Link:  Pharm/Biotech Resources

Title:  Multiple antigenic peptides immunogenic against Streptococcus pneumonia

United States Patent:  6,903,184

Issued:  June 7, 2005

Inventors:  Ades; Edwin W. (Atlanta, GA); Johnson; Scott E. (Lilburn, GA); Jue; Danny L. (Tucker, GA); Sampson; Jacquelyn S. (College Park, GA); Carlone; George M. (Stone Mountain, GA)

Assignee:  The United States of America as represented by the Secretary of the Department (Washington DC)

Appl. No.:  613092

Filed:   July 10, 2000

The invention provides a nucleic acid encoding the 37-kDa pneumococcal surface adhesion A protein (PsaA) from Streptococcus pneumoniae. The invention also provides purified polypeptides encoded by the nucleic acid encoding the 37-kDa protein from and the nucleic acids comprising unique fragment of at least 10 nucleotides of the 37-kDa protein. Additionally, multiple antigenic peptides that provide protection against S. pneumoniae challenge are provided. These multiple antigen peptides comprise the peptides that immunospecifically bind to the monoclonal antibodies. Also provided are vaccines comprising such immunogenic peptides, and methods of conferring protective immunity against Streptococcus pneumoniae infection by administering therapeutic composition comprising the immunogenic peptides of the invention. Also provided are methods of detecting the presence of Streptococcus pneumoniae in a sample using antibodies or antigens and methods of preventing and treating Streptococcus pneumoniae infection in a subject.

FIELD OF THE INVENTION

This invention relates to preventing infection by Streptococcus pneumoniae. More specifically, the invention relates to peptides derived from a peptide library that are related to the S. pneumoniae pneumococcal surface adhesion A protein (PsaA) and that are immunogenic in a subject. The invention also relates to pharmaceutical and therapeutic compositions containing these peptide fragments and methods of conferring protection against infection by S. pneumoniae. Even more specifically, this invention relates to multiple antigenic peptides immunogenic against Streptococcus pneumoniae.

BACKGROUND OF THE INVENTION

Pneumococcal disease continues to be a leading cause of sickness and death in the United States and throughout the world. The currently used polysaccharide vaccines have limited efficacy in children under 2 years of age and exhibit variable serotype-specific efficacy among vaccinated individuals. For these reasons, alternative vaccine formulations have been investigated that do not require the use of multiple capsular polysaccharides. One current approach under consideration is the use of immunogenic species-common proteins as vaccine candidates. These proteins could be used in combination with other immunogenic proteins or as protein carriers in a protein, polysaccharide, or oligosaccharide conjugate vaccine. An effective vaccine that includes a common protein could eliminate the need for formulations based on multiple capsular polysaccharides (as in the current 23-valent polysaccharide vaccine) by offering a broader range of protection against a greater number of serotypes. Additionally, a protein-based vaccine would be T-cell dependent and provide a memory response, thereby resulting in a more efficacious vaccine.

