Note: Descriptions are shown in the official language in which they were submitted.
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PEPTIDES
FIELD OF THE INVENTION
The invention relates to peptides and their use in vaccines against
streptococcal
infection.
BACKGROUND TO THE INVENTION
There are many Streptococcus species, responsible for causing disease states
in
humans and other animals.
Infections caused by streptococci can vary from uncomplicated suppurative
diseases like pharyngitis and skin infections to severe diseases such as
sepsis and toxic
shock syndrome. Rheumatic fever and glomerulonephritis are serious
nonsuppurative
sequelae following acute S. pyogenes infections. Other diseases caused by
streptococci
include scarlet fever, impetigo, erypsipelas, myositis, necrotizing fasciitis,
septic
arthritis, cellulitis, colonization and destruction of heart valves
(endocarditis), neonatal
infections, conjunctivitis, sinusitis, perotonitis, omphalitis, meningitis,
abortion and
chorioamnionitis, post-partum sepsis, upper respiratory tract disease in
humans,
pneumonia, otitis media, wound infections, abscesses, empyema, mastitis,
urinary tract
infections, osteomyelitis, strangles in horses, dental pathogens in humans,
renal
infections in humans.
Many bacterial infections such as acute pharyngitis are treatable with
antibiotics.
However, in certain areas of the world, antibiotic resistance, particularly to
erythromycin, is becoming more common. Recently, there has been an increase in
the
incidence of acute rheumatic fever linked to streptococcal pharyngitis. Acute
rheumatic
fever has been associated with at least six different M-types of S. pyogenes.
In addition,
the number of cases of severe streptococcal infection has been rising, leading
to
bacteraemia and sepsis, necrotising fasciitis and myositis, puerperal sepsis
and
streptococcal toxic shock syndrome (STSS).
Prompt and aggressive treatment is essential in severe streptococcal
infection.
Presently, this treatment may encompass surgical debridement, antibiotic
treatment,
intravenous fluid, oxygen or ventilator support, dialysis, vasoconstrictives
to elevate
blood pressure, steroids and anti-thrombolytics. Even so, severe streptococcal
infection
can be fatal.
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Present vaccine strategies for S. pyogenes have been focused on the outer
membrane M-proteins. These proteins are considered to be key virulence factors
in
view of their ability to confer resistance to phagocytosis on the bacteria.
Furthermore,
these proteins provoke harmful host immune responses through their
superantigenicity
S and their ability to induce cross-reactive antibody responses in humans.
However, the
variability and number of M-proteins and their ability to produce cross-
reactive
antibody responses have led to problems in the formulation of an effective
vaccine
against various serotypes of S. pyogenes.
There is a continuing need to develop additional treatment strategies for
severe
streptococcal infection and create new vaccine formulations which may be used
to
combat not only Streptococcus pyogenes infection but also infection caused by
other
streptococcus species.
SUMMARY OF THE INVENTION
The inventors have now established a link between clinical infection and
virulence of S. pyogenes and the ability of S. pyogenes to aggregate. The
ability to
aggregate has been linked to an immunogenic region of protein H of S.
pyogenes.
Amino acid sequences corresponding to the amino acid sequence of this region
can be
found in the outer membrane proteins not only of a wide variety of S. pyogenes
serotypes but also of many other streptococcal strains.
Accordingly, the present invention provides a polypeptide of up to 50 amino
acids in length, suitable for use as a vaccine against streptococcal
infection, comprising:
(a) the amino acids 150-168 of protein H of S. pyogenes having the
sequence QKQQQLETEKQISEASRKS;
(b) an amino acid sequence of an outer membrane protein of a streptococcal
strain corresponding to sequence (a);
(c) a fragment of sequence (a} or sequence (b) of 6 or more amino acids; or
(d) a sequence comprising the sequence (a), (b} or (c) modified by deletion,
insertion, substitution or rearrangement.
The present invention also provides a chimeric protein comprising a first
polypeptide having the sequence (a), (b), (c), (d) above and a second
polypeptide which
is not naturally contiguous to the first polypeptide.
