FI81452C - Diagnostic system for determining the pilus protein of Neisseria gono rrhoeae bacterium - Google Patents

Description

1 81452

This invention relates to a diagnostic system for the detection of gonorrhea based on antibodies to antigens or immunogens in the form of a polypeptide.

An estimated two million cases of inflammation caused by Neisseria gonorrhoeae are reported each year. Gonococcal inflammation in men usually results in relatively simple urinary tract and genital inflammation. 1-3% of cases of gonorrhea are such that gonococcal infection has spread over a wide area, but the incidence of this disease with current treatments is low.

On the other hand, in women infected with gonorrhea, 10-20% of cases lead to urethritis, and even if properly treated, this infection can lead to recurrent urethritis, miscarriages, and a sterile cause. It is estimated that 1.8 million cases of urethritis in the United States are attended by private doctors each year and 220,000 are hospitalized.

Numerous studies to develop an effective vaccine have identified gonococcal surface components such as lipopolysaccharide (LPS), peptide glycan, outer membrane proteins, capsules, IgA1 protease, and cilia. Of the above, cilia are suspected to be essential components of the gonorrhea vaccine, and these cilia are shown to be immunogenic in humans and non-toxic to humans. A general presentation can be found in Schoolnik et al., "A Pilus Peptide Vaccine For The Prevention Of Gonorrhea," Prog. Allergy 33: 314-331 (1983).

2 81 452

Bacterial cilia are protein-like parts associated with the surface of a bacterium that promote the ability of the bacterium to infect by facilitating the attachment of the bacterial cell to the epithelial tissues of the host. In the case of N. gonorrhoeae, the cilia protein is the major surface antigen.

Each cilia is masculine in structure and consists of repetitive, similar subunits (pilin), each with a molecular weight of about 18,000 daltons. There are several serotypes of N. gonorrhoeae cilia; however, studies of the peptide map of antigenically and immunogenically distinct pilin proteins indicate that they all share a common region at the amino terminus of the protein. On the other hand, the carboxy-terminal region of the protein has been found to vary from one serotype to another. An overview of this cilia protein can be found in Meyer et al., Cell 30:45:52 (1982).

In the above-mentioned Schoolnik et al. discloses the use of an immunogenic cilia peptide comprising a constant region at the amino terminus of a protein.

Contrary to the above, it has been found that the variable region of the N. gonorrhoeae pilin protein located close to the carboxy terminus of the protein comprises both highly variable peptide chains and stable peptide chains defining antigenic determinants, which determinants can be used to prepare antigens and / or immunogens. antigens and / or immunogens mimic these antigenic determinants and that induce the production of antibodies in host mammals.

The present invention contemplates a polypeptide that is smaller than the naturally occurring gonococcal cilia protein, and a pharmaceutically acceptable polypeptide of this polypeptide.

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3,841,452 acceptable salts with which the peptide and salts can mimic an immunologically stable antigenic determinant site in the carboxy-terminal variable region of the cochlear protein of a gonococcal bacterium, and are thus capable of acting as an antigen or immunogen against gonococcal infection. The present invention further contemplates a diagnostic assay utilizing a polypeptide and / or receptor of the present invention, such as an antibody formed by the action of this polypeptide.

The essential features of the invention are set out in the appended claims.

The polypeptide of the present invention comprises at least one chain of amino acid residues consisting of at least five amino acid residues and up to about 60 amino acid residues, which defines a chain capable of immunologically mimicking the antigenic determinant site of a gonococcal pilin protein. This chain of amino acid residues may be repeated one or more times in the same polypeptide molecule. More than one type of repeating unit as well as more than one similar repeating unit may be present in one polypeptide molecule according to an embodiment of the present invention.

Such a protein can be prepared as a fusion protein synthesized by recombinant DNA techniques, or it can be formed from separate amino acid residues or assemblies of amino acid residues.

4,81452 The polypeptides of this invention may be shown to be peptides having the sequence of amino acid residues, written from left to right and in the direction from the amino terminus to the carboxy terminus, corresponding to the formula 1 2 3 4 5 -X -X -X -X-X - wherein X 1 is an amino acid residue the side chain is positively charged and belongs to the group consisting of histidine (HIS), lysine (LYS) and arginine (ARG), 2 3 X and X may be the same or different and are non-polar amino acid residues and belong to the corresponding leucine (LEU), proline (PRO), tryptophan (TRP), phenylalanine (PHE), valine (VAL), alanine (ALA) and isoleucine (ILE), and X and X may be the same or different and are polar but uncharged amino acid residues belonging to the corresponding groups of serine (SER), threonine (THR), cysteine (CYS) and glycine (GLY) systems.

The most preferred chains of amino acid residues that define the determinant region of the desired antigenicity are included in the chains of the following formulas written from left to right and in the amino-to-carboxy-terminal direction: -LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP-; -Thr-LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP LYS-ALA-SER-ASP-ALA-LYS-; and -GLY-SER-VAL-LYS-TRP-PHE-CYS-GLY-GLN-PRO-VAL-THR-ARG-, as well as similar modifications of their antigenicity. Also preferred are the corresponding polypeptides themselves, i.e., the polypeptides 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 H-GLY-SER-VAL-LYS-TRP-PHE-CYS-GLY-GLN-PRO-VAL-THR-ARG-OH,

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5,81452 their pharmacologically acceptable salts and similar variants in terms of their antigenicity.

The polypeptides of this invention are smaller than the naturally occurring gonococcal cilia protein (pilin) and comprise a chain of at least five and at most about 60 amino acid residues, preferably five to twenty amino acid residues, corresponding to an immunologically stable antigenic gland. in the carboxy-terminal variable region of the protein. These polypeptides are useful as such, or as pharmaceutically acceptable salts, as an active component of a vaccine, as an inoculum, or in a diagnostic assay.

As used herein, the term "antigenicity determinant" refers to a structural component of a molecule that elicits a specific reaction with corresponding antibody molecules (immunoglobulin) formed by this same or similar antigen. Antigenic determinants in the accompanying polypeptides comprise chemically active surface groupings of amino acid residues.

As used herein, the term "antigen" refers to an entity that is bound by an antibody.

As used herein, the term "immunogen" refers to an entity that induces the production of antibodies in a host animal. In some cases, the antigen and the immunogen are the same entity, while in some cases the two entities are different.

As used herein, the phrase "immunologically mimics" means that the immunogenic polypeptide of the present invention is not a natural protein or a fragment cleaved from a natural protein, but is a synthetically produced polypeptide prepared by solid phase 681452 or recombinant DNA techniques that induces such antibodies. the production of agents that bind to this inducing polypeptide and the corresponding pilin or a polypeptide portion of the pilin protein.

