U.S. patent application number 10/299636 was filed with the patent office on 2004-04-22 for pneumococcal genes, portions thereof, expression products therefrom, and uses of such genes, portions and products.
Invention is credited to Briles, David E., Brooks-Walter, Alexis, Crain, Marilyn J., Hollingshead, Susan, McDaniel, Larry S., Swiatlo, Edwin, Tart, Rebecca, Yother, Janet.
Application Number | 20040077847 10/299636 |
Document ID | / |
Family ID | 24108319 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077847 |
Kind Code |
A1 |
Briles, David E. ; et
al. |
April 22, 2004 |
Pneumococcal genes, portions thereof, expression products
therefrom, and uses of such genes, portions and products
Abstract
The present invention relates to pneumococcal genes, portions
thereof, expression products therefrom and uses of such genes,
portions and products; especially to genes of Streptococcus
pneumoniae, e.g., the gene encoding pneumococcal surface protein A
(PspA), i.e., the pspA gene, the gene encoding pneumococcal surface
protein A-like proteins, such as pspA-like genes, e.g., the gene
encoding pneumococcal surface protein C (PspC), i.e., the pspC
gene, portions of such genes, expression products therefrom, and
the uses of such genes, portions thereof and expression products
therefrom.
Inventors: |
Briles, David E.;
(Birmingham, AL) ; McDaniel, Larry S.; (Ridgland,
MS) ; Swiatlo, Edwin; (Birmington, AL) ;
Yother, Janet; (Birmingham, AL) ; Crain, Marilyn
J.; (Birmingham, AL) ; Hollingshead, Susan;
(Birmingham, AL) ; Tart, Rebecca; (Benson, NC)
; Brooks-Walter, Alexis; (Birmingham, AL) |
Correspondence
Address: |
Michael L. Goldman
NIXON PEABODY LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603-1051
US
|
Family ID: |
24108319 |
Appl. No.: |
10/299636 |
Filed: |
November 19, 2002 |
Related U.S. Patent Documents
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10299636 |
Nov 19, 2002 |
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08714741 |
Sep 16, 1996 |
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08714741 |
Sep 16, 1996 |
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08529055 |
Sep 15, 1995 |
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10299636 |
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May 18, 1992 |
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Current U.S.
Class: |
536/23.1 ;
G9B/20.009; G9B/25.003 |
Current CPC
Class: |
C12Q 1/689 20130101;
A61P 31/00 20180101; A61P 31/04 20180101; A61K 38/00 20130101; A61K
39/00 20130101; C12N 15/746 20130101; G06K 15/02 20130101; C07K
14/3156 20130101; G01N 33/56911 20130101; C07K 2319/00 20130101;
C12N 15/74 20130101; G11B 20/10 20130101; C07K 2319/02 20130101;
C07K 14/7151 20130101; A61K 39/092 20130101; G11B 25/043 20130101;
G11B 5/012 20130101; G06T 9/005 20130101; C07K 14/28 20130101 |
Class at
Publication: |
536/023.1 |
International
Class: |
C07H 021/02; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 15, 1993 |
JP |
5-88369 |
Nov 16, 1993 |
JP |
5-287079 |
Claims
What is claimed:
1. An isolated nucleic acid molecule encoding a pneumoccocal
surface protein (PspC) gene of S. pneumoniae or epitope
thereof.
2. A vector or plasmid comprising the isolated nucleic acid
molecule of claim 1.
3. An immunological composition comprising the nucleic acid
molecule of claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 08/714,741, filed Sep. 16, 1996, now U.S. Pat. No.
6,500,613, issued Dec. 31, 2002, which is a continuation-in-part of
U.S. patent application Ser. No. 08/529,055, filed Sep. 15, 1995,
which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] 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, e.g., 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 therefrom. Such uses
include uses of the genes and portions thereof for obtaining
expression products by recombinant techniques, as well as for
detecting the presence of Streptococcus pneumoniae or strains
thereof by detecting DNA thereof by hybridization or amplification
(e.g., PCR) and hybridization techniques (e.g., obtaining
DNA-containing sample, contacting same with genes or fragment under
PCR, amplification and/or hybridization conditions, and detecting
presence of or 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 (including antibody or other immunological
response by administering composition to a suitable host); or, the
expression product can be for use in detecting the presence of
Streptococcus pneumoniae by detecting antibodies to Streptococcus
pneumoniae protein(s) or antibodies to a portion thereof in a host,
e.g., by 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
[0003] 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.
[0004] 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.
[0005] 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. 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.
[0006] 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 or as carriers
for polysaccharides.
[0007] McDaniel et al. (I), 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.
[0008] McDaniel et al. (II), 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.
[0009] McDaniel et al. (III), J. Exp. Med. 165:381-394, 1987,
relates to immunization of X-linked immunodeficient (XID) mice with
non-encapsulated pneumococci expressing PspA, but not isogenic
pneumococci lacking PspA, which protects mice from subsequent fatal
infection with pneumococci.
[0010] McDaniel et al. (IV), 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.
[0011] Crain et al, Infect. Immun., 56:3293-3299, 1990, relates to
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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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:
[0016] 1. Abstracts of 89th Annual Meeting of the American Society
for Microbiology, p.125, item D-257, May 1989;
[0017] 2. Abstracts of 90th Annual Meeting of the American Society
for Microbiology, p. 98, item D-106, May 1990;
[0018] 3. Abstracts of 3rd International ASM Conference on
Streptococcal Genetics, p. 11, item 12, June 1990;
[0019] 4. Talkington et al, Infect. Immun. 59:1285-1289, 1991;
[0020] 5. Yother et al (I), J. Bacteriol. 174:601-609, 1992;
[0021] 6. Yother et al (II), J. Bacteriol. 174:610-618, 1992.
[0022] 7. McDaniel et al (V), Microbiol. Pathogenesis,
13:261-268.
[0023] 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 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 represent in
such compositions (as well as how to test for, detect or diagnose
one strain from another).
[0024] 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
[0025] The invention provides an isolated amino acid molecule
comprising residues 1 to 115, 1 to 260, 192 to 588, 192 to 299, or
residues 192 to 260 of pneumococcal surface protein A of
Streptococcus pneumoniae.
[0026] The invention further provides an isolated DNA molecule
comprising a fragment of a pneumococcal surface protein A gene of
Streptococcus pneumoniae encoding the isolated amino acid
molecule.
[0027] The invention also provides PCR primers or hybridization
probes comprising the isolated DNA molecule.
[0028] The invention additionally provides an antigenic, vaccine or
immunological composition comprising the amino acid molecule.
[0029] The invention includes an isolated DNA molecule comprising
nucleotides 1 to 26, 1967 to 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.
[0030] The invention also includes an isolated DNA molecule
comprising a fragment having homology with a portion of a
pneumococcal surface protein A gene of Streptococcus pneumoniae.
The DNA preferably is 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):
1 CCGGATCCAGCTCCTGCACCAAAAAC; (SEQ ID NO:1)
GCGCGTCGACGGCTTAAACCCATTCACCATTGG; (SEQ ID NO:2)
CCGGATCCTGAGCCAGAGCAGTTGGCTG; (SEQ ID NO:3)
CCGGATCCGCTCAAAGAGATTGATGAGTCTG; (SEQ ID NO:4)
GCGGATCCCGTAGCCAGTCAGTCTAAAGCTG; (SEQ ID NO:5)
CTGAGTCGACTGGAGTTTCTGGAGCTGGAGC; (SEQ ID NO:6)
CCGGATCCAGCTCCAGCTCCAGAAACTCCAG; (SEQ ID NO:7)
GCGGATCCTTGACCAATATTTACGGAGGAGGC; (SEQ ID NO:8)
GTTTTTGGTGCAGGAGCTGG; (SEQ ID NO:9) GCTATGGGCTACAGGTTG; (SEQ ID
NO:10) CCACCTGTAGCCATAGC; (SEQ ID NO:11)
CCGCATCCAGCGTGCCTATCTTAGGGGCTGGTT; (SEQ ID NO:12) and
GCAAGCTTATGATATAGAAATTTGTAAC (SEQ ID NO:13)
[0031] (thus, the invention broadly comprehends DNA homologous to
portions of pspA; preferably further including restriction
sequences).
[0032] 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.
[0033] 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.
[0034] Additionally, the invention includes 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 further comprising purifying PspA by isolation on
a choline-Sepharose affinity column. These processes are also
included in the invention.
[0035] An immunological composition comprising these extracts is
comprehended by the invention, as well as an immunological
composition comprising the full length PspA.
[0036] A method for enhancing the inmmunogenicity of a
PspA-containing immunological composition comprising, in said
composition, the C-terminal portion of PspA, is additionally
comprehended, as well.
[0037] 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, e.g., FIGS. 13A-13T).
[0038] Further, the invention provides an isolated amino acid
molecule comprising pneumococcal surface protein C, PspC, of
Streptococcus pneumoniae 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 hybrization probes consisting essentially of the
isolated DNA molecule.
[0039] Still further, an isolated amino acid molecule comprising
pneumococcal surface protein C, PspC, of Streptococcus pneumoniae
is provided, having an alpha-helical, proline rich and repeat
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 hybrization probes consisting essentially of
the isolated DNA molecule are provided by the present
invention.
[0040] Additionally, the present invention provides immunological
compositions comprising PspC.
[0041] These and other embodiments are disclosed or are obvious
from the following detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIGS. 1A and 1B show: Evaluation of digested plasmid
constructs. FIG. 1A: 1% agarose gel electrophoresis of plasmids
isolated from transformed E. coil 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: DEL5 pspA insert, lane 5:
pRCT113, lane 6: BG9739 pspA insert, lane 7: pRCT1117, and lane 8:
L81905 pspA insert. FIG. 1B: Corresponding Southern blot of gel in
FIG. 1A probed with full-length Rx1 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 pspA
fragments from the pneumococcal donor strains used in cloning.
[0043] FIG. 2 shows: Evaluation of strain RCT105 cell fractions
containing 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.
[0044] FIG. 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 (A) or
immunoblotted with PspA MAb XiR278 (B). The PspA isolated from R36A
shows the characteristic monomer (84 kDa) and dimer bands.
[0045] FIG. 4 shows: Cell lysates of pneumococcal isolates MC27 and
MC28 were subjected to SDS-PAGE and 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.
[0046] FIG. 5 (FIGS. 5A and 5B) shows: Southern blot of HindIII
digest of MC25-MC28 chromosomal DNA developed at a stringency
greater than 95 percent. A digest of Rx1 DNA was used as a
comparison. The blot was probed with LSNpspA13/2, a full length Rx1
probe (FIG. 5) and LSMpspA12/6 a 5' probe of Rx1 pspA (FIG. 5). The
same concentration of Rx1 DNA was used in both panels, but the
concentrations of MC25-MC28 DNA in FIG. 5B were half that used in
FIG. 5A to avoid detection of partial digests.
[0047] FIG. 6 shows: RFLP of amplified pspA. PspA 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 SmaI, Lane 8
KpnI.
[0048] FIG. 7 shows: A depiction of PspA showing the relative
location and orientation of the oligonucleotides.
[0049] FIG. 8 shows: Derivatives of the S. pneumoniae D39-Rx1
family.
[0050] FIGS. 9 to 10 show: Electrophoresis of pspA or amplified
pspA product with HhaI (FIG. 9), Sau3AI (FIG. 10).
[0051] FIG. 11 shows: RFLP pattern of two isolates from six
families.
[0052] FIG. 12 shows: RFLP pattern of two isolates from six
families (using products from amplification with SKH2 and
LSM13)
[0053] FIGS. 13A-13P show: Sequence primarily in the N-terminal
half of PspA for a variety of strains are shown, corresponding to
(SEQ ID NO: 32) through (SEQ ID NO: 86), respectively. FIGS. 13Q-T
show the complete DNA sequence (SEQ ID NO: 87) and amino acid
sequence (SEQ ID NO: 88), respectively, for EF5668 pspA.
[0054] FIG. 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).
[0055] FIGS. 15A and 15B show: a Southern blot of HindIII digest of
MC25-28 chromosomal DNA, using a digest of Rx1 DNA as a comparison;
the blot was probed with LSMpspA13/2, a full length Rx1 probe (A),
and LSMpspA12/6, a 5' probe of Rx1 pspA (B); the same concentration
of Rx1 DNA was used in both panels, but the concentrations of
MC25-28 DNA in B were half that used in A to avoid detection of
partial digests.
[0056] FIGS. 15C, 15D and 15E show: the nucleotide sequences of
primers LSM13 (SEQ ID NO: 13); LSM2 (SEQ ID NO: 2), LSM12 (SEQ ID
NO: 12) and LSM6 (SEQ ID NO: 6), and that of probes LSpspA13/2 (SEQ
ID NO: 89)and LSMpspA12/6 (SEQ ID NO: 90).
[0057] FIG. 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; BclI was used in lane 1; BamHI was
used in lane 2; BstN I was used in lane 3; PstI 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.
[0058] FIG. 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 Rx1, and arrows represent the relative position (not to
scale) and orientation of oligonucleotides.
[0059] FIG. 18 shows: a restriction map of the pZero vector.
[0060] FIG. 19 shows: the nucleotide sequences of SKH2 (SEQ ID NO:
107), LSM13 (SEQ ID NO: 20), N192 (SEQ ID NO: 91) and C588 (SEQ ID
NO: 92).
[0061] FIG. 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
alpha-helical regions which are homologous between PspA and PspC
are indicated by the striped pattern; and PCR primers are indicated
by the arrows.
[0062] FIGS. 21A-21E show: the amino acid (SEQ ID NO: 94) and
nucleotide sequence (SEQ ID NO: 93) of PspC, wherein the putative
-10 and -35 regions are underlined, and the ribosomal binding site
is in lower case.
[0063] FIGS. 22A-22C show: the Bestfit analysis of PspA and PspC
percent identity is 69% and percent similarity is 77%; amino acids
of PspA are on the bottom line (1-588) (SEQ ID NO: 96); and amino
acids of PspC are on the top line (249-891) (SEQ ID NO: 95), and a
dashed line indicated identity.
[0064] FIGS. 23A-23E show: the coiled coil motif of the alpha-helix
of PspC (SEQ ID NO: 97); amino acids that are not in the coiled
coil motif are in the right column.
[0065] FIG. 24 shows: a matrix plot comparison of the repeat
regions of the alpha-helical region of PspC.
[0066] FIGS. 25A-25C show: the sequence of the alpha helical and
proline regions of LXS532 (PspC.D39) (SEQ ID NO: 98);.
[0067] FIGS. 26A-26B show: a comparison of nucleotides of pspA.RX1
(SEQ ID NO: 99); topspC.D39 (SEQ ID NO: 100).
[0068] FIGS. 27A-27D show: a BESTFIT analysis of pspC.EF6797 (SEQ
ID NO: 101) and pspc.D39 (SEQ ID NO: 101).
[0069] FIGS. 28A-28B show: the amino acid comparison of PspC of
EF6797 (SEQ ID NO: 103) and D39 (SEQ ID NO: 104).
[0070] FIGS. 29A-29B show: the amino acid comparison of PspC.D39
(SEQ ID NO: 105) and PspA.Rx1 (SEQ ID NO: 106).
DETAILED DESCRIPTION
[0071] Knowledge of and 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 compositions containing PspA.
[0072] As discussed above and in the following Examples, the
invention relates to truncated PspA, e.g., PspA C-terminal to
position 192 such as aa. 192-588 ("BC100") 192-299 and 192-260 of
PspA eliciting cross-protection, as well as to DNA encoding such
truncated PspA (which amplify the coding for these amino acid
regions homologous to most PspAs).
[0073] The invention further relates to a pspA-like gene, or a pspC
gene and portions thereof (e.g., probes, primers) which can
hybridize thereto and/or amplify that gene, as well as to DNA
molecules which hybridize to 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).
[0074] Indeed, the invention further relates to oligonucleotide
probes and/or primers which react with 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
cross-reactive epitopes of interest.
[0075] 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.
[0076] The invention demonstrates that the pspC gene is homologous
to the pspA gene in the leader sequence, 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 C-terminal
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 products, is therefore a target for intervention of
pneumococcal infection.
[0077] 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 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.
[0078] 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 about 1.4%, preferably 1.2%)
choline chloride, elution of live pneumococci with a salt solution
containing choline chloride, e.g., about 1% about 3%, preferably 2%
choline 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, e.g., C.sub.1-C.sub.6,
preferably C.sub.2 alkanolamine, i.e., preferably C.sub.2
alkanolamine, i.e., preferably ethanolamine (e.g., 0.0000005% to
0.0000015%, preferably 0.000001% choline chloride plus 0.02% to
0.04% alkanolamine (ethanolamine), preferably 0.03%). PspA from
such pneumococci is then preferably isolated from a
choline-sepharose affinity column, thereby providing highly
purified PspA. Such isolated and/or purified PspA is highly
immunogenic and is useful in antigenic, immunological or vaccine
composition.
[0079] 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, e.g., 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 ore 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).
[0080] 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, 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.
[0081] The invention further demonstrates that there 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 of PspA.
And, the invention thus provides oligonucleotides which permit
amplification of most, e.g., a majority, if not all of S.
pneumoniae and thereby permit RFLP analysis of a majority, if not
all, S. pneumoniae.
[0082] The invention also provides PspC, having an approximate
molecular weight of 105 kD, with an estimated .mu.l 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 C-terminal 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 cross-protective 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, i.e., 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.
[0083] An epitope of interest is an antigen or immunogen or
immunologically active fragment thereof from a pathogen or toxin of
veterinary or human interest.
[0084] 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 composition containing the pneumococcal epitope of
interest, elicits an immunological response--local or systemic. The
response can, but need not be, protective. An 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).
[0085] The invention therefore also provides a method of inducing
an immunological response in a host mammal comprising administering
to the host an immunogenic, immunological or vaccine composition
comprising the pneumococcal epitope of interest, and a
pharmaceutically acceptable carrier or diluent.
[0086] The DNA encoding the pneumococcal epitope of interest can be
DNA which codes for full length PspA, 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.
[0087] Regions of PspA and PspC have been identified from the Rx1
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 overlapping fragments of PspA, e.g., fragment 192 to 588
includes 192 to 260, and fragment 1 to 314 includes 1 to 115 and
192 to 260.
[0088] 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 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
(e.g., size, charge, etc.) and the codon dictionary, without undue
experimentation.
[0089] 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 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.
[0090] 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 of
a gene which are transcribed but which are subsequently excised
from the primary RNA transcript).
[0091] At a minimum, the DNA sequence can code for a peptide at
least 8 or 9 amino acids long. This is the 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, i.e., an antibody or
T cell response; but, for a protective response (as from a vaccine
composition), a longer peptide is preferred.
[0092] 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 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 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 from the tertiary
structure of the folded three dimensional protein. Janis Kuby,
Immunology, (1992) pp. 79-80.
[0093] 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.
[0094] Yet another method for determining an epitope of interest is
to perform an X-ray crystallographic analysis of the antigen (full
length)-antibody complex. Janis Kuby, Immunology, (1992) p. 80.
[0095] 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 motifs which are
peptide sequences which are known to be likely to bind to the MHC
molecule.
[0096] The 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
MHC molecules, and should bind with high enough affinity to
generate an 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 occurrence of the peptide
sequence in other vital cells.
[0097] 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 MHC" located on another cell's
surface. 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`.
[0098] Class I MHC complexes 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 lymphokines. The lymphokines stimulate a response; cells
arrive and kill the viral infected cell.
[0099] 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.
[0100] 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 generate an immune response (See Bocchia, M. et al,
Specific Binding of Leukemia Oncogene Fusion Protein Peptides to
HLA Class I Molecules, Blood 85:2680-2684; Englehard, V H,
Structure of peptides associated with class I and class II MHC
molecules Ann. Rev. Immunol. 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 presented on the cell surface in
association with class I or class II MHC molecules.
[0101] 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 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.
[0102] Even further, another method is simply to generate or
express portions of a protein of interest, 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 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 expressing portions of a protein of
interest for analysis as to whether antibodies thereto inhibit
growth in vitro. For example, the skilled artisan can generate
portions of a protein of interest by: selecting 8 to 9 or 13 to 25
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 complex, 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.
[0103] 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 map, using the general methods
described above, or other mapping methods known in the art.
[0104] 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. Pat. No. 5,019,384, issued May 28, 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. 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.")
[0105] Further, the invention demonstrates that more than one
serologically complementary PspA molecule can be in an antigenic,
immunological or vaccine composition, so as to elicit better
response, e.g., 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
cross-protection evaluation so as to provide a maximally
efficacious composition.
[0106] The determination of the amount of antigen, e.g., PspA or
truncated portion thereof and optional adjuvant in the inventive
compositions and the preparation of those compositions can be in
accordance with standard techniques well known to those skilled in
the pharmaceutical or veterinary arts. In particular, the amount of
antigen and adjuvant in the inventive compositions and the dosages
administered are determined by techniques well known to those
skilled in the medical or veterinary arts taking into consideration
such factors as the particular antigen, the adjuvant (if present),
the age, sex, weight, species and condition of the particular
patient, and the route of administration. For instance, dosages of
particular PspA 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, e.g., the
Examples), as is the amount of any adjuvant typically administered
therewith. Thus, the skilled artisan can readily determine the
amount of antigen and optional adjuvant in compositions and to be
administered in methods of the invention. Typically, an adjuvant is
commonly used as 0.001 to 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, e.g., Examples below or in applications cited
herein).
[0107] Typically, however, the antigen is present in an amount on
the order of micrograms to milligrams, or, about 0.001 to about 20
wt %, preferably about 0.01 to about 10 wt %, and most preferably
about 0.05 to about 5 wt % (see, e.g., Examples below).
[0108] 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
LD.sub.50 in a suitable animal model e.g., rodent such as mouse;
and, the dosage of the composition(s), concentration of components
therein and timing of administering the composition(s), which
elicit a suitable immunological response, such as by titrations of
sera and analysis thereof for antibodies or antigens, e.g., by
ELISA and/or RFFIT analysis. Such determinations do not require
undue experimentation from the knowledge of the skilled artisan,
this disclosure and the documents cited herein. And, the time for
sequential administrations can be ascertained without undue
experimentation.
[0109] Examples of compositions of the invention include liquid
preparations for orifice, e.g., oral, nasal, anal, vaginal,
peroral, intragastric, mucosal (e.g., perlingual, alveolar,
gingival, olfactory or respiratory mucosa) etc., administration
such as suspensions, syrups or elixirs; and, preparations for
parenteral, subcutaneous, intradermal, intramuscular or intravenous
administration (e.g., 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 be lyophilized. 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
"REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985,
incorporated herein by reference, may be consulted to prepare
suitable preparations, without undue experimentation.
[0110] Compositions of the invention, are conveniently provided as
liquid preparations, e.g., 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, e.g., gelatin covered liquid, whereby the gelatin is
dissolved in the stomach for delivery to the gut. 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.
[0111] Compositions 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 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.
[0112] Liquid preparations are normally easier to 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.
[0113] 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, e.g., liquid dosage form
[e.g., whether the composition is to be formulated into a solution,
a suspension, gel or another liquid form], or solid dosage form
[e.g., whether the composition is to be formulated into a pill,
tablet, capsule, caplet, time release form or liquid-filled
form].
[0114] Solutions, suspensions and gels, normally contain a major
amount of water preferably purified water) in addition to the
antigen, lipoprotein and optional adjuvant. Minor amounts of other
ingredients such as pH adjusters (e.g., a base such as NaOH),
emulsifiers or dispersing agents, buffering agents, preservatives,
wetting agents, jelling agents, (e.g., methylcellulose), colors
and/or flavors may also be present. The compositions can be
isotonic, i.e., it can have the same osmotic pressure as blood and
lacrimal fluid.
[0115] 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.
[0116] 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 the agent
selected. The important point is to use an amount which will
achieve the selected viscosity. Viscous compositions are normally
prepared from solutions by the addition of such thickening
agents.
[0117] A pharmaceutically acceptable preservative can be employed
to increase the shelf-life of the compositions. Benzyl alcohol may
be suitable, although a variety of preservatives including, for
example, parabens, thimerosal, chlorobutanol, or benzalkonium
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.
[0118] Those 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 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.
[0119] The immunologically effective compositions of this invention
are prepared by mixing the ingredients following generally accepted
procedures. For example the selected components may be simply mixed
in a blender, or other standard device to produce a concentrated
mixture which may then be adjusted to the final concentration and
viscosity by the addition of water or thickening agent and possibly
a buffer to control pH or an additional solute to control tonicity.
Generally the pH may be from 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 (e.g., solid vs. liquid). Dosages for humans or
other mammals can be determined without undue experimentation by
the skilled artisan, from this disclosure, the documents cited
herein, the Examples below (e.g., from the Examples involving
mice).
[0120] Suitable regimes for initial administration and booster
doses or for sequential administrations also are variable, may
include an initial administration followed by subsequent
administrations; but nonetheless, may be ascertained by the skilled
artisan, from this disclosure, the documents cited herein, and the
Examples below.
[0121] PCR techniques for amplifying sample DNA for diagnostic
detection or assay methods are known from the 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 known in
the art.
[0122] The following Examples are provided for illustration and are
not to be considered a limitation of the invention.
EXAMPLES
Example 1
Truncated Streptococcus pneumoniae PspA Molecules Elicit
Cross-Protective Immunity Against Pneumococcal Challenge
[0123] 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 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.
[0124] The immunogenic nature of proteins makes them 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 LD.sub.50. PspA is unique among these macromolecules in
that it can elicit antibodies in animals that protect against
inoculums 100-fold greater than the LD.sub.50.
[0125] 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 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 immunogenic and protection-eliciting
properties of PspA suggest that it may be a good candidate molecule
for a protein-based pneumnococcal vaccine.
[0126] Four distinct domains of PspA have been identified based on
DNA sequence. They include a N-terminal highly charged
alpha-helical region, a proline-rich 82 amino acid stretch, a
C-terminal repeat segment comprised of ten 20-amino acid repeat
sequences, and a 17-amino acid tail. A panel of MAbs to Rx1 PspA
have been produced and the binding sites of nine of these Mabs were
recently localized within the Rx1 pspA sequence in the
alpha-helical region. Five of the Rx1 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 the alpha-helical domain of Rx1 PspA.
[0127] Truncated PspAs containing amino acids 192-588 or 192-299,
from pneumococcal strain Rx1 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 Rx1 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 Rx1 was
comparable to that elicited by full-length Rx1 PspA. Therefore,
cross-protective 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 Rx1 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
PspAs from capsular type 4 and 5 strains collectively protect
against early death caused by challenge with capsular type 4 and 5
parental strains as well as type 3, 6A, and 6B S. pneumoniae.
[0128] Bacterial strains and culture conditions. All pneumococci
were from the culture collection of this laboratory, and have been
described (Yother, J. et al., Infect. Immun. 1982; 36: 184-188;
Briles, D. E., et al., Infect. Immun. 1992; 60: 111-116; McDaniel,
L. S., et al., 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, WJU2), 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, N.J. unless indicated
otherwise.
[0129] S. pneumoniae were grown in Todd Hewitt broth (Difco,
Detroit, Mich.) supplemented with 5% yeast extract (Difco).
Mid-exponential phase cultures were used for seeding inocula in
Lactated Ringer's (Abbott laboratories, North Chicago, Ill.) for
challenge studies. For pneumococcal strains used in challenge
studies, inocula ranged from 2.8 to 3.8 log.sub.10 CFU (verified by
dilution plating on blood agar). Plates were incubated overnight in
a candle jar at 37.degree. C.
[0130] E. coli DH1 and BL21(DE3) were cultured in LB medium (1%
Bacto-tryptone (Difco), 0.5% Bacto Yeast (Difco), 0.5% NaCl, 0.1%
dextrose). For the preparation of cell lysates, recombinant E coli
were grown in minimal E medium supplemented with 0.05 M thiamine,
0.2% glucose, 0.1% casamino acids (Difco), and 50 mg/ml kanamycin.
Permanent bacterial stocks were stored at -80.degree. C. in growth
medium containing 10% glycerol.
[0131] Construction of plasmid-based strains. pET-9a (Novagen,
Madison, Wis.) was used for cloning truncated pspA genes from
fourteen S. pneumoniae strains: DBL5, DBL6A, WU2, BG9739, EF5668,
L81905, 0922134, BG8090, BG8743, BG9163, DBL1, EF3296, EF6796, and
EF10197 (Table 1). pspA gene fragments, from fifteen strains, were
amplified by PCR using two primers provided by Connaught
Laboratories, Swiftwater, Pa. Primer
N192-5'GGAAGGCCATATGCTCAAAGAGATTGATGAGTCT3' (SEQ ID NO: 13) and
primer C588-5'CCAAGGATCCTTAAACCCATTCACCATT GGC3' (SEQ ID NO: 14)
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 Bio-chemical 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.
[0132] E. coli DH1 cells were transformed by the method of Hanahan
(Hanahan, D. J. Mol. Biol. 1983; 166: 557-580). Stable
transfonnants were identified by screening on LB-kanamycin plates.
Plasmid constructs, isolated from each of these strains, were
electroporated (Electro Cell Manipulator 600, BTX Electroporation
System, San Diego, Calif.) 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 2). All plasmid
constructs and PCR-amplified pspA gene fragments were evaluated by
agarose gel electrophoresis (with 1 kb DNA ladder, Gibco BRL,
Gaithersburg, Md.). Next, Southern analysis was performed using
LMpspA1, a previously described full-length pspA probe (McDaniel.
L. S. et al., Microb. Pathog. 1992; 13: 261-269) random primed
labeled with digoxigenin-11-dUTP (Genius System, Boehringer
Mannheim, Indianapolis, Ind.). Hybridization was detected with
chemiluminescent sheets according to the manufacturer's
instructions (Schleicher & Schuell, Keene, N.H.).
[0133] 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 cultures containing kanamycin and grown
overnight at 37.degree. C. Next, an 80 ml LB culture, inoculated
with 1:100 dilution of the overnight culture, was grown at
37.degree. C. to mid-exponential phase (A.sub.600 of ca. 0.5) and a
1 ml sample was harvested and resuspended (uninduced cells) prior
to 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.times.g, 10 min, DuPont Sorvall RC 5B Plus), and the
supernatant discarded. One pellet was resuspended in 5 ml of 20 mM
Tris-HCl ph 7.4 200 mM NaCl, 1 mM (ethylenedinitrilo)-tetraacetic
acid disodium salt (EDTA) and frozen at -20.degree. C. overnight.
Cells were thawed at 65.degree. C. for 30 min, placed on ice, and
sonicated for vive 10-sec pulses (0.4 relative output, Fisher Sonic
Dismembrator, Dynatech Laboratories, Inc. Chantilly, Va.). Next,
the material was centrifuged (9000.times.g, 20 min) and the
supernatant was designated the crude extract-cytoplasmic fraction.
The pellet was resuspended in Tris-NaCl-EDTA buffer and labeled the
insoluble cell wall and membrane fraction. The other pellet, from
the divided induced culture, was resuspended in 10 ml of 30 mM
Tris-HCl pH 8.0 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.sub.4 (10 ml, 10 min., shaking 4.degree. 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.
[0134] 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.
[0135] SDS-PAGE and immunoblot analysis. E. coli cell fractions
containing recombinant PspA proteins and biotinylated molecular
weight markers (low range, Bio-Rad, Richmond, Calif.) were
separated by sodium dodecyl sulfate-polyacrylamide (10%; Bethesda
Research Laboratories, Gaithersburg, Md.) gel electrophoresis
(SDS-PAGE) by the method of Laenmuli (Laemmli, U.K. Nature 1970;
227: 680-685). Samples were first boiled for 5 min in sample buffer
containing 60 mM Tris pH 6.8, 1% 2-B-mercaptoethanol (Sigma, St.
Louis, Mo.), 1% SDS, 10% glycerol, and 0.01% bromophenol blue. Gels
were subsequently transferred (1 hr, 100 volts) to nitrocellulose
(0.45 mM pores, Millipore, Bedford, Mass.) 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 NaCl, pH 7.4 for 30 min prior to incubating with
PspA-specific monoclonal antibodies diluted in PBST for 1 hr at
25.degree. C. Next, the blot was washed 3 times with PBST before
incubating with alkaline phosphatase-labeled goat anti-mouse
immunoglobulin (Southern Biotechnology Associates, Inc.,
Birmingham, Ala.) for 1 hr at 25.degree. C. Washes were performed
as before and blots were developed with 0.5 mg/ml
5-bromo-4-chloro-3-indolyl phosphate and 0.01% nitro blue
tetrazolium (Sigma) first dissolved in 150 .mu.l of dimethyl
sulfoxide and then diluted in 1.5 M Tris-HCl pH 8.8. Dot blots were
analyzed similarly. Lysate samples (2 .mu.l) were spotted on
nitrocellulose filters (Millipore), allowed to dry, blocked, and
detected as just described.
[0136] 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.degree. C.). Cultures were harvested by centrifugation (10 min
at 9000.times.g), resuspended in Tris-acetate pH 6.9, and frozen at
-80.degree. C. overnight. Samples were thawed at 65.degree. C. for
30 min, cooled on ice, and sonicated. Next the samples were treated
with 0.2 mM AEBSF (Calbiochem, La Jolla, Calif.) at 37.degree. 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.degree. C. until
subsequent immunizations were performed.
[0137] Mouse immunization and challenge. CBA/CAHN-XID/J mice
(Jackson Laboratories, Bar Harbor, Me.), 6-12 weeks old, were
employed for protection studies. These mice carry a X-linked
immunodeficiency that 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 dH.sub.2O. Inmunized
and nonimmunized mice (groups of 2 to 5 animals) were 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 3-week evaluation period were designated protected against
death and scored as surviving 22 days for statistical 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)
[0138] 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 immunosorbent assay (ELISA) using native, parental
PspAs isolated from pneumococcal strains DBL5, BG9739, and L81905.
PspAs were immobilized on microtiter plates by incubating in 0.5
NaHCO.sub.3, 0.5 M NaCO.sub.3pH9.5 at 4.degree. 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.sub.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).
[0139] Statistics. The one-tailed Fisher exact and two sample rank
tests were used to evaluate protection against death and extension
of life in the mouse model.
[0140] 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.
[0141] Fourteen of the amplified pspA genes were cloned and three
clones containing truncated PspA molecules from pneumococcal
strains DBL5, BG9739, and L81905 were further studied (Table 2). To
verify the constructions, plasmids from recombinant E. coli strains
(RCT105, RCT113, RCT 117, and RCT125 (Table 2) were isolated,
digested with NdeI and BAMHI restriction endonucleases, and
electrophoresed in 1% agarose side-by-side with the PCR products
used in their respective constructions (FIG. 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.
FIG. 1B depicts the corresponding Southern blot probed with
full-length Rx1pspA 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, FIG. 1B) as
well as the partially undigested pRCT113 and pRCT117 (lane 5 and
7), confirming successful cloning 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.
[0142] Expression of recombinant PspA in E. coli B121 (De3).
Transformed E. coli strains RCT105, RCT113, PCT117, 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.
[0143] FIG. 2 reveals that both the cytoplasmic (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 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 7), 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 kb DBL5pspA gene product (43.6 kDa; FIG. 1A, lane 4). Like
full-length Rx1 PspA, the observed and predicted molecular weights
for truncated PspAs do not agree precisely. In addition, immunoblot
analysis was performed for recombinant E. coil strains RCT113, and
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.
[0144] Evaluating the protective capacity of recombinant, truncated
PspAs. The truncated PspA proteins 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 sham-immunized) and
recombinant PspA-immunized mice were challenged with mouse-virulent
strains A66.3, BG7322, DBL6A, WU2, DBL5, BC9739, and L81905. Table
3 presents the day of death for each infected mouse.
[0145] Immunization with truncated PspA from RCT113, RCT117, and
RCT105 conferred protection against death for all mice challenged
with capsular type 3 strains (A66.3 and WU2) (Table 3). 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 3). Truncated BG9739 PspA
elicited protection against all challenge strains (100%) evaluated
in this study, while recombinant L81905 and DBL5 truncated PspAs
conferred protection against death with 71% and 57% of S.
pneumoniae challenge strains, respectively.
[0146] Anti-PspA antibody titers elicited by the three immunogens
vary over approximately a 10-fold range (Table 3). 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 times 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).
[0147] In summary, immunization with RCT113 and RCT117 PspAs
protected mice against fatal challenge with capsular 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 no protection against
type 4 and 5 strains. These data demonstrate that truncated PspAs
from capsular type 4 and 5 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.
[0148] PspA has been shown to be a protection-eliciting molecule of
S. pneumoniae. Immunization with 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 Rx1 PspA extending from amino acid residue 192 to
the C-terminus at residue 588 is demonstrated.
[0149] 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 Rx1 pspA.
Successful PCR amplification extended to all capsule types
analyzed. Thus, the C-terminal two-thirds of PspA may be amplified
from many, if not all, pneumococcal capsule types with Rx1
pspA-specific primers. This technique is thus applicable to the
development of antigenic immunological or vaccine compositions
containing multiple PspA or fragments thereof.
[0150] 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 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 observed in four of the seven challenge strains. In
fact, in all instances except for one (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 Rx1 PspA (amino
acids 192-588) cross-protects mice against fatal S. pneumoniae WU2
challenge. More importantly, these data show that the homologous
regions of diverse PspAs demonstrate comparable cross-protective
abilities.
[0151] 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 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 5
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.
[0152] There may be several reasons why the truncated PspAs from
capsular type 4 and type 5 strains failed to 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 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 5 is apparent from the
fact that in immunologically normal mice these strains have lower
LD.sub.50s and/or are more consistently fatal than strains of
capsular types 3, 6A, or 6B.
[0153] Another possibility is that epitopes critical to
protection-eliciting capacity with capsular type 4 and 5 strains
are not present in the C-terminal two-thirds of PspA (amino acids
192-588), the truncated fragments used for immunization. The
critical epitopes for these strains 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 Rx1 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-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 or
surface-exposed PspA. However, it is 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.
2TABLE 1 pspA recombinant strains categorized by pneumococcal
capsular type. Capsular Parent Respective Type Strains Recombinant
Strains 3 WU2, EF10197 RCT111, RCT137 4 BG9739, EF5668 RCT113,
RCT115 L81905, EF3296 RCT117, RCT133 5 DBL5 RCT105 6A DBL6A, EF6796
RCT109, RCT135 6B BG9163, DBL1 RCT129, RCT131 14 TJO893 none* 19
BG8090 RCT121 23 0922134, BG8743 RCT119, RCT123 *Truncated pspA
amplified recently, not yet cloned
[0154]
3TABLE 2 Description of recombinant strains used in evaluating the
protection-eliciting capacity of truncated PspAs in mice.
Recombinant Capsule Type Strain Description of Parent PspA RCT 105
BL21(DE3) E. coli with pET-9a: DBL5 5 RCT 113 BL21(DE3) E. coli
with pET-9a: BG9739 4 RCT 117 BL21(DE3) E. coli with pET-9a: L81905
4 RCT 125 BL21(DE3) E. coli with pET-9a (vector only)
[0155]
4Table 3 Evaluation of the protection elicited by truncated S.
pneumoniae PspA molecules in mice by days to death post-challcnge*.
Challenge Strain [capsular type] (log.sub.10 dose in CFU)
Immunizing Reciprocal A66.3 WU2 DBL6A BG7322 DBL5 BG9739 L81905
recombinant PspA/ anti-PspA [type 3] [type 3] [type 6A] [type 6B]
[type 5] [type 4] [type 4] PspA donor strain titer.sup..dagger.
(2.78) (3.57) (3.24) (3.11) (3.81) (3.56) (3.62) RCT113/BG9739
5590-50,300 4x > 21.sup..dagger-dbl. 4x > 21.sup..sctn. 15,
3x > 21.sup..dagger-dbl. 12, 13, 16, >21.sup..dagger-dbl. 3,
3, 4, 5.sup..sctn. 5, 5, 5, 7.sup..sctn. 5, 6, 8,
8.sup..dagger-dbl. RCT117/L81905 5590-150,900 4x >
21.sup..dagger-dbl. 4x > 21.sup..sctn. 7, 16, 2x >
21.sup..dagger-dbl. 10, 12, 13, >21.sup..sctn. 3, 3, 4,
4.sup..paragraph. 4, 5, 13, >21.sup..sctn. 3, 4, 6, 8
RCT105/DBL5 1860-16,770 4x > 21.sup..dagger-dbl. 4x >
21.sup..sctn. 8, 10, 13, 21.sup..dagger-dbl. 4x >
21.sup..dagger-dbl. 2, 2, 2, >21 2, 2, 2, 4 4, 5, 5, 5
RCT125/vector only 20-620 3, 6, 6, >21 2, 3, 3, >21 3, 6, 6,
6 7, 8, 8, 14 2, 2, 2, 2 2, 2, 3, 4, 5 2, 3, 5, 5 none 0 2, 2, 2 2,
3 3, 3, 4 6, 7, 9 2, 5 3, 5 2, 5 *Animals surviving the 3-week
evaluation period were sacrificed and days to death recorded as
>21 days. For statistical analysis, P values were calculated at
22 days for these fully protected mice. .sup..dagger.Range of four
sera per group of mice; titers measured against native donor PspAs
.sup..dagger-dbl.P .ltoreq. 0.012 .sup..sctn.P .ltoreq. 0.035
.sup..paragraph.P .ltoreq. 0.057 Note: One-tailed Fisher exact and
two sample rank tests were used for statistical analysis.
Example 2
Localization of Protection-Eliciting Epitopes and PspA of S.
pneumoniae
[0156] 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 Rx1 in 15 other diverse pneumococcal
strains. The DNA encoding these regions was then amplified and
cloned. The recombinant PspA fragments expressed were evaluated for
their ability to elicit cross-protection against a panel of
virulent pneumococci.
[0157] Bacterial strains and media conditions. S. pneumoniae
strains were grown in Todd Hewitt broth with 0.5% yeast extract
(THY) (both from Difco Laboratories, Detroit, Mich.) at 37.degree.
C. or on blood agar plates containing 3% sheep blood at 37.degree.
C. under reduced oxygen tension. E. coli strains were grown in
Luria-Bertani medium or minimal E medium. Bacteria were stored at
-80.degree. 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 .mu.g/ml for E. Coli.
[0158] 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).
[0159] The pMAL-p2 vector (New England Biolabs, Protein Fusion
& Purification System, catalog #800) was used for cloning pspA
gene fragments to amino acids 192-299 from strain Rx1 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
polymerase chain reaction (PCR) as described previously (McDaniel,
L. S. et al., Microb. Pathog. 1994; 17: 323) using primers
5'CCGGATCCGCTCAAAGAGATTGATGAGTCTG3' [LSM4] (SEQ ID NO: 16) and
5'CTGAGTCGACTGAGTITCTGGAGCTGGAGC3' [LMS6) (SEQ ID NO: 17) made with
BamHI and SalI restriction endonuclease sites, respectively.
Primers were based on the sequence of Rx1 PspA. PCR products and
the pMAL vector were digested with BAMHI and SalI, and ligated
together. Clones were transformed into E. Coli DH5.alpha. by the
methods of Hanahan. Stable transformants were selected on LB plates
containing 100 .mu.g/ml Ap. These clones were screened on LB plates
containing 0.1 mM IPTG, 80 .mu.g/ml X-gal and 100 .mu.g/ml Ap and
replica LB plates with 100 .mu.g/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 full-length pspA
probe (McDaniel, L. S. et al., Microb. Pathog. 1994; 17: 323),
randomly primed labeled with digoxigenin-11-dUTP (Genius System,
Boehdinger Mannheim, Indianapolis, Ind.) and detected by
chemiluminescence.
[0160] 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.degree. 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.degree. C., harvested, and the periplasmic contents
released by osmotic shock. For strains BAR36A, BAR39, BAR66,
BAR5668, BAR9739, BARL5, BAR6A and BAR100, bacteria were 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
#800). Briefly, amylose resin was poured into a 10 mL column and
washed with column buffer. The diluted osmotic shock extract was
loaded at a flow rate of approximately 1 mL/minute. The column was
then washed again with column buffer and the fusion protein eluted
off the column with column buffer containing 10 mM maltose. Lysates
were stored at -20.degree. C. until further use.
[0161] Characterization of truncated PspA proteins used for
immunization. The truncated PspA molecules, controls and molecular
weight markers (Bio-Rad, Richmond, Calif.) were electrophoresed in
a 10% sodium dodecyl (SDS)--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.
[0162] 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 .mu.g/ml in phosphate buffered saline (PBS) and
100 .mu.l 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 duplicate 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, Ala.)
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 for each antibody
with each preparation.
[0163] Immunization and Protection Assays. Six to nine week old
CBA/CAHN-XID/J (CBA/N) mice were obtained from the Jackson
Laboratory, Bar Harbor, Me. 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 .mu.g of protein per mouse. Fourteen days
later the mice were boosted intraperitoneally with 3 .mu.g of
antigen diluted in Ringer's lactate without adjuvant. Control mice
were 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.
[0164] Mice that were immunized with the homologues of Rx1 BAR416
were immunized as described above. The 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.
[0165] Serum analysis. Mice were retro-orbitally bled with a 75
.mu.l 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 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).
[0166] Intravenous infection of mice. Pneumococcal cultures were
grown to late log phase in THY. Pneumococci were diluted to
10.sup.4 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 LD.sub.50 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.
[0167] Statistical analysis. Statistical significance 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 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.
[0168] Cloning into PMAL vector. Using primers based on the
sequence of Rx1 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 6). 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, BAR66, BAR9739, BARL15, BAR6A and BAR100
were isolated, digested with BamHI and SalI restriction
endonucleases and electrophoresed on a 0.7% TSE 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
Rx1 pspA DNA at high stringency conditions. The cloning of R36A,
D39, A66, BG9739, DBL5, DBL6A and LM100 pspA DNA into pMal-p2 was
confirmed by the recognition of all BamHI and SalI digested DNA
inserts by the Rx1 probe.
[0169] Expression and conformation of truncated recombinant
proteins. The transformed E. coli strains BAR36A, BAR39, BAR66,
BAR9739, BARL5, 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, 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 space. 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 were recognized by a rabbit polyclonal
antibody made to strain BC100 PspA. The apparent M.sub.r of
full-length PspA from WU2 is 91.5 kD. The M.sub.r of maltose
binding protein is 42 kD and the expected M.sub.r 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 quantitatively confirmed the
reactivities that were observed in the western blots with all
protein extracts.
[0170] Protection and cross-protection against fatal pneumococcal
infection elicited by cloned PspA fragments. CBA/N mice were
immunized with the truncated PspA fragment encoded by pBC100, which
is composed of amino acids 192 to 588 of Rx1 PspA, and challenged
with 13 different S. pneumoniae strains representing 7 different
capsular types (Table 4). 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.
[0171] The BC100 immunization studies made it clear 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 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 5). 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 fill-length Rx1 PspA of 750. Mice immunized with BC100 had
geometric mean reciprocal titers of close to 2000, while
non-immunized mice had anti-PspA titers of <10.
[0172] The above data indicates that the BAR416 fragment from Rx1
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 BAR416 homologous regions from the 7 different
clones and then challenging with strain WU2 (Table 6). 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 protein had anti-PspA reciprocal titers of <10.
[0173] 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-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 from the
cross-reactivity of a panel of nine MAbs all of which were made by
immunization with Rx1 PspA. The epitopes of four of the antibodies
in the panel reacted 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, L. S., et al., Microb.
Pathog. 1994; 17, 323). Each of these 9 Mabs were tested for its
ability to react with 8 different PspAs in addition to Rx1. 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
whose epitopes map between 192 and 260 amino acids each
cross-reacted with an average of 4 of the 8 non-Rx1 PspAs, and all
of them reacted with at least two non-Rx1 PspAs. Thus, based on
this limited section of individual epitopes, it would appear that
epitopes in the region from 192-260 amino acids are generally much
more cross-reactive than epitopes in the region from 1-115 amino
acids.
[0174] The BC100 fragment of Rx1 PspA 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, M. J., et al., Infect. Immun. 1990; 58:
3293). The earlier finding made it clear that epitopes
cross-protective between Rx1 and WU2 PspAs exist. The importance of
cross-reactions in the region C-terminal to residue 192 is
demonstrated in this Example where 13 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 Rx1
PspA, could protect against infection with WU2. Moreover,
immunization with PspA fragments from 192-588 and 192-299 were able
to elicit protection against infection against WU2. This Example
shows that the BC100 Rx1 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 cross-protection, permits
the conclusion that epitopes in this 107 amino acid region of PspA
are sufficient to elicit significant cross-protection against a
number of different strains.
[0175] Evidence that a comparable region of other PspAs is also
able to elicit cross-protection came from the studies where
sequences homologous to the 192-299 region of Rx1 PspA were made
from 5 other PspAs. All 5 of these fragments elicited significant
protection against challenge with strain WU2. These data provide
some suggestion for serologic differences in cross-protection
elicited by the 192-299 region.
[0176] Based on present evidence, without wishing to be bound by
any one particular theory, it is submitted that the PspAs in
strains D39, Rx1 and R36A are identical. All of the 9 mice
immunized with the 192-299 fragments from R36A and D39 survived
challenge with WV2. Only LM100, one of the non-R36A/D39 PspAs,
protected as high a percentage of mice from WU2. The difference in
survival elicited by the R36A/D39 PspAs and the non-Rx1 related
PspAs was statistically significant.
[0177] 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 Rx1,
protected against death in 100% of the mice challenged with 7
different strains of S. pneumonia, but only delayed death with
strain D39, which is thought to have the same PspA as strain Rx1.
Thus, some of the differences in cross-protection are undoubtedly
related to factors other than PspA cross-reactivity. 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
known.
[0178] These results suggest that a vaccine containing only the
recombinant PspA fragments homologous with Rx1 amino acids 192-299
is effective against pneumococcal 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 to the amino acid or to the
DNA, or which is amplified by the DNA, e.g., 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 aa. 192-260 can be used to elicit
antibodies for use in antibody- binding kits assays or tests.
5TABLE 4 Protection of mice by immunization with BC100 from Rx1
PspA BC100 Immunogen Controls Challenge Capsule PspA # alive/ %
Median days # alive/ % Median days P Strain* type type # dead
Survival alive # dead Survival alive Value.sup..sctn. D39 2 25 0/5
0% 5 0/3 0% 2 0.02 WU2 3 1 4/0 100% >21 0/3 0% 3 0.002 ATCC6303
3 7 5/0 100% >21 0/5 0% 7 0.004 A66 3 13 4/0 100% >21 0/3 0%
1 0.03 EF10197 3 18 5/0 100% >21 0/3 0% 2 0.02 EF5668 4 12 1/3
25% 9 0/3 0% 4 N.S. EF3296 4 20 1/3 25% 5 0/3 0% 3 N.S. L81905 4 23
1/4 20% 4 0/6 0% 2 0.02 BG9739 4 26 0/4 0% 6.5 0/3 0% 2 N.S. DBL5 5
33 0/5 0% 5 0/3 0% 2 0.02 BG7322 6 24 4/0 100% >21 1/2 33.3% 6
0.03 EF6796 6A 1 4/0 100% >21 0/3 0% 1 0.03 DBL6A 6A 19 5/0 100%
>21 0/3 0% 7 0.03 *Mice were challenged with approximately
10.sup.3 CFU/mL of each strain .sup..sctn.P values were based on
comparison of days alive by a one-tailed Wilcoxon 2 sample-rank
test
[0179]
6TABLE 5 Protection of mice by immunization with BAR416 from Rx1
PspA BAR416 Immunogen Controls Challenge Capsule PspA # alive/ %
Median days # alive/ % Median days P Strain.sup.+ type type # dead
Survival alive # dead Survival alive Value.sup..sctn. WU2 3 1 4/1
80% >21 0/3 0% 1 0.002 ATCC6303 3 7 2/3 40% 13 1/4 20% 4 0.048
A66 3 13 5/0 100% >21 0/5 0% 2 0.004 BG7322 6 24 3/2 60% >21
0/4 0% 7 0.02 EF6796 6A 1 3/2 60% >21 0/5 0% 5 0.004 DBL6A 6A 19
0/5 0% 7 0/5 0% 2 0.008 Note, mice were challenged with about
10.sup.3 CFU of each strain .sup..sctn.P values were based on
comparison of days alive by a one-tailed Wilcoxon 2 sample-rank
test
[0180]
7TABLE 6 Protection of mice against S. pneumoniae WU2 by
immunization with BAR416 Analogs of 7 PspAs Parent Capsule PspA %
Median days P value* Immunogen Strain type type # alive/total #
Survival alive vs. MBP BAR36A R36A -- 25 4/4 100% >21 0.002
BAR39 D39 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 BARL5 DBL5 5
33 4/8 50% 21 0.03 BAR6A DBL6A 6A 19 3/5 60% >21 0.05 BAR100
LM100 22 ND 5/5 100% >21 0.0008 MBP -- -- -- 0/8 0% 2 -- *P
values were based on comparison of days alive by a one-tailed
Wilcoxon 2 sample-rank test Note, the PspA fragments used for
immunization were cloned from products amplified with primers LSM4
and LSM6. In addition to the strains listed above, PCR reactions
with LSM4 and LSM6 amplified products of the appropriate size from
strains BG9163, WU2, L81905, EF6796, EF5668, BG7376, BG7322, and
BG5-8A.
[0181]
8TABLE 7 Reactivity of MAbs with PspAs of Different Pneumococci
Donor of test PspA MAb mapping to 1-115 amino acids MAb mapping to
192-260 amino acids Capsule PspA Xi126 XiR1224 XiR1526 XiR35 XiR16
XiR1323 Xi64 XiR278 XiR1325 Strain Type Type IgG2b IgM IgG2b IgG2a
IgG2a IgM IgM IgG1 IgG2a Rx1 rough 25 ++ ++ ++ ++ ++ ++ ++ ++ ++
ATCC101813 3 3 ++ - - - - ++ ++ ++ ++ EF10197 3 18 - - - - - - - ++
+/- BG9739 4 26 - - - - - ++ - + ++ L81905 4 23 - - - - - - - - -
BG-5-8A 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 against a panel of nine different pneumococcal strains.
Rx1 served as a positive control. The results are presented as ++
(strong reaction), + (weak, but clearly positive reaction), +/-
(difficult #to detect), and - (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 Rx1 PspA
Example 3
Isolation of PspA and Truncated Forms Thereof, and Immunization
Thereby
[0182] 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 (CC), or
by growth in a chemically defined medium (CDM) containing 1.2
percent CC (CDM-CC) or medium in which the choline had been
replaced by ethanolamine (CDM-ET). Since CDM-ET supernatants lack
high concentrations of choline, the PspA released into them can be
adsorbed to a choline (or choline analog) column and isolated by
elution from the column with 2 percent choline chloride (CC).
[0183] 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
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 Rx1. Strain Rx1 was derived from strain
R36A, which was in turn derived from the encapsulated type 2
strain, D39. PspAs from these three strains appears to be
indentical based on serologic and molecular weight analysis.
Molecular studies have shown no differences in the pspA genes of
strains D39, Rx1, and R36A. The third strain that provided PspA in
this Example is the mouse virulent capsular type 3 strain WU2. Its
PspA is highly cross-reactive with that from R36A and Rx1, and
immunization with Rx1 and D39 PspA can protect against otherwise
fatal infections with strain WU2.
[0184] S. pneumoniae
[0185] Strains off S. pneumoniae used in this study have been
described previously (Table.sup.8). Bacteria were grown in either
Todd-Hewitt broth with 0.5 percent yeast extract (THY), or a
chemically defined medium (CDM) described previously.sup.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.
[0186] 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.sup.36. To release PspA from
pneumococci three procedures were used. One approach was to grow
pneumococci in 100 ml of THY and collect the cells by
centrifugation at mid-log phase. The pellet was washed three times
in lactated Ringer's solution (Abbot Lab. North Chicago, Ill.),
suspended in a small volume of 2 percent choline chloride in
phosphate buffered saline (PBS) (pH 7.0), incubated for 10 minutes
at room temperature, and centrifuged to remove the whole
pneumococci. From immunoblots with anti-PspA MAb Xi126.sup.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.sup.36.
[0187] The CDM used in the remaining two procedures was modified
from that of Van der Rijn.sup.43. For normal growth it contained
0.03% CC. To cause PspA to be released during bacterial growth, the
pneumococci were grown in CDM containing 1.2 percent choline
chloride (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.sup.49. In
CDM-CC and CDM-ET, PspA is released from the pneumecoccal surface
because of its inability to bind to the cholines in the
lipoteichoic acids.sup.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.sup.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 grow in long
chains.sup.36,49. As the pneumococci reach stationary phase they
die, cease making PspA, and rapidly settle out. Preliminary
studies, using serial dilution dot blots to 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 pneumcocci
began to settle out, the medium was recovered by centrifugation at
2900.times.g for 20 minutes, and filtered with a low
protein-binding filter (0.45.mu. Nalgene Tissue Culture Filter
#158-0045).
[0188] For growth in CDM-CC or CDM-ET, the pneumococci were first
adapted to the defined medium and then, in the case of CDM-ET, to
very low choline concentrations. To do this, strains were first
inoculated into 1 part of THY 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 ethanolamine (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 percent ethanolamine). These steps
were repeated until the strain would grow in CDM-ET containing
0.000,001 percent choline and 0.03 percent ethanolamine. It was
critical that cultures be passed while in exponential growth phase
(at about 10.sup.7 CFU/ml). Even trace contamination of the medium
by exogenous choline resulted in failure of the PspA to be released
from the pneumococcal surface.sup.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 frozen in 80
percent CDM-ET and 20 percent glycerol at -80.degree. C. When grown
subsequently the strain was inoculated directly into the
CDM-ET.
[0189] Isolation of native (fill-length) PsvA. PspA was isolated
from the medium of cells grown in CDM-ET using choline-Sepharose
prepared by conjugating choline to epoxy-activated
Sepharose.sup.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, Calif.). The column was monitored
by quantitative dot blot. The loading material, washes, and the
eluted material were dot blotted (1 .mu.l) as undiluted, 1/4,
{fraction (1/16)}, {fraction (1/64)}, {fraction (1/256)}, and
{fraction (1/1024)} on nitrocellulose. The membranes were then
blocked with 1 percent BSA in PBS, incubated for 1 hr with
PspA-specific MAbs Xi126 or XiR278, and developed with biotinylated
goat-anti-mouse Ig, alkaline phosphatase conjugated streptavidin
(Southern Biotechnology Associates Inc. Birmingham, Ala.), and
nitrobluetetrazolium substrate with 5-bromo 4chloro-3-indoyl
phosphate p-toluidine salt (Fisher Scientific, Norcross
Ga.).sup.17. The purity of eluted PspA was assessed by
silver-stained (silver stain kit, Bio Rad, Hercules, Calif.)
SDS-PAGE gels run as described previously.sup.32. Immunoblots of
SDS-PAGE gels were developed with MAbs Xi126 and XiR278.sup.17.
[0190] 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.sup.31,37. An overnight culture of JY4306 was grown in 100
ml of Luria Broth (LB) containing 50 .mu.g/ml ampicillin. The
culture was grown at 37.degree. C. 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 a wet paste, were harvested at 4.degree.
C. at 12,000.times.g. The pellet was washed in 10 volumes of 25 mM
Tris pH 7.7 at 0.degree. 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.times.g) and rapidly suspended in 900 ml of 1 percent sucrose
with 1 mM Pefabloc SC hydrochloride (Boehringer Mannheim Corp.,
Indianapolis, Ind.) at 0.degree. C. The suspension was pelleted at
8000.times.g at 4.degree. C. for 15 minutes and the PspA-containing
supernatant (periplasmic extract).sup.51 recovered. The recombinant
PspA was precipitated from the periplasmic extract by 70 percent
saturated ammonium sulfate overnight at 4.degree. C. The
precipitated material was collected by centrifugation at
12,000.times.g at 4.degree. C. for 30 minutes. The precipitated
protein was resuspended in 35 ml of 20 mM histidine 1 percent
sucrose at pH 6.6 (HSB). Insoluble materials were removed at
1,000.times.g at 4.degree. C. for 10 minutes. The clarified
material was dialyzed versus HSB, passed through a 0.2 .mu.g filter
and further purified on a 1 ml MonoQ HR 5/5 column (Pharmacia
Biotech, Inc., Piscataway, N.J.) 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 NaCl. By SDS-PAGE the material was
approximately 90 percent pure. The yield from 6 liters of culture
was 2 mg (Bio-Rad protein assay) of recombinant PspA.
[0191] 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. To 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 5.times.10.sup.8 CFU per ml/O.D. and confirmed by
colony counts (on blood agar) of serial dilutions of the
inoculum.
[0192] Immunization challenge, and bleeding of mice. CBA/CAHN/XID/J
(CBA/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 intraperitoneally 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 Mich.). 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
dilution or concentration, the dose was described as 100 .mu.l. If
the material was first diluted {fraction (1/10)}, or concentrated
10 fold, the dose was referred to as 10 or 1000 .mu.l
respectively.
[0193] ELISA for antibodies to PspA. Specific modifications of
previously reported ELISA conditions.sup.17, are described.
Microtitration plates (Nunc Maxisorp, P.G.C. Scientific,
Gaithersburg Md.) were coated with undiluted supernatants of Rx1
and WG44.1 pneumococci grown in CDM-ET or 1 percent BSA in PBS.
Mice were bled retro-orbitally (75 .mu.l) in a heparanized
capillary tube (Fisher Scientific, Fair Lawn, N.J.) The blood was
immediately diluted in 0.5 ml of one percent bovine serum albumin
in PBS. The dilution of the resultant sera was {fraction (1/15)}
based on an average hematocrit of 47 percent. The sera were diluted
in 7 three fold dilution in microtitration wells starting at
{fraction (1/45)}. Mab Xi126 was used as a positive control. The
maximum reproducible O.D. observed with Xi126 was defined as
"maximum O.D." 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 33 percent of maximum O.D. The binding to
the Rx1 CDM-ET coated plates was shown to be PspA-specific, since
in no case did we observe .gtoreq.33 percent of maximum binding of
immune sera or Xi126 on plates coated with WG44.1 CDM-ET or
BSA.
[0194] 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 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
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, Calif.).
[0195] 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.
[0196] PspA-containing preparations from pneumococci were able to
protect mice from fatal sepsis following i.v. challenge with
3.times.10.sup.3 (100.times.LD.sub.50) capsular type 3 S.
pneumoniae (Table 9). Comparable preparations from the strains
unable to make PspA (WG44.1 and JY1119), or unable to make full
length PspA (LM34 and JY2141) were unable to elicit protection.
Regardless of the method of isolation the minimum protective dose
was derived from pneumococci grown in from 10-30 .mu.l of medium.
We also observed.sup.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.sup.36,37 indicating the PspA is not normally released in
quantity into the medium of growing pneumococci.
[0197] 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.
[0198] 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 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
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.sub.j.sup.10 are typical of isolations from
300 ml of CDM-ET medium from R36A grown pneumococci. We isolated 84
.mu.g of PspA from 300 ml of medium, or about 280 .mu.g /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 .mu.g/liter of PspA, or about 0.4 .mu.g/ml.
[0199] 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 or
CDM-ET from a WG44.1 culture, indicating that PspA is the only
protein that could be isolated by this procedure. Moreover by
silver stained SDS PAGE gel the PspA isolated from R36A appeared to
be homogenous (FIG. 3). Although autolysin can also be isolated on
choline-Sepharose.sup.20,50, we did not expect it to be isolated by
this procedure since autolysin is not released from pneumococci
grown in choline deficient medium.sup.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.
[0200] 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 non-specific
trapping of aggregates in the column matrix. The elution buffer
contains 50 mM Tris acetate 0.25 M NaCl 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.
[0201] Immunization of mice 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
.mu.g of R36A 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
antibody were seen with the 1 .mu.g dose of PspA. In the presence
of CFA, however, the 0.1 .mu.g dose was as immunogenic as the 1
.mu.g dose.
[0202] To test the ability 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 10.sup.2, the A66 strain is several
logs more virulent when BALB/c mice are used.sup.47,52. The
difference in virulence of A66 and WU2, was partially compensated
for by challenging the immunized CBA/N mice with lower doses of
strain A66 than WU2.
[0203] After immunization of CBA/N mice with 1 and 0.1 .mu.g doses
of PspA we observed protection against WU2 challenge regardless of
whether or not CFA was used as an adjuvant (Table 4). At the lowest
dose, 0.01 .mu.g 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.sup.47,53,
survivors were only observed among mice immunized with the 1 and
0.1 .mu.g 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 death of mice infected
with A66 we observed a statistically significant delay in the time
to death in each immunized group as compared to the pooled
controls.
[0204] The 29 kDa N-terminal fragment of PspA can elicit protection
against infection when injected with CFA. We have compared the
immunogenicity, with and without CFA, of an isolated 29 kDa
fragment composed of the first 260 amino acids of PspA. Unlike the
case with FL-PspA, adjuvant was 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
.mu.g/mouse, or about 100 and 300 times the minimum dose of FL-PspA
that can elicit protection in the absence of adjuvant.
[0205] Injection with CFA revealed the presence of additional
protection eliciting antigen(s) in CDM-CC, and CDM-ET growth medium
but not in the 2 percent choline chloride eluates of live cells
[0206] The observation that Freund's adjuvant could have such a
major effect on the immunogenicity of the 29 kDa fragment (Table
12), prompted us to reexamine the 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 dose of PspA-containing growth
medium (CDM-CC and CDM-ET) required to elicit protection was
reduced from 10-30 .mu.l (Table 9) down to 1 to 3 .mu.l (Table 13).
When CFA was used as an adjuvant with CDM-CC and CDM-ET from
PspA-strains WG44.1 and JY1119 we were able to elicit protective
immune responses if material from .gtoreq.100 .mu.l 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 the presence of adjuvant.
[0207] 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 82 l. It should
be noted that although 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 has the potential to elicit
PspA-specific immunity. In contrast to these results, the material
eluted 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.sup.37, and would be expected to be washed
away prior to the elution with 2 percent CC. Extension of studies
to BALB/c mice and i.p. challenge route
[0208] The studies above all involve i.v. challenge of CBA/N mice
expressing with the XID genetic defect. The i.v. route, used in the
present studies provides a relevant model for bacteremia and
sepsis, but pneumococci have higher LD.sub.50s when injected i.v.
than i.p. CBA/N mice are hypersusceptible to pneumococcal infection
because of the XID defect. This genetic defect prevents them from
having circulating naturally occurring antibody to phosphocholine.
The absence of these antibodies has 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, 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.
[0209] 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 fact that
the C-terminal half of PspA binds to cell surface choline.sup.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 required to elicit protection against otherwise fatal
sepsis with greater than 100 LD.sub.50 of capsular type 3 S.
pneumoniae. When the first injection was given with adjuvant, doses
as small as 10 ng could elicit protective response. The potent
immunogenicity of PspA, and the ability to isolate it on
choline-Sepharose columns provides a demonstration for the possible
use of PspA as a vaccine in humans.
[0210] A large body of published.sup.17,29,37 as well as
unpublished evidence indicates that the major protection eliciting
epitopes of PspA are located in the .alpha.-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 amino acid a-helical region are able to elicit
protection when given with CPA. 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 immunogenic for FLP spA failed to
elicit a protective response. This result is consistent with
previous results showing that a fragment composed of the N-terminal
245 amino acids.sup.31,37 could elicit protection against otherwise
fatal pneumococcal infection of mice when the immunization was
given with CFA.sup.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
sequences important in the inmmunogenicity of the molecule as a
whole, since the fill 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 be especially immunogenic because they exhibit more
extensive aggregation than is seen with fragments expressing only
the .alpha.-helical region.sup.38. Protein aggregates are known to
generally be more antigenic and less tolerogenic than individual
free molecules.sup.54.
[0211] PspA is the major protection eliciting component of our
pneumococcal extractsu. 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 mutation blocking the production of some other antigen.
Strains WG44.1 and JY1119 contain identical deletions that include
the 5' end of the pspA genes and extend about 3 kb upstream of
pspA.sup.37. WG44.1 is a mutant of the non-encapsulated strain Rx1
and JY1119 was made by transforming capsular type 3 strain WU2 with
the WG44.1 pspA mutation. In no case were preparations from WG44.1
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 3 kb deletion linked to the
mutant pspA genes of WG44.1and JY1119, we also used strains JY2141
and LM34.sup.26,37. In these strains the Rx1 pspA gene has been
insertionally inactivated causing the production of N-terminal
fragments of 115 and 245 amino acids respectively. These strains
have no other known mutations. Although Rx1 and R36A are closely
related non-encapsulated strains, some of the studies included Rx1
as the PspA+ control since it is the isogenic partner 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.sup.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. LM34 was
tested without CFA in only 3 mice, but gave results consistent with
those obtained with JY2141.
[0212] Anticapsular antibodies are known to be protective against
pneumococcal infection.sup.5,19. However, in these studies it is
unlikely that 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.sup.39,41. The R36A strain has been recently demonstrated to
lack detectable type 3 capsule on the surface or in its
cytoplasm.sup.55. Furthermore, the CBA/N mice used in most of the
studies are unable to make antibody responses to capsular type 3
polysaccharide.sup.56.
[0213] Non-PspA protection eliciting components. The observation
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 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
protection eliciting molecules in the WG44.1 supernatant are
unknown. In this regard, it is of interest that unlike R36A, strain
Rx1 has been shown to contain a very small amount of cytoplasmic
type 3 polysaccharide (but totally lacks surface type 3
polysaccharide.sup.55). This difference from Rx1 apparently came
about through genetic manipulations in the construction of Rx1 from
R36A.sup.39,41. Thus, preparations made from Rx1 or from its
daughter strains WG44.1, LM34, or JY2141 could potentially contain
small amounts of capsular polysaccharide. For a 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.sup.57 that would be
required to release the small amount of type 3 polysaccharide from
the cytoplasm of the Rx1 family of strains; 2) CBA/N mice made
protective responses to the non-PspA antigens, but express the XID)
immune response deficiency which permits responses to proteins, but
blocks antibody to most polysaccharides.sup.46, including type 3
capsular polysaccharide.sup.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.
[0214] A number of non-PspA protection eliciting pneumococcal
proteins have been identified: pneumolysin, autolysin,
neuraminidase, and PsaA which are 52, 36.5, 101 and 37 kDa
respectively.sup.21,58,59,60. The non-PspA protection eliciting
components reported here could be composed of a mixture of these
and/or other non-identified proteins. Attempts to identify lambda
clones producing non-PspA protection eliciting proteins as
efficacious as PspA have not been successful.sup.25.
[0215] Isolation of PspA. The protective capacity of the 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 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 the
pneumococci 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
the PspA contained in it can be adsorbed directly to a
choline-Sepharose column for affinity purification.
[0216] One liter of CDM-ET growth medium contains about 400 .mu.g
of PspA, and we were able to isolate about 3/4 of it to very high
purity. At 0.1 .mu.g/dose, a liter of CDM-ET contains enough PspA
to immunize about 4,000 mice; or possibly 40-400 humans. Our
present batch size 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 volumes of R36A
CDM-ET.
9TABLE 8 Pneumococcal Strains Capsule PspA Parent Construction
Strain type expressed strain technique References D39 2 full length
-- clinical isolate 26, 44 R36A non- full length D39
non-encapsulated 23, 44, 45 encapsulated mutant Rx1 non- full
length R36A derived from R36A 26, 39, 41 encapsulated WG55.1 non-
none Rx1 aberrant insertion 26, 37 encapsulated inactivation with
pKSD300 LM34 non- aa 1-245 of Rx1.sup.a Rx1 insertional 26, 37, 42
encapsulated inactivation with pKSD300 JY2141 non- aa 1-115 of
Rx1.sup.a Rx1 insertional 37 encapsulated inactivation with pJY4208
WU2 3 full length -- clinical isolate 25, 46 JY1119 3 none WU2
transformation with 37 WG44.1 DNA A66 3 full length -- clinical
isolate 44, 47 .sup.aLM34 and LY2141 express fragments containing
the first 245 and first 115 amino acids of Rx1 PspA
respectively.
[0217]
10TABLE 9 PspA is the major protection-eliciting component in
antigen preparations made by three different methods Dose as Strain
volume Median (PspA of media Days Alive: P versus Preparation
status) in .mu.l.sup.a Alive Dead controls.sup.b 2% CC R36A 1000
>21 2:0 eluate from (PspA.sup.+) 200 >21 2:0 live cells 20
>21 2:0 2 1.5 0:2 all R36A >21 6:2 0.03 JY2141 1000 3, >21
1:1 (aa 1-115) 200 1 0:2 20 1 0:2 CDM/CC Rx1 100 >21 9:0
<0.0001 clarified (PspA.sup.+) 30 >21 2:1 medium 10 2 1:2 3 2
0:3 ALL 2, >21 12:6 0.0004 LM34 100 2, 2, >21 1:2 WG44.1 100
2 0:9 (PspA.sup.+) 30 2 0:3 10 2 0:3 4 2 0:3 WU2 1000 >21 3:0
0.05 (PspA.sup.+) 100 >21 1:0 ALL >21 4:0 0.03 JY1119 1000 4
0:3 (PspA.sup.-) CDM-CC 100 2 0:2 CDM-ET R36A 100 >21 8:0
<0.0001 clarified (PspA.sup.+) 10 3, >21 5:5 0.004 medium 1
1.5 3:5 0.1 2 0:2 ALL >21 16:12 0.006 JY2141 100 1.5 0:2 (aa
1-115) 10 1.5 0:2 WG44.1 100 3 0:2 (PspA.sup.-) 10 1.5 0:2 None --
2 0:14 -- .sup.aAntigen dose is given as the volume of growth media
from which the 0.1 ml of injected material was derived. Each mouse
was injected twice i.p. with the indicated dose diluted as
necessary in lactated Ringer's injection solution. .sup.bControls
used for statistical comparisons: 2% CC, all JY2141; CDM-CC Rx1,
all WG44.1; CDM-CC WU2, JY1119; CDM-ET, all WG44.1 + all
JY2141.
[0218]
11TABLE 10 Isolation of PspA from 300 ml of CDM-ET media after the
growth of R36A or WG44.1 pneumococcia R36A WG44.1 max. total dot
max. .mu.g total .mu.g reciprocal blot .mu.g protein total .mu.g
reciprocal fraction protein/ml protein.sup.b dot blot.sup.c
units.sup.b,d per/ml protein.sup.b dot blot.sup.c 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 1.sup.st wash <1 <1 10.sup.th wash <1 <1
elution #1 26 78 256 770 <1 -- <1 elution #2 2 6 16 48 <1
-- <1 elution #3 <1 -- 4 12 <1 -- <1 total eluted 84
830 -- <1 .sup.aThe 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 TBA. Elution was with
TBA plus 2 percent CC. .sup.bTotal .mu.g protein or total dot blot
units reflect the total protein in the 300 ml of the loading
material or the 3 ml size of the eluted fractions. .sup.cMAb XiR278
was used in the immunoblots to detect PspA in dot blots. .sup.dDot
blot units were calculated as the reciprocal dot blot titer times
the volume in ml.
[0219]
12TABLE 11 Purified full-length PspA is able to elicit protection
against fatal sepsis in mice. Challenge with Challenge with
10.sup.5.1 WU2 10.sup.4.2 A66 Adjuvant Anti-PspA Median P vs.
Median P vs. or Titer.sup.b Alive: Days pooled Alive: Days pooled
Antigen Dose.sup.a Diluent (Log mean .+-. S.E.) Dead Alive
control.sup.c Dead Alive controls.sup.c R36A 1 .mu.g Ringer's 3.3
.+-. 0.2 5:0 >21 0.015 2:3 4 0.002 (PspA.sup.+) 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 .mu.g 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 .mu.l Ringer's <1.6 n.d. n.d. 1:4 3 n.s. (PspA.sup.-) 360
Ringer's <1.6 n.d. n.d. 0:5 2 n.s. 36 Ringer's <1.6 n.d. n.d.
0:5 2 n.s. 3600 .mu.l 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 .sup.aFor comparison with the data in Table 2,
it should be noted that the 1, 0.1, and 0.01 .mu.g doses were
derived from 3600, 360, and 36 .mu.l of R36A growth media.
Equivalent dilutions of the PspA.sup.- eluate from strain WG44.1
were injected as controls. The amount of the WG44.1 preparations
injected is listed as 3600, 360, and 36 .mu.l and corresponds to
the volume original growth medium from which the doses of WG44.1
was prepared. .sup.bAntibody values were expressed as reciprocal
ELISA titer. .sup.cP 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 at
P < 0.0001.
[0220]
13TABLE 12 The 29 kDa N-terminal fragment of Rx1 PspA must be
injected with adjuvant to elicit protection against WU2.sup.a .mu.g
29 kDa Adjuvant Median Days P versus PspA or diluent Alive
Alive:Dead none.sup.b 30 CFA >21 3:0 0.0006 3 CFA >21 3:0 30
Ringer's 2 0:3 3 Ringer's 2 1:2 none CFA 2 0:7 none Ringer's 2 0:7
.sup.aThe 29 kDa fragment comprises the first 260 amino acids of
PspA. .sup.bFor the calculation of P values the 30 .mu.g and 3
.mu.g 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 Ringer'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.
[0221]
14TABLE 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 Median (PspA (as volume
Day Alive: Preparation status) in .mu.l) Alive Dead P values.sup.a
P vs. all JY2141 2% CC R36A 1000 >21 2:0 eluate from
(PspA.sup.+) 200 >21 5:0 0.02 live cells 20 >21 5:0 0.02 2
>21 5:0 0.02 all R36A >21 17:0 0.001 JY2141 1000 >21 2:0
(aa 1-115) 200 1 0:2 20 1 0:2 2 1 0:2 all JY2141 1 2:6 P vs pooled
cont. CDM-CC Rx1 1000 >21 3:0 0.002 clarified (PspA.sup.+) 100
>21 3:0 0.002 medium + CFA WU2 1000 >21 3:0 0.002
(PspA.sup.+) 100 >21 3:0 0.002 3 >21 3:0 0.002 WG44.1 1000
>21 5:1 <0.0001 (PspA.sup.+) 100 2.5 2:4 0.002 JY1119
(PspA.sup.+) 1000 >21 3:0 0.002 100 >21 3:0 0.002 CDM-ET R36A
1000 >21 3:1 0.004 clarified (PspA.sup.+) 10 >21 4:0 0.004
medium + CFA 1 >21 3:1 0.004 0.2 2 0:4 JY2141 10 >21 2:0 (aa
1-115) 1 >21 2:0 all JY2141 -- >21 4:0 0.004 WG44.1 100
>21 2:0 (PspA.sup.+) 10 2 0:2 CDM-ET only +CFA 2 0:9 None none
1.5 0:4 Pooled Controls.sup.b 2 0:13 .sup.aIn cases where there
were not statistically significant results no P value was shown.
.sup.b"Pooled Controls" refers to "CDM-ET only" Data and "None"
data.
[0222]
15TABLE 14 Immunization of BALB/c mice with isolated PspA elicits
protection against WU2 S. pneumoniae Challenge P vs. Antigen
Adjuvent Log Days to controls Source Dose.sup.a or diluent CFU
Route Death TSR/FE.sup.b R36A 1 .mu.g CFA 4 i.p. 2, >21, >21,
0.06/0.03 (PspA.sup.+) >21 WG44.1 100 .mu.l CFA 4 i.p. 2, 3
(PspA.sup.-) None -- CFA 4 i.p. 2, 2, 2, 4 R36A 1 .mu.g none 6 i.v.
2, >21, >21, 0.06/0.03 (PspA.sup.+) >21 WG44.1 100 .mu.l
none 6 i.v. 5, 7 (PspA.sup.-) None -- none 6 i.v. 2, 2, 2, 3 Pooled
i.v. iv. or 0.008/ and i.p. i.p. 0.0007 results .sup.aThe 1 .mu.g
dose of R36A PspA was isolated from 100 .mu.l of CDM-ET medium. As
a control mice were injected with a corresponding volume of
choline-column effluent from a mock isolation of PspA from the
PspA.sup.-strain WG44.1. The dose of WG44.1 material is expressed
as 100 .mu.l since this is the volume CDM-ET from which the
injected column effluent was derived. .sup.bP values calculated by
Wilcoxon two-sample rank test, TSR, or Fisher exact, FE versus
pooled controls for each group. "Pooled controls" include data
obtained by injection of "WG44.1" and "none". The i.p. and i.v.
studies gave comparable results. When the data from the two studies
were pooled the P values by both tests were .ltoreq.0.008. In cases
# where there were not statistically significant results no P value
was shown.
[0223] References
[0224] 1. Anonymous. Pneumococcal polysaccharide vaccine. MMWR
1981, 30, 410-419
[0225] 2. Farley, J. J. King, J. C., Nair, P., al., e. Invasive
pneumococcal disease among infected and uninfected children of
mothers with immunodeficiency virus infection. J. Pediatr. 1994,
124, 853-858
[0226] 3. Schwartz B., Cove, S., Lob-Lovit, J., Kirkwood, B R.
Potential interactions for the prevention of childhood pneumonia in
developing countries; etiology of acute lower respiratory
infections among young children in developing countries. Ped.
Infect. Dis. in Press
[0227] 4. Avery, O. T., Goebel, W. F. Chemoimmunological studies of
the soluable specific substance of pneumococcus. I. The isolation
and properties of the acetyl polysaccharide of pneumococcus type 1.
J. Exp. Med. 1933, 58, 731-755
[0228] 5. Austrian, R. Pneumococcal Vaccine: Development and
Prospects. Am. J. Med 1979, 67, 547-549
[0229] 6. Shapiro, E. D., Berg, A. T., Austrian, R., Schroeder, D.,
Parcells, V., Margolis, A., Adair, R. K., Clemmens, J. D.
Protective efficacy of polyvalent pneumococcal polysaccharide
vaccine. N. Engl. J. Med 1991, 325, 1453-1460
[0230] 7. Fedson, D. S. Pneumococcal vaccination in the prevention
of community-acquired pneumonia: an optimistic view of
cost-effectiveness. Sem. Resp. Infect. 1993, 8, 285-293
[0231] 8. Robbins, J. B., Austrian R., Lee, C -J., Rastogi, S. C.,
Schiffnan, G., Henrichsen, J., Makela, P. M., Broome, C. V.,
Facklam, R. R., Tiesjema, R. H., Parke, J. C., Jr. Considerations
for formulating the second-generation pneumococcal capsular
polysaccharide vaccine with emphasis on the cross-reactive types
within groups. J Infect Dis 1983, 148, 1136-1159
[0232] 9. Gotschlich, E. C., Goldschneider, I., Lepow, M. L., Gold,
R. The immune response to bacterial polysaccharides in man.
Antibodies in human diagnosis and therapy. New York, Raven, 1971,
391-402.
[0233] 10. Cowan, M. J., Ammann, A. J., Wara, D. W., Howie, V. M.,
Schultz, L., Doyle, N., Kaplan, M. Pneumococcal polysaccharide
immunization in infants and children. Pediatrics 1978, 62,
721-727
[0234] 11. Mond, J. J., Lees, A., Snapper, C. M. T cell-independent
antigens type 2. Ann. Rev. Immunol. 1995, 13, 655-692
[0235] 12. Chiu, S. S., Greenberg, P. D., Marcy, S. M., Wong, V. K,
Chang, S. J., Chiu, C. Y., Ward, J. I. Mucosal antibody responses
in infants following immunization with Haemophilus influenzae.
Pediatric Res. Abstracts 1994,35, 10A
[0236] 13. Kauppi, M., Eskola, J., Katlity, H. H. influenzae type b
(Hib) conjugate vaccines induce mucosal IgA1 and IgA2 antibody
responses in infants and children. ICAAC Abstracts 1993, 33,
174
[0237] 14. Dagen, R., Melamed, R., Abramson, O., Piglansky, L.,
Greenberg, D., Mendehnan, P. M., Bohidar, N., Ter-Minassian, D.,
Cvanovich, N., Lov, D., Rusk, C., Donnelly, J., Yagupsky, P. Effect
of heptavalent pneumococcal-OMPC conjugate vaccine on
nasopharyngeal carriage when administered during the 2nd year of
life. Pediat. Res. 1995, 37, 172A.
[0238] 15. Fattom, A., Vann, W. F., Szu, S. C., Sutton, A., Bryla,
D., Shiffman, G., Robbins, J. B., Schneerson, R. Synthesis and
physiochemical and immunological characterization of pneumomcoccus
type 12F polysaccharide-diptheria toxoid conjugates. Infect. Immun.
1988, 56, 2292-2298
[0239] 16. Kennedy, D., Derousse, C., E., A. Immunologic response
of 12-18 month children to licensed pneumococcal polysaccharide
vaccine primed with Streptococcus pneumoniae 19F conjugate vaccine.
ICAAC 1994, Abstract, G89
[0240] 17. McDaniel, L. S., Ralph, B. A., McDaniel, D. O., Briles,
D. E. Localization of protection-eliciting epitopes on PspA of
Streptococcus pneumoniae between amino acid residues 192 and 260.
Microbial Pathogenesis 1994, 17, 323-337
[0241] 18. Langermann, S., Palaszynski, S. R., Burlein, J. E.,
Koenig, S., Hanson, M. S., Briles, D. E., Stover, C. K. Protective
humoral response against pneumococcal infection in mice elicited by
recombinant Bacille Calmette-Gurin vaccines expressing PspA. J.
Exp. Med. 1994, 180, 2277-2286
[0242] 19. Siber, G. R. Pneumococcal Disease: Prospects for a New
Generation of Vaccines. Science 1994, 265, 1385-1387
[0243] 20. Lock, R. A., Hansman, D., Paton, J. C. Comparative
efficacy of autolysin and pneumolysin as immunogens protecting mice
against infection by Streptococcus pneumoniae. Microbial
Pathogenesis 1992, 12, 137-143
[0244] 21. Sampson, J. S., O'Connor, S. P., Stinson, A. R., Tharpe,
J. A., Russell, H. Cloning and nucleotide sequence analysis of
psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton
protein homologus to previously reported Streptococcus sp.
adhesins. Infect. Immun. 1994, 62, 319
[0245] 22. Paton, J. C., Lock, R. A., Lee, C. -J., Li, J. P.,
Berry, A. M., Mitchell. Purification and immunogenicity of
genetically obtained pneumolysin toxoids and their conjugation to
Streptococcus pneumoniae type 19F polysaccharide. Infect Immun.
1991, 59, 2297-2304
[0246] 23. McDaniel, L. S., Scott, G., Kearney, J. F., 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
[0247] 24. Briles, D. E., Forman, C., Horowitz J. C., Volanakis, J.
E., Benjamin, W. H., Jr., McDaniel, L. S., Eldridge, J., Brooks, J.
Antipneumococcal effects of C-reactive protein and monoclonal
antibodies to pneumococcal cell wall and capsular antigens. Infect.
Immun. 1989, 57, 1457-1464
[0248] 25. McDaniel, L. S., Sheffield, J. S., Delucchi, P., Briles,
D. E. PspA, a surface protein of Streptococcus pneumoniae, is
capable of eliciting protection against pneumococci of more than
one capsular type. Infect. Immun. 1991, 59, 222-228
[0249] 26. McDaniel, L. S., Yother, J., Vijayakumar, M., McGarry,
L., Guild, W. R., Briles, D. E. Use of insertional inactivation to
facilitate studies of biological properties of pneumococcal surface
protein A (PspA). J. Exp. Med. 1987, 165, 381-394
[0250] 27. Yother, J., McDaniel, L. S., Crain, M. J., Tallington,
D. F., Briles, D. E. Pneumococcal surface protein A: Structural
analysis and biological significance In: Dunny, G. M., Cleary, P.
P., McKay, L. L. ed. Genetics and Molecular Biology of
Streptococci, Lactococci, and Enterococci. Washington, DC: American
Society for Microbiology, 1991, 88-91
[0251] 28. Waltman, W. D., II, McDaniel, L. S., Gray, B. M.,
Briles, D E. Variation in the molecular weight of PspA
(Pneumococcal Surface Protein A) among Streptococcus pneumoniae.
Microb. Pathog. 1990, 8, 61-69
[0252] 29. Crain, M. J., Waltman, W. D., II, Turner, J. S., Yother,
J., Talkington, D. E., McDaniel, L. M., Gray, B. M., Briles, D. E.
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
[0253] 30. McDaniel, L. S., Scott, C., Widenhofer, K., Carroll,
Briles, D. E. Analysis of a surface protein of Streptococcus
pnemoniae recognized by protective monoclonal antibodies. Microb.
Pathog. 1986, 1, 519-531
[0254] 31. Yother, J., Briles, D. E. Structural properties and
evolutionary relationships of PspA, a surface protein of
Streptococcus pneumoniae, as revealed by sequence analysis. J.
Bact. 1992, 174, 601-609
[0255] 32. Talkington, D. R, Crimnmins, D. L., Voellinger, D. C.,
Yother, J., Briles, D. E. A 43-kilodalton pneumococcal surface
protein, PspA: isolation, protective abilities, and structural
analysis of the amino-terminal sequence. Infect. Immun. 1991, 59,
1285-1289
[0256] 33. McDaniel, L. S., McDaniel, D. O. Genetic analysis of the
gene encoding type 12 PspA of Streptococcus pneumoniae strain
EF5668 In: Feretti, J. J., Gilmore, M. S., Khenhammer, T. R.,
Brown, F. ed. Genetics of the streptococci, enterococci, and
lactococci. Basel: Dev. Biol. Stand. Basel Krager, 1995,
283-286
[0257] 34. Fischetti, V. A., Pancholi, V., Schneewind, O.
