WO1985004654A1 - A peptide vaccine or diagnostic, and a polypeptide useful therefor - Google Patents

A peptide vaccine or diagnostic, and a polypeptide useful therefor Download PDF

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Publication number
WO1985004654A1
WO1985004654A1 PCT/US1985/000565 US8500565W WO8504654A1 WO 1985004654 A1 WO1985004654 A1 WO 1985004654A1 US 8500565 W US8500565 W US 8500565W WO 8504654 A1 WO8504654 A1 WO 8504654A1
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Prior art keywords
polypeptide
amino acid
lys
thr
ser
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PCT/US1985/000565
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English (en)
French (fr)
Inventor
Magdalene Y. H. So
Carolyn D. Deal
Per O. Hagblom
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Scripps Clinic And Research Foundation
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Publication of WO1985004654A1 publication Critical patent/WO1985004654A1/en
Priority to DK565285A priority Critical patent/DK565285A/da
Priority to FI854839A priority patent/FI81452C/fi

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    • 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)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • 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

  • This invention relates to antigens, immunogens, and to vaccines utilizing such immunogens. More particularly, this invention relates to polypeptide antigens or immunogens, antibodies raised by such immunogens, and a vaccine suitable for the prevention of gonorrhea.
  • Background Art The annual incidence of reported infections by Neisseria gonorrhoeae is estimated to be about two million cases. A gonococcal infection in men usually results in a relatively uncomplicated urogenital infection. Disseminated gonococcal infection is reported to occur in 1 to 3% of those with gonorrhea, but the morbidity of this disease with current therapy is slight.
  • Penicillinase-produc- ing strains of gonococci have also emerged. Ith a view toward developing an efficacious vaccine, several studies have examined surface components of the gonococcus, including lipopolysaccharide (LPS) , peptideglycan, outer membrane proteins, capsules, IgAl protease, and pili. Of the foregoing, pili have been proposed as essential constituents of a gonorrhea vaccine, and it has been reported that pili are immunogenic and nontoxic for humans. See, generally, Schoolnik et al. , "A Pilus Peptide Vaccine For The Prevention Of Gonorrhea," Prog. Allergy 33_:314-331 (1983).
  • LPS lipopolysaccharide
  • the pili are proteinaceous surface appendages of bacteria which promote infectivity by facilitating the attachment of the bacterial cell to host epithelial tissues.
  • the pilus protein is a primary surface antigen.
  • Each pilus is a fimbriak structure composed of repeating identical subunits (pilin) , each having a molecular weight of approximately 18,000 daltons.
  • serotypes of N. gonorrhoeae pili There are many serotypes of N. gonorrhoeae pili? however, peptide mapping studies of antigenically and immunogenically different pilin indicate that they all share a conserved region located at the amino terminal end of the protein. On the other hand, the carboxy terminal region of the protein has been found to be variable from serotype to serotype.
  • a general description of the pilus protein can be found in Meyer et al.. Cell 30:45:52 (1982).
  • variable region of N. gonorrhoeae pilin protein near the carboxy terminal end of the protein contains both hypervariable sequences as well as conserved sequences defining antigenic determinant sites that can be utilized to prepare antigens and/or immunogens that immunologically mimi ⁇ k these antigenic determinant sites, and that elicit antibody production in a mammalian host.
  • the present invention contemplates a polypeptide that is smaller than a naturally-occurring gonococcal pilin protein, and the pharmaceutically acceptable salts thereof, that are capable of immunologically mimicking a conserved antigenic determinant site within a variable region of the carboxy-terminal half of the gonococcal pilin and thus are capable of being an antigen, or an immunogen, against a gonococcal infection. Additionally, the present invention contemplates a vaccine that contains such an immunogen as well as a method of immunization against a gonococcal infection. This invention further contemplates a diagnostic assay utilizing a polypeptide of this invention and/or a receptor such as an antibody elicited by such a polypeptide.
  • the polypeptide of this invention comprises at least one amino acid residue sequence, containing at least five amino acid residues and up to about 60 amino acid residues, that defines a sequence capable of immunologically mimicking an antigenic determinant site of a gonococcal pilin.
  • This amino acid residue sequence can repeat as a unit one or more times in the same polypeptide molecule. More than one type of such repeating unit, and more than one repeating unit of the same type, can be present in a single polypeptide molecule that embodies the present invention.
  • Such polypeptide can be made as a fusion protein synthesized by genetic engineering techniques or it can be built-up from individual amino acid residues, or amino acid residue blocks.
  • Polypeptides embodying this invention can be defined as including the amino acid residue sequence, taken from left to right and in the direction from the amino-terminus to the carboxy-terminus, of the formula wherein X is an amino acid residue having a positively charged side chain and is a member "of the group consisting of histidine (HIS) , lysine (LYS) and arginine (ARG) , X 2 and X3 can be the same or different and are non-polar amino acid residues that are members of the respective groups consisting of leucine (LEU) , proline (PRO) , tryptophan (TRP) , phenylalanine (PHE) , valine (VAL) , alanine (ALA) ' and isoleucine (ILE) , and X 4 and X5 can be the same * or different and are polar but uncharged amino acid residues that are members of the respective groups consisting of serine (SER) , threonine (THR) , cyst
  • Preferred amino acid residue sequences that define a desired antigenic determinant site are
  • VAL-THR-ARG- • VAL-THR-ARG-, and antigenically related variants thereof. Also preferred are the corresponding polypeptides themselves, that is. H-LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP-OH ; H-THR-LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP- LYS-ALA-SER-ASP-ALA-LYS-OH; and
  • Polypeptides of the present invention are smaller than the naturally-occurring gonococcal pilin protein and include an amino acid residue sequence of at least five to about 60 amino acid residues, preferably five to twenty amino acid residues, that immunologically mimicks a conserved antigenic determinant site in the variable region of the carboxy-terminal half of gonococcal pilin protein.
  • the present polypeptides are useful by themselves, or as pharmaceutically acceptable salts, as the active constituent in a vaccine, as an inoculum, or in a diagnostic assay.
  • antigenic determinant designates the structural component of a molecule that is responsible for specific interaction with corresponding antibody (immunoglobulin) molecules elicited by the same or related antigen.
  • Antigenic determinants in the present polypeptides comprise chemically active surface groupings of amino acid residues.
  • antigen means an entity that is bound by an antibody.
  • immunogen describes an entity that induces antibody production in the host animal. In some instances the antigen and the immunogen are the same entity, while in other instances the two entities are different.
  • immunologically mimicks is used herein to mean that an immunogenic polypeptide of this invention is not a natural protein or a cleaved fragment of a natural protein, but a manufactured polypeptide, as by solid phase synthesis or genetic engineering techniques, which polypeptide induces production of antibodies that bind to the inducing polypeptide and also to a corresponding pilin or pilin polypeptide portion. All amino acid residues identified herein are in the natural or L-configuration unless otherwise specified. In keeping with standard peptide nomenclature, abbreviations for amino acid residues that have been used herein are as follows:
  • pharmaceutically acceptable salts refers to the non-toxic alkali metal, alkaline earth metal and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, and ammonium salts and the like which are prepared by methods well known in the art.
  • pharmaceutically acceptable salts refers to the non-toxic alkali metal, alkaline earth metal and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, and ammonium salts and the like which are prepared by methods well known in the art.
  • non-toxic acid addition salts which are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid.
  • Representative salts include the hydrochloride, hydrobro ide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, ' succinate, tartrate, and the like.
  • polypeptides meeting the foregoing conditions elicit antibodies in a mammalian host and are believed to be capable of forming a beta-turn structure that. ermits the polypeptide to immunologically mimick a desired conserved antigenic determinant site within a variable region of the carboxy-terminal half of the pilin protein.
  • the polypeptide is no more than about 60 amino acid residues long and contains in each defined antigenic determinant site at least one amino acid residue having a positively charged side chain.
  • polypeptides containing one or more such amino acid residue sequences can be formed into relatively larger synthetic moieties by joining the individual polypeptides head-to-tail or by interpolypeptide cysteine disulfide bonds. These polypeptides can be characterized as
  • X designates an amino acid residue.
  • X is an amino acid residue having a positively charged side chain such as HIS, LYS or ARG.
  • X 2 and X3 can be the same or different but are non-polar amino acid residues selected from the group consisting of LEU, PRO, TRP, PHE, VAL, ALA and ILE.
  • X 4 and X5 can be the same or different but are polar, uncharged amino acid residues selected from the group consisting of SER,
  • the charges referred to above relate to ionic charges on the amino acid residues when those residues are present in an aqueous solution at pH 7.0. See for example, Lehninger, Short Course in Biochemistry, p. 37, Worth Publishers, Inc., New York, New York (1973).
  • polypeptide containing one or more of the foregoing amino acid residue sequences can also include a further amino acid residue sequence that immunologically mimicks an i munorecessive antigenic determinant site of a gonococcal pilin.
  • polypeptides embodying the present invention are represented by polypeptides that are individually constituted by only one of the amino acid residue sequences specifically defined hereinabove, i.e., H-LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP-OH; .H-THR-LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP- •
  • Biochemical evidence from immunoassay and from analogy with conserved protein-protein interaction in solved X-ray crystallographic structures with differing sequences such as in the dimer contacts of oligomeric enzymes indicates that the conservation of protein-protein recognition does not require a strict conservation of sequence, for relatedness. While single amino acid residue changes may affect such recognition to a wide degree depending upon the nature of the substitution, in general terms the relatedness of two differing amino acid sequences with respect to protein-protein (and antigenic and/or immunogenic) recognition can be expressed in terms of seven basic amino acid parameters:
  • a consensus matrix can be used to assign numerical values for each amino acid pair in the sequences being considered for relatedness.
  • the following consensus matrix wherein the individual amino acid residues are designated by a one-letter code in the interests of conciseness, can be used: -
  • Sequence comparison using the foregoing consensus matrix involves the determination of all possible alignments and the subsequent scoring of these alignments by the matrix. Two sequences are then aligned by computing the maximum match score from the consensus matrix. An alignment score in standard deviation units can be determined by taking the difference between the maximum matched score and the average maximum matched score for random permutation of the two sequences and then dividing by the standard deviation of the random score.
  • a consensus matrix score greater than three standard deviations shows significant relatedness at a confidence level of more than 99.7%. This is a restrictive criterion since it gives a frequency of 0.005 for all 5-residue peptides and 0.0014 for all 13-residue peptides occurring in 2222 known protein sequences.
  • a consensus matrix score greater than two standard deviations shows significant relatedness at a confidence level of more than 95.4%.
  • the. consensus matrix score is calculated by ascertaining the matrix value for each aligned amino acid residue pair under consideration and then summing the individual values for each such pair. The obtained sum is then compared against the number of standard deviations signifying the desired confidence level. If the obtained sum is greater than the product of the selected number of standard deviations and the number of amino acid residue pairs under consideration, then the amino acid residue sequences being compared are antigenically related to the indicated confidence level. For example, to ascertain the antigenic relatedness of the amino acid residue sequences - S-TRP-PHE-CYS-GLY- and -ARG-ILE-PHE-CYS-GLY- the consensus matrix yields the following values
  • the consensus matrix score must exceed the number of amino acid residue pairs under consideration times 3, i.e., 5 x 3 or 15. Inasmuch as 27 > 15, the desired antigenic relatedness is indeed present.