All amino acid residues mentioned herein are in the natural L-configuration unless otherwise indicated. The following abbreviations are used for amino acid residues in accordance with the common names of the peptides: _Abbreviation_ Amino acid 1-letter 3-letter Y TYR -L-tyrosine G GLY -glycine F PHE -L-phenylalanine M MET -L-methionine A ALA -L-alanine S SER -L-serine I ILE -L-isoleucine L LEU -L-leucine T THR -L-threonine V VAL -L-valine P PRO -L-proline K LYS -L-lysine N ASN -L-asparagine H HIS -L-histidine Q GLN -L-glutamine E GLU -L-glutamic acid W TRP -L-tryptophan R ARG -L-arginine D ASP -L-aspartic acid C CYS -L-cysteine

As used herein, the term "pharmaceutically acceptable salts" refers to non-toxic alkali metal and alkaline earth metal salts commonly used in the pharmaceutical industry, as well as ammonium salts such as sodium,

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7 81 452 Potassium, lithium, calcium and magnesium salts as well as ammonium salts and the like prepared by methods well known in the art. This term also includes non-toxic acid addition salts which are usually prepared by reacting the compounds of this invention with a suitable organic or inorganic acid. Such salts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, and the like.

Polypeptides that meet the above conditions produce antibodies in a host mammal and are expected to be capable of forming a beta-inverse structure that allows this polypeptide to immunologically mimic a desired permanent antigenicity determinant in the carboxy-terminal variable region of a pilin protein. Preferably, this polypeptide is no longer than about 60 amino acid residues, and contains at least one amino acid residue with a positively charged side chain at each particular antigenic determinant site.

One or more chains of amino acid residues meeting the above conditions may be present in the polypeptide as repeating units. In addition, polypeptides containing one or more such amino acid residue chains can be formed into relatively larger synthetic entities by attaching separate polypeptides at their ends (the carboxy terminus of one peptide to the amino terminus of another peptide) or by linking them with cysteine disulfide bonds between polypeptides.

These polypeptides can be characterized in that they contain chains of amino acid residues written from left to right and in the direction from the amino terminus to the carboxy terminus corresponding to the formula -X1-X2-X3-X4-X5-881452 wherein each X represents an amino acid residue. In addition, X 1 is an amino acid residue having a positively charged side chain, such as HIS, LYS or ARG. X and X may be the same or different, but are non-polar amino acid residues selected from the group consisting of amino acid residues LEU, PRO, TRP, PHE, VAL, ALA and ILE. Likewise, X and X may be the same or different, but are polar, uncharged amino acid residues selected from the group consisting of amino acids SER, THR, CYS and GLY. The above charges refer to the ionic charges of amino acid residues when these residues are in aqueous solution at pH 7.0. See, e.g., Lehninger, Short Course in Biochemistry, page 37, Worth Publishers, Inc., New York, New York (1973).

Particularly preferred chains of amino acid residues belonging to the above group, written from left to right and in the direction from the amino terminus to the carboxy terminus, are as follows: -LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP-; -Thr-LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP LYS-ALA-SER-ASP-ALA-LYS-; and -GLY-SER-VAL-LYS-TRP-PHE-CYS-GLY-GLN-PRO-VAL-THR-ARG-, whose amino acid residue chains have been found to remain unchanged in the carboxy-terminal variable region of pilin proteins, which were derived from six different , From the MS 11 serotype of N. gonorrhoeae strain as described in Meyer et al., Proc. Nat11 Acad. Sci.

USA (1984): 81: 6110-6114, as well as antigenically similar variants of these peptides, as discussed below.

More than one such chain of amino acid residues as described above, usually located at a certain distance from each other, may be present in the same polypeptide, with chains of other types of amino acid residues located between them. In addition, this same polypeptide contains one or more of the above

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9 81452, may also comprise other chains of amino acid residues that immunologically mimic the immunoresessive antigenic determinant site in the cochlear protein of a gonococcal bacterium.

Another grouping of preferred and intrachain disulfide loop polypeptides of the present invention can be described by polypeptides each consisting of only one chain of amino acid residues as specifically defined above, i.e., the peptides 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 H-GLY-SER-VAL-LYS-TRP-PHE-CYS-GLY-GLN-PRO-VAL-THR-ARG-OH.

The biochemical results obtained from the immunoassays, as well as the X-ray crystal structures elucidated by analogy of the permanent interaction between the two proteins using different chains, such as dimeric couplings of oligomeric enzymes, indicate that the maintenance of the unrecognized reaction between the two proteins is not required. Alteration of a single amino acid residue can affect in very different ways in such identification, depending on the nature of this substitution, in general, the similarity of two different amino acid chains for protein-protein identification (and antigen and / or immunogen identification) can be represented by seven basic amino acid parameters: 1 2 3 4 5 hydrophobicity; 2 changes in known chains due to development; 3 side chain size; 4 charge and polarity; 5 tendency to inverse secondary structure; 10 81 452 (6) tendency to the secondary structure of the beta strand; and (7) a tendency to a helical secondary structure.

To determine the degree of chain identity associated with antigenic and / or immunogenic identification, and thus antigenically similar variants, an auxiliary matrix can be used in which each amino acid pair of the chains under consideration for similarity is assigned a numerical value. For the purposes of the present invention, the following auxiliary matrix may be used, in which the individual amino acid residues are represented as single-letter codes for brevity:

ARNDCQEGHILKKFPSTWYV

A 7 -5 -1 -2 0 0 -1 2 -1 0 1 -2 2 -1 0 0 0 -3 -3 1 r -5 10 0 -1 -3 2 -1 -5 5 -4 -4 5 -3 -2 -3 0 0 -1 -1 -4 N -1 0 6 3 1 3 0 1 3 -2 -2 2 -1 -3 1 4 2 -3 0 -2 D -2 -1 3 7 - 2 1 4 0 0 -3 -3 0 -2 -4 0 1 0 -5 -2 -3 CO -3 1 -2 71 -2 1000 -2 00034 -2 20 Q0231162 -1 400200013 -1 00 E -1 - 1 0 4 -2 2 7 -3 1 -3 -2 0 -1 -3 -2 0 0 -5 -3 -3 G 2 -5 1 0 1 -1 -3 3 -2 -3 -3 -2 - 2 -5 2 3 1 -6 -2 -2 H -1 530041 -2 8 -1 040001202 -1 1 0 -4 -2 -3 0 0 -3 -3 -1 5 4 -3 2 2 -2 -2 0 0 0 4 L 1 -4 -2 -3 0 0 -2 -3 0 4 6 -2 4 3 -1 -2 0 1 0 3 K -2 5 2 0 -2 2 0 -2 4 -3 -2 8 -1 -3 -1 0 0 -4 -2 -3 M 2 -3 -1 -2 0 0 -1 -2 0 2 4 -1 6 2 0 -1 0 0 -1 2 T -1 -2 - 3 -4 0 0 -3 -5 0 2 3 -3 2 7 -2 -3 0 4 3 2 p 0 -3 1 0 0 0 -2 2 0 -2 -1 -1 0 -2 7 2 1 -4 -1 -1 S 0 0 4 1 3 1 0 3 1 -2 -2 0 -1 -3 2 5 3 -3 0 -1 T00204301200000136 -2 10 W -3 -1 -3 -5 -2 -1 -5 -6 0 0 1 -4 0 4 -4 -3 -2!) 2 0 Y -3 -1 0 -2 2 0 -3 -2 2 0 0 -2 -1 3 -1 0 1 2 8 0

V 1 -4 -2 -3 0 0 -3 -2 -1 4 3 -3 2 2 -1 -1 0 0 0 S

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n 81452

The comparison of chains using the auxiliary matrix described above involves determining all possible rows, after which these rows are scored using the matrix. The two chains are then compared by calculating the maximum possible compatibility score from this auxiliary matrix. The reference score in units of standard deviation can be determined by determining the difference between the maximum match score and the average, maximum match score in a random permutation of the two chains, which is then divided by the standard deviation of this random score.

In the context of the present invention, a score obtained with an auxiliary matrix greater than three standard deviations (mean value is approximately about 3 residues) indicates significant similarity at a level of greater than 99.7% confidence.

This is a limiting criterion as it gives a frequency of 0.005 for all 5 residue peptides and 0.0014 for all 13 residue peptides present in 2222 known protein chains. Similarly, a score obtained with an auxiliary matrix greater than two standard deviations (mean value is approximately about 2 for each residue) indicates significant similarity at a confidence level greater than 95.4%.

When similarity is determined in the context of the present invention, the score of the auxiliary matrix is calculated by confirming the matrix value of each pair of amino acid residues in the row under consideration, after which the individual values of each such pair are added together. This sum obtained is then compared to the number of standard deviations, which standard deviations are significant to the desired level of reliability. If the sum obtained is greater than the product of the selected number of standard deviations and the number of pairs of amino acid residues under consideration, then the chains of amino acid residues to be compared are antigenically similar at said level of confidence.

ia 81452

For example, to determine the antigenic similarity of the amino acid residue sequences -LYS-TRF-PHE-CYS-GLY and -ARG-ILE-PHE-CYS-GLY, the following values are obtained from the helper matrix

Value -LYS- & -ARG- or K & R 5 -TRP- & -ILE- or W&I 0 -PHE- & -PHr.- or F & F 7 -CYS- & -CYS- or C&C 7 -GLY- & -GLY- or G&G 8

A total of 27

Antigenic similarity at the 99.7% confidence level requires that the helper rice score exceeds the number obtained by multiplying the number of pairs of amino acid residues considered by three, i.e., 5x3, or 15. Since indeed 27> 15, the desired antigenic similarity holds.

·. ·. For purposes of this invention, antigenic similarity between the polypeptides of this invention preferably applies to a level of confidence of at least about 95%, and more preferably to a level of confidence of at least about 99%.

Mixtures of the aforementioned polypeptides, including those having antigenically similar regions as defined above, may also be used to prepare a vaccine against gonococcal infection or a diagnostic test for such infection, and / or as a graft to produce antibodies.

Il 13 8 1 452 Thus, the term "vaccine" as used herein in its various grammatical forms refers to the protection of the host mammal. As used herein in its various grammatical forms, the term "graft" is intended to describe a mixture comprising a polypeptide of the present invention as an active ingredient and used to prepare antibodies that immunologically bind to the protein of the gonococcal bacterium. Thus, the vaccine and the inoculum may contain the same ingredients, but their use is different.

Polypeptides that are suitable antigens or immunogens, or both, in this invention may be prepared synthetically or by recombinant DNA techniques, and may be in monomeric or multimeric forms when used in vaccines, inoculations, or diagnostic agents. When used in a vaccine or inoculum, the polypeptide may be used alone, as in the case of an oligomer or multimer, or may be used as a conjugate bound to a carrier moiety. When the polypeptide of this invention is used as such as an immunogen, it typically contains a total of about 20 to 35 amino acid residues. Shorter polypeptides are preferably coupled to a carrier.

Particularly useful carriers for conjugates include, for example, keyhole limpet snail hemocyanin (KLH), tetanus toxoid, poly-L- (LYS: GLU), peanut agglutinin, ovalbumin, soybean agglutinin, bovine serum albumin (BSA), human serum and the like.

The synthetic polypeptides used herein are preferably coupled to keyhole limpet snail hemocyanin (KLH) using the following well-known method. The KLH support is first activated with an ester of m-maleimidobenzoyl-N-hydroxysuccinimide, followed by attachment to the polypeptide via a cysteine residue added to the amino terminus 14,81452 or carboxy terminus of the polypeptide by the Michael addition reaction as described by Biiu et al., Liu et al. 80: 690 (1979).

The polypeptide of this invention may also be coupled to a carrier in some other manner, and may be coupled to carriers other than KLHr, as mentioned above. For example, a polypeptide can be coupled to a carrier to form a tetanus toxoid via free amino groups, using a 0.04% glutaraldehyde solution in a known manner. See, e.g., Klipstein et al. J. Infect. Dis. 147: 318 (1983).

Cysteine residues added to the amino and carboxy termini of a synthetic polypeptide have been found to be particularly useful in forming conjugates by disulfide bonds and Michael addition reaction products, but other methods known in the art can be used to prepare conjugates. Examples of other binding (coupling) procedures include the use of dialdehydes such as glutaraldehyde (set forth above) and the like, or the use of a carbodiimide technique, such as in the case of amide bonds between a carrier and a polypeptide in a water-soluble carbodiimide, e.g. 3- (3-dimethylaminopropyl) carbodiimide.

As is well known in the art, it is often advantageous to bind a synthetic polypeptide to a carrier via a mediating linking group. The aforementioned glutaraldehyde is one such linking group.

However, if cysteine is used to attach the peptide to the carrier, then the mediating linking group is preferably, as mentioned above, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The MBS is typically first attached to the support by an ester-amide exchange reaction. This may be followed by the aforementioned Michael reaction, or the addition of MBS may be followed by the addition of a protected mercapto group such as thiolacetic acid (CH 2 Cl 2) by a Michael addition reaction across the maleimido double bond. After removal of the protecting acyl group

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is 81452 a disulfide bond is formed between the unprotected linking mercaptan group and the mercaptan of the cysteine residue added to the synthetic polypeptide.

