EP1162880A1 - Human sperm surface antigens - Google Patents
Human sperm surface antigensInfo
- Publication number
- EP1162880A1 EP1162880A1 EP00919555A EP00919555A EP1162880A1 EP 1162880 A1 EP1162880 A1 EP 1162880A1 EP 00919555 A EP00919555 A EP 00919555A EP 00919555 A EP00919555 A EP 00919555A EP 1162880 A1 EP1162880 A1 EP 1162880A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- sperm
- antigens
- amino acid
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/16—Masculine contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/18—Feminine contraceptives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention is directed to sperm cell surface antigens that are recognized by antisperm antibodies present in the serum of infertile men and women. More particularly the invention is directed to sperm-specific surface protein/peptide epitopes as well as derivatives and antibodies directed to those protein epitopes.
- ASA antisperm antibodies
- the present invention is directed to proteins identified by high resolution 2-D electrophoresis.
- the sperm antigens are separated in the first dimension by either isoelectric focusing or nonequilibrium pH gradient electrophoresis (to screen a range of acidic and basic proteins) followed by PAGE and a sensitive western blotting method.
- the subject sera were initially screened for the presence of antisperm antibodies by the immunobead test (IBT, as described in Bronson R, Cooper G, Rosenfeld D. In : Jagiello G and Vogel H (Eds), Bioregulators of reproduction, academic press, New York 1981 : 521-527) and only those sera showing significant reactivity for the presence of antisperm antibodies were utilized to identify the immunodominant antigens.
- IBT immunobead test
- sperm antigens unique to infertile patients were identified by excluding those antigens recognized by sera from clinically fertile subjects using computerized comparison of 2-D immunoblots.
- a database of 2-D gel images of silver stained proteins (sperm proteome) and a database of vectorially labeled sperm surface proteins (sperm surface index, Biol Reprod 1997; 56: 771-787) allowed the definition of a subset of sperm surface antigens relevant to antibody mediated infertility.
- the present invention is directed to therapeutic and diagnostic compositions and methods of using those compositions in procedures relating to fertility.
- the compositions of the present invention comprise sperm cell surface antigens that are recognized by antisperm antibodies present in the serum of infertile men and women.
- the invention further relates to therapeutic compositions and methods of diagnosis and therapy, including compositions and methods for modulating fertility (i.e. inhibiting or promoting). Brief Description of the Drawings
- Figs. 1A-1D represent a Western blot analysis by serial incubation of a single blot with 5 sera from fertile males (Fig. 1A) and fertile females (Fig. IB) compared with 5 infertile males (Fig. IC) and infertile females (Fig. ID) (Dil:l :2000). Note major auto- and iso-antigens (arrows) which are uniquely recognized by the infertile subjects.
- Figs. 2A-2B represent computer generated images showing the major auto-antigens (Fig. 2A) and iso-antigens (Fig. 2B), recognized by infertile sera.
- the computer scanned images of western blots were compared between fertile and infertile groups using common protein spots across two images as anchors.
- the protein spots recognized by the fertile subject sera (both male and female) were subtracted from the image representing the infertile male and female subjects and an image depicting all antigens uniquely recognized by the infertile males (Fig. 2A) and infertile females (Fig. 2B) was generated. Note that there are more auto-antigens than iso-antigens and that there is a differential pattern of immunoreactivity between the sexes. Arrows indicate the protein spots recognized by both male and female subjects.
- Fig. 3. represents a computer generated composite image of two dimensional IEF/PAGE of vectorially iodinated human sperm proteins.
- the sperm were radioiodinated, and solubilized as described in Example 1 and separated by two dimensional eletrophoresis by IEF/PAGE.
- the composite image was generated after scanning the autoradiograms of the surface proteins immobilized on nitrocellulose membrane and comparing the images to the locus of auto-antigens and iso-antigens (Fig. 1). Arrows point to the locus of immunodominant surface auto- and iso- antigens.
- nucleic acid As used herein, “nucleic acid,” “DNA,” and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone.
- nucleic acid analogs i.e. analogs having other than a phosphodiester backbone.
- peptide nucleic acids which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
- an effective amount means an amount sufficient to produce a selected effect.
- an effective immunizing amount of the sperm surface protein is an amount sufficient to induce an immune response in the host to which the vaccine preparation is administered.
- the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
- the present invention is directed to sperm surface antigens that are recognized by sera isolated from infertile patients but not by sera isolated from clinically fertile subjects.
- the sperm antigens of the present invention are unique to infertile patients and were identified by excluding those antigens recognized by sera from clinically fertile subjects using computerized comparison of 2-D immunoblots.
- a database of 2-D gel images of silver stained proteins (sperm proteome) and a database of vectorially labeled sperm surface proteins (sperm surface index, Biol Reprod 1997; 56: 771-787) allowed the definition of a subset of sperm surface antigens relevant to antibody mediated infertility.
- SSPs sperm surface proteins
- the SSP proteins, polypeptides and peptide fragments thereof can be prepared for a variety of uses. For example, such molecules can be used for the generation of antibodies, for use in diagnostic and therapeutic assays, for the identification of other sperm gene products involved in sperm motility, or for the identification of compounds that modulate sperm motility.
- One embodiment of the present invention is directed to a pharmaceutical composition comprising a sperm surface peptide epitope and a pharmaceutically acceptable carrier.
- the pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like as well as buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
- Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
- Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
- Suitable pharmaceutical excipients for use in the compositions of the present invention include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
- the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
- composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
- Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
- Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
- the formulation should suit the mode of administration.
- the SSP containing compositions of the present invention are used to modulate fertility in a mammalian species.
- the method comprises administering a composition comprising a therapeutically effective amount of an SSP antigen.
- the composition is formulated as a contraceptive vaccine comprising one or more SSP proteins (or SSP protein fragments or analogs thereof) and administered to a mammalian species to induce an immune response.
- the SSP-containing composition is used to treat a subject for a fertility-related disorder by administering an effective amount of the sperm surface protein or fragment thereof.
- the SSP compositions of the present invention can be used in a method for treating immunological infertility.
- the method comprises obtaining a serum sample form the individual exhibiting immunological infertility, determining the sperm surface proteins targeted by the antibodies present in the individual's serum and administering a composition comprising one or more peptides (or peptide derivatives or peptide mimetics) targeted by the individual's antibodies.
- the composition is administered topically to a female to bind to the individual's anti-sperm antibodies and thus enhance the fertility of the female.
- the SSPs of the present invention can be used to screen for molecules that interact with sperm surface proteins, including those molecules that bind to the surface proteins.
- the method comprises contacting the sperm specific protein/peptide with one or more molecules under conditions conducive to complex formation between the sperm specific protein/peptide and the molecule, and recovering the molecule that specifically binds to the sperm surface protein/peptide.
- the invention provides polypeptides and their respective fragments and analogs that have at least one of the following activities: capable of modulating sperm's ability to fertilize an egg, function as sperm marker, have the ability to bind to sera isolated from an infertile individual, and are immunogenic.
- the sperm surface protein is a human protein that comprises an amino acid sequence selected from the group: He Asn Ser Gin Trp Nal Nal Pro Leu Arg (SEQ ID NO: 1); Leu Asn Ser Gin Trp Val Val Pro Leu Arg (SEQ ID NO: 2); Met Val Asn He Met He He He He Arg (SEQ ID NO: 3);
- compositions comprising the sperm surface peptide epitopes of the present invention are administered as a contraceptive vaccine formulation.
- the sperm surface peptide epitope is an isolated or recombinant polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1-23.
- the epitope is a peptide consisting of a sequence selected from SEQ ID NOS 1-23.
- the present invention provides a method for modulating fertility in a mammalian species.
- the method comprises administering a composition comprising a therapeutically effective amount of an isolated or recombinant polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1-23.
- the composition may comprise a fragment of that polypeptide wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1 -23 or the polypeptide.
- the composition comprises a peptide having a sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS 1 -23.
- a composition for modulating fertility in a mammalian species comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1 and 2.
- the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 3-17.
- the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 18-21.
- the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence of SEQ ID NOS 22.
- the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence of SEQ ID NOS 23.
- the present invention also encompasses the nucleic acid sequences that encode the peptide sequences represented by SEQ ID NOS: 1-23. Nucleic acid sequences encoding these peptides can be synthesized using standard techniques known to those skilled in the art. In addition, the gene sequences encoding the full length polypeptides that contain these amino acid sequences can be isolated from eukaryotic genomes, including mammalian genomes, using standard techniques known to those skilled in the art. In a preferred embodiment the nucleic acid sequences encoding the SSPs are derived from the human genome.
- the SSPs of the present invention may include proteins that represent derivatives of the natural proteins, i.e. proteins that contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the amino acid sequences described above, including the amino acid sequences of SEQ ID NOS: 1- 23, that result in a "silent" change, in that the change produces a functionally equivalent peptide.
- Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
- nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral anlino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- deletion or non- conservative alterations can be engineered into the nucleic acid sequences encoding the SSPs to produce altered SSP gene products.
- Such alterations can, for example, alter one or more of the biological functions of the SSPs. Further, such alterations can be selected so as to generate products that are better suited for expression, scale up, etc. in the host cells chosen.
- cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
- tyrosine residues can be deleted or substituted with another amino acid residue in order to eliminate tyrosine phosphorylation.
- Fusion proteins in which an SSP protein or a portion of an SSP protein (such as the amino acid sequences of SEQ ID NOS 1-23) is fused to an unrelated protein are also within the scope of this invention.
- Such proteins and peptides can be designed on the basis of the synthetic nucleotide sequence that encode amino acid sequences of SEQ ID NOS 1 -23.
- Fusion proteins include, but are not limited to, IgFc fusions which stabilize the SSP protein or peptide and prolong half life in vivo; or fusions to any amino acid sequence that allows the fusion protein to be anchored to the cell membrane; or fusions of SSP proteins or peptide fragments to an enzyme, fluorescent protein, luminescent protein, or a flag epitope protein or peptide which provides a marker function.
- the SSP polypeptides of the invention can further comprise posttranslational modifications, including, but not limited to stearation, myristylations, palmitation, glycosylations, acetylations, and phosphorylations. If a native SSP does not have recognition motifs that allow such modifications, it would be routine for one skilled in the art to introduce into an SSP gene nucleotide sequences that encode motifs such as enzyme recognition signals so as to produce a modified SSP gene product.
- the present invention also encompasses the synthesis of peptides and peptide analogs of the native protein and peptide sequences disclosed herein.
- Such peptides or analogues thereof may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues.
- the peptides may be prepared in linear form using conventional solution or solid phase peptide syntheses and cleaved from the resin followed by purification procedures (Creighton, 1983, Protein Structures And Molecular Principles, W .H. Freeman and Co., N.Y.). Suitable procedures for synthesizing the peptides described herein are well known in the art.
- the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).
- analogues and derivatives of the peptides can be chemically synthesized.
- the linkage between each amino acid of the peptides of the invention may be an amide, a substituted amide or an isostere of amide.
- Nonclassical amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the sequence.
- Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogues in general.
- amino acid can be D (dextrorotary) or L (levorotary).
- Cyclized peptides may be formed by the addition of Cys residues to the termini of linear peptides. Formation of disulfide linkages, if desired, is generally conducted in the presence of mild oxidizing agents. Chemical oxidizing agents may be used, or the compounds may simply be exposed to atmospheric oxygen to effect these linkages. Various methods are known in the art, including those described, for example, by Tarn, J.P. et al, 1979, Synthesis 955-957; Stewart et al, 1984,
- An additional alternative is described by Kamber et al, 1980, Helv Chim-Acta 63:899-915.
- a method conducted on solid supports is described by Albericio, 1985, Int.-J. Peptide Protein Res . 26:92-97. Any of these methods may be used to form disulfide linkages in the peptides of the invention.
- the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques.
- a polynucleotide sequence encoding a linear form of the peptide can be synthesized and inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
- the expression vehicle is then transfected into a suitable target cell which will express the peptide.
- the expressed peptide is then isolated by procedures well-established in the art.
- Methods for recombinant protein and peptide production are well known in the art (see, e.g., Maniatis et al, 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al, 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY).
- Methods for introducing codon substitutions to the native sequence in order to encode an antagonistic peptide based on the disclosure herein are well known to those skilled in the art.
- the SSP gene products, peptide fragments thereof and fusion proteins thereof may be produced by recombinant DNA technology using techniques well known in the art.
- the SSPs of the present invention are used in a method of diagnosing or screening for the presence of, or a predisposition for, developing a fertility-related disorder associated with the presence of antibodies directed to sperm surface proteins. More particularly, in one embodiment the method is used to diagnosing or screening for immunological infertility by detecting the presence of antibodies to polypeptides that comprise an amino acid sequence selected from SEQ ID NOS: 1-23. The method comprises the steps of obtaining a blood sample from an individual, isolating the serum, contacting the serum sample with a composition comprising a sperm surface antigen and detecting the presence of antibodies in the serum sample that specifically bind to the sperm surface antigens.
- the presence of such antibodies indicates the presence or disposition of a fertility-related disorder.
- the detection of antibodies in the serum sample that specifically bind to the sperm surface antigens can be conducted using standard techniques known to those skilled in the art such as mobility shift assays and the use of labeled components.
- the SSPs can be covalently bound to an inert solid substrate and contacted with the sample serum. The SSPs are then washed to remove any unbound and non-specifically bound serum components. The SSPs are then contacted with labeled secondary antibody that is specific for the antibodies of the organism that provided the serum sample (i.e. if the serum sample is human than the labeled secondary antibody is an anti-human antibody).
- SSPs can be bound to a variety of solid support (e.g. agarose,
- Sepharose polystyrene or other chromatography beads
- primary amines can be linked to NHS, cyanogen bromide activated or maleimide activated resins.
- cyanogen bromide activated or maleimide activated resins are commercially available such as Pharmacia Corporation's CH-activated Sepharose.
- SSPs can be linked through their carboxylic acid residues using, for example the commercially available resin Pharmacia EAH-activated Sepharose.
- the sperm surface proteins of the present invention are used as vaccines in human and non- human animals. More particularly, the present invention encompasses the use of SSP- based vaccines for contraception.
- SSPs are delivered to a subject to elicit an active immune response, that acts as a temporary and reversible antagonist of conception.
- an antigen could be administered at a certain period of the month, for example during ovulation to raise the anti-sperm antibody titer of a female subject to block fertilization.
- SSPs are useful as vaccines for permanent sterilization of a subject.
- Such vaccines can be used to elicit a T-cell mediated attack on sperm cells useful as a method for irreversible sterilization.
- Methods for generating T-cell specific responses, such as adoptive immunotherapy, are well known in the art (see, for example, Vaccine Design, Michael F. Powell and Mark J. Newman Eds., Plenum Press, New York, 1995, pp 847-867). Such techniques may be particular useful for vetinary contraceptive or sterilization purposes, where a single dose vaccination may be desirable.
- Sperm surface protein antigens can be produced in large amounts and purified for use in vaccine preparations.
- the sperm surface proteins of the invention also have utility in immunoassays, eg, to detect or measure in a sample of body fluid from a vaccinated subject the presence of antibodies to the antigen, and thus to diagnose and/or to monitor immune response of the subject subsequent to vaccination.
- immunoassays eg, to detect or measure in a sample of body fluid from a vaccinated subject the presence of antibodies to the antigen, and thus to diagnose and/or to monitor immune response of the subject subsequent to vaccination.
- the preparation of vaccines containing an immunogenic polypeptide as the active ingredient is known to one skilled in the art (see, for example, Vaccine Design, Michael F. Powell and Mark J. Newman Eds., Plenum Press, New York, 1995, pp 821-902)
- the immunopotency of sperm surface protein antigens can be determined by monitoring the immune response in test animals following immunization with the sperm surface protein antigen, or by use of any immunoassay known in the art. Generation of a humoral (antibody) response and/or cell-mediated immunity, may be taken as an indication of an immune response.
- Test animals may include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and eventually human subjects.
- Methods of introducing the vaccine may include oral, intravaginal, intradennal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and via scarification ( scratching through the top layers of skin, e.g. , using a bifurcated needle) or any other standard routes of immunization.
- the immune response of the test subjects can be analyzed by various approaches such as: the reactivity of the resultant immune serum to the egg protein antigen, as assayed by known techniques, e.g., immunosorbent assay (ELISA), immunoblots, radioimmunoprecipitations, etc., or in the case where the egg protein antigen displays antigenicity or immunogenicity, by protection of the immunized host against fertilization in the immunized host.
- ELISA immunosorbent assay
- the vaccine of the invention may be tested in rabbits for the ability to induce an antibody response to the sperm surface protein antigen.
- Male specific-pathogen- free (SPF) young adult New Zealand White rabbits may be used.
- the test group each receives a fixed concentration of the vaccine.
- a control group receives an injection of 1 mM Tris-HCl pH 9.0 without the sperm surface protein antigen.
- Blood samples may be drawn from the rabbits every one or two weeks, and serum analyzed for antibodies to the sperm surface protein. The presence of antibodies specific for the antigen may be assayed, eg, using an ELIS A. Effective doses (immunizing amounts) of the vaccines of the invention may be extrapolated from dose-response curves derived from animal model test systems.
- Suitable preparations of such vaccines include injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared.
- the preparation may also be emulsified, or the polypeptides encapsulated in liposomes.
- the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
- the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
- adjuvants which may be effective, include, but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-nor-muramyl- L-alanyl-D- isoglutamine, N-acetylmuramyl-L-alanyl- D-isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine and surface active substances such as lysolecithin, pluronic polyols; polyanions; peptides; oil emulsions; alum, and MDP..
- the effectiveness of an adjuvant may be determined by measuring the induction of antibodies directed against an immunogenic polypeptide containing an egg surface protein polypeptide epitope, the antibodies resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants.
- the immunogen may include one or more polypeptides that comprise the amino acid sequences of SEQ ID NOS: 1-23.
- the immunogen can also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation.
- the recombinant antigen is a hapten, i.e., a molecule that is antigenic in that it can react selectively with cognate antibodies, but not immunogenic in that it cannot elicit an immune response
- the hapten may be covalently bound to a carrier or immunogenic molecule; for instance, a large protein such as serum albumin will confer immunogenicity to the hapten coupled to it.
- the hapten-carrier may be formulated for use as a vaccine.
- the polypeptides may be formulated into the vaccine as neutral or salt forms.
- Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
- the vaccines of the invention may be multivalent or univalent. Multivalent vaccines are made from recombinant viruses that direct the expression of more than one antigen.
- the patient to which the vaccine is administered is preferably a mammal, most preferably a human, but can also be a non-human animal including but not limited to cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats.
- the vaccine formulations of the invention comprise an effective immunizing amount of the sperm surface protein and a pharmaceutically acceptable carrier.
- Vaccine preparations comprise an effective immunizing amount of one or more antigens and a pharmaceutically acceptable carrier.
- the carrier is preferably sterile.
- the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
- the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
- Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. The formulation should suit the mode of administration.
- the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration.
- the invention also provides a pharmaceutical pack or kit comprising one or more containers comprising one or more of the ingredients of the vaccine formulations of the invention.
- One aspect of the present invention is directed to the antibodies
- the present invention encompasses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of an anti-sperm surface protein antibody effective to inhibit fertilization.
- the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the art and will be administered by reference to the immune response and antibody titers of the subject.
- a method for preparing an contraceptive antiserum, wherein the antiserum comprises an antibody against a sperm surface antigen.
- the sperm surface antigen is a protein unique to male gametes and more particularly to sperm cells.
- the method comprises the steps of immunizing and animal with a sperm surface protein or an immunogenic fragment thereof, obtaining a serum sample from the immunized animal, screening the serum for the ability to bind to a sperm surface protein (or immunogenic fragment thereof) and recovering serum based on its ability to bind the sperm surface protein.
- the resulting serum may include polyclonal antibodies or may comprise one or more monoclonal antibodies.
- antibody-secreting cells can be obtained from the immunized animal and those cells can be immortalized and screened, using standard techniques, for cells that secrete antibodies that bind to sperm surface proteins. Those cells that secrete antibodies that bind to sperm surface proteins can be isolated and used to produce antibodies.
- the generated antibodies may be isolated by standard techniques known in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays.
- the antibodies may also be used to monitor treatment and/or disease progression.
- any immunoassay system known in the art may be used for this purpose including but not limited to competitive and noncompetitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich” immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays.
- radioimmunoassays ELISA (enzyme-linked immunosorbent assays), "sandwich” immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays.
- the vaccine formulations of the present invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of a host is achieved by the administration of pre-formed antibody directed against a heterologous organism.
- the antibodies generated by the vaccine formulations of the present invention can also be used in the production of antiidiotypic antibody.
- the antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne, 1974, Ann. Immunol, (paris) 125c:373; Jerne, et al., 1982, EMBO J. 1 :234).
- a therapeutic of the invention e.g. , encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc.
- Methods of introduction include but are not limited to intradennal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
- the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
- compositions of the invention may be desirable to administer locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
- the therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317- 327; see generally ibid. )
- the therapeutic can be delivered in a controlled release system.
- a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
- polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.
- a controlled release system can be placed in proximity of the therapeutic target, i.e., the testes, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138).
- the present invention also provides pharmaceutical compositions.
- the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier.
- the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
- compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
- the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
- the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- the amount of the therapeutic of the invention which will be effective in the treatment of a particular or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
- in vitro assays may optionally be employed to help identify optimal dosage ranges.
- the precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the practitioner and each patient's circumstances.
- suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight.
- Suppositories generally contain active ingredient in the range of 0.5% to 10%o by weight; oral formulations preferably contain 10% to 95% active ingredient.
- Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
- Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
- Antisperm antibodies in the infertile female patient sera were detected by indirect immunobead binding as described in Manual of clinical laboratory immunology, 4th edition, American society for microbiology, Washington, D.C 1992; 1013-1017.
- the antisperm antibodies bound to the spermatozoa of male patients were detected by direct immunobead binding. All the serum and sperm samples were tested for the presence of IgG, IgM and IgA specific antisperm antibodies and included 18 infertile male subjects, 9 infertile female subjects, 5 fertile male subjects and 5 fertile female subjects.
- Sera were chosen for further study based on a high IBT score: i.e., more than 60% of the spermatozoa were observed to bind beads indicative of IgG and/or IgM specific antisperm antibodies. All the selected sera contained antibodies directed against the sperm head or the entire spermatozoa. A total of 15 serum samples were chosen from infertile men and 6 serum samples were chosen from infertile women for the western blot analysis.
- sperm antigens Semen specimens were obtained from fertile donors with normal sperm quality. Only ejaculates with normal semen characteristics were used in this study. After liquefaction of the semen the mature sperm were separated from the seminal plasma, immature germ cells and non-sperm cells (mainly white blood cells and epithelial cells) by Percoll (Pharmacia Biotech, Uppsala, Sweden) density centrifugation as described by Naaby-Hansen et al. (Biol Reprod 1997; 56: 771-787). Prior to the last centrifugation, the cells were counted and all samples showed >90% motility. The spermatozoa obtained from 8-12 individuals were pooled and frozen immediately until further use. All samples were obtained under informed consent using forms approved by the University of Virginia Human Investigation Committee.
- Radio-iodination was performed according to the procedure adopted and standardized by Naaby-Hansen et al (Biol Reprod 1997; 56: 771-787).
- Percoll- purified spermatozoa were suspended in Ham's F-10 medium to a final concentration of 20 x lOVml.
- Washed Iodo-beads (Pierce Chemical Co., Rockford, IL) (one bead per 8 x 10 6 spermatozoa) and carrier-free 125 I-Na (10 ⁇ Ci/10 6 spermatozoa) were added to the sample. Radiolabeling was performed by incubating the sample for 10 min at 20°C on a rocking table.
- the cells were removed from the iodo-beads by pipetting and immediately were subjected to a second Percoll density gradient centrifugation, before being washed three times in Ham's F-10 medium.
- 'Surface' proteins studied here are predominantly plasma membrane proteins accessible to external labeling on ejaculated sperm, which, following the preparation methods employed, consist almost entirely of cells that have not undergone the acrosome reaction.
- the cells were counted before the final wash, and the resulting pellet was used for extraction of sperm proteins.
- Autoradiography was performed using a sandwich of components in the following order; intensifying screen, blot, two layers of film, and intensifying screen. X-ray films were routinely exposed for 3 weeks.
- Isoelectric focusing was performed using the gel composition as described earlier (Naaby-Hansen et al., Biol Reprod 1997; 56: 771-787).
- Carrier ampholine compositions were 20% pH 5-7, 20% pH 7-9 and 60% pH 3.5 -10.
- Sixty- five microliters of sperm extract ( ⁇ 0.15 mg of protein) were applied per rod.
- the tubes were filled by gently overlaying the sample with a buffer containing 5% NP-40, 1% ampholines (pH 3.5-10), 8 M urea and 100 mM DTT . Focusing was conducted for a total of 17,700 volt-h using voltage stepping: 2h at 200v, 5h at 500v, 1 lh at 800 v and 3h at 2000 v.
- the membranes were rinsed twice for 5 min in PBS (pH 7.4), and excess binding sites were blocked by incubation for 1 hour at room temperature in PBS (pH 7.4) containing 5% dry milk and 0.05% Tween 20.
- the blots were incubated with the serum at 1 :2000 dilution at 4°C overnight under constant slow rocking. In some cases a given blot was incubated with more than one serum. In these instances the blots were washed in PBS (pH 7.4) containing 0.05% Tween 20 following incubation with the first serum at 4°C overnight and incubated with another serum under the same conditions as described above.
- the anchors were chosen based on the electrophoretic mobility , the constellation of the protein spots and the shape of the spots.
- the resulting composite image of the separated protein spots was compared and matched with the images corresponding to infertile male and female groups to subtract the protein spots recognized by the fertile subjects from the protein spots recognized by the infertile subjects, thus identifying the major auto- and iso-antigens which are uniquely recognized by the infertile subjects.
- a database of the sperm surface proteins (sperm surface encyclopedia) was created following the matching of auto radiograms obtained after surface radio- iodination. Care was taken to include only those spots which were consistently labeled.
- the computer generated images containing the auto-antigens and the iso- antigens were matched with the sperm surface encyclopedia to identify the surface auto- and iso-antigens.
- the protein pattern was highly reproducible although minor variations between donors were observed with regard to the relative abundance and charge of same proteins which were found to vary when individual samples were compared by computer analysis.
- the identities of some of the major protein spots were identified by microsequencing individual spots or immunoblotting with antisera to specific proteins. For example, different heat shock protein family members were resolved. These proteins are abundant on the sperm membrane.
- PH-20 a sperm hyaluronidase, is a membrane bound protein represented by three 53 kDa isoforms.
- the major cytosolic proteins, ⁇ -tubulin and ⁇ -tubulin were also detected as was human sperm calreticulin.
- 2D western blots of sperm proteins resolved by IEF or NEPHGE were probed with serum from a fertile male subject or with serum from an infertile male subject.
- the serum from the infertile subject showed stronger immuno-reactivity to some spots than the fertile subject although the importance of such differences in intensity are not presently understood.
- a number of antigenic spots among both acidic and basic proteins were reactive with infertile but not fertile sera.
- differences in immunological reactivity were noted in comparing western blots probed with an infertile female serum to those reactive with fertile female serum; the infertile subject's serum recognized more antigenic spots and also showed stronger reactivity to some spots than did the serum from the fertile female.
- FIG. 1A A comprehensive overview of the repertoire of immunoreactive sperm proteinsrecognized by fertile and infertile sera from both sexes was obtained by serially incubating blots with 5 sera from each group of subjects (Fig. 1A to ID). The sera from the infertile subjects were selected based on their high immunoreactivity , heterogeneity in the immunoreactivity, and unique recognition of certain protein spots following individual western blot analysis of the sera.
- Figures 1 A and IB present the repertoire of sperm proteins bound by sera from 5 fertile male (A) and 5 fertile female (B) subjects.
- the molecular weights of prominent protein spots uniquely recognized by male patients are 34 kDa (pi 4.2), 42 kDa (pi 4.3), 88.5 kDa ( pi 4.0), 106 kDa (pi 4.2), 41.8 kDa (pi 5.3, pi 5.4, pi 5.5),61.5 kDa (pi 5.62), 60.4 kDa (pi 5.75) and 60 kDa (pi 6.6 and 6.7).
- Major iso- antigens recognized by infertile females are at the molecular weights of 60 kDa (pi 6.2, pi 6.4, pi 5.82, pi 5.88),32.3 kDa (pi 6.7, pi 6.8, pi 6.85) and 41.2 kDa (pi 5.1 ).
- Figure 3 presents a computer generated composite image of human sperm surface proteins obtained following computer scanning of autoradiograms obtained from 2D IEF-PAGE of vectorially iodinated sperm proteins (Biol Reprod 1997; 56: 771-787). 103 proteins were identified on the sperm surface by this method. Computer aided matching of the sperm auto- and iso-antigens with the composite image of the sperm surface proteins provided for the identification of a subset of sperm surface auto- and iso-antigens. The location of six sperm surface auto-antigens and iso-antigens are depicted in the Fig. 3 (arrows).
- Two of the sera also recognized two sperm surface proteins at molecular weights of34 kDa and 38 kDa, both at a PI of 4.2.
- Table 1 lists the masses and pi of the sperm surface iso- and auto- antigens.
- Table 1 Sperm surface auto- and iso-antigens correlated with the IBT results of the cognate patient sera.
- two dimensional electrophoresis was employed for the separation of the proteins based on the iso-electric points of the individual proteins in the first dimension and on the molecular weight in the second dimension, which offers better resolution of the proteins and may serve as a prelude to microsequencing.
- the high resolution 2-D gel techniques which were optimized for sperm protein separation are suitable for screening sera for antisperm antibodies.
- Initial screening of the sera from the infertile patients for the presence of sperm surface antibody by IBT provided additional focus by directing attention to sera containing antisperm antibodies which are relevant in antibody mediated events of agglutination, cytotoxicity or blocking at the sperm surface and thus functionally related to infertility.
- male sera recognized many more antigens than female sera (310 versus 205) although a comprehensive evaluation with a greater number of samples from female subjects is required to statistically validate the conclusion.
- a longer constant exposure of sperm proteins to the immune system among the male patients may explain the higher reactivity among males.
- a distinct difference in the pattern of immunoreactivity between male and female patients was discernible.
- the male patient sera was reactive to several acidic proteins ( pi 4.0 to 5.0) which were not recognized by any of the analyzed sera from females (see Fig. IC and ID).
- major iso-antigens recognized by the female patients in the pi range of 6.0 to 7.5 were poorly reactive to sera from the male patients (Fig. IC and ID).
- a rational basis for identifying fertility related sperm antigens relies on the identification of those antigens which are on the cell surface. Hence it is especially critical to distinguish between immunity to sperm surface antigens and the internal antigens of spermatozoa. For this reason the sperm surface proteins of live sperm were labeled, rather than labeling the surface proteins of fixed sperm (which have damaged membranes aloowing the labeling of internal proteins). The sperm surface proteins were radiolabeling with 125 I followed by IEF /PAGE as well as NEPHGE/PAGE separation (see Naaby-Hansen et al., Biol Reprod 1997; 56: 771 - 787).
- a composite image was obtained consisting of 103 surface labeled proteins in IEF /PAGE gels.
- This computer image enabled the categorization a subset of 6 sperm surface antigens which may have role in immunoinfertility.
- One of these antigens is a 88.2 kDa protein at a pi of 4.0. , recognized by two male infertile patients. Naaby-Hansen et al (Biol Reprod 1997; 56: 771-787) have earlier shown this protein to be a sperm surface protein, phosphorylated on tyrosine residues.
- two women's sera showed striking similarity in the immunoreactivity, recognizing the same protein spots at 60 kDa and pi of 6.2 and 6.4.
- both these sperm proteins were found to be on the surface of the sperm as demonstrated by surface labeling of the sperm with radioactive I25 I. Further, both sera contained IgG antibodies directed towards the head of the sperm as shown by immunobead test, confirming that the antigens are exposed on the sperm surface, probably located on the sperm head. Two male patients' sera strongly recognized 2 protein spots at 34 kDa and 38 kDa which were identified on the sperm surface.
- the computer digitization of information on the 2-D gel patterns of surface labeled sperm proteins opens the possibility for a new tool in the diagnosis of antisperm antibodies and possible treatment of antibody mediated infertility.
- 2-D immunoblotting with patients sera can now be employed to ascertain those patients with antibodies to protein antigens accessible at the cell surface and hence obtain more specific information on a patient's repertoire of auto or iso- antibodies.
- An upper and lower band ran at approximately 36 kDa with a pi of 4.4.
- the band was cut from the gel as closely as possible to minimize excess polyacrylamide, divided into a number of smaller pieces and washed and destained in 500 ⁇ L 50% methanol overnight.
- the gel pieces were dehydrated in acetonitrile, rehydrated in 50 ⁇ L of 10 mM dithiolthreitol in 0.1 M ammonium bicarbonate and reduced at 55 °C for 1 h.
- the DTT solution was removed and the sample alkylated in 50 ⁇ L 50 mM iodoacetamide/0.1 M ammonium bicarbonate at room temperature for 1 h in the dark.
- the reagent was removed and the gel pieces washed with 100 ⁇ L 0.1 M ammonium bicarbonate and dehydrated in 100 ⁇ L acetonitrile for 5 min.
- the acetonitrile was removed and the gel pieces rehydrated in 100 ⁇ L 0.1 M ammonium bicarbonate.
- the pieces were rehydrated in 100 ⁇ L acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation.
- the gel pieces were rehydrated in 12.5 ng/ ⁇ L trypsin in 50 mM ammonium bicarbonate and incubated on ice for 45 min. Any excess trypsin solution was removed and 20 ⁇ L 50 mM ammonium bicarbonate added.
- the sample was digested overnight at 37° C and the peptides formed extracted from the polyacrylamide in two 200 ⁇ L 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to ⁇ 20 ⁇ L for LC-MS analysis.
- the LC-MS system consisted of Finnigan-MAT TSQ7000 system with an electrospray ion source interfaced to a 10 cm x 75 um id POROS 10 RC reversed phase capillary column.
- One ⁇ L volumes of the extract are injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.6 ⁇ L/min.
- the electrospray ion source is operated at 4.5 kV with a 1.2 ⁇ L/min coaxial sheath liquid flow of 70% methanol/30% water/0.125%) acetic acid and a coaxial nitrogen flow adjusted as needed for optimum sensitivity.
- the digest was analyzed by capillary LC-electrospray mass spectrometry to measure the molecular weight of the peptides present in the digest.
- Peptide sequences for the peptides detected were determined by collisionally activated dissociation using LC- electrospray-tandem mass spectrometry with argon as the collision gas.
- both bands produce similar series of peptides. Both bands contain two peptides that match the database sequence of human hyaluronic acid binding protein. Both bands contain additional peptides that cannot be matched to any database peptide and therefore appear to be from a second, unknown protein.
- X designates I or L which cannot be distinguished by low energy CAD
- Mo designates an oxidized M
- lower case letters designate tentative assignments
- _ designates a single unknown amino acid
- — designates an unknown number of unknown amino acids.
- VYTS represents amino acid sequence
- GGWEXEXNGTEAK represents amino acid sequences Gly Gly Trp Glu He Glu He Asn Gly Thr Glu Ala Lys (SEQ ID NO: 32), Gly Gly Trp Glu He Glu Leu Asn Gly Thr Glu Ala Lys (SEQ ID NO: 33), Gly Gly Trp Glu Leu Glu He Asn Gly Thr Glu Ala Lys (SEQ ID NO: 34), and Gly Gly Trp Glu Leu Glu Leu Asn Gly Thr Glu Ala Lys (SEQ ID NO : 35);
- AFVDFXSDEXKEER represents amino acid sequences Ala Phe Val Asp Phe He Ser Asp Glu He Lys Glu Glu Arg (SEQ ID NO: 36), Ala Phe Val Asp Phe He Ser Asp Glu Leu Lys Glu Glu Arg (SEQ ID NO: 37), Ala Phe Val Asp Phe Leu Ser Asp Glu He Lys Glu Glu Arg (SEQ ID NO: 38), and Ala Phe Val Asp Phe Leu Ser Asp Glu Leu Lys Glu Glu Arg (SEQ ID NO: 39); and
- SGGWELELNGTEAK represents amino acid sequences Ser Gly Gly Trp Glu Leu Glu Leu Asn Gly Thr Glu Ala Lys (SEQ ID NO: 40).
- the gel piece was transferred to a siliconized tube and washed and destained in 200 ⁇ L 50% methanol overnight.
- the gel pieces were dehydrated in acetonitrile, rehydrated in 30 ⁇ L of 10 mM dithiolthreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h.
- the DTT solution was removed and the sample alkylated in 30 ⁇ L 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h.
- the reagent was removed and the gel pieces dehydrated in 100 ⁇ L acetonitrile.
- the acetonitrile was removed and the gel pieces rehydrated in 100 ⁇ L 0.1 M ammonium bicarbonate.
- the pieces were dehydrated in 100 ⁇ L acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation.
- the gel pieces were rehydrated in 20 ng/ ⁇ L trypsin in 50 mM ammonium bicarbonate on ice for 10 min. Any excess trypsin solution was removed and 20 ⁇ L 50 mM ammonium bicarbonate added.
- the sample was digested overnight at 37 °C and the peptides formed extracted from the polyacrylamide in two 30 ⁇ L aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 25 ⁇ L for LC-MS analysis.
- the LC-MS system consisted of a Finnigan LCQ ion trap mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm x 75 um id Phenomenex Jupiter 10 um C18 reversed-phase capillary column. 0.5-5 ⁇ L volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.25 ⁇ L/min.
- the nanospray ion source was operated at 2.8 kV.
- the digest was analyzed using the double play capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in sequential scans. This mode of analysis produces approximately 400 CAD spectra of ions ranging in abundance over several orders of magnitude. Not all CAD spectra are derived from peptides.
- M(o) designates oxidized M
- C is carbamidomethyl modified unless noted as C a (acrylamide)
- _ designates a single unknown residue
- — designates an unknown number of unknown residues.
- XQQT represents amino acid sequences He Gin Gin Thr (SEQ ID NO: 41) and Leu Gin Gin Thr (SEQ ID NO: 42);
- XNSQWVVPLR represents amino acid sequences He Asn Ser Gin Trp Val Val Pro Leu Arg (SEQ ID NO: 1); and Leu Asn Ser Gin Trp Val Val Pro Leu Arg (SEQ ID NO: 2);
- MVNXMXXR represents amino acid sequences Met Val Asn He Met He He He He Arg (SEQ ID NO: 3), Met Val Asn Leu Met He He He Arg (SEQ ID NO: 4), Met Val Asn He Met Leu He He Arg (SEQ ID NO: 5), Met Val Asn He Met He Leu He Arg (SEQ ID NO: 6), Met Val Asn He Met He Leu Arg (SEQ ID NO: 7), Met Val Asn He Met He Leu Leu Arg (SEQ ID NO: 8), Met Val Asn He Met Leu Leu He Arg (SEQ ID NO: 9), Met Val Asn He Met Leu He Leu Arg (SEQ ID NO: 10), Met Val Asn Leu Met Leu He He Arg (SEQ ID NO: 11), Met Val Asn Leu Met Leu He He Arg (SEQ ID NO: 12), Met Val Asn Leu Met He Leu He Arg (SEQ ID NO: 13), Met Val Asn Leu Met He He Leu Arg (SEQ
- Peptides from band 2 were isolated as described in Examples 3 and 4.
- the peptides shown in Table 4 were detected in the band 2 digest and could not be matched to any database sequence.
- the peptides were low abundance and the digest contained a number of higher abundance peptides that all matched keratin sequences.
- PXSS represents amino acid sequences Pro He Ser Ser (SEQ ID NO: 43) and Pro Leu Ser Ser (SEQ ID NO: 44);
- MXPVXE represents amino acid sequences Met He Pro Val He Glu (SEQ ID NO: 18), Met Leu Pro Val He Glu (SEQ ID NO: 19), Met He Pro Val Leu Glu (SEQ ID NO: 20), and Met Leu Pro Val He Glu (SEQ ID NO: 21);
- HGQSAEYEF represents amino acid sequence His Gly Gin Ser Ala Glu Tyr Glu Phe (SEQ ID NO: 22).
- FQQDGGAS represents amino acid sequence Phe Gin Gin Asp Gly Gly Ala Ser (SEQ ID NO: 23).
- YIGEFDR represents amino acid sequence Tyr He Gly Glu Phe Asp Arg (SEQ ID NO: 45);
- SLQYLNLR represents amino acid sequence Ser Leu Gin Tyr Leu Asn Leu Arg (SEQ ID NO: 46); DNQIDTSLGFSR represents amino acid sequence
- MXPVXE represents amino acid sequences Met He Pro Val He Glu (SEQ ID NO: 18), Met Leu Pro Val He Glu (SEQ ID NO: 19), Met He Pro Val Leu Glu (SEQ ID NO: 20), and Met Leu Pro Val He Glu (SEQ ID NO: 21);
- HGQSAEYEF represents amino acid sequence His Gly Gin Ser Ala Glu Tyr Glu Phe (SEQ ID NO: 22).
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Abstract
The present invention relates to sperm surface proteins and the use of such proteins to modulate fertility in a mammalian species. More particularly the invention is directed to purified sperm-specific surface peptide epitopes as well as derivatives and antibodies directed to those protein epitopes.
Description
Human Sperm Surface Antigens
US Government Rights
This invention was made with United States Government support under Grant No. HD-29099, P30-28934 and D543-TW00654, awarded by the
National Institute of Health. The United States Government has certain rights in the invention.
Field of the Invention The present invention is directed to sperm cell surface antigens that are recognized by antisperm antibodies present in the serum of infertile men and women. More particularly the invention is directed to sperm-specific surface protein/peptide epitopes as well as derivatives and antibodies directed to those protein epitopes.
Background of the Invention
The correlation of antisperm antibodies (ASA) with some cases of unexplained infertility suggests a role for these antibodies in blocking fertilization. The incidence of immunity to sperm in infertile couples is estimated to be 9 to 36%. In contrast, the prevalence of AS As in the general population is approximately 0 to 2%. Antisperm antibodies are thought to impair fertility by inhibiting sperm motility, sperm penetration of the cervical mucus, capacitation, or the acrosome reaction, or they may invoke the complement cascade resulting in sperm lysis.
A complete understanding of the mechanism behind immunologic infertility as well as improved diagnosis and treatment are dependent on knowledge of the identities of specific sperm antigens capable of eliciting the production of functionally relevant sperm antibodies. For example, were a cocktail of recombinant sperm surface antigens available, it might serve as the target for an immunoassay of patient sera to detect the presence of specific antibodies mediating infertility, thus improving diagnosis of immunological infertility. Further, antisperm antibodies and their cognate antigens may provide the basis for immunologic control of fertility in the form of a birth control vaccine.
Several studies have reported the identification of sperm antigens
recognized by systemic and/or local auto- and iso-antibodies from infertile individuals using immunoblotting techniques. In most cases, however, the reactions of sperm antigens with control sera from fertile individuals were not described, so the functional relevance of these antigens in fertility remains unclear. Furthermore, most of the earlier reports used unidimensional gel electrophoresis for the separation of sperm proteins.
The present invention is directed to proteins identified by high resolution 2-D electrophoresis. The sperm antigens are separated in the first dimension by either isoelectric focusing or nonequilibrium pH gradient electrophoresis (to screen a range of acidic and basic proteins) followed by PAGE and a sensitive western blotting method. In addition, the subject sera were initially screened for the presence of antisperm antibodies by the immunobead test (IBT, as described in Bronson R, Cooper G, Rosenfeld D. In : Jagiello G and Vogel H (Eds), Bioregulators of reproduction, academic press, New York 1981 : 521-527) and only those sera showing significant reactivity for the presence of antisperm antibodies were utilized to identify the immunodominant antigens. Moreover, sperm antigens unique to infertile patients were identified by excluding those antigens recognized by sera from clinically fertile subjects using computerized comparison of 2-D immunoblots. A database of 2-D gel images of silver stained proteins (sperm proteome) and a database of vectorially labeled sperm surface proteins (sperm surface index, Biol Reprod 1997; 56: 771-787) allowed the definition of a subset of sperm surface antigens relevant to antibody mediated infertility.
Summary of the Invention The present invention is directed to therapeutic and diagnostic compositions and methods of using those compositions in procedures relating to fertility. The compositions of the present invention comprise sperm cell surface antigens that are recognized by antisperm antibodies present in the serum of infertile men and women. The invention further relates to therapeutic compositions and methods of diagnosis and therapy, including compositions and methods for modulating fertility (i.e. inhibiting or promoting).
Brief Description of the Drawings
Figs. 1A-1D represent a Western blot analysis by serial incubation of a single blot with 5 sera from fertile males (Fig. 1A) and fertile females (Fig. IB) compared with 5 infertile males (Fig. IC) and infertile females (Fig. ID) (Dil:l :2000). Note major auto- and iso-antigens (arrows) which are uniquely recognized by the infertile subjects.
Figs. 2A-2B represent computer generated images showing the major auto-antigens (Fig. 2A) and iso-antigens (Fig. 2B), recognized by infertile sera. The computer scanned images of western blots were compared between fertile and infertile groups using common protein spots across two images as anchors. The protein spots recognized by the fertile subject sera (both male and female) were subtracted from the image representing the infertile male and female subjects and an image depicting all antigens uniquely recognized by the infertile males (Fig. 2A) and infertile females (Fig. 2B) was generated. Note that there are more auto-antigens than iso-antigens and that there is a differential pattern of immunoreactivity between the sexes. Arrows indicate the protein spots recognized by both male and female subjects.
Fig. 3. represents a computer generated composite image of two dimensional IEF/PAGE of vectorially iodinated human sperm proteins. The sperm were radioiodinated, and solubilized as described in Example 1 and separated by two dimensional eletrophoresis by IEF/PAGE. The composite image was generated after scanning the autoradiograms of the surface proteins immobilized on nitrocellulose membrane and comparing the images to the locus of auto-antigens and iso-antigens (Fig. 1). Arrows point to the locus of immunodominant surface auto- and iso- antigens.
Detailed Description of the Invention
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
As used herein, "nucleic acid," "DNA," and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within
the scope of the present invention.
As used herein, "effective amount" means an amount sufficient to produce a selected effect. For example, an effective immunizing amount of the sperm surface protein is an amount sufficient to induce an immune response in the host to which the vaccine preparation is administered.
As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
The present invention is directed to sperm surface antigens that are recognized by sera isolated from infertile patients but not by sera isolated from clinically fertile subjects. The sperm antigens of the present invention are unique to infertile patients and were identified by excluding those antigens recognized by sera from clinically fertile subjects using computerized comparison of 2-D immunoblots. A database of 2-D gel images of silver stained proteins (sperm proteome) and a database of vectorially labeled sperm surface proteins (sperm surface index, Biol Reprod 1997; 56: 771-787) allowed the definition of a subset of sperm surface antigens relevant to antibody mediated infertility. These identified sperm surface proteins are referred to herein generically as SSPs and include the isolated natural proteins and fragments thereof as well as recombinant derivatives and synthetic analogs of the native proteins. The SSP proteins, polypeptides and peptide fragments thereof, can be prepared for a variety of uses. For example, such molecules can be used for the generation of antibodies, for use in diagnostic and therapeutic assays, for the identification of other sperm gene products involved in sperm motility, or for the identification of compounds that modulate sperm motility. One embodiment of the present invention is directed to a pharmaceutical composition comprising a sperm surface peptide epitope and a pharmaceutically acceptable carrier. The pharmaceutical carriers can be sterile
liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like as well as buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients for use in the compositions of the present invention include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In accordance with one embodiment the SSP containing compositions of the present invention are used to modulate fertility in a mammalian species. The method comprises administering a composition comprising a therapeutically effective amount of an SSP antigen. In one preferred embodiment the composition is formulated as a contraceptive vaccine comprising one or more SSP proteins (or SSP protein fragments or analogs thereof) and administered to a mammalian species to induce an immune response.
In one embodiment, the SSP-containing composition is used to treat a subject for a fertility-related disorder by administering an effective amount of the
sperm surface protein or fragment thereof. In particular, the SSP compositions of the present invention can be used in a method for treating immunological infertility. The method comprises obtaining a serum sample form the individual exhibiting immunological infertility, determining the sperm surface proteins targeted by the antibodies present in the individual's serum and administering a composition comprising one or more peptides (or peptide derivatives or peptide mimetics) targeted by the individual's antibodies. In one embodiment the composition is administered topically to a female to bind to the individual's anti-sperm antibodies and thus enhance the fertility of the female. In accordance with another embodiment the SSPs of the present invention can be used to screen for molecules that interact with sperm surface proteins, including those molecules that bind to the surface proteins. The method comprises contacting the sperm specific protein/peptide with one or more molecules under conditions conducive to complex formation between the sperm specific protein/peptide and the molecule, and recovering the molecule that specifically binds to the sperm surface protein/peptide.
In accordance with one embodiment, the invention provides polypeptides and their respective fragments and analogs that have at least one of the following activities: capable of modulating sperm's ability to fertilize an egg, function as sperm marker, have the ability to bind to sera isolated from an infertile individual, and are immunogenic. In a preferred embodiment, the sperm surface protein is a human protein that comprises an amino acid sequence selected from the group: He Asn Ser Gin Trp Nal Nal Pro Leu Arg (SEQ ID NO: 1); Leu Asn Ser Gin Trp Val Val Pro Leu Arg (SEQ ID NO: 2); Met Val Asn He Met He He He Arg (SEQ ID NO: 3);
Met Val Asn Leu Met He He He Arg (SEQ ID NO: 4); Met Val Asn He Met Leu He He Arg (SEQ ID NO: 5); Met Val Asn He Met He Leu He Arg (SEQ ID NO: 6); Met Val Asn He Met He He Leu Arg (SEQ ID NO: 7); Met Val Asn He Met He Leu Leu Arg (SEQ ID NO: 8);
Met Val Asn He Met Leu Leu He Arg (SEQ ID NO: 9); Met Val Asn He Met Leu He Leu Arg (SEQ ID NO: 10);
Met Val Asn Leu Met Leu He He Arg (SEQ ID NO: 11);
Met Val Asn Leu Met Leu He He Arg (SEQ ID NO: 12);
Met Val Asn Leu Met He Leu He Arg (SEQ ID NO: 13);
Met Val Asn Leu Met He He Leu Arg (SEQ HO NO: 14); Met Val Asn Leu Met He Leu Leu Arg (SEQ ID NO: 15);
Met Val Asn Leu Met Leu Leu He Arg (SEQ ID NO: 16);
Met Val Asn Leu Met Leu He Leu Arg (SEQ ID NO: 17);
Met He Pro Val He Glu (SEQ ID NO: 18);
Met Leu Pro Val He Glu (SEQ ID NO: 19); Met He Pro Val Leu Glu (SEQ HO NO: 20);
Met Leu Pro Val He Glu (SEQ ID NO: 21);
His Gly Gin Ser Ala Glu Tyr Glu Phe (SEQ ED NO: 22); and
Phe Gin Gin Asp Gly Gly Ala Ser (SEQ ID NO: 23).
