IMPROVED METHOD OF PRODUCTION OF PNEUMOCOCCAL SURFACE PROTEINS

Abstract: The present invention provides improved methods for preparation of immunoligical or vaccine compositions comprising PspA-like polypeptides. Specifically, the present invention provides vaccine compositions wherein PspA or PspA-like polypeptide from Rx1 or other representatives of clade 2 or family 1 have been produced by recombinant methods. Recombinant constructs are provided which express a substantially homogeneous, truncated form of immunologically active PspA-like polypeptides in host cells transformed with such constructs.

Description: IMPROVED METHOD OF PRODUCTION OF PNEUMOCOCCAL SURFACE PROTEINS BACKGROUND OF THE INVENTION Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneumonia, and a leading cause of fatal infections in the elderly and persons with underlying medical conditions, such as pulmonary disease, liver disease, alcoholism, sickle cell, cerebrospinal fluid leaks, acquired immune deficiency syndrome (AIDS), and patients undergoing immunosuppressive therapy. It is also a leading cause of morbidity in young children. Pneumococcal infections cause approximately 40,000 deaths in the U.S. yearly. The most severe pneumococcal infections involve invasive meningitis and bacteremia infections, of which there are 3,000 and 50,000 cases annually, respectively; despite the use of antibiotics and vaccines, the prevalence of pneumococcal infections has declined little over the past twenty-five years. It is generally accepted that immunity to Streptococcus pneumoniae can be mediated by specific antibodies against the polysacchande capsule of the pneumococcus. However, neonates and young children fail to make adequate immune response against most capsular polysaccharide antigens and can have repeated infections involving the same capsular serotype. Moreover, there are over ninety known capsular serotypes of S. pneumoniae, of which twenty-three account for about 95% of the disease. Pneumococcal vaccines have been developed by combining the 23 different capsular polysaccharides that are the prevalent types of human pneumococcal disease. These 23 polysaccharide types have been used in a licensed pneumococcal vaccine since 1983 (D.S. Fedson and D. M. Musher, Vaccines. S.A. Plotkin and J.E.A. Montimer, eds., 1994, pp. 517- 564). The licensed 23-valent polysaccharide vaccine has a reported efficacy of approximately 60% in preventing bacteremia caused vaccine type pneumococci in healthy adults. An alternative approach for protecting children, and also the elderly, from pneumococcal infection would be to identify protein antigens that could elicit protective immune responses. Such proteins may serve as a vaccine by themselves, may be used in conjunction with successful polysaccharide-protein conjugates, or as carriers for polysaccharides. McDaniel et al. (I), J. Exp. Med. 160:386-397, 1984, describes the production of hybridoma antibodies that recognize cell surface polypeptide(s) on S. pneumoniae and protect mice from infection by encapsulated pneumococci. On the basis of these results, a major surface protein antigen, designated "pneumococcal surface protein A", or "PspA", was identified. McDaniel et al. (II), Microbial Pathogenesis 1:519-531, 1986, performed studies to characterize PspA. Considerable diversity in the PspA molecule in different strains was found, as were differences in the epitopes recognized by different antibodies. McDaniel et al. (Ill), J. Exp. Med. 165:381-394, 1987, demonstrated that immunization of X-linked immunodeficient (XID) mice with non-encapsulated pneumococci expressing PspA, but not isogenic pneumococci lacking PspA, protects mice from subsequent fatal infection with pneumococci. McDaniel et al. (IV), Infect. Immun., 59:222-228, 1991, demonstrated that immunization of mice with a recombinant full length fragment of PspA was able to elicit protection against pneumococcal strains of capsular types 6A and 3. Analyses of the nucleotide and amino acid sequences indicate that the PspA molecule comprises three major regions. See, e.g., Briles, et al, U.S. Patent No. 5,476,929, the teachings of which are expressly incorporated by reference. The first 288 amino acids at the amino terminal end of the protein are predicted to have a strong alpha helical structure. The adjacent region of 82 amino acids (289 to 370 of PARxl PspA) has a high density of proline residues; based on similar regions in other prokaryotic proteins, this region is believed to traverse the bacterial cell wall. The remaining 201 amino acids at the carboxyl-terminal end of the molecule (371 to 571 of PARxl PspA) have a repeated amino acid sequence that binds to phosphocholine and lipoteichoic acids followed by a seventeen (17) amino acid tail (572- 588). Based on this structure, the PspA molecule is thought to associate with the inner membrane and lipoteichoic acids via the repeated region in the middle of the carboxyl- terminal end of the protein. The pro line region in the middle of the protein is thought to traverse the cell wall placing the alpha helical region on the outer surface of the S. pneumoniae cells. This model is consistent with the demonstration that the alpha helical region, which extends from the surface of the cell, contains the protective epitopes (Yother, J. et al, J. Bacteriol. 1992: 174:601-609; Yother, J. et al, J. Bacteriol. 1994:176:2976-2985; McDaniels, L.S. et al, Microbial. Pathog. 1994; 17:323-337; and Ralph, B.A., et al, Ann. N.Y. Acad. Sci. 1994; 730:361-363). In addition to the published literature specifically referred to above, the inventors, in conjunction with co-workers, have published further details concerning PspA's, as follows: 1. Abstracts of 89 th Annual Meeting of the American Society for Microbiology, p. 125, item D-257, May, 1989; 2 2.. AAbbssttrraaccttss ooff 9910 th Annual Meeting of the American Society for Microbiology, p. 98, item D-106, May 1990 3. Abstracts of 3 rd International ASM Conference on Streptococcal Genetics, p. 11, item 12, June 1990; 4. Talkington et al, Infect. Immun. 59:1285-1289, 1991; 5. Yother et al, (I), J. Bacteriol. 174:601-609, 1992; and 6. Yother et al, (II), J. Bacteriol. 174:610-618, 1992. 7. McDaniel et al, (V), Microbiol. Pathogenesis, 13:261-268, 1994. In addition to the above studies, 08/529,055, filed September 15, 1995, 08/470,626, filed June 6, 1995, 08/467, 852, filed June 6, 1995, 08/469,434, filed June 6, 1995, 08/468,718, filed June 6, 1995, 08/247,491, filed May 23, 1994, 08/214,222, filed March 17, 1994, 08/214,164, filed March 17, 1994, 08/246,636, filed May 20, 1994, and 08/319,795, filed October 7, 1994, and U.S. Patent No. 5,476,929, relate to vaccines comprising PspA and fragments thereof, methods for expressing DNA encoding PspA and fragments thereof, DNA encoding PspA and fragments thereof, the amino acid sequences of PspA and fragments thereof, compositions containing PspA and fragments thereof and methods of using such compositions. Each of these applications, as well as each document or reference cited in these applications and each document or reference provided above, is hereby incorporated herein by reference. Documents or references are also cited in the following text, either in the Reference List before the claims or in the text itself; each of these documents or references is hereby expressly incorporated herein by reference. The prior art fails to provide an immunological composition or vaccination regimen which would elicit protection against various diversified pneumococcal strains, without having to combine a large number of possibly competitive antigens within the same formulation. The present invention fulfills that need by employing recombinant DNA technology to more reproducibly prepare immunogenic polypeptides designed to confer broad protection against diverse pneumococcal strains. In the specification which follows and the drawings accompanying the same, there are utilized certain accepted abbreviations with respect to the amino acids represented thereby. The following Table 1 identifies those abbreviations: TABLE I AMINO ACID ABBREVIATIONS A=Ala-Alanine M=Met=Methionine C=Cys=Cysteine N=Asn=Asparagine D=Asp=Aspartic Acid P=Pro=Proline E-Glu=Glutamic Acid Q=Gln=Glutamine F=Phe=Phenylalanine R=Arg=Arginine G=Gly=Glycine S=Ser=Serine H=His=Histidine T=Thr=Threonine I=Ile=Isoleucine V=Val=Valine K=Lys=Lysine W=Trp=Tryptophan L=Leu=Leucine Y=Tyr=Tyrosine SUMMARY OF THE INVENTION The present invention relates to improved recombinant PspA constructs which allows for higher yield and more efficient purification of PspA-like polypeptides when expressed in prokaryotic hosts. (As used herein, the term "recombinant construct" or "construct" includes vectors and plasmid encoding, and capable of expressing, foreign genes in transformed host cells.) The wild type pspA gene from strain Rxl of S. pneumoniae contains an ATG at codon position 96, which upon expression produces two forms of PspA polypeptide, a first resulting from initiation of translation at position 1 and a second truncated form of PspA, resulting from initiation of translation at position 96. The wild-type pspA gene has been truncated and transformed into E. coli where it can produce two forms of a truncated PspA polypeptide; a first, 314 aa (50 kD) form, resulting from initiation of translation at position 1 and a second, further truncated form of PspA, resulting from initiation of translation at position 96 (i.e., aa96-314, corresponding to an approximate ms of 35 kD). Recombinants comprising the modified and truncated pspA gene, in which the ATG at position 96 was altered to no longer encode methionine, expressed only one form of PspA, the 50kD aa molecule, PARxl-MI. Rxl is a representative of one of four families of S. pneumoniae, and vaccine compositions containing PspA-like polypeptides can now be more conveniently prepared using the recombinant PspA construct of the present invention. By eliminating the second, truncated form of PspA at the genetic level, the yield of the recombinant product of interest is improved, and this product can be more readily purified following expression in recombinant hosts. The immunodominant epitopes of native PARxl are retained in the modified PspA- like polypeptide of the present invention, making this polypeptide a suitable component in immunological compositions designed to elicit an immune response to representatives of clade 2 of S. pneumoniae. Genes derived from a variety of strains of S. pneumoniae, including representatives of other families or clades (as hereinafter defined) expressing PspA or PspA-like polypeptides (including the recently described PspC polypeptides) may contain internal translational initiation sites similar to the ATG sequence at codon position 96 of the pspA gene of Rxl. Such sites, regardless of their specific codon position relative to codon position 96 of the pspA gene from strain Rxl, will likely complicate developments of homogeneous preparations of such polypeptides by recombinant methods. The skilled artisan can readily adopt the teachings of the present invention to modify such sites and thereby improve yield and ease of purification of the immunologically active products of interest. While the examples contained herein refer to modifications of recombinant pspA from Rxl, this is meant to be merely illustrative of the more general teachings of the present invention 1. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows the DNA sequence of the modified pspA gene of the present invention and the deduced amino acid sequence of the PspA-like polypeptide encoded by the modified pspA gene. DETAILED DESCRIPTION A comparison of the amino acid sequences in the C-terminal region of the alpha helix of PspAs from 24 strains of S. pneumoniae has revealed that the PspA strains can be grouped into 6 clades with greater than 75% homology, and these clades can be grouped into 4 families with greater than 50% homology. A clade is defined herein as comprising PspAs which exhibit greater than 75% sequence homology in aligned sequences of the C-terminal region of the alpha helix, and a family is defined herein as those clades which exhibit greater than or equal to 50% homology between member PspA sequences in aligned sequences of the C-terminal region of the alpha helix. PspAs of strains within the same PspA clade exhibit reciprocal cross-protection from immunization and challenge experiments. The present invention contemplates vaccine compositions comprising two or more, preferably no more than 10, and more preferably a minimum of 4 and a maximum of 6 recombinant forms of PspA-like polypeptide representing a single clade each, and a pharmaceutically acceptable carrier or diluent. In a preferred embodiment, a homogeneous form of a recombinant PspA-like polypeptide from Rxl, a member of clade 2, is the preferred representative of clade 2 and or family 1 which is optionally included in the vaccine composition of the present invention. Recombinant methods are preferred since a high yield is desired. The basic steps in the recombinant production of modified PspA include: a) construction of a synthetic or semi-synthetic DNA encoding the PspA-like polypeptide, b) integrating said DNA into an expression vector in a manner suitable for the expression of the PspA-like polypeptide either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with said expression vector, d) culturing said transformed or transfected host cell, and e) recovering and purifying the recombinantly produced PspA-like polypeptide. For recombinant expression, the sequence coding for a PspA-like polypeptide may be wholly synthetic, semi-synthetic or the result of modification of the native pspA gene. Synthetic genes, the in vitro or in vivo transcription and translation of which will result in the production of PspA-like polypeptides may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode PspA-like polypeptides. The gene encoding the PspA-like polypeptide may be created by synthetic methodology. Such methodology of synthetic gene construction is well known in the art. Brown, E.L., Belagaje, R., Ryan, M.J., and Khorana, H.G. (1979) in Methods in Enzvmology. Academic Press, N.Y., Vol. 68, pgs. 109-151, the entire teaching of which is hereby incorporated by reference. The DNA segments corresponding to the PspA gene, or fragments thereof, are generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). The synthetic pspA gene may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into expression and amplification plasmids. The choice of restriction sites are chosen so as to properly orient the sequence coding for the PspA-like polypeptide with control sequences to achieve proper in- frame reading and expression of the PspA-like polypeptide. A variety of other such cleavage sites may be incorporated depending on the particular recombinant constructs employed and may be generated by techniques well known in the art. DNA sequences designed for recombinant production of PspA are well known. For example, 08/529,055, filed September 15, 1995, 08/470,626, filed June 6, 1995, 08/467,852, filed June 6, 1995, 08/469,434, filed June 6, 1995, 08/468,718, filed June 6, 1995, 08/247,491, filed May 23, 1994, 08/214,222, filed March 17, 1994 and 08/214,164, filed March 17, 1994, 08/246,636, filed May 20, 1994, and 08/319,795, filed October 7, 1994, and U.S. Patent No. 5,476,929, relate to vaccines comprising PspA and fragments thereof, methods for expressing DNA encoding PspA and fragments thereof, DNA encoding PspA and fragments thereof, the amino acid sequences of PspA and fragments thereof, compositions containing PspA and fragments thereof and methods of using such compositions. The "polymerase chain reaction" or "PCR" refers to a procedure or technique in which amounts of a preselected piece of nucleic acid, RNA and or DNA, are amplified as described in U.S. Patent No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51, 263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). PCR can also be used to conveniently introduce any desired sequence change genes of interest. See generally, Ausubel et al, eds, Current Protocols in Molecular Biology. s8.5.1 (John Wiley & Sons, 1995). Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to form the plasmids required. To effect the translation of the desired PspA-like polypeptide sequence, one inserts the engineered DNA sequence coding for the PspA-like polypeptide in any of a plethora of appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases. A synthetic version of the DNA coding sequence is designed to posses restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification plasmids. The coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence into the desired cloning vectors by techniques well known in the art. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The choice of restriction sites are chosen so as to properly orient the DNA coding sequence with control sequences to achieve proper in- frame reading and expression of the PspA-like polypeptide. In general, plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell are used with these hosts. The vector ordinarily carries a replication site as well as marker sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar, et al, Gene 2:95 [1977]), pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA construction. The DNA sequence coding for the PspA-like polypeptide must be positioned so as to be in proper reading frame with the promoter and ribosome binding site of the expression vector, both of which are functional in the host cell in which the DNA coding sequence for the PspA-like polypeptide is to be expressed. In the preferred practice of the invention, the promoter-operator region is placed in the same sequential orientation with respect to the ATG start codon of DNA sequence encoding the PspA-like polypeptide as the promoter-operator occupies with respect to the ATG-start codon of the gene from which it was derived. Synthetic or modified promoter-operator regions such as the tac promoter are well known in the art. When employing such synthetic or modified promoter-operator regions they should be oriented with respect to the ATG start codon of the DNA sequence coding for the PspA- like polypeptide as directed by their creators. The present invention relates to the alteration of a nucleotide sequence such that internal start sites within the nucleotide sequence are substituted, deleted or otherwise altered to eliminate the internal ATG start sites. Elimination of the internal start sites enables the expression of a single polypeptide rather than multiple polypeptides from a single nucleotide sequence. The single polypeptide expressed can be a fusion protein, can contain a signal sequence or a truncated form of the native polypeptide. The DNA sequence for a truncated form of the PspA-like polypeptide of strain Rxl contains at least one such internal ATG sequence. This sequence appears at codon position 127 of the deduced sequence of this PspA-like polypeptide. See Briles, U.S. Patent No. 5,476,929 Figure 3a. Following cleavage of a "leader sequence" corresponding to 31 amino acids at the amino-terminus of the PspA-like polypeptide, a "mature" sequence, beginning with the codon Glu, corresponds to the expressed polypeptide of interest. An internal start site appears at codon position 96 counting from the first amino acid, Glu, of the native pspA polypeptide after cleavage of the leader sequence. (In figure 1, the ATG appears at codon 97 because the truncated pspA molecule represented in that figure has an additional amino acid at the N terminus which is an artifact of expression in a prokaryotic host). In a preferred embodiment of the present invention the internal start site within the pspA gene or truncated forms of the pspA gene that include an internal ATG start site are altered such that when the gene or its truncated forms are expressed, a single modified pspA polypeptide or truncated modified pspA polypeptide is produced. For example, the internal ATG start site could be deleted such that codons 95 and 97 (coding for lys and ile respectively) would be adjacent after deletion. Alternatively, multiple codons surrounding and including the internal start site could be deleted. For example, the codons 92-100 could be deleted such that in the resultant pspA gene the codons 91 and 101 would then be adjacent. The deletion should preferably not affect the three dimensional structure of the molecule such that important immunologic epitopes are altered. In a preferred embodiment of the present invention codons 95-97 are deleted from the pspA gene. In a more preferred embodiment codon 96 is deleted. Alternatively, the internal ATG start codon could be substituted by a different codon that is not recognized by polymerases as a start site for transcription initiation. Therefore any of the 60 codons that are not ATG or stop codons (TGA, TAA, TAG) could be used to replace the ATG internal start site. Therefore the polypeptide produced from such a recombinant DNA molecule could be expressed to produce a recombinant form of the polypeptide wherein the internal met encoded by ATG is replaced with one of nineteen other naturally occurring amino acids. Preferred embodiments include codons that code for amino acids that share similar size and charge properties as methionine, such as isoleucine, valine, leucine, and phenylalanine. In general, prokaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention. For example, E. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains which may be used include E. coli B and E. coli XI 776 (ATCC No. 31537), E. coli W3110 (phototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with prokaryotic hosts include the β-lactamase (vector pGX2907 [ATCC 39344] contains the replicon and b-lactamase gene) and lactose promoter systems (Chang et al, [1978] Nature 275:615; and Goeddel et al, [1979] Nature 281:544), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpΕ fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282). However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding PspA-like polypeptides using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding the PspA-like polypeptide. These examples are illustrative rather than limiting. While the discussion above and the examples provided herein refer to prokaryotic expression, those having skill in the art can readily appreciate that the PspA-like polypeptides of the instant invention may also be recombinantly produced in eukaryotic expression systems. Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes. 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. The techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, et al (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or Current Protocols in Molecular Biology (1989) and supplements. The PspA-like polypeptide of the present invention may be made either by direct expression or as fusion protein comprising the PspA-like polypeptide followed by enzymatic or chemical cleavage. It is often observed in the production of certain peptides in recombinant systems that expression as a fusion protein prolongs the lifespan and/or increases the yield of the desired peptide. A variety of peptidases (e.g. trypsin) which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi- synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., Carter P. Site Specific Proteolysis of Fusion Proteins, Ch. 13 in Protein Purification: From Molecular Mechanisms to Large Scale Processes. American Chemical Soc, Washington, D.C. (1990). The present invention provides an immunogenic, immunological or vaccine composition containing recombinant polypeptides derived from pneumococcal strain(s), and a pharmaceutically acceptable carrier or diluent. An immunological composition containing the PspA-like polypeptides elicits an immunological response - local or systemic. The response can, but need not be, protective. An immunogenic composition containing the PspA-like polypeptides likewise elicits a local or systemic immunological response which can, but need not be, protective. A vaccine composition elicits a local or systemic protective response. Accordingly, the terms "immunological composition" and "immunogenic composition" include a "vaccine composition" (as the two former terms can be protective compositions). The invention therefore also provides a method of inducing an immunological response in a host mammal comprising administering to the host an immunogenic, immunological or vaccine composition comprising a recombinant PspA-like polypeptide and a pharmaceutically acceptable carrier or diluent. The determination of the amount of antigen, e.g., PspA. PspA-like polypeptide or truncated portion thereof and optional adjuvant in the inventive compositions and the preparation of those compositions can be in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary arts. In particular, the amount of antigen and adjuvant in the inventive compositions and the dosages administered are determined by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as the particular antigen, the adjuvant (if present), the age, sex, weight, species and condition of the particular patient, and the route of administration. For instance, dosages of particular PspA-like polypeptide antigens for suitable hosts in which an immunological response is desired, can be readily ascertained by those skilled in the art from this disclosure, as is the amount of any adjuvant typically administered herewith. Thus, the skilled artisan can readily determine the amount of antigen and optional adjuvant in compositions and to be administered in methods of the invention. Typically, an adjuvant is commonly used as 0.001 to 50wt% solution in phosphate buffered saline, and the antigen is present on the order of micrograms to milligrams, such as about 0.0001 to about 5 wt%, preferably about 0.0001 to about 1 wt%, most preferably about 0.0001 to about 0.05 wt% (see, e.g., Examples below or in applications cited herein). Typically, however, the antigen is present in an amount on the order of micrograms to milligrams, or, about 0.001 to about 20 wt%, preferably about 0.01 to about 10 wt%, and most preferably about 0.05 to about 5 wt%. Of course, for any composition to be administered to an animal or a human, including the components thereof, and for any particular method of administration, it is preferred to determine therefor: toxicity, such as by determining the lethal dose (LD) and LD 50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response, such as by titrations of sera and analysis thereof for antibodies or antigens, e.g., by ELISA and or RFFIT analysis. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation. Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert with respect to the PspA-like polypeptide antigen and optional adjuvant. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments, from this disclosure and the documents cited herein. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17 th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations of PspA polypeptides of the present invention in a form suitable for administration to animals or humans, without undue experimentation. A pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions. Compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient or animal, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals can be determined without undue experimentation by the skilled artisan, from this disclosure and from the documents cited herein. Suitable regimes for initial administration and booster doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure and the documents cited herein. Methods of treating or preventing infection by immunization of susceptible or infected subjects with PspA-like polypeptides have been described above. Such methods induce "active" protection in which the subject, following immunization, mounts an immune response to PspA-like polypeptides which is capable of hastening recovery from, or preventing disease caused by infection with S. pheumoniae. It is also possible to treat infected subjects "passively". Passive protection entails the production of antibodies in one host, followed by administration of those antibodies to a second, infected host. In the context of S. pheumoniae infection, for example, the PspA-like polypeptides of the present invention can be used to raise antibodies to PspA. Such antibodies could then be administered to infected individuals to hasten recovery from infection. The following Examples are provided for illustration and are not to be considered a limitation of the invention. EXAMPLES Example 1 : Construction of PARxl -MI expression vector pPARxl-MI (A) Generation of recombinant host cells containing the PARXl-MI coding sequence pPARxl-MI is a recombinant construct encoding the N-terminal region (aal-314) of the PspA polypeptide of Rxl. This sequence was engineered to change aa96 (ATG-Met) to ATA-Ile using mutagenesis by the polymerase chain reaction (PCR) technique. Two PCR reactions were performed to introduce the sequence change, each using pKSD1014 plasmid DNA containing Rxl PspA as the template. The first reaction used a pair of Rxl specific oligonucleotide primers (5 '-CCCTAGCATCTGC AT ATGGAAGAATCTCCCGT-AGCC-3' SEQ ID NO.: 1 and 5'-AGCTTCATCTATTATCTTATCTGCTGCGTC-3' SEQ ID NO: 2) to prepare a sequence corresponding to aal-100 of Rxl PspA. The second reaction used a pair of Rxl specific oligonucleotide primers (5'-GACGCAGCAGATAAGATAATAGATGA- AGCT-3' SEQ ID NO: 3 and 5'-CGCATGGATCCTTAAGGAGCCGGCGCTGGTTTTGG- 3' SEQ ID NO: 4) to prepare a sequence corresponding to aa91 to 314 of Rxl PspA. Restriction sites for Ndel and BamHl, respectively, are highlighted in bold. The products of these two PCR reactions were then purified and used as templates to construct the full length (0.95 kb) sequence encoding a modified form of aal-314 of Rxl-MI in which He has been substituted for Met at codon position 96. This third PCR reaction used two specific oligonucleotide primers (5'-CCCTAGCATCTGCATATGGAAG- AATCTCCCGTAGCC-3' SEQ ID NO: 1 and 5'-CGCATGGATCCTTAAGGAGCCGGCG- -CTGGTTTTGG-3 " SEQ ID NO: 4. This construct was generated such that 5' and 3' ends (with respect to the PspA sense sequence) were bracketed with Ndel and BamHl restriction site sequences (noted in bold in the sequences above). A stop codon was introduced immediately upstream from the BamHl restriction site. The product was purified by agarose gel electrophoresis and digested with Ndel and BamHl restriction enzymes. The restricted DNA was purified and ligated with Ndel-BamHl digested pET-9a (Novagen). The ligated DNA was transformed into Subcloning Efficiency DH5 α competent cells (Gibco BRL) and plated for isolated colonies. Plasmid DNA was recovered from isolated colonies by conventional methods and analyzed to confirm the presence of the construct of interest. A large-scale plasmid purification was performed from one such colony and the construct sequenced using the dideoxy sequencing method. This plasmid, designated pPARxl-MI, contained a gene fragment identical to the amino-terminal 314 aa of the wild type-Rxl sequence except for an ATG to ATA change at amino acid position 96. Example 2: Expression and characterization of PARxl-MI. pPjARxl-MI was transformed into BL21 (DE3) competent cells (Novagen). Transformed cells were grown to mid-exponential phase and induced with 0.5mM IPTG (Sigma, Inc.). Cells were harvested approximately 2 hours after induction by centrifugation. Cell pellets were resuspended in a PBS-lysozyme lysis buffer (0.5 mg/ml lysozyme in PBS). The cells were lysed by freeze thaw and the lysate was centrifuged. The supernatant was decanted and adjusted to 70% ammonium sulfate (J.T. Baker) to precipitate proteins. This solution was centrifuged and the supernatant was discarded. The pellet was resuspended in 30% ammonium sulfate and centrifuged. The supernatant was extensively dialyzed with a buffer consisting of lOOmM sodium acetate, ImM EDTA, lOmM NaCl, pH 4.5. The dialysate was centrifuged and the supernatant was applied to a Q Sepharose Fast Flow (Pharmacia Biotech) column. The column flow through and wash were collected and dialyzed against 50mM Tris, 2 mM EDTA, lOmM NaCl, pH 7.5. The dialysate was centrifuged and the supernatant was applied to a Q Sepharose Fast Flow column. The column was washed with 50mM Tris, 2mM EDTA, lOOmM NaCl, pH 7.5, then PARxl-MI was eluted with 50mM Tris, 2mM EDTA, 200 mM NaCl, pH 7.5. This elute was analyzed by SDS-PAGE; proteins were visualized by Coomassie Blue staining and PspA products were identified by Western immunoblot analysis with a monoclonal anti-PspA antibody (XiR278). Two major bands, migrating at approximately 50 kD and 35 kD, respectively, were observed in lanes loaded with PARxl. In contrast, lanes conesponding to PARxl-MI contained a single major band migrating at approximately 50kD, corresponding to the aal-314 PspA molecule. The identity of this band was confirmed by Western immunoblot analysis. The immunoblot was probed with monoclonal anti-PARxl followed by phosphate-labeled goat anti-mouse IgG, then developed with BCIP/NBT. PARxl contains two bands, migrating at approximately 50 kD and 35 kD, respectively, corresponding to the aal-314 PspA molecule and the 97-314 form resulting from initiation of translation at position 97. Substitution of He for Met at this position in PARxl-MI results in a substantially homogeneous, single form of PspA migrating at approximately 50 kD. Certain physical properties of PARxl-MI were also examined and compared to those of PARxl . In particular, circular dichroism (CD) and sedimentation analyses were performed. The CD spectropolarimetric method is routinely used to examine the secondary structure or conformation of proteins. The CD technique is based upon the way optically active substances absorb right or left circularly polarized light. See, e.g., Yant, J.T., et al, (1986). Methods in Enzymol. 130:208-269 and Change, C.T., et al, Anal. Biochem. (1978) 91:13-31. The percentages of the protein secondary-structures could not be determined for the PARxl preparation because this preparation contains two different forms of the PspA molecule. Analysis of PARxl-MI revealed that the protein has 61.5±5% alpha-helix, no beta- sheet and 37.5% unordered form. These results compare favorably to the predicted structure of the aal-314 form of PARxl, indicating that the Met-Ile substitution did not result in any pronounced secondary structure differences. This finding is consistent with the results of immunological analyses, described hereinbelow, which demonstrate that no major immundominant epitopes were destroyed by the substitution, and no new immunodominant epitopes were created. The sedimentation coefficient of PARxl-MI was determined by velocity sedimentation analysis using conventional techniques. See, e.g., Ralson, G. (1993), Introduction to Analytical Ultracentrifugation, p. 1-87, Beckman Instruments, Inc., Fullerton, CA; Stafford, W.F. III. (1992) Anal. Biochem/ 203:295-301. The sedimentation coefficient calculated for PARxl-MI was 2.15S. The value obtained for PARxl was 2.13. The results of this analysis indicate that the three-dimensional structure of these molecules is comparable, again consistent with the results of the immunological analyses described hereinbelow. Example 3: Immunological characterization of PARxl-MI product (A) Analysis of PARxl and PARxl-MI by nolvclonal anti-PARxl To determine whether PARxl-MI retained all the immunodominant epitopes presented by PARxl the following experiment was performed. Competitive inhibition of anti-PARxl binding to PARxl antigen was analyzed using a BIAcore sensory chip coated with PARxl antigen. Rabbit polyclonal anti-PARxl (1200 ng/ml) was allowed to react to the chip either alone or in the presence of increasing concentration of PARxl antigen or PARxl-MI antigen. The concentration of uninhibited antibody able to bind to the PARxl antigen on the sensory chip surface was measured using mass transport measurements on the BIAcore instrument. The mouse monoclonal IgG anti-PspA antibody, p81-122F10.Al 1, was used as a standard for these measurements. Comparison of the inhibition curves generated by this analysis indicated that there was no significant difference in immunological activity of PARxl-MI relative to the wild-type PARxl antigen. Thus, the substitution of He for Met at position 96 in PARxl had no demonstrable effect on the immunodominant epitopes within PARxl . (B) Analysis of PARxl and PARxl-MI by nolvclonal anti-PARxl-MI To determine whether the amino acid substitution within PARxl-MI (He for Met at position 96) created any new immunodominant epitopes the following experiment was performed. Rabbit polyclonal antibody was generated following immunization with PARxl- MI. This polyclonal anti-PARxl-MI (1200 ng/ml) was allowed to react with a BIAcore sensory chip coated with PARxl-MI antigen, either alone or in the presence of increasing concentration of PARxl antigen or PARxl-MI antigen. The concentration of uninhibited antibody able to bind to the PARxl antigen on the sensory chip surface was measured using mass transport measurements on the BIAcore instrument. As before, the mouse monoclonal IgG anti-PspA antibody P81-122F10.A11 was used as a standard control. The inhibition curves resulting from this analysis revealed no significant difference between the two recombinant PspA products. Thus, the amino acid substitution within PARxl-MI did not result in the generation of any new immunodominant epitopes not present on PARxl. Having thus described in detail certain preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above description, as many apparent variations thereof are possible without departing from the spirit or scope thereof. REFERENCES 1. Center of Disease Control. 19484. Pneumococcal polysaccharide vaccine usage, United States, MMWR 33:273-276, 281. 2. Mufson, M.A., G. Oley and D. Hughey, 1982. Pneumococcal disease in a medium- sized community in the United States. JAMA 248:1486-1489. 3. Hook, E.W., CA. Horton and D.R. Schaberg. 1983. Failure of intensive care unit support to influence mortality from pneumococcal bacteremia. JAMA 249:1055-1057. 4. Breiman, R.F., J.S. Spika, V.J. Navarro, P.M. Darden and CP. Darby. 1990. Pneumococcal bacteremia in Charleston County, South Carolina. Arch. Intern. Med. 150:1401-1405. 5. Afessa, B., W.L. Greaves and W.R. Frederick. 1995. 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Claims: WE CLAIM: 1. An isolated DNA sequence encoding a PspA molecule wherein internal, naturally occurring translational initiation sites have been modified or eliminated, such that expression of said DNA sequence results in a single form of PspA. 2. The DNA sequence of claim 1, wherein said DNA sequence was derived from strain Rxl of S. pneumoniae. 3. The DNA sequence of claim 1, wherein said DNA sequence is as set forth in SEQ. ID No.: 5. 4. A recombinant construct, comprising the DNA sequence of claim 1 operably linked to an expression control sequence. 5. A unicellular host transformed with the recombinant construct of claim 1. 6. The unicellular host of claim 5, wherein said host is a prokaryote. 7. The unicellular host of claim 6, wherein said prokaryote is E. coli. 8. A method of producing PspA, comprising the steps of culturing the host of claim 7 and recovering PspA from the culture. 9. PspA produced by the method of claim 8. 10. An immunological composition comprising a therapeutically effective amount of the PspA-like polypeptide of claim 9 and a pharmaceutically acceptable carrier. 11. The immunological composition of claim 10, further comprising an adjuvant. 12. A method of treating or preventing S. pneumoniae infection in a susceptible animal, which method comprises inoculation with an immunoeffective amount of the immunological composition of claim 11. 13. The method of claim 12, wherein said animal is human. 14. The method of claim 13, wherein said human is a child. 15. A method of producing antibodies reactive with PspA, which method comprises vaccination of an animal with an immunoeffective amount of the immunological composition of claim 11. 16. Antibodies to PspA produced by the method of claim 11. 17. A method of treating S. pheumoniae infection in an infected animal, which method comprises administration of a therapeutic amount of the PspA antibodies of claim 18. The DNA sequence of claim 1 , herein the internal, naturally occurring translational initiation site at position 96 (met codon) has been replaced by a codon from the group consisting of ATA, ATC, ATT, TTG, CTG or GTG. 19. The DNA sequence of claim 18, wherein the met codon (ATG) at position 96 has been replaced by ATA (ile). 20. The PspA of claim 9, wherein said PspA is comprised of amino acids 1-115 of the mature PspA molecule and in which isoleucine has been substituted for methionine at position 96.

Inventors: BECKER ROBERT, GRAY MARY-ANN, PYLE DEREK, BECKER, ROBERT, GRAY, MARY-ANN, PYLE, DEREK
Applicants: BECKER ROBERT, GRAY MARY ANN, PASTEUR MERIEUX CONNAUGHT, PYLE DEREK, BECKER, ROBERT, GRAY, MARY-ANN, PYLE, DEREK
Application year: 1998
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N1, C12N15