  • antigenic relatedness among polypeptides within the scope of the invention preferably is present at least to about 95% confidence level, and more preferably to at least about 99% confidence level.
  • a mixture of the foregoing polypeptides can also be used to make up a vaccine against or a diagnostic assay for a gonococcal infection, and/or an inoculum for raising antibodies.
  • Vaccines containing effective amounts of the present polypeptides induce production of antibodies in a sufficient amount to protect the vaccinated individual from infection with gonococcus and thus prevent gonorrhea.
  • Booster injections can be given if needed.
  • the word “vaccine” in its various ' grammatical forms is used herein in relation to the protection of a host mammal.
  • the word “inoculum” in its various grammatical forms is used herein to describe a composition containing a polypeptide of this invention as an active ingredient used for the preparation of antibodies that immunologically bind to gonococcal pili.
  • a vaccine and an inoculum may thus contain the identical ingredients, but their uses are different.
  • polypeptides suitable as antigens or immunogens, or both, for the present purposes can be produced synthetically or by genetic engineering techniques, and can be in monomeric as well as multimeric forms for use in vaccines, inocula, or as diagnostics.
  • the polypeptide When used in a vaccine or inoculum, the polypeptide may be used alone, as in the case of an ' oligomer or a multimer, or used linked to another carrier. moiety as a conjugate.
  • a polypeptide of this invention typically contains a total of about 20 to about 35 amino acid residues. Shorter polypeptides are preferably linked to a carrier.
  • Particularly useful conjugate carriers include keyhole limpet hemocyanin (KLH) , tetanus toxoid, poly-L-(LYS:GLU) , peanut agglutinin, ovalbumin, soybean agglutinin, bovine serum albumin (3SA) , human serum albumin, and the like.
  • KLH keyhole limpet hemocyanin
  • tetanus toxoid poly-L-(LYS:GLU)
  • peanut agglutinin ovalbumin
  • soybean agglutinin ovalbumin
  • bovine serum albumin (3SA) bovine serum albumin
  • human serum albumin and the like.
  • the synthetic polypeptides utilized herein are preferably coupled to keyhole limpet hemocyanin (KLH) using the following well known method.
  • KLH carrier is first activated with m-maleimidobenzoyl-N- hydroxysuccinimide ester, and is subsequently coupled to the polypeptide through a cysteine residue added to the amino-terminus or carboxy-terminus of the polypeptide by a Michael addition reaction, as described in Liu et al., Biochem. 8 : 690 (1979).
  • a polypeptide of this invention may also be coupled to a carrier through different means, and may be coupled to carriers other than KLH as noted before.
  • a polypeptide may be coupled to a tetanus toxoid carrier through free amino groups, using a 0.04 percent glutaraldehyde solution as is well known. See, for example, Klipstein et al. J. Infect. Dis. 147: 318 (1983).
  • Cysteine residues added at the amino- or carboxy-terminii of the synthetic polypeptide have been found to be particularly useful for forming conjugates via disulfide bonds and Michael addition reaction products, but other methods well known in the art for preparing conjugates can also be used.
  • Exemplary additional binding (linking) procedures include the use of dialdehydes such as glutaraldehyde (discussed above) and the like, or the use of carbodiimide technology as in the use of a water-soluble carbodiimide, e.g. l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, to form amide links between the carrier and polypeptide.
  • dialdehydes such as glutaraldehyde (discussed above) and the like
  • carbodiimide technology as in the use of a water-soluble carbodiimide, e.g. l-ethyl-3-(3-dimethylaminopropyl) carbod
  • the intermediate linking group is preferably an m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) , also discussed before.
  • MBS is typically first added to the carrier by an ester-amide interchange reaction. Thereafter, the above Michael reaction can be followed, or the MBS addition may be followed by a Michael addition of a blocked mercapto group such as thiolacetic acid (CH 3 COSH) across the maleimido-double bond. After cleavage of the acyl blocking group, a disulfide bond is formed between the deblocked linking group mercaptan and the mercaptan of the added cysteine residue of the synthetic polypeptide.
  • a blocked mercapto group such as thiolacetic acid (CH 3 COSH)
  • the choice of carrier is more dependent upon the ultimate intended use of the immunogenic polypeptide than upon the determinant portion of the immunogen, and is based upon criteria not particularly involved in the present invention. For example, if an inoculum is to be used in animals, as for the production of anti-polypeptide antibodies to be used to assay for the presence of gonococcal pilii, a carrier that does not generate an untoward reaction in the particular animal should be selected. If a vaccine against Neisseria gonorrheae is to be used in man, then the overriding concerns involve the lack of immunochemical or other side reaction of the carrier and/or the resulting immunogen, safety and efficacy—the same considerations that apply to any vaccine intended for human use.
  • manufactured means that the polypeptide molecule or polypeptide repeating unit has been built up synthetically by chemical means, i.e., chemically synthesized or by human-mediated biological means, e.g., by genetic engineering techniques.
  • the manufactured polypeptides embodying the present invention are free from naturally occurring proteins and fragments thereof.
  • the well-known solid phase chemical synthesis in which blocked amino acid residues are added in a serial manner to obtain the desired polypeptide is the preferred method of synthesis, and is discussed in greater detail hereinbelow.
  • polypeptides suitable for the purposes of the present invention can be synthesized by the well-known solid phase method. See, for example, Houghten et al.. Int.
  • the amino acid residues are linked to a resin (solid phase) through an ester linkage from the carboxy-terminal residue.
  • a resin solid phase
  • the polypeptide is to be linked to a carrier via a CYS residue, it is convenient to utilize that CYS residue as the carboxy-terminal residue that is ester-bonded to the resin.
  • the alpha-a ino group of each added amino acid typically is protected by a tertiary-butoxycarbonyl (t-BOC) group prior to the - amino acid being added into the growing polypeptide chain.
  • the t-BOC group is then removed prior to addition of the next amino acid to the growing polypeptide chain.
  • Reactive amino acid side chains are also protected during synthesis of the polypeptide.
  • Usual side-chain protecting groups used for the remaining amino acid residues are as follows: 0-(p_-bromobenzyoxycarbonyl) for tyrosine, O-benzyl for threonine, serine, aspartic acid and glutamic acid, and S-methoxy-benzyl for cysteine.
  • Protected amino acids are recrystallized from appropriate solvents to give single spots by thin layer chromatography. Couplings are typically carried out using a ten-fold molar excess of both protected amino acid and dicyclohexyl carbodiimide over the number of milliequivalents of initial N-terminal amino acid. A two molar excess of both reagents can also be used. For asparagine, an equal molar amount of N-hydroxy-benzotriazole is added to the protected amino acid and dimethyl-formamide is used as the solvent. All coupling reactions are typically more than 99% complete by the picric acid test of Gisin, Anal. Chem. Acta, Si8_:248-249 (1972).
  • a portion of the resulting, protected, resin-bonded polypeptide (1 gram) is treated with two milliliters of anisole, and anhydrous hydrogen fluoride, 20 milliliters, is condensed into the reaction vessel at dry ice temperature.
  • the resulting mixture is stirred, at 4° for 1.0 hour to cleave the protecting groups and remove the polypeptide from the resin.
  • the residue is extracted with anhydrous diethyl ether three times to remove the anisole, and the residue is dried in vacuo.
  • the vacuum dried material is first extracted with 5% aqueous acetic acid (3 times 50 milliliters each) followed by extractions using 50% aqueous acetic acid (4 times 50 milliliters).
  • the first extraction removes low molecular weight polypeptides and tyrosine that is used in some preparations to protect the CYS mercapto groups.
  • the second extraction separates the free polypeptide from the resin. After dilution with water to a concentration of 10-20% acetic acid, the resulting solution is lyophilized to provide a monomeric, unoxidized, polypeptide.
  • Polypeptide multimers can be prepared by bonding together the synthesized polypeptide monomers in a head-to-tail manner using the aforementioned solid phase method, i.e., one complete polypeptide sequence can be synthesized on the resin, followed by one or more of the same or different polypeptide sequences, with the entire multimeric unit thereafter being cleaved from the resin and used as described herein.
  • synthesized polypeptide monomers that contain added cysteine residues at both the amino- and carboxy-termini can be bonded together by intramolecular, interpolypeptide cystine disulfide bonds utilizing an oxidation procedure to form an immunogenic polymer.
  • the polymer so prepared contains a plurality of the polypeptides of this invention as repeating.units. Those repeating units are bonded together by the above-discussed oxidized cysteine residues.
  • CYS residues in a polypeptide of this invention for the purposes of binding the polypeptide to a carrier or preparing a polymer is not to be construed as altering the amino acid sequence of the polypeptide repeating units from a sequence that immunologically mimicks an antigenic determinant site of a gonococcal pilin.
  • the present vaccines and inocula include one or more of the polypeptides described hereinabove together with a pharmaceutically acceptable diluent such as physiological saline, phosphate-buffered saline (PBS), or other injectable liquid.
  • a pharmaceutically acceptable diluent such as physiological saline, phosphate-buffered saline (PBS), or other injectable liquid.
  • Additives customarily used in vaccines or inocula may also be present, if desired.
  • Illustrative of such additives are stabilizers such as lactose or sorbitol, and adjuvants such as aluminum hydroxide, sulfate or phosphate, an alum, or an alginate.
  • Precipitated aluminum phosphate is a particularly suitable adjuvant for a vaccine, while complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) are preferred for use in inocula.
  • Vaccines and inocula of the present invention may be administered by injection, usually intramuscularly or subcutaneously, orally by means of an enteric capsule or tablet, as a suppository, as a nasal spray, and by other suitable routes of administration.
  • a suitable dose of the polypeptide depends, in part, upon the chosen route of administration and a number of other factors. Included among those factors are the body weight of the mammal to be immunized, the carrier when used, the adjuvant when used, and the number of innoculations desired to be used.
  • Individual innoculations for a human patient typically contain unit doses of about 10 micrograms to about 100 milligrams of polypeptide, exclusive of any carrier to which the polypeptide may be linked. If desired, a series of doses may be administered over a period of time for optimum immunity.
  • Unit dosage forms of the vaccine can also be provided, if desired, containing the aforementioned amounts,of the polypeptide.
  • the immunogen contained in a vaccine or an inoculum is present in an "effective amount," which amount depends upon a variety of factors as is well known in the immunological arts, e.g., the body weight of the mammal to be immunized, the carrier moiety used, the adjuvant used, the duration of protection sought, and the desired immunization protocol.
  • Typical inoculum stock solutions are prepared with CFA, IFA or alum as follows: An amount of the polypeptide, synthetic polypeptide-conjugate or polymeric polypeptide sufficient to provide the desired, effective amount of polypeptide per inoculation is dissolved in PBS at a pH value of 7.2. Equal volumes of CFA or IFA are then mixed with the polypeptide solution to provide an inoculum containing polypeptide, water and adjuvant in which the water-to-oil ratio is about 1:1. The mixture is thereafter homogenized to provide the inoculum stock solution. When alum is used, about 200 micrograms of conjugate is absorbed onto about 4 milligrams of alum to prepare the stock inoculum.
  • Rabbits can be utilized herein to raise • anti-polypeptide antibodies.
  • the host rabbit is typically injected subcutaneously with an inoculum comprising 200 micrograms of a polypeptide conjugate (polypeptide plus carrier) emulsified in CFA; 200 micrograms of polypeptide conjugate in IFA; and 200 micrograms of polypeptide conjugate with 4 milligrams alum injected intraperitoneally on days 0, 14 and 21, respectively, of the immunization schedule.
  • Each inoculation (immunization) consists of four injections of the inoculum. Mice may be immunized in a similar way using about one tenth of the above dose per injection. Animals are typically bled 4 and 15 weeks after the first injection. Control pre-immune serum is obtained from each animal by bleeding just before the initial immunization.
  • Control inoculum stock solutions can also be prepared with keyhole limpet hemocyanin (KLH) , KLH in CFA or IFA, KLH-alum absorbed, KLH-alura absorbed-pertussis, edestin, thyroglobulin, tetanus toxoid, tetanus toxoid in IFA, cholera toxoid and cholera toxoid in IFA, and the like.
  • KLH keyhole limpet hemocyanin
  • KLH in CFA or IFA KLH-alum absorbed
  • KLH-alura absorbed-pertussis edestin
  • thyroglobulin tetanus toxoid
  • cholera toxoid cholera toxoid
  • cholera toxoid cholera toxoid in IFA
  • Sera that provide anti-polype tide antibody titers (dilutions) of at least about 1:160 are considered useful in providing the antibodies of this invention.
  • the typically utilized ELISA is described in greater detail in Bittle et al.. Nature .298: 30-33 (1982), which is incorporated herein by reference.
  • Suitable monoclonal receptors may also be prepared using hybridoma ' technology as described by Niman et al., Proc. Natl. Acad. S ⁇ i., USA 80_: 4949-4953 (1983), which description is incorporated herein by reference.
  • Monoclonal receptors need not only be obtained from hybridoma supernatants, but may also be obtained in generally larger quantities from as ⁇ ites fluid of mammals into which the desired hybridoma has been introduced. Production of monoclonal antibodies using ascites fluid is well known and will not be dealt with further herein.
  • a receptor of this invention binds both to the polypeptide to which it was raised and also to the corresponding pilin protein whose antigenic determinant site the polypeptide of this invention immunologically mimmicks.
  • a polypeptide of this invention may be both an immunogen and an antigen.
  • the receptors of this invention are at least oligoclonal as compared to naturally occur ng polyclonal antibodies since they are raised to an immunogen having relatively few epitopes as compared to the epitopes of an intact pilin molecule.
  • receptors of this invention bind to epitopes of the polypeptide while naturally occurring antibodies raised to a gonococcal pilin bind to epitopes throughout the pilin molecule.
  • the polypeptides, antibodies, and antibody combining sites provided by these polypeptides, and methods of the present invention may also be used for diagnostic tests, such as immunoassays.
  • diagnostic techniques include, for example, enzyme immune assay, enzyme multiplied immunoassay technique (EMIT) , enzyme-linked immunosorbent assay (ELISA) , radio-immune assay (RIA) , fluorescence immune assay, either single or double antibody techniques, and other techniques in which either the antibody combining site or the. antigen is labeled with some detectable tag.
  • An illustrative diagnostic system embodying the present invention to detect N. gonorrhoeae contains receptor molecules such as antibodies, substantially whole antibodies, or antibody combining . sites, raised to a polypeptide of this invention.
  • the system also includes an indicating means for signaling the presence of an immunoreaction between the receptor and the antigen.
  • the indicating means allows the immunoreaction to be detected.
  • a body sample such as a cervical or urogenital smear
  • the receptor molecule immunoreacts with the pilin antigen to form an immunoreactant, and the indicating means present then signals the immunoreaction.
  • One such exemplary embodiment is an immunofluorescent assay in which a cervical or urogenital smear is acetone-fixed to a plain microscope slide.
  • a second antibody such as a goat-antirabbit antibody can then incubated on the test slide, if desired.
  • the second antibody is labeled by being linked to a fluorochrome dye such as fluorscein isothiocyanate (FITC) .
  • FITC fluorscein isothiocyanate
  • FITC-labeled goat-antirabbit antibodies that bound to the first antibodies on the test slide. Presence of the FITC-labeled antibodies may be detected using fluorescent microscopy and thereby signal the presence of a N. gonorrhoeae infection.
  • Fab and F(ab') 2 antibody portions that are prepared by methods well known in the art.
  • Another diagnostic method of this invention is an ELISA assay.
  • a polypeptide antigen of this invention is bound on a solid support such as the walls of a microtiter plate. Non-specific binding sites on the microtiter well walls are thereafter blocked with a protein such as BSA. Un-bound polypeptide and BSA are removed from the microtiter well as by rinsing.
  • a body sample such as those above is admixed with an excess of an antibody of this, invention in an aqueous solution, and the admixture is maintained for a -time sufficient to form an immunoreaction between the antibody and any gonococcal pili antigen present. That liquid admixture is then mixed with the above-described polypeptide-bound solid support to form a second admixture containing solid and liquid phases. The solid/liquid phase admixture is maintained for a time sufficient for previously unreacted antibodies to immunoreact with the polypeptide antigen. The liquid phase is thereafter separated from the solid phase.
  • a solution of a second, labeled antibody that reacts with the first-named antibody is then admixed with the solid phase.
  • An examplary second antibody is a peroxidase-linked goat anti-rabbit antibody where the first-named antibodies are raised in rabbits.
  • the admixture formed from the solid phase and the second, labeled antibody solution is maintained for a time period sufficient to form an immunoreaction between the two antibodies.
  • the solid and liquid phases are thereafter separated.
  • a solution containing a substrate for the enzyme such as hydrogen peroxide and a color-forming dye precursor such as o-phenylenediamine is thereafter admixed with the solid phase.
  • the optical density at a preselected wave length (e.g., 405 nanometers) may then by determined after a predetermined time period has elapsed and compared to the optical density of a control to determine whether the gonococcal antigen was present in the body sample.
  • Polypeptide conjugation to tetanus toxoid carrier using the gluteraldehyde method was performed by admixing equal amounts of peptide and tetanus toxoid in PBS to final concentrations of 2 mg/ml. When difficulty in dissolving a particular peptide was encountered, the pH value of the produced admixture was raised to about 8.0.
  • a fresh glutaraldehyde working dilution was prepared prior to each coupling by diluting a 25% (w/v in PBS) glutaraldehyde stock solution 1:65 in ice cold PBS.
  • the fresh glutaraldehyde solution was then added to the above obtained peptide-carrie.r- solution at a ratio of 124 microliters to 1 milliliter, respectively.
  • the resulting reaction composition was incubated with stirring overnight at room temperature.
  • the reacation product was dialyzed for at least 6 hours against distilled H 2 0, and then lyophilized.
  • EXAMPLE 3 Screening of Rabbit Sera for Anti-Polypeptide Antibodies Rabbit anti-sera were screened for the presence of anti-polypeptide antibodies using an enzyme linked i munosorbent assay (ELISA) .
  • ELISA enzyme linked i munosorbent assay
  • Non-specific binding sites on the microtiter well walls were threafter blocked by incubating 50 ⁇ 1 of 3% (w/v) BSA/PBS in each well for 4 hours at 37°C. in a humidified chamber. After incubation, excess BSA was removed by inverting and shaking the plates. Polypeptide bound to a solid support whose non-specific binding sites had been blocked was thus provided for use as target antigen.
  • the rabbit antisera were further screened to determine their ability to immunoreact with gonococcal pilin protein in a Western blot assay. Gonococcal pilin protein was isolated from the
  • N. gonorrhoeae strain MS-11 (described in Meyer et al., supra) by the procedure outlined below.
  • N. gonorrhoeae MS-11 was plated and grown to confluency on GCB base medium (Diffco Co., Detroit, MI) in 15X100 mm petri dishes (American Scientific Products, McGaw, IL) .
  • the cultures from 50 dishes were swabbed into 35 ml of 50mM ethanolamine, pH 10.5.
  • the pili were sheared from the bacteria by vortexing for 30 seconds and immersing for 30 seconds in a 0°C. bath 4 times in seriatim.
  • the sheared pili were separated from the bacteria by centrifugation at 10,000 rpm for 20 minutes in a JA20 rotor (Beckman Instruments, Fullerton, CA) .
  • the supernatant containing pilin protein was then dialyzed against pili buffer (.15M NaCl, 0.05M Tris, pH 7.5) for about 16 hours. During dialysis a majority of the pilin protein present formed insoluble aggregates. These aggregates were subsequently isolated by centrifugation at 10,000 rpm for 15 minutes in the above-described rotor. The pilin protein pellet was then resuspended to a concentration of 1 mg/ l in 50mM ethanolamine, pH 10.5. The pilin was further isolated by ' eletrophoresis in a 15% SDS-polyacrylamide gel, and transferred to nitrocellulose (Schleicher & Schuel, ' Catalogue No.
  • the gonococcal pilin protein bound to the nitrocellulose solid support thus obtained was then used as target antigen in a Western blot assay to detect anti-pilin antibodies.
  • the rabbit anti-polypeptide antisera were diluted approximately 1:100 in 3% (w/v) BSA/PBS or BLOTTO [PBS containing 50 g/1 nonfat dry milk, 0.1 ml/1 antifoam A emulsion (Sigma Chemical Co., St. Louis, Mo.) and 1 ml/1 thimerosal] .
  • a Western blot was then incubated in 25 ml of each diluted antiserum for about 16 hours at 4°C. with constant agitation so as to contact the anti-polypeptide antibodies present in the antiserum with the solid phase bound pilin protein.
  • the blots were washed twice with 25 ml of TBS buffer (0.9% NaCl, lO M Tris, pH 7.4) containing 0.5% octylphenoxy polyethoxy ethanol (Triton X100) , washed three times with 25 ml of TBS alone, and then washed with 25 ml of 3% (w/v) BSA/TBS.
  • TBS buffer 0.9% NaCl, lO M Tris, pH 7.4
  • Triton X100 octylphenoxy polyethoxy ethanol
  • Anti-polypeptide antibodies bound to gonococcal pilin protein were detected by first reacting the blots with peroxidase labeled goat anti-rabbit IgG (Boehringer Mannheim) diluted about 1:2000 in 3% (w/v) BSA/TBS or BLOTTO for 16 hours at 4°C. with constant agitation. The blots were then washed as previously described and developed by incubation with 25 ml of the ABTS developing solution described in Example 3, above, for about 45-60 minutes.
  • anti-polypeptide antibodies that immunorea ⁇ ted with gonococcal pilin protein in the Western blot assay of Example 4, above, were examimed for their ability to immunoreact with whole bacteria used as solid phase target antigen in an ELISA.
  • Neisseria Eleven strains of Neisseria, including ten pathogenic gonococcal strains (Table 2, below) were obtained from J. Knapp, Neisseria Reference Lab, Seattle, WA. The strains were grown on GC chocolate agar at 35°C. in a 5% CO2 humidified incubator and confirmed by Gram stain, oxidase reaction, colony morphology and sugar fermentation patterns as is well known in the art.
  • Neisseria antigen stocks were prepared by swabbing cultures propagated as described above into PBS (pH 7.4). The optical density of each bacterial suspension was then adjusted to 0.1 at 750 nm with PBS (pH 7.4), aliquoted and frozen.
  • Solid phase bound Neisseria antigen was prepared by adding 50ul of Neisseria stock suspension to microtiter ("Immulon 1" stripwells) . The bacteria were pelleted from solution onto the walls of the wells by centrifugation for 5 minutes at 1000 g in a 4°C. tabletop centrifuge.
  • the Neisseria antigen was crosslinked to the solid phase by admixing 200 ⁇ ⁇ l of 0.25% glutaraldehyde in PBS at 4°C. in each well. After a 5 minute incubation at 4°C, the wells were washed 4 times with PBS. Non-specific binding sites were blocked by incubating 350ul of 5% BSA/PBS containing 0.05% polyoxyethylene (20)sorbitan monolaurate ("Tween 20”) for 2 hours at 37°C. in each well. The BSA blocking solution was subsequently removed by aspiration, thus providing Neisseria as target antigen bound to the walls of microtiter wells.
  • BSA blocking solution was subsequently removed by aspiration, thus providing Neisseria as target antigen bound to the walls of microtiter wells.
  • Rabbit antipolypeptide antisera to be examined for anti-Neisseria activity were serially diluted two-fold in 1% (w/v) BSA/PBS. 200
  • Milliliters of each dilution were adm ' ixed in a well prepared as described above forming a solid/liquid phase immunoreaction composition. Contact was maintained by incubating the wells for 1 hour at 37°C, thus allowing the anti-polypeptide antibodies present in the serum dilutions to immunoreact with solid phase bound Neisseria antigen. After incubation, the solid and liquid phases were separated by subjecting the wells to three one-minute washes with PBS containing 0.05% "Tween 20.”
  • Anti-polypeptide antibodies immunologically bound to the solid phase Neisseria were detected with a peroxidase-labeled goat anti-rabbit IgG indicating means obtained from Cappel, A Division of Cooper Biomedical, Malvern, PA.
  • the goat anti-rabbit IgG was diluted 1:2000 in 1% (w/v) BSA/PBS and 200 l were admixed in each well. The admixture was incubated for 1 hour at 37°C. thereby allowing immunoreaction between the goat anti-rabbit IgG antibodies and any rabbit anti-polypeptide antibodies present as bound to the solid phase Neisseria. After incubation the wells were washed five times for one minute each as described above.
  • the above described ELISA was used to examine the anti-gonococcal pilin polypeptide antibodies for their ability to (1) differentiate between pathogenic and commensal Neisseria species; and (2) immunoreact with members of a representative panel of N ⁇ gonorrhoeae pathogenic strains.
  • the anti-polypeptide antibodies were immunoreacted against the pathogenic ⁇ qonorrhoeae strain POMP 1 serovar (NRL 7122) and the commensal N ⁇ meningitidis B strain (NRL 9206).
  • the ability to differentiate between the pathogenic and commensal strains was then examined by calculating the ratio of the absorbance produced in the pathogenic strain ELISA versus the absorbance in the commensal strain ELISA.
  • Table 3 The results of examining the anti-polypeptide antibodies by this procedure are shown in Table 3, below.
  • antibodies induced by polypeptides made in accordance with the present invention demonstrate significant immunospecificity for the pathogenic ⁇ qonorrhoeae strain as opposed to the commensal ⁇ _ meningitidis strain.
  • the polypeptides of the present invention are suitable vaccine and inocula components because they direct the host's immune response towards disease-producing Neisseria species.
  • the second series of experiments utilizing the Neisseria antigen ELISA examined the ability of anti-polypeptide antibodies made in accordance with the present invention to immunoreact with members of a representative panel of N ⁇ gonorrhoeae pathogenic strains.
  • Antiserum GC4-9307 contained a sufficient titer of anti-polypeptide antibodies to be able to recognize 9 out of the 10-pathogenic strains screened (i.e., all but NRL 8035) when used at a 1:1600 dilution and compared to the pre-immune control serum.
  • antiserum GC6-9310 recognized 7 out of the 10 pathogenic strains (i.e., all but NRL 8035, NRL 5766 and NRL 8038) at a 1:1600 dilution.

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PCT/US1985/000565 1984-04-06 1985-04-04 A peptide vaccine or diagnostic, and a polypeptide useful therefor WO1985004654A1 (en)

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DK565285A DK565285A (da) 1984-04-06 1985-12-05 Peptidvaccine eller diagnosticeringsmiddel og et polypeptid til anvendelse hertil
FI854839A FI81452C (fi) 1984-04-06 1985-12-05 Diagnostiskt system foer bestaemning av pilusproteinet av neisseria gonorrhoeae-bakterie.

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0161095A2 (en) * 1984-04-30 1985-11-13 The Board Of Trustees Of The Leland Stanford Junior University Vaccine against urinary infection
WO1986006635A1 (en) * 1985-05-15 1986-11-20 Biotechnology Australia Pty. Ltd. Oral vaccines
WO1994008013A1 (en) * 1992-10-07 1994-04-14 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University Pilin variants and uses thereof
US5411732A (en) * 1986-04-25 1995-05-02 Kabi Pharmacia Aktiebolag Preparation of fused proteins, antibodies and processes therefore
WO1999055875A2 (en) * 1998-04-29 1999-11-04 American Cyanamid Company VACCINES CONTAINING RECOMBINANT PILIN AGAINST NEISSERIA GONORRHOEAE OR $i(NEISSERIA MENINGITIDIS)
US7189405B1 (en) * 1999-10-29 2007-03-13 Rice Peter A Peptide mimics of conserved gonococcal epitopes and methods and compositions using them

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4584195A (en) * 1984-06-21 1986-04-22 The Board Of Trustees Of The Leland Stanford Junior University Broad spectrum vaccine against gonorrhea

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US4186182A (en) * 1975-02-28 1980-01-29 Research Corporation Serological test for Neisseria gonorrhoeae antibodies
US4188371A (en) * 1977-09-28 1980-02-12 Corning Glass Works Immunological detection of Neisseria bacteria via labelled antibodies
US4241045A (en) * 1978-05-15 1980-12-23 Research Corporation Purified antigen to test for Neisseria gonorrheae antibodies
US4330623A (en) * 1980-02-19 1982-05-18 Merck & Co., Inc. Process for solubilization of gonococcal antigen
US4351761A (en) * 1978-05-15 1982-09-28 Research Corporation Purified antigen to test for Neisseria gonorrhoeae antibodies
US4386066A (en) * 1981-08-20 1983-05-31 Merck & Co., Inc. Immunogenic complex from N. gonorrhoeae
US4461838A (en) * 1975-04-25 1984-07-24 Bactex, Inc. Gonococcal Pili processes for the preparation thereof and the use thereof for the detection of and prevention of infections caused by Neisseria gonorrhoeae
US4497900A (en) * 1982-04-12 1985-02-05 Abbott Laboratories Immunoassay for Neisseria gonorrhoeae antigens

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US4186182A (en) * 1975-02-28 1980-01-29 Research Corporation Serological test for Neisseria gonorrhoeae antibodies
US4461838A (en) * 1975-04-25 1984-07-24 Bactex, Inc. Gonococcal Pili processes for the preparation thereof and the use thereof for the detection of and prevention of infections caused by Neisseria gonorrhoeae
US4188371A (en) * 1977-09-28 1980-02-12 Corning Glass Works Immunological detection of Neisseria bacteria via labelled antibodies
US4241045A (en) * 1978-05-15 1980-12-23 Research Corporation Purified antigen to test for Neisseria gonorrheae antibodies
US4351761A (en) * 1978-05-15 1982-09-28 Research Corporation Purified antigen to test for Neisseria gonorrhoeae antibodies
US4330623A (en) * 1980-02-19 1982-05-18 Merck & Co., Inc. Process for solubilization of gonococcal antigen
US4386066A (en) * 1981-08-20 1983-05-31 Merck & Co., Inc. Immunogenic complex from N. gonorrhoeae
US4497900A (en) * 1982-04-12 1985-02-05 Abbott Laboratories Immunoassay for Neisseria gonorrhoeae antigens

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Title
CHEMICAL ABSTRACT, HANSEN, et al. Vol. 100, page 137079, issued 1984 *
CHEMICAL ABSTRACT, JUDD Vol. 97, page 125478, issued 1982 *
CHEMICAL ABSTRACT, JUDD Vol. 99, page 101900, issued 1983 *
CHEMICAL ABSTRACT, SCHOOLNIK, et al. Vol. 98, page 196097, issued 1983 *
CHEMICAL ABSTRACT, SWANSON Vol. 90, page 199948, issued 1979 *
CHEMICAL ABSTRACT, WONG, et al. Vol. 96, page 50283, issued 1982 *
See also references of EP0177583A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0161095A2 (en) * 1984-04-30 1985-11-13 The Board Of Trustees Of The Leland Stanford Junior University Vaccine against urinary infection
EP0161095A3 (en) * 1984-04-30 1987-06-03 The Board Of Trustees Of The Leland Stanford Junior University Vaccine against urinary infection
WO1986006635A1 (en) * 1985-05-15 1986-11-20 Biotechnology Australia Pty. Ltd. Oral vaccines
US5411732A (en) * 1986-04-25 1995-05-02 Kabi Pharmacia Aktiebolag Preparation of fused proteins, antibodies and processes therefore
WO1994008013A1 (en) * 1992-10-07 1994-04-14 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University Pilin variants and uses thereof
WO1999055875A2 (en) * 1998-04-29 1999-11-04 American Cyanamid Company VACCINES CONTAINING RECOMBINANT PILIN AGAINST NEISSERIA GONORRHOEAE OR $i(NEISSERIA MENINGITIDIS)
WO1999055875A3 (en) * 1998-04-29 2000-04-13 American Cyanamid Co VACCINES CONTAINING RECOMBINANT PILIN AGAINST NEISSERIA GONORRHOEAE OR $i(NEISSERIA MENINGITIDIS)
US7189405B1 (en) * 1999-10-29 2007-03-13 Rice Peter A Peptide mimics of conserved gonococcal epitopes and methods and compositions using them
US7871628B2 (en) 1999-10-29 2011-01-18 University Of Massachusetts Peptide mimics of conserved gonococcal epitopes and methods and compositions using them

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FI81452C (fi) 1990-10-10
FI81452B (fi) 1990-06-29
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AU4159085A (en) 1985-11-01
JPS61501777A (ja) 1986-08-21
NZ211715A (en) 1989-01-27
FI854839A (fi) 1985-12-05
EP0177583A1 (en) 1986-04-16
ZA852629B (en) 1985-11-27
FI854839A0 (fi) 1985-12-05
IL74829A0 (en) 1985-07-31
AU582358B2 (en) 1989-03-23

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