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In a further aspect, the present invention provides a polynucleotide encoding
a
polypeptide of the present invention.
The present invention also relates to expression vectors comprising a
polynucleotide in the invention and regulatory sequences operably linked to
said
polynucleotide for expression of a polypeptide encoded by the polynucleotides
and to
host cells transfected with such expression vectors.
The present invention also provides a pharmaceutical composition comprising a
polypeptide of the invention together with a pharmaceutical composition
comprising a
polypeptide of the invention together with a pharmaceutically acceptable
carrier and a
vaccine composition comprising a polypeptide of the invention, an adjuvant in
a
pharmaceutically acceptable carrier or a polynucleotide of the invention
together with a
pharmaceutically acceptable carrier for the polynucleotide.
The invention also relates to an antibody against a polypeptide according to
the
invention.
1 S In a further aspect, the invention provides a polypeptide of up to 50
amino acids
in length comprising:
(a) the amino acids 150-168 of protein H of S pyogenes having the sequence
QKQQQLETEKQISEASRKS;
(b) an amino acid sequence of an outer membrane protein of a streptococcal
strain corresponding to sequence (a), said sequence having the ability to
interfere with
aggregation or adhesion of said streptococcal strain;
{c} a fragment of sequence (a) or (b) of six or more amino acids, which
retains the ability to interfere with streptococcal aggregation or adhesion;
(d) a sequence comprising a sequence (a), (b) or (c) modified by deletion,
insertion, substitution or rean~angement, the sequence retaining the ability
to interfere
with streptococcal aggregation or adherence.
DESCRIPTION OF THE FIGURES
Figure 1. Sedimentation analysis of AP 1 bacteria
APl bacteria were grown at 37°C over night in TH (O), TH
containing 10%
human plasma (~) or TH containing 1.4 mg/ml human IgG (o). Bacteria were
resuspended and left to settle at room temperature. The sedimentation rate was
obtained
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by plotting values of optical density at 620 nm against various time points.
Figure 2. Solubilization of API surface proteins
{A) API bacteria were incubated with PBS (O), papain {~), streptococcal
cysteine proteinase (e) or CNBr (1). Following digestion bacteria were
centrifuged,
washed, resuspended and analyzed for sedimentation.
(B) Bacterial cells as in (A) above, at a concentration of 2x109 bacteria/ml,
were serially diluted and tested for binding of'ZSI-labelled IgG.
(C) SDS-PAGE analysis of CNBr-solubilized material from AP1 bacteria.
The fragments generated by CNBr are indicated in the schematic representations
of
protein H and M1 protein. The NHZ-terminal signal sequences (Ss) are
indicated, and
the proteins are associated with the bacterial cell wall through the COOH-
terminal D
domains. The sequences in the Ss, C and D domains show a high degree of
homology.
IgGFc-binding is located in the A-B domains of protein H and in the S domain
of M 1
protein. Numbers in the figure refer to amino acid residue positions. The
protein H
used herein is a truncated form (42-349) lacking the 27 COOH-terminal amino
acid
residues associated with the bacterial cell wall, whereas the M1 protein used
covers
residues 42-484
Figure 3. Protein H binds to AP I bacteria and to purif ed protein H
(A) AP 1 and AP6 bacteria, 2x 1 O9 cells/ml, were serially diluted and tested
for binding of'zsI-labelled protein H (~) or M1 protein (o). 'Z'I-labelled IgG
(O} was
used as a positive control.
(B) Various amounts of protein H and M1 protein were applied to PVDF
filters. Filters were incubated with'~'I-labelled protein H or M1 protein
(2x105 cpm/ml)
for 3 h and autoradiographed for 3 days.
Figure 4. Mapping of the self associating region in protein H
(A) Binding of'~'I-labelled protein H (O), fragment AB (e) or fragment A of
protein H (~) to API bacteria.
(B) The binding of'~'I-labelled protein H to API bacteria (2x109 cells/ml)
was inhibited with various amounts of unlabelled protein H (O) or with
fragments AB
(e) and A (O) of protein H.
