 50 bp
- NMB300 300 bp
- the display of the MenB peptides on the surface of E. coli required transfer of the inserts from the frame selection vector pMAL4.31 to the display plasmids pMAL9.1 (LamB) or pHIEl l (FhuA). MenB genome-derived DNA fragments were excised by Fsel and Noil restriction enzymes. Ligation of inserts with the respective plasmid DNA resulted in approximately 5 x 10 5 individual clones. The E. coli clones were scraped off the LB plates and frozen without further amplification. Successful transfer of the libraries was proven by colony PCR analysis of 24 randomly picked clones from both libraries. Almost all clones contained an insert of the expected size.
- the antigen identification screens have discovered numerous immunogenic peptide sequences (i.e. 184 antigens as defined in Table 3) of Neisseria meningitidis serogroup B.
- the antigens were selected from 12 different bacterial surface display screens with 6 different IgG screening reagents prepared from pooled sera and two different display libraries (LamB and FhuA). All of the antigens and those peptides derived from the respective sequences depicted in Tables 3 to 5 have been confirmed either by Western blotting or by FACS analysis of selected E. coli clones.
- the list of antigens identified with human sera and genomic libraries displaying MenB peptides provides the basis for the selection of candidate vaccine antigens for prevention or treatment of meningococcal infections.
- Example 3 Epitope serology of synthetic peptides derived from MenB antigens with human sera
- Biotin-labeled peptides were coated on Streptavidin ELISA plates (EXICON) at 10 ⁇ g/ml concentration according to the manufacturer's instructions. Highly specific Horse Radish Peroxidase (HRP)-conjugated anti-human IgG secondary antibodies (Southern Biotech) were used according to the manufacturers' recommendations (dilution: 1,000 X). Sera were tested at two serum dilutions, 1 :400 and 1 :2,000. Following manual coating, peptide plates were processed and analyzed by the Gemini 160 ELISA robot (TECAN) with a built-in ELISA reader (GENIOS, TECAN).
- HRP Horse Radish Peroxidase
- Peptides were synthesized in small scale (4 mg resin; up to 288 in parallel) using standard F-moc chemistry on a Rink amide resin (PepChem, Tubingen, Germany) using a SyroII synthesizer (Multisyntech, Witten, Germany). After the sequence was assembled, peptides were elongated with Fmoc-epsilon-aminohexanoic acid (as a linker) and biotin (Sigma, St. Louis, MO; activated like a normal amino acid). Peptides were cleaved off the resin with 93% TFA, 5% triethylsilane, and 2% water for one hour.
- Peptides were dried under vacuum and freeze dried three times from acetonitrile/water (1 :1). The presence of the correct mass was verified by mass spectrometry on a Reflex III MALDI-TOF (Bruker, Bremen Germany). The peptides were used without further purification.
- the identified antigens were further characterized in order to select the most promising vaccine candidates for further animal studies.
- the validation steps included epitope serology with synthetic peptides and human sera (Example 3) and in vitro bactericidal activity with epitope induced hyperimmune mouse sera (Example 4).
- human serum samples from infected and exposed individuals for the presence of epitope-specific antibodies. Based on the bioinformatic analysis of DNA sequences derived from bacterial clones selected by the encoding immunogenic epitopes, corresponding peptides were designed and synthesized.
- the peptides i.e. antigens
- peptides were synthesized with an N-terminal biotin-tag and used as coating reagents on Streptavidin-coated ELISA plates. Epitope serology was performed in two steps. First, peptides were analysed with 21 serum samples that were also used for antigen identification (labeled by an * in Table 2, column "serum-no.”). 14 samples were from convalescing MenB, 4 from convalescing MenC infected patients, and 3 from exposed, but not colonized family members. Peptide-specific antibodies were detected by standard ELISA at two different serum dilutions (40Ox and 2,00Ox).
- E. coli clones harboring plasmids encoding the platform protein fused to a MenB peptide were grown in LB medium supplemented with 50 ⁇ g/ml Kanamycin at 37°C. Overnight cultures were diluted 1 :10, grown until an OD 60O of 0.5 and induced with 0.2 raM IPTG for 2 hours. Pelleted bacterial cells were suspended in PBS buffer and disrupted by sonication on ice, generating a crude cell extract. According to the OD 600 measurement, an aliquot corresponding to 5xlO 7 cells was injected into NMRI mice i.v., followed by a boost after 2 weeks. Serum was taken 1 week after the second injection. Epitope specific antibody levels were measured by Western blotting and peptide ELISA.
- Immunogenicity was assessed by visual detection of immunoreactive bands corresponding to the platform proteins LamB or FhuA with altered mobility due to the presence of inserted MenB epitopes (representative examples are shown in Figs. 4 and 5).
- peptide ⁇ LISA performed with synthetic peptides corresponding to epitopes expressed in the E. coli platform proteins. The levels of antibodies were assessed by ELISA readings at two different serum dilutions (200 X and 1,000 X) measured in five individual mouse sera (representative examples are shown in Fig. 5).
- Serum samples that showed epitope specific antibody levels were further analysed in the in vitro colorimetric bactericidal assay described above. Due to the low signal background ratio obtained with whole serum, we purified total IgG in order to provide higher amounts of antibodies in this assay. 100 cfu of Meningococcus serogroup B MC58 strain were incubated with 10 or 20 ⁇ g total IgGs in the presence of guinea pig serum as complement source. In this assay the same amount of total IgGs purified from mouse sera generated with total MenB lysate showed 100% killing activity.
- IgGs induced by "empty" LamB and FhuA platform protein carrying E. coli bacteria were used as background controls that resulted in about 10% killing activity (Fig. 7). All of the sera that induced >20% bactericidal activity were considered positive, and were quantified based on the percent of reduction in bacterial growth. The results are also shown in Table 4, column BA.
- Table 2 Summary of immune characterization of selected serum samples.
- ELISA reactivity ELISA reactivity of peptides with human sera.
- NMBl 523.4 aaeapaaeapaaeapateapaae +++++ +++++ 34
- NMB1523.5 eapaaeapaaeapaaeapaaeapaaea +++++ +++++ 35
- NMB1533.5 apaseapaaeaapadaaeapaagnca ++++ +++++ 39
- NMB 1768.6 taspavsyaigqhfkdlagqnang ++ +++ 42
- NMB0030.3 dlieankqsiaaaaapaaeegkyekvae +++ n.d. 113
- NMB0249.2 fayeglyhesrlknpkikqggewmdv +++ n.d. 127 Peptide Amino acid (aa) seq ELISA SeroSEQ ID NO: name reactivity conversion
- NMB1368.2 kpskegfggktrgegfkkegfkrd +++ n.d. 157
- NMB 1768.9 hesgekinysirrnlsldkademi ++ n.d. 174
- WB Western Blot with antiserum generated in mice against the indicated bacterial clones with N. meningitidis grown in RPMI or BHI medium; if different results for RPMI and BHI were obtained, 1 and 2 indicate results in RPMI and BHI, respectively
- BA Bactericidal activity measured with antiserum generated in mice against the indicated bacterial clones (at least 1 bacterial clone)
- Clone WB Western Blot with antiserum generated in mice against the indicated bacterial clones with E. coli lysate expressing the respective peptide encoded by the bacterial clone
- ELISA react. ELISA reactivity of peptides with antiserum generated in mice against the indicated bacterial clones.
- NMB0045.3 1 16 +++ n.d. NMB0045.4 10 +++++ +
- MBFBW 18 Easrgkgkigttgrgigpayedkvarrairaadllhpeklrekldavlayynvqlqhlhnaepv 205 + NMB0815.2 70 ++ +/- kaed MBFBS81 Nicotgyelpdggktdilpcgsdavetckpiyetmpgwrestfgvkdygalpenakaylkrie 206 evcgap
- MBFBM80 gklvtgkdkgengsstdegeglvtakevidavnkagwrmktttangqtgqadkfetvtsgtn 208 ++ +++ MBFBN66 tagtngdttvhlngigstltdtllntgattnvtndnvtddekkraasvkdvlnagwnikgvkpgtt 209 + NMB0992.6 26 ++ +
- MBFAA62 aaeapaaeapaaeapateapaaeapaaeapaaeapaaeaaateapaaeaaateap 217 ++ NMB 1523.4 34 ++++ ++ ++++ +++++ NMB1523.5 35 ++++ +++++ NMB 1523.6 36 ++++ +++++++
- Group III Bactericidal activity: positive Seroconversion: not determined (n.d.)
- Group IV Bactericidal activity: negative Seroconversion: positive (incl. weak)
- Group VII Bactericidal activity: not determined (n.d.) Seroconversion: positive (incl. weak)
- Group IX Bactericidal activity: not determined (n.d.) Seroconversion: not determined (n.d.)
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