The choice of carrier depends more on the end use of this immunogenic polypeptide than on the determinant portion of the immunogen, and this choice of carrier is based on criteria not specifically related to this invention. For example, if the inoculum is to be used in animals, e.g., to produce antibodies to this polypeptide that are used to test for the presence of gonococcal pilin protein, then a carrier should be selected that does not cause adverse reactions in that particular animal.

As used herein, the term "synthetically produced" means that the polypeptide molecule or repeating unit formed by the polypeptide has been synthesized by chemical means, i.e., synthesized chemically or by human-developed biological methods, e.g., using recombinant DNA technology. Thus, the synthetically prepared polypeptides of this invention do not contain naturally occurring proteins or protein fragments. The most preferred synthetic method, described in more detail below, is the well-known solid phase chemical synthesis in which protected amino acid residues are added one after the other to obtain the desired polypeptide.

As discussed above, polypeptides suitable for the purposes of this invention can be synthesized by the well-known solid phase IE 81452 method. See, e.g., Houghten et al.,

Int. J. Pept. Proc. Res. 16: 311-320 (1980), and Merrifield, J. Am. Chem. Soc. 85 ^: 2149-2154 (1963), which publications are incorporated herein by reference. Synthesis of polypeptides by the solid phase method can be performed on a Beckman peptide synthesizer, Model 990B, commercially available from Beckman Instruments Co., Berkeley, CA, USA.

When the synthetic polypeptides of the present invention are prepared by the above-mentioned solid phase method, the amino acid residues are coupled to the resin (solid phase) from their carboxy-terminal residue by an ester bond. If the polypeptide is to be attached to the support via a CYS residue, then it is common to use this CYS residue as this carboxy-terminal residue, which is further bound by an ester bond to the resin.

The alpha-amino group of each amino acid is typically protected with a tertiary butoxycarbonyl group (t-BOC) prior to attachment of the amino acid to the growing polypeptide chain. This t-BOC group is then removed before the next amino acid is added to this growing polypeptide chain. Likewise, the reactive side chains of amino acids are protected during polypeptide synthesis. Common groups used for side chain protection of the remaining amino acid residues are: O- (p-bromobenzoxycarbonyl) for tyrosine, O-benzyl for threonine, serine, aspartic acid and glutamic acid, and S-methoxybenzyl for cysteine. The protected amino acids are recrystallized from the appropriate solvents to give a single spot by thin layer chromatography. Coupling reactions are usually performed using a ten-fold molar excess of both the protected amino acid and dicyclohexylcarbodiimide over the number of milliequivalents of the original N-terminal amino acid. A double molar excess of both reagents can also be used.

Il 17 81 452

In the case of asparagine, an equal molar amount of N-hydroxybenzotriazole is added to the protected amino acid, and dimethylformamide is used as the solvent. All coupling reactions are typically more than 99% complete based on the picrin acid assay, see Gisin, Anal. Chem. Acta, 58_: 248-24 9 (1972).

A portion of the resulting protected, resin-bound polypeptide (1 gram) is treated with two milliliters of anisole, and 20 milliliters of anhydrous hydrogen fluoride are concentrated in a reaction vessel at the temperature of carbonic acid. The resulting mixture is stirred at 4 ° C for 1.0 hour to remove the protecting groups, as well as to remove the polypeptide from the resin. After evaporation of the hydrogen fluoride at 4 ° C with a stream of nitrogen, the residue is extracted three times with anhydrous diethyl ether 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 ml each), followed by extraction with 50% aqueous acetic acid (4 times 50 ml). The first extraction removes the low molecular weight polypeptides as well as tyrosine, which is used in some preparations to protect the mercapto groups of the CYS residue. The second extraction separates the free polypeptide from the resin. After this mixture is diluted with water to an acetic acid content of 10-20%, the resulting solution is lyophilized to give a monomeric, non-oxidizing polypeptide.

Multimers of this polypeptide can be prepared by sequentially binding the monomers of the synthesized polypeptide to the carboxy terminus ("head-to-tail") of the amino terminus of one polypeptide using the above solid phase method, i.e., one complete polypeptide chain can be synthesized attached to a resin followed by synthesis of one or more ie 81452 a similar or different polypeptide chain, after which this whole multimeric unit is cleaved from the resin and used as described below.

Alternatively, synthetic polypeptide monomers having a cysteine residue attached to both the amino and carboxy termini (diCYS polypeptides) can be linked together by intramolecular, interpolar cysteine disulfide bonds using an oxidation method to yield an immunogenic polymer. This polymer thus prepared contains numerous polypeptides of this invention in their repeating units. These repeating units are bound to each other via the aforementioned oxidized cysteine residues.

The presence of one or two CYS residues attached to the end or ends of a polypeptide of this invention that allow binding of this polypeptide to a carrier, or the preparation of a polymer, should not be construed as an amino acid chain change in repeating units of the polypeptide compared to a chain that immunologically mimics the gonococcal antigenic determinant.

In a typical laboratory preparation, 10 milligrams of diCYS polypeptide (having an unoxidized cysteine residue at the amino and carboxy termini) is dissolved in 250 ml of 0.1 M ammonium bicarbonate buffer at a pH of about 8. The dissolved diCYS polypeptide is then oxidized by stirring with air. cautiously for about 18 hours, or until the Ellman experiment no longer detects free mercapto groups. / See Ellman, Arch. Biochem. Biophys. 82: 70-77 (1959-7. Thereafter, the polymer thus prepared is typically isolated by lyophilization, redissolution, and chromatographic purification.

Typical methods that can be used to prepare fusion proteins using recombinant DNA technology are described in Berman, BioTechniques 1 (4): 178-183 (1983), Silhavy et al., Microbiological Reviews 47 (3): 313-344

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19 81 452 (1983), and Young et al., Proc. Natl. Acad. Sci. USA 80: 1194-1198 (1984).

The compositions of this invention comprise one or more of the polypeptides described above and a pharmaceutically acceptable diluent, e.g., physiological saline, phosphate buffered saline (PBS), or other injectable fluid. If desired, they may also contain additives commonly used in grafts. Examples of such additives are stabilizers, such as lactose or sorbitol, and excipients, such as aluminum hydroxide, sulphate or phosphate, alum or alginate. Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant are preferably used in inoculations.

20 81 452

The inoculum contains an "effective amount" of this immunogen, the effective amount of which depends on several factors well known in immunology, such as the body weight of the mammal to be immunized, the carrier used, the excipient used, the duration of protection sought, and the desired schedule for immunization.

In addition, whole antibodies formed by the action of a polypeptide of the present invention, as well as substantially whole antibodies, and antibody binding sites derived from such antibodies, form another aspect of the present invention. These molecules are collectively referred to as the "receptor".

Receptors can be generated in mammals, such as rabbits, goats, horses, and the like, by immunizing them with the inoculations described above. The methods used for immunization are essentially the same as those used for vaccinations, except that strong adjuvants such as CFA and / or IFA may be used in inoculations given to animals, which are not acceptable for human use.

Typical inoculum stock solutions are prepared using CFAr, IFA, or alum as follows: The polypeptide, synthetic polypeptide conjugate, or polymeric polypeptide is dissolved in PBS pH 7.2 in an amount to provide the desired effective amount of polypeptide in the inoculum.

This polypeptide solution is then mixed with an equal volume of CFAr or IFAr to provide an inoculum containing the polypeptide, water, and excipient, with a water to oil ratio of about 1: 1. This mixture is then homogenized to give an inoculum stock solution. When alum is used, about 200 micrograms of conjugate is absorbed into about 4 milligrams of alum to provide an inoculum stock mixture.

. Rabbits can be used to produce antibodies against these polypeptides. When using rabbits

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81452 A host rabbit is usually injected subcutaneously with an inoculum comprising 200 micrograms of CFArha emulsified polypeptide conjugate (polypeptide plus carrier); 200 micrograms of polypeptide conjugate in IFA; and 200 micrograms of the polypeptide conjugate along with 4 milligrams of alum is injected intraperitoneally on days 0, 14, and 21 of the immunization schedule, respectively. Each inoculation (immunization) consists of four injections of inoculation.

Mice can be immunized in the same manner using one-tenth of the above doses for each injection.

Animals are typically bled 4 and 15 weeks after the first injection. The control pre-immunization serum is obtained from each animal by collecting blood immediately before the first immunization.

As a control, inoculum stock solutions can also be prepared using keyhole limpet snail hemocyanin (KLH), KLH in CFAR or lFA, alum absorbed KLH, alum absorbed KLH, and pertussis toxin, edestine, thyroglobulin, tetanus toxoid In IFA, cholera toxoid as well as cholera toxoid in IFA, and the like.

The efficacy of the above immunization method is typically determined by an ELISA assay in which the immunogenic polypeptide of this invention is used as an antigen to determine the amount of antibodies present in the diluted serum obtained from the aforementioned blood samples. Sera that produce anti-polypeptide antibody titers (dilutions) of at least about 1: 160 are considered useful in the production of the antibodies of this invention. The ELISA assay typically used is described in more detail in Bittle et al., Nature 298: 30-33 (1982), which is incorporated herein by reference.

22 81 4 52

Suitable monoclonal receptors, typically whole antibodies, can also be prepared using hybridoma techniques, as described in Niman et al., Proc. Natl. Acad. Sci., USA 80: 4949-4953 (1983), the disclosure of which is incorporated herein by reference. It is not necessary that the monoclonal receptors be obtained from the supernatant of the hybridoma, but they may also be obtained, usually in larger amounts, from the serum-derived fluid collected in the stomach of the mammals into which the desired hybridoma was implanted. The production of mono-clonal antibodies using fluid accumulated in the stomach is well known and will not be discussed further herein.

The receptor of the present invention binds both to the polypeptide which caused the receptor to form and to to the corresponding pilin protein whose antigenic determinant is immunologically mimicked by the polypeptide of the present invention. Thus, a polypeptide of the present invention may be both an immunogen and an antigen.

The receptors of this invention are at least oligoclonal to naturally occurring polyclonal antibodies because they are formed by an immunogen having relatively few epitopes compared to epitopes of a complete pilin molecule. Thus, the receptors of this invention bind to epitopes of the polypeptide, whereas naturally occurring antibodies formed by the action of the gonococcal pilin protein bind to all epitopes of the pilin molecule.

The polypeptides of this invention, as well as the antibodies and antibody binding sites obtained from these polypeptides, as well as the methods of this invention, may also be used in diagnostic assays, such as immunoassays. Such diagnostic methods include, for example, enzymatic immunoassays, enzymatic multiple immunoassay methods (EMIT), enzyme-linked immunosorbent assay.

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23 81452 (ELISA), radioimmunoassays (RAI), fluorescence-based irane assays, either single or double antibody methods, and other techniques in which either the antibody binding site or antigen is labeled with a detectable substance or moiety. A general presentation can be found in Maggio, Enzyme Immunoassay, CRC Press, Cleveland, Ohio (1981), and Goldman, M., Fluorescent Antibody Methods, Academic Press, New York, N.Y. (1980).

A diagnostic system for detecting N. gonorrhoeae that illustrates the present invention comprises receptor molecules that have been generated by a polypeptide of the present invention, such as antibodies, substantially whole antibodies, or antibody binding sites. This system likewise comprises an indicator of the occurrence of an immunoreaction between the receptor and the antigen. The immune response can be expressed by this indicator. When a body sample, such as a paste from the cervix or ureteral genitals, is mixed with the receptor molecules, these receptor molecules immunoreact with the pilin antigen to form the product of the immunoreaction, and the indicator present then signals the occurrence of the immunoreaction.

One such illustrative embodiment is an immunofluorescence assay in which a paste obtained from the cervix or urinary tract is attached to a standard microscope plate with acetone. Samples of antibodies raised in accordance with this invention, for example, generated in rabbits, usually from about 10 micrograms to about 500 micrograms, are incubated on these plates using known methods.

After all non-immunoreacted antibodies have been rinsed off and the non-specific binding sites on the plate have been protected with a protein, such as BSA, another 24,81452 antibody, such as a goat anti-rabbit antibody, can then be incubated on this assay plate, if desired. . This second antibody is labeled with a fluorochromatic dye such as fluorescein isothiocyanate (FITC).

After this second incubation, the excess of the second antibody is rinsed off, leaving FITC-labeled goat anti-rabbit antibodies bound to the first antibody on the test plate. The presence of FITC-labeled antibodies can be detected under a fluorescent microscope to detect the presence of N. gonorrhoeae infection.

The use of whole, undamaged, biologically active antibodies corresponding to the receptor molecules is not necessary in many diagnostic systems, such as the immunofluorescence assay described above. Only the immunologically active, idiotype-containing, antigen-binding and recognizing receptor site in the antibody molecule, i.e., the antibody binding site, can be used in these experiments. Examples of such antibody binding sites include antibody portions known in the art by the abbreviations Fab and F (ab ') 2, which can be prepared by methods well known in the art.

Another diagnostic method of the present invention is an ELISA assay. In this experiment, the antigen formed by the polypeptide of this invention is bound to a solid support, such as the wells of a microtiter plate. The non-specific binding sites on the walls of the wells of the microtiter plate are then protected with a protein such as BSA. Unbound polypeptide and BSA are removed from the wells of the microtiter plate by rinsing.

For example, a body sample such as the one mentioned above is mixed with an excess of an aqueous solution of the antibody of this invention, and this mixture is allowed to stand until the immuno-

II

The reaction between the antibody and the gonococcal pilin antigen present has time to occur. This liquid mixture is then poured onto the solid support-bound polypeptide described above to form a second mixture comprising a solid and a liquid phase. This mixture of solid and liquid phases is allowed to stand until the antibodies unreacted in the previous step have time to immunoreact with the antigen formed by the polypeptide. The liquid phase is then separated from the solid phase.

A solution containing a second labeled antibody that reacts with the first antibody is then mixed into this solid phase. An example of such a second antibody is a peroxidase-linked goat anti-rabbit antibody, wherein the former antibodies have been generated in rabbits. The mixture of solid phase and this second labeled antibody solution is allowed to stand until an immunoreaction takes place between the two antibodies. The solid and liquid phases are then separated.

A solution containing a substrate of said enzyme, such as hydrogen peroxide, and a precursor of a color-forming dye, such as o-phenylenediamine, are then mixed into this solid phase. The optical density of the sample can be determined at a preselected wavelength (e.g., 405 nanometers) after a predetermined period of time, and this optical density is compared to the optical density of the reference to determine whether or not gonococcal antigen was present in the body sample.

The present invention is further illustrated by the following detailed examples.

26 81 452

Example 1

Synthesis of polypeptides

A series of short synthetic polypeptides with amino acid residue sequences corresponding to small portions of the gonococcal pilin protein were synthesized in Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963), as described in Houghten et al., Int. J. Pept. Proc. Res., 16: 311-320 (1980), using a Beckman peptide synthesizer, Model 990B (Beckman Instruments Co., CA, USA). The description of the polypeptide, as well as the location of the chains of their corresponding amino acid residues in the gonococcal hair protein, is shown in Table I below.

Table I

Synthetic polypeptides corresponding to portions of the gonococcal pilin protein 2

Description Location Amino acid residue chain

441 140-159 KEIDTKHLPSTCRDKASDA

GC41 145-153 KHLPSTCRD

GC51 144-159 TKHLPSTCRDKASDAK

GC61 115-127 GSVKWFCGQPVTR

The polypeptides are coupled to the tetanus toxoid using the glutaraldehyde method described in Example 2.

2

The location corresponds to the positions of the amino acid residues in the pilin protein chain of the gonococcal bacterium described in Meyer et al., Proc. Nat'1 Acad. Sci. USA (1984) 81: 6110-6114.

Example 2

Coupling of the polypeptide to the carrier

Conjugation of the polypeptide to the tetanus toxoid carrier using the glutaraldehyde method was accomplished by mixing equal amounts of peptide and tetanus toxoid in PBS to a final concentration of 2 mg / ml. If difficulties were found in dissolving any of the peptides, the pH of the resulting mixture was raised to approximately 8.0.

Il 27 8 1 4 52

Prior to each coupling reaction, a new working dilution of glutaraldehyde was prepared by diluting a 25% stock solution of glutaraldehyde (w / v in PBS) 1:65 with ice-cold PBS. This new glutaraldehyde solution was then added to the peptide and carrier solution prepared above in a ratio of 124 microliters to 1 milliliter, respectively. The resulting reaction mixture was incubated with stirring overnight at room temperature.

After incubation, the reaction product was dialyzed against distilled water for at least 6 hours, after which the product was lyophilized.

Example 3

Screening for Anti-Polypeptide Antibodies in Rabbit Sera The presence of anti-polypeptide antibodies in rabbit antisera was determined using an enzyme-linked immunosorbent assay (ELISA). The antigen formed by the polypeptide prepared as described in Example 1 above was adsorbed to the well walls of a microtiter plate to obtain a solid phase-bound target antigen.

To prepare the solid phase bound polypeptide, 25 μl of a 0.1% (w / v) BSA / PBS mixture containing about 5 picomoles of polypeptide was placed in the well of a microtiter plate and incubated at 37 ° C until the liquid had completely evaporated. The antigen thus formed by the deposited polypeptide was attached to this solid phase by incubating 50 μl of methanol in each well for 5 minutes at room temperature. After incubation, the methanol was removed by inverting and shaking the plate and allowing the plates to air dry for 5-10 minutes.

The non-specific binding sites on the walls of the wells of the microtiter plate were then protected by incubating 50 μl of 3% (w / v) BSA / PBS in each well for 4 hours at 37 ° C in a humid chamber. After incubation, 28 81 452 excess BSA was removed by inverting and shaking the plates. Thus, a polypeptide bound to a solid support with protected non-specific binding sites was obtained, which could be used as a target antigen.

To determine the presence of anti-polypeptide antibodies in rabbit sera, a series of dilutions were prepared from each sample obtained by diluting the sample in duplicate with 1% (w / v) BSA / PBS. 25 microliters of each dilution was contacted with the solid phase bound polypeptide by mixing this 25 microliters into the appropriate well of the microtiter plate prepared above. This contact was maintained by incubating these wells for about 16 hours at 37 ° C in a humidified chamber, allowing any anti-polypeptide antibodies present in the serum dilutions to immunoreact with the target antigen formed by the solid phase-bound polypeptide. After incubation, the solid and liquid phases were separated by filling the wells with distilled water, inverting the wells and shaking them 10 times in succession.

To detect the presence of an immunoreaction between anti-polypeptide antibodies and solid-phase bound antigen, 25 ul of goat anti-rabbit IgG labeled with indicator peroxidase (Boehringer Mannheim Biochemicals; Indianapolis, Indiana) was mixed into each well. After incubation for 1.5 hours at 37 ° C in a humidified chamber, they were washed 10 times with distilled water as described above. Each well was then mixed with 50 microliters of developer solution / 55 mg of ABTS (2,2'-azino-di- (3-ethyl-benzthiazoline sulfonate); Boehringer Mannheim) and 100 of 30% hydrogen peroxide. Rated to 100 ml of 0.1 M sodium citrate (pH 4.2), and each well was incubated for about 40-60 minutes at room temperature.The indicator reaction was stopped by mixing 50 μl of 5% (w / v) sodium dodecyl sulfate (SDS) in water in each well. The magnitude of the indicator reaction (color development) was determined by measuring the absorbance of each well at 414 nm.