(C) Schematic figure of protein H. The various protein H fragments are
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indicated below the figure together with the sequence for the aggregative
protein H
peptide (APP). Numbers refer to amino acid residues.
Figure 5. Radiolabelled protein H has affinity for protein H-Sepharose.
'~SI-labelled protein H ( 1 O6 cpm) was applied to a protein H-Sepharose
column.
'The column was extensively washed with PBSAT, eluted with human IgG (5 mg/ml)
and, after a second wash with PBSAT, finally eluted with 3 M KSCN. The
radioactivity of the fractions were plotted against the volume.
Figure 6. APP is cross-linked to protein H.
Protein H (300 pmol) was crosslinked with DSS in the presence of'2sI-labelled
APP (approximately 450 pmol). Samples were analyzed by SDS-PAGE, one gel was
stained and one gel was dried and autoradiographed. Lane 1: protein H without
crosslinker; Lane 2: protein H with crosslinker; Lane 3: protein H crosslinked
in the
presence of "SI-APP; Lane 4: protein H crosslinked in the presence of'ZSI-APP
and
excess amount of unlabelled APP (450 nmol); Lane 5: protein H crosslinked in
the
presence of'25I-labelled B1 domain of protein L.
Figure 7. APP-related sequences are found in several M and M-like
proteins
Alignment of sequences related to APP found in the data base. Identity to APP
at the amino acid and the nucleotide level is given together with the M
serotype from
which the protein originates. Indicated is also the position of the APP-
related sequence
in the various proteins M49, Sir22, ML2.1, M1, M5, MI2, Arp4 and M6.
Figure 8. Analysis of S. pyogenes sedimentation and adherence
(A) Wild-type AP1, mutant lacking protein H (BM27.6), mutant lacking both
protein H and M1 protein (BMJ71), wild-type M6 strain (JRS4) (which does not
express protein H), and mutant lacking M6 protein (JRS145), were tested for
aggregation. The sedimentation was measured as decrease in optical density at
620 nm
after 1 h. Mean values t SD are given.
(B) Wild-type strains and mutants were tested for their adherence to human
epithelial cells. One hundred per cent adherence correspond to 1.49 x 1 O6 t
0.5 AP 1
bacteria/ epithelial cell layer or 0.27 x 106 t 0.04 x 106 JRS4
bacteria/epithelial cell
layer. The AP1 mutants are compared to API, the JRS145 mutant is compared to
JRS4.
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Values are mean f SD.
Figure 9. Results of ELISA using antiserum prepared by injecting APP
conjugated to KLH together with adjuvant into rabbits.
Figure 10. Nucleotide sequence of APP-peptide (33-mer) and homology to
other M proteins.
Figure 11. Alignment of APP-related sequences using APP-peptide (33-
mer)
Figure 12. Effect of pre-adsorption with peptides on detection of antibodies
induced by Spy-PH-YQE33 peptide. Sheep were primed and boosted with Protein H-
derived peptide Spy-PH-YQE33 conjugated to KLH carrier protein. Post-immune
sera
(I:10000 dilution) were incubated (60 min, 37°C} with 100pg/ml
irrelevant peptide
(Spn-LP-KEY17) or 100 ~g/ml relevant peptide (Spy-PH-QKQ19). Control samples
were incubated in the absence of peptide. Antibodies against Spy-PH-YQE33
epitopes
were then determined by ELISA. Results are expressed as optional density (OD)
measured at 450 nm (mean, n=4 wells).
DETAILED DESCRIPTION OF THE INVENTION
A polypeptide of the invention is one of up to 50 amino acids in length,
suitable
for use as a vaccine against a streptococcal infection. The polypeptide may
consist
essentially of (a) the amino acids 150-168 of Protein H of S pyogenes having
the
sequence QKQQQLETEKQISEASRKS. A polypeptide of the invention may also
comprise (b) an amino acid sequence of an outer membrane protein of a
streptococcal
strain corresponding to sequence (a), a fragment of either sequence (a) or (b)
of six or
more amino acids in length; or (d) a sequence comprising a sequence (a), (b)
or (c)
modified by deletion, insertion, substitution or rearrangement.
Streptococci have been classified into various groups under the Lancefield
classification. This classification is based on group-specific antigens in the
cell wall
polysaccharide. Key Lancefield groups are Group A comprising Streptococcus
pyogenes, Group B comprising Streptococcus agalactiae, Group C and G including
Streptococcus equi and Streptococcus equisimilis, Group D including
Enterococcus
faecalis and Group Viridian including Streptococcus mutans and Streptococcus
sanguis.
A non-groupable species is Streptococcus pneumoniae.
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Within each group, bacteria may be further classified according to their
semtype. For Group A Streptococcus, serotyping is carried out by reference to
additional surface antigens, the M proteins. These proteins divide the group A
Streptococcus into more than 80 serotypes. Some M serotypes are associated
with
increased virulence and pathogenicity. In particular, MI, 2, 3, 4, 5, 6, 7, 8,
10, 12, 14,
16, 17, 18, 19, 22, 24, 49, 55, 57 and 60 serotypes are associated with more
serious
infection in humans.
S.pyogenes can express a number of other outer membrane proteins including
immunoglobulin binding proteins. Protein H is an example of an outer membrane
protein expressed by M1 serotypes ofS. pyogenes. Protein H, along with other
IgG
binding proteins expressed by S. pyogenes, is structurally related to M
proteins which
may also bind IgG, and now considered to be part of the same family. Nielson
et al
Biochemistry 1995 Vol. 34 No. 41 13688-13698 describes the structure of
protein H
and M 1 protein of AP 1 strain of S pyogenes and sets out the full sequence
and domain
structure of these proteins.
Some streptococcal strains have been shown to aggregate, with a greater
ability
to aggregate being associated with an increase in the virulence of the
bacteria. The
examples set out below demonstrate the role of protein H in aggregation,
through
protein H - protein H interactions. This aggregative activity has been mapped
to the
sequence (a) above comprising residues 1 SO-168 of protein H and referred to
herein as
APP. APP is suitable for use as a vaccine against strepococci in view of its
role in the
aggregative properties associated with virulence of the bacteria. Similarly
the sequence
of residues 145-177 of protein H encompassing the sequence (a), namely
sequence
YQEQLQKQQQLETEKQISEASRKSLSRDLEASR is of particular interest, and is
referred to herein as a 33-mer.
Sequence comparison studies with outer membrane proteins of a broad range of
S. pyogenes serotypes show that there are related sequences to be found in
many other
S pyogenes strains. Furthermore, similar sequences were observed in non-group
A
streptococci. Accordingly the invention relates to the sequence (b} comprising
a
corresponding sequence of an outer membrane protein of a streptococcal strain.
This
includes those proteins expressed on the surface of Groups A, B, C, D, G and
Viridian
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streptococcus. In a preferred embodiment of the invention, the sequence (b) is
derived
from an outer membrane protein of Group A Streptococcus, S pyogenes. In a
preferred
embodiment, the amino acid sequence is derived from one of the more virulent
serotypes of S.pyogenes namely M1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 17,
18, 19, 22, 24,
49, 55, 57 and 60. The outer membrane protein may for example comprise one of
the
M proteins of Group A Streptococcus. Examples of other proteins include
protein Arp
and protein Sir22 expressed by some streptococcal strains which are associated
with
binding of plasma proteins. The relevant outer membrane proteins and sequences
can
be elucidated by using APP or the 33-mer and establishing the best possible
alignment
when looking at the sequences of outer membrane proteins of streptococci.
Examples of these sequences are given in Figures 7 and 11. Examples of these
sequences are set out below:
QKQQQLEKEKQISEASRKS, IEKAKLEEEKQISDASRQS,
AEQQKLEEQNKISEA SRKG, GQIKQLEEQNKISEASRKG,
AEHQKLKEEKQISDASRQG, AELDKVKEEKQISDASRQG,
YQEQLQKQQQLEKEKQISEASRKSLSRDLEASR,
YQEQLQKQQQLEKEKQISEASRKSLRRDLEASR,
YKEQLHKQQQLETEKQISEASRKSLSRDLEASR,
KKELEAEHQKLKEEKQISDASRQGLSRDLEASR,
KEQLTIEKAKLEEEKQISDASRQSLRRDLDASR,
KKQLEAEQQKLEEQNKISEASRKGLRRDLDASR,
LAEKDGQIKQLEEQNKISEASRKGTARDLEAVR,
KKQLEAEHQKLEEQNKISEASRQSLRRDLDASR, or
LANLTAELDKVKEEKQISDASRQGLRRDLDASR.
As can be seen from the examples given in Figure 7, some of the more virulent
strains of streptococci identified above have sequences which correspond to
sequence
(a) which emphasises the suitability of the sequences as vaccines. Other
corresponding
sequences may include sequences within the C2 and C3 repeat domains of protein
H
having a high degree of homology with APP.
Preferably, a polypeptide of sequence (b) or (d) of the invention will be at
least
40% homologous to the sequence (a) or the 33-mer identified above over its
entire
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length, based on amino acid identity. More preferably, the sequence (b) or (d)
of the
invention is at least 50% and more preferably at least 60%, 70% or 80%
homologous to
the sequence (a). More preferably, the sequence (b) or (d) comprises the
sequence that
is at least 90% and more preferably at least 95%, 97% or 98% homologous to the
sequence (a) or the 33-mer identified above.
As an alternative approach, the presence of a corresponding sequence may be
established through hybridisation using the nucleotide sequences set out in
Figure 10, as
discussed in more detail below.
The invention also relates to fragments of the sequences (a) and (b) of six or
more amino acids in length, preferably 8 or more amino acids or 10 or more
amino
acids in length. This fragments may be up to 10 amino acids in length
preferably up to
12, 16 or 18 amino acids in length.
Amino acid substitutions, modifications deletions or rearrangements may be
made to the sequences (a), (b) or (c) for example at from 1, 2, 3, 4, 5 up to
9 amino
acids of sequence (a) preferably, at from 1, 2 or 3 amino acids. Conservative
substitutions may be made, for example according to the following Table. Amino
acids
in the same block in the second column and preferably in the same line in the
third
column may be substituted for each other.
ALIPHATIC Non-polar G A P
ILV
Polar-uncharged C S T M
NQ
Polar-charged D E
KR
AROMATIC H F W Y
Other preferred substitutions can be established for example by comparison of
the conserved sequences as defined above. Additionally, other substitutions
seen in
more than one of the conserved sequences set out above may be considered to be
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preferred.
Except where specified to the contrary, the polypeptide sequences described
herein are shown in the conventional one-letter code and in the N-terminal to
C-
terminal orientation. The amino acid polypeptides of the invention may also be
S modified to include non naturally-occurring amino acids or to increase the
stability of
the compound in vivo. When the compounds are produced by synthetic means, such
amino acids may be introduced during production. The compound may also be
modified following either synthetic or recombinant production.
Polypeptides of the invention may also be made synthetically using D-amino
acids. In such cases, the amino acids will be linked in a reverse sequence in
the C to N
orientation. This is conventional in the art for producing such peptides.
A number of side-chain modifications for amino acids are known in the art and
may be made with the side-chains of polypeptides of the present invention.
Such
modifications include for example, modifications of the amino acid group by
reductive
alkylation by reaction with an aldehyde followed by reduction with NaBH"
amidination
with methyl acetimidate, for acylation with acetic anhydride. The carboxy
terminus and
any other carboxy side-chains may be blocked in the form of an ester group,
e.g. a C,_6
alkyl ester.
The above examples and modifications to amino acids are not exhaustive. Those
of skill in the art may modify amino acid side-chains where desired using
chemistry
known per se in the art.
The polypeptide of the invention may consist essentiahy of the sequences (a),
(b), (c) or (d).
In general, the polypeptides of the present invention are selected or modified
to
maintain their suitability as vaccine compositions. As will be well
appreciated by those
skilled in the art, a polