EP1301530A2 - Multiple antigenic peptides immunogenic against streptococcus pneumoniae - Google Patents

Multiple antigenic peptides immunogenic against streptococcus pneumoniae

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Publication number
EP1301530A2
EP1301530A2 EP01950993A EP01950993A EP1301530A2 EP 1301530 A2 EP1301530 A2 EP 1301530A2 EP 01950993 A EP01950993 A EP 01950993A EP 01950993 A EP01950993 A EP 01950993A EP 1301530 A2 EP1301530 A2 EP 1301530A2
Authority
EP
European Patent Office
Prior art keywords
seq
peptide
arm
peptides
multiple antigenic
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Withdrawn
Application number
EP01950993A
Other languages
German (de)
English (en)
French (fr)
Inventor
Edwin W. Ades
Scott E. Johnson
Danny L. Jue
Jacquelyn S. Sampson
George M. Carlone
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
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US Department of Health and Human Services
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Filing date
Publication date
Priority claimed from US09/613,092 external-priority patent/US6903184B1/en
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Publication of EP1301530A2 publication Critical patent/EP1301530A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to multiple antigenic peptides (MAPs) which can be administered as a vaccine to confer immunity against Streptococcus pneumoniae.
  • MAPs multiple antigenic peptides
  • 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 two years of age and exhibit variable serotype-specific efficacy among vaccinated individuals. For these reasons, alternative vaccine formulations that do not require the use of multiple capsular polysaccharides have been sought.
  • One potential strategy involves 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.
  • Patent 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.
  • 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).
  • the present invention provides novel immunogenic peptides obtained from a random library by selecting for high affinity binding to monoclonal antibodies specific for PsaA epitopes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the multiple antigenic peptide can also be a bipeptide that contains any combination of SEQ ID NOs:5-10 or immunogenic fragments thereof.
  • 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.
  • 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.
  • 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: 8.
  • 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.
  • 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:6 and at least one second arm comprising SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. 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.
  • the multiple antigenic peptide can also be a tripeptide that contains any combination of SEQ ID NOs:5-10 or immunogenic fragments thereof.
  • 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.
  • 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.
  • the multiple antigenic peptide has a first arm comprising SEQ ID NO:5, a second arm comprising SEQ ID NO: 10, and a third arm comprising SEQ ID NO:7, 8, or 9; or the multiple antigenic peptide has a first arm comprising SEQ ID NO:5, a second arm comprising SEQ ID NO: 6, and a third arm comprising SEQ ID NO: 8 or SEQ ID NO: 9; or the multiple antigenic peptide has a first arm comprising SEQ ID NO: 6, a second arm comprising SEQ ID NO: 7, and a third arm comprising SEQ ID NO:8, 9, or 10; or the multiple antigenic peptide has a first arm comprising SEQ ID NO:7, a second arm comprising SEQ ID NO:8, and a third arm comprising SEQ ID NO:9 or 10, and so on.
  • any arm can be an immunogenic fragment of SEQ ID NO: 5, 6, 7, 8, 9, or 10.
  • 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.
  • the peptide is lipidated with monopalmitic acid.
  • the invention furthermore provides a therapeutic composition and/or vaccine 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 in the subject.
  • the invention additionally provides a therapeutic composition and/or vaccine 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 in the subject.
  • the invention additionally provides a therapeutic composition and/or vaccine in which the immunogenic peptide is a multiple antigenic peptide of the current invention as described above.
  • the invention additionally provides a therapeutic composition and/or vaccine 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.
  • the peptide is lipidated with monopalmitic acid.
  • the invention still further describes a method of conferring protective immunity against infection by S. pneumoniae in a subject in which the therapeutic compositions and/or vaccines 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.
  • Fig. 1 A is a diagram showing a bi-peptide heterogeneous MAP with arms of alternating peptides with sequences of either SEQ ID NO: 5 or SEQ ID NO: 6.
  • Fig. IB is a diagram showing a bi-peptide heterogeneous MAP with arms of alternating peptides with sequences of either SEQ ID NO: 5 or SEQ ID NO:9.
  • Fig. 1C is a diagram showing a tri-peptide heterogeneous MAP with a first arm with the sequence of SEQ ID NO:5, a second arm with the sequence of SEQ ID NO: 10, and a third arm with the sequence of SEQ ID NO:9.
  • the carboxy terminal lysine (i.e., K) residue in each peptide is shared between two arms of the MAP.
  • NLe is norleucine.
  • Figs. 2 A and 2B are diagrams showing the basic structure of non-lipidated MAPs (Fig. 2A) and of lipidated MAPs (Fig. 2B), in which "B” represents three 15- amino acid base peptides designated as either Pl, P2, or P3; "J” represents the palmitoyl (palmitic acid) group, which is linked to the N-terminal of the base peptides (Pl, P2, and P3) through the 3 -amino acid chain linker (-cysteine-serme-serine-) designated as "U” (Tarn JP, In: Multiple antigenic peptide system: A novel design of synthetic peptide vaccines and immunoassay, Tarn, JP and Kaiser, ET, eds, New York: Alan R.
  • Fig. 3 is a diagram showing the basic structure of bipeptides P79 and P80, in which the base peptides are linked together by the 2-amino acid linker lysine-norleucine (K-NLe) to form a 4-branched bipeptide (Srivastava N, et al. Hybridoma 19:23-31, 2000; and Tarn JP, supra).
  • P79 consists of two units each of base peptides Pl and P2.
  • hi bipeptide P80, P2 units are replaced with P3 units.
  • FIG. 4 is a diagram showing the basic structure of tripeptide P81, in which base peptides are linked together by the 2-amino acid linker lysine-norleucine (K-NLe) to form a 3-branched tripeptide consisting of base peptides Pl, P2, and P3 (Srivastava N, supra, and Tarn, JP, supra).
  • K-NLe 2-amino acid linker lysine-norleucine
  • immunogenic peptide refers to a peptide which, upon being administered to a subject, or taken up by the subject in other ways, elicits an immune response.
  • the immune response includes at least the generation of antibodies which specifically bind the immunogenic substance (i.e., a humoral response).
  • An immunogenic substance may in addition elicit a cellular immunological response.
  • Such an immunogen is any of the immunogenic peptides obtained by screening a library of random peptides using monoclonal antibodies that immunospecifically react with PsaA from S. pneumoniae.
  • immunogenic response and “immunogenic response” may include at least a humoral response, that is, the generation of antibodies which specifically bind the immunogenic substance.
  • An immunogenic response may, either alternatively or in addition, refer to a cellular immunological response.
  • protective immunity refers to a state in which a subject has generated antibodies, at least some of which are neutralizing antibodies, in response to exposure to a pathogen-related immunogen.
  • Neutralizing antibodies bind the immunogenic component of the pathogen in such a way that proliferative infection by the pathogen is inhibited or abrogated, such that the subject remains essentially free of symptomatic disease.
  • Protective immunity may also arise from an alternative immunogenic response which leads to inactivation, loss, or destruction of the pathogenic agent.
  • immunogenic carrier relates to any of a variety of immunogenic biological polymers which themselves elicit immune responses when introduced into a subject. Immunostimulatory carriers, when employed in conjunction with an immunogen of interest, such as the peptides of the present invention, provide enhanced immunogenic response in the subject to the immunogen of interest.
  • adjuvant relates to a composition that enhances the immunogenic activity of an immunogenic substance when administered in conjunction with that substance.
  • a "library” refers to a set of fragments derived from a biological macromolecule, wherein each member of the set is a candidate for possessing a desired biological activity expressing a desired biological function.
  • a library is either a peptide library or a library of oligonucleotide fragments, each member of which contains a nucleotide sequence which encodes a particular member of the peptide library.
  • the peptide library is a set of peptides which are coded for by a random oligonucleotide library.
  • the desired activity for a given peptide is that the peptide be immunogenic in a subject against S. pneumoniae.
  • a "subject" is a mammal in whom it is desired to elicit an immune response to the pathogenic organism S. pneumoniae.
  • a principal class of subjects of the present invention is human beings, especially infants and elderly people, in whom S. pneumoniae is in fact pathogenic. In human subjects, therefore, the immune response is intended to be a protective immune response.
  • S. pneumomae may or may not be inherently pathogenic.
  • Such non-human subjects employed as experimental animals which provide an immune response can be useful in characterizing and optimizing the compositions and methods of the invention.
  • Such mammals include, by way of non-limiting example, mice, rats, and non-human primates.
  • An additional class of subjects includes animals served in veterinary practice, including pets and livestock animals. If S. pneumoniae is pathogenic in such subjects, eliciting protective immunity is desirable.
  • Purified protein as used herein means that the protein or fragment is sufficiently free of contaminants or cell components with which the protein normally occurs as to distinguish the protein from the contaminants or cell components. It is not contemplated that "purified” necessitates having a preparation that is technically totally pure (homogeneous), but purified as used herein means the protein or polypeptide fragment is sufficiently separated from contaminants or cell components with which it normally occurs to provide the protein in a state where it can be used in an assay, such as immunoprecipitation or ELISA. For example, the purified protein can be in an electrophoretic gel.
  • washing conditions refers to the washing conditions used in a nucleic acid hybridization protocol.
  • the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5-20°C below the calculated T m of the nucleic acid hybrid under study.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein coding nucleic acid of interest and then washed under conditions of different stringencies.
  • the T m of such an oligonucleotide can be estimated by allowing about 2°C for each A or T nucleotide and about 4°C for each G or C. For example, an 18 nucleotide probe of 50% G-C would, therefore, have an estimated T m of 54°C.
  • a "therapeutic composition” relates to a composition which may be administered to a subject in order to elicit a protective immune response, and which contains one or more of the immunogenic peptides of the present invention alone or in conjunction with an immunostimulatory carrier or an adjuvant.
  • the therapeutic compositions contain the peptide and the carrier in either a mixture or as a chemical conjugate. Together these constitute the active agent. If more than one peptide is employed and the composition is a conjugate, each peptide can be either alone or conjugated to an immunostimulatory carrier.
  • the therapeutic composition generally contains the components of a pharmaceutical formulation in which the active agent is suspended or dissolved.
  • the components of pharmaceutical formulations are well known to those who are skilled in immunology or pharmaceutical science. The formulation should be suitable to administer the active agent to a subject in order to elicit an immune response and confer protective immunity against the pathogen related to the immunogenic peptide.
  • allelic variation refers to an immunogenic PsaA peptide or protein obtained from a serotype of S. pneumoniae other than that of a reference serotype such as serotype 2.
  • An allelic variant describes the same 37-kDa pneumococcal surface adhesin protein, or a similar protein that is diverged from the 37-kDa Streptococcus pneumoniae protein set forth in the Sequence Listing as SEQ ID NO:2 by less than 15% in its corresponding amino acid identity.
  • this allelic variant is less than 10% divergent in its corresponding amino acid identity, more preferably less than 7% divergent, more preferably less than 5% divergent, more preferably less than 3% divergent, more preferably less than 2% divergent, and most preferably less than 1% divergent in their corresponding amino acid identity.
  • These amino acids can be substitutions within the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:2, or the variants can be either deletions from or additions to the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:2.
  • 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.
  • isolated refers to a nucleic acid which is essentially separated from other genes that naturally occur in S. pneumoniae.
  • 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.
  • 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:l is also provided.
  • Unique fragments 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.
  • 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, WI), 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.
  • nucleotides 1-10 represent a unique fragment.
  • 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:l.
  • 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.
  • primers for amplifying nucleic acids from other strains of Streptococcus pneumoniae in order to isolate allelic variants of the 37-kDa protein
  • primers for reverse transcription of 37-kDa protein RNA or as probes for use in detection techniques such as nucleic acid hybridization.
  • nucleic acid hybridization such as nucleic acid hybridization.
  • 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:l encoding the 37-kDa protein.
  • the corresponding nucleic acids are less than 15% divergent in their sequence identity.
  • 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.
  • 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.
  • 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 United States Patent Application Serial No. 08/715,131, filed September 17, 1996 (now U.S. Patent No. 5,854,416), which is a continuation-in-part of United States Patent Application Serial No.
  • Amplification procedures such as amplification using the polymerase chain reaction, or "PCR”
  • PCR polymerase chain reaction
  • 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.
  • 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 United States Patent Application Serial No. 08/715,131, filed September 17, 1996 (now U.S. Patent No. 5,854,416), which is a continuation-in-part of United States Patent Application Serial No. 08/222,179, filed April 4, 1994 (now abandoned), which is a continuation-in-part of United States Patent Application Serial No.
  • 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.
  • 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 loss in the respective activity.
  • 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.
  • internal amino acids can be sequentially removed and the immunoreactivity tested for each of the deletions.
  • 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 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.
  • 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:l, 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, New York, (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 biologies.
  • 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.
  • the monoclonal antibodies (MAbs) employed in the present invention are MAb 1E7A3D7C2, or a fragment thereof which retains the characteristics of antibody 1E7A3D7C2, such as its binding specificity and its binding affinity; MAb 1B6E12H9, or a fragment thereof which retains the characteristics of antibody 1B6E12H9; MAb 3C4D5C7, or a fragment thereof wliich 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
  • hybridoma 1E7A3D7C2 hybridoma 1B6E12H9
  • hybridoma 3C4D5C7 hybridoma 4E9G9D3
  • hybridoma 4H5C10F3 hybridoma 6F6F9C8
  • hybridoma 8G12G11B10 hybridoma 1E7A3D7C2
  • hybridoma 1B6E12H9 hybridoma 3C4D5C7
  • hybridoma 4E9G9D3 hybridoma 4H5C10F3
  • hybridoma 6F6F9C8 hybridoma 8G12G11B10.
  • a therapeutic composition comprising an immunogenic polypeptide encoded by the nucleic acid as set forth in the Sequence Listing as SEQ ID NO:l, 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 peptide that immunospecifically binds to a monoclonal antibody obtained in response to immunizing an animal with Streptococcus pneumoniae PsaA. Examples of such immunogenic peptides are set forth in SEQ ID NOs: 5-10.
  • 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.
  • 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 the organism from which the polypeptide was derived.
  • an immune response especially a cell-mediated immune response
  • a 37-kDa pneumococcal surface adhesin protein from a specific strain can provide later protection from re-infection or from infection from a closely related strain.
  • the 37-kDa protein provided by the present invention 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.
  • 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.
  • ELISA enzyme-linked immunosorbent assays
  • 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 for the particular subject to be immunized.
  • 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, Florida, 1987).
  • 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 by injection.
  • 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. Patent No 3,710,795). In addition, powders or aerosols may be formulated for administration by inhalation.
  • 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:l, 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.
  • 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:l, or an immunogenic polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:l, or the immunogenic peptides of the invention (for example, those set forth in SEQ ID NOs: 5-10), either alone or with a pharmaceutically acceptable carrier.
  • 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:l, or an immunogenic polypeptide encoded by a nucleic acid comprising
  • 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 as set forth in the Sequence Listing as SEQ ID NO:l, 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.
  • Epitopic Immunogenic Peptides discloses novel epitopic immunogenic peptides, obtained as the peptides encoded by a random oligonucleotide library, by selecting for high affinity binding of the peptides by monoclonal antibodies specific for epitopes on the PsaA antigen.
  • 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 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-after peptide or protein, such as an antibody that recognizes a particular epitope. Once identified by capture, the heterologous sequence, either the nucleic acid or the protein, can be obtained and identified.
  • a filamentous bacteriophage such as Ml 3, 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.
  • 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.
  • 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 (a) providing a library comprised of random oligonucleotides, wherein the oligonucleotides are about 30-45 nucleotides in length;
  • step (e) sequencing the gene for the coat protein of any bacteriophage particle obtained in step (d) thereby yielding the nucleotide sequence of that member of the oligonucleotide library whose translation product has the sequence of a peptide potentially capable of eliciting protective immunity against Streptococcus pneumoniae.
  • the method described above is directed against S.
  • the coat protein is the gene III protein which is the tail protein of a filamentous bacteriophage such as Ml 3, fl, or fd, and the monoclonal antibody is obtained in response to immunizing an animal with Streptococcus pneumoniae pneumococcal surface adhesion A protein (PsaA).
  • the peptides are isolated using a procedure that emphasizes capturing only those peptides that have a high affinity for the antibodies. This increases the likelihood that a selected peptide will effectively induce a protective humoral immune response.
  • the sequences of the peptides which bind to the antibodies can be identified by sequencing the gene III fusion of the bacteriophage particle obtained in the biopanning process.
  • the actual immunogenic peptides can then be synthesized using a conventional peptide synthesizer. These peptides are then incorporated into a therapeutic composition in which the immunogenic peptides are combined with an immunostimulatory carrier to be administered to a subject. Upon being administered in effective amounts, the subject elicits the production of antibodies against S. pneumoniae. This results in conferring protective immunity against infection by S. pneumoniae on the subject.
  • PsaA is a 37-kDa species-common protein from S. pneumoniae (pneumococcus) which is effectively immunogenic. It is common to all the serotypes whose polysaccharides are components of the pneumococcal vaccine currently in use (Russell et al., 1990, "Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae” , J. Clin. Microbiol. 28:2191-2195). The sequence of the PsaA gene cloned from serotype R36A has been described (U.S. Patent 5,422,427 to Russell et al.), and the sequence of PsaA protein was deduced.
  • the peptides of the present invention contain immunogenic epitopes selected by binding to PsaA-specific monoclonal antibodies.
  • the peptide is about 10-25 amino acid residues in length. More preferably, the peptide is about 12-22 amino acid residues in length, and most preferably about amino acid 15 residues in length.
  • the peptides of the invention have the sequences provided in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
  • the invention encompasses immunogenic peptides which may be shorter than these sequences.
  • immunogenic fragments of SEQ ID NO: 5, immunogenic fragments of SEQ ID NO: 6, immunogenic fragments of SEQ ID NO:7, and immunogenic fragments of SEQ ID NO:8 are also encompassed by the present invention.
  • the invention therefore encompasses an allelic immunogenic peptide which, for example, was obtained by a biopanning procedure in which the monoclonal antibodies were raised by immunizing with an allelic variant, or in other ways known to those skilled in the relevant arts.
  • sequence of such a peptide is at least 80% identical to any of the following sequences: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, immunogenic fragments of SEQ ID NO:5, immunogenic fragments of SEQ ID NO:6, immunogenic fragments of SEQ ID NO:7, and immunogenic fragments of SEQ ID NO: 8.
  • the monoclonal antibodies (MAbs) disclosed above were used further in procedures of the present invention.
  • the specific MAbs that were used are designated 1E7 (1E7A3D7C2), 6F6 (6F6F9C8), 4E9 (4E9G9D3), 8G12 (8G12G11B10), and 1B6 (1B6E12H9).
  • MAbs were obtained as a result of immunization of an animal with PsaA; such antibodies therefore represent molecules whose antigen-binding domains bind immunogenic epitopes of the invention.
  • Identification of immunogenic epitopes related to PsaA may be achieved in any of a number of ways.
  • Methods to identify immunogenic epitopes may employ any MAb obtained in response to primary immunization with PsaA. Any procedure which narrows down the overall molecular structure of PsaA to moieties or fragments thereof may be employed in identifying immunogenic epitopes thereof.
  • chemical modification of specific residues of PsaA yields modified products whose reactivity with a ligand such as an anti-PsaA MAb may be impaired. Knowledge of which residue or residues were modified in products with impaired binding may be used to identify those residues as potentially being a portion of the epitope. Additionally, biopanning, described above, may be used.
  • fragments of PsaA may be synthesized chemically by peptide synthesis.
  • a set of peptides are synthesized which represents a systematic progression along the entire sequence of the protein from its N-terminus to its C-terminus. Windows of predetermined lengths may be "walked” along the protein sequence generating a set of peptides which encompasses most or all of the original sequence.
  • Methods of peptide synthesis are well-known to workers of skill in the fields of peptide chemistry, protein chemistry, and immunology. Commercial instruments are available for the automated synthesis of peptides once their sequences are specified.
  • a set of peptides obtained in this way may be subjected to assays which establish whether they bind to PsaA-specific ligands, such as anti-PsaA MAbs.
  • Immunoassay methods are preferred for such determinations and are well-known to workers of skill in immunology. They include procedures such as enzyme-linked immunosorbent assays (ELISA), using, for example, competitive formats or direct heterogeneous formats.
  • ELISA enzyme-linked immunosorbent assays
  • Peptides found to bind with high affinity to the PsaA-specific ligands are presumed to contain or encompass an immunogenic epitope of PsaA.
  • the immunogenic peptides of the invention are identified in the selection or screening procedures described in the preceding paragraphs.
  • the sequences of the peptides positively selected next need to be obtained.
  • the location of inhibitory modifications in the sequence yields peptides centered on, or containing, that modified residue.
  • the sequence is immediately available from the identity of the positive sample.
  • the positive bacteriophages are isolated and the nucleic acid is amplified, either by expansion of the phage particles in culture or by amplification of the nucleic acid itself. The nucleic acid is then isolated and sequenced to identify the coding sequence for the heterologous peptide and the coding sequence translated to yield the peptide sequence.
  • peptides are synthesized in order to serve as immunogenic peptides in a subject.
  • Methods for synthesizing peptides are well-known to skilled workers in the art of immunochemistry, immunology, and/or protein chemistry.
  • peptides can be synthesized using solid phase F-moc chemistry according to the method of Stewart et al., " 'Solid peptide synthesis," 2 nd ed., Pierce Chemical Co., Rockford, IL (1984).
  • an automated peptide synthesizer such as an automated synthesizer available from Advanced ChemTech (Advanced ChemTech, Inc., Louisville, KY).
  • synthesizer that can be used for synthesizing peptides according to the current invention is the Advanced ChemTech ACT model 396MPS. Once synthesized, sequences are typically verified using an automated peptide sequencer such as a Porton model 2090 (Beckman Instruments Inc., Mountain View, CA).
  • peptides of the current invention 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.
  • the phrase "consisting essentially of with respect to peptides of the current invention is intended to cover such modified peptides which, as illustrated in the Examples section below, can be identified and routinely generated by those of skill in the art.
  • peptides of the current invention are combined with an immunostimulatory carrier and/or with an adjuvant prior to administration to a subject, as is well-known to those of skill in the art of immunochemistry or immunology and discussed herein in the "Therapeutic Compositions" section.
  • immunostimulatory carriers are proteins such as keyhole limpet hemocyanin, bovine serum albumin, thyroglobulin, diphtheria toxoid, and the like.
  • the immunogenic peptides and the carrier may be combined either noncovalently or covalently. When combined noncovalently, they are mixed together so that they comprise components in a therapeutic composition to be administered to a subject.
  • alum, proteosomes, certain lipids, such as palmitic acid (see below), QS21, or alhydrogel (2%; #A1090BS, Accurate Chemical and Scientific Company, Westbury, NY) could be used as an adjuvant in the present invention (da Fonseca, D. P., et al., "Identification of new cytotoxic T-cell epitopes on the 38-kilodalton lipoglycoprotein of Mycobacterium tuberculosis by using lipopeptides," Infect. Immun. 66:3190 (1998); Sheikh, N.
  • immunogenicity of peptides of the current invention can be enhanced by attachment of the peptides to proteosomes or by addition of a cysteine residue.
  • a spacer such as a CYGG spacer
  • a lauroyl group can be attached to the peptide' s amino terminal end (Bio-Synthesis, Lewisville, TX).
  • the lauroyl group enhances the hydrophobic complexing of peptide groups to proteosomes (Lowell, G.
  • Proteosomes can be prepared from the outer membrane complex vesicles from Group B meningococci, strain 99M as described by Zollinger (Zollinger, et al., (1990)).
  • Synthetic lipopeptides can be complexed to proteosomes on a 1 : 1 (w/w) ratio by combining the components in the presence of detergent.
  • the detergent can be removed by extensive dialysis (Lowell, G. H., et ai., (1988)).
  • peptides of the current invention are lipidated with, for example, but not limited to, monopalmitic acid, to stimulate an immune response to the peptide (Verhaul et al., "Monopalmitic acid-peptide conjugates induce cytotoxic T cell responses against malarial epitopes: importance of spacer amino acids," J Immunol. Methods, 182:219 (1995)).
  • Lipidated versions of the peptides of the current invention containing monopalmitic acid can be synthesized by coupling palmitic acid (Sigma Chemicals, St.
  • the tripeptide cysteine- serine-serine is added to the amino terminus of the peptides of the current invention to facilitate attachment of a lipid such as monopalmitic acid.
  • lipidated versions of the peptides of the current invention can be produced using recombinant DNA technology using methods known in the art.
  • constructs can be developed that contain a heterologous leader sequence joined to nucleic acids encoding the peptides of the current invention.
  • the heterologous leader sequence is lipidated by a host organism.
  • the leader sequence may, for example, be derived from the ospA gene of Borrelia burgdorferi. (Ades et al., "Recombinant lipidated PsaA protein, methods of preparation and use," PCT Publication WO 99/40200 (1999)).
  • compositions of the present invention are described in the "Therapeutics Compositions" section above.
  • immunogenic peptides are formulated with a pharmaceutically acceptable vehicle for administration to a subject.
  • a pharmaceutically acceptable vehicle for administration to a subject.
  • Such vehicles are well known to those of skill in the pharmaceutical sciences, and include preparations in liquid, gel, or solid forms for administration by oral, sublingual, or parenteral routes, including, but not limited to, intravenous, subcutaneous, intramuscular, mucosal, and inhalation.
  • These dosage forms may be conventional preparations such as solutions or suspensions having immediate bioavailability, or they may be controlled release formulations or devices having the property of releasing the active immunogenic peptide slowly over an extended time period.
  • therapeutic compositions comprising peptides of the present invention confer protective immunity against S. pneumoniae in subjects, preferably human subjects, to whom they are administered.
  • immunogenic fragments of such peptides are also encompassed within the present invention.
  • An immunogenic fragment is any peptide shorter than the peptide from which it is derived (the parent) whose sequence is identical to the sequence of a portion of the parent peptide and which retains immunogenicity. It is generally understood in the field of immunochemistry that such peptides must be at least about six residues long in order to be antigenic. Thus any fragment should be at least six residues in length and may have a maximum length one residue less than the parent peptide. Identifying immunogenic fragments can be accomplished using any method which will identify immunogenicity.
  • These methods include, for example, the biopanning procedure described above, as well as direct demonstration of immunogenicity by combining the candidate peptide with an immunostimulatory carrier to form the active component of a pharmaceutical composition, administering the pharmaceutical composition to a subject and assessing whether an immunogenic response has occurred.
  • a peptide fragment which has been positively identified as being immunogenic may also be assessed for its ability to elicit protective immunity in a subject. This is carried out using methods described herein for determining whether an experimental subject animal exhibiting an immunogenic response to a PsaA peptide fragment resists a challenge by S. pneumoniae.
  • peptides of the current invention are administered in conjunction with one another to enhance the effectiveness of the immunization.
  • Administration "in conjunction with” encompasses simultaneous and sequential administration, as well as administration in combined form or separately.
  • the compositions may include multiple peptides having the sequences given by SEQ ID NO: 5, or an immunogenic fragment thereof, SEQ ID NO:6, or an immunogenic fragment thereof, SEQ ID NO:7, or an immunogenic fragment thereof, SEQ ID NO:8, or an immunogenic fragment thereof, SEQ ID NO:9, or an immunogenic fragment thereof, SEQ ID NO: 10, or an immunogenic fragment thereof, or a fragment of SEQ ID NO:2 whose length is 10-25 residues, preferably 12- 22 residues, or more preferably about 15 residues.
  • compositions include peptides having the sequence given by SEQ ID NO: 5 and peptides having the sequence given by SEQ ID NO:6. In another embodiment, the compositions include peptides having the sequence given by SEQ ID NO: 5 and SEQ ID NO:9. Multiple Antigenic Peptides
  • the peptides are administered as multiple antigenic peptides (MAPs) (Figs. 1-4).
  • MAPs multiple antigenic peptides
  • Methods for synthesizing and administering multiple antigenic peptides are known in the art (see, e.g., Reynolds et al., "T and B epitope determination and analysis of multiple antigen peptides for the Schistosoma mansoni experimental vaccine triose- phosphate isomerase," J Immunol, 152: 193 (1994); Basak et al., "Application of the multiple antigenic peptides (MAP) strategy to the production of prohormone convertases antibodies: synthesis, characterization and use of 8-branched immunogenic peptides," J Pept.
  • multiple antigenic peptides are synthesized by branching two peptides from lysine residues according to the methods of Tarn, J. P., "Multiple antigenic peptide system: A novel design for synthetic peptide vaccines and immunoassay.
  • Synthetic Peptides Approaches to Biological Problems, J. P. Tarn and E. T. Kaiser, eds., Alan R. Liss, Inc., New York, 3- 18 (1989).
  • MAPs of the current invention comprise at least 2, more preferably at least 3, and most preferably at least 4 copies of one of the peptides of the current invention.
  • Homogeneous MAPs are MAPs which contain the same peptide on each of its arms.
  • Heterogeneous MAPs are MAPs which contain different peptides on its arms.
  • the homogeneous four-arm MAPs comprise SEQ ID NO:5.
  • the homogeneous four-arm MAPs comprise SEQ ID NO:6.
  • the homogeneous four-arm MAPs comprise SEQ ID NO:7.
  • the homogeneous four-arm MAPs comprise SEQ ID NO:8.
  • the homogeneous four-arm MAPs comprise SEQ ID NO:9.
  • the homogeneous four-arm MAPs comprise SEQ ID NO: 10.
  • MAPs of the current invention are three-arm MAPs having as arm sequences any of the peptides of the current invention, including peptides having the sequences given by SEQ ID NO:5, or an immunogenic fragment thereof, SEQ ID NO: 6, or an immunogenic fragment thereof, SEQ ID NO: 7, or an immunogenic fragment thereof, SEQ ID NO: 8, or an immunogenic fragment thereof, SEQ ID NO: 9, or an immunogenic fragment thereof, SEQ ID NO: 10, or an immunogenic fragment thereof, or a fragment of SEQ ID NO:2 whose length is 10-25 residues, preferably 12- 22 residues, or more preferably about 15 residues.
  • a first arm of the three-arm MAP comprises a peptide has the sequence given by SEQ ID NO: 5
  • a second arm of the three-arm MAP has the sequence given by SEQ ID NO:9
  • a third arm of the three-arm MAP has the sequence given by SEQ ID NO: 10.
  • the arms comprise, respectively, SEQ ID NOs:5, 6, and 7.
  • immunogenic amounts of the peptides of the current invention can be determined using standard procedures.
  • initial immunizations may contain between about 1 ⁇ g and 10 mg, preferably between about 10 ⁇ g and 1 mg, and more preferably between about 50 ⁇ g and 500 ⁇ g of one or more of the peptides of the current invention.
  • Booster immunizations are typically given to the animal receiving the initial immunization. The general timing requirements of booster administrations are known in the art. In one embodiment, booster administrations are given at 3 and 6 weeks after the initial administration. Booster administrations typically contain about one-half of the amount of peptide as the initial immunizations. Standard techniques may be used for monitoring the immunologic response to the immunizations, as described in the "Therapeutic Compositions" section above.
  • immunological response may be determined using a nasopharyngeal (NP) challenge, as demonstrated in the Examples section below.
  • NP challenge a subject or animal, for example a mouse, may be challenged intranasally (IN) with 10 6 colony forming units (cfu) of Streptococcus pneumoniae suspended in 0.85% physiological saline. After sufficient time for bacterial multiplication, (e.g., 5 days after intranasal challenge), animals are sacrificed and nasal washes are performed and cultured by the method of Wu, H.Y., et al., ("Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice," Microb. Pathog.
  • the wash can be diluted 3x out to a final dilution of 1 :486.
  • Fifty microliters of each dilution can be cultured on blood agar + gentamicin plates (Trypticase soy agar supplemented with 5% defibrinated sheep blood and 0.5% gentamicin).
  • Data from NP colonization and carriage in immunized mice and placebo (PBS)-immunized controls can be analyzed using standard statistical tests such as the t-test or the Mann- Whitney rank sum test.
  • Nasopharyngeal carriage is the number of colony forming units per nose. Nasopharyngeal colonization is either positive or negative for a mouse depending on whether at least 1 cfu forms in 25 ⁇ l of nasal wash.
  • Klebsiella pneumoniae Haemophilus influenzae (types a-f), Legionella micdadei, L. pneumophila, Mycoplasma pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Streptococcus agalactiae, S. equisimilis, S. pyogenes, and group G streptococci.
  • mice Female B ALB/c mice were immunized with whole cell suspensions of S. pneumoniae R36A, a rough derivative of the capsular type 2 strain D39 (Avery et al. (1944) J Exp. Med. 79:137-157). The mice were immunized by intravenous injection three times and once by intraperitoneal injection. The maximum number of cells injected at any time was about 10 6 . Fusion was done on day 25 by using standard procedures (Clafin et al. (1978) Curr Top, Microbiol. Immunol 81:107-109). Spleen cells of 4 mice were fused with Sp2/0-Agl4 myeloma cells (Schulman et al.
  • ELISA Screening of hybridoma culture supernatants was done by ELISA.
  • U-bottom microtitration plates (Costar, Cambridge, MA) were sensitized with 50 ⁇ l of S. pneumoniae whole cell suspension (10 9 cfu/ml) diluted 1:4,000 in 0.1 M carbonate buffer, pH 9.6, and kept for 16 h at 4°C. The plates were washed 5 times with 0.9% NaCl containing 0.05% Tween 20 (NaCl-T). Culture supernatants (50 ⁇ l) from the fusion plates were added to 50 ⁇ l of a solution containing 2% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • SDS-PAGE SDS-PAGE and immunoblot analysis.
  • Sodium dodecyl sulfate-poiyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Tsang et al. ((1983) Methods Enzymol, 92:377-391), using an 8% acrylamide resolving gel.
  • Equal volumes of sample buffer (5% SDS- 10% 2-merca ⁇ toethanol-20% glycerol in 0.01 M Tris HCl, pH 8.0) and cell suspension containing 2.4 ⁇ g protein per ⁇ l were mixed, heated at 100°C for 5 min, and a 5- ⁇ l sample was applied to 1 of 15 wells.
  • Protein concentrations were determined by the method of Markwell et al. ((1978), Anal. Biochem. 87:206-210), with BSA as the standard. Proteins separated by SDS-PAGE were either silver stained by the method of Morrissey ((1981) Anal. Biochem. 117:307-310) or electroblotted onto nitrocellulose (Schleicher & Schuell, Inc., Keene, N.H.). The immunoblot procedure was done according to the method of Tsang et al. (1983) with slight modifications.
  • the blots were given three 5-min washes with PBS, pH 7.2, containing 0.3% Tween-20 and were gently agitated overnight (16 h) at 25°C. The blots were blocked for 1 h with casein-thimerosal buffer (CTB) (Kenna et al. (1985) J. Immunol Metk, 85:409-419). After three rinses with CTB, the blots were exposed to goat anti-mouse immunoglobulin horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond, Calif) for 2 h at 25°C. Conjugate dilutions (1 :2,000) were made in CTB.
  • CTB casein-thimerosal buffer
  • the cells were transferred to fresh and undiluted Lowicryl K4M two times during the next 24-hour period.
  • the Lowicryl K4M-treated cells were imbedded in gelatin capsules and placed in a box lined with aluminum foil.
  • the capsules were hardened using a short-wave UV light source (35 cm distance for 72 h at -20 °C).
  • the box was brought to room temperature, and the capsules were allowed to continue hardening for up to 14 days.
  • Samples of the capsule were cut into 100- ⁇ m thin sections and picked up on nickel grids. Grids containing the sample were placed on a droplet of ovalbumin solution in PBS containing sodium azide (E.Y.
  • the grids were rinsed two times and post-stained with osmium tetroxide, uranyl acetate, and lead citrate. The grids were examined with a Philips 410 transmission electron microscope.
  • MAbs were produced by the method of Kohler et al. (1975, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature 256:495-497) as modified by the method of Zola et al. (1982, "Techniques for production and characterization of monoclonal hybridoma antibodies," in J. G. Hurrell (ed.), Monoclonal hybridoma antibodies: techniques and applications, CRC Press Inc., Boca Raton, FL, pp 1-57.)
  • the 37-kDa purified PsaA used for immunization of mice was from S. pneumoniae serotype 22F, and had been purified according to the method of Tharpe et al.
  • PsaA pneumococcal surface adhesin A
  • All the MAbs were produced by immunizing with purified PsaA from serotype 22F except for 1E7 (1E7A3D7C2), which was produced by immunizing with a nonencapsulated strain of S. pneumoniae, R36A (Russell et al., 1990, "Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae," J. Clin. Microbiol 28: 2191-2195).
  • the PsaA was isolated using procedures set forth in Examples 3 and 5 below.
  • mice were initially immunized intraperitoneally with purified protein at a final concentration of 180 ⁇ g/ml in a 1 : 1 emulsion with Freund's incomplete adjuvant (Sigma Chemical Co., St. Louis, MO) and phosphate buffered saline pH 7.2. One month later, the mice were boosted with 110 ⁇ g/ml purified PsaA without adjuvant.
  • the hybridoma fusion was performed using standard procedures (Clafin et al., 1978, "Mouse myeloma-spleen cell hybrids: enhanced hybridization frequencies and rapid screening procedures," Curr. Top Microbiol. Immunol 81:107-109).
  • Spleen cells from two mice were fused with Sp 2/0-Agl4 myeloma cells (Schulman et al., 1978. "A better cell line for making hybridomas secreting specific antibodies," Nature 276:269-270).
  • Sera from immunized mice and tissue culture supernatant from hybridized cells were screened for reactivity against PsaA by ELISA using a goat anti-mouse immunoglobulin-horseradish peroxidase conjugate, and by SDS-PAGE combined with Western blotting to standard PsaA, in conventional procedures.
  • Hybridomas yielding positive results in the screen were expanded and used in the identification of the peptides; these were 6F6 (6F6F9C8), 4E9 (4E9G9D3), 8G12 (8G12G11B10), and 1B6 (1B6E12H9). These MAbs, along with 1E7, were used in this investigation.
  • MAb 1E7 correspondingly reacted with all pneumococcal strains tested (24 serotypes) to yield a sensitivity of 100%.
  • all pneumococcal strains tested 24 serotypes
  • sensitivity of 100% For specificity, none of 55 different nonpneumococcal strains of bacteria (representing 19 genera and 36 species) reacted, thus yielding a specificity of 100%.
  • the MAbs were biotinylated by incubating 1 mg of the protein in 0.1 M NaHCO 3 , pH 8.4, with 100 ⁇ g of N-hydroxysuccinimidyl-biotin ester (initially dissolved in DMSO).
  • Streptococcus pneumoniae DNA digested with restriction enzyme Sau3Al was ligated to BamHI digested pUC 13 and transformed into E. coli TB1. Recombinant clones were identified by colony immunoblot using the 37-kDa monoclonal antibody.
  • the plasmid pSTR3-l is an example of the pneumococcal surface adhesin A gene cloned into pUC13.
  • PsaA for use as an antigen or immunogen can be purified using any known method for protein purification.
  • Streptococcus pneumoniae can be conventionally cultured and the cells harvested. Purified 37-kDa protein can then be isolated from the Streptococcus pneumoniae cell mass by extraction with a non-ionic detergent and further purified by ammomum sulfate fractionation and isoelectric focusing. (Tharpe et al, 1996, "Purification and seroreactivity of pneumococcal surface adhesin A (PsaA)." Clin. Diagn. Lab. Immunol 3: 227-229).
  • coli strain TB1 containing a expression vector encoding the 37-kDa protein can be cultured and harvested using conventional techniques.
  • suitable expression vectors are those that contain a bacteriophage ⁇ PL Promoter (e.g., pKKl 773-3), a hybrid trp-lac promoter (e.g., pET-3a) or a bacteriophage T7 promoter.
  • the 37-kDa protein (PsaA) can then to be extracted from the cells using conventional methods for protein purification.
  • mice carrying the xid mutation were used in this protection study. They were tested for protection against challenge with a virulent capsular type 3 Streptococcus pneumoniae strain,
  • mice were anesthetized with Ketamine/Rompun and bled infraorbitally to obtain pre-immunization sera.
  • 37-kDa protein (pneumococcal surface adhesin A) was emulsified in complete Freund's adjuvant (CFA) to a protein concentration of 54 ⁇ g per ml.
  • CFA complete Freund's adjuvant
  • mice were injected subcutaneously into 2 axillary and 2 inguinal sites at 0.1 ml per site, delivering approximately 22 ⁇ g protein/mouse.
  • Ten control mice were treated identically with CFA and buffer substituting for protein.
  • IP intraperitoneally
  • IV intravenously
  • mice carrying the xid mutation were injected according to the following protocol:
  • mice were bled prior to immunization to establish baseline immunity.
  • Ten test mice were immumzed subcutaneously in four sites with a total of 21 ⁇ g of 37- kDa protein antigen (PsaA) emulsified in Complete Freund's adjuvant (CFA).
  • PsaA protein antigen
  • CFA Complete Freund's adjuvant
  • mice were boosted intraperitoneally (IP) with 100 ⁇ g of the 37-kDa protein antigen (test mice) or with buffer (controls). No adjuvant was used with this booster immunization.
  • mice were bled via the infraorbital sinus and they were collected and pooled into the two groups (immunized and controls). At the same time, blood was collected from individual mice to assay for antibody responses.
  • mice One day later, two additional mice were injected intraocularly with 0.1 ml of pooled immune sera to attempt to passively transfer immunity.
  • Three additional mice were injected intraperitoneally (IP) with 0.1 ml of pooled control mouse sera.
  • IP intraperitoneally
  • mice Only five mice were injected at this step because of the small amount of sera obtained from the immunized mice.
  • mice immunized with the 37-kDa protein were protected from fatal challenge with strain WU2; this immunity could be passively transferred with sera from immunized mice. (Originally 10 test mice were used. However, two of these mice died of other causes prior to being challenged with WU2.)
  • ELISA enzyme-linked immunosorbent assay
  • Conjugates can be prepared by use of a carrier protein bound to the 37-kDa protein, or an immunogenic peptide or polypeptide derived from the 37-kDa protein, via a linker, to elicit a T-cell-dependent response.
  • the carrier protein can be any immunogenic protein, for example, but not limited to, keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, diphtheria toxoid, or bacterial outer membrane proteins.
  • bacterial outer membrane proteins useful as conjugates, include outer membrane proteins of Neisseria meningitidis and Haemophilus influenzae.
  • Neisseria meningitidis can be selected from Neisseria meningitidis, group A, B, or C.
  • the 37-kDa protein or polypeptides thereof can be used in a conjugate in which the 37-kDa protein or a polypeptide thereof is the T-cell dependent immunogenic carrier for a polysaccharide antigen that is a B-cell stimulator. This is based on the theory that polysaccharide antigens are B-cell stimulators and that protective immunity is usually generated by a combination of B-cell and T-cell stimulation. Immune responses induced by protein antigens exhibit T-cell dependent properties (i.e., booster and carrier priming).
  • T-cell dependent stimulation is important because most children less than two years of age do not respond to T-cell independent antigens.
  • the attachment or conjugation of antigens can be accomplished by conventional processes, such as those described in U.S. Patent No. 4,808,700, involving the addition of chemicals that enable the formation of covalent chemical bonds between the carrier immunogen and the immunogen.
  • the 37-kDa protein antigen of this invention can be administered to mammals, especially humans, in a variety of ways.
  • Exemplary methods include parenteral (subcutaneous) administration given with a nontoxic adjuvant, such as an alum precipitate, or peroral administration given after reduction or ablation of gastric activity, or in a pharmaceutical form that protects the antigen against inactivation by gastric juice (e.g., a protective capsule or microsphere).
  • a pharmaceutical form that protects the antigen against inactivation by gastric juice e.g., a protective capsule or microsphere.
  • the dose and dosage regimen will depend mainly upon whether the antigen is being administered for therapeutic or prophylactic purposes, the patient, and the patient's history.
  • the total pharmaceutically effective amount of antigen administered per dose will typically be in the range of about 2 ⁇ g to 50 ⁇ g per patient.
  • the antigen will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently non-toxic and non-therapeutic. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Non- aqueous vehicles such as fixed oils and ethyl oleate, may also be used.
  • Liposomes may be used as vehicles.
  • the vehicle may contain minor amounts of additives, such as substances which enhance isotonicity and chemical stability (e.g., buffers and preservatives).
  • Bacterial strains All isolates of S. pneumoniae were provided and serotyped by the Streptococcal Reference Laboratory, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC).
  • the pneumococcal serotype 6B strain used for cloning and sequencing was a CDC reference strain (SP-86).
  • E. coli DH5 ⁇ (Bethesda Research Laboratories, Gaithersburg, MD) was used as the recipient host for plasmids (pUC19 and its derivatives).
  • S. pneumoniae strains were grown on Trypticase soy agar plates with 5% sheep blood cells or, where indicated, in Todd-Hewitt broth containing 0.5% yeast extract.
  • E. coli cultures were grown in Luria broth which, when required, was supplemented with 100 ⁇ g/ml of ampicillin (Sigma Chemical Co., St Louis, Mo.).
  • DNA sequencing was performed using the ABI PRISM Dye Terminator Cycle Sequencing kit and procedure (Perkin-Elmer, Cetus, Foster City, Calif). Sequence data were analyzed with the DNASTAR software program (DNASTAR, Inc., Madison, Wis.) and the Wisconsin Genetics Computer Group sequence analysis software program (Fenno et al., 1989, "Nucleotide sequence analysis of a type 1 fimbrial gene of Streptococcus sanguis FW213," Infect. Immun. 57:3527-3533). Preparation of genomic DNA for polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.
  • PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism
  • High molecular weight pneumococcal DNA was prepared by the procedure of Graves et al., 1993, "Universal bacterial DNA isolation procedure,” p.617-621, in D.H. Pershing et al. (ed), Diagnostic molecular biology, American Society for Microbiology, Washington, D.C.) with modifications. Sixteen-hour cultures of type specific S. pneumoniae were grown in 50 ml of Todd-Hewitt broth containing 0.5%> yeast extract in screw cap flasks at 37°C without shaking. Cultures were pelleted at 8000 x g for 15 min at room temperature and washed with phosphate-buffered saline (10 mM, pH 7.2).
  • the cell pellet was solubilized in 2.5 ml of buffer composed of 10 mM Tris, 1.0 mM EDTA, pH 8.0, and 0.4% SDS. Fifteen microliters of proteinase K (20 mg/ml) was added, and the lysate was incubated at 37°C for 1 h. The mixture was adjusted to 0.48 M NaCl with the addition of 500 ⁇ l of 5M NaCl and, after mixing by inversion, 400 ⁇ l of 10% hexadecyltrimethylammonium bromide in 0.7% NaCl was added. This suspension was mixed as before, incubated for 30 min at 65°C, and extracted with an equal volume of phenol-chloroform-isoamyl alcohol.
  • the upper aqueous phase was separated by centrifugation at 1500 x g and extracted with chloroform-isoamyl alcohol. DNA was precipitated from the upper aqueous phase with 2.5 volumes of ethanol at -70°C for 30 min. It was pelleted and dried in a desiccator, resuspended in water and quantitated by measuring absorbance at 260 nm. PCR-RFLP. Restriction enzymes EcoRI, Hinfl, Maelll, Mboll, Mnll, and Nhel were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN); Rsa ⁇ , Tsp509l, Eco57l. and Xmnl were purchased from New England Biolabs (Beverly, Mass.).
  • Primer sequences for the amplification reaction were selected from the N-terminal (nucleotides 181-201) and C-terminal (nucleotides 1106-1126) sequences of the S. pneumoniae serotype 6B gene (Pl, AGGATCTAATGAAAAAATTAG (SEQ ID NO:3); P2. TCAGAGGCTTATTTTGCCAAT (SEQ ID NO:4)) and flanking regions.
  • the primers were synthesized using standard procedures.
  • the reactions were performed with the Perkin-Elmer PCR amplification kit. Reaction volumes were 100 ⁇ l and contained the standard lx reaction buffer without Mg, 1 ⁇ M of each primer, 2.0 mM MgCl 2 , 0.2 mM dNTPs, template DNA, and 2.5 UT of Taq DNA polymerase.
  • the source of the template DNA was either extracted purified chromosomal DNA or a bacterial colony.
  • Conditions for amplification were as follows: 30 cycles of denaturation 94°C, 1 min, annealing 52°C, 0.5 min, and extension 72°C, 1.5 min. Amplified products were separated on a 1% agarose gel and visualized with ethidium bromide.
  • a direct colony amplification procedure was adapted, which shortened template preparation by eliminating the necessity of extracting chromosomal DNA.
  • the procedure consisted of adding a single bacterial colony directly from the plate into the PCR reaction mixture and heating at 95°C for 10 minutes. The remaining PCR steps were performed as outlined for extracted chromosomal DNA and are given above.
  • Genomic DNA was partially digested by Sau3Al was ligated to it ⁇ mHl-digested pUC18 and used to transform E. coli DH5 ⁇ . Recombinant colonies were selected for resistance to ampicillin and the formation of white colonies in the presence of isopropyl- ⁇ -galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside.
  • Colony immunoblot screening using anti-PsaA MAb of approximately 2,500 colonies yielded two positive clones, which were selected, purified, and rescreened by Western blot analysis using the same MAb.
  • the single open reading frame (ORF) present encodes the PsaA gene sequence.
  • This ORF is 930 nucleotides long and when amplified and subcloned into vector systems such as pGEM (Promega, Madison, Wis.) and BAC-to-BACTM expression system (Bethesda Research Laboratories, Gaithersburg, Md.) expresses full-length PsaA, reactive with anti-PsaA MAb antibodies.
  • This ORF encodes a peptide of 309 amino acids with a deduced molecular weight of 34,598 and an isoelectric point of 5.23.
  • Sequence identities were 81%, 81%, 77%, 82%, and 57%, respectively, for PsaA (S. pneumoniae strain R36A), SsaB (S. sanguis), FimA (S. parasanguis), ScaA (S. gordonii) and EfaA (E. faecalis). Additionally, all six sequences showed great similarity in organization. Each contains a hydrophobic leader peptide containing the prolipoprotein consensus sequence LXXC (for signal peptidase II cleavage) within the first 17-20 amino acids. This N-terminal leader sequence appears to represent the area of greatest variability. It is followed by a region of high similarity from amino acids 36 to 150. The region from amino acid position 150 to 198 is a variable region and is followed by another conserved region (amino acid position 198 to 309).
  • PsaA S. pneumoniae strain R36A
  • SsaB S. sanguis
  • FimA S. parasanguis
  • PCR-RFLP analysis of chromosomal DNA from the 23 serotype strains in a 23-valent vaccine was used to examine the degree of conservation of the PsaA gene among 23 S. pneumoniae serotypes, representing the 23 serotypes in a 23-valent vaccine. Since previous attempts to amplify pneumococcal type strains with primers corresponding to strain R36A were unsuccessful, PCR primers were selected based on the nucleotide sequence of the PsaA gene from serotype 6B, specifically, the regions of the gene that encode the N-terminal and C-terminal regions of the PsA polypeptide. Using primers complementary to the PsaA gene from S.
  • pneumoniae serotype 6B the PsaA gene from all 23 serotypes and subtypes represented in the 23-valent vaccine was amplified from chromosomal DNA. A total often enzymes were chosen that had restriction endonuclease digestion sites throughout the entire length of the serotype 6B PsaA gene. Nine of the ten enzymes give identical patterns for all 23 PsaA genes analyzed. The one exception, restriction enzyme Tsp509l, had six sites within the gene and generated seven fragments upon digestion with sizes of 7, 30, 68, 146, 151,166,and 362 bp.
  • the Tsp509l patterns of 3 to 4 additional strains of each of 23 serotype strains were analyzed. All strains analyzed were random clinical isolates from the United States that had been submitted to CDC for serotyping. The majority of the 80 strains were blood isolates; exceptions were two strains from cerebrospinal fluid, two strains from pleural fluid, and one strain each from the eye and nose. Of the strains analyzed, 10% had the extra Tsp509l site, resulting in the altered RFLP pattern. This modification was seen only in types 4, 8, 1 IF, and 33F.
  • This analysis discloses the cloning and sequencing of the gene encoding PsaA from S. pneumoniae serotype 6B and a subsequent analysis of the gene in the 23 pneumococcal polysaccharide vaccine serotypes. Sequence analysis revealed that the serotype 6B sequence and the previously published strain R36A were less similar than expected. The nucleotide sequence and its flanking regions were only 73% homologous to the original strain R36A PsaA, whereas the actual PsaA coding sequences had a computed homology of 78%. Protein sequence similarity between the two sequences was only 81%.
  • the 37-kDa protein from serotype 22F was used to generate monoclonal antibodies 1B6E12H9, 3C4D5C7, 4E9G9D3, 4H5C10F3, 6F6F9C8, and 8G12G11B10 (disclosed in United States Patent Application Ser. No. 08/715,131 , now U.S. Patent No. 5,854,416, incorporated herein by reference).
  • the MAbs were analyzed for their ability to confer protection from infection by Streptococcus pneumoniae. Table 2 shows that of 5 monoclonal antibodies tested, one in particular gave efficient protection from subsequent S. pneumoniae challenge (8G12G11B10). The protection from S. pneumoniae was dose-responsive, demonstrating that the monoclonal antibody was responsible for the protection (Table 3).
  • mice/group 10 mice/group.
  • Example 10 Phage Display Library.
  • a phage display library containing inserts of 15 amino acid residues located at the N-terminal part of the pill coat protein was constructed in the phage FUSE 5 as vector.
  • the library was made by ligating a synthetic 33 bp Bgll fragment into FUSE 5 and transfecting E. coli Kqll/kan+ cells by electroporation.
  • the phage progeny contain the display library.
  • the eluted phage were titrated and amplified, and then subjected to two further rounds of selection performed as above.
  • the amount of biotinylated MAb used was 1 nM and 1 pM, respectively, in the second and third rounds, such that only high affinity peptides were bound by the end of the last cycle.
  • Example 11 were propagated and sequenced. For each MAb, ten phage specimens resulting from the selection process were sequenced. Approximately 1 ⁇ g of single-stranded DNA was purified by phenol and chloroform extraction, ethanol- precipitated and resuspended in 7 ⁇ l water. Sequencing reactions were performed using a 27-mer primer complementary to the FUSE 5 vector sequence derived from a region in wild-type pill common to all fd-tet derived vectors and 35 S Sequenase version 2 (U.S. Biochemicals, Cleveland, OH). The sequences obtained are shown in Table 4.
  • Peptides having the sequences set forth in SEQ ID NOs. 5, 6, 7, and 8 can be synthesized by conventional methods, for example, using an automated peptide synthesizer.
  • the peptides can be purified by reversed phase HPLC, and the principal peak can be collected.
  • Peptide sequences can be verified by automated peptide sequencing, using an automated sequencing apparatus such as that manufactured by Beckman Instruments, Inc., Mountain View, CA.
  • Each peptide can be conjugated to an adjuvant such as keyhole limpet hemocyanin using coupling mediated by water-soluble carbodiimide reagent.
  • the resulting conjugate can be dissolved at a final concentration of about 180 ⁇ g/ml in phosphate buffered saline pH 7.2 and combined at an approximate 1 : 1 ratio in emulsion with Freund's incomplete adjuvant (Sigma Chemical Co., St. Louis, MO).
  • BALB/c mice can be initially immunized intraperitoneally with this suspension, and one month later, the mice can be boosted with about 110 ⁇ g/ml conjugate without adjuvant.
  • Example 14 Immunization of Mice with a Consensus Peptide Identified by Phage Display.
  • a peptide having a consensus sequence (SEQ ID NO: 8) derived from the sequences of several peptides identified by phage display was tested for its ability to elicit an immunological response against PsaA.
  • the consensus peptide (L V R R F V H R R P H V E S Q; SEQ ID NO:7) derived from the peptides which bound monoclonal antibodies 8G12, 6F6 and 1E7 in the phage display experiments described in Example 12 above, was synthesized with a CYGG spacer and a lauroyl group to its amino terminal end (Bio-Synthesis, Lewisville, TX).
  • the lauroyl group enhances the hydrophobic complexing of peptide groups to proteosomes (Lowell, G. H., et al, "Proteosomes, hydrophobic anchors, iscoms, and liposomes for improved presentation of peptide and protein vaccines," in: New Generation Vaccines, Woodrow, G.M., Levine, M.M. (Ed.), Marcel Deklcer, Inc., New York, pp. 141 - 160 (1990); Lowell, G. H., et al., "Peptides bound to proteosomes via hydrophobic feet become highly immunogenic without adjuvants," J Exp. Med. 167:658 (1988); and Zollinger, W.
  • the negative controls consisted of mice primed with 10, 25, or 50 ⁇ g of proteosomes at Week 0 and boosted with 10, 25, or 50 ⁇ g of proteosomes at Weeks 2 and 3. Sera was collected every week for 4 weeks and the anti-PsaA specific antibodies were tested by enzyme-linked immunoadsorbent assay (ELISA).
  • Anti-PsaA specific ELISA was performed as follows: Nuiic immuno Maxi Sorb plates were coated with 5 ⁇ g/ml of purified native PsaA protein at 4°C overnight. Plates were washed with PBS/Tween buffer (PBS containing 0.01% Tween-20) and blocked with PBS/Tween buffer containing 1% BSA. Serial dilutions of mouse sera starting at 1 : 10 in PBS/Tween/BSA were incubated for 1 h at 37°C. The plates were washed four times with PBS/Tween. Anti-mouse IgG and IgM conjugated to horseradish peroxidase (Sigma, St.
  • mice were immunized with 10, 25, or 50 ⁇ g of the consensus peptide complexed to proteosomes.
  • the sera from peptide immunized mice exhibited an anti-PsaA-response. Results of this study are shown in Tables 5 and 6.
  • the peptide immunized mice developed an IgG response where the titers ranged from 159 to 252 at Week 4. The titers were calculated after normalizing the data for proteosome background values.
  • the immune response to the 50 ⁇ g peptide dose group was the highest.
  • the anti-PsaA response to the peptide is significantly lower (p ⁇ 0.05) than immune response to the PsaA protein.
  • the data is represented as 25% titer of the control sera from mice immunized with 20 ⁇ g PsaA.
  • Example 15 Immunization of Mice with a Lipidated and Unlipidated form of a Peptide Identified by Phage Display (data from Srivastava, N., et al. Hybridoma 19;23. 2000 .
  • a peptide having the amino acid sequence of SEQ ID NO: 5 (T V S R V P W T A W A F H G Y) and which had been selected by phage display using MAb 4E9, was tested for its ability to protect mice from S. pneumoniae challenge.
  • Two forms of the peptide were synthesized: one with a palmitoyl residue at its N-terminal end (“SEQ ID NO: 5 -lipidated”) and another without such derivatization (“SEQ ID NO:5- unlipidated”).
  • peptides were synthesized using an ACT model 396 HPS peptide synthesizer (Advanced ChemTech, Louisville, KY) using solid phase F-moc chemistry as described by Stewart et al., "Solid peptide synthesis, 2 nd ed., Pierce Chemical Co., Rockford, IL (1984).
  • Lipidated versions of the peptides described above containing monopalmitic acid were synthesized by coupling palmitic acid (Sigma Chemicals, St. Louis, MO) to the deprotected amino-terminus of the resin-bound peptide employing the same reaction conditions as for the standard amino acid couplings described above (Verhaul et al. (1995)).
  • the peptide SEQ ID NO:5-lipidated was suspended in 100 ⁇ l PBS 0.01 M, pH 7.2, whereas the unlipidated peptide (SEQ ID NO:5-unlipidated), was mixed with the adjuvant alhydrogel (2%; #A1090BS, Accurate Chemical and Scientific Company, Westbury, NY) at a concentration of 6.3 mg/ml in PBS to enhance the immunogenicity of the peptide.
  • Control mice (n - 12) were similarly immunized but without peptide. Each mouse was immunized subcutaneously between the shoulders.
  • mice were challenged with 4.9 x 10 6 cfu of S pneumoniae, strain PLN-D39 (kindly provided by James Paton, Women's and Children's Hospital, North Sydney, S.A. Australia), a pneumolysin- negative derivative of D39. This was followed 5 days later by euthanasia and culturing of nasal washes. PBS nasal washes were done by the method of Wu, H.Y., et al., ("Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice," Microb. Pathog. 23:127 (1997)). Each wash was diluted 3x out to a final dilution of 1 :486.
  • the lipidated peptides contained the amino acid sequence CSS bound to the amino terminus of the amino acid sequence from the sequence listing indicated such that the cysteine (C) residue is at the amino terminus of the peptide.
  • mice were challenged intranasally (IN) with 10 6 cfu of serotype 2, 4, or 6B of Streptococcus pneumoniae suspended in 10 ⁇ l of 0.85% physiological saline. This was followed 5 days later by euthanasia and culturing of nasal washes. Nasopharyngeal (NP) colonization and carriage were analyzed as described in Example 15. Immunizations with non-lipidated and lipidated, mono-peptide (i.e., homogeneous) and bi-peptide MAPs (i.e., heterogeneous MAPs comprising two different peptide sequences) resulted in an immunological response against S.
  • PsaA peptides used in this experiment are immunogenic and reduce carriage of the 3 serotypes of S. pneumoniae tested. Furthermore, bi-peptide MAPs were more effective in reducing carriage than mono-peptide MAPs. Table 8. Percent reduction in naso-pharyngeal (NP) colonization: non-lipidated four-arm homogeneous MAPs compared to controls ( -value)
  • Peptide Type* 2(PLN-D39) 4 6B
  • the lipidated peptides contained the amino acid sequence CSS bound to the amino terminus of the amino acid sequence from the sequence listing indicated such that the cysteine (C) residue is at the amino terminus of the peptide.
  • Homogenous (monopeptide) and heterogenous (di- or tripeptide) three-arm MAPs can also be used to provide protection against S. pneumoniae challenge.
  • tripeptide three-arm MAPs containing any combination of SEQ ID NOs: 5-10 or immunogenic fragments thereof e.g., a homogenous tripeptide MAP containing three arms, each having the amino acid sequence set forth in SEQ ID NO:5, or a heterogeneous tripeptide MAP which contains a first arm having the sequence of SEQ ID NO:5, a second arm having the sequence of SEQ ID NO: 9, and a third arm having the sequence of SEQ ID NO: 10; see, e.g., Fig.
  • Examples 15 and 16 can be synthesized and tested using similar reagents, equipment, and procedures as described in Examples 15 and 16.
  • Other such examples of tripeptide combinations are (but are not limited to): a first arm comprising SEQ ID NO: 5, a second arm comprising SEQ ID NO: 6, and a third arm comprising SEQ ID NO: 7, 8, or 9; a first arm comprising SEQ ID NO:5, a second arm comprising SEQ ID NO: 10, and a third arm comprising SEQ ID NO:7, 8, or 9; a first arm comprising SEQ ID NO: 6, a second arm comprising SEQ ID NO: 7, a third arm comprising SEQ ID NO: 9 or 10, and so on.
  • Example 18 Inhibition of Pneumococcal Carriage in Mice by Subcutaneous Immunization with Peptides from PsaA.
  • mice Animals.
  • ND-4 Female; age 8 weeks old mice were obtained from Harlan Sprague Dawley, Inc., Indianapolis, LN. Upon arrival, mice were arranged in groups of 8 per cage. All animals were housed under standard conditions (25 °C, relative humidity ⁇ 40%) with food and water available ad libitum. All mice were allowed to acclimate to their new environment for 1 week before experimentation.
  • MAPs homologous, multi-antigenic peptides
  • the bi- and tripeptide constructs were synthesized by the differential deprotection of Fmoc- Lys(Dde) or Fmoc-Lys(ivDde) (Novabiochem, San Diego, CA) with piperidine and hydrazine using the method of Tarn (Tarn JP, In: Multiple antigenic peptide system: A novel design of synthetic peptide vaccines and immunoassay, Tarn, JP and Kaiser, ET, eds, New York: Alan R. Liss, Inc., 1989, pp. 3-18).
  • the crude peptides were analyzed for fidelity of synthesis by matrix-assisted-laser-desorption/ionization-time of flight mass spectroscopy, followed by one or more of the following procedures: amino acid analysis, high performance liquid chromatography (HPLC), capillary electrophoresis, and/or peptide sequencing.
  • HPLC high performance liquid chromatography
  • capillary electrophoresis capillary electrophoresis
  • peptide sequencing When necessary the peptides were purified by semi-prep HPLC, using acetonitrile/water/trifluoroacetic acid buffer systems.
  • the MAPs contain four branches that are homologous to each other and consist of either Pl, P2 or P3. Lipidated forms (designated as P43, P44, and P45) and nonlipidated forms (designated as P46, P47, and P48) are identical in structure except the former have a mono-substituted palmitoyl residue attached at the N-terminal of each base peptide unit by the 3 -amino acid linker, cysteine-serine-serine (Figs. 2 A and 2B).
  • the MAPs were lipidated with palmitic acid to study the effect of an adjuvant on peptide immunogenicity and subsequent influence on NP carriage.
  • Bipeptides are a combination of the base peptides (Pl and P2 or Pl and P3) (Fig. 3).
  • P79 contains 2 units each of Pl and P2, while in P80, P2 is replaced with P3.
  • the tripeptide identified as P81 and consistmg of three branches, is made up of one unit each of the base peptides (Fig. 4). In all cases, the peptide branches were synthesized on either a 3-arm or 4-arm lysine-norleucine core (Figs. 1-3).
  • the lipidated MAPs were suspended in 100 ⁇ L of sterile 0.01 M, pH 7.2, phosphate-buffered saline (PBS), while all other peptides were mixed with 100 ⁇ L of the adjuvant alum (alhydrogel-2%, #A1090B, Accurate Chemical and Scientific Company, Westbury, NY) at 6.3 mg/mL in PBS to enhance the immunogenicity of the peptide.
  • One week following the final boost all mice were challenged intranasally with 1-3 X 10 6 colony forming units (cfu) of the appropriate S. pneumoniae isolate suspended in 10 ⁇ L of 0.9% sodium chloride buffer (SCB; Abbott Laboratories, North Chicago, IL). Challenge inoculum was diluted serially 10-fold and plated on BA plates to determine challenge dose.
  • SCB 0.9% sodium chloride buffer
  • Carriage for a particular mouse was defined as the number of cfu in 50 ⁇ L of collected nasal wash after counts for each dilution were adjusted to a nondiluted level and averaged. Sensitivity of carriage assay was 40 cfu/ml of nasal wash.
  • P80 288 (74) .02 b P P8811 7 79966 ((3399)) .14 a Data represent the median number of colony forming units (cfu)/mouse (n 8) recovered in 100 ⁇ l of wash from the nasal-pharyngeal cavity. Mice were challenged with 10 6 cfu intranasally. b Significant difference, P ⁇ .05 vs. control mice injected with either phosphate buffered saline or alum. Mann- Whitney rank sum test was used for calculation of significant difference between groups.

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US6903184B1 (en) 1998-03-02 2005-06-07 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Multiple antigenic peptides immunogenic against Streptococcus pneumonia
EP2371843B1 (en) * 2005-05-19 2014-09-17 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by the Secretary, Department of Health and Human Services Functional epitopes of streptococcus pneumoniae psaA antigen and uses thereof
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WO2002004497A2 (en) 2002-01-17
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