Il 29 81 452

Rabbit antisera with a titer found to be 4-fold higher than in the negative control were considered positive for the presence of anti-polypeptide antibodies. Antibodies that immunoreacted with their inducing polypeptides were generated in rabbits using all of the peptides listed in Table I above.

Example 4

Western * in spotting test

Rabbit antisera were further examined to determine their ability to immunoreact with gonococcal pilin protein by Western blotting. Gonococcal pilin protein was isolated from N. gonorrhoeae strain MS-11 (described in Meyer et al., Supra) by the following method.

N. gonorrhoeae strain MS-11 was plated and grown as a unitary bacterial layer on GCB medium (Diffco Co., Detroit, MI) in 15 x 100 mm petri dishes (American Scientific Products, McGaw, IL). Cultures from 50 plates were transferred to 35 ml of 50 mM ethanolamine, pH 10.5. The cilia were removed from the bacteria by rotating the bacterial suspension for 30 seconds, and immersing it in a bath at 0 ° C for 30 seconds four times in a row.

The detached cilia were separated from the bacteria by centrifugation of the suspension at 10,000 rpm for 20 minutes in a JA20 rotor (Beckman Instruments, Fullerton, CA.). The pilin protein-containing supernatant was then dialyzed against cilia buffer (0.15 M NaCl, 0.05 M Tris, pH 7.5) for approximately 16 hours. During dialysis, most of the pilin protein present formed insoluble aggregates. These pellets were then isolated by centrifugation at 10,000 rpm for 15 minutes in the above rotor. This pellet of pilin protein was then resuspended at 1 mg / ml in 50 mM ethanolamine, pH 10.5.

30 8 1 4 5 2

The pilin protein was further isolated electrophoretically on a 15% SDS-polyacrylamide gel, and transferred to nitrocellulose (Schleicher & Schuel, Cat. No. BA85, Keene, NH) using an electronic spotting apparatus (CBS Scientific, Del Mar, CA) and transfer buffer comprising 25 mM Tris, 192 mM glycine, 20% methanol and 0.01% NaN 2.

The resulting gonococcal pilin protein (Western blot) bound to a solid support to form a nitrocellulose was then used as a target antigen in a Western blot assay to detect anti-pilin antibodies.

Anti-polypeptide antisera from rabbits were diluted approximately 1: 100 with 3% (w / v) BSA / PBS or BLOTTO / PBS containing 50 g / l skim milk powder, 0.1 ml / l antifoam antifoam A- emulsion (Sigma Chemical Co., St. Louis, Mo.) and 1 ml / L thimerosal7 · The Western blot was then incubated in 25 ml of each diluted antiserum for approximately 16 hours at 4 ° C with constant agitation, whereby the anti-serum polypeptide antibodies came into contact with solid phase-bound pilin protein.

After incubation, the spots were washed twice with 25 ml of TBS buffer (0.9% NaCl, 10 mM Tris, pH 7.4) containing 0.5% octylphenoxy-polyethoxyethanol (Triton X100), washed three times with 25 ml of TBS alone, and then washed with 25 ml of 3% (w / v) BSA / TBS solution.

Anti-polypeptide antibodies bound to gonococcal pilin protein were detected by first reacting the spots with peroxidase-labeled goat anti-rabbit IgG (Boehringer Mannheim) diluted approximately 1: 2000 3% (w / v). BSA / TBS or BLOTTO solution, and the reaction was allowed to proceed for 16 hours at 4 ° C with constant stirring. The spots were then washed as described above, and developed by incubating them in 25 ml of the above

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si 81452 of the ABTS developing solution described in Example 3 for about 45-60 minutes.

The results from the Western spotting experiment indicate that all anti-polypeptide antisera generated in rabbits immunized with the polypeptides listed in Table I also contain antibodies that immunoreact with the gonococcal bacterial hair protein.

Example 5 Neisseria ELISA

Anti-polypeptide antibodies that immunoreacted with the pilin protein of the gonococcal bacterium in the Western blot assay described in Example 4 were tested for their ability to immunoreact with the whole bacterium used in the ELISA as a solid phase-bound target antigen. Eleven Neisseria strains, ten of which were pathogenic gonococcal strains (Table 2 below) were obtained from J. Knapp, Neisseria Reference Lab, Seattle, WA. Strains were grown on GC succinic agar at 35 ° C in 5% CC in a humidified incubator and confirmed by Gram staining, oxidase reaction, colony morphology and sugar fermentation progress, as is standard in the art.

Table 2 Tests tested

Strain number (NRL) Description 7122 N. gonorrhoeae POMP 1 * 8658 N. gonorrhoeae POMP 2 7929 N. gonorrhoeae POMP 3 6611 N. gonorrhoeae POMP 4 5767 N. gonorrhoeae POMP 5 8035 N. gonorrhoeae POMP 6 5766 N. gonorrhoe N. gonorrhoeae POMP 8 8660 N. gonorrhoeae POMP 9 1955 N. gonorrhoeae Will

9206 N. meningitidis B

32 81 452

A stock solution of Neisseria antigen was prepared by transferring cultures grown as described above to PBS (pH 7.4). The optical density of each bacterial suspension was then set to 0.1 using 750 nm in PBS (pH 7.4), aliquoted and frozen.

Solid-phase bound Neisseria anticene was prepared by adding 50 of a stock suspension of Neisseria bacteria to the wells of a microtiter plate ("Immunolon 1"). Cell pellets different from solution were formed on the walls of these wells by centrifugation at 1000 g for 5 minutes at 4 ° C in a table fug.

After centrifugation, Neisseria antigen was cross-linked to the solid phase by mixing 200 μl of PBS in each well.

O

prepared 0.25% glutaraldehyde at 4 ° C. After incubating the wells for 5 minutes at 4 ° C, the wells were washed Four times with PBS. Non-specific binding sites were protected by incubating in each well for 2 hours at 37 ° C 350 μl of a 5% BSA / PBS solution containing 0.05% polyoxyethylene (20) sorbitan monolaurate ("Tween 20"). The BSA solution used for protection was then removed by suction to obtain Neisseria bacteria bound to the walls of the wells of the microtiter plate, which acted as target antigens.

Rabbit anti-polypeptide antisera tested for anti-Neisserial activity were serially diluted by doubling each time with 1% (w / v) BSA / PBS. 200 microliters of each dilution was taken and mixed into the wells prepared as described above to give a mixture of solid and liquid phase immuno-reactions. Contact was maintained by incubating the wells for 1 hour at 37 ° C, during which time the anti-polypeptide antibodies present in the serum dilutions had time to immunoreact with the solid phase-bound Neisseria antigen. After incubation, the solid and liquid phases were separated by washing the wells three times with PBS containing 0.05% "Tween 20".

Il 33 81 452

Anti-polypeptide antibodies immunologically bound to solid-phase attached Neisseria antigen were detected by an indicator formed by peroxidase-labeled goat anti-rabbit IgG from Cappel, A Division of Cooper Biomedical, Malvern, PA. Goat anti-rabbit IgG was diluted 1: 2000 with 1% (w / v) BSA / PBS and 200 μl of the dilution was transferred to each well. The mixtures were incubated for 1 hour at 37 ° C to allow an immunoreaction between goat anti-rabbit IgG antibodies and any rabbit antipolypeptidic antibodies bound to solid phase-attached Neisseria antigen. After incubation, the wells were washed five times, each time lasting one minute, as described above.

The presence of goat anti-rabbit IgG antibodies bound to the products of the immunoreaction between rabbit anti-polypeptide antibodies and Neisseria was detected using O-phenylenediamine as a peroxidase substrate. 200 microliters of developing O-phenylenediamine (ODSI-Carpinter, Carpinter, CA) was mixed in each well, and the wells were incubated for 30 minutes at room temperature.

The color development reaction was stopped by adding 50 μl of 2 N HCl to each well. The magnitude of the indicative reaction present was determined immediately by measuring the absorbance of each well at 490 nm using a Dynatech ELISA plate analyzer.

The ELISA assay described above determined the ability of antibodies against gonococcal pilin polypeptide to (1) differentiate between pathogenic and host-only Neisseria strains; and (2) immunoreact with representatives of pathogenic strains of N. gonorrhoeae.

In the first set of experiments, anti-polypeptide antibodies were immunoreacted against the pathogenic N. gonorrhoeae strain against POMP 1 serovar (NRL 7122) and against host-only N. meningitidis strain B (NRL 9206).

The ability of the antibody to distinguish between pathogenic and host-only strains was then determined by calculating the ratio of the absorbance obtained in the pathogenic strain ELISA and the absorbance obtained in the host-only ELISA. The results obtained with anti-polypeptide antibodies in this study are shown in Table 3 below.

Table 3

Specificity study of antibodies against rabbit N. gonorrhoeae against pilin peptide_

Peptide Rabbit Absorbance at 490 nm ID no. antiser-.,, ...

.. N. gonorrhoeae N. meningitidis B.

T_. mi (strain 7122) (strain 9206) Ratio1 ID number 94312 0.037 0.136 94 322 0.068 0.013 44 T4 4 0.848 0.599 1.4 GC4 GC4-9307 0.661 0.324 2.0 GC4 GC4-9309 0.239 0.283 0.8 GC6 GC6-9310 0.274 0.119 2.3 N. gonorrhoeae / N. the ratio of meningitidis absorbance values; dilution of antisera 1: 3200.

2

Negative comparisons

As can be seen from the previous table, the antibodies induced by the polypeptides of this invention, in particular the antibodies against the polypeptides GC4 and GC6 shown in Table 1, can be found to have significant immunospecificity against a pathogenic N. gonorrhoeae strain, utilizing only N. compared to the meningitidis strain. The polypeptides of this invention are suitable components for use in vaccines and grafts because they direct the host's immune response against disease-causing Neisserial species.

Il 35 81 452

In a second series of experiments using a Neisseria antigen-based ELISA, the ability of anti-polypeptide antibodies produced in accordance with this invention to immunoreact with representatives of pathogenic N. gonorrhoeae strains was examined. The results from these experiments, shown in Table 4 below, indicate that the antibodies formed by the more preferred polypeptides GC4 and GC6 have broad immunospecificity against pathogenic strains of N. gonorrhoeae. The antiserum GC4-9307 contained a high enough titer of anti-polypeptide antibodies to be able to identify nine of the ten pathogenic strains studied (i.e., all but no strain NRL 8035) when used at a 1: 1600 dilution and compared to pre-immunization. compared to the reference serum. Similarly, antiserum GC6-9310 identified seven of ten pathogenic strains (i.e., all but no strains NRL 8035, NRL 5766, and NRL 8038) at a 1: 1600 dilution.

Table 4

Effect of rabbit antisera on the Neisseria bacterial series

Strain Absorbance at 490 nm λ NRL n ° 'GC4-9307 GC6-9310 94322 7122 0.678 0.133 0.021 8658 0.541 0.229 0.003 7929 0.616 0.205 0, 6611 0.697 0.197 0.060 5767 0.659 0.191 0.098 8035 0.690 573 0.83 0.83 8038 1,069 0.169 0.220 8660 0.833 0.273 0.12 1955 0.550 0.220 0.084 9206 0.626 0.120 0.020 ^ 1: 1600 dilution of antisera 2

Reference antiserum 36 81452

The immunospecificity (cross-reactivity) of the various pathogenic strains shown in Table 4 above indicates that the polypeptides of this invention induce broad-spectrum antibodies. The ability to induce broad-spectrum antibodies is a highly desirable feature in vaccine development because many pathogens, including N. gonorrhoeae, remain unidentified and destroyed by the host immune system due to antigenic (strain) modification.

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Claims (4)

37 81 452 A diagnostic system suitable for detecting Neisseria gonorrhoeae pilus protein, characterized in that the system comprises (a) receptor molecules formed in a host animal by a polypeptide smaller than the naturally occurring gonococcal cilia protein comprising a chain of amino acid residues consisting of at least five amino acid residues and up to about 60 amino acid residues, which is LYS-HIS-LEU-PRO-SER-THR-CYS-ARG-ASP-, or -THR-LYS-HIS-LEU-PRO-SER- THR-CYS-ARG-ASP-LYS-ALA-SER-ASP-ALA-LYS-, GLY-SER-VAL-LYS-TRP-PHE-CYS-GLY-GLN-PRO-VAL-THR-ARG-, or antigen-like variants and capable of mimicking an immunologically stable antigenic determinant site in the carboxy-terminal variable region of a gonococcal pilin protein, and (b) an indicator capable of indicating said receptor an immunoreaction between rim molecules and a pilus protein. Diagnostic system according to Claim 1, characterized in that the polypeptide has the formula 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 or H-GLY-SER-VAL-LYS-TRP-PHE-CYS-GLY-GLN-PRO-VAL-THR-ARG OH. Diagnostic system according to Claim 1, characterized in that the receptor is a whole antibody. The diagnostic system of claim 1, wherein the receptor is an antibody binding site. 38 81 452