Conservation of a hexapeptide sequence in the anchor region of
surface proteins from gram-positive cocci. Molec. Microbial. 1990,
4, 1603-1605
[0258] 35. Schneewind, O., Fowler, A., Faull, K. F. Structure of
cell wall anchor of cell surface proteins in Staphylococcus aureus.
Science 1995, 263, 103-106
[0259] 36. Yother, J., White, J. M. Novel surface attachment
mechanism for the streptococcus pneumoniae protein PspA. J. Bact.
1994, 176,2976-2985
[0260] 37. Yother, J., Handsome, G. L., 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, 610-618
[0261] 38. Talkington, D. F., Voellinger, D. C., McDaniel, L. S.,
Briles, D. E. Analysis of pneumococcal PspA microheterogeneity in
SDS polyacrylamide gels and the association of PspA with the cell
membrane. Microbial Pathogenesis 1992, 13, 343-355
[0262] 39. Smith, M. D., Guild, W. R. A plasmid in Streptococcus
pneumoniae. J. Bacteriol. 1979,137, 735-739
[0263] 40. Shoemaker, N B., Guild, W. R. Destruction of low
efficiency markers is a slow process occurring at a heteroduplex
stage of transformation. Mol. Gen. Genet. 1974, 128, 283-290
[0264] 41. Raven, A. W. Reciprocal capsular transformations of
pneumococci. J. Bact. 1959, 77, 296-309
[0265] 42. McDaniel, L. S., Sheffield, J. S., Swiatlo, E., Yother,
J., Crain, M. J., Briles, D. E. Molecular localization of variable
and conserved regions of pspA, and identification of additional
pspA homotogous sequences in Streptococcus pneumoniae. Microbial
Pathogenesis 1992, 13, 261-269
[0266] 43. Rijn, V. D., Kessler, R. E. Growth characteristics of
Group A Streptococci in a new chemically defined medium. Infec.
Immun. 1980, 27,444-448
[0267] 44. Avery, O. T., MacLeod, C. M., McCarty, M. 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
[0268] 45. McCarty, M. The transforming principle. New York,
Norton, 1985, 252.
[0269] 46. Briles, D. E., Nahm, M., Schroer, K., Davie, J., Baker,
P., Kearney, J., 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
[0270] 47. Briles, D. E., Crain, M. J., Gray, B. M., Forman, C.,
Yother, J. A strong association between capsular type and mouse
virulence among human isolates of Streptococcus pneumoniae. Infect.
Immun. 1992, 60, 111-116
[0271] 48. Waltman, W. D., II, McDaniel, L. S., Andersson, B.,
Bland, L., Gray, B. M., Svanborg-Eden, C., Briles, D. E. Protein
serotyping of Streptococcus pneumoniae based on reactivity to six
monoclonal antibodies. Microb. Pathog. 1988, 5, 159-167
[0272] 49. Tomasz, A. Surface components of Streptococcus
pneumoniae. Rev. Infect. Dis 1981, 3, 190-211
[0273] 50. Garcia, J. L., Garcia, E., Lopez, R. Overproduction and
rapid purification of the amidase of Streptococcus pneumoniae.
Arch. Microbial. 1981, 149, 52-56
[0274] 51. Osbom, M. J., Munson, J. Separation of the inner
(cytoplasmic) and outer membranes of gram negative bacteria.
Methods Enzymol. 1974, 31A, 642-653
[0275] 52. Briles, D. E., Horowitz J., McDaniel, L. S., Benjamin,
W. H., Jr., Claflin, J. L., Booker, C. L., Scott, G., Forman, C.
Genetic control of susceptibility to pneumococcal infection. Curr.
Top. Microbiol. Immunol. 1986, 124, 103-120
[0276] 53. Briles, D. E., Forman, C., Crain, M. Mouse antibody to
phosphocholine can protect mice from infection with mouse-virulent
human isolates of Streptococcus pneumoniae. Infect. Immun. 1992,
60, 1957-1962
[0277] 54. Weigle, W. O. Immunological unresponsiveness. Academic
Press, New York, N.Y., 1973.
[0278] 55. Dillard, J. P., Yother, J. Genetic and molecular
characterization of capsular polysaccharide biosynthesis in
Streptococcusp pneumoniae type 3. Molec. Microbial 1994, 12,
959-972
[0279] 56. Amsbaugh, D. F., Hansen, C. T., Prescott, B., Stashak,
P. W., Barthold, D. R., Baker, P. J. 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
[0280] 57. 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
[0281] 58. Paton, J. C., Lock, R. A., Hansman, D. C. Effect of
immunization with pneumolysin on survival time of mice challenged
with Streptococcus pneumoniae. Infect. Immun. 1983, 40, 548-552
[0282] 59. Berry, A. M., Lock, R. A., Hansman, D., Paton, J. C.
Contribution of autolysin to virulence of streptococcus pneumoniae.
Infect. Immun. 1989, 57, 2324-2330
[0283] 60. Lock, R. A., Paton, J. C., Hansman, D. Purification and
immunologic characterization of neuraminidase produced by
Streptococcus pneumoniae. Microbial Pathogenesis 1988, 4, 33-43
[0284] 61. Tuomanen, E., Liu, H., Hengstler, B., Zak, O., Tomasz,
A. The Induction of meningeal inflammation by components of the
pneumococcal cell wall. 1985, 151, 859-868
[0285] 62. Tuomanen, E., Tomasz, A., Hengsfler, B., Zak, O. The
relative role of bacterial cell wall and capsule in the induction
of inflammation in pneumococcal meningitis. J. Infect. Dis. 1985,
151, 535-540
[0286] 63. Paton, J. C. Pathogenesis of pneumococcal disease. 1993,
363-368
[0287] 64. Briese, T., Hakenbeck, R. Interaction of the
pneumococcal amidase with lipoteichoic acid and choline. 1985, 146,
417-427
Example 4
Evidence For Simultaneous Expression of Two PspAs
[0288] 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 Rx1 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. 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.
[0289] 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
antibodies (MAbs).
[0290] A serotyping system for PspA has been developed, which uses
a panel of seven MAbs. PspA serotypes are 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.
[0291] While most pneumococcal strains appear to have two DNA
sequences homologous with both the 5' and 3' halves of pspA,
site-specific truncation mutations of Rx1 have revealed that one
these, pspA, encodes PspA. The other sequence has been
provisionally designated as the 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
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 2 Kb in size from
70% of pneumococci. For the remaining 30% of pneumococci no
amplification was observed with the primers used.
[0292] Evidence for Two PspAs:
[0293] 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. 4). 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 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 XiI26, 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.
[0294] Simultaneous Production of Both PspAs:
[0295] 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.
[0296] Number of psDA Genes:
[0297] 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
(FIG. 5A). In comparison, when Rx1 DNA was digested with Hind III
and hybridized with plSMpspA13.2, homologous sequences were
detected on 9.1 and 4.2 Kb fragments as expected from previous
studies (9) (FIG. 5A). Results consistent with only two
pspA-homologous genes in MC25-28 were also obtained with digestion
using four additional enzymes (Table 15).
[0298] In previous studies it has been reported that probes for the
5' half of pspA (encoding the alpha-helical 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 5' Rx1 probe of pLSMpspA12/6 bound both pspA homologous bands
at a stringency of greater than 95 percent. The same probe bound
only the pspA containing fragment Rx1 at a stringency above 95
percent (FIG. 5B).
[0299] 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 approximately equal to the size of the 2.1 Kb amplified
product (FIG. 6). These results suggest that the 2.1 Kb amplified
DNA represents the amplified product of only a single DNA sequence.
Rx1, by comparison, produced an amplified product of 2.0 Kb and
five fragments of 0.76, 0.468, 0.390, 0.349 and 0.120, when
digested with Hha 1 as expected from its known pspA sequence.
[0300] The 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 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 since they share PspA serotypes, amplified
pspA RFLPs, chromosomal pspA RFLPs, capsule type, and resistance to
penicillin.
[0301] The interpretation of studies presented here, showing the
existence of two PspAs in the four strains MC25-28, must be set in
the context of what is known 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 Rx1 are
made by 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.
[0302] PspA was originally defined by reciprocal absorption studies
demonstrating that a panel of MAbs to Rx1 surface proteins each
reacted with some protein and later by studies using Rx1 and WU2
derivatives expressing 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 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.
[0303] 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 the
dimeric or monomeric status of the protein, seems unlikely since
whenever MAbs react with the PspA of a stain, 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.
[0304] 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 (perhaps in this isolate there has been a recombination
between pspA DNA and the pspA-like locus, thus allowing 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 does not occur
under in vitro growth conditions. 3) The pspA-like locus is
normally a nonfunctional pseudogene sequence that for an
unexplained reason has become functional in these isolates.
[0305] It 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.
[0306] 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 pspA-like sequence of Rx1. This finding indicates that the
pspA-like sequence of MC25-28 is more similar to the Rx1 pspA
sequence than is the Rx1 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 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
conclusive detecting, determining or isolating of pneumococci.
[0307] Isolates and Bacterial Cell Culture:
[0308] Pneumococcal isolates described in these studies were
cultured from patients in Barcelona, Spain (one adult at Bellvitge
Hospital, and three children at San Juan de Dios) between 1986 and
1988 (Table 2). These penicillin resistant pneumococci originally
in the collection of Dr. Brian Spratt were shared with applicants
by Dr. Alexander Tomasz at the Rockefeller Institute. Rx1 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 0.5% 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.
[0309] Bacterial Lysates:
[0310] Cell lysates were prepared by incubating the 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, Ill.)
[0311] PspA Serotyping:
[0312] 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 assigned based on the particular combination of
MAbs with which each PspA was reactive.
[0313] Colony Immunoblotting:
[0314] A ten ml tube of Todd-Hewitt broth with 0.5% yeast extract
was inoculated with overnight growth of MC23 from a blood agar
plate. The isolate was allowed to grow to a concentration of
10.sup.7 cells/ml as determined by an O.D. of 0.07 at 590 nm. MC23
was serially diluted and 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 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.
[0315] Chromosomal DNA Preparation:
[0316] Pneumococcal chromosomal DNA was prepared as in Example 9.
The cells were harvested, washed, lysed, and digested with 0.5%
(wt/vol) SDS and 100 .mu.g/ml proteinase K at 37.degree.0 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.degree. C. for 20 minutes, then
extracted with chlorofomn/isoamyl alcohol. DNA was precipitated
with 0.6 volumes isopropanol, washed, and resuspended in 10 mM
Tris-HCL, 1 mM EDTA, pH 8.0. DNA concentration was determined by
spectrophotometric analysis at 260 nm.
[0317] Probe Preparation:
[0318] 5' and 3' oligonucleotide primers homologous with
nucleotides 1 to 26 and 1967 to 1990 of Rx1 pspA (LSM 13 and LSM2,
respectively) were used to amplify the full length pspA and
construct probe LSMpspA13/2 from Rx1 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
LSMpspA 12/6. PCR generated DNA was purified by Gene Clean (Bio101
Inc., Vista, Calif.) and random prime-labeled with
digoxigenin-11-dUTP using the Genius 1 Nonradioactive DNA Labeling
and Detection Kit as described by the manufacturer (Boehringer
Mannheim, Indianapolis, Ind.).
[0319] DNA Electrophoresis:
[0320] For Southern blot analysis, approximately 10 .mu.g 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, Sul 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 cases, 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.
[0321] Southern Blot Hybridization
[0322] The DNA in the gel was depurinated in 0.25N HCL for 10
minutes, denatured in 0.5M NaOH and 1.5M NaCl for 30 minutes, and
neutralized in 0.5M Tris-HCl (pH 7.2), 1.5M NaCl and 1 mM disodium
EDTA for 30 minutes. DNA was transferred to a nylon membrane
(Micron Separations INC, Massachusetts) using a POSIBLOT pressure
blotter (Strategene, La Jolla, Calif.) for 45 minutes and fixed by
UV irradiation. The membranes were prehybridized for 3 hours at
42.degree. C. in 50% formamide, 5.times.SSC, 5.times. Denhardt
solution, 25 mM sodium phosphate (pH 6.5), 0.5% SDS 3% (wt/vol)
dextran sulfate and 500 g/ml of denatured salmon containing 45%
formamide, 5.times.SSC, 1.times. Denhardt solution, 20 mM sodium
phosphate (pH 6.5), 0.5% SDS, 3% dextran sulfate, 250 .mu.g/ml
denatured sheared salmon sperm DNA and about 20 ng of
heat-denatured diogoxigenin-labeled probe DNA. After hybridization,
the membranes were washed twice in 0.1% SDS and 2.times.SSC for 3
minutes at room temperature. The membranes were washed twice to a
final stringency of 0.1% SDS in 0.3.times.SSC at 65.degree. C. for
15 minutes. This procedure yields a stringency greater than 95
percent. The membranes were developed using the Genius 1
Nonradioactive DNA Labeling and Detection Kit as described by the
manufacturer (Boehringer Mannheim, Indianapolis, Ind.). To perform
additional hybridization with other probes, the membranes were
stripped in 0.2N NaOH/0.1% SDS at 40.degree. C. for 30 minutes and
then washed twice in 2.times.SSC.
[0323] Polymerase Chain Reaction (PCR):
[0324] 5' and 3' primers homologous with the DNA encoding the N-
and C-terminal ends of PspA (LSM13 and LSM2, respectively) were
used in these experiments. Amplifications were made using Taq DNA
polymerase, MgCl.sub.2 and 10.times. reaction buffer obtained from
Promega (Madison, Wis.). DNA used for PCR was prepared using the
method previously described in this paper. Reactions were conducted
in 50 ml volumes containing 0.2 mM of each dNTP, and 1 ml of each
primer at a working concentration of 50 mM. MgCl.sub.2 was used at
an optimal concentration of 1.75 mM 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 (M.J. Research, Inc.) using the following three step
program. Step 1 consisted of a denaturing temperature of 94.degree.
C. for 2 minutes. Step 2 consisted of 9 complete cycles of a
donaturinq temperature of 94.degree. C. for 1 minute, an annealing
temperature of 50.degree. C. for 2 minutes, and an extension
temperature of 72.degree. C. for 3 minutes. Step 3 cycled for 19
times with a denaturing temperature of 94.degree. C. for 1 minute,
an annealing temperature of 60.degree. C. for 2 minutes, and an
extension temperature of 72.degree. C. for 3 minutes. At the end of
the last cycle, the samples were held at 72.degree. C. for 3
minutes to ensure complete extension.
[0325] Band Size Estimation:
[0326] 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 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,
Wash.) in order to calculate molecular weights based on migration
distances observed in the Southern blot.
16Table 15 Restriction Fragments Restriction Strains Examined
(sizes in kilobases) Enzyme MC25 MC26 MC27 MC28 RX1 MC25-MC28 RX1
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
Pst I + + >14, 6.1 10.0, 4.0
[0327]
17TABLE 16 Penicillin Resistant Capsular Serogroup 6 Strains from
Spain MIC Isolate Penicillin (.mu.g/ml) Year Site Hospital MC25 1
1986 sputum Bellvitge MC26 4 1988 ear San Juan de Dios MC27 1 1988
ear San Juan de Dios MC28 2 1988 ? San Juan de Dios
[0328] References
[0329] 1. Grain M. J., W. D. Waltman II, J. S. Turner, J. Yother,
D. E. Talkington, L. S. McDaniel, B. M. Gray and D. E. 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.
[0330] 2. Briles D. E., 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.
[0331] 3. McDaniel L. S., J. Yother, M. Vijayakumar, L. McGarry, W.
R. Guild and D. E. Briles. Use of insertional inactivation to
facilitate studies of biological properties of pneumococcal surface
protein A (PspA). J Exp Med 1987; 165:381-394.
[0332] 4. Grain M. J. Unpublished data.
[0333] 5. 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.
[0334] 6. Talkington D. F., D. L. Crienis, D. C. Voellinger, J.
Yother and D. E. 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.
[0335] 7. McDaniel L. S., B. A. Ralph, D. O. McDaniel and D. E.
Briles. Localization of protection-eliciting epitopes on PspA of
Streptococcus pneumoniae between amino acid residues 192 and 260.
Microb Pathogen 1994; 17:323-337.
[0336] 8. Yother J., G. L. Handsome and D. E. Briles. 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; 174:610-618.
[0337] 9. McDaniel L. S., J. S. Sheffield, E. Swiatlo, J. Yother,
M. J. Crain and D. E. Briles. Molecular localization of variable
and conserved regions of pspA, and identification of additional
pspA homologous sequences in Streptococcus pneumoniae. Microb
Pathogen 1992; 13:261 -269.
[0338] 10. Waltman W. D. II, L. S. McDaniel, B. M. Gray and D. E.
Briles. Variation in the molecular weight of PspA (Pneumococcal
Surface Protein A) among Streptococcus pneumoniae. Microb Pathogen
1990; 8:61-69.
[0339] 11. Munoz R., J. M. Musser, M. Crain, D. E. Briles, A.
Marton, A. J. Parkinson, U. Sorensen and A. Tomasz. Geographic
distribution of penicillin-resistant 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.
[0340] 12. Brooks-Walter A. and L. S. McDaniel. 1994. Unpublished
data.
[0341] 13. Talkington D. F., D. C. Voelkinger, L. S. McDaniel and
D. E. Briles. Analysis of pneumococcal PspA microheterogeneity in
SDS polyacrylamide gels and the association of PspA with the cell
membrane. Microb Pathogen 1992; 13:343-355.
[0342] 14. McDaniel L. S., G. Scott, K. Widenhofer, Carroll and D.
E. Briles. Analysis of a surface protein of Streptococcus
pneumoniae recognized by protective monoclonal antibodies. Microb
Pathogen 1986: 1:519-531.
[0343] 15. Sheffield J. S., 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.
[0344] 16. Briles D. E, 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 pneumoniae. Infect
Imunn 1992; 60:111-116.
Example 5
Southern Blot Analysis of pspAs and Fragments of pspA
[0345] In this example, Applicants used oligonucleotides derived
from the DNA sequence of pspA of S. pneumoniae Rx1 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 .alpha.-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.
[0346] In the previous Example, it was shown that strain Rx1 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. 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
Rx1 and most other strains hybridized with the pspA probes. When
the genome of Rx1 was digested with HindIII and hybridized with
these, two pspA-homologous sequences were found to be in 4.0 and
9.1 kb fragments. Using derivative of Rx1 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-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.
[0347] 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 and are
therefore useful as both hybridization probes and as primers for
polyrmerase chain reaction (PCR) analysis.
[0348] Hybridization with Oligonucleotide Probes.
[0349] The oligonucleotides used in this study were based on the
previously determined sequence of Rx1 PspA. Their position and
orientation relative to the structural domains of Rx1 PspA are
shown in FIG. 7. The reactivity of these oligonucleotide probes
with the pspA and pspA-like sequences was examined by hybridization
with a HindIII digest of Rx1 genomic DNA (Table 17). As expected,
each of the eight probes recognized the pspA-containing 4.0 kb
fragment of the HindIII digested Rx1 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 4.0
kb.
[0350] 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 Rx1 is a laboratory 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 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.
[0351] The only strain not giving at least two pspA-homologous
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 Rx1
pspA. Even at 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 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 Rx1 pspA. These results are consistent with earlier findings
that the pairs of HindIII fragments from individual isolates
generally include two separate but homologous sequences, rather
than fragments of a single pspA gene.
[0352] 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 2.times.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.
[0353] The last column in Table 18 give the ratio of strains that
showed one reactive HindHIII 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 pspA-like 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 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 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.
[0354] 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 60% 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 fraction of strains of any
oligonucleotide (29-35 percent), suggesting that the portion of
pspA encoding the .alpha.-helical region is the least conserved
region of pspA.
[0355] 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 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. The probes
most reactive with these additional sequences are those for the
leader, the .alpha.-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 the LSM3 (.alpha.-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.
[0356] Amplification of pspA
[0357] 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 12 different capsular types using primers based on the
Rx1 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
proline-rich region (Table 19). LSM2 was also used in combination
with four other 5' primers LSM1, 3, 7, 8 and 12. LSM8 is located 5'
of the pspA start site (near the -35 region).
[0358] If a predominant sequence of the expected length was
amplified that could be detected on a Southern blot with a
fill-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
.alpha.-helical sequence was found to be less conserved than the
primers 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 Rx1 sequence 5' of the pspA start site.
This oligonucleotide was not used in the hybridization studies.
[0359] 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 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 .alpha.-helical, proline-rich, and repeats were
also amplified from the same isolates. As seen in Table 20, the
variation in size of pspA appeared to come largely from variation
in the size of pspA encoding encodes the .alpha.-helical
region.
[0360] Using probes that consisted of approximately the 5' and 3'
halves of pspA it has been determined that the portion of pspA that
encodes the .alpha.-helical regions is less conserved than the
portion of pspA that encodes the C-terminal half of the molecule.
This Example shows using 4 oligonucleotide probes from within each
half of the DNA encoding PspA. Since a larger number of smaller
probes 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 of likely differences
and similarities between pspA and the pspA-like sequences.
[0361] The only strains in which the pspA gene has been identified
by molecular mutations are Rx1, D39 and WL2. Rx1 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 fill length-pspA as a probe, it is not possible to
distinguish between the pspA and pspA-like 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 Rx1 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 would be expected to identify the known Rx1 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
that reacted with Rx1 pspA and LSM3 reacted with WU2 pspA. Each of
these probes reacted with 4 of the other 15 strains. When these
probes identified a band, however, the band was generally also
detected by all other Rx1 probes reactive with that strain's DNA.
Additional evidence that the LSM3 and LSM4 were restricted to
reactivity with pspA was that they reacted with the same bands in
all three non-Rx1 strains. Each probe identifies pspA in certain
strains and even when used in combination they recognized pspA in
over 40 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 pspA-homologous sequence at high
stringency. Collectively 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 probes.
[0362] By making the assumption that in different strains the Rx1
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
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 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.
[0363] Using these assumptions the most variable portion of the
pspA gene was observed to be the -35 region and the portion
encoding .alpha.-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 Rx1 sequence
makes it likely that other probes for the repeat regions give
similar results.
[0364] The portion of the pspA-like sequence most similar to Rx1
pspA was that encoding the leader sequence, the 5' portion of the
proline rich region, and the repeat region, and those portions
encoding the N-terminal end of the proline-rich and repeat regions.
The repeat region of PspA has been shown to be involved in the
attachment to PspA to the pneumococcal surface. The conservation of
the repeat region among both pspA and pspA-like genes suggests that
if a PspA-like protein is produced, 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-pneumococcal drug
target. The conservation in the leader sequence between pspA and
the pspA-like sequence was also not surprising since similar
conservation has been reported for the leader sequence of other
gram positive proteins, such as M protein of group A streptococci.
It is noteworthy, 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.
[0365] Although the region encoding the C-terminus of pspA (LSM12)
or the 3' portion of the proline-rich sequence (LSM1) appear to be
highly conserved within pspA genes, corresponding regions in the
pspA-like sequences are either lacking, or very distinct from those
in pspA. The reason for conservation at these sites is not
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
differences from pspA portend 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 between all
pspA and pspA-like genes.
[0366] With two strains, some of the oligonucleotide probes
identified more than two pspA-homologous sequences. 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 the 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 share areas of homology with the
leader, .alpha.-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 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 component of a pneumococoal antigenic,
imumunological vaccine compositions.
[0367] Bacterial Strains, Growth Conditions and Isolation of
Chromosomal DNA.
[0368] S. pnemoniae 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.degree. C. to an approximate density of
5.times.10.sup.8 cells/ml. Following harvesting of the cells by
centrifugation (2900.times.g, 10 minutes), the DNA was isolated as
previously described and stored at 4.degree. C. in TE (10 mM Tris,
1 mM EDTA, pH 8.0).
[0369] Amplification of pspA Sequences.
[0370] 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
Rx1. These oligonucleotides were obtained from Oligos Etc.
(Wilsonville, Oreg.) and are listed in Table 22.
[0371] PCRs were done with a MJ Research, Inc., Programmable
Thermal Cycler (Watertown, Mass.) 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
.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,
200 mM of each deoxynucleotide 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
minutes. Then the samples were subjected to 10 cycles consisting of
1 minute at 94.degree. C., 2 minutes at 50.degree. C., and 3
minutes at 72.degree. C. followed by another 20 cycles of 1 minute
at 94.degree. C., 2 minutes at 50.degree. C., and 3 minutes at
72.degree. C. followed by another 20 cycles of 1 minute at
94.degree. C., 2 minutes at 60.degree. C., and 3 minutes at
72.degree. C. After all 30 cycles, the samples were held at
72.degree. C. for an additional 5 minutes prior to cooling to
4.degree. C. The PCR products were analyzed by agarose gel
electrophoresis.
[0372] DNA Hybridization Analysis.
[0373] Approximately 5 .mu.g of chromosomal DNA was digested with
HindIll according to the manufacturer's instructions (Promega,
Inc., Madison, Wis.). 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, N.H.)
[0374] Labeling of oligonucleotide with and detection of
probe-target hybrids were both performed with the Genius System
according to the manufacturer's instructions (Mannheim,
Indianapolis, Ind.). All hybridizations were done for 18 hours at
42.degree. C. without formamide. By assuming that 1% base-pair
mismatching results in a 1.degree. 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 defined as 90% or greater
homology, and low stringency is 80-85% sequence homology.
18TABLE 17 Hybridization of oligonucleotides with HindIII
restriction fragments of Rx1 DNA. Stringency Oligonucleotide Region
Low High LSM12 Leader N.D. 4.0, 9.1 LSM5 .alpha.-helix N.D. 4.0
LSM3 .alpha.-helix 4.0, 9.1 4.0 LSM4 .alpha.-helix 4.0 4.0 LSM7
Proline 4.0, 9.1 4.0, 9.1 LSM1 Proline 4.0, 9.1 4.0, 9.1 LSM10
Repeats N.D. 4.0, 9.1 LSM2 C-terminus 4.0, 9.1 4.0 Note. Values
indicated are the sizes of restriction fragments expressed as
kb.
[0375]
19TABLE 18 Summary of Hybridization at High and Low Stringency of 8
Oligonucleotides with HindIII Restriction Fragments of the 17
Pneumococcal Isolates Listed in FIG. 2 Percent with Percent with
Percent with 1 band/ Oligonucle- .gtoreq.1 band .gtoreq.2 bands 1
band .gtoreq.1 band otide Low High Low High Low High Low High LSM12
82 59 24 0.29 LSM5 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.
[0376]
20TABLE 19 Amplification of Pneumococcal Isolates using the
Indicated 5' Prime Combination with the 3' Primer LSM2 at the 3'
end of pspA Nucleotide Amplified/ Percent 5' Primer Region Position
Tested Amplified LSM8 -35 47 to 70 2/14 14 LSM12 leader 162 to 188
8/14 57 LSM3 .alpha.-helical 576 to 598 3/14 21 LSM7 proline 1093
to 1117 12/14 86 LSM1 proline 1312 to 1331 14/14 100 Note, by 2
.times. 5 chi square analysis the differnet primers 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.
[0377]
21TABLE 20 Size of amplified pspA fragments in kilobases Number
pspA Primer pspAs Region Pairs examined Size Range S.D. Full length
LSM12 + LSM2 9 1.9-2.3 0.4 0.17 .alpha.-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.
[0378]
22TABLE 21 Pneumococcal strains Strain Relevant characteristics WU2
Capsular type 3, PspA type 1 D39 Capsular type 2, PspA type 25 R36A
Nonencapsulated mutant of D39, PspA type 25 Rx1 Nonencapsulated
variant of R36A, PspA type 25 DBL5 Capsular type 5, PspA type 33
DBL6A 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 AC40 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 D109-1B Capsular type 23, PspA type 12 BG9709
Capsular type 9, PspA type 0 BG58C Capsular type 6A, PspA type ND
L81905 Capsular type 4, PspA type 25 L82233 Capsular type 14, PspA
type 0 L82006 Capsular type 1, PspA type 0
[0379]
23TABLE 22 PCR primers. Primer Sequence (5' to 3') LSM1
CCGGATCCAGCTCCTGCACCAAAAAC LSM2 GCGCGTCGACGGCTTAAACCCATTCACCATTGG
LSM3 CCGGATCCTGAGCCAGAGCAGTTGGCTG LSM4
CCGGATCCGCTCAAAGAGATTGATGAGTCTG LSM5
GCGGATCCCGTAGCCAGTCAGTCTAAAGCTG LSM6
CTGAGTCGACTGGAGTTTCTGGAGCTGGAGC LSM7
CCGGATCCAGCTCCAGCTCCAGAAACTCCAG ISM8
GCGGATCCTTGACCAATATTTACGGAGGAGGC LSM9 GTTTTTGGTGCAGGAGCTGG LSM10
GCTATGGCTACAGGTTG LSM11 CCACCTGTAGCCATAGC LSM12
CCGGATCCAGCGTGCCTATCTTAGGGGCTGGTT LSM13
GCAAGCTTATGATATAGAAATTTGTAAC
[0380]
24TABLE 23 Hybridization at high stringency of eight different PspA
probes with HindIII digests of 18 strains of Streptococcus
pneumoniae Strain Probe Rx1/D39 WU2 DBL5 DBL6A A66 AC94 AC17 AC40
AC107 FL- 4.0, 9.1 3.8 3.7, 5.8 3.0, 3.4 3.6, 4.3 3.6, 6.3 3.6, 6.3
3.2, 3.6 3.6, 6.3 Rx1 LSM12 4.0, 9.1 3.8 3.7, 5.8 3.0, 3.4 4.3 3.6,
6.3 3.2, 3.6 LSM5 4.0 3.6, 6.3 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 3.2, 3.6 LSM1 4.0, 9.1 3.8 3.7, 5.8 3.4
6.3 3.2 3.6 LSM10 4.0, 9.1 3.8 3.7 3.4 3.6, 4.3 3.6, 6.3 3.2 3.6,
6.3 10 LSM2 4.0 3.7 3.6 3.6 3.6, 6.3 Strain Probe AC100 AC140 DC109
BG9709 BG58C L81905 L82233 L82006 FL- 4.0, 8.0 3.0, 4.0 3.3, 4.7
2.2, 9.6 1.4, 3.2 3.6, 5.2 3.7, 4.3, 6.4 Rx1 3.6 8.2 LSM12 4.0, 8.0
4.0 3.3, 4.7 2.2, 9.6 1.4, 3.2, 3.6 1.3, 3.7 3.6 LSM5 2.2, 9.6 3.6
1.2, 2.3 3.6 LSM3 2.2 3.6 3.6 LSM4 2.2 3.6 3.6 3.7 LSM7 3.0, 4.0
3.3, 4.7 2.2, 9.6 3.6 2.3, 3.7 3.6 LSM1 4.0 4.0 2.2 5.2 LSM10 4.0
4.0 3.3, 4.7 2.2, 9.6 3.2, 3.6, 5.2 1.3, 3.7 4.3, 6.4 3.6 LSM2 4.0
3.0, 4.0 4.7 4.3 Note: All probes were tested versus HindIII
digests of all strains. If no bands are listed none were detected.
Strains Rx1 and D39 gave identical results and are shown in a
singel column. The full name of strain AC109 is AC109-1B
Example 6
Restriction Fragment Length Polymorphisms of pspA Reveals
Grouping
[0381] Pneumococcal surface A (PspA) is a protection eliciting
protein of Streptococcus pneumoniae. The deduced amino acid
sequence of PspA predicts three distinct domains; an .alpha.
helical coiled-coil region, followed by two adjacent proline-rich
regions, and ten 20 amino acid repeats. Almost all PspA molecules
are cross-reactive 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 Rx1 were used to amplify the full-length pspA gene and
the 5' portion of the gene including the .alpha.-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 family.
[0382] Numerous techniques have been employed in epidemiological
surveillance of pneumococci which include serotyping, ribotyping,
pulsed field electrophoresis, 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 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 most
serologically diverse protein known 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.
[0383] 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 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.
[0384] A less ambiguous typing system that takes advantage of the
diversity of PspA was therefore necessary to develop and was used
to examine the 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.
[0385] 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 length polymorphism (RFLP) were (outside
of 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
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.
[0386] Bacterial Strains
[0387] Derivatives of the S pneumoniae D39-Rx1 family were kindly
provided by Rob Massure and Sanford Lacks (FIG. 8). Eight clinical
isolates from Spain and four isolates from Hungary, a gift from
Alexander Tomasz. Seventy-five random clinical isolates from
Alabama, Sweden, Alaska, and Canada were also studied.
[0388] PCR Amplifications
[0389] 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. Amplification was accomplished in a 50 .mu.l
reaction containing approximately 50 ng template DNA, 0.25U Taq, 50
.mu.M of each primer, 175 .mu.M MgCl.sub.2, and 200 .mu.M dNTP in a
reaction buffer containing 10 .mu.M Tris-HCl, pH 9.0, 50 .mu.M KCl,
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 of an initial denaturation step at
94.degree. C., followed by 29 cycles of 94.degree. C. for 1 min,
55.degree. C. for 2 min, and 72.degree. C. for 3 min. The final
cycle included an incubation at 72.degree. for 5 min.
[0390] Restriction Fragment Analysis of PCR-Amplified Product
[0391] Aliquots of the PCR mixtures were digested with Hha I or
Sau3A I in a final volume of 20 .mu.l according to manufacturer's
protocols. After digestion the DNA fragments were electrophoresed
on a 1.3% TBE agarose gel and stained with et idium bromide.
Fragment sizes were estimated by comparison to a 1 kb DNA ladder
(Gibco BRL)
[0392] 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 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 25).
[0393] Stability of Amplified RFLP Pattern Within Clonally Related
Pneumococci
[0394] 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-Rx1 family. Rx1 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 into D39 by different laboratories (FIG. 8). During this
period unencapsulated strains were maintained in vitro, and D39 was
passed both in vivo and in vitro passage. All the derivatives of
D39, including Rx1, R6, RNC, and R36A, produced a 1.9 kb fragment
upon PCR 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 0.83, 0.58, 0.36 and a 0.27
kb fragment in all of the D39-RX1 derivatives of the family.
Digesting the full length pspA with Hha I resulted in bands which
were 0.76, 0.47, 0.39, 0.35, and 0.12 kb (FIG. 9 or Table 26).
[0395] The stability of pspA polymorphism was also investigated
using pneumococcal isolates which had 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 2.0 kb. After digesting full length pspA with
Hha I, four fragments were visualized, 89, 0.48, and 0.28 kb.
Digestion with Sau3A I yielded five fragments 0.880, 0.75, 0.35,
0.34, and 0.10 kb. Capsule type 6B pneumococcal strains, DB 1, DB2,
DB3, and DB4, were obtained from an outbreak in Spain. Full length
pspA from these strains were approximately 1.9 kb Digestion of the
PCR-amplified fragment with Hhs I resulted in four fragments which
were 0.83, 0.43, 0.33, and 0.28 kb. Sau3A I digestion yield a 0.88,
0.75, 0.34, and 0.10 kg fragments. DB6, DB8, 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 0.90, 0.52, 0.34, and 0.30 kb
and Sau3A I fragments were 0.75, 0.52, 0.39, 0.22, 0.20 and 0.10 kb
in size FIG. 10). DB7 had a 19A capsular serotype and was not
identical to DB6, DB8, and DB9. In the D39Rx1 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 expected if the amplified product represents a single pspA
sequence.
[0396] Diversity of RFLP Pattern of Amplified pspA from Random
Pneumococcal Isolates
[0397] PCR amplification of the pspA gene from 70 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 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 seemingly 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.
[0398] 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 of which restriction enzyme was used. The
subfamilies represent situations where two families share most but
not all the restriction fragments. With certain strains an RFLP
pattern was observed where detectable fragment size differed from
the pattern of the established family by less than 100 bp. Since
the 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 FIG. 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).
[0399] Previous DNA hybridization studies have demonstrated that
the pspA gene of different isolates are the most conserved in their
3' region of the gene and more variable in the 5' region of the
gene. Thus, it 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 amplify this fragment which is
approximately 1.6 kb. Examination of this region of pspA afforded
two things.
[0400] This primer pair permitted amplification of 90% 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 of the
gene
[0401] FIG. 12 contains the same strains which were examined in
FIG. 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 region confirms the original designated
families. However, these primers amplify a smaller portion of the
psaA and therefore the difference in the families is not as
dramatic as the RFLP patterns obtained from the RFLP pattern of the
full length gene.
[0402] 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 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 pattern is stable over long passages in
vitro.
[0403] These findings suggest that the population of 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 within a population. Further, these
findings enable techniques to diagnose S. pneumoniae via PCR or
hybridization by primers on probes to regions of pspA common within
groupings.
[0404] The sequence studies divide the known strains into several
families based on sequence homologies. 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 for analysis. "Families" or
"groupings" identified by the 5' half of the alpha-helical region,
the 3' half of the .alpha.-helical region and the proline rich
region are each distinct and differ slightly from each other In
addition there is considerable evidence of other diversify
(including base substitutions and deletions and insertions in the
sequences) among otherwise closely related molecules.
[0405] This result indicates that it is expected that there will be
a continuum of overlapping sequences of PspAs, rather than a
discrete set of sequences.
[0406] The findings indicate that there is the greatest
conservation of sequence in 3' half of the .alpha.-helical region
and in the immediate 5' tip. Because the diversity in the mid-half
of the .alpha.-helical region is 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
alpha-helical region and the 5' tip of the coding sequence are
likely to the critical sequences for predicting PspA
cross-reactions and vaccine composition.
[0407] 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 conserved. The reason for this is
presumably because antibodies to epitopes in this region are not
surface exposed.
[0408] Based on our present sequences of 27 diverse pspAs we have
found that there are 4 families of the 3' half of the
.alpha.-helical region and 2-3 families of the very 5' tip of the
.alpha.-helical region. Together these form 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.
25TABLE 29 Relationship of Capsular type and RFLP family.
RELATIONSHIP BETWEEN CAPSULAR TYPE AND RFLP FAMILY pspA Capsule
Type family 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 A 3 B 1 1 C 2 1 2 2 1 D 1 1 DD 2 E 1 2 1 F 1 1 3
1 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 2 1 1 1 2 2 1
K 1 1 1 KK 1 1 1 1 1 L 1 1 M 1 1 MM 1
[0409]
26TABLE 24 Oligonucleotides used in this study. Nucleotide
Designation Sequence 5'-3' position LSM2 GCG CGT CGA CGG CTT 1990
to 1967 (SEQ ID NO:18) AAA CCC ATT CAC CAT TGG LSM1 CCG GAT CCA GCT
CCT 1312 to 1331 (SEQ ID NO:19) GCA CCA AAA AC LSM13 GCA AGC TTA
TGA TAT 1 to 26 (SEQ ID NO:20) AgA AAT TTG TAA C SKH2 CCA CAT ACC
GTT TTC 1333 to 1355 (SEQ ID NO:21) TTG TTT CCA GCC
[0410]
27TABLE 25 Amplification of pspA from a panel of 72 independent
isolates* of S. pneumoniae. LSM13 AND LSM13 AND NUMBER OF LSM2 SKH2
CAPSULE STRAINS % OF STRAINS % OF STRAINS TYPE EXAMINED AMPLIFIED
AMPLIFIED 1 3 100 100 2 1 100 100 3 8 50 87 4 6 67 100 5 1 100 100
6 7 29 86 6A 2 100 100 6B 6 100 100 7 2 50 100 8 1 100 100 9V 3 100
100 9A 2 100 100 9L 1 100 100 9N 3 100 100 10 1 100 100 11 2 50 100
12 2 0 100 13 1 100 100 14 4 0 75 15 2 50 50 19 5 100 100 22 3 33
100 23 1 100 100 33 1 0 100 35 1 0 100 nd 3 100 100 *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 one representative isolate from
such clonal groups.
[0411]
28TABLE 36 Rx1-D39 derivatives SIZE OF Hha I DIGESTS SIZE OF Sau3AI
ISOLATE (Kb) DIGESTS (Kb) D39 .76, .47, .39, .35, .12 .83, .58,
.36, .27 Rx1 .76, .47, .39, .35, .12 .83, .58, .36, .27 R800 .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, .36, .27
R6X .76, .47, .39, .35, .12 .83, .58, .36, .27 R36NC .76, .47, .39,
.35, .12 .83, .58, .36, .27 R36A .76, .47, .39, .35, .12 .83, .58,
.36, .27
[0412]
29TABLE 27 Strain Information and family designation of independent
isolates- PspA SIZE OF Hha I SIZE OF Sau3AI STRAIN CAPSULE TYPE
TYPE FAMILY FRAGMENTS FRAGMENTS BG9163 6B 21 C 1.55, .35 1.05, .35,
.22 EF6796 6A 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, .35
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, .35
BG7941 11 ND F 1.3, .40, .29, .10 .82, .76, .35 BG7813 14 8 H 1.05,
.70, .36 .90, .77, .35 BG7736 8 ND H 1.05, .70, .36 .90, .77, .35
AC113 9A ND I 1.4, .34, .28 1.2, .80 AC99 9V 5 I 1.4, .34, .28 1.2,
.80
[0413]
30TABLE 28 Relationship of RFLP family and PspA type. RELATIONSHIP
BETWEEN PSPA TYPE AND RFLP FAMILY pspA PspA Type FAMILY 0 1 3 5 8
12 13 16 18 19 20 21 24 25 26 30 33 34 36 37 ND A 1 1 B 1 1 1 C 2 1
1 4 D 1 1 DD 2 E 1 1 1 F 1 1 4 FF 1 3 G 1 1 H 1 1 1 1 5 I 3 1 2 2 1
II 1 J 4 1 1 1 3 K 1 1 1 KK 1 1 3 L 1 1 M 1 1 1 1 MM 1
Example 7
Ability of PspA Immunogens to Protect Against Individual Challenge
Strains
[0414] CBA/N or BALB cJ mice were given 1 injection of 0.5-.mu.g
PspA in CPA, followed 2 weeks later by a boost in saline, and
challenged between 7 and 14 (average 10) 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 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 10.sup.3 to
10.sup.4 pneumococci in inoculum, but in all cases the challenge
was at least 100 times LD.sub.50.
[0415] The results are shown in the following Tables 30 to 60, and
the conclusions set forth therein.
[0416] From the data, it appears that an antigenic, immunological
or vaccine composition can contain any two to seven, preferably
three to five PspA, e.g., 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
than did R36a/Rx1/D39, and that also surprisingly PspA from WU2
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
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 the tests. Similarly, one can select PspAs on the basis
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.
[0417] A note about use of medians rather than averages.
[0418] 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 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.
31TABLE 30 Relative ability of different PspAs to Protect against
each challenge strains of S. pneumoniae (Summary of statistically
significant protection) Vaccine PspA R36A, JD908/ JS1020/ JS5010.3
JS3020 All best Challenge Caps PspA pspA Rx1, D39 WU2 BG9739 EF3296
EF5668 L81905 DBL5 DBL6A immune protect Strain type type family K a
b E DD b II D -- -- D39 2 25 K ++ +++ + ++ +++ WU2 3 1 a +++ +++
+++ +++ +++ +++ +++ +++ +++ A66 3 13 a +++ +++ +++ +++ +++ +++
+.+-. +++ +++ EF10197 3 18 M +++ +++ +++ +++ ATCC6303 3 7 a +++ +++
+++ BG9739 4 26 b + +++ + 0+ 0 +.+-. 0 0 ++ +++ EF3296 4 20 E +.+-.
+.+-. 0+ 0 0 0 +.+-. EF5668 4 12 DD + 0 +++ 0+ +++ 0+ + 0+ ++ +++
L81905 4 23 b + + ++ ++ 0 + +.+-. +.+-. ++ ++ DBL5 5 33 II + + + +
++ 0 ++ ++ EF6796 6A 1 C +++ +++ +++ DBL6A 6A 19 D +++ +.+-. ++ +=
+++ +.+-. +.+-. +++ ++ +++ BG9163 6B 21 C +++ +++ +++ +++ BG7322 6B
24 C +++ +++ +.+-. 0 +++ +.+-. +++ +.+-. +++ +++ Note: Empty cells
indicate that no experiment has been done. Bold means significant
at P < 0.05, Small font bold (+) means 0.02 .ltoreq. P <
0.05. Large font bold means P < 0.02. For this table statistical
significance refers to delay in time to death except as indicate in
the (+) footnote below. When "all immune" showed significant
protection against death but individual data cells did not, the
result for # "all immune" is presented under best protection on the
assumption that if more mice were done in each data cell one or
more of them would have exhibited significant protection against
death. +++ = statistically significant protection against death;
.gtoreq.50% protection from death ++ = statistically significant
protection against death; <50% protection from death +.+-. =
statistically significant delay in death; .gtoreq.20 protection
from death + = statistically significant delay in death; <20
protection from death, (or significant protection against death but
not a significant delay in death) 0++ = Not statistically delay in
time to death; but .gtoreq.50% protection from death 0+ = Not
statistically delay in time to death; but >1.5 day extension in
median time to death or .gtoreq.20% protection from death. 0 = No
apparent extension in time to death or protection from death.
[0419]
32TABLE 31 Relative ability of different PspAs to Protect against
each challenge strains of S. pneumoniae (Expressed as Median days
Alive post challenge) Vaccine PspA R36A, JD908 JS1010/ JS5010.3
JS3020 All All Challenge Caps PspA pspA Rx1, D39 WU2 BG9739 EF3296
EF5668 L81905 DBL5 DBL6A immune control Strain type type family K a
b E DD b II D -- -- D39 2 25 K 4.5 >21 4 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 18 M
>21 >21 >21 2 ATCC6303 3 7 a >21 >21 5 BG9739 4 26 b
3 >21 6 3 3 5, 13 2 2 3 2 EF3296 4 20 E 5 5 4.5 2 2 3 2 EF5668 4
12 DD 6 2 >21 13 >21 4 >21 5 8 3 L81905 4 23 b 5 5 8 6 3 5
3 3.5 5 2 DBL5 5 33 II 4 3 3 3.5 6 2 3.5 2 EF6796 6A 1 C >21
>21 1 DBL6A 6A 19 D >21 8.5 13 9 >21 8 12 >21 12.5 5.5
BG9163 6B 21 C >21 >21 >21 8.5 BG7322 6B 24 C >21
>21 14.5 6 >21 12.5 >21 11 >21 7 Note: Bold denotes
statistically significant extension of life at P < 0.05. Small
font denotes 0.02 .ltoreq. P < 0.05; large font denotes P <
0.02. Median times to death indicated was 8, >21, are situations
where the medium 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 calculated median.
Fractional values such as 3.5, indicate that the median is halfway
# between two numbers, in this case 3 and 4. As indicated in the
original data (S103B), some experiments were terminated prior to 21
days post infection. There is little reason to assume, however,
that results would have been significantly effected by the early
terminations since very few mice infected with the strains used in
those studies, have ever been observed to die later than 10 or 15
days post challenge. For statistical purposes all mice alive at the
# end of experiments were assumed to have been completely
protected, and for the sake of calculations all surviving mice were
assigned values of >21.
[0420]
33TABLE 32 Ability of different PspAs to Protect Against Each
Challenge strain of S. pneumoniae (Expressed as increase in
survival time in days) (A denotes .gtoreq.50% immune mice alive)
Vaccine PspA R36A, JD908 JS1020/ JS5010.3 JS3020 Challenge Caps
PspA pspA Rx1, D39 WU2 BG9739 EF3296 EF5668 L81905 DBL5 DBL6A All
Best Strain type type family K a b E DD b II D immune Result D39 2
25 K 2.5 A 2 3 A WU2 3 1 a A A A A A A A A A A66 3 13 a A A A A A A
2 A A EF10197 3 18 M A A A A ATCC6303 3 7 a A A A BG9739 4 26 b 1 A
4 1 1 3, 11 0 0 1 A 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 L81905 4 23 b 3 3 6 4 1 3 1 1.5 3 6 DBL5 5 33
II 2 1 1 1.5 4 0 1.5 4 EF6796 6A 1 C A A A DBL6A 6A 19 D A 3 7.5
3.5 A 2.5 6.5 A 7 A BG9163 6B 21 C A A A A BG7322 6B 24 C A A 7.5
-1 A 5.5 A 4 A A R36A WU2 BG9739 EF3296 EF5668 L81905 DBL5 DBL6A
All Best Note: Bold denotes statistically significant extension of
life at P < 0.05. Small font denotes 0.02 .ltoreq. P < 0.05;
large font denotes P < 0.02. Median increases in survival listed
as 3, 9 or 1, A denote groups where the median does not fall within
a continuum of values. In these cases the values given are those
closest to the calculated median. Fractional values such as 3.5,
indicate that the median is halfway between two numbers, in this
case 3 and 4.
[0421]
34TABLE 33 Relative ability of different PspAs to Protect against
each challenge strain of S. pneumoniae (expressed % alive at 21
days post challenge) Vaccine PspA R36A, JD908 JS1020/ JS5010.3
JS3020 Challenge Caps PspA pspA Rx1, D39 WU2 BG9739 EF3296 EF5668
L81905 DBL5 DBL6A All immune Strain type type family K a b E DD b
II D -- All control D39 2 25 K 38 60 30 38 3 WU2 3 1 a 100 100 100
100 100 100 100 100 1.5 A66 3 13 a 75 100 80 75 100 60 20 76 5
EF10197 3 18 M 100 80 90 0 ATCC6303 3 7 a 100 100 0 BG9739 4 26 b
11 60 13 25 0 25 0 0 12 0 EF3296 4 20 E 25 20 10 0 0 8 0 EF5668 4
12 DD 22 25 60 40 100 40 60 0 41 9 L81905 4 23 b 10 0 31 40 0 0 14
0 14 0 DBL5 5 33 II 10 14 0 0 29 0 4 0 EF6796 6A 1 C 100 100 0
DBL6A 6A 19 D 67 25 33 0 60 25 0 80 35 4 BG9163 6B 21 C 89 80 86 20
BG7322 6B 24 C 100 60 25 0 89 25 80 25 55 6 Bold, denotes
statistically significant protection against death at P < 0.05.
Bold small font, indicates significant protection against death at
0.02 .ltoreq. P < 0.05. Bold large font, indicates significant
protection against death at P < 0.02.
[0422]
35TABLE 34 Relative ability of different PspAs to Protect against
each challenge strain of S. pneumoniae (% protected from death at
21 days post challenge) Vaccine PspA R36A, WU2 BG9739 DBL5 DBL6A
Challenge Caps PspA pspA Rx1, D39 JD908 JS1020 EF3296 EF5668 L81905
JS5010.3 JS3020 All immune Best Strain type type family K a b E DD
b II D -- result 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 80 90 100 ATCC6303 3 7 a 100 100 100 BG9739 4 26
b 11 60 13 25 0 25 0 0 12 60 EF3296 4 20 E 25 20 10 0 0 8 25 EF5668
4 12 DD 14 18 56 34 100 34 56 -10 35 100 L81905 4 23 b 10 0 31 40 0
0 14 0 14 40 DBL5 5 33 II 10 14 0 0 29 0 4 29 EF6796 6A 1 C 100 100
100 DBL6A 6A 19 D 66 22 30 -4 58 22 -4 79 33 79 BG9163 6B 21 C 86
75 83 86 BG7322 6B 24 C 100 57 22 0 88 22 79 22 52 100 Bold,
denotes statistically significant protection against death at P
< 0.05. Bold small font, indicates significant protection
against death at 0.02 .ltoreq. P < 0.05. Bold large font,
indicates significant protection against death at P < 0.02. %
protected has been corrected for any survivors in the control mice.
% protected = 100 .times. (% alive in immune - % alive in
control)/(100 - % alive in control). Thus, if there were any mice
alive in the control animals, the calculated "% protected" is less
than the observed "% alive" listed in the # previous table. The
only exceptions to this are if 100% of immunized mice lived.
Negative numbers mean that less immunized mice lived than did
control mice. #Please note that none of these negative numbers are
significant even though we are using a one tailed test.
[0423]
36TABLE 35 Recommended Immunoge_Protection against the indicated
challenge strains of S. pneumoniae Based on Protection Score Based
on median days alive and percent protected (numbers refer to
preference as a vaccine strain with respect to the indicated
challenge strain, 1 = best) Vaccine PspA R36A, WU2 BG9739 DBL5
DBL6A Challenge Caps PspA pspA Rx1, D39 JD908 JS1020 EF3296 EF5668
L81905 JS5010.3 JS3020 Strain type type family K a b E DD b II D
D39 2 25 K 2 1 3 WU2 3 1 a 1 1 1 1 1 1 1 A66 3 13 a 2 1 2 2 1 3 0
EF10197 3 18 M 1 2 ATCC6303 3 7 a 1 BG9739 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 L81905 4 23
b 2 0 1 1 0 0 0 0 DBL5 5 33 II 2 3 0 3 1 0 EF6796 6A 1 C 1 DBL6A 6A
19 D 2 0 3 0 2 0 0 1 BG9163 6B 21 C 1 1 BG7322 6B 24 C 1 2 3 1 3 1
3 Number of #1's 7 5 3 1 3 2 3 2 Bold, denotes statistically
significant protection against death at P < 0.05. Where more
than one PspA were equally protective, the same values were given
to each. Recommendations are based on days to death with %
protection dividing ties, especially among those where greater than
50% of mice lived to 21 days. "0" indicates test were conducted but
compared to the other PspAs this one is not recommended.
Conclusions: Statistically significant protection against death
with >50% protection; 11/14 of the strains = 79% Statistically
significant protection against death; 13/14 strains = 93%
Statistically significant extension of life in 14/14 or 100% of
strains.
[0424]
37TABLE 36 Best Choice for Vaccine Components as of 95/8/27 Vaccine
Component (cumulative strains protected) % maximally protected
Criterion 1 2 3 4 5 6 .gtoreq.#1 PspA for R36A WU2 BG9739* EF5668
DBL5 DBL6A each challenge (7) (10) (11) (12) (13) (14) strain 50%
71% 79% 86% 93% 100% .gtoreq.#2 PspA for R36A BG9739 each challenge
(12) (12) strain 86% 100% Max score R36A WU2 BG9739 DBL5 (+) type
(9) (11) (13) (14) score 64% 79% 92% 100% Max R36A WU2 BG9739 DBL5
increase in (9) (11) (13) (14) Days alive 64% 79% 92% 100% %
protected R36A WU2 DBL5 EF5668 DBL6A EF3296 (7) (10) (11) (12) (13)
(14) 50% 64% 79% 86% 92% 100% Theoretical R36A BG9739 DBL5 EF3296
mixture based (10) (12) (13) (14) on a few 64% 86% 92% 100%
testable assumptions (see below) *This is not a unique combination.
See table 37 below.
[0425]
38TABLE 37 Combinations where all Challenge Strains have a Vaccine
strain with a score of .gtoreq. #2 Number of Total PspAs in Number
of #1s Combination Combination #1 strains Total #1s and #2s 2 R36A
+ BG9739 8 10 20 3 R36A + BG9739 + WU2 11 15 25 3 R36A + WU2 + DBL5
11 15 21 3 R36A + WU2 + EF5668 11 15 23 3 R36A + WU2 + DBL5 11 15
22
[0426]
39TABLE 38 Pooled Data for Protection against D39 by various PspAs;
Days alive for each mouse Log Days to Death/immunogen CFU Rx1/R36A
JD908 Exp. D39 Mice D39 (WU2) EF5668 All Immune control 143 4.5
CBA/N 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 BALB/c 3, 3x > 21 2, 2, 2, 4 BCG E143 3.0
CBA/N 2, 6, 3x > 10 3, 3, 3, 5, 5 E140 2.81 CBA/N 4, 4, 5, 7, 15
2, 2, 2 BC100 E169 2.7 CBA/N 2, 4x > 21 2, 5, 3x > 21 1, 2,
2, 2, 3 E154 2.6 CBA/N 2, 2, 3, 2x > 21 4x 2, 5, > 21 All
.ltoreq. 2, 3, 3, 3, 4, 4, 4, 1, 1, 2, 2, 2 4x 1, 6x 2, 3.0 5, 7,
15 3, 3, 4 All 4x 2, 5x 3, 2, 5, 3x > 21 1, 1, 2, 2, 2, 2, 6 1,
1, 9x 2, 5x 1, 16x 2, 3x 4, 5, 7, 3x > 21 5x 3, 3x 4, 6x 3, 4,
4, 15, 9x > 21 5, 5, 6, 7, 15, 5, 5, 5, > 21 15x > 21
[0427]
40TABLE 39 Pooled Data for Protection against D39 by various PspAs
Median Days Alive & alive:dead with corresponding P values. Log
Rx1/R36A JD908 All CFU D39 (WU2) EF5668 Immune Control Exp.1 D39
Mice med a:d med a:d med a:d med a:d med a:d 143 4.5 CBA/N 2 0:5 2
0:5 n.s. E145 4.0 CBA/N 3 0:5 2 0:5 n.s. E028 BCG 5.93 BALB/c
>21.029 3:1 2 0:4 n.s. E143 3.0 CBA/N >21 3:2 3 0:5 n.s. n.s.
E140 BC100 2.81 CBA/N 50.018 0:5 2 0:3 E169 2.7 CBA/N >21.016
4:1.024 >21 3:2 2 0:5 E154 2.6 CBA/N 3 2:3 2 1:5 n.s. n.s. All
.ltoreq. 3.0 4.0008 0:10 2 0:5 2 0:13 n.s. All 4.50057 9:15.001
>21.006 3:2.0045 4 3:7.034 5.0001 15:24.0002 2 1:32 ++ +++ (2,
6) ++ n.s. ++ +++ + ++ % alive 38 60 30 38 3 36 59 28 36 Rx1/D39
WU2 EF5668 All immune controls
[0428]
41TABLE 40 Pooled Data for Protection against WU2. by various PspAs
Days to Death/immunogen CFU Rx1 JD108 JS1020 BG9739 L81905 DBL5
JS3020 Exp. WU2 Mice FL-R36A BC100 (WU2) (BG9739) bc100 EF5668
bc100 bc100 (DBL6A) control Dr. Ed, expt +++ lots of prior expts.
+++ E012 3.0 CBA/N 15x 21 1, 1, 11x 2, 7x 3, 4 E028 6.01 BALB/c 4x
> 21 4, 6, 6, >21 0.05/n.s. E084 3.75.sup.1 CBA/N 3x > 15
1, 2, 2, 2, 3, 3, >15 E125 3.57 CBA/N 4x > 21 4x > 21 4x
> 21 2, 2, 3, 3, 3, bc100 >21 E129 3.18 CBA/N 5x > 23 2,
2, 2, 2, 3 E140 3.43 CBA/N 4x > 21 1, 5x 2, 3, 4 BC100 E143 3.0
CBA/N 8x > 10 1, 1, 2, 2, 2, 3 E144 3.9 CBA/N 5x > 21 5x 2
E172 3.98 CBA/N 5x > 21 5x 3 All 19x > 21 4x > 21 5x >
21 8x > 21 4x > 21 8x > 21 4x > 21 4x > 21 5x >
21 6x 1, 33x 2, 20x 3, 4, 4, 4, 6, 6, > 21 All Immune 61 >
21
[0429]
42TABLE 41 Pooled Data for Protection against WU2 by various PspAs
Median days Alive Alive:Dead P value based on Alive:Dead P value
calculated compared to pooled controls (in this case 65 control
mice) CFU Rx1 JD108 JS1020 BG9739 L81905 DBL5 JS3020 Exp. WU2 Mice
FL-R36A BC100 (WU2) (BG9739) bc100 EF5668 bc100 bc100 (DBL6A)
control Dr. Ed, expt +++ lots of prior expts. +++ E012 .sup.-3.0
CBA/N >21 1, 1, 11x 2, 15:0 7x 3, 4 E028 6.01 BALB/c 4x > 21
4, 6, 6, >21 E084 3.75.sup.1 CBA/N 3x > 15 1, 2, 2, 2, 3, 3,
>15 E125 3.57 CBA/N 4x > 21 4x > 21 4x > 21 2, 2, 3, 3,
3, bc100 >21 E129 3.18 CBA/N 5x > 23 2, 2, 2, 2, 3 E140 3.43
CBA/N 4x > 21 1, 5x 2, 3, 4 BC100 E143 3.0 CBA/N 8x > 10 1,
1, 2, 2, 2, 3 E144 3.9 CBA/N 5x > 21 5x 2 E172 3.98 CBA/N 5x
> 21 5x 3 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 FL-R36A Rx1 JD108 JS1020 BG9739
EF5668 L81905 DBL5 JS3020 control BC100 (WU2) (BG9739) bcc100 bc100
bc100 (DBL6A) P value P value WU2 median days based on days based
on % Challenge days of death of death _aq to death alive:dead Score
alive prot. All 61x > 21 >21 61:0 <.0001 <.0001 +++ 100
100 immune All 6x 1, 33x 2, 20x 2 1:64 2 2 controls 3, 4, 4, 4, 6,
6, >21
[0430]
43TABLE 42 Pooled Data for Protection against A66. by various PspAs
Days to Death/immunogen JS5010.3 CFU FL-R36A/ Rx1 JD908 JS1020
BG9739 L81905 L1905 FL DBL5 JS3020 Exp. A66 Mice D39 BC100 (WU2)
(BG9739) bc100 EF5668 FL bc100 (DBL5) bc100 (DBL6A) control 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 3x 2, 3, bc100 6, 6, >21 E104 3.0
CBA/N 2, 8, 3, 4, 4, 2, 4, 4, 2, 2, 2, 3x > 22 2x > 22 5,
> 22 2, 3 E143 3.0 CBA/N 4, 2, 2, 3, 4x > 10 E140 3.43 CBA/N
4x > 21 1, 1, 1 E172 3.94 CBA/N 5x > 21 E145 3.97 CBA/N 13,
1, 2, 2, 4x > 21 2, 4 E121 4.16 CBA/N 3x 3, 2x 4, 1, 8x 2, 5x
> 21 > 21 All 3x 3, 4x > 21 5x > 21 2, 8, 4x > 21 4,
5x > 21 4x > 21 3, 4, 4, 4x > 21 2, 4, 4, 7x 1, 2x 4, 13,
3x > 21 4x > 21 2x > 21 5, > 21 22x 2, 14x > 21 3x
3, 4, 3x 6, 2x > 21 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 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 <0.0001 0.0075 <0.0001 0.006 <0.0001 0.0001 n.s.
0.0001 n.s. DBL5 Mini Pools R36A/Rx1/WG44.1 JD908 BG9739 EF5668
L81905 3, 4, 4, 4, 6x > 21 DBL6A Control >21 >21 >21
>21 >21 >21 4 2 18:6 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. Score +++ +++ +++ +++ +++ +++ +.+-. %
alive 72 100 80 75 100 60 20 5 71 100 79 74 100 58 16 0 A66
R36A/Rx1/WG44.1 JD908 BG9739 EF5668 L81905 DBL5 DBL6A A66 challenge
days of death median days alive alive:dead P-days to death
P-alive:dead Score % alive % protected All immune 2, 2, 4x 3, 7x 4,
5, >21 50:16 <0.0001 <0.0001 +++ 76 75 8, 13, 50x > 21
All controls 7x 1, 22x 2, 3x 3, 2 2:36 5 0 4, 3x 6, 2x > 21
[0431]
44TABLE 43 Pooled Data for Protection against EF10197. by various
PspAs CFU Days to Death/immunogen EF Rx1 JS1020 JS3020 JS5010.3 FL
Exp. 10197 Mice BC100 (BG9739) L81905 (DBL6A) EF5668 (DBL5)0
control E140 3.00 CBA/N 5x > 21 2, 2, 2 MI BCG 2.70 CBA/N * 2,
2, 2, 2, 2 E129 3.34 CBA/N 8, 4x > 23 2, 2, 2, 2, 9 *This was a
passive protection study. Its controls have been included to
increase the numbers of control mice.
[0432]
45TABLE 44 Pool of Pools for protection agaisnt EF10197 Group Delay
in death and/or survival Survival line Description days to death
(medain) P values etc. alive:dead P values etc. 1a Rx1 (E140) 5x
> 21 0.017 vs 1b 5:0 0.018 vs 1b 0.0013 vs 4b 0.008 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 1b Rx1
controls (E140) 2, 2, 2 0:3 2b MI BCG 2, 2, 2, 2, 2 0:5 3b JS1020
cont. (E129) 2, 2, 2, 2, 9 0:5 4b all controls (without MI BCG) 2,
2, 2, 2, 2, 2, 2, 9 0:8
[0433]
46TABLE 45 Summary 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 -- -- -- *+++ = statistically
significant protection against death with .gtoreq.50%
protected.
[0434]
47TABLE 46 Pooled Data for Protection against ATCC6303. by various
PspAs CFU Days to Death/immunogen ATCC Rx1 JS1020 JS3020 JS5010.3
FL Exp. 6303 Mice BC100 (BG9739) L81905 (DBL6A) EF5668 (DBL5)0
control E140 2.30 CBA/N 5x > 21 4, 4x 5 E129 3.80 CBA/N n.v.
[0435]
48TABLE 47 Pool of Pools for protection agaisnt ATCC6303 Group
Delay in death and/or survival Survival line Description days to
death (medain) P values etc. alive:dead P values etc. 1a Rx1 (E140)
5x > 21 (>21) 0.0040 5:0 0.004 1b RX1 controls (E140) 4, 4x 5
5 -- 0:5 --
[0436]
49TABLE 48 Summary of protection against ATCC6303 Immunogen
alive:dead % alive % protected median DOD P time alive P alive:dead
Score* Rx1 5:0 100 100 >21 0.004 0.004 +++ Rx1 controls 0:5 0 0
5 -- -- -- *+++ = statistically significant protection against
death with .gtoreq.50% protected.
[0437]
50TABLE 49 Pooled Data for Protection against BG9739. by various FL
PspAs Days to Death/immunogen JS5010.3 CFU R36A BC100 JD908 JS1020
bc100 EF3296 EF5668 bc100 FL bc100 JS3020 Exp. BG9739 Mice FL (Rx1)
(WU2) (BG9739) (BG9739) FL FL (L81905) (DBL5) (DBL5) (DBL6A)
control E140 2.76 CBA/N 3, 3, 2, 2, 3 10, 11 E104 2.89 Xid 6, 6, 7,
2, 2, 2, 2, 2, 2, 2, 2, 3, 8, 8 3, 4 2, 3 5, 5 E125 3.56 CBA/N 5,
5, 4, 5, 13, 2, 2, 3, 3, 4, 5, 7 >21 2, 4 4, 5, 6 E172 3.71
CBA/N 6, 7, 3, 4, 6, 3x > 21 6, 7 E124 3.76 Xid 2, 2, 2, 2, 2,
2, 2, 2, 2, 2, 3 2, 9 2, 2 E084 4.05 BALB/c 4x 2, 9x 2 2x > 14
E144 4.09 Xid 2, 3, 2, 3, 3, 2, 3, 3, 2, 2, 2, 3, 6, 7, >10 3, 4
3, 3 >21 All 2, 3, 3, 3, 6, 7, 4x 2, 6, 6, 5, 5, 2, 3, 2, 3, 3,
7x 2, 3, 8x 2, 21x 2, 3, 6, 10, 11 3x > 21 7, 8, 8, 5, 7 3, 7 3,
4 3, 4 3, 9 7x 3, >21 2x > 21 >21 4, 3x 5, 3x 6, 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:d
[0438]
51TABLE 50 Pooled Data for Protection against BG9739. by bc100s and
FL PspAs Days to Death/immunogen JS5010.3 CFU R36A BC100 JD908
JS1020 bc100 EF3296 EF5668 bc100 FL bc100 JS3020 con- Exp. BG9739
Mice FL (Rx1) (WU2) (BG9739) (BG9739) FL FL (L81905) (DBL5) (DBL5)
(DBL6A) trol E140 2.76 CBA/ 3, 3, 2, 2, 3 N 10, 11 E104 2.89 Xid 6,
6, 7, 2, 2, 2, 2, 2, 2, 2, 2, 3, 8, 8 3, 4 2, 3 5, 5 E125 3.56 CBA/
5, 5, 4, 5, 13, 2, 2, 3, 3, 4, N 5, 7 >21 2, 4 4, 5, 6 E172 3.71
CBA/ 6, 7, 3, 4, 6, N 3x > 21 6, 7 E124 3.76 Xid 2, 2, 2, 2, 2,
2, 2, 2, 2, 2, 3 2, 9 2, 2 E084 4.05 BALB/ 4x2, 9x 2 c 2x > 14
E144 4.09 Xid 2, 3, 2, 3, 2, 3, 3, 2, 2, 2, 3, 6, 3, 7, 3, 4 3, 3
>21 >10 FL + bc100 BG9739 R36A/Rx1/D39 WU2 BG9739 EF3296
EF5668 L81905 DBL5 DBL6A Cont. All 2, 4x 3, 6, 10, 6, 7, 4x 2, 3x
5, 2x 6, 2, 3, 2, 3x 3, 4, 5, 13, 10x 2, 3, 3, 4, 4 8x 2, 3, 9 21x
2, 11, >21 3x > 21 2x 7, 2x 8, 2x > 21 3, 7 4 >21 7x 3,
3x >21 4, 3x 5, 3x 6, 7 median days alive 3 >21 6 3 3 5, 13 2
2 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 <0.0001 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 % alive 11 60 13 25 0 25 0 0 0 % protected 11 60 13
25 0 25 0 0 0 BG9739 challenge R36A/Rx1/D39 WU2 BG9739 EF3296
EF5668 L81905 DBL5 DBL6A Cont. P value P value based based median
on on days of days of alive: days to alive: % BG9739 death death
dead death dead Score % alive protected All immune 3 8:59 0.009
0.023 ++ 12 12 All controls 2 0:38
[0439]
52TABLE 51 Pooled Data for Protection against EF3296. by various
PspAs Days to Death/immunogen CFU Rx1 JD908 JS1020 JS5010.3 FL
JS3020 Exp. EF3296 Mice BC100 WU2 (BG9739) (DBL5) (DBL6A) control
E84.sup.1 3.99 BALB/c 4x 2, >14 9x 2 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, 4x 6 All 3, 4, 6, >21 3, 3, 5, 5,
>21 4x 2, 4, 1, 1, 5x 2, 3, 3 1, 1, 5x 2, 3, 1, 1, 15x 2, 3x 5,
6, >21 4, 5 5x 3 4, 4x 6 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 0 % alive 25 20 10 0 0 0 % prot. 25 20 10 0
0 0 Best EF3296 Rx1 JD908 JS1020 JS5010.3 FL JS3020 control
challenge BC100 WU2 (BG9739) (DBL5) (DBL6A) EF3296 median P - days
P - alive: % % challenge days alive alive:dead to death dead Score
alive prot All immune 3 3:35 n.s. n.s. 0 8 8 All control 2 0:27
[0440]
53TABLE 52 Pooled Data for Protection against EF5668. by various
FL-PspAs and bc100s Days to Death/immunogen JS5010.3 CFU Rx1 JD908
JS1020 FL JS3020 Exp. EF5668 Mice R36A BC100 (WU2) (BG9739) EF3296
EF5668 L81905 DBL5 DBL6A control E143 3.0 CBA/N 5x > 10 1, 1, 2,
2, >10 E140 3.59 CBA/N 4, 6, 12, 2, 4, 6 >21 E171 3.69 CBA/N
2, 2, 2, 3, 3, 3, 4, 1, 3, 6, 6, >21 2x > 21 7 E124 3.90
CBA/N 3, 3, 3x > 15 3, 4, 5, 6, 6 3, 3, 3, 4, 9 E145 3.94 CBA/N
3, 4, 4, 2, 10, 2, 4, 13, 2, 3, 3, 4, 16, >19 3x > 19 2x >
19 >21 Pool 3, 3x 4, 6, 12, 16, 2, 2, 2, 3, 2, 10, 2, 4, 13, 5x
> 21 3, 3, 4, 3, 3, 3x > 3, 4, 5, 6, 6 3x 1, 4x 2, 2x > 21
>21 3x > 21 2x > 21 2x > 21 21 6x 3, 3x 4, 3x 6, 7, 9,
2x > 21 median days alive 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 - days alive
0.013 n.s. 0.0187 n.s. 0.001 n.s. n.s. n.s. P - alive:dead n.s.
n.s. 0.027 n.s. 0.0002 n.s. 0.027 n.s. Score + 0 +++ 0+ +++ 0+ + 0+
% alive 22 25 60 40 100 40 60 0 9 % prot 14 18 56 34 100 34 56 -10
9 EF5668 R36A/Rx1/D39 WU2 BG9739 EF3296 EF5668 L81905 DBL5 DBL6A
control Summary of protection against EF6796 Immunogen alive:dead %
alive % protected median DOD P-time alive P alive vs dead Rx1 4:0
100 100 >21 0.029 0.029 controls 0:3 0 0 1 -- -- +++ =
statistically significant protection from death with .gtoreq.50%
protected;
[0441]
54TABLE 53 Pooled Data for Protection against DBL6A. by various FL
PspAs and bc100 PspAs Days to Death/immunogen CFU BC100 JD908
JS1020 bc100 L81905 bc100 JS5010.3 bc100 JS3020 Exp. DBL6A Mice Rx1
R36A WU2 BG9739 BG9739 EF3296 EF5668 FL L81905 DBL5 DBL5 DBL6A
control E171 2.69 CBA/N 6, 7, 8, 3, 3, 7, 2, 3, 4, 9, >21 9,
>21 6, 6 E152 3.24 CBA/N 15, 7, 16, 8, 10, 3x 3, 4, 3x > 21
2x > 21 13, 21 3x 6 E140 3.25 CBA/N 4x > 21 4, 7, 7 E146 3.57
CBA/N 7, 8, 6, 8, 9, 10, 13, 7, 8, 12, 9, 4, 4, 5, 10, 10, 10 3x
> 21 13, 13 4x > 21 5, 18 2x > 21 E129 4.14 CBA/N 3, 6, 4,
5, 6, 8, 10, 8, >23 13 Total Name of Pools R36A/Rx1/D39 WU2
BG9739 EF3296 EF5668 L81905 DBL5 DBL6A controls Pooled data 7, 8,
10, 6, 8, 9, 3, 6, 8, 10, 13, 6, 8, 9, 10, 13, 3, 3, 7, 7, 9, 16,
7, 8, 8, 10, 12, 9, 2, 4x 3, 6x > 21 >21 15, 3x > 21 10,
10 3x > 21 2x > 21 3x 13, 21 4x > 21 6x 4, 3x 5, 6x 6, 7,
7, 8, 18, >21 median days alive >21 8.5 13 9 >21 8 12
>21 5 alive:dead 6:3 1:3 3:6 0:5 3:2 2:6 0:9 4:1 1:24 P - days
alive <0.0001 0.0082 0.0025 0.0036 0.0001 0.037 0.002 <0.0001
P - alive:dead 0.0019 n.s. 0.048 n.s. 0.0093 n.s. n.s. 0.0009 Score
+++ +.+-. ++ +.+-. +++ +.+-. +.+-. +++ 67 25 33 0 60 25 0 80 4 66
22 30 -4 58 22 -4 79 0 DBL6A challenge R36A/Rx1/D39 WU2 BG9739
EF3296 EF5668 L81905 DBL5 DBL6A controls median P value P value
DBL6A days of days of alive: based on days based on % % challenge
death death dead to death alive:dead Score alive prot. All immune
12.5 19:35 <0.0001 0.0019 ++ 35 33 All control 5 1:24
[0442]
55TABLE 54 Pooled Data for Protection against BG9163 by various
PspAs Days to Death/immunogen CFU JS1020 Exp. BG9163 Mice Rx1
Rx1.BCG (BG9739) all immune control E169 2.67 CBA/N 5x > 24 4,
5, 8, 8, >24 E140 3.14 CBA/N n.v. E129 4.0 CBA/N 12, 4x > 23
7, 9, 9, 13, >23 E028 6.217 CBA/N 6, 3x > 21 5, 6, 8, 10
Immunogens Rx1/R36A/D39 BG9739 all immune control Pooled Data 6, 8x
> 21 12, 4x > 21 6, 12, 4, 5, 7, 8, 8, 9, 12x > 21 9, 12,
2x > 21 median days alive >21 >21 >21 8.5 alive:dead
8:1 4:1 12:2 2:8 P - days alive 0.0086 0.0097 0.0027 P - alive:dead
0.0045 0.047 0.0022 % alive 89 80 86 20 % prot. 86 75 83 0 score
+++ +++ +++ BG9163 Challenge Rx1/R36A/D39 BG9739 all immune control
median P value P value days of days of alive: based on days based
on % % EF5668 death death dead to death alive:dead Score alive
prot. All immune 8 18:26 0.0015 0.005 ++ 41 35 All control 3
2:21
[0443]
56TABLE 55 Pooled Data for Protection against L81905. by various
FL-PspAs Days to Death/immunogen CFU BC100 JD908 JS1020 bc100 bc100
JS5010.3 bc100 JS3020 Exp. L81905 Mice R36A (Rx1) (WU2) (BG9739)
BG9739 EF3296 EF5668 L81905 (DBL5) (DBL5) (DBL6A) control E172 2.45
CBA/N 3, 4, 5, 3, 3, 4, 6, 6 4, 4 E140 3.11 CBA/N 2, 5, 5, 2, 2, 2,
6, 8 3, 3 E084 3.86 BALB 2, 2, 1, 8x 2 5x > 14 E104 -3.5 CBA/N
3, 7, 8, 3, 3, 3, 3, 4, 5, 2, 4, 4, 8, 11 2x > 22 5, 6 4, 5 E124
-3.5 CBA/N 2, 2, 2, 2, 2, 2, 1, 2, 2, 2, 3 3, 5 2, 2 E125 3.6 CBA/N
5, 6, 3, 4, 4, 5, 2, 2, 3, 8, 8 6, 8 5, 5 5, 5, 5 E144 4.11 CBA/N
3, 3, 6, 6, 6, 2, 2, 3, 2, 2, 3x 3 5, 6, 2x > 10 3, 3 >10 All
3, 3, 3, 4, 5, 2, 2, 3, 7, 5, 6, 6, 6, 6, 2, 2, 3, 3, 4, 4x 2, 4x
3, 4, 5, 3x 2, 3, 3, 1, 1, 20x 2 5, 6, 6, 6 8, 8, 11, 8, 8 2x >
10 3, 3 6, 8 2x > 21 5, 5 4, 8x 3. 6x 4. >21 5x > 21 3x 5,
6 4x 5 medi- 5 5 5 >21 7 6 3 5 3 5 3.5 2 an 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 dead P rank P a:d score
[0444]
57TABLE 56 Protection against L81905. by various bc100s &
FL-PspAs pooled together Days to Death/immunogen CFU BC100 JD908
JS1020 bc100 bc100 JS5010.3 bc100 JS3020 Exp. L81905 Mice R36A
(Rx1) (WU2) (BG9739) BG9739 EF3296 EF5668 L81905 (DBL5) (DBL5)
(DBL6A) control E172 2.45 CBA/N 3, 4, 5, 3, 3, 4, 6, 6 4, 4 E140
3.11 CBA/N 2, 5, 5, 2, 2, 2, 6, 8 3, 3 E084 3.86 BALB 2, 2, 1, 8x 2
5x > 14 E104 -3.5 CBA/N 3, 7, 8, 3, 3, 3, 3, 4, 5, 2, 4, 4, 8,
11 2x > 22 5, 6 4, 5 E124 -3.5 CBA/N 2, 2, 2, 2, 2, 2, 1, 2, 2,
2, 3 3, 5 2, 2 E125 3.6 CBA/N 5, 6, 3, 4, 4, 5, 2, 2, 3, 8, 8 6, 8
5, 5 5, 5, 5 E144 4.11 CBA/N 3, 3, 5, 6, 6, 6 2, 2, 3, 2, 2, 3x 3
6, >10 2x > 10 3, 3 Pooled 2, 3, 3, 3x 5, 3, 4, 5, 2, 2, 3,
5, 6, 7, 6, 6, 6, 2, 2, 3, 3, 4, 4x 2, 4x 3, 4, 5, 3x 2, 3, 3, 1,
1, 20x 2 6, 6, 8, >21 6, 6 4x 8, 11, 5x > 21 2x > 10 3, 3
6, 8 5, 5, 2x > 21 4, 3x 5, 6 8x 3. 6x 4. 4x 5 Median days alive
5 5 8 6 3 5 3 3.5 2 alive:dead 1:9 0:5 5:11 2:3 0:5 0:4 2:12 0:10
0:40 P - days alive 0.0005 0.0035 <0.0001 0.0002 n.s. 0.01 0.035
0.044 P - alive:dead n.s. n.s. 0.0001 0.01 n.s. n.s. n.s. n.s.
score + + ++ ++ 0 + + + % alive 10 0 31 40 0 0 14 0 0 % protected
challenge with L81905 R36A/Rx1/D39 WU2 BG9739 EF3296 EF5668 L81905
DBL5 DBL6A controls median P value P value L81905 days of days of
alive: based on days based on % % challenge death death dead to
death alive:dead Score alive prot. All immune 5 10:59 <0.0001
0.008 ++ 14 14 All control 2 0:40
[0445]
58TABLE 57 Pooled Data for Protection against DBL5 by various
FL-PspAs & bc100s Days to Death/immunogen CFU BC100 JS1020
bc100 bc100 JS5010.3 bc100 JS3020 Exp. DBL5 Mice R36A Rx1 BG9739
JS1020 EF5668 L81905 DBL5 DBL5 DBL6A control E84.sup.1 3.90 BALB/c
6x 2 9x 2 E140 3.27 CBA/N 4, 4, 5, 2, 2, 2 5, 5 E104 3.39 Xid 3, 3,
6, 7, 7, 2, 2, 4, 5, 5 2, 4x 3 >22, >22 15, >22, >22
E124 3.76 Xid 2, 2, 2, 5, >15 5x 2 1, 1, 2, 2, 2 E125 3.81 CBA/N
3, 3, 3, 3, 4, 4 2, 2, 5x 2, 5 4, 5 2, >21 E144 4.13 XID 3, 3,
3, 2, 2, 3, 4, 4 5x 2 3, >10 total name of pool R36A/Rx1/D39
BG9739 EF5668 L81905 DBL5 DBL6A controls pooled data 4x 3, 2x 4, 3x
6x 2, 4x 3, 4, 2, 2, 3, 4, 4 3, 3, 4, 4 6x 2, 5, 7, 7, 15, 7x 2, 4,
5, 5 1, 1, 26x 2, 5, >21 5, >21, >21 4x > 21 4x 3, 5
median days alive 4 3 3 3.5 6 2 2 alive:dead 1:9 2:12 0:4 0:4 4:10
0:10 0:32 P - days alive <0.0001 0.0063 .041 0.001 0.0025 n.s. P
- alive:dead n.s. n.s. n.s. n.s. 0.0056 n.s. Score + + + + ++ 0 %
alive 10 14 0 0 29 0 0 % protected 10 14 0 0 29 0 0 DBL5 challenge
R36A/Rx1/D39 BG9739 EF5668 L81905 DBL5 DBL6A controls median P
value P value DBL5 days of days of alive: based on days based on %
% challenge death death dead to death alive:dead Score alive Prot.
All immune 3.5 7:49 <0.0001 0.034 ++ 3.6 3.6 All control 2 0:33
.sup.1This immunization was with cell eluted PspA. Note BALB/cJ
mice were used. Also note 10.sup.4 Challenge CFU.
[0446]
59TABLE 58 Pooled Data for Protection against EF6796 by various
PspAs Days to Death/immunogen CFU Rx1 JS1020 JS3020 JS5010.3 FL
DBL5 Exp. WU2 Mice BC100 (BG9739) L81905 (DBL6A) (DBL5) bc100
control E140 3.75 CBA/N 4x > 21 1, 1, 1 E28 ? BALB n.v.
[0447]
60TABLE 59 Pool of Pools for protection against EF6796 Group Delay
in time to death and/or survival Protection against death line
Description days to death (medain DOD) P values etc. alive:dead P
values etc. 1a Rx1 4x > 21 (>21) 0.029 4:0 0.029 1b Rx1
controls 1, 1, 1 (1) -- 0:3 --
[0448]
61TABLE 60 Pooled Data for Protection against BG7322. by various
FL-PspAs and bc100s Days to Death/immunogen CFU D39/ Rx1 JD908
bc100 bv100 JS5010.3 bc100 JS3020 Exp. BG7322 Mice R36A BC100 (WU2)
BG9739 EF3296 EF5668 L81905 DBL5 DBL5 DBL6A control E171 2.78 CBA/N
10, 15, 1, 3, 6, 6, 7 3x > 21 S143 3.0 CBA/N 7, 8x > 10 2, 2,
4, 5, 7, 7, 8, 8 E140 3.14 CBA/N 4x > 21 3, 6, 6, >21 BC100
E152 3.11 CBA/N 12, 13, 10, 12, >21, >21, 6, 7, 7, 8, 8, 16,
>21 13, >21 >21, >21 9, 14 E146 3.57 CBA/N 18, 20, 5,
3x 6, 10 6, 10, 11, 4, 8, 11, 4, 5, 5, 3x > 21 11, 19 18, >21
6, >21 E169 3.94 CBA/N 5x > 21 2, 5, 5, 6, 7 Immunogens
R36A/Rx1/D39 JD908 BG9739 EF3296 EF5668 L81905 DBL5 DBL6A Cont.
Pools 18, 20, 12x > 21 10, 15, 12, 13, 5, 3x 6, 10 7, 8x > 21
10, 12, 6, 10, 11, 4, 8, 11, 1, 3x 2, 3, 3, 3x > 21 16, >21
13, >21 11, 19 18, >21 4, 4, 5x 5, >21, >21, >21,
>21 7x 6, 6x 7, 4x 8, 9, 14, 2x > 21 median day alive >21
>21 14.5 6 >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.
<0.0001 0.0013 0.0002 0.028 P - alive:dead <0.0001 0.004 n.s.
n.s. <0.0001 n.s. 0.0076 n.s. % alive 100 60 25 0 89 25 80 25 6
% protected 100 57 22 0 88 22 79 22 6 Score +++ +++ +.+-. 0 +++
+.+-. +++ +.+-. BG7322 Challenge R36A/Rx1/D39 JD908 BG9739 EF3296
EF5668 L81905 DBL5 DBL6A Cont. P value P value BG7322 median days
of alive: based on days based on % % Challenge death dead to death
alive:dead Score alive Prot. All immune >21 30:25 <0.0001
<0.0001 +++ 55 52 All controls 6 2:32
Example 8
Ability of PspA Immunogens to Protect Against Individual Challenge
Strains
[0449] In example 7 some of the capsular type 2, 4, and 5 strains
were not completely protected from death by 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
type 2, 4, and 5 pneumococci. This result is shown in the table
below.
[0450] 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.
62TABLE 60A Days of death of BALB/cByJ mice after immunization with
monovalent and polyvalent vaccine. Challenge Strains Immunogen pspA
Log Days to Death strain caps PspA B region Challenge 4-5 valent
mixture name type type clade dose 1 mg R36A + CFA (0.5 .mu.g each)
+ CFA JY2141 + CFA None D39 2 25 2 4.76 3, 4x > 21 3, 4x > 21
3, 4, 5, 3, 3, 11, >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, 2, 3x > 21
2, 2, 3, 4 2, 3, 4, 4 3, >21, >21 BG9739 4 26 1 4.07-4.4 7,
8x > 21 3, 8x > 21 1, 5, 6, 6, 9, 3, 3, 3, 4, 6, 4x > 21
7, 7, 2x > 21 L81905 4 23 1 6.90-6.96 2, 2, 2, 2, 5, 2, 6, 8, 9,
1, 1, 1, 1, 2, 1, 4x 2, 3x, 3, 5, 6x > 21 3, 4, 5, 4, >21 4x
> 21 2x > 21 EF5668 4 12 4 6.10-6.93 3, 3, 4, 3x 3, 6x >
21 4x 3, 4, 4, 6, 3, 5x 4, 7x > 21 6, >21 6, >21 DBL5 4 33
2 3.30 7, 14, 3, 5, 5, 2, 2, 2, 4, 6 4, 5, 5, 6, 9 3x > 21 2x
> 21 DBL6A 6A 19 1 4.34 6, 9, 10, 10, 11, 12, 3, 11, 11, 13, 8,
9, 11, 11, >21 13, >21 16 21, >21 BG7322 6B 21 ? 3.9 8, 8,
3x > 21 5x > 21 6, 6, 7, 8, 10 2, 5, 6, 8, 8 .sup.Note,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. .sup.14 valent vaccine mixture
R36A, BG9739, EF5668, and DBL5 - all E180 .sup.24 valent vaccine
mixture R36A, BG9739, DBL5, EF3296 D39 and DBL6A .sup.35 valent
vaccine mixture R36A, BG9739, DBL5, EF3296, EF5668
Example 9
Characterization OP PspA Epitopes Within Pneumococcal Strains
MC25-28
[0451] 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 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. S, et al., J. Infect.
Dis. 1993; 168: 158-163).
[0452] The following techniques were used to characterize the
location of difference PspA epitopes:
[0453] Bacterial cell culture. Bacteria were grown in Todd-Hewitt
broth with 0.5% yeast extract or on blood agar plates overnight at
37.degree. 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.
[0454] 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 6.8). Total
pneumococcal protein in the lysates was quantitated by the
bicinchoninic acid method (BCA Protein Assay Reagent; Pierce
Chemical Company, Rockford, Ill.),
[0455] PspA serotyping. 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 assigned based on the particular combination of MAbs with
which each PspA was reactive.
[0456] Colony immunoblotting. A ten mL tube of Todd-Hewitt broth
with 0.5% yeast extract was inoculated with overnight growth of
MC25 from a blood agar plate. The isolate was allowed to grow to a
concentration of 10.sup.7 cells/mL as determined by an O.D of 0.07
at 590 nm. MC25 was serially diluted and 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 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 blood agar plate. The membranes were developed as Western
blots according to PspA serotyping methods.
[0457] 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 (FIG. 14). The MAbs
XiR278 and 2A4 detected a PspA molecule with an apparent molecular
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 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 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 conditions. All colonies on the
plate, as well as all of the progeny from a single colony, reacted
with MAbs XiR278. 2A4, and 7D2.
Example 10
Southern Blot Analysis of Chromosomal DNA Isolated from
Pneumococcal Strains MC25-28
[0458] Pneumococcal chromosomal DNA was prepared by the Youderian
method (Sheffield, J. S., et al., Biotechniques, 1992; 12:
836-839). Briefly, for a 500 ml culture in THY or THY with 1%
choline, cells were centrifuged at 8000 rpm in GSA rotor for 30
minutes at 4.degree. 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 centritued twice at 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.degree. C. for 15 minutes if sodium deoxycholate was used. If
SDS was used, incubation at 37.degree. C. was not necessary. The
cells were incubated at 65.degree. C. for 15 minutes, and 1/5
volume of 5.0 M potassium acetate was added, and the cell
suspension was incubated for 30 minutes at 65.degree. C.
[0459] The cells were placed on ice for 60 minutes, and centrifuged
at 12,000 rpm in an SS-34 rotor for 10 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.
[0460] 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 aqueous phase was extracted by adding 2 volumes
95% 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, e.g., 500 ul.
[0461] The cells were harvested, washed, lysed, and digested with
0.5% (st/vol) SDS and 100 .mu.g/mL proteinase K at 37.degree. C.
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 65.degree. C. for 20 min., and then
extracted with chloroform/isoamyl alcohol. DNA was precipitated
with 0.6 volumes isopropanol, washed, and resuspended in 10 mM
Tris-HCl, 1 mM 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 Spring Harbor: Cold Spring Harbor
Laboratory, 1984; and Murray, M. G., et al., Nucleic Acids Res.
1980; 8 4321-1325).
[0462] Probe preparation 5' and 3' oligonucleotide primers
homologous with nucleotides to 26 and 1967 to 1990 of Rx1 pspA
(LSM13 and LSM2, respectively) were used to amplify the full length
pspA and construct probe LSMNpspA13/2 from Rx1 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 amplify the variable .alpha.-helical region to construct probe
LSMpspA12/6. PCR generated DNA was purified by Gene Clean (Bio101
Inc., Vista, Calif.) and random prime-labeled with
digoxigenin-11-dUTP using the Genius 1 Nonradioactive DNA Labeling
and Detection Kit as described by the manufacturer (Boehringer
Mannheim, Indianapolis, Ind.).
[0463] DNA electrophoresis. For Southern blot analysis,
approximately 10 .mu.g of chromosomal DNA was digested to
completion with a single restriction endonuclease (Hind III, Kpn 1,
EcoRI, Dra I, or Pst I), then electrophoresed on a 0.7% agarose gel
for 16-48 h at 35 volts. For PCR analysis, 5 .mu.L 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 I), 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.
[0464] Southern blot hybridization. The DNA in the gel was
depurinated in 0.25N HCl for 10 min, denatured in 0.5M NaOH and
1.5M NaCl for 30 min, and neutralized in 0.5M fris-HCl (pH 7.2),
1.5M NaCl and 1 mM disodium EDTA for 30 min. DNA was transferred to
a nylon membrane (Micron Separations INC, Mass.) using a POSIBLOT
pressure blotter (Stratagene, LaJolla, Calif.) for 45 min and fixed
by UV irradiation. The membranes were prehybridized for 3 h at
42.degree. C. in 50% fornamide, 5.times.SSC, 5.times. Denhardt
solution, 25 mM sodium phosphate (pH 6.5), 0.5% SDS, 3% (wt/vol)
dextran sulfate and 500 g/mL of denatured salmon sperm DNA. The
membranes were then hybridized at 42.degree. C. for 18 h in a
solution containing 45% formamide, 5.times.SSC, 1.times. Denhardt
solution, 20 mM sodium phosphate (pH 6.5), 0.5% SDS, 3% dextran
sulfate, 250 .mu.g/mL denatured sheared salmon sperm DNA, and about
20 ng of heat-denatured digoxigenin-labeled probe DNA. After
hybridization, the membranes were washed twice in 0.1% SDS and
2.times.SSC for 3 min at room temperature. The membranes were
washed twice to a final stringency of 0.1% SDS in 0.3.times.SSC at
65.degree. C. for 15 min. This procedure yielded a stringency
greater than 95 percent. The membranes were developed using the
Genius 1 Nonradioactive DNA Labeling and Detection Kit as described
by the manufacturer (Boehringer Mannheim, Indianapolis, Ind.). To
perform additional hybridization with other probes, the membranes
were stripped in 0.2N NaOH/0.1% SDS at 40.degree. C. for 30 min and
then washed twice in 2.times.SSC.
[0465] PCR. 5' 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 50 .mu.L volumes containing 0.2
mM of each dNTP, and 1 .mu.L of each primer at a working
concentration of 50 mM. MgCl.sub.2 was used at an optimal
concentration of 1.75 mM 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 (M.J.
Research, Inc.) using the following three step program: Step 1
consisted of a denaturing temperature of 94.degree. C. for 2 min;
Step 2 consisted of 9 complete cycles of a denaturing temperature
of 94.degree. C. for 1 min, an annealing temperature of 50.degree.
C. for 2 min, and an extension temperature of 72.degree. C. for 3
min; Step 3 cycled for 19 times with a denaturing temperature
94.degree. C. for 1 min, an annealing temperature of 60.degree. C.
for 2 min, and an extension temperature of 72.degree. C. for 3 min,
and at the end of the last cycle, the samples were held at
72.degree. C. for 5 min to ensure complete extension.
[0466] Band size estimation. 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 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, Wash.) in order to calculate molecular
weights based on migration distances observed in the Southern
blot.
[0467] 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 (FIGS. 15A, 15C and 15D. In
comparison, when Rx1 DNA was digested with Hind III and hybridized
with LSMpspA13/2, homologous sequences were detected on 9.1 and 4.2
kb fragments as expected from previous studies with PspA (FIG.
15A). Results consistent with two pspA-homologous genes in MC25-28
were obtained with two pspA-homologous genes in MC25-28 digested
using four additional enzymes (Table 61).
63TABLE 61 Chromosomal RFLPs with probe LSMpspA13/2 for isolates
MC25-26 and Rx1 Restriction Fragments (sizes Restric- in kilobases)
tion Strains Examined MC25- Enzyme MC25 MC26 MC27 MC28 RX1 28 Rx1
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
Pst I + + >14, 6.1 10.0, 4.0
[0468] 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, i.e.,
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 Rx1 are made of a single gene
capable of encoding a 69 kDa protein, supporting the assertion that
two PspAs have been observed, i.e., PspA and PspC.
[0469] It has been observed that probes for the 5' half of pspA
(encoding the .alpha.-helical half of the protein) bind the pspC
sequence of most strains only at a stringency of around 90%. With
chromosomal digests of MC25-28, it was observed that the 5' Rx1
probe LSMpspA12/6 (FIG. 15E) bound two pspA homologous bands at
even higher stringency. The same probe bound only the pspA
containing fragment of Rx1 at the higher stringency (FIG. 15B).
[0470] 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 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
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 (FIG. 16). These results suggested that the
2.1 kb amplified DNA represents the amplified product of only a
single pspA gene. Rx1 produced an amplified product of 2.0 kb and
five fragments of 0.76, 0.468, 0.390, 0.349 and 0.120 kb when
digested with Hha I as expected from its known pspA sequence.
[0471] 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, 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
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 second PspA is these
isolates is due to the pspC gene not being turned off during in
vitro growth conditions.
[0472] 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 of the .alpha.-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 Rx1 pspA sequence than the Rx1
pspC sequence. If the pspC 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
[0473] 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.degree.
C. in 100 ml of Todd-Hewitt broth supplemented with 0.5% yeast
extract to an approximate density of 5.times.10.sup.8 cells/ml.
After harvesting of the cells by centrifugation (2900 g, 10 min),
the DNA was isolated and stored at 4.degree. C. in TE (10 mM Tris,
1 mM EDTA, pH8.0). Mannhein, Indianapolis, Ind.). All
hybridizations were done for 18 hours at 42.degree. C. without
formamide. By assuming that 1% base-pair mismatching results in a
1.degree. C. decrease in T.sub.m 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 T.sub.m by
established methods. High stringency is defined as .gtoreq.90%, and
low stringency is .ltoreq.85% base-pair matching.
[0474] 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 Rx1. These
oligonucleotides were synthesized by Oligos, Etc. (Wilson, Oreg.),
and are listed in Table 63.
64TABLE 63 Oligonucleotide sequences. Primer 5' .fwdarw. 3' LSM111
CCGGATCCAGCTCCTGCACCAAAAC LSM2 GCGCGTCGACGCTTAAACCCATTCACCATTGG
LSM3 CCGGATCCTGAGCCAGAGCAGTTGGCTG LSM4
CCGGATCCGCTCAAAGAGATTGATGAGTCTG LSM5
GCGGATCCCGTAGCCAGTCAGTCTAAAGCTG LSM6
CTGAGTCGACTGGAGTTTCTGGAGCTGGAGC LSM7
CCGGATCCAGCTCCAGCTCCAGAAACTCCAG LSM9 GTTTTTGGTGCAGGAGCTGG LSM10
GCTATGGCTACAGGTTG LSM12 CCGGATCCAGCGTGCCTATCTTAGGGGCTGGT LSM112
GCGGATCCTTGACCAATARRRACGGAGGAGGC
[0475] PCR was done with an MJ Research, Inc., Programmable Thermal
Cycler (Watertown, Mass.), using approximately 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 .mu.l
containing a final concentration of 50 mM KCl, 10 mM Tris-HCl (pH
8.3), 1.5 mM MgCl.sub.2, 0.01% gelatin, 0.5 .mu.M of each primer,
200 .mu.M of each deoxynucleotide triphosphate, and 2.5 U of Taq
DNA polymerase. The samples were denatured at 94.degree. C. for 2
minutes and 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 20 cycles of: 1 min at 94.degree. C., 2 min at
60.degree. C., and 3 min at 72.degree. C. After 30 total cycles,
the samples were held at 72.degree. C. for an additional 5 min
prior to cooling to 4.degree. C. The amplicons were then analyzed
by agarose gel electrophoresis.
[0476] Oligonucleotides 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 Rx1 is a laboratory
derivative of the clinical isolate D39 and consequently, both
strains showed identical hybridization patterns and are a single
column in Table 64.
65TABLE 64 Summary of hybridization of oligonucleotides with
HINDIII chromosomal restriction fragments. Strains Rx1/ Probe D39
WU2 DBL5 DBL6A A66 AC94 AC17 AC40 AC107 FL- 4.0, 9.1.sup.b 3.8 3.7,
5.8 3.0, 3.4 3.6, 4.3 3.6, 6.3 3.6, 6.3 3.2, 3.6 3.2, 3.6 Rx1.sup.a
LSM12 4.0, 9.1 3.8 3.7, 5.8 3.0, 3.4 4.3 --.sup.c 3.6, 6.3 3.2, 3.6
-- LSM5 4.0 -- -- -- -- 3.6, 6.3 -- -- -- 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 -- -- 3.2, 3.6 -- LSM111 4.0, 9.1 3.8 3.7, 5.8 3.4 --
6.3 -- 3.2 LSM10 4.0, 9.1 3.8 3.7 3.4 3.6, 4.3 -- 3.6, 6.3 3.2 3.6,
3.3 LSM2 4.0 0 3.7 -- -- 3.6 3.6 -- 3.6, 6.3 Strains Probe AC100
AC140 DB109 BG9709 BG58C L81905 L82233 L82006 FL- 4.0, 8.0 3.0, 4.0
3.3, 4.7 3.3, 4.7 1.4, 3.2 3.6, 5.2 8.2, 3.7 4.3, 6.4 Rx1.sup.a 3.6
LSM12 4.0, 8.0 4.0 3.3, 4.7 2.2, 9.6 1.4, 3.2, 3.6 1.3, 3.7 -- 3.6
LSM5 -- -- -- 2.2, 9.6 3.6 1.2, 2.3, -- -- 3.6 LSM3 -- -- -- 2.2
3.6 3.6 -- -- LSM4 -- -- -- 2.2 3.6 3.6 3.7 -- LSM7 -- 3.0, 4.0
3.3, 4.7 2.2, 9.6 3.6 3.6, 2.3 3.7 -- LSM111 4.0 4.0 -- 2.2 -- 5.2
-- -- LSM10 4.0 4.0 3.3, 4.7 2.2, 9.6 3.6, 3.2 3.6, 5.2 1.3, 3.7
4.3, 6.4 LSM2 4.0 3.0, 4.0 4.7 -- -- -- -- 4.3 .sup.aFull-length
pspA of strain Rx1. .sup.bnumbers are size in kilobase pairs.
.sup.cno hybridization observed with corresponding probe.
[0477] 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 pspA probe. Moreover, the same
pairs of fragments were frequently detected by probes derived from
the 3' as well as the 5' region of Rx1 pspA. These results
suggested that the HindIII fragments from different isolates
include two separate but homologous sequences, rather 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
location of HindIII recognition sites.
[0478] Oligonucleotides which hybridize with-a single restriction
fragment 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 homology 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 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 C-terminus (LSM2)
appeared to be relatively specific for pspA since they generally
detect only one of the HindIII fragments of each strain.
[0479] 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 cross-reactive, 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 pspC sequences. LSM3, 4, and 5
hybridize with the smallest number of strains of any
oligonucleotides (29-35 percent), suggesting that the
.alpha.-helical domain is the least conserved region within pspA.
In strains BG58C and L81905 oligonucleotides detect more than two
HindIII 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 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 sequences are those for the leader,
the alpha-helical region, and the proline-rich region.
[0480] The oligonucleotides used as hybridization probes were also
tested for their utility as primers in the polymerase chain
reaction (PCR). Amplification of 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 amplifying sequences (Table 63). The lowest
frequency of amplification was observed with LSM112 which was
derived from the Rx1 sequence 5' to the pspA start site. This
oligonucleotide was not used in the hybridization studies. DNA
fragments generated by PCR were blotted and hybridized with a
full-length pspA probe to confirm homology to pspA.
[0481] Further evidence for variability at the pspA locus comes
from the differences in the sizes of the 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 .alpha.-helical, proline-rich, and repeat domains were
amplified from corresponding strains and variation in pspA appears
to come from sequences within the .alpha.-helical coding
region.
66TABLE 65 Amplification of pspA by PCR using the indicated
oligonucleotides as 5' primers in combination with the 3' - primer
LSM2. Amplified/ Percent 5' - primer Domain Tested Amplified LSM112
-35 (upstream) 2/14 14 LSM12 leader 8/14 57 LSM3 .alpha.-helical
3/14 21 LSM7 proline 12/14 86 LSM111 proline 14/14 100
[0482] 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 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 and around these
sequences.
[0483] A useful pspA-specific DNA probe would identify Rx1 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 Rx1 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 Rx1-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 16 of the 17 isolates and in each
case revealed a consensus DNA fragment recognized by most or all of
the oligonucleotide probes.
[0484] 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 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
.alpha.-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 Rx1 sequence makes it likely that other
probes within the repeat sequences would give similar results.
[0485] The portion of Rx1 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).
[0486] In two strains, some oligonucleotide probes identified more
than two pspA-homologous sequences. In 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, .alpha.-helical, and proline region, and they have no
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.
[0487] 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
[0488] Chromosomal DNA from S. pneumoniae EF6796, serotype 6A
clinical isolate, was isolated by methods including purification
through a cesium chloride gradient, as described in Example 8. The
HindII-EcoRI fragment of EF6796 was cloned in modified pZero vector
(Invitrogen, San Diego, Calif.) in which the Zeocin-resistance
cassette was replaced by a kanamycin cassette (shown in FIG. 18).
Recombinant plasmids were electroporated into Escherichia coli
TOP10F' cells (F' {lacI.sup.qTet.sup.R} mcrA
.DELTA.(mrr-hsdRMS-mcrBC) .o slashed.80lacZ.DELTA.M15 .DELTA.lacX74
deoR recAl araD139 .DELTA.(ara-leu)7967 gal/U gal/K rpsL endAl
nupG) (Invitrogen).
[0489] The 5' region of pspA.Rx1 does not hybridize to pspC
sequence at high stringencies by Southern analysis. Utilizing both
the full-length Rx1 pspA probe, and a probe containing the sequence
encoding .alpha.-helical region of PspA, it was possible to
identify which DNA fragment contained pspA and which fragment
contained the pspC locus. The pspC locus and the pspA gene of
EP6796 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, Bio101,
Inc., Vista, Calif.). The size-fractionated DNA was then ligated in
HindIII-EcoRI-digested pZero, and electroplated into E. coli
TOP10F' cells. Kanamycin-resistant 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.
[0490] Escherichia coli strain LXS200 which contains the cloned
PspC gene from Streptococcus pneumoniae strain EF6796 was deposited
on Jul. 24, 2001 under the terms of the Budapest Treaty with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md., 20852, USA, under accession number ATCC No.
PTA-3526.
[0491] 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 Computer Group (GCG) programs supported by the Center for
AIDS Research (P30 A127767), 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 analyzed using Matcher.
[0492] A gene probe for cloning the pspC locus Two oligonucleotide
primers, N192 and C558 (shown in FIG. 19), have been used
previously to clone fragments homologous to the region of Rx1 pspA
encoding 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.Rx1 (FIG. 17); this includes
approximately 100 amino acids of the .alpha.-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.
[0493] Independently, a larger pspA fragment from strain EF6796 was
made using primers LSM13 and SKH2 (shown in FIG. 19) for the
purpose of direct sequencing of serologically diverse pspA
genes.
[0494] 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 .alpha.-helical, and the proline-rich
regions (amino acid -15 to 450) (FIG. 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 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.R1. The cloned fragment in pRCT135 was
assumed to represent the 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 Rx1. The second gene locus was
called pspC, in recognition of its distinct chromosomal location,
not sequence differences from the prototype pspA gene.
[0495] Analysis of the nucleotide and amino acid sequence of pspC
EF6796. To test the hypothesis that pRCT135 represented pspC of
EF6796, and to further investigate pspC, the entire EF6796 pspC
gene was cloned as a 3.4 kb HindIII-EcoRI fragment forming pLXS200.
DNA sequence of the pspC-containing clone pLXS200 revealed an open
reading frame of 2782 nucleotides based on the analysis of putative
transcriptional and translation start and stop sites (FIGS.
21A-21E). The predicted open reading frame encodes a 105 kDa
protein which has an estimated pI of 6.09.
[0496] PspA.Rx1 and PspC.EF6796 are similar in that they both
contain an .alpha.-helical region followed by a proline-rich domain
and repeat region (FIG. 20). However, there are several features of
the amino acid 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.Rx1 are 76% similar and 68%
identical based on GCG Bestfit program for this region (FIGS.
22A-22C). 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 (FIGS.
21A-21E). A potential transcriptional terminator occurs at a stein
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 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.
[0497] 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 the similar regions of S. agalactiae .beta. 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 1 antigen, it is
clearly a distinct protein.
[0498] The N-terminus of PspC is quite different from the
N-terminus of PspA. Prediction of the secondary structure utilizing
Chou-Fausman analysis (Chao, P. Y. et at., Adv. Enzymol. Relat
Areas Mol. Biol. 1978: 47: 45-148), suggests that the structure of
amino acids 16 to 589 of PspC is predominately .alpha.-helical. The
Matcher program was used to examine periodicity in the
.alpha.-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 coiled-coil region of the
.alpha.-helix of PspC (between amino acid 32 to 600) has three
breaks in the heptad repeat (FIGS. 23A-23E). These disturbances in
the 7 residue periodicity occur at amino acids 99 to 104, 224 to
267 and 346 to 350. The .alpha.-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.
[0499] The sequence encoding the .alpha.-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 conserved both at the nucleotide and
amino acid level (amino acids 188 to 348 and 438 to 598) (FIG. 24).
PspA lacks evidence for any repeats this prominent within the
.alpha.-helical region. These repeat regions could provide a
mechanism for recombination that could alter the N-terminal 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 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 the two repeats. Perhaps some deviation
from coiled-coil structure between the two repeats is critical to
maintain the a-helical structure.
[0500] Previous studies have shown that a major cross-protective
region of PspA comprises the C-terminal 1/3 of the .alpha.-helical
region (between residues 192 and 260 of PspA.Rx1). This region
accounts for the binding of 4 of 5 cross-protective immunity in
mice. Homology between PspC and PspA begins at amino acid 148 of
PspA, thus 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 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 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 Rx1 is no greater than the
differences between many additional PspAs, which have been shown to
be highly cross-protective.
[0501] A proline-rich domain exists between amino acid 590 to 652.
The sequence, PAPAPEK (SEQ ID NO: 108) is repeated six times in
this region. This region is very similar to the proline-rich region
of PspA.Rx1 which contains the sequence PAPAP (SEQ ID NO: 109)
repeated eight times in two proline-rich regions. These two regions
of PspA.Rx1 are separated by 27 charge amino acids; no such spacer
region is present in PspC.
[0502] 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 protein is cell-associated. For example, in M
proteins of S. pyogenes this domain contains both a Pro- and
Gly-rich regions. The fibronectin-binding protein of S. pyogenes,
S. dysgalactiae, and Staphylococcus aureus contains a proline-rich
region with a three-residue 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 (SEQ ID NO: 110). This region lacks glycine
normally found in the proline regions of M-proteins. The last
proline repeat region of this molecule is PAPAK (SEQ ID NO: 111),
which is more similar to the proline-region of PspA and PspC than
it is to M-proteins.
[0503] 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 responsible for aberrant migration of these proteins
through SDS-polyacrylamide gels.
[0504] The repeat region of PspC is a common motif found among
several proteins in gram positive organisms. Autolysin of S.
pneumoniae, toxins A and B of Clostridium difficile,
glucosyltransferases from S. downel 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 PspA and PspC is much greater than that of PspC
to the other proteins (Table 66).
[0505] 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 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 not known whether PspC is
attached to the cell surface or secreted.
[0506] PspA and PspC proteins both have .alpha.-helical coiled-coil
regions, proline-rich central regions, repeat regions, with a
choline binding motifs, and the C-terminal 17 amino acid tail. PspA
and PspC share three regions of high sequence identity. One of
these is a protection-eliciting region present within the
.alpha.-helical domain. The other two regions are the proline-rich
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
.alpha.-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 repeat in the .alpha.-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 protection-eliciting and elicit
cross-protective antibodies.
[0507] The sequence similarity between the promoter 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 the .beta.
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.
[0508] Using analogous procedures, a second PspC sequence was
isolated from strain D39 of S. pneumoniae. FIGS. 25A-25C, 26A-26B,
27A-27D, 28A-28B to 29A-B show the sequence data of PspC from
strain D39, complete from upstream of the promoter through the
proline-rich region. Strain D39 has the same genetic background as
strains Rx1, from which pspA was sequenced. D39 and Rx1 have the
same pspC gene based on Southern blot analysis.
[0509] The alpha-helical encoding region of the D39pspC gene is one
third of the size of the homologous region from the EF6796pspC
gene. The proline-rich region of the D39pspC gene was more similar
to Rx1 pspA than to EF6796 pspC. Even so, the two pspC genes were
86% identical at the nucleotide sequence, and 67% identical at the
amino acid level.
[0510] In the alpha-helical sequence of EF6797 pspC a strong repeat
was observed. This was absent in the pspC sequence of D39. The
D39pspC sequence also lacks a leader sequence, found in the EF6797
pspC sequence.
[0511] 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 extent of variability appears to
be even greater than that which has been observed for pspA.
67TABLE 66 PERCENT HOMOLOGY OF CHOLINE BINDING REGIONS Percent
similarity/identity Protein Organism PspA PspC PspC S. pneumoniae
86/60 100/100 Bacteriophage Cp-1 S. pneumoniae 56/30 56/28 LytA S.
pneumoniae 57/33 61/32 PspA C. perfringens 64/45 59/42 alpha toxin
C. novyi 54/29 57/33 CspB C. acetobutylicum 58/36 61/45
[0512] Having thus described in detail certain preferred
embodiments of the present invention, it is to be understood that
the invention defined by the appended claims is not to be limited
by particular details set forth in the above description, as many
apparent variations thereof are possible without departing from the
spirit or scope thereof.
REFERENCES
[0513] Mufson M A. Streptococcus pneumoniae. In: Mandell G L,
Douglas R G, Jr, Bennett J E, (eds.) Principles and Practice of
Infectious Diseases. New York: Churchill Livingston,
1990:1539-50.
[0514] Cohen C, Parry D A D. alpha-helical coiled coils: more facts
and better predictions. Science 1994;236:488-9.
[0515] Shapiro E D, Berg A T, Austrian R, et al. Protective
efficacy of polyvalent pneumococcal polysaccharide vaccine. N Engl
J Med 1991;325:1453-60.
[0516] Feldman C, Munro N C, Jeffery P K, 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.
[0517] Lock R A, Paton J C, Hansman D. Comparative efficacy of
pneumococcal neurarninidase and pneumolysin as immunogens
protective against Streptococcus pneumoniae. Microb Pathog
1988;5:461-7.
[0518] Sampson J S, O'Connor S P, Stinson A R, Tharpe J A, Russell
H. Cloning and nucleotide sequence analysis of psaA, the
Streptococcus pneumoniae gene encoding a 37-kilodalton protein
homologus to previously reported Streptococcus sp. adhesins. Infect
Immun 1994;62:319-24.
[0519] McDaniel L S, Sheffield J S, Delucchi P, Briles D E. PspA, a
surface protein of Streptococcus pneumoniae, is capable of
eliciting protection against pneumococci of more than one capsular
type. Infect Immun 1991 ;59:222-8.
[0520] Berry A M, Lock R A, Hansman D, Paton J C. Contribution of
autolysin to virulence of Streptococcus pneumoniae. Infect Immun
1989;57:2324-30.
[0521] Berry A M, Yother J, Briles D E, Hansman D, Paton J C.
Reduced virulence of a defined pneumolysin-negative mutant of
Streptococcus pneumoniae. Infect Immun 1989;57:2037-42.
[0522] McDaniel L S, Yother J, Vijayakumar M, McGarry L, Guild W R,
Briles D E. Use of insertional inactivation to facilitate studies
of biological properties of pneumococcal surface protein A (PspA).
J Exp Med 1987;165:381-94.
[0523] Waltman W D II, McDaniel L S, Gray B M, Briles D E.
Variation in the molecular weight of PspA (Pneumococcal Surface
Protein A) among Streptococcus pneumoniae. Microb Pathog
1990;8:61-9.
[0524] Crain M J, Waltman W I II, Turner J S, et al. Pneumococcal
surface protein A (PspA) is serologically highly variable and is
expressed by all clinically important capsular serotypes of
Streptococcus pneumoniae. Infect Immun 1990;58:3293-9.
[0525] Yother J, Briles D E. Structural properties and evolutionary
relationships of PspA, a surface protein of Streptococcus
pneumoniae, as revealed by sequence analysis. J Bact 1992;174:
601-9.
[0526] McDaniel L S, Scott G, Kearney J F, Briles D E. Monoclonal
antibodies against protease sensitive pneumococcal antigens can
protect mice from fatal infection with Streptococcus pneumoniae. J
Exp Med 1984;160:386-97.
[0527] McDaniel L S, Ralph B A, McDaniel D O, Briles D E.
Localization of protection-eliciting epitopes on PspA of
Streptococcus pneumoniae between amino acids residues 192 and 260.
Microb Path 1994; 17:323-37.
[0528] Yother J, Forrnan C, Gray B M, Briles D E. Protection of
mice from infection with Streptococcus pneumoniae by
anti-phosphocholine antibody. Infect Immun 1982;36:184-8.
[0529] Briles D E, Crain M J, Gray B M, Forman C, Yother J. Strong
association between capsular type and virulence for mice among
human isolates of Streptococcus pneumoniae. Infect Immun 1 992;60:
111-6.
[0530] McDaniel L S, Sheffield J S, Swiatlo E, Yother J, Crain M J,
Briles D E. Molecular localization of variable and conserved
regions of pspA, and identification of additional pspA homologous
sequences in Streptococcus pneumoniae. Microb Pathog
1992;13:261-9.
[0531] McDaniel L S, McDaniel D O. Analysis of the gene encoding
type 12 PspA of S. pneumoniae EF5668. In: Ferretti J J, Gilmore M
S, Klaenhammer T R, Brown F ed. Genetics of Streptococci,
Enterococci and Lactococci. Basel: Karger, 1995:283-6.
[0532] Briles D E, Forman C, Crain M. Mouse antibody to
phosphocholine can protect mice from infection with mouse-virulent
human isolates of Streptococcus pneumoniae. Infect Immun
1992;60:1957-62.
[0533] Davis R W, Boststein D, Roth J R. A manual for genetic
engineering: advanced bacterial genetics. Cold Spring Harbor, N.Y.:
Cold Spring Harbor Laboratory Press, 1980.
[0534] Studier F W, Moffatt B A. Use of baceriophage T7 RNA
polymerase to direct selective high-level expression of cloned
genes. J Mol Biol 1986;189:113-30.
[0535] Hanahan D. Studies on transformation of Escherichia coli
with plasinids. J Mol Biol 1983;166:557-80.
[0536] Laemmli U K. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature
1970;227:680-5.
[0537] Towbin H, Stachelin T, Gordon J. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. PNAS 1979;76:4350-4.
[0538] Amsbaugh D F, Hansen C T, Prescott B, Stashak P W, Barthold
D R, Baker P J. 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;136:931-49.
[0539] Briles D E, Nahm M, Schroer K, et al. Antiphosphocholine
antibodies found in normal mouse serum are protective against
intravenous infection with type 3 Streptococcus pneumoniae. J Exp
Med 1981;153:694-705.
[0540] Zar J H. Biostatistical Analysis. 2nd Ed. Englewood Cliffs,
N.J.: Prentice-Hall, Inc., 1984:718.
[0541] Yother J, Handsome G L, 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;174:610-8.
[0542] Talkington D F, Voellinger D C, McDaniel L S, Briles D E.
Analysis of pneumococcal PspA microheterogeneity in SDS
polyacrylamide gels and the association of PspA with the cell
membrane. Microb Pathog 1992;13:343-55.
[0543] Talkington D F, Crimmins D L, Voellinger D C, Yother J,
Briles D E. A 43-kilodalton pneumococcal surface protein, PspA:
isolation, protective abilities, and structural analysis of the
amino-terminal sequence. Infect Immun 1991;59:1285-9.
[0544] Schneewind O, Model P, Fischetti V A. Sorting of protein A
to the staphylococcal cell wall. Cell 1992;70:267-81.
[0545] Yother J, White J M. Novel surface attachment mechanism for
the streptococcus pneumoniae protein PspA. J Bact
1994;176:2976-85.
[0546] Gray B M. Pneumococcal infection in an era of multiple
antibiotic resistance. Adv Ped Inf Dis 1995; In press.
[0547] Filice G. A., L. L. Van Etta, C. P. Darby and D. W. Fraser.
1986. Bacteremia in Charleston County, South Carolina. Am. J.
Epidemiol. 123:128.
[0548] Gillespie S. H. 1989. Aspects of pneumococoal infection
including bacterial virulence, host response and vaccination. J.
Med Microbiol. 28:237.
[0549] Musher D. M. 1992. Infections caused by Streptococcus
pneumoniae: Clinical spectrum, pathogenesis, immunity, and
treatment. Cur. Infect. Dis. 14:801.
[0550] Nordenstam G, B. Anderson, D. E. Briles, J. Brooks, A. Oden,
A. Svanborg and C. S. Eden. 1990. High anti-phosphorylcholine
antibody levels and mortality associated with pneumonia. Scand. J.
Infect. Dis. 22:187.
[0551] Giebink G. S. 1989. The microbiology of otitis media.
Pediatr. Infect. Dis. J. 8:S18.
[0552] Giebink G. S. 1985. Preventing pneumococcal disease in
children: recommendations for using pneumococcal vaccine. Pediatr.
Infect Dis. 4:343.
[0553] Siber G. R. 1994. Pneumococcal disease: prospects for a new
generation of vaccines. Science 265:1385.
[0554] Cadoz M., J. Armand, F. Arminjon, J. -P. Michel, M. Michel,
F. Denis and G. Schiffman. 1985. A new 23 valent pneumococcal
vaccine: immunogenicity and reactogenicity in adults. J. Biol.
Stand. 13:261.
[0555] Robbins J. B., R. Austrian, C. -J. Lee, S. C. Rastogi, G.
Schiffman, J. Henrichsen, P. H. Makela, C. V. Broome, R. R.
Facklam, R. H. Tiesjema and J. C. Parke Jr. 1983. Considerations
for fornulating the second-generation pneumococcal-capsular
polysaccharide vaccine with emphasis on the cross-reactive types
within groups. J. Infect. Dis. 148:1136.
[0556] Forrester H. L., D. W. Jahigen and F. M. LaForce. 1987.
Inefficacy of pneumococcal vaccine in a high-risk population. Am.
J. Med. 83:425.
[0557] Douglas R. M. and H. B. Miles. 1984. Vaccination against
Streptococcus pneumoniae in childhood: lack of demonstrable benefit
in young Australian children. J. Infect. Dis. 149:861.
[0558] Douglas R. M., J. C. Paton, S. J. Duncan and D. J. Hansman.
1983. Antibody response to pneumococcal vaccination in children
younger than five years of age. J. Infect. Dis. 148:131.
[0559] Leinonen M., 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. 5:39.
[0560] Makela P. H., M. Leinonen, S. Pukander and P. Karma. 1981. A
study of the pneumococcal vaccine in prevention of clinically acute
attacks of recurrent otitis media Rev. Infect. Dis. 3:S124.
[0561] Riley I. D. and R. M. Douglas. 1981. An epidemiologic
approach to pneumococcal disease. Rev. Infect. Dis. 3:233.
[0562] Wright P. F., S. H. Sell, W. K. Vaughn, C. Andrews, K B.
McConnell and G. Schiffman. 1981. Clinical studies of pneumococcal
vaccines in infants. II. Efficacy and effect on nasopharyngeal
carriage. Rev. Infect. Dis. 3:S108.
[0563] Lock R. A., S. C. Paton and D. Hansman. 1988. Comparative
efficacy of pneumococcal neuraminidase and pneumolysin as
immunogens protective against Streptococcus pneumoniae. Microbial
Pathogenesis 5:461.
[0564] McDaniel L. S. and D. E. Briles. 1986. Monoclonal antibodies
against bacteria Orlando, Fla.: Academic Press, Inc., 143.
[0565] Paton J. C., R. A. Lock and D. J. Hansman. 1983. Effect of
immunization with pneumolysin on survival time of mice challenged
with Streptococcus pneumoniae. Infect. Immun. 40:548.
[0566] Talkington D. F., D. L. Crimins, D. C. Voellinger, J. Yother
and D. E. Briles. 1991. A 43-kilodalton pneumococcal surface
protein, PspA: isolation, protective abilities, and structural
analysis of the amino-terminal sequence. Infect. Immun.
59:1285.
[0567] Yother J., C. Forman, B. M. Gray and D. E. Briles. 1982.
Protection of mice from infection with Streptococcus pneumoniae by
anti-phosphocholine antibody. Infect. Immun. 36:184.
[0568] Crain M. J., W. D. Waltman, J. S. Turner, J. Yother, D. F.
Talidngton, L. S. McDaniel, B. M. Gray and C. E. Briles. 1990.
Pneumococcal surface protein A (PspA) is serologically highly
variable and is expressed by all clinically important capsular
serotypes of Streptococcus pneumoniae. Infect. Immun. 58:3293.
[0569] Briles D. E., J. Yother and L. S. McDaniel. 1988. Role of
pneumococcal surface protein A in the virulence of Streptococcus
pneumoniae. Rev. Infect. Dis. 10:S372.
[0570] McDaniel L. S., J. Yother, M. Vijayakumar, L. McGarry, W. R.
Guild and D. E. Briles. 1987. Use of insertional inactivation to
facilitate studies of biological properties of pneumocoocal surface
protein A (PspA). J. Exp. Med. 165:281.
[0571] Waltman W. D., L. S. McDaniel, B. M. Gray and D. E. Briles.
1990. Variation in the molecular weight of PspA (pneumococcal
surface protein A) among Streptococcus pneumoniae. Microbial
Pathogenesis 8:61.
[0572] Yother J. and D. E. Briles. 1992. Structural properties and
evolutionary relationships of PspA, a surface protein of
Streptococcus pneumoniae, as revealed by sequence analysis. J.
Bacterial. 174:601.
[0573] Yother J. and J. M. White. 1994. Novel surface attachment
mechanism for the Streptococcus pneumoniae protein PspA J. Bact.
176:2976.
[0574] Yother J., G. L. Handsome and D. E. Briles. 1992. Truncated
forms of PspA that are secreted from Streptococcus pneumoniae and
their use in functional studies and cloning of the pspA gene. J.
Bacteriol. 174:610.
[0575] McDaniel L. S., J. S. Sheffield, P. DeLucchi and D. E.
Briles. 1991. PspA, a surface protein of Streptococcus pneumoniae,
is capable of eliciting protection against pneumococci of more than
one capsular type. Infect. Immun. 59:222.
[0576] McDaniel L. S., B. A. Ralph, D. O. McDaniel and D. E.
Briles. 1994. Localization of protection-eliciting epitopes on PspA
of Streptococcus pneumoniae between amino acid residues 192 and
260. Micro. Pathogenesis 17:323.
[0577] McDaniel L. S., K. Scott, J. F. Kearney and D. E. Briles.
1984. Monoclonal antibodies against protease sensitive pneumococcal
antigens can protect mice from fatal infection with Streptococcus
pneumoniae. J. Exp. Med. 160:386.
[0578] Davis R. W., W. D. Boststein and J. R. Roth. 1980. A manual
for genetic engineering: Advanced bacterial genetics. Cold Spring
Harbor, N.Y.: Cold Spring Harbor Laboratory, 201.
[0579] Hanahan D. 1983. Studies on transformation of Escherichia
coli with plasmids. J. Mol. Biol. 166:557.
[0580] Birnboim B. C. and J. Doly. 1979. A rapid alkaline
extraction procedure for screening recombinant plasmid DNA. Nuc.
Acids Res. 7:1513.
[0581] Osborn M. J. and J. Munson. 1974. Separation of the inner
(cytoplasmic) and outer membranes of gram negative bacteria Methods
Enzymol. 31A:642.
[0582] Wicker L. S. and I. Scher. 1986. X-linked immune deficiency
(Xid) of CBA/N mice. New York: Apringer-Verlag, 86.
[0583] Ansbaugh D. F., C. T. Hansen, B. Prescott, P. W. Stasbak, D.
R. Barthold and P. J. Baker. 1972. Genetic control of the antibody
response to type III pneumococcal polysaccharide in mice. I.
Evidence that an X4inked gene plays a decisive role in determining
responsiveness. J. Exp. Med. 136:931.
[0584] Briles D. E., M. Nahm, K. Schoer, J. Davie, P. Baker, J. F.
Kearney and R. Barletta. 1981. Anti-phosphocholine antibodies found
in normal mouse serum are protective against intravenous infection
with type 3 S. pneumoniae. J. Exp. Med. 153:694.
[0585] McDaniel L. S., J. S. Sheffield, E. Swiatlo, J. Yother, M.
J. Crain and D. E. Briles. 1992. Molecular localization of variable
and conserved regions of pspA and identification of additional pspA
homologous sequences in Streptococcus pneumoniae. Microbial
Pathogenesis 13:261.
[0586] Alexander, J. E., Lock, R. A., Peeters, C. C. A. M.,
Poolman, J. T., Andrew, P. W., Mitchell, T. J., Hansman, D., and
Paton, J. C. (1994) Immunization of mice with pneumolysin toxoid
confers a significant degree of protection against at least nine
serotypes of Streptococcus pneumoniae. Infect Immun 62:
5683-5688.
[0587] Avery, O. T., McLeod, C. M., and McCarty, M. (1944) 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 79: 137-158.
[0588] Briles, D. E., Nahm, M., Schroer, K., Davie, J., Baker, P.,
Keamey, J. F., and Barletta, R. (1981) Anti-phosphocholine
antibodies found in normal mouse serum are protective against
intravenous infection with type 3 S. pneumoniae. J Exp Med 153:
694-705.
[0589] Crain, M. J., Waltman, W. D. II, Tumer, J. S., Yother, J.,
Talkington, D. E., McDaniel, L. M., Gray, B. M., and Briles, D. E.
(1990) Pneumococcal surface protein A (PspA) is serologically
highly variable and is expressed by all clinically important
capsular serotypes of Streptococcus pneumoniae. Infect Immun 58:
3293-3299.
[0590] Haanes-Fritz, E., Kraus, W., Burdett, V., Dale, J. B.,
Beachey, E. H., and Cleary, P. (1988) Comparison of the leader
sequences of four group A streptococcal M protein genes. Nucl Acids
Res 16: 4667-4677.
[0591] McDaniel, L. S., Yother, J., Vijayakumar, M., McGarry, L.,
Guild, W. R., and Briles, D. E. (1987) Use of insertional
inactivation to facilitate studies of biological properties of
pneumococcal surface protein A (PspA). J Exp Med 165: 381-394.
[0592] McDaniel, L. S., Sheffield, J. S., Delucchi, P., and Briles,
D. E. (1991) PspA, a surface protein of Streptococcus pneumoniae,
is capable of eliciting protection against pneumococci of more than
one capsular type. Infect Immun 59: 222-228.
[0593] McDaniel, L. S., Sheffield, J. S., Swiatlo, E., Yother, J.
Crain, M. J., and Briles, D. E. (1992) Molecular localization of
variable and conserved regions of pspA, and identification of
additional pspA-homologous sequences in Streptococcus pneumoniae.
Microbial Pathogenesis 13: 261-269.
[0594] McDaniel, L. S., Ralph, B. A., McDaniel, D. O., and Briles,
D. E. (1994) Localization of protection-eliciting epitopes on PspA
of Streptococcus pneumoniae between amino acid residues 192 and
260. Microbial Pathogenesis 17: 323-337.
[0595] Meinkoth, J., and Wahl, G. (1984) Hybridization of nucleic
acids immobilized on solid supports. Anal Biochem 138: 267-284.
[0596] Sampson, J. S., O'Connor, S. P., Stinson, A. R., Tharpe, J.
A., and Russell, H. (1994) Cloning and nucleotide sequence analysis
of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton
protein homologous to previously reported Streptococcus sp.
adhesins. Infect Immun 62: 319-324.
[0597] Shoemaker, N. B., and Guild, W. R. (1974) Destruction of low
efficiency markers is a slow process occurring at a heteroduplex
stage of transformation. Mol Gen Genet 128: 283-290.
[0598] Siber, G. R. (1994) Pneumococcal disease: prospects for a
new generation of vaccines. Science 265:1385-1387.
[0599] Talkington, D. F., Crimnins, D. L, Voellinger, D. C.,
Yother, J., and Briles, D. E. (1991) A 43-kilodalton pneumococcal
surface protein, PspA: isolation, protective abilities, and
structural analysis of the amino-terminal sequence. Infect Immun
59:1285-1289.
[0600] Waltman, W. D. II, McDaniel, L. S., Gray, B. M., and Briles,
D. E. (1990) Variation in the molecular weight of PspA
(pneumococcal surface protein A) among Streptococcus pneumoniae.
Microbial Pathogenesis 8: 61-69.
[0601] Yother, J., McDaniel, L. S., and Briles, D. E. (1986)
Transformation of encapsulated Streptococcus pneumoniae. J
Bacteriol 168: 1463-1465.
[0602] Yother, J., and Briles, D. E. (1992) Structural properties
and evolutionary relationships of PspA, a surface protein of
Streptococcus pneumoniae, as revealed by sequence analysis. J
Bacteriol 174: 601-609.
[0603] Yother, J., Handsome, G. L., and Briles, D. E. (1992)
Truncated forms of PspA that are secreted from Streptococcus
pneumoniae and their use in functional studies and cloning of the
pspA gene. J Bacteriol 174: 610-618.
[0604] Yother, J., and White, J. M. (1994) Novel surface attachment
mechanism of the Streptococcus pneumoniae protein PspA. J Bacterial
176: 2976-2985.
[0605] Anonymous. Pneumococcal polysaccharide vaccine. MMWR 1981,
30, 410-419
[0606] Farley, J. J., King, J. C., Nair, P., Hines, S. E.,
Tressler, R. L., Vink, P. E. Invasive pneumococcal disease among
infected and uninfected children of mothers with immunodeficiency
virus infection. J. Pediatr. 1994,124, 853-858
[0607] Schwartz, B., Gove, S., Lob-Lovit, J., 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,
[0608] Avery, O. T., Goebel, W. F. Chemoimmunological studies of
the soluble specific substance of pneumococcus. I. The isolation
and properties of the acetyl polysaccharide of pneumococcus type 1.
J. Exp. Med. 1933, 58, 731 - 735
[0609] Austrian, R. Pneumococcal Vaccine: Development and
Prospects. Am. J. Med 1979, 67, 547-549
[0610] Shapiro, E. D., Berg, A. T., Austrian, R., Schroeder, D.,
Parcells, V., Margolis, A., Adair, R. K, Clemmens, J. D. Protective
efficacy of polyvalent pneumococcal polysaccharide vaccine. N.
Engl. J. Med 1991, 325, 1453-1460
[0611] Fedson, D. S. Pneumococcal vaccination in the prevention of
community-acquired pneumonia: an optimistic view of
cost-effectiveness. Sem. Resp. Infect. 1993, 8, 285-293
[0612] Gotschlich, E. C., Goldschneider, I., Lepow, M. L., Gold, R.
The immune response to bacterial polysaccharides in man. In:
Antibodies in human diagnosis and therapy, (Ed. Haber, E., Krause,
R. M.) Raven, N.Y., 1977, 391-402.
[0613] Cowan, M. J., Anmnann, A. J., Wara, D. W., Howie, V. M.,
Schultz, L., Doyle, N., Kaplan, M. Pneumococcal polysaccharide
immunization in infants and children. Pediatrics 1978, 62,
721-727
[0614] Mond, J. J., Lees, A., Snapper, C. M. T cell-independent
antigens type 2. Ann. Rev. Immunol. 1995, 13, 655-692
[0615] Stein, K. E. Thymus-independent and thymus-dependent
responses to polysaccharide antigens. J Infect. Dis. 1992, 162,
S49
[0616] Chiu, S. S., Greenberg, P. D., Marcy, S. M., Wong, V. K.,
Chang, S. J., Chiu, C. Y., Ward, J. I. Mucosal antibody responses
in infants following immunization with Haemophilus influenzae.
Pediatr. Res. Abstracts 1994, 35, 10A
[0617] Kauppi, M., Eskola, J., Kathty, H. H. influenzae type b
(Hib) conjugate accines induce mucosal IgA1 and IgA2 antibody
responses in infants and hildren. ICAAC Abstracts 1993, 33, 174
[0618] Dagen, R., Melamed, R., Abramson, O., Piglansky, L.,
Greenberg, D., Mendelman, P. M., Bohidar, N., Ter-Minassian, D.,
Cvanovich, N., Lov, D., Rusk, C., Donnelly, J., Yagupsky, P. Effect
of heptavalent pneumococcal-OMPC conjugate vaccine on
nasopharyngeal carriage when administered during the 2nd year of
life. Pediatr. Res. 1995, 37, 172A
[0619] Fattom, A., Vann, W. F., Szu, S. C., Sutton, A., Bryla, D.,
Shiffinan, G., Robbins, J. B., Schneerson, R. Synthesis and
physiochemical and immunological characterization of pneumococcus
type 12F polysaccharide-diptheria toxoid conjugates. Infect Immun.
1988,56,2292-2298
[0620] Kennedy, D., Derousse, C., E., A. 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
[0621] McDaniel, L. S., Ralph, B A., McDaniel, D. O., Briles, D. E.
Localization of protection-eliciting epitopes on PspA of
Streptococcus pneumoniae between amino acid residues 192 and 260.
Microb. Pathog 1994, 17, 323-337
[0622] Langermann, S., Palaszynski, S. R., Burlein, J. E., Koenig,
S., Hanson, M. S., Briles, D. E, Stover, C. K. Protective humoral
response against pneumococcal infection in mice elicited by
recombinant Bacille Calmette-Gurin vaccines expressing PspA. J.
Exp. Med. 1994, 180, 2277-2286
[0623] Siber, G. R. Pneumococcal disease: prospects for a new
generation of vaccines. Science 1994, 265, 1385-1387
[0624] Lock, R. A., Hansman, D., Paton, J. C. Comparative efficacy
of autolysin and pneumolysin as immunogens protecting mice against
infection by Streptococcus pneumoniae. Microb. Pathog. 1992, 12,
137-143
[0625] Sampson, J. S., O'Connor, S. P., Stinson, A. R., Tharpe, J.
A., Russell, H. Cloning and nucleotide sequence analysis of psaA,
the Streptococcus pneumoniae gene encoding a 37-kilodalton protein
homologus to previously reported Streptococcus sp. adhesins. Infect
Immun. 1994, 62, 319
[0626] Paton, J. C., Lock, R. A., Lee, C. -J., Li, J. P., Berry, A.
M., Mitchell. Purification and immunogenicity of genetically
obtained pneumolysin toxoids and their conjugation to Streptococcus
pneumoniae type 19F polysaccharide. Infect. Immun. 1991, 59,
2297-2304
[0627] McDaniel, L. S., Scott, G., Kearney, J. F., 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
[0628] Briles, D. E., Forman, C., Horowitz, J. C., Volanakis, J.
E., Benjamin, W. H., Jr., McDaniel, L. S., Eldridge, J., Brooks, J.
Antipneumococcal effects of C-reactive protein and monoclonal
antibodies to pneumococcal cell wall and capsular antigens. Infect.
Immun. 1989, 57, 1457-1464
[0629] McDaniel, L. S., Sheffield, J. S., Delucchi, P., Briles, D.
E. PspA, a surface protein of Streptococcus pneumoniae, is capable
of eliciting protection against pneumococci of more than one
capsular type. Infect. Immun. 1991, 59, 222-228
[0630] McDaniel, L. S., Yother, J., Vijayakumar, M., McGarry, L.,
Guild, W. R., Briles, D. E. Use of insertional inactivation to
facilitate studies of biological properties of pneumococcal surface
protein A (PspA). J. Exp. Med. 1987, 165, 381-394
[0631] Yother, J., McDaniel, L. S., Crain, M. J., Talkington, D.
F., Briles, D. E. Pneumococcal surface protein A: Structural
analysis and biological significance In: Genetics and Molecular
Biology of Streptococci, Lactococci, and Enterococci, (Ed. Dunny,
G. M., Cleary, P P., McKay, L. L.) American Society for
Microbiology, Washington, DC, 1991, 88-91.
[0632] Waltman, W. D., II, McDaniel, L. S., Gray, B. M., Briles, D.
E. Variation in the molecular weight of PspA (Pneumococcal Surface
Protein A) among Streptococcus pneumoniae. Microb. Pathog. 1990, 8,
61-69
[0633] Crain, M. J., Waltman, W. D., II, Turner, J. S., Yother, J.,
Talkington, D. E., McDaniel, L. M., Gray, B. M., Briles, D. E.
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
[0634] McDaniel, L. S., Scott, G., Widenhofer, K., Carroll, Briles,
D. E. Analysis of a surface protein of Streptococcus pneumoniae
recognized by protective monoclonal antibodies. Microb. Pathog.
1986, 1, 519-531
[0635] Tart, R. C., McDaniel, L. S., Ralph, B. A., Briles, D. E.
Truncated Streptocccus pneumoniae PspA molecules elicit
cross-protective immunity against pneumococcal challenge in mice.
J. Infect Dis. 1995, In Press,
[0636] Yother, J., Briles, D. E. Structural properties and
evolutionary relationships of PspA, a surface protein of
Streptococcus pneumoniae, as revealed by sequence analysis. J.
Bact. 1992, 174, 601-609
[0637] Talkington, D. F., Crimmins, D. L., Voellinger, D. C.,
Jother, J., Briles, D. E. A 43-kilodalton pneumococcal surface
protein, PspA: isolation, protective abilities, and structural
analysis of the amino-terminal sequence. Infect. Immun. 1991, 59:,
1285-1289
[0638] McDaniel, L. S., McDaniel, D. O. Genetic analysis of the
gene encoding type 12 PspA of Streptococcus pneumoniae strain
EF5668 In: Genetics of the streptococci, enterocococci, and
lactococci, (Ed. Feretti, J. J., Gilmore, M. S., Khenhammer, T. R.,
Brown, F.) Dev. Biol. Stand. Basel Krager, Basel, 1995,
283-286.
[0639] Fischetti, V. A., Pancholi, V., Schneewind, O. Conservation
of a hexapeptide sequence in the anchor region of surface proteins
from gram-positive cocci. Mol. Microbiol. 1990, 4, 1603-1605
[0640] Schneewind, O., Fowler, A., Faull, K. F. Structure of cell
wall anchor of cell surface proteins in Staphylococcus aureus.
Science 1995, 268, 103-106
[0641] Yother, J., White, J. M. Novel surface attachment mechanism
for the Streptococcus pneumoniae protein PspA. J. Bact. 1994, 176,
2976-2985
[0642] McDaniel, L. S., Brooks-Walter, A., Briles, D. E., Swiatlo,
E. Oligonucleotides identify conserved and variable regions of pspA
and pspA-like sequences of Streptococcus pneumoniae. Mol.
Microbiol. Submitted,
[0643] Yother, J., Handsome, G. L., 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, 610-618
[0644] Talkington, D. F., Voellinger, D. C., McDaniel, L. S.,
Briles, D. E. Analysis of pneumococcal PspA microheterogeneity in
SDS polyacrylamide gels and the association of PspA with the cell
membrane. Microb. Pathog. 1992, 13, 343-355
[0645] Smith, M. D., Guild, W. R. A plasmid in Streptococcus
pneumoniae. J. Bacterial. 1979, 137, 735-739
[0646] Shoemaker, N. B., Guild, W. R. Destruction of low efficiency
markers is a slow process occurring at a heteroduplex stage of
transformation. Mol. Gen. Genet. 1974, 128, 283-290
[0647] Raven, A. W. Reciprocal capsular transformations of
pneumococci. J. Bact. 1959, 71, 296-309
[0648] McDaniel, L. S., Sheffield, J. S., Swiatlo, E., Yother, J.,
Crain, M. J., Briles, D E. Molecular localization of variable and
conserved regions of pspA, and identification of additional pspA
homologous sequences in Streptococcus pneumoniae. Microb. Pathog.
1992, 13, 261-269
[0649] Brooks-Walter, A., McDaniel, L. S., Hollingshead, S. K.,
Briles, D. E. Restriction fragment length polymorphisms of pspA of
Streptococcus pneumoniae reveal a genetic polymorphism.
Submitted
[0650] van de Rijn, I., Kessler, R E. Growth characteristics of
Group A Streptococci in a new chemically defined medium. Infec.
Immun. 1980, 27, 444-448
[0651] Waltman, W. D., II, McDaniel, L. S., Andersson, B., Bland,
L., Gray, B. M., Svanborg-Eden, C., Briles, D. E. Protein
serotyping of Streptococcus pneumoniae based on reactivity to six
monoclonal antibodies. Microb. Pathog. 1988, 5, 159-167
[0652] Tomasz, A. Surface components of Streptococcus pneumoniae.
Rev. Infect. Dis 1981, 3, 190-211
[0653] Garcia, J. L., Garcia, E., Lopez, R. Overproduction and
rapid purification of the amidase of Streptococcus pneumoniae.
Arch. Microbiol. 1987, 149, 52-56
[0654] Osborn, M. J., Munson, J. Separation of the inner
(cytoplasmic) and outer membranes of gram negative bacteria.
Methods Enzymol. 1974, 31A, 642-653
[0655] Briles, D. E., Horowitz, J., McDaniel, L. S., Benjamin, W.
H., Jr., Claflin, J. L., Booker, C. L., Scott, G., Forman, C.
Genetic control of susceptibility to pneumococcal infection. Curr.
Top. Microbiol. Imnmunol. 1986, 124, 103-120
[0656] Briles, D. E., Crain, M. J., Gray, B. M., Forman, C.,
Yother, J. A strong association between capsular type and mouse
virulence among human isolates of Streptococcus pneumoniae. Infect
Immun. 1992, 60, 111-116
[0657] Musher, D. M., Raizan, K. R., Weinstein, L. The effect of
Listeria monocytogenes on resistance to pneumococcal infection.
Soc. Exp. Bio., and Med. 1970, 135, 557-560
[0658] Roberts, P., Jeffery, P. K., Mitchell, T. J., Andrew, P. W.,
Boulnois, G. J., Feldman, C., Cole, P. J., Wilson, R. Effect of
immunization with Freund's adjuvant and pneummolysin on histologic
features of pneumococcal infection in the rat lung in vivo. Infect.
Immun. 1992, 60, 4969-4972
[0659] Weigle, W. O. Immunological unresponsiveness In: Adv.
Immunol., (Ed. Dixon, J. F., Kunkel, H. G.) Academic Press, New
York, N.Y., 1973, 61-162.
[0660] Alexander, J. E., Lock, R. A., Peeters, C. C. A. M.,
Poolman, J. T., Andrew, P. W., Mitchell, T. J., Hansman, D., Paton,
J. C. 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
[0661] Berry, A. M., Lock, R. A., Hansman, D., Paton, J. C.
Contribution of autolysin to virulence of Streptococcus pneumoniae.
Infect. Immun. 1989, 57, 2324-2330
[0662] Lock, R. A., Paton, J. C., Hansman, D. Purification and
immunologic characterization of neuraminidase produced by
Streptococcus pneumoniae. Microb. Pathog. 1988, 4, 33-43
[0663] Talkingtou, D., Koenig, A., Russell, H. The 37 kDa protein
of Streptococcus pneumoniae protects mice against fatal challenge.
American Society of Microbiology Abstracts 1992, 149
[0664] Dillard, J. P., Yother, J. Genetic and molecular
characterization of capsular polysaccharide biosynthesis in
Streptococcus pneumoniae type 3. Mol. Microbiol. 1994, 12,
959-972
[0665] 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
[0666] Briles, D. E., Nahm, M., Schroer, K, Davie, J., Baker, P.,
Keamey, J., 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
[0667] Amsbaugh, D. F., Hansen, C. T., Prescott, B., Stashak, P.
W., Barthold, D. R., Baker, P. J 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
[0668] Avery, O. T., MacLeod, C. M., McCarty, M. 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
[0669] McCarty, M. The transforming principle. Norton, N.Y., 1985,
252.
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