Epitopic regions of pneumococcal surface protein A

A region of the PspA protein of the Rx1 strain of Streptococus pneumoniae has been identified as containing protection-eliciting epitopes which are cross-reactive with PspAs of other S.pneumoniae strains. The region comprises the 68-amino acid sequence extending from amino acid residues 192 to 260 of the Rx1 PspA strain.

FIELD OF INVENTION 
This invention relates to recognition of epitopic regions of pneumococcal 
surface protein A (PspA), the major virulence factor of Streptococcus 
pneumoniae. 
BACKGROUND TO 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. 
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 to make an immune 
response against polysaccharide antigens and can have repeated infections 
involving the same capsular serotype. 
One approach to immunizing infants against a number of encapsulated 
bacteria is to conjugate the capsular polysaccharide antigens to proteins 
to make them immunogenic. This approach has been successful, for example, 
with Haemophilus influenzae b (see U.S. Pat. No. 4,496,538 to Gordon and 
U.S. Pat. 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 
adults. Furthermore, such a vaccine would probably be much more expensive 
to produce than any of the other childhood vaccines in routine use. 
An alternative approach for protecting children, and also the elderly, from 
pneumococcal infection would be to identify protein antigens that could 
elicit protective immune responses. Such proteins may serve as a vaccine 
by themselves, may be used in conjunction with successful 
polysaccharide-protein conjugates, or as carriers for polysaccharides. 
In McDaniel et al (I), J.Exp.Med. 160:386-397, 1984, there is described the 
production of hybridoma antibodies that recognize cell surface proteins 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. 
In McDaniel et al (II), Microbial Pathogenesis 1:519-531, 1986, there are 
described studies on the characterization of the PspA. From the results of 
McDaniel (II), McDaniel (III), J.Exp. Med. 165:381-394, 1987, Waltman et 
al., Microb. Pathog. 8:61-69, 1990 and Crain et al., Infect. Immun. 
58:3293-3299, 1990, it was also apparent that the PspAs of different 
strains frequently exhibit considerable diversity in terms of their 
epitopes, and apparent molecular weight. 
In McDaniel et al (III), there is disclosed that immunization of X-linked 
immunodeficient (XID) mice with non-encapsulated pneumococci expressing 
PspA, but not isogenic pneumococci lacking PspA, protects mice from 
subsequent fatal infection with pneumococci. 
In McDaniel et al (IV), Infect. Immun., 59:222-228, 1991, there is 
described 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. 
In Crain et al, (supra) there is described a rabbit antiserum that detects 
PspA in 100% (n=95) of clinical and laboratory isolates of strains of S. 
pneumoniae. When reacted with seven monoclonal antibodies to PspA, 
fifty-seven S. pneumoniae isolates exhibited thirty-one different patterns 
of reactivity. Accordingly, although a large number of 
serologically-different PspAs exist, there are extensive cross-reactions 
between PspAs. 
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 molecular weights, which range from 67 to 99 kD. The observed 
differences are stably inherited and are not the result of protein 
degradation. 
Immunization with a partially purified PspA from a recombinant .lambda. 
gt11 clone, elicited protection against challenge with several S. 
pneumoniae strains representing different capsular and PspA types, as 
described in McDaniel et al (IV), 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 
lysis was not possible. 
While the protein is variable in structure between different pneumococcal 
strains, numerous cross-reactions 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. 
In addition to the published literature specifically referred to above, the 
inventors, in conjunction with coworkers, 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; 
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 (I), J. Bacteriol. 174:601-609, 1992; 
6. Yother et al (II), J. Bacteriol. 174:610-618, 1992; and 
7. McDaniel et al (V), Microbiol Pathogenesis, 13:261-268. 
In the aforementioned copending United States patent applications Ser. Nos. 
656,773, now abandoned and 835,698, now abandoned as well as in Yother et 
al (I) and (II), there are described the preparation of mutants of S. 
pneumomiae that secrete an immunogenic truncated form of the PspA protein, 
and the isolation and purification of the secreted protein. The truncated 
form of PspA was found to be immunoprotective and to contain the 
protective epitopes of PspA. The PspA protein described therein is soluble 
in physiologic solution and lacks at least the functional cell membrane 
anchor region. 
In the specification which follows and the drawings accompanying the same, 
there are utilized certain accepted abbreviations with respect to the 
amino acids represented thereby. The following Table I identifies those 
abbreviations: 
TABLE I 
______________________________________ 
AMINO ACID ABBREVIATIONS 
______________________________________ 
A = Ala = Alanine M = Met = Methionine 
C = Cys = Cysteine N = Asn = Asparagine 
D = Asp = Aspartic Acid 
P = Pro = Proline 
E = Glu = Glutamic Acid 
Q = Gln = Glutamine 
F = Phe = Phenylalanine 
R = Arg = Arginine 
G = Gly = Glycine S = Ser = Serine 
H = His = Histidine 
T = Thr = Threonine 
I = Ile = Isoleucine 
V = Val = Valine 
K = Lys = Lysine W = Try = Tryptophan 
L = Leu = Leucine Y = Tyr = Tyrosine 
______________________________________ 
SUMMARY OF INVENTION 
In accordance with the present invention, there has been identified a 
68-amino acid region of PspA from the Rx1 strain of Streptococcus 
pneumoniae which not only contains protection-eliciting epitopes, but also 
is sufficiently cross-reactive with other PspA's from other S. pneumoniae 
strains so as to be a suitable candidate for the region of PspA to be 
incorporated into a recombinant PspA vaccine. 
The 68-amino acid sequence extends from amino acid residues 192 to 260 of 
the Rx1 PspA protein. While the disclosure herein refers specifically to 
the specific 68 amino acid sequence of the Rx1 PspA protein, any region of 
a PspA protein from any other S. pneumoniae strains which is homologous to 
this sequence of the Rx1 PspA protein is included within the scope of the 
invention, for example, from strains D39 and R36A. 
Accordingly, in one aspect, the present invention provides an isolated PspA 
protein fragment comprising amino acid residues 192 to 260 of the PspA 
protein of the Rx1 strain of Streptococcus pneumoniae and containing at 
least one protection-eliciting epitope. 
The protein fragment may be one containing an amino acid sequence 
corresponding to or homologous to the amino acid residues 192 to 260 of 
the PspA protein of the Rx1 strain and hence may comprise fragments larger 
than ones containing the specific amino acid sequence. 
The protein fragment of the invention may be produced recombinantly in the 
form of a truncated C-terminal deleted product containing the protein 
fragment, specifically a truncated C-terminal-deleted product containing 
the approximately C-terminal third of an .alpha.-helical region of the 
native PspA protein. 
The present invention also includes an isolated protein fragment comprising 
an amino acid sequence corresponding to that of a protein-eliciting 
epitope contained in amino acid residues 192 to 260 of the PspA protein of 
the Rx1 strain of Streptococcus pneumoniae.

GENERAL DESCRIPTION OF INVENTION 
As described in the prior U.S. patent applications referred to above and in 
Yother et al (I) and (II), the pspA gene of strain Rx1 encodes a 65 kDa 
molecule composed of 588 amino acids. The nucleotide sequence (SEQ ID No: 
1) of the pspA gene and derived amino acid sequence (SEQ ID No: 2) are set 
forth in FIGS. 1A-C. The N-terminal half of the molecule is highly charged 
and its DNA sequence predicts an .alpha.-helical coiled-coil protein 
structure for this region (288 amino acids), as seen in FIG. 2. The 
C-terminal half of PspA, which is not .alpha.-helical, includes a 
proline-rich region (83 amino acids) and a repeat region containing the 
highly conserved twenty amino acid repeats, as well as a slightly 
hydrophobic sequence of 17 amino acids at the C-terminus. It is known that 
PspA is anchored to S. pneumoniae by its C-terminal half and it is likely 
that the proline-rich region serves to tangle the molecule in the cell 
wall. In addition, it is anticipated that the highly-charged 
.alpha.-helical region begins at the cell wall and extends into and 
possibly through the capsule. This model is supported by the observation 
that the s-helical domain contains all the surface exposed epitopes 
recognized by monoclonal antibodies (MAbs) reactive with PspA on the 
pneumococcal surfaces. 
The PspA protein of S. pneumoniae strain Rx1 has been mapped to locate 
protection-eliciting epitopes. Such mapping has been effected by employing 
antibodies to PspA protein and recombinant fragments of PspA. This mapping 
technique, described in detail in the Examples below, has identified an 
amino acid sequence corresponding to the C-terminal third of the 
.alpha.-helical region of PspA as containing protection-eliciting 
epitopes, specifically the amino acid residues 192 to 260 of the Rx1 PspA 
protein. The amino acid sequence from residues 192 to 260 is the 
C-terminal third of the .alpha.-helical sequence, expected to be near the 
cell wall surface. 
Since the portion of the sequence from residues 192 to 260 contains only 68 
amino acids, individual PspA protein fragments of this size may not be 
optimally antigenic. This difficulty is overcome by producing recombinant 
proteins containing tandem fragments of different PspAs expressed by gene 
fusions of the appropriate portions of several pspA genes. 
Accordingly, in a further aspect of the invention, there is provided a PspA 
protein fragment comprising a plurality of conjugated molecules, each 
molecule comprising amino acid residues 192 to 260 of the PspA protein of 
the Rx1 strain of Streptococcus pneumoniae and containing at least one 
protection-eliciting epitope, each molecule being derived from a different 
strain of S. pneumoniae. 
Such tandem molecules can be engineered to maintain proper coiled-coil 
structure at the points of junction and to be large enough to be 
immunogenic and to express an array of protection-eliciting epitopes that 
may cross-react with a wide spectrum of PspAs. Alternatively, individual 
recombinantly-produced peptides may be attached by chemical means to form 
a complex molecule. 
A further alternative is to attach the PspA fragment to a larger carrier 
protein or bacterial cell, either as a recombinant fusion product or 
through chemical attachment, such as by covalent or ionic attachment. 
The protein fragments and peptide analogs thereof provided herein are 
useful components of a vaccine against disease caused by pneumococcal 
infection. Accordingly, the present invention provides, in a yet further 
aspect, a vaccine comprising the PspA protein fragments defined herein as 
an immunologically-active component thereof. 
BIOLOGICAL MATERIALS 
The Examples which follow as well as in the accompanying drawings, 
reference is made to certain plasmid materials containing whole or 
truncated pspA gene sequences. The following Table II provides a summary 
of such materials: 
TABLE II 
______________________________________ 
Plasmid Identification 
Gene Product 
______________________________________ 
pKSD1014 whole gene amino acids 1 to 588 
pJY4284 or 5' terminal region 
amino acids 1 to 115 
pJY4285 
pJY4310 5'-terminal region 
amino acids 1 to 192 
pJY4306 5'-terminal region 
amino acids 1 to 260 
pBC207 3'-terminal region 
amino acids 119 to 588 
pBC100 3'-terminal region 
amino acids 192 to 588 
______________________________________ 
EXAMPLES 
Example 1 
This Example describes the bacterial strains, plasmids and hybridoma 
antibodies used herein. 
S. pneumoniae strains, identified in Table III below, were grown in Todd 
Hewitt broth with 0.5% yeast extract at 37.degree. C. or on blood agar 
plates containing 3% sheep blood in a candle jar. E. coli strain DH1 
(Hanahan, J. Mol. Biol. 166:557) was grown in LB medium or minimal E 
medium. Plasmids included pUC18 (Gene 33:103), pJY4163 (Yother et al 
(II)), and pIN-III-ompA (EMBO J. 3:2437). 
All antibody-secreting hybridoma lines were obtained by fusions with 
non-antibody-secreting myeloma cell line P3-X63-Ag.8.653 (J. Immunol. 
123:1548). The specific antibodies employed are identified in Table III 
below. The anti-PspA hybridoma cell lines Xi64, Xi126 and XiR278 have 
previously been described in McDaniel et al (I) and Crain et al (supra). 
The remaining cell lines were prepared by immunizing CBA/N mice with 
recombinant D39 PspA expressed in .lambda.gtII by the technique described 
in McDaniel et al (I). The cell lines producing antibodies to PspA were 
all identified using an ELISA in which microtitration plates were coated 
with heat-killed (60.degree. C., 30 mins) S. pneumoniae R36A or Rx1, which 
would select for those MAbs that react with surface exposed epitopes on 
PspA. The heavy chain isotypes of the MAbs were determined by developing 
the ELISA with affinity purified goat antibody specific for .mu. and 
.lambda. heavy chains of IgM and IgG mouse immunoglobulin. The specificity 
of the MAbs for PspA was confirmed by immunoblot analysis. 
All six newly-produced MAbs, identified in Table III as XiR 1526, XiR 35, 
XiR 1224, XiR 16, XiR 1325 and XiR 1323, detected a protein of the 
expected size (apparent molecular weight of 84 kDa) in an immunoblot of 
strains Rx1 and D39. No reactivity was observed for any of the MAbs in an 
immunoblot of strain WG44.1, a PspN variant of Rx1 (see McDaniel et al 
(III) and Yother et al (II)). 
TABLE III 
__________________________________________________________________________ 
Reactivities of MAbs with PspAs from Streptococcus pneumoniae 
Monocional Antibody (Isotype) 
Streptococcus pneumoniae 
XiR1526 
XiR35 
XiR1224 
Xi126 
XiR16 
Xi64 
XiR1325 
XiR278 
XiR1323 
Strain Capsule type 
PspA type 
Ref. # 
(1gG2b) 
(1gG2a) 
(1gM) 
(1gG2b) 
(1gG2a) 
(1gM) 
(1gG2a) 
(1gG1) 
(1gM) 
__________________________________________________________________________ 
Rx1 rough 25 36 ++ ++ ++ ++ ++ ++ ++ ++ ++ 
ATCC101813 
3 3 37 - - - ++ - ++ ++ ++ ++ 
EF10197 3 18 38 - - - - - - -/+ ++ - 
BG9739 4 26 38 - - - - - - ++ + ++ 
L81905 4 23 38 - - - - - - - - - 
BG-5-8A 6A 0 38 - - + -/+ - - - + + 
BG9163 6B 21 38 - - - - - - - + - 
LM100 22 ND * - - - -/+ - - - - - 
WU2 3 1 39 - - - ++ - ++ ++ ++ ++ 
Protection against WU2 - - - + - + + + + 
__________________________________________________________________________ 
Example 2 
This Example describes the provision of the pspA gene from pneumococcal 
strain Rx1 by polymerase chain reaction (PCR). 
PCR primers were designed based on the sequence of the pspA gene from 
pneumococcal strain Rx1 (see FIG. 1 SEQ ID NO: 1). The 5'-primers were 
LSM3 and LSM4. LSM3 was 28 bases in length and started at base 576 and 
LSM4 was 31 bases in length and started at base 792, and both contained an 
additional BamHI site. The 3' pspA primer was LSM2 which was 33 bases in 
length and started at base 1990 and contained an additional SalI site. 
The nucleotide sequences for the primers are as follows: 
LSM2 5'-GCGCGTCGACGGCTTAAACCCATTCACCATTGG-3' (SEQ ID NO:3) 
LSM3 5'-CCGGATCCTGAGCCAGAGCAGTTGGCTG-3' (SEQ ID NO: 4) 
LSM4 5'-CCGGATCCGCTCAAAGAGATTGATGAGTCTG-3' (SEQ ID NO: 5) 
Approximately 10 ng of genomic Rx1 pneumococcal DNA was amplified using a 
5' and 3' primer pair. The sample was brought to a total volume of 50 
.mu.l containing a final concentration of 50 mM KCl, 10 mM tris-HCl (pH 
8.3), 1.5 mM MgCl.sub.2, 0.001% gelatin, 0.5 mM each primer and 200 mM of 
each deoxynucleoside triphosphate and 2.5 U of Taq DNA polymerase. 
Following overlaying of the samples with 50 .mu.l of mineral oil, the 
samples were denatured at 94.degree. C. for 2 mins and then subjected to 
10 cycles consisting of 1 min. at 94.degree. C., 2 min. at 50.degree. C. 
and 3 min. at 72.degree. C., followed by another 20 cycles of 1 min. at 
94.degree. C., 2 min. at 60.degree. C. and 3 min. at 72.degree. C. After 
completion of the 30 cycles, the samples were held at 72.degree. C. for an 
additional 5 min., prior to cooling to 4.degree. C. 
Example 3 
This Example describes expression of truncated PspA molecules. 
3'-deleted pspAs that express N-terminal fragments in E. coli and which 
secrete the same fragments from pneumococci were constructed as described 
in the aforementioned U.S. patent applications Ser. Nos. 835,698 and 
656,773 (see also Yother et al (II), supra). 
For expression of 5'-deleted pspA constructs, the secretion vector 
pIN-III-ompA was used. Amplified pspA fragments were digested with BamHI 
and SalI and ligated into the appropriately BamHI/SalI- digested 
pIN-III-ompA vector, providing the inserted fragment fused to the ompA 
leader sequence in frame and under control of the lac promoter. 
Transformants of E. coli DH1 were selected on minimal E medium 
supplemented with casamino acids (0.1%), glucose (0.2%) and thiamine (0.05 
mM) with 50 .mu.g/ml of ampicillin. 
For induction of lac expression, bacteria were grown to an optical density 
of approximately 0.6 at 660 nm at 37.degree. C. in minimal E medium and 
IPTG was added to a concentration of 2 mM. The cells were incubated for an 
additional two hours at 37.degree. C., harvested and the periplasmic 
contents released by osmotic shock. An immunoblot of the truncated PspA 
proteins produced by the various plasmids is shown in FIG. 4. 
By these procedures, there were provided, for the 3'- deleted pspAs, 
plasmids pJY4284, pJY4285, pJY4310 and pJY4306 and for the 5'-deleted 
pspAs, plasmids pBC207 and pBC100. Plasmid pJY4284 and pJY4285 contain an 
insert of 564 base pairs, nucleotides 1 to 564 and encoded a predicted 13 
kDa PspA C-terminal-deleted product corresponding to amino acids 1 to 115. 
Plasmid pJY4310 contains an insert of 795 base pairs, nucleotides 1 to 795 
and encoded a predicted 21 kDa C-terminal-deleted product corresponding to 
amino acid 1 to 192. However pJY4306 contained an insert of 999 base 
pairs, nucleotides 1 to 999 and encoded a predicted 29 kDa 
C-terminal-deleted product corresponding to amino acids 1 to 260. Plasmid 
pBC100 contained an insert of 1199 base pairs, nucleotides 792 to 1990, 
and encoded a predicted 44 kDa PspA N-terminal deleted product containing 
amino acids 192 to 588. pBC207 contained an insert of 1415 base pairs, 
nucleotide 576 to 1990, and encoded a predicted 52 kDa PspA N-terminal 
deleted product containing amino acids 119 to 588. 
The pspA gene sequences contained in these plasmids code for and express 
amino acids as identified in FIG. 2 SEQ ID NO: 2. 
Example 4 
This Example describes the procedure of effecting immunoassays. 
Immunoblot analysis was carried out as described in McDaniel et al (IV). 
The truncated PspA molecules prepared as described in Example 3 or 
pneumococcal preparations enriched for PspA (as described in McDaniel et 
al (II)) were electrophoresed in a 10% sodium dodecyl sulfate 
polyacrylamide gel and electroblotted onto nitrocelluloses. The blots were 
probed with individual MAbs, prepared as described in Example 1. 
A direct binding ELISA procedure was used to quantitatively confirm 
reactivities observed by immunoblotting. In this procedure, osmotic shock 
preparations were diluted to a total protein concentration of 3 .mu.g/ml 
in phosphate buffered saline (PBS) and 100 .mu.l was added to wells of 
Immulon 4 microtitration plates. After blocking with 1% bovine serum 
albumin in PBS, unfractionated tissue culture supernates of individual 
MAbs were titered in duplicate by 3-fold serial dilution through 7 wells 
and developed as described in McDaniel et al (IV) using a goat anti-mouse 
immunoglobulin alkaline phosphate conjugated secondary antibody and 
alkaline phosphate substrate. Plates were read in a DYNATECH (trademark) 
plate reader at 405 nm, and the 30% end point was calculated for each 
antibody with each preparation. 
The protective capacity of the MAbs was tested by injecting three CBA/N 
mice i.p. with 0.1 ml of 1/10 dilution (about 5 to 30 .mu.g) of each 
hybridoma antibody 1 hr prior to i.v. injection of 10.sup.3 CFU of WU2 or 
D39 pneumococci (&gt;100.times.LD.sub.50). Protection was judged as the 
ability to prevent death of all mice in a group. All non-protected mice 
died of pneumococcal infection within 48 hours post challenge. 
Example 5 
This Example describes mapping of the epitopes on PspA using the monoclonal 
antibodies described in Example 1. 
The six newly-produced monoclonal antibodies described in Example 1 and 
identified in Table III were used along with the previously-described 
monoclonal antibodies Xi64, Xi126 and XiR278 to map epitopes on PspA. 
To determine whether each of the MAbs recognized different epitopes, each 
of them was reacted with eight additional S. pneumoniae strains, as 
identified in Table III, in immunoblots of SDS-PAGE separated proteins. 
Seven different patterns of activity were observed. Three antibodies, 
XiR16, XiR35 and XiR1526, appeared to recognize epitopes found on Rx1 PspA 
but none of the other PspAs. Accordingly, it was possible that these three 
antibodies might all react with the same epitope as Rx1 PspA. 
MAb Xi64 and Xi126 both reacted strongly only with epitopes on ATCC 101813, 
WU2 and Rx1 PspAs, but not with PspAs of the other strains. However, it is 
known from studies of larger panels of PspAs (as described in McDaniel et 
al (III) and Crain et al) that Xi126 and Xi64 recognize different 
determinants. 
The remaining four antibodies each exhibited unique patterns of reactivity 
with the panel of PspAs. Accordingly, the nine antibodies tested 
recognized at least seven different epitopes on PspA. 
For reasons which are not clear, the type 2 strain D39 appeared to be 
uniquely able to resist the protective effects of antibodies to PspA 
(McDaniel et al (IV)). As described in McDaniel et al (I), greater than 
forty times the amount of Xi126 was required to passively protect against 
the D39 strain as compared to the WU2 strain. None of the six 
newly-produced monoclonal antibodies protected against the D39 strain. In 
contrast, immunization of mice with Rx1 PspA elicits protection against 
A66, WU2 and EF6796 strains (mouse virulent pneumococci of capsular types 
3, 3 and 6A respectively), all of which have PspA types that are different 
from those of Rx1 and D39 (see McDaniel et al (IV)). In view of the close 
serologic similarity between the type 25 PspA of Rx1 and type 1 PspA of 
WU2 (Crain et al), WU2 pneumococci were used to challenge mice that had 
been passively protected with the MAbs. All five of the MAbs that were 
observed to bind WU2 PspA were able to protect against infection with 1000 
CFU of WU2. Protective antibodies were found in IgM, IgG1, IgG2b and Ig2a 
heavy chain isotype classes. 
Example 6 
This Example describes mapping of the epitopes of PspA using the 
recombinant truncated PspA molecules formed in Example 3. 
The five-overlapping C-terminal or N-terminal deleted PspA fragments, 
prepared as described in Example 3 and shown in FIG. 2, were used to map 
epitopes on PspA. The general location of the epitopes detected by each of 
the mice MAbs, as described in Example 5, was determined using the five 
C-terminal-deleted and two N-terminal deleted PspA molecules. As a 
positive control, the reactivity of each antibody was examined with a 
clone, pKSD1014, expressing full-length PspA. 
As noted earlier, the reactivity of the MAb was determined by two methods. 
In one method, reactivity between the fragments and MAb was evaluated in 
immunoblots of the fragment preparations after they had been separated by 
SDS-PAGE. In the second method, a direct ELISA was used to quantify the 
reactivity of the MAbs with non-denatured PspA fragment. 
The reactivities observed and the quantification of such activity is set 
forth in the following Table IV: 
TABLE IV 
__________________________________________________________________________ 
Reactivity of PspA Fragments with Monclonal Antibodies.sup.1 
PspA Monoclonal Antibodies 
Fragments 
Xi126 
XiR35 
XiR1526 
XiR1224 
XiR16 
XiR1323 
Xi64 XiR1325 
XiR278 
__________________________________________________________________________ 
pJY4285 
++ 
72 ++ 
5 ++ 
&lt;3 + &lt;3 + 4 - &lt;3 - &lt;3 - &lt;3 - &lt;3 
pJY4310 
++ 
116 
++ 
4 ++ 
&lt;3 + 5 ++ 
16 - 31 - &lt;3 - &lt;3 - &lt;3 
pJY4306 
++ 
1127 
++ 
78 
++ 
554 
++ 
805 
++ 
2614 
++ 
&lt;3 ++ 
643 
++ 
717 
+ &lt;3 
pBC207 
- &lt;3 - &lt;3 
- &lt;3 - &lt;3 + &lt;3 ++ 
61 ++ 
&lt;3 ++ 
&lt;3 ++ 
4527 
pBC100 
- &lt;3 - &lt;3 
- &lt;3 - &lt;3 - &lt;3 ++ 
15 ++ 
709 
++ 
4401 
++ 
4746 
Rx1 ++ 
63 ++ 
15 
++ 
42 ++ 
48 ++ 
118 
++ 
44 ++ 
64 ++ 
111 
++ 
468 
pIN-III 
- &lt;3 - &lt;3 
- &lt;3 - &lt;3 - &lt;3 - &lt;3 - &lt;3 - &lt;3 - &lt;3 
__________________________________________________________________________ 
.sup.1 Antibodies were reacted with the indicated PspA fragments in 
immunoblot of SDSPAGE separations, or by ELISA using microtitration plate 
coated with preparations enriched for the indicated PspA fragments. Rx1 
PspA serves as a positive control, and pINIII-ompA(vector alone) serves a 
a negative control. The results of the immunoblot are presented as ++ 
(strong reaction), + (weak but clearly positive reaction) and - (no 
reaction). ELISA values are given as the reciprocal dilution of each 
monoclonal antibody that gave 30% of maximum binding with wells coated 
with the indicate fragment preparation. 
The asterisk (*) after some of the antibodies denotes those which are abl 
to protect against fatal pneumococcal infection with strain WU2 or D39 S. 
pneumoniae. 
The deduced locations of the epitopes are indicated in FIG. 3. 
As can be seen from the data in Table IV, three of the antibodies, Xi126 
and XiR35 and XiR1526, react strongly with all three C-terminal-deleted 
clones in immunoblot analysis, indicating that the sequence required to 
form the epitope(s) detected by all three lies within the first 115 amino 
acids of PspA. This map position is in agreement with the failure of these 
antibodies to react with either of the N-terminal-deleted clones that lack 
the first 119 and 191 amino acids. 
MAbXiR1224 reacted strongly by immunoblot with the longest 
C-terminal-deleted fragment (pJY4306), but showed substantially weaker 
reactions with the shorter two C-terminal-deleted fragments. This result 
indicates that, while the binding site of the antibody may be in the first 
115 amino acids, residues beyond amino acid 192 may be important for the 
conformation or stability of the epitope. 
By immunblot, the three antibodies Xi64, XiR1325 and XiR278, all reacted 
with the longest C-terminal-deleted fragment and both of the 
N-terminal-deleted fragments, thus locating their determinants between 
amino acid positions 192 and 260. Generally confirmatory results were 
obtained in ELISAs with the native molecules. However, in a few cases, 
reactions were observed in ELISAs with full length PspA but not with a 
truncated molecule even though the same truncated fragment was reactive 
with the antibody by immunoblot. These observations may have resulted from 
an altered conformation of the truncated fragments under physiologic 
conditions that masked or prevented the formation of determinant present 
in full-length PspA and in the denatured fragments. 
Two antibodies XiR216 and XiR1323 showed what, at first appeared to be 
anomalous reactions, indicating that epitopes detected by the antibodies 
might be in more than one portion of PspA. In view of this unexpected 
result, the assays were repeated multiple times with two sets of 
preparations of the truncated fragments. The results of the additional 
assays confirmed the two-position mapping of epitopes for these two MAbs. 
By immunoblot, MAb XiR16 reacted strongly with the two longest 
C-terminal-deleted fragments and failed to react with the shortest 
N-terminal-deleted fragment. Accordingly, the epitope detected must be 
N-terminal to position 192. Unexpectedly, MAbXiR16 reacted weakly in 
immunoblots with both the longest N-terminal-deleted fragment (residues 
119 to 158) and the shortest C-terminal-deleted fragment (residues 1 to 
115). Since the fragments do not overlap, and if the weak immunoblot 
reactivities with fragments (reactivities not seen by ELISA) are not an 
artifact, the MAb XiR16 must recognize epitopes on both fragments. 
In the case of MAb XiR1323, the immunoblot data clearly places the detected 
epitope between positions 192 and 260. In the ELISA studies, however, 
XiR1323 reacted strongly and reproducibly with the C-terminal-deleted 
fragment pJY4310 (amino acid residues 1 to 192) as well as the shortest 
N-terminal-deleted fragment pBC100 (amino acid residues 192 to 588). 
Curiously, an ELISA reaction was not observed between MAb XiR1323 and 
pJY4306 (amino acid residues 1 to 260), even though MAb XiR1323 reacted 
strongly with this fragment by immunoblot. 
These findings provide additional evidence for distal conformation effects 
on antigenic determinants of PspA. They also indicate that, on the native 
fragments, MAb XiR1323 sees epitopes on both sides of position 192. The 
relationship between expression of the epitopes in other PspAs and their 
position in Rx1 PspA is demonstrated in Table IV in which is listed the 
antibodies in accordance with their apparent map position in PspA. The 
five antibodies (including XiR16) that clearly recognize epitopes 
N-terminal to position 116 are listed at the left side of Table IV. The 
four antibodies that clearly recognize epitopes C-terminal to position 192 
are listed on the right side of Table IV. Three of the five epitopes 
N-terminal of position 192 (those recognized by XiR1526, XiR35, and XiR16) 
were not found on any of the other eight PspAs tested. One epitope 
(recognized by XiR 1224) was weakly expressed by one other strain and 
another (recognized by Xi126) was expressed on two other strains. In 
contrast, the four epitopes present in the C-terminal third of the PspA 
.alpha.-helical region were each present in from two to six other strains. 
The greater conservation of the region C-terminal to position 192, as 
compared to the region N-terminal to position 192 was significant at 
P&lt;0.05 by both the Chi-square and the two sample rank tests. Based on the 
mapping results (Table III) and the strain distribution results (Table 
IV), it is apparent that all of the antibodies except possibly XiR35 and 
XiR1526 must recognize different PspA determinants. 
Example 7 
This Example contains a discussion of the mapping results achieved in 
Example 6. 
The results set forth in Example 6 clearly demonstrate that the protection 
eliciting epitopes of PspA are not restricted to the N-terminal end of the 
surface exposed .alpha.-helical half of the molecule. In fact, four of the 
five antibodies protective against S. pneumoniae WU2 reacted with the 
C-terminal third of the .alpha.-helical region of PspA. This portion of 
the .alpha.-helical region is thought to closest to the cell wall (see 
Yother et al (II)). 
About half of the MAbs recognized determinants N-terminal to amino acid 115 
and the other half recognized epitopes C-terminal to residue 192. Since 
the nine antibodies were selected for their ability to bind native PspA on 
the surface of heat-killed whole pneumococci, the distribution of the 
epitopes they recognize suggests that determinants between positions 115 
and 192 are either not immunogenic or are not exposed on the native 
molecule as expressed on pneumococci. 
Curiously two MAbs (XiR16 and XiR1323) appeared to possibly react with 
epitopes in more than one position on PspA. Although the bulk of the data 
for XiR16 placed its epitope N-terminal of position 115, weak immunoblot 
patterns suggested that a reactive epitope(s) may also exist C-terminal to 
residue 115. In the case of XiR1323, the bulk of the data indicated that 
its epitope is between positions 192 and 260. However, the ELISA assay 
showed significant reactivity of the antibody with a C-terminal-deleted 
PspA fragment extending from residues 1 to 192. Although there are no 
extensive repeats in the N-terminal half of PspA, there are a few short 
repeated sequences that occur more than once in the coiled-coil motif. One 
such sequence is glu-glu-ala-lys which starts at amino acid positions 156, 
164, and 178 and another is lys-ala-lys-leu starting at positions 181 and 
251 (see FIG. 1). In the case of XiR1323, the antibody reacted with the 
epitope on the 1 to 192 fragment under natured but not denatured 
conditions. This may indicate that the epitope is conformational and may 
not have the same exact sequence as the epitope recognized (under both 
natured and denatured conditions) between residues 192 and 260. 
One mechanism that may account for the lack of exposure of epitopes between 
amino acid 115 and 192 would be a folding back of this portion of the 
.alpha.-helical sequence on itself or other parts of PspA to form a 
coiled-coil structure more complex than a simple coiled-coil dimer. If 
this occurred, it could explain how PspA tertiary structure can sometimes 
be dependent on distant PspA structures. A suggestion that this might, in 
fact, be the case comes from the observation that some of the truncated 
forms did not express certain epitopes under physiologic conditions that 
were detected on the whole molecule under the same conditions and were 
shown to be present in the fragment after denaturation in SDS. 
Since a PspA vaccine may need to contain fragments of several serologically 
different PspAs, it would be desirable to include in a vaccine only those 
portions of each PspA that are most likely to elicit cross-protective 
antibodies. Based on the results presented herein with Rx1 PspA, it 
appears likely that the portion of the PspA sequences corresponding to 
residues 192 to 260 of Rx1 PspA is the best portion of PspA to include in 
a recombinant PspA vaccine. The epitopes in this portion of PspA were 
three and a half times as likely to be present in the PspAs of other 
strains as the epitopes in the residue 1 to 115 portion of the sequence, 
and none of the 9 antibodies studied clearly reacted with the middle third 
of the .alpha.-helical region. 
Example 8 
This Example shows protection of mice by PspA fragments. Five mice were 
immunized with purified fragment produced by pBC207 in E. coli and five 
with purified fragment produced by pBC100 in E. coli. In both cases, the 
fragments were injected in Freund's complete adjuvant. All mice immunized 
with each fragment survived challenge with 100.times.LD.sub.50 of WU2 
capsular type 3 S.pneumoniae. 
Five additional mice were injected with adjuvant plus an equivalent 
preparation on non-PspA producing E. coli. All mice died when challenged 
with the same dose of WU2. 
The data presented in this Example conclusively proves that epitopes 
C-terminal to amino acids 119 and 192 respectively are capable of 
eliciting protective immunity. This result is consistent with the findings 
presented in the earlier Examples that the region of PspA from amino acids 
192 to 260 contain protection-eliciting epitopes. 
SEQUENCE IDENTIFICATIONS 
SEQ ID NO: 1 DNA sequence for pspA gene (FIG. 1) 
SEQ ID NO: 2 Deduced amino acid sequence for PspA protein (FIG. 1) 
SEQ ID NO: 3 Nucleotide sequence for PCR primer LSM 2 
SEQ ID NO: 4 Nucleotide sequence for PCR primer LSM 3 
SEQ ID NO: 5 Nucleotide sequence for PCR primer LSM 4 
SUMMARY OF THE DISCLOSURE 
In summary of this disclosure, the present invention provides a PspA 
protein fragment which contains protection-eliciting epitopes and which is 
cross-reactive and can be incorporated into a vaccine against disease 
caused by pneumococcal infection. Modifications are possible within the 
scope of this invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2085 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Streptococcus pneumoniae 
(B) STRAIN: Rx1 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: JY4313 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 1..2085 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join(127..1984) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AAGCTTATGATATAGAAATTTGTAACAAAAATGTAATATAAAACACTTGACAAATATTTA60 
CGGAGGAGGCTTATACTTAATATAAGTATAGTCTGAAAATGACTATCAGAAAAGAGGTAA120 
ATTTAGATGAATAAGAAAAAAATGATTTTAACAAGTCTAGCCAGCGTC168 
MetAsnLysLysLysMetIleLeuThrSerLeuAlaSerVal 
1510 
CCTATCTTAGGGGCTGGTTTTGTTGCGTCTCAGCCTACTGTTGTAAGA216 
AlaIleLeuGlyAlaGlyPheValAlaSerGlnProThrValValArg 
15202530 
GCAGAAGAATCTCCCGTAGCCAGTCAGTCTAAAGCTGAGAAAGACTAT264 
AlaGluGluSerProValAlaSerGlnSerLysAlaGluLysAspTyr 
354045 
GATGCAGCGAAGAAAGATGCTAAGAATGCGAAAAAAGCAGTAGAAGAT312 
AspAlaAlaLysLysAspAlaLysAsnAlaLysLysAlaValGluAsp 
505560 
GCTCAAAAGGCTTTAGATGATGCAAAAGCTGCTCAGAAAAAATATGAC360 
AlaGlnLysAlaLeuAspAspAlaLysAlaAlaGlnLysLysTyrAsp 
657075 
GAGGATCAGAAGAAAACTGAGGAGAAAGCCGCGCTAGAAAAAGCAGCG408 
GluAspGlnLysLysThrGluGluLysAlaAlaLeuGluLysAlaAla 
808590 
TCTGAAGAGATGGATAAGGCAGTGGCAGCAGTTCAACAAGCGTATCTA456 
SerGluGluMetAspLysAlaValAlaAlaValGlnGlnAlaTyrLeu 
95100105110 
GCCTATCAACAAGCTACAGACAAAGCCGCAAAAGACGCAGCAGATAAG504 
AlaTyrGlnGlnAlaThrAspLysAlaAlaLysAspAlaAlaAspLys 
115120125 
ATGATAGATGAAGCTAAGAAACGCGAAGAAGAGGCAAAAACTAAATTT552 
MetIleAspGluAlaLysLysArgGluGluGluAlaLysThrLysPhe 
130135140 
AATACTGTTCGAGCAATGGTAGTTCCTGAGCCAGAGCAGTTGGCTGAG600 
AsnThrValArgAlaMetValValProGluProGluGlnLeuAlaGlu 
145150155 
ACTAAGAAAAAATCAGAAGAAGCTAAACAAAAAGCACCAGAACTTACT648 
ThrLysLysLysSerGluGluAlaLysGlnLysAlaProGluLeuThr 
160165170 
AAAAAACTAGAAGAAGCTAAAGCAAAATTAGAAGAGGCTGAGAAAAAA696 
LysLysLeuGluGluAlaLysAlaLysLeuGluGluAlaGluLysLys 
175180185190 
GCTACTGAAGCCAAACAAAAAGTGGATGCTGAAGAAGTCGCTCCTCAA744 
AlaThrGluAlaLysGlnLysValAspAlaGluGluValAlaProGln 
195200205 
GCTAAAATCGCTGAATTGGAAAATCAAGTTCATAGACTAGAACAAGAG792 
AlaLysIleAlaGluLeuGluAsnGlnValHisArgLeuGluGlnGlu 
210215220 
CTCAAAGAGATTGATGAGTCTGAATCAGAAGATTATGCTAAAGAAGGT840 
LeuLysGluIleAspGluSerGluSerGluAspTyrAlaLysGluGly 
225230235 
TTCCGTGCTCCTCTTCAATCTAAATTGGATGCCAAAAAAGCTAAACTA888 
PheArgAlaProLeuGlnSerLysLeuAspAlaLysLysAlaLysLeu 
240245250 
TCAAAACTTGAAGAGTTAAGTGATAAGATTGATGAGTTAGACGCTGAA936 
SerLysLeuGluGluLeuSerAspLysIleAspGluLeuAspAlaGlu 
255260265270 
ATTGCAAAACTTGAAGATCAACTTAAAGCTGCTGAAGAAAACAATAAT984 
IleAlaLysLeuGluAspGlnLeuLysAlaAlaGluGluAsnAsnAsn 
275280285 
GTAGAAGACTACTTTAAAGAAGGTTTAGAGAAAACTATTGCTGCTAAA1032 
ValGluAspTyrPheLysGluGlyLeuGluLysThrIleAlaAlaLys 
290295300 
AAAGCTGAATTAGAAAAAACTGAAGCTGACCTTAAGAAAGCAGTTAAT1080 
LysAlaGluLeuGluLysThrGluAlaAspLeuLysLysAlaValAsn 
305310315 
GAGCCAGAAAAACCAGCTCCAGCTCCAGAAACTCCAGCCCCAGAAGCA1128 
GluProGluLysProAlaProAlaProGluThrProAlaProGluAla 
320325330 
CCAGCTGAACAACCAAAACCAGCGCCGGCTCCTCAACCAGCTCCCGCA1176 
ProAlaGluGlnProLysProAlaProAlaProGlnProAlaProAla 
335340345350 
CCAAAACCAGAGAAGCCAGCTGAACAACCAAAACCAGAAAAAACAGAT1224 
ProLysProGluLysProAlaGluGlnProLysProGluLysThrAsp 
355360365 
GATCAACAAGCTGAAGAAGACTATGCTCGTAGATCAGAAGAAGAATAT1272 
AspGlnGlnAlaGluGluAspTyrAlaArgArgSerGluGluGluTyr 
370375380 
AATCGCTTGACTCAACAGCAACCGCCAAAAGCTGAAAAACCAGCTCCT1320 
AsnArgLeuThrGlnGlnGlnProProLysAlaGluLysProAlaPro 
385390395 
GCACCAAAAACAGGCTGGAAACAAGAAAACGGTATGTGGTACTTCTAC1368 
AlaProLysThrGlyTrpLysGlnGluAsnGlyMetTrpTyrPheTyr 
400405410 
AATACTGATGGTTCAATGGCGACAGGATGGCTCCAAAACAACGGTTCA1416 
AsnThrAspGlySerMetAlaThrGlyTrpLeuGlnAsnAsnGlySer 
415420425430 
TGGTACTACCTCAACAGCAATGGTGCTATGGCTACAGGTTGGCTCCAA1464 
TrpTyrTyrLeuAsnSerAsnGlyAlaMetAlaThrGlyTrpLeuGln 
435440445 
TACAATGGTTCATGGTATTACCTCAACGCTAACGGCGCTATGGCAACA1512 
TyrAsnGlySerTrpTyrTyrLeuAsnAlaAsnGlyAlaMetAlaThr 
450455460 
GGTTGGGCTAAAGTCAACGGTTCATGGTACTACCTCAACGCTAATGGT1560 
GlyTrpAlaLysValAsnGlySerTrpTyrTyrLeuAsnAlaAsnGly 
465470475 
GCTATGGCTACAGGTTGGCTCCAATACAACGGTTCATGGTATTACCTC1608 
AlaMetAlaThrGlyTrpLeuGlnTyrAsnGlySerTrpTyrTyrLeu 
480485490 
AACGCTAACGGCGCTATGGCAACAGGTTGGGCTAAAGTCAACGGTTCA1656 
AsnAlaAsnGlyAlaMetAlaThrGlyTrpAlaLysValAsnGlySer 
495500505510 
TGGTACTACCTCAACGCTAATGGTGCTATGGCTACAGGTTGGCTCCAA1704 
TrpTyrTyrLeuAsnAlaAsnGlyAlaMetAlaThrGlyTrpLeuGln 
515520525 
TACAACGGTTCATGGTACTACCTCAACGCTAACGGTGCTATGGCTACA1752 
TyrAsnGlySerTrpTyrTyrLeuAsnAlaAsnGlyAlaMetAlaThr 
530535540 
GGTTGGGCTAAAGTCAACGGTTCATGGTACTACCTCAACGCTAATGGT1800 
GlyTrpAlaLysValAsnGlySerTrpTyrTyrLeuAsnAlaAsnGly 
545550555 
GCTATGGCAACAGGTTGGGTGAAAGATGGAGATACCTGGTACTATCTT1848 
AlaMetAlaThrGlyTrpValLysAspGlyAspThrTrpTyrTyrLeu 
560565570 
GAAGCATCAGGTGCTATGAAAGCAAGCCAATGGTTCAAAGTATCAGAT1896 
GluAlaSerGlyAlaMetLysAlaSerGlnTrpPheLysValSerAsp 
575580585590 
AAATGGTACTATGTCAATGGTTTAGGTGCCCTTGCAGTCAACACAACT1944 
LysTrpTyrTyrValAsnGlyLeuGlyAlaLeuAlaValAsnThrThr 
595600605 
GTAGATGGCTATAAAGTCAATGCCAATGGTGAATGGGTTTAAGCCGAT1992 
ValAspGlyTyrLysValAsnAlaAsnGlyGluTrpVal* 
610615 
TAAATTAAAGCATGTTAAGAACATTTGACATTTTAATTTTGAAACAAA2040 
GATAAGGTTCGATTGAATAGATTTATGTTCGTATTCTTTAGGTAC2085 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 619 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetAsnLysLysLysMetIleLeuThrSerLeuAlaSerValAlaIle 
151015 
LeuGlyAlaGlyPheValAlaSerGlnProThrValValArgAlaGlu 
202530 
GluSerProValAlaSerGlnSerLysAlaGluLysAspTyrAspAla 
354045 
AlaLysLysAspAlaLysAsnAlaLysLysAlaValGluAspAlaGln 
505560 
LysAlaLeuAspAspAlaLysAlaAlaGlnLysLysTyrAspGluAsp 
65707580 
GlnLysLysThrGluGluLysAlaAlaLeuGluLysAlaAlaSerGlu 
859095 
GluMetAspLysAlaValAlaAlaValGlnGlnAlaTyrLeuAlaTyr 
100105110 
GlnGlnAlaThrAspLysAlaAlaLysAspAlaAlaAspLysMetIle 
115120125 
AspGluAlaLysLysArgGluGluGluAlaLysThrLysPheAsnThr 
130135140 
ValArgAlaMetValValProGluProGluGlnLeuAlaGluThrLys 
145150155160 
LysLysSerGluGluAlaLysGlnLysAlaProGluLeuThrLysLys 
165170175 
LeuGluGluAlaLysAlaLysLeuGluGluAlaGluLysLysAlaThr 
180185190 
GluAlaLysGlnLysValAspAlaGluGluValAlaProGlnAlaLys 
195200205 
IleAlaGluLeuGluAsnGlnValHisArgLeuGluGlnGluLeuLys 
210215220 
GluIleAspGluSerGluSerGluAspTyrAlaLysGluGlyPheArg 
225230235240 
AlaProLeuGlnSerLysLeuAspAlaLysLysAlaLysLeuSerLys 
245250255 
LeuGluGluLeuSerAspLysIleAspGluLeuAspAlaGluIleAla 
260265270 
LysLeuGluAspGlnLeuLysAlaAlaGluGluAsnAsnAsnValGlu 
275280285 
AspTyrPheLysGluGlyLeuGluLysThrIleAlaAlaLysLysAla 
290295300 
GluLeuGluLysThrGluAlaAspLeuLysLysAlaValAsnGluPro 
305310315320 
GluLysProAlaProAlaProGluThrProAlaProGluAlaProAla 
325330335 
GluGlnProLysProAlaProAlaProGlnProAlaProAlaProLys 
340345350 
ProGluLysProAlaGluGlnProLysProGluLysThrAspAspGln 
355360365 
GlnAlaGluGluAspTyrAlaArgArgSerGluGluGluTyrAsnArg 
370375380 
LeuThrGlnGlnGlnProProLysAlaGluLysProAlaProAlaPro 
385390395400 
LysThrGlyTrpLysGlnGluAsnGlyMetTrpTyrPheTyrAsnThr 
405410415 
AspGlySerMetAlaThrGlyTrpLeuGlnAsnAsnGlySerTrpTyr 
420425430 
TyrLeuAsnSerAsnGlyAlaMetAlaThrGlyTrpLeuGlnTyrAsn 
435440445 
GlySerTrpTyrTyrLeuAsnAlaAsnGlyAlaMetAlaThrGlyTrp 
450455460 
AlaLysValAsnGlySerTrpTyrTyrLeuAsnAlaAsnGlyAlaMet 
465470475480 
AlaThrGlyTrpLeuGlnTyrAsnGlySerTrpTyrTyrLeuAsnAla 
485490495 
AsnGlyAlaMetAlaThrGlyTrpAlaLysValAsnGlySerTrpTyr 
500505510 
TyrLeuAsnAlaAsnGlyAlaMetAlaThrGlyTrpLeuGlnTyrAsn 
515520525 
GlySerTrpTyrTyrLeuAsnAlaAsnGlyAlaMetAlaThrGlyTrp 
530535540 
AlaLysValAsnGlySerTrpTyrTyrLeuAsnAlaAsnGlyAlaMet 
545550555560 
AlaThrGlyTrpValLysAspGlyAspThrTrpTyrTyrLeuGluAla 
565570575 
SerGlyAlaMetLysAlaSerGlnTrpPheLysValSerAspLysTrp 
580585590 
TyrTyrValAsnGlyLeuGlyAlaLeuAlaValAsnThrThrValAsp 
595600605 
GlyTyrLysValAsnAlaAsnGlyGluTrpVal 
610615 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GCGCGTCGACGGCTTAAACCCATTCACCATTGG33 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CCGGATCCTGAGCCAGAGCAGTTGGCTG28 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CCGGATCCGCTCAAAGAGATTGATGAGTCTG31 
__________________________________________________________________________