AU739604B2 - Pneumococcal surface proteins, genes encoding these and expression of these or portions thereof - Google Patents

Description

S F Ref: 412594D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: UAB Research Foundation UAB Station Birmingham Alabama 35294 UNITED STATES OF AMERICA Actual Inventor(s): David E. Briles, Larry S. McDaniel, Edwin Swlatlo, Janet Yother, Marilyn J. Crain, Susan Hollingshead, Rebecca Tart and Alexis Brooks-Walter Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Invention Title: Pneumococcal Surface Proteins, Genes Encoding These and Expression of These or Portions Thereof The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 Pneumococcal Surface Proteins, Genes Encoding These and O Expression of These or Portions Thereof Field of the Invention This invention relates to pneumococcal genes, portions thereof, expression products therefrom and uses of such genes, portions and products; especially to genes of Streptococcus pneumoniae, the gene encoding pneumococcal surface protein A (PspA) (said gene being "pspA"), pspA-like genes, pneumococcal surface protein C (PspC) (said gene being "pspC"), portions of such genes, expression products therefrom, and the uses of such genes, portions thereof and expression products [n:\libc]03437:DKM therefrom. Such uses include uses of the genes and portions thereof for obtaining expression products by recombinant techniques, a well as for detecting the presence of Streptococcus pneumoniae or strains thereof by detecting DNA thereof by hybridization or amplification PCR) and hybridization techniques obtaining DNA-containing sample, contacting same with genes or fragment under PCR, amplification and/or hybridization conditions, and detecting presence of or I isolating hybrid or amplified product). The expression product uses include use in preparing antigenic, immunological or vaccine compositions, for eliciting antibodies, an immunological response (other than or additional to antibodies) or a protective response 'i (including antibody or other immunological response by administering composition to a suitable host); or, the expression product can be for use in detecting the Spresence of Streptococcus pneumoniae by detecting antibodies to Streptococcus pneumoniae protein(s) or go0 antibodies to a portion thereof in a host, by S obtaining an antibody-containing sample from a relevant host, contacting the sample with expression product and detecting binding (for instance by having the product labeled); and, the antibodies generated by the aforementioned compositions are useful in diagnostic or detection kits or assays. Thus, the invention relates to varied compositions of matter and methods for use thereof.

BACKGROUND OF THE INVENTION Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneumonia.

Despite the use of antibiotics and vaccines, the prevalence of pneumococcal infections has declined little over the last twenty-five years.

,3 It is generally accepted that immunity to Streptococcus pneumoniae can be mediated by specific antibodies against the polysaccharide capsule of the pneumococcus. However, neonates and young children fail 0 to make an immune response against polysaccharide antigens and can have repeated infections involving the same capsular serotype.

S One approach to immunizing infants against a number of encapsulated bacteria is to conjugate the capsular polysaccharide antigens to protein to make them immunogenic. This approach has been successful, for example, with Haemophilus influenzae b (see U.S. Patent o0 no. 4,496,538 to Gordon and U.S. Patent no. 4,673,574 to Anderson). However, there are over eighty known capsular serotypes of S. pneumoniae of which twenty-three account for most of the disease. For a pneumococcal polysaccharide-protein conjugate to be successful, the capsular types responsible for most pneumococcal infections would have to be made adequately immunogenic.

This approach may be difficult, because the twenty-three polysaccharides included in the presently-available vaccine are not all adequately immunogenic, even in 1o 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 .oo* conjugates, or as carriers for polysaccharides.

McDaniel et al. J. Exp. Med. 160:386-397, 1984, relates to the production of hybridoma antibodies that recognize cell surface polypeptide(s) on S.

pneumoniae and protection of mice from infection with certain strains of encapsulated pneumococci by such antibodies. This surface protein antigen has been termed "pneumococcal surface protein A" or PspA for short.

g3 McDaniel et al. Microbial Pathogenesis 1:519-531, 1986, relates to studies on the characterization of the 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. (III), J. Exp. Med. 165:381- S 394, 1987, relates to 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. Infect. Immun., 59:222- 228, 1991, relates to immunization of mice with a recombinant full length fragment of PspA that is able to elicit protection against pneumococcal strains of capsular types 6A and 3.

Crain et al, Infect.Immun., 56:3293-3299, 1990, relates to a rabbit antiserum that detects PspA in 100% 9 (n 95) of clinical and laboratory isolates of strains S of S. pneumoniae. When reacted with seven monoclonal antibodies to PspA, fifty-seven S. pneumoniae isolates exhibited thirty-one different patterns of reactivity.

The PspA protein type is independent of capsular type. It would seem that genetic mutation or exchange in the environment has allowed for the development of a large pool of strains which are highly diverse with respect to capsule, PspA, and possibly other molecules with variable structures. Variability of PspA's from different strains also is evident in their ee* molecular weights, which range from 67 to 99 kD. The observed differences are stably inherited and are not the 3O result of protein degradation.

Immunization with a partially purified PspA from a recombinant X gtll clone, elicited protection against challenge with several S. pneumoniae strains representing different capsular and PspA types, as S described in McDaniel et al. Infect. Immun. 59:222- 228, 1991. Although clones expressing PspA were constructed according to that paper, the product was insoluble and isolation from cell fragments following S lysis was not possible.

While the protein is variable in structure between different pneumococcal strains, numerous crossreactions exist between all PspA's,- suggesting that sufficient common epitopes may be present to allow a single PspA or at least a small number of PspA's to elicit protection against a large number of S. pneumoniae strains.

Ic 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 89th Annual Meeting of the American Society for Microbiology, p. 125, item D-257, May 1989; 2. Abstracts of 90th Annual Meeting of the American Society for Microbiology, p. 98, item D-106, May 1990; t 3. Abstracts of 3rd 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 J. Bacteriol. 174:601- 609;, 1992; and 6. Yother et al J. Bacteriol. 174:610- 618, 1992.

7. McDaniel et al Microbiol.

Pathogenesis, 13:261-268.

It would be useful to provide PspA or fragments thereof in compositions, including PspA's or fragments from varying strains in such compositions, to provide antigenic, immunological or vaccine compositions; and, it ;3 is even further useful to show that the various strains can be grouped or typed, thereby providing a basis for cross-reactivities of PspA's or fragments thereof, and thus providing a means for determining which strains to 28.AUG.2001 18:25 SPRUSON FERGUSON 61 2 92615486 N0.323 P.6/22 6 represent in such compositions (as well as how to test for, detect or diagnose one strain from another), Further, it would be advantageous to provide a pspA-like gene or a pspC gene in certain strains, as well as primers (oligonucleotides) for identification of such a gene, as well as of conserved regions in that gene and in pspA; for instance, for detecting, determining, isolating, or diagnosing strains of S. pneumonia. These uses and advantages, it is believed, have not heretofore been provided in the art.

Objects and Summary of the Invention Herein disclosed is an isolated amino acid molecule comprising residues 1 to 115, 1 to 260, o1 192 to 588, 192 to 299, or residues 192 to 260 of pneumococcal surface protein A of Streptococcus pneumoniae.

Also disclosed herein is an isolated DNA molecule comprising a fragment of a pneumococcal surface protein A gene of Streptococcus pneumoniae encoding the isolated amino acid molecule.

1 Also disclosed herein are PCR primers or hybridization probes comprising the isolated DNA molecule.

Also disclosed herein is an antigenic, vaccine or immunological composition comprising the amino acid molecule.

Also disclosed herein is an isolated DNA molecule comprising nucleotides 1 to 26, 1967 to 20 1990, 161 to 187, 1093 to 1117, or 1312 to 1331 or 1333 to 1355 of a pneumococcal surface protein A gene of Streptococcus pneumoniae. The DNA molecule can be used as a PCR primer or hybridization probe; and therefore the invention comprehends a PCR primer or hybridization probe comprising the isolated DNA molecule,---, Also disclosed herein is an isolated DNA molecule comprising a fragment having homology with a portion of a pneumococcal surface protein A gene of Streptococcus pneumoniae. The DNA may be one of the following (which include the portion having homology and restriction sites, and selection of other restriction sites or sequences for such DNA is within the ambit of the skilled artisan from this disclosure):

CCGGATCCAGCTCCTGCACCAAAAC;

GCGCGTCGACGGCTTAAACCCATTCACCATTGG;

CCGGATCCTGAGCCAGAGCAGTTGGCTG;

jA CCGGATCCGCTCAAAGAGATTGATGAGTCTG; and

GCGGATCCCGTAGCCAGTCAGTCTAAAGCTG;

SCTGAGTCGACTGGAGTTTCTGGAGCTGGAGC;

rr'\nI h\T m77AnAn I lAA- 28.AUG.2001 18:25 SPRUSON FERGUSON 61 2 92615486 NO.323 P.7/22 7

CCGGATCCAGCTCCAGCTCCAGAAACTCCAG;

GCGGATCCTTGACCAATATTTACGGAGGAGGC;

GTTTTTGGTGCAGGAGCTGG;

GCTATGGCTACAGGTTG;

CCACCTGTAGCCATAGC;

CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT; and

GCAAGCTTATGATATAGAAATTTGTAAC

(thus, the invention broadly comprehends DNA homologous to portions of pspA; preferably further including restriction sequences).

1 0 These DNA molecules can be used as PCR primers or probes; and thus, the invention comprehends a primer or probe comprising and of these molecules.

The invention further still provides PCR probe(s) which distinguishes between pspA and pspA-like nucleotide sequence, as well as PCR probe(s) which hybridizes to both pspA and pspA-like nucleotide sequences.

1s Additionally, herein disclosed is a PspA extract prepared by a process comprising: growing pneumococci in a first medium containing choline chloride, eluting live pneumococci with a choline chloride containing salt solution, and growing the pneumococci in a second medium containing an alkanolamine and substantially no choline; as well as a PspA extract prepared by that process and :44: further comprising purifying PspA by isolation on a choline-Sepharose affinity column. These 20 processes are also included in the invention, An immunological composition comprising these extracts is also herein disclosed, as well as an immunological composition comprising the full length PspA.

A method for enhancing the immunogenicity of a PspA-containing immunological composition comprising, in said composition, the C-terminal portion of PspA, is further disclosed, as well.

Further, there is disclosed an isolated amino acid molecule comprising pneumococcal surface protein C, PspC, of Streptococcus pneumaniae having an alpha-helical, proline rich and repeat regions, an isolated DNA molecule comprising a pneumococcal surface protein C gene encoding the aforementioned PspC, and primers and hybridisation probes consisting essentially of the isolated DNA molecule.

Also disclosed is an immunological composition comprising at least two PspAs. The latter immunological composition can have the PspAs from different groups or families; the groups or families can be based on RFLP or sequence studies (see, Fig. 13).

Still further, an isolated amino acid molecule comprising pneumococcal surface protein C, 'Rp PspC, of Streptococcus pneumoniae is disclosed, having an alpha-helical, proline rich and repeat i nvT ih\r Ir771Wtidltre A-^nn regions, having substantial homology with a protection eliciting region of PspA, and an isolated DNA molecule comprising a pneumococcal surface protein C gene encoding the aforementioned PspC, and primers and hybridisation probes consisting essentially of the isolated DNA molecule are disclosed by the present invention.

Additionally, there are disclosed immunological compositions comprising PspC.

According to a first embodiment of the invention, there is provided an isolated DNA molecule comprising a pspA-like nucleotide sequence which includes regions which are substantially homologous with at least the leader, 5' portion of the proline-rich-region-encoding and repeat region-encoding portions of the pspA gene from S. pneumoniae, or a fragment of said sequence capable of acting as a probe or PCR primer in respect of said sequence, wherein said sequence or fragment thereof does not hybridise under conditions of high stringency with at least any one of the following: the a-helical region-encoding sequences of the pspA gene from S. pneumoniae; 5 the C-terminus region-encoding sequences of the pspA gene from S. pneumoniae; or the 3' portion of the proline-rich-encoding sequences of the pspA gene from S. pneumoniae.

0 According to a second embodiment of the invention, there is provided an isolated DNA 20 molecule according to the first embodiment which does not hybridise under conditions of high stringency with the following sequences which do hybridise with the pspA nucleotide sequence under conditions of high stringency:

CCGGATCCAGCTCCTGCACCAAAAAC;

GCGCGTCGACGGCTTAAACCCATTCACCATTGG;

CCGGATCCTGAGCCAGAGCAGTTGGCTG;

CCGGATCCGCTCAAAGAGATTGATGAGTCTG; and

GCGGATCCCGTAGCCAGTCAGTCTAAAGCTG.

According to a third embodiment of the invention, there is provided an isolated DNA molecule according to the first or second embodiments which does hybridise under conditions of high stringency with the following sequences which also hybridise with the pspA nucleotide sequence under conditions of high stringency:

CCGGATCCAGCTCCAGCTCCAGAAACTCCAG;

GCTATGGGCTACAGGTTG; and

CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT.

[1:\DayLib\LIBZZ]06041 spec.doc:gcc 2e.AUG.2001 18:26 SPRUSON FERGUSON 61 2 92615486 NO.323 P.9/22 8a According to a fourth embodiment of the invention, there is provided an isolated DNA molecule according to any one of the preceding embodiments, which can be amplified by the following PCR primer pairs of oligonucleotides: CCGGATCCAGCTCCTGCACCAAAAAC and

CCGGATCCAGCTCCAGCTCCAGAAACTCCAG;

CCGGATCCAGCTCCTGCACCAAAAAC and GCTATGGGCTACAGGTTG; or CCGGATCCAGCTCCTGCACCAAAAC and

CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT.

o According to a fifth embodiment of the invention, there is provided a PCR primer for :amplifying a pspA-like nucleotide sequence, consisting essentially of an isolated DNA molecule of any one of the preceding embodiments.

According to a sixth embodiment of the invention, there is provided a PCR primer according to the fifth embodiment, which is selected from the following sequences: 1 5

CCGGATCCAGCTCCTGCACCAAAAAC;

CCGGATCCAGCTCCAGCTCCAGAAACTCCAG.

GCTATGGGCTACAGGTTG; or

CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT

According to a seventh embodiment of the invention, there is provided a PCR amplification 20 product from a PCR primer according to the fifth or sixth embodiments.

According to an eighth embodiment of the invention, there is provided an isolated polypeptide comprising a PspA-like polypeptide, encoded by a nucleotide sequence according to ite invention, or a PCR amplification product according to the seventh embodiment.

According to a ninth embodiment of the invention, there is provided an immunological composition comprising a polypeptide according to the eighth embodiment.

According to a tenth embodiment of the invention, there is provided a method for immunising a patient against infection with Streptococcus pneumonias, comprising administering to said patient an isolated polypeptide, a PspA-like extract, or an immunological composition according to the invention. Also provided is: an isolated polypeptide, PspA-like extract, or an immunological composition according to the invention, when used for immunising a patient against infection with Streptococcus pneumoniae; and the use of an isolated polypeptide, a PspA-like extract, or an immunological composition according to the invention for the manufacture of a medicament for immunising a patient against infection with Streplococcus pneumoniae, r.T l.lt*I hi rn 28.FlUG.2001 18:26 SPRUSON FERGUSON 61 2 92615486 NO-323 P.10/22 8b According to an eleventh embodiment of the invention, there is provided an antibody specific for a PspA-like polypeptide, or an epitope thereof, wherein said antibody does not bind to PspA from Streptococcus pneumoniae, or to the a-helical, C-terminus, or the C-terminal portion of the proline-rich regions thereof.

s According to a twelfth embodiment of the invention, there is provided a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable carrier.

According to a thirteenth embodiment of the invention, there is provided a method for immunising a patient against infection with Streptococcus pneumoniae, comprising administering to t o said patient an antibody according to the invention or a pharmaceutical composition comprising it.

Also provided is: an antibody according to the invention or a pharmaceutical composition omprising it, when used to immunise a patient against infection with Streptococcus pneumoniae; S. 9 and the use of an antibody according to the invention for the manufacture of a medicament for S* immunising a patient against infection with Streptococcus pneumoniae, 15 According to a fourteenth embodiment of the invention, there is provided a method for diagnosing the presence of Streptococcus pneumoniae comprising the use of a detection agent selected from: a DNA molecule; a PCR primer a probe; an isolated polypeptide; or an antibody according to the invention. Also provided is: a detection agent selected from: a DNA molecule; a PCR primer a probe; an isolated polypeptide; or an antibody according to the invention; when 20 used for diagnosing the presence of Streptococcus pneumoniae; and the use of a detection agent selected from: a DNA molecule; a PCR primer; a probe; an isolated polypeptide; or an antibody according to the invention; for the manufacture of a composition for diagnosing the presence of Streptcoccus pneumoniae.

According to a fifteenth embodiment of the invention, there is provided a method for detecting pspA-like nucleotide sequences andlor distinguishing between pspA and pspA-like nucleotide sequences, said method comprising: probing a portion of a sample suspected of including one or both of said sequences with at least one probe according to the third embodiment; and probing another portion of said sample with at least one probe according to the second embodiment; whereby any sequences detected by step but not by step are pspA-like nucleotide sequences, and any sequences detected by steps and are pspA nucleotide sequences.

There is also provided: a probe according to the third embodiment, when used for detecting pspA- A 'A Z like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide frhAvT ThV .R771fAf41 trr -in r'wr 28.AUG.2001 18:27 SPRUSON FERGUSON 61 2 92615486 NO.323 P.11/22 8c sequences; a probe according to the second embodiment, when used for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences; at least one probe according to the third embodiment, and at least one probe according to the second embodiment, when used for detecting pspA-like nucleotide sequences and/or distinguishing s between pspA and pspA-like nucleotide sequences; and the use of at least one probe according to the third embodiment, and at least one probe according to the second embodiment, for the manufacture of a diagnostic assay for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences, ;According to a sixteenth embodiment of the invention, there is provided a method for 10o detecting pspA-like nucleotide sequences comprising amplifying DNA in a sample by PCR, using a S. PCR primer according to the invention. Also provided is: a PCR primer according to the invention, when used for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences; and the use of a PCR primer according to the invention for the manufacture of a diagnostic assay for detecting pspA-like nucleotide sequences and/or s distinguishing between pspA and pspA-like nucleotide sequences.

PspA-like nucleotide sequences and pneumococcal strains detected by the methods according to the fifteenth or sixteenth embodiments are also provided.

These and other embodiments are disclosed or are obvious from the following detailed description, 9 20 Brief Description of the Figures Figures 1A and 1B show: Evaluation of digested plasmid constructs. Fig. 1A: 1% agarose gel electrophoresis of plasmids isolated from transformed E o, %1JIGIt d IWs L-U II LIaIIaII IIU iE.

coli BL21(DE3) strains stained with ethidium bromide.

Lane 1: 1 kb DNA ladder (sizes noted in kb), lane 2: pRCT125; lane 3: pRC105, lane 4: DBL5 PsyA insert, lane pRCT113, lane 6: BG9739 pspA insert, lane 7: pRCT117, S and lane 8: L81905 DPDA insert. Fig. IB: Corresponding Southern blot of gel in Fig. 1A probed with full-length Rxl PspA and hybridization detected as described in Example 1. The arrow indicates the 1.2 kb pspA digested inserts from plasmid constructs and the PCR-amplified 'o psPA fragments from the pneumococcal donor strains used in cloning.

Figure 2 shows: Evaluation of strain RCT105 cell fractions containinQ truncated DBL5 PspA. Proteins from E. coli cell fractions were resolved by 10% SDS- PAGE, transferred to NC, and probed with MAb XiR278.

Lane 1: molecular weight markers (noted in kDa), lane 2: full-length, native DBL5 PspA, lane 3: uninduced cells, lanes 4-6: induced cells; 1 hr, 2 hr, and 3 hr of IPTG induction respectively, lane 7: periplasmic proteins, lane 8: cytoplasmic proteins, and lane 9: insoluble cell wall/membrane material.

Figure 3 shows: SDS-PAGE of R36A PspA (80 ng) column isolated from CDM-ET and an equal volume of an equivalent WG44.1 prep. Identical gels are shown stained with Bio-Rad silver kit or immunoblotted with PspA MAb XiR278(B). The PspA isolated from R36A shows the characteristic monomer (84 kDa) and dimer bands.

Figure 4 shows: Cell lysates of pneumococcal isolates MC27 and MC28 were subjected to SDS-PAGE and So transferred to nitrocellulose for Western blotting with seven MAb to PspA. 7D2 detected a protein of 82 kDa in each isolate and XiR278 and 2A4 detected a protein of 190 kDa in each isolate. MAb Xi64, Xi126, 1A4 and SR4W4 were not reactive. Strains MC25 and MC26 yielded identical results.

Figure 5 (Figs. 5A and 5B) shows: Southern blot of Hind III digest of MC25-MC28 chromosomal

DNA

developed at a stringency greater than 95 percent.

A

digest of Rxl DNA was used as a comparison. The blot was probed with LSMpspA13/2, a full length Rxl probe (Fig. and LSMpspA12/6 a 5' probe of Rx1 pspA (Fig. The S same concentration of Rxl DNA was used in both panels, but the concentrations of MC25-MC28 DNA in Fig. SB were half that used in Fig. 5A to avoid detection of partial digests.

Figure 6 shows: RFLP of amplified pspA. PspA ic from MC25 was amplified by PCR using 5' and 3' primers for pspA (LSM13 and LSM, respectively). The amplified DNA was digested with individual restriction endonucleases prior to electrophoresis and staining with ethidium bromide. Lane 1 BclI, Lane 2 BAMHI, Lane 3 BstNI, Lane 4 PstI, Lane 5 SacI, Lane 6 EcoRI, Lane 7 aSmaI, Lane 8 KpnI.

Figure 7 shows: A depiction of PspA showing the relative location and orientation of the oligonucleotides.

Figure 8 shows: Derivatives of the S.

pneumoniae D39-Rxl family.

Figures 9 to 10 show: Electrophoresis of pspA or amplified pspA product with HhaI (Fig. Sau3AI (Fig. Figure 11 shows: RFLP pattern of two isolates from six families.

Figure 12 shows: RFLP pattern of two isolates from six families (using products from amplification with SKH2 and LSM13).

o0 Figure 13 shows: Sequence primarily in the Nterminal half of PspA.

Figure 14 shows: Cell lysates of pneumococcal isolates MC27 and MC28, subjected to SDS-PAGE and Western blotting with seven MAbs to PspA; 7D2 detected a protein of 82 kDa in each isolate, and Xi278 and 2A4 detected a protein of 190 kDa in each isolate; MAbs Xi64, Xi126, 1A4 and SR4W4 were not reactive; strains MC25 and MC26 yielded identical results (not shown).

Figure 15A and 5B show: a Southern blot of Hind III digest of MC25-28 chromosomal DNA, using a digest of Rxl DNA as a comparison; the blot was probed with LSMpspA13/2, a full length Rxl probe and LSMpspAl2/6, a 5' probe of Rxl pspA the same concentration of Rxl DNA was used in both panels, but the concentrations of MC25-28 DNA in B were half that used in '0 A to avoid detection of partial digests.

Figures 15C and 15D show: the nucleotide sequences of primers LSM13, LSM2, LSM12 and LSM6, and that of probes LSMpspA13/2 and LSMpspAl2/6.

Figure 16 shows: RFLP of amplified pspA, wherein PspA from MC25 was amplified by PCR using 5' and 3' primers for pspA (LSM13 and LSM2, respectively); the amplified DNA was digested with individual restriction endonucleases prior to electrophoresis and staining with ethidium bromide; Bcl I was used in lane 1; BamH I was o used in lane 2; BstN I was used in lane 3; Pst I was used in lane 4; Sac I was used in lane 5; EcoR I was used in lane 6; Sma I was used in lane 7; and Kpn I was used in lane 8.

Figure 17 shows: position and orientation of oligonucleotides relative to domains encoded by pspA; numbers along the bottom of the Figure represent amino acids in the mature PspA polypeptide from strain Rxl, and arrows represent the relative position (not to scale) and orientation of oligonucleotides.

Figure 18 shows: a restriction map of the pZero vector.

Figure 19 shows: the nucleotide sequences of SKH2, LSM13, N192 and C588.

Figure 20 shows: a comparison of the structural motifs of PspA and PspC; PspA has a smaller alpha-helical region, and does not contain the direct repeats within the alpha-helix (indicated by the dashed lines); the alpha-helical regions which are homologous between PspA and PspC are indicated by the dashed lines); the alphahelical regions which are homologous between PspA and PspC are indicated by the striped pattern; and PCR primers are indicated by the arrows.

Figure 21 shows: the amino acid and nucleotide sequence of PspC, wherein the putative -10 and regions are underlined, and the ribosomal binding site is in lower case.

ic Figure 22 shows: the Bestfit analysis of PspA and PspC; percent identity is 69% and percent similarity is 77%; amino acids of PspA are one the bottom line (1- 588) and amino acids of PspC are on the top line (249- 891), and a dashed line indicated identity.

Figure 23 shows: the coiled coil motif of the alpha-helix of PspC; amino acids that are not in the coiled coil motif are in the right column.

S.

5 0* S 0 *5

S

o Figure 24 shows: the repeat regions of the -c Figure 25 shows: helical and proline region Figure 26 shows: of pspA.Rxl to pspC.D39.

Figure 27 shows: 4 pspC.EF6797 and pspC.D39.

Figure 28 shows: PspC of EF6797 and D39.

Figure 29 shows: PspC.D39 and PspA.Rxl.

3o DETAILED DESCRIPTION Knowledge of and a matrix plot comparison of alpha-helical region of PspC.

the sequence of the alpha s of LXS532 (PspC.D39).

a comparison of nucleotides a BESTFIT analysis of the amino acid comparison of the amino acid comparison of familiarity with the applications incorporated herein by reference is assumed; and, those applications disclose the sequence of pspA as well as certain portions thereof, and PspA and 31 compositions containing PspA.

As discussed above and in the following Examples, the invention relates to truncated PspA, e.g., PspA C-terminal to position 192 such as a.a. 192-588 ("BC100") 192-299 and 192-260 of PspA eliciting crossprotection, as well as to DNA encoding such truncated PspA (which amplify the coding for these amino acid g regions homologous to most PspAs).

The invention further relates to a pspA-like gene, or a pspC gene and portions thereof probes, primers) which can hybridize thereto and/or amplify that gene, as well as to DNA molecules which hybridize to 'o pspA, so that one can, by hybridization assay and/or amplification, ascertain the presence of a particular pneumococcal strain; and, the invention provides that a PspC can be produced by the pspA-like or pspC sequence **(which PspC can be used like PspA).

Indeed, the invention further relates to S oligonucleotide probes and/or primers which react with S: pspA and/or pspC of many, if not all, strains, so as to permit identification, detection or diagnosis of any pneumococcal strain, as well as to expression products of such probes and/or primers, which can provide crossreactive epitopes of interest.

The repeat region of pspA and/or pspC is highly conserved such that the present invention provides oligonucleotide probes or primers to this region reactive with most, if not all strains, thereby providing diagnostic assays and a means for identifying epitopes of interest.

The invention demonstrates that the pspC gene is homologous to the pspA gene in the leader sequence, Ao first portion of the proline-rich region and in the repeat region; but, these genes differ in the second portion of their proline-rich regions and at the very 3' end of the gene encoding the 17 amino acid tail of PspA.

The product of the pspC gene is expected to lack a Cterminal tail, suggesting different anchoring than PspA.

Drug interference with functions such as surface binding of the coding for repeat regions of pspA and the pspC genes, or with the repeat regions of the expression O products, is therefore a target for intervention of pneumococcal infection.

Further still, the invention provides evidence of additional pspA homologous sequences, in addition to pspA and the pspC sequence. The invention, as mentioned above, includes oligonucleotide probes or primers which distinguish between pspA and the pspC sequence, e.g., LSM1 and LSM2, useful for diagnostic detecting, or 'O isolating purposes; and LSM1 and LSM10 or LSM1 and LSM7 which amplify a portion of the pspC gene, particularly the portion of that gene which encodes an antigenic, immunological or protective protein.

The invention further relates to a method for the isolation of native PspA by growth of pneumococci medium containing high concentrations of(about 0.9% to Sabout preferably choline chloride, elation of live pneumococci with a salt solution containing choline chloride, about 1% about preferably 2% choline .0 chloride, and growth of pneumococci in medium in which the choline in the medium has been almost or substantially completely replaced with a lower alkanolamine, c,-C 6 preferably

C

2 alkanolamine, preferably

C

2 alkanolamine, preferably Sethanolamine 0.0000005% to 0.0000015%, preferably 0.000001% choline chloride plus 0.02% to 0.04% alkanolamine (ethanolamine), preferably PspA from such pneumococci is then preferably isolated from a choline-sepharose affinity column, thereby providing 3D highly purified PspA. Such isolated and/or purified PspA is highly immunogenic and is useful in antigenic, immunological or vaccine composition.

Indeed, the growth media of the pneumococci grown in the presence of the alkanolamine (rather than choline) contains PspA and is itself highly.immunogenic and therefore useful as an antigenic, immunological or vaccine composition; and, is rather inexpensive to produce. Per microgram of PspA, the PspA in the alkanolamine medium is much more protective than PspA isolated by other means, from extracts. Perhaps, without wishing to necessarily be bound by any one particular theory, there is a synergistic effect upon PspA by the other components present prior to isolation, or simply PspA is more protective (more antigenic) prior to isolation and/or purification (implying a possibility of some loss of activity from the step of isolation and/or purification).

The invention further relates to the N-terminal 115 amino acids of PspA, which is useful for compositions comprising an epitope of interest, immunological or vaccine compositions, as well as the DNA coding therefor, i' which is useful in preparing these N-terminal amino acids by recombination, or for use as probes and/or primers for hybridization and/or amplification for identification, detection or diagnosis purposes.

The invention further demonstrates that there 2 is a grouping among the pspA RFLP families. This provides a method of identifying families of different PspAs based on RFLP pattern of pspAs, as well as a means for obtaining diversity of PspAs in an antigenic, immunological or vaccine composition; and, a method of characterizing clonotypes of PspA based on RFLP patterns S* of PspA. And, the invention thus provides oligonucleotides which permit amplification of most, a majority, if not all of S. pneumoniae and thereby permit RFLP analysis of a majority, if not all, S.

pneumoniae.

The invention also provides PspC, having an approximate molecular weight of 105 kD, with an estimated pi of 6.09, and comprising an alpha-helical region, followed by a proline-rich domain and repeat region.

A

major cross-protective region of PspA comprises the Cterminal third of the alpha-helical region (between residues 192 and 260 of PspA), which region accounts for the binding of 4 of 5 cross-protective MAb, and PspA fragments comprising this region can elicit crossprotective immunity in mice. Homology between PspC and PspA begins at amino acid 148 of PspA, thus including the region from 192 to 299, and including the entire PspC sequence C-terminal of amino acid 486. Due to the substantial sequence homology between PspA and PspC in a region comprising the epitopes of interest, known to be protection eliciting, PspC is likely to comprise epitopes of interest similar to those found in PspA. Antibodies specific for this region of PspA, between amino acids 148 and 299, should cross-react with PspC, and thus afford protection by reacting with PspC and PspA.

Similarly, immunization with PspC would be expected to elicit antibodies cross-protective against PspA.

An epitope of interest is an antigen or immunogen or immunologically active fragment thereof from a pathogen or toxin of veterinary or human interest.

The present invention provides an immunogenic, immunological or vaccine composition containing the pneumococcal epitope of interest, and a pharmaceutically acceptable carrier or diluent. An immunological S. composition containing the pneumococcal epitope of interest, elicits an immunological response local or systemic. The response can, but need not be, protective.

Am immunogenic composition containing the pneumococcal epitope of interest, 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 3- of inducing an immunological response in a host mammal comprising administering to the host an immunogenic, immunological or vaccine composition comprising the O pneumococcal epitope of interest, and a pharmaceutically acceptable carrier or diluent.

The DNA encoding,the pneumococcal epitope of interest can be DNA which codes for full length PspA, S PspC, or fragments thereof. A sequence which codes for a fragment of PspA or PspC can encode that portion of PspA or PspC which contains an epitope of interest, such as a protection-eliciting epitope of the protein.

Regions of PspA and PspC have been identified tO from the Rxl strain of S. pneumoniae which not only contain protection-eliciting epitopes, but are also sufficiently cross-reactive with other PspAs from other S. pneumoniae strains so as to be suitable candidates for the region of PspA to be incorporated into a vaccine, immunological or immunogenic composition. Epitopic regions of PspA include residues 1 to 115, 1 to 314, 192 to 260 and 192 to 588. DNA encoding fragments of PspA can comprise DNA which codes for the aforementioned epitopic regions of PspA; or it can comprise DNA encoding c overlapping fragments of PspA, fragment 192 to 588 includes 192 to 260, and fragment 1 to 314 includes 1 to 115 and 192 to 260.

As to epitopes of interest, one skilled in the art can determine an epitope of immunodominant region of a peptide or polypeptide and ergo the coding DNA therefor *se* from the knowledge of the amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as from the nature of particular amino acids size, charge, etc.) and the codon dictionary, without undue Si experimentation.

A general method for determining which portions of a protein to use in an immunological composition focuses on the size and sequence of the antigen of interest. "In general, large proteins, because they have 9- more potential determinants are better antigens than small ones. The more foreign an antigen, that is the less similar to self configurations which induce tolerance, the more effective it is in provoking an immune response." Ivan Roitt, Essential Immunology, 1988.

As to size, the skilled artisan can maximize the size of the protein encoded by the DNA sequence to be inserted into the viral vector (keeping in mind the packaging limitations of the vector). To minimize the DNA inserted while maximizing the size of the protein expressed, the DNA sequence can exclude introns (regions ,6 of a gene which are transcribed but which are subsequently excised from the primary RNA transcript).

At a minimum, the DNA sequence can code for a peptide at least 8 or 9 amino acids long. This is the S minimum length that a peptide needs to be in order to stimulate a CD4+ T cell response (which recognizes virus infected cells or cancerous cells). A minimum peptide length of 13 to 25 amino acids is useful to stimulate a CD8+ T cell response (which recognizes special antigen presenting cells which have engulfed the pathogen). See Kendrew, supra. However, as these are minimum lengths, these peptides are likely to generate an immunological response, an antibody or T cell response; but, for a protective response (as from a vaccine composition), a longer peptide is preferred.

With respect to the sequence, the DNA sequence preferably encodes at least regions of the peptide that generate an antibody response or a T cell response. One method to determine T and B cell epitopes involves epitope mapping. The protein of interest "is fragmented 2t. into overlapping peptides with proteolytic enzymes. The individual peptides are then tested for their ability to bind to an antibody elicited by the native protein or to induce T cell or B cell activation. This approach has been particularly useful in mapping T-cell epitopes since S; the T cell recognizes short linear peptides- complexed with MHC molecules. The method is less effective for determining B-cell epitopes" since B cell epitopes are often not linear amino acid sequence but rather result f rom, the tertiary structure of the f olded three dimensional protein. Janis Kuby, Immaunologyr, (1992) pp.

79-80.

Another method for determining an epitope of interest is to choose the regions of the protein that are hydrophilic. Hydrophilic residues are often on the surface of the protein and therefore often the regions of the protein which are accessible to the antibody. Janis Kuby, Immunology, (1992) P. 81.

Yet another method for determining an epitope of interest is to perform an X-ray cyrstallographic analysis of the antigen (full length) -antibody complex.

Janis Kuby, Immnunologyv, (1992) p. Still another method for choosing an epitope of interest which can generate a T cell response is to identify from the protein sequence potential HLA anchor .:*binding motif s which are peptide sequences which are known to be likely to bind to the MIIC molecule.

*~OThe peptide which is a putative epitope, to generate a T cell response, should be presented in a MHC complex. The peptide preferably contains appropriate anchor motifs for binding to the MHiC molecules, and should bind with high, enough af finity to generate an ~K immune response. Factors which can be considered are: the HLA type of the patient (vertebrate, animal or human) expected to be immunized, the sequence of the protein, the presence of appropriate anchor motifs and the occurance of the peptide sequence in other vital cells.

An immune response is generated, in general, as follows: T cells recognize proteins only when the protein has been cleaved into smaller peptides and is presented in a complex called the "major histocompatability complex MHCI" located on another cell's ~,surf ace. There are two classes of MHC complexes class I and class II, and each class is made up of many different alleles. Different patients have different types of MHC complex alleles; they are said to have a 'different HLA type'.

Class I MHC comp;exes are found on virtually every cell and present peptides from proteins produced inside the cell. Thus, Class I MHC complexes are useful for killing cells which when infected by viruses or which have become cancerous and as the result of expression of an oncogene. T cells which have a protein called CD4 on their surface, bind to the MHC class I cells and secrete *c lymphokines. The lymphokines stimulate a response; cells arrive and kill the viral infected cell.

Class II MHC complexes are found only on antigen- presenting cells and are used to present peptides from circulating pathogens which have been endocytosed by the antigen- presenting cells. T cells which have a protein called CD8 bind to the MHC class II cells and kill the cell by exocytosis of lytic granules.

Some guidelines in determining whether a protein is an epitopes of interest which will stimulate a T cell response, include: Peptide length the peptide should be at least 8 or 9 amino acids long to fit into the MHC class I complex and at least 13-25 amino acids long to fit into a class II MHC complex. This length is a minimum for the peptide to bind to the MHC complex. It is preferred for the peptides to be longer than these lengths because cells may cut the expressed peptides.

The peptide should contain an appropriate anchor motif which will enable it to bind to the various class I or class II molecules with high enough specificity to .o generate an immune response (See Bocchia, M. et al, Specific Bindin of Leukemia Oncoene Fusion Protein Peptides to HLA Class I Molecules, Blood 85:2680-2684; Englehard, VH, Structure of peptides associated with class I and class II MHC molecules Ann. Rev. Immunol.

3! 12:181 (1994)). This can be done, without-undue experimentation, by comparing the sequence of the protein of interest with published structures of peptides associated with the MHC molecules. Protein epitopes recognized by T cell receptors are peptides generated by enzymatic degradation of the protein molecule and are prestnted on the cell surface in association with class I or class II MHC molecules.

Further, the skilled artisan can ascertain an epitope of interest by comparing the protein sequence with sequences listed in the protein data base. Regions of the protein which share little or no homology are (0 better choices for being an epitope of that protein and are therefore useful in a vaccine or immunological composition. Regions which share great homology with widely found sequences present in vital cells should be avoided.

SEven further, another method is simply to generate or express portions of a protein of interest, e generate monoclonal antibodies to those portions of the protein of interest, and then ascertain whether those antibodies inhibit growth in vitro of the pathogen from o which the from which the protein was derived. The skilled artisan can use the other guidelines set forth in this disclosure and in the art for generating or o expressing portions of a protein of interest for analysis as to whether antibodies thereto inhibit growth in vitro.

l For example, the skilled artisan can generate portions of a protein of interest by: selecting 8 to 9 or 13 to amino acid length portions of the protein, selecting hydrophilic regions, selecting portions shown to bind from X-ray data of the antigen (full length)-antibody Scomplex, selecting regions which differ in sequence from other proteins, selecting potential HLA anchor binding motifs, or any combination of these methods or other methods known in the art.

Epitopes recognized by antibodies are expressed on the surface of a protein. To determine-the regions of a protein most likely to stimulate an antibody response one skilled in the art can preferably perform an epitope O map, using the general methods described above, or other mapping methods known in the art.

As can be seen from the foregoing, without undue experimentation, from this disclosure and the knowledge in the art, the skilled artisan can ascertain the amino acid and corresponding DNA sequence of an epitope of interest for obtaining a T cell, B cell and/or antibody response. In addition, reference is made to Gefter et al., U.S. Patent No. 5,019,384, issued May 28, ,o 1991, and the documents it cites, incorporated herein by reference (Note especially the "Relevant Literature" section of this patent, and column 13 of this patent which discloses that: "A large number of epitopes have been defined for a wide variety of organisms of interest.

I, Of particular interest are those epitopes to which neutralizing antibodies are directed. Disclosures of such epitopes are in many of the references cited in the Relevant Literature section.") than Further, the invention demonstrates that more i 0 than one serologically complementary PspA molecule can be in an antigenic, immunological or vaccine composition, so as to elicit better response, protection, for instance, against a variety of strains of pneumococci; and, the invention provides a system of selecting PspAs for a multivalent composition which includes crossprotection evaluation so as to provide a maximally efficacious composition.

The determination of the amount of antigen, PspA or truncated portion thereof and optional 3 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 ;K 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 antigens for suitable hosts in which an immunological response is desired, can be readily ascertained by those skilled in the art from this disclosure (see, the Examples), as ,is the amount of any adjuvant typically administered therewith. Thus, the skilled artisan can readily determine the amount of *o 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 50 wt% 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, Examples below or in applications cited herein).

Typically, however, the antigen is present in J 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 wt% (see, Examples below).

O: Of course, for any composition to be administered to an animal or 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 LDS in a suitable animal model 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, by ELISA and/or RFFIT analysis. Such determinations do not 3 require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administration .s can be ascertained without undue experimentation.

Examples of com ositions of the invention include liquid preparations for orif ice, oral, nasal, anal, vaginal, peroral, intragastric, mucosal perlingual,' alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or C intravenous administration injectable administration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions can also bel1yophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as t REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

Compositions of the invention, are conveniently provided as liquid preparations, isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the invention can be in the "solid" form of pills, tablets, capsules, caplets and the like, including "solid" preparations which are time-released or which have a liquid filling, gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the CS' ut. If nasal or respiratory (mucosal) administration is desired, compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or,, a dose having a particular particle size.

SCompositions of the invention can contain pharmaceutically acceptable flavors and/or colors for rendering them more appealing, especially if they are administered orally. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like Gc and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity preferably of 2500 to 5000 cps, since above that range they become more difficult to administer.

However, above that range, the compositions can approach solid or gelatin forms which are then easily administered as a swallowed pill for oral ingestion.

Liquid preparations are normally easier to ac prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection or orally, to animals, children, particularly small. children, and others who may have difficulty swallowing a pill, tablet, capsule or the like, or in multi-dose situations. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.

Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, liquid dosage form whether the composition is to be formulated into a solution, a suspension, gel or another liquid form], or solid dosage form whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form O or liquid-filled form].

Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the antigen, iipoprotein and optional adjuvant. Minor amounts of other ingredients such as pH adjusters a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, ic methylcellulose), colors and/or flavors may also be present. The compositions can be isotonic, it can have the same osmotic pressure as blood and lacrimal fluid.

The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon *see the agent selected. The important point is to use an 3G amount which will achieve the selected viscosity.

Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increase the shelf-life of the 31 compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium O chloride may also be employed. A suitable concentration -of the preservative will be from 0.02% to 2% based on the total weight although there may be. appreciable variation depending upon the agent selected.

SThose skilled in the art will recognize that the components of the compositions must be selected to be chemically inert with respect to the PspA antigen and optional adjuvant. This will present no problem to those skilled in chemical and pharmaceutical principles, or 'o problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

The immunologically effective compositions of Sthis invention are prepared by mixing the ingredients Sfollowing generally accepted procedures. For example the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture 0 which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent r and possibly a buffer to control pH or an additional solute to control tonicity. Generally the pH may be from S about 3 to 7.5. 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 solid vs. liquid).

Dosages for humans or other mammals can be determined jo without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, the Examples below from the Examples involving mice).

Suitable regimes for initial administration and booster doses or for sequential administrations also are j o variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the Examples below.

PCR techniques for amplifying sample DNA for diagnostic detection or assay methods are known from the 6 art cited herein and the documents cited herein (see Examples), as are hybridization techniques for such methods. And, without undue experimentation, the skilled artisan can use gene products and antibodies therefrom in diagnostic, detection or assay methods by procedures *O known in the art.

The following Examples are provided for illustration and are not to be considered a limitation of the invention.

EXAMPLES

6 EXAMPLE 1 TRUNCATED STREPTOCOCCUS PNEUMONIAE PspA MOLECULES ELICIT CROSS-PROTECTIVE

IMMUNITY

AGAINST PNEUMOCOCCAL

CHALLENGE

Since the isolation of S. pneumoniae from human saliva in 1881 and its subsequent connection with lobar pneumonia two years later, human disease resulting from pneumococcal infection has been associated with a significant degree of morbidity and mortality. A recent survey of urgently needed vaccines in the developing and developed world places an improved pneumococcal vaccine among the top three vaccine priorities of industrialized countries. The currently licensed vaccine is a 23-valent composition of pneumococcal capsular polysaccharides that is only about 60% effective in the elderly and due to poor efficacy is not recommended for use in children 30 below two years of age. Furthermore the growing frequency of multi-drug resistant strains of S.

pneumoniae being isolated accentuates the need for a more effective vaccine to prevent pneumococcal infections.

The immunogenic nature of proteins makes them ;3 prime targets for new vaccine strategies. Pneumococcal molecules being investigated as potential protein vaccine candidates include pneumolysis, neuraminidase, autolysin and PspA. All of these proteins are capable of eliciting immunity in mice resulting in extension of life and protection against death with challenge doses near the LDso. PspA is unique among, these macromolecules in that is can elicit antibodies in animals that protect against inoculums 100-fold greater than the LD 5 PspA is a surface-exposed protein with an apparent molecular weight of 67-99 kDa that is expressed by all clinically relevant S. pneumoniae strains examined to date. Though PspAs from different pneumococcal (o strains are serologically variable, many PspA antibodies exhibit cross-reactivities with PspAs from unrelated strains. Upon active immunization with PspA, mice generate PspA antibodies that protect against subsequent challenge with diverse strains of S. pneumoniae. The 6immunogenic and protection-eliciting properties of PspA suggest that it may be a good candidate molecule for a protein-based pneumococcal vaccine.

Four distinct domains of PspA have been identified based on DNA sequence. They include a Nterminal highly charged alpha-helical region, a prolinerich 82 amino acid stretch, a C-terminal repeat segment S0 comprised of ten 20-amino acid repeat sequences, and a 17-amino acid tail. A panel of MAbs to Rxl PspA have been produced and the binding sites of nine of these Mabs 6 were recently localized within the Rxl pspA sequence in the alpha-helical region. Five of the Rxl Mabs were protective in mice infected with a virulent pneumococcal strain, WU2. Four of these five protective antibodies were mapped to the distal third (amino acids 192-260) of 30 the alpha-helical domain of Rxl PspA.

Truncated PspAs containing amino acids 192-588 or 192-299, from pneumococcal strain Rxl were cloned and the recombinant proteins expressed and evaluated for their ability to elicit protection against subsequent challenge with S. pneumoniae WU2. As with full-length Rxl PspA, both truncated PspAs containing the distal alpha-helical region protected mice against fatal WU2 pneumococcal infection. However, the recombinant PspA fragment extending from amino acid 192 to 588 was more immunogenic than the smaller fragment, probably due to its larger size. In addition, the protection elicited by the amino acid fragment 192-588 of Rxl was comparable to that elicited by full-length Rxl PspA. Therefore, crossprotective epitopes of other PspAs were also sought in the C-terminal two-thirds of the molecule. As discussed below, PspAs homologous to amino acids 192-588 of strain i Rxl were amplified by PCR, cloned, and expressed in E.

coli. Then three recombinant PspAs, from capsule type 4 and 5 strains, were evaluated for their ability to confer cross-protection against challenge strains of variant capsular types. The data demonstrate that the truncated *C PspAs from capsular type 4 and 5 strains collectively protect against or early death caused by challenge with capsular type 4 and 5 parental strains as well as type 3, 6A, and 6B S. pneumoniae.

Bacterial strains and culture conditions. All -o pneumococci were from the culture collection of this laboratory, and have been described .(Yother, J. et al., S" Infect. Immun. 1982; 16: 184-188; Briles, et al., Infect. Immun. 1992; 60: 111-116; McDaniel, et al., S Microb. Pathog. 1992; 13: 261-269; and McDaniel, L.S, et al., In: Ferretti, J.J. et al., eds. Genetics of streptococci, enterococci, and lactococci. 1995; 283- 286), with the exception of clinical isolates TJ0893, 0922134 and BG8740. Pneumococcal strains TJ0893 and 0922134 were recovered from the blood of a 43-year old male and an elderly female, respectively. S. pneumoniae BG8743 is a blood isolate from an 8-month old infant.

Strains employed in this study included capsular type 3 (A66.3, EF10197, WU2), type 4 (BG9739, EF3296, EF5668, L81905), type 5 (DBL5), type 6A (DBL6A, EF6796), type 6B (BG7322, BG9163, DBL1), type 14 TJ0893), type 19 (BG8090), and type 23 (0922134, BG8743). In addition, strain WG44.1, which expresses no detectable PspA, was employed in PspA-specific antibody analysis. All chemicals were purchased from Fisher Scientific, Fair Lawn, New Jersey unless indicate otherwise.

S. pneumoniae were grown in Todd Hewitt broth (Difco, Detroit, Michigan) supplemented with 5% yeast extract (Difco). Mid-exponential phase cultures were used for seeding inocula in Lactated Ringer's (Abbott laboratories, North Chicago, Illinois) for challenge studies. For pneumococcal strains used in challenge X studies, inocula ranged from 2.8 to 3.8 log 10

CFU

(verified by dilution plating on blood agar). Plates were incubated overnight in a candle jar at 370C.

E. coli DH1 and BL21(DE3) were cultured in LB medium Bacto-tryptone (Difco), 0.5% Bacto Yeast (Difco), 0.5% NaC1, 0.1% dextrose). For the preparation of cell lysates, recombinant E. coil were grown in minimal E medium supplemented with 0.05 M thiamine, 0.2% glucose, 0.1% casamino acids (Difco), and 50 mg/ml Skanamycin. Permanent bacterial stocks were stored at 80 0 C in growth medium containing 10% glycerol.

Construction of plasmid-based strains. pET-9a (Novagen, Madison, Wisconsin) was used for cloning truncated pspA genes from fourteen S. pneumoniae strains: DBL5, DBL6A, WU2, BG9739, EF5668, L81905, 0922134, 5 BG8090, BG8743, BG9163, DBL1, EF3296, EF6796, and EF10197 (Table pspA gene fragments, from fifteen strains, were amplified by PCR using two primers provided by Connaught Laboratories, Swiftwater, Pennsylvania Primer N192- 5'GGAAGGCCATATGCTCAAAGAGATTGATGAGTCT3' and primer 3 C588 5'CCAAGGATCCTTAAACCCATTCACCATTGGC 3 were engineered with NdeI and BamHI restriction endonuclease sites, respectively. PCR-amplified gene products were digested with BamHI and NdeI, and ligated to linearized pET-9a digested likewise and further treated with bacterial alkaline phosphatase United States Biochemical Corporation, Cleveland, Ohio) to prevent recircularization of the cut plasmid. Clones were first established in E. coli BL21(DE3) which contained a chromosomal copy of the T7 RNA polymerase gene under the control of an inducible lacUV5 promoter., E. coli DH1 cells were transformed by the method of Hanahan (Hanahan, D. J. Mo1. Biol. 1983; 166: 557-580). Stable transformants were identified by screening on LB-kanamycin plates. Plasmid constructs, isolated from each of these strains, were electroporated (Electro Cell Manipulator 600, BTX Electroporation 0o System, San Diego, California) into E. coli BL21(DE3) and their respective strain designations are listed in Table 1. The pET-9a vector alone was introduced into E. coli BL21(DES) by electroporation to yield strain RCT125 (Table All plasmid constructs and PCR-amplified pspA :6 gene fragments were evaluated by agarose gel electrophoresis (with 1 kb DNA ladder, Gibco

BRL,

Gaithersburg, Maryland). Next, Southern analysis was performed using LMpspAl, a previously described fulllength pspA probe (McDaniel. L.S. et al., Microb. Pathog.

2 1992; l3: 261-269) random primed labeled with S digoxigenin-ll-dUTP (Genius System, Boehringer Mannheim, Indianapolis, Indiana). Hybridization was detected with chemiluminescent sheets according to the manufacturer's instructions (Schleicher Schuell, Keene, New 2 Hampshire).

Cell fractionation of recombinant E. coli strains. Multiple cell fractions from transformed

E.

coli were evaluated for the expression of truncated PspA molecules. Single colonies were inoculated into 3 ml LB o cultures containing kanamycin and grown overnight at S370C. Next, an 80 ml LB culture, inoculated with 1:100 dilution of the overnight culture, was grown at 370C to mid-exponential phase

(A

0 o of ca. 0.5) and a 1 ml sample was harvested and resuspended (uninduced cells) prior to 3- induction with isopropylthiogalactoside (IPTG, 0.3 mM final concentration). Following 1, 2, and 3 hr of induction, 0.5 ml of cells were centrifuges, resuspended, and labeled induced cells. The remaining culture was divided into two aliquots, centrifuged (4000 x g, 10 min, DuPont Sorvall RC 5B Plus), and the supernatant discarded. One pellet was resuspended in 5 ml of 20 mM S Tris-HC1 ph 7.4 200 mM NaC1, 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt (EDTA) and frozen at -20 0

C

overnight. Cells were thawed at 65 0 C for 30 min, placed on ice, and sonicated for vive 10-sec pulses (0.4 relative output, Fisher Sonic Dismembrator, Dynatech ic Laboratories, Inc. Chantilly, Virginia). Next, the material was centrifuged (9000 x g, 20 min) and the supernatant was designed the crude extract-cytoplasmic fraction. The pellet was resuspended in Tris-NaCl-EDTA buffer and labeled the insoluble cell well and membrane r' fraction. The other pellet, from the divided induced culture, was resuspended in 10 ml of 30 mM Tris-HC1 pH containing 20% sucrose and 1 mM EDTA and incubated at room temperature for 10 min with agitation. Cells were then centrifuged, the supernatant removed, and the pellet resuspended in 5 mM MgSO 4 (10 ml, 10 min, shaking 4 0

C

bath). This material was centrifuged and the supernatant was designated osmotic shock-periplasmic fraction. Cell fractions were evaluated by SDS-PAGE and immunoblot analysis.

MAbs to PspA. PspA-specific monoclonal antibodies (MAbs) XiR278 and 1A4 were used as previously described (Crain, M.j. et al., 1990, Infect. Immun.; 58: 3293-3299). MAb P50-92D9 was produced by immunization with DBL5 PspA. The PspA-specificity of MAb P50-92D9 was confirmed by Western Analysis by its reactivity with native PspAs from S. pneumoniae DBL5, BG9739, EF5668, and L81095 and its failure to recognize the PspA-control strain WG44.1.

SDS-PAGE and immunoblot analysis. E. coli cell fractions containing recombinant PspA proteins and biotinylated molecular weight markers (low range, Bio- Rad, Richmond, California) were separated by sodium O dodecyl sulfate-polyacrylamide Bethesda Research Laboratories, Gaithersburg, Maryland) gel electrophoresis (SDS-PAGE) by the method of Laemmli (Laemmli, U.K. Nature 1970; 222: 680-685). Samples were first boiled for 5 min in sample buffer containing 60 mM Tris pH 6.8, 1% 2-Bmercaptoethanol (Sigma, St. Louis, Missouri), 1% SDS, glycerol, and 0.01% bromophenol blue. Gels were subsequently transferred (1 hr, 100 volts) to nitrocellulose (0.45 mM pores, Millipore, Bedford, ¢o Massachusetts) as per the method of Towbin et al. Blots were blocked with 3% casein, 0.05% Tween 20 in 10 mM Tris, 0.1 M NaC1, pH 7.4 for 30 min prior to incubating with PspA-specific monoclonal antibodies diluted in PBST for 1 hr at 25 0 C. Next, the blot was washed 3 times with K PBST before incubating with alkaline phosphatase-labeled goat anti-mouse immunoglobulin (Southern Biotechnology Associates, Inc., Birmingham, Alabama) for 1 hr at 25 0

C.

Washes were performed as before and blots was developed with 0.5 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.01% nitro blue tetrazolium (Sigma) first dissolved in S: 150 Al of dimethyl sulfoxide and then diluted in 1.5 M Tris-HC1 pH 8.8. Dot blots were analyzed similarly.

Lysate samples (2 Al) were spotted on nitrocellulose filters (Millipore), allowed to dry, blocked, and detected as just described.

Preparation of cell lysates containing recombinant PspA proteins. Transformed E. coli strains RCT105, RCT113, RCT117, and RCT125 (Table 2) were grown in mid-exponential phase in minimal E medium before IPTG induction (2 mM final concentration, 2 hours, 37 0

C).

Cultures were harvested by centrifugation (10 min at 9000 x resuspended in Tris-acetate pH 6.9, and frozen at 0 C overnight. Samples were thawed at 65 0 C for 30 min, cooled on ice, and sonicated. Next the samples were treated with 0.2 mM AEBSF (Calbiochem, La Jolla, California) at 37 0 C for 30 min and finally centrifuged to remove cell wall and membrane components. Dot blot analysis was performed using PspA-specific MAbs to validate the presence of recombinant, truncated PspA molecules in the lysates prior to their use as immunogens in mice. Unused lysate material was stored at -20 0

C

s until subsequent immunizations were performed.

Mouse immunization and challenge.

CBA/CAHN-

XID/J mice (Jackson Laboratories, Bar Harbor, Maine), 6- 12 weeks old, were employed for protection studies.

These mice carry a X-linked immunodeficiency that is prevents them from generating antibody to polysaccharide components, thus making them extremely susceptible to pneumococcal infection. Animals were immunized subcutaneously with cell lysates from E coli recombinant strains RCT105, RCT113, RCT117, and RCT125 (Table 2) in complete Freund's adjuvant for primary immunizations.

Secondary injections were administered in incomplete adjuvant and subsequent boosts in dH20. Immunized and nonimmunized mice (groups of 2 to 5 animals) were S challenged with S. pneumoniae strains A66.3, BG7322, DBL6A, WU2, DBL5, BG9739, and L81905 intravenously (tail vein) to induce pneumococcal sepsis. Infected animals were monitored for 21 days and mice that survived the 3week evaluation period were designated protected against death and scored as surviving 22 days for statistical M analysis. Protection that resulted in extension of life was calculated as a comparison between mean number of days to death for immunized versus pooled control mice (nonimmunized and RCT125 sham-immunized; total of 6-7 animals).

30 Determination of PspA serum levels. Mice were bled retro-orbitally following the secondary boost and again prior to challenge. Representative mouse titers were evaluated by enzyme-linked immunorsorbent assay (ELISA) using native, parental PspAs isolated from S: pneumococcal strains DBL5, BG9739, and L81905. PspAs were immobilized on microtiter plates by incubating in NaHCO 3 0.5 M Na 2

CO

3 pH9.5 at 4 0 C overnight. Alkaline phosphatase-labeled goat anti-mouse immunoglobulin (Southern Biotechnology Associates, Inc.) was used to detect mouse serum antibodies. Color development was with p-nitrophenyl phosphate (Sigma, 1 mg/ml) in 0.5 m MgCL 2 pH 9.8 with 10% diethanolamine and absorbance was read at 405 nm after a 30 min incubation. Reciprocal titers were calculated as the last dilution of, antibody that registered an optical density value of 0.1. Sera from individual mice within a particular immunogen group were evaluated separately and then the respective titers from four mice per group were combined to obtain titer range (Table 3).

Statistics. The one-tailed Fisher exact and two sample rank tests were used to evaluate protection Sagainst death and extension of life in the mouse model.

Cloning of truncated pspA genes. Using primers N192 and C588, truncated pspA genes from fifteen diverse pneumococcal strains representing eight different capsular types (Table 1) were amplified by PCR. Even though variability exists in pspA genes from different strain, this result demonstrates that sufficient conservation exists between variant pspA genes to allow sequence amplification in all strains examined to date.

Successful pspA PCR-amplification extended to all capsule types evaluated.

.:Fourteen of the amplified pspA genes were cloned and three clones containing truncated PspA molecules from pneumococcal strains DBL5, BG9739, and L81905 were further studies (Table To verify the o constructions, plasmids from recombinant E. coli strains *3 (RCT105, RCT113, RCT117, and RCT125 (Table 2) were isolated, digested with NdeI and BAMHI restriction endonucleases, and electrophoresed in 1% agarose side-byside with the PCR products used in their respective constructions (Figure 1A). The digestion reaction was complete for pRCT105, while pRCT113 and pRCT117 digestions were incomplete (lanes 5 and 7, respectively).

This gel was denatured and DNA transferred to nylon for Southern analysis. Figure 1B depicts the corresponding Southern blot probed withfull-length RxlpspA DNA. Lane 1 contains pRCT125, digested vector alone, which does not react with the pneumococcal DNA-specific probe, as expected. The pspA-specific probe hybridized with the PCT products and the digested plasmid inserts (see arrow, Figure 1B) as well as the partially undigested pRCT113 and pRCT117 (lane 5 and confirming successful cloning o of DBL5, BG9739, and L81905 pspA DNA. Constructions were similarly confirmed with the eleven additional recombinant strains containing truncated pspA genes from S. pneumoniae strains of different capsular and PspA types.

Expression of recombinant PspA in E. coli B121(De3). Transformed E. coli strains RCT105, RCT113, RCT117, and RCT125 were cultured to mid-exponential phase prior to the addition of IPTG to induce expression of the cloned, truncated pspA gene in each strain. A cell fractionation experiment was performed to identify the location of recombinant PspA proteins in transformed

E.

coli strains. Samples representing uninduced cells, included cells (1 hr, 2 hr, and 3 hr time intervals), the periplasmic fraction, the cytoplasmic fraction, and insoluble cell wall/membrane material were resolved by SDS-PAGE. Proteins were then transformed to nitrocellulose and Western analysis was performed using monoclonal antibodies specific for PspA epitopes.

Figure 2 reveals that both the cytoplasmic 0 (lane 8) and the insoluble matter fractions (lane 9), from recombinant strain RCT 105, contain a protein of approximately 53.7 kDa that is recognized by MAb XiR278 that is not seen in the uninduced cell sample (lane 3).

This protein increases in quantity in direct correlation 53 with the length of IPTG induction (lanes 4=6; 1 hr, 2 hr, and 3 hr respectively). No truncated RCT105 PspA was found in the periplasmics fraction (lane which was expected since the pET-9a vector lacks a signal sequence that would be necessary for directing proteins to the periplasm. The observed molecular weight (ca. 53.5 kDa) is larger than the predicted molecular weight for the 1.2 Skb DBL5pspA gene product (43.6 kDa; Figure 1A, lane 4).

Like full-length Rxl PspA, the observed and predicted molecular weights for truncated PspAs do not agree precisely. In addition, immunoblot analysis was performed for recombinant E. coli strains RCT113, and vo RCT117 (using MAbs 1A4 and P50-92D, respectively) and similar results were obtained, while no cell fractions from control strain RCT125 were recognized by MAb XiR278.

Evaluating the protective capacity of recombinant, truncated PspAs. The truncated PspA Sproteins from strains RCT113, RCT117, and RCT105 were expressed and analyzed for their ability to generate cross-protection against a battery of seven S. pneumoniae strains. Control mice (non-immunized and RCT125 shamimmunized) and recombinant PspA-immunized mice were o challenged with mouse-virulent strains A66.3, BG7322, S DBL6A, WU2, DBL5, BG9739, and L81905. Table 3 presents the day of death for each infected mouse.

Immunization with truncated PspA from RCT113, S RCT117, and RCT105 conferred protection against death for all mice challenged with capsular type 3 strains (A66.3 and WU2 (Table The three truncated PspAs also provided significant protection against death with DBL6A, and BG7322 pneumococci (capsular types 6A and 6B, respectively). In addition, immunization with recombinant RCT113 PspA extended days to death in mice challenged with strains DBL5, BG9739, and L81905, while RCT117 PspA prolonged the lives of mice inoculated with BG9739 pneumococci (Table Truncated BG9739 PspA elicited protection against all challenge strains (100%) evaluated in this study, while recombinant L81905 and truncated PspAs conferred protection against death with 71% and 57% of S. pneumoniae challenge strains, respectively.

Anti-PspA antibody titers elicited by the three immunogens vary over approximately a 10-fold range (Table The lowest antibody levels were elicited by RCT105 and this truncated PspA also elicited protection against the fewest number of challenge strains. RCT113 and RCT117 elicited three and nine time as much anti-PspA antibody, respectively. As expected, no antibody to PspA was detected in nonimmunized mice nor was specific-PspA antibody measured in mice immunized with the vector-only control strain (RCT125).

In summary, immunization with RCT113 and RCT117 PspAs protected mice against fatal challenge with Scapsular type 3 and 6A strains and extended life for mice inoculated with type 4, 5, and 6B pneumococci. RCT105 PspA immunization protected against fatal infection with capsular type 3 and 6B strains and prolonged time to death for type 6A S. pneumoniae but offered not protection against type 4 and 5 strains. These data demonstrate that truncated PspAs from capsular type 4 and pneumococci collectively protect mice and ergo other hosts, such as humans, against or delay death caused by each of the seven challenge strains. In general, however, more complete protection was observed against strains of capsular type 3, 6A, and 6B than against type 4 and 5 S. pneumoniae.

PspA has been shown to be a protectioneliciting molecule of S. pneumoniae. Immunization with 3 C PspA has also been shown to be cross-protective, although eliciting more complete protection against certain strains than others. Thus, it is possible that a broadly protective PspA vaccine might need to contain PspAs of more than one pneumococcal strain. The distal third of the alpha-helical region of PspA has been identified as a major protective region of PspA. Moreover, this region is presented in a very antigenic form when expressed with the intact C-terminal half of the molecule. In this Example, the ability to use truncated PspA proteins homologous to the region of Rxl PspA extending from amino acid residue 192 to the C-terminus at residue 588 is demonstrated.

The C-terminal two-thirds of PspA was cloned from fourteen strains by PCR amplification of a gene fragment of the appropriate size (1.2 kb) which hybridized with full-length Rxl pspA. Successful

PCR

io amplification extended to all capsule types analyzed.

Thus, the C-terminal two-third of PspA may be amplified from many, if not all, pneumococcal capsule types with Rxl pspA-specific primers. This technique is thus applicable to the development of antigenic immunological If or vaccine compositions containing multiple PspA or fragments thereof.

Of these clones, three truncated PspA proteins **were expressed and evaluated in mouse immunization studies to determine their ability to cross-protect against challenge with a variety of pneumococcal capsular types. All three recombinant PspAs elicited antibody reactive with their respective donor PspA and all three elicited protection against pneumococcal infection. Of S the two truncated PspA proteins that elicited the highest antibody responses, 100% and 71% of the challenge strains were protected. RCT105 PspA, which elicited the lowest titers of PspA-specific antibody, yielded protection against 57% of S. pneumoniae strains evaluated. With all truncated PspAs, significant levels of protection were 3 observed in four of the seven challenge strains. In fact, in all instances except for on (RCT105-immunized mice challenged with strain BG9739) the trend was for truncated PspA-immunization to elicit protection against pneumococcal challenge. These results demonstrate that truncated Rxl PspA (amino acids 192-588) cross-protects mice against fatal S. pneumoniae WU2 challenge. More importantly, these data show that the homologous regions e of diverse PspAs demonstrate comparable cross-protective abilities.

Strains of capsular type 4 and 5 were more difficult to protect against than were type 3, 6A and 6B pneumococcal strains. Serological differences in PspAs might affect cross-protection in some cases. Yet the difficulty in protecting against the type 4 and 5 strains used herein could not be explained on this basis, since the truncated PspA immunogens were cloned from the same ;o three type 4 and 5 strains used for challenge. Both PspAs from the type 4 strains delayed death caused by one or both type 4 challenge strains but neither could prevent death caused by either type 4 pneumococcal strain. Moreover, the truncated PspA from the type strain DBL5 elicited protection against death or delayed death with strains of capsular types 3, 6A and 6B but failed to protect against infection with its donor strain or either type 4 challenge strain.

There may be several reasons why the truncated 2 0 PspAs from capsular type 4 and type 5 strains failed to o protect against death even with their homologous donor S.

pneumoniae strains. One possibility is that the type 4 and 5 strains chosen for study are especially virulent in the XID mouse model. XID mice fail to make antibodies to polysaccharides and are therefore extremely susceptible S to pneumococcal infection with less than 100 CFU of most strains, including those of capsular type 3, 4, 5, 6A, and 6B. The increased mouse virulence of types 4 and i s apparent from the fact that in immunologically normal mice these strains have lower LD 0 ,s and/or are more consistently fatal than strains of capsular types 3, 6A, or 6B.

Another possibility is that epitopes critical to protection-eliciting capacity with capsular type 4 and 33 5 strains are not present in the C-terminar two-thirds of PspA (amino acids 192-588), the truncated fragments used for immunization. The critical epitopes for these Sstrains may be located in the N-terminal two thirds of the alpha-helical region of their PspA molecules.

Finally, it is also possible that PspA may be less exposed on some S. pneumoniae strains than others.

Strain Rxl PspA amino acid sequence does not contain the cell wall attachment motif LPXTGX described by Schneewind et al. found in many gram-positive bacteria. Rather, PspA has a novel anchoring mechanism that is mediated by choline interactions between pneumococcal membrane- 0 associated lipoteichoic acid and the repeat region in the C-terminus of the molecule. Electron micrographic examination has confirmed the localization of PspA on the pneumococcal surface and PspA-specific MAb data supports the accessibility of surface-exposed PspA. However, it Sis.not known whether S. pneumoniae strains differ substantially in the degree to which different PspA regions are exposed to the surrounding environment. Nor is it known if the quantity of PspA expressed on the bacterial cell surface differs widely between strains.

e o* e ee e .o S e Table 1. PsPA recombinant strains categorized by pneumococcal capsular type.

Capsular Type Parent Strains 3 WU2, EF10197 4 BG9739, EF5668 L81905, EF3296 Respective Recombinant Strains RCTl11, RCT137 RCT1l3, RCTl17, RCT133 RCTl105 DBL6A, EF6796 BG9163, DBLl TJO8 93* BG8090 0922134, BG8743 RCT1O9, RCT135 RCTl29, RCT131 none RCT121 RCT119, RCT123 Truncated PsPA amplified recently, not yet cloned Table 2. Description of recombinant strains used in evaluating the protection-eliciting capacity of truncated PspAs in mice.

Recombinant Strain Description Capsule Type ofL r±rn LtsJ1 RCT 105 BL2l(DE3) E_-coli with pET-9a: RCT 113 BL2l(DE3) E.coli 4 with pET-9a:BG9739 RCT 117 BL2l(DE3) E.colj with 4 pET-9a: LB1905 RCT 125 BL21(DE3) E.coli with only) 0 0 .0 0* .0 .0 00.

Table 3. Evaluation of the protection elicited by trun cated S. pneumoniae PspA molecules in mice by days to death post-challenge Challenge Strain [capsular type] log,, dose in CFU) Immunizing Reciproca A66.3 WU2 OBLISA BG7322 DBL.S BG9739 1131905 recombinant ant-PspA Ntpe 31 1"yp 31 ["yp 6AI [type 69) [1yp 5] [type 41 [lp41 PspA/PspkA ttter (2.78) (3.57) (3.24) (3.11) (3.81) (3.56) (3.82) donor strain RCT113/BG9739 5590-50.300 4x>21' 4x>21§ 15.3x>21t 12.13A.6>21t 3.3.4.5' 5,5.5,71 5.6,8.8' 9CT117/1.81905 5590-150.900 4x>21' 4x>21§ 7,16.Z(>21' 10.12.13>21' 3,3,4.41 4.,13>21' 3,4.6.8 1860-16,770 4x>21' 4x>21§ 8.10.13.21 t 4x>21t 21 2.2.2,41 RCT12S/ 2G-620 21 21 3,1.6,156 7.8.8.14 2.2.2,2 2.2,3.4.5 2.3.5.5 vector only none 0 2,2.2 12.3 13,3.4 16.7.9 12.5 3.5 Animals surviving the 3-week evaluation period were sacrificed and days to death recorded as >21 at 22 days for these fully protected mice.

t Range of tour sera per group of mice; titers measured against native donor PspAs P<0.012 P<0.035 I P<O.057 Note: One-tailed Fisher exact and two sample rank tests were used for statistical analysis.

days. For statistical analysis, E values were calculated S EXAMPLE 2 LOCALIZATION OF PROTECTION-ELICITING EPITOPES AND PspA OF S. PNEUMONIAE This Example, the ability of PspA epitopes on two PspA fragments (amino acids 192-588 and 192-299) to elicit cross-protection against a panel of diverse pneumococci is demonstrated. Also, this Example identifies regions homologous to amino acids 192-299 of Rxl in 15 other diverse pneumococcal strains. The DNA encoding these regions was then amplified and cloned.

i. The recombinant PspA fragments expressed were evaluated for their ability to elicit cross-protection against a panel of virulent pneumococci.

Bacterial strains and media conditions.

S.

pneumoniae strains were grown in Todd Hewitt broth with yeast extract (THY) (both from Difco Laboratories, Detroit, Michigan) at 37 0 C or on blood agar plates containing 3% sheep blood at 37 0 C under reduced oxygen tension. E. coli strains were grown in Luria-Bertani medium or minimal E medium. Bacteria were stored at 0 C in growth medium supplemented with 10% glycerol.

E.

coli were transformed by the methods of Hanahan (Hanahan, D. J. Mol. Biol. 1983; 166: 557). Ampicillin (Ap) was used at a concentration of 100 gg/ml for E. Coli.

Construction of pIN-III-ompA3 and pMAL-based

E.

Coli strains. Recombinant plasmids pBC100 and pBAR416 that express and secrete pspA fragments from E. Coli were constructed with PIN-III-ompA3 as previously described (McDaniel, L.S. et al., Microb. Pathog. 1994; 17: 323).

The pMAL-p2 vector (New England Biolabs, 6c Protein Fusion Purification System, catalog #800) was used for cloning pspA gene fragments to amino acids 192- 299 from strain Rxl and from 7 other S. pneumoniae strains: R36A, D39, A66, BF9739, DBL5, DBL6A, and LM100.

Amplification of the pspA gene fragments was done by the .3 polymerase chain reaction (PCR) as described previously (McDaniel, L.S. et al., Microb. Pathog. 1994; 17: 323) using primers 5'CCGGATCCGCTCAAAGAGATTGATGAGTCTG 3 [LSM4] and 5'CTGAGTCGACTGAGTTTCTGGAGCTGGAGC 3 [LMS6] made with BamHI and SalI restriction endonuclease sites, respectively. Primers were based on the sequence of Rxi PspA. PCR products and the pMAL vector were digested with BAMHI and Sail, and ligated together. Clones were transformed into E. Coli DH5a by the methods of Hanahan.

Stable transformants were selected on LB plates containing 100pg/ml Ap. These clones were screened on LB plates containing 0.1 mM IPTG, 80 gg/ml X-gal and 100 Ag/ml Ap and replica LB plates with 100 Mg/ml Ap according to the manufacturer's instructions. The strain designations for these constructs are listed in Table 6.

Positive clones were evaluated for the correct pspA gene fragment by agarose gel electrophoresis following plasmid isolation by the methods of Birnboim and Doly (Birnboim, H.C. et al., Nucl. Acids Res. 1979, 7: 1513). Southern analysis was done as previously described using a fulllength pspA probe (McDaniel, L.S. et al., Microb. Pathog.

1994; 17: 323), randomly primed labeled with digoxigenin- 11-dUTP (Genius System, Boehdinger Mannheim, Indianapolis, Indiana) and detected by chemiluminescence.

Expression of recombinant PspA protein fragments. For induction of expression of strains BC100 and BAR416, bacteria were grown to an optical density of approximately 0.6 at 660 nm at 37 0 C in minimal media, and IPTG was added to a final concentration of 2 mM. The cells were incubated for an additional 2 hours at 37 0

C,

harvested, and the periplasmic contents released by osmotic shock. For strains BAR36A, BAR39, BAR66, BAR5668, BAR9739, BARL5, BAR6A and BAR100, bacteria were *:So grown and induced as above except LB media 10 mM.

glucose was the culture medium. Proteins from these strains were purified over an amylose resin column according to the manufacturer's instructions (New England Biolabs, Protein Fusion Purification System, Catalog 3o #800). Briefly, amylose resin was poured into a 10 mL column and washed with column buffer. The diluted S osmotic shock extract was loaded at a flow rate of approximately 1 mL/minute. The column was then washed again with column buffer ang the fusion protein eluted off the column with column buffer containing 10 mM Smaltose. Lysates were stored at -200C until further use.

Characterization of truncated PspA proteins used for immunization. The truncated PspA molecules, controls and molecular weight markers (Bio-Rad, Richmond, CA) were electrophoresed in a 10% sodium dodecyl (SDS) f, polyacrylamide gel and electroblotted onto nitrocellulose. Rabbit polyclonal anti-PspA serum and rabbit antimaltose binding protein were used as the primary antibodies to probe the blots.

A direct binding ELISA procedure was used to quantitatively confirm reactivities observed by immunoblotting. For all protein extracts, osmotic shock preparations were diluted to a concentration of 3 pg/ml in phosphate buffered saline (PBS), and 100 pl was added to the wells of Immulon 4 microtitration plates (Dynatech Laboratories, Inc., Chantilly, VA). After blocking with 1.5% bovine serum albumin in PBS, unfractionated tissue culture supernates of individual MAbs were titered in duplicated by three-fold serial dilution through seven wells and developed using an alkaline phosphatase-labeled goat anti-mouse immunoglobulin secondary antibody (Southern Biotech Associates, Birmingham, AL) and alkalinephosphatase substrate (Sigma, St. Louis, MO).

The plates were read at 405 nm in a Dynatech plate reader after 25 minutes, and the 30% end point was calculated So for each antibody with each preparation.

Immunization and Protection Assays. Six to nine week old CBA/CAHN-XID/J (CBA/N) mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. CBA/N mice carry an X-linked immunodeficiency trait, which renders them relatively unable to respond to polysaccharide antigens, but they do respond with normal levels of antibodies against protein antigens. Because of, the absence of antibodies reactive with the phosphocholine determinant of C-polysaccharide in their serum, the mice are highly ,susceptible to pneumococcal infection. Mice immunized with the BC100 fragment were injected inguinally with antigen emulsified in CFA, giving an approximate dose of 3 Ag of protein per mouse.

Fourteen days later the mice were boosted intraperitoneally with 3 pg of antigen diluted in Ringer's lactate without adjuvant. Control mice were io immunized following the same protocol with diluent and adjuvant, but no antigen. Mice immunized with the BAR416 fragment were injected with 0.2 ml at two sites in the sublinguinal area with antigen emulsified in CFA. The mice were boosted inguinally fourteen days later with antigen emulsified in IFA and were boosted a second time fourteen days later intraperioneally with 0.2 ml of antigen diluted in Ringer's lactate without adjuvant.

Mice that were immunized with the homologues of Rxl BAR416 were immunized as described above. The :2o control animals followed the same immunization protocol but received maltose binding protein (MBP) diluted 1:1 in CFA for their immunization and were also boosted with

MBP.

Serum analysis. Mice were retro-orbitally bled with a 75 Ml heparinized microhematocrit capillary tube (Fisher Scientific) before the first immunization and then once approximately 2 hours before challenge with virulent pneumococci. The serum was analyzed for the S. presence of antibodies to PspA by an enzyme-linked immunosorbent assay (ELISA) using native full-length R36A PspA as coating antigen as previously described (McDaniel, L.S. Microb. Pathog. 1994; 17: 323).

Intravenous infection of mice. Pneumococcal cultures were grown to late log phase in THY.

3 Pneumococci were diluted to 104 CFU based on-the optical density at 420 nm into lactated Ringer's solution. Seven days following the last boost injection for each group, diluted pneumococci were injected intravenously (tail vein) in a volume of 0.2 ml and plated on blood agar plates to confirm the numbers of CFU per milliliter. The final challenge dose was approximately 50-100 times the

SLD

5 s of each pneumococcal strain listed in Tables 4-6.

The survival of the mice was followed for 21 days.

Animals remaining alive after 21 days were considered to have survived the challenge.

Statistical analysis. Statistical significance 'i of differences in days to death was calculated with the Wilcoxon two-sample rank test. Statistical significance of survival versus death was made using the Fisher exact test. In each case, groups of mice immunized with PspA containing preparations were compared to unimmunized I6 controls, or controls immunized with preparations lacking PspA. One-tailed, rather than two-tailed, calculations were used since immunization with PspA or fragments of PspA has never been observed to cause a statistically significant decrease in resistance to infection.

Cloning into pMAL vector. Using primers based on the sequence of Rxl PspA, LSM4 and LSM6, pspA gene fragments were amplified by PCR from fifteen out of fifteen pneumococcal strains examined. Seven of the eleven gene fragments were cloned into pMAL-p2 and transformed into E. coli (Table The correct insert for each new clone was verified by agarose gel electrophoresis and Southern hybridization analysis.

Plasmids from recombinant E. coli strains BAR36A, BAR39, S BAR66, BAR9739, BARL5, BAR6A and BAR100 were isolated, digested with BamHI and Sail restriction endonucleases and electrophoresed on a 0.7% TBE agarose gel. The gel was then denatured and the DNA transferred to a nylon membrane for southern hybridization. The blot was probed with full-length Rxl pspA DNA at high stringency .4 conditions. The cloning of R36A, D39, A66, BG9739, DBL6A and LM100 pspA DNA into pMal-p2 was confirmed by the recognition of all BamHI and SalI digested

DNA

inserts by the Rxl probe.

Expression and conformation of truncated recombinant proteins. The transformed E. coli strains BAR36A, BAR39, BAR66, BAR9739, BARLS, BAR6A and BAR100 were grown in LB media supplemented with 10 mM glucose and induced with 2 mM IPTG for expression of the truncated PspA protein fused with maltose binding protein. Transformed E. coli strains BC100 and BAR416, 'O which express PspA fragments fused to the ompA leader sequence in the pIN-III-ompA3 vector, were grown in minimal medium and induced with 2 mM IPTG for expression.

Both vectors, pIN-III-ompA3 and pMal-p2, are vectors in which fusion proteins are exported to the periplasmic Sspace. Therefore, an osmotic shock extract from the pMal-p2 containing bacteria was then run over an amylose column for purification and resolved by SDS-PAGE western blotting. The western blot of the protein extracts from BAR36A, BAR39, BAR66, BAR9739, BARL5, BAR6A and BAR100 o were recognized by a rabbit polyclonal antibody made to strain BC100 PspA. The apparent Mr of full-length PspA S from WU2 is 91.5 kD. The Mr of maltose binding protein is S 42 kD and the expected Mr for the PspA portion of the fusion is 12 kD. All extracts exhibited molecular weights that ranged from 54 to 80 kD. This range of molecular weights can be attributed to the variability of pspA among different pneumococcal strains. An ELISA, with plates coated with the various cloned fragments o quantitatively confirmed the reactivities that were observed in the western blots with all protein extracts.

Protection and cross- rotection against fatal Pneumococcal infection elicited by cloned PspA frarments.

CBA/N mice were immunized with the truncated PspA fragment encoded by pBC100, which is composed of amino 3 acids 192 to 588 of Rxl PspA, and challenged with 13 different S. pneumoniae strains representing 7 different capsular types (Table With all 13 strains, the immunization resulted in protection from death or an extended time to death. With 10 of the strains the difference was statistically significant. With strains of capsular types 3, 6A, and 6B, all immunized mice were protected against death. Although there were fewer survivors in the case of capsular types 2, 4, and 5, the immunization with BC100 resulted in significant increases in times to death.

The BC100 immunization studies made it clear /V that epitopes C-terminal to residue 192 could elicit cross-protection. The BAR416 fragment, which includes amino acids 192-299, could elicit protection from fatal infection with a single challenge strain WU2. This Example shows the ability of BAR416 immunization to 6 protect against the 6 strains that had been best protected against by immunization with BC100.

Immunization with the BAR416 construct resulted in increased time to death for all 6 challenge. strains examined (Table BAR416 provided significant protection against death with WU2, A66, BG7322 and EF6796 pneumococci (capsular types 3, 3, 6B and 6A respectively). It also prolonged the lives of mice challenged with ATCC6303 and DBL6A pneumococci (capsular types 3 and 6A respectively). Serum from mice immunized with the BAR416 fragment yielded a geometric mean reciprocal anti-PspA ELISA titer to full-length Pxi PspA of 750. Mice immunized with BClOO had geometric mean reciprocal titers of close to 2000, while non-immunized mice had anti-PspA titers of 30The above data indicates that the BAR416 fragment from Rxl elicits adequate cross-reactive immunity to protect against diverse pneumococci and suggests that this region must be serologically conserved among PspAs. This hypothesis was confirmed by immunized with recombinant BA.R4 16 homologous regions from the 7 different clones and then challenging with strain WU2 (Table All 7 immunogens elicited significant protection. PspA fragments from capsular types 2 and 22 and the rough R36A strain elicited complete protection against death with all challenged mice. All of the other immunogens were able to extend the time to death of all the mice with the median days to death being 21 days or >21 days. Serum from mice immunized with the BAR416 homologous fragments had anti-PspA reciprocal titers that ranged from 260 to 75,800 with an average of 5700 while control animals immunized with only maltose binding 'o protein had anti-PspA reciprocal titers of Antibody reactivities. All of the above immunization studies attest to the cross-reactivity of epitopes encoded by amino acids from position 192-299.

This region includes the C-terminal third of the a- 1S helical region and the first amino acids of the proline rich region. Other evidence that epitopes within this region are cross-reactive among different PspAs comes form the cross-reactivity of a panel of nine MAbs all of S which were made by immunization with Rxl PspA. The epitopes of four of the antibodies in the panel reacted S with epitopes mapping between amino acids 192-260. The epitopes of the other five MAbs in the panel map between amino acids 1 and 115 (McDaniel, et al., Microb.

S Pathog. 1994; 17: 323). Each of these 9 MAbs were tested S- for its ability to react with 8 different PspAs in addition to Rxl. The 5 MAbs whose epitopes were located within the first 115 amino acids, reacted on average with only 1 other PspA. Three of the 5 in fact, did not react with any of the other 8 PspAs. In contrast the MAbs 0 whose epitopes map between 192 and 260 amino acids each cross-reacted with an average of 4 of the 8 non-Rxl PspAs, and all of them reacted with at least two non-Rxl PspAs. Thus, based on this limited section of individual epitopes, it would appear that epitopes in the region 3 from 192-260 amino acids are generally much more crossreactive than epitopes in the region from 1-115 amino acids.

The BC100 fragment of Rxl PspaA can elicit protection against the encapsulated type 3 strain WU2.

Although the PspAs of the two strains can be distinguished serologically they are also cross-reactive (Crain, et al., Infect. Immun. 1990; 58: 3293).

The earlier finding made it clear that epitopes crossprotective between Rxl and WU2 PspAs exist. The importance of cross-reactions in the region C-terminal to residue 192 is demonstrated in this Example where 13 iv mouse virulent challenge strains have been used to elicit detectable protection against all of them. The first indication that epitopes C-terminal to position 192 might be able to elicit cross-protection came from our earlier study where we showed the MAbs Xi64, XiR278, XiR1323, and XiR1325, whose epitopes mapped between amino acids 192 and 260 of strain Rxl PspA, could protect against infection with WU2. Moreover, immunization with PspA fragments from 192-588 and 192-299 were able to elicit rotection against infection against WU2. This Example shows that the BC100 Rxl fragment (192-588) elicits significant protection against each of 13 different mouse virulent pneumococci, thereby firmly establishing the ability of epitopes C-terminal to position 192 to elicit .a protective response. The observation that a fusion protein containing amino acids 192-299 fused C-terminal to maltose binding protein could also elicit crossprotection, permits the conclusion that epitopes in this 107 amino acid region of PspA are sufficient to elicit significant cross-protection against a number of 0 different strains.

Evidence that a comparable region of other PspAs is also able to elicit cross-protection cam from the studies where sequences homologous to the 192-299 region of Rxl PspA were made from 5 other PspAs. All 3< of these fragments elicited significant protection against challenge with strain WU2. These data provide some suggestion for serologic differences in crossprotection elicited by the 192-299 region.

Based on present,evidence, without wishing to be bound by any one particular theory, it is submitted S that the PspAs in strains D39, Rxl and R36A are identical. All of the 9 mice immunized with the 192-299 fragments from R36A and D39 survived challenge with WU2.

Only LM100, one of the non-R36A/D39 PspAs, protected as high a percentage of mice from WU2. The difference in 'i survival elicited by the R36A/D39 PspAs and the non-Rxi related PspAs was statistically significant.

The data does indicate however, that all of the differences in protection against different strains are not due to differences in serologic cross-reactivity.

BC100, which is made from Rxl, protected against death in 100% of the mice challenged with 7 different strains of S. pneumonia, but only delayed death with strain D39, Swhich is thought to have the same PspA as strain Rxl.

Thus, some of the differences in cross-protection are undoubtedly related to factors other than PspA crossreactivity. Whether such factors are related to differences in virulence of the different strains in the hypersuceptible Xid mouse, or differences in requirements for epitopes N-terminal to amino acid 192, or difference in the role of PspA in different strains is not yet S known.

These results suggest that a vaccine containing only the recombinant PspA fragments homologous with Rxl amino acids 192-299 is effective against pneumococcal ,o infection. Moreover, the results demonstrate that utility of PspA a.a. 192-299, a.a. 192-260 and DNA coding therefor, e.g. primers N192 or 588 (variants of LSM4 and LSM2) as useful for detecting the presence of pneumococciae by detecting presence of that which binds 3 to the amino acid or to the DNA, or which is amplified by the DNA, by using that DNA as a hybridization probe, or as a PCR primer, or by using the amino acids in antibody-binding kits, assays or tests; and, the results demonstrate that a.a. 192-299 and a.a. 192-260 can be used to elicit antibodies for use in antibody-binding kits assays or tests.

ee Table 4 Protection of mice by immunization with BC100 from Rxl PspA BC100 Immunogen Controls Challenge Strain* Capsule PspA 0 alive Median days 0 alive Median days /0 dead Survival alive

P

Value' 0.02 0.002 Idead Srival le 25 0% 5 0% 1 4 1 i 0% 2 WU2 1 10o0 0/3 0/3 nil0 3A 1 ATCC6303 7 100% 100% 0% 13 100% 0/3 7 1 I00% I EF10197 100%

I

100% EFS668 4 12 1/3 2 5 9 0/3 (NO 4 EF3296 4 2 1/3 25% 5 0/3 Oro/ 31 L81905 0 6 I I BG9739 4 26 0/4 0% 6.5 0/3 5 33 0/5 0% 5 0/3 0% 0% 0% 2 2 2 6 0.004 0.03 0.02

N.S.

N.S.

0.02

N.S.

0.02 0.03 0.03 0.03 BG7322 100% 1/2 100% I I 1 33.3% EF6796 100% 100% t t I- I L 0/3 DBL6SA 6A 19 5/0 1 100% >21 1 5/ 0% >1 i O Mice were challenged with approximately 10 3 CFU/mL of each strain §P values were based on comparison of days alive by a one-tailed Wilcoxon 2 sample-rank test 9 *9 9 9 *99 9 9 9 9** 9* .9 9* 9 9 *9 *9 9*9 *9 99 9 9* *9 9 9 9* 9C** 9* 9*9 *9 9 Table 5 Protecion of mice by immunization with BAR416 from Rxl PspA BCI00 ImmunoWei Controls Challenge Capsule PspA 0 alive %Median dlays .9alive %Median days Strain* type ye 1dead survival allve /Idead Survival a]"v Value' WU2 3 1 4/1 80% >21 0/3 00/ 1 0.0021 ATCC6303 3 7 2/3 40% 13 1/4 20% 4 0.048 A66 3 13 5/0 100% >21 0/5 2 0.004 BG7322 6 24 3/2 60% >21 0/4 7 0.02 EF6796 6A 1 3/2 60% >21 0/5, 00/ 5 0.004 DBL6A 6A 19 0/5 0% 7 0/5 1 2 0.008 Note, mice were challenged with about 10 3 CFU of each strain.

§P values were based on comparison of da alive by a one-tailed Wilcoxon 2 sample-rank test 0 0 0 0 0 0* 0 00 0 0** *0 00 0*0 00 0 *0 0 00 *4 0 0 *0 0 0* 0 0* *0 0 Table 6 Protection of mice against S. pneumoniae WU2 by immunization with BAR416 Analogs of 7 PspAs Immunogen Parent Capsule PspA alive %Median days P value* Strain type type Itotal# Survival alive vs. Map BAR36A R36A 25 4/4 100% 21 0.002 BAR39 039 2 25 5/5 100% >21 0.0008 BAR66 A66 3 13 7/8 88% 21 <0.0001 BAR9739 BG9739 4 26 5/8 63% 21 0.0002 DBL5 5 33 4/8 50% 21 0.03 BARGA DBL6A 6A 19 3/8 60% >21 BAR100 LMV .100 22 ND 5/5 100% 21 0.00018 MBP 0/8 0% 2 P values were based on comparison of days alove by one-tailed Wilcoxon 2 sample-rank test Note, the PspA fragments used for immunization were cloned from products amplified with primers LSMV4 and LSMV6. In addition to the strains listed above, PCR reactions with LSM4 and LSM6 amplified products of the appropriate size strains BG9163, WU2, L81905,, EF6796, EF5668, BG7376, 13(7322 and BG5-8A.

0* 0 0** 0S *0 0* C 0* C 0 0 *0

C

0* 0 *0 9 Table 7 Reactivity of MAbs with PspAs of Different Pneumnococci Donor of test PspA MAb mapping to 1-115 amino acids MAb mapping to 192-260 amino acids Strain Capsule PspA X1126 XiR1224 XiR1526 XIR35 XIR16 X1R1323 X164 XiR278 XIR1325 Type Type lgG 2b 1gM IgG2b IgG2a IgG2a 1gM 1gM IgGi lgG2a RKI rough 25 44 ATCCIO 1813 3 3 EF10197 3 18 BG9739 4 26 L81905 4 23 BG-5-8-A 6A 0 BG9163 6B 21 LM100 22 N.D. WU2 3 1 Note, immunoblot analysis was carried out with the nine MAbs from this study from this study against a panel of nine different pneumococcal, strains. Rxl served as a positive control. The results are presented as (strong reaction), (weak, but clearly positive reation), (difficult to detect), (no reaction). The PspA of all strains gave a positive reaction with rabbit antiserum against PspA. N.D. means not determined. Mapping of epitopes was to fragments of strain Axi PspA.

EXAMPLE 3 ISOLATION OF PspA AND TRUNCATED

FORMS

THEREOF AND IMMUNIZATION

THEREBY

PspA is attached,to the pneumococcal surface through a choline binding site on PspA. This allows for successful procedures for the isolation of FL-PspA. PspA can be released from the surface of pneumococci by elution with 2 percent choline chloride or by growth in a chemically defined medium (CDM) contained 1.2 percent CC (CDM-ET). Since CDM-ET superatants lack high v concentrations of choline, the PspA released into them can be adsorbed to a choline (or choline analog) column and isolated be elution from the column with 2 percent choline chloride

(CC).

This Example describes the ability to obtain PspA by these procedures, and the ability of PspA obtained by these procedures to elicit protection in mice against otherwise fatal pneumococcal sepsis. Native PspA from strains R36A, RX1, and WU2 was used because these strains have been used previously in studies of the o ability of PspA to elicit protective immunity (see, e.g., Examples infra and supra). The first MAbs to PspA were made against PspA from strain R36A and the first cloned fragments of PspA and PspA mutants came from strain Rxl.

Strain Rxl was derived from strain R36A, which was in S2 turn derived from the encapsulated type 2 strain, D39.

PspAs from these three strains appears to be identical based on serologic and molecular weight analysis.

Molecular studies have shown no differences in the pspA genes of strains D39, Rxl, and R36A. The third strain that provided PspA in this Example is the mouse virulent capsular type 3 strain WU2. Its PspA is highly crossreactive with that from R36A and Rxl, and immunization with Rxl and D39 PspA can protect against otherwise fatal infections with strain WU2.

'3 S. pneumoniae Strains of S. pneumoniae used in this study have been described previously (Table Bacteria were grown in either Todd-Hewitt broth with 0.5 percent yeast Sextract (THY), or a chemically defined medium (CDM) described previously 32, 43 Serial passage of stock cultures was avoided. Strains were maintained frozen in THY 20 percent glycerol and cultured from a scraping of the frozen culture.

Recovery of PspA from pneumococci PspA is not found in the medium of growing pneumococci unless they have reached stationary phase and autolysis has commenced 36. To release PspA from *O pneumococci three procedures were used. In one approach were grow pneumococci 100 ml of THY and collect the cells by centrifugation at mid-log phase. The pellet was washed three time in lactated Ringer's solution (Abbot Lab. North Chicago, IL), suspended in a small volume of 2 percent choline chloride in phosphase buffered saline (PBS) (pH incubated for 10 minutes at room temperature, and centrifuged to remove the whole pneumococci. From immuboblots with anti Pspa MAb Xil26 48 at serial dilutions of the original culture, the suspended pellet, and the supernatant, it was evident that this procedure released about half of the PspA originally present on the pneumococci. Analysis of silver stained polyacrylamide gels showed this supernatant to contain proteins in addition to PspA 36 The CDM used in the remaining two procedures was modified from that of Van der Rijn 4 For normal growth it contained 0.03% CC. To cause PspA to be e released during bacterial growth, the pneumococci were grown in CDM containing 1.2 percent choline chloride 0 (CDM-CC), or in CDM containing 0.03 percent ethanolamine and only 0.000,001 percent choline (CDM-ET). In media lacking a normal concentration of choline the F-antigen and-C-polysaccharide contain phosphoethanolamine rather than phosphocholine 49. In CDM-CC and CDM-ET, PspA is released from the pneumococcal surface because of its inability to bind to the cholines in the lipoteichoic acids 36. In addition to releasing PspA from the pneumococcal surface, growth in CDM-CC or CDM-ET facilitates PspA isolation by its other effects on the cell wall. In these media pneumococci do not autolyse 49 thus permitting them to be grown into stationary phase to maximize the yield of PspA. In these media septation does not occur and the pneumococci grown in long chains 36 49 As the pneumococci reach stationary phase they die cease making Psp^A, and rapidly settle out.

Preliminary studies, using serial dilution dot blots to ,i quantitate PspA, indicated that the production of PspA ceases at about the time the pneumococci begin to settle out, with the formation of visible strands of the condensed pneumococcal chains. When the pneumococci began to settle out, the medium was recovered by i centrifugation at 2900 x g for 20 minutes, and filtered with a low protein-binding filter (.45y Nalgene Tissue Culture Filter #158-0045).

For growth in CDM-CC or CDM-ET, the pneumococci were first adapted to the defined medium and then, in the c case of CDM-ET, to very low choline concentrations. To do this, strains were first inoculated into 1 part of THY 0 and 9 parts of CDM medium containing 0.03 percent choline and 0.03 percent ethanolamine. After two subsequent subcultures in CDM containing 0.03 percent choline and 0.03 percent ethanoline (0.1 ml of culture 0.9 ml of pre-warmed fresh medium), the culture was used to inoculate CDM with only 0.003 percent choline (and 0.03 0" percent ethanolamine). These steps was repeated until the strain would grow in CDM-ET containing 0.000,001 3 percent choline and 0.03 percent ethanolamine. It was 0 critical that cultures be passed while in exponential growth phase (at about 10 7 CFU.ml). Even trace contamination of the medium by exogenous choline resulted in the failure of the PspA to be released from the 3~ pneumococcal surface 36 Thus, disposable plastic ware was used for the preparation of CDM-ET media and for growth of cultures. Once a strain was adapted to CDM-ET it was froze in 80 percent CDM-ET and 20 percent glycerol at When grown subsequently the strain was inoculated directly into the CDM-ET. Isolation of native (full-lenqth) PspA SPspA was isolated from the medium of cells grown in CDM-ET using choline-Sepharose prepared by conjugating choline to epoxy-activated Sepharose 50

A

separate column was used for media from different strains to avoid cross-contamination of their different PspAs.

For isolation of PspA from clarified CDM-ET, we used a 0.6 ml bed volume of choline-Sepharose. The column bed was about 0.5 cm high and 1.4 cm in diameter. The flow rate during loading and washing was approximately 3 ml/min. After loading 300 ml CDM-ET supernatant, the column was washed 10 times with 3 ml volumes of 50 mM Tris acetate buffer, pH 6.9 containing 0.25 M NaCl (TAB).

The washed column was eluted with sequential 3 ml volumes of 2 percent CC in TAB. Protein eluted from the column was measured (Bio-Rad protein assay, Bio-Rad, Hercules, The column was monitored by quantitative dot blot.

The loading material, washes, and the eluted material were dot blotted (1 Al) as undiluted, 1/4, 1/16, 1/64, 1/256, and 1/1024 on nitrocellulose. The membranes were then blocked with 1 percent BSA in PBS, incubated for 1 hr with PspA-specific MAbs Xil26 or XiR278, and developed with biotinylated goat-anti-mouse Ig, alkaline phosphatase conjugated streptavidin (Southern Biotechnology Associates Inc. Birmingham, AL), and nitrobluetetrazolium substrate with 5-bromo 4-chloro-3- J indoyl phosphate p-toluidine salt (Fisher Scientific, Norcross

GA)

17 The purity of eluted PspA was assessed by silver-stained (silver stain kit, Bio Rad, Hercules, CA) SDS-PAGE gels run as described previously 2 Immunoblots of SDS-PAGE gels were developed with MAbs Xil26 and XiR2781 7 Isolation of 29 kDa PspA The 29 kDa fragment comprising the N-terminal 260 amino acids of PspA was produced in DH1 E. coli from pJY4306 3 37. An overnight culture of JY4306 was grown in S 100 ml of Luria Broth (LB) containing 50g/ml ampicillin.

The culture was grown at 370C in a shaker at 225 rpm.

This culture was used to inoculate 6 one liter cultures that were grown under the same conditions. When the culture O.D. at 600 nm reached 0.7, 12 grams of cells, as (o a wet paste, were harvested at 4 0 C at 12,000 xg. The pellet was washed in 10 volumes of 25 mM Tris pH 7.7 at 0°C and suspended in 600 ml of 20% sucrose, 25 mM Tris pH 7.7 with 10 mM ethylenediamine tetraacetic acid (EDTA) for 10 minutes. The cells were pelleted by centrifugation (8000 xg) and rapidly suspended in 900 ml of 1 percent sucrose with 1 mM Pefabloc SC hydrochloride (Boehringer Mannheim Corp., Indianapolis, IN.) at 00 C.

The suspension was pelleted at 8000 xg at 4°C. The precipitated from the periplasmic extract by 70 percent AD saturated ammonium sulfate overnight at 4 0 C for S* minutes. The precipitated protein was resuspended in ml of 20 mM histidine 1 percent sucrose at pH 6.6 (HSB).

o Insoluble materials were removed at 1,000 xg at 4°C for 10 minutes. The clarified material was dialyzed versus HSB, passed through a 0.2gm filter and further purified S"a on a 1 ml MonoQ HR 5/5 column (Pharmacia Biotech, Inc., Piscataway, equilibrated with HSB. The clarified material was loaded on the column at 1 ml/min, and the column was washed with 10 column volumes of HSB. The column was then eluted with a gradient change to 5 mM NaCl per minute at a flow rate of 1 ml/min. As detected by immuno blot with Xi126, SDS-PAGE and absorbance, PspA eluted as a single peak at approximately 0.27 to 0.30 M NaC1. By SDA-PAGE the material was approximately 3S percent pure. The yield from 6 liters of culture was 2 mg (Bio-Rad protein assay) of recombinant PspA.

Growth of pneumococci for challenge Mice were challenged with log-phase pneumococci grown in THY. For challenge, the pneumococci were diluted directly into lactated Ringer's without prior washing or centrifugation. In inject the desired numbers of pneumococci, their concentration in lactated Ringer's solution was adjusted to an O.D. of about 0.2 at 420 nM (LKB Ultrospec III spectrophotometer). The number of pneumococci present was calculated at 5x10 8 CFU per ml/O.D. and confirmed by colony counts (on blood agar) of serial dilutions of the inoculum.

Immunization, challenge, and bleeding of mice CBA/CAHN/XID/J (CB A/N) and BALB/cByJ (BALB/c) mice were purchased from Jackson Laboratory Bar Harbor, ME. Mice were given two injections two weeks apart and challenged i.v. two weeks later. Injections without

CFA

were given intrapertioneally in a 0.1 ml of Ringers.

Where indicated, the first injection was given in complete Freund's adjuvant (CFA) consisting of approximately a 1:1 emulsion of antigen solution and CFA oil (Difco, Detroit MI). Antigen in CFA was injected inguinally in 0.2 ml divided between the two hind legs.

All mice were boosted i/p. without adjuvant. When mice were injected with media supernatants or 2 percent choline chloride eluates of whole bacteria, the amounts of material injected were expressed as the volume of media from which the injected material was derived. For example, if the clarified medium from pneumococci grown in CDM-CC or CDM-ET was used for immunization without j dilution or concentration, the dose was described as 100 Al. If the material was first diluted 1/10, or concentrated 10 fold, the dose was referred to as 10 or 1000 pl respectively.

ELISA. for antibodies of PspA Specific modifications of previously reported ELISA conditions 1 7 are described. Microtitration plates (Nunc Maxisorp, P.G.C. Scientific, Gaithersburg MD.) were coated with undiluted supernatants of Rxl and WG44.1 pneumococci grown in CDM-ET or 1 percent BSA in PBS.

Mice were bled retro-orbitally (75 Al) in a heparanized capillary tube (Fisher Scientific, Fair Lawn, The blood was immediately diluted in 0.5 ml of one percent bovine serum albumin in PBS. The dilution of the resultant sera was 1/15 based on an average hematocrit of 47 percent. The sera were diluted in 7 three fold dilution in microtitration wells starting at 1/45. Mab ;o Xi126 was used as a positive control. The maximum reproducible O.D. observed with Xil26 was defined as "maximum The O.D. observed in the absence of immune sera or MAb was defined as "minimum O.D." Antibody titers were defined as the dilution that gives ,4 33 percent of maximum O.D. The binding to the Rxl CDM-ET coated plates was shown to be PspA-specific, since in no case did we observe 33 percent of maximum binding of immune sera or Xil26 on plates coated with WG44.1 CDM-ET or BSA.

Statistical analysis Unless otherwise indicated P values refer to comparisons using the Wilcoxin two-sample rank test to compare the numbers of days to death in different groups.

Mice alive at 21 days were assigned a value of 22 for the a sake of calculation. P values of >0.05 have been regarded as not significant. Since we have never observed immunization with PspA or other antigens to make pneumococci more susceptible to infection the P values have been calculated as single tailed tests. To 0 determine what the P value would have been if a two tailed test had been used the values given should be multiplied by two. In some cases p values were given for comparisons of alive versus dead. These were always calculated using the Fisher exact test. All statistical calculations were carried out on a Macintosh computer using InStat(San Diego, CA). PspA is the major protection-eliciting component released from pneumococci grown in CDM-ET or CDM-CC, or released from conventionally grown pneumococci by elution with 2% CC.

PspA-containing preparations from pneumococci were able to protect mice from fatal sepsis following i.v. challenge with 3 x 10 3 (100 x LDso capsular type 3 S. pneumoniae (Table Comparable preparations from the strains unable to elicit protection. Regardless of the method of isolation the minimum protective dose was derived from pneumococci grown in from 10-30 Al of c medium. We also observed 9 that supernatants of log phase pneumococci grown in normal THY or CDM media could not elicit protection (data not shown). This finding is consistent with earlier studies 36 37 indicating the PspA is not normally released in quantity into the medium of growing pneumococci.

Isolated PspA can elicit protection against fatal infection Although PspA was necessary for these preparations to elicit protection it was possible that it did not act alone. Mice were thus, immunized with purified FL-PspA to address this question.

Isolation of FL-PspA from CDM-ET growth medium We isolated the FL-PspA from CDM-ET rather than from CDM-CC medium or a 2 percent choline chloride a6 elution of live cells, because the high levels of choline present in the latter solutions prevents adsorption of the PspA to the choline residues on the choline-Sepharose column. PspA for immunization was isolated from strain R36A, as the strain is non-encapsulated and the isolated o PspA could not be contaminated with capsular polysaccharide. As a control we have conducted mock isolations from WG44.1 since this strain has an inactivated pspA gene and produces no PspA. The results shown in Table 10 are typical of isolations from 300 ml i3 of CDM-ET medium from R36A grown pneumococci. We isolated 84 Ag of PspA from 300 ml of medium, or about 280 Ag/liter. Based on the dot blot results this appears to be about 75% of the PspA in the original medium, and that CDM-ET from R36A cultures contains about 400 Ag/liter of PspA, or about,0.4 Ag/ml.

No serologically detectable PspA was seen in the CDM-ET from WG44.1 cultures. More significantly there was undetectable protein recovered from the choline-Sepharose column after adsorption of CDM-ET from a WG44.1 culture, indicating that PspA is the only protein that could be isolated by this procedure.

Io Moreover, by silver stained SDS PAGE gel the PspA isolated from R36A appeared to be homogenous (Figure 3).

Although autolysin can also be isolated on choline- Sepharose 20 50 we did not expect it to be isolated by this procedure since autolysin is not released from 'S pneumococci grown in choline deficient medium 36 The immunologic purity of the isolated PspA was emphasized by the fact that immunization with it did not elicit any antibodies detectable on plates coated with CDM-ET supernatants of WG44.1.

o Loading more than 300 ml on the 0.6 ml bed volume column did not result in an increased yield, which suggested that the column capacity had been reached.

However, increasing the depth of the choline-Sepharose bed to greater than 0.5 cm, decreased the amount of PspA eluted from the column, presumably because of nonspecific trapping of aggregates in the column matrix.

The elution buffer contains 50 mM Tris acetate 0.25 M NaCI and 2% choline chloride. Elution without added NaCl or with 1M NaCl resulted in lower yields. Elution with less than 1% CC also reduced yields.

Immunization o fmice with purified R36A PspA For immunization we used only the first 3 ml fraction of the R36A column. Mice were immunized with two injections of 1, 0.1, or 0.01 Ag o fR36A PspA, spaced two weeks apart. As controls, some mice were inoculated with comparable dilutions of the first 3 ml fraction from the WG44.1 column. Purified FL-PspA elicited antibody to PspA at all doses regardless of whether CFA was used as an adjuvant (Table 11). In the absence of CFA the highest levels of antibodywere seen with the 1 Ag dose of PspA. In the presence of CFA, however, the 0.1 Ag Sdose was as immunogenic as the 1 Mg dose.

To test the ability of the different doses of the different doses of PspA to elicit protection against challenge we infected the immunized mice with two capsular type 3 strains, WU2 and A66. Although both of these strains are able to kill highly susceptible

CBA/N

XID mice at challenge doses of less than 102, the A66 strain is several logs more virulent when BALB/c mice are used 47,52. The difference in virulence ofA66 and WU2, was partially compensated for by challenging the immunized CBA/N mice with lower doses of strain A66 than WU2.

After immunization of CBA/N mice with 1 and 0.1 Ag doese of PspA we observed protection against WU2 challenge regardless of whether or not CFA was used as an adjuvant (Table At the lowest dose, 0.01 gg PspA, most of the mice immunized with PspA CFA lived whereas most immunized with PspA alone did not; however, the difference was not statistically significant. When immunized mice were challenged with the more virulent strain A66 survivors were only observed among mice immunized with the 1 and 0.1 Mg doses. There was slightly more protection against fatal A66 infection among mice immunized with CFA than without, but the difference was not statistically significant. When the two sample rank test was used to analyze the time to 3. death of mice infected with A66 we observed a statistically significant delay in the time to death ineach immunized group as compared to the pooled controls.

The 29 kDa N-terminal frarment of PsPA can elicit s protection against infection when inected With CFA We have compared the immunogenicity, with and without CFA, of an isolated 29 kDa fragment composed of O the first 260 amino acids of PspA. Unlike the case with FL-PspA, adjuvant ws required for the 29 kDa fragment to elicit a protective response. This was observed even though the immunizing doses of the 29 kDa antigen used were 10 and 30 Ag/mouse, or about 100 and 300 times the minimum does of FL-PspA that can elicits protection in the absence of adjuvant.

Injection with CFA revealed the presence of additional protection eliciting antigen(s) in CDM-CC. and CDM-ET io growth medium but not in the 2 percent choline chloride eluates of live cells The observation that Freud's adjuvant could have such a major effect on the immunogenicity of the 29 kDa fragment (Table 12), prompted us to reexamine the '6 immunogens described in Table 2 to determine if immunization with adjuvant might enhance protection elicited by PspA-containing preparations or provide evidence for protection eliciting antigens in addition to PspA. By using CFA with the primary injection, the does of PspA-containing growth medium (CDM-CC and CDM-ET) required to elicit protection was reduced fro 10-30 il (Table 9) down to 1 to 3 Al (Table 13). When CFA was used as an adjuvant with CDM-CC and CDM-ET from PspAstrains WG44.1 and JY1119 we were able to elicit protective immune responses if material from 100gl or more of media were injected. Thus, although there were apparently some protection eliciting components other than PspA in CDC-CC and CDM-ET growth media, PspA remained the major protection eliciting component even in 3 the presence of adjuvant.

One of the media used for injection was CDM-ET in which JY2141 had been grown. This medium elicited protection against WU2 challenge even when injected at doses as low as 1 1l. It should be noted that although 3~ this strain does not make full-length PspA,-it secretes a truncated molecule comprising the first 115 amino acids of PspA into the growth medium. Thus, unlike CDM-ET from WG44.1 and JY1119, CDM-ET from JY2141 with 2 percent cc was relatively non-immunogenic even when emulsified with CFA. This result is consistent with the fact that the 115 amino acid N-terminal PspA fragment of JY2141 is not surface attached 3 and would be expected to be washed away prior to the elution with 2 percent

CC.

Extension of studies to BALB/c mice i.p. challenqe route The studies above all involve i.v. challenge of CBA/N mice expressing with the XID genetic defect. The I i.v. route, used in the present studies provides a relevant model for bacteremia and sepsis, but pneumococci have higher

LD

50s when injected i.v. than i.p. CBA/N mice are hypersusceptible to pneumococcal infection because of the XIC defect. This genetic defect prevents them from having circulating naturally occurring antibody to phosphocholine. The absence of these antibodies have been shown to make XID mice several logs more susceptible to pneumococci than isogenic mice lacking the immune defect. From the data in Table 14 it is clear, however, o that immunization with PspA can protect against infection in mice lacking the XID defect even when the challenge is by the i.p. route. Thus, there is no reason to suspect :'that the results presented are necessarily dependent on the use of the CBA/N XID mouse or the i.v. route.

S PspA is highly immunogenic These studies provide the first quantitative data on the amount of purified FL-PspA that is required to elicit protective immunity in mice. The isolated PspA for these studies was obtained by taking advantage of the 3. 30 fact that the C-terminal half of PspA binds to cell surface choline 36 The isolated FL-PspA was found to be highly immunogenic in the mouse. Only two injections of 100 ng of PspA in the absence of adjuvant were reqired to elicit protection against otherwise fatal sepsis with greater than 100 Ld 5 s of capsulat type 3 S. pneumoniae.

When the first injection was given with adjuvant, doses as small as 10 ng could elicit protective response The S potent immunogenicity of PspA, and the ability to isolate it on choline-Sehparose columns provides a demonstration for the possible use of PspA as a vaccine in humans.

A large body of published 17 29, 37 as well as unpublished evidence indicates that the major protection eliciting epitopes of PspA are located in the a-helical (N-terminal) half of the molecule. From the present studies, it is clear that immunization with N-terminal fragments containing the first 115 or 260 of the 288 io amino acid a-helical region are able to elicit protection when given with CFA. However, these fragments were not able to elicit protective responses without CFA. In the case of the both the 115 and 260 amino acid fragments, even immunization at 100 times the minimum dose that is 1* immunogenic for FL-PspA failed to elicit a protective response. This result is consistent with previous results showing that a fragment composed of the Nterminal 245 amino acids 3' 37 could elicit protection against otherwise fatal pneumococcal infection of mice 2o when the immunization was given with CFA 32 In that study no immunization without CFA was attempted. Even though the C-terminal half of PspA may not contain major protection-eliciting epitopes it appears to contain sequence important in the immunogenicity of the molecule 2< as a whole, since the full length molecule elicited much greater protection than the N-terminal fragments. The effect of the C terminal half on antigenicity may be in part that it doubles the size of the immunogen.

Molecules containing the C-terminal half of PspA may also ~5 be especially immunogenic because they exhibit more extensive aggregation than is seen with fragments expressing only the a-helical region 38. Protein aggregates are known to generally be more antigenic and less tolerogenic than individual free molecules 54 O PspA is the maior protection eliciting component of our pneumococcal extracts Evidence that PspA is the major protection eliciting component of the CDM-ET, CDM-CC growth media and the two percent CC eluates was dependent on the use of mutant pneumococci that lacked the ability to produce FL-PspA. More than one pspA mutant strain was used to insure that the failure to elicit protection in the absence of FL-PspA was not a spurious result of non-PspA ,o mutation blocking the production of some other antigen.

Strains WG44.2 and JY1119 contain identical deletions that include the 5' end of the pspA genes and extend about 3 kb upstream of pspA 37 WG44.1 is a mutant of the non-encapsulated strain Rxl and JY1119 was made by transforming capsular type 3 strain WU2 with the WG44.1 pspA mutation. In no case were preparations from WG4.1 and JY1119 as efficient at eliciting protection as those from the PspA+ strains. To rule out the possibility that protection elicited by preparations from the PspA+ strains was elicited by some non-PspA molecule also encoded by a 3kb deletion linked to the mutant pspA genes of WG44.1 and JY1119, we also used strains JY2141 and LM34 26 37 In these strains the Rxl pspA gene has been insertionally inactivated causing the production of N- 2 terminal fragments of 115 and 245 amino acids respectively. These strains (have no other known mutations. Although Rxl and R36A are closely related non-encapsulated strains, some of the studies included Rxl as the PspA+ control since it is the isogenic partner o to WG44.1, LM34, and JY2141. The N terminal fragments produced by JY2141 and LM34 lack the surface anchor and are secreted into the medium 36. Two percent CC eluates of JY2141 were non-protection eliciting even in the presence of adjuvant. In the absence of adjuvant, CDM-ET from JY2141 was not protection-eliciting. iM34 was tested without CFA in only 3 mice, but gave results consistent with those obtained witli J2141.

Anticapsular antibodies are known to be protective against pneumococcal infection 5,19 However, in these studies it is unlikely they account for any of the protection we attributed to PspA. Our challenge strain bore the type 3 capsular polysaccharide and our primary source of PspA was strain R36A, which is a spontaneous non-encapsulated mutant of a capsular type 2 strain 39.4. The 36A strain has been recently demonstrated to lack detectable type 3 capsule on the C surface or in its cytoplasm 55 Furthermore, the CBA/N mice used in most of the studies are unable to make antibody responses type 3 polysaccharide 56 Non-PspA protection components The observaiton that CDM-CC and CDM-ET supernatants of WG44.1 could elicit protection when injected in large amounts with adjuvant, suggested that these supernatants contained at least trace amounts of non-PspA protection eliciting molecules. In the case of preparations containing PspA eluted from the surface of 0 live washed pneumococci with 2 percent CC, there was no evidence for any protection eliciting components other :.:than PspA, presumably because the protection-eliciting non-PspA proteins released into the media were removed by the previous washing step. The identity of the 2 protection eliciting molecules in the WG44.1 supernatant are unknown. In this regard, it is of interest that unlike R36A, strain Rxl has been shown to contain a very S. small amount of cytoplasmic type 3 polysaccharide (but totally lacks surface type 3 polysaccharide 55). This S do difference from Rxl apparently came about through genetic manipulations in the contruction of Rxl from R36A 39 Thus, preparations made from Rxl or from its daughter strains WG44.1, LM34, or JY2141, could potentially contain small amounts of capsular polysaccharide. For a C number of reasons however, it seems very unlikely that the non-PspA protection-eliciting material identified in these studies was type 3 capsular polysaccharide (expressed by the WU2 challenge strain: 1) growth of these strains was either in CDM-CC or CDM-ET, each of which prevent autolysin activity and lysis 57 that would be required to release the small amount of type 3 polysaccharide from the cytoplasm of the Rxl family strains; 2) CBA/N mice made protective responses to the non-PspA antigens, but express XID immune response deficiency which permits responses to proteins, but blocks antibody to most polysaccharides 46, including type S3 capsular polysaccharide 56; and 3) immunogenicity of the non-PspA component required CFA, an adjuvant known to stimulate T-dependent (protein) rather than T-independent (polysaccharide) antibody responses.

A number of non-PspA protection eliciting 'S pneumococcal proteins have been identified: pneumolysin, autolysin, neuraminidase, and PspA which are 52, 36.5, 107 and 37 kDa respectively 2158,59,60. The non-PspA protection eliciting components reported here could be composed of a mixture of these and/or other non- 0 identified proteins. Attempts to identify lambda clones producing non-PspA protection eliciting proteins as efficacious as PspA have not been successful 25 Isolation of PsPA The protection capacity CDM-CC, CDM-ET and material eluted from live cells with 2% CC were similar in. terms of the volume of the original culture from which the injected dose was derived. The major advantage of eluting the PspA from the surface of pneumococci with 2 percent CC is that the pneumococci may be grown in any G standard growth medium, and do not have to be first adapted to a defined medium. Moreover, concentration of PspA can be accomplished by centrifugation of th epneumococci prior to the elution of the PspA. An advantage of using either CDM-CC and CDM-ET media was that these media prevented lysis and pneumococci could be grown into stationary phase without contaminating the preparations with cytoplasmic contents and membrane and wall components. A particular advantage of CDM-ET growth medium is that since it lacks high concentrations of choline of PspA contained in it can be adsorbed directly to a choline-Sepharose column for affinity purification.

One liter of CDM-ET growth medium contains about 400 Ag of PspA, and we were able to isolate about 3/4 of it to very high purity. At 0.1 pg/dose, a liter of CDM-ET contains enough PspA to immunize about 4,000 mice or possibly 40-400 humans. Our present batch size .o for a single column run is only 300 ml of CDM-ET. This could presumably be increased by increasing the amount of the adsorbent surface by increasing the diameter of the column. Using our present running buffer we have found that a choline-Sepharose resin depth of 0.5 cm was optimal; increases beyond 0.5 cm caused the overall yield to decrease rather than increase, even in the presence of larger loading of R36A CDM-ET.

o* *~o

C

0@O 0 ~e 0 See

B

C 'I S 0 0 p *S Ce 0 0. 65 V..C C 0 0 0. 0 S Table 8 Pneumococcal Strains strain capsule Type: PspA Expressed Patent Construction References [strain -technique D39 2 full length clinical isolate 26,44 R36A non-encapsulated full length D39 non-encapsulated 23,44,45 mutuant Rxl non-encapsulated full length R36A derived from 26,39,41 WG44.1 non-encapsulated none Rxl aberrant263 insertion263 activation with pKSD300 LM34 non-encapsulated aa-l-245 of Rxl 8 Rxl insertional 2,74 inactivation 2,74 with pKSD300 JY2141 non-encapsulated aa-l-l1S of Rxla Rxl insertional 37 inactivation with pJY42O8 WLJ2 3 full length clinical isolate 25,46 JY1l9 3 none WU2 transformation 37 WG44.lDNA A66 3 full length clinical isolate 1 44,47 LM])34 and LY2141 express fragments containing the first 245 and first 115 amino acids of Rxl PspA respectively.

Table 9 PspA is the major protection-eliciting component in antigen preparations made by three different methods Preparation Strain (PupA Status) Dose as volume of media in Median Days Alive Alive: P versus Dead controlsb ul* 2% CC eluate from live cells R3 6A (PsPA+) JY2141 (aal-115)

CDH-CC

clarif ied medium Rx 1 (PepA+) LM3 4 WG44. I (PsPA*) WU2 (pspA+) JY1l19 (pspA*)

CDM-CC

R3 6A (papA+) JY2 141 (aal-115) WG44. 1 (pspA*) 1000 200 20 2 all R36S 1000 200 100 30 10 3 All 100 100 30 4 1000 100

ALL

1000 100 100 10 100 10 >21 >21 >21 1.5 >21 3, >21 1 1 >21 >21 2 2 2, >21 2 ,2 ,>21 2 2 2 2 >21 >21 >21 4 2 >21 3, >21 1.5 2 >21 1.5 1.5 3 1.5 9: 0 1:2 0:3 12:6 1:2 <0.0001 0.0004 0.2 6:2 0.03

CDM-ET

clarif ied medium 3:0 4:0 0:3 0:2 8:0 5:5 0:2 16:12 0:2 0: 2 0:2 0: 2 0.05 0.03 0001 0.004 0.006 None Antigen dose is given as the volume of growth media from which the 0.1 ml of injected material was derive. Each mouse was injected twice i-p. with the indicate doe diluted as necessary in lactated Ringer's injection solution.

Controls used for statistical comparisons: A% CC, all JY2141; CDM-CC Rxl, all WG44.l; CDM-Cc WU2, JY2141 all JY2141.

*9 Table 10 Isolation of PspA from 300.1 of CDM-ET media after growth of R36A or W044.1 pneumococci' R3 6A W044.1 Fraction Pg total p max total Pg total p max protein/mi protein reciprocal dot blot protein Protein reciprocal dot blote unlits'," per/mi dot blee growth media 13.3 3,990 4 1200 13.7 4,110 <1 fall-through 13.6 4,080 1 300 13.5 4,050 <1 lst wash <1 wash <1 elution 11 26 78 256 770 <1 <1 elution 12 2 6 16 48 <1 <1 elution 13 <1 -4 12 <1 <1 total eluted 84 830 <1 The columns were loaded with 300 ml of clarified CDM-ET medium after the growth of R36A or WG44.1. The column was washed with 10 sequential 3 ml fractions of TRA. Elution was with TBA plus 2 percent CC.

b Total pig protein or total dot blot units reflect the total in the 300 ml of the loading material or the 3 ml size of the eluted fractions.

C Ab XiR278 was used in the irnmunobiota to detect PspA in dot blots.

d Dot blot units were calculated as the reciprocal as the reciprocal dot titer times the volume in mi.

4 .4 4 *4 4* *4 4 4 4 4 .4 4 4* 4 4* 4 Table 11 Purified full-length PspA is able to elicit protection against fatal sepsis in mice Anti-PspA Challenge with Challenge with s WU2 1042 A.66 Antigen Dose' Adjuvant or titer b Alive: Median P vs. Alive: Median P vs.

Diluent (Log mean Dead Days pooled Dead Days pooled Alive controlc Alive controls' R36A 1 pg Ringer's 3.3 0.2 5:0 >21 0.015 2:3 4 0.002 (PspA*) 0.1 Ringer's 2.6 0.2 4:0 >21 0.041 1:4 4 0.0032 0.01 Ringer's 2.7 0.2 1:4 4 n.s. 0:5 3 0.0058 1 pg CFA 3.5 0.2 5:0 >21 0.027 3:2 >21 0.0012 0.1 CFA 3.6 0.1 5:0 >21 0.013 2:3 4 0.0012 0.01 CFA 3.1 0.2 4:1 >21 0.015 0:5 3 0.0058 WG44.1 3600 pl Ringer's <1.6 n.d n.d 1:4 3 n.s (PspA') 360 Ringer's <1.6 n.d n.d 0:5 2 n.9 36 Ringer's <1.6 n.d n.d 0:5 2 n.s 3600 pl CFA <1.6 n.d n.d 0:5 2 n.s 360 CFA <1.6 n.d n.d 1:4 2 n.s 36 CFA <1.6 n.d n.d 0:5 2 n.s saline CFA <1.6 1:5 4 n.d. n.d.

pooled <1.6 1:5 4 2:28 2 controls For comparison with the data in Table 2, it should be noted that the 1, 0.1 and 0.01 pg doses were derived from 3600, 360, and 36 pl of R36A growth media. Equivalent dilutions of the PspAeluate from strain WG44.1 were injected as controls. The amount of the WG44.1 preparations injected is listed as 3600, 360, and 36 pl and corresponds to the volume original growth medium from which the doses of WG44.1 was prepared.

b Antibody values were expressed as reciprocal ELISA titer.

C P values calculated by the Wilcoxon two sample rank test. By Kruskal-Wallis nonparametric

ANOVA

for the WU2 challenge was significant at P=0.01, for A66 significance was a P<0.0001.

Table 12. The 29 kDa N-terminal fragment of Rxl PspA must be injected with aojuvant to elicit against WU2" ug 29 kDa Adjuvant or Median Days P versus PspA diluent Alive Alive: Dead noneb CFA >21 3 0 0.0006 3 CFA >21 Ringer's 2 0 3 3 Ringer's 2 1: 2 none CFA 2 0: 7 none Ringer's 2 0: 7 The 29 kDa fragment comprises the first 260 amino acids of PspA.

b For the calculation of P values the 30ug and 3 ug data were pooled; mice immunized with PspA CFA were compared to CFA controls; mice immunized with PspA Ringer's were compared to controls immunized with Ringers's.

Only the statistically significant P values are shown. The calculated P value of PspA CFA versus CFA alone, was 0.0006 by both the Wilcoxon two sample rank test and the Fisher exact test.

Table 13 PspA is not the only protection eliciting molecule released from pneumococci by interference with binding to choline on the surface of pneumococci Strain Dose (as Median Alive: P values" Preparation (PspA volume Day Dead status) in ul) Alive 2% CC eulate from live calls R36A (PspA*) JY2141 (aa 1-115)

CDM-CC

clarified medium

CFA

Rxl (PepA') WU2 (PapA WG44.1

(PSPA-)

JYI11I9

(PSPA*)

R36A (PspA*) 1000 200 2 all R36A 1000 200 2 all JY2141 1000 100 1000 100 3 1000 100 1000 100 1000 10 0.2 10 10 100 2 :0 5: 0 5:0 5:0 17: 0 2 :0 0: 2 0: 2 0:2 2:6 P vs. all JY2141 0.02 0.02 0.02 0.001

CDM-ET

clarified medium

CFA

3: 0 3: 0 3 :0 3 :0 3: 0 5:1 2:4 3: 0 3: 0 3:1 4:0 3:1 0: 4 4 :0 0:2 0:9 0: 4 P versus pooled cont.

0.002 0.002 0.002 0.002 0.002 0.0001 0.002 0.002 0.002 0.004 0.00.4 0.004 0.004 JY2141 (aa 1-115) all JY2141 WG44.i (PspA')

CFA

none CDM-ET only None Pooled Controls' 2 0 :13 in Cases where there were not statistically significant results no P value was shown.

"*Pooled Controls' refers to *CDM-ET only' Data and "None' data.

*e.

C

C C C C e*e

C

C

Ce.

C. Ce Ce C.

.C C C be C C C C C C Ce *4 Ce C Ce. C Table 14 Immunication of BALB/c mice with isolated PspA WU2 S. Pneumoniae elicits protection against Antigen Adjuvant Challenge Days to Death -P VTSS~ftls' Source Dose' doren Log CFU Route R36A (PspA+) lug CFA 4 i.p. 2,>21,>2l,>2 0.06./0.03 WG44.l1 (PspA-) iQ0ul CFA 4 i.p. 1 None CFA 4 i.p. 2,3 R36A (PspA+) lug none 6 i.v. 2,>21,>21,>2 0.06./0.03 WG44.l (PspA*) lC)ul none 6 1 None none 6 iLv. 5,7 Pooled I.V and i.v. or l.p. 0.008/0.0007 iLp. results The lug dose of 1136A PspA was isolated from 100 ul of COM-ET medium. As a control mice were Injected with an corresponding volume of choline-column effluent from a mock isolation of PspA from the PspA- strain WG44.l. The dose of WG344.11 material Is expressed as 100 ul since this Is the volume COM- ET from which the Injected column effluent was derived.

bP values calculated byt Wilcoxon two-sample rank test, TSR, or Fisher exact, FE versus pooled controls for each groupd. "Pooled controls" include data obtained with by injection of "WG44IC" and "none'. The I.p.

and lv. studies EXAMPLE 4 EVIDENCE FOR SIMULTANEOUS EXPRESSION OF TWO PspAs From Southern blot analysis there has been an issue as to whether most isolates of S. pneumoniae has two DNA sequences that hybridize with both 5' and 3' halves of Rxl pspA, or whether this is an artifact of Southern blot. When bacterial lysates have been examined by Western blot, the results have always been consistent with the production of a single PspA by each isolate.

iv This Example provides evidence for the first time that two PspAs of different apparent molecular weights and different serotypes can be simultaneously expressed by the same isolate.

Different PspAs frequently share cross-reactive epitopes, and an immune serum to one PspA was able to recognize PspAs on all pneumococci. In spite of these similarities, PspAs of different strains can generally be distinguished by their molecular weights and by their reactivity with a panel of PspA-specific monoclonal "o antibodies (MAbs).

SA serotyping system for PspA has been developed which uses a panel of seven MAbs. PspA serotypes are S: designated based on the pattern of positive or negative reactivity in immunoblots with this panel of MAbs. Among a panel of 57 independent isolates of 9 capsular groups/types, 31 PspA serotypes were observed. The large diversity of PspA was substantiated in a subsequent study of 51 capsular serotype 6B isolates from Alaska, provided by Alan Parkinson at the Arctic Investigations Laboratory of the Centers for Disease Control and Prevention. Among these 51 capsular type 6B isolates were observed 22 different PspAs based on PspA serotype and molecular weight variations of PspA.

While most pneumococcal strains appear to have 3j two DNA sequences homologous with both the 5' and 3' halves of pspA, site-specific truncation mutations of Rxl have revealed that one these, pspA, encodes PspA. The other sequence has been provisionally designated as the A pspA-like sequence. At present whether the pspA-like sequence makes a gene product is unknown. Evidence that the pspA and pspA-like genes are homologous but distinct groups of alleles comes from Southern blot analysis at I high stringencies. Additional evidence that pspA and the pspA-like loci are distinct comes from studies using PCR primers that permit amplification of a. single product approximately 2Kb in size from 70% of pneumococci. For the remaining 30% of pneumococci no amplification was 1 observed with the primers used.

Evidence for two PspAs: When the strains of MC25-28 were examined with the panel of seven MAbs specific for different PspA epitopes, all four demonstrated the same patterns of reactivity (Fig. The MAbs XiR278 and 2A4 detected a PspA molecule with an apparent molecular weight of 190 KDa in each isolate. In accordance with the previous PspA serotyping system, the 190 KDa molecule was designated as PspA type 6 because of its reactivity with o: XiR278 and 2A4, but none of the five other MAbs in the typing system. Each isolate also produced a second PspA molecule with an apparent molecular weight 82 KDa. The 82 KDs PspA in each isolate was detected only with the MAb 7D2 and was designated as type 34. No reactivity was detected with MAbs Xil26, Xi64, 1A4, or SR4W4. The fact that. all four capsular 6B strains exhibit two PspAs, based on both molecular weights and PspA serotypes, suggested that they might be members of the same clone.

Simultaneous production of both PspAs: 3 0 Results from the colony immunobloting showed that both PspAs were present simultaneously in each colony of these isolates when grown in vitro. All colonies on each plate of the original culture, as well as all of the progeny colonies from a single colony, reacted with MAbs XiR278, 2A4, and 7D2.

Number of pspA genes: O One explanation for the second PspA molecule was that these strains contained an extra pspA gene.

Since most strains contain a pspA gene and a pspA-like gene it was expected that if an extra gene were present one might observe at least three pspA homologous loci in isolates MC25-28. In Hind III digests of MC25-28 each strain revealed a 7.7 and 3.6 Kb band when probed with plSMpspA13/2 (Figure 5A). In comparison, when Rxl DNA was digested with Hind III and hybridized with v plSMpspA13.2, homologous sequences were detected on 9.1 and 4.2 Kb fragments as expected from previous studies (Figure 5A). Results consistent with only two pspAhomologous genes in MC25-28 were also obtained with digestion using four additional enzymes (Table In previous studies it has been reported that probes for the 5' half of pspA (encoding the alphahelical half of the protein) bind the pspA-like sequence of most strains only at a stringency of around 90%. With chromosomal digests of MC25-28 we observed that the So Rxl probe of pLSMpspA12/6 bound both pspA homologous S bands at a stringency of greater than 95 percent. The same probe bound only the pspA containing fragment Rxl at a stringency above 95 percent (Figure Further characterization of the pspA gene was done by RFLP analysis of PCR amplified pspA from each strain. Since previous studies indicated that individual strains yielded only one product, and since the amplification is carried out with primers based on a known pspA sequence, it seems likely that in each case the amplified products represent the pspA rather than the pspA-like gene. When MC25-28 were subjected to this procedure, an amplified pspA product of 2.1 Kb was produced in each case. When digested with Hha 1 digest the sum of the fragments obtained with each enzyme was zx approximately equal to the size of the 2.1 Kb amplified product (Figure These results suggest that the 2.1 Kb amplified DNA represents the amplified product of only O a single DNA sequence. Rxl, by comparison, produced an amplified product of 2.0 Kb and five fragments of 0.76, 0.468, 0390, 0.349 and 0.120, when digested with Hha 1 as expected from its known pspA sequence.

SThe four isolates examined in this Example are the first in which two PspAs have unambiguously been observed. The interpretation that two PspAs are simultaneously expressed by a single pneumococcal isolate is based on the observation that bands of different io molecular weights were detected by different MAbs to PspA. Isolates used in this study were from a group originally selected for study by Brian Spratt because of their resistance to penicillin. It is very likely that all four of the isolates making two PspAs are related f since they share PspA serotypes, amplified pspA RFLPs, chromosomal pspA RFLPs, capsule type, and resistance to penicillin.

The interpretation of studies presented here, showing the existence of two PspAs in the four strains 20 MC25-28, must be set in the context of what is know about the serology PspA as detected by Western blots. PspAs of different strains have been shown previously to exhibit apparent molecular weight sizes ranging from 60 to 200 KDa as detected by Western blots. At least part of this difference in size is attributable to secondary structure. Even for the PspA of a single isolate, band of several sizes are generally observed. Mutation and immunochemistry studies have demonstrated, however, that all of the different sized PspA band from Rxl are made by 3O a single gene capable of encoding a 69 KDa protein. The heterogeneity of band size on Western blots of PspA made by a single strain appears to be due to both degradation and polymerization.

PspA was originally defined by reciprocal 3 absorption studies demonstrating that a panel of MAbs to Rxl surface proteins each reacted with some protein and later by studies using Rxl and WU2 derivatives expressing e various truncated forms of PspA. In both cases it was clear that each MAbs to the PspA of a given strain reacted with the same protein. Such detailed studies have not been done with each of the several hundred human isolates. It is possible that with some isolates, reactivity of the MAbs with two PspAs may have gone unnoticed. This could have happened if all reactive antibodies detected both PspAs of the same isolate, or if the most prominent migration bands from each of the two o PspAs co-migrated. With isolates MC25-28 the observation of two PspAs was possible because clearly distinguishable bands of different molecular weights reacted preferentially with different MAbs.

Applicants favor the interpretation that isolates MC25-28 each make two PspAs, because an alternative possibility, namely, that the 190 KDa PspA detected by MAbs XiR278 and 2A4 might be a dimer of the 84 KDa monomer detected by MAb 7D2, if the epitopes recognized by the different MAbs were dependent on either 2o the dimeric or monomeric status of the protein, seems unlikely since whenever MAbs react with the PspA of a strain, they usually detect both the monomeric and the dimeric forms. No other isolates have been observed where some MAbs detected only the apparent dimeric form of PspA while others detected only the monomeric form.

There could be several possible explanations for the failure to observe two PspAs produced by most strains. 1) All pneumococci might make two pspAs in culture, but MAbs generally recognize only one of them So (perhaps in this isolate there has been a recombination between pspa DNA and the pspA-like locus, thus allowing S that locus to make a product detected by MAb to PspA).

2) All pneumococci can have two pspAs but the expression of one of them generally does not occur under in vitro j growth conditions. 3) The pspA-like locus is normally a nonfunctional pseudogene sequence that for an unexplained reason has become functional in these isolates.

SIt seems unlikely that the expression of only a single PspA by most strains is the result of a phase shift that permits the expression of only the pspA or pspA-like gene at any one time, since many of the strains examined repeatedly and consistently produce the same PspA. In the case of strains MC25-28, the appearance of two PspAs is apparently not the result of a phase switch, since individual colonies produced both the type 6 and the type 34 PspAs.

o Presumably in these four strains, the second PspA protein is produced by the pspA-like DNA sequence.

At high stringency, the probe comprising the coding region of the alpha-helical half of PspA recognized both pspA homologous sequences of MC25-28 but not the pspAi like sequence of Rxl. This finding indicates that the pspA-like sequence of MC25-28 is more similar to the Rxl pspA sequence than is the Rxl pspA-like sequence. If the pspA-like sequence of these strains is more similar to pspA than most pspA-like sequences, it could explain why AO we were able to see the products of pspA-like genes of these strains with our MAbs. The finding of two families of PspAs made in vivo by pneumococci, allows for use of the second PspA in compositions, as well as the use of DNA primers or probes for the second gene for more SQ conclusive detecting, determining or isolating of pneumococci.

Isolates and Bacterial Cell Culture: Pneumococcal isolates described in these studies were cultured from patients in Barcelona, Spain (one adult at Bellvitge Hospital, and three children at an Juan de Dios) between 1986 and 1988 (Table These penicillin resistant pneumococci originally in the collection of Dr. Brian Spratt were shared with applicants by Dr. Alexander Tomasz at the Rockefeller 3- Institute. Rxl is a rough pneumococcus used in previous studies, and it is the first isolate in which pspA was sequenced. Bacteria were grown in Todd-Hewitt broth with yeast extract or on blood agar plates overnight in a candle jar. Capsular serotype was confirmed by cell agglutination using Danish antisera (Statens Seruminstitut, Copenhagen, Denmark) as previously described. The isolates were subsequently typed as 6B by Quellung reaction, utilizing rabbit antisera against 6A or 6B capsule antigen prepared by Dr. Barry Gray.

Bacterial lysates: Cell lysates were prepared by incubating the *o bacterial cell pellet with 0.1% sodium deoxycholate, 0.01% sodium dedecylsulfate (SDS), and 0.15 M sodium citrate, and then diluting the lysate in 0.5M Tris hydrochloride (pH 6.8) as previously described. Total pneumococcal protein in the lysates was quantitated by the bicinchonic acid method (BCA Protein Assay Reagent; Pierce-Chemical Company, Rockford, IL).

PspA serotyping: Serotyping of PspA was performed according to previously published methods. Briefly, pneumococcal cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and developed as Western blots using a panel of seven MAbs to PspA. PspA serotypes were 9 assigned based on the particular combination of MAbs with which each PspA was reactive.

Colony Immunoblottina: A ten ml tube of Todd-Hewitt broth with yeast extract was inoculated with overnight growth of MC23 from a blood agar plate. The isolate was allowed to grow to a concentration of 107 cells/ml as determined by an O.D. of 0.07 at 590nm. MC23 was serially diluted and spread-plated on blood agar plates to give approximately 1 00 cells per plate. The plates were allowed to grow overnight in a candle jar, and a single block agar plate with well-defined colonies was selected. Four nitrocellulose membranes were consecutively placed on the plate. Each membrane was lightly weighted and left in place for 5 minutes. In order to investigate the possibility of phase-variation between the two proteins detected on Western blots a single colony was picked from the plate, resuspended in ringers, and spread-plated onto a blood agar plate. The membranes were developed as Western blots according to PspA serotyping methods.

Chromosomal DNA Preparation: Pneumococcal chromosomal DNA was prepared as in Example 9. The cells were harvested, washed, lysed, and digested with 0.5% (wt/vol) SDS and 100g/ml proteinase K io at 37 0 C for 1 hour. The cell wall debris, proteins, and polysccharides were complexed with 1% hexadecyl trimethyl ammonium bromide (CTAB) and 0.7M sodium chloride at 65 0

C

for 20 minutes, then extracted with chloroform/isoamyl alcohol. DNA was precipitated with 0.6 volumes /1 isopropanol, washed, and resuspended in 10mM Tris-HCL, 1mM EDTA, pH 8.0. DNA concentration was determined by spectrophotometric analysis at 260nm.

Probe preparation: 5' and 3' oligonucleotide primers homologous !0 with nucleotides 1 to 26 and 1967 to 1990 of Rxl pspA (LSM 13 and LSM2, respectively) were used to amplify the S full length pspA and construct probe LSMpspA13/2 from Rxl S. genomic DNA. 5' and 3' oligonucleotide primers homologous to nucleotides 161 to 187 and nucleotides 1093 to 1117 (LSM 12 and LSM 6, respectively) were used to amplify the variable alpha-helical region to construct probe LSMpspA12/6. PCR generated DNA was purified by Gene Clean (BiolOl Inc., Vista, CA) and random primelabeled with digoxigenin-ll-dUTP using the Genius 1 3o Nonradioactive DNA Labeling and Detection Kit as described by the manufacturer (Boehringer Mannheim, Indianapolis, IN).

DNA electrophoresis: For Southern blot analysis, approximately of chromosomal DNA was digested to completion with a single restriction endonuclease, (Hind III, Kpn 1, EcoR 1, Dra 1, or Pst 1) then electrophoresed on a 0.7% agarose gel for 16-18 hours at 35 volts. For PCR analysis, 5ul of product were incubated with a single restriction endonuclease,, (Bcl 1, BamH 1, Pst 1, Sac 1, EcoR 1 Sma 1, and Kpn 1) then electrophoresed on a 1.3% agrose gel for 2-3 hours at 90 volts. In both case, 1 Kb DNA ladder was used for molecular weight makers

(BRL,

Gaithersburg, MD) and gels were stained with ethidium bromide for 10 minutes and photographed with a ruler.

Southern blot hybridization 'o The DNA in the gel was depurinated in 0.25N HCL for 10 minutes, denatured in 0.5M NaOH and 1.5M NsCl for minutes, and neutralized in 0.5M Tric-HC1 (pH 7.2), NaCl and ImM disodium EDTA for 30 minutes. DNA was transferred to a nylon membrane (Micron Separations

INC,

K MA) using a POSIBLOT pressure blotter (Strategene, La Jolla, CA for 45 minutes and fixed by UV irradiation.

The membranes were prehybridized for 3 hours at 42 0 C in formamide, 5X SSC, 5X Denhardt solution, 25mM sodium phosphate (pH 0.5% SDS 3% (wt/vol) dextran sulfate and 500Og/ml of denatured salmon containing formamide, 5X SSC, IX Denhardt solution, 20mM sodium phosphate (pH 0.5% SDS, 3% dextran sulfate, 250Ag/ml denatured sheared salmon sperm DNA and about of heat-denatured diogoxigenin-labeled probe DNA.

5 1 After hybridization, the membranes were washed twice in 0.1% SDS and 2X SSC for 3 minutes at room temperature.

The membranes were washed twice to a final stringency of 0.1% SDS in 0.3X SSC at 65 0 C for 15 minutes. This p::rocedure yields a stringency greater than 95 percent.

So The-membranes were developed using the Genius 1 Nonradioactive DNA Labeling and Detection Kit as described by the manufacturer (Boehringer Mannheim, Indianapolis, IN). To perform additional hybridization with other probes, the membranes were stripped in 0.2N NaOH/0.1%SDS at 40 0 C for 30 minutes and then washed twice in 2X SSC.

Polymerase Chain Reaction

(PCR):

and 3' primers homologous with the DNA encoding the N- and C-terminal ends of PspA (LSMl3 and LSM2, respectively) were used in these experiments.

Amplifications were made using Taq DNA polymerase, MgCI 2 and 1oX reaction buffer obtained from Promega (Madison, WI). DNA used for PCR was prepared using the method previously described in this paper. Reactions were conducted in 50ml volumes containing 0.2mM of each dNTP, and iml of each primer at a working concentration of je 50mM MgCl 2 was used at an optimal concentration of 1.75mM with 0.25 units of Taq DNA polymerase. Ten to thirty ng of genomic DNA was added to each reaction tube.

The amplification reactions were performed in a thermal cycler Research, Inc.) using the following three step program. Step 1 consisted of a denaturing temperature of 94 0 C for 2 minutes. Step 2 consisted of 9 complete cycles of a denaturing temperature of 94 0 C for 1 minute, an annealing temperature of 50 0 C for 2 minutes, and an extension temperature of 72 0 C for 3 minutes. Step 12( 3 cycled for 19 times with a denaturing temperature of :0 94 0 C for 1 minute, an annealing temperature of 60 0 C for 2 minutes, and an extension temperature of 72 0 C for 3 0 0. minutes. At the end of the last cycle, the samples were 00 held at 72 0 C for 5 minutes to ensure complete extension.

9 Band size estimation: 6..4 Fragment sizes in the molecular weight standard and in. the Southern blot hybridization patterns were calculated from migration distances. The standard molecular sizes were fitted to a logarithmic regression o model using Cricket Graph (Cricket Software, Malvern, PA). The molecular weights of the detected bands were 6 estimated by entering the logarithmic line equation obtained by Cricket Graph into Microsoft Excel (Microsoft Corporation, Redmond, WA) in order to calculate molecular 3 weights based in migration distances observed in the Southern blot.

I

'0 00 0 :0 .0 .0 TABLE Restriction Enzyme Strains Examined Restriction Fragments (sizes In kilobases) MC26 MC27 MC28 RX1 H-ind III 7.7, 3.6 9.1, 4.2 Kpn I 11.6, 10.6 10.6, 9.8 EcoR I 8.4, 7.6 7.8, 6.6 Dra I 2.1, 1.1 1.9,0.9 Pst I 14, 6.1 10.0,4.0 TABLE 16 Penicillin Resistant Capsular Serogroup 6 Strains from Spain Isolate Penicillin MIC (Ug/ml) Year Site Hospital 1 1986 sputum Bellvitge MC26 4 1958 ear San Juan de Dios MC27 1 1988 ear San Juan de Dios MC28 2 1988 ?San Juan de Dios ZXAMPLE 5 SOUTHERN BLOT ANALYSIS OF pspAs AND FRAGMENTS OF pspA In this example,, Applicants used oligonucleotides derived from the DNA sequence of pspA of C S. pneumoniae Rxl both as hybridization probes and as primers in the polymerase chain reaction to investigate the genetic variation and conservation of the different regions of pspA and pspA-like sequences. The probes used ranged in size from 17 to 33 bases and included sequences representing the minus 35, the leader, the a-helical region, the proline-rich regions, the repeat regions, and the C-terminus. Applicants examined 18 different isolates representing 12 capsular and 9 PspA serotypes.

The proline-rich, repeat, and leader, regions were highly conserved among pspA and pspA-like sequence.

In the previous Example, it was shown that strain Rxl and most other strains of S. pneumoniae had two homologous sequences that could hybridize with probes encoding the N terminal and C terminal halves of PspA.

2o This conclusion that these were separate sequences was supported by the fact that no matter which restriction enzymes was used there were always at least two (generally two sometimes three or four) restriction fragments of Rxl and most other strains hybridized with Q the pspA probes. When the genome of Rxl was digested with HindIII and hybridized with these, two pspAhomologous sequences were found to be in 4.0 and 9.1 kb fragments. Using derivative of Rxl which had insertion mutations in pspA, it was possible to determine that the 4.0 kb fragment contained the functional pspA sequence.

The pspA-homologous sequence included within the 9.1 kb band was referred to as the pspA-like sequence. Whether or not the pspA-like sequences makes a product is not know, and none has been identified in vitro. Since pspA- 3 specific mutants can be difficult to produce in most strains, and exist for only a limited number of pneumococcal isolates, this Example identifies oligonucleotide probes that could distinguish between the pspA and pspA-like sequences.

The purpose of this Example was to further define both the conserved and variable regions of pspA, and to determine whether the central proline-rich region is variable or conserved, and identify those domains of pspA that are most highly conserved in the pspA-like sequence (and ergo, provide oligonucleotides that can distinguish between the two). Oligonucleotides were used i° and are therefore useful as both hybridization probes and as primers for polymerase chain reaction (PCR) analysis.

Hybridization with oliqonucleotide probes The oligonucleotides used in this study were based on the previously determined sequence of Rxl PspA.

Their position and orientation relative to the structural domains of Rxl PspA are shown in Figure 7. The reactivity of these oligonucleotide probes with the pspA and pspA-like sequences was examined by hybridization with a HindIII digest of Rxl genomic DNA (Table 17). As Zc expected, each of the eight probes recognized the pspAcontaining 4.0 kb fragment of the HindIII digested Rxl DNA. Five of the 8 probes (LSM1, 2, 3, 7, and 12) could also recognize the pspA-like sequence of the 9.1 kb band at least at low stringency. At high stringency four of the probes (LSM2, 3, 4 and 5) were specific for the kb.

These 8 probes were used to screen HindIII digest of the DAN from 18 strains of S. pneumoniae at low and high stringency. For comparison to earlier studies each of the strains was also screened using a full-length pspA probe. Table 23 illustrates the results obtained with each strain at high stringency. Table 18 summarizes the reactivities of the probes with the strains at high and low stringency. Strain Rxl is a laboratory 3. derivative of the clinical isolate, D39. The results obtained with both strains were identical. They are listed under a single heading in Table 23 and are counted 9 as a single strain in Table 28. Although AC17 and AC94 are related clinical isolates, they have distinguishable pspAs and are listed separately. All of the other strains represent independent isolates.

The only strain not giving at least two pspAhomologous HindIII fragments was WU2. This observation was expected since WU2 was previously shown to have only one pspA-homologous sequence and to give only a single HindIII fragment that hybridizes with Rxl pspA. Even at '0 high stringency 6 of the 8 probes detected more than one fragment in at least one of the 18 strains Tables 18 and 23. Probes LSM7, 10 and 12 reacted with DNA from a majority of the strains and detected two fragments in over 59% of the strains they reacted with. In almost /T every case the fragments detected by the oligonucleotide probes were identical in size to those detected by the full-length pspA probe. Moreover, the same pairs of fragments were frequently detected by probes from the 3' as well as the 5' half Rxl pspA. These results are Ac consistent with earlier findings that the pairs of HindIII fragments from individual isolated generally include two separate but homologous sequences, rather than fragments of a single pspA gene.

The differences in the frequency with which the oligonucleotides reacted with (at least one fragment) of the strains in the panel was significant at P 0.0001 by S 2 x 8 chi square). When the oligonucleotides were compared in terms of their ability to react with both fragments of each strain the P value was also 0.0001.

Table 18 gives the percentage of strains reactive with each probe, the percentage in which only one fragment was reactive, and the percentage in which two (or more) fragments were reactive.

The last column in Table 18 give the ratio of 3: strains that showed one reactive HindIII fragment at high stringency divided by the total number of reactive strains. In this column values of 1 were obtained with probes that only reacted with one band in each reactive strain. Such probes are assumed to be those that are most specific for pspA. 'The lowest values were obtained with probes that generally see two bands in each strain.

Such probes are assumed to be those that represent regions relatively conserved between the pspA and pspAlike sequences. At high stringency, probes LSM3 and LSM4 detected only a single HindIII fragment in the DAN of strains they reacted with. These findings suggested .e probes LSM3 and LSM4 were generally detecting alleles of pspA rather than the pspA-like sequence. The observation that the fragments detected by LSM3 or LSM4 were also detected by all of the other reactive probes, strengthened the conclusion that these probes generally detected the pspA rather than the pspA-like sequence.

WU2 has only one pspA-homologous DNA sequence and secretes a serologically detectable PspA. The fact that LSM3 reacts with the single HindIII fragment of WU2 is consistent with the interpretation that LSM3 detects the 2o pspA sequences. Sequences representing the second proline region (LSM1) and the C-terminus (LSM2) appeared to also be relatively specific for the pspA sequences since they were generally detected in only one of the HindIII fragments of each strain.

a Oligonucleotides, LSM12, and LSM10 detected the most conserved epitopes of pspA and generally reacted with both pspA-homologous fragments of each strain (Table 18). LSM7 was not quite as broadly cross-reactive but detected two PspAs in 41% of strains including almost of the strains it reacted with. Thus, sequences representing the leader, first proline region, and the repeat region appear to be relatively conserved not only within pspA but between the pspA and pspA-like sequences.

LSM3, 4, and 5 reacted with the DNA from the smallest 33 fraction of strains of any oligonucleotide (29 percent), suggesting that the portion of pspA encoding 100 the a-helical region is the least conserved region of pspA.

With two strains BG85C and L81905, the oligonucleotides detected more than two HindIII fragments containing pspA-homologous sequences. Because of the small size of the oligonucleotide probes and the absence of HindIII restriction sites within any of them, it is very unlikely that these multiple fragments were the results of fragmentation of the target DNA within the ;0 probed regions. In almost every case the extra oligonucleotides were detected at high stringency by more than one oligonucleotide. These data strongly suggest that at least in these two strains there are 3 or 4 sequences homologous to at least portions of the pspA.

i- The probes most reactive with these additional sequences are those for the leader, the a-helical region and the proline rich region. The evidence for the existence of these additional pspA-related sequences was strengthened by results with BG58C and L81905 at low stringency where ~o the LSM3 (a-helical) primer picked up the extra 1.2 kb "band of L81905 (in addition to the 3.6 kb band) and the LSM7 (proline-rich) primer picked up the extra 3.2 and 1.4 kb bands (in addition to the 3.6 kb band) of BG58C.

Amplification of pspA The utility of these oligonucleotides as PCR primers was examined by determining if they could amplify fragments of pspA from the genomic DNA of different pneumococcal isolates. Applicants attempted to amplify pspAs from 14 diverse strains of S. pneumoniae comprising Av 12 different capsular types using primers based on the Rxl pspA sequence. Applicants observed that the 3' primer LSM2, which is located at the 3' end of pspA, would amplify an apparent pspA sequence from each of the 14 pneumococcal strains when used in combination with LSM1 located in the region of pspA encoding the prolinerich region (Table 19). LSM2 was also used in combination with four other 5' primers LSM1, 3, 7, 8 and 101 12. LSM8 is located 5' of the pspA start site (near the region).

If a predominant sequence of the expected length was amplified that could be detected on a Southern blot with a full-length pspA probe, we assumed that pspA gene of the amplified DNA had homologous sequences similar to those of the pspA primers used. Based on these criteria the primer representing the a-helical sequence was found to be less conserved than the primers ro representing the leader, proline, and C-terminal sequences. These results were consistent with those observed for hybridization. The lowest frequency of amplification was observed with LSM8 which is from the Rxl sequence 5' of the pspA start site. This oligonucleotide was not used in the hybridization studies.

Further evidence for variability comes from differences in the sizes of the amplified pspA gene. The Example showed that when PCR primers LSM12 and LSM2 were do used to amplify the entire coding region of PspA, PCR products from different pneumococcal isolates ranged in size from 1.9 and 2.3 kb (Table 20). The regions within pspA encoding the a-helical, proline-rich, and repeats were also amplified from the same isolates. As seen in 4' Table 20, the variation in size of pspA appeared to come largely from variation in the size of pspA encoding encodes the a-helical region.

Using probes that consisted of approximately the 5' and 3' halves of pspA it has been determined that 3o the portion of pspA that encodes the a-helical regions is less conserved than the portion of pspA that encodes the C-terminal half of the molecule. This Example show using 4 oligonucleotide probes from within each half of the DNA encoding PspA. Since a larger number of smaller probes S3 were used, Applicants have been able to obtain a higher resolution picture of conserved and variable sequences within pspA and have also been able to identify regions f 102 O of likely differences and similarities between pspA and the pspA-like sequences.

The only strains in which the pspA gene has been identified by molecular mutations are Rxl, D39 and WU2. Rxl and D39 apparently have identical pspA molecules that are the result of the common laboratory origin of these two strains. WU2 lacks the pspA-like gene. Thus, when most pneumococci are examined by Southern blotting using full length-pspA as a probe, it 1o is not possible to distinguish between the pspA and pspAlike loco, since both are readily detected. A major aim of these studies was to attempt to identify conserved and variable regions within the pspA and pspA-like loci. A related aim was to determine whether probes based on the Rxl pspA could be identified that, would permit one to differentiate pspA from the pspA-like sequence. Ideally such probes would be based on relatively conserved portion of the pspA sequence that was quite different in the pspA-like sequence. A useful pspA specific probe do would be expected to identify the known Rxl and WU2 pspA genes and identify only a single HindIII fragment in most other strains. Two probes (LSM3 and LSM4) never reacted with more than one pspA-homologous sequence in any particular strain. Both of reacted with Rxl pspA and 2 LSM3 reacted with WU2 pspA. Each of these probes reacted w i t h 4 o f t h e other 15 strains. When these probes S" identified a band, however, the band was generally also detected by all other Rxl probes reactive with that strain's DNA. Additional evidence that the LSM3 and LSM4 350 were restricted to reactivity with pspA was that they reacted with the same bands in all three non-Rxl strains.

Each.probe identifies pspA in certain strains and even when used in combination they recognized pspA in over percent of strains. Probes for the second proline-rich region (LSM1) and the C-terminus of pspA (LSM2) generally, but not always, identified only one pspAhomologous sequence at high stringency. Collectively 103 LSM1, 2, 3, and 4 reacted with 16 of the 17 isolates and in each case revealed a consensus band recognized by most to all of the reactive pr6bes.

By making the assumption that in different strains the Rxl pspA probes are more likely to recognize pspA than the pspA-like sequences, it is possible to make some predictions about areas of conservation and variability within the pspA and pspA-like sequences.

When a probe detected only a single pspA-homologous 1c sequence in an isolate, it was assumed that it was pspA.

If the probe detected two pspA-homologous sequences, it was assumed that it was reacting with both the pspA and pspA-like sequence. Thus, the approximate frequency with which a probe detects pspA can be read from Table 18 as /6 the percent of strains where it detects at least one pspA-homologous band. The approximate frequency with which the probes detect the pspA-like sequence is the percent.of strains in which two or more pspA-homologous band are detected.

Using these assumptions the most variable portion of portion of the pspA gene was observed to be the -35 region and the portion encoding a-helical region.

The most conserved portion of pspA was found to be the repeat region, the leader and the proline rich region.

Although only one probe from the region was used, the high degree of conservation among the 10 repeats in the Rxl sequence makes it likely that other probes for the repeat regions give similar results.

The portion of the pspA-like sequence most o similar to Rxl pspA was that encoding the leader sequence, the 5' portion of the proline rich region, and the repeat region, and those portions encoding the Nterminal end of the proline-rich and repeat regions. The repeat region of PspA has been shown to be involved in 3, the attachment to PspA to the pneumococcal surface. The conservation of the repeat region among both pspA and pspA-like genes suggests that if is PspA-like protein is 28.AUG.2001 18:27 SPRUSON FERGUSON 61 2 92615486, NO.323 P.12/22 104 produced, that it may have a surface attachment mechanism similar to that of PspA. The need for a functional attachment site may explain the conservation of the repeat region. Moreover, the conservation in DNA encoding the repeat regions of the pspA and pspA-like genes suggests that the repeat regions may serve as a potential anti-pneumccoccal drug target. The conservation in the leader sequence between pspA and the pspA-like sequence was also not surprising since similar C conservation has been reported for the leader sequence of other gram positive proteins, such as M protein of group A streptococci. It is noteworthily, however, that there is little evidence at the DNA level that the PspA lead is shared by many genes other than PspA and the possible gene product of the pspA-like locus.

Although the region encoding the C-terminus of pspA (LSM2) or the 3' portion of the proline-rich 9sequence (LSMI) appear to be highly conserved within pspA 9 genes, corresponding regions in the pspA-like sequences p are- either lacking, or very distinct from those in pspA.

The reason for conservation at these sites is not *9 apparent. In the case of the PspA, its C-terminus does not appear to be necessary for attachment, since mutants :lacking the C-terminal 49 amino acids are apparently as tightly attached to the cell surface as those with the complete sequence. Whether these difference from osoA portends a subtle difference in the mechanism of attachment of proteins produced by these two sequences in unknown. If the C-terminal end of the pspA-like sequence, or the 3' portion of the proline-rich sequence in the pspA-like sequence are as conserved within the pspA-like family of genes as it is within pspA, then this region of pspA and the pspA-like sequence serve as targets for the development of probes to distinguish 36 between all pspA and pspA-like genes.

With two strains, some of the oliganucleotide probes identified more than two pspA-homologous 105 Ssequences. In the case of each of these strains, there was a predominant sequence recognized by almost all of the probes, and two or three additional sequences that were each recognized by at least two of the probes. One interpretation of the data is that there may be more than two pspA-homologous genes in some strains. The significance of such sequences is far from established.

It is of interest however, that although the additional sequences is far from established. It is of interest a however, that although the additional sequences share areas of homology with the leader, a-helical, and proline region, they exhibited no homology with the repeat region of the C-terminus of pspA. These sequences, thus, might serve as elements that can recombine with pspA and/or the i pspA-like sequences to generate sequence diversity.

Alternatively the sequences might produce molecules with very different C-terminal regions, and might not be surface attached. If these pspA-like sequences make products, however, they, like PspA, may be valuable as a SO component of a pneumococcal antigenic, immunological vaccine compositions.

Bacterial strains, growth conditions and isolation of chromosomal DNA S. pneumoniae strains used in this study are listed in Table 5. Strains were grown in 100 ml of Todd- Hewitt broth with 0.5% yeast extract at 37 0 C to an approximate density of 5x10 8 cells/ml. Following *harvesting of the cells by centrifugation (2900xg, minutes), the DNA was isolated as previously described 3o and stored at 4°C in TE (10mM Tris, 1mM EDTA, pH Amplification of pspA sequences Polymerase chain reaction (PCR) primers, which were also used as oligonucleotide probes in Southern hybridizations, were designed based on the sequence of pspA from pneumococcal strain Rxl. These oligonucleotides were obtained from Oligos Etc.

(Wilsonville, OR) and are listed in Table 22.

106 PCRs were done with a MJ Research, Inc., Programmable Thermal Cycler (Watertown, MA) as previously described using approximately 10 ng of genomic pneumococcal DAN with appropriate 5' and 3' primer pair.

The sample was brought to a total volume of 50 Al containing a final concentration of 50mM KC1, 10mM Tris- HC1 (PH 1.5 mM MgCl 2 0.001% gelatin, 0.5 mM each primer, 200mM of each deoxynucleotide triphosphate, and U of Taq DNA polymerase. Following overlaying of the ,O samples with 50 Al of mineral oil, the samples were denatured at 94 0 C for 2 minutes. Then the samples were subjected to 10 cycles consisting of 1 minute at 94 0 C, 2 minutes at 50 0 C, and 3 minutes at 720C followed by another 20 cycles of 1 minute at 94 0 C, 2 minutes at and 3 minutes at 72 0 C followed by another 20 cycles of 1 minute at 94 0 C, 2 minutes at 60 0 C, and 3 minutes at 720C.

After all 30 cycles, the samples were held at 72°C for an additional 5 minutes prior to cooling to 4°C. The PCR products were analyzed by agarose gel electrophoresis.

o DNA hybridization analysis Approximately 5ig of chromosomal DNA was digested with HindIII according to the manufacturer's instructions (Promega, Inc., Madison, WI). The digested DNA was electrophoresesed at 35 mV overnight in a 0.8% agarose gels and then vacuum-blotted onto Nytran membranes (Schleicher Schuell, Keene, NH).

Labeling of oligonucleotide with and detection of probe-target hybrids were both performed with the Genius System according to the manufacturer's so instructions (Mannheim, Indianapolis, IN). All hybridizations were done for 18 hours at 42 0 C without formamide. By assuming that 1% base-pair mismatching results in a 1 0 C decrease in Tm designations of "high" and "low" stringency were defined by salt concentration and temperature of post-hybridization washes. Homology between probe and target sequences was derived using calculated Tm the established method. High stringency is 107 defined as 90% or greater homology, and low stringency is 80-85% sequence homology.

108 Table 17. Hybridization of oligonucleotides with Hindlill restriction fragments of'Rxl DNA.

Stringency Oligonucleotide Region Low High LSM12 Leader N.D. 4.0, 9.1 a-helix N.D. LSM3 a-helix 4.0, 9.1 LSM4 a-helix 4.0 LSM7 Proline 4.0, 9.1 4.0, 9.1 LSM1 Proline 4.0, 9.1 4.0, 9.1 LSM1O Repeats N.D. 4.0, 9.1 LSM2 C-terminus 4.0, 9.1 Note. Values indicated are the sizes of restriction fragments expressed as kb.

109

I

Table 18. Summary of Hybridization at High and Low Stringency of 8 Oligonucleotides with Hindll Restriction Fragments of the 17 Pneumococcal Isolates Listed in Figure 2 Oligonucleotide Percent Percent Percent 1 band/ with l1 band with 2 bands with 1 band 2 1 band Low High Low High Low High Low High LSM12 82 59 24 0.29 29 18 12 0.40 LSM3 65 35 41 0 24 35 0.36 1.00 LSM4 35 29 0 0 35 29 1.00 1.00 LSM7 94 71 71 41 24 29 0.25 0.42 LSM1 100 65 53 12 47 53 0.47 0.82 LSM10 94 59 35 0.37 LSM2 88 53 41 12 47 41 0.53 0.78 Note, for all values listed all 17 strains were examined.

if no value is listed, then no strains were examined.

28.AUG.2081 18:27 SPRUSON FERGUSON 61 2 92615486 N0.323 P.13/22 110 Table Amplification of Pneumococcal Isolates using the Indicated 5' Prime Combination with the 3' Primer LSM2 at the 3' end of pspA Primer Region Nucleotide Position Amplified/ Percent Tested Amplified LSM8 -35 47 to 70 2/14 14 LSM12 leader 162 to 188 8/14 57 LSM3 -helical 576 to 598 3/14 21 LSM6 proline 1093 to 1117 12/14 86 LSM1 proline 1312 to 1331 14/14 100 Note, by 2x5 chi square analysis the different primers amplified different amplified different frequencies of pspAs (P 0.0001). The tendency for there to be more amplification with the 3' most primers was significant at P <0.0001.

9* a 9*@ a a* a.

a a a a a ft e o *o oo *o*oo 111 Table 20. Size of amplified pspA fragments in kilobases pspA Primer Pairs number of pspAs Size Range S.D.

Region examined Full length LSM12 LSM2 9 1.9 2.3 0.4 0.17 a-helical LSM12 LSM6 6 1.1 1.5 0.4 0.17 Proline LSM7 LSM9 3 0.23 0 0 Repeats LSM1 LSM2 19 0.6-0.65 0.05 0.01 Note: amplification was attempted with each set of primers on a panel of 19 different pspAs. Data is shown only for pspAs that could be amplified with the indicated primer pairs.

112 Table 21 Pneumnococcal Strains Strain Relevant characteristics WU2 Capsular type 3, PspA type 1 D39 Capsular type 2, PspA type R36A Nonencapsulated mutant of D39, PspA type Rxl Nonencapsulated variant of R36A, PspA type 06L5 Capsular type 5, PspA type 33 DBLGA Capsular type 6A, PspA type 19 A66 Capsular type 3, PspA type 13 AC94 Capsular type 9L, PspA type 0 AC17 Capsular type 9L, PspA type 0 Capsular type 9L, PspA type 0 AC107 Capsular type 9V, PspA type 0 AC100 Capsular type 9V, PspA type 0 AC140 Capsular type 9N, PspA type 18 D109-11B Capsular type 23, PspA type 12 B G9709 Cpua ye9 sAtp BG58C Capsular type 9, PspA type ND L8190 Capsular type 6, PspA type ND LB 195Ca**j~rtn.APzAt r L82233 Capsular type 14, PspA type L82006 Capsular type 1, PspA type 0 113 Table 22 PCR Primers Primer Sequence to 3') LSM 1 CCGGATCCAGCTCCTGCACCAAAAAC LSM2 GCGCGTCGACGGC77AAACCCA1-rCACCATTGG LSM3

CCGGATCCTGAGCCAGAGCAGTTGGCTG

LSM4

CCGGATCCGCTCAAAGAGATTGATGAGTCTG

GCGGATCCCGTAGCCAGTCAGTCTAPAGCTG

LSM6

CTGAGTCGACTGGAGFFVCTGGAGCTGGAGC

LSM7

CCGGATCCAGCTCCAGCTCCAGAAACTCCAG

LSM8 GCGGATCCTTGACCAATATrTACGGAGGAGGC LSM9 G1TFFVrGGTGCAGGAGCTGG LSM 10

GCTATGGCTACAGGTTG

LSM1 1

CCACCTGTAGCCATAGC

LSM 12

CCGGATCCAGCGTGCCTATCUTAGGGGCTGGUT

LSM 13

GCAAGCTTATGATATAGAAATTGTAAC

V

V

V

V Table 23 Hybridization at high strength of eight different PspA probes with Hind III digests of 18 strains of Streptococcus pneumoniae SProbe train Rx1/ WU2 DBL5 0BL6A A66 AC94 AC17 AC40 AC107 AC100 AC140 DC109 BG9709 BG58C L81905 LB2233 LB2006 D39 _0 ri.-RA 4.0.U.1 3.7,5.8 3.0.3.4 3.6,6.3 3.6.6.3 3.2.

I 3.6.6.3 4.0,8.0 3.0.4.0 I '0 'I I LSM12 4.0.9.1 3.7,5.8 3.0.3.4 3.6.6.3 4.0,8.0 4.0 3.3,4.7 3.3,4.7 2.2.

9.6 2.2.

9.6 2.2.

1.4,3.2.

3.6 1.4,3.2.

3.6 3.6,5.2 3.6 1.2.23.

4.0 3.6,6.3 9 3.6 1 3.6 LSM3 4.0 3.8 6.3 LSM4 4.0 LSM7 4.0,9.1 3.8 3.7 3.0,3.4 3.6 32. 3.040 1 3.6 LSM1 4.0.9.1 3.8 3.7,5.8 3.4 6.3 3.2 3.6 4.0 4.0 4.0.9.1 3.8 3.7 3.4 3.6. 3.6.63 3.2 3.6,6.3 4.0 4.0 4.3, LSM2 4.0 3.7 36 3.6 3.6,6.3 4.0 3.0.4.0 2.2 3.8 2.2 3.8 3.3,4.7 2.2Z 0.8 3.8 2.2 3.3,4.7 2.2. 3.2 9.6 3.6 4.7 3.6 3.8 3.6 2.3 3.6 5.2 3.6.5.2 3.7 8.2 1.3,3.7 3.7 3.7 1.3.3.7 4.3, 6.4 4.3, 6.4 I An Note: All probes were tested versus Hindill digests of all strains. If no bands are listed none were detected. Strains Rxl and D39 gave identical results and are shown in a single column. The full name of strain AC109 is AC109-1B 43 115 EXAMPLE 6 RESTRICTION FRAGMENT LENGTH POLYMORPHISMS OF pspA REVEALS GROUPING Pneumococcal surface A (PspA) is a protection eliciting protein of Streptococcus pneumoniae. The S deduced amino acid sequence of PspA predicts three distinct domains; an a helical coiled-coil region, followed by two adjacent proline-rich regions, and ten amino acid repeats. Almost all PspA molecules are crossreactive with each other in variable degrees. However, using a panel of monoclonal antibodies specific for individual epitopes, this protein has been shown to exhibit considerable variability even within strains of the same capsular type. Oligonucleotide primers based on the sequence of pspA from S. pneumoniae Rxl were used to 0 amplify the full-length pspA gene and the 5' portion of the gene including the a-helical and the proline-rich region. PCR-amplified product were digested with Hha I or Sau3A I to visualize restriction fragment length polymorphism of pspA. Although strains were collected from around the world and represented 21 different capsular types, isolates could be grouped into 17 families or subfamilies based on their RFLP pattern. The validity of this approach was confirmed by demonstrating that pspA of individual strains which are known to be clonally related were always found within a single pspA Ifamily.

Numerous techniques have been employed in epidemiological surveillance of pneumococci which include serotyping, ribotyping, pulsed field electrophoresis, 3 multilocus enzyme electrophoresis, penicillin-binding protein patterns, and DNA fingerprinting. Previous studies have also utilized the variability of pneumococcal surface protein A (PspA) to differentiate pneumococci. This protein, which can elicit protective 35 antipneumococcal antibodies, is a virulence factor found on all pneumococcal isolates. Although PspA molecules are commonly cross-reactive, they are seldom antigenically identical. This surface protein is the 116 most serologically diverse protein know on pneumococci; therefore, it is an excellent market to be used to follow individual strains. Variations in PspA and the DNA surrounding its structural gene have proven useful for differentiation of S. pneumoniae.

When polyclonal sera are used to identify PspA, cross-reaction is observed between virtually all isolates. Conversely, when panels of monoclonal antibodies are used to compare PspA of independent io isolation they are almost always observed to express different combinations of PspA epitopes. A typing system based on this approach has limitations because it does not easily account for differences in monoclonal binding strength to different PspA molecules. Moreover, some strains are weakly reactive with individual monoclonal antibodies and may not always give consistent results.

A less ambiguous typing system that takes advantage of the diversity of PspA was therefore necessary to develop and was used to examine the Jc clonality of strains. This method involves examination of the DNA within and adjacent to the pspA locus.

Southern hybridizations of pneumococcal chromosomal DNA digested with various endonucleases, such as Hind III, Dra I, or Kpn I, and probed with labeled pspA provided a means to study the variability of the chromosome surrounding pspA. When genomic DNA is probed, the pspA and the pspA-like loci are revealed. In most digests the pspA probe hybridizes to 2-3 fragments and, digests of independent isolates were generally dissimilar.

o Like the monoclonal typing system, the Southern hybridization procedure permitted the detection of clones of pneumococci. However, it did not provide a molecular approach for following pspA diversity. Many of the restriction sites defining the restriction fragment 3 length polymorphism (RFLP) were outside 6f the pspA gene, and it was difficult to differentiate the pspA gene from the pspA-like locus. In an effort to develop a system to r 117 follow pspA diversity Applicants examined the RFLP of PCR-amplified pspA. Amplified pspA was digested with Sau3A I and Hha I, restriction enzymes with four base recognition sites. To evaluate the utility of this approach pspA from clinical and laboratory strains known to be clonally related as well as random isolates were examined.

Bacterial strains Derivatives of the S. pneumoniae D39-Rx1 family *o were kindly provided by Rob Massure and Sanford Lacks (Figure Eight clinical isolates from Spain and four isolates from Hungary, a gift from Alexander Tomasz.

Seventy-five random clinical isolates from Alabams, Sweden, Alaska, and Canada were also studied.

S PCR amplifications The oligonucleotide primers used in this study are listed in Table 24. Chromosomal DNA, which was isolated according to procedures described by Dillard et al., was used as template for the PCR reactions.

;16- Amplification was accomplished in a 50 .l reaction containing approximately 50 ng template DNA, .25U Taq, AM of each primer, 175 AM MgCl 2 and 200 gM dNTP in a reaction buffer containing 10 AM Tris-HC1, pH 9.0, KC1, 0.1% Triton X-100, 0.01% wt/vol. gelatin. The mixture was overlaid with mineral oil, and placed in a DNA thermal cycler. The amplification program consisted on an initial denaturation step at 94oC, followed by 29 cycles opf 94°C for 1 min, 55 0 C for 2 min, and 72oC for 3 min. The final cycle included an incubation at 72 0 C for o 5 min.

Restriction fragment analysis of PCR-amplified product Aliquots of the PCR mixtures were digested with a Hha I or Sau3A I in a final volume of 20g1 according to manufacturer's protocols. After digestion the DNA fragments were electrophoresed on a 1.3% TBE agarose gel and stained with ethidium bromide. Fragment sizes were estimated by comparison to a ikb DNA ladder (Gibco BRL).

118 W Because of the variability of pspA, and the fact that the entire pspA sequence is known for only one gene, it has not been possible to design primers which amplify pspA from 100% of pneumococcal strains. However, oligonucleotide primers, LSM2 and LSM1, can amplify an 800 bp region of the C-terminal end in 72 of the 72 stains tested. Based on hybridizations at different stringencies, this region was found to be relatively conserved in pneumococcal strains, and thus would not be IC expected to be optimal for following restriction polymorphisms within the pspA molecule.. LSM13 and LSM2, primers which amplify the full length pspA gene, can amplify pspA from approximately 79% 55/75 of the strains tested (Table Stability of amplified RFLP pattern within clonally related pneumococci To -determine the stability of pspA during long passages in vitro, we examined the RFLP pattern of the pspA gene of the derivatives of the S. pneumoniae D39-Rxl family. Rxl is an acapsular derivative of S. pneumoniae D39, the prototypical pneumococcal laboratory strain isolated by Avery in 1914. Throughout the 1900's "spontaneous and chemical mutations have been introduced oe into D39 by different laboratories (Figure During S* this period unencapsulated strains were maintained in Se vitro, and D39 was passed both in vivo and -ro passage. All the derivatives of D39, including Rxl, R6, RNC, and R36A, produced a 1.9 kb fragment upon PCR oze ~amplification of full length pspA. All members of the family exhibited the RFLP pattern. Digestion with Sau3A I of PCR amplified full length pspA revealed a .83, .58, 36 and a .27 kb fragment in all of the D39-rXl derivatives of the family. Digesting the full length pspA with Hha I resulted in bands which were .76. .47, .39, .35, and .12 kb (Figure 9 or Table 26).

The stability of pspA polymorphism was also investigated using pneumococcal isolates which had 119 previously been shown to be clonally related by other criteria, including capsule type, antibiotic resistance, enzyme electromorph, and PspA serotype. Three sets of isolates, all of which were highly penicillin resistant, were collected from patients during an outbreak in Hungary and two separate outbreaks in Spain. PCR amplified full length pspA from the capsular type 19A pneumococcal strains from the outbreak in Hungary, DB18, DB19, DB20, and DB21, resulted in a band approximately 1o 2.0 kb. After digesting full length pspA with Hha I, four fragments were visualized., 89, .48, and .28 kb.

Digestion with Sau3A I yielded five fragments .880, .34, and .10kb. Capsule type 6B pneumococcal strains, DB1, DB2, DB3, and DB4, were obtained from an Soutbreak in Spain. Full length pspA from these strains were approximately 1.9 kb. Digestion of the PCRamplified fragment with Hhs I resulted in four fragments which were .83, .43, .33, and .28 kb. Sau3A I digestion yield a .88, .75, .34, and .10 kg fragments. DB6, DB8, 0 and DB9, which are capsular serotype 23F strains, were isolated from a second outbreak in Spain. DB6, DB8, and DB9 had an amplified pspA product which was 2.0 kb. Hha I digested fragments were .90, .52, .34, and .30 kb and Sau3A I fragments were .75, .52, .39, .22, .20, and kb in size (Figure 10). DB7 had a 19A capsular serotype o and was not identical to DB6, DB8, and DB9. In the D39/Rxl family and in each of the three outbreak families the size of the fragments obtained from the Hha I and the Sau3A I digests totaled approximately 2.0 kb which is ?0 expected if the amplified product represents a single pspA sequence.

Diversity of RFLP pattern of amplified pspA from random pneumococcal isolates PCR amplification of the pspA gene from 5 random clinical pneumococcal isolates yielded full-length pspA ranging in size from 1.8 kb to 2.3 kb. RFLP analysis of PCR-derived pspA revealed two to six DNA 120 9 fragments ranging in size from 100 bp to 1.9 kb depending on the strain. The calculated sum of the fragments never exceeded the size of the 'original amplified fragment.

Not all pneumococcal strains had a unique pspA, and some Sseemingly unrelated isolates from different geographical regions and different capsular types exhibited similar RFLP, patterns. Isolates were grouped into families based on the number of fragments produced by Hha I and Sau3A I digests and the relative size of these fragments.

Based on the RFLP patterns it was possible to identify 17 families with four of the families containing pairs of subfamilies. Within families all of the restriction fragments were essentially the same regardless which restriction enzyme was used. The 5 subfamilies represent situations where two families share most but not all the restriction fragments. With certain strains an FRLP pattern was observed where detectable fragment size differed from the pattern of the established family by less than 100 bp. Since the Zo differences were considered small compared to the differences in the fragment size and the number of fragments between families, they were not considered in family designation. The RFLP pattern of two isolates from six of the families is pictured in Figure 11, Table 27. These families were completely independent of the capsular type or the protein type as identified by monoclonal antibodies (Table 28 and 29).

Previous DNA hybridization studies have demonstrated that the pspA gene of different isolates are JO the most conserved in their 3' region of the gene and more variable in the 5' region of the gene. Thus, if seemed likely that the differences in the pspA families reflected primarily differences in the 5' end of the gene. To confirm this theory, the a helical and proline region of pspA was examined without the amino acid repeats. Nucleotide primers LSM13 and KSH2 were used to 121 amplify this fragment which is approximately 1.6 kb.

Examination of this region of pspA afforded two things.

This primer pair permitted amplification of of the strains which is greater than the 75% of the strains which can be amplified with oligonucleotides which amplify the full length gene. Second, it allowed Applicants to examine if the original groupings which were based on the full length gene coincide with the fingerprint patterns obtained by looking at the 5' half lC of the gene.

Figure 12 contains the same strains which were examined in Figure 11 but the PCR products were amplified with SKH2 and LSM13. The RFLP patterns obtained from digestion of the amplified a helical and proline rich Ri region confirms the original designated families.

However, these primers amplify a smaller portion of the psaA and therefore the difference is the families is not as dramatic as the RFLP patterns obtained from the RFLP pattern of the full length gene.

2O The polymerase chain reaction has simplified the process of analyzing pspA gene and have provided a means of using pspA diversity to examine the epidemiology of S. pneumoniae. Because not all strains contained a unique fingerprint of pspA, RFLP patterns of pspA cannot 2 be used alone to identify the clonality of a strain.

These results indicate the RFLP of PCR-amplified pspA from pneumococcal strains in conjunction with other techniques may be useful for identifying the clonal relatedness among pneumococcal isolates, and that this IC pattern is stable over long passages in vitro.

These findings suggests that the population of S..pspA is not as diverse as originally believed. PCR-RFLP of pspA may perhaps represent a relatively simplistic technique to quickly access the variability of the gene 3 within a population. Further, these findings enable techniques to diagnose. S. pneumoniae via PCR or 122 hybridization by primers on probes to regions of pspA common within groupings.

The sequence studies divide the known strains into several families based on sequence homologies.

S Sequence data demonstrates that there have been extensive recombinations occurring in nature within pspA genes.

The net effect of the recombination is that the "families" identified by specific sequences differ depending upon which part of the pspA molecule is used io for analysis. "Families" or "grouping identified by the half of the alpha-helical region, the 3' half of the a-helical region and the proline rich region are each distinct and differ slightly from each other. In addition there is considerable evidence of other diversity (including base substitute and deletions and insertions in the sequences) among otherwise closely related molecules.

This result indicates that it is expected that there will be a continuum of overlapping sequences of uo PspAs, rather than a discrete set of sequences.

The findings indicate that there is the greatest conservation of sequence in the 3' half of a a- *helical region and in the immediate 5' tip. Because the diversity in the mid half of the a-helical region is 3 greater, this region is of little use in predicting cross-reactivity among vaccine components and challenge strains. Thus, the sequence of 3' half of the alphahelical region and the 5' tip of the coding sequence are likely to the critical sequence for predicting PspA C cross-reactions and vaccine composition.

The sequence of the proline-rich region may not be particularly important to composition of a vaccine because this region has not been shown to be able to elicit cross-protection even though it is highly 3 conserved. The reason for this is presumably because *a antibodies to epitopes in this region are not surface exposed.

123 Based on our present sequences of 27 diverse pspAs we-have found that there are 4 families of the 3' half of the a-helical region and 2-3 families of the very tip the a-helical region. Together these form 6 6 combinations of the 3' and 5' families. This approach therefore should permit us to identify a panel of pspAs with 3' and 5 helical sequences representative of the greatest number of different pspAs. See Fig. 13.

S S S Table 29. Relationship of Capsular type and RFLP family.

RELATIONSHIP BETWEEN CAPSULAR TYPE AND RFLP FAMILY Capsule Type pspA 1 2 3 4 5 6 6A 6B 7 8 9A 9L 9N 9V 10 11 12 13 14 15 19 22 23 31 33 35 ND family A 3 B 1 1 C 2 1 2 2 1 D 1 1 DD 2 E 1 2 1 F 1 1 31 FF 1 1 1 1 G 1 1 H 1 1 2 1 1 1 1 1 I 2 2 4 II 1 J 2 1 1 1 2 2 K 1 1 1 KK 1 1 1 1 L L1 1 M 1 1

MM

Table 24. Oligonucleotides used in this study.

125 Designation Sequence Nucleotide position LSM2 GCG CGT CGA CGG CUT 1990 to 1967 AAA CCC AlT CAC CAT

TGG

LSM1 CCG GAT CCA GCT CCT 1312 to 1331 CCA AAA AC LSM13 GCA AGC TTATGA TAT 1 to 26 AgA AlT TTG TMA C SKH2 CCA CAT ACC GUT TTC TUG lT CCA GCC 1 1333 to 1355 126 Table 25. Amplification of pspA from a panel of 72 independent isolates* of S.

pneumoniae.

LAPSULE TYPE 1 2 3 4 6 6A 6B 7 8 9V 9A 9L 9N 11 12 13 14 19 22 23 33 nd NUMBER OF

STRAINS

EXAMINED

3 1 8 6 1 7 2 6 2 1 3 2 1 3 1 2 2 1 4 2 5 3 1 1 1 3 LSM13 AND LSM2 OF STRAINS

AMPLIFIED

100 100 50 67 100 29 100 100 50 100 100 100 100 100 100 50 0 100 0 50 100 33 100 0 0 100U OF STAINS

AMPLIFIED

100 100 87 100 100 86 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 LSM13 AND SKH2 *.1 100 Our strain collection contains several groups of isolates known to be previously to be clonal and collected for that purpose. The data reported in the table includes only representative isolate from such clonal groups.

127 Table 26. Rxl-D39 derivatives ISOLATE SIZE OF HMA I DIGESTS SIZE OF Sau3A I D39 IET .7,(7K.9 3,.1b8,.8,.6)2 D39 .76,1.479 .39,.35,.12 .83, .58, .36, .27 R800 .76, .47, .39, .35, .12 .83, .58,.36,.27 R0 .76,.47,.39,.35,.12 .83,.58,.36,.27 R6 .76, .47, .39, .35, .12 .83,.58,.36,.27 R61 .76,.47,.39,.35,.12 .83, .58,8.6,.27 R6NC .76,1.47, .39, .35, .12 .83, .58, .36, .27 R36NC .76, .47,39,35,12 .83, .58, .36, .27 Table 27. Strain information and family designation of independent isolates.

STRAIN CAPSULE PspA FAMILY SIZE OF Hha I SIZE OF Sau3A I TYPE TYPE FRAGMENTS

FRAGMENTS

BG9163 GB 21 c 1.55, .35 1.05,.35,.22 EF6796 GA 1 C 1.5, .35 1.05, .35, .22 EF5668 4 12 DD 1.25, .49, .32 1.0,.80,.35 EF8616A 4 ND DD 1.25, .49, .32 1.0, .80, EF3296 4 20 E 1.0, .40, .33 1.15, .50, .34 EF4135 4 ND E 1.0, .40, .33 1.15, .50, .34 BG7619 10 ND F 1.3, .40, .29, .10 .82, .76, BG7941 11 ND F 1.3, .40, .29, .10 .82, .76, BG7813 14 8 H 1.05, .70, .36 .90, .77, BG7736 8 ND H 1.05, .70, .36 .90,.77,.35 IAC1 13 9A ND 1 1.4, .34, .28 AC99 9V 5 1 1.4, .34, .28 1.2,.80 Table 28. Relationship of RFLP family and PsnA tne RELATIONSHIP BETWEEN PSPA TYPE AND RFLP FAMILY PspA Type PSpA 0 1 3 5 8 12 13 16 18 19 20 21 24 25 26 30 33 34 36 37 ND

FAMILY

A1

B

C 2 1 14 D11 DD2 F 114 FF13 G11 H 1 I3 12 21 4I 1 KK 11 3 L11 M1 111

MMI

129 EXAMPLE 7 ABILITY OF PspA IMMUNOGENS TO PROTECT AGAINST INDIVIDUAL CHALLENGE STRAINS CBA/N or BALB cJ mice were given 1 injection of Mg PspA in CFA, followed 2 weeks later by a boost in saline, and challenged between 7 and 14 (average days post boost. Control mice were administered a similar immunization regimen, except that the immunization came from an isogeneic strain unable to make PspA. The PspA was either full length, isolated from i0 pneumococci or cloned full length or BC100 PspA, as little statistical significance has been seen in immunogenicity between full length PspA and BC100. The challenge doses ranged from about 103 to 104 pneunocci in inoculum, but in all cases the challenge was at least 100 i< times LD 50 The results are shown in the following Tables to 60, and the conclusions set forth therein.

From the data, it appears that an antigenic, immunological or vaccine composition can contain any two QC to.seven, preferably three to five PspA, PspAs from R36A and BG9739, alone, or combined with any or all of PspAs from Wu2, Ef5668, and DB15. Note that surprisingly WU2 PspA provided better protection against D39 that did R36a/Rxl/D39, and that also surprisingly PspA from Wu2 2 protected better against BG9739 than did PspA from BG9739. Combinations containing R36A, BG9739 and WU2 PspAs were most widely protective; and therefore, a preferred composition can contain any three PspA, preferably R36A, BG9739 and WU2. The data in this 3. 0 Example shows that PspA from varying strains is protective, and that it is possible to formulate protective compositions using any PspA or any combination of the PspAs from the eight different PspAs employed in Sthe. tests. Similarly, one can select PspAs on the basis 3" of the groupings in the previous Example.- Note additionally that each of PspA from R36A, BG9739, EF5668 and DBL5 are, from the data, good for use in compositions.

130 A note about use of medians rath an averages. Applicants have chosen to express data as median (a non-parametric'parameter) rather than averages because the times to death do not follow a normal distribution. In fact there are generally two peaks.

One is around day 3 or 6 when most of the mice die and the other is at 21 for mice that live. Thus, it becomes nonsensical to average values like 21 or 22 with values like 3 or 6. One mouse that lives out of 5 has a '0 tremendous effect on such an average but very little effect on the median. Thus, the median becomes the most robust estimator of time to death of most of the mice.

Co.o o *o *o 0 *oooo *o *o o *oooo *oo a. a.

a **a TABLE Relative abilily of different PspAs to Protect against each challenge strains of S. pneurnoniae (Summary of statistically significant protection) Vaccine Callenge Caps PspA p3P4 R36A, RxI. JD908fW1I2 JS1020/BG EF3296 EF5668 LB19os .155010.3 JS53020 All Immune besw D39 9739 0815 DBIBA pagedc Straln fye tp amily K a b E DD b if 0D 039 2 25 K WU2 3 1 a .4 .4 +4 .4 .4 A66 3 13 a .4 ENI 01 97 3 is M ATCC6303 3 7 a BG9739 4 26 b 0+ 0 0 0 EF3296 4 1 40 E 0+ 1 1 1 0 1 0 0 +4 EF5668 4 1 2 f DD_ 0 0. 1 0 181905 4 231 b0

}I

+4 1 .4 081-5 5~ 33 11 EF6796 6A I J C DBL6A 6A 19 D SG9163 6B8 21 C 11 BG7322 68 1 24._ C I 0 j indicate in the I I footnote bobw When 'a mnonr showed eianificent potptno egwn death but indivda deta ooab dd mot. the Ar for -a in e 1. Peeoad under beat protona heeStotthtI mwe n, i aw rn eet data ee stetiticey sigrdfcutrt protectiont ageiu: death; 3t 50% protection Ioni death tittCa Oign tti fcnr tOon aae1iut dath: -C 50% protection Iron death s tatieftcay significant delay in death; protection from death e teticat atgnfaant deay in death; 20 protection Irn death. lt a tgifiant protection against death but ntot a igrnticarit delay in death) Not 1tittCal delaGY in fire to death; butit a50% rotection Iron death 0+ W1o eereol delay In tira to death; it 1.5 day .erwiona in medihan timea to death wo z 20% protection hon death.

0 No 60ormatt seterson in t*re 1o death tor poteecin from death.

C**

C

C

C C C CC C. CC C CC C C C C C C.

C C C C C C C *C CCC C TABLE 31 Relative ability of different PspAs to Protect against each challenge strains of S. pneumoniae, (Expressed as Median days Alive post challenge) ~Vaccine Challenge Caps Psp psp4 R3&k. RKI, J09G8/WU2 JSIO20/BG EF3296 EFS668 181906 JS501O.3 JS3020 All Immune All D39 9739 0615 DBLGA control strain typo typo family K a b E DD b 11 D 039 2 25 K 4.5 >21 4 1 5 2 WU2 3 1 a >21 >21 >21 >21 >21 >21 >21 >21 2 A66 3 13 a >21 >21 >21 >21 >21 >21 4 >21 2 EF10197 3 1s M >21 21 >21 2 ATCC6303 3 7 a >21 >21 BG9739 4 26 b 3 >21 6 3. 3 5.13 2 2 3 2 EF329 4 20 e 5 5 4.5 2 2 3 2 EF5M6 4 12 DD 6 2 >21 13 >21 4 >21 5 8 3 181905 4 23 b 5 5 8 6 35 3 3.5 5 2 0815 5 33 11 4 3 3 3.5 6 2 3.5 2 EF6796 6A I C >21 >21 I DB16A' 6A 19 0 >21 8.5 13 9 >21 8 12 >21 las BG9163 68 21 C >21 >21 >21 8832 6 4 C>1>114.6 1 6 >21 12.5 >21 11 1 >217 Note: Bold denotes statIstically significant extension of life at P 0.05. Snmnl font denvotes 0.02 :5 P 0.05; large font denotes P 0.02.

4Median times to death Indicated as 8. >21. are situations where. the mediumn as not within a continuum of values. In those cases the numbers shown are those closest to the median. In these cases the values give are those closest to the calculted median. Fractional values such as 3.6. Indicate that the median Is halfway between two numbers. In this case 3 and 4. As Indicated In the original data (5103B). some exp>eriments were terminated prior to 21 days post Infection. There Is ittle reason to assume, however. that results would have been aigniticanily effected by the early terminatlon's since very few mice Infected with the strains used In those studies, have ever been observed to die later Mhan or 15 days post chlltenge. For statistical purposes all mice alive. at the end of experiments were assumed to have been compietely protected, and for the sake of caicutailons all surviving mice were assigned values of >21.

9 9. 9 S 99 9** 9* 9*9 9* S 9* 9 9 99 *9 S 9 9* 9* TABLE 32 Ability of different PspAs to Protect Against Each Challenge strain of S. pneumoniae (Expressed as increase in survival time in days) (A denotes 50% Immune mice alive) Vaccine PspA Challenge Caps'I PspA IpspA4 R36A, I JD908/WU2 JS102O/ IEF3296 IEF5668 L 8190.5 1JS50lo.3 I IRxiWD 3 9j I BG9739 I I fl I DBL-5 j S3020 DBL6A All immune Best Result Strain family D39 2 25 K_ 2.5 A 3_

A

11 a A A IA i A Al Al 2 ATCC6303 1 31 7~ a I fA I i

A,

A

[-A

A

A

BG973941261 bil A i 4 V K 1 10, 1 1 U tj EF3296 4 20 E 3 3 2.5 0 0 1 3 EF5668 4 12 DD 3 -1 A 10 A 1 A 2 5 A L181905 4 23 b 3 3 6 4 1 3 1 1.536 5 33 1I 2 1 1 1.5 4 0 154 EF6795 6A 1 C AA DI3LBA 6A 19 D A 3 7.5 3.5 A 2.5 6.5 A 7 A BG9163 613 21 C A A

___AA

4 BG7322 6B 24 C A A 7.5 -1 A 5.5 A 4 A A R36A WU2 BG9739 EF3296 EF56568 L81905 DBL5 DB6 l est "ol dontoo olfethicy sigifcant 9,FtwiWCn of Its at P 0.'05. Smal font doinots 0.02<1o <0.05; tog. font deno, P 0.02. Medi incresen "~mOsoW hatd 9 or 1,A dernott, groups w eft dS ndion dons "of fol 1tfn a ontinuum of vsku~. to ihe cene tho tralu give we thoot. eI,..t to t cohe~asW Rationef waluZe eocl w 3.5. Indicate that Out noodion Be hdtfw betwn two nunb., in Noe cao 3 end 4.

0 6 0.

a. a a.

a a a. a *a a a a a a *aa a 0 TABLE 33 Relative ability of different PspAs to Protect against each challenge strains of S. pneumoniae 01 alie '.Oy d JJ~ L.I Id Vaccine PspA Challenge Caps PspA pspA R36A, JD908/WU2 JS1020/ EF3296 EF5668 L81905 JS5010.3 JS3020 All All I Rxl, D39 BG9739 _815 DBL6A immune control Strain type type family K a b E DD b It D D39 2 25 K 38 60 30 38 3 WU2 3 1 a 100 100 100 100 100 100 100 100 A66 3 13 a 75 100 80 75 100 60 20 76 EF10197 3 18 M 100 80 AIL UL-biU 3 f7{ a 1 0 0 8G9739 4 26 b 11 60 13 25 EF3296 4 20 E 25 20 10 EF5668 4 12 DD 22 25 40 L81905 4 23 b in0110 r 0 100 5 f 33 111 10 14 0 EF6795 6A I f C 100 100 0 25 0 0 12 0 0 0 8 0 40 60 0 41 9 0 14 0 14 0 0 29 0 4 0 100 0 0 80 35 4 86 25 80 25 55 6 DBL6A o BG9163 68 21 C 89 80 8G7322 6B 24 C 1 100 60 25 0 8- Bold. demioma siftcary ggnithCnt potectin agakt death at F 0.05. Bold aeom l ftot. rdiaa ,iOnficnt paotaetin eg t death at 0.02 P. 0.05. Bold Ifg. oot. iooatea inncent Wctection a*nt death .t P 0.02.

.0 1%.

*0 0*0 0.: 4.4 e.

44 *4 4 @4 4 4* 4 4 S. 4* 4* 4 0 TABLE 34 Relative ability of different PspAs to Protect against each challenge strains of S. pneumoniae (%pLr!!ected from death at 21 days post challenge) PspA Challenge Caps PspA psp4 R36A, JD908/WU2 JS 1020/ EF3296 EF566B 18 1905 JS5010.3 JS3020 All Best BG9__ DBL5 DBL6A Immune result Strain type type family K a b E DD b 11 D D39 2 25 K 36 59 28 36 59 WU2 3 1 a 100 100 100 100 100 100 100 100 100 A66 3 13 a 71 100 79 74 100 58 16 75 100 EF10197 3 18 M 100 8BO___ 90 100 ATCC6303 3 7 a 100 100 100 BG39739 4 26 b 11 60 13 25 0 25 0 0 12 EF3296 4 20 E 25 20 10 0 0 8 EF5668 4 12 DD 14 18 56 34 100 34 56 -10 35 100 181905 4 23 b 10 0 31 40 0 0 14 0 14 D1315 5 33 11 10 14 0 0 29 0 4 29 EF6796 6A 1 C 100 100 100 DBL6A 6A 19 D GO 22 30 -4 58 22 -4 79 33 79 BG9163 GB3 21 C GO 75836 BG7322 61B 24 C 100 57 22 0 88 22 79 22 52 100

W

wn Goid, denot"o .ttaticlly significant rotectton against death at P 0.05. Bold osoet font, iridiestasignifiowit protecton agart.' deatht .10.02 S P 0.05. gold largo font. indictes inficet protactio against deatho ot P< 0.02.

1 :otocted has bon corrected for my .sviww, Inthel cortol mice4.

neotast 100 a adne im immurs In ort,offflOO% ar-. i onnf. Thu., it daaw. mice We. it do cotrof uu~td. ft.e caiculted protected* i. bee du the obeerved 'S aft. Feted in flu prarin al. The ot* exceto, to thi.ar .it 100% of immnized mice 1h.d. Nagetine numes new, that immnized mim lived thmv did cometol mies. Plo.. tnte ftal u of th-a negative romber. ars. sgnificent "an though we weoofrk a ott tafod toot.

900 0 S 0 0 S 0 @0 0 0 0 00 S SOS 0 0 000 @500 @5 50 S 0S *0 @50 @0 0 *0 00 *S @55 0 5 *0 00 0 @0 0000 @0 005 0 0 TABLE Recommende Immunogens to Protection against the indicated chlallenge strains of S. ptnrlraa Based on projection Score Based on median daps alive and percent protected (numbers refer 12 preference as a vaccine strain with respect to the indicated challenge strain, I1-best) Vaccine PspA Challenge Cap PSpkA psp4 P36A. AxI. JD908/WU2 JS1020/BG EF3296 EFS668 181905 DBLS OBLEA 39 9739 JS501 0.3 JS3020 slrain tp tye family K a b E DD b ii D D39 2 25 K 2 1 3 WU2 3 1 a 1 1 I 1 I I 1 A66 3 13 a 2 1 2 2 1 3 0 EF10197 3 18. M 1 2 ATCC6303 3 7 a 1 869739 4 26 b 3 1 2 3 3 2 0 0 EF3296 4 20 E 1 1 2 0 0 EF5668 4 12 DD 0 0 2 3 1 0 2 0 (.81905 4 23 b 2 0 1 1 0 0 0 0 D815 5 33 it 2 3 0 3 1 0 EF6796 GA I C I DBL6A 6A 19 0 2 0 3 0 2 0 0 1 BG9163 68 21 C I 1 BG7322 68 24 C 1 2 3 13 13 Number of 11 7 1 5 1 3 1 3 232 Sed dsoto Statitically signifiant probection agaist death at P <0.05. Whro twor Ma~ owne PopA -we oqudy protecive. the we given to Rown at er a.w booed on days to death with Protection dividing tie -O'pci.1ty awotg thoe. vvhog gretr than 50% of rroo. 0,.d to 21 day@. indic.to est wwoo Cnductd but wotpod to the other P.PAs the. ons Is rot tooor-*rdetd.

C.-Wi~ena. Swtesty sigifant protectione egeouat death wvith 50% protaction: 11114 of the 79% Stestoaf lqnifircent peootiho ogeiu dsot 13114 a%"n 93% Stetitlceffy a~gnifeent extemion ot rf.e In 14114 or 100% of soi.

137 TABLE 36 Best Choice for Vaccine Components as of 95/8/27 Vaccine Component (cumulative strains protected) maximally protected____ Criterion 1 2 -34 5 6 a# 1 PspA for R36A WU2 BG9739* EF5668 DBL5 DBL6A each challenge (10) (11) (12) (13) (14) strain 50% 71% 79% 86% 93% 100%/ S#2 PspA for R36A BG9739 each challenge (12) (12) strain 86%O 1000% Max score R36A WU2 BG9739 0815 type score (11) (13) (14) 64% 79% 92% 100%/ Max Increase in R36A WU2 BG9739 Days alive (11) (13) (14) 64% 79% 92% 100%/ protected R36A WU2 DBI-5 EF5668 DBLSA EF3296 (10) (11) (12) (13) (14) 64% 79% 88% 92'% 100%/ Theoretical mixture R36A BG9739 DBL5 EF3296 based on a few (10) (12) (13) (14) testable 64% 86% 92% 100%/ assumptions (see below) *This is not a unique combination. See table below.

0* TABLE 37 00 Combinations where all Challenge Strains have a Vaccine strain a score of 2: #2 Number of Combination Number Total #Is Total PspAs in of #ls and Combination I strains #2s 2 R36A +BG9739 8 10 3 R36A +BG9739 11 15 ___WU-2 3 R36A +WU2 11 15 21 DBI-5 3 R36A +WU2 11 15 23 3 R36A +WU2 11 15 22 TABLE 38 138 Pooled Data for Protection againt 039 by various PopAs: alive for each mouse Day, to Dahinuoe Exp. Log CFU MiCe AxI/R8A JOSOB IWU2) EF6a88 AN bywvnuns control 039 039 143 4.5 CBAIN 1.1.2.2.2 1.1.2.2.3 E145 4.0 CBA/N 2.3.3.3.4 1.1.2.3,4 E028 5.93 SALS/c 3,3x 21 2.2.2.4 8CG E143 3.0 C8AIN 2.6.3x> 10 3.3.3.5.5 E140 2.61 CBA/N 4.4.5.7.15 2.2.2 SCI 00 E169 2.7 CSAIN 2,4x >21 2.5.3x >21 1.2.2.2.3 E154 2.8 CBA.JN 2.2.3.2z >2 401. 6x2.3.3,4 All 2,3.3.3,4,4, 1.1.2.2.2 401, 6x2,3.3.4 zc. 4,5.7,15 All 4x2. 5x3. 2.5.3x >21 1.1.2.2.2.2.6. l.1.9x2.5x3.3 5x 1. Il x2.6a3.4.

3x4.5.7. 15, 3x 21 x4.5.5,8.7,1 5. 4.5.5.5, 21 9x>21 15x >21

S

a. a a

S.

S. a S. a *aa a a a a. a a a a..

a a a C a a a a a a a .a a *a C. aC C a a a C. TABLE 39 Pooled Dots for Protection against 039 by various PapA.

Median Days Alive It ofive:doad with, corresponding P values Exp. Log Mice RxI/R36A 039 JD908 (WU21 EF5668 Anl Immvune Control

CFUI

D39 med ad mad a:d med a:d mad s:d mod ad 143 4.5 CBA/N 2 0:5 2 nis El145 4.0 CBAfN 3 0:5 2 n,,a.

E028 acG 5.93 8ALfl/c 21 3:1 2 0:4 .029 nat E143 3.0 CBAIN >21 3:2 3 flu. nas E140 BCIOO 2.81 CEA/N 5 0:5 2 0:3 0.018 69 2.7 CBA/N >21 4:1 >21 3:2 2 .018 .024 .018 n.es E154 2.8 C8A/N 3 2.3 2 n.s. n.e.

Anl s3.0 4 0:10 2 0:5 2 0:13 .0008 ns All 4.5 9:15 >21 3:2 4 2M) 3:7 5 15:24 2 1:32 .0057 .001 .0006 .0045 n.s. .034 .0001 .0002 alive 38 60 30 38 3 38 59 28 38 RxIID39 WU2 EF5688 All immune controls TABLE Data for Protection against W1.2. by various PspAs CFU Days to Death/immunogen Exp. WIJ2 MICE FL-R36A Rx1 JD1O8 JS1020 BG9739 EF5668 181905 DBL5 JS3020 control ____BC11OO (WU2) (BG9739) bc1OO bc1OO bclO00 (DBL6A) Dr. Ed, expt. lots of prior expts. E012 '3.0 CBA/N 15x>21 ____1,1,11x2,7x3,4 E028 6.01 BALB/c 4x>21 4,6,6, 21 E084 3.75' CBA/N 3x> 15 E125 3.57 CBA/N 4x>21 4x>21 4x>21 2.2,3,3,3, >21 E129 3.18 CB3A/N 5x>23 E140 3.43 CBA/N 4x>21 1 ,5x2,3,4 BC1 00 E143 3.0 CBA/N 8x> 10 _____112223 E144 3.9 CB3A/N 5x2 E172 3.98 CBAI/N 5x>21 All 19x >21 4x>21 5x>21 8x>21 4x>21 8x>21 4x>21 4x>21 5x>21 6xl,33x2,20x3, IIIIIIII1_ 4,4,4,6.6, >21 All Immune I61x>21 01 TABLE 41 Pooled Data for Protection against WU2 by various PspAs Exp. CFU MICE Median days Alive Alive:Dead P value based on Alive:Dead WU2 P value calculated compared to pooled controls (in this case 65 control mice) SCORE FL-R36A Rxi JD108 JS1020 BG9739 EF5668 LB 1905 DBL-5 JS3020 control ___BCi100 (U2) (BG9739) bclOO bclOO bc1OO (DBLBA) Dr. Ed, expt. lots of prior expts. E012 '3.0 CBA/N 15x >21 E028 6.01 BALB/c 4x>21 4,6,6, 21 E084 3.75' CBA/N 15 E125 3.57 CBA/N 4x>21 4x>21 4x>21 2,2,3,3,3, >21 bcl 00 E129 3.18 CBA/N 5x>23 E140 3.43 CBA/N 4x> 21 1,5x2,3,4 E143 3.0 CBA/N 8x> 10 1,1,2,2,2,3 E144 3.9 CBA/N x>21 5x2 E172 3.98 CBA/N All >21 >21 >21 >21 >21 >21 >21 >21 >21 2 19:0 4:0 5:0 8:0 4:0 8:0 4:0 4:0 4:0 1:64 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 alive 100 100 100 100 100 100 100 100 100 2 S. S S S S *S S 55

S

S

Data for Protection against WU2 by various PspAs Exp. CFU MICE Median days Alive Alive:Dead P value based on Alive:Dead WU2 P value calculated compared to pooled controls (in this case 65 control mice) FL-R336A Rxl JD108 JS1020 BG9739 EF5668 18 1905 DBL5 JS3020 control _____BC100 (WU2) (BG9739) WcOO bclOO bc1OO (DBL6A) FL-R36A RxlI JD108 JS1020 BG9739 EF5668 L81905 DBLS5 JS3020 control BCI00 (WU2)_ (BG9739) WcOO bc1OO bclOO (DBLBA) WU2 days of median days alive: P value P value Score alive %protected Challenge death of death dead based on based on days to alive:dead All immune 61x>21 >21 61:0 <.0001 <.0001 100 100 All controls 6x1 ,33x2,20 2 1:64 2 2 x3,4 ,4 ,4,6 ,6, I >21

C

C

C

TABLE 42 ______Pooled Data for Protecion against ASS. by various PspAs CFU Days to Death/immunogen Exp. A66 MICE FL-R36A/ Rxl JD908 JS1020 BG9739 EF5668 181905 L81905 JS5O1O.3 DBLS JS3020 control D39 BC100 (WU2) (BG9739) bc1OO FL WcOO FL bclOO (DBLSA) I_ (D1315)_ E169 2.60 CBA/N 5x>21 5x>21 1,1,2,2, 6 E152 2.78 CBA/N 4x>21 4x>21 4x>21 3x2,3,6, WcOO E104 3.0 CBA/N 2,8,3x >22 3,4,4,2x 2,2,2,2.

>22 3 E143 3.0 CBA/N 10 E140 3.43 CBA/N 4x>21 E172 3.94 CBA/N E145 3.97 CBA/N 13,4x >21 1,2,2,2, E121 4.16, CBA/N 3x3,2x4,5 1,8x2,> x>21 All 3x3,2x4.1 4x>21 5x>21 2,8,3x >21 4x>2i 4,4x>21 5x>21 4x>21 3,4,4,2x 4x>21 2,4,4,5, 7x1,22x 3,14x>21 >21 >21 2,3x3,4, 3x6,2x median; >21 >21 >21 >21 >21 >21 >21 >21 4 >21 4 2 A:D 14:6 4:0 5:0 3:2 5:0 4:1 5:0 4:0 2:3 4:0 1:4 2:36 a a a a a a a.

a .a a a. a a a a.

a a a a. a. a a a. _____Pooled Data for Protection against A66. by various PspAs CFU Days to Death/immunogen Exp. A66 MICE FL-R36A/ Rx1 JD908 JS1O2O 8G9739 EFS668 18 1905 L81905 JS5OIO.3 DBI.5 JS3020 control D39 BC100 (WU2) (BG9739) WcOO F L WcOO FIL bclOO (DBL6A) (DBL5) P values <0.0001 0.0002 <0.0001 0.004 0.0002 0.0006 <0.0001 0.0002 0.0025 0.0002 0.015 <0.0001 0.0001 0.000 1 0.0075 <0.0001 0.006 <0.0001 0.0001 n.s. 0.0001 n.s Mini Pools A36A/Rxl/WG44.1 JD908 BG9739 EF5668 181905 DBL5 DBL6A Control 3,4,4,4,6x>21 >21 21 >21 21 21 >21 4 2 5:0 8:2 4:1 9:0 6:4 1:4~ 2:36 P values <0.0001 0.0006 0.015 rank/a:d <0.0001 <0.0001 <0.0001 0.006 <0.0001 0.0004 n.s.

alive 72 100 80 75 100 60 20 71 100 79 74 100 58 '16 0 A66chlngeI R36A/Rx1J/WG4A.1 -JD908 BG9739 EF5668 18 1905 DBL5 DBLSA 9 9 ~9* 9** 9 9 9* 0% 0 A66 days of median days alive: P days to P Score alive protected challenge death alive dead death alive:dead All immune 2,2,4x3,7x4, 21 50:16 <0.0001 <0.0001 76 5,8,13,50x All controls 7x1 ,22x2,3x 2 2:36 5 0 3,4,3x6,2x> TABLE 43 Lii Data for Protection against EF10197. by various PspAs Exp. CFU Mice Days to Death/immunogen 10197 Rxl JS1020 L81905 JS3020 EF5660 JS5O10.3 control BC 100 (BG9739) (DBL6A)

FL'

E140 3.00 CBA/N 5x >21 2,2,2 Ml BCG 2.70 CBA/N *2,2,2,2,2 E129 3.34 CBA/N 8,4x >23 *This was a passive protection study. Its controls have been Included to increase the numbers of control mice.

a 0 a a a a a a a *0 a..

a a a a a a a. *0 a a a a a *a a a a a a a a. a .a a TABLE 44 Pool of Pools for protection against EF10197 Group Delay in death and/or survival S-urvival line Description days to death (median) P values etc. alive:dead P values etc.

la Rxl (E140) 5x 21 0.017 vs lb 5:0 0.018 vs lb vs 4b 0.0008 vs 4b 3a JS1020 (E129) 8, 4x 23 0.0007 vs 3b 4:1 0.024 vs 3b 4a all immune 8, 9x 21 0.0001 vs 4b 9:1 0.0002 vs 4b l b Rxi controls (E140) 2.2,2 2b Ml BCG 2,2,2,2,2 3b JS1020 cont. (E129) 2,2,2,2,9 4b all control (without Ml 2,2,2,2,2,2,2,9 0:8 BCG) TABLE of protection against EF10197 Immunogen alive:dead alive protected median DOD P time alive P alive:Dead Score* Rx1 5:0 100 100 >21 0.017 0.018+++ JS1020 4:1 80 80 21 0.0007 0.024+++ all immune 9:1 90 90 >21 <0.0001 0.0002...

all controls 0:8 0 0 2 0 0 0 0 0 0 I I. *0 I I A *00 0 .0 ~0* 0 00 0 00 a 0 0 0 00* 00 TABLE 46 Data for Protection against ATCC6303. by various PspAs Exp. CFU Mice Days to Death/immunogen

ATCC

6303 Rx1 JS1020 181905 JS3020 EF5668 JS5O1O.3FL control (BG9739) E140 2.30 CBA/N 5 x >21 4,4x5 E129 3.80 CBA/N Ln.y.

TABLE 47 Pool of Pools for protection against ATCC6303 Delay In death and/or survival Sria line Description days to death (median) P values etc. alive: dead P values etc.

1a RxI(E140) 5x,,.21 0.0040 Ib Rx1 controls (E140) 4,4,,5 5 -0: TABLE 48 of protection against ATCC6303 Immunogen alive:dead allve protete median DOD P time alive P alive: dead Score* Rx1 5:0 100 100 210.4004+++ Rx1 controls J 0:5 0 0 statistically significant protection against death with >50% protected.

S S

S

S S S S

S.

S *S*

S

S

S S S S S S. *5 55 5 S S S *5 S S S S S S TABLE 49 Data for Protecion against B09739, by FIL PspAs CFU Mice toDeath/immunogen____ Exp. BG9739 R36A BC1D) JD908 JS1020 WcOO EF329 EF566 WOO0 Js5OlO0 bclOO JS3020 control FIL (Rxl) (WU2) (BG9739 (BG9739) 6 FIL 8 FIL (181905) .3 FL (DBL5) (DBL6A) E140 2.76 CBA/N 2,2.3 E104 2.89 Xid 6.6,7,8,8 2,2,2.3 2,2,2,2, 2,2,3,5,5 3 E125 3.56 C8A/N 5,5,5,7 4,5,13, 2,2,2,4 3,3,4,4,5 ,6 E172 3.71 CBA/N 6,7,3 x 3,4,6,6,7 __>21 E124 3.76 Xid 2,2,2,2 2,2,2,2, 2,2,2,2,2 1 9 E084 4.05 BALB/c 4x2,2x> 1W E144 4.09 Xid 2,3,6, 2.3,3,7, 2,3.3.3 2,2,2,3,3 >10 ,4 All 2,3,3,6 3,3,10,11 6,7,3 x 4X2,6,6,7 5,5,5,7 2,3,3,7, 2,3,3,3 7x2,3, 8x2,3,9 21x2,7x2 2,1 21 ,8,8,2X> >21 .4 3,4 ,3x4,3x5, _2 3x6,7 median 3 3,10 21 6 5 3 3 5,13 2 2 2 2 a:d 1:4 -0:4 3:2 2:9 0:4 1:4 0:5 1:3 0:10 0:4 0:10 0:38 P rank P a:dI

I

0e* 4 4 4 4 4 4** 4 .4 4* *4 *44 44 4 .4 4 4 4* 4* 4*4 4 TABLE Pooled Data for Protection against BG9739, by Wcoos and FL PspAs CFU Mice ____Days toDeath/immunogen________ Exp. BG9739 R36A BC100 JD908 JS 1020 WcOO EF3296 EF5668 WcOO JS501 bclOO JS3020 control FL (RXl) (WU2) (BG9739 (BG9739) FL FL (181905) 0.3 FL (DBL5) (DBL6A) E140 2.76 CBA/N 3,3,10,1 2,2,3 1 E104 2.89 Xid 6,6,7,8,8 2,2,2,3, 2,2,2,2, 2,2,3,5,5 _4 3 E125 3.58 CBA/N 5,5,5,7 4,5,13, 2,2,2,4 3,3,4,4,5 21 ,6 E172 3.71 CBA/N 6,7,3 x 3.4,6,6,7 >21 E124 3.76 Xid 2,2,2,2, 2,2.2,2, 2,2,2,2,2 3 9 E084 4.05 BALB/c 4x 2,2x 1 4 E144 4.09 Xid 2,3,6, 2,3,3,7, 2,3,3,3, 2.2.2,3,3 1_ 0 1 1_ 2,1 1 >10 411 S S S S S S S

S

S.

S..

S

0 5 55 .55 5* 5 S a S 55 S 55 5 55 .5 5 *S5. S S *S S S Pooled Data for Protection against BG9739, by bclo~s and FL PspAs CFU Mice ___Days toDeath/immunogen Exp. BG9739 R36A BC100 JD908 JS1020 bcIOO EF3296 EF5668 bclO00 JS501 WcOO JS3020 control FL (Rxl) (WU2) (BG9739 (BG9739) FIL FL (181905) 0.3 FIL (DBL5) (DBL6A) FIL bclOO BG9739 R36A/Rxl/D39 WU2 BG9739 EF3296 EF5668 L81905 DBLS DBICA Cont.

All 2,4x3,6, 10,11, >21 6,7,3x 4x2,3x5,2x6,2x7,2x8,2 2,3,3,7t 2,3x3,4 4,5,13, I 0x2,3,3,4,4 8x2,3,9 21 x2,7x3 21 x>21 >21 21 ,3x4,3x5, 3467 median days alive 3 >21 6 3 3 5,13 2 2 1 2 alive:dead 1:8 3:2 2:13 1:4 0:5 1:3 0:14 0:10 0:38 P days alive 0.0096 0.000 0.0013 n.s. n.s. 0.0022 n.s. n.s.

P alive:dead n.s. 0.0008 n.s n.s. n.s. n.s. n.s. n.s.

Score 0+ 0 0 0 0 alive 11 60 13 25 0 0 0 0 0 protected 11 60 1 3 25 0 0 0 0 1G73 challenge R36A/RxI/D39 WU2 BG9739 EF32-96 EF5668 0815 DBL5 OBISA. Cont.

BG9739 days of death median days alive:dead P value based P value based Score Alive% of death on days to on alive:dead death All immune 3 8.59 0.0009 0.023 12 12 All controls 2 0:38 J 000 S S S S S S S S

I'

*5 S 55

S..

S .5 S @56 *5* *S S. .5 :0 *o S0 :0 0 I I TABLE 51 Pooled Data for Protection against EF3296. by various PspAs Exp. CFU Mice Days to Death/immunogen EF26Rxl BC100 JD908 WU2 JS1O2O JS5O1O.3FL JS3020 control (BG9739) (DBL5) (DBL6A) E84' 3.99 BALB/c 14 E140 2.92 CBA/N 3,4,6, >21 3,3,3 E104 3.11 CBA/N 4,5,5,5,6 2,2,2,3,3 2,2,3,4,5 2,2,2,3,4 E124 3.94 CBA/N 1,1,2,2,2 1,1,2,2,2 1,1,2,2,2 E172 4.06 CBA/N 3,46 All 3,4,6, >21 3,3,5,5, >21 4X2,4,3x5, 1, 1,5x2,3,3 1,1,5x2,3, 1, 1, 15x2, 6, >21 4,5 5x3,4,4x6 median days to death 5 5 4.5 2 2 2 alive:dead 1:3 1:4 1:9 0:9 0:10 0:27 P-days to death 0.0077 0.0094 n.s. n.s. n.s.

P-alive:dead n.s. n.s. n.s. n.s. n.s. n.s.

Score 0 0 "%alive 25 20 10 0 0 0 Best EF3296 I median alive: P -days: P alive: Score%%% challenge days alive dead to death dead alive prot f All immune 3 3:35 n.s. n.s. 0 8 8 All -control 0:27' 1 a a a a a a a. a..

TABLE 52 Pocl.d Date For Potcion agamnst EF568. by various FL-PpA a d bel 00 Exp. CFJ e* Days to Dthlirrmnogan EF568 836A Rx1 JD908 JS1020(G9 F3298 EF5668 L81905 JS501.3 53020 control BC100 WU2I 7391 FL DSLOA E143 3.0 CBAIN 5x>10 1.1.2.2, E140 3.59 CSAIN 4.6. 2.4.6 12, >21 E171 3.69 C8A/N 2.2.2.3 3.3,4. 1.3,86.7 >21 2x>21 El 24 3.90 C8AJN 3.3,3x 3.4,5.6, 3.3.34.9 E145 3.94 CABIN 3.4.418>19 2.10 2.4.,>21 2.3.34, 3x>19 13. >21 2x>19 Pool 3.3x4..12,16 2.2.2 2.10. 2.4. 5x<21 3.3,4 3,3, 3,4. 3x1.42 2x>21 3. <21 3x >?21 13, 2x>21 3x 5.6 6x3.3x4 2x>21 >21 3 x,7,9, 2x>21 maliandaysalive 6 2 <21 13 <21 4 <21 5 3 alive:dead 2:7 1:4 3:2 2:3 5:0 2:3 3:2 0:5 2:21 P. day salive 0.01,3 n.s. 0.0187 n.m. 0.001 n.s. n.s. n..

P- ahiv:dead ns. n. 0.027 n.s. 0.0002 n.s. 0.027 n.s.

Score 0 0+ 1 0+ 0+ %alive 25 s0 40 100 40 60 0 9 "prot. 14. 18 1 34 100 34 56 -10 9

S.

S S EF5088 R36AISkl/D39 WU2 BG9739 I EF3298 I EF5668 L81905 DBL5 DBLBA CC"d Summary of protection against EF6796 Immunogen alive:dead alive protected median DOD P time alive P alive vs. dead RXII 4:0 100 1100 21 0.029 03.0291 controls 0:3 0 0 C a C C C *a

C

TABLE 53 Pooled Data for Protection against D13L6A. by various FL PspAs and bclOO PspAs Exp. CEU PMco Days to Doethfrrmnogan D81.6A 8CIOO M3A JD908 JS1O2O WcOO EF3296 EFS608 L81905 bclOO is bcIOO JS2030 control RXl I M2 8 G BG9739 Fl. 181905 5010.3 081-5 DSLOA 9739 061-5 E171 2.89 CBAIN a.7.8 3.3.7.9, 2.3.4,8.6 >21 >21 E152 3.24 MBAIN 15,3x >2 7,18, 8.10. 3 x3,4,3x8 1. 2x>21 13.

E140 3.25 CBAIN 4x >2 4.737 E148 3.57 C13A/N 7,8.10 8.8.9.1 10.13.3 7.8.12. 9,4x >2 4.45.5,18 10.10 X>21 13.13 1 2x>2 E129 4.14 CMAIN 3,6.8,10,1 23 Tota3 Name of Pools R30AIRx1 1D39 OG9739 EF9738 EF5608 I a IO I t I Pooled date 7, 8.10. Bx 21 3.6,8.101 3.15,3x 21 8.8.9.

10 10. 13.3 x>21 3.3.7.7,9., 1 6,2x 21 7. 8.8. 10. 12.

3x 13,21 9,4x >2 I~ I .1 1. .i I median days alive x.0 4 12 afive:doad a:3 1:3 3:6 0:5 3:2 2:80: days alive <0.0001 0200 0.0025 t0.0038 0.0001 0.037 -0.002 >21 4:1 <0.000 0.009 conltrols 8K4 Bx8. 7.7.8. 18.

>21 1:24 P alive:dad 0.0019 n.s.

I~m I I ns I It I I x*' DBL6A days of median days alive:dead P value P value Score alive protected death of death based on based on days to alive:dead death All immune 12.5 19:35 000OO1 0.0019 T35 33 All control 5j 1:24 1

C

C C C *C* a C. I.

C a C C C C C C C CC. C TABLE 54 Data for Protection against BG9163 by various PspAs Exp. CFU Mice toDeath/immunogen BG9163 RXl Rxl.BCG JS 1020 all immune control (BG9739 E169 2.67 CBA/N 5x>24 >24 E140 3.14 CBA/N n.y. E129 4.0 CBA/N 12,4x >23 7,9,9,13, >23 E028 6.217 CBN 6,3x >21 Irnrnu~nogens RxIIRt38AID39 OG9739 all irriune control Pooled Data 6,8x>21 I 2,4x >21 8. 12,12x >21 4,5.7.8.8,9,9.12.

>21 mdian dvys alive >21 21 >21 alivo:dead 8:1 4:1 12:2 2:8 days alive 0.0086 0.0097 0.0027 P. live:deaI 0.0045 0.0647 0.0022 alive 89 80 8e %port. 86 75 83 0 score.

BG9163 Challenge Rxl/R36A1039 BG9739 all immrune control EF5668 days of median days. alive:dead P value P value Score alive protected death of death based on based on days to alive:dead 1 All Immune T 8 18:26 0.0015 1 0.005 41 I I All control 2:21 2:2 I a a a a. a a..

a. a a a a .a a a a .a a TABLE Poolod Dote for Protection againat L81905. by various R.-PspAt Exp. CFU Dwy, to Doathfirrunuinogen L8 0 38A S.CI00 JD908 JS1020 WcOO EF3298 EF5868 WeOO it WcOO JS3020 control JRK II (WU21 186 L81905 5010.3 (08L1 (DBLBA) 9739) (08151 El 72 2.45 CLAIN 3,4,5,8. 3.3.4,4,4 E140 3.11 CBAIN 2.5,5.6. 2.2,2,3,3 E084 3.88 BALB 2.2,5x> 1,8x2 141 E104 .3.5 CB.A/N 3,7,8.8, 3.3,3.2x 3,4.5.5, 2,4.4,4,5 __Ill. 11 >22 a El124 -3.5 CELAIN 2.2,2.2. 2.2.2.3. 1.2,2.2,2 3 E125 3.6 CBA/N 5'e.8'8 3,4.8.8 4,5.5,5 2.2.3.5.5,5 P144 4.11 M8AIN 3.3.5.6. 8.6,6.2x 2.2.3.3. 2.2.303 >10 31 All 3.4,5.0. 2.2.3.7. 5.8.8.8 6,6,6.2x 2.2.3.3. 3,4,6.8 4.x2,4x3 4,5,55 3x.3.3. 1. l,2x2.gx3.8x4,4x 21 6 15>10 3 2x>21 4.3x5.8 media n 5 5 5 >21 7 63 5 3 5 3.5 2 alive: 1:4 0-:5 0:5 5:7 0:4 2:3 0:5 0:4 2:8 0:4 0:10 0:40 [deed P rank Pea:d Scer. o 9 0 9 0 0 Ce C C C C~ *C C C C ,b* C CC ,S C* 9 C C S 0*4 TABLE 56 Pvotoctbon against L81905. by variouso 00, FL.PspAs pooled together Exp. CFIJ "~CO ast eteruo 181905- -DvtoDthrrruoe A36 00io JD908 .JS1020 WcOO EF3296 EF5668 bclO 00 Js bc1lOO JS3020 control IRx1) (WIJ21 (BG 18 1905 5010.3 (OfiL5) (DBLOAI 06.1 E172 2.45 CMAIN 3,4.5.6, 34 E140 3.11 CSAIN 2,5.5A6 E084 3.80 BALS 2.,.5x 8x 1 El 04 .3.5 CBAIN 3.7,8.8. 3 ,33 A2. 3,4.5,5. 2,4,4,4,5 1 >2 6 El124 -3.5 CBAfN 2. 2.2. 2.2.2.3. 1,2.2.2.2 E125 3.6 CSAIN 5.6.8.8 3.4.6.8 4..52.2.3.5.5.5 E144 4.11 CBA/N 3.3.5,6. 6 ,6.8.2x 2.2.3,3. 2 .x >10 >10 3 223 Poof.d 2.3.3.3x5.O.6.8.>2l 3,4,5,6. 2 2 3 ,5,6,7.4x8,1 1, 6 ,6,6.2x 2.2.3.3. 3,4.8,8 42,40,4.55.5, 3x2.3.3, 1,1.20x2 6 5x>21 >10 3 2x>21 4 ,3x56 8x3.6o4, I median dayslive 5 5 863 5 3 3.5 2 elive:dlead 1:9 0:5 5:11 2:3 0:5 0:4 2:12 0:10 0:40 P days alive 0.000S 0.0035 <0.0001 0.0002 nos.. 0.01 0.035 0.044 P alive:deed nos. n.e. 0.0001 0.01 n. I n.s. n.s. nos scare live 10 0 31 40 0 0 1 protected 1 challenge with L81905 A36AjRxI/D39 WIJ2 BG9739 EF3296 EF5668 L81905 061.5 DOM8 onttole 00 00* be* be 0 0.

L81905 days of median days- alive:dead P value P value Score alive protected challenge death of death based on based on days to alive:dead All Immune 5 10:59 <0.0001 0.008 14 14 All control 0:40 see

C

S a*.C* *a 0.

00 0 V a V.

TABLE 57 ______Pooled Data for Protection against 0815 by various FL-PapAs bclOOs Exp. CFU lMce D~rV to Death/irrrnogai, 0915 MOOA JC00 S1020 bclOO EF5668 bclOO JS5O1O.3 belO10 JS3020 control P11 G9739 is 1020 161905 0815 0815 DBA E84' 3.90 BALB/c Ox2

W.

E140 3.27 MBAIN 4,4.5.5. 2.2,2 E104 3.39 Xid 3,3.6, 7,7,15, 2.2,4,5, 2,4x3 22 22,> 22 E124 3.78 Xid 2.2.2,5. 5x2 1.1.2.2.2 El125 3.81 CBAI]N 3.3A4, 3.3.4.4 2.2,2. 5x2.5 21 E144 4.13 XID 3.3.3,3, 2.2.3.4. 5x2 4 total Name of Pool R38A/Rx1fD39 BG9739 EF5888 181905 0615 DSLOA controls Pooled Data 4 x3,2x 4 .3,t5. 21 8x2.4x3.4.5, 2,2.3.4. 3,3,4.4 6x2,5.7.7,1 5,4x 21 7x2,4., 1.1 ,2Wx,403,5 >21.>21 4 median days alive 4 3 3 3.5 62 2 aIivo:dead 1:9 2:12 0:4 0:4 4:10 0:10 0:32 days alive <0.0001 0.0063 .041 0.001 0.0025 n.s.

P aliva:deed n.a. n.a. n.s. n.2. 0.0055 n.e.

score 0 alive 10 14 0 0 29 0 0 protected 10 14 0 0 29 0 0 I BL llv 136AP.D39 BG 89739 -EF5668 L 81905 081 DSOA controls This immunization was with cell eluted PspA. Note BALB/cJ mice were used. Also note 104 Challenge CFU.

0 0@ 9 ~0* 0** *0 4 0 *00 *0 *0 0 0* 0** 0 0 00 0 *0 000 0 9* 0~ 0* *9 0 D1315 days of median days alive:dead P value P value Score alive protected challenge death of death based on based on days to alive:dead death All Immune 3.5 7:49 1 0.0001 0.034 :3.6 3.6 All control 2 _:3 TABLE 58 :oled Data for Protection against EF6796 by various PSpAs Exp. CFU Mice to Death/immunogen WU2 Rx1 JS1020 L81905 JS3020 JS5O10.3R. DBL5 control 13 C100 (BG9739) (DBL6A) bc1O E140 3.75 CBA/N -4x >21 E28 BALB n.y.

TABLE 59 Pool of Pools for protection against EF6796 j line Group Delay in time to death and/or survival Protection against death Dsrtin jdays to death (median DOD) IP values etc. alive:dead 1 P values etc.

4x >21 k21) 0.029 I i I Rxl controls 1,1,1 4:0 0.029 0:3 A I CO C 9 C i C.

.0

C

CC

*iC C C C C C SC C C C C C .i *C C *C TABLE Pooled Dae fo Protectron inant 807322. by vriours F.PspAs bclOOs Exp. FU FAcS Days to Dsthlifwnmunogen 7322 D391 Rxl JD908 bclOO EF3298 EF5668 bvOO JSO1. bclOO JS3020 control IBC100 (WJ21 8G9739 L81905 3 D81.5 DOLOA

DSLS

E171 2.78 CBAIN 10.15.3 1.3.0667 x>21 S143 3.0 CBA/N 7.8x> 1 2.2.3,4.7.7 E140 3.14 CBAN 4x>21 >21 6C1 00 E152 3.11 CBAIN 12.13.1 10,12.1 >21, 6,7,7.8.8.9 6.>21 3, >21 >21, .14 >21 E148 3.57 CSa"N 18.20,3 5.3xx. 6.10.11. 4.8.11.1 4.5.5.8.>2 x>21 10 11.19 8.>21 1 E169 3.94 CBAIN 5x>21 2.5.5.0.7 Invmunogens R36AIlxlID39 J0908 809739 EF329e EF568 L81905 DBL5 DBLGA Cont.

Pools 18.20.12x> 21 10.15,3 12.131 5.3.x6. 7.8x> 2 10.12. 6.10,11.11.19>21. 4.8.11.1 1.3x2.3.3.

x>21 21 10 1 13>21 >21.>21.>21 8.>21 4 4 .x5.7.x 15.80,4x%* 9.14.2x>2 median days alive >21 >21 14.5 e >21 12.5 >21 11 6 alive:dead 9:0 3:2 1:3 0:5 8:1 1:3 4:5 1:3 2:32 p. days alive <0.0001 0.0007 0.001 n.s. <0.000 0.0013 0.0002 0.028 Paliv:deed <0.0001 0.004 n. s. n.s. <0.000 n.s. 0.0076 n.s.

alive 100 so 25 0 89 25 80 25 6 protected 100 1 57 2? 0 88 22 79 22 Score +1 0 1 4± I .0 0 OG7322 Chn:-= IR3*AIfbt1/39 JD908 IBG9739 I E3296 EF5OBs 1895 65DOLGA Cr *9* 9* 9 9* 9** 9*9 9. 8G7322 median days allve:dead P value P value Score alive protected Challenge of death based on based on days to alive:dead death All immune >21 30:25 0.01 <0.0001 55 52 All controls 61 2:32 i 167 SEXAMPLE 8 ABILITY OF PspA IMMUNOGENS TO PROTECT AGAINST INDIVIDUAL CHALLENGE STRAINS In Example 7 some of the capsular type 2, 4, and 5 strains were not completely protected from death by S immunization. In these studies the BALB/cByJ mouse was used instead of the hypersusceptible, immunodeficient CBA/N mouse used for the Example 7 studies. With the BALB/cJ mouse it was observed that immunization with PspA was in fact able to protect against death with capsular to type 2, 4, and 5 pneumococci. This result is shown in the table below.

The data from Table 60A also demonstrates that a mixture of 4 5 full length PspAs was as effective, or more effective than immunization with a single PspA.

ee a'.

0 6 p so.

6 0 0 0 0 0 0 0 0 0 000 0 00 00a 0 0600 0* S 0 00 *00 60 0 0 006. 0 0 Table GOA. Days of death of BALB/cByJ nice after inwanization with imonovalent and polyu-lent vaccine.

Challenge Str-ains Immunogen Strain m Np.A PrM. Lot Drys to IDijz nAme tym typ 13 ,im CbkoP I rmt R36A S .k nmiuw IY2141 N ed d-o CFA CFA CFA D39 2 25 2 4.76 3, 4x >21 3, 4x >21 3,4,5, 11, 3,3, >21 4, 4, 8 WU2 3 1 2 4.8 4x >21 4x >21 6, 3x >21 3,4,2x >21 A66 3 13 4.7 3, 3, >21, 2, 3x >21 2, 2,3, 4 2, 3,4,4 21 BG9739 4 26 1 4.07- 7,8x >21 3, 8x >21 1,5,6,6,9, 3,3,3,4,6, 4.4 4x >21 7, 7, 2x 21 L81905 4 23 1 6.90- 2,2,2,2,5, 2,6,8,9, 1, 1, 1,1,2, 1, 4 x 2.3x3, 6.96 5, 6x 21 3, 4, 5, 2x 4, 21 4x 21 21 a a a..

a a. .a a a 0 EF5668 4 12 4 6.10- 3 4,7x 3x 3, 6x >21 4x 3, 4, 4,6, 3, 5x4, 6.

6.93 21 6, 21 >21 4 33 2 3.30 7, 14, 3x >21 3 5, 5,2x 2, 2, 2,4, 6 4, 5,5,6, 9 >21 DBL6A 6A 19 1 4.34 6,9, 10, 11, 10,11,12,13, 3, 11, 11, 13, 8, 9, 11, 21, 21 >21 16 >21 BG7322 6B 21 3.9 8, 8, 3x 21 Sx >21 6, 6, 7, 8,10 2,5, 6,8, 8 Note, numbers such as Ox 21 indicate tha 8 mice lied greater than 21 days.

Noe,JY2141 is a preparation from a strain that lacks PspA. None no immunization.

Note, mice were given two immunizations with PspA two weeks apart and challenged intravenously 2 weeks after the last immunization. The first immunization was given with complete Freund's adjuvant (CFA) subcutaneously, the second immunization was given intraperitoneally in saline.

'4 valent vaccine mixture R36A, BG9739, EF5668, and DBL.5 all E180 24 valent vaccine mixture R36A, BG9739, DBL5, EF3296, D39 and DI3L6A 3 5 valent vaccine mixture R36A, BG9739, DBL5, EF3296, EF5668 170 w EXAMPLE 9 CHARACTERIZATION OF PspA EPITOPES WITHIN PNEUMOCOCCAL STRAINS MC25-28 The strains examined came from a group of 13 capsular serotype 6B strains which have been identified that are members of a multiresistant clone, having resistance to penicillin, chloramphenicol, tetracycline, and some have acquired resistance to erythromycin. The pneumococcal isolates described in the following studies (MC25-28) are members of this 6B clone. Although 1o previously thought to be geographically restricted to Spain (unlike the widespread multiresistant Spanish serotype 23F clone), members of this clone have been shown to be responsible for an increase in resistance to penicillin in Iceland (Soares, et al., J. Infect.

S Dis. 1993; 168: 158-163).

The following techniques were used to characterize the location of difference PspA epitopes: Bacterial cell culture Bacteria were grown in Todd-Hewitt broth with Qc 0.5% yeast extract or on blood agar plates overnight at 37 0 C in a candle jar. Capsular serotype was confirmed by cell agglutination using Danish antisera (Statens Seruminstitut, Copenhagen, Denmark). The isolates were subtyped as 6B by Quellung reaction, utilizing rabbit antisera against 6A or 6B capsule antigen.

Bacterial lysates Cell lysates were prepared by incubating the bacterial cell pellet with 0.1% sodium deoxycholate, 0.01% sodium dodecylsufate (SDS), and 0.15 M sodium citrate, and then diluting the lysate in 0.5M Tris hydrochloride (pH Total pneumococcal protein in the lysates was quantitated by the bicinchoninic acid method (BCA Protein Assay Reagent; Pierce Chemical Company, Rockford, IL).

PspA serotypinq Pneumococcal cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and developed as Western blots using a panel of seven MAbs to 171 w PspA. PspA serotypes were assigned based on the particular combination of MAbs with which each PspA was reactive.

Colony immunoblotting A ten mL tube of Todd-Hewitt broth with yeast extract was inoculated with overnight growth of from a blood agar plate. The isolate was allowed to grow to a concentration of 10 7 cells/mL as determined by an O.D. of 0.07 at 590nm. MC25 was serially diluted and (o spread-plated on blood agar plates to give approximately 100 cells per plate. The plates were allowed to grow overnight in a candle jar, and a single blood agar plate with well-defined colonies was selected. Four nitrocellulose membranes were consecutively placed on the 16 plate. Each membrane was lightly weighted and left in place for 5 min. In order to investigate the possibility of phase-variation between the two proteins detected on Western blots a single colony was picked from the plate, resuspended in ringer's solution, and spreadplated onto a 2o blood agar plate. The membranes were developed as Western blots according to PspA serotyping methods.

When the strains MC25-28 were examined with the panel of seven MAbs specific for different PspA epitopes, all four demonstrated the same patterns of reactivity I: (Fig. 14). The MAbs XiR278 and 2A4 detected a PspA olecule wit apparent mlecular weight of 190 kDa in each isolate. In accordance with the PspA serotyping system, the 190 kDa molecule was designated as PspA type 6 because of its reactivity with XiR278 and 2A4, but none 3o of the five other MAbs in the typing system. Each isolate also produced a second PspA molecule with an apparent molecular weight of 82 kDa. The 82 kDa PspA of each.isolate was detected only with the MAb 7D2 and was designated as type 34. No reactivity was detected with S MAbs Xi126, Xi64, 1A4, or SR4Wr. Results from the colony immunoblotting showed that both PspAs were present simultaneously in these isolates under in vitro growth 172 conditions. All colonies on the plate, as well as all of the progeny form a single colony, reacted with MAbs XiR278, 2A4, and 7D2.

EXAMPLE 10 SOUTHERN BLOT ANALYSIS OF CHROMOSOMAL

DNA

ISOLATED FROM PNEUMOCOCCA STRAINS MC25-28 Pneumococcal chromosomal DNA was prepared by the Youderian method (Sheffield, et al., Biotechniques, 1992; 12: 836-839). Briefly, for a 500 ml culture in THY or THY with 1% choline, cells were tO centrifuged at 8000 rpm in GSA rotor for 30 minutes at 4 0 C. The supernatant was decanted, and the cells were washed with 1 to 2 volumes of sterile water to remove choline, if used. This step was only necessary when sodium deoxycholate was used. The wasted cells were i/ centrifued twice a 8000 rpm in GSA rotor for 10 minutes.

Cells were resuspended in 3.5 ml TE buffer, containing 1% SDS or 1% sodium deoxycholate, and incubated at 37 0 C for minutes if sodium deoxycholate was used. If SDS was used, incubation at 37 0 C was not necessary. The cells 0o were incubated at 65 0 C for 15 minutes, and 1/5 volume of M potassium acetate was added, and the cell suspension was incubated for 30 minutes at 65 0

C.

The cells were placed on ice for 60 minutes, and centrifuged at 12,000 rpm in an SS-34 rotor for minutes. The supernatant was transferred to a clean centrifuge tube, and 2 volumes of cold 95% ethanol was added. After mixing, DNA was spooled on to a glass pasteur pipet, and air dried. The DNA was resuspended in 4 ml TE, and 4.0 g cesium chloride was added. The solution was split into two aliquots in ultracentrifuge tubes, and the tubes were filled to their maximum capacity using 1.0 g/ml cesium chloride in TE. Before closing the tubes, 300 ml of 10 ug/ml ethidium bromide was added.

3 The solution was centrifuged at-45,000 rpm overnight, or for 6 hours at 55,000 rpm. The chromosomal band was extracted using a gradient, at least 6 times with 1 volume'each salt-saturated isopropanol. The 173 W aqueous phase was extracted by adding 2 volumes ethanol. The DNA came out of solution immediately, and it was spooled on to a pasteur pipet. The DNA pellet was washed by dipping the spooled DNA in 5 ml 70% ethanol.

The DNA was air dried, and resuspended in the desired volume of TE, 500 ul.

The cells were harvested, washed, lysed, and digested with 0.5% (st/vol) SDS and 100g/mL proteinase

K

at 370C for 1 h. The cell wall debris, proteins, and polysaccharides were complexed with 1% hexadecyl trimethyl ammonium bromide (CTAB) and 0.7M sodium chloride at 650C for 20 min., and then extracted with chloroform/isoamyl alcohol. DNA was precipitated with 0.6 volumes isopropanol, washed, and resuspended in Tris-HC1, 1mM EDTA, pH 8.0. DNA concentration was determined by spectrophotometric analysis at 260 nm (Meade, H.M. et al., J. Bacteriol 1982; 149: 114-122; Silhavy, T.J. et al., Experiments with Gene Fusion, Cold S Spring Harbor: Cold Spring Harbor Laboratory, 1984; and Murray, et al., Nucleic Acids Res. 1980; 8 4321- 4325).

Probe preparation and 3' oligonucleotide primers homologous with nucleotides to 26 and 1967 to 1990 of Rxl pspA S (LSM13 and LSM2, respectively) were used to amplify the ful l lengh ppA and construct probe LSMpspA13/2 from Rxl genomic DNA. 5' and 3' oligonucleotide primers homologous to nucleotides 161 to 187 and nucleotides 1093 to 1117 (LSM12 and LSM6, respectively) were used to o amplify the variable a-helical region to construct probe LSMpspA12/6. PCR generated DNA was purified by Gene Clean (Bioll01 Inc., Vista, CA) and random prime-labeled with digoxigenin-ll-dUTP using the Genius 1 Nonradioactive DNA Labeling and Detection Kit as described by the manufacturer (Boehringer Mannheim, Indianapolis,

IN).

174 DNA electrophoresis For Southern blot analysis, approximately of chromosomal DNA was digested to completion with a single restriction endonuclease (Hind III, Kpn 1, EcoRI, Dra I, or Pst then electrophoresed on a 0.7% agarose gel for 16-48 h at 35 volts. For PCR analysis, 5gL of product were incubated with a single restriction endonuclease (Bcl 1, BamH I, Bst I, Pst I, Sac I, EcoR I, Sma I, and Kpn then electrophoresed on a 1.3% agarose gel for 2-3 h at 90 volts. In both cases, 1 kb DNA ladder was used for molecular weight markers

(BRL,

Gaithersburg, MD), and gels were stained with ethidium bromide for 10 min and photographed with a ruler.

Southern blot hybridization The DNA in the gel was depurinated in 0.25N HC1 for 10 min, denatured in 0.5M NaOH and 1.5M NaCI for min, and neutralized in 0.5M Tris-HCl (pH 1.5M NaCl and 1mM disodium EDTA for 30 min. DNA was transferred to a nylon membrane (Micron Separations INC, MA) using a POSIBLOT pressure blotter (Stratagene, LaJolla, CA) for min and fixed by UV irradiation. The membranes were prehybridized for 3 h at 42oC in 50% formamide, 5X SSC, Denhardt solution, 25mM sodium phosphate (pH 0.5% SDS, 3% (wt/vol) dextran sulfate and 500/g/mL of denatured salmon sperm DNA. The membranes were then S hybridized at 42 0 C for 18 h in a solution containing formamide, 5X SSC, lX Denhardt solution, 20mM sodium phosphate (pH 0.5% SDS, 3% dextran sulfate, 250,g/mL denatured sheared salmon sperm DNA, and about 0o 2Ong of heat-denatured digoxigenin-labeled probe DNA.

After hybridization, the membranes were washed twice in 0.1% SDS and 2X SSC for 3 min at room temperature. The membranes were washed twice to a final stringency of 0.1% SDS in 0.3X SSC at 65°C for 15 min. This procedure 53 yielded a stringency greater than 95 percent. The membranes were developed using the Genius 1 Nonradioactive DNA Labeling and Detection Kit as 175 described by the manufacturer (Boehringer Mannheim, Indianapolis, IN). To perform additional hybridization with other probes, the mebranes were stripped in 0.2N NaOH/0.1% SDS at 40 0 C for 30 min and then washed twice in 7 2X SSC.

PCR

and 3' primers homologous with the DNA encoding the N- and C-terminal ends of PspA (LSM13 and LSM2, respectively) were used. Reactions were conducted in 50AL volumes containing 0.2mM of each dNTP, and IL of each primer at a working concentration of 50mM. MgCl 2 was used at an optimal concentration of 1.75mM with 0.25 units of Taq DNA polymerase. Ten to thirty ng of genomic DNA was added to each reaction tube. The amplification 7 reactions were performed in a thermal cycler (M.J.

Research, Inc.) using the following three step program: Step 1 consisted of a denaturing temperature of 94 0 C for 2 min; Step 2 consisted of 9 complete cycles of a denaturing temperature of 94 0 C for 1 min, an annealing *o temperature of 50 0 C for 2 min, and an extension temperature of 72 0 C for 3 min; Step 3 cycled for 19 times with a denaturing temperature 94 0 C for 1 min, an annealing temperature of 60 0 C for 2 min, and an extension temperature of 72 0 C for 3 min; and at the end of the last Z4 z< cycle, the samples were held at 72 0 C for 5 min to ensure Band size estimation Fragment sizes in the molecular weight standard and. in the Southern blot hybridization patterns were 30 calculated from migration distances. The standard molecular sizes were fitted to a logarithmic regression model using Cricket Graph (Cricket Software, Malvern, PA). The molecular weights of the detected bands were estimated by entering the logarithmic line equation obtained by Cricket Graph into Microsoft Excel (Microsoft Corporation, Redmond, WA) in order to calculate molecular 176 weights based on migration distances observed in the Southern blot.

Since most strains contain a pspA gene and a pspc gene, it was expected that if an extra gene were present one might observe at least three pspA homologous loci in isolates MC25-28. In Hind III digests of MC25-28 each strain revealed 7.7 and 3.6 kb bands when probed with LSMpspA13/2 (Figure 15A and 15C). In comparison, when Rxl DNA was digested with Hind III and hybridized ;o with LSMpspA13/2, homologous sequences were detected on 9.1 and 4.2 kb fragments, as expected from previous studies with PspA (Figure 15A). Results consistent with two pspA-homologous genes in MC25-28 were obtained with two pspA-homologous genes in MC25-28 digested using four iS additional enzymes (Table 61).

Table 61. Chromosomal RFLPs with probe LSMpspA13/2 for isolates MC25-28 and Rxl

S

S

Restriction Strains Examined Restriction Fragments Enzyme (sizes in kilobases) MC26 MC27 MC28 RX1 MC25-28 Rxl Hind III 7.7, 3.6 9.1, 4.2 Kpn I 11.6, 10.6 10.6, 9.8 EcoR I 8.4, 7.6 7.8, 6.6 Dra I 2.1, 1.1 1.9, 0.9 Pat I >14, 6.1 10.0, The four isolates examined are all members of a single clone of capsular type 6B pneumococci isolated from Spain. These four isolates are the first in which two PspAs have been observed, PspA and PspC, based on the observation that bands of different molecular weights were detected by different MAbs to PspA.

Mutation and immunochemistry studies have demonstrated that all of the different sized PspA bands from Rxl are made of a single gene capable of encoding a-69kDa protein, supporting the assertion that two PspAs have been observed, PspA and PspC.

177 PIt has been observed that probes for the half of pspA (encoding the a-helical half of the protein) bind the pspC sequence of most strains only at a stringency of around 90%. With chromosomal digests of SMC25-28, it was observed that the 5' Rxl probe LSMpspAl2/6 (Figure 15D) bound two pspA homologous bands at even higher stringency. The same probe bound only the pspA containing fragment of Rxl at the higher stringency (Figure io Further characterization of the pspA gene was done by RFLP analysis of PCR amplified pspA from each strain. Since previous studies indicated that individual strains yielded only one product, and since the amplification was conducted with primers based on a known S pspA sequence, it was assumed that the product amplified from each strain represented the pspA rather than the pspC gene. When MC25-28 were subjected to this procedure, an amplified pspA product of 2.1 kb was obtained from each of the four strains. When digested d o with Hha I, this fragment yielded bands of 1.1, 0.46, 0.21 and 0.19 kb for each of the four isolates. A single isolate, MC25, was analyzed with eight additional enzymes. Using each restriction enzyme, the sum of the fragments was always approximately equal to the size of whole pspA (Figure 16). These results suggested that the 2.1 kb amplified DNA represents the amplified product of only a single pspA gene. Rxl produced an amplified .product of 2.0 kb and five fragments of 0.76. 0.468, 0.3.90, 0.349 and 0.120 kb when digested with Hha I as S: o expected from its known pspA sequence.

There are several possible explanations for the observation of PspA and PspC in these strains but not in other strains. All isolates might make PspA and PspC in culture, but MAbs generally recognize only PspA (perhaps, 53 in this isolate there has been a recombination between pspC DNA and the pspC locus, allowing that locus to make a product detected by MAb to PspA). All isolates can 178 have PspA and PspC, but the expression of one of them generally does not occur under in vitro growth conditions. The pspC locus is normally a nonfunctional pseudogene sequence that, for an unexplained reason, has become functional in these isolates. Results from the colony immunoblotting of these isolates failed to show a detectable in vitro phase shift between either PspA type 6 (XIR278 and 2A4) or PspA type 34 (7D2) protein. This strengthens the second explanation, and suggests that the I~ second PspA is these isolates is due to the pspC gene not being turned off during in vitro growth conditions.

Presumably, in these four strains, the second PspA protein is provided by the pspC DNA sequence. At high stringency, the probe comprising the coding region S of the a-helical half of PspA recognized both pspA homologous sequences of MC25-18, but not the pspC sequence of Rx1. The finding indicated that the pspC sequence of MC25-28 is more similar to the Rxl pspA sequence than the Rxl pspc sequence. If the pspC w sequence of these strains is more similar to pspA than most pspc sequences, it could explain why the products of pspC genes cannot generally be identified by MAbs.

EXAMPLE 11 IDENTIFICATION OF CONSERVED AND VARIABLE REGIONS OF pspA AND pspC SEQUENCES OF S. PNEUMONIAE The S. pneumoniae strains used in this study are listed in Table 62. The strains are human clinical isolates representing 12 capsular and 9 PspA serotypes.

All strains were grown at 37 0 C in 100ml of Todd-Hewitt S c broth supplemented with 0.5% yeast extract to an approximate density of 5 x 108 cells/ml. After harvesting of the cells be centrifugation (2900 g, 10min), the DNA was isolated, and stored at 4 0 C in TE (10 mM Tris, 1mM EDTA, 179 Table 62. Streptococcus pneurnoniae strains used.

Strain IWU2 D3 9 R36A Rx 1 DBL6A A66 AC9 4 AC 17 AC4 0 AC107 AC100 AC140 D109-1B BG9709 L8 1905 L8 2233 L82 006 Relevant phenotype Ref ArA1'n capsular type 3, PspA type 1 Capsular type 2, PspA type 25 Nonencapsuated m~utant of D39, papA type 25 Derivatitve of R36A, PopA type 25 Capsular type 5, PspA type 33 Capsular type 6A, PapA type 19 Capsular type 3, PapA type 13 Capsular type 9L, PapA type 0 Capsular type 9L, PapA type 0 Capsular type 9L, PapA type 0 Capsular type 9V, PspA type 0 Capsular type 9V, PspA type 0 Capsular type 9N, PapA type 18 capsular type 23, PspA type 12 Capsular type 9, PapA type 0 Capsular type 4, PapA type 25 Capsular type 14, PspA type 0 Capsular type 1, PspA type 0 Approximately 5,4tg of chromosomal Briles et al., 1981 Avery et al., 1944 Avery et al., 1944 Shoemaker arnd Guild, 1974 Yother et al., 1986 Yother et al., 1986 Avery et al., 1944 Waltman et al., 1992 Waltman et al., 1992 Waltman et al., 1992 Waltman et al., 1992 Waltman et al., 1992 Waltman et al., 1992 McDaniel et al., 1992 McDaniel et al., 1992 McDaniel et al., McDaniel et al., McDaniel et al., 1992 1992 1992 DNA was *0 6 digested with HindIll according to the manufacturer's instructions (Promega, Inc. Madison, WI) The digested DNA was subjected to electrophoresis at 35 mV overnight in 0.8% agarose gels and then vacuum-blotted onto Nytrans membranes (Schleicher Schuell, Keene, NH).

The oligonucleotides uses were based on the previously determined sequence of Rxl pspA. Their position and orientation relative to the structural domains of Rxi pspA are shown .in Figure 17. Labeling of ol1igonucleot ides and detection of probe-target hybrids 180 O were both performed with the Genius System® according to manufacturer's instructions (Boehringer-Mannhein, Indianapolis, IN). All hybridizations were done for 18 hours at 42 0 C without formamide. By assuming that 1% S base-pair mismatching results in a 1 0 C decrease in T arbitrary designations of "high" and "low" stringency were defined by salt concentration and temperature of post-hybridization washes. Homology between probe and target sequences was derived using calculated Tm by io established methods. High stringency is defined as and low stringency is 5 85% base-pair matching.

PCR primers, which were also used as oligonucleotide probes in Southern blotting and hybridizations, were designed based on the sequence of pspA from pneumococcal strain RXl. These oligonucleotides were synthesized by Oligos, Etc.

(Wilson, OR), and are listed in Table 63.

S**

000* oe f 0**o* 28.AUG.2001 18:27 SPRUSON FERGUSON 61 2 92615486 NO.323 P.14/22 181 Table 63. Oligonucleatide sequences, Primer 5' 3' LSH1

CCGATCCAGCTCCTGCACCAAAAC

LSMZ2

GCCCTCGACCOTTAAACCCTTACCATTGG

LSM3

COGGATCTGAGCGAGCAGTTGGCTG

LS84

CCGGATCCCVTCAAAGAGATTGATGAGTCTG

LSMiS

GCGGATCCCGTAGCAGTCAGTTAAACTC

LSM6

CTGAGTCGACTGTTCGGATCTGCAGCTGGAGC

LSX7 CCCGATCCACGCTCCAGCTCChGAAACTCCAG .O LSM9

GTTTTTGGTCCAGGCCAGCG

LSM1O CCTATGGCTAcAGGTTG LH12 CCGcATCCAGCGTCCTATCTTAGGGGCTGCT

T

LSM112

CCGGATCCTTGACCJIATADYRACCAGCC

S.9 *9 PCR was done with an MJ Research, Inc., SProgrammable Thermal Cycler (Watertown, MA), using Sapproximately 10 ng of genomic pneumococcal DNA as template with designated 5' and 3' primer pairs. The sample was brought to a total volume of 50 gl containing a final concentration of 50 mM KC1, 10 mM Tris-Hcl (pH 1.5mM MgCl, 0.01% gelatin, 0.5 AM Of each primer, 200 AM of each deoxynucleoside triphosphate, and 2.5 U of Tag' DNA polymerase. The samples were denatured at 94 0

C

S9for 2 minutes and subjected to 10 cycles consisting of: 1 min at 949C, 2 min at 50C, and 3 min at 72-C, followed by 20 cycles of: 1 min at 940C, 2 min at 60 0 C, and 3 min at 726C. After 30 total cycles, the samples were held at 729C for an additional 5 min prior to cooling to The amplicons were then analyzed by agarose gel electrophoresis.

Sc Oligonucleosides were used to probe HindIII digests of DNA from 18 strains of S. pneumoniae under conditions of low and high stringency. Each strain was also Screened using a full-length pspA probe. Table 64 summarizes the results for each strain under conditions of high stringency. Strain Rxl is a laboratory derivative of the clinical isolate D39 and consequently, 182 both strains showed identical hybridization patterns and are a single column in Table 64.

Table 64. Summary of hybridization of oligonucleotides with HINDIII chromosomal restriction fragments.

strains Probe POOl/ WU 0815 DBL8 ABO AC94 AC17 AC40 ACIO AC10 AC14 D810 BG970 BG58 18190 L8223 19200 D39 2 A 7 0 0 9 5 38 FL-Rxl' 4.0.9. 3.8 3.7.5. 3.0,3. 3604. 386(13 3.88. 3.2.3. 3.2.3. 4.0,8. 3.0.4. 3.3,4.7 1.4.3.2 3.8,6. 8.2.3. 4.3.8.4 I8 4 330 0 73.0 2 7 LSM12 4.0.9. 3.8 3.0,3. 4.3 c 3.880 40.8. 4.0 3.3,4. 2.298 1.4,3.2. 3.8 1.3.3. 1 8 4 3 8 0 7 3.8 7 4.0 3.8.3 2.2,9.6 3.8 1.2.2.

3.

3.8 LSM3 4.0 3.8 3 2.2 3.8 3.8 LSM4 4.0 2.2 3.8 38 3.7 ISM7 4.0.9. 3.8 3.7 3.0.3. 3.6 3.2,3. 3.04. 3.3,4. 2.2.9.0 3.8 382. 3.7 1- 4 8 a 0 7 3 LSM1 1 4.0.9. 3.8 3.7,5. 3.4 0.3 3.2 380 4.0 4.0 .2.2 5.2 4.0,9. 3.8 3.7 3.4 3804. 3.86 3.2 3863. 4.0 4.0 3.3.4. 2.2.9.8 3.8.3.2 3.8.5. 1.3.3. 4.3864 1 3 3 3 727 LSM2 4.0 0 3.7 386 386 a 3880. 4.0 3.0.4. 4.7 4.3 3 1-0 "Full-length pspA of strain Rxl.

OnIurnbers are size in kflobase pairs.

'no hybridizetion obeerved with corresponding probe.

184 I The only strain which did not have more than one pspA-homologous HindIII fragment was WU2, which was previously shown using a full-length pspA probe. Even at high stringency, six of the eight probes detected more than one fragment in at least one of the 18 strains (Table 64). LSM7, 10 and 12 hybridized with two fragments in more than one-half of the strains, and the fragments detected by the oligonucleotide probes were identical in size to those detected by the full-length :o pspA probe. Moreover, the same pairs of fragments were frequently detected by probes derived from the 3' as well as the 5' region of Rxl pspA. These results suggested that the HindIII fragments from different isolates include two separate but homologous sequences, rather tS than fragments of a single pspA gene. Based on the diversity of the hybridization patterns and the size of restriction fragments, it is clear that pspA and pspC sequences are highly diverse and that these loci have considerable sequence variability as determined by S location of HindIII recognition sites.

Oligonucleotides which hybridize with a single restriction fragments in each strain were assumed to be specific for pspA. At high stringency, LSM3 and LSM4 detected only a single HindIII fragment in the strains with which they reacted. Restriction fragments containing hmnlogy to LSM3 or LSM4 were the same as those which hybridize with all of the other homologous probes. This suggested that LSM3 and LSM4 specifically detect pspA rather than the pspC sequence. That LSM3 0 hybridizes with a single restriction fragment of WU2 further confirmed that this oligonucleotide is specific for pspA. Sequences from the portion of the gene encoding the second proline region (LSM111) and the Cterminus (LSM2) appeared to be relatively specific for S pspA since they generally detect only one of the HindIII fragments of each strain.

185 1 Oligonucleotides LSM12 and LSM10 were able to detect the most conserved epitopes of pspA and generally hybridize with multiple restriction fragments of each strain (Table 65). LSM7 was not as broadly crossreactive, but detected two pspAs in 41% of strains including almost 60% of the strains with which it reacts.

Thus, sequences representing the leader, first proline region, and the repeat region appear to be relatively conserved not only within pspA but between the pspA and o pspC sequences. LSM3, 4, and 5 hybridize with the smallest number of strains of any oligonucleotides (29-35 percent), suggesting that the a-helical domain is the least conserved region within pspA. In strains BG58C and L81905 oligonucleotides detect more than two HindIII C, fragments containing sequences with homology to pspA.

Because of the absence of HindIII restriction sites within any of the oligonucleotides it was unlikely that these .multiple fragments result from the digestion of chromosomal DNA within the target regions. Also, the So additional restriction fragments were detected at high stringency by more than one oligonucleotide. Possibly, in these two strains, there are three or four sequences with DNA homology to some portions of pspA. The probes most consistently reactive with these additional 2 sequences are those for the leader, the alpha-helical region, and the proline-rich region.

.The oligonucleotides used as hybridization probes were also tested for their utility as primers in the polymerase chain reaction (PCR). Amplification of ;o pspA from 14 strains of S. pneumoniae comprising 12 different capsular types was attempted with the primers listed in Table 63. LSM2, derived from the 3' end of pspA, were able to amplify an apparent pspA sequence from each of 14 pneumococcal strains when used in combination with LSM111, which is within the sequence of pspA encoding the proline-rich region. Combinations of LSM2 with primers upstream in pspA were variably successful in 186 amplifying sequences (Table 65). The lowest frequency of amplification was observed with LSM112 which was derived from the Rxl sequence 5' to the pspA start site. This oligonucleotide was not used in the hybridization S studies. DNA fragments generated by PCR were blotted and hybridized with a full-length pspA probe to confirm homology to pspA.

Further evidence for variability at the pspA locus comes from the differences in the sizes of the ,o amplified pspA gene. When PCR primers LSM12 and LSM2 were used to amplify the entire coding region of PspA, PCR products from different pneumococcal isolates ranged in size from 1.9 to 2.3 kbp. The regions of pspA which encode the a-helical, proline-rich, and repeat domains were amplified from corresponding strains and variation in pspA appears to come from sequences within the ahelical coding region.

Table 65. Amplification of pspA by PCR using the indicated oligonucleotides as 5' primers in 4 combination with the 3' primer LSM2.

«e o• primer Domain Amplified/ Percent Tested Amplified LSM112 -35 (upstream) 2/14 14 LSM12 leader 8/14 57 LSM3 a-helical 3/14 21 LSM7 proline 12/14 86 LSMll1 proline 14/14 100 These studies have provided a finer resolution map of the location of conserved and variable sequences within pspA. Additionally, regions of divergence and identity between pspA and the pspc sequences have been identified. This data confirmed serological studies, and demonstrated that pspA and pspC sequences are highly 187 O variable at the DNA sequence level. The diversity of HindIII restriction fragment polymorphisms contained pspA and the pspC sequence supported earlier data using larger probes that detected extensive variability of the DNA in f and around these sequences.

A useful pspA-specific DNA probe would identify Rxl and WU2 pspA genes, in which restriction maps are known, and would identify only a single restriction fragment in most strains. Two probes, LSM3 and LSM4, do not hybridize with more than one HindIII restriction fragment in any strain of pneumococcus. Both of these oligonucleotides hybridize with Rxl pspA and LSM3 hybridizes with WU2 pspA. However, each of these probes hybridize with only four of the other 15 strains. When these probes identify a fragment, however, it is generally also detected by all other Rxl-derived probes.

Oligonucleotides from the second proline-rich region (LSM111) and the C-terminus of pspA (LSM2) generally identify only one pspA-homologous sequence at high stringency. Collectively, LSM111, 2, 3 and 4 react with e 16 of the 17 isolates and in each case revealed a consensus DNA fragment recognized by most or all of the oligonucleotide probes.

S* When an oligonucleotide probe detected only a single DNA fragment it was presumed to be pspA. If the probe detected multiple fragments, it was-presumed to hybridize with pspA. If the probe detected multiple fragments, it was presumed to hybridize with pspA and the 188 pspc sequence. Based on these assumptions the most variable portion between pspA and pspc is the region immediately upstream from the -35 promoter region and that portion encoding the a-helical region. The most conserved portion between pspA and pspC was found to be the repeat region, the leader and the proline-rich region sequences. Although only one probe from within the repeat region was used, the high degree of conservation among the 10 repeats in the Rxl sequence makes it likely io that other probes within the repeat sequences would give similar results.

The portion of Rxl pspA most similar to the pspC sequence was that encoding the leader peptide, the upstream portion of the proline-rich region, and the repeat region. The repeat region of PspA has been shown to be involved in the attachment of this protein to the pneumococcal cell surface. The conservation of the repeat region within pspc sequences suggests that if these loci encode a protein, it may have a similar functional attachment domain. The conservation of the leader sequence between pspA and the pspC sequence was also not surprising since similar conservation has been reported for the leader sequence of other proteins from gram positive organisms, such as M protein of group A streptococci (Haanes-Fritz, E. et al., Nucl. Acids Res.

1988; 16: 4667-4677).

In two strain, some oligonucleotide probes identified more than two pspA-homologous sequences. In 189 these strains, there was a predominant sequence recognized by almost all of the probes, and two or three additional sequences share homology with DNA encoding the leader, a-helical, and proline region, and they have no S homology with sequences encoding the repeat region in the C-terminus of PspA. These sequences might serve as cassettes which can recombine with pspA and/or the pspc sequences to generate antigenic diversity.

Alternatively, the sequences might encode proteins with very different C-terminal regions and might not be surface attached by the mechanism of PspA.

Oligonucleotides which hybridize with a single chromosomal DNA fragment were used as primers in PCR to examine the variability of domains within pspA. These results demonstrate that full-length pspA varies in size among strains of pneumococci, and that this variability is almost exclusively the result of sequences in the alpha-helix coding region.

EXAMPLE 12 CLONING OF PSpC Chromosomal DNA from S. pneumoniae EF6796, serotype 6A clinical isolate, was isolated by methods including purification through a cesium chloride S gradient, as described in Example 8. The HindII-EcoRI fragment of EF6796 was cloned in modified pZero vector (Invitrogen, San Diego, CA) in which the Zeocinresistance cassette was replaced by a kanamycin cassette (shown in Figure 18). Recombinant plasmids were electroporated into Escherichia coli TOPF cells F electroporated into Escherichia coli TOP1OF' cells (F' 190 O {lacIqTetR} mcrA A(mrr-hsdRMS-mcrBC) 0801acZAM15 AlacX74 deoR recAl araD139 A(ara-leu)7967 galU galK rpsL endAl nupG] (Invitrogen).

The 5' region of pspA.Rxl does not hybridize to pspC sequence at high stringencies by Southern analysis.

Utilizing both the full-length Rxl pspA probe, and a probe containing the sequence encoding a-helical region of PspA, it was possible to identify which DNA fragment contained pspA and which fragment contained the pspc ,0 locus. The pspC locus and the pspA gene of EF6796 were mapped using restriction enzymes. After digestion of chromosomal DNA with HindIII, the pspc locus was localized to a fragment of approximately 6.8 kb.

Following a double digest with HindIII and EcoRI, the pspC locus was located in a 3.5 kb fragment. To obtain the intact pspC gene of EF6796, chromosomal DNA was digested with HindIII, separated by agarose gel electrophoresis, the region between 6 and 7.5 kb purified, and subsequently digested with EcoRI. This digested DNA was analyzed by electrophoresis, and DNA fragments of 3.0 to 4.0 kb were purified (GeneClean, BiolOl, Inc., Vista, CA). The size-fractionated DNA was then ligated in HindIII-EcoRI-digested pZero, and electroplated into E. coli TOP1OF' cells. Kanamycinresistant transformants were screened by colony blots and probed with full-length pspA. A transformant, LXS200, contained a vector with a 3.5 kb insert which hybridized to pspA.

*9* 191 Sequencing of pspC in pLXS200 was completed using automated DNA sequencing on an ABI 377 (Applied Biosystems, Inc., PLACE). Sequence analyses were performed using the University Of Wisconsin Genetics 6 Computer Group (GCG) programs supported by the Center for AIDS Research (P30 AI27767), MacVector 5.0, Sequencer 2.1, and DNA Strider programs. Sequence similarities of pspC were determined using the NCBI BLAST server. The coiled-coil structure predicted by pspC sequence was ic analyzed using Matcher.

A gene probe for cloning the pspC locus Two oligonucleotide primers, N192 and C558 (shown in Figure 19), have been used previously to clone fragments homologous to the region of Rxl pspA encoding S amino acids 192-588 from various pneumococcal strains.

These primers are modifications (altered restriction sites) of LSM4 and LSM2 which were previously shown to amplify DNA encoding the C-terminal 396 amino acids of PspA.Rxl (Figure 17); this includes approximately 100 o amino acids of the a-helical region, the proline rich region, and the C-terminal choline-binding repeat region.

Using primers N192 and C558, a 1.2 kb fragment from strain EF6796 was amplified by PCR, and subsequently cloned in pET-9A (designated PRCT135). This insert was then partially sequenced.

Independently, a larger pspA fragment from strain EF6796 was made using primers LSM13 and SKH2 °ooo 192 O (shown in Figure 19) for the purpose of direct sequencing of serologically diverse pspA genes.

The LSM13 and SKH2 primer pair result in the amplification of the 5' end of most pspA gene(s) encoding the .upstream promoter, the leader peptide, the a-helical, and the proline-rich regions (amino acid -15 to 450) (Figure 20). From the strain EF6796, the LSM13 and SKH2 primers amplified a 1.3 kb fragment (pspA.EF6796), which was sequenced. The sequence from pRCT135 and the S LSM13/SKH2 PCR-generated fragment pspA.EF6796 was not identical. The fragment obtained by PCR using primers LSM13 and SKH2 was designated pspA based on its location within the same chromosomal location as pspA.Rxl. The cloned fragment in pRCT135 was assumed to represent the S sequence of the second gene locus, pspC, known to be present-from Southern analysis. Both genes have significant similarity to the corresponding regions of the prototype pspA gene from strain Rxl. The second gene locus was called pspC, in recognition of its distinct £0 chromosomal location, not sequence differences from the prototype pspA gene.

Analysis of the nucleotide and amino acid sequence of psDC EF6796 To test the hypothesis that pRCT135 represented 2. pspC of EF6796, and to further investigate pspC, the entire EF6796 pspC gene was cloned as a 3.4 kb HindlII- EcoRl fragment forming pLXS200. DNA sequence of the pspC-containing clone pLXS200 revealed an open reading 193 fram of 2782 nucleotides based on the analysis of putative transcriptional and translation start and stop sites (Figure 21). The predicted open reading frame encodes a 105 kDa protein which has an estimated pi of 4 6.09.

PspA.Rxl and PspC.EF6796 are similar in that they both contain an a-helical region followed by a proline-rich domain and repeat region (Figure However, there are several features of the amino acid is sequence of PspC which are quite distinct from PspA.

From comparisons at the nucleotide as well as the predicted amino acid sequence, it is apparent that the region of strong homology between PspC and PspA begins at amino acid 458 of PspC (amino acid 147 of PspA) and extends to the C-terminus of both proteins (positions 899 and 588 respectively). The predicted amino acid sequence of.PspC.EF6796 and PspA.Rxl are 76% similar and 68% identical based on GCG Bestfit program for this region (Figure 22). The nucleotide sequence identity between pspC and pspA is 87% for the same region. Eight bases upstream of the ATG start site is putative ribosomal binding site, TAGAAGGA. The proposed transcriptional start -35 (TATACA) and -10 (TATAGT) regions are located between 258 to 263 and 280 to 285, respectively (Figure Z 21). A potential transcriptional terminator occurs at a stem loop between nucleotides 3237 through 3287. The putative signal sequence of PspC is typical of other gram positive bacteria. This region consists of a charged e 194 region followed by a hydrophobic core of amino acids. A potential cleavage site of the signal peptide occurs at amino acid 37 following the Val-His-Ala. The first amino acid of the mature protein is a Glu residue.

Other than features similar to all signal sequences, there is no homology in this region between pspA and pspC. This confirms that pspC is present in a separate chromosomal locus from that of pspA. The signal sequence and upstream region have striking similarity to to the similar regions of S. agalactiae 8 antigen (accession number X59771). The Bantigen of Group B streptococci is a cell surface receptor that binds IgA. Similarity to the bac gene ends with the start of the mature protein of PspC, and the nucleotides are 75% identical in this region. Thus, although pspC is in a very similar chromosomal locus to the B antigen, it is clearly a distinct protein.

The N-terminus of PspC is quite different from the N-terminus of PspA. Prediction of the secondary s structure utilizing Chou-Fausman analysis (Chao, P.Y. et al., Adv. Enzymol. Relat Areas Mol. Biol. 1978: 47: 148), suggests that the structure of amino acids 16 to 589 of PspC is predominately a-helical. The Matcher program was used to examine periodicity in the a-helical region of PspA. The characteristic seven residue periodicity is maintained by having hydrophobic residues at the first and fourth positions (a and d) and hydrophobic residues at the remaining positions. The r

I

195 coiled-coil region of the a-helix of PspC (between amino acid 32 to 600) has three breaks in the heptad repeat (Figure 23). These disturbances in the 7 residue periodicity occur at amino acids 99 to 104, 224 to 267 and 346 to 350. The a-helical region of PspA has seven breaks in the motif, each break ranging from a few amino acids to 23 amino acids each. In contrast, the three breaks in the coiled-coil motif of PspC involve 5, 43 and 4 amino acids, respectively.

;c The sequence encoding the a-helical region of PspC contains two direct repeats 483 nucleotides (160 amino acids) long which are 88% percent identical at the nucleotide level. These repeats, which occur between nucleotides 562 to 1045 and nucleotides 1312 to 1795, are 1i conserved both at the nucleotide and amino acid level (amino acids 188 to 348 and 438 to 598) (Figure 24).

PspA lacks evidence for any repeats this prominent within the a-helical region. These repeat regions could provide a mechanism for recombination that could alter the Nterminal half of the PspC molecule. Although repeat motifs are common in bacterial surface proteins, a direct repeat this large or separated by a large spacer region is novel. The evolutionary significance of this region is not known. A Blast search of the repeat region and A the 267 nucleotide bases between them revealed no sequence with significant homology at the nucleotide or amino acid level. However, one of the structural breaks in the coil-coiled region of PspC is the region between ooo* *°eo *oooo *go* 196 the two repeats. Perhaps some deviation from coiled-coil structure between the two repeats is critical to maintain the a-helical structure.

Previous studies have shown that a major crossprotective region of PspA comprises the C-terminal 1/3 of the a-helical region (between residues 192 and 260 of PspA.Rxl). This region accounts for the binding of 4 of cross-protective immunity in mice. Homology between PspC and PspA begins at amino acid 148 of PspA, thus 1 including the region from 192 299. The homology between PspA and the PspC includes the entire PspC sequence C-terminal of amino acid 486. Based on the fact that PspA and PspC are so similar in this region known to be protection-eliciting, PspC is also likely to be a 6 protection-eliciting molecule. Because of close sequence and conformational similarity of the proteins in this region, antibodies specific for the region of PspA between amino acid 148 and 299 should cross-react with PspC and thus afford protection by reacting with PspC and -2 PspA. Likewise, immunization with the PspC would be "expected to elicit antibodies cross-protective against PspA. The differences between PspC of strain EF6796 and PspA of strain Rxl is no greater than the differences between many additional PspAs, which have been shown to 25 be highly cross-protective.

A proline-rich domain exists between amino acid 590 to 652. The sequence, PAPAPEK, is repeated six times in this region. This region is very similar to the °oe 197 proline-rich region of PspA.Rxl which contains the sequence PAPAP repeated eight times in two proline-rich regions. These two regions of PspA.Rxl are separated by 27 charge amino acids; no such spacer region is present S in PspC.

Many cell surface proteins of other gram positive bacteria contain proline-rich regions. These are often associated with a domain of protein that is predicted to be near the cell wall murein layer when the ,c protein is cell-associated. For example, in M proteins of S. pyogenes this domain contains both a Pro- and Glyrich regions. The fibronectin-binding protein of S.

pyogenes, S. dysgalactiae, and Staphylococcus aureus contains a proline-rich region with a three-residue 'i periodicity (pro-charged-uncharged) that is not found in PspA or PspC. An M-like protein of S. equi contains a proline-rich region that is comprised of the tetrapeptide PEPK. This region lacks glycine normally found in the proline regions of M-proteins. The last proline repeat region of this molecule is PAPAX, which is more similar to the proline-region of PspA and PspC than it is to Mproteins.

Proline-rich regions of gram positive bacterial proteins have been reported previously to transit the cell wall. The differences in proline-rich regions of proteins from diverse bacteria may reflect differences in protein function or possibly subtle differences in cell wall function. Proline-rich regions are thought to be *e *°lo *e 198 responsible for aberrant migration of these proteins through SDS-polyacrylamide gels.

The repeat region of PspC is a common motif found among several proteins in gram positive organisms.

6 Autolysin of S. pneumoniae, toxins A and B of Clostridium difficile, glucosyltransferases from S. downei and S.

mutans, and CspA of C. acetobiltylicum all contain similar regions. In PspA these repeats are responsible for binding to the phosphatidylcholine of teichoic acid and lipoteichoic acid in cell wall of pneumococci.

However, bacterial proteins containing C-terminal repeats are secreted, which may imply either a lost or gained function. Although all of these proteins have similar repeat regions the similarity of the repeat regions of ,r PspA and PspC is much greater than that of PspC to the other proteins (Table 66).

Interestingly, PspC like PspA has a 17 amino acid partially hydrophobic tail. The function of this 17 amino acid region is unknown. In the case of PspA it has 20 been shown that mutants lacking the tail bind the surface of pneumococci as well as PspAs in which the tail is expressed. Presently, it is now known whether PspC is attached to the cell surface or secreted.

PspA and PspC proteins both have a-helical coiled-coil regions, proline-rich central regions, repeat regions, with a choline binding motifs, and the Cterminal 17 amino acid tail. PspA and PspC share three i 199 regions of high sequence identity. One of these is a protection-eliciting region present within the a-helical domain. The other two regions are the prolinerich domain and a repeat domain shared with other choline binding proteins and thought to play a role in cell surface association. The similarity throughout most of the structure of the PspA and the PspC molecules raises the possibility that the two molecules may play at least slightly redundant functions. However, the fact that the N-terminal half of the protein is not homologous to any of the a-helical sequence of PspA suggests the PspC and PspA may have evolved for at least somewhat different roles on the cell surface. One of the most striking differences between the two molecules is the single i- repeat in the a-helical region of PspC. Although neither the exact function of PspA nor of PspC are known, the observation that a major cross-protective region of PspA is highly homologous with a similar region of PspC, raises the possibility that both molecules are -siciti and ±licit cross-protective antibodies.

The sequence similarity between the promoter o region of the pspC gene and the bac gene from group B streptococci is very intriguing. It implies that an interspecies recombination event has occurred and, this interspecies recombination has contributed to the evolution of the pspC. The pspc gene thus has a chimeric structure, being partially like pspA and partially like 200 the 6 antigen. In the latter case, all protein similarity is limited to the signal sequence. Similar interspecies recombination events have contributed to the evolution of the genes encoding penicillin binding protein.

Using analogous procedures, a second PspC sequence was isolated from strain D39 of S. pneumoniae.

Figures 25 to 29 show the sequence data of PspC from strain D39, complete from upstream of the promoter S through the proline-rich region. Strain D39 has the same genetic background as strains Rxl, from which pspA was sequenced. D39 and Rxl have the same pspc gene based on Southern blot analysis.

The alpha-helical encoding region of the D39 pspo gene is one third of the size of the homologous region from the EF6796 pspC gene. The proline-rich region of the D39 pspC gene was more similar to Rxl pspA than to EF6796 pspC. Even so, the two pspC genes were 86% identical at the nucleotide sequence, and 67% o identical at the amino acid level.

s ro In the alpha-helical sequence of EF6797 pspC a strong repeat was observed. This was absent in the pspc sequence of D39. The D39 pspC sequence also lacks a leader sequence, found in the EF6797 pspC sequence.

"This data strongly indicates that there is variability in the structure of pspc, similar to previous observations for pspA. In the case of pspC, however, the *o• ooo 201 extent of variability appears to be even greater than that which has been observed for pspA.

202 Table 66.

PERCENT HOMOLOGY OF CHOLINE BINDING REGIONS Percent similarity/identity Protein Organism PspA PspC PspC S. pneumoniae 86/60 100/100 Bacteriophage Cp- S. pneumoniae 56/30 56/28 1 LytA S. pneumoniae 57/33 61/32 0 PspA C. perfringens 64/45 59/42 alpha toxin C. novyi 54/29 57/33 CapB C. 58/36 61/45 acetobutylicum Having thus described in detail certain S 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.

0 0 0 0000 203 V REFERENCES 1. Alexander, Lock, Peeters,

C.C.A.M.,

Poolman, Andrew, Mitchell, T.J., Hansman, Paton, Immunization of mice with pneumolysin toxoid confers a significant degreee of protection against at least nine serotypes of Streptococcus pneumoniae, Infection and Immunity 1994, 62:5683-5688.

2. Amsbaugh, Hansen, Prescott, Stashak, Barthold, Baker, Genetic control of the antibody response to type III pneumococcal polysaccharide in mice. I. Evidence that an X-linked gene plays a decisive role in determining responsiveness, J. Exp. Med. 1972, 136:931-949.

3. Anonymous, Pneumococcal polysaccharide vaccine, MMWR 1981, 30:410-419.

4. Austrian, Pneumococcal Vaccine: Development and Prospects, Am. J. Med. 1979, 67:547-549.

Avery, McLeod, and McCarty, Studies on the chemical nature of the substance inducing transformation of pneumococcal types, Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III., J. Exp. Med.

1944, 79:137-158.

6. Avery, Goebel, Chemoimmunological studies of the soluble specific substance of pneumococcus. The isolation and properties of the acetyl polysaccharide of pneumococcus type 1, J.

Exp. Med. 1933, 58:731-755.

7. Badenes, and Dan E. Parfitt, Reducing background and interference on Southern Blots probed w irth nonriadioactive chemiluminescent probes, BioTechniques 1994, 17:622-624.

8. Berry, Yother, Briles, Hansman D., Paton, Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae, Infect. Immun. 1989, 57:2037-42.

9. Berry, Lock, Hansman, Paton, J.C., Contribution of autolysin to virulence of Streptococcus pneumoniae, Infect Immun 1989, 57:2324-30.

Birnboim H.C. and J. Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nuc. Acids Res., 1979, 7:1513.

oOeO 204 9 11. Briese, Hakenbeck, Interaction of the pneumococcal amidase with lipoteichoic acid and choline, 1985, 146:417-427.

12. Briles, J. Yother and L.S. McDaniel, Role of pneumococcal surface protein A in the virulence of Streptococcus pneumoniae, Rev. Infect. Dis. 1988, 10:S372-374.

13. Briles, Horowitz, McDaniel, L.S., Benjamin, Jr., Claflin, Booker, C.L., Scott, Forman, C. Genetic control of susceptibility to pneumococcal infection, Curr. Top.

Microbiol. Immunol. 1986, 124:103-120.

14. Briles, Forman, Horowitz, Volanakis, Benjamin, Jr., McDaniel, Eldridge, Brooks, Antipneumococcal effects of Creactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens, Infect. Immun. 1989, 57:1457-1464.

Briles, Nahm, Schroer, Davie, J., Baker, Kearney, Barletta, R., Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 Streptococcus pneumoniae, J. Exp. Med.

1981, 153:694-705.

16. Briles, M.J. Crain, B.M. Gray, C. Forman and J. Yother, A strong association between capsular type and mouse virulence among human isolates of Streptococcus pheumoniae, Infect. Immun. 1992, 60:111-116.

17. Briles Forman, Crain, Mouse antibody to phosphocholine can protect mice from infection with mouse-virulent human isolates of Streptococcus pneumoniae, Infect. Immun. 1992, 60:1957-62.

S" 18. Brooks-Walter, McDaniel, Hollingshead, Briles, D.E. Restriction fragment length polymorphisms of pspA of Streptococcus pneumoniae reveal a genetic polymorphism, Submitted.

19. Brooks-Walter A. and L.S. McDaniel, 1994, Unpublished data.

20. Cadoz J. Armand, F. Arminjon, Michel, M.

Michel, F. Denis and G. Schiffman, A new 23 valent pneumococcal vaccine: immunogenicity and reactogenicity in adults, J. Biol. Stand. 1985, 13:261.

21. Chiu, Greenberg, Marcy, Wong, Chang, Chiu, Ward, Mucosal 205 antibody responses in infants following immunization with Haemophilus influenzae, Pediatr. Res.

Abstracts 1994, 35:10A.

22. Cohen Parry alpha-helical coiled coils: more facts and better predictions, Science 1994, 236:488-9.

23. Cowan, M.J. Ammann, Wara, Howie, V.M.

Schultz, Doyle, Kaplan, Pneumococcal polysaccharide immunization in infants and children, Pediatrics 1978, 62:721-727.

24. Crain Unpublished data.

Crain, Waltman, II, Turner, J.S., Yother, Talkington, McDaniel, Gray, Briles, Pneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae, Infect.

Immun. 1990, 58:3293-3299.

26. Dagen, Melamed, Abramson, Piglansky,

L.,

Greenberg, Mendelman, Bohidar, Ter- Minassian, Cvanovich, Lov, Rusk, C., Donnelly, Yagupsky, Effect of heptavalent pneumococcal-OMPC conjugate vaccine on nasopharyngeal carriage when administered during the 2nd year of life, Pediatr. Res. 1995, 37:172A.

27. Davis Boststein Roth In: A manual for genetic engineering advanced bacterial genetics, Cold Spring Harbor, NY (Cold Spring Harbor Laboratory Press) 1980.

28. Dillard, Yother, J. Genetic and molecular characterization of capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 3, Mol. Microbiol. 1994, 12:959-972.

29. Douglas R.M. and H.B. Miles, Vaccination against Streptococcus pneumoniae in childhood: lack of demonstrable benefit in young Australian children, J. Infect. Dis. 1984, 149:861.

Douglas J.C. Paton, S.J. Duncan and D.J.

Hansman, Antibody response to pneumococcal vaccination in children younger than five years of age, J. Infect. Dis., 1983, 148:131.

31. Farley, King, Nair, Hines, S.E., Tressler, Vink, Invasive pneumococcal disease among infected and uninfected children of mothers with immunodeficiency virus infection,

J.

Pediatr. 1994, 124:853-858.

**oo 206 32. Fattom, Vann, Szu, Sutton, A., Bryla, Shiffman, Robbins, Schneerson R. Synthesis and physiochemical and immunological characterization of pneumococcus type 12F polysaccharide-diphtheria toxoid conjugates, Infect Immun. 1988, 56:2292-2298.

33. Fedson, Pneumococcal vaccination in the prevention of community-acquired pneumonia: an optimistic view of cost-effectiveness, Sem. Resp.

Infect. 1993, 8:285-293.

34. Feldman, Munro Jeffery et al.

Pneumolysin induces the salient histologic features of pneumococcal infection in the rat lung in vivo, Am. J. Respir. Cell. Mol. Biol. 1992, 5:416-23.

Filice, L.L. Van Etta, C.P. Darby and D.W.

Fraser, Bacteremia in Charleston County, South Carolina. Am. J. Epidemiol., 1986, 123:128.

36. Fischetti, Pancholi, Schneewind,

O.

Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-positive cocci, Mol. Microbiol., 1990, 4:1603-1605.

37. Forrester D.W. Jahigen and F.M. LaForce, Inefficacy of pneumococcal vaccine in a high-risk population, Am. J. Med. 83:425.

38. Garcia, Garcia, Lopez, Overproduction and rapid purifcation of the amidase of Streptococcus pneumoniae, Arch. Microbiol. 1987, 149:52-56.

39. Giebink Preventing pneumococcal disease in children: recommendations for using pneumococcal vaccine, Pediatr. Infect Dis., 1985, 4:343.

40. Giebink The microbiology of otitis media, Pediatr. Infect. Dis. 1989, 8:S18.

41. Gillespie Aspects of pneumococcal infection including bacterial virulence, host response and vaccination, J. Med. Microbiol., 1989, 28:237.

42. Gotschlich, Goldschneider, Lepow,

M.L.,

Gold, R. The immune response to bacterial polysaccharides in man, In: Antibodies in human Sdiagnosis and therapy, (Ed. Haber, Krause, R.M.) Raven, New York, 1977, 391-402.

43. Gray Pneumococcal infection in an era of multiple antibiotic resistance, Adv. Ped. Inf. Dis.

1995; In press.

207 44. Haanes-Fritz, Kraus, Burdett, Dale, Beachey, and Cleary, Comparison of the leader sequences of four group A streptococcal

M

protein genes. Nucl. 'Acids. Res., 1988, 16:4667- 4677.

Hanahan D. Studies on transformation of Escherichia coli with plasmids, J. Mol. Biol. 1983, 166:557-80.

46. Kauppi, Eskola, Kathty, Influenza type b (Hib) conjugate vaccines induce mucosal IgAl and IgA2 antibody responses in infants and children, ICAAC Abstracts 1993, 33:174.

47. Kennedy, Derousse, Immunologic response of 12 -18 month old children to licensed pneumococcal polysaccharide vaccine primed with Streptococcus pneumoniae 19F conjugate vaccine, ICAAC 1994, 34th annual meeting, 236.

48. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 1970, 227:680-5.

49. Langermann, S. Palaszynski, Burlein,

J.E.,

Koenig, Hanson, Briles, Stover, Protective humoral response against pneumococcal infection in mice elicited by recombinant Bacille Calmette-Guein vaccines expressing PspA, J. Exp. Med. 1994, 180:2277-2286.

Leinonen A. Sakkinen, R. kalliokoski,

J.

Luotenen, M. Timonen and P.H. Mekela, Antibody response to 14-valent pneumococcal capsular polysaccharide vaccine in preschool age children, Pediatr. Infect. Dis. 1986, 5:39.

51. Lock, Patent, Hansman, Purification anu immunologic characterization of neuraminidase produced by Streptococcus pneumoniae, Microbial Pathogenesis 1988, 4:33-43.

52. Lock, Hansman, Paton, Comparative S: efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae, Microbial Pathogenesis 1992, 12:137-143.

53. Lock, Paton, Hansman, Comparative efficacy of pneumococcal neuraminidase and neumolysin as immunogens protective against Streptococcus pneumoniae, Microb. Pathog. 1988, 5:461-7.

54. Makela, M. Leinonen, J. Pukander and P. Karma, A study of the pneumococcal vaccine in prevention of 208 9 clinically acute attacks of recurrent otitis media, Rev. Infect. Dis. 1981, 3:S124.

McCarty, In: The transforming principle, New York, Norton, 1985, 252.

56. McDaniel, Ralph, McDaniel, Briles, Localization of protection-eliciting epitopes on PspA of Streptococcus pneumoniae between amino acid residues 192 and 260, Microb. Pathog. 1994, 17:323-337.

57. McDaniel, Scott, Widenhofer, Carroll, Briles, Analysis of a surface protein of Streptococcus pneumoniae recognized by protective monoclonal antibodies, Microb. Pathog. 1986, 1:519- 531.

58. McDaniel, Brooks-Walter, Briles, D.E., Swiatlo, E. Oligonucleotides identify conserved and variable regions of pspA and pspA-like sequences of Streptococcus pneumoniae, Mol. Microbiol., Submitted.

59. McDaniel L.S. and D.E. Briles, In: Monoclonal antibodies against bacteria, (Orlando, FL, Academic Press, Inc.), 1986, 143.

McDaniel, Yother, Vijayakumar, M., McGarry, Guild, Briles, Use of insertional inactivation to facilitate studies of biological properties of pneumococcal surface protein A (PspA), J. Exp. Med. 1987, 165:381-394.

61. McDaniel, Sheffield, Delucchi, and Briles, D.E. (1991) PspA, a surface protein of Streptococcus pneumoniae, is capable of eliciting protection against pneumococci of more than one S. capsular type, Infect Immun 59:222-228.

62. McDaniel, Scott, Kearney, Briles, D.E. Monoclonal antibodies against protease sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae, J. Exp. Med. 1984, 160:386-397.

63. McDaniel, McDaniel, In: Analysis of the gene encoding type 12 PspA of S. pneumoniae EF5668, (Ferretti Gilmore Klaenhammer T.R., Brown F. Genetics of Streptococci, Enterococci and Lactococci (Basel: Karger) 1995, 283-6.

64. McDaniel, Sheffield, Swiatlo, E., Yother, J. Crain, and Briles, Molecular localization of variable and conserved regions of pspA, and identification of additional pspA- *o o 209 homologous sequences in Streptococcus pneumoniae, Microbial Pathogenesis, 1992, 13:261-269.

Meade, S.R. Long, C.B. Ruvkin, S.E. Brown and F.M. Ausubel, Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium melioti induced by transposon Tn5 mutagenesis,

J.

Bacteriol 1982, 149:114-122.

66. Meinkoth, and Wahl, Hybridization of nucleic acids immobilized on solid supports, Anal. Biochem.

(1984) 138:267-284.

67. Mond, Lees, Snapper, T cellindependent antigens type 2, Ann. Rev. Immunol.

1995, 13:655-692.

68. Mufson, Streptococcus pneumoniae. In: Principles and Practice of Infectious Diseases, Mandell Douglas Jr., Bennett eds.

(New York: Churchill Livingston) 1990, 1539-50.

69. Munoz, J.M. Musser, M. Crain, D.E. Briles, A.

Marton, A.J. Parkinson, U. Sorensen and A. Tomasz, Geographic distribution of penicillin-resistent clones of Streptococcus pneumoniae: characterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis, Clinic. Infect. Dis. 1992, 15:112-118.

Murray, and W.F. Thompson, Rapid isolation of high molecular weight plant DNA, Nucl. Acids. Res.

1980, 8:4321-4325.

71. Musher Infections caused by Streptococcus pneumoniae: Clinical spectrum, pathogenesis, immunity, and treatment, Clin. Infect. Dis., 1992, 14:801.

S 72. Musher, Raiza, Weinstein, L. The effect of Listeria monocytogenes on resistance to pneumococcal infection. Soc. Exp. Bio. and Med., 1970, 135:557-560.

73. Nordenstam B. Anderson, D.E. Briles, J. Brooks, A. Oden, A. Svanborg and C.S. Eden, High antiphosphorylcholine antibody levels and mortality associated with pneumonia, Scand. J. Infect. Dis., 1990, 22:187.

74. Osborn, Munson, Separation of the inner (cytoplasmic) and outer membranes of gram negative bacteria, Methods Enzymol., 1974, 31A:642-653.

75. Paton, Pathogenesis of pneumococcal disease, 1993, 363-368.

210 76. Paton, Lock, Lee, Li, J.P., Berry, Mitchell. Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide, Infect. Immun.

1991, 59:2297-2304.

77. Paton, Lock, Hansman, Effect of immunization with pneumolysin on survival time of mice challenged with Streptococcus pneumoniae, Infect. Immun. 1983, 40:548-552.

78. Raven, Reciprocal capsular transformations of pneumococci, J. Bact. 1959, 77:296-309.

79. Rijn, MacLeod, McCarty, Studies on the chemical nature of the substance inducing transformation of pneumococcal types, Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III, J. Exp. Med.

1944, 79:137-158.

Riley, I.D. and R.M. Douglas, An epidemiologic approach to pneumococcal disease, Rev. Infect. Dis., 1981, 3:233.

81. Robbins, Austrian, Lee, Rastogi, Schiffman, Henrichsen, Makela,

P.H.,

Broome, Facklam, Tiesjema, Parke, Jr., Considerations for formulating the second-generation pneumococcal capsular polysaccharide vaccine with emphasis on the crossreactive types within groups, J. Infect Dis 1983 148:1136-1159.

82. Roberts, Jeffery, Mitchell, Andrew, Boulnois, Feldman, Cole, P.J., Wilson, R. Effect of immunization with Freund's adjant and pneummolysin on histologic features of pneumococcal infection in the rat lung in vivo, Infect. Immun. 1992, 60:4969-4972.

83. Sampson, O'Connor, Stinson, A.R., Tharpe, Russell, Cloning and nucleotide Ssequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp.

adhesions, Infect. Immun. 1994, 62:319-24.

84. Scheewind, Fowler, Faull, Structure of S* cell wall anchor of cell surface proteins in Staphylococcus aureus, Science 1995, 268:103-106.

85. Schneewind Model Fischetti Sorting of protein A to the staphylococcal cell wall, Cell 1992, 70:267-81.

211 9 86. Schwartz, Gove, Lob-Lovit, Kirkwood, B.R. Potential interactions for the prevention of childhood pneumonia in developing countries: etiology of accute lower respiratory infections among young children in developing countries, Ped.

Infect. Dis., in Press.

87. Shapiro, Berg, Austrian, Schroeder, Parcells, Margolis, Adair, R.K., Clemmens, J.D. Protective efficacy of polyvalent pneumococcal polysaccharide vaccine, N. Engl. J.

Med., 1991, 325:1453-1460.

88. Sheffield, W.H. Benjamin and L.S. McDaniel.

Detection of DNA in Southern Blots by Chemiluminescence is a sensitive and rapid technique, Biotechniques 1992, 12:836-839.

89. Shoemaker, Guild, Destruction of low efficiency markers in a slow process occurring at a heteroduplex stage of transformation, Mol. Gen.

Genet. 1974, 128:283-290.

Siber, G.R. Pneumococcal disease: prospects for a new generation of vaccines, Science 1994, 265:1385- 1387.

91. Sihavy, M.L. Berman and L.W. Enquist, In: Experiments with gene fusions, (Cold Spring Harbor: Cold Springs Harbor Laboratory), 1984.

92. Smith, I. Krohn, G.T. Hermanson, A.K. Malia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M.

Goeke, B.J. Olson and D.C. Klenk, Measurement of Protein using Bicinchoninic Acid, Anal Biochem 1985, 150:76-85.

93. Smith, Guild, W.R. A plasmid in Streptococcus pneumoniae, J. Bacteriol. 1979, 137:735-739.

94. Southern, Detection of specific sequences S* among DNA fragments separated by gel electrophoresis, J. Mol. Biol. 1975, 98:503-517.

95. Stein, Thymus-independent and thymus-dependent responses to polysaccharide antigens, J. Infect.

Dis. 1992, 162:S49.

96. Studier Moffatt Use of baceriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986, 189:113-30.

97. Talkington, Koenig, Russell, The 37 kDa S. protein of Streptococcus pneumoniae protects mice *e.

*oo *ee 212 against fatal challenge, American Society of Microbiology Abstracts 1992, 149.

98. Talkington, Voellinger, McDaniel,

L.S.

Briles, Analysis of pneumococcal PspA microheterogeneity in SDS polyacrylamide gels and the association of PspA with the cell membrane, Microb. Pathog. 1992, 13:343-355.

99. Talkington, Crimmins, Voellinger,

D.C.,

Jother, Briles, A 43-kilodalton pneumococcal surface protein, PspA: isolation protective abilities, and structural analysis of the amino-terminal sequence, Infect. Immun. 1991, 59:1285-1289.

100. Tart, McDaniel, Ralph, Briles, D.E. Truncated Streptocccus pneumoniae PspA molecules elicit cross-protective immunity against pneumococcal challenge in mice, J. Infect. Dis.

1995, In Press.

101. Tomasz, A. Surface components of Streptococcus pneumoniae. Rev. Infect. Dis. 1981, 3:190-211.

102. Tomasz, A. Biological consequences of the replacement of choline by ethanolamine in the cell wall of pneumococcus: chain formation, loss of transformability, and loss of autolysis, Proc. Natl.

Acad. Sci. USA 1968, 59:86-93.

103. Towbin H, Staehelin T, Gordon Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, PNAS 1979, 76:4350-4.

104. Tuomanen, Liu, Hengstler, Zak, 0., Tomasz, The Induction of meningeal inflammation by components of the pneumococcal cell wall. 1985, 151, 859-868.

105. Tuomanen, Tomasz, Hengstler, Zak, 0.

The relative role of bacterial cell wall and capsule in the induction of inflammation in pneumococcal meningitis, J. Infect. Dis. 1985, 151:535-540.

106. van de Rijn, Kessler, R.E. Growth characteristics of Group A Streptococci in a new Schemically defined medium, Infec. Immun. 1980, 27:444-448.

107. Waltman, W.D. II, McDaniel, Gray, and Briles, Variation in the molecular weight of PspA (pneumococcal surface protein A) among SStreptococcus pneumoniae, Microbial Pathogenesis 1990, 8:61-69.

213 108. Waltman, II, McDaniel, Andersson, B., Bland, Gray, Svanborg-Eden, Briles, Protein serotyping of Streptococcus pneumoniae based on reactivity to six monoclonal antibodies, Microb. Pathog. 1988, 5:159-167.

109. Weigle, W.O. Immunological unresponsiveness In: Adv.

Immunol., (Ed. Dixon, Kunkel, Academic Press, New York, New York, 1973, 61-162.

110. Wicker L.S. and I. Scher, X-linked immune deficiency (Xid) of CBA/N mice, New York: Apringer-Verlag 1986, 86.

111. Wright S.H. Sell, W.K. Vaughn, C. Andrews, K.B. McConnell and G. Schiffman, Clinical studies of pneumococcal vaccines in infants, II, Efficacy and effect on nasopharyngeal carriage, Rev. Infect. Dis.

1981, 3:S108.

112. Yother, McDaniel, Crain, Talkington, Briles, Pneumococcal surface protein A: Structural analysis and biological significance In: Genetics and Molecular Biology of Streptococci, Lactococci, and Enterococci, (ed. Dunny, G.M., Cleary, McKay, American Society for Microbiology, Washington, DC, 1991, 88-91.

113. Yother J. and D.E. Briles, Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequences analysis, J. Bact. 1992, 174:601-609.

114. Yother, McDaniel, and Briles, D.E., Transformation of encapsulated Streptococcus pneumoniae, J. Bacteriol. 1986, 168:1463-1465.

115. Yother J, White JM. Novel surface attachment mechanism for the strept s pumoniae prot PspA, J. Bact. 1994, 176:2976-85.

116. Yother, Handsome, Briles, D.E. Truncated forms of PspA that are secreted from Streptococcus pneumoniae and their use in functional studies and cloning of the pspA gene, J. Bact., 1992, 174, 610- S618.

117. Yother J, Forman C, Gray BM, Briles DE. Protection of mice from infection with Streptococcus pneumoniae b* y anti-phosphocholine antibody, Infect. Immun.

1982, 36:184-8.

118. Zar Biostatistical Analysis, 2nd Ed., Englewood Cliffs, NJ, Prentice-Hall, Inc., 1984, 718.

Claims (71)

1. An isolated DNA molecule comprising a pspA-like nucleotide sequence which includes regions which are substantially homologous with at least the leader, portion of the proline-rich-region-encoding and repeat region-encoding portions of the pspA gene from S. pneumoniae, or a fragment of said sequence capable of acting as a probe or PCR primer in respect of said sequence, wherein said sequence or fragment thereof does not hybridise under conditions of high stringency with at least any one of the following: the (-helical region-encoding sequences of the pspA gene from S. pneumoniae; the C-terminus region-encoding sequences of the pspA gene from S. pneumoniae; or the 3' portion of the proline-rich-encoding sequences of the pspA gene from S. pneumoniae.

2. An isolated DNA molecule according to claim 1, wherein said nucleotide sequence 15 encodes an amino acid sequence comprising a PspA-like polypeptide or an antigenic, immunological or protective portion thereof.

3. An isolated DNA molecule according to claim 2, wherein said PspA-like polypeptide has alpha-helical, proline rich and repeat regions.

4. An isolated DNA molecule according to claim 3, wherein the alpha-helical region of 20 the PspA-like polypeptide comprises a C-terminus having substantial homology with a protection- eliciting region of PspA.

5. An isolated DNA molecule according to any one of claims 1 to 4 which does not hvhiid Ilntlrr rnntrinn'o M k r;,k 31 ,ll hybridise under conditions of high stringency with the following sequences which do hybridise with the pspA nucleotide sequence under conditions of high stringency: CCGGATCCAGCTCCTGCACCAAAAAC; GCGCGTCGACGGCTTAAACCCATTCACCATTGG; CCGGATCCTGAGCCAGAGCAGTTGGCTG; CCGGATCCGCTCAAAGAGATTGATGAGTCTG; and GCGGATCCCGTAGCCAGTCAGTCTAAAGCTG.

6. An isolated DNA molecule according to any one of claims 1 to 4 which does not hybridise under conditions of high stringency with the following sequences which do hybridise with the pspA nucleotide sequence under conditions of high stringency: CCGGATCCTGAGCCAGAGCAGTTGGCTG; or CCGGATCCGCTCAAAGAGATTGATGAGTCTG. [I:\DayLib\LIBZZ]06041 spec.doc:gcc 215

7. An isolated DNA molecule according to any one of claims 1 to 6 which does hybridise under conditions of high stringency with the following sequences which also hybridise with the pspA nucleotide sequence under conditions of high stringency: CCGGATCCAGCTCCAGCTCCAGAAACTCCAG; GCTATGGGCTACAGGTTG; and CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT.

8. An isolated DNA molecule according to any one of claims 1 to 7, which can be amplified by the following PCR primer pairs of oligonucleotides: CCGGATCCAGCTCCTGCACCAAAAAC and CCGGATCCAGCTCCAGCTCCAGAAACTCCAG; CCGGATCCAGCTCCTGCACCAAAAAC and GCTATGGGCTACAGGTTG; or CCGGATCCAGCTCCTGCACCAAAAAC and CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT. 1 5

9. An isolated DNA molecule according to claim 8, which encodes an antigenic, immunological or protective protein. An isolated DNA molecule as defined in any one of claims 1 to 9, substantially as hereinbefore described with reference to any one of the examples.

11. A PCR primer for amplifying a pspA-like nucleotide sequence, consisting essentially of 20 an isolated DNA molecule of any one of claims 1 to

12. A PCR primer according to claim 11, which is selected from the following sequences: CCGGATCCAGCTCCTGCACCAAAAAC; CCGGATCCAGCTCCAGCTCCAGAAACTCCAG. C IA TUC IAUAU I TG; or CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT.

13. A PCR primer as defined in claim 11 or claim 12, substantially as hereinbefore described with reference to any one of the examples.

14. A PCR amplification product from a PCR primer according to any one of claims 11 to 13.

15. A PCR amplification product according to claim 14, which comprises at least a portion of a pspA-like nucleotide sequence.

16. A PCR amplification product according to claim 14 or claim 15, wherein said portion encodes an antigenic, immunological or protective protein.

17. A PCR amplification product from a PCR primer according to any one of claims 11 to 13, substantially as hereinbefore described with reference to any one of the Examples. [I:\DayLib\LIBZZ]06041 spec.doc:gcc S 0. 0* S 216

18. A hybridisation probe consisting essentially of an isolated DNA molecule according to any one of claims 1 to

19. A hybridisation probe consisting essentially of an isolated DNA molecule according to any one of claims 1 to 10, substantially as hereinbefore described with reference to any one of the Examples. A PCR probe which distinguishes between pspA and pspA-like nucleotide sequences.

21. A PCR probe according to claim 20, complementary to a fragment"of the pspA nucleotide sequence from the a-helical region-encoding, the C-terminus region-encoding or the 3' portion of the proline-rich-encoding sequences of the pspA gene from S. pneumoniae. 1o 22. A PCR probe according to claim 20 or claim 21, which can be amplified by the PCR primer pairs of oligonucleotides: CCGGATCCAGCTCCTGCACCAAAAAC and CCGGATCCAGCTCCAGCTCCAGAAACTCCAG; CCGGATCCAGCTCCTGCACCAAAAAC and GCTATGGGCTACAGGTTG; or CCGGATCCAGCTCCTGCACCAAAAAC and CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT.

23. A PCR probe according to claim 20 or claim 21, selected from the following sequences: CCGGATCCTGAGCCAGAGCAGTTGGCTG; and CCGGATCCGCTCAAAGAGATTGATGAGTCTG.

24. A PCR probe which distinguishes between pspA and pspA-like nucleotide sequences, substantially as hereinbefore described with reference to any one of the Examples. r, A 9' L:-L L- A PCR probe which hybridises to both pspA and pspA-iike nucleotide sequences.

26. A PCR probe according to claim 25, complementary to a fragment of the pspA nucleotide sequence from the leader, 5' portion of the proline-rich-region-encoding, or repeat region-encoding portions of the pspA gene from S. pneumoniae.

27. A PCR probe according to claim 25 or claim 26, selected from the following sequences: 3o CCGGATCCAGCTCCAGCTCCAGAAACTCCAG; GCTATGGGCTACAGGTTG; and CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT.

28. A PCR probe which hybridises to both pspA and pspA-like nucleotide sequences, substantially as hereinbefore described with reference to any one of the Examples. [I:\DayLib\LIBZZ]0604lspec.doc:gcc 217

29. An isolated polypeptide comprising a PspA-like polypeptide, encoded by a nucleotide sequence according to any one of claims 1 to 10, or a PCR amplification product according to any one of claims 14 to 17. An isolated polypeptide according to claim 29, having alpha-helical, proline rich and repeat regions.

31. An isolated polypeptide according to claim 29 or claim 30, wherein the alpha-helical region comprises a C-terminus having substantial homology with a protection-eliciting region of PspA.

32. An isolated polypeptide according to claim 30 or claim 31, wherein the alpha-helical to region further comprises a seven residue periodicity and a coiled coil region having three breaks in a heptad repeat.

33. An isolated polypeptide according to any one of claims 29 to 32, having strong homology with pneumococcal surface protein A, PspA, of S. pneumoniae from amino acid 147 of PspA, extending to the C-terminus of PspA. 15 34. An isolated polypeptide according to any one of claims 29 to 33, further comprising a signal sequence consisting essentially of a charged region followed by a hydrophobic core of amino acids. An isolated polypeptide according to any one of claims 29 to 34, further comprising a .17 amino acid, partially hydrophobic tail. 20

36. An isolated polypeptide according to any one of claims 29 to 35, further comprising an epitope of interest.

37. An isolated polypeptide comprising a PspA-like polypeptide, substantially as hereinbefore described, with reference to any one of the examples.

38. A PspA-iike extract, substantially as hereinbefore described.

39. An immunological composition comprising a polypeptide according to any one of claims 29 to 37. An immunological composition comprising a polypeptide according to any one of claims 29 to 37, substantially as hereinbefore described.

41. A method for immunising a patient against infection with Streptococcus pneumoniae, comprising administering to said patient an isolated polypeptide according to any one of claims 29 to 37, a PspA-like extract according to claim 38, or an immunological composition according to claim 39 or claim

42. A method according to claim 41, substantially as hereinbefore described. [I:\DayLib\LIBZZ]06041 spec.doc:gcc 218

43. An isolated polypeptide according to any one of claims 29 to 37, a PspA-like extract according to claim 38, or an immunological composition according to claim 39 or claim 40, when used for immunising a patient against infection with Streptococcus pneumoniae.

44. An isolated polypeptide when used according to claim 43, substantially as hereinbefore described. Use of an isolated polypeptide according to any one of claims 29 to 37, a PspA-like extract according to claim 38, or an immunological composition according to claim 39 or claim for the manufacture of a medicament for immunising a patient against infection with Streptococcus pneumoniae.

46. A use according to claim 45, substantially as hereinbefore described.

47. An antibody specific for a PspA-like polypeptide, or an epitope thereof, wherein said antibody does not bind to PspA from Streptococcus pneumoniae, or to the a-helical, C-terminus, or the C-terminal portion of the proline-rich regions thereof.

48. An antibody according to claim 47, raised against a polypeptide according to any one 5 of claims 29 to 37. S49. An antibody specific for a PspA-like polypeptide, or an epitope thereof, wherein said .i antibody does not bind to PspA from Streptococcus pneumoniae, substantially as hereinbefore described, with reference to any one of the examples.

50. A pharmaceutical composition comprising an antibody according to any one of claims 47 to 49 and a pharmaceutically acceptable carrier.

51. A method for immunising a patient against infection with Streptococcus pneumoniae, comprising administering to said patient an antibody according to any one of claims 47 to 49 or a pharmaceutical composition according to claim

52. An antibody according to any one of claims 47 to 49 or a pharmaceutical composition according to claim 50, when used to immunise a patient against infection with Streptococcus pneumoniae.

53. Use of an antibody according to any one of claims 47 to 49 for the manufacture of a medicament for immunising a patient against infection with Streptococcus pneumoniae.

54. A method for diagnosing the presence of Streptococcus pneumoniae in a sample comprising the use of a detection agent selected from: a DNA molecule according to any one of claims 1 to 10; a PCR primer according to any one of claims 11 to 13; a probe according to any one of claims 18 to 28; an isolated polypeptide according to any one of claims 29 to 37; or an antibody according to any one of claims 47 to 49. A method according to claim 54, wherein the diagnosis is that of infection of a patient with Streptococcus pneumoniae. [I:\DayLib\LIBZZ]06041 spec.doc:gcc 219

56. A method according to claim 54 or claim 55, substantially as hereinbefore described.

57. A detection agent selected from: a DNA molecule according to any one of claims 1 to 10; a PCR primer according to any one of claims 11 to 13; a probe according to any one of claims 18 to 28; an isolated polypeptide according to any one of claims 29 to 37; or an antibody according to any one of claims 47 to 49; when used for diagnosing the presence of Streptococcus pneumoniae in a sample.

58. A detection agent when used according to claim 57, wherein the diagnosis is that of infection of a patient with Streptococcus pneumoniae.

59. A detection agent when used according to claim 57 or claim 58, substantially as hereinbefore described. Use of a detection agent selected from: a DNA molecule according to any one of claims 1 to 10; a PCR primer according to any one of claims 11 to 13; a probe according to any one of claims 18 to 28; an isolated polypeptide according to any one of claims 29 to 37; or an antibody according to any one of claims 47 to 49; for the manufacture of a composition for 1 5 diagnosing the presence of Streptococcus pneumoniae in a sample.

61. A use according to claim 60, wherein the diagnosis is that of infection of a patient with Streptococcus pneumoniae.

62. A use according to claim 60 or claim 61, substantially as hereinbefore described.

63. A method for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences, said method comprising: probing a portion of a sample suspected of including one or both of said sequences with at least one probe according to any one of claims 25 to 28; and probing another portion of said sample with at least one probe according to any III UI U~ldllllI LU LU LQ one of claims 20 to 24; whereby any sequences detected by step but not by step are pspA-like nucleotide sequences, and any sequences detected by steps and are pspA nucleotide sequences.

64. A method for detecting pspA-like nucleotide sequences comprising amplifying DNA in a sample by PCR, using a PCR primer according to any one of claims 11 to 13.

65. A method according to claim 63 or claim 64, wherein said method comprises detection and/or diagnosis of infection of a patient with Streptococcus pneumoniae.

66. A method according to claim 63 or claim 64, wherein said method comprises detection or diagnosis of a particular pneumococcal strain or grouping of pneumococcal strains. [I:\DayLib\LIBZZ]06041 spec.doc:gcc k- 220

67. A method for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences, said method being substantially as hereinbefore described, with reference to any one of the examples.

68. A pspA-like nucleotide sequence detected by a method according to any one of claims 63, 64 or 67.

69. A pspA-like nucleotide sequence detected by a method according to any one of claims 63, 64 or 67, substantially as hereinbefore described, with reference to any one of the examples. An isolated pneumococcal strain detected by a method according to any one of claims 63 to 67.

71. An isolated pneumococcal strain according to claim 70 which is a PspA-like polypeptide-presenting pneumococcal strain.

72. An isolated pneumococcal strain detected by a method according to any one of claims 63 to 67, substantially as hereinbefore described, with reference to any one of the 15 examples.

73. A probe according to any one of claims 25 to 28, when used for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences.

74. A probe according to any one of claims 20 to 24, when used for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences.

75. At least one probe according to any one of claims 25 to 28, and at least one probe according to any one of claims 20 to 24, when used for detecting pspA-like nucleotide sequences Sand/or distinguishing between pspA and pspA-like nucleotide sequences.

76. A PCR primer according to any one of claims 11 to 13, when used for detecting pspA- Slike nu.eotide sequences andior distinguishing between pspA and pspA-like nucleotide sequences.

77. A probe when used according to any one of claims 73 to 75, or a PCR primer when used according to claim 76, wherein said use comprises detection and/or diagnosis of infection of a patient with Streptococcus pneumoniae.

78. A probe when used according to any one of claims 73 to 75, or a PCR primer when used according to claim 76, wherein said use comprises detection or diagnosis of a particular pneumococcal strain or grouping of pneumococcal strains.

79. At least one probe according to any one of claims 25 to 28, when used for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences, substantially as hereinbefore described, with reference to any one of the examples. [I:\DayLib\LIBZZ]06041 spec.doc:gcc 221 At least one probe according to any one of claims 20 to 24, when used for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences, substantially as hereinbefore described, with reference to any one of the examples.

81. A PCR primer according to any one of claims 11 to 13, when used for detecting pspA- like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences, substantially as hereinbefore described, with reference to any one of the examples.

82. Use of at least one probe according to any one of claims 25 to 28, arfd at least one probe according to any one of claims 20 to 24, for the manufacture of a diagnostic assay for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences.

83. Use of a PCR primer according to any one of claims 11 to 13 for the manufacture of a diagnostic assay for detecting pspA-like nucleotide sequences and/or distinguishing between pspA and pspA-like nucleotide sequences.

84. A use according to claim 82 or claim 83, wherein said diagnostic assay is for detection I15 and/or diagnosis of infection of a patient with Streptococcus pneumoniae.

85. A use according to claim 82 or claim 83, wherein said diagnostic assay is for detection or diagnosis of a particular pneumococcal strain or grouping of pneumococcal strains. Dated 30 August, 2001 S 20 UAB Research Foundation S oS Patent Attorneys for the Applicant/Nominated Person SPRI SON FEP.Rc' CTGUSO [1:\DayLib\LIBZZ]06041spec.doc:gcc