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Title:  Protein-based Streptococcus pneumoniae vaccines
United States Patent: 
7,504,110
Issued: 
March 17, 2009

Inventors:
 Mizrachi Nebenzahl; Yaffa (Beer Sheva, IL)
Assignee:
  Ben-Gurion University of the Negev Research and Development Authority (Beer-Sheva, IL)
Appl. No.:
 10/953,513
Filed:
 September 30, 2004


 

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Abstract

The present invention is primarily directed to a method for preventing infection of a mammalian subject with S. pneumoniae, wherein said method comprises administering to said subject an effective amount of one or more S. pneumoniae cell wall and/or cell membrane proteins and/or immunogenically-active fragments, derivatives or modifications thereof, wherein said proteins are selected from a defined group of immunogenic proteins. The present invention further provides vaccine compositions containing said cell wall and/or cell membrane proteins.

Description of the Invention

SUMMARY OF THE INVENTION

It has now been found that it is possible to protect individuals against infection with S. pneumoniae by means of administering to said individuals a vaccine composition comprising one or more proteins isolated from the outer layers of the aforementioned bacteria and/or one or more immunogenically-active fragments, derivatives or modifications thereof. Unexpectedly, it was found that such vaccine compositions are effective against a wide range of different S. pneumoniae serotypes, and in all age groups, including those age groups which do not produce anti-S. pneumoniae antibodies following inoculation with polysaccharide-based vaccines.

It is to be noted that in the context of the present invention, the terms "fragments", "derivatives" and "modifications" are to be understood as follows:

"Fragment": a less than full length portion of the native sequence of the protein in question, wherein the sequence of said portion is essentially unchanged as compared to the relevant part of the sequence of the native protein.

"Derivative": a less than full length portion of the native sequence of the protein in question, wherein either the sequence further comprises (at its termini and/or within said sequence itself) non-native sequences, i.e. sequences which do not form part of the native protein in question. The term "derivative" also includes within its scope molecular species produced by conjugating chemical groups to the amino residue side chains of the native proteins or fragments thereof, wherein said chemical groups do not form part of the naturally-occurring amino acid residues present in said native proteins.

"Modification": a full length protein or less than full length portion thereof comprising non-native amino acid residues and sequences of such non-native residues, which have been introduced as a consequence of mutation of the native sequence (by either random or site-directed processes).

The term "immunogenically-active" is used herein in ordinary sense to refer to an entity (such as a protein or its fragment or derivative) that is capable of eliciting an immune response when introduced into a host subject.

The present invention is primarily directed to a method for preventing infection of mammalian subjects with S. pneumoniae, wherein said method comprises administering to a subject in need of such treatment an effective amount of one or more S. pneumoniae cell wall and/or cell membrane proteins and/or immunogenically-active fragments, derivatives or modifications thereof, wherein said proteins are selected from the group consisting of: phosphoenolpyruvate protein phosphotransferase, phospho-mannomutase, trigger factor, elongation factor G, tetracycline resistance protein (tetO), DNA directed RNA polymerase alpha-chain, NADH oxidase, glutamyl-tRNA amidotransferase subunit A, N utilization substance protein A homolog, XAA-HIS dipeptidase, cell division protein ftsz, zinc metalloproteinase in SCAA 5' region (ORF 6), L-lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase, UDP-glucose 4-epimerase, GTP binding protein typA/BipA, GMP synthase, glutamyl-tRNA synthetase, NADP-specific glutamate dehydrogenase, Elongation factor TS, phosphoglycerate kinase (cell wall protein), pyridine nucleotide-disulfide oxido-reductase, 40S ribosomal protein S1, 6-phosphogluconate dehydrogenase, aminopeptidase C, carbomyl-phosphate synthase (large subunit), PTS system mannose-specific IIAB components, ribosomal protein S2, dihydroorotate dehydrogenase, aspartate carbamoyltransferase, elongation factor Tu, Pneumococcal surface immunogenic protein A (PsipA), phosphoglycerate kinase (cell membrane protein), ABC transporter substrate-binding protein, endopeptidase O, Pneumococcal surface immunogenic protein B (PsipB) and Pneumococcal surface immunogenic protein C (PsipC).

The means used to identify the aforementioned S. pneumoniae proteins, and their unique public access database accession codes will be disclosed and described hereinbelow.

In a preferred embodiment of the method of the present invention, one or more adjuvants may be optionally administered to the subject together with one or more of the aforementioned S. pneumoniae cell wall and/or cell membrane proteins or fragments thereof.

In one particularly preferred embodiment, the method of the present invention for preventing infection of mammalian subjects by S. pneumoniae comprises administering to a subject in need of such treatment an effective amount of S. pneumoniae glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

In another particularly preferred embodiment, the method of the present invention for preventing infection of mammalian subjects by S. pneumoniae comprises administering to a subject in need of such treatment an effective amount of S. pneumoniae fructose-biphosphate aldolase.

In a further particularly preferred embodiment, the method of the present invention for preventing infection of mammalian subjects by S. pneumoniae comprises administering to a subject in need of such treatment an effective amount of one or more immunogenically-active fragments of the aforementioned S. pneumoniae fructose-biphosphate aldolase protein. Although many such active fragments may be generated from this protein and used in the presently-disclosed method, in an especially preferred embodiment, the active fragment used corresponds to the peptide encoded by the first 294 nucleotides of the fructose biphosphate aldolase gene (SP0605 Streptococcus pneumoniae TIGR4), referred to herein as SEQ ID no.1, and defined in the sequence listing that forms an integral part of the present disclosure.

In one preferred embodiment of the method of the invention, the cell wall and/or cell membrane proteins are S. pneumoniae proteins that are associated with an age-related immune response.

The term "age-related immune response" (as used throughout this application) indicates that the ability of subjects to produce antibodies to the protein or proteins causing said immune response increases with age. In the case of human subjects, said ability is measured over a time scale beginning with neonates and ending at approximately age four years and adults. In non-human mammalian subjects, the "age-related immune response" is measured over an age range extending from neonates to an age at which the immune system of the young mammal is at a stage of development comparable to that of a pre-puberty human child and adults.

In another preferred embodiment of the method of the invention, the cell wall and/or cell membrane proteins are lectins.

In another preferred embodiment of the method of the invention, the cell wall and/or cell membrane proteins are non-lectins.

In another preferred embodiment of the method of the invention, the cell wall and/or cell membrane proteins are a mixture of lectins and non-lectins.

The term "lectins" is used hereinabove and hereinbelow to indicate proteins having the ability to specifically bind to polysaccharides or oligosaccharides. Conversely, the term "non-lectins" is used to refer to proteins lacking the aforementioned saccharide-binding property.

In one preferred embodiment of the method of the invention, the mammalian subject is a human subject.

In another aspect, the present invention is directed to a vaccine composition comprising as the active ingredient one or more S. pneumoniae cell wall and/or cell membrane proteins and/or immunogenically-active fragments, derivatives or modifications thereof, wherein said proteins are selected from the group consisting of: phosphoenolpyruvate protein phosphotransferase, phospho-mannomutase, trigger factor, elongation factor G, tetracycline resistance protein (tetO), DNA directed RNA polymerase alpha-chain, NADH oxidase, glutamyl-tRNA amidotransferase subunit A, N utilization substance protein A homolog, XAA-HIS dipeptidase, cell division protein ftsz, zinc metalloproteinase in SCAA 5' region (ORF 6), L-lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase, UDP-glucose 4-epimerase, GTP binding protein typA/BipA, GMP synthase, glutamyl-tRNA synthetase, NADP-specific glutamate dehydrogenase, Elongation factor TS, phosphoglycerate kinase (cell wall protein), pyridine nucleotide-disulfide oxido-reductase, 40S ribosomal protein S1, 6-phosphogluconate dehydrogenase, aminopeptidase C, carbomyl-phosphate synthase (large subunit), PTS system mannose-specific IIAB components, ribosomal protein S2, dihydroorotate dehydrogenase, aspartate carbamoyltransferase, elongation factor Tu, Pneumococcal surface immunogenic protein A (PsipA), phosphoglycerate kinase (cell membrane protein), ABC transporter substrate-binding protein and endopeptidase O, Pneumococcal surface immunogenic protein B (PsipB) and Pneumococcal surface immunogenic protein C (PsipC).

The vaccine compositions of the present invention may also contain other, non-immunologically-specific additives, diluents and excipients. For example, in many cases, the vaccine compositions of the present invention may contain--in addition to the S. pneumoniae cell-wall and/or cell-membrane protein(s)--one or more adjuvants.

In one particularly preferred embodiment, the vaccine composition of the present invention comprises an effective amount of S. pneumoniae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the active ingredient.

In another particularly preferred embodiment of the present invention, the vaccine composition comprises an effective amount of S. pneumoniae fructose-biphosphate aldolase as the active ingredient.

In a further particularly preferred embodiment, the vaccine composition of the present invention for preventing infection of mammalian subjects by S. pneumoniae comprises an effective amount of one or more immunogenically-active fragments of the aforementioned S. pneumoniae fructose-biphosphate aldolase protein. Although many such active fragments may be generated from this protein and incorporated into the presently-disclosed composition, in an especially preferred embodiment, the active fragment used corresponds to the peptide encoded by the first 294 nucleotides of the fructose biphosphate aldolase gene (SP0605 Streptococcus pneumoniae TIGR4), herein referred to as SEQ ID no.1.

The aforementioned vaccine compositions may clearly be used for preventing infection of the mammalian subjects by S. pneumoniae. However, said vaccine composition is not restricted to this use alone. Rather it may be usefully employed to prevent infection by any infectious agent whose viability or proliferation may be inhibited by the antibodies generated by a host in response to the inoculation therein of the one or more S. pneumoniae proteins provided in said composition.

In one preferred embodiment of the vaccine composition of the present invention, the cell wall and/or cell membrane proteins are S. pneumoniae proteins that are associated with an age-related immune response.

In another preferred embodiment of the vaccine composition of the present invention, the cell wall and/or cell membrane proteins are lectins.

In another preferred embodiment of the vaccine composition of the present invention, the cell wall and/or cell membrane proteins are non-lectins.

In a further preferred embodiment of the vaccine composition of the present invention, the cell wall and/or cell membrane proteins are a mixture of lectins and non-lectins.

The present invention also encompasses the use of one or more S. pneumoniae cell wall and/or cell membrane proteins and/or immunogenically-active fragments, derivatives or modifications thereof in the preparation of a vaccine for use in the prevention of diseases and carrier states caused by said S pneumoniae, wherein said proteins are selected from the group consisting of: phosphoenolpyruvate protein phosphotransferase, phospho-mannomutase, trigger factor, elongation factor G, tetracycline resistance protein (tetO), DNA directed RNA polymerase alpha-chain, NADH oxidase, glutamyl-tRNA amidotransferase subunit A, N utilization substance protein A homolog, XAA-HIS dipeptidase, cell division protein ftsz, zinc metalloproteinase in SCAA 5' region (ORF 6), L-lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase, UDP-glucose 4-epimerase, GTP binding protein typA/BipA, GMP synthase, glutamyl-tRNA synthetase, NADP-specific glutamate dehydrogenase, Elongation factor TS, phosphoglycerate kinase (cell wall protein), pyridine nucleotide-disulfide oxido-reductase, 40S ribosomal protein S1, 6-phosphogluconate dehydrogenase, aminopeptidase C, carbomyl-phosphate synthase (large subunit), PTS system mannose-specific IIAB components, ribosomal protein S2, dihydroorotate dehydrogenase, aspartate carbamoyltransferase, elongation factor Tu, Pneumococcal surface immunogenic protein A (PsipA), phosphoglycerate kinase (cell membrane protein), ABC transporter substrate-binding protein endopeptidase O, Pneumococcal surface immunogenic protein B (PsipB) and Pneumococcal surface immunogenic protein C (PsipC).

In one particularly preferred embodiment, the protein used in the preparation of the vaccine is S. pneumoniae glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

In another particularly preferred embodiment of the present invention, the protein used in the preparation of the vaccine is S. pneumoniae fructose-biphosphate aldolase.

In a further particularly preferred embodiment of this aspect of the present invention, an immunogenically-active fragment of the aforementioned S. pneumoniae fructose-biphosphate aldolase protein is used in the preparation of the vaccine. Although many such active fragments may be generated from this protein and used in the preparation of the vaccine, in an especially preferred embodiment, the active fragment used corresponds to the peptide encoded by the first 294 nucleotides of the fructose biphosphate aldolase gene (SP0605 Streptococcus pneumoniae TIGR4), herein referred to as SEQ ID no.1.

Preferably, the cell wall and/or cell membrane proteins used in the preparation of said vaccine are S. pneumoniae proteins that are associated with an age-related immune response.

In another preferred embodiment, the cell wall and/or cell membrane proteins used in the preparation of the abovementioned vaccine are lectins.

In yet another preferred embodiment the cell wall and/or cell membrane proteins used in the preparation of the abovementioned vaccine are non-lectins.

In a further preferred embodiment, the cell wall and/or cell membrane proteins used in the preparation of the abovementioned vaccine are a mixture of lectins and non-lectins.

The present invention is also directed to one or more S. pneumoniae cell wall and/or cell membrane proteins and/or immunogenically-active fragments, derivatives or modifications thereof for use as a vaccine for the prevention of diseases and carrier states caused by said S pneumoniae, wherein said proteins are selected from the group consisting of: phosphoenolpyruvate protein phosphotransferase, phospho-mannomutase, trigger factor, elongation factor G, tetracycline resistance protein (tetO), DNA directed RNA polymerase alpha-chain, NADH oxidase, glutamyl-tRNA amidotransferase subunit A, N utilization substance protein A homolog, XAA-HIS dipeptidase, cell division protein ftsz, zinc metalloproteinase in SCAA 5' region (ORF 6), L-lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase, UDP-glucose 4-epimerase, GTP binding protein typA/BipA, GMP synthase, glutamyl-tRNA synthetase, NADP-specific glutamate dehydrogenase, Elongation factor TS, phosphoglycerate kinase (cell wall protein), pyridine nucleotide-disulfide oxido-reductase, 40S ribosomal protein S1, 6-phosphogluconate dehydrogenase, aminopeptidase C, carbomyl-phosphate synthase (large subunit), PTS system mannose-specific IIAB components, ribosomal protein S2, dihydroorotate dehydrogenase, aspartate carbamoyltransferase, elongation factor Tu, Pneumococcal surface immunogenic protein A (PsipA), phosphoglycerate kinase (cell membrane protein), ABC transporter substrate-binding protein, endopeptidase O, Pneumococcal surface immunogenic protein B (PsipB) and Pneumococcal surface immunogenic protein C (PsipC).

In one particularly preferred embodiment, the protein for use as said vaccine is S. pneumoniae glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

In another particularly preferred embodiment of the present invention, the protein for use as said vaccine is S. pneumoniae fructose-biphosphate aldolase.

In a further particularly preferred embodiment of this aspect of the present invention, an immunogenically-active fragment of the aforementioned S. pneumoniae fructose-biphosphate aldolase protein is provided for use as said vaccine. Although many such active fragments may be generated from this protein and used in the preparation of the vaccine, in an especially preferred embodiment, the active fragment used corresponds to the peptide encoded by the first 294 nucleotides of the fructose biphosphate aldolase gene (SP0605 Streptococcus pneumoniae TIGR4), herein referred to as SEQ ID no.1.

In one preferred embodiment, the cell wall and/or cell membrane proteins for use as described above are S. pneumoniae proteins associated with an age-related immune response.

In another preferred embodiment, the aforementioned cell wall and/or cell membrane proteins are lectins.

In a further preferred embodiment, the aforementioned cell wall and/or cell membrane proteins are non-lectins.

In a still further preferred embodiment, the aforementioned cell wall and/or cell membrane proteins are a mixture of lectins and non-lectins.

It is to be noted that when the S. pneumoniae proteins and/or fragments, derivatives or modifications thereof used in the aforementioned methods, compositions and vaccines are lectins, said methods, compositions and vaccines are particularly efficacious in the prevention of localized S. pneumoniae infections. In one preferred embodiment, the localized infections are infections of mucosal tissue, particularly of nasal and other respiratory mucosa.

Conversely, when the S. pneumoniae proteins and/or their fragments, derivatives or modifications used in the aforementioned methods, compositions and vaccines are non-lectins, said methods, compositions and vaccines are particularly efficacious in the prevention of intra-peritoneal and systemic S. pneumoniae infections.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Vaccination protects individuals (and by extension, populations) from the harmful effects of pathogenic agents, such as bacteria, by inducing a specific immunological response to said pathogenic agents in the vaccinated subject.

Vaccines are generally, but not exclusively, administered by means of injection, generally by way of the intramuscular, intradermal or subcutaneous routes. Some vaccines may also be administered by the intravenous, intraperitoneal, nasal or oral routes.

The S. pneumoniae-protein containing preparations of the invention can be administered as either single or multiple doses of an effective amount of said protein. The term "effective amount" is used herein to indicate that the vaccine is administered in an amount sufficient to induce or boost a specific immune response, such that measurable amounts (or an increase in the measurable amounts) of one or more antibodies directed against the S. pneumoniae proteins used may be detected in the serum or plasma of the vaccinated subject. The precise weight of protein or proteins that constitutes an "effective amount" will depend upon many factors including the age, weight and physical condition of the subject to be vaccinated. The precise quantity also depends upon the capacity of the subject's immune system to produce antibodies, and the degree of protection desired. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. However, for the purposes of the present invention, effective amounts of the compositions of the invention can vary from 0.01-1,000 .mu.g/ml per dose, more preferably 0.1-500 .mu.g/ml per dose, wherein the usual dose size is 1 ml.

The vaccines of the present invention will generally comprise an effective amount of one or more S. pneumoniae proteins as the active component, suspended in an appropriate vehicle. In the case of intranasal formulations, for example, said formulations may include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline may also be added. The nasal formulations may also contain preservatives including, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa. An additional mode of antigen delivery may include an encapsulation technique, which involves complex coacervation of gelatin and chondroitin sulfate (Azhari R, Leong K W. 1991. Complex coacervation of chondroitin sulfate and gelatin and its use for encapsulation and slow release of a model protein. Proc. Symp. Control. Rel. 18:617; Brown K E, Leong K, Huang C H, Dalal R, Green G D, Haimes H B, Jimenez P A, Bathon J. 1998. Gelatin/chondroitin 6-sulfate microspheres for delivery of therapeutic proteins to the joint. Arthritis Rheum 41:2185-2195).

The present invention also encompasses within its scope the preparation and use of DNA vaccines. Vaccination methods and compositions of this type are well known in the art and are comprehensively described in many different articles, monographs and books (see, for example, chapter 11 of "Molecular Biotechnology: principles and applications of recombinant DNA" eds. B. R. Glick & J. J. Pasternak, ASM Press, Washington, D.C., 2.sup.nd edition, 1998). In principle, DNA vaccination is achieved by cloning the cDNAs for the desired immunogen into a suitable DNA vaccine vector, such as the pVAC vector (Invivogen). In the case of pVAC, the desired immunogenic proteins are targeted and anchored to the cell surface by cloning the gene of interest in frame between the IL2 signal sequence and the C-terminal transmembrane anchoring domain of human placental alkaline phosphatase. Such DNA vaccine vectors are specifically designed to stimulate humoral immune responses by intramuscular injection. The antigenic peptide produced on the surface of muscle cells is taken up by antigen presenting cells (APCs) and processed through the major histocompatibility complex (MHC) class II pathway.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be presented dry in tablet form or a product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservative.

However, in general, the vaccines of the present invention would normally be administered parenterally, by the intramuscular, intravenous, intradermal or subcutaneous routes, either by injection or by a rapid infusion method. Compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Besides the abovementioned inert diluents and solvents, the vaccine compositions of the invention can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

The aforementioned adjuvants are substances that can be used to augment a specific immune response. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the subject being vaccinated. Adjuvants that may be usefully employed in the preparation of vaccines include: oil adjuvants (for example, Freund's complete and incomplete adjuvants, that will be used in animal experiments only and is forbidden from use in humans), mineral salts, alum, silica, kaolin, and carbon, polynucleotides and certain natural substances of microbial origin. An additional mode of antigen delivery may include an encapsulation technique, which involves complex coacervation of gelatin and chondroitin sulfate (Azhari R, Leong K W. 1991. Complex coacervation of chondroitin sulfate and gelatin and its use for encapsulation and slow release of a model protein. Proc. Symp. Control. Rel. 18:617; Brown K E, Leong K, Huang C H, Dalal R, Green G D, Haimes H B, Jimenez P A, Bathon J. 1998. Gelatin/chondroitin 6-sulfate microspheres for delivery of therapeutic proteins to the joint. Arthritis Rheum 41:2185-2195).

Further examples of materials and methods useful in the preparation of vaccine compositions are well known to those skilled in the art. In addition, further details may be gleaned from Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton, Pa., USA (1980).

The S. pneumoniae cell-wall and/or cell-membrane proteins for use in working the present invention may be obtained by directly purifying said proteins from cultures of S. pneumoniae by any of the standard techniques used to prepare and purify cell-surface proteins. Suitable methods are described in many biochemistry text-books, review articles and laboratory guides, including inter alia "Protein Structure: a practical approach" ed. T. E. Creighton, IRL Press, Oxford, UK (1989).

However, it is to be noted that such an approach suffers many practical limitations that present obstacles for producing commercially-viable quantities of the desired proteins. The S. pneumoniae proteins of the present invention may therefore be more conveniently prepared by means of recombinant biotechnological means, whereby the gene for the S. pneumoniae protein of interest is isolated and inserted into an appropriate expression vector system (such as a plasmid or phage), which is then introduced into a host cell that will permit large-scale production of said protein by means of, for example, overexpression.

As a first stage, the location of the genes of interest within the S. pneumoniae genome may be determined by reference to a complete-genome database such as the TIGR database that is maintained by the Institute for Genomic Research (web site: http colon double slash www dot tigr dot org slash). The selected sequence may, where appropriate, be isolated directly by the use of appropriate restriction endonucleases, or more effectively by means of PCR amplification. Suitable techniques are described in, for example, U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, as well as in Innis et al. eds., PCR Protocols: A guide to method and applications.

Following amplification and/or restriction endonuclease digestion, the desired gene or gene fragment is ligated either initially into a cloning vector, or directly into an expression vector that is appropriate for the chosen host cell type. In the case of the S. pneumoniae proteins, Escherichia coli is the most useful expression host. However, many other cell types may be also be usefully employed including other bacteria, yeast cells, insect cells and mammalian cell systems.

High-level expression of the desired protein within the host cell may be achieved in several different ways (depending on the chosen expression vector) including expression as a fusion protein (e.g. with factor Xa or thrombin), expression as a His-tagged protein, dual vector systems, expression systems leading to incorporation of the recombinant protein inside inclusion bodies etc. The recombinant protein will then need to be isolated from the cell membrane, interior, inclusion body or (in the case of secreted proteins) the culture medium, by one of the many methods known in the art.

All of the above recombinant DNA and protein purification techniques are well known to all skilled artisans in the field, the details of said techniques being described in many standard works including "Molecular cloning: a laboratory manual" by Sambrook, J., Fritsch, E. F. & Maniatis, T., Cold Spring Harbor, N.Y., 2.sup.nd ed., 1989, which is incorporated herein by reference in its entirety.

As disclosed and explained hereinabove, each of the abovementioned embodiments of the invention may be based on the use of one or more intact, full length, cell wall and/or cell membrane proteins or, in the alternative, or in addition thereto, fragments, derivatives and modifications of said full length proteins. Fragments may be obtained by means of recombinant expression of selected regions of the cell wall protein gene(s). Alternatively, such fragments may be obtained by means of controlled, site-specific cleavage of the cell-wall proteins using one or more enzymes such as factor X, trypsin, chymotrypsin etc., as are well known in the art. Derivatives of the full length proteins or fragments thereof may be obtained by introducing non-native sequences within the DNA sequences encoding said proteins, followed by expression of said derivatized sequences. Derivatives may also be produced by conjugating non-native groups to the amino residue side chains of the cell wall proteins or protein fragments, using standard protein modification techniques. Modified cell wall proteins and protein fragments for use in the present invention may also be obtained by the use of site-directed mutagenesis techniques. Such techniques are well known in the art and are described, for example, in "Molecular cloning: a laboratory manual" by Sambrook, J., Fritsch, E. F. & Maniatis, T., Cold Spring Harbor, N.Y., 2.sup.nd ed., 1989. Of particular interest is the use of one or more of the preceding techniques to create fragments or derivatives possessing the desired epitopic sites, but lacking other domains which are responsible for adverse effects such as suppression of cellular immune responses. It is to be emphasized that all of the immediately preceding discussion of fragments, derivatives and mutants of the cell wall proteins disclosed herein are to be considered as an integral part of the present invention.
 

Claim 1 of 6 Claims

1. A vaccine composition comprising an isolated immunogenically active fragment of Streptococcus pneumoniae fructose-biphosphate aldolase, wherein the fragment consists of the amino acid sequence encoded by the first 294 nucleotides of the coding sequence of said Streptococcus pneumoniae fructose-biphosphate aldolase as set forth in SEQ ID NO: 1.

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