An immunogenic species-common protein has been identified from Streptococcus pneumoniae (Russell et al., 1990, "Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae. "J. Clin. Microbiol., 28:2191-2195; and U.S. Pat. No. 5,422,427). A 37-kDa S. pneumoniae protein has been the focus of several studies and is now designated pneumococcal surface adhesin protein A (PsaA). (This 0.37-kDa protein was referred to as pneumococcal fimbrial protein A in U.S. Pat. No. 5,422,427; the terms are used interchangeably in the present specification.) Immunoblot analysis studies using anti-PsaA monoclonal antibody showed that PsaA is common to all 23 pneumococcal vaccine serotypes (Russell et al., 1990). Enzyme-linked-immunosorbent assay studies have indicated that patients with pneumococcal disease show an antibody increase in convalescent-phase serum to PsaA compared with acute-phase serum antibody levels (Tharpe et al., 1995, "Purification and seroreactivity of pneumococcal surface adhesin A (PsaA)," Clin. Diagn. Lab. Immunol. 3:227-229; and Tharpe et al., 1994, "The utility of a recombinant protein in an enzyme immunoassay for antibodies against Streptococcus pneumoniae," Abstr. V-2, p 617, 1994, American Society for Microbiology, Washington, D.C.). Additionally, a limited in vivo protection study showed that antibodies to the 37-kDa protein protect mice from lethal challenge. (Talkington et al., 1996, "Protection of mice against fatal pneumococcal challenge by immunization with pneumococcal surface adhesin A (PsaA)," Microbial Pathogenesis 21:17-22). The gene encoding PsaA from S. pneumoniae strain R36A (an unencapsulated strain) has been cloned in Escherichia coli and sequenced; this strain, however, does not contain a 37-kDa protein encoding nucleic acid that is highly conserved among the various serotypes, (Sampson et al., 1994, "Cloning and nucleotide sequence analysis of PsaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins," Infect. Immun. 62:319-324). This particular nucleic acid and the corresponding polypeptide, therefore, are of limited value for use as diagnostic reagents, in preventing infection, in treating infection, or in vaccine development. In U.S. patent application Ser. No. 08/715,131, filed Sep. 17, 1996, (now U.S. Pat. No. 5,854,416) which is a continuation-in-part of U.S. patent application Ser. No. 08/222,179, filed Apr. 4, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 07/791,377, filed Sep. 17, 1991 (now U.S. Pat. No. 5,422,427), all of which are hereby incorporated by reference in their entirety, an isolated nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae, unique fragments of at least 10 nucleotides of this nucleic acid which can be used in methods to detect the presence of Streptococcus pneumoniae in a sample and as immunogenic vaccines have been disclosed. Furthermore, a purified polypeptide encoded by this nucleic acid, encoding the 37-kDa protein of Streptococcus pneumoniae, which can be used in immunogenic vaccines, has been disclosed. Additionally, purified antibodies which bind to the 37-kDa protein of Streptococcus pneumoniae or fragments thereof, which can be used in methods to detect the presence of Streptococcus pneumoniae, and in therapeutic and prophylactic methods, have been disclosed. Sequence conservation is a necessary requirement for a candidate species-common vaccine. The sequence conservation of the PsaA gene among pneumococcal types, and specifically among encapsulated pneumococci which cause the vast majority of cases of serious disease, remains under investigation. There exists a need to identify characteristic epitopes related to S. pneumoniae PsaA in order to provide polypeptides which can serve as a vaccine for multiple strains of Streptococcus pneumoniae. The present invention addresses this need by determining effective epitopic peptides related to S. pneumoniae PsaA, and employing those peptides in therapeutic compositions directed against Streptococcus pneumoniae infection.

SUMMARY OF THE INVENTION

The present invention describes novel immunogenic peptides obtained from a random library by selecting for high affinity binding to monoclonal antibodies specific for PsaA epitopes. In addition, the peptides of the invention have the capability of serving as immunogens in a subject, thereby effectively eliciting the production of antibodies by the subject and additionally conferring protective immunity against infection by S. pneumoniae on the subject. The invention also relates to a selection method employed to obtain such peptides.

The peptides of the invention include peptides comprising residues whose sequence is chosen from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.10, and immunogenic fragments thereof. In certain embodiments, the peptides consist essentially of a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.10, and immunogenic fragments thereof. In certain embodiments the peptides consist of a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.10, and immunogenic fragments thereof.

The invention additionally provides peptides as described above, wherein the peptides are multiple antigenic peptides. In one embodiment, the multiple antigenic peptide has at least one arm comprising a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ. ID. NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.10, and immunogenic fragments thereof. In another embodiment, the multiple antigenic peptide has at least one arm consisting essentially of a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.10, and immunogenic fragments thereof. In-another embodiment, the multiple antigenic peptide has at least one arm consisting of a sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ. ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO.10, and immunogenic fragments thereof.

In one embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:5 and at least one second arm comprising SEQ ID NO:6. In another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:5 and at least one second arm comprising SEQ ID NO:7. In another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:5 and at least one second arm comprising SEQ ID NO:9. In another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:6 and at least one second arm comprising SEQ ID NO:7. In another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:10 and at least one second arm comprising SEQ ID NO:9. In yet another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:10 and at least one second arm comprising SEQ ID NO:7. In still another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:5 and at least one second arm comprising SEQ ID NO:10. In another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:6 and at least one second arm comprising SEQ ID NO:7. In another embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:5, at least one second arm comprising SEQ ID NO:6, and at least one third arm comprising SEQ ID NO:7. In another preferred embodiment, the multiple antigenic peptide has at least one first arm comprising SEQ ID NO:5, at least one second arm comprising SEQ ID NO:9, and at least one third arm comprising SEQ ID NO:10.

In another aspect, the current invention is a peptide that immunospecifically binds to a monoclonal antibody obtained in response to immunizing an animal with Streptococcus pneumoniae PsaA, wherein the peptide is lipidated. In one embodiment, the peptide is lipidated with monopalmitic acid.

The invention furthermore provides a therapeutic composition in which the immunogenic peptides are combined with an immunostimulatory carrier to be administered to a subject in order to elicit an immune response which confers protective immunity against infection by S. pneumoniae on the subject.

The invention additionally provides a therapeutic composition in which the immunogenic peptides are combined with an adjuvant to be administered to a subject in order to elicit an immune response which confers protective immunity against infection by S. pneumoniae on the subject.

The invention additionally provides a therapeutic composition in which the immunogenic peptide is multiple antigenic peptide of the current invention as described above.

The invention additionally provides a therapeutic composition in which the immunogenic peptide is a peptide that immunospecifically binds to a monoclonal antibody obtained in response to immunizing an animal with Streptococcus pneumoniae PsaA, wherein the peptide is lipidated. In one embodiment, the peptide is lipidated with monopalmitic acid.

The invention still further describes a method of conferring protective immunity against infection by S. pneumoniae on a subject in which the therapeutic compositions of the invention are administered to the subject.

A further aspect of the invention presents a method for identifying a peptide incorporating PsaA or a fragment thereof (i.e., an immunogenic peptide) that elicits an immunogenic response in a subject directed against S. pneumoniae. The method entails preparing a random peptide library, screening the peptide library in order to identify immunogenic peptides, and obtaining the amino acid sequence of the immunogenic peptide.

The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully to describe the state of the art to which this application pertains.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic Acids

In one aspect, the invention provides an isolated nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae whose amino acid sequence is set forth in the Sequence Listing as SEQ ID NO:2. The term "isolated" refers to a nucleic acid which is essentially separated from other genes that naturally occur in S. pneumoniae. In one embodiment, the present invention provides an isolated nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae wherein the nucleic acid is the nucleic acid whose nucleotide sequence is set forth in the Sequence Listing as SEQ ID NO:1. An isolated nucleic acid comprising a unique fragment of at least 10 nucleotides of the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1 is also provided. "Unique fragments," as used herein, means a nucleic acid of at least 10 nucleotides that is not identical to any other known nucleic acid sequence at the time the invention was made. Examples of the sequences of at least 10 nucleotides that are unique to the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1 can be readily ascertained by comparing the sequence of the nucleic acid in question to sequences catalogued in GenBank, or other sequence database, using computer programs such as DNASIS (Hitachi Engineering, Inc.), or Word Search or FASTA of the Genetics Computer Group (GCG) (Madison, Wis.), which search the catalogued nucleotide sequences for similarities to the nucleic acid in question. If the sequence does not match any of the known sequences, it is unique. For example, the sequence of nucleotides 1-10 can be used to search the databases for an identical match. If no matches are found, then nucleotides 1-10 represent a unique fragment. Next, the sequence of nucleotides 2-11 can be used to search the databases, then the sequence of nucleotides 3-12, and so on up to nucleotides 1321 to 1330 of the sequence set forth in the Sequence Listing as SEQ ID NO:1. The same type of search can be performed for sequences of 11 nucleotides, 12 nucleotides, 13 nucleotides, etc. The possible fragments range from 10 nucleotides in length to 1 nucleotide less than the sequence set forth in the Sequence Listing as SEQ ID NO:1. These unique nucleic acids, as well as degenerate nucleic acids can be used, for example, as primers for amplifying nucleic acids from other strains of Streptococcus pneumoniae in order to isolate allelic variants of the 37-kDa protein, or as primers for reverse transcription of 37-kDa protein RNA, or as probes for use in detection techniques such as nucleic acid hybridization. One skilled in the art will appreciate that even though a nucleic acid of at least 10 nucleotides is unique to a specific gene, that nucleic acid fragment can still hybridize to many other nucleic acids and therefore be used in techniques such as amplification and nucleic acid detection.

Also provided are nucleic acids which encode allelic variants of the 37-kDa protein of S. pneumoniae set forth in the Sequence Listing as SEQ ID NO:2. The homology between the protein coding region of the nucleic acid encoding the allelic variant of the 37-kDa protein is preferably less than 20% divergent from the region of the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1 encoding the 37-kDa protein. Preferably, the corresponding nucleic acids are less than 15% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 10% divergent in their sequence identity, more preferably less than 7% divergent, more preferably less than 5% divergent, more preferably less than 4% divergent, more preferably less than 3% divergent, more preferably less than 2% divergent, and most preferably less than 1% divergent in their corresponding nucleotide identity. In particular, the nucleic acid variations can create up to about 15% amino acid sequence variation from the protein set forth in the Sequence Listing as SEQ ID NO:2.

One skilled in the art will appreciate that nucleic acids encoding homologs or allelic variants of the 37-kDa protein set forth in the Sequence Listing as SEQ ID NO.2 can be isolated from related gram-positive bacteria. The nucleic acid encoding a 37-kDa protein may be obtained by any number of techniques known to one skilled in the art. Methods of isolating nucleic acids of the invention, including probes and primers that may be used, are set forth in U.S. patent application Ser. No. 08/715,131, filed Sep. 17, 1996 (now U.S. Pat. No. 5,854,416), which is a continuation-in-part of U.S. patent application Ser. No. 08/222,179, filed Apr. 4, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 07/791,377, filed Sep. 17, 1991 (now U.S. Pat. No. 5,422,427). General methods that may be employed for these purposes are set forth in Sambrook et al., "Molecular Cloning: a Laboratory Manual," Cold Spring Harbor Laboratory Press (1989), and Ausubel et al. "Current Protocols in Molecular Biology," John Wiley and Sons, New York, 1987 (updated quarterly). Amplification procedures that may be employed in the nucleic acid isolation protocols are well known to those skilled in the art (see, for example, Innis et al., 1990, "PCR Protocols: A Guide to Methods and Applications," Academic Press, Inc. An example of amplification of a nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae serotype 6B is discussed in the Example contained herein.

37-kDa Protein

The present invention also provides a purified polypeptide as set forth in the Sequence Listing as SEQ ID NO:2 and a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1. The protein can be used as a vaccine component as well as a reagent for identifying subject antibodies raised against Streptococcus pneumoniae during infection. The purified protein can also be used in methods for detecting the presence of Streptococcus pneumoniae.

Unique fragments of the 37-kDa protein can be identified in the same manner as that used to identify unique nucleic acids. For example, a sequence of 3 amino acids or more, derived from the sequence of the 37-kDa protein, as set forth in the Sequence Listing as SEQ ID NO:2, can be used to search the protein sequence databases. Those that do not match a known sequence are therefore unique. Methods of preparing these proteins and protein fragments are set forth in U.S. patent application Ser. No. 08/715,131, filed Sep. 17, 1996, (now U.S. Pat. No. 5,854,416) which is a continuation-in-part of U.S. patent application Ser. No. 08/222,179, filed Apr. 4, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 07/791,377, filed Sep. 17, 1991 (now U.S. Pat. No. 5,422,427).

The present invention provides peptide fragments related to the 37-kDa pneumococcal surface adhesin protein. The polypeptide fragments of the present invention can be recombinant polypeptides obtained by cloning nucleic acids encoding fragments of the polypeptide in an expression system capable of producing the polypeptide fragments thereof, as described above for the 37-kDa protein. For example, one can identify an immunoreactive peptide related to the 37-kDa pneumococcal surface adhesin protein which can cause a significant immune response by using antibodies raised against the adhesin protein, cloning the nucleic acid encoding that polypeptide into an expression vector, and isolating that particular polypeptide for further uses, such as diagnostics, therapy, and vaccination. Amino acids which do not contribute to the immunoreactivity and/or specificity can be deleted without a a loss in the respective activity. For example, amino or carboxy-terminal amino acids can be sequentially removed from any peptide identified using the procedure outlined above, and the immunoreactivity tested in one of many available assays. Alternatively, internal amino acids can be sequentially removed and the immunoreactivity tested for each of the deletions.

In another example, a peptide fragment related to a 37-kDa pneumococcal surface adhesin protein can comprise a modified polypeptide wherein at least one amino acid has been substituted for the amino acid residue originally occupying a specific position, or a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the polypeptide, can be replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified 37-kDa pneumococcal surface adhesin protein.

Immunoreactive peptide fragments related to a 37-kDa pneumococcal surface adhesin protein can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acid residues, provided the immunoreactivity of the peptide is not significantly impaired compared to the 37-kDa pneumococcal surface adhesin protein. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, and the like. In any case, the peptide must possess a bioactive property, such as immunoreactivity, comparable to the 37-kDa pneumococcal surface adhesin protein.

Antibodies

The present invention employs a purified antibody which selectively binds with the polypeptide encoded by the nucleic acid set forth in the sequence listing as SEQ ID NO:1, or a polypeptide encoded by a unique fragment of at least 10 nucleotides of SEQ ID NO: 1. The antibody (either polyclonal or monoclonal) can be raised to the 37-kDa pneumococcal surface adhesin protein or a unique fragment thereof, in its naturally occurring form or in its recombinant form. The antibody can be used in a variety of techniques or procedures such as diagnostics, treatment, or immunization. Antibodies can be prepared by many well-known methods (see, e.g., Harlow and Lane, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can be injected into an animal in an amount and at intervals sufficient to elicit an immune response. Antibodies can be purified directly, to yield polyclonal antibodies. Alternatively, spleen cells can be obtained from the animal. The cells can be then fused with an immortal cell line and screened for antibody secretion to yield monoclonal antibodies. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced (see, e.g., Kelly et al., Bio Technology, 1992, 10:163-167: Bebbington et. al., 1992, Bio Technology, 10:169-175).

The phrase "selectively binds" with the polypeptide refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and-other biologics. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample. Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies which selectively bind with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding. In some instances, it is desirable to prepare monoclonal antibodies from various subjects. A description of techniques for preparing such monoclonal antibodies may be found in Stites et al., editors, "Basic and Clinical Immunology," (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited therein, and in Harlow and Lane ("Antibodies: A Laboratory Manual," Cold Spring Harbor Publications, New York, (1988)).

The monoclonal antibodies (MAbs) employed in the present invention (disclosed in U.S. patent application Ser. No. 08/715,131, filed Sep. 17, 1996 (now U.S. Pat. No. 5,854,416), incorporated herein by reference) are MAb 1 E7A3D7C2, or a fragment thereof which retains the characteristics of antibody 1 E7A3D7C2, such as its binding specificity and its binding affinity; MAb 1 B6E12H9, or a fragment thereof which retains the characteristics of antibody 1 B6E12H9; MAb 3C4D5C7, or a fragment thereof which retains the characteristics of antibody 3C4D5C7; MAb 4E9G9D3, or a fragment thereof which retains the characteristics of antibody 4E9G9D3; MAb 4H5C10F3, or a fragment thereof which retains the characteristics of antibody 4H5C10F3; MAb 6F6F9C8, or a fragment thereof which retains the characteristics of antibody 6F6F9C8; and MAb 8G12G11 B10, or a fragment thereof which retains the characteristics of antibody 8G12G11B10.

The hybridomas used to produce the respective monoclonal antibodies employed in the present invention (disclosed in U.S. patent application Ser. No. 08/715,131, filed September 17, 1996 (now U.S. Pat. No. 5,854,416), incorporated herein by reference) are hybridoma 1 E7A3D7C2, hybridoma 1B6E12H9, hybridoma 3C4D5C7, hybridoma 4E9G9D3, hybridoma 4H5C10F3, hybridoma 6F6F9C8, and hybridoma 8G12G11B10.

Therapeutic Compositions

Also provided by the present invention is a therapeutic composition comprising an immunogenic polypeptide encoded by the nucleic acid as set forth in the Sequence Listing as SEQ ID NO:1, or a unique fragment of at least 10 nucleotides of SEQ ID NO:1. The invention also provides therapeutic compositions comprising at least one immunogenic polypeptide that immunospecifically binds to a monoclonal antibody obtained in response to immunizing an animal with Streptococcus pneumoniae PsaA. The therapeutic composition is preferably combined with an immunostimulatory carrier. The therapeutic composition confers protective immunity against S. pneumoniae infection when administered to a subject.

The polypeptides provided by the present invention can be used to vaccinate a subject for protection from a particular disease, infection, or condition caused by the organism from which the 37-kDa pneumococcal surface adhesin protein (or a unique fragment thereof) was derived. Polypeptides of a 37-kDa pneumococcal surface adhesin protein of serotype 6B, or a unique fragment thereof, can be used to inoculate a subject organism such that the subject generates an active immune response to the presence of the polypeptide or polypeptide fragment which can later protect the subject from infection by organism from which the polypeptide was derived. One skilled in the art will appreciate that an immune response, especially a cell-mediated immune response, to a 37-kDa pneumococcal surface adhesin protein from a specific strain can provide later protection from reinfection or from infection from a closely related strain. The 37-kDa protein provided by the present invention, however, is relatively conserved among the 90 serotypes of S. pneumoniae and can, therefore, serve as a multivalent vaccine. Immunization with the 37-kDa pneumococcal surface adhesin protein or with the immunogenic peptides of the invention can be achieved by administering to subjects the 37-kDa pneumococcal surface adhesin protein either alone or with a pharmaceutically acceptable carrier, (Kuby, J. 1992 "Immunology," W. H. Freeman and Co., New York). Immunogenic amounts of the 37-kDa pneumococcal surface adhesin protein or of the immunogenic peptides of the invention can be determined using standard procedures. Briefly, various concentrations of the present polypeptide are prepared, administered to subjects, and the immunogenic response (e.g., the production of antibodies to the polypeptide or cell mediated immunity) to each concentration is determined. Techniques for monitoring the immunogenic response, both cellular and humoral, of patients after inoculation with the polypeptide, are well known in the art. For example, samples can be assayed using enzyme-linked immunosorbent assays (ELISA) to detect the presence of specific antibodies, such as serum IgG (Hjelt et al., J. Med. Virol., 21:39-47, (1987)); lymphocyte or cytokine production can also be monitored. The specificity of a putative immunogenic antigen of any particular polypeptide can be ascertained by testing sera, other fluids, or lymphocytes from the inoculated patient for cross-reactivity with other closely related 37-kDa pneumococcal surface adhesin proteins. The amount of a polypeptide of the 37-kDa pneumococcal surface adhesin protein or of the immunogenic peptides of the invention to be administered will depend on the subject, the condition of the subject, the size of the subject, and the like, but will be at least an immunogenic amount. The polypeptide can be formulated with adjuvants and with additional compounds, including cytokines, with a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier or adjuvant in the therapeutic composition of the present invention can be selected by standard criteria (Arnon, R. (Ed.) "Synthetic Vaccines," 1:83-92, CRC Press, Inc., Boca Raton, Fla., 1987). By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable (i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in an undesirable manner with any of the other components of the pharmaceutical composition in which it is contained). The carrier or adjuvant may depend on the method of administration and the particular patient. Methods of administration can be parenteral, oral, sublingual, mucosal, inhaled, absorbed, or injection. Actual methods of preparing the appropriate dosage forms are known, or will be apparent, to those skilled in this art: see, for example, Remington's Pharmaceutical Sciences (Martin, E. W. (ed.) latest edition Mack Publishing Co., Easton, Pa.). Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Another approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained (see, e.g., U.S. Pat. No. 3,710,795). In addition, powders or aerosols may be formulated for administration by inhalation.

Detection Methods

The present invention provides methods of detecting the presence of Streptococcus pneumoniae in a subject, based on several variations of immunoassays, using either a purified polypeptide encoded by the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1, a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, an antibody which selectively binds the purified polypeptide encoded by the nucleic acid set forth in the Sequence Listing as SEQ ID NO: 1, or an antibody which selectively binds a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, and detecting the binding of the antibody with the polypeptide, the binding indicating the presence of Streptococcus pneumoniae in the subject. There are numerous immunodiagnostic methods that can be used to detect antigen or antibody as the following non-inclusive examples illustrate. These methods, as well as others, can not only detect the presence of antigen or antibody, but quantitate antigen or antibody as well. These methods are set forth in U.S. patent application Ser. No. 08/715,131, filed Sep. 17, 1996 (now U.S. Pat. No. 5,854,416), which is a continuation-in-part of U.S. patent application Ser. No. 08/222,179, filed Apr. 4, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 07/791,377, filed Sep. 17, 1991 (now U.S. Pat. No. 5,422,427). In general, the detection methods that may be employed in practicing the present invention are described in, for example, Harlow et al., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988).

Methods of Treating and Preventing Infection

The present invention also provides a method of preventing Streptococcus pneumoniae infection in a subject at risk of infection by S. pneumoniae, comprising administering to the subject an effective amount of a therapeutic composition comprising an immunogenic polypeptide encoded by the nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae as set forth in the Sequence Listing as SEQ ID NO:1, or an immunogenic polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, or the immunogenic peptides of the invention either alone or with a pharmaceutically acceptable carrier.

The present invention further provides a method of treating a Streptococcus pneumoniae infection in a subject, comprising administering to the subject an effective amount of an antibody to the polypeptide encoded by the nucleic acid asset forth in the Sequence Listing as SEQ ID NO:1, or a polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, either alone or with a pharmaceutically acceptable carrier. Treating a subject already infected with a particular organism by administering to the subject an antibody against the organism is well known in the art. For example, immune globulin isolated from animals or humans previously exposed to rabies virus is currently a therapy for rabies virus infection. Better treatment of infected individuals can be achieved by administering to those individuals monoclonal antibodies since those monoclonals react or bind more specifically than the polyclonals, (see, e.g., Kaplan et al., "Rabies," Sci. Am. 242:120-134 (1980)).

Epitopic Immunogenic Peptides

The present invention discloses novel epitopic immunogenic peptides obtained as the peptides coded in a random oligonucleotide library by selecting for high affinity binding of the epitopes to monoclonal antibodies specific for epitopes on the PsaA antigen.

In an additional method, a procedure known as "biopanning" or "panning", a target protein or peptide is selected from a library expressed as a heterologous insert on an external surface of a microorganism. A bacterium or virus, for example, may have a nucleotide sequence encoding a heterologous peptide or protein sequence incorporated into its chromosomal nucleic acid in such a way that a fusion or chimera is created. The fusion represents a natural protein of the microorganism directly linked with the heterologous peptide or protein. Once expressed on the surface of the microorganism, it can be probed by a ligand specific for the sought peptide or protein, such as an antibody. Once identified by capture, the heterologous sequence, either the nucleic acid or the protein, can be obtained and identified.

A common implementation of this procedure is well known to those of skill in the fields of protein chemistry, immunology, and virology. A filamentous bacteriophage such as M13, fl, or fd is employed. These bacteriophages have two well-known structural proteins on their surfaces: the gene III protein and the gene VIII protein. The nucleic acid of the phage is altered by incorporating a fusion sequence of the heterologous peptide in frame with the gene for one or the other of these structural proteins. When one is seeking a target peptide from among a large set, or library, of such peptides, the corresponding library of heterologous nucleotide sequences coding for the members of the peptide library is incorporated into the structural protein gene. The resulting bacteriophage population (termed a phage display library) is subjected to procedures which optimize selection of only those virus particles expressing members of the peptide library for which the PsaA-specific ligand, such as an MAb, has a high affinity. The bacteriophage particles so selected may then be amplified by further culture, or their nucleic acids may be amplified by methods such as polymerase chain reaction. In this way the nucleic acid of the captured particle may be isolated and sequenced to provide the coding sequence for the high affinity epitope bound to the MAb or other ligand. Biopanning is described for example, in Smith, G. P. and K. K. Scott (1993, "Libraries of Peptides and Proteins Displayed on Filamentous Phage", Meth. Enzymol. 217: 228-257).

The immunogenic peptides of the invention were obtained using a biopanning procedure that has general applicability for identifying the sequence of a peptide potentially capable of eliciting protective immunity against a pathogenic microorganism. The method includes the steps of

Claim 1 of 1 Claim

1. An isolated multiple antigenic peptide, wherein the multiple antigenic peptide has at least one first arm comprising the amino acid sequence of SEQ ID NO: 5, at least one second arm comprising the amino acid sequence of SEQ ID NO: 6, and at least one third arm comprising the amino acid sequence of SEQ ID NO: 7.

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