Derivatives of choline binding proteins for vaccines Number:7,435,421 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides bacterial immunogenic agents for administration to humans and non-human animals to stimulate an immune response. It particularly relates to the vaccination of mammalian species with pneumococcal derived polypeptides that include an alpha helix but exclude a choline binding region as a mechanism for stimulating production of antibodies that protect the vaccine recipient against infection by pathogenic bacterial species. In another aspect the invention provides antibodies against such proteins and protein complexes that may be used as diagnostics and/or as protective/treatment agents for pathogenic bacterial species.<H Derivatives, vaccines, Derivatives, of, , 7435421.html, Patent, pto, 7435421

Title: Derivatives of choline binding proteins for vaccines

Abstract: The present invention provides bacterial immunogenic agents for administration to humans and non-human animals to stimulate an immune response. It particularly relates to the vaccination of mammalian species with pneumococcal derived polypeptides that include an alpha helix but exclude a choline binding region as a mechanism for stimulating production of antibodies that protect the vaccine recipient against infection by pathogenic bacterial species. In another aspect the invention provides antibodies against such proteins and protein complexes that may be used as diagnostics and/or as protective/treatment agents for pathogenic bacterial species.

Patent Number: 7,435,421 Issued on 10/14/2008 to Wizemann, et al.

Inventors: Wizemann; Theresa M. (Potomac, MD), Koenig; Scott (Rockville, MD), Johnson; Leslie S. (Germantown, MD)
Assignee: MedImmune, Inc. (Gaithersburg, MD)

Appl. No.: 11/062,080

Filed: February 18, 2005

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
10254995	Sep., 2002	6863893
09286981	Apr., 1999	6503511
60085743	May., 1998
60080878	Apr., 1998

Current U.S. Class: 424/244.1 ; 424/184.1; 424/190.1; 424/234.1; 424/237.1; 514/2; 514/8

Current International Class: A61K 39/00 (20060101); A01N 37/18 (20060101); A61K 39/38 (20060101); A61K 38/00 (20060101); A61K 39/02 (20060101); A61K 39/09 (20060101); A61K 39/085 (20060101); A61K 38/16 (20060101)

Field of Search: 424/190.1,184.1,234.1,237.1,244.1 514/2,8

References Cited [Referenced By]

U.S. Patent Documents


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2003/0096950	May 2003	Tuomanen et al.
2003/0138447	July 2003	Wizemann et al.
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Foreign Patent Documents


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Primary Examiner: Minnifield; N. M
Attorney, Agent or Firm: Olstein; Elliot M. Grant; Alan J.

Parent Case Text

This application is a Divisional of U.S. application Ser. No. 10/254,995, filed 25 Sep. 2002 now U.S. Pat. No. 6,863,893 which is a divisional of U.S. application Ser. No. 09/286,981, filed 6 Apr. 1999, now U.S. Pat. No. 6,503,511, which claims the benefit of U.S. Provisional Application Ser. No. 60/085,743, filed May 15, 1998 and U.S. Provisional Application Ser. No. 60/080,878, filed Apr. 7, 1998, the disclosures of all of which are hereby incorporated by reference in their entirety.

Claims

What is claimed is:

1. A method for protecting against S. pneumoniae infections in a host comprising immunizing said host with a vaccine comprising a polypeptide having a plurality of alpha helical portions wherein at least one of said alpha helical portions is at least 90% identical to the sequence of SEQ ID NO: 1, wherein said polypeptide does not comprise a choline binding portion and is present in an amount sufficient to protect against infection by S. pneumoniae when said vaccine is administered to a mammal.

2. The method of claim 1, wherein said polypeptide is protective against otitis media, meningitis, and sepsis infections caused by S. pneumoniae.

3. The method of claim 1, wherein said S. pneumoniae is one or more of types 1-5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F.

4. The method of claim 1, wherein said polypeptide does not comprise an HPS region.

5. The method of claim 1, wherein said polypeptide further comprises a proline rich region.

6. The method of claim 5, wherein said plurality of alpha helical portions are between the N-terminus of said polypeptide and said proline rich region.

Description

This invention relates generally to the field of bacterial antigens and their use, for example, as immunogenic agents in humans and animals to stimulate an immune response. More specifically, it relates to the vaccination of mammalian species with a polypeptide comprising an alpha helix-forming polypeptide obtained from a choline binding polypeptide as a mechanism for stimulating production of antibodies that protect the vaccine recipient against infection by pathogenic bacterial species. Further, the invention relates to antibodies and antagonists against such polypeptides useful in diagnosis and passive immune therapy with respect to diagnosing and treating such pneumococcal infections.

In a particular aspect, the present invention relates to the prevention and treatment of pneumonococcal infections such as infections of the middle ear, nasopharynx, lung and bronchial areas, blood, CSF, and the like, that are caused by pneumonococcal bacteria. In this regard, certain types of Streptococcus pneumoniae are of particular interest.

S. pneumoniae is a gram positive bacteria which is a major causative agent in invasive infections in animals and humans, such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen, et al. NEJM 322:1280-1284 (1995)). As part of the infective process, pneumococci readily bind to non-inflamed human epithelial cells of the upper and lower respiratory tract by binding to eukaryotic carbohydrates in a lectin-like manner (Cundell et al., Micro. Path. 17:361-374 (1994)). Conversion to invasive pneumococcal infections for bound bacteria may involve the local generation of inflammatory factors which may activate the epithelial cells to change the number and type of receptors on their surface (Cundell, et al., Nature, 377:435-438 (1995)). Apparently, one such receptor, platelet activating factor (PAF) is engaged by the pneumococcal bacteria and within a very short period of time (minutes) from the appearance of PAF, pneumococci exhibit strongly enhanced adherence and invasion of tissue. Certain soluble receptor analogs have been shown to prevent the progression of pneumococcal infections (Idanpaan-Heikkila et al., J. Inf. Dis., 176:704-712 (1997)).

A family of choline binding proteins (CBPs), which are non-covalently bound to phosphorylcholine, are present on the surface of pneumococci and have a non-covalent association with teichoic acid or lipoteichoic acid. An example of such family is choline binding protein A (CbpA), an approximately 75 kD weight type of CBP which includes a unique N-terminal domain, a proline rich region, and a C-terminal domain comprised of multiple 20 amino acid repeats responsible for binding to choline. A segment of the N-terminal portion of CbpA protein forms an alpha helix as part of its three-dimensional structure.

Accordingly, it is an object of the present invention to provide a polypeptide having broad protection against pneumococcal infections.

Definitions

In order to facilitate understanding of the description below and the examples which follow certain frequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 .mu.g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 .mu.l of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 .mu.g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37.degree. C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).

"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar amounts of the DNA fragments to be ligated.

"HPS portion" as used herein refers to an amino acid sequence as set forth in SEQ ID NO:2 for a choline binding protein ("CBP") of a pneumococcal bacteria that may be located amino terminal with respect to the proline rich portion of the overall amino acid sequence for such CBP.

The terms "identity", "% identity" or "percent identity" as utilized in this application refer to a calculation of differences between two contiguous sequences which have been aligned for "best fit" (to provide the largest number of aligned identical corresponding sequence elements, wherein elements are either nucleotides or amino acids) and all individual differences are considered as individual difference with respect to the identity. In this respect, all individual element gaps (caused by insertions and deletions with respect to an initial sequence ("reference sequence")) over the length of the reference sequence and individual substitutions of different elements (for individual elements of the reference sequence) are considered as individual differences in calculating the total number of differences between two sequences. Individual differences may be compared between two sequences where an initial sequences (reference sequence) has been varied to obtain a variant sequence (comparative sequence) or where a new sequence (comparative sequence) is simply aligned and compared to such a reference sequence. When two aligned sequences are compared all of the individual gaps in BOTH sequences that are caused by the "best fit" alignment over the length of the reference sequence are considered individual differences for the purposes of identity. If an alignment exists which satisfies the stated minimum identity, then a sequence has the stated minimum identity to the reference sequence. For example, the following is a hypothetical comparison of two sequences having 100 elements each that are aligned for best fit wherein one sequence is regarded as the "reference sequence" and the other as the comparative sequence. All of the individual alignment gaps in both sequences are counted over the length of the reference sequence and added to the number of individual element substitution changes (aligned elements that are different) of the comparative sequence for the total number of element differences. The total number of differences (for example 7 gaps and 3 substitutions) is divided by the total number of elements in the length of the reference sequence (100 elements) for the "percentage difference" (10/100). The resulting percentage difference (10%) is subtracted from 100% identity to provide a "% identity" of 90% identity. For the identity calculation all individual differences in both sequences are considered in the above manner over a discrete comparison length (the length of the reference sequence) of two best fit aligned sequences to determine identity. Thus, no algorithm is necessary for such an identity calculation.

"Isolated" in the context of the present invention with respect to polypeptides and/or polynucleotides means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to a vaccine for treating or preventing pneumococcal bacterial infections which utilizes as an immunogen at least one polypeptide truncate of a pneumococcal surface-binding protein, analog, or variant having a highly conserved immunogenic alpha-helical portion (corresponding generally to a "consensus" amino acid sequence as set forth in SEQ ID NO:1) with respect to different types of pneumococcal bacteria, which polypeptide does not include a choline-binding portion. Preferably, the C-terminal choline-binding portion is absent from such polypeptides. More preferred are such polypeptides wherein the HPS amino acid sequence is also absent. Even further preferred are polypeptides wherein the highly conserved immunogenic alpha-helical portion corresponding generally to a "consensus" amino acid sequence as set forth in SEQ ID NO:1 also corresponds generally to the amino acid sequence as set forth in SEQ ID NO:19 (amino acids 1 to 103 of SEQ ID NO:19 are identical to amino acids 1 to 103 of SEQ ID NO:1). Also preferred as vaccines are recombinantly-produced, isolated polypeptides that are missing both an HPS portion and the choline-binding portion.

More preferred as vaccines are one or more polypeptide truncates of pneumococcal surface-binding proteins, analogs or variants including a single highly conserved alpha-helix immunogenic portion with respect to different types of pneumococci, which polypeptides do not include a C-terminal choline-binding portion. Further preferred are isolated recombinantly produced polypeptides having such structure. Also preferred are such polypeptides that do not include either a C-terminal choline-binding portion or a HPS portion.

The present invention further provides a vaccine comprising a polypeptide including an immunogenic portion that is capable of forming an alpha helix, which polypeptide includes a sequence that has at least 85% identity and preferably at least 87% identity to the amino acid sequence of SEQ ID NO:1, wherein the isolated polypeptide does not include a C-terminal choline-binding portion. Further preferred are such polypeptides that comprise a polypeptide sequence that has at least 85% identity and preferably at least 87% identity to an amino acid sequence according SEQ ID NO:19. Preferably, the sequence of the isolated polypeptide includes neither an HPS portion (SEQ ID NO:2) nor a C-terminal choline-binding portion. Further preferred are isolated recombinantly produced polypeptides having such structure. In particular, such polypeptides corresponding to alpha helical structures of different types of S. pneumoniae bacteria are contemplated. Particularly preferred are the serotypes 1-5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F of such S. pneumoniae bacteria. Examples of such serotypes of bacteria are readily available from standard ATCC catalogs.

In an additional aspect, the present invention further provides a vaccine against S. pneumoniae comprising a synthetic or recombinant polypeptide comprising a plurality of alpha-helical portions, each derived from different naturally occurring S. pneumoniae choline-binding polypeptides wherein such alpha-helical portions have at least 85% identity to the amino acid sequence of SEQ ID NO:1, and wherein the isolated polypeptide does not include a choline-binding portion. Further preferred are those wherein the amino acid sequence for the alpha-helix areas is at least 85% identical to the amino acid sequence of SEQ ID NO:19. Preferably, such synthetic polypeptide includes neither a HPS portion nor a choline-binding portion. Analogs and variants of such chain structure polypeptides wherein such alpha helical portions may be synthetic variant amino acid sequences (or may be a mixture of naturally occurring and variant sequences) are also contemplated and embraced by the present invention. In a preferred aspect, chain vaccines polypeptides having at least ten different alpha helical structures corresponding to S. pneumoniae serotypes 1-5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F are provided. Further preferred are polypeptides including at least fifteen of such alpha-helical structures, more preferred are polypeptides including at least 20 such alpha-helical structures and more preferred are polypeptides including at least one alpha-helical structure corresponding to each of the S. pneumoniae serotypes 1-5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F. Another preferred polypeptide comprises each of the alpha helical structures from the amino acid sequences of SEQ ID NOS:3-18 which correspond to SEQ ID NO:1.

In another aspect, the invention relates to passive immunity vaccines formulated from antibodies against a polypeptide including a highly conserved immunogenic portion with respect to different types of pneumococcal bacteria which portion is capable of forming an alpha-helix having the hereinbefore described identity to the amino acid sequence of SEQ ID NO:1, which polypeptide does not include a C-terminal choline-binding portion, wherein said antibodies will bind to at least one S. pneumoniae species. Preferably, if such polypeptide is a truncate of a native pneumococcal surface-binding protein both its HPS portion (where applicable) and its choline-binding portion are absent from such polypeptide. Such passive immunity vaccines can be utilized to prevent and/or treat pneumococcal infections in immunocompromised patients, patients having an immature immune system (such as young children) or patients who already have an ongoing infection. In this manner, according to a further aspect of the invention, a vaccine can be produced from a synthetic or recombinant polypeptide wherein the polypeptide includes the conserved alpha helical portions of two or more different choline binding polypeptides of S. pneumoniae.

This invention also relates generally to the use of an isolated polypeptide having a highly conserved immunogenic portion with respect to different types of pneumococcal bacteria which portion is capable of forming an alpha-helix (corresponding generally to SEQ ID NO:1 or to SEQ ID NO:19) wherein the isolated polypeptide does not include a choline-binding portion, to raise antibodies in non-human mammalian species useful, for example, as diagnostic reagents and vaccines.

In yet another aspect, the present invention relates to the production of a polypeptide including a highly conserved immunogenic portion with respect to different types of pneumococcal bacteria which portion is capable of forming an alpha-helix whose sequence corresponds generally to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:19, wherein the isolated polypeptide does not include a choline-binding portion. Preferably, such recombinant production is of a truncated native pneumococcal surface-binding polypeptide wherein both the HPS portion (where applicable) and the choline-binding portion are absent.

In still another aspect, the present invention provides an isolated choline-binding polypeptide, wherein the non-choline binding region of such polypeptide has at least 90% identity to the corresponding amino acid sequence portion of a naturally occurring pneumococcal surface-binding protein which is a member selected from the group consisting of SEQ ID NOS:3-18. The invention relates to fragments of such polypeptides which include at least the conserved alpha-helical portion corresponding generally to SEQ ID NO:1, and which has at least 85% identity thereto, wherein the isolated polypeptide preferably is free of a choline binding region.

In another aspect the present invention provides an isolated polypeptide comprising an amino acid sequence which has at least 90% identity to one of the amino acid sequences selected from the group consisting of SEQ ID NO:3-18. Preferably, such isolated polypeptide comprises an amino acid sequence which has at least 95% identity, and more preferably 97% identity, to one of the amino acid sequences selected from the group consisting of SEQ ID NO:3-18. The invention further relates to fragments of such polypeptides.

In a yet further aspect, the present invention provides a S. pneumoniae CBP polypeptide encoded by a polynucleotide that will hybridize under highly stringent conditions to the complement of a polynucleotide encoding a polypeptide having an amino acid selected from the group consisting of SEQ ID NOS:1 and 3-18. Particularly preferred are polypeptides comprising an amino acid sequence segment that is at least 90% identical to the amino acid sequence of SEQ ID NO:1. Further preferred are such polypeptides comprising a contiguous amino acid sequence that has at least 95% identity with respect to the amino acid sequence of SEQ ID NO:1. And, even more preferred are polypeptides comprising an amino acid sequence that has at least 97% identity with respect to the amino acid sequence of SEQ ID NO:1.

In another aspect the present invention provides polynucleotides which encode the hereinabove described polypeptides of the invention. The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The polynucleotides which encode polypeptides including the amino acid sequences of at least one of SEQ ID NOS:3-18 (or polypeptides that have at least 90% identity to the amino acid sequences of such polypeptides) may be one of the coding sequences shown in SEQ ID NOS:20-35 or may be of a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the DNA of SEQ ID NOS:20-35.

The polynucleotides which encode the polypeptides of SEQ ID NOS:3-18 may include: only the coding sequence for the polypeptide; the coding sequence for the polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the polypeptide. The polypeptides encoded may comprise just a single alpha-helical portion or multiple alpha-helical portion and may independently or collectively include N-terminal sequences 5' of such alpha helical areas and/or sequences corresponding to the "X" structures or proline rich areas (as set forth in FIG. 1, for example).

The invention further relates to a polynucleotide comprising a polynucleotide sequence that has at least 95% identity and preferably at least 97% identity to a polynucleotide encoding one of the polypeptides comprising SEQ ID NO:3-18. The invention further relates to fragments of such polynucleotides which include at least the portion of the polynucleotide encoding the polypeptide sequence corresponding to SEQ ID NO:1.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. In particular, the polypeptides may include any or all of the types of structures set forth schematically in FIG. 1.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides including the amino acid sequences of SEQ ID NOS:3-18. The variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides. Complements to such coding polynucleotides may be utilized to isolate polynucleotides encoding the same or similar polypeptides. In particular, such procedures are useful to obtain alpha helical coding segments from different serotypes of S. pneumoniae, which is especially useful in the production of "chain" polypeptide vaccines containing multiple alpha helical segments.

Thus, the present invention includes polynucleotides encoding polypeptides including the same polypeptides as shown in the Sequence Listing as SEQ ID NOS:3-18 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of SEQ ID NOS:3-18. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in the Sequence Listing as SEQ ID NOS:20-35. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides (hybridization target sequences) which hybridize to the complements of the hereinabove-described sequences if there is at least 70% and preferably 80% identity between the target sequence and the complement of the sequence to which the target sequence hybridizes, preferably at least 85% identity. More preferred are such sequences having at least 90% identity, preferably at least 95% and more preferably at least 97% identity between the target sequence and the sequence of complement of the polynucleotide to which it hybridizes. The invention further relates to the complements to both the target sequence and to the polynucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:3 to 18. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the complements of the hereinabove-described polynucleotides as well as to those complements. As herein used, the term "stringent conditions" means hybridization will occur with the complement of a polynucleotide and a corresponding sequence only if there is at least 95% and preferably at least 97% identity between the target sequence and the sequence of complement of the polynucleotide to which it hybridizes. The polynucleotides which hybridize to the complements of the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain an immunogenic portion that will cross-react with an antibody to at least one of the polypeptides having a sequence according to SEQ ID NOS:3-18, or to a polypeptide that includes an amino acid sequence which has at least 85% identity to that of SEQ ID NO:1.

In a still further aspect, the present invention provides for the production of such polypeptides and vaccines as set forth above having a histidine label (or other suitable label) such that the full-length proteins, truncates, analogs or variant discussed above can be isolated due to their label.

In another aspect the present invention relates to a method of prophylaxis and/or treatment of diseases that are mediated by pneumococcal bacteria that have surface-binding CBP proteins. In particular, the invention relates to a method for the prophylaxis and/or treatment of infectious diseases that are mediated by S. pneumoniae that have a CBP surface-binding protein that forms an alpha helix (comprising a sequence that has at least an 85% identity to the amino acid sequence of SEQ ID NO:1). In a still further preferred aspect, the invention relates to a method for the prophylaxis and/or treatment of such infections in humans.

In still another aspect the present invention relates to a method of using one or more antibodies (monoclonal, polyclonal or sera) to the polypeptides of the invention as described above for the prophylaxis and/or treatment of diseases that are mediated by pneumococcal bacteria that have CBP surface-binding proteins. In particular, the invention relates to a method for the prophylaxis and/or treatment of infectious diseases that are mediated by S. pneumoniae CBP proteins which include an alpha helical portion having the hereinbefore described identity to the consensus sequence of SEQ ID NO:1. In a still further preferred aspect, the invention relates to a method for the prophylaxis and/or treatment of otitis media, nasopharyngeal, bronchial infections, and the like in humans by utilizing antibodies to the alpha-helix containing immunogenic polypeptides of the invention as described above.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of a pneumococcal CBP protein which shows from the N-terminal to the C-terminal, respectively, (a) a N-terminal sequence, (b) one of a potential alpha-helical forming area conserved segment (R1) that may not be present in some CBP polypeptides, (c) an optional small bridging sequence of amino acids that may bridge two conserved alpha-helical segments (X), (d) a second of a potential alpha-helical forming area consensus sequence (R2) related to the first consensus sequence (which corresponds to SEQ ID NO:1), (e) a proline rich area sequence, (f) a choline binding repeats area, and (e) a C-terminal tail sequence. Where relevant, an optional HPS sequence may naturally occur 5' of the proline rich sequence and 3' of the R1, X, and/or R2 areas.

FIG. 2 reports the results for passive immunity protection against 1600 cfu virulent serotype 6B S. pneumoniae SP317 (in mice) that was provided by day 31 rabbit antisera to a pneumococcal CBP truncate polypeptide, NR1XR2 (truncate missing both the proline and the choline binding areas, but including two conserved alpha-helical areas R1 and R2). Eighty percent of the mice immunized with the truncate antisera prior to challenge survived the 14 day observation period. By contrast, all mice immunized with a control sera (pre-immune rabbit sera) were dead by day 7.

FIG. 3 reports the results for passive immunity protection against 3450 cfu virulent serotype 6B S. pneumoniae SP317 (in mice) that was provided by day 52 rabbit antisera to a pneumococcal CBP truncate polypeptide, NR1XR2 (truncate missing both the proline and the choline binding areas, but including two conserved alpha-helical areas R1 and R2). One hundred percent of the mice immunized with the truncate antisera prior to challenge survived the 10 day observation period. By contrast, ninety percent of the mice immunized with a control sera (pre-immune rabbit sera) were dead at day 10.

FIG. 4 reports the results for passive immunity protection against 580 cfu virulent serotype 6B S. pneumoniae SPSJ2 (in mice) that was provided by day 31 rabbit antisera to a pneumococcal CBP truncate polypeptide, NR1XR2 (truncate missing both the proline and the choline binding areas, but including two conserved alpha-helical areas R1 and R2). Fifty percent of the mice immunized with the truncate antisera prior to challenge survived the 10 day observation period. By contrast, all mice immunized with a control sera (pre-immune rabbit sera) were dead by day 8.

FIG. 5 reports the results for active immunity protection against 560 cfu virulent serotype 6B S. pneumoniae SPSJ2 (in mice) that was provided by immunization with a pneumococcal CBP truncate polypeptide, NR1X (truncate missing the second conserved alpha-helical area R2, as well as both the proline and the choline binding areas). Eighty percent of the mice actively immunized with the NR1X CBP truncate prior to challenge survived the 14 day observation period. By contrast, all mice immunized with a control (sham mice) of PBS and adjuvant were dead by day 8.

FIG. 6 reports the results for active immunity protection against 680 cfu virulent serotype 6B S. pneumoniae SPSJ2 (in mice) that was provided by immunization with a pneumococcal CBP truncate polypeptide, NR1XR2 (truncate missing both the proline and the choline binding areas, but including two conserved alpha-helical areas R1 and R2). Fifty percent of the mice actively immunized with the NR1XR2 CBP truncate prior to challenge survived the 14 day observation period. By contrast, all mice immunized with a control (SP90) protein and adjuvant were dead by day 9.

FIG. 7 is an alignment report of the amino terminus of CBP polypeptides from various types of S. pneumoniae (Norway 4 (SEQ ID NO: 9), ATCC33400(1) (SEQ ID NO: 3), ATCC11733(2) (SEQ ID NO: 4), ATCC2 (SEQ ID NO: 5), ATCC4 (SEQ ID NO: 6), ATCC6B (SEQ ID NO: 7), ATCC18C-3 (SEQ ID NO: 8), R6X(2) (SEQ ID NO: 10), SPB105(6B) (SEQ ID NO: 11), SPB328(23F) (SEQ ID NO: 12), SPB331(14) (SEQ ID NO: 13), SPB365(23F) (SEQ ID NO: 14), SPR332(9V) (SEQ ID NO: 15), SPSJ2(6B) (SEQ ID NO: 16), SPSJ9(14) (SEQ ID NO: 17), and SPSJ12(19A) (SEQ ID NO: 18) and a consensus sequence is reported at the top of each row (sets of lines) of the comparison. The consensus sequence for the comparison is listed as the "Majority" sequence (SEQ ID NO:36). One letter codes are utilized to represent the sequences which are aligned for a "best fit" comparison wherein dashes in a sequence indicate spacing gaps of the contiguous sequence.

FIG. 8 shows the sequence pair distances for the amino acid sequences as described for FIG. 7 and set forth therein. A Clustal method with identity residue weight table is used. The percent similarity for such a comparison is reported for the amino acid sequences set forth in FIG. 7.

FIG. 9 is an alignment report for a first helical region in the amino acid sequences of CBP polypeptides from various types of S. pneumoniae (Norway 4 (SEQ ID NO: 9), ATCC33400(1) (SEQ ID NO: 3), ATCC11733(2) (SEQ ID NO: 4), ATCC2 (SEQ ID NO: 5), ATCC4 (SEQ ID NO: 6), ATCC6B (SEQ ID NO: 7), ATCC18C-3 (SEQ ID NO: 8), R6X(2) (SEQ ID NO: 10), SPB105(6B) (SEQ ID NO: 11), SPB328(23F) (SEQ ID NO: 12), SPB331(14) (SEQ ID NO: 13), SPB365(23F) (SEQ ID NO: 14), SPR332(9V) (SEQ ID NO: 15), SPSJ2(6B) (SEQ ID NO: 16), SPSJ9(14) (SEQ ID NO: 17), and SPSJ12(19A) (SEQ ID NO: 18) and a consensus sequence is reported at the top of each row (sets of lines) of the comparison. The consensus sequence for the comparison is listed as the "Majority" sequence (SEQ ID NO:38). One letter codes are utilized to represent the sequences which are aligned for a "best fit" comparison wherein dashes in a sequence indicate spacing gaps of the contiguous sequence.

FIG. 10 shows the sequence pair distances for the amino acid sequences as described for FIG. 9 and set forth therein. A Clustal method with identity residue weight table is used. The percent similarity for such a comparison is reported for the amino acid sequences set forth in FIG. 9.

FIG. 11 is an alignment report for the region X in the amino acid sequences of CBP polypeptides from various types of S. pneumoniae (Norway 4 (SEQ ID NO: 9), ATCC33400(1) (SEQ ID NO: 3), ATCC11733(2) (SEQ ID NO: 4), ATCC2 (SEQ ID NO: 5), ATCC4 (SEQ ID NO: 6), ATCC6B (SEQ ID NO: 7), ATCC18C-3 (SEQ ID NO: 8), R6X(2) (SEQ ID NO: 10), SPB105(6B) (SEQ ID NO: 11), SPB328(23F) (SEQ ID NO: 12), SPB331(14) (SEQ ID NO: 13), SPB365(23F) (SEQ ID NO: 14), SPR332(9V) (SEQ ID NO: 15), SPSJ2(6B) (SEQ ID NO: 16), SPSJ9(14) (SEQ ID NO: 17), and SPSJ12(19A) (SEQ ID NO: 18) and a consensus sequence is reported at the top of each row (sets of lines) of the comparison. The consensus sequence for the comparison is listed as the "Majority" sequence (SEQ ID NO:37). One letter codes are utilized to represent the sequences which are aligned for a "best fit" comparison wherein dashes in a sequence indicate spacing gaps of the contiguous sequence.

FIG. 12 shows the sequence pair distances for the amino acid sequences as described for FIG. 11 and set forth therein. A Clustal method with identity residue weight table is used. The percent similarity for such a comparison is reported for the amino acid sequences set forth in FIG. 11.

FIG. 13 is an alignment report for the second helical region A in the amino acid sequences of CBP polypeptides from various types of S. pneumoniae (Norway 4 (SEQ ID NO: 9), ATCC33400(1) (SEQ ID NO: 3), ATCC11733(2) (SEQ ID NO: 4), ATCC2 (SEQ ID NO: 5), ATCC4 (SEQ ID NO: 6), ATCC6B (SEQ ID NO: 7), ATCC18C-3 (SEQ ID NO: 8), R6X(2) (SEQ ID NO: 10), SPB105(6B) (SEQ ID NO: 11), SPB328(23F) (SEQ ID NO: 12), SPB331(14) (SEQ ID NO: 13), SPB365(23F) (SEQ ID NO: 14), SPR332(9V) (SEQ ID NO: 15), SPSJ2(6B) (SEQ ID NO: 16), SPSJ9(14) (SEQ ID NO: 17), and SPSJ12(19A) (SEQ ID NO: 18) and a consensus sequence is reported at the top of each row (sets of lines) of the comparison. The consensus sequence for the comparison is listed as the "Majority" sequence (SEQ ID NO:1). One letter codes are utilized to represent the sequences which are aligned for a "best fit" comparison wherein dashes in a sequence indicate spacing gaps of the contiguous sequence.

FIG. 14 shows the sequence pair distances for the amino acid sequences as described for FIG. 13 and set forth therein. A Clustal method with identity residue weight table is used. The percent similarity for such a comparison is reported for the amino acid sequences set forth in FIG. 13.

FIG. 15 is an alignment report for the second helical region B in the amino acid sequences of CBP polypeptides from various types of S. pneumoniae (Norway 4 (SEQ ID NO: 9), ATCC33400(1) (SEQ ID NO: 3), ATCC11733(2) (SEQ ID NO: 4), ATCC2 (SEQ ID NO: 5), ATCC4 (SEQ ID NO: 6), ATCC6B (SEQ ID NO: 7), ATCC18C-3 (SEQ ID NO: 8), R6X(2) (SEQ ID NO: 10), SPB105(6B) (SEQ ID NO: 11), SPB328(23F) (SEQ ID NO: 12), SPB331(14) (SEQ ID NO: 13), SPB365(23F) (SEQ ID NO: 14), SPR332(9V) (SEQ ID NO: 15), SPSJ2(6B) (SEQ ID NO: 16), SPSJ9(14) (SEQ ID NO: 17), and SPSJ12(19A) (SEQ ID NO: 18) and a consensus sequence is reported at the top of each row (sets of lines) of the comparison. The consensus sequence for the comparison is listed as the "Majority"0 sequence (SEQ ID NO:19). One letter codes are utilized to represent the sequences which are aligned for a "best fit" comparison wherein dashes in a sequence indicate spacing gaps of the contiguous sequence.

FIG. 16 shows the sequence pair distances for the amino acid sequences as described for FIG. 15 and set forth therein. A Clustal method with identity residue weight table is used. The percent similarity for such a comparison is reported for the amino acid sequences set forth in FIG. 15.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention there is provided a vaccine to produce a protective response against S. pneumoniae infections which employs a polypeptide which comprises a member selected from the group consisting of:

(a) an amino acid sequence which produces an alpha helical structure and which is at least 85% identical to the amino acid sequence of SEQ ID NO:1 and which is free of a choline binding region, and

(b) an isolated truncate of a naturally occurring S. pneumoniae polypeptide that comprises an alpha helical portion that has at least 85% identity to the amino acid sequence of SEQ ID NO:1 and is free of a choline binding region,

(c) an isolated truncate of a naturally occurring S. pneumoniae polypeptide that comprises an alpha helical portion that has at least 90% identity to the amino acid sequence of SEQ ID NO:19 and is free of a choline binding region. In a preferred aspect, such isolated truncate polypeptide is a member selected from the group consisting of SEQ ID NOS:3-18 and said isolated polypeptide is free of a choline binding region and, if relevant, a HPS region; or a fragment thereof which includes at least the alpha helical segment which corresponds to the consensus sequence of SEQ ID NO:1. Particularly preferred are vaccines which utilize such truncate polypeptides that include at least such alpha helical area or utilize a recombinant immunogen polypeptide comprising at least two of such alpha-helical segments. Such polypeptide may be a recombinant polypeptide containing multiple alpha-helical areas from one or more trucates. Further preferred are recombinant immunogen polypeptides comprising at least two alpha-helical areas corresponding to the alpha helical areas of two or more truncates from different types of pneumococcal bacteria. Such polypeptide may be a recombinant polypeptide containing multiple alpha-helical areas from one or more different types of pneumococcal bacteria.

In accordance with the present invention, there is provided an isolated polypeptide comprising a truncated surface-binding polypeptide derived from S. pneumoniae, said isolated polypeptide containing an alpha-helical area whose amino acid sequence corresponds generally to the amino acid sequence of SEQ ID NO:1, but free of a choline binding area. Preferably, said isolated polypeptide also omits any naturally occurring repeats of the alpha-helical forming area and omits any HPS amino acid sequence that may be present.

It is an object of the present invention to utilize as immunogenic composition for a vaccine (or to produce antibodies for use as a diagnostic or as a passive vaccine) comprising an immunogenic polypeptide comprising a pneumococcal surface-binding polypeptide with an alpha helical portion from which a choline binding region has been omitted. In one embodiment, such truncated proteins (naturally or recombiantly produced, as well as functional analogs) from S. pneumoniae bacteria are contemplated. Even more particularly, S. pneumoniae polypeptides having a single alpha helical portion that omit any HPS areas that occur and choline binding areas of the native protein are contemplated.

A particularly preferred embodiment of such an immunogenic composition is for use as a vaccine (or as an immunogen for producing antibodies useful for diagnostics or vaccines) wherein the active component of the immunogenic composition is an isolated polypeptide comprising at least one member selected from the group consisting of:

(a) an amino acid sequence which is selected from SEQ ID NOS:3-19,

(b) a polypeptide which has at least 90% identity to (a), preferably at least 95% identity to (a), and even more preferred at least 97% identity to (a), or

(c) a fragment of (a) or (b) wherein such fragment includes at least one alpha helical portion that corresponds to the consensus sequence which is SEQ ID NO:1 and said fragment does not comprise a choline binding region. Preferably, such vaccines utilize a polypeptide that contains neither a choline binding region nor an HPS region that occurs as part of the amino acid sequences in the native proteins.

In another preferred embodiment, there is provided a vaccine which includes at least one isolated polypeptide which includes an amino acid sequence which has at least 85% identity (preferably 87% identity and more preferably at least 90% identity) to SEQ ID NO:1, which isolated polypeptide is free of a choline binding portion and, where applicable, is also preferably free of an HPS portion. The preferred polypeptide may also include one or more of the N-terminal sequences that are located 5' of the alpha helical areas in the polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOS:3-18, or the like. The polypeptide truncate may also include one or more of the proline regions (region "P" in FIG. 1) and/or the spanning region (region "X" in FIG. 1).

In another aspect of the invention, such an immunogenic composition may be utilized to produce antibodies to diagnose pneumococcal infections, or to produce vaccines for prophylaxis and/or treatment of such pneumococcal infections as well as booster vaccines to maintain a high titer of antibodies against the immunogen(s) of the immunogenic composition.

While other antigens have been contemplated to produce antibodies for diagnosis and for the prophylaxis and/or treatment of pneumococcal infections, there is a need for improved or more efficient vaccines. Such vaccines should have an improved or enhanced effect in preventing bacterial infections mediated pneumococci having surface-binding polypeptides. Further, to avoid unnecessary expense and provide broad protection against a range of pneumococcal serotypes there is a need for polypeptides that comprise an immunogenic amino acid sequence corresponding to a portion of pneumococcal surface-binding polypeptides that is a highly conserved portion among various types of pneumococci. Preferably, such polypeptides avoid the inclusion of amino acid sequences corresponding to other portions of the native polypeptides, such as the choline binding region and/or the HPS region.

There is a need for improved antigenic compositions comprising highly conserved portions of polypeptides that bind to the surface of pneumococcal bacteria for stimulating high-titer specific antisera to provide protection against infection by pathogenic pneumococcal bacteria and also for use as diagnostic reagents.

In such respect, truncated polypeptides, functional variant analogs, and recombinantly produced truncated polypeptides of the invention are useful as immunogens for preparing vaccine compositions that stimulate the production of antibodies that can confer immunity against pathogenic species of bacteria. Further, preparation of vaccines containing purified proteins as antigenic ingredients are well known in the art.

Generally, vaccines are prepared as injectables, in the form of aqueous solutions or suspensions. Vaccines in an oil base are also well known such as for inhaling. Solid forms which are dissolved or suspended prior to use may also be formulated. Pharmaceutical carriers are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use. Examples of such carriers include, but are not limited to, water, saline solutions, dextrose, or glycerol. Combinations of carriers may also be used.

Vaccine compositions may further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents. or emulsifying agents, which can serve to improve the effectiveness of the vaccine.

Vaccines are generally formulated for parenteral administration and are injected either subcutaneously or intramuscularly. Such vaccines can also be formulated as suppositories or for oral administration, using methods known in the art.

The amount of vaccine sufficient to confer immunity to pathogenic bacteria is determined by methods well known to those skilled in the art. This quantity will be determined based upon the characteristics of the vaccine recipient and the level of immunity required. Typically, the amount of vaccine to be administered will be determined based upon the judgment of a skilled physician. Where vaccines are administered by subcutaneous or intramuscular injection, a range of 50 to 500 .mu.g purified protein may be given.

The term "patient in need thereof" refers to a human that is infected with, or likely, to be infected with, pathogenic pneumococcal bacteria that produce CbpA, or the like, preferably S. pneumoniae bacteria (however a mouse model can be utilized to simulate such a patient in some circumstances).

In addition to use as vaccines, the polypeptides of the present invention can be used as immunogens to stimulate the production of antibodies for use in passive immunotherapy, for use as diagnostic reagents, and for use as reagents in other processes such as affinity chromatography.

The polynucleotides encoding the immunogenic polypeptides described above may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The identification of multiple coil structures of alpha helical amino acid segments in the S. pneumoniae polypeptides according to the invention may be determined by the location of proline rich areas with respect to one another. Further the "X" area optionally located between two or more alpha-helical structures can play a part in the formation of a coil within a coil structure. Standard three-dimensional protein modeling may be utilized for determining the relative shape of such structures. An example of a computer program, the Paircoil Scoring Form Program ("PairCoil program"), useful for such three-dimensional protein modelling is described by Berger et al. in the Proc. Natl. Acad. of Sci. (USA), 92:8259-8263 (August 1995). The PairCoil program is described as a computer program that utilizes a mathematical algorithm to predict locations of coiled-coil regions in amino acid sequences. A further example of such a computer program is described by Wolf et al., Protein Science 6:1179-1189 (June 1997) which is called the Multicoil Scoring Form Program ("Multicoil program"). The MultiCoil program is based on the PairCoil algorithm and is useful for locating dimeric and trimeric coiled coils.

In a preferred aspect, the invention provides for recombinant production of such polypeptides in a host bacterial cell other than a S. pneumoniae species host to avoid the inclusion of native surface-binding polypeptides that have a choline binding region. A preferred host is a species of such bacteria that can be cultured under conditions such that the polypeptide of the invention is secreted from the cell.

The present invention also relates to vectors which include polynucleotides encoding one or more of the polypeptides of the invention that include the highly conserved alpha-helical amino acid sequence in the absence of an area encoding a choline binding amino acid sequence, host cells which are genetically engineered with vectors of the invention and the production of such immunogenic polypeptides by recombinant techniques in an isolated and substantially immunogenically pure form.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors comprising a polynucleotide encoding a polypeptide comprising the highly conserved alpha-helical region but not having a choline binding region, or the like of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides which encode such polypeptides. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA s