Note: Descriptions are shown in the official language in which they were submitted.
CA 0222401~ 1997-12-08
WO9~'4~28 PCT/CA96/00322
STREPTOCOCCAL ~EAT S~OCR PROTEINS
Ml;!MRl;'.R.~ OF T~E HSP70 FANILY
L~NlCA~ FIELD OF T~E lNVL~.~lON
This invention relates to novel heat shock
proteins of Streptococcus pneumoniae, Streptococcus
pyogenes and Streptococcus agalactiae and immunologically
related polypeptides, which provide the basis for new
immunotherapeutic, prophylactic and diagnostic agents
useful in the treatment, prevention and diagnosis of
disease. More particularly, this invention relates to
heat shock proteins of S. pneumoniae, S. pyogenes and S.
agalactiae, members of the HSP70 family which have an
apparent molecular mass of 70-72 kilodaltons, to the
corresponding nucleotide and derived amino acid sequences,
to recombinant DNA methods for the production of
HSP70/HSP72 and immunologically related polypeptides, to
antibodies that bind to these HSP's, and to methods and
compositions for the diagnosis, prevention and treatment
of diseases caused by S. pneumoniae and related bacteria,
such as Streptococcus pyogenes and Streptococcus
agalactiae
BACKGROUND OF THE INVENTION
S. pneumoniae is an important agent of disease
in humans, especially among infants, the elderly and
immunocompromised persons. It is a bacterium frequently
isolated from patients with invasive diseases such as
bacteraemia/septicaemia, pneumonia, and men;ngitis with
high morbidity and mortality throughout the world.
Although the advent of antimicrobial drugs has reduced the
overall mortality from pneumococcal diseases, the presence
of resistant pneumococcal organisms has become a major
problem in the world today. Effective pneumococcal
vaccines could have a major impact on the morbidity and
mortality associated with S. pneumoniae disease. Such
CA 0222401~ 1997-12-08
W096/40928 PCT/CA96/00322
vaccines would also potentially be useful to prevent
otitis media in infants and young children.
It is clear that a number of pneumococcal
factors are potentially important in the pathogenesis of
disease [G.J. Boulnois, J. Gen. Microbiol., 138, pp. 249-
259 (1992); C.J. Lee et al., Crit. Rev. Microbiol., 18,
pp. 89-114 (1991)]. The capsule of the pneumococcus,
despite its lack of toxicity, is considered to be the sine
qua non of pneumococcal virulence. More than 80
pneumococcal capsular serotypes are identified on the
basis of antigenic differences. Antibodies are the
mechanism of protection and the importance of anticapsular
antibodies in host defenses against S. pneumoniae is well
established [R. Austrian, Am. J. Med., 67, pp. 547-549
(1979)]. Nevertheless, the currently available
pneumococcal vaccine, comprising 23 capsular
polysaccharides that most frequently caused disease, has
significant shortcomings such as the poor ;mmllnogenicity
of capsular polysaccharides, the diversity of the
serotypes and the differences in the distribution of
serotypes over time, geographic areas and age groups. In
particular, the failure of existing vaccines to protect
young children against most serotypes has spurred
evaluation of other S. pneumoniae components. Increasing
2s evidence indicates that certain pneumococcal proteins may
play an active role both in terms of protection and
pathogenicity [J.C. Paton, Ann. Rev. Microbiol., 47,
pp. 89-115 (1993)]. So far, however, only a few S.
pneumoniae proteins have been studied. This might result
from the lack of protein-specific antibodies which renders
difficult the study of the role of protein antigens in
protection and pathogenicity. It is believed that the
pneumococcal protein antigens are not very immunogenic and
that most antibody responses are to the phosphocholine and
the capsular polysaccharides [L.S. McDaniel et al., J.
Exp. Med., 160, pp. 386-397 (1984); R.M. Krause, Adv.
Immunol., 12, pp. 1-56 (1970); D.G. Braun et al., J. Exp.
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
Med., 129, pp. 809-830 (1969)]. In a study using X-linked
immunodeficient mice, which respond poorly to carbohydrate
antigens and to phosphocholine, but make relatively normal
responses to protein antigens, the frequency for obtaining
monoclonal antibodies reactive with pneumococcal protein
antigens was less than 10%, thus suggesting that S.
pneumoniae proteins are poor immunogens [McDaniel et al.,
supra].
Streptococcus agalactiae, also called Group B
Streptococcus (GBS),is the most common cause of sepsis
(blood infection) and meningitis in newborns. GBS is also
a frequent cause of newborn pneumonia. Approximately
8,000 babies in the United States get GBS disease each
year; 5%-15% of these babies die. Babies that survive,
particularly those who have meningitis, may have long-term
problems, such as hearing or vision loss or learning
disabilities. In pregnant women, GBS can cause urinary
tract infections, womb infections (amnionitis,
endometritis), and stillbirth. Among women who are not
pregnant and men, the most common diseases caused by GBS
are blood infections, skin or soft tissue infections, and
pneumonia. Approximately 20% of men and nonpregnant women
with GBS disease die of the disease. GBS infections in
both newborns and adults are usually treated with
antibiotics (e.g., penicillin or ampicillin) given
intravenously. Most GBS disease in newborns can be
prevented by giving certain pregnant women antibiotics
intravenously during labor. Vaccines to prevent GBS
disease are being developed. In the future, it is
expected that women who will be vaccinated will make
antibodies that cross the placenta and protect the baby
during birth and early infancy.
Since the 1980s, Streptococcus pyogenes, also
called Group A Streptococcus (GAS) is reemerging as a
cause of severe diseases which would be due to an increase
CA 0222401~ 1997-12-08
W0~ 0~28 PCT/CA96/00322
in virulence of the organism. GAS causes pharyngitis,
commonly called "strep throat", and skin infections
(impetigo, erysipelas/cellulitis). "Strep throat" and
impetigo can lead to glomerulonephritis (kidney damage).
s Approximately 3~ of "strep throat" infections result into
rheumatic fever (migrating arthritis) whose complications
include chorea (neurological symptoms) and, in 50% of the
cases, rheumatic heart disease (heart valve damage) with
endocarditis as a possible long term consequence. It is
important to treat impetigo and "strep throat" with
antibiotics to prevent the development of complications.
Infection with toxin-producing strains can result in
scarlet fever (diffuse rash and fever) or in the extremely
severe streptococcal toxic shock syndromes (TSS; GAS have
been termed ~flesh eating bacteria') which are
characterized by the rapid development of shock and
multiple organ system failure. TSS have a 30 to 70%
fatality rate in spite of aggressive treatment involving
the removing of the focus of bacterial infection and
antibiotic therapy. The incidence of TSS is 10 to 20
cases per 100,000. No vaccine against GAS is presently
available.
Heat shock or stress proteins ("HSPs") are among
the most highly conserved and abundant proteins found in
2s nature [F.C. Neidhardt et al., Ann. Rev. Genet., 18,
pp. 295-329 (1984); S. Lindquist, Ann. Rev. Biochem., 55,
pp. 1151-1191 (1986)]. They are produced by all cells in
response to various physiological and nonphysiological
stimuli. The heat shock response, in which a sudden
increase in temperature induces the synthesis of HSPs, is
the best studied of the stress responses. Other
environmental conditions such as low pH, iron deficiency
and hydrogen peroxyde can also induce HSPs. The HSPs have
been defined by their size, and members of hsp90, hsp70,
and hsp60 families are among the major HSPs found in all
prokaryotes and eukaryotes. These proteins fulfill a
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W O 9f'~:C328 PCT/CA96/00322
variety of chaperon functions by aiding protein folding
and assembly and assisting translocation across membranes
[C. Georgopoulos and W.J. Welch, Ann. Rev. Cell. Biol., 9,
pp. 601-634 (1993)i D. Ang et al., J. Biol. Chem., 266,
s pp. 24233-24236 (1991)]. As molecular chaperons and
possibly via other mechanisms, HSPs are likely involved in
protecting cells from the deleterious effects of stress.
The fact that several virulence factors are regulated by
environmental conditions suggests a role for HSPs in
microbial pathogenicity [J.J. Mekalanos, J. Bacteriol.,
174, pp. 1-7 (1992); P.J. Murray and R.A. Young, J.
Bacteriol., 174, pp. 4193-4196 (1992)]. In that respect,
recent studies on Salmonella species suggest that the
stress response might be critically linked to the ability
of intracellular pathogens to initiate and sustain an
infection [N.A. Buchmeir and F. Heffron, Science, 248,
pp. 730-732 (1990); K.Z. Abshire and F.C. Neidhardt,
J. Bacteriol., 175, pp. 3734-3743 (1993); B.B. Finlay
et al., Science, 243, pp. 940-943 (1989)]. Others have
demonstrated that lysteriolysin, an essential virulence
factor in L. monocytogenes, is induced under heat shock
conditions [Z. Sokolovic and W. Goebel, Infect. Immun.,
57, pp. 295-298 (1989)].
Evidence is now accumulating that HSPs are major
antigens of many pathogens. Members of the hsp60 family,
also called GroEL-related proteins for their similarity to
the E. coli GroEL protein, are major antigens of a variety
of bacterial pathogens including Mycobacterium leprae and
Mycobacterium tuberculosis [D. Young et al., Proc. Natl.
Acad. Sci. USA, 85, pp. 4267-4270 (1988)], Legionella
pneumophila [B.B. Plikaytis et al., J. Clin. Microbiol.,
25, pp. 2080-2084 (1987)], Borrelia burgdorferi [B.J. Luft
et al., J. Immunol., 146, pp. 2776-2782 (1991)], and
Chlamydia trachomatis [E.A. Wagar et al., J. Infect. Dis.,
162, pp. 922-927 (1990)]. This antigen is a homologue of
the ubiquitous "common antigen", and is believed to be
present in every bacterium [J.E. Thole et al., Microb.
CA 0222401~ 1997-12-08
WO9.l4C928 PCT/CA96/00322
Pathogen., 4, pp. 71-83 (1988). Antibodies to the members
of the hsp70 family, or DnaK-related proteins, have also
been described for several bacterial and parasitic
infections [Young et al., supra; Luft et al., supra; D.M.
s Engman et al., J. Immunol., 144, pp. 3987-3991 (1990);
N.M. Rothstein et al., Molec. Biochem. Parasitol., 33,
pp. 229-235 (1989); V. Nussenzweig and R.S. Nussenzweig,
Adv. Immunol., 45, pp. 283-334 (1989)]. HSPs can elicit
strong B- and T- cell responses and it was shown that 20%
of the CD4' T-lymphocytes from mice inoculated with M.
tuberculosis were reactive to the hsp60 protein alone
[S.H.E. Kaufman et al., Eur. J. Immunol., 17, pp. 351-357
(1987)]. Similarly, 7 out of a collection of 24
monoclonal antibodies to M. leprae proteins recognized
determl~nts on hsp60 [H.D. Engers et al., Infect. Immun.,
48, pp. 603-605 (1985)]. It seems that the immune
response to stress proteins might play an important role
in protection against infection. Consistent with that is
the demonstration that antibodies and T cells reactive
with microbial HSPs can exhibit neutralizing and
protective activities [A. Noll et al., Infect. Immun., 62,
pp. 2784-2791 (1994); and S.L. Danilition et al., Infect.
Immun., 58, pp. 189-196 (1990)]. The immunological
properties of stress proteins make them attractive as
2s vaccine components and several HSPs are presently being
considered for preventing microbial infection and treating
cancer. So far, however, studies have focused on
intracellular pathogens such as Mycobacteria, Salmonella,
Chlamydia and several parasites. Information concerning
the heat shock protein antigens in extracellular gram-
positive bacteria is far less documented. In S.
pneumoniae, S. pyogenes and S. agalactiae, neither the
heat shock proteins nor their gene structures have been
identified.
DISCLOSURE OF THE INVENTION
The present invention addresses the problems
referred to above by providing novel heat shock proteins
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W O 96/40928 PCT/CA96/00322
from S. pneumoniae, S. pyogenes and S. agalactiae, and
immunologically related polypeptides. Also provided are
DNA sequences that code for the foregoing polypeptides,
vectors containing the polypeptides, unicellular hosts
transformed with those vectors, and a process for making
substantially pure, recombinant polypeptides. Also
provided are antibodies specific to the foregoing
polypeptides. The polypeptides, DNA sequences and
antibodies of this invention provide the basis for novel
methods and pharmaceutical compositions for the detection,
prevention and treatment of disease. Particularly, this
invention provides a novel vaccine based on fragments of
these polypeptides that are specific to streptococcal
strains.
The novel heat shock protein is the
approximately 72 kDa heat shock protein of Streptococcus
pneumoniae ("HSP72") (SEQ ID NO:5), the approximately 70
kDa heat shock protein of Streptococcus pyogenes ("HSP70")
(SEQ ID NO:20)and the approximately 70 kDa heat shock
protein of Streptococcus agalactiae ("HSP70") (SEQ ID
NO:22), including analogues, homologues, and derivatives
thereof, and fragments of the foregoing polypeptides
containing at least one immunogenic epitope. Preferred
fragments of HSP70/72 include the C-terminal portion of
the HSP70/72 polypeptides. More particularly,it includes
the C_terminal 169-residue fragment ("C-169") (residues
439-607, SEQ ID NO:5), the C-terminal 151-residue fragment
("C-151") (residues 457-607, SEQ ID No:5),and smaller
fragments consisting of peptide epitopes within the C-169
region. Particularly preferred fragments within the C-169
region of HSP72 include the peptide sequences
GFDAERDAAQAALDD (residues 527-541 of SEQ ID NO:5) and
AEGAQATGNAGDD W (residues 586-600 of SEQ ID NO:5), which
are exclusive to HSP72 of Streptococcus pneumoniae. Even
more preferred are fragments that elicit an immune
reaction against S. pneumoniae, S. pyogenes and S.
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W O g~'4D328 PCT/CA96/00322
agalactiae but do not provoke auto-immune reaction in a
human host. Such fragments may be selected from the
following peptides: CS870, CS873, CS874, CS875, CS876,
CS877, CS878, CS879, CS880, CS882, MAPl, MAP2, MAP3 and
s MAP4 (see TABLE 5, supra).
Preferred antibodies of this invention are the
Fl-Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 monoclonal
antibodies ("MAbs"), which are specific to HSP72.
More preferred antibodies are the F2-Pn3.2 and
F2-Pn3.4 monoclonal anibodies that are specific to both
HSP 70 and HSP72. Even more preferred are the Fl-Pn3.1
antibodies that are specific for Streptococcus pneumoniae.
The preferred polypeptides and antibodies of
this invention provide the basis for novel methods and
ls pharmaceutical compositions for the detection, prevention
and treatment of pneumococcal diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a fluorogram, which shows the
effect of heat shock on S. pneumoniae protein synthesis.
The cell extracts in panel A are S. pneumoniae type 6
strain 64. The cell extracts in panel B are S. pneumoniae
type 4 strain 53. The cell extracts in the odd numbered
lanes were incubated at 37~C. The cell extracts in the
even numbered lanes were incubated at 45~C for 5 minutes.
The cell extracts were then labeled with [35S]methionine
for 10 minutes (lanes 1, 2 and 7, 8), 30 minutes (lanes 3,
4 and 9, 10), or 60 minutes (lanes 5, 6). Molecular mass
markers in kilodaltons are shown to the left. The
positions of HSP80, HSP72 and HSP62 are shown by arrows at
the right-hand side of each panel.
FIG. 2 is a graphical depiction of a comparison
of the electrophoretic profiles of [35S]methionine-labeled
proteins in S. pneumoniae in the presence (----) or
absence ( _ ) of exposure to heat shock. Densitometric
tracings were determined by measuring the relative optical
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W096/40928 PCT/CA9~0~322
density (Y axis) vs. the mobility of labeled protein bands
(X axis). The densitometric scans of the SDS PAGE of FIG.
1, lanes 1 and 2, is shown.
FIG. 3 depicts a fluorogram, which shows the
S S. pneumoniae protein antigens immunoprecipitated by sera
from mice ;mmlln;zed with detergent-soluble S. pneumoniae
protein extract. [35S]methionine-labeled proteins from
S. pneumoniae grown at 37~C and incubated at 37~C (lanes 3,
5, 7 and 9) or heat-shocked at 45~C ( lanes 4, 6, 8 and 10)
were immunoprecipitated with sera from mouse 1 (lanes 3 to
6) or mouse 2 (lanes 7 to 10) and then analyzed by SDS-
PAGE and fluorography. The sera were tested after the
first (lanes 3,4 and 7,8) and after the second (lanes 5,6
and 9,10) ;mmllnization. Cell lysates from [35S]methionine-
labeled non heat-shocked and heat-shocked S. pneumoniae
are shown in lanes 1 and 2, respectively. The position of
HSPs is indicated by the arrows at the left of the
fluorogram.
FIG. 4 depicts a fluorogram, which shows the
20 S. pneumoniae protein antigens immunoprecipitated by sera
from mice ;mmlln;zed with heat-killed S. pneumoniae
bacteria. [35S]methionine-labeled proteins from
S. pneumoniae grown at 37~C and incubated at 37~C (lanes 3,
5 and 7) or heat-shocked at 45~C ( lanes 4, 6 and 8) were
25 immunoprecipitated with sera from mouse 1 (lanes 3,4),
mouse 2 (lanes 5, 6) or mouse 3 ( lanes 7, 8) and then
analyzed by SDS-PAGE and fluorography. Sera were tested
after the second immunization only. Cell lysates from
[35S]methionine-labeled non heat- and heat-shocked
S. pneumoniae are shown in lanes 1 and 2, respectively.
The position of HSPs is indicated by the arrows at the
left of the fluorogram.
FIG. 5 depicts a photograph, which shows the
S. pneumoniae antigens detected by Western blot analysis.
Whole cell extracts were probed with sera from 15 mice
(lanes 1-15) ;mmlln;zed with heat-killed S. pneumoniae
bacteria. Lane 16 shows the HSP72 protein detected by MAb
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W096/40928 PCT/CA96/00322
Fl-Pn3.1. In panel A, the sera were tested after the
second immnnization. In panel B, the reactivity of 4 out
of 15 sera tested after the first immllnization is shown.
The positions of 53.5 kDa- and 47 kDa-protein bands are
indicated by the bars at the left. The position of HSP72
is shown by the arrows at the right of each panel.
FIG. 6 depicts a fluorogram showing the
specificity of MAb Fl-Pn3.1 for HSP72. [35S]methionine-
labeled proteins of S. pneumoniae in the absence (lanes 1,
3 and 5) or presence (lanes 2, 4 and 6) of exposure to
heat shock were immunoprecipitated with IgG2a-control MAb
(lane 3,4) or Fl-Pn3.1 (lane 5,6) and then analyzed by
SDS-PAGE and fluorography. Cell lysates from
[35S]methionine-labeled non heat-shocked and heat-shocked
S. pneumoniae are shown in lanes 1 and 2, respectively.
The position of HSPs (all three) is shown by the arrows at
the left of the fluorogram.
FIG. 7, panel A, depicts an ;mmnnohlot~ which
shows the reaction of heat-shocked and non heat-shocked
[35S]methionine-labelled S. pneumoniae cell extracts with
MAb Fl-Pn3.1. Lane 1 contains heat-shocked cell lysates
(45~C). Lane 2 contains non heat-shocked cell lysates
(37~C). Panel B depicts a fluorogram of the immunoblot
shown in panel A.
FIG. 8 depicts a Western Blot, which shows
subcellular localization of S. pneumoniae HSP72. Sample
containing 15 ~g protein of membrane fraction (lane 1) and
cytoplasmic fraction (lane 2) of S. pneumoniae were
electrophoresced on SDS-PAGE transferred to nitrocellulose
and probed with MAb Fl-Pn3.1.
FIG. 9 is a photograph of an immunoblot showing
the reactivity of recombinant fusion proteins cont~;n;ng
the C-169 region of S. pneumoniae HSP72 with MAb Fl-Pn3.1.
Lane 1 contains whole cell extracts from S. pneumoniae
strain 64 probed with HSP72-specific MAb Fl-Pn3.1.
Lanes 2 and 3 contain phage lysates from E. coli infected
with ~JBD17 cultured in the presence (+) or absence (-) of
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W O 96/40928 PCT/CA96/00322
IPTG and probed with HSP72-specific MAb Fl-Pn3.1. Lanes 4
and 5 contain phage lysates from E. coli infected with
~JBD7 cultured in the presence (+) or absence (-) of IPTG
and probed with HSP72-specific MAb Fl-Pn3.1. Molecular
s mass markers are shown to the left. The positions of the
74kDa- and 160 kDa-reactive proteins are shown on the left
and on the right, respectively.
FIG. 10 is a schematic representation of the
restriction map of the HSP72(DnaK) and Fuc loci and
inserts of recombinant clones. The relationships between
DNA fragments are shown with respect to each other.
FIGS. lOA and lOC illustrate the restriction map of the
HSP72(DnaK) and Fuc loci, respectively. FIG lOB
illustrates the inserts of the various phages and plasmids
described in Example 3. H(HindIII); E(EcoRI); V(EcoRV);
P(PstI); and X(XhoI) indicate positions of restriction
endonuclease sites. DNA fragments on the HSP72/DnaK locus
(-); the Fuc locus (///); and fragments used as probes in
the Southern blot analyses ( ) are indicated.
FIG. 11 depicts the SDS-PAGE and Western blot
analyses of the recombinant 74 kDa protein. Whole cell
extracts from E. coli transformed with plasmids pJBD179
(lane 1), pJBDf51 (lanes 2 and 3) and pJBDf62 (lane 4 and
5) and cultured in presence (+) or absence (-) of IPTG
were subjected to 10% polyacrylamide gel electrophoresis.
The proteins were then visualized by Coomassie Blue
staining (A) or Western blotting (B) using HSP-specific
MAb Fl-Pn3.1. Molecular mass markers in kilodaltons are
shown to the left. The arrow at the left-hand side of
each panel marks the 74 kDa protein marker.
FIG. 12 depicts the detection of native and
recombinant HSP72 antigens by Western blot analysis.
Whole cell lysates from E. coli transformed with plasmids
pJBDk51 (lanes 1 and 3) and pJBD291 (lane 2) and cell
lysates from S. pneumoniae strain 64 (lane 4) were
subjected to 10% polyacrylamide gel electrophoresis and
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WO 96/40928 PCT/CA9~ r~
were electrotransferred to nitrocellulose. The immunoblot_
was probed with HSP72-specific MAb Fl-Pn3.1.
FIGS. 13A-13D depict a comparison of the
predicted amino acid sequence of the S. pneumoniae HSP72
s open reading frame (HSP72 SPNEU) with those previously
reported for the following HSP70/DnaK proteins: ECOLI,
Escherichia colii BORBU, Borrelia burgdorferi; BRUOV,
Brucella ovis; CHLPN, Chlamydia pneumonia; BACME, Bacillus
megatorium; BACSU, Bacillus subtilis; STAAU,
10 Staphylococcus aureus; LACLA, Lactococcus lactis; and
MYCTU, Mycobacterium tuberculosis. Only mismatched amino
acids are indicated. Identical and conserved amino acids
are boxed and shadowed, respectively.
FIG. 14 depicts a photograph of an SDS-PAGE,
which shows the recombinant S. pneumoniae HSP72 purified
by affinity chromatography. Supernatant fractions from
E. coli (pJBDk51) lysates (lane 2) and 20 ~g of
immunoaffinity-purified HSP72r.c (lane 3) were subjected to
10% polyacrylamide gel electrophoresis. The proteins were
then visualized by Coomassie Blue staining. Lane 1 shows
the migration of molecular mass markers (106 kDa, 80 kDa,
49.5 kDa, 32.5 kDa, 27.5 kDa and 18.5 kDa).
FIG. 15 depicts a photograph of SDS-PAGE, which
shows the recombinant S. pneumoniae C-169 fragment
purified by solubilization of inclusion bodies. Various
amounts of purified C-169 protein (lane 1, 5 ~ugi lane 2,
2.5 ~ug; and lane 3, 1 ,ug) and whole cell lysates from
E. coli transformed with plasmids pDELTAl (lane 4) and
pJBD~l (lane 5) were subjected to 10% polyacrylamide gel
electrophoresis. The proteins were then visualized by
Coomassie Blue staining.
FIG. 16 is a graphical depiction of the survival
curve of Balb/c mice protected from S. pneumoniae
infection by immnn;zation with HSP72r.C. Data are
presented as the per cent (%) survival over a period of
14 days for a total of 10 mice per experimental group.
CA 0222401~ 1997-12-08
WO~ 928 PCT/CA96/00322
FIG. 17 is a graphical depiction of the survival
curve of Balb/c mice protected from S. pneumoniae
infection by ;mmlln;zation with C-169r.~. Data are
presented as the per cent (%) survival over a period of 14
days for a total of 10 mice per experimental group.
FIG. 18 is a map of plasmid pURV3 cont~;ning C-
151reC, the coding region for the 151 amino acids at the
carboxyl end of the HSP72 of S. pneumoniae; AmpiR,
ampicillin-resistance coding region; ColE1 ori, origin of
replicationi cI857, bacteriophage ~ cI857 temperature-
sensitive repressor gene; ~ PL, bacteriophage ~
transcription promoter; Tl, Tl transcription terminator.
The direction of transcription is indicated by the arrows.
BglII and BamHI are the restriction sites used to insert
the coding region for the C-151reC of the HSP72 of S.
pneumoniae. FIG. 19 illustrates the
distribution of anti-S. pneumoniae titers in sera from
Balb/c mice ;mmlln;zed with HSP72reC. Sera were collected
after the first, second and third injection with 1 ,ug (O)
or 5 ~g (-) of HSP72reC and evaluated individually for
anti-S. pneumoniae antibody by ELISA. Titers were defined
as the highest dilution at which the A410 values were 0.1
above the background values. Plain lines indicate the
median reciprocal of antibody titers for each group of
2s mice while the dashed line indicates the median value for
preimmune sera.
FIG. 20 illustrates the distribution of anti-S.
pneumoniae titers in sera from Balb/c mice ;mmlln;zed with
C-169reC. Sera were collected after the first, second and
third injection with 1 ,ug (O) or 5 ,ug (-) of C-169reC and
evaluated individually for anti-S. pneumoniae antibody by
ELISA. Titers were defined as the highest dilution at
which the A410 values were 0.1 above the background
values. Plain lines indicate the median reciprocal of
antibody titers for each group of mice while the dashed
line indicates the median value for preimmune sera.
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W O 96/40928 PCT/CA96/00322
FIG. 21 illustrates the distribution of anti-S.
pneumoniae titers in sera from Balb/c mice ;mml~n;zed with
C-l5lreC. Sera were collected after the first, second and
third injection with 0.5 ,ug of C-l5lreC and evaluated
individually for anti-S. pneumoniae antibody by ELISA.
Titers were defined as the highest dilution at which the
A410 values were 0.l above the background values. Plain
lines indicate the median reciprocal of antibody titers
for each group of mice while the dashed line indicates the
median value for preimmune sera.
FIG. 22 illustrates the antibody response of
cynomolgus monkeys ;mmllnlzed with recombinant HSP72
antigens. Groups of two monkeys were ;mml~n;zed with
either HSP72reC or C-l69reC protein at day l, day 22 and
day 77. Sera were collected regularly during the course of
the ;mmllnization and evaluated individually for
pneumococcal HSP72 specific antibody by Western blot
analysis. Titers were defined as the highest dilution at
which the HSP72 band was visualized.
FIG. 23 illustrates the binding of hyperimmune sera
to peptides in a solid-phase ELISA. Rabbit, mouse and
monkey sera from animals imml~nized with either HSP72reC or
C-l69reC protein were tested for their reactivity to
peptides. Optical density values were obtained with sera
tested at a dilution of l:l00 except for the values
corresponding to the reactivity of rabbit sera to peptide
MAP2 and murine sera to peptides MAP2 and MAP4 which were
obtained with sera diluted l:l000.
FIG. 24 depicts the consensus sequence established
from the DNA sequences of the hsp70/dnak open reading
frames of Streptococcus pneumoniae (spn-orf),
Streptococcus pyogenes ( sga-orf) and Streptococcus
agalactiae ( sgb-orf) and indicates the substitutions and
insertions of nucleotides specific to each species.
FIG. 25 depicts the consensus sequence established
from the protein sequences of the Hsp70 of Streptococcus
pneumoniae (spn-prot), Streptococcus pyogenes (sga-prot)
14
CA 0222401~ 1997-12-08
W O 9~t4'928 PCT/CA96/00322
and Streptococcus agalactiae ( sgb-prot) and indicates the
substitutions and insertions of amino acids specific to
each species.
FIG. 26 depicts a fluorogram, which shows the effect
of heat shock on S. agalactiae protein synthesis and the
S. agalactiae protein antigen immunoprecipitated by MAb
F2-Pn3.4. Cell lysates from [35S]methionine-labeled
proteins from S. agalactiae grown at 37~C and incubated at
37~C (odd numbered lanes) or heat-shocked at 43~C (even
numbered lanes) were analysed by SDS-PAGE and
fluorography. Lanes 3 and 4 show the immunoprecipitates
obtained using MAb F2-Pn3.4.
DETAILED DESCRIPTION OF THE INVENTION
According to one aspect of the invention, we
provide novel heat shock proteins of S. pneumoniae, S.
pyogenes and S. agalactiae, and analogues, homologues,
derivatives and fragments thereof, cont~;n;ng at least one
immunogenic epitope. As used herein, a "heat shock
protein" is a naturally occurring protein that exhibits
preferential transcription during heat stress conditions.
The heat shock protein according to the invention may be
of natural origin, or may be obtained through the
application of recombinant DNA techniques, or conventional
chemical synthesis techniques.
As used herein, ~immunogenic~ means having the
ability to elicit an immune response. The novel heat
shock proteins of this invention are characterized by
their ability to elicit a protective immune response
against Streptococcal infections, more particularly
against lethal S. pneumoniae, S. pyogenes and S.
agalactiae.
The invention particularly provides a
Streptoccus pneumoniae heat shock protein of approximately
72 kDa ("HSP72"), having the deduced amino acid sequence
of SEQ ID NO: 5, and analogues, homologues, derivatives and
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fragments thereof, containing at least one immunogenic
epitope.
As used herein, "analogues" of HSP72 are those
S. pneumoniae proteins wherein one or more amino acid
residues in the HSP72 amino acid sequence (SEQ ID NO:5) is
replaced by another amino acid residue, providing that the
overall functionality and immunogenic properties of the
analogue protein are preserved. Such analogues may be
naturally occurring, or may be produced synthetically or
by recombinant DNA technology, for example, by mutagenesis
of the HSP72 sequence. Analogues of HSP72 will possess at
least one antigen capable of eliciting antibodies that
react with HSP72, e.g. Streptococcus pyogenes and
Streptococcus agalactiae.
As used herein, "homologues" of HSP72 are
proteins from Streptococcal species other than pneumoniae,
pyogenes or agalactiae, or genera other than Streptococcus
wherein one or more amino acid residues in the HSP72 amino
acid sequence (SEQ ID NO:5) is replaced by another amino
acid residue, providing that the overall functionality and
immunogenic properties of the homologue protein are
preserved. Such homologues may be naturally occurring, or
may be produced synthetically or by recombinant DNA
technology. Homologues of HSP72 will possess at least one
antigen capable of eliciting antibodies that react with
HSP72, e.g. Enterococcus faecalis.
As used herein, a "derivative" is a polypeptide
in which one or more physical, chemical, or biological
properties has been altered. Such alterations include,
but are not limited to: amino acid substitutions,
modifications, additions or deletions; alterations in the
pattern of lipidation, glycosylation or phosphorylation;
reactions of free amino, carboxyl, or hydroxyl side groups
of the amino acid residues present in the polypeptide with
other organic and non-organic molecules; and other
alterations, any of which may result in changes in
primary, secondary or tertiary structure.
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The "fragments" of this invention will have at
least one immunogenic epitope. An "immunogenic epitope"
is an epitope that is instrumental in eliciting an immune
response. The preferred fragments of this invention will
elicit an immune response sufficient to prevent or lessen
the severity of infection, e.g., S. pneumoniae infection.
Preferred fragments of HSP72 include the C-terminal region
of the polypeptides. More preferred fragment include the
C-terminal 169-residue fragment ("C-169") (SEQ ID NO:5,
residues 439-607), the C-terminal 151-residue ("C-151")
(SEQ ID No:5, residues 457-607) and smaller fragments
consisting of peptide epitopes within the C-169 region.
Particularly preferred fragments within the C-169 region
of HSP72 include the peptide sequences GFDAERDAAQAALDD
(residues 527-541 of SEQ ID NO:5) and AEGAQATGNAGDDW
(residues 586-600 of SEQ ID NO:5), which are exclusive to
HSP72 of Streptococcus pneumoniae, or corresponding
degenerate fragments from S. pyogenes or S. agalactiae
(see FIG. 25). Even more preferred are fragments that
elicit a specific immune reaction against Streptococcal
strains. Such fragments may be selected from the
following peptides: CS870, CS873, CS874, CS875, CS876,
CS877, CS878, CS879, CS880, CS882, MAP1, MAP2, MAP3 and
MAP4 (see TABLE 5, supra), or homologues thereof.
In a further aspect of the invention, we provide
polypeptides that are immunologically related to HSP70/72.
As used herein, "immunologically related" polypeptides are
characterized by one or more of the following properties:
(a) they are immunologically reactive with
antibodies generated by infection of a mAmm~lian host with
Streptococcus pneumoniae cells, which antibodies are
immunologically reactive with HSP72 (SEQ ID NO:5) and
HSP70 (SEQ ID NO:20 and SEQ ID NO:22);
(b) they are capable of eliciting antibodies that
3s are immunologically reactive with HSP72 (SEQ ID NO:5) and
HSP70 (SEQ ID NO:20 and SEQ ID NO:22);
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(c) they are immunologically reactive with
antibodies elicited by ;mml]n;zation of a mammal with HSP72
(SEQ ID NO:5).
By definition, analogues, homologues and
derivatives of HSP70/72 are immunologically related
polypeptides. Moreover, all immunologically related
polypeptides contain at least one HSP70/72 antigen.
Accordingly, "HSP70/72 antigensN may be found in HSP70/72
itself, or in immunologically related polypeptides.
In a further aspect of the invention, we provide
polypeptides that are immunologically related to HSP72.
As used herein, "immunologically related" polypeptides are
characterized by one or more of the following properties:
(a) they are immunologically reactive with
antibodies generated by infection of a mammalian host with
Streptococcus pneumoniae cells, which antibodies are
immunologically reactive with HSP72 (SEQ ID NO:5);
(b) they are capable of eliciting antibodies that
are immunologically reactive with HSP72 (SEQ ID NO:5);
(c) they are immunologically reactive with
antibodies elicited by imm~nl zation of a m~mm~l with HSP72
(SEQ ID NO:5).
By definition, analogues, homologues and
derivatives of HSP72 are immunologically related
polypeptides. Moreover, all immunologically related
polypeptides contain at least one HSP72 antigen.
Accordingly, "HSP72 antigens" may be found in HSP72
itself, or in immunologically related polypeptides.
As used herein, "related bacteria" are bacteria
that possess antigens capable of eliciting antibodies that
react with HSP72. Examples of related bacteria include
Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus mutans, Streptococcus sanguis, Streptococcus
agalactiae and Enterococcus faecalis.
It will be understood that by following the
examples of this invention, one of skill in the art may
determine without undue experimentation whether a
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particular analogue, homologue, derivative,
immunologically related polypeptide, or fragment would be
useful in the diagnosis, prevention or treatment of
disease. Useful polypeptides and fragments will elicit
antibodies that are immunoreactive with HSP72 (Example 4).
Preferably, useful polypeptides and fragments will
demonstrate the ability to elicit a protective immune
response against lethal bacterial infection (Example 5).
Also included are polymeric forms of the
polypeptides of this invention. These polymeric forms
include, for example, one or more polypeptides that have
been crosslinked with crosslinkers such as avidin/biotin,
glutaraldehyde or dimethylsuberimidate. Such polymeric
forms also include polypeptides containing two or more
1S tandem or inverted contiguous protein sequences, produced
from multicistronic mRNAs generated by recombinant DNA
technology.
This invention provides substantially pure HSP72
and immunologically related polypeptides. The term
"substantially pure" means that the polypeptides according
to the invention, and the DNA sequences encoding them, are
substantially free from other proteins of bacterial
origin. Substantially pure protein preparations may be
obtained by a variety of conventional processes, for
example the procedures described in Examples 3 and 5.
In another aspect, this invention provides, for
the first time, a DNA sequence coding for a heat shock
protein of S. pneumoniae, specifically, HSP72 (SEQ ID
NO:4, nucleotides 682-2502).
The DNA sequences of this invention also include
DNA sequences coding for polypeptide analogues and
homologues of HSP72, DNA sequences coding for
immunologically related polypeptides, DNA sequences that
are degenerate to any of the foregoing DNA sequences, and
3s fragments of any of the foregoing DNA sequences. It will
be readily appreciated that a person of ordinary skill in
the art will be able to determine the DNA sequence of any
19
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of the polypeptides of this invention, once the
polypeptide has been identified and isolated, using
conventional DNA sequencing techniques.
Oligonucleotide primers and other nucleic acid
probes derived from the genes encoding the polypeptides of
this invention may also be used to isolate and clone other
related proteins from S. pneumoniae and related bacteria
which may contain regions of DNA bacteria that are
homologous to the DNA sequences of this invention. In
addition, the DNA se~uences of this invention may be used
in PCR reactions to detect the presence of S. pneumoniae
or related bacteria in a biological sample.
The polypeptides of this invention may be
prepared from a variety of processes, for example by
protein fractionation from appropriate cell extracts,
using conventional separation techniques such as ion
exchange and gel chromatography and electrophoresis, or by
the use of recombinant DNA techniques. The use of
recombinant DNA techniques is particularly suitable for
preparing substantially pure polypeptides according to the
invention.
Thus according to a further aspect of the
invention, we provide a process for the production of
HSP72, immunologically related polypeptides, and fragments
2s thereof, comprising the steps of (1) culturing a
unicellular host organism transformed with a vector
containing a DNA sequence coding for said polypeptide or
fragment and one or more expression control sequences
operatively linked to the DNA sequence, and (2) recovering
a substantially pure polypeptide or fragment.
As is well known in the art, in order to obtain
high expression levels of a transfected gene in a host,
the gene must be operatively linked to transcriptional and
ranslational expression control sequences that are
functional in the chosen expression host. Preferably, the
expression control sequences, and the gene of interest,
will be contained in an expression vector that further
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comprises a bacterial selection marker and origin of
replication. If the expression host is a eukaryotic cell,
the expression vector should further comprise an
expression marker useful in the eukaryotic expression
s host.
The DNA sequences encoding the polypeptides of
this invention may or may not encode a signal seguence.
If the expression host is eukaryotic, it generally is
preferred that a signal sequence be encoded so that the
mature protein is secreted from the eukaryotic host.
An amino terminal methionine may or may not be
present on the expressed polypeptides of this invention.
If the terminal methionine is not cleaved by the
expression host, it may, if desired, be chemically removed
by standard techniques.
A wide variety of expression host/vector
combinations may be employed in expressing the DNA
sequences of this invention. Useful expression vectors
for eukaryotic hosts include, for example, vectors
comprising expression control sequences from SV40, bovine
papilloma virus, adenovirus, adeno-associated virus,
cytomegalovirus, and retroviruses. Useful expression
vectors for bacterial hosts include bacterial plasmids,
such as those from E. coli, including pBluescript, pGEX2T,
2s pUC vectors, col El, pCRl, pBR322, pMB9 and their
derivatives, wider host range plasmids, such as RP4, phage
DNAs, e.g., the numerous derivatives of phage lambda, e.g.
~gtlO and ~gtll, NM989, and other DNA phages, such as M13
and filamentous single stranded DNA phages. Useful
expression vectors for yeast cells include the 2,u plasmid
and derivatives thereof. Useful vectors for insect cells
include pVL 941.
In addition, any of a wide variety of expression
control sequences may be used in these vectors to express
3s the DNA sequences of this invention. Useful expression
control sequences include the expression control sequences
associated with structural genes of the foregoing
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WO9C'4-~28 PCT/CA96/00322
expression vectors. Examples of useful expression control
sequences include, for example, the early and late
promoters of SV40 or adenovirus, the lac system, the trp
system, the TAC or TRC system, the T3 and T7 promoters the
major operator and promoter regions of phage lambda, the
control regions of fd coat protein, the promoter for 3-
phosphoglycerate kinase or other glycolytic enzymes, the
promoters of acid phosphatase, e.g., Pho5, the promoters
of the yeast alpha-mating system and other constitutive
and inducible promoter sequences known to control the
expression of genes of prokaryotic or eukaryotic cells or
their viruses, and various combinations thereof. The T7
RNA polymerase promoter ~lO is particularly useful in the
expression of HSP72 in E. coli (Example 3).
Host cells transformed with the foregoing
vectors form a further aspect of this invention. A wide
variety of unicellular host cells are useful in expressing
the DNA sequences of this invention. These hosts may
include well known eukaryotic and prokaryotic hosts, such
as strains of E. coli, Pse7l~o~on~.s, Bacillus,
Streptomyces, fungi, yeast, insect cells such as
Spodoptera frugiperda (SF9), ~n;m~l cells such as CHO and
mouse cells, African green monkey cells such as COS l, COS
7, BSC l, BSC 40, and BMT lO, human cells, and plant cells
2s in tissue culture. Preferred host organisms include
bacteria such as E. coli and B. subtilis, and m~mm~l ian
cells in tissue culture.
It should of course be understood that not all
vectors and expression control sequences will function
equally well to express the DNA sequences of this
invention. Neither will all hosts function equally well
with the same expression system. However, one of skill in
the art may make a selection among these vectors,
expression control sequences and hosts without undue
experimentation and without departing from the scope of
this invention. For example, in selecting a vector, the
host must be considered because the vector must replicate
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in it. The vector's copy number, the ability to control
that copy number, and the expression of any other proteins
encoded by the vector, such as antibiotic markers, should
also be considered. In selecting an expression control
sequence, a variety of factors should also be considered.
These include, for example, the relative strength of the
sequence, its controllability, and its compatibility with
the DNA sequences of this invention, particularly as
regards potential secondary structures. Unicellular hosts
should be selected by consideration of their compatibility
with the chosen vector, the toxicity of the product coded
for by the DNA sequences of this invention, their
secretion characteristics, their ability to fold the
protein correctly, their fermentation or culture
requirements, and the ease of purification from them of
the products coded for by the DNA sequences of this
invention. Within these parameters, one of skill in the
art may select various vector/expression control
sequence/host combinations that will express the DNA
sequences of this invention on fermentation or in large
scale animal culture.
The polypeptides encoded by the DNA sequences of
this invention may be isolated from the fermentation or
cell culture and purified using any of a variety of
2s conventional methods including: liquid chromatography such
as normal or reversed phase, using HPLC, FPLC and the
like; affinity chromatography (such as with inorganic
ligands or monoclonal antibodies); size exclusion
chromatography; immobilized metal chelate chromatography;
gel electrophoresisi and the like. One of skill in the
art may select the most appropriate isolation and
purification techniques without departing from the scope
of this invention.
In addition, the polypeptides of this invention
3s may be generated by any of several chemical techniques.
For example, they may be prepared using the solid-phase
synthetic technique originally described by R. B.
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Merrifield, "Solid Phase Peptide Synthesis. I. The
Synthesis Of A Tetrapeptide", J. Am. Chem. Soc., 83,
pp. 2149-54 (1963), or they may be prepared by synthesis
in solution. A summary of peptide synthesis techniques
s may be found in E. Gross & H. J. Meinhofer, 4 The
Peptides: Analysis, Synthesis, Biology; Modern Techniques
of Peptide And Amino Acid Analysis, John Wiley & Sons,
(1981) and M. Bodanszky, Principles Of Peptide Synthesis,
Springer-Verlag (1984).
The preferred compositions and methods of this
invention comprise polypeptides having enhanced
immunogenicity. Such polypeptides may result when the
native forms of the polypeptides or fragments thereof are
modified or subjected to treatments to enhance their
immunogenic character in the intended recipient.
Preferred polypeptides are fragments that are specific to
Streptococcal species such as fragments selected from the
C-terminal portion of thenative polypeptides. Numerous
techniques are available and well known to those of skill
in the art which may be used, without undue
experimentation, to substantially increase the
immunogenicity of the polypeptides herein disclosed. For
example, the polypeptides may be modified by coupling to
dinitrophenol groups or arsanilic acid, or by denaturation
with heat and/or SDS. Particularly if the polypeptides
are small polypeptides synthesized chemically, it may be
desirable to couple them to an immunogenic carrier. The
coupling of course, must not interfere with the ability of
either the polypeptide or the carrier to function
appropriately. For a review of some general
considerations in coupling strategies, see Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, ed. E.
Harlow and D. Lane (1988). Useful immunogenic carriers
are well known in the art. Examples of such carriers are
keyhole limpet hemocyanin (KLH); albumins such as bovine
serum albumin (BSA) and ovalbumin, PPD (purified protein
derivative of tuberculin); red blood cells; tetanus
24
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WOg~C3~8 PCT/CA96/00322
toxoid; cholera toxoidi agarose beads; activated carbon;
or bentonite.
Modification of the amino acid sequence of the
polypeptides disclosed herein in order to alter the
lipidation state is also a method which may be used to
increase their immunogenicity and biochemical properties.
For example, the polypeptides or fragments thereof may be
expressed with or without the signal sequences that direct
addition of lipid moieties.
In accordance with this invention, derivatives
of the polypeptides may be prepared by a variety of
methods, including by in vi tro manipulation of the DNA
encoding the native polypeptides and subsequent expression
of the modified DNA, by chemical synthesis of derivatized
DNA sequences, or by chemical or biological manipulation
of expressed amino acid sequences.
For example, derivatives may be produced by
substitution of one or more amino acids with a different
natural amino acid, an amino acid derivative or non-native
amino acid, conservative substitution being preferred,
e.g., 3-methylhistidine may be substituted for histidine,
4-hydroxyproline may be substituted for proline, 5-
hydroxylysine may be substituted for lysine, and the like.
Causing amino acid substitutions which are less
2s conservative may also result in desired derivatives, e.g.,
by causing changes in charge, conformation and other
biological properties. Such substitutions would include
for example, substitution of a hydrophilic residue for a
hydrophobic residue, substitution of a cysteine or proline
for another residue, substitution of a residue having a
small side chain for a residue having a bulky side chain
or substitution of a residue having a net positive charge
for a residue having a net negative charge. When the
result of a given substitution cannot be predicted with
certainty, the derivatives may be readily assayed
according to the methods disclosed herein to determine the
presence or absence of the desired characteristics.
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The polypeptides may also be prepared with the
objective of increasing stability or rendering the
molecules more ~mPn~hle to purification and preparation.
One such technique is to express the polypeptides as
fusion proteins comprising other S. pneumoniae or non-
S. pneumoniae sequences. It is preferred that-the fusion
proteins comprising the polypeptides of this invention be
produced at the DNA level, e.g., by constructing a nucleic
acid molecule encoding the fusion, transforming host cells
with the molecule, inducing the cells to express the
fusion protein, and recovering the fusion protein from the
cell culture. Alternatively, the fusion proteins may be
produced after gene expression according to known methods.
An example of a fusion protein according to this invention
is the FUCI/HSP72 (C-169) protein of Example 3, infra.
The polypeptides of this invention may also be
part of larger multimeric molecules which may be produced
recombinantly or may be synthesized chemically. Such
multimers may also include the polypeptides fused or
coupled to moieties other than amino acids, including
lipids and carbohydrates.
The polypeptides of this invention are
particularly well-suited for the generation of antibodies
and for the development of a protective response against
disease. Accordingly, in another aspect of this
invention, we provide antibodies, or fragments thereof,
that are immunologically reactive with HSP72. The
antibodies of this invention are either elicited by
;mml]nization with HSP72 or an immunologically related
polypeptide, or are identified by their reactivity with
HSP72 or an immunologically related polypeptide. It
should be understood that the antibodies of this invention
are not intended to include those antibodies which are
normally elicited in an animal upon infection with
3s naturally occurring S. pneumoniae and which have not been
removed from or altered within the animal in which they
were elicited.
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The antibodies of this invention may be intact
immunoglobulin molecules or fragments thereof that contain
an intact antigen binding site, including those fragments
known in the art as F(v), Fab, Fab' and F(ab')2. The
antibodies may also be genetically engineered or
synthetically produced. The antibody or fragment may be
of ~n;m~l origin, specifically of m~mm~lian origin, and
more specifically of murine, rat, monkey or human origin.
It may be a natural antibody or fragment, or if desired, a
recombinant antibody or fragment. The antibody or
antibody fragments may be of polyclonal, or preferably, of
monoclonal origin. They may be specific for a number of
epitopes but are preferably specific for one.
Specifically preferred are the monoclonal antibodies F1-
Pn3.1, F2-Pn3.~, F2-Pn3.3 and F2-Pn3.4 of Example 2,
infra. One of skill in the art may use the polypeptides
of this invention to produce other monoclonal antibodies
which could be screened for their ability to confer
protection against S. pneumoniae , S. pyogenes, S.
agalactiae or other Streptococcal related bacterial
infection when used to imml]n;ze naive animals. Once a
given monoclonal antibody is found to confer protection,
the particular epitope that is recognized by that antibody
may then be identified. Methods to produce polyclonal and
monoclonal antibodies are well known to those of skill in
the art. For a review of such methods, see Antibodies, A
Laboratory Manual, supra, and D.E. Yelton, et al., Ann.
Rev. of Biochem., 50, pp. 657-80 (1981). Determination of
immunoreactivity with a polypeptide of this invention may
be made by any of several methods well known in the art,
including by immunoblot assay and ELISA.
An antibody of this invention may also be a
hybrid molecule formed from immunoglobulin sequences from
different species (e.g., mouse and human) or from portions
of immunoglobulin light and heavy chain sequences from the
same species. It may be a molecule that has multiple
binding specificities, such as a bifunctional antibody
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prepared by any one of a number of techniques known to
those of skill in the art including: the production of
hybrid hybridomasi disulfide exchange; chemical cross-
linking; addition of peptide linkers between two
s monoclonal antibodies; the introduction of two sets of
immunoglobulin heavy and light chains into a particular
cell line; and so forth. The antibodies of this
invention may also be human monoclonal antibodies, for
example those produced by immortalized human cells, by
SCID-hu mice or other non-human ~n;m~l S capable of
producing "human" antibodies, or by the expression of
cloned human immunoglobulin genes.
In sum, one of skill in the art, provided with
the teachings of this invention, has available a variety
of methods which may be used to alter the biological
properties of the antibodies of this invention including
methods which would increase or decrease the stability or
half-life, immunogenicity, toxicity, affinity or yield of
a given antibody molecule, or to alter it in any other way
that may render it more suitable for a particular
application.
The polypeptides, DNA sequences and antibodies
of this invention are useful in prophylactic, therapeutic
and diagnostic compositions for preventing, treating and
diagnosing disease.
Standard immunological techniques may be
employed with the polypeptides and antibodies of this
invention in order to use them as immunogens and as
vaccines. In particular, any suitable host may be
injected with a pharmaceutically effective amount of
polypeptide to generate monoclonal or polyvalent
antibodies or to induce the development of a protective
immunological response against disease. Preferably, the
polypeptide is selected from the group consisting of
HSP72 (SEQ ID NO:5), HSP70 (SEQ ID NO:20 and SEQ ID NO:22)
or fragments thereof.
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As used herein, a "pharmaceutically effective
amount" of a polypeptide or of an antibody is the amount
that, when administered to a patient, elicits an immune
response that is effective to prevent or lessen the
severity of Streptococcal or related bacterial
infections.
The administration of the polypeptides or
antibodies of this invention may be accomplished by any of
the methods described in Example 10, infra, or by a
variety of other standard procedures. For a detailed
discussion of such techniques, see Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, ed.
E. Harlow and D. Lane (1988). Preferably, if a
polypeptide is used, it will be administered with a
pharmaceutically acceptable adjuvant, such as complete or
incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or
ISCOM (immunostimulating complexes). Preferably, the
composition will include a water-in-oil emulsion or
aluminum hydroxide as adjuvant and will be administered
intramuscularly. The vaccine composition may be
administered to the patient at one time or over a series
of treatments. The most effective mode of administration
and dosage regimen will depend upon the level of
immunogenicity, the particular composition and/or adjuvant
used for treatment, the severity and course of the
expected infection, previous therapy, the patient~s health
status and response to imml~n;zation, and the judgment of
the treating physician. For example, in an
immunocompetent patient, the more highly immunogenic the
polypeptide, the lower the dosage and necessary number of
;mml]n;zations. Similarly, the dosage and necessary
treatment time will be lowered if the polypeptide is
administered with an adjuvant.
Generally, the dosage will consist of an initial
3s injection, most probably with adjuvant, of about 0.01 to
10 mg, and preferable 0.1 to 1.0 mg, HSP72 antigen per
patient, followed most probably by one or maybe more
29
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booster injections. Preferably, boosters will be
administered at about 1 and 6 months after the initial
injection.
Any of the polypeptides of this invention may be
used in the form of a pharmaceutically acceptable salt.
Suitable acids and bases which are capable of forming
salts with the polypeptides of the present invention are
well known to those of skill in the art, and i~clude
inorganic and organic acids and bases.
To screen the polypeptides and antibodies of
this invention for their ability to confer protection
against diseases caused by S. pneumoniae or related
bacteria, or their ability to lessen the severity of such
infection, one of skill in the art will recognize that a
number of animal models may be used. Any animal that is
susceptible to infection with S. pneumoniae or related
bacteria may be useful. The Balb/c mice of Example 5,
infra, are the preferred An;mAl model for active
immunoprotection screening, and the severe-combined
immunodeficient mice of Example 5 are the preferred animal
model for passive screening. Thus, by administering a
particular polypeptide or antibody to these animal models,
one of skill in the art may determine without undue
experimentation whether that polypeptide or antibody would
~5 be useful in the methods and compositions claimed herein.
According to another e-mbodiment of this
invention, we describe a method which comprises the steps
of treating a patient with a vaccine comprising a
pharmaceutically effective amount of any of the
polypeptides of this invention in a manner sufficient to
prevent or lessen the severity, for some period of time,
of Streptococcal or related bacterial infection. Again,
the preferred polypeptide for use in such methods is
HSP70/HSP72, or fragments thereof.
The polypeptides, DNA sequences and antibodies
of this invention may also form the basis for diagnostic
methods and kits for the detection of pathogenic
CA 0222401~ 1997-12-08
WO9''1C32h PCT/CA96/00322
organisms. Several diagnostic methods are possible. For
example, this invention provides a method for the
detection of Streptococcus pneumoniae, Streptococcus
pyogenes, Streptococcus agalactiae or related bacteria in
a biological sample comprising the steps of:
(a) isolating the biological sample from a
patient;
(b) incubating an antibody of this invention,
or fragment thereof with the biological sample to form a
mixture; and
(c) detecting specifically bound antibody or
fragment in the mixture which indicates the presence of
Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus agalactiae or related bacteria. Preferable
antibodies for use in this method include monoclonal
antibodies Fl-Pn3.1, F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4.
Alternatively, this invention provides a method
for the detection of antibodies specific to Streptococcus
pneumoniae or related bacteria in a biological sample
comprising:
(a) isolating the biological sample from a
patienti
(b) incubating a polypeptide of this invention
or fragment thereof, with the biological sample to form a
mixture; and
(c) detecting specifically bound polypeptide in
the mixture which indicates the presence of antibodies
specific to Streptococcus pneumoniae or related bacteria.
HSP72 (SEQ ID NO:5), the C-169 fragment thereof (residues
439-607 of SEQ ID NO:5), the C-151 fragment thereof
(residues 457-607 of SEQ ID NO j5) and peptide fragments
GFDAERDAAQAALDD ( residues 527-541 of SEQ ID NO: 5) and
AEGAQATGNAGDDW ( residues 586-600 of SEQ ID NO: 5) are the
preferred polypeptide and fragments in the above method
for the detection of antibodies.
One of skill in the art will recognize that
these diagnostic tests may take several forms, including
CA 0222401~ 1997-12-08
W 0 96/40928 PCT/CA9~ F~7
an enzyme-linked immunosorbent assay (ELISA), a
radioimmunoassay or a latex agglutination assay.
The diagnostic agents may be included in a kit
which may also comprise instructions for use and other
5 appropriate reagents, preferably a means for detecting
when the polypeptide or antibody is bound. For example,
the polypeptide or antibody may be labeled with a
detection means that allows foE the detection of the
polypeptide when it is bound to an antibody, or for the
detection of the antibody when it is bound to
S. pneumoniae or related bacteria. The detection means
may be a fluorescent labeling agent such as fluorescein
isocyanate (FIC), fluorescein isothiocyanate (FITC), and
the like, an enzyme, such as horseradish peroxidase (HRP),
glucose oxidase or the like, a radioactive element such as
125I or s1Cr that produces gamma ray emissions, or a
radioactive element that emits positrons which produce
gamma rays upon encounters with electrons present in the
test solution, such as 11C, 150, or 13N. B;~;ng may also
be detected by other methods, for example via avidin-
biotin complexes. The linking of the detection means is
well known in the art. For instance, monoclonal antibody
molecules produced by a hybridoma may be metabolically
labeled by incorporation of radioisotope-containing amino
acids in the culture medium, or polypeptides may be
conjugated or coupled to a detection means through
activated functional groups.
The DNA sequences of this invention may be used
to design DNA probes for use in detecting the presence of
Streptococcus pneumoniae or related bacteria in a
biological sample. The probe-based detection method of
this invention comprises the steps of:
(a) isolating the biological sample from a
patient;
(b~ incubating a DNA probe having a DNA
sequence of this invention with the biological sample to
form a mixture; and
CA 0222401~ 1997-12-08
WO9~ 28 PCT/CA96/00322
(c) detecting specifically bound DNA probe in
the mixture which indicates the presence of Streptococcus
pneumoniae or related bacteria.
The DNA probes of this invention may also be
used for detecting circulating nucleic acids in a sample,
for example using a polymerase chain reaction, as a method
of diagnosing Streptococcus pneumoniae or related
bacterial infections. The probes may be synthesized using
conventional techniques and may be immobilized on a solid
0 phase, or may be labeled with a detectable label. A
preferred DNA probe for this application is an oligomer
having a sequence complementary to at least about 6
contiguous nucleotides of HSP72 (SEQ ID NO: 4, nucleotides
682-2502).
The polypeptides of this invention may also be
used to purify antibodies directed against epitopes
present on the protein, for example, using immunoaffinity
purification of antibodies on an antigen column.
The antibodies or antibody fragments of this
invention may be used to prepare substantially pure
proteins according to the invention for example, using
immunoaffinity purification of antibodies on an antigen
column.
EXAMPLES
In order that this invention may be better
understood, the following examples are set forth. These
examples are for purposes of illustration only, and are
not to be construed as limiting the scope of the invention
in any manner.
Example l describes the identification of HSP72,
an immunoreactive heat shock protein according to the
invention. Example 2 describes the isolation of
monoclonal antibodies against epitopes of HSP72. Example
3 describes the preparation of recombinant HSP72 and
fragments of HSP72 according to the invention. Example 4
describes the antigenic specificity and immunoreactivity
CA 0222401~ 1997-12-08
WO9~ 32& PCT/CA96/00322
of monoclonal antibodies directed against HSP72, and the
identification of immunologically related proteins
according to the invention. Example 5 describes processes
for obtaining substantially pure HSP72, and the use of
HSP72 or antibodies against it to protect against
experimental S. pneumoniae infection. Example 6 describes
the preparation of recom.binant C-151 fragment of HSP72
according to the invention. Example 7 describes the
humoral immune response following the ;mmlln;zation with
recombinant HSP72 or fragments of HSP72 according to the
invention. Example 8 describes the localization of linear
B-cell epitopes on the HSP72. Example 9 describes the
hsp70 genes and HSP70 proteins from S. agalactiae and S.
pyogenes. Example 10 describes the use of HSP72 antigen
in a human vaccine.
EXAMPLE 1 - Identification of Tmml~noreactive
S. pneumoniae Heat Shock Proteins
A. Procedures
Unless otherwise noted, the following procedures
were used throughout the Examples herein.
1. Bacteria
S. pneumoniae strains were provided by the
Laboratoire de la Santé Publique du Québec, Sainte-Anne de
Bellevue. S. pneumoniae strains included type 4 strain 53
and type 6 strain 64. If not specified, S. pneumoniae
type 6 strain 64 was used. Bacterial strains were grown
overnight at 37~C in 5% CO2 on chocolate agar plates.
Antigen Preparations
Various S. pneumoniae antigens were prepared for
imm~nization and immunoassays. Heat-killed whole cell
antigens were obtained by incubating bacterial suspensions
34
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
in a water bath prewarmed at 56 C for 20 minutes.
Detergent-soluble proteins were extracted from
S. pneumoniae as follows. Heat-killed bacteria were
suspended in 10 m~M Hepes buffer (4-(2-Hydroxyethyl)-l-
piperazinethan-sulfonsaure) (Boehringer M~nnheim GmbH~
Germany) at pH 7.4 and sonicated at 20,000 Kz/second, four
times for 30 seconds. Intact cells and large debris were
removed by centrifugation at 1,700 g for 20 minutes. The
supernatant was collected and centrifuged at 100,000 g for
60 minutes. The pellet was resuspended in 1 ml of Hepes
buffer, and 1 ml of 2% N-lauroyl sarcosine (Sigma Chemical
Co., St. Louis, Mo.) was added. The mixture was incubated
for 30 minutes at room temperature and the detergent-
soluble fraction was harvested by centrifugation at
100,000 g for 60 minutes.
3. Heat Shock Treatment
S. pneumoniae bacteria (type 4, strain 53 and
type 6, strain 64) were resuspended in Eagle's M;n;m~l
Essential Medium lacking methionine (ICN Biomedicals Inc.,
Costa Mesa, CA) and supplemented with 1% BIO-X~ (Quelab
Laboratories, Montreal, Canada~ for 15 minutes at 37~C and
then divided into fractions of equal volume. The samples
were incubated at either 37~C or 45~C for 5 minutes and
then labeled with 100 ~Ci/ml [35S]methionine (ICN) for 10,
30, or 60 minutes at37~C. The bacteria were harvested and
cell extracts were prepared using Tris-HCl lysis buffer as
described above, or SDS-PAGE sample buffer.
4. Imm~unization Of Mice
Female Balb/c mice (Charles River Laboratories,
St-Constant, Québec, Canada) were imm~lnized with
S. pneumoniae antigens. Immune sera to S. pneumoniae
type 6 strain 64 were obtained from mice ;mmllnized, at
two-week intervals, by subcutaneous injections of 107 heat-
killed bacteria or 20 ,ug of detergent-soluble pneumococcal
CA 0222401~ 1997-12-08
W O 96/40928 PCT/C A96/00322
proteins absorbed to alllm;nt~m hydroxide adjuvant
(Alhydrogel~; Cedarlane Laboratories Ltd., Horny, Ontario,
Canada). Blood samples were collected prior to
imml~nization and at seven days following the first and
5 second imm~lnization.
5. SDS-PAGE and Immunoassays
Cell extracts were prepared for SDS-PAGE,
Western blot analysis and radioimmunoprecipitation assay
by incubating bacterial suspensions in Tris-HCl lysis
buffer (50mM Tris, 150 mM NaCl, 0.1% Na dodecyl sulfate,
0.5% Na deoxycholate, 2% Triton~ X-100, 100 ,ug/ml
phenylmethylsulfonylfluoride, and 2,ug/ml aprotinin) at pH
8.0 for 30 minutes on ice. Lysed cells were cleared by
centrifugation and the supernatants were aliquoted and
kept frozen at -70 C.
SDS-PAGE were performed on a 10% polyacrylamide
gel according to the method of Laemmli [Nature, 227,
pp. 680-685 (1970)], using the Mini Protean~ system (Bio-
Rad Laboratories Ltd., Mississauga, Canada). Samples were
denatured by boiling for 5 minutes in sample buffer
containing 2% 2-mercaptoethanol. Proteins were resolved
by staining the polyacrylamide gel with PhastGel Blue~
(Pharmacia Biotech Inc., Baie d~Urfé, Canada~. The
radiolabeled products were visualized by fluorography.
Fluorograms were scanned using a laser densitometer.
Immunoblot procedures were performed according
to the method of Towbin et al. [Proc. Natl. Acad. Sci.
USA, 76, pp. 4350-4354 (1979)]. The detection of antigens
reactive with antibodies was performed by an indirect
antibody immunoassay using peroxidase-labeled anti-mouse
immunoglobulins and the o-dianisidine color substrate.
Radioimmunoprecipitation assays were performed
as described by J.A. Wiley et al. [J. Virol., 66,
pp. 5744-5751 (1992)]. Briefly, sera or hybridoma culture
supernatants were added to radiolabeled samples cont~i n; n~
36
CA 0222401~ 1997-12-08
W O 9~'1r328 PCT/CA96/00322
equal amounts of [35S]methionine. The mixtures were
allowed to incubate for 90 minutes at 4 C with constant
agitation. The immune complexes were then precipitated
with bovine serum albumin-treated protein A Sepharose
(Pharmacia) for 1 hour at 4 C. The beads were pelleted
and washed three times in Tris buffered saline at pH 8.0,
and the antigen complexes were then dissociated by boiling
in sample buffer. The antigens were analyzed by
electrophoresis on SDS-PAGE. The gels were fixed,
enhanced for fluorography using Amplify~ (Amersham Canada
Limited, Oakville, Ontario, Canada), dried, and then
exposed to X-ray film.
B. Characterization of the Heat
Shock Response in S. pneumoniae
We studied the heat shock response of
S. pneumoniae by ex~m'n;~ the pattern of protein
synthesis before and after a shift from 37~C to 45~C.
FIG. 1 shows the results when S. pneumoniae type 6 strain
64 (panel A) and type 4 strain 53 (panel B) were grown at
37~C, incubated at 37~C (lanes 1,3,5,7 and 9) or at 45~C
(lanes 2, 4, 6, 8 and 10) for 5 minutes, and then labeled
with [~5S]methionine for 10 minutes (lanes 1,2 and 7,8), 30
2s minutes (lanes 3,4 and 9,10), or 60 minutes (lanes 5,6).
The fluorogram derived from SDS-PAGE indicated
that the synthesis of at least three proteins was
increased by increasing the temperature (FIG. 1). The
most prominent induced protein was about 72 kDa (HSP72),
whereas the other two were approximately 80 kDa (HSP80)
and 62 kDa (HSP62). Increased protein synthesis was
already apparent after 10 minutes of labeling (FIG. 1,
lanes 1, 2 and 7, 8) and became more significant when the
labeling period was prolonged to 30 minutes (FIG. 1,
lanes 3, 4 and 9, 10) and 60 minutes (FIG. 1, lanes 5, 6).
The effect of elevated temperature on the protein
synthesis profile of two different S. pneumoniae strains
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
was similar, with HSPs of similar molecular mass being
synthesized (compare Panel A (type 6 strain 64) to Panel B
(type 4 strain 53) in FIG. 1).
Analysis of the densitometric tracings from
scanning the protein synthesis profiles allowed the
estimation of the relative amounts of proteins. For
example, with respect to heat-shocked S. pneumoniae type 6
strain 64, after 10 minutes of labeling, HSP80 and HSP62
made up 2.9% and 6.8% of the labeled proteins,
respectively, compared to less than 0.1% at 37~C (FIG. 2).
Labeled proteins having an apparent molecular mass of 72
kDa were detected at both 37~C and 45~C conditions
(FIG. 2). Radioimmunoprecipitation analysis revealed,
however, that HSP72 was undetectable at 37~C (supra; and
FIGS. 3, 4 and 6) thus indicating that peak 9 from FIG. 2
corresponds to protein component(s) comigrating with
HSP72. Assuming no variation in the labeling of this
material, these results would suggest that the amount of
HSP72 represents 8.7% of the total labeled cell protein
after heat shock treatment. A comparison of the
densitometric tracings revealed that cellular proteins
corresponding to peaks 4, 10, 13, 17, 19, and 21 were
synthesized at almost the same rate irrespective of heat
shock treatment (FIG. 2). However, the synthesis of
several proteins (peaks 1, 2, 3, 15, 20, 22, 24, and 26)
declined considerably in response to heat shock (FIG. 2).
C. Immune Responses to S. pneumoniae HSPs
In order to assess the antibody response to
pneumococcal HSPs, mouse sera were first assayed by
radioimmunoprecipitation. The repertoire of labeled
proteins recognized by sera from mice ;mmlln;zed with
5. pneumoniae antigen preparations are shown in FIGS. 3
and 4. FIG. 3 relates to detergent soluble protein
preparations. FIG. 4 relates to heat-killed bacterial
preparation. Although many bands were detected by most
antisera, HSP72 was a major precipitation product. The
38
CA 0222401~ 1997-12-08
W096/40928 PCT/CA96/00322
specificity of antibodies for HSP72 was demonstrated by _
the detection of proteins among heat-shocked products only
(FIG. 3, lanes 4, 6, 8 and 10; FIG. 4, lanes 4, 6 and 8).
Interestingly, all immnnized mice consistently recognized
HSP72. The antibodies reactive with the HSP72 were not
specific to the strain used during the ;mmllnization since
strong reactivities were observed with heterologous
S. pneumoniae HSP72. It should be noted that in addition
to HSP72, one sera precipitated comigrating product
labeled at both 37~C and 45~C (FIG. 4, lane 4). This 72
kDa-product probably corresponds to component from peak 9
in FIG. 2 and was ~ot detected in immunoblots. HSP62 is
another immune target which was precipitated by some but
not all imm.une sera (FIG. 3, lane 6 and, FIG. 4, lanes 4
and 6). None of the sera tested reacted with HSP80. No
proteins were precipitated when preim.mune sera taken from
the mice used in this study were tested for the presence
of antibodies reactive with the labeled products.
As depicted in FIGS. 3 and 5, antibodies to
HSP72 could be detected after one immllnization with either
detergent-soluble proteins or whole cells extracts of
S. pneumoniae. In addition, a marked increase in the
antibody response to HSP72 was observed after a second
imm~1nization (FIG. 3, compare 4 and 6, and lanes 8 and
10).
The immunoblot patterns of 15 mice immllnized
with heat-killed S. pneumoniae bacteria were remarkably
consistent with the results of the previously descri~ed
radioim.munoprecipitation. Although antibody response
variation occurred to a variety of proteins, HSP72 was a
major immunoreactive antigen with 8 (53%) positive sera
after the first immunization (FIG. 5). Antibodies to
HSP72 were detected in 13 out of 15 (87%) immune sera
tested after the second immllnization. Two other prominent
antigens having apparent molecular mass of 53.5 and 47 kDa
were detected in 5 (33%) and 7 (47%) sera, respectively
39
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
(FIG. 5). The 72 kDa-reactive band was confirmed as the -
pneumococcal HSP72 by using recombinant HSP72 antigens
(Example 3, infra) in an immunoblot assay. Preimmune sera
failed to detect any pneumococcal proteins.
EXAMPLE 2 - Isolation of Monoclonal Antibodies
Against Epitopes of HSP72
A. Procedures
1. Tmml~;zation of Mice And Fusion
Female Balb/c mice (Charles River Laboratories~
were imml]nized with S. pneumoniae antigens. One set of
mice (fusion experiment 1) were imml~nized by peritoneal
injection with 107 formalin-killed whole cell antigen from
strain MTL suspended in Freund's complete adjuvant, and
were boosted at two-week intervals with the same antigen
and then with a sonicate from heat-killed bacteria in
Freund's incomplete adjuvant. A second group of mice
(fusion experiment 2) were imm~ni zed three times at three-
week intervals with 75 ,ug of detergent-soluble
pneumococcal antigens extracted from strain 64 (type 6) in
25 ~ug of Quil A adjuvant (Cedarlane Laboratories Ltd.,
Hornby, Ontario, Canada). Three days before fusion, all
mice were injected intraperitoneally with the respective
antigen suspended in PBS alone. Hybridomas were produced
by fusion of spleen cells with nonsecreting SP2/0 myeloma
cells as previously described by J. Hamel et al. [J. Med.
Microbiol., 23, pp. 163-170 (1987)]. Specific hybridoma
were cloned by sequential limiting dilutions, expanded and
frozen in liciuid nitrogen. The class, subclass, and
light-chain type of MAbs were determined by ELISA as
described by D. Martin et al., [Eur. J. Immunol., 18,
pp. 601-606 (1988)] using reagents obtained from Southern
Biotechnology Associates Inc. (Birmingham, AL).
CA 0222401~ 1997-12-08
WO9~/~D~28 PCT/CA96/00322
2. Subcellular Fractionation
Pneumococci were separated into subcellular
fractions according to the technique described by Pearce
et al. [Mol. Microbiol., 9, pp. 1037-1050 (1993)].
Briefly, S. pneumoniae strain 64 (type 6) was grown in
Todd Hewitt broth supplemented with 0.5~ (w/v) yeast
extract for 6 hours at 37~C and isolated by centrifugation.
Cell pellets were resuspended in 25 mM Tris-HCl pH 8.0, 1
mM EDTA, 1 mM phenylmethylsulphonylfluoride (PMSF) and
sonicated for 4 minutes with 15 second bursts. Cellular
debris were removed by centrifugation. The bacterial
membranes and cytoplasmic contents were separated by
centrifugation at 98,000 g for 4 hours. The cytoplasmic
(supernatant) and the membrane (pellet) fractions were
adjusted to 1 mg protein per ml and subjected to SDS-PAGE
and immunoblot analyses.
B. Identification and Characterization
of MAbs to the HSP72 of S. pneumoniae
Culture supernatants of hybridomas were
initially screened by dot enzyme immunoassay using whole
cells from S. pneumoniae strain 65 (type 4) according to
the procedures described in D. Martin et al. (supra).
Positive hybridomas were then retested by immunoblotting
in order to identify the hybridomas secreting MAbs
reactive with the HSP72. Of 26 hybridomas with anti-
S. pneumoniae reactivity in immunoblot, four were found torecognize epitopes present on a protein band with an
apparent molecular mass of 72 kDa. The four hybridomas
were designated F1-Pn3.1 (from fusion experiment 1) and
F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4 (from fusion experiment
2). Isotype analysis revealed that hybridoma F1-Pn3.1
(from fusion experiment 1) secreted IgG2~ immunoglobulins,
whereas hybridomas F2-Pn3.2, F2-Pn3.3, and F2-Pn3.4 (from
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
fusion experiment 2) all secreted IgG1~. The specificity
of the MAbs for HSP72 was clearly demonstrated by the lack
of radioimmunoprecipitation activity against
[35S]methionine-labeled S. pneumoniae proteins obtained
from cultures incubated at 37~C and the immunoprecipitation
of a 72kDa-protein with heat shock-derived lysates
incubated at 45~C. FIG. 6, (lanes 5 and 6) demonstrates the
results obtained for MAb Fl-Pn3.l. The same results were
obtained with MAbs F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4
[35S]methionine-labelled lysates from nonheat-
shocked and heat-shocked S. pneumoniae cells probed with
the MAbs were electrophoresed on SDS-PAGE gels and then
subjected to Western blot analysis. The resulting
immunoblots revealed the presence of HSP72 antigen in both
samples. FIG. 7, panel A, shows the results obtained for
MAb Fl-Pn3.l. The same results were obtained with MAbs
F2-Pn3.2, F2-Pn3.3 and F2-Pn3.4. Accordingly, the heat
shock stress did not significantly increase the reactivity
of anti-HSP72 monoclonal antibodies. The fluorograph of
the immunoblots, however, clearly showed that the heat
shock response had occurred (FIG. 7, panel B). These
experiments revealed that the rate of synthesis of
S. pneumoniae HSP72 increases in response to heat shock,
but that the absolute amounts of HSP72 do not increase
after heat shock.
C. Cellular localization of HSP72
In order to investigate the cellular location of
HSP72, S. pneumoniae cell lysates were fractionated by
differential centrifugation resulting in a soluble
fraction and a particulate fraction, enriched in membrane
proteins, supra. Sample containing 15 ,ug protein of
membrane fraction (lane l) and cytoplasmic fraction (lane
2) of S. pneumoniae were electrophoresed on SDS-PAGE,
transferred to nitrocellulose and probed with MAb Fl-
42
CA 02224015 1997-12-08
WO 96/40928 PCT/CA96/00322
Pn3.1. In the resulting Western blots, HSP72 was found in
both fractions, with the majority of the protein
associated with the cytoplasmic fraction (FIG. 8).
S EXAMPLE 3 - Molecular Cloning, Sequencing
and Expression of Genes Coding
for HSP72 Antigens
A. Procedures
1. Strains and Plasmids
Strains and plasmids used in this study are listed
in Table 1.
43
CA 02224015 1997-12-08
W 09~'4~92& PCT/CA96/00322
TA8LE 1: ~IACTERIAI, ST~2aTr~c~ P~IAGES AND pT.~QMTn~:
Strain, Relevant Characteristics Reference
Phage Plasmid or Source
E . col i Strains
JMlO9 ~( l ac -proAB ) [ F I traD proAB BRL
lacI~ Z~I15]
Y1090 r~-m~- lon supF [pMC9] Amersham
BL21(DE3) lac W 5-T7 RNA polymerase Studier
et al. (infra)
Phages
~gtll cI8 57 S10O cloning vector Amersham
~JBD7 LacZ-HSP72 fusion; 2.3 kb This study
EcoRI fragment in ~gtll
~JBD17 FucI-HSP72 chimeric; 2.4 This study
kb EcoRI and
2.3 kb EcoRI fragments in
~gtll
Plasmids
pWSK29 Ampr; low copy number Wang et al.
cloning vector (infra)
pWKS30 same as pWSK29 but Wang et al.
opposite multi cloning (infra)
site
pJBD171 same as ~JBD17 but in This study
pWSK29
pJBD177 2. 8 kb XhoI-EcoRI This study
fragment in pWKS30
no recombinant HSP72
protein expressed
pJBD179 FucI-HSP72 fusion; 2.4 kb This study
EcoRI and O. 8 kb EcoRI-
EcoRV fragments in pWSK29
pT7-5 Ampr; T7 promoter ~10 Tabor et al.
( infra )
pT7-6 same as pT7-5 but Tabor et al.
opposite multi cloning (infra)
site
pJBDf51 same as pJBD179 but in This study
pT7-5
pJBDf62 same as pJBD179 but in This study
pT7-6
pDELTAl Ampr; Tn 1000 BRL
pJBD~l same as pJBD179 but in This study
pDELTAl
44
CA 0222401~ 1997-12-08
W096/40928 PCT/CA96/00322
pJBD291 HSP72; 3.2 kb HindIII This study _
fragment in pWSK29
pJBDk51 same as pJBD291 but in This study
pT7-5
pJBD~4 same as pJBD291 but in This study
pDELTAl
E. coli strains were grown in L broth or on L
agar at 37~C. When necessary, ampicillin was added to the
media at the concentration of 50 ~g/ml. Plasmids were
isolated by using the Magic/Wizard~ Mini-Preps kit
(Promega, Fisher Scientific, Ottawa, Canada).
2. General Recombinant DNA Techniques
Restriction endonucleases, T4 DNA ligase, and
DNA molecular weight standards were purchased from
Boehringer Mannheim Canada, Laval, Quebec or Pharmacia
Biotech, Uppsala, Sweden. DNA restriction endonuclease
digestion and ligation were performed as described by
J. Sambrook et al. [Molecular cloning. A laboratory
manual. Cold Spring Harbor Laboratory Press, N.Y.
(1989)]. Agarose gel electrophoresis of DNA fragments was
performed following the procedure of J. Sambrook et al.
(supra) using the TAE buffer (0.04 M Tris-acetate; 0.002 M
EDTA) from Boehringer Mannheim. DNA fragments were
purified from agarose gel by using the Prep-A-Gene~ DNA
purification kit (Bio-Rad Laboratories Ltd., Mississauga,
Ontario). Transformation was carried out by
electroporation with the Gene Pulser~ (Bio-Rad) following
the protocol provided by the manufacturer.
3. Construction and Screening
of Genomic Library
A genomic S. pneumoniae DNA library was
generated in the bacteriophage expression vector ~gtll
(~gtll cloning system, Amersham) according to the
CA 0222401~ 1997-12-08
W 096/40928 PCT/CA96/00322
procedure provided by the manufacturer. Chromosomal DNA_
of S. pneumoniae type 6 strain 64 was prepared by
following the procedure of J.C. Paton et al. [Infect.
Immun., 54, pp. 50-55 (1986)]. The S. pneumoniae
chromosomal DNA was partially digested with EcoRI, and the
4- to 7-kb fragments were fractionated and purified from
agarose gel. The fragments were ligated into ~gtll arms,
packaged, and the resulting phage mixtures used to infect
E. coli Y1090. Immunoscreening of plaques expressing
recombinant HSP72 antigens was performed using HSP72-
specific monoclonal antibody Fl-Pn3.1, supra. Plaque
clones expressing peptides recognized by MAb Fl-Pn3.1 were
isolated and purified. Liquid lysates were prepared and
DNA was purified from a Promega LambdaSorb phage adsorbent
lS according to the manufacturer's directions followed by
conventional DNA purification procedures.
4. Southern Blot Analysis
The nonradioactive DIG DNA Labelling and
Detection kit, obtained from Boehringer M~nnheim, was used
to perform Southern blot analysis in this example. The
DNA fragments selected for use as probes (infra) were
purified by agarose gel electrophoresis and then labelled
~5 with digoxigenin (DIG)~ dUTP. Pneumococcal chromosomal
DNA was digested with HindIII and the digests were
separated by electrophoresis on an 0.8% SDS-PAGE gel and
transformed onto positive charged nylon membranes
(Boehringer Mannheim) as described by J. Sambrook et al.
(supra). The membrane was then blotted with the DIG-
labelled DNA probes according to the protocol of the
manufacturer.
5. DNA Sequencing and Sequence Analysis
The DNA fragments sequenced in this example were
first cloned into plasmid pDELTA 1 (GIBCO BRL Life
46
CA 0222401~ 1997-12-08
W 096/40928 PCT/CA96/00322
Technologies, Burlington, Ontario). A series of nested
deletions were generated from both strands by in vi~o
deletion mediated by Tn 1000 transposon transposition
(Deletion Factory System, GIBCO BRL) following the
s procedures provided by the supplier. These deletions were
sized by agarose gel electrophoresis and appropriate
deletion derivatives were selected for sequencing by the
dideoxynucleotide chain terminating method of F. Sanger
et al. [Proc. Natl. Acad. Sci. USA, 74, pp. 5463-5467
~1977)]. To sequence the gaps between deletion templates,
oligonucleotides were synthesized by oligonucleotide
synthesizer 392 (ABI, Applied Biosystems Inc., Foster
City, CA). The sequencing reaction was carried out by PCR
(DNA Thermal Cycler 4803, Perkin Elmer) using the Taq
DyeDeoxy Terminator Cycle Sequencing kit (ABI), and DNA
electrophoresis was performed on automated DNA sequencer
373A (ABI).
6. Expression of Cloned Gene in
E. coli T7 RNA pol/promoter system
High level expression of the cloned gene in this
example was achieved by employing the bacteriophage T7 RNA
polymerase/promoter system in E. coli. The DNA fragment
specifying the recombinant protein was ligated into
plasmids pT7-5 or pT7-6 [S. Tabor and C.C. Richardson,
Proc. Natl. Acad. Sci. USA, 82, PP. 1074-1078 (1985)], in
a proper orientation in which the gene to be expressed was
placed under the control of phage T7 RNA polymerase
specific promoter ~10. The resulting plasmid was
transformed into E. coli strain BL21 (DE3) [F.W. Studier,
and B.A. Moffatt, J. Mol. Biol., 189, pp. 113-130 (1986)]
which carries the T7 RNA polymerase structural gene on its
chromosome under the control of the inducible lacW5
promoter. Upon IPTG induction, the T7 RNA polymerase
induced in the BL21 (DE3) transformants specifically
47
CA 0222401~ 1997-12-08
W O 9f'4'928 PCT/CA96/00322
transcribed the gene under the control of T7 promoter ~lQ.
The overexpressed recombinant proteins were visualized by
either Western blotting or Coomassie Blue staining.
7. N-terminal Amino Acid Sequence
Analysis of HSP72
Pneumococcal HSP72 was purified by
immunoprecipitation using MAb F1-Pn3.1 (supra) and samples
of cell wall extracts of S. pneumoniae strain 64 prepared
as described by L.S. Daniels et al. [Microb. Pathogen., 1,
pp. 519-531 (1986)] as antigen. The immune precipitates
were resolved by SDS-PAGE and then transferred to
polyvinylidene difluoride (PVDF) membrane by the method of
P. Matsudaira [J. Biol. Chem., 262, pp. 10035-10038
(1987)]. PVDF membrane was stained with Coomassie Blue,
the HSP72 band excised and then analyzed in an automated
protein sequencer (ABI), according to standard procedures.
B. Construction of Plasmids Cont~ining
S. pneumoniae HSP72 Gene Fragments
Corresponding to C-169
The ~gtll S. pneumoniae genomic DNA library was
screened with the HSP72-specific MAb F1-Pn3.1. Seventeen
(17) immunoreactive clones were isolated and purified from
a total of 1500 phages tested. To confirm the specificity
of the proteins expressed by the recombinant phages,
Western blot analysis of the recombinant phage lysates was
performed. Two groups of clones were identified among the
17 positive clones recognized by MAb F1-Pn3.1 and their
representatives were designated as ~JBD7 and ~JBD17 for
further characterization. As shown in FIG. 9, whole cell
extracts from S. pneumoniae strain 64 (lane 1) and phage
lysates from ~. coli infected with ~JBD17 (lanes 2 and 3)
or ~JBD7 (lanes 4 and 5) cultured in the presence (+) or
absence (-) of IPTG were subjected to 10% polyacrylamide
48
CA 0222401~ 1997-12-08
W O gC'1~328 PCT/CA96/00322
gel electrophoresis and were electrotransferred to
nitrocellulose. The immunoblot was probed with HSP72-
specific MAb Fl-Pn3.1. Clone ~JBD17 had two EcoRI-EcoRI
insert fragments of 2.4 kb and 2.3 kb (FIG. 10), and
s expressed a chimeric recombinant protein having an
apparent molecular mass of 74 kDa on SDS-PAGE gel (FIG. 9,
lanes 2 and 3). Clone ~JBD7 was found to contain a 2.3 kb
EcoRI insert fragment and produced an apparent fusion
protein consisting of LacZ and the 74 kDa chimeric.protein
expressed from clone ~JBD17. The fusion protein had an
apparent molecular mass of 160 kDa as estimated by SDS-
PAGE (FIG. 9, lane 5). The expression of the chimeric
recombinant protein encoded by phage ~JBD17 was
independent of IPTG induction (FIG. 9, lanes 2 and 3)
while the expression of the recombinant fusion protein
encoded by phage ~JBD7 was dependent on induction of the
lac promoter (FIG. 9, lanes 4 and 5).
In an attempt to subclone the HSP72 gene, the
pneumococcal DNA insert from clone ~JBD17 was extracted,
purified and ligated into a low copy plasmid pWSK29 [R.F.
Wang and S.R. Kushner, Gene, 100, pp. 195-199 (1991)] to
generate plasmid pJBD171. The insert from pJBD171 was
characterized by restriction mapping (Fig. lOB), and a
series of subcloning and immunoblotting was carried out to
define the boundaries of the gene coding for the antigen
reactive with MAb Fl-Pn3.1. The region responsible for
expression of the 74 kDa chimeric protein was found to
localize on the 3.2 kb EcoRI-EcoRV fragment, which
consists of the intact 2.4 kb EcoRI-EcoRI fragment and the
0.8 kb EcoRI-EcoRV portion of the 2.3 kb EcoRI-EcoRI
fragment. The plasmid carrying the 3.2 kb EcoRI-EcoRV
insert was designated pJBD179.
49
CA 0222401~ 1997-12-08
WO 96/40928 PCT/CA96/00322
C. Expression and DNA Sequence
Analysis of a Ch;meric Gene
Coding for C-169
To further determine the transcriptional
direction of the gene coding for the 74 kDa chimeric
protein on the 3.2 kb EcoRI-EcoRV fragment, and to
increase the yield of the 74 kDa chimeric protein for
immunological study, we decided to express the 74 kDa
chimeric protein in the E. coli T7 RNA and T7 promoter
system. The 3.2 kb EcoRI-EcoRV fragment, derived from
pJBD179, was ligated into plasmids pT7-5 and pT7-6 in
which the multi-cloning sites were placed in opposite
orientation with respect to the T7 RNA polymerase specific
T7 promoter ~10. The ligation mixture was used to
transform E. col i JM109 and positive transformants
reactive with MAb F1-Pn3.1 were identified by the colony
lifting method described by J. Sambrook et al. [supra].
The resulting recombinant plasmids, derived from pT7-5 and
pT7-6, were designated pJBDf51 and pJBDf62, respectively.
The intact 3.2 kb EcoRI-EcoRV insert in these recombinant
plasmids and their orientation was determined by
restriction mapping. To achieve overexpression of the
74 kDa chimeric protein, pJBDf51 and pJBDf62 were
transformed, separately, into E. coli BL21(DE3). The
transformants were induced with IPTG (1 mM) for 3 hours at
37~C. The cells were harvested, washed, resuspended in
1% SDS and boiled for 10 minutes. The lysates were then
used for SDS-PAGE and immunoblot analysis. As expected,
both transformants produced the 74 kDa chimeric protein
readily detected by Western blotting with MAb F1-Pn3.1
(FIG. 11). However, under the IPTG induction condition,
only transformants BL21(DE3)(pJBDf51) overexpressed the 74
kDa chimeric protein (FIG. llA and B, lane 2) indicating
that the transcriptional direction of the gene on the 3.2
CA 0222401~ 1997-12-08
WO 9G/4v~28 PCT/CA96/00322
kb EcoRI-EcoRV fragment is from the EcoRI end towards the
EcoRV end (FIG. 10A).
The 3.2 kb EcoRI-EcoRV fragment was cloned into
plasmid pDELTA 1 to yield plasmid pJBD~l. A series of
overlapping deletions were generated and used as DNA
sequencing templates. The DNA sequence of the entire 3.2
kb EcoRI-EcoRV insert is SEQ ID NO:l. Two open reading
frames ("ORFs"~ were found and their orientation is
indicated in FIG. 10B ("ORF27" and "FucI-HSP72 (C-169)").
In front of these two ORFs, putative ribosome-b;~;ng
sites were identified (SEQ ID NO:l, nucleotides 18-21 and
760-763). No obvious -10 and -35 promoter sequences were
detected. ORF27 spans nucleotides 30-755 (SEQ ID NO:l) and
encodes a protein of 242 amino acids with a calculated
molecular weight of 27,066 daltons. The deduced amino
acid sequence of this protein is SEQ ID NO:2. We
designated this gene orf27, and compared it to other known
sequences. No homologous gene or protein was found. The
large ORF (nucleotides 771-2912, SEQ ID NO:l) specifies a
protein of 714 amino acids with a predicted molecular mass
of 79,238 daltons. The deduced amino acid sequence of
this protein is SEQ ID NO:3. This ORF was compared with
other known sequences to determine its relationship to
other amino acid sequences. This analysis revealed a high
degree of similarity of the encoded protein to the
sequence of E. coli fucose isomerase (FucI) and to several
HSP70 gene family members, also known as DnaK genes.
Alignment of SEQ ID NO:3 and those of the E. coli FucI and
HSP70 (Dnak~ proteins indicated that the N-terminal
portion corresponding to amino acids 1 to 545 (SEQ ID
NO:3) of the 74 kDa chimeric protein is highly homologous
to E. coli FucI, while the C-terminal portion
corresponding to amino acids 546-714 (SEQ ID NO:3) is
similar to HSP70 (DnaK) proteins. It is noteworthy that
there is an EcoRI restriction site lying in the junction
of these two portions of the gene coding for the 74 kDa
protein (SEQ ID NO:l, between nucleotides 2404 and 2405).
CA 0222401~ 1997-12-08
WO 9~ 28 PCT/CA96/00322
Other restriction sites exist between nucleotides 971 and
972 (Pst I), nucleotides 1916 and 1917 (Pst I),
nucleotides 1978 and 1979 (Xho I), and nucleotides 3164
and 3165 (EcoRV). From these data we concluded that the
5 74 kDa protein was a chimeric protein encoded by two
pieces of S. pneumoniae chromosomal DNA, a 2.4 kb EcoRI-
EcoRI fragment derived from the FucI homologous gene and a
2.3 kb EcoRI-EcoRI fragment derived from the HSP72 gene.
D. Southern Blot Analysis
Southern blotting was performed in order to
confirm that the 74 kDa protein is a chimeric protein and
to attempt to clone the entire pneumococcal HSP72 gene.
15 Chromosomal S. pneumoniae DNA was digested with HindIII to
completion, separated on a 0.8% agarose gel, and
transferred onto two positively charged nylon membranes
(Boehringer MAnnheim). The membranes were then blotted
with either the 0.8 kb EcoRI-EcoRV probe, derived from the
20 2.3 kb EcoRI-EcoRI fragment, or the 1 kb PstI-PstI probe,
obtained from the 2.4 kb EcoRI-EcoRI fragment. Both
probes had been previously labelled with digoxigenin-dUTP.
These two probes hybridized two individual HindIII
fragments of different sizes (FIGS. 10B and 10C). The 0.8
25 kb EcoRI-EcoRV probe recognized the 3.2 kb HindIII
fragment and the 1 kb PstI-PstI probe reacted with the 4
kb HindIII fragment. This result further indicated that
the gene responsible for the expression of the 74 kDa
chimeric protein was generated by fusion, in frame, of two
30 pieces of EcoRI fragments, one originated from the
fragment containing the 5' portion of the S. pneumoniae
FucI homologue, the other derived from the segment
carrying the C-169 fragment of the pneumococcal HSP72
gene. The fact that the 0.8 kb EcoRI-EcoRV probe
35 hybridized a single 3.2 kb fragment suggested that there
is only a single HSP72 gene copy in S. pneumoniae.
CA 0222401~ 1997-12-08
W096/40928 PCT/CA96/00322
E. Production of Recombinant HSP72
A partial pneumococcal genomic library was
generated by ligation of the pool of HindIII digests of
chromosomal DNA, with sizes ranging from 2.8 to 3.7 kb,
into plasmid pWSK29/HindIII. The ligation mixture was
used to transform E. coli strain JM 109 and the
transformants were screened by hybridization with the 0.8
kb EcoRI-EcoRV probe. One representative plasmid from
four positive hybridizing clones was named pJBD291.
Restriction analysis of the insert and Western blot of the
cell lysate of transformants were employed to verify that
the plasmid pJBD291 indeed carries the 3.2 kb HindIII
fragment containing the HSP72 gene expressing the
recombinant HSP72 protein (FIG. 10B). The HSP72 protein
expressed by the transformants (pJBD291) migrated on the
SDS-PAGE gel at the same position as the native HSP72
protein (FIG. 12). To sequence the entire HSP72 gene and
to overexpress the full-length HSP72 protein, the 3.2 kb
HindIII fragment was isolated from plasmid pJBD291, and
subcloned into plasmids pDELTA 1 and pT7-5 to generate
pJBD~4 and pJBDk51, respectively.
The entire 3.2 kb HindIII DNA fragment carried
on the plasmid pJBD~4 and the 2.3 kb EcoRI-EcoRI DNA
fragment contained on the plasmid pJBD177 were sequenced.
Altogether, the nucleotide sequence comprised 4320 base
pairs and revealed two ORFs (SEQ ID NO:4). The first ORF,
starting at nucleotide 682 and ending at nucleotide 2502
(SEQ ID NO:4), was identified as the pneumococcal HSP72
gene, and the second ORF, spanning from nucleotide 3265 to
nucleotide 4320 (SEQ ID NO:4), was located 764 base pairs
downstream from the HSP72 structural gene and was
identified as the 5' portion of the pneumococcal Dna~
gene. The putative ribosome binding site ("AGGA") was
located 9 base pairs upstream from the start codon of the
HSP72 structural gene, while the typical ribosome binding
CA 0222401~ 1997-12-08
WO 96/40928 PCT/CA96/00322
site ("AGGA") was found 66 base pairs upstream from the -
start codon of the Dna~ structural gene. No typical 5
regulatory region was identified in front of these two
genes. Restriction sites are located between nucleotides
1 and 2 (HindIII), nucleotides 1318 and 1319 (EcoRI),
nucleotides 1994 and 1995 (EcoRI), nucleotides 3343 and
3344 (HindIII), and nucleotides 4315 and 4316 (EcoRI).
The gene organization of HSP72 (DnaK) and DnaJ in
S. pneumoniae is similar to that of E. coli [Saito, H. and
Uchida, Mol. Gen. Genet. 164, 1-8 (1978)] as well as
several other Gram positive bacteria [Wetzstein, M.
et al., J. Bacteriol. 174, 3300-3310 (1992)]. However,
the intragenic region of S. pneumoniae is significantly
larger and no ORF for the grpE gene was found upstream of
the HSP72 (DnaK) structural gene.
The predicted HSP72 protein has 607 amino acids
and a calculated molecular mass of 64,755 daltons, as
compared to the 72 kDa molecular mass estimated by SDS-
PAGE. The predicted HSP72 protein is acidic with an
isoelectric point (pI) of 4.35. Automated Edman
degradation of the purified native HSP72 protein extracted
from S. pneumoniae strain 64 revealed SKIIGIDLGTTN-AVAVLE
as the 19 amino acid N-terminal sequence of the protein.
The amino-terminal methionine was not detected, presumably
due to in situ processing which is known to occur in many
proteins. No amino acid residue was identified on
position 13. The 19 amino acid N-terminal sequence
obtained from the native HSP72 protein is in full
agreement with the 19 amino acid N-terminal sequence
deduced from the nucleotide sequence of the recombinant
S. pneumoniae HSP72 gene (SEQ ID NO:5) thus confirming the
cloning. This N-terminal sequence showed complete
identity with the DnaK protein from Lactococcus lactis and
68.4% identity with the DnaK protein from Escherichia
Coli. Similarly, the alignment of the predicted amino
acid sequence of HSP72 (SEQ ID NO:5) with those from other
bacterial HSP70 (DnaK) proteins also revealed high
54
CA 0222401~ 1997-12-08
W 096140928 PCT/CA96100322
homology (FIGS. 13A-13D). For example, HSP72 showed 54% -
identity with the E. coli DnaK protein. The highest
identity value was obtained from comparison with the Gram
positive bacterium Lactococcus lactis, showing 85%
identity with HSP72. Like other HSP70 proteins of Gram
positive bacteria, HSP72 misses a stretch of 24 amino
acids near the amino terminus when compared with DnaK
proteins from Gram negative bacteria (FIGS. 13A-13D).
Although HSP72 shares homology with HSP70 (DnaK)
proteins from other organisms, it does possess somë unique
features. Sequence divergence of the HSP70 (DnaK)
proteins is largely localized to two regions (residues 244
to 330 and 510 to 607, SEQ ID NO:5). More specifically,
the peptide sequences GFDAERDAAQAALDD (residues 527 to
541, SEQ ID NO:5) and AEGAQATGNAGDD W (residues 586 to
600, SEQ ID NO:5) are exclusive to HSP72. The fact that
the C-terminal portion of HSP72 is highly variable
suggests that this portion carries antigenic determ;n~nts
specific to S. pneumoniae. Consistent with this
hypothesis, monoclonal antibodies directed against the C-
169 fragment of HSP72 (infra), were not reactive with
~. coli and S. aureus, which are known to express DnaK
proteins similar to HSP72.
The truncated DnaJ protein of S. pneumoniae (SEQ
~5 ID NO:6) has 352 amino acids, which show a high degree of
similarity with the corresponding portions of the L.
lactis DnaJ protein (72% identity) and the E. coli DnaJ
protein (51% identity)~ The predicted truncated DnaJ
protein contains high glycine content (15%). Four Gly-,
Cys-rich repeats, each with the Cys-X-X-Cys-X-Gly-X-Gly
motif characteristic of DnaJ proteins [P.A. Silver and
J.C. Way, Cell, 74, pp. 5-6 (1993)], were identified
between amino acids 148 and 212 of the S. pneumoniae DnaJ
protein (SEQ ID No 6). Three repeated GGFGG sequences
( residues 75-79, 81-85, and 90-94) were found near the N-
terminus.
CA 0222401~ 1997-12-08
WO 96/40928 PCT/CA~G/003?~
F. Reactivity of MAbs Against
Recombinant Antigens
The ~our HSP72 specific MAbs (F1-Pn3.1, F2-
Pn3.2, F2-Pn3.3 and F2-Pn3.4, supra) were tested for their
reactivity against proteins expressed by E. coli infected
or transformed with recombinant phages and plasmids
containing HSP72 sequences. The four individual MAbs
reacted with the lacZ-HSP72 fusion protein expressed by
the clone ~JBD7, thus localizing the epitopes recognized
by these MAbs to the C-terminal 169 residues.
Surprisingly, the proteins encoded by the pneumoccocal
inserts in ~JBD17 and pJBD~1 were recognized by only 3 of
lS 4 Mabs. These results suggest that although the C-169
fragments synthesized in E. coli infected with ~JBD7 and
~JBD17 have the same primary structure, they have distinct
conformation. The lack of reactivity of MAb F2-Pn3.2 with
some recombinant proteins raised the possibility that this
particular MAb recognizes a more complex epitope.
- Although complex, F2-Pn3.2 epitopes are still recognizable
on Western immunoblots. The complete HSP72r~C protein
expressed by E. coli containing the recombinant plasmid
pJBD~4 was reactive with all four MAbs.
~5
EXAMPLE 4 - Antigenic Specificity and
Reacti~ity of HSP72-Specific
Monoclonal Antibodies
The reactivity of MAbs F1-Pn3.1, F2-3.2., F2-
Pn3.3 and F2-Pn3.4 to a collection of bacterial strains
including 20 S. pneumoniae strains representing 16
capsular serotypes (types 1, 2, 3, 4, 5, 6, 8, 9, 10, 11,
12, 14, 15, 19, 20, and 22) and the 17 non-pneumococcal
bacterial strains listed in Table 2, was tested using a
dot enzyme immunoassay as described by D. Martin et al.
[supra] and immunoblotting. For dot enzyme immunoassay,
the bacteria were grown overnight on chocolate agar plates
56
CA 0222401~ 1997-12-08
WO 96/~928 PCT/CA9('0~
and then suspended in PBS, pH 7.4. A volume of 5 ,ul of a
suspension containing approximately 109 CFU/ml was applied
to a nitrocellulose paper, blocked with PBS containing 3%
bovine serum albumin, and then incubated sequentially with
S MAbs and peroxydase-labeled secondary antibody. Whole
cell extracts were prepared for Western blot analysis by
boiling bacterial suspensions in sample buffer for 5
minutes.
TABLE 2:LIST OF NON-PNE~MOi~'O~ T- ISO~ATES
TESTED BY DOT ENZYNE INM~NO~-C,e~Y
Strain
Designation Genus species group or type
C-2 Streptococcus pyogenes group A
C-3 Streptococcus agalactiae group B
C-7 Enterococcus faecalis group D
C-9 Streptococcus bovis group D
C-14 Streptococcus mutans
C-15 Streptococcus salivarius
C-19 Streptococcus sanguis
C-20 Streptococcus sanguis
C-21 Streptococcus sanguis
C-22 Streptococcus sanguis II
C-23 Streptococcus sanguis II
C-24 Streptococcus sanguis II
C-25 Streptococcus sanguis II
C-27 Gemella morbillorum
C-30 Staphylococcus aureus
C-33 Bacillus
C-36 Escherichia coli
When tested by dot enzyme immunoassay, each MAb
reacted with each of the S. pneumoniae strains and none of
the non-pneumococcal isolates. These results were
unexpected since comparison studies revealed that HSP72 is
CA 0222401~ 1997-12-08
WO 96/40928 PCT/CA96i'~ ?
very similar to other known bacterial HSP70 (DnaK)
proteins, for example those from E. coli and S. aureus.
Immunoblots were then performed to further
investigate the immunoreactivities of our MAbs. As shown
in Table 3, each MAb exhibited some reactivity. Although
the percent identity of the E. coli amino acid seguence
and the HSP72 amino acid sequence (SEQ ID NO:5) is 54%,
the four HSP72-specific MAbs did not recognize the E. coli
HSP70 (DnaK) protein. Similarly, the HSP72-specific MAbs
did not react with the C. trachomatis HSP70 (DnaK)
protein, which has 56% amino acid identity with the amino
acid sequence of HSP72. High amino acid sequence homology
is observed between HSP72 and the HSP70 (DnaK) proteins
from gram positive bacterial species. However, again,
none of the HSP72-specific MAbs reacted with S. aureaus or
Bacillus gram positive species, which exhibit 74% and 76
amino acid sequence homology, respectively, with HSP72.
From these data it is clear that although HSP70 (DnaK)
proteins may be structurally related to HSP72, they are
immunologically distinct. Among the non-pneumococcal
isolates that reacted with at least one MAb, there is S.
pyogenes, Enterococcus faecalis, S. mutans and S. sanguis,
which all belong to the Streptococcus or Streptococcus-
related Enterococcus genus. So far, neither the HSP70
protein, nor the gene structure has been identified in
these Streptococcus or Enterococcus species. Altogether,
these observations indicate that hypervariable amino acid
sequences or residues within HSP70 (DnaK) proteins are
involved in antigenicity. Interestingly, immunoblotting
analysis revealed that there was no significant variation
in the molecular mass of the HSP70 (DnaK) proteins among
both S. pneumoniae isolates and immunoreactive non-
pneumococcal isolates.
58
CA 022240l5 l997-l2-08
W O 96/40928 PCT/CA96~ 322
TABLE 3: REA~.lv~ OF MABS WITH NON-PNEIn ~OCr~T~ ISOLATES IN
W~:S.~:nN IN~UN03LOTTING
Bacterial Strain MA~8
Designation genu~/~pecie~ type Fl- F2- F2- F2-
PN3.1 Pn3.2 PN3.3 Pn3.4
C-2 Streptococcus group A - + - +
pyogenes
C3 Streptococcus group B
agalactiae
C-7 Enterococcus group D - +
faecalis
C-9 Streptococcus group D
bovis
C-14 Streptococcus - + - +
mutans
C-15 Streptococcus - - - ~
salivarius
C-l9 Streptococcus I + +
sanguis
C-20 Streptococcus I + + - +
sanguis
C-21 Streptococcus I + + + +
sanguis
C-22 Streptococcus II + +
sanguis
C-23 Streptococcus II + +
sanguis
C-24 Streptococcus II + + + +
sanguis
C-25 Streptococcus II + + + +
sanguis
C-27 Gemella
morbillorum
C-30 Staphylococcus
aureus
C-33 ~acillus
C-36 rscheric~;a - - - -
coli
C-RP Chlamydia L2
~rachoma~lsb
a _ indicates a weak signal compared to the reactivity
observed with 5. pneumoniae antigens
b C. trachomatis purified elementary bodies were tested.
59
CA 022240l~ l997-l2-08
WO 96/40928 PCT/CA96/00322
EXAMPLE 5 - Purification of HSP72 And Its
Use As An Immunogen to Protect
Against Lethal S. Pneumoniae In~ection
A. Procedures
1. Preparation of Purified
Recombinant HSP72 Protein
and Recombinant C-169
High level exclusive expression of the HSP72
gene was achieved by employing the bacteriophage T7 RNA
polymerase/T7 promoter system in E. coli . The 3.2 kb
HindIII fragment was cloned in both orientations in front
of the T7 promoter ~10 in the plasmid pT7-5. The
resulting plasmid pJBDk51 was then transformed into
E. coli strain BL21 (DE3). Overexpression of the
recombinant HSP72 protein (HSP72r.C) was induced by
culturing in broth supplemented with antibiotics for a 3-
hour period after the addition of IPTG to a final
concentration of 1 mM. E. coli expressing high levels of
HSP72r.C were concentrated by centrifugation and lysed by
mild sonication in 50 mM Tris-Cl (pH 8.0), 1 mM EDTA and
100 mM NaCl lysis buffer containing 0.2 mg/ml lysozyme.
The cell lysates were centrifuged at 12,000 g for 15
minutes and the supernatants were collected. HSP72r,c was
purified by immunoaffinity using monoclonal antibody Fl-
Pn3.1 immobilized on sepharose 4B beads (Pharmacia). The
purity of eluates was assessed on SDS-PAGE.
The recombinant C-169 protein (C~169r,c) was
expressed in the form of insoluble inclusion bodies in
E. coli strain JM109 transformed with the plasmid pJBD~l.
Protein inclusion bodies were recovered from pelleted
bacterial cells disrupted by sonication as described
before. The pellets were washed in lysis buffer
containing 1 mg/ml of deoxycholate to remove contaminating
materials, and the protein inclusion bodies were then
solubilized in urea 6 M. The protein solution was
CA 0222401~ 1997-12-08
W O 96/40928 PC~r/CA96/00322
centrifuged at 100,000 g and the cleared supernatant
collected and dialysed against phosphate-buffered saline.
After purification, the protein content was determined by
the Bio-Rad protein assay (Bio-Rad Laboratories,
Mississauga, Ontario, Canada).
2. Active Immunoprotection Studies
Two groups of 10 female Balb/c mice (Charles
River Laboratories) were ; mmlln; zed subcutaneously three
times at two-week intervals with 0.1 ml of purified
HSP72r.C or C- 169r~c antigens absorbed to Alhydrogel
adjuvant. Two antigen doses, approximately 1 and 5 ~ug,
were tested. A third group of 10 control mice were
immllnized identically via the same route with Alhydrogel
adjuvant alone. Blood samples were collected from the
orbital sinus prior to each ;mmml~n;zation and five to
seven days following the third injection. The mice were
then challenged with approximately 106 CFU of the type 3
S. pneumoniae strain WU2. Samples of the S. pneumoniae
challenge inoculum were plated on chocolate agar plates to
determine the CFU and to verify the challenge dose.
Deaths were recorded at 6-hour intervals for the first 3-4
days post-infection and then at 24-hour intervals for a
period of 14 days. On days 14 or 15, the surviving mice
were sacrificed and blood samples tested for the presence
of S. pneumoniae organisms. Antibody responses to the
recombinant HSP72 antigens are described in Example 7.
3. Passive Immunoprotection Studies
One NZW rabbit (Charles River Laboratories) was
;mmlln;zed subcutaneously at multiple sites with
approximately 50 ug of the purified C-169r.c protein
adsorbed to Alhydrogel adjuvant. The rabbit was boosted
three times at two-week intervals with the same antigen
and blood samples collected 7 and 14 days following the
61
CA 0222401~ 1997-12-08
WO 9~ 28 PCT/CA96/00322
last ;mmllnization. The serum samples were pooled and
antibodies were purified by precipitation using 40%
saturated ammonium sulfate.
Severe-combined immunodeficient SCID mice were
injected intraperitoneally with 0.25 ml of the purified
rabbit antibodies l hour before intravenous challenge with
5000 or 880 CFU of the type 3 S. pneumoniae strain WU2.
Control SCID mice received sterile buffer or antibodies
purified from non;mml1ne rabbit sera. Samples of the
S. pneumoniae challenge inoculum were plated on chocolate
agar plates to determine the CFU and to verify the
challenge dose. The SCID mice were chosen because of
their high susceptibility to S. pneumoniae infection.
Blood samples (20 ,ul each) obtained 24 hours post-
challenge were plated on chocolate agar and tested for thepresence of S. pneumoniae organisms. The level of
detection was 50 CFU/ml. Deaths were recorded at 24-hour
intervals for a period of 5 days.
B. Results
The availability of cloned S. pneumoniae DNA
inserts encoding the complete or partial (C-169) HSP72
protein and the expression of recombinant proteins in
E. coli allowed the obtention of purified proteins useful
for the investigation of the vaccinogenic potential of
HSP72 protein. Both HSP72r.C and C-l69r.C proteins were
obtained in a relatively pure state with no contaminants
detected on Coomassie Blue-stained SDS polyacrylamide gels
(FIGS. 14 and 15, respectively).
To evaluate the vaccinogenic potential of HSP72,
we first examined the ability of HSP72r.C to elicit a
protective immune response. Groups of lO mice were
immunized with full-length HSP72r.C (l ~ug or 5 ,ug dose) and
challenged with 4.2 million CFU of S. pneumoniae type 3
strain WU2. Eighty percent (80%) of the mice dosed with 1
,ug HSP72r.C survived the challenge, as did 50% of the mice
62
CA 0222401~ 1997-12-08
W O 95'4~28 PCT/CA96/00322
dosed with 5 ~g HSP72 . None of the naive mice immlln;zed
with Alhydrogel adjuvant alone without antigen survived
the challenge (FIG. 16). No S. pneumoniae organisms were
detected in any of the blood samples collected on days 14
or 15 from mice surviving infection. The observation that
HSP72r~C elicited protection against type 3 strain WU2
pneumococci indicated that HSP72 derived from DNA
extracted from a type 6 strain contains epitopes capable
of eliciting protection against a heterologous strain
having a different capsular type.
We further examined the immune response to the
HSP72 protein by using recombinant protein fragments
expressed from E. coli transformed with a chimeric fucI-
HSP72 gene. Mice lmmnnized with purified C~169r~c were
protected from fatal pneumococcal challenge, thus
demonstrating that some, if not all, epitopes eliciting
protection are present in the C-terminal region of the
HSP72 molecule comprising the last 169 residues. Groups
of 10 mice were ;mml-~ized with C~169r~c (1 ~g or 5 ~g
doses) and challenged with 6 million CFU of S. pneumoniae
type 3 strain WU2 . Sixty percent ( 60~ ) of the mice dosed
with 1 ,ug C-169r.C survived the challenge, as did 70% of
the mice dosed with 5 ~g C~169r,c (FIG. 17 ) . In contrast,
all of the naive mice were dead by 2 days post-challenge.
Therefore, the C-terminal portion of S. pneumoniae HSP72,
which includes the region of maximum divergence among DnaK
proteins, is a target for the protective immune response.
As illustrated in Table 4 below, two independent
experiments demonstrated that SCID mice passively
transferred with rabbit anti-C-169r.c antibodies were
protected from fatal infection with S. pneumoniae WU2. In
contrast, none of the 15 control mice survived. The
control mice received antibodies from non;mmllne rabbit
sera or received sterile buffer alone. In addition, all
3s mice from the control groups had positive S. pneumoniae
hemoculture 24 hours post-challenge, while S. pneumoniae
63
CA 02224015 1997-12-08
W O 96/40928 PCT/C A96/00322
organisms were detected in only 2 out of a total of l0
imm1lnized SCID mice.
TABLE 4: PASSIVE INM~NIZATION S~T~DIES SHOWING PRG ~-~lON
OF SCID MICE FROM EXP~r~ AL S. PNEUMONIAE l~r~llON BY
ANTI-C-169r.C RABBIT ANTIBODIES
~xperiment Injection No. of Mice No. of Mice
Surviving Testing
Challenge Positive for
after S days the Presence of
S. pneumoniae
l sterile 0/5 5/5
buffer
anti-C-l69r~c 4/5 2/5
control 0/5 5/5
antibodies
2 sterile 0/5 5/5
buffer
anti-C-l69r~c 5/5 0/5
s
In experiments l and 2 (Table 4), mice were
challenged with 5000 and 880 CFU of type 3 S. pneumoniae
strain WU2, respectively. Results in Table 4 are
expressed as the number of mice surviving challenge, or
testing positive for the presence of S. pneumoniae,
compared to the total number of mice in each group.
Demonstration of the anti-HSP72 specificity of
the antibody elicited by imml]nization with recombinant
HSP72 or C-169 proteins came from Western Blot analyses
using S. pneumoniae cell lysates as antigens. A single
band corresponding to HSP72 was detected by all rabbit and
mouse antisera tested. These serologic results suggested
that the protection following the immllnization with
recombinant proteins was due to the production of
antibodies reactive with S. pneumoniae HSP72.
EXAMPLE 6 - Heat-Inducible Expression System for High
Level Production of the C-l5l Terminal Portion of the
HSP72 Protein
64
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
A. Construction of Plasmid pURV3 Containing the C-
151 terminal coding region of the HSP72 of S.
pneumoniae
The DNA region coding for 151 amino acids at
the carboxyl end of the HSP72 of S. pneumoniae was
inserted downstream of the promoter ~ PL into the
translation vector p629 [H. J. George et al.,
Bio/Technology 5, pp. 600-603 (1987)]. This vector
contains a cassette of the bacteriophage ~ cI857
temperature sensitive repressor gene from which the
functional PR promoter has been deleted. The inactivation
of the cI857 repressor by a temperature increase from the
ranges of 30-37~C to 37-42~C results in the induction of
the gene under the control of ~ PL. The induction of gene
expression in E. coli cells by a temperature shift is
advantageous for large scale fermentation since it can
easily be achieved with modern fermenters. However, it
should be understood that while E. coli was the
microorganism of choice in the experiments herein
described, other host organisms, such as yeast, are
intended to be included within the scope of this
invention.
A fragment of 477 nucleotides, including the
region of 457 bases between 2050 to 2506 in HSP72 gene of
5. pneumoniae (see SEQ ID NO 4), was amplified by the
polymerase chain reaction (PCR) from the S. pneumoniae
type 6 strain 64 genomic DNA using the oligonucleotide
primers OCRR26 (5' -GGCAGATCTATGAAGGCCAAAGACCTTGGAAC)
and OCRR27 (5'-CGCGGATCCTTACTTTTCCGTAAACTCTCCGT).
Chromosomal DNA was prepared from a 90 ml culture of
exponentionally growing cells of S. pneumoniae in heart
infusion broth using the method of Jayarao et al. [J.
Clin. Microbiol., 29, pp. 2774-2778 (1991)]. DNA
amplification reactions were made using a DNA Thermal
Cycler, Perkin Elmer, San Jose, CA. In OCRR26, an ATG
start codon is present in frame just upstream of the
CA 0222401~ 1997-12-08
WO 96/40928 PCT/CA96/00322
coding region for the amino-terminus region of the C-151
The primers OCRR26 and OCRR27 contain, respectively, a
BglII (AGATCT) and a BamHI (GGATCC) recognition site in
order to facilitate the cloning of the PCR product into
5 the dephosphorylated restriction sites BglII and Ba/nHI of
p629. The PCR product was purified from agarose gels by
the method of phenol freeze [S. A. Benson, Biotechniques
2, pp. 67-68 (1984) ] and digested with the restriction
enzymes BglII and BaznHI. The BglII-Ba~ I fragment of 471
10 base pairs was then ligated into the BglII and BamHI
recognition sites dephosphorylated of p629. A partial map
of the resulting plasmid pURV3 is shown in FIG. 18. This
plasmid was transformed by the method of Simanis [~n~hi~n,
D. In D. M. Glover (ed.), DNA Cloning, pp. 109-135,
IS (1985) ] into the E. coli strain XLI Blue MRF' (~ (mcrA)183
~(mcrCB-hsdSMR-mrr)173 endAl supE44 thi-l recAl gyrA96
relAl lac [F' proAB lacI~Z~M15 TnlO (Tetr)]c ) which was
obtained from Stratagene, La Jolla, CA. The transformants
grown at 37~C were screened by colony immunoblot [J.
Sambrook et al. (supra)] using the MAb F1-Pn3.1 reactive
with C-169rec. Plasmid DNA was purified from a selected
transformant and the DNA insert was sequenced by PCR using
the Taq Dye Deoxy Terminator Cycle Sequencing kit of
Applied Biosystems Inc. (ABI) and DNA electrophoresis was
'5 performed on automated DNA sequencer 373A (ABI). The
nucleotide sequence of the insert perfectly matched the
nucleotide sequence of the C-151 coding region of the
HSP72 gene. (See SEQ ID No: 25 and corresponding amino
acid sequence at SEQ ID No: 26.) The plasmid was
transformed into the prototrophic E. coli strain W3110
(ATCC 27325) for the production of C-151reC.
B. Expression of C-151rec and Antigen
Preparation
The recombinant C-151rec was synthesized with a
methionine residue at its amino end in E. coli strain
W3110 harboring the plasmid pURV3. E. coli cells were
66
CA 0222401~ 1997-12-08
WO 96/40928 PCT/CA96/00322
grown at 30~C in LB broth containing 100 ,ug of ampicillin-
per ml until the A600 reached a value of 0.6. The cells
were then cultivated at 40~C for 18 hours to induce the
production of C-151reC protein. A semi-purified C-151reC
protein was prepared using the following procedures. The
bacterial cells were harvested by centrifugation and the
resulting pellet was washed and resuspended in phosphate-
buffered saline. Lysozyme was added and the cells were
incubated for 15 min on ice before disruption by pulse
sonication. The cell lysates were cleared by ~
centrifugation and the supernatants were collected and
subjected to separation using an Amicon's ultrafiltration
equipment (stirred cells series 8000, Amicon Canada Ltd.
Oakville, Ontario). The ultrafiltrate not retained by a
YM30 membrane was recovered, analysed by SDS-PAGE and
stained with Coomassie blue R-250. Protein concentrations
were estimated by comparing the staining intensity of the
C-151reC protein with those obtained with defined
concentrations of soybean trypsin inhibitor.
C Reactivity of MAbs Against C-15lrec
A panel of 10 monoclonal antibodies selected
for their reactivity with the_S. pneumoniae HSP72 protein
were tested for their reactivity to C-151rec by Western
'5 blot analysis using YM30-ultrafiltrates prepared as
described above. The MAbs included a series of six
monoclonal antibodies raised to the HSP72reC protein (F3-
Pn3.5 to F3-Pn3.10) and monoclonal antibodies Fl-Pn3.1,
F2-Pn3.2, F2-Pn3.3, F2-Pn3.4. The three MAbs Fl-Pn3.1, F2-
Pn3.3 and F2-Pn3.4 that were reactive with C-169reC also
recognized the C-151reC fragment. All other MAbs were only
reactive with HSP72reC thus indicating that they may be
directed against epitopes present in the amino terminal
region of the HSP72 protein.
67
CA 0222401~ 1997-12-08
W096/40928 PCT/CA96/00322
EXAMPLE 7 - Antibody Response of Balb/c Mice and Macaca-
Fascicularis (cynomolgus) Monkeys to Recombinant HSP72
Antigens _
A. Procedures
1. Tmmllnization of ~n i mA 1 S
Groups of 10 female Balb/c mice were immllnized
subcutaneously with either HSP72 rec or C-169 rec as
described in Example 5. In order to assess the antibody
response to C-151reC, a group of 6 mice were ;mmlln;zed
three times at two-week intervals with 0.5 ,ug of C-151reC
absorbed to Alhydrogel adjuvant by intraperitoneal
injection. Sera from blood samples collected prior each
immllnization and four to seven days after the third
immllnization were tested for antibody reactive with S.
pneumoniae by ELISA using plates coated with S. pneumoniae
cell wall extracts.
Female cynomolgus monkeys were ;mm~ln;zed
intramuscularly at Day 1, 22 and 77 with 0.5 ml cont~;ning
150 ~ug of purified HSP72reC or C-169rQC antigens absorbed
to Alhydrogel adjuvant. Blood samples were collected
regularly before and after each imm~lnization and the sera
were tested for antibody reactive with S. pneumoniae HSP72
antigen by Western blot analysis.
The specificity of the raised antibodies for_S.
pneumoniae HSP72 was confirmed by Western blot analyses to
S. pneumoniae cell extracts and purified recombinant
antigens.
B. Results
The results previously described in Example 5
clearly demonstrate the protective nature of the antibody
response elicited following immllnization with recombinant
HSP72 antigens. Here we monitored the appearance of serum
antibody response in mice (FIG. 19, 20 and 21) and in
monkeys (FIG. 22) during the immllnization schedule. Both
species responded strongly to the full-length and
truncated recombinant HSP72 proteins used as immunogens
68
CA 0222401~ 1997-12-08
WO 9~'~D~28 PCT/CA96/00322
with average titers of 1:64000 after the third injection.-
Detailed analysis of individual sera revealed that each
animal responded to the imm~lnization in developping
antibodies reactive with S. pneumoniae HSP72.
In mice immllnized with C-169reC~ the two doses
tested, i.e. 1 and 5 ug, were similarly efficient with the
induction of similar antibody titers (FIG. 20). A strong
boost response was observed after the second injection
with C-169reC with no enhancement in the antibody titers
after a third injection. In contrast to this, we observed
that the immune response to the HSP72reC was dose-
dependent. Increases in the specific antibody titers were
observed after a second and a third injection with either
HSP72rec or C-151rec (FIG. 19 and 21).
Study of the immune response of monkeys clearly
indicated that the immunogenicity of recombinant HSP72
antigens is not restricted to rodents such as rabbit and
mouse. The humoral response following the second
injection with either antigen is characterized by a strong
increase in HSP72-specific antibody titers that can
persist for several weeks without any detectable decrease
in their antibody titers (FIG. 22). In addition, specific
serum antibodies were detectable in the sera of each
monkey after a single injection of recombinant antigens.
EXAMPLE 8 - B-Cell Epitope Mapping of HSP72 Stress
Protein
In Example 3, it was shown that significant
variability in the primary sequence of the HSP70 proteins
was mainly localized to two regions corresponding to amino
acid residues 244 to 330 and 510 to 607 of the S.
pneumoniae HSP72 protein. These variable regions may
contain B-cell epitopes responsable for the antigenic
heterogeneity reported in Example 4. To investigate this
possibility, the reactivity of polyclonal and monoclonal
69
CA 0222401~ 1997-12-08
WO96/40s28 PCT/CA96/00322
antibodies to S. pneumoniae HSP72 were tested against
fourteen peptides selected to cover most of these regions.
A. Procedures
Fourteen peptides of 14 to 30 amino acids
residues were synthesized. The peptide sequences and
their locations in the protein are sum.marized in Table 5.
Peptides CS870, CS873, CS874, CS875, CS876, CS877, CS878,
CS879, CS880 and CS882 were synthesized by Biochem
Immunosystem Inc. (Montreal, CAn~) using an automated
peptide synthesizer. Peptides MAPl, MAP2, M~P3 and MAP4
were synthesized onto a branching lysine core as Multiple
Antigenic Peptides (MAP) by the Service de Séquence de
Peptides de 1~Est du Québec, Centre de recherche du CHUL
~Sainte-Foy, Canada). Peptides were purified by reverse-
phase high-pressure liquid chromatography. Peptides were
solubilized in distilled water except for peptides CS874
and CS876 which were solubilized in a small volume of
either 6M guanidine-HCl or dimethyl sulfoxide and then
adjusted to 1 mg/ml with distilled water.
Peptide ELISA were performed by coating synthetic
peptides onto Immunolon 4 microtitration plates (Dynatech
Laboratories, Inc., Chantilly, VA) at a concentration of
50 ug/ml according to the prodedures described in J. Hamel
et al. [supra]. To confirm the reactivity of MAbs with
~5 peptides, the ability of fluid-phase peptides to inhibit
MAb binding to solid HSP72 was determined. For the
inhibition assay, microtitration plates were coated with
S. pneumoniae cell wall extracts. Hybridoma culture
supernatants containing the HSP72-specific MAbs were
incubated overnight at 4~C with several concentrations of
peptide. Peptide treated and control supernatants were
then tested by ELISA as described above.
Immune sera were from ~nim~ls imm~ln;zed three
times with recombinant HSP72 antigens. One rabbit was
immunized with 37.5 ,ug of purified HSP72reC according to
the immunization protocol described in Example 5. Pool
murine sera were from three Balb/c mice imml~nized with
CA 02224015 1997-12-08
WO9Gl4r9~8 PCT/CA96/00322
HSP72reC from Example 5 and monkey pool sera were from
groups of two animals lmml~n;zed with either HSP72reC or C-
169rec -
5 TABLE 5: SE~u~N~S AND LOCATIONS OF ~ lC ~ v~S
COPR~CPONDING TO S. PNE~MONIAE HSP72
AMINO ACID RESID~ES
Peptide Location Sequence
ID No.
CS876 247-261 TSTQISLPFITAGEA 7
CS877 257-271 TAGEAGPLHLEMTLT 8
CS878 268-281 MTLTRAKFDDLTRD 9
CS879 276-290 DDLTRDLVERTKVPV 10
CS880 286-299 TKVPVRQALSDAGL 11
CS882 315-333 RIPA W EAVKA~l~K~:~NK 23
CS873 457-471 KAKDLGTQKEQTIVI 12
CS874 467-481 QTIVIQSNSGLTDEE 24
CS875 477-491 LTDEIDRMMKDAEA 13
MAP 1 487-510 KDAEANAESDKKRKEEVDLRNEVD 14
CS870 507-521 NEVDQAIFAl~K~ K 15
MAP 2 517-544 EKTIKETEGKGFDAERDAAQAALD 16
DLKK
MAP 3 544-573 KAQEDNNLDDMKAKLEALNEKAQG 17
LAVKLY
MAP 4 583-607 QEGAEGAQATGNAGDD W DGEFTE 18
K
B. Identification and Localization of Linear B-
Cell Epitopes
The results presented in FIG. 23 revealed that
most of the immunological reactivity was observed with the
CA 0222401~ 1997-12-08
WO9G/1~328 PCT/CA96/00322
peptides localized within amino acid residues 457 and 607-
corresponding to the C-151 fragment of HSP72. Rabbit,
mice and monkey sera antibody from ~nim~l S ;mml~n; zed with
either recombinant HSP72reC of C-169reC were reactive with
s both, peptide MAP2 and peptide MAP4. Interestingly, the
sequence of peptides MAP2 and MAP4 spans the
hypervariable carboxyl-terminal region containing the
sequences GFDAERDAAQAALDD (residues 527 to 541) and
AEGAQATGNAGDDW (residues 586 to 600) defined as exclusive
to S. pneumoniae HSP72 based on the comparison of HSP70
protein sequences available in the data banks. Our data
thus revealed that both peptide sequences contain linear
B-cell epitopes. In addition, the peptide MAP4 alone was
also recognized by the MAb Fl-Pn3.1. This reactivity was
confirmed by fluid-phase inhibition assays in which 10
~g/ml of MAP4 caused complete inhibition of Fl-Pn3.1
binding to HSP72. Polyclonal antisera from ~nim~l S
;mml~nized with the complete HSP72 recombinant protein also
recognized B-cell epitopes localized on peptides CS875,
MAPl and MAP3. All together these data indicate that the
hypervariable C-151 terminal fragment of the HSP72
stimulates B-cell responses and possibly constitutes the
immunodom;n~nt portion of the HSP72 protein. The lack of
reactivity of MAbs F2-Pn3.3 and F2-Pn3.4 with the
synthetic peptides suggest that they react with
conformational determinants present on the C-terminal
region of the HSP72. The existence of protective epitopes
in the C-151 region was strongly suggested in Example 5
where mice imm~ ed with purified C-169rec were protected
from fatal infection with a virulent strain of S.
pneumoniae thus suggesting that the carboxyl-terminal
fragments C-169 or C-151 of_S. pneumoniae HSP72 or even
smaller fragments thereof may prove very useful for the
development of a future vaccine.
The variable region comprised within the amino
acid residues 244 to 330 also constitutes an antigenic
domain. Linear epitopes located on overlapping peptides
CA 0222401~ 1997-12-08
WO9S'~D328 PCT/CA9~'~C~22
CS877 (amino acids 257 to 271) and CS878 (amino acids 26
to 281), peptides CS880 (amino acis 286-299) and peptides
CS882 (amino acids 315-333) were identified by hyperimmune
sera.
s
EXAMPLE 9 - HSP70 (DnaK) from Streptococcus pyogenes and
Streptococcus agalactiae: Molecular Cloning and DNA
Sequencing of the hsp70 Genes; Nucleotide and Protein
Sequence Analyses; Antigenic Relatedness to S. pneumoniae;
Increased Streptococcus agalactiae HSP70 synthesis''in
response to heat.
A. Procedures
1. Bacterial Strains and Plasmid Vector
The strains of S. pyogenes (Group A
Streptococcus) and S. agalactiae (Group B Streptococcus)
used in this study were provided by the Laboratoire de la
Santé Publique du Québec (LSPQ), Sainte-Anne de Bellevue,
Québec, Canada. S. agalactiae type II strain V8
corresponds to the ATCC strain 12973. S. pyogenes strain
Bruno corresponds to the ATCC strain 19615. The E. coli
strain XLI Blue MRF' was obtained from Stratagene.
Streptococcal strains were grown at 37~C in a 5
% C~2 incubator. The streptococci were streaked on
tryptic soy agar plates containing 5 % sheep blood (Les
Laboratoires Quélab, Montréal, Canada), liquid cultures
were made in heart infusion broth (Difco Laboratories,
Detroit, MI) without agitation. The E. coli strain was
grown at 37~C in L-broth with agitation at 250 rpm or on L-
agar.
The general cloning phagemid pBluescript KS(-)
was purchased from Stratagene.
2. Recombinant DNA Techniques
Restriction enzymes, T4 DNA ligase, and calf
intestinal phosphatase were used as recommended by the
suppliers (Pharmacia [Canada] Inc., Baie d~Urfe, Canada;
and New England Biolabs Ltd., Mississauga, Canada).
CA 0222401~ 1997-12-08
W096/40928 PCT/CA96/00322
Preparation of plasmids by equilibrium centrifugation in-
CsCl-ethidium bromide gradients, agarose gel
electrophoresis of DNA fragments, Southern hybridization,
and colony DNA hybridization were performed as described
by J. Sambrook et al.[ supra]. Chromosomal DNA of the
streptococcal bacteria was prepared using the procedure of
B. M. Jayarao et al. [J. Clin. Microbiol., 29, pp. 2774-
2778 (1991)] adapted for bacterial cultures of 90 ml.
Rapid plasmid preparations were made accordingly to D.
Ish-Horowicz et al. [Nucl. Acids Res. 9, pp. 2989-2998
(1981)]. Plasmids used for DNA sequencing were purified
using plasmid kits from Qiagen Inc. (Chatsworth, CA). DNA
fragments were purified from agarose gels by the method of
phenol freeze [S. A. Benson, Biotechniques 2, pp. 67-68
(1984)]. DNA probes were labeled with a32P-dCTP or
digoxigenin (DIG)~ dUTP using the random primer labeling
kits of Boehringer M~nnheim (Laval, C~n~). Plasmid
transformations were carried out by the method of S;mAn;s
[~An~h~n, D. In D. M. Glover (ed.), DNA Cloning, pp. 109-
135, (1985)]. The sequencing of genomic DNA inserts inplasmids was done using synthetic oligonucleotides. The
sequencing reactions were carried out by the polymerase
chain reaction (PCR) using the Taq Dye Deoxy Terminator
Cycle Sequencing kit (ABI) and DNA electrophoresis was
performed on automated DNA sequencer 373A (ABI). The
assembly of the DNA sequence was performed using the
program Sequencher 3.0 from the Gene Codes Corporation
(Ann Arbor, MI). Analysis of the DNA sequences and their
predicted polypeptides were performed with the program
Gene Works version 2.45 from Intelligenetics, Inc.
(Mountain View, CA). DNA amplification reactions were
made using a DNA Thermal Cycler 480, Perkin Elmer.
Oligonucleotides were synthesized by oligonucleotide
synthesizer model 394 (ABI).
CA 0222401~ 1997-12-08
W O 96/40928 PCTICA96/00322
3. Molecular Cloning of the Genes hsp70/dnak-
of S. agalactiae and S. pyogenes
Chromosomal DNA from S. agalactiae and S.
pyogenes was digested to completion with various
restriction enzymes with palindromic hexanucleotide
recognition sequences. The digests were analysed by
Southern hybridization using a labeled PCR-amplified DNA
probe corresponding to a 782 base-pairs region starting at
base 332 downstream from the ATG initiation codon of the
HSP72 gene of S. pneumoniae (see SEQ ID NO 4). This DNA
region was selected because it is relatively well
conserved among the hsp70 genes of Gram-positive bacteria
that have been characterized. The PCR amplification was
done on the genomic DNA of S. pneumoniae using the
oligonucleotides OCRR2 (5'-AAG~ ATCACAGTTCCGG) and
OCRR3 (5'-GATACCAAGTGACAATGGCG). Hybridizing genomic
restriction fragments of sufficient size to code for a 70-
kDa polypeptide (>1.8 kb) were partially purified by
extraction of genomic fragments of corresponding size from
agarose gel. Verification of the presence of the hsp70
gene among the purified genomic restriction fragments was
done by Southern hybridization using the labeled 782-bp S.
pneumoniae DNA probe.
The purified genomic DNA restriction fragments
were cloned into dephosphorylated compatible restriction
sites of pBluescript KS(-) and transformed into the E.
coli strain XLI Blue MRF~. The colonies were screened by
DNA hybridization using the labeled 782-bp S. pneumoniae
DNA probe. Extracted plasmids were digested with various
restriction enzymes to evaluate the size of the inserts
and to verify the presence of the hsp70 gene by Southern
hybridization using the labeled 782-bp S. pneumoniae DNA
probe. Plasmid pURV5 contains a 4.2-kb HindIII insert of
the genomic DNA of S. agalactiae. Plasmid pURV4 contains
a 3.5-kb HindIII fragment of the genomic DNA of S.
pyogenes .
CA 0222401~ 1997-12-08
wo g~a~2~ PCT/CA96/00322
4. Heat Shock and Protein Labeling
The stress response of S. agalactiae to an heat
shock was assayed by pulse-labeling with [35S]methionine
as described before in Example 1. S. agalactiae bacteria
grown overnight in SMAM (Methionine assay Medium
supplemented with 1 mg/l methionine, 1~ (v/v) Isovitalex
and 1 mg/l choline chloride) were pelleted by
centrifugation and then resuspended in the methionine-free
SMAM medium. The bacteria were incubated at 37~C for 1 h
and then divided into two fractions of equal volume. The
samples were either incubated at 37 or 43~C for 10 minutes
and then labeled with 100 ~Ci/ml [35S]methionine for 30
minutes at 37~C. The bacteria were extensively washed with
PBS and cell extracts were prepared by treatment with
mutanolysine and lysozyme as described for the DNA
isolation (M.Jayarao et al., supra) followed by
sonication.
5. Immunological Characterization
A series of six monoclonal antibodies raised to
the HSP72reC protein (F3-Pn3.5 to F3-Pn3.10) and the
monoclonal antibodies Fl-Pn3.1, F2-Pn3.2, F2-Pn3.3, F2-
Pn3.4 were tested for their reactivity to HSP70 antigens
from S. pyogenes and S._ agalactiae_ by Western blot
analysis. Cell lysates from S. pyogenes and_S. agalactiae
were obtained from treatment with mutanolysine and
lysozyme (M.Jayarao et al., supra)., sonication and
boiling in SDS-PAGE sample buffer. Cell lysates from E.
coli transformed with either pURV4 or pURV6 producing
truncated S._ pyogenes HSP70 antigens were tested after
boiling in SDS-PAGE sample buffer.
B. DNA Sequence Analysis of the hsp70 /dnak Genes
of Streptococcus pyogenes, Streptococcus agalactiae and
Streptococcus pneumoniae_
A region of 2438 bases in the 4.2-kb HindIII
insert of plasmid pURV5 was sequenced. This sequence
76
CA 0222401~ 1997-12-08
WO~ 28 PCT/CA96/00322
contains an open reading frame (ORF) of 1830 nucleotides~
coding for a polypeptide of 609 amino acids with a
molecular weight of 64907 ( see SEQ ID NO: 7) . The ORF has
an ATG start codon beginning at position 248 and TAA stop
codon ending at position 2077. The ATG start codon is
preceeded by the sequence GAGG, starting at position 237,
which is complementary to 16S rRNA and serves as a
ribosome bi n~i ng site in E. coli [G. D. Stormo et al.,
Nucleic Acids Res. 10, pp. 2971-2996 (1982) ] . The ORF and
the polypeptide of the HSP70 of S. agalactfae are,
respectively, identical at 85 and 95 % to the ORF and
polypeptide of the HSP72 of S. pneumoniae.
Prelimin~ry sequence comparisons with the HSP72
of S. pneumoniae showed that the 3.5-kb HindIII insert in
plasmid pURV4 lacks the 3'-end coding region of the hsp70
of S. pyogenes. An attempt to clone a 3-kb SalI genomic
fragment cont~;nin~ the entire coding region of hsp70 of
S. pyogenes yielded plasmid pURV6 contAi n i ng a 3.1-kb
insert lacking the 5'-end coding region of the gene. The
assembly of the hsp70 gene regions present in plasmids
pURV4 and pURV6 gave a 2183 nucleotide region containing
an ORF of 1824 bases coding for a polypeptide of 608 amino
acids with a molecular weight of 64847 (see SEQ ID NO:
20). The ATG start codon begins at position 204 and the
TAA stop codon extends to position 2030. Similarly to the
hsp70 of S. agalactiae, the ATG start codon is preceeded
by a putative ribosome bi n~ site sequence GAGG starting
at position 193[G. D. Stormo, supra]. The ORF and the
deduced polypeptide of the hsp70 of S. pyogenes are,
respectively, identical at 85 and 94 % to the ORF and
polypeptide of the HSP72 of S. pneumoniae. The ORF of
plasmid pURV4 lacks 125 base pairs coding for 41 amino
acids at the carboxyl end of the HSP70 of S. pyogenes;
the ORF thus codes for the 567 amino acids of the amino
end of that HSP70 (N-567reC). The ORF of plasmid pURV6
lacks 114 base pairs coding for 38 amino acids at the
amino end of the HSP70 of S. pyogenes ; the ORF thus codes
CA 0222401~ 1997-12-08
W09~ 328 PCT/CA96/00322
for the 570 amino acids of the carboxyl end of that HSP7
(C-570rec ) -
The global comparison of the DNA open reading frames
(FIG. 24) and amino acid sequences (FIG. 25) of the
HSP70/DnaK of S. pyogenes, S. agalactiae, and S. pneumoniae
gave percentages of identity of 82 and 93 %, respectively.
C. Increased Synthesis of HSP70 by S. agalactiae
in Response to Heat
One ~;men~ional SDS-polyacrylamide gel
electrophoretic analysis of cell extracts of heat~shocked
and control S. agalactiae pulse-labeled with
[35S]methionine revealed that the synthesis of a 70 kDa-
protein was significantly increased after a thermal stress
(FIG. 26, lanes l and 2). Radioimmunoprecipitation
analysis revealed that the heat inducible 70kDa-protein
was easily detected at 43~C using monoclonal antibody F2-
Pn3.4 thus indicating that the protein belongs to the heat
shock protein 70 (hsp70/DnaK) family (FIG. 26, lanes 3 and
4)
D. Antigenic Relatedness of HSP70 Proteins in
S. pneumoniae,_S. pyogenes and S. agalactiae
In this study, a panel of MAbs were used to
investigate the antigenic relatedness of S. pyogenes, S.
agalactiae and S. pneumoniae HSP70 proteins. Eight of ten
MAbs reacted with all three Streptoccocus species thus
indicating that some B-cell epitopes are widely
distributed among S. pneumoniae , S. pyogenes and S.
agalactiae. The MAb Fl-Pn3.1 which is directed against an
epitope located between amino acid residues 584 and 607 of
HSP72 from_S. pneumoniae did not react with HSP70
antigens from either S.pyogenes or S. agalactiae.
Comparison of this region among the three Streptococcus
species revealed differences in 5 to 8 amino acids located
between amino acids 589 and 596. The MAb F2-Pn3.3 which
78
CA 0222401~ 1997-12-08
W O ~'4~328 PCT/CA96/00322
was also directed against epitopes present in the C-151 _
region was reactive with S. agalactiae but not wih S.
pyogenes. These data clearly indicate that HSP70 proteins
from Streptococcus species are structurally and
immunologically related. There is however immunological
distinction.
Analysis of the reactivity of MAbs F3-Pn3.5, F3-
Pn3.6, F3-Pn3.7 and F3-Pn3.10 with truncated recombinant
S. pyogenes HSP70 antigens allowed the identification of
an antigenic region near the amino-terminal end on the S.
pneumoniae HSP72. These MAbs reacted with constructs
expressing the N-terminal 567 amino acid residues but
failed to react with constructs expressing the C-570
fragment. These data localized the epitopes recognized by
the MAbs F3-Pn3.5, F3-Pn3.6, F3-Pn3.7 and F3-Pn3.10 to
between residues 1 and 38 of the HSP72 protein.
EXAMPLE 10 - Use of HSP70/HSP72 As A Human Vaccine
To formulate a vaccine for human use,
appropriate HSP72 antigens may be selected from the
polypeptides described herein. For example, one of skill
in the art could design a vaccine around the HSP70/HSP72
polypeptide or fragments thereof containing an imml~nogenic
epitope. The use of molecular biology techniques is
particularly well-suited for the preparation of
substantially pure recombinant antigens.
The vaccine composition may take a variety of
forms. These include, for example solid, semi-solid and
liquid dosage forms, such as powders, liquid solutions or
suspensions, and liposomes. Based on our belief that the
HSP70/HSP72 antigens of this invention may elicit a
protective immune response when administered to a human,
the compositions of this invention will be similar to
3s those used for immllnizing humans with other proteins and
polypeptides, e.g. tetanus and diphtheria. Therefore, the
79
CA 0222401~ 1997-12-08
W O gC'4C928 PCT/CA96/00322
compositions of this invention will preferably comprise
pharmaceutcially acceptable adjuvant such as incomplete
Freund's adjuvant, aluminum hydroxide, a muramyl peptide,
a water-in oil emulsion, a liposome, an ISCOM or CTB, or a
5 non-toxic B subunit from cholera toxin. Most preferably,
the compositions will include a water-in-oil emulsion or
aluminum hydroxide as adjuvant.
The composition would be a~m;n;stered to the
patient in any of a number of pharmaceutically acceptable
forms including intramuscular, intradermal, subcutaneous
or topic. Preferrably, the vaccine will be administered
intramuscularly.
Generally, the dosage will consist of an initial
injection, most probably with adjuvant, of about 0.0l to
l0 mg, and preferably 0.l to l.0 mg HSP72 antigen per
patient, followed most probably by one or more booster
injections. Preferably, boosters will be administered at
about l and 6 months after the initial injection.
An important consideration relating to
pneumococcal vaccine development is the ~uestion of
mucosal imm~lnity~ The ideal mucosal vaccine will be
safely taken orally or intranasally as one or a few doses
and would elicit protective antibodies on the appropriate
surfaces along with systemic ;mmlln;ty. The mucosal
vaccine composition may include adjuvants, inert
particulate carriers or recombinant live vectors.
The anti-HSP72 antibodies of this invention are
useful for passive immunotherapy and immunoprophylaxis of
humans infected with S. pneumoniae, S. pyogenes, S.
agalactiae or related bacteria. The dosage forms and
regimens for such passive ;mml~nization would be similar to
those of other passive immunotherapies.
An antibody according to this invention is
exemplified by a hybridoma producing MAb Fl-Pn3.l
deposited in the American Type Culture Collection in
Rockville, Maryland, USA on July 21, 1995, and identified
CA 0222401~ 1997-12-08
WO 9G,~4'328 PCT/CA9~ 'OC372
as Murine Hybridoma Cell Line, Fl-Pn3.1. This deposit wa-s
assigned accession number HB 11960.
While we have described herein a number of
embodiments of this invention, it is apparent that our
basic embodiments may be altered to provide other
embodiments that utilize the compositions and processes of
this invention. Therefore, it will be appreciated that
the scope of this invention includes all alternative
embodiments and variations that are defined in the
foregoing specification and by the claims appended hereto;
and the invention is not to be limited by the specific
embodiments which have been presented herein by way of
example.
81
CA 0222401~ 1997-12-08
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
s
(i) APPLICANT: Hamel, Josee
Brodeur, Bernard R
Martin, Denis
Rioux, Clement
(ii) TITLE OF INVENTION: STREPTOCOCCAL HEAT SHOCK PROTEINS
M~MR~R.~ OF T~E HSP70 FAMILY
(iii) NUMBER OF ~u~S: 26
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Goudreau Gage Dubuc & Martineau Walker
(B) STREET: 800 Place Victoria, Suite 3400, Stock
F.Yrh~n~e Tower
(C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: CANADA
(F) ZIP: HqZlE9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/472,534
(B) FILING DATE: 07-JUN-1995
(vii) PRIOR APPLICATION DATA: .
(A) APPLICATION NUMBER: US (PROVIS)60/001,805
(B) FILING DATE: 04-AUG-1995
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Leclerc/Dubuc/Prince, Alain/Jean/Gaetan
(C) REFERENCE/DOCKET NUMBER: BIOVAC2-PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHOWE: (514) 397-7400
(B) TELEFAX: (514) 397-4382
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3167 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus pneumoniae
(ix) FEATURE:
(A) NAME/KEY: CDS
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CA 0222401~ 1997-12-08
WO 9G/1 r 328 PCT/CA96/00322
(B) LOCATION: 30... 755
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 771.. .2912
(D) OTHER INFORMATION: /product= ~FucI/HSP72 (C-169)"
-
(xi) ~yu~ DESCRIPTION: SEQ ID NO:1:
GAACTTCATT TTTAGAAAGG AGTGAG m ATG TCT CAA GAT GAA AAA TTA ATT 53
Met Ser Gln Asp Glu Lys Leu Ile
1 5
15 CGT GAA CAG ATT TGT GAT GTT TGT CAT AAG ATG TGG CAA CTT GGT TGG 101
Arg Glu Gln Ile Cys Asp Val Cys His Lys Met Trp Gln Leu Gly Trp
10 15 20
GTT GCT GCT AAC GAT GGG AAT GTA TCT GTT CGA TTA GAT GAG GAT ACC 149
20 Val Ala Ala Asn Asp Gly Asn Val Ser Val Arg Leu Asp Glu Asp Thr
25 30 35 40
ATT CTT GCA ACA CCT ACT GGT ATC AGC AAA AGT TTT ATT ACA CCA GAA 197
Ile Leu Ala Thr Pro Thr Gly Ile Ser Lys Ser Phe Ile Thr Pro Glu
45 50 55
AAG CTG GTG AAG TTA AAT CTT AAA GGA GAG ATT TTA GAA GCA GAA GGT 245
Lys Leu Val Lys Leu Asn Leu Lys Gly Glu Ile Leu Glu Ala Glu Gly
60 65 70
GAT TAC TGT CCT TCT AGT GAA ATT AAA ATG CAC ATT CGG TGC TAC GAA 293
Asp Tyr Cys Pro Ser Ser Glu Ile Lys Met His Ile Arg Cys Tyr Glu
75 80 85
35 GAA CGT GAG GAT GTT CGT TCA GTT GTT CAC GCG CAT CCA CCG ATT GCA 341
Glu Arg Glu Asp Val Arg Ser Val Val His Ala His Pro Pro Ile Ala
90 95 100
ACA GGA TTT GCT CTT GCA CAC ATT CCT TTA GAT ACT TAT TCA CTA ATT 389
40 Thr Gly Phe Ala Leu Ala His Ile Pro Leu Asp Thr Tyr Ser Leu Ile
105 110 115 120
GAG AGC GCG ATT GTG GTT GGG GCA ATT CCT ATT ACC CCA TTT GGA GTA 437
Glu Ser Ala Ile Val Val Gly Ala Ile Pro Ile Thr Pro Phe Gly Val
125 130 135
CCG TCT ACA ATG GAA GTG CCA GAA GCA ATT ACA CCT TAT CTG CCC GAT 485
Pro Ser Thr Met Glu Val Pro Glu Ala Ile Thr Pro Tyr Leu Pro Asp
lg0 145 150
CAT GAT GTC ATG CTA TTA GAA AAT CAT GGA GCT CTG ACT GTC GGA AGC 533
His Asp Val Met Leu Leu Glu Asn His Gly Ala Leu Thr Val Gly Ser
155 160 165
55 GAT GTC ATT ACA GCA TAC TAC CGT ATG GAA ACT TTA GAA TTA GTC GCA 581
Asp Val Ile Thr Ala Tyr Tyr Arg Met Glu Thr Leu Glu Leu Val Ala
170 175 180
AAG ACA ACC TTC CAC GGA AGA ATG TTA CTT TCT ACA AAG GGC ATT GAG 629
60 Lys Thr Thr Phe His Gly Arg Met Leu Leu Ser Thr Lys Gly Ile Glu
185 190 195 200
GAG CAA GAA ATT GCT CGT CCG ACT TTA GAA CGT CTA TTC TCA ATG CGA 677
Glu Gln Glu Ile Ala Arg Pro Thr Leu Glu Arg Leu Phe Ser Met Arg
205 210 215
GAA AAT TAT AAG GTT ACA GGT CGT CAC CCA GGC TAC CGT AAA TAT AAT 725
Glu Asn Tyr Lys Val Thr Gly Arg His Pro Gly Tyr Arg Lys Tyr Asn
220 225 230
83
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WO 9.'4C928 PCT/CA96/00322
GGC GAT GGT AGT ATA AAA GAA ACA AAA AAA TAAGAGGAAA GTATT ATG ATC 776
Gly Asp Gly Ser Ile Lys Glu Thr Lys Lys Met Ile
235 240
CAA CAT CCA CGT ATT GGG ATT CGT CCG ACT ATT GAT GGT CGT CGT CAA 824
Gln His Pro Arg Ile Gly Ile Arg Pro Thr Ile Asp Gly Arg Arg Gln
5 lO 15
10 GGT GTA CGC GAA TCA CTT GAA GTA CAA ACA ATG AAC ATG GCT AAA AGT 872
Gly Val Arg Glu Ser Leu Glu Val Gln mr Met Asn Met Ala Lys Ser
20 25 30
GTG GCA GAT TTG ATT TCA AGC ACA TTG AAA TAT CCA GAT GGG GAA CCT 920
15 Val Ala Asp Leu Ile Ser Ser mr Leu Lys Tyr Pro Asp Gly Glu Pro
35 40 45 50
GTG GAA TGT GTG ATT TCT CCA TCT ACC ATT GGT CGT GTT CCA GAG GCT 968
Val Glu Cys Val Ile Ser Pro Ser m r Ile Gly Arg Val Pro Glu Ala
55 60 65
GCA GCT TCC CAT GAG TTG TTT AAA AAA TCA AAT GTT TGC GCA ACA ATT 1016
Ala Ala Ser His Glu Leu Phe Lys Lys Ser Asn Val Cys Ala Thr Ile
70 75 80
ACA GTT ACA CCA TGC TGG TGT TAT GGT AGT GAA ACT ATG GAT ATG TCT 1064
m r Val m r Pro Cys Trp Cys Tyr Gly Ser Glu mr Met Asp Met Ser
85 90 95
30 CCA GAT ATT CCT CAT GCT ATT TGG GGA TTT AAT GGG ACA GAA CGC CCA 1112
Pro Asp Ile Pro His Ala Ile Trp Gly Phe Asn Gly Thr Glu Arg Pro
100 105 110
GGA GCT GTC TAT CTT GCA GCT GTA CTA GCT TCA CAT ACT CAA AAA GGG 1160
35 Gly Ala Val Tyr Leu Ala Ala Val Leu Ala Ser His mr Gln Lys Gly
115 120 125 130
ATT CCA GCC TTT GGG ATT TAT GGT AGA GAT GTT CAG GAA GCT AAT GAT 1208
Ile Pro Ala Phe Gly Ile Tyr Gly Arg Asp Val Gln Glu Ala Asn Asp
135 140 145
ACA GCT ATT CCA GAA GAT GTC AAA GAA AAA CTT TTA CGT TAT GCG CGG 1256
m r Ala Ile Pro Glu Asp Val Lys Glu Lys Leu Leu Arg Tyr Ala Arg
150 155 160
GCA GTT CTT GCA ACT GGC TTG ATG AGA GAC ACT GCT TAC CTA TCA ATG 1304
Ala Val Leu Ala Thr Gly Leu Met Arg Asp Thr Ala Tyr Leu Ser Met
165 170 175
50 GGT AGT GTT TCG ATG GGG ATT GGT GGT TCT ATT GTA AAT CCA GAT TTC 1352
Gly Ser Val Ser Met Gly Ile Gly Gly Ser Ile Val Asn Pro Asp Phe
180 185 190
TTC CAA GAA TAC TTA GGA ATG CGA AAT GAA TCG GTA GAT ATG ACG GAG 1400
55 Phe Gln Glu Tyr Leu Gly Met Arg Asn Glu Ser Val Asp Met Thr Glu
195 200 205 210
TTC ACG CGC CGT ATG GAC CGT GGT ATT TAC GAC CCT GAA GAG TTC GAA 1448
Phe Thr Arg Arg Met Asp Arg Gly Ile Tyr Asp Pro Glu Glu Phe Glu
215 220 225
CGT GCG CTC AAA TGG GTG AAA GAA AAC GTA AAA GAA GGA TTC GAC CAT 1496
Arg Ala Leu Lys Trp Val Lys Glu Asn Val Lys Glu Gly Phe Asp His
230 235 240
AAC CGT GAA GAC CTT GTT TTA AGC CGT GAA GAA AAA GAT AGA CAA TGG 1544
Asn Arg Glu Asp Leu Val Leu Ser Arg Glu Glu Lys Asp Arg Gln Trp
245 250 255
84
CA 0222401~ 1997-12-08
WO ~"4'928 PCT/CA96100322
GAA TTT GTT ATT AAG ATG TTC ATG ATT GGA CGT GAC TTA ATG GTT GGT 1592
Glu Phe Val Ile Lys Met Phe Met Ile Gly Arg Asp Leu Met Val Gly
260 265 270
AAC CCA AGA CTT GCT GAA CTT GGT TTT GAG GAA GAA GCA GTT GGT CAC 1640
Asn Pro Arg Leu Ala Glu Leu Gly Phe Glu Glu Glu Ala Val Gly His
275 280 285 290
10 CAT GCT TTA GTA GCT GGT TTC CAA GGT CAA CGT CAG TGG ACA GAC CAT 1688
His Ala Leu Val Ala Gly Phe Gln Gly Gln Arg Gln Trp Thr Asp His
295 300 305
TTT CCA AAT GGG GAC TTT ATG GAA ACT TTC CTC AAT ACT CAG m GAC 1736
lS Phe Pro Asn Gly Asp Phe Met Glu Thr Phe Leu Asn Thr Gln Phe Asp
310 315 320
TGG AAT GGT ATT CGA AAA CCA TTT GTA m GCG ACA GAG AAT GAT TCA . 1784
Trp Asn Gly Ile Arg Lys Pro Phe Val Phe Ala Thr Glu Asn Asp Ser
325 330 335
CTA AAT GGT GTG TCT ATG CTC TTT AAT TAT CTA TTA ACA AAT ACT CCA 1832
Leu Asn Gly Val Ser Met Leu Phe Asn Tyr Leu Leu Thr Asn Thr Pro
340 345 350
CAA ATC TTT GCT GAT GTG CGT ACT TAT TGG AGT CCA GAG GCT GTT GAA 1880
Gln Ile Phe Ala Asp Val Arg Thr Tyr Trp Ser Pro Glu Ala Val Glu
355 360 365 370
30 CGT GTA ACA GGA TAT ACT TTA GAG GGT CGT GCT GCA GCT GGA TTC TTA 1928
Arg Val Thr Gly Tyr Thr Leu Glu Gly Arg Ala Ala Ala Gly Phe Leu
375 380 385
CAT CTA ATC AAC TCT GGA TCT TGT ACA TTG GAT GGT ACA GGT CAA GCT 1976
35 His Leu Ile Asn Ser Gly Ser Cys Thr Leu Asp Gly Thr Gly Gln Ala
390 395 400
ACT CGA GAT GGC AAA CCT GTT ATG AAA CCA TTC TGG GAG TTG GAT GAA 2024
Thr Arg Asp Gly Lys Pro Val Met Lys Pro Phe Trp Glu Leu Asp Glu
405 410 415
AGT GAA GTA CAG GCT ATG CTT GAA AAT ACA GAC TTC CCA CCA GCA AAC 2072
Ser Glu Val Gln Ala Met Leu Glu Asn Thr Asp Phe Pro Pro Ala Asn
420 425 430
CGC GAA TAC TTC CGT GGA GGA GGA TTC TCA ACT CGT TTC TTG ACG AAG 2120
Arg Glu Tyr Phe Arg Gly Gly Gly Phe Ser Thr Arg Phe Leu Thr Lys
435 440 445 450
S0 GGG GAT ATG CCA GTA ACA ATG GTA CGT CTC AAT CTT TTA AAA GGG GTT 2168
Gly Asp Met Pro Val Thr Met Val Arg Leu Asn Leu Leu Lys Gly Val
455 460 465
GGT CCA GTG CTA CAA ATT GCA GAA GGT TAC ACA CTT GAA CTT CCT GAA 2216
55 Gly Pro Val Leu Gln Ile Ala Glu Gly Tyr Thr Leu Glu Leu Pro Glu
470 475 480
GAT GTT CAC CAT ACT TTA GAT AAT CGT ACA GAT CCA GGA TGG CCA ACT 2264
Asp Val His His mr Leu Asp Asn Arg Thr Asp Pro Gly Trp Pro mr
485 490 495
ACT TGG TTT GCT CCA CGT TTG ACA GGA AAA GGT GCT TTC AAG TCT GTC 2312
mr Trp Phe Ala Pro Arg Leu Thr Gly Lys Gly Ala Phe Lys Ser Val
500 505 510
TAT GAC GTC ATG AAT AAT TGG GGA GCT AAT CAC GGA GCC ATA ACA TAT 2360
Tyr Asp Val Met Asn Asn Trp Gly Ala Asn His Gly Ala Ile mr Tyr
515 520 525 530
CA 0222401~ 1997-12-08
W O 96~4~928 PCT/CA96/00322
GGA CAC ATT GGA GCA GAC TTG ATT ACC TTG GCT TCT ATG TTG AGA ATT 2408
Gly His Ile Gly Ala Asp Leu Ile Thr Leu Ala Ser Met Leu Arg Ile
535 540 545
CCT CAA ATC GAA GTA ACA TTT GAC ATC GAC AAG AAC GGT ATC GTG TCT 2456
Pro Gln Ile Glu Val Thr Phe Asp Ile Asp Lys Asn Gly Ile Val Ser
550 555 560
10 GTT AAG GCC AAA GAC CTT GGA ACT CAA AAA GAA CAA ACT ATT GTC ATC 2504
Val Lys Ala Lys Asp Leu Gly Thr Gln Lys Glu Gln m r Ile Val Ile
565 570 575
CAA TCG AAC TCA GGT TTG ACT GAC GAA GAA ATC GAC CGC ATG ATG AAA 2552
15 Gln Ser Asn Ser Gly Leu Thr Asp Glu Glu Ile Asp Arg Met Met Lys
580 585 590
GAT GCA GAA GCA AAC GCT GAA TCC GAT AAG AAA CGT AAA GAA GAA GTA 2600
Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val
595 600 605 610
GAC CTT CGT AAT GAA GTG GAC CAA GCA ATC TTT GCG ACT GAA AAG ACA 2648
Asp Leu Arg Asn Glu Val Asp Gln Ala Ile Phe Ala Thr Glu Lys Thr
615 620 625
ATC AAG GAA ACT GAA GGT AAA GGC TTC GAC GCA GAA CGT GAC GCT GCC 2696
Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala
630 635 640
30 CAA GCT GCC CTT GAT GAC CTT AAG AAA GCT CAA GAA GAC AAC AAC TTG 2744
Gln Ala Ala Leu Asp Asp Leu Lys Lys Ala Gln Glu Asp Asn Asn Leu
645 650 655
GAC GAC ATG AAA GCA AAA CTT GAA GCA TTG AAC GAA AAA GCT CAA GGA 2792
35 Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gln Gly
660 665 670
CTT GCT GTT AAA CTC TAC GAA CAA GCC GCA GCA GCG CAA CAA GCT CAA 2840
Leu Ala Val Lys Leu Tyr Glu Gln Ala Ala Ala Ala Gln Gln Ala Gln
675 680 685 690
GAA GGA GCA GAA GGC GCA CAA GCA ACA GGA AAC GCA GGC GAT GAC GTC 2888
Glu Gly Ala Glu Gly Ala Gln Ala Thr Gly Asn Ala Gly Asp Asp Val
695 700 705
GTA GAC GGA GAG TTT ACG GAA AAG TAAGATGAGT GTATTGGATG AAGAGTATCT 2942
Val Asp Gly Glu Phe Thr Glu Lys
710
50 AAAAAATACA CGAA~GTTT ATAATGATTT TTGTAATCAA GCTGATAACT ATAGAACATC 3002
AAAAGATTTT ATTGATAATA TTCCAATAGA ATATTTAGCT AGATATAGAG AAATTATATT 3062
AGCTGAGCAT GATAGTTGTG TCAAAAATGA TGAAGCGGTA AGGAATTTTG TTACCTCAGT 3122
All~l~l~lCT GCATTTGTAT CGGCGATGGT ATCAGCTATG ATATC 3167
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Gln Asp Glu Lys Leu Ile Arg Glu Gln Ile Cys Asp Val Cys
1 5 10 15
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His Lys Met Trp Gln Leu Gly Trp Val Ala Ala Asn Asp Gly Asn Val
Ser Val Arg Leu Asp Glu Asp Thr Ile Leu Ala Thr Pro Thr Gly Ile
Ser Lys Ser Phe Ile Thr Pro Glu Lys Leu Val Lys Leu Asn Leu Lys
Gly Glu Ile Leu Glu Ala Glu Gly Asp Tyr Cys Pro Ser Ser Glu Ile
Lys Met His Ile Arg Cys Tyr Glu Glu Arg Glu Asp Val Arg Ser Val
85 90 95
Val His Ala His Pro Pro Ile Ala Thr Gly Phe Ala Leu Ala His Ile
100 105 110
Pro Leu Asp mr Tyr Ser Leu Ile Glu Ser Ala Ile Val Val Gly Ala
115 120 125
Ile Pro Ile Thr Pro Phe Gly Val Pro Ser Thr Met Glu Val Pro Glu
130 135 140
Ala Ile Thr Pro Tyr Leu Pro Asp His Asp Val Met Leu Leu Glu Asn
lg5 150 155 160
His Gly Ala Leu Thr Val Gly Ser Asp Val Ile Thr Ala Tyr Tyr Arg
165 170 175
Met Glu Thr Leu Glu Leu Val Ala Lys Thr Thr Phe His Gly Arg Met
180 185 190
~5 Leu Leu Ser Thr Lys Gly Ile Glu Glu Gln Glu Ile Ala Arg Pro Thr
195 200 205
Leu Glu Arg Leu Phe Ser Met Arg Glu Asn ~yr Lys Val Thr Gly Arg
210 215 220
His Pro Gly Tyr Arg Lys Tyr Asn Gly Asp Gly Ser Ile Lys Glu Thr
225 230 235 240
Lys Lys
(2) INFORMATION FOR SEQ ID No:3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 714 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ile Gln His Pro Arg Ile Gly Ile Arg Pro Thr Ile Asp Gly Arg
1 5 10 15
Arg Gln Gly Val Arg Glu Ser Leu Glu Val Gln Thr Met Asn Met Ala
~5 Lys Ser Val Ala Asp Leu Ile Ser Ser Thr Leu Lys Tyr Pro Asp Gly
Glu Pro Val Glu Cys Val Ile Ser Pro Ser Thr Ile Gly Arg Val Pro
87
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Glu Ala Ala Ala Ser His Glu Leu Phe Lys Lys Ser Asn Val Cys Ala
Thr Ile Thr Val Thr Pro Cys Trp Cys Tyr Gly Ser Glu Thr Met Asp
Met Ser Pro Asp Ile Pro His Ala Ile Trp Gly Phe Asn Gly Thr Glu
100 105 110
Arg Pro Gly Ala Val Tyr Leu Ala Ala Val Leu Ala Ser His Thr Gln
115 120 125
Lys Gly Ile Pro Ala Phe Gly Ile Tyr Gly Arg Asp Val Gln Glu Ala
130 135 140
Asn Asp Thr Ala Ile Pro Glu Asp Val Lys Glu Lys Leu Leu Arg Tyr
145 150 155 160
Ala Arg Ala Val Leu Ala Thr GlY Leu Met Arg Asp Thr Ala Tyr Leu
165 170 175
Ser Met Gly Ser Val Ser Met Gly Ile Gly Gly Ser Ile Val Asn Pro
180 185 190
Asp Phe Phe Gln Glu Tyr Leu Gly Met Arg Asn Glu Ser Val Asp Met
195 200 205
Thr Glu Phe Thr Arg Arg Met Asp Arg Gly Ile Tyr Asp Pro Glu Glu
210 215 220
Phe Glu Arg Ala Leu Lys Trp Val Lys Glu Asn Val Lys Glu Gly Phe
225 230 235 240
Asp His Asn Arg Glu Asp Leu Val Leu Ser Arg Glu Glu Lys Asp Arg
245 250 255
Gln Trp Glu Phe Val Ile Lys Met Phe Met Ile Gly Arg Asp Leu Met
260 265 270
Val Gly Asn Pro Arg Leu Ala Glu Leu Gly Phe Glu Glu Glu Ala Val
275 280 285
Gly His His Ala Leu Val Ala Gly Phe Gln Gly Gln Arg Gln Trp Thr
290 295 300
Asp His Phe Pro Asn Gly Asp Phe Met Glu Thr Phe Leu Asn Thr Gln
305 310 315 320
Phe Asp Trp Asn Gly Ile Arg Lys Pro Phe Val Phe Ala Thr Glu Asn
325 330 335
Asp Ser Leu Asn Gly Val Ser Met Leu Phe Asn Tyr Leu Leu mr Asn
340 345 350
Thr Pro Gln Ile Phe Ala Asp Val Arg Thr Tyr Trp Ser Pro Glu Ala
355 360 365
Val Glu Arg Val Thr Gly Tyr Thr Leu Glu Gly Arg Ala Ala Ala Gly
370 375 380
Phe Leu His Leu Ile Asn Ser Gly Ser Cys Thr Leu Asp Gly Thr Gly
385 390 395 400
~5 Gln Ala Thr Arg Asp Gly Lys Pro Val Met Lys Pro Phe Trp Glu Leu
405 410 415
Asp Glu Ser Glu Val Gln Ala Met Leu Glu Asn Thr Asp Phe Pro Pro
420 425 430
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Ala Asn Arg Glu Tyr Phe Arg Gly Gly Gly Phe Ser Thr Arg Phe Leu
435 440 445
mr Lys Gly Asp Met Pro Val mr Met Val Arg Leu Asn Leu Leu Lys
450 455 460
Gly Val Gly Pro Val Leu Gln Ile Ala Glu Gly Tyr mr Leu Glu Leu
465 470 475 480
Pro Glu Asp Val His His mr Leu Asp Asn Arg mr Asp Pro Gly Trp
485 490 495
Pro Thr mr Trp Phe Ala Pro Arg Leu mr Gly Lys Gly Ala Phe Lys
500 505 510
Ser Val Tyr Asp Val Met Asn Asn Trp Gly Ala Asn His Gly Ala Ile
515 520 525
Thr Tyr Gly His Ile Gly Ala Asp Leu Ile mr Leu Ala Ser Met Leu
530 535 540
Arg Ile Pro Gln Ile Glu Val mr Phe Asp Ile Asp Lys Asn Gly Ile
545 550 555 560
Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gln Lys Glu Gln mr Ile
565 570 575
Val Ile Gln Ser Asn Ser Gly Leu mr Asp Glu Glu Ile Asp Arg Met
580 585 590
Met Lys Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu
595 600 605
Glu Val Asp Leu Arg Asn Glu Val Asp Gln Ala Ile Phe Ala m r Glu
610 615 620
Lys m r Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp
625 630 635 640
Ala Ala Gln Ala Ala Leu Asp Asp Leu Lys Lys Ala Gln Glu Asp Asn
645 650 655
Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala
660 665 670
Gln Gly Leu Ala Val Lys Leu Tyr Glu Gln Ala Ala Ala Ala Gln Gln
675 680 685
Ala Gln Glu Gly Ala Glu Gly Ala Gln Ala Thr Gly Asn Ala Gly Asp
690 695 700
Asp Val Val Asp Gly Glu Phe Thr Glu Lys
705 710
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4320 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus pneumoniae
89
CA 022240l~ 1997-12-08
WO 9~'~1C~28 PCT/CA96/00322
(ix) FEATURE: _
(A) NAME/KEY: CDS
(B) LOCATION: 6822502
(D) OTHER INFORMATION: /product= ""Heat-shock protein 72
(ix) FEATURE:
(A) NANE/KEY: CDS
(B) LOCATION: 3265..4320
(D) OTHER INFORMATION: /product= ""NH2-terminal portion of
DNA J
(ix) FEATURE:
(A) NANE/KEY: ~at_peptide
(B) LOCATION: 682.. 2502
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
20 AAGCTTGATT CACGC'llIGA AA~.~Ar.AA~G AATTGAAGAA ATCGCAGCAG ATGGCGAATT 60
TGACCATAAC TACCATATGG CCATCCAAAC TCTCCCAGCA GACGATGAAC ACCCAGTAGA 120
TACCATCGCC CAA~l~lllC AAAAAGGCTA CAAACTCCAT GACCGCATCC TACGCCCAGC 180
AATGGTAGTG GTGTATAACT AAGATACAAA GCCCGTAAAA AGCTCGCAGT AAAAATAGGA 240
GATTGACGAA ~l~lCGATG AACACAAGM AATCTATCTT TTTTACTCAG AGCTTAGGGC 300
GTGTTCGATT CGGCAATTCT GACGGTAGCT AAAGCAACTC GTCAGAAAAC GGCAGTCGCT 360
ATGGCGTTTG TCTAGCTTCC TTACTAACTC ~~ CGAAA TAAAATCGAT TTCGACTCTT 420
CGTGTCGCAA TTTA~ATAAT AGAA~ACTTG TCC~-AAACrA rAATAAAcTA T~AAr-AAAr~A 480
TAAAAT~TGT TTGGCTTTGT AATAGTGAGC GAAGCGAACC AAAGACGATA CTCTTCGCTG 540
TGGCGCTATT TGCGCAAATT TTGAGACCTT AGGCTCAAAG TTTAGTCAAA GAGATTGACA 600
AAGTCAAGCT CTGACGGCGT CGCCACTTAA GAAGAGTATC AAAAAr.AAAA ATAr.AAAATT 660
AACTAACAAG GAGAAAAACA C ATG TCT AAA ATT ATC GGT ATT GAC TTA GGT 711
Met Ser Lys Ile Ile Gly Ile Asp Leu Gly
1 5 10
ACA ACA AAC TCA GCA GTT GCA GTT CTT GAA GGA ACT GAA AGC AAA ATC 759
Thr Thr Asn Ser Ala Val Ala Val Leu Glu Gly Thr Glu Ser Lys Ile
15 20 25
50 ATC GCA AAC CCA GAA GGA AAC CGC ACA ACT CCA TCT GTA GTC TCA TTC 807
Ile Ala Asn Pro Glu Gly Asn Arg Thr Thr Pro Ser Val Val Ser Phe
30 35 40
AAA AAC GGA GAA ATC ATC GTT GGT GAT GCT GCA AAA CGT CAA GCA GTT 855
55 Lys Asn Gly Glu Ile Ile Val Gly Asp Ala Ala Lys Arg Gln Ala Val
45 50 55
ACA AAC CCA GAT ACA GTT ATC TCT ATC AAA TCT AAG ATG GGA ACT TCT 903
Thr Asn Pro Asp Thr Val Ile Ser Ile Lys Ser Lys Met Gly Thr Ser
6060 65 70
GAA AAA GTT TCT GCA AAT GGA AAA GAA TAC ACT CCA CAA GAA ATC TCA 951
Glu Lys Val Ser Ala Asn Gly Lys Glu Tyr Thr Pro Gln Glu Ile Ser
75 80 85 90
GCT ATG ATC CTT CAA TAC TTG AAA GGC TAC GCT GAA GAC TAC CTT GGT 999
Ala Met Ile Leu Gln Tyr Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly
95 100 105
CA 022240l~ l997-l2-08
WO 9~'~0328 PCT/CA96/00322
GAG AAA GTA ACC AAA GCT GTT ATC ACA GTT CCG GCT TAC TTC AAC GAC 1047
Glu Lys Val mr Lys Ala Val Ile Thr Val Pro Ala Tyr Phe Asn Asp
110 115 120
GCT CAA CGT CAA GCA ACA AAA GAC GCT GGT AAA ATT GCT GGT CTT GAA 1095
Ala Gln Arg Gln Ala mr Lys Asp Ala Gly Lys Ile Ala Gly Leu Glu
125 130 135
10 GTA GAA CGT ATT GTT AAC GAA CCA ACT GCA GCA GCT CTT GCT TAT GGT 1143
Val Glu Arg Ile Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly
140 145 150
TTG GAC AAG ACT GAC AAA GAA GAA AAA ATC TTG GTA TTT GAC CTT GGT 1191
15 Leu Asp Lys Thr Asp Lys Glu Glu Lys Ile Leu Val Phe Asp Leu Gly
155 160 165 170
GGT GGT ACA TTC GAC GTC TCT ATC CTT GAA TTG GGT GAC GGT GTC TTC 1239
Gly Gly m r Phe Asp Val Ser Ile Leu Glu Leu Gly Asp Gly Val Phe
175 180 185
GAC GTA TTG TCA ACT GCA GGG GAC AAC AAA CTT GGT GGT GAC GAC TTT 1287
Asp Val Leu Ser mr Ala Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe
190 195 200
GAC CAA AAA ATC ATT GAC CAC TTG GTA GCA GAA TTC AAG AAA GAA AAC 1335
Asp Gln Lys Ile Ile Asp His Leu Val Ala Glu Phe Lys Lys Glu Asn
205 210 215
30 GGT ATC GAC TTG TCT ACT GAC AAG ATG GCA ATG CAA CGT TTG AAA GAT 1383
Gly Ile Asp Leu Ser mr Asp Lys Met Ala Met Gln Arg Leu Lys Asp
220 225 230
GCG GCT GAA AAA GCG AAG AAA GAC CTT TCT GGT GTA ACT TCA ACA CAA 1431
35 Ala Ala Glu Lys Ala Lys Lys Asp Leu Ser Gly Val Thr Ser m r Gln
235 240 245 250
ATC AGC TTG CCA TTT ATC ACT GCA GGT GAG GCT GGA CCT CTT CAC TTG 1479
Ile Ser Leu Pro Phe Ile mr Ala Gly Glu Ala Gly Pro Leu His Leu
255 260 265
GAA ATG ACT TTA ACT CGT GCG AAA TTT GAT GAT TTG ACT CGT GAC CTT 1527
Glu Met mr Leu mr Arg Ala Lys Phe Asp Asp Leu mr Arg Asp Leu
270 275 280
GTT GAA CGT ACA AAA GTT CCA GTT CGT CAA GCC CTT TCA GAT GCA GGT 1575
Val Glu Arg m r Lys Val Pro Val Arg Gln Ala Leu Ser Asp Ala Gly
285 290 295
50 TTG AGC TTG TCA GAA ATC GAC GAA GTT ATC CTT GTT GGT GGT TCA ACT 1623
Leu Ser Leu Ser Glu Ile Asp Glu Val Ile Leu Val Gly Gly Ser Thr
300 305 310
CGT ATC CCT GCC GTT GTT GAA GCT GTT AAA GCT GAA ACT GGT AAA GAA 1671
55 Arg Ile Pro Ala Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys Glu
315 320 325 330
CCA AAC AAA TCA GTA AAC CCT GAT GAA GTA GTT GCT ATG GGT GCG GCT 1719
Pro Asn Lys Ser Val Asn Pro Asp Glu Val Val Ala Met Gly Ala Ala
335 340 345
ATC CAA GGT GGT GTG ATT ACT GGT GAT GTC AAG GAT GTT GTC CTT CTT 1767
Ile Gln Gly Gly Val Ile mr Gly Asp Val Lys Asp Val Val Leu Leu
350 355 360
GAT GTA ACG CCA TTG TCA CTT GGT ATC GAA ACA ATG GGT GGA GTA TTT 1815
Asp Val mr Pro Leu Ser Leu Gly Ile Glu mr Met Gly Gly Val Phe
365 370 375
91
CA 022240l~ l997-l2-08
WO g6~'928 PCT/cA96/00322
ACA AAA CTT ATC GAT CGC AAC ACT ACA ATC CCA ACA TCT AAA TCA CAA 1863
Thr Lys Leu Ile Asp Arg Asn Thr Thr Ile Pro Thr Ser Lys Ser Gln
380 385 390
s
GTC TTC TCA ACA GCA GCA GAC AAC CAA CCA GCC GTT GAT ATC CAC GTT 1911
Val Phe Ser Thr Ala Ala Asp Asn Gln Pro Ala Val Asp Ile His Val
395 400 405 410
1O CTT CAA GGT GAA CGC CCA ATG GCA GCA GAT AAC AAG ACT CTT GGA CGC 1959
Leu Gln Gly Glu Arg Pro Met Ala Ala Asp Asn Lys Thr Leu Gly Arg
415 420 425
TTC CAA TTG ACT GAT ATC CCA GCT GCA CCT CGT GGA~ ATT CCT CAA ATC 2007
15 Phe Gln Leu Thr Asp Ile Pro Ala Ala Pro Arg Gly Ile Pro Gln Ile
430 435 490
GAA GTA ACA TTT GAC ATC GAC AAG AAC GGT ATC GTG TCT GTT AAG GCC 2055
Glu Val Thr Phe Asp Ile Asp Lys Asn Gly Ile Val Ser Val Lys Ala
445 450 455
AAA GAC CTT GGA ACT CAA AAA GAA CAA ACT ATT GTC ATC CAA TCG AAC 2103
Lys Asp Leu Gly Thr Gln Lys Glu Gln Thr Ile Val Ile Gln Ser Asn
460 465 470
TCA GGT TTG ACT GAC GAA GAA ATC GAC CGC ATG ATG AAA GAT GCA GAA 2151
Ser Gly Leu Thr Asp Glu Glu Ile Asp Arg Met Met Lys Asp Ala Glu
475 480 485 490
30 GCA AAC GCT GAA TCC GAT AAG AAA CGT AAA GAA GAA GTA GAC CTT CGT 2199
Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val Asp Leu Arg
495 S00 SOS
AAT GAA GTG GAC CAA GCA ATC TTT GCG ACT GAA AAG ACA ATC AAG GAA 2247
35 Asn Glu Val Asp Gln Ala Ile Phe Ala m r Glu Lys Thr Ile Lys Glu
510 515 520
ACT GAA GGT AAA GGC TTC GAC GCA GAA CGT GAC GCT GCC CAA GCT GCC 2295
Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala Gln Ala Ala
525 530 535
CTT GAT GAC CTT AAG AAA GCT CAA GAA GAC AAC AAC TTG GAC GAC ATG 2343
Leu Asp Asp Leu Lys Lys Ala Gln Glu Asp Asn Asn Leu Asp Asp Met
540 545 550
AAA GCA AAA CTT GAA GCA TTG AAC GAA AAA GCT CAA GGA CTT GCT GTT 2391
Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gln Gly Leu Ala Val
SSS 560 565 570
S0 AAA CTC TAC GAA CAA GCC GCA GCA GCG CAA CAA GCT CAA GAA GGA GCA 2439
Lys Leu Tyr Glu Gln Ala Ala Ala Ala Gln Gln Ala Gln Glu Gly Ala
575 580 585
GAA GGC GCA CAA GCA ACA GGA AAC GCA GGC GAT GAC GTC GTA GAC GGA 2487
55 Glu Gly Ala Gln Ala Thr Gly Asn Ala Gly Asp Asp Val Val Asp Gly
590 595 600
GAG TTT ACG GAA AAG TAAGATGAGT GTATTGGATG AAGAGTATCT AAAAAATA~A 2542
Glu Phe Thr Glu Lys
605
CGAAAAGTTT ATAATGATTT TTGTAATCAA GCTGATAACT ATAGAACATC AAAA~ATTTT 2602
ATTGATAATA TTCCAATAGA ATATTTAGCT AGATATAGAG AAATTATATT AGCTGAGCAT 2662
GATAGTTGTG TCAAAAATGA TGAAGCGGTA AGGAATTTTG TTACCTCAGT A~ l 2722
GCATTTGTAT CGGCGATGGT ATCAGCTATG ATATCATTAG AAATACAAAC ATATAAATTT 2782
GTAATACCGT TCATAATTGG TATGATTTGG ACAGTAGTTG TATTTCTTAT GATCAATTGG 2842
92
CA 022240l~ l997-l2-08
WO 9~'4C~28 PCT/CA96/00322
AATTATATAG GCAAATACTA A~AA~A~CA AAAATATATA AATATTTCTG TACTTATAGG 2902
ATATTTAAAA TC~AATAAA GTTAATTTAC TTATTTGCAG AGGTTGCAAC CCAGCCTCTG 2962
TTTTTCGATA AAAAGGGACG GAATCTCATT TGTTTGGGTT T~ ATC AATAGAAAGG 3022
AACAAAGAGT GTTCGTAACT GAACACGGGT TTCAGAATTT CTTACTAAAT ATAAAA~.AAA 3082
GGAATTGAAC CCGACCTAAA TGGTGGTTCG ATTCAGAACA TCAATAGAAA GGAATAAGGG 3142
'l~'l-l'C~'l'AAC TGAACACGGG CTACGGACTG TGCCAAAAAG ATA~lll~ l CTAGGACGTA 3202
AGCGTCCGTC GTCAAAACTC CTAGATGGCT GTGTCCGTTT GACGCCCTTT GTATCTTGAA 3262
lS TT ATG AAC AAT ACT GAA TTT TAT GAT CGT CTG GGG GTA TCC AAA AAC 3309
Met Asn Asn Thr Glu Phe Tyr Asp Arg Leu Gly Val Ser Lys Asn
1 5 10 lS
GCT TCG GCA GAC GAA ATC AAA AAG GCT TAT CGT AAG CTT TCC AAA AAA 3357
20 Ala Ser Ala Asp Glu Ile Lys Lys Ala Tyr Arg Lys Leu Ser Lys Lys
20 25 30
TAT CAC CCA GAT ATC AAC AAG GAG CCT GGT GCT GAG GAC AAG TAC AAG 3405
Tyr His Pro Asp Ile Asn Lys Glu Pro Gly Ala Glu Asp Lys Tyr Lys
35 40 45
GAA GTT CAA GAA GCC TAT GAG ACT TTG AGT GAC GAC CAA AAA CGT GCT 3453
Glu Val Gln Glu Ala Tyr Glu Thr Leu Ser Asp Asp Gln Lys Arg Ala
50 55 60
GCC TAT GAC CAG TAT GGT GCT GCA GGC GCC AAT GGT GGT TTT GGT GGA 3501
Ala Tyr Asp Gln Tyr Gly Ala Ala Gly Ala Asn Gly Gly Phe Gly Gly
65 70 75
35 GCT GGT GGT TTC GGC GGT TTC AAT GGG GCA GGT GGC TTC GGT GGT TTT 3549
Ala Gly Gly Phe Gly Gly Phe Asn Gly Ala Gly Gly Phe Gly Gly Phe
80 85 90 95
GAG GAT ATT TTC TCA AGT TTC TTC GGC GGA GGC GGT TCT TCG CGC AAT 3597
40 Glu Asp Ile Phe Ser Ser Phe Phe Gly Gly Gly Gly Ser Ser Arg Asn
100 1~5 110
CCA AAC GCT CCT CGC CAA GGA GAT GAT CTC CAG TAT CGT GTC AAT TTG 3645
Pro Asn Ala Pro Arg Gln Gly Asp Asp Leu Gln Tyr Arg Val Asn Leu
115 120 125
ACC TTT GAA GAA GCT ATC TTC GGA ACT GAG AAG GAA GTT AAG TAT CAT 3693
Thr Phe Glu Glu Ala Ile Phe Gly Thr Glu Lys Glu Val Lys Tyr His
130 135 140
CGT GAA GCT GGC TGT CGT ACA TGT AAT GGA TCT GGT GCT AAG CCA GGG 3741
Arg Glu Ala Gly Cys Arg Thr Cys Asn Gly Ser Gly Ala Lys Pro Gly
145 150 155
55 ACA AGT CCA GTC ACT TGT GGA CGC TGT CAT GGC GCT GGT GTC ATT AAC 3789
Thr Ser Pro Val Thr Cys Gly Arg Cys His Gly Ala Gly Val Ile Asn
160 165 170 175
GTC GAT ACG CAG ACT CCT CTT GGT ATG ATG CGT CGC CAA GTA ACC TGT 3837
60 Val Asp Thr Gln Thr Pro Leu Gly Met Met Arg Arg Gln Val Thr Cys
180 185 190
GAT GTC TGT CAC GGT CGA GGA AAA GAA ATC AAA TAT CCA TGT ACA ACC 3885
Asp Val Cys His Gly Arg Gly Lys Glu Ile Lys Tyr Pro Cys Thr Thr
195 200 205
TGT CAT GGA ACA GGT CAT GAG AAA CAA GCT CAT AGC GTA CAT GTG AAA 3933
Cys His Gly Thr Gly His Glu Lys Gln Ala His Ser Val His Val Lys
210 215 220
93
CA 022240l~ l997-l2-08
WO gf '~328 PCT/cA96/00322
ATC CCT GCT GGT GTG GAA ACA GGT CAA CAA ATT CGC CTC GCT GGT CAA 3981
Ile Pro Ala Gly Val Glu Thr Gly Gln Gln Ile Arg Leu Ala Gly Gln
225 230 235
GGT GAA GCA GGC TTT AAC GGT GGA CCT TAT GGT GAC TTG TAT GTA GTA 4029
Gly Glu Ala Gly Phe Asn Gly Gly Pro Tyr Gly Asp Leu Tyr Val Val
240 245 250 255
10 GTT TCT GTG GAA GCT AGT GAC AAG TTT GAA CGT GAA GGA ACG ACT ATC 4077
Val Ser Val Glu Ala Ser Asp Lys Phe Glu Arg Glu Gly Thr Thr Ile
260 265 270
TTC TAC AAT CTC AAC CTC AAC TTT GTC CAA GCG GCT CTT GGT GAT ACA 4125
15 Phe Tyr Asn Leu Asn Leu Asn Phe Val Gln Ala Ala Leu Gly Asp Thr
275 280 285
GTA GAT ATT CCA ACT GTT CAC GGT GAT GTT GAA TTG GTT ATT CCA GAG 4173
Val Asp Ile Pro Thr Val His Gly Asp Val Glu Leu Val Ile Pro Glu
290 295 300
GGA ACT CAG ACT GGT AAG AAA TTC CGC CTA CGT AGT AAG GGG GCA CCG 4221
Gly Thr Gln Thr Gly Lys Lys Phe Arg Leu Arg Ser Lys Gly Ala Pro
305 310 315
AGC CTT CGT GGC GGT GCA GTT GGT GAC CAA TAC GTT ACT GTT AAT GTC 4269
Ser Leu Arg Gly Gly Ala Val Gly Asp Gln Tyr Val Thr Val Asn Val
320 325 330 335
30 GTA ACA CCG ACA GGC TTG AAC GAC CGC CAA AAA GTA GCC TTG AAA GAA 4317
Val Thr Pro Thr Gly Leu Asn Asp Arg Gln Lys Val Ala Leu Lys Glu
340 345 350
TTC 4320
Phe
(2) INFORMATION FOR SEQ ID No:5:
- 40
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 607 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ser Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Ala Val
1 5 10 15
Ala Val Leu Glu Gly Thr Glu Ser Lys Ile Ile Ala Asn Pro Glu Gly
20 25 30
Asn Arg Thr Thr Pro Ser Val Val Ser Phe Lys Asn Gly Glu Ile Ile
35 g0 45
Val Gly Asp Ala Ala Lys Arg Gln Ala Val Thr Asn Pro Asp Thr Val
50 55 60
Ile Ser Ile Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser Ala Asn
Gly Lys Glu Tyr Thr Pro Gln Glu Ile Ser Ala Met Ile Leu Gln Tyr
Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Thr Lys Ala
100 105 110
94
CA 022240l~ l997-l2-08
WO 9''4D97& PCT/CA96/00322
Val Ile Thr Val Pro Ala Tyr Phe Asn Asp Ala Gln Arg Gln Ala Thr
115 120 125
S Lys Asp Ala Gly Lys Ile Ala Gly Leu Glu Val Glu Arg Ile Val Asn
130 135 140
Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Leu Asp Lys Thr Asp LYs
145 150 155 160
Glu Glu Lys Ile Leu Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val
165 170 175
Ser Ile Leu Glu Leu Gly Asp Gly Val Phe Asp Val Leu Ser Thr Ala
180 185 190
Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe Asp Gln Lys Ile Ile Asp
195 200 205
His Leu Val Ala Glu Phe Lys Lys Glu Asn Gly Ile Asp Leu Ser Thr
210 215 220
Asp Lys Met Ala Met Gln Arg Leu Lys Asp Ala Ala Glu Lys Ala Lys
225 230 235 240
Lys Asp Leu Ser Gly Val Thr Ser Thr Gln Ile Ser Leu Pro Phe Ile
245 250 255
Thr Ala Gly Glu Ala Gly Pro Leu His Leu Glu Met mr Leu Thr Arg
260 265 270
Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr Lys Val
275 280 285
Pro Val Arg Gln Ala Leu Ser Asp Ala Gly Leu Ser Leu Ser Glu Ile
290 295 300
Asp Glu Val Ile Leu Val Gly Gly Ser Thr Arg Ile Pro Ala Val Val
305 310 315 320
Glu Ala Val Lys Ala Glu Thr Gly Lys Glu Pro Asn Lys Ser Val Asn
325 330 335
Pro Asp Glu Val Val Ala Met Gly Ala Ala Ile Gln Gly Gly Val Ile
340 345 350
Thr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro Leu Ser
355 360 365
Leu Gly Ile Glu Thr Met Gly Gly Val Phe Thr Lys Leu Ile Asp Arg
370 375 380
Asn Thr Thr Ile Pro Thr Ser Lys Ser Gln Val Phe Ser Thr Ala Ala
385 390 395 400
Asp Asn Gln Pro Ala Val Asp Ile His Val Leu Gln Gly Glu Arg Pro
405 410 415
Met Ala Ala Asp Asn Lys Thr Leu Gly Arg Phe Gln Leu Thr Asp Ile
420 425 430
Pro Ala Ala Pro Arg Gly Ile Pro Gln Ile Glu Val Thr Phe Asp Ile
- 435 440 445
Asp Lys Asn Gly Ile Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gln
450 455 460
Lys Glu Gln Thr Ile Val Ile Gln Ser Asn Ser Gly Leu Thr Asp Glu
465 470 475 480
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
Glu Ile Asp Arg Met Met Lys Asp Ala Glu Ala Asn Ala Glu Ser Asp
485 490 495
Lys Lys Arg Lys Glu Glu Val Asp Leu Arg Asn Glu Val Asp Gln Ala
500 505 510
Ile Phe Ala Thr Glu Lys Thr Ile Lys Glu Thr Glu Gly Lys Gly Phe
515 520 525
Asp Ala Glu Arg Asp Ala Ala Gln Ala Ala Leu Asp Asp Leu Lys Lys
530 535 540
Ala Gln Glu Asp Asn Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala
15545 550 555 560
Leu Asn Glu Lys Ala Gln Gly Leu Ala Val Lys Leu Tyr Glu Gln Ala
565 570 575
~0 Ala Ala Ala Gln Gln Ala Gln Glu Gly Ala Glu Gly Ala Gln Ala Thr
580 585 590
Gly Asn Ala Gly Asp Asp Val Val Asp Gly Glu Phe Thr Glu Lys
595 600 605
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 352 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Asn Asn Thr Glu Phe Tyr Asp Arg Leu Gly Val Ser Lys Asn Ala
1 5 10 15
Ser Ala Asp Glu Ile Lys Lys Ala Tyr Arg Lys Leu Ser Lys Lys Tyr
20 25 30
His Pro Asp Ile Asn Lys Glu Pro Gly Ala Glu Asp Lys Tyr Lys Glu
4535 40 45
Val Gln Glu Ala Tyr Glu Thr Leu Ser Asp Asp Gln Lys Arg Ala Ala
Tyr Asp Gln Tyr Gly Ala Ala Gly Ala Asn Gly Gly Phe Gly Gly Ala
65 70 75 80
Gly Gly Phe Gly Gly Phe Asn Gly Ala Gly Gly Phe Gly Gly Phe Glu
85 90 95
Asp Ile Phe Ser Ser Phe Phe Gly Gly Gly Gly Ser Ser Arg Asn Pro
100 105 110
Asn Ala Pro Arg Gln Gly Asp Asp Leu Gln Tyr Arg Val Asn Leu Thr
60115 120 125
Phe Glu Glu Ala Ile Phe Gly Thr Glu Lys Glu Val Lys Tyr His Arg
130 135 140
Glu Ala Gly Cys Arg Thr Cys Asn Gly Ser Gly Ala Lys Pro Gly Thr
145 150 155 160
Ser Pro Val Thr Cys Gly Arg Cys His Gly Ala Gly Val Ile Asn Val
165 170 175
96
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
Asp m r Gln Thr Pro Leu Gly Met Met Arg Arg Gln Val mr Cys Asp
180 185 190
Val Cys His Gly Arg Gly Lys Glu Ile Lys Tyr Pro Cys m r mr Cys
195 200 205
His Gly mr Gly His Glu Lys Gln Ala His Ser Val His Val Lys Ile
210 215 220
Pro Ala Gly Val Glu mr Gly Gln Gln Ile Arg Leu Ala Gly Gln Gly
225 230 235 240
Glu Ala Gly Phe Asn Gly Gly Pro Tyr Gly Asp Leu Tyr Val Val Val
2g5 250 255
Ser Val Glu Ala Ser Asp Lys Phe Glu Arg Glu Gly mr Thr Ile Phe
260 265 270
Tyr Asn Leu Asn Leu Asn Phe Val Gln Ala Ala Leu Gly Asp mr Val
275 280 285
Asp Ile Pro m r Val His Gly Asp Val Glu Leu Val Ile Pro Glu Gly
290 295 300
mr Gln mr Gly Lys Lys Phe Arg Leu Arg Ser Lys Gly Ala Pro Ser
305 310 315 320
Leu Arg Gly Gly Ala Val Gly Asp Gln Tyr Val mr Val Asn Val Val
325 330 335
mr Pro mr Gly Leu Asn Asp Arg Gln Lys Val Ala Leu Lys Glu Phe
340 345 350
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Thr Ser Thr Gln Ile Ser Leu Pro Phe Ile Thr Ala Gly Glu Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(8) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Thr Ala Gly Glu Ala Gly Pro Leu His Leu Glu Met Thr Leu Thr
1 5 10 15
- (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
97
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~h~U~N~ DESCRIPTION: SEQ ID NO:9:
lû Met Thr Leu Thr Arg Ala Lys Phe Asp Asp Leu Thr Arg Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr Lys Val Pro Val
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:11:
U~N~ CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Thr Lys Val Pro Val Arg Gln Ala Leu Ser Asp Ala Gly Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Lys Ala Lys Asp Leu Gly Thr Gln Lys Glu Gln Thr Ile Val Ile
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
98
CA 022240l~ l997-l2-08
W O 96/40928 PCT/CA96/00322
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Leu Thr Asp Glu Ile Asp Arg Met Met Lys Asp Ala Glu Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Lys Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu
1 5 10 15
Val Asp Leu Arg Asn Glu Val Asp
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) ~Qu~N~: DESCRIPTION: SEQ ID NO:15:
Asn Glu Val Asp Gln Ala Ile Phe Ala Thr Glu Lys Thr Ile Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Glu Lys Thr Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg
1 5 10 15
Asp Ala Ala Gln Ala Ala Leu Asp Asp Leu Lys Lys
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
~ (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
99
CA 0222401~ 1997-12-08
W O 96/40928 PCT/CA96/00322
Lys Ala Gln Glu Asp Asn Asn Leu Asp Asp Met Lys Ala Lys Leu Glu
1 5 10 15
S Ala Leu Asn Glu Lys Ala Gln Gly Leu Ala Val Lys Leu Tyr
(2) INFORNATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
15 (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Gln Glu Gly Ala Glu Gly Ala Gln Ala Thr Gly Asn Ala Gly Asp Asp
1 5 10 15
Val Val Asp Gly Glu Phe Thr Glu Lys
20 25
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2183 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptococcus pyogenes
(ix) FEAT~'RE:
(A) NAME/KEY: CDS
(B) LOCATION: 20g..2030
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
CAGCGATGGT A~ll~lllAT AACTAAGGTA AATGAGTTTT C~lllll~lC CGTAATGACA 60
GT~AACTAGA TAGCAAGTTA GAAGCTATTT CGCTTGCTGA TTAAACTATA GTGATTGCTT 120
AGAATTGGAA GTAAAATAAT TCGAGTGCTT ACTAAGATAA ATT~AATAA AAAGTAATAA 180
AGTATAAAAT AAGAGGTATT AAC ATG TCT AAA ATT ATT GGT ATT GAC TTA 230
Met Ser Lys Ile Ile Gly Ile Asp Leu
GGT ACA ACA AAC TCA GCA GTA GCA GTT CTT GAA GGG ACT GAA TCA AAA 278
Gly Thr Thr Asn Ser Ala Val Ala Val Leu Glu Gly Thr Glu Ser Lys
10 15 20 25
65 ATC ATT GCT AAC CCA GAA GGC AAT CGT ACA ACT CCT TCA GTA GTA TCA 326
Ile Ile Ala Asn Pro Glu Gly Asn Arg Thr Thr Pro Ser Val Val Ser
30 35 40
TTC AAA AAT GGT GAA ATT ATC GTG GGT GAT GCT GCA AAA CGC CAA GCA 374
70 Phe Lys Asn Gly Glu Ile Ile Val Gly Asp Ala Ala Lys Arg Gln Ala
45 50 55
100
CA 022240l~ l997-l2-08
WO 9~ 328 PCT/cA96tOO322
GTG ACA AAC CCA GAA ACA GTA ATC TCT ATT AAA TCT AAA ATG GGA ACT 422
Val Thr Asn Pro Glu Thr Val Ile Ser Ile Lys Ser Lys Met Gly Thr
60 65 70
TCT GAA AAA GTT TCT GCA AAT GGT AAA GAA TAT ACT CCT CAA GAA ATT 470
Ser Glu Lys Val Ser Ala Asn Gly Lys Glu Tyr Thr Pro Gln Glu Ile
75 80 85
10 TCA GCA ATG ATT CTT CAA TAC CTT AAA GGT TAT GCT GAA GAC TAT CTT 518
Ser Ala Met Ile Leu Gln Tyr Leu Lys Gly Tyr Ala Glu Asp Tyr Leu
90 95 100 105
GGA GAA AAA GTA GAA AAA GCA GTT ATT ACT GTT CCA GCT TAT TTC AAC 566
IS Gly Glu Lys Val Glu Lys Ala Val Ile Thr Val Pro Ala Tyr Phe Asn
110 115 120
GAT GCA CAA CGT CAA GCA ACT AAA GAC GCT GGT AAA ATT GCA GGT CTT 614
Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Lys Ile Ala Gly Leu
125 130 135
GAA GTA GAA CGT ATC GTT AAT GAA CCA ACA GCA GCT GCA CTT GCT TAT 662
Glu Val Glu Arg Ile Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr
140 145 150
GGT ATG GAC AAG ACT GAC AAG GAT GAA AAA ATC TTA GTT TTT GAC CTT 710
Gly Met Asp Lys Thr Asp Lys Asp Glu Lys Ile Leu Val Phe Asp Leu
155 160 165
30 GGT GGT GGT ACA TTT GAC GTA TCA ATC CTT GAA TTA GGT GAT GGT GTC 758
Gly Gly Gly Thr Phe Asp Val Ser Ile Leu Glu Leu Gly Asp Gly Val
170 175 180 185
TTC GAC GTT CTT GCA ACA GCA GGT GAT AAC AAA CTT GGT GGT GAC GAC 806
35 Phe Asp Val Leu Ala Thr Ala Gly Asp Asn Lys Leu Gly Gly Asp Asp
190 195 200
TTT GAC CAA AAA ATT ATT GAT TTC TTA GTG GCT GAA TTT AAG AAA GAA 854
Phe Asp Gln Lys Ile Ile Asp Phe Leu Val Ala Glu Phe Lys Lys Glu
205 210 215
AAT GGT ATT GAC TTA TCA CAA GAT AAG ATG GCA CTT CAA CGC TTG AAA 902
Asn Gly Ile Asp Leu Ser Gln Asp Lys Met Ala Leu Gln Arg Leu Lys
220 225 230
GAT GCT GCT GAA AAA GCT AAA AAA GAT CTT TCA GGT GTG ACA CAA ACA 950
Asp Ala Ala Glu Lys Ala Lys Lys Asp Leu Ser Gly Val Thr Gln Thr
235 240 245
50 CAA ATT TCA TTA CCG TTC ATC ACT GCT GGT TCT GCT GGT CCT CTT CAC 998
Gln Ile Ser Leu Pro Phe Ile Thr Ala Gly Ser Ala Gly Pro Leu His
250 255 260 265
TTA GAG ATG AGC TTA TCT CGT GCT AAA TTT GAC GAT CTC ACT CGT GAC 1046
55 Leu Glu Met Ser Leu Ser Arg Ala Lys Phe Asp Asp Leu Thr Arg Asp
270 275 280
CTT GTT GAA CGT ACG AAA ACT CCA GTT CGT CAA GCT CTT TCA GAT GCA 1094
Leu Val Glu Arg Thr Lys Thr Pro Val Arg Gln Ala Leu Ser Asp Ala
285 290 295
GGA TTG TCA TTG TCA GAA ATT GAT GAA GTT ATC CTT GTT GGT GGA TCA 1142
Gly Leu Ser Leu Ser Glu Ile Asp Glu Val Ile Leu Val Gly Gly Ser
300 305 310
ACT CGT ATC CCA GCA GTT GTC GAA GCT GTA AAA GCT GAA ACT GGT AAA 1190
Thr Arg Ile Pro Ala Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys
315 320 325
101
CA 022240l~ l997-l2-08
W 0 96/40928 PCT/CA96tO0322
GAA CCA AAT AAA TCT GTA M C CCT GAT GAA GTG GTT GCT ATG GGT GCT 1238
Glu Pro Asn Lys Ser Val Asn Pro Asp Glu Val Val Ala Met Gly Ala
330 335 340 345
GCT ATC CAA GGT GGG GTT ATC ACT GGG GAT GTG AAA GAC GTT GTC CTT 1286
Ala Ile Gln Gly Gly Val Ile mr Gly Asp Val Lys Asp Val Val Leu
3S0 35S 360
0 CTT GAC GTA ACA CCA TTG TCA CTT GGT ATT GAA ACA ATG GGT GGT GTC 1334
Leu Asp Val mr Pro Leu Ser Leu Gly Ile Glu mr Met Gly Gly Val
365 370 37S
TTC ACT AAA TTG ATC GAC CGC AAT ACA ACT ATC CCA ACA TCT AAA TCA 1382
15 Phe mr Lys Leu Ile Asp Arg Asn mr mr Ile Pro mr Ser Lys Ser
380 38S 390
CAA GTC TTC TCA ACA GCA GCA GAC AAC CAA CCA GCC GTT GAT ATC CAT 1430
Gln Val Phe Ser mr Ala Ala Asp Asn Gln Pro Ala Val Asp Ile His
395 400 405
GTT CTT CAA GGT GAA CGC CCA ATG GCA GCA GAT AAC AAG ACT CTT GGT 1478
Val Leu Gln Gly Glu Arg Pro Met Ala Ala Asp Asn Lys m r Leu Gly
410 415 420 425
CGC TTC CAA TTG ACT GAT ATC CCA GCT GCA CCT CGT GGA ATC CCA CAA 1526
Arg Phe Gln Leu mr Asp Ile Pro Ala Ala Pro Arg Gly Ile Pro Gln
430 435 440
30 ATT GAA GTA ACA TTT GAT ATC GAT AAA AAC GGT ATT GTT TCT GTA AAA 1574
Ile Glu Val mr Phe Asp Ile Asp Lys Asn Gly Ile Val Ser Val Lys
44S 450 455
GCT AAA GAC CTT GGT ACG CAA AAG GAA CAA CAC ATC GTT ATC AAA TCA 1622
35 Ala Lys Asp Leu Gly mr Gln Lys Glu Gln His Ile Val Ile Lys Ser
460 465 470
AAC GAC GGA CTT TCT GAA GAA GAA ATT GAT CGC ATG ATG AAA GAC GCT 1670
Asn Asp Gly Leu Ser Glu Glu Glu Ile Asp Arg Met Met Lys Asp Ala
475 480 48S
GAA GCT AAT GCC GAA GCC GAT GCG AAA CGT AAA GAA GAA GTT GAC CTT 1718
Glu Ala Asn Ala Glu Ala Asp Ala Lys Arg Lys Glu Glu Val Asp Leu
490 495 500 505
AAA AAC GAA GTT GAC CAA GCT ATC TTT GCT ACT GAA AAA ACA ATC AAA 1766
Lys Asn Glu Val Asp Gln Ala Ile Phe Ala Thr Glu Lys mr Ile Lys
510 515 520
S0 GAA ACT GAA GGT AAA GGC TTT GAC ACA GAA CGC GAT GCA GCG CAA TCA 1814
Glu Thr Glu Gly Lys Gly Phe Asp mr Glu Arg Asp Ala Ala Gln Ser
525 530 535
GCT CTT GAC GAG TTA AAA GCT GCG CAA GAA TCT GGC AAC CTT GAC GAC 1862
55 Ala Leu Asp Glu Leu Lys Ala Ala Gln Glu Ser Gly Asn Leu Asp Asp
540 545 550
ATG AAA GCT AAA CTT GAA GCA TTA AAT GAA AAA GCG CAA GCT TTG GCT 1910
Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gln Ala Leu Ala
SSS 560 565
GTT AAA ATG TAC GAG CAA GCT GCA GCA GCT CAA CAA GCA GCA CAA GGT 1958
Val Lys Met Tyr Glu Gln Ala Ala Ala Ala Gln Gln Ala Ala Gln Gly
570 575 580 585
GCA GAA GGT GCA CAA GCT AAT GAT TCA GCA AAT AAT GAT GAT GTT GTA 2006
Ala Glu Gly Ala Gln Ala Asn Asp Ser Ala Asn Asn Asp Asp Val Val
590 595 600
102
CA 022240l~ l997-l2-08
W O 96/40928 PCT/CA96/00322
GAT GGC GAA TTT ACA GAA AAG TAATGATTTA GTTATCTAGT AACATTAATA 2057
Asp Gly Glu Phe Thr Glu Lys
605
TCCGAATTCA GAGGTTGTAC CAAACCTCTG TTTTTGGCTA AATAAAATGT AAAAATGCTG 2117
ACGTCAAAAT ATTTTAAGAA AG~AATA~AA GTTCGATTAT TCGAACACAG GCTAAAGCGT 2177
l0 GTAAAG 2183
(2) INFORMATION FOR SEQ ID No:20:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 608 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met Ser Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Ala Val
1 5 10 15
Ala Val Leu Glu Gly Thr Glu Ser Lys Ile Ile Ala Asn Pro Glu Gly
Asn Arg Thr Thr Pro Ser Val Val Ser Phe Lys Asn Gly Glu Ile Ile
35 90 45
Val Gly Asp Ala Ala Lys Arg Gln Ala Val Thr Asn Pro Glu Thr Val
50 55 60
Ile Ser Ile Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser Ala Asn
65 70 75 80
Gly Lys Glu Tyr Thr Pro Gln Glu Ile Ser Ala Met Ile Leu Gln Tyr
85 90 95
Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Glu Lys Ala
100 105 110
Val Ile Thr Val Pro Ala Tyr Phe Asn Asp Ala Gln Arg Gln Ala mr
115 120 125
Lys Asp Ala Gly Lys Ile Ala Gly Leu Glu Val Glu Arg Ile Val Asn
130 135 140
Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Met Asp Lys Thr Asp Lys
145 150 155 160
Asp Glu Lys Ile Leu Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val
165 170 175
Ser Ile Leu Glu Leu Gly Asp Gly Val Phe Asp Val Leu Ala Thr Ala
180 185 190
Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe Asp Gln Lys Ile Ile Asp
195 200 205
Phe Leu Val Ala Glu Phe Lys Lys Glu Asn Gly Ile Asp Leu Ser Gln
210 215 220
Asp Lys Met Ala Leu Gln Arg Leu Lys Asp Ala Ala Glu Lys Ala Lys
225 230 235 240
Lys Asp Leu Ser Gly Val Thr Gln Thr Gln Ile Ser Leu Pro Phe Ile
245 250 255
103
CA 022240l~ l997-l2-08
WO g~'4~g28 PCT/CA96/00322
Thr Ala Gly Ser Ala Gly Pro Leu His Leu Glu Met Ser Leu Ser Arg
260 265 270
Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu Val Glu Arg mr Lys Thr
S 275 280 285
Pro Val Arg Gln Ala Leu Ser Asp Ala Gly Leu Ser Leu Ser Glu Ile
290 295 300
0 Asp Glu Val Ile Leu Val Gly Gly Ser Thr Arg Ile Pro Ala Val Val
305 310 315 320
Glu Ala Val Lys Ala Glu Thr Gly Lys Glu Pro Asn Lys Ser Val Asn
325 330 335
Pro Asp Glu Val Val Ala Met Gly Ala Ala Ile Gln Gly Gly Val Ile
340 34S 3S0
Thr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro Leu Ser
3SS 360 36S
Leu Gly Ile Glu ~hr Met Gly Gly Val Phe Thr Lys Leu Ile Asp Arg
370 375 380
Asn Thr Thr Ile Pro Thr Ser Lys Ser Gln Val Phe Ser Thr Ala Ala
385 390 395 400
Asp Asn Gln Pro Ala Val Asp Ile His Val Leu Gln Gly Glu Arg Pro
40S 410 41S
Met Ala Ala Asp Asn Lys Thr Leu Gly Arg Phe Gln Leu Thr Asp Ile
420 425 430
Pro Ala Ala Pro Arg Gly Ile Pro Gln Ile Glu Val Thr Phe Asp Ile
435 440 445
Asp Lys Asn Gly Ile Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gln
4S0 4S5 460
Lys Glu Gln His Ile Val Ile Lys Ser Asn Asp Gly Leu Ser Glu Glu
465 470 475 480
Glu Ile Asp Arg Met Met Lys Asp Ala Glu Ala Asn Ala Glu Ala Asp
485 490 49S
Ala Lys Arg Lys Glu Glu Val Asp Leu Lys Asn Glu Val Asp Gln Ala
500 505 510
Ile Phe Ala Thr Glu Lys Thr Ile Lys Glu Thr Glu Gly Lys Gly Phe
515 520 525
Asp Thr Glu Arg Asp Ala Ala Gln Ser Ala Leu Asp Glu Leu Lys Ala
530 535 540
Ala Gln Glu Ser Gly Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala
545 550 SSS S60
Leu Asn Glu Lys Ala Gln Ala Leu Ala Val Lys Met Tyr Glu Gln Ala
565 570 S7S
Ala Ala Ala Gln Gln Ala Ala Gln Gly Ala Glu Gly Ala Gln Ala Asn
580 585 590
Asp Ser Ala Asn Asn Asp Asp Val Val Asp Gly Glu Phe Thr Glu Lys
595 600 605
104
CA 022240l~ l997-l2-08
WO g~'~C328 PCT/CA96/00322
(2) INFORMATION FOR SEQ ID NO 21
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 2438 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS double
(D) TOPOLOGY linear
10(ii~ MOLECULE TYPE DNA (genomic)
(iii) HYPOTHETICAL NO
(iv) ANTI-SENSE NO
(vi) ORIGINAL SOURCE
(A) ORGANISM Streptococcus agalactiae
(ix) FEATURE
20(A) NAME/KEY CDS
(B) LOCATION 248 2077
(xi) SEQUENCE DESCRIPTION SEQ ID NO 21
CTTTCAAAAG GGATATAAAT TGCACGAGCG TCTGCTAAGA CCAGCGATGG TA~~ lA 60
TAACTAAGGT AAATGAGTTT TC~rllll~l CCGTAATGAC AGTAAACTAG ATAGCAAGTT 120
AGAAGCTATT CAGCTTGCTG ATTAAACTAT AGTGATTGCT TAGAATTGGA AGTAAAATAA 180
TTCGAGTGCT TACTAA~.A~A AATTGAAATA AAAAGTAATA AAGTATTATA AAATAA~.A~G 240
TA~TAAC ATG TCT AAA ATT ATT GGT ATT GAC TTA GGT ACA ACA AAC TCA 289
35Met Ser Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser
1 5 10
GCA GTA GCA GTT CTT GAA GGG ACT GAA TCA AAA ATC ATT GCT AAC CCA 337
Ala Val Ala Val Leu Glu Gly Thr Glu Ser Lys Ile Ile Ala Asn Pro
4015 20 25 30
GAA GGC AAT CGT ACA ACT CCT TCA GTA GTA TCA TTC AAA AAT GGT GAA 385
Glu Gly Asn Arg Thr mr Pro Ser Val Val Ser Phe Lys Asn Gly Glu
35 40 45
ATT ATC GTG GGT GAT GCT GCA AAA CGT CAA GCG GTA ACA AAT CCA GAT 433
Ile Ile Val Gly Asp Ala Ala Lys Arg Gln Ala Val Thr Asn Pro Asp
50 55 60
50 ACT GTT ATC TCT ATC AAA TCA AAG ATG GGA ACT TCT GAA AAA GTT TCT 481
Thr Val Ile Ser Ile Lys Ser Lys Met Gly Thr Ser Glu Lys Val Ser
65 70 75
GCA AAT GGT AAA GAA TAT ACT CCT CAA GAA ATT TCA GCA ATG ATT CTT 529
55 Ala Asn Gly Lys Glu Tyr Thr Pro Gln Glu Ile Ser Ala Met Ile Leu
80 85 90
CAA TAC CTT AAA GGT TAT GCT GAA GAC TAT CTT GGA GAA AAA GTA GAA 577
Gln Tyr Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Glu
6095 100 105 110
AAA GCA GTT ATT ACT GTT CCA GCT TAC TTC AAC GAT GCA CAA CGT CAG 625
Lys Ala Val Ile Thr Val Pro Ala Tyr Phe Asn Asp Ala Gln Arg Gln
115 120 125
GCA ACT AAA GAC GCT GGT AAA ATT GCA GGT CTT GAA GTA GAA CGT ATC 673
Ala Thr Lys Asp Ala Gly Lys Ile Ala Gly Leu Glu Val Glu Arg Ile
130 135 140
105
CA 022240l~ l997-l2-08
W O 96/40928 PCT/CA96/00322
GTT AAC GAA CCA ACA GCA GCC GCA CTT GCT TAT GGT ATG GAC AAG ACT 721
Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Met Asp Lys Thr
145 150 155
GAC AAG GAT GAA AAA ATC TTA GTT TTT GAC CTT GGT GGT GGT ACA TTT 769
Asp Lys Asp Glu Lys Ile Leu Val Phe Asp Leu Gly Gly Gly Thr Phe
160 165 170
10 GAC GTA TCA ATC CTT GAA TTA GGT GAT GGT GTC TTC GAC GTT CTT GCA 817
Asp Val Ser Ile Leu Glu Leu Gly Asp Gly Val Phe Asp Val Leu Ala
175 180 185 190
ACA GCA GGT GAT AAC AAA CTT GGT GGT GAC GAC TTT GAC CAG AAA ATT 865
15 Thr Ala Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe Asp Gln Lys Ile
195 200 205
ATT GAT TTC TTG GTA GAA GAA TTC AAG AAA GAA AAT GGT ATT GAT CTT 913
Ile Asp Phe Leu Val Glu Glu Phe Lys Lys Glu Asn Gly Ile Asp Leu
210 215 220
TCT CAA GAC AAA ATG GCT CTT CAA CGC TTG AAA GAT GCT GCT GAA AAA 961
Ser Gln Asp Lys Met Ala Leu Gln Arg Leu Lys Asp Ala Ala Glu Lys
225 230 235
GCT AAA AAA GAC CTT TCA GGT GTA ACT CAA ACT CAA ATT TCA TTA CCG 1009
Ala Lys Lys Asp Leu Ser Gly Val Thr Gln Thr Gln Ile Ser Leu Pro
240 245 250
30 TTC ATC ACT GCT GGT TCT GCT GGT CCT CTT CAC TTG GAG ATG AGC TTA 1057
Phe Ile Thr Ala Gly Ser Ala Gly Pro Leu His Leu Glu Met Ser Leu
255 260 265 270
TCA CGT GCT AAA TTT GAC GAT CTC ACT CGT GAC CTT GTT GAA CGT ACG 1105
35 Ser Arg Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu Val Glu Arg Thr
275 280 285
AAA ACT CCA GTT CGT CAA GCT CTT TCA GAT GCA GGC TTG TCA TTG TCA 1153
Lys Thr Pro Val Arg Gln Ala Leu Ser Asp Ala Gly Leu Ser Leu Ser
290 295 300
GAA ATT GAT GAA GTT ATC CTC GTT GGT GGA TCA ACA CGT ATC CCA GCA 1201
Glu Ile Asp Glu Val Ile Leu Val Gly Gly Ser Thr Arg Ile Pro Ala
305 310 315
GTT GTT GAA GCT GTA AAA GCT GAA ACT GGT AAA GAA CCA AAT AAA TCT 1249
Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys Glu Pro Asn Lys Ser
320 325 330
50 GTT AAC CCT GAT GAA GTG GTT GCC ATG GGT GCT GCT ATC CAA GGT GGT 1297
Val Asn Pro Asp Glu Val Val Ala Met Gly Ala Ala Ile Gln Gly Gly
335 340 345 350
GTT ATC ACT GGG GAT GTG AAA GAC GTT GTA CTT CTT GAC GTA ACA CCA 1345
55 Val Ile Thr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro
355 360 365
TTG TCA CTT GGT ATT GAA ACA ATG GGT GGT GTC TTC ACT AAA TTG ATC 1393
Leu Ser Leu Gly Ile Glu Thr Met Gly Gly Val Phe mr Lys Leu Ile
370 375 380
GAC CGC AAC ACA ACT ATC CCA ACA TCT AAA TCA CAA GTC TTC TCA ACA 1441
Asp Arg Asn Thr Thr Ile Pro Thr Ser Lys Ser Gln Val Phe Ser Thr
385 390 395
GCA GCA GAC AAC CAA CCA GCC GTT GAT ATC CAT GTT CTT CAA GGT GAA 1489
Ala Ala Asp Asn Gln Pro Ala Val Asp Ile His Val Leu Gln Gly Glu
400 405 410
106
CA 022240l~ l997-l2-08
W O 96/40928 PCT/CA96/00322
CGC CCA ATG GCA GCA GAT AAC AAA ACA CTC GGT CGC TTC CAA TTG ACT 1537
Arg Pro Met Ala Ala Asp Asn Lys mr Leu Gly Arg Phe Gln Leu mr
415 420 425 430
GAT ATC CCA GCT GCA CCT CGT GGA ATC CCA CAA ATT GAA GTA ACA TTT 1585
Asp Ile Pro Ala Ala Pro Arg Gly Ile Pro Gln Ile Glu Val mr Phe
435 440 445
10 GAT ATC GAT AAA AAT GGT ATT GTA TCT GTT AAA GCT AAA GAT CTC GGT 1633
Asp Ile Asp Lys Asn Gly Ile Val Ser Val Lys Ala Lys Asp Leu Gly
450 455 460
ACT CAA AAA GAA CAA CAC ATT GTT ATC CAA TCT AAT TCA GGA TTA ACT 1681
15 mr Gln Lys Glu Gln His Ile Val Ile Gln Ser Asn Ser Gly Leu mr
465 470 475
GAT GAA GAA ATT GAT AAA ATG ATG AAA GAT GCT GAA GCA AAT GCT GAA 1729
Asp Glu Glu Ile Asp Lys Met Met Lys Asp Ala Glu Ala Asn Ala Glu
480 485 490
GCA GAT GCA AAA CGT AAA GAA GAA GTT GAT CTT AAA AAT GAA GTT GAC 1777
Ala Asp Ala Lys Arg Lys Glu Glu Val Asp Leu Lys Asn Glu Val Asp
495 500 505 510
CAA GCC ATC TTT GCA ACA GAA AAA ACT ATT AAA GAA ACT GAA GGC AAA 1825
Gln Ala Ile Phe Ala Thr Glu Lys mr Ile Lys Glu mr Glu Gly Lys
515 520 525
30 GGT TTT GAT ACA GAA CGC GAT GCA GCG CAA TCA GCA CTT GAT GAG TTG 1873
Gly Phe Asp mr Glu Arg Asp Ala Ala Gln Ser Ala Leu Asp Glu Leu
530 535 540
AAA AAA GCT CAA GAA TCA GGT AAC CTT GAC GAC ATG AAA GCT AAA CTT 1921
35 Lys Lys Ala Gln Glu Ser Gly Asn Leu Asp Asp Met Lys Ala Lys Leu
545 550 555
GAA GCT CTT AAC GAA AAA GCA CAA GCT CTT GCA GTT AAA CTT TAC GAA 1969
Glu Ala Leu Asn Glu Lys Ala Gln Ala Leu Ala Val Lys Leu Tyr Glu
560 565 570
CAA GCG GCT GCA GCA CAA CAA GCA GCT CAA GGG GCT GAA GGT GCA CAA 2017
Gln Ala Ala Ala Ala Gln Gln Ala Ala Gln Gly Ala Glu Gly Ala Gln
575 580 585 590
TCA GCT GAT TCA TCA AGC AAG GGT GAT GAT GTT GTA GAT GGC GAA TTC 2065
Ser Ala Asp Ser Ser Ser Lys Gly Asp Asp Val Val Asp Gly Glu Phe
595 600 605
50 ACT GAG AAA TAATTATTAA TATTGTTCAG ATTCATTTGA ATATAAGCAT 2114
m r Glu Lys
610
GAAAACTATA CTAGCATAGT AAAGTTCTTC GTGATAGGGA TTGCTCAATA ATCTAGATAA 2174
GTTTCAGATT ACATAAGCTA ATTTCGCTAT CACTAAATAA AAACATATTA ATAATAAATA 2234
GGCGGGGCGC CTCGCTCCGT ~ llATT AAGTGTCATA TATATGTTAA CTATTTAGAG 2294
60 CTGTAACTGG GCAAGAATAA TTGTTAATCT CTTCAAGTGT AGTATATGAA CAAAATATAA 2354
AGGATTAGAT AATGAACAAT ACAGAATTTT ATGATCGTCT TGGCGTTTCA AAAGATGCTT 2414
CTCAGGACGA AATAAAAAAA GCTT 2438
(2~ INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 609 amino acids
(B) TYPE: amino acid
107
CA 022240l~ l997-l2-08
W O 9G/4~328 PCT/CA96/00322
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met Ser Lys Ile Ile Gly Ile Asp Leu Gly Thr mr Asn Ser Ala Val
1 5 10 15
Ala Val Leu Glu Gly Thr Glu Ser Lys Ile Ile Ala Asn Pro Glu Gly
20 25 30
Asn Arg m r mr Pro Ser Val Val Ser Phe Lys Asn Gly Glu Ile Ile
35 40 45
Val Gly Asp Ala Ala Lys Arg Gln Ala Val mr Asn Pro Asp m r Val
50 55 60
Ile Ser Ile Lys Ser Lys Met Gly m r Ser Glu Lys Val Ser Ala Asn
65 70 75 80
Gly Lys Glu ~yr mr Pro Gln Glu Ile Ser Ala Met Ile Leu Gln Tyr
Leu Lys Gly Tyr Ala Glu Asp Tyr Leu Gly Glu Lys Val Glu Lys Ala
100 105 110
Val Ile mr Val Pro Ala Tyr Phe Asn Asp Ala Gln Arg Gln Ala mr
115 120 125
Lys Asp Ala Gly Lys Ile Ala Gly Leu Glu Val Glu Arg Ile Val Asn
130 135 140
Glu Pro m r Ala Ala Ala Leu Ala Tyr Gly Met Asp Lys mr Asp Lys
145 150 155 160
Asp Glu Lys Ile Leu Val Phe Asp Leu Gly Gly Gly mr Phe Asp Val
165 170 175
~0 Ser Ile Leu Glu Leu Gly Asp Gly Val Phe Asp Val Leu Ala m r Ala
180 - 185 190
Gly Asp Asn Lys Leu Gly Gly Asp Asp Phe Asp Gln Lys Ile Ile Asp
195 200 205
Phe Leu Val Glu Glu Phe Lys Lys Glu Asn Gly Ile Asp Leu Ser Gln
210 215 220
Asp Lys Met Ala Leu Gln Arg Leu Lys Asp Ala Ala Glu Lys Ala Lys
225 230 235 240
Lys Asp Leu Ser Gly Val Thr Gln Thr Gln Ile Ser Leu Pro Phe Ile
245 250 255
Thr Ala Gly Ser Ala Gly Pro Leu His Leu Glu Met Ser Leu Ser Arg
260 265 270
Ala Lys Phe Asp Asp Leu Thr Arg Asp Leu Val Glu Arg mr Lys mr
275 280 285
Pro Val Arg Gln Ala Leu Ser Asp Ala Gly Leu Ser Leu Ser Glu Ile
290 295 300
Asp Glu Val Ile Leu Val Gly Gly Ser mr Arg Ile Pro Ala Val Val
305 310 315 320
Glu Ala Val Lys Ala Glu mr Gly Lys Glu Pro Asn Lys Ser Val Asn
325 330 335
Pro Asp Glu Val Val Ala Met Gly Ala Ala Ile Gln Gly Gly Val Ile
340 345 350
108
CA 022240l~ l997-l2-08
W O 96/40928 PCT/CA96/00322
mr Gly Asp Val Lys Asp Val Val Leu Leu Asp Val Thr Pro Leu Ser
355 360 365
Leu Gly Ile Glu mr Met Gly Gly Val Phe mr Lys Leu Ile Asp Arg
370 375 380
Asn mr Thr Ile Pro mr Ser Lys Ser Gln Val Phe Ser mr Ala Ala
385 390 395 400
Asp Asn Gln Pro Ala Val Asp Ile His Val Leu Gln Gly Glu Arg Pro
405 410 415
Met Ala Ala Asp Asn Lys m r Leu Gly Arg Phe Gln Leu mr Asp Ile
420 425 430
Pro Ala Ala Pro Arg Gly Ile Pro Gln Ile Glu Val Thr Phe Asp Ile
435 440 445
Asp Lys Asn Gly Ile Val Ser Val Lys Ala Lys Asp Leu Gly Thr Gln
450 455 460
Lys Glu Gln His Ile Val Ile Gln Ser Asn Ser Gly Leu mr Asp Glu
465 470 475 480
Glu Ile Asp Lys Met Met Lys Asp Ala Glu Ala Asn Ala Glu Ala Asp
485 490 495
Ala Lys Arg Lys Glu Glu Val Asp Leu Lys Asn Glu Val Asp Gln Ala
500 505 510
Ile Phe Ala Thr Glu Lys mr Ile Lys Glu Thr G1U Gly Lys Gly Phe
515 520 525
Asp mr Glu Arg Asp Ala Ala Gln Ser Ala Leu Asp Glu Leu Lys Lys
530 535 540
Ala Gln Glu Ser Gly Asn Leu Asp Asp Met Lys Ala Lys Leu Glu Ala
545 550 555 560
Leu Asn Glu Lys Ala Gln Ala Leu Ala Val Lys Leu Tyr Glu Gln Ala
565 570 575
Ala Ala Ala Gln Gln Ala Ala Gln Gly Ala Glu Gly Ala Gln Ser Ala
580 585 590
Asp Ser Ser Ser Lys Gly Asp Asp Val Val Asp Gly Glu Phe m r Glu
595 600 605
Lys
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Arg Ile Pro Ala Val Val Glu Ala Val Lys Ala Glu Thr Gly Lys Glu
1 5 10 15
Pro Asn Lys
109
CA 022240l~ l997-l2-08
WO 96/40928 PCT/CA96/00322
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Gln Thr Ile Val Ile Gln Ser Asn Ser Gly Leu Thr Asp Glu Glu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOVRCE:
(A) OR~ANT~M: Streptococcus pneumoniae
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..456
(D) OTHER INFORMATION: /product= "C-terminal 151-residue
fragment (C-151) of HSP72"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
ATG AAG GCC AAA GAC CTT GGA ACT CAA AAA GAA CAA ACT ATT GTC ATC 48
Met Lys Ala Lys Asp Leu Gly mr Gln Lys Glu Gln Thr Ile Val Ile
1 5 10 15
CAA TCG AAC TCA GGT TTG ACT GAC GAA GAA ATC GAC CGC ATG ATG AAA 96
Gln Ser Asn Ser Gly Leu Thr Asp Glu Glu Ile Asp Arg Met Met Lys
20 25 30
GAT GCA GAA GCA AAC GCT GAA TCC GAT AAG AAA CGT AAA GAA GAA GTA 144
Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val
35 40 45
GAC CTT CGT AAT GAA GTG GAC CAA GCA ATC TTT GCG ACT GAA AAG ACA 192
Asp Leu Arg Asn Glu Val Asp Gln Ala Ile Phe Ala mr Glu Lys mr
50 55 60
60 ATC AAG GAA ACT GAA GGT AAA GGC TTC GAC GCA GAA CGT GAC GCT GCC 240
Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala
65 70 75 80
CAA GCT GCC CTT GAT GAC CTT AAG AAA GCT CAA GAA GAC AAC AAC TTG 288
65 Gln Ala Ala Leu Asp Asp Leu Lys Lys Ala Gln Glu Asp Asn Asn Leu
85 90 95
GAC GAC ATG AAA GCA AAA CTT GAA GCA TTG AAC GAA AAA GCT CAA GGA 336
Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gln Gly
100 105 110
110
CA 022240l~ l997-l2-08
WO 9~/lD328 PCT/CA9G/00
CTT GCT GTT AAA CTC TAC GAA CAA GCC GCA GCA GCG CAA CAA GCT CAA 384
Leu Ala Val Lys Leu Tyr Glu Gln Ala Ala Ala Ala Gln Gln Ala Gln
115 120 125
5 GAA GGA GCA GAA GGC GCA CAA GCA ACA GGA AAC GCA GGC GAT GAC GTC 432
Glu Gly Ala Glu Gly Ala Gln Ala Thr Gly Asn Ala Gly Asp Asp Val
130 135 140
GTA GAC GGA GAG TTT ACG GAA AAG TAAG 460
10 Val Asp Gly Glu Phe Thr Glu Lys
1g5 150
-
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 152 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Met Lys Ala Lys Asp Leu Gly Thr Gln Lys Glu Gln mr Ile Val Ile
1 5 10 15
Gln Ser Asn Ser Gly Leu Thr Asp Glu Glu Ile Asp Arg Met Met Lys
20 25 30
Asp Ala Glu Ala Asn Ala Glu Ser Asp Lys Lys Arg Lys Glu Glu Val
35 40 45
Asp Leu Arg Asn Glu Val Asp Gln Ala Ile Phe Ala Thr Glu Lys Thr
50 55 60
Ile Lys Glu Thr Glu Gly Lys Gly Phe Asp Ala Glu Arg Asp Ala Ala
Gln Ala Ala Leu Asp Asp Leu Lys Lys Ala Gln Glu Asp Asn Asn Leu
85 90 95
Asp Asp Met Lys Ala Lys Leu Glu Ala Leu Asn Glu Lys Ala Gln Gly
l00 105 110
Leu Ala Val Lys Leu Tyr Glu Gln Ala Ala Ala Ala Gln Gln Ala Gln
115 120 125
Glu Gly Ala Glu Gly Ala Gln Ala Thr Gly Asn Ala Gly Asp Asp Val
130 135 140
Val Asp Gly Glu Phe Thr Glu Lys
145 150
111