In one aspect of the invention compositions comprising the sperm surface peptide epitopes of the present invention are administered as a contraceptive vaccine formulation. More particularly the sperm surface peptide epitope is an isolated or recombinant polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1-23. In one embodiment the epitope is a peptide consisting of a sequence selected from SEQ ID NOS 1-23.
In accordance with one embodiment, the present invention provides a method for modulating fertility in a mammalian species. The method comprises administering a composition comprising a therapeutically effective amount of an isolated or recombinant polypeptide, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1-23.
Alternatively, the composition may comprise a fragment of that polypeptide wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1 -23 or the polypeptide. In one embodiment, the composition comprises a peptide having a sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS 1 -23.
In one embodiment a composition for modulating fertility in a mammalian species comprises a polypeptide antigen, wherein the polypeptide
comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 1 and 2. In another embodiment the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 3-17. In another embodiment the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 18-21. In another embodiment the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence of SEQ ID NOS 22. In another embodiment the composition comprises a polypeptide antigen, wherein the polypeptide comprises an amino acid sequence of SEQ ID NOS 23.
The present invention also encompasses the nucleic acid sequences that encode the peptide sequences represented by SEQ ID NOS: 1-23. Nucleic acid sequences encoding these peptides can be synthesized using standard techniques known to those skilled in the art. In addition, the gene sequences encoding the full length polypeptides that contain these amino acid sequences can be isolated from eukaryotic genomes, including mammalian genomes, using standard techniques known to those skilled in the art. In a preferred embodiment the nucleic acid sequences encoding the SSPs are derived from the human genome.
The SSPs of the present invention may include proteins that represent derivatives of the natural proteins, i.e. proteins that contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues within and/or adjacent to the amino acid sequences described above, including the amino acid sequences of SEQ ID NOS: 1- 23, that result in a "silent" change, in that the change produces a functionally equivalent peptide. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral anlino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
Alternatively, where alteration of function is desired, deletion or non- conservative alterations can be engineered into the nucleic acid sequences encoding the SSPs to produce altered SSP gene products. Such alterations can, for example, alter one or more of the biological functions of the SSPs. Further, such alterations can be selected so as to generate products that are better suited for expression, scale up, etc. in the host cells chosen. For example, in one embodiment, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. In another embodiment, tyrosine residues can be deleted or substituted with another amino acid residue in order to eliminate tyrosine phosphorylation.
Fusion proteins in which an SSP protein or a portion of an SSP protein (such as the amino acid sequences of SEQ ID NOS 1-23) is fused to an unrelated protein are also within the scope of this invention. Such proteins and peptides can be designed on the basis of the synthetic nucleotide sequence that encode amino acid sequences of SEQ ID NOS 1 -23. Fusion proteins include, but are not limited to, IgFc fusions which stabilize the SSP protein or peptide and prolong half life in vivo; or fusions to any amino acid sequence that allows the fusion protein to be anchored to the cell membrane; or fusions of SSP proteins or peptide fragments to an enzyme, fluorescent protein, luminescent protein, or a flag epitope protein or peptide which provides a marker function.
The SSP polypeptides of the invention can further comprise posttranslational modifications, including, but not limited to stearation, myristylations, palmitation, glycosylations, acetylations, and phosphorylations. If a native SSP does not have recognition motifs that allow such modifications, it would be routine for one skilled in the art to introduce into an SSP gene nucleotide sequences that encode motifs such as enzyme recognition signals so as to produce a modified SSP gene product.
The present invention also encompasses the synthesis of peptides and peptide analogs of the native protein and peptide sequences disclosed herein. Such peptides or analogues thereof, may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues. For example, the peptides may be prepared in linear form using conventional solution or solid phase
peptide syntheses and cleaved from the resin followed by purification procedures (Creighton, 1983, Protein Structures And Molecular Principles, W .H. Freeman and Co., N.Y.). Suitable procedures for synthesizing the peptides described herein are well known in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).
In addition, analogues and derivatives of the peptides can be chemically synthesized. The linkage between each amino acid of the peptides of the invention may be an amide, a substituted amide or an isostere of amide. Nonclassical amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogues in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). Cyclized peptides may be formed by the addition of Cys residues to the termini of linear peptides. Formation of disulfide linkages, if desired, is generally conducted in the presence of mild oxidizing agents. Chemical oxidizing agents may be used, or the compounds may simply be exposed to atmospheric oxygen to effect these linkages. Various methods are known in the art, including those described, for example, by Tarn, J.P. et al, 1979, Synthesis 955-957; Stewart et al, 1984,
SoiidPhase Peptide Synthesis, 2d Ed., Pierce Chemical Company Rockford, IL; Ahmed et al, 1975, J. Bioi. Chem. 250:8477-8482; and Pennington et al, 1991 Peptides 1990 164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. An additional alternative is described by Kamber et al, 1980, Helv Chim-Acta 63:899-915. A method conducted on solid supports is described by Albericio, 1985, Int.-J. Peptide Protein Res . 26:92-97. Any of these methods may be used to form disulfide linkages in the peptides of the invention.
When the peptide is composed entirely of gene-encoded amino acids (such as with SEQ ID NOS: 1-23), or a portion of it is so composed, the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques. For recombinant production, a polynucleotide sequence encoding a linear form of the peptide can be synthesized and inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation. The expression vehicle is then transfected into a suitable target cell which will express the peptide. Depending on the expression system used, the expressed peptide is then isolated by procedures well-established in the art. Methods for recombinant protein and peptide production are well known in the art (see, e.g., Maniatis et al, 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al, 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY). Methods for introducing codon substitutions to the native sequence in order to encode an antagonistic peptide based on the disclosure herein are well known to those skilled in the art. The SSP gene products, peptide fragments thereof and fusion proteins thereof, may be produced by recombinant DNA technology using techniques well known in the art. In accordance with one embodiment, the SSPs of the present invention are used in a method of diagnosing or screening for the presence of, or a predisposition for, developing a fertility-related disorder associated with the presence of antibodies directed to sperm surface proteins. More particularly, in one embodiment the method is used to diagnosing or screening for immunological infertility by detecting the presence of antibodies to polypeptides that comprise an amino acid sequence selected from SEQ ID NOS: 1-23. The method comprises the steps of obtaining a blood sample from an individual, isolating the serum, contacting the serum sample with a composition comprising a sperm surface antigen and detecting the presence of antibodies in the serum sample that specifically bind to the sperm surface antigens. The presence of such antibodies indicates the presence or disposition of a fertility-related disorder.
The detection of antibodies in the serum sample that specifically bind to the sperm surface antigens can be conducted using standard techniques known to those skilled in the art such as mobility shift assays and the use of labeled components. For example the SSPs can be covalently bound to an inert solid substrate and contacted with the sample serum. The SSPs are then washed to remove any unbound and non-specifically bound serum components. The SSPs are then contacted with labeled secondary antibody that is specific for the antibodies of the organism that provided the serum sample (i.e. if the serum sample is human than the labeled secondary antibody is an anti-human antibody). SSPs can be bound to a variety of solid support (e.g. agarose,
Sepharose, polystyrene or other chromatography beads) using protocols available to those skilled in the art. For example, primary amines can be linked to NHS, cyanogen bromide activated or maleimide activated resins. Many of these resins are commercially available such as Pharmacia Corporation's CH-activated Sepharose. Alternatively, SSPs can be linked through their carboxylic acid residues using, for example the commercially available resin Pharmacia EAH-activated Sepharose.
In accordance with one embodiment of the present invention the sperm surface proteins of the present invention are used as vaccines in human and non- human animals. More particularly, the present invention encompasses the use of SSP- based vaccines for contraception. In one aspect of the invention, SSPs are delivered to a subject to elicit an active immune response, that acts as a temporary and reversible antagonist of conception. For example, such vaccines could be used for active immunization of a subject, to raise an antibody response to temporarily block the sperm's motility or access to the egg. In one aspect of the invention, an antigen could be administered at a certain period of the month, for example during ovulation to raise the anti-sperm antibody titer of a female subject to block fertilization.
In another embodiment of the invention, SSPs, are useful as vaccines for permanent sterilization of a subject. Such vaccines can be used to elicit a T-cell mediated attack on sperm cells useful as a method for irreversible sterilization. Methods for generating T-cell specific responses, such as adoptive immunotherapy, are well known in the art (see, for example, Vaccine Design, Michael F. Powell and Mark J. Newman Eds., Plenum Press, New York, 1995, pp 847-867). Such
techniques may be particular useful for vetinary contraceptive or sterilization purposes, where a single dose vaccination may be desirable.
Sperm surface protein antigens can be produced in large amounts and purified for use in vaccine preparations. The sperm surface proteins of the invention also have utility in immunoassays, eg, to detect or measure in a sample of body fluid from a vaccinated subject the presence of antibodies to the antigen, and thus to diagnose and/or to monitor immune response of the subject subsequent to vaccination. The preparation of vaccines containing an immunogenic polypeptide as the active ingredient is known to one skilled in the art (see, for example, Vaccine Design, Michael F. Powell and Mark J. Newman Eds., Plenum Press, New York, 1995, pp 821-902)
The immunopotency of sperm surface protein antigens can be determined by monitoring the immune response in test animals following immunization with the sperm surface protein antigen, or by use of any immunoassay known in the art. Generation of a humoral (antibody) response and/or cell-mediated immunity, may be taken as an indication of an immune response. Test animals may include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and eventually human subjects.
Methods of introducing the vaccine may include oral, intravaginal, intradennal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and via scarification ( scratching through the top layers of skin, e.g. , using a bifurcated needle) or any other standard routes of immunization. The immune response of the test subjects can be analyzed by various approaches such as: the reactivity of the resultant immune serum to the egg protein antigen, as assayed by known techniques, e.g., immunosorbent assay (ELISA), immunoblots, radioimmunoprecipitations, etc., or in the case where the egg protein antigen displays antigenicity or immunogenicity, by protection of the immunized host against fertilization in the immunized host.
As one example of suitable animal testing of an sperm surface protein vaccine, the vaccine of the invention may be tested in rabbits for the ability to induce an antibody response to the sperm surface protein antigen. Male specific-pathogen- free (SPF) young adult New Zealand White rabbits may be used. The test group each
receives a fixed concentration of the vaccine. A control group receives an injection of 1 mM Tris-HCl pH 9.0 without the sperm surface protein antigen.
Blood samples may be drawn from the rabbits every one or two weeks, and serum analyzed for antibodies to the sperm surface protein. The presence of antibodies specific for the antigen may be assayed, eg, using an ELIS A. Effective doses (immunizing amounts) of the vaccines of the invention may be extrapolated from dose-response curves derived from animal model test systems.
Suitable preparations of such vaccines include injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, suspension in, liquid prior to injection, may also be prepared. The preparation may also be emulsified, or the polypeptides encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
Examples of adjuvants which may be effective, include, but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-nor-muramyl- L-alanyl-D- isoglutamine, N-acetylmuramyl-L-alanyl- D-isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine and surface active substances such as lysolecithin, pluronic polyols; polyanions; peptides; oil emulsions; alum, and MDP.. The effectiveness of an adjuvant may be determined by measuring the induction of antibodies directed against an immunogenic polypeptide containing an egg surface protein polypeptide epitope, the antibodies resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants.
The immunogen may include one or more polypeptides that comprise the amino acid sequences of SEQ ID NOS: 1-23. In addition the immunogen can also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation. In instances where the recombinant antigen is a hapten, i.e., a molecule that is antigenic in that it can react selectively with
cognate antibodies, but not immunogenic in that it cannot elicit an immune response, the hapten may be covalently bound to a carrier or immunogenic molecule; for instance, a large protein such as serum albumin will confer immunogenicity to the hapten coupled to it. The hapten-carrier may be formulated for use as a vaccine. The polypeptides may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases, such as, for example, sodium potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
The vaccines of the invention may be multivalent or univalent. Multivalent vaccines are made from recombinant viruses that direct the expression of more than one antigen. The patient to which the vaccine is administered is preferably a mammal, most preferably a human, but can also be a non-human animal including but not limited to cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats.
The vaccine formulations of the invention comprise an effective immunizing amount of the sperm surface protein and a pharmaceutically acceptable carrier. Vaccine preparations comprise an effective immunizing amount of one or more antigens and a pharmaceutically acceptable carrier. The carrier is preferably sterile. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. The formulation should suit the mode of administration. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is administered by
injection, an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration. The invention also provides a pharmaceutical pack or kit comprising one or more containers comprising one or more of the ingredients of the vaccine formulations of the invention. One aspect of the present invention is directed to the antibodies
(monoclonal or polyclonal) that bind to the SSPs. In particular, those antibodies that immunospecifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-23. In one preferred embodiment the antibody is a monoclonal antibody. The antibodies generated against the SSP antigens of the present invention also have potential uses in vaccination against fertilization, sterilization, diagnostic immunoassays, passive immunotherapy, and generation of antiidiotypic antibodies. Thus the present invention encompasses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of an anti-sperm surface protein antibody effective to inhibit fertilization. In immunization procedures, the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the art and will be administered by reference to the immune response and antibody titers of the subject.
In accordance with one embodiment, a method is provided for preparing an contraceptive antiserum, wherein the antiserum comprises an antibody against a sperm surface antigen. In a preferred embodiment the sperm surface antigen is a protein unique to male gametes and more particularly to sperm cells. The method comprises the steps of immunizing and animal with a sperm surface protein or an immunogenic fragment thereof, obtaining a serum sample from the immunized animal, screening the serum for the ability to bind to a sperm surface protein (or immunogenic fragment thereof) and recovering serum based on its ability to bind the sperm surface protein. The resulting serum may include polyclonal antibodies or may comprise one or more monoclonal antibodies. Alternatively, antibody-secreting cells can be obtained from the immunized animal and those cells can be immortalized and screened, using standard techniques, for cells that secrete antibodies that bind to sperm surface proteins. Those cells that secrete antibodies that bind to sperm surface proteins can be isolated and used to produce antibodies.
The generated antibodies may be isolated by standard techniques known in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays. The antibodies may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art may be used for this purpose including but not limited to competitive and noncompetitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich" immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays.
The vaccine formulations of the present invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of a host is achieved by the administration of pre-formed antibody directed against a heterologous organism. The antibodies generated by the vaccine formulations of the present invention can also be used in the production of antiidiotypic antibody. The antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen of the pathogenic microorganism (Jerne, 1974, Ann. Immunol, (paris) 125c:373; Jerne, et al., 1982, EMBO J. 1 :234).
Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g. , encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradennal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
In another embodiment, the therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317- 327; see generally ibid. )
In yet another embodiment, the therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neural. 25:351 (1989); Howard et al., J. Neurosurg. 71 :105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the testes, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138).
The present invention also provides pharmaceutical compositions. The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include
a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The amount of the therapeutic of the invention which will be effective in the treatment of a particular or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suppositories generally contain active ingredient in the range of 0.5% to 10%o by weight; oral formulations preferably contain 10% to 95% active ingredient. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
Example 1 Materials: Human sera
The sera were obtained from infertile men and women with "unexplained" infertility. The infertile subjects did not have any previously diagnosed hormonal, infective or physical causes for their infertility . The male subjects did not undergo vasectomy.
Immunobead binding
Antisperm antibodies in the infertile female patient sera were detected by indirect immunobead binding as described in Manual of clinical laboratory immunology, 4th edition, American society for microbiology, Washington, D.C 1992; 1013-1017. The antisperm antibodies bound to the spermatozoa of male patients were detected by direct immunobead binding. All the serum and sperm samples were tested for the presence of IgG, IgM and IgA specific antisperm antibodies and included 18 infertile male subjects, 9 infertile female subjects, 5 fertile male subjects and 5 fertile female subjects.
Criteria for choosing the sera for the western blot analysis
Sera were chosen for further study based on a high IBT score: i.e., more than 60% of the spermatozoa were observed to bind beads indicative of IgG and/or IgM specific antisperm antibodies. All the selected sera contained antibodies directed against the sperm head or the entire spermatozoa. A total of 15 serum samples were chosen from infertile men and 6 serum samples were chosen from infertile women for the western blot analysis.
Preparation of sperm antigens Semen specimens were obtained from fertile donors with normal sperm quality. Only ejaculates with normal semen characteristics were used in this study. After liquefaction of the semen the mature sperm were separated from the seminal plasma, immature germ cells and non-sperm cells (mainly white blood cells and epithelial cells) by Percoll (Pharmacia Biotech, Uppsala, Sweden) density centrifugation as described by Naaby-Hansen et al. (Biol Reprod 1997; 56: 771-787). Prior to the last centrifugation, the cells were counted and all samples showed >90% motility. The spermatozoa obtained from 8-12 individuals were pooled and frozen immediately until further use. All samples were obtained under informed consent using forms approved by the University of Virginia Human Investigation Committee.
Radio-iodination
Radio-iodination was performed according to the procedure adopted
and standardized by Naaby-Hansen et al (Biol Reprod 1997; 56: 771-787). Percoll- purified spermatozoa were suspended in Ham's F-10 medium to a final concentration of 20 x lOVml. Washed Iodo-beads (Pierce Chemical Co., Rockford, IL) (one bead per 8 x 106 spermatozoa) and carrier-free 125I-Na (10 μCi/106 spermatozoa) were added to the sample. Radiolabeling was performed by incubating the sample for 10 min at 20°C on a rocking table. The cells were removed from the iodo-beads by pipetting and immediately were subjected to a second Percoll density gradient centrifugation, before being washed three times in Ham's F-10 medium. 'Surface' proteins studied here are predominantly plasma membrane proteins accessible to external labeling on ejaculated sperm, which, following the preparation methods employed, consist almost entirely of cells that have not undergone the acrosome reaction. The cells were counted before the final wash, and the resulting pellet was used for extraction of sperm proteins. Autoradiography was performed using a sandwich of components in the following order; intensifying screen, blot, two layers of film, and intensifying screen. X-ray films were routinely exposed for 3 weeks.
Solubilization Procedures
Spermatozoa were solubilized in a lysis buffer containing 2% octyl-β gluco-pyranoside (OBG) containing 100 mM dithiothreitol (DTT), urea (9.8 M), and the protease inhibitors: 2 mM PMSF, 5 mM iodoacetamide, 5 mM EDTA, 3 mg/ml L- l-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride (TLCK), 1.46 mM pepstatin A, and 2.1 mM leupeptin. Five x 108 cells per milliliter were solubilized by constant shaking at 4° C for 45 min. Insoluble material was removed by centrifugation at 10,000 x g for 2 min, and the supernatant was applied to the first electrophoretic dimension.
Electrophoresis
Isoelectric focusing was performed using the gel composition as described earlier (Naaby-Hansen et al., Biol Reprod 1997; 56: 771-787). Carrier ampholine compositions were 20% pH 5-7, 20% pH 7-9 and 60% pH 3.5 -10. Sixty- five microliters of sperm extract (~0.15 mg of protein) were applied per rod. The tubes were filled by gently overlaying the sample with a buffer containing 5% NP-40,
1% ampholines (pH 3.5-10), 8 M urea and 100 mM DTT . Focusing was conducted for a total of 17,700 volt-h using voltage stepping: 2h at 200v, 5h at 500v, 1 lh at 800 v and 3h at 2000 v.
Nonequilibrium pH gradient electrophoresis (NEPHGE) was performed in 14 x 0.15 -cm acrylamide rods using the gel composition described by Celis et al., Electrophoresis 1992; 13:893- 959) and Naaby-Hansen et al (Biol Reprod 1997; 56: 771-787). The carrier ampholine composition was 13% pH 3.5-10,37% pH 5-8,13% pH 6.5-9 and 37% pH 8-10.5. 55μl (-0.13 mg of protein) of spermatozoa extract was applied per rod. Electrophoresis was conducted for a total of 8600 volt-h using 400 volts for 5 h and 550 volts for 3 hours.
Two dimensional SDS-P AGE was carried out in 0.15 -cm- thick, 16 x 16-cm slab gels using linear gradient gels (T= 9-15%; T= concentration of acrylamide in the gel) in a Protean II xi Multi-cell apparatus (Bio-Rad, Richmond, CA) (Biol Reprod 1997; 56: 771-787). Electrotransfer to nitrocellulose membranes was carried out as described in Biol Reprod 1997; 56: 771-787.
Immunoblotting Analysis
Following electrophoretic transfer of 2-D separated polypeptides, the membranes were rinsed twice for 5 min in PBS (pH 7.4), and excess binding sites were blocked by incubation for 1 hour at room temperature in PBS (pH 7.4) containing 5% dry milk and 0.05% Tween 20. The blots were incubated with the serum at 1 :2000 dilution at 4°C overnight under constant slow rocking. In some cases a given blot was incubated with more than one serum. In these instances the blots were washed in PBS (pH 7.4) containing 0.05% Tween 20 following incubation with the first serum at 4°C overnight and incubated with another serum under the same conditions as described above. 15 sera were analyzed from infertile men and 6 sera were analyzed from infertile women which contained antisperm antibodies as shown by the IBT. Secondary enzyme-conjugated antibodies (Jackson ImmunoResearch Lab., WestGrove, PA) (goat antihuman IgG+lgM) were diluted 1 :5000 in PBS containing 0.05% Tween 20 and the blots were incubated for 1 hour at 20°C. The secondary antibody alone did not bind to blotted human sperm proteins (data not shown). Horseradish peroxidase conjugates were visualized by enhanced
chemiluminescence (ECL) using the manufacturer's protocol (Amersham Corp., U.K).
Scanning and computer analysis
X-ray films from chemiluminescence preparations and the autoradiograms from the radio-iodination experiments were scanned with a Kodak camera (Eastman Kodak, Rochester, NY). The resulting 2-D images were analyzed using the Bio Image "2D analyzer" version 6.1 (Bio Image, Ann Arbor, MI). This software automates the identification, quantification and comparison of 2D -gel separated protein spots. A composite image representing the aggregate protein spots recognized by the fertile subjects of both sexes was obtained after matching the computer scanned images of the ECL blots corresponding to fertile male and female individuals by using protein spots common to each image as reference points or "anchors". The anchors were chosen based on the electrophoretic mobility , the constellation of the protein spots and the shape of the spots. The resulting composite image of the separated protein spots was compared and matched with the images corresponding to infertile male and female groups to subtract the protein spots recognized by the fertile subjects from the protein spots recognized by the infertile subjects, thus identifying the major auto- and iso-antigens which are uniquely recognized by the infertile subjects. A database of the sperm surface proteins (sperm surface encyclopedia) was created following the matching of auto radiograms obtained after surface radio- iodination. Care was taken to include only those spots which were consistently labeled. The computer generated images containing the auto-antigens and the iso- antigens were matched with the sperm surface encyclopedia to identify the surface auto- and iso-antigens.
Results
All the infertile sera studied (15 men, and 6 women) contained IgG and/or IgM antisperm antibodies with more than 60% of the sperm binding beads in the IBT. The control sera used from the fertile individuals (5 males and 5 females) were negative for the IBT with less than 10% of spermatozoa bound by immunobeads. Silver stained 2-D gels (16 x 15 cm) of nonionic detergent/urea-
solubilized human sperm proteins resolved more than 1300 protein spots by IEF/PAGE and NEPHGE/PAGE. These gels represent the repertoire of protein spots obtained from a pool of 10 individuals' sperm. The protein pattern was highly reproducible although minor variations between donors were observed with regard to the relative abundance and charge of same proteins which were found to vary when individual samples were compared by computer analysis. The identities of some of the major protein spots were identified by microsequencing individual spots or immunoblotting with antisera to specific proteins. For example, different heat shock protein family members were resolved. These proteins are abundant on the sperm membrane. PH-20, a sperm hyaluronidase, is a membrane bound protein represented by three 53 kDa isoforms. The major cytosolic proteins, α-tubulin and β-tubulin were also detected as was human sperm calreticulin.
2D western blots of sperm proteins resolved by IEF or NEPHGE were probed with serum from a fertile male subject or with serum from an infertile male subject. The serum from the infertile subject showed stronger immuno-reactivity to some spots than the fertile subject although the importance of such differences in intensity are not presently understood. In addition, a number of antigenic spots among both acidic and basic proteins were reactive with infertile but not fertile sera. Similarly, differences in immunological reactivity were noted in comparing western blots probed with an infertile female serum to those reactive with fertile female serum; the infertile subject's serum recognized more antigenic spots and also showed stronger reactivity to some spots than did the serum from the fertile female.
Analysis of the acidic antigens Analysis of individual infertile sera showed marked variability in the range of specific proteins recognized but also demonstrated that some antigens were common to different individuals. This observation of a range of recognized sperm antigens implies considerable diversity in the antisperm antibody repertoire in the human male. On the other hand, similarity in the patterns of immunoreactivity for certain sperm antigens was frequently noted. Computer analysis and comparison of blots reacted with infertile and fertile sera indicated that 4 protein spots at approximately 60 kDa recognized by both infertile individuals belong to a subset of
proteins not recognized by the fertile subjects.
A comprehensive overview of the repertoire of immunoreactive sperm proteinsrecognized by fertile and infertile sera from both sexes was obtained by serially incubating blots with 5 sera from each group of subjects (Fig. 1A to ID). The sera from the infertile subjects were selected based on their high immunoreactivity , heterogeneity in the immunoreactivity, and unique recognition of certain protein spots following individual western blot analysis of the sera. Figures 1 A and IB present the repertoire of sperm proteins bound by sera from 5 fertile male (A) and 5 fertile female (B) subjects. Comparison of the sperm antigens recognized by fertile male (A) and fertile female (B) subjects reveals a remarkable overall similarity in the repertoire between the two sexes, although the fertile male sera recognized a greater number of antigens than did the fertile female sera. Figures IC and ID show the western blots serially probed with sera from 5 infertile male (C) and 5 infertile female subjects (D) respectively. Comparison of the western blots probed with fertile patient sera and infertile patient sera (Fig 1A+B to C+ D) demonstrates that several discrete sperm antigens were recognized by the infertile patients (arrows). The molecular weights of prominent protein spots uniquely recognized by male patients are 34 kDa (pi 4.2), 42 kDa (pi 4.3), 88.5 kDa ( pi 4.0), 106 kDa (pi 4.2), 41.8 kDa (pi 5.3, pi 5.4, pi 5.5),61.5 kDa (pi 5.62), 60.4 kDa (pi 5.75) and 60 kDa (pi 6.6 and 6.7). Major iso- antigens recognized by infertile females are at the molecular weights of 60 kDa (pi 6.2, pi 6.4, pi 5.82, pi 5.88),32.3 kDa (pi 6.7, pi 6.8, pi 6.85) and 41.2 kDa (pi 5.1 ).
Variation in the immunoreactivity between male and female patient sera
The computer image analysis of the immunoblots showed that a total of 310 antigens were recognized by the infertile male patient sera whereas 205 antigens were recognized by the infertile female patient sera. The results suggest that the immunoreactivity to sperm antigens shown by infertile males is probably higher than that of infertile females. Further, a distinct variation in the pattern of immunoreactivity is discernible between the two sexes, each of these groups showing specificity for different sets of proteins (Fig. IC and ID) even though proteins common to both subject groups were noted.
Computer aided subtraction of the antigens recognized by both male
and female fertile subjects from the total repertoire of antigens recognized by the infertile male groups revealed 86 protein antigens which were exclusively recognized by infertile male subjects (Fig 2 A) compared to the fertile subjects. Of these 86 antigens which were uniquely recognized by the infertile male patients, 72 were specific to only infertile male patient sera and were not recognized bv infertile female sera. Similar computer aided analysis of the 2-D blots yielded 26 protein spots which were unique to the infertile female subjects (Fig. 2B). 12 of these antigens were specific to only infertile female patients and were not recognized by infertile male patients. Among the sperm antigens which were exclusively recognized by the infertile patients sera (both males and females) 14 antigens were commonly recognized by both male and female patient sera (arrows).
Sperm surface antigens exclusively recognized by the ASA positive patients
Figure 3 presents a computer generated composite image of human sperm surface proteins obtained following computer scanning of autoradiograms obtained from 2D IEF-PAGE of vectorially iodinated sperm proteins (Biol Reprod 1997; 56: 771-787). 103 proteins were identified on the sperm surface by this method. Computer aided matching of the sperm auto- and iso-antigens with the composite image of the sperm surface proteins provided for the identification of a subset of sperm surface auto- and iso-antigens. The location of six sperm surface auto-antigens and iso-antigens are depicted in the Fig. 3 (arrows). Prominent are two 60 kDa proteins with pis of 6.2 and 6.4 which were recognized by 2 infertile womens' sera. Among the sperm antigens recognized by the infertile male sera, 2 sera recognized a 88.2 kDa sperm surface protein with a PI of 4.0. Two of the sera also recognized two sperm surface proteins at molecular weights of34 kDa and 38 kDa, both at a PI of 4.2. Table 1 lists the masses and pi of the sperm surface iso- and auto- antigens.
Table 1. Sperm surface auto- and iso-antigens correlated with the IBT results of the cognate patient sera.
Correlation of the western blot results with the IBT results It was interesting to note that those sera that gave a high score in the immunobead test for binding to various surface domains contained antibodies which were shown in the 2D analysis to recognize sperm surface proteins on the western blots. Table 1 also notes the immunobead pattern of these subjects' sera. Each of these sera showed IBT reactivity towards IgG and/or IgM specific antibody. The antibodies were directed towards the head or the entire surface of the sperm.
Discussion
Specific sperm proteins were recognized and identified on 2-D gels by the serum from antisperm antibody positive infertile subjects. These antigens are likely to play a significant role in causing immunoinfertility and may play a role in events of the fertilization cascade. Many studies carried out in the past using western blotting of unidimensional gels yielded variable results regarding the molecular weights of the spermatozoal antigens recognized by serum containing antisperm antibodies. Most of those studies were performed on infertile individuals and the reaction of sperm antigens with fertile control sera was often not evaluated. Further, contradictory reports regarding the molecular weights of the reactive sperm antigens may be due to variations in the sperm extraction procedure. In the present invention
two dimensional electrophoresis was employed for the separation of the proteins based on the iso-electric points of the individual proteins in the first dimension and on the molecular weight in the second dimension, which offers better resolution of the proteins and may serve as a prelude to microsequencing. The high resolution 2-D gel techniques which were optimized for sperm protein separation are suitable for screening sera for antisperm antibodies. Initial screening of the sera from the infertile patients for the presence of sperm surface antibody by IBT provided additional focus by directing attention to sera containing antisperm antibodies which are relevant in antibody mediated events of agglutination, cytotoxicity or blocking at the sperm surface and thus functionally related to infertility.
Immunoblot analysis of individual sera imply that there is considerable diversity in the antisperm immunoglobulin repertoire. The finding in the present study that many of the patients did not recognize the same set of antigens may be due in part to the complexity of sperm architecture and diversity of antigens involved in the autoimmune response. It perhaps also suggests that the mechanism and pattern of infertility in these patients might differ. Antibodies recognizing different sperm surface proteins may influence the ability of sperm to fertilize by acting at different stages of the fertilization process. Differences in the antibody repertoire might also relate to differences in the mode of immunization. Previous studies have shown that the subclasses of antisperm IgA antibodies vary in men who have autoimmunity to sperm (Branson et al., Human sperm antibodies and their detection. In: Manual of clinical laboratory immunology, 4th edition, American society for microbiology, Washington, D.C 1992; 1013-1017). Western blot data (see Fig. 1 A and IB) confirm the presence of sperm auto-antibodies in a high proportion of both fertile and infertile individuals, although the fertile individuals recognized many fewer sperm antigens and the relative intensity of the reactivity was weaker compared to the infertile individuals. These data imply that most of the sperm antigens recognized by fertile sera are internal because each of these sera was negative for antisperm antibodies in the immunobead test. Several of these intracellular antigens have been microsequenced and identified as components of sperm specific structures such as the fibrous sheath and outer dense fibers. In the
present invention, systematic identification of a subset of these antigens has aided in the identification of sperm antigens which elicit production of functionally relevant and/or irrelevant antibodies which may have a role in the impairment of reproductive function. The serial incubation of sera for immunoblotting combined with computer analysis succeeded in providing information on the major auto- and iso- antigens unique to the infertile patients. Male sera recognized many more antigens than female sera (310 versus 205) although a comprehensive evaluation with a greater number of samples from female subjects is required to statistically validate the conclusion. A longer constant exposure of sperm proteins to the immune system among the male patients may explain the higher reactivity among males. In addition, a distinct difference in the pattern of immunoreactivity between male and female patients was discernible. The male patient sera was reactive to several acidic proteins ( pi 4.0 to 5.0) which were not recognized by any of the analyzed sera from females (see Fig. IC and ID). Conversely, major iso-antigens recognized by the female patients in the pi range of 6.0 to 7.5 were poorly reactive to sera from the male patients (Fig. IC and ID).
A rational basis for identifying fertility related sperm antigens relies on the identification of those antigens which are on the cell surface. Hence it is especially critical to distinguish between immunity to sperm surface antigens and the internal antigens of spermatozoa. For this reason the sperm surface proteins of live sperm were labeled, rather than labeling the surface proteins of fixed sperm (which have damaged membranes aloowing the labeling of internal proteins). The sperm surface proteins were radiolabeling with 125I followed by IEF /PAGE as well as NEPHGE/PAGE separation (see Naaby-Hansen et al., Biol Reprod 1997; 56: 771 - 787). Utilizing computer analysis a composite image was obtained consisting of 103 surface labeled proteins in IEF /PAGE gels. This computer image enabled the categorization a subset of 6 sperm surface antigens which may have role in immunoinfertility. One of these antigens is a 88.2 kDa protein at a pi of 4.0. , recognized by two male infertile patients. Naaby-Hansen et al (Biol Reprod 1997; 56: 771-787) have earlier shown this protein to be a sperm surface protein, phosphorylated on tyrosine residues. Interestingly, two women's sera showed striking
similarity in the immunoreactivity, recognizing the same protein spots at 60 kDa and pi of 6.2 and 6.4. Both these sperm proteins were found to be on the surface of the sperm as demonstrated by surface labeling of the sperm with radioactive I25I. Further, both sera contained IgG antibodies directed towards the head of the sperm as shown by immunobead test, confirming that the antigens are exposed on the sperm surface, probably located on the sperm head. Two male patients' sera strongly recognized 2 protein spots at 34 kDa and 38 kDa which were identified on the sperm surface. Although the current analysis of the sera based on both IBT and western blotting to diagnose immunity to sperm is able to detect the presence of antisperm antibodies and identify the cognate antigens on the sperm surface, identification of the epitopes against which these antibodies are directed should improve the reliability of the analysis. Recognition of multiple antigens which appear to have a potential role in fertility could occur due to common epitopes.
Earlier reports have shown that monoclonal antibodies of the same iso- type but directed against different antigens present on the sperm head could block sperm attachment to the zona pellucida or sperm-egg membrane fusion. Further, antibodies directed against different epitopes of the same sperm surface antigen may also affect sperm fertilizing ability in different ways.
The computer digitization of information on the 2-D gel patterns of surface labeled sperm proteins opens the possibility for a new tool in the diagnosis of antisperm antibodies and possible treatment of antibody mediated infertility. Based on the sperm proteome and the index of sperm surface proteins, 2-D immunoblotting with patients sera can now be employed to ascertain those patients with antibodies to protein antigens accessible at the cell surface and hence obtain more specific information on a patient's repertoire of auto or iso- antibodies. As these databases expand with the identification of additional iso- and auto-antigens, and as micro sequence information and micro sequence based cloning provides characterization of the relevant proteins, the possibility may emerge for a rational basis for immunotherapy of antibody mediated infertility using defined native or recombinant sperm antigens. Further, diagnosis of antibody mediated infertility may be simplified by using 2-D immunoblots with a patient's serum and referencing the result to the database of surface antigens.
Example 2
Sequence analysis of two 2D gel bands. Identification of hyaluronic acid binding protein (peptide no. 1, Table 1)
Methods:
An upper and lower band ran at approximately 36 kDa with a pi of 4.4. The band was cut from the gel as closely as possible to minimize excess polyacrylamide, divided into a number of smaller pieces and washed and destained in 500 μL 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 50 μL of 10 mM dithiolthreitol in 0.1 M ammonium bicarbonate and reduced at 55 °C for 1 h. The DTT solution was removed and the sample alkylated in 50 μL 50 mM iodoacetamide/0.1 M ammonium bicarbonate at room temperature for 1 h in the dark. The reagent was removed and the gel pieces washed with 100 μL 0.1 M ammonium bicarbonate and dehydrated in 100 μL acetonitrile for 5 min. The acetonitrile was removed and the gel pieces rehydrated in 100 μL 0.1 M ammonium bicarbonate. The pieces were rehydrated in 100 μL acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 12.5 ng/μL trypsin in 50 mM ammonium bicarbonate and incubated on ice for 45 min. Any excess trypsin solution was removed and 20 μL 50 mM ammonium bicarbonate added. The sample was digested overnight at 37° C and the peptides formed extracted from the polyacrylamide in two 200 μL 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to <20 μL for LC-MS analysis.
The LC-MS system consisted of Finnigan-MAT TSQ7000 system with an electrospray ion source interfaced to a 10 cm x 75 um id POROS 10 RC reversed phase capillary column. One μL volumes of the extract are injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.6 μL/min. The electrospray ion source is operated at 4.5 kV with a 1.2 μL/min coaxial sheath liquid flow of 70% methanol/30% water/0.125%) acetic acid and a coaxial nitrogen flow adjusted as needed for optimum sensitivity. The digest was analyzed by capillary LC-electrospray mass spectrometry to measure the molecular weight of the peptides present in the digest. Peptide sequences for the peptides
detected were determined by collisionally activated dissociation using LC- electrospray-tandem mass spectrometry with argon as the collision gas.
Results: The molecular weights determined by LC-MS analysis and amino acid sequences determined by LC-tandem MS of peptides these two digests are shown in Table 2. Six peptides were detected in the darker stained band, LB3-83-2 (Anne lower) and four of those same peptides (peptides #2, 3, 4, and 5) were detected in the lighter stained band, LB3-83-1 (Anne upper). Sequence information was obtained for five of the six peptides. Database searches using CAD spectral information
(SEQUEST) and partial peptide sequences (BLAST) identified two peptides, numbers 3 and 4, in the database sequence of human hyaluronic acid binding protein (NCBInr.6.8.97 accession number 730772, calculated MW=31.4 kDa, calculated pl=4.3). The other peptides could not be found in this or any other database sequence. In summary, both bands produce similar series of peptides. Both bands contain two peptides that match the database sequence of human hyaluronic acid binding protein. Both bands contain additional peptides that cannot be matched to any database peptide and therefore appear to be from a second, unknown protein.
Table 2. Peptide sequences from two 2D gel bands (LB3-83-1 and LB3-83-2).
1 X designates I or L which cannot be distinguished by low energy CAD, Mo designates an oxidized M, lower case letters designate tentative assignments, _ designates a single unknown amino acid, — designates an unknown number of unknown amino acids. Abbreviations are as follows: "X" = He or Leu; SVXXSXK represents amino acid sequences
Ser Val He Leu Ser Leu Lys (SEQ ID NO: 24), Ser Val Leu He Ser Leu Lys (SEQ ID NO: 25),
Ser Val Leu Leu Ser He Lys (SEQ ID NO: 26),
Ser Val He He Ser Leu Lys (SEQ ID NO: 27),
Ser Val He He Ser He Lys (SEQ ID NO: 28),
Ser Val He Leu Ser He Lys (SEQ ID NO: 29), and Ser Val Leu Leu Ser Leu Lys (SEQ ID NO: 30); VYTS represents amino acid sequence
Val Tyr Thr Ser (SEQ ID NO: 31);
GGWEXEXNGTEAK represents amino acid sequences Gly Gly Trp Glu He Glu He Asn Gly Thr Glu Ala Lys (SEQ ID NO: 32), Gly Gly Trp Glu He Glu Leu Asn Gly Thr Glu Ala Lys (SEQ ID NO: 33), Gly Gly Trp Glu Leu Glu He Asn Gly Thr Glu Ala Lys (SEQ ID NO: 34), and Gly Gly Trp Glu Leu Glu Leu Asn Gly Thr Glu Ala Lys (SEQ ID NO : 35);
AFVDFXSDEXKEER represents amino acid sequences Ala Phe Val Asp Phe He Ser Asp Glu He Lys Glu Glu Arg (SEQ ID NO: 36), Ala Phe Val Asp Phe He Ser Asp Glu Leu Lys Glu Glu Arg (SEQ ID NO: 37), Ala Phe Val Asp Phe Leu Ser Asp Glu He Lys Glu Glu Arg (SEQ ID NO: 38), and Ala Phe Val Asp Phe Leu Ser Asp Glu Leu Lys Glu Glu Arg (SEQ ID NO: 39); and
SGGWELELNGTEAK represents amino acid sequences Ser Gly Gly Trp Glu Leu Glu Leu Asn Gly Thr Glu Ala Lys (SEQ ID NO: 40).
Example 3 Sequence analysis of 3 CM stained 2D-gel bands (peptide no. 2, Table 1).
Methods:
The gel piece was transferred to a siliconized tube and washed and destained in 200 μL 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 μL of 10 mM dithiolthreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 μL 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 μL acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 μL 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 μL acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/μL trypsin in 50 mM ammonium bicarbonate on ice for 10 min. Any excess trypsin solution was removed and 20 μL 50 mM ammonium bicarbonate added. The sample was digested overnight at 37 °C and the peptides formed extracted from the polyacrylamide in two 30 μL aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 25 μL for LC-MS analysis.
The LC-MS system consisted of a Finnigan LCQ ion trap mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm x 75 um id Phenomenex Jupiter 10 um C18 reversed-phase capillary column. 0.5-5 μL volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.25 μL/min. The nanospray ion source was operated at 2.8 kV. The digest was analyzed using the double play capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in sequential scans. This mode of analysis produces approximately 400 CAD spectra of ions ranging in abundance over several orders of magnitude. Not all CAD spectra are derived from peptides.
The data were analyzed by database searching using the SEQUEST search algorithm. Peptides that were not matched by this algorithm were interpreted manually and searched versus the EST databases using the SEQUEST algorithm.
Results:
The peptides shown in Table 3 were detected in band C48. None of these peptides match any protein or EST entries. The peptides appear completely novel.
Table 3. Peptide sequences from Band C48 (LB6-52-3).
1 1 and L cannot be distinguished by low energy CAD but are inferred by the database sequence, M(o) designates oxidized M, C is carbamidomethyl modified unless noted as Ca (acrylamide), _ designates a single unknown residue, — designates an unknown number of unknown residues. Abbreviations are as follows: "X" = He or Leu;
XQQT represents amino acid sequences He Gin Gin Thr (SEQ ID NO: 41) and Leu Gin Gin Thr (SEQ ID NO: 42);
XNSQWVVPLR represents amino acid sequences He Asn Ser Gin Trp Val Val Pro Leu Arg (SEQ ID NO: 1); and Leu Asn Ser Gin Trp Val Val Pro Leu Arg (SEQ ID NO: 2);
MVNXMXXXR represents amino acid sequences Met Val Asn He Met He He He Arg (SEQ ID NO: 3), Met Val Asn Leu Met He He He Arg (SEQ ID NO: 4), Met Val Asn He Met Leu He He Arg (SEQ ID NO: 5), Met Val Asn He Met He Leu He Arg (SEQ ID NO: 6), Met Val Asn He Met He He Leu Arg (SEQ ID NO: 7), Met Val Asn He Met He Leu Leu Arg (SEQ ID NO: 8), Met Val Asn He Met Leu Leu He Arg (SEQ ID NO: 9), Met Val Asn He Met Leu He Leu Arg (SEQ ID NO: 10), Met Val Asn Leu Met Leu He He Arg (SEQ ID NO: 11), Met Val Asn Leu Met Leu He He Arg (SEQ ID NO: 12), Met Val Asn Leu Met He Leu He Arg (SEQ ID NO: 13), Met Val Asn Leu Met He He Leu Arg (SEQ ID NO: 14),
Met Val Asn Leu Met He Leu Leu Arg (SEQ ID NO: 15), Met Val Asn Leu Met Leu Leu He Arg (SEQ ID NO: 16), Met Val Asn Leu Met Leu He Leu Arg (SEQ ID NO: 17);
Example 4
Peptides from band 2 were isolated as described in Examples 3 and 4. The peptides shown in Table 4 were detected in the band 2 digest and could not be matched to any database sequence. The peptides were low abundance and the digest contained a number of higher abundance peptides that all matched keratin sequences.
Table 4. Peptide sequences from Band 2 (LB5-83-10).
1 1 and L cannot be distinguished by low energy CAD but are inferred by the database sequence, Mo designates oxidized M, C is carbamidomethyl modified unless noted, _ designates a single unknown residue, designates an unknown number of unknown residues. 2 Molecular weights for +2 ions are monoisotopic, molecular weights for >+3 ions are average. Abbreviations are as follows: "X" - He or Leu;
PXSS represents amino acid sequences Pro He Ser Ser (SEQ ID NO: 43) and Pro Leu Ser Ser (SEQ ID NO: 44);
MXPVXE represents amino acid sequences Met He Pro Val He Glu (SEQ ID NO: 18), Met Leu Pro Val He Glu (SEQ ID NO: 19), Met He Pro Val Leu Glu (SEQ ID NO: 20), and
Met Leu Pro Val He Glu (SEQ ID NO: 21);
HGQSAEYEF represents amino acid sequence His Gly Gin Ser Ala Glu Tyr Glu Phe (SEQ ID NO: 22).
FQQDGGAS represents amino acid sequence Phe Gin Gin Asp Gly Gly Ala Ser (SEQ ID NO: 23).
Band 3. The peptides shown in Table 5 were detected in the band 3 digest. Analysis of this data was complicated by the low abundance of the protein being digested. Peptides 4 and 6 were identified in the database sequence of human B7 protein (NCBI.nr.Ol .04.99 accession number 1732415, calculated MW = 36.1 kDa, calculated pi 5.0). Peptide 3 was identified in the database sequence of bacillus type I signal peptidase (NCBI.nr.Ol.04.99 accession number 126186, calculated MW = 21.0 kDa, calculated pi = 9.4). No database matches were found for any of the other peptides and none of those peptides could be matched to the EST database. One may also note that peptides 8 and 9 in band 3 are the same as peptides 2 and 3 in band 2.
Table 5. Peptide sequences from Band 3 (LB5-83-11).
1 1 and L cannot be distinguished by low energy CAD but are inferred by the database sequence, Mo designates oxidized M, C is carbamidomethyl modified unless noted, _ designates a single unknown residue, designates an unknown number of unknown residues. 2 Molecular weights for +2 ions are monoisotopic, molecular weights for >+3 ions are average. Abbreviations are as follows: "X" = He or Leu;
YIGEFDR represents amino acid sequence Tyr He Gly Glu Phe Asp Arg (SEQ ID NO: 45);
SLQYLNLR represents amino acid sequence Ser Leu Gin Tyr Leu Asn Leu Arg (SEQ ID NO: 46); DNQIDTSLGFSR represents amino acid sequence
Asp Asn Gin He Asp Thr Ser Leu Gly Phe Ser Arg (SEQ ID NO: 47);
MXPVXE represents amino acid sequences Met He Pro Val He Glu (SEQ ID NO: 18), Met Leu Pro Val He Glu (SEQ ID NO: 19), Met He Pro Val Leu Glu (SEQ ID NO: 20), and Met Leu Pro Val He Glu (SEQ ID NO: 21);
HGQSAEYEF represents amino acid sequence His Gly Gin Ser Ala Glu Tyr Glu Phe (SEQ ID NO: 22).
Claims
1. An isolated or recombinant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 22 and SEQ ID NO: 23.
2. The polypeptide of claim 1 wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 18 through SEQ ID NO: 22 and SEQ ID NO: 23.
3. A method of diagnosing immunological infertility, said method comprising the step of obtaining a serum sample from an individual; contacting the sample with a composition comprising a sperm surface antigen; and screening for antigen/antibody reactions.
4. The method of claim 3 wherein the step of screening for antigen antibody reactions comprises the use of a labeled secondary antibody.
5. The method of claim 4 wherein the sperm surface antigen is bound to a solid substrate.
6. The method of claim 5 wherein the sperm surface antigen is a protein recognized by sera isolated from infertile patients and wherein said protein is not recognized by sera from fertile patients.
7. The method of claim 5 wherein the sperm surface antigen is a polypeptide comprising an amino acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 22 and SEQ ID NO: 23.
8. A pharmaceutical composition comprising a peptide and a pharmaceutically acceptable carrier, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 22 and SEQ ID NO: 23.
9. The pharmaceutical composition of claim 8 further comprising an adjuvant.
10. A method for modulating fertility in a mammalian species, said method comprising administering to said mammalian species a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 22 and SEQ ID NO: 23.
11. The method of claim 10 wherein the composition further comprises an adjuvant.
12. The method of claim 10 wherein the composition is administered by intravenous injection.
13. An antibody that immunospecifically binds a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 22 and SEQ ID NO: 23.
14. A contraceptive composition comprising an antibody according to claim 13 and a pharmaceutical carrier.
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US5830472A (en) * | 1996-06-28 | 1998-11-03 | The University Of Virginia Patent Foundation | Purified sperm surface antigen, monoclonal antibody therefor and applications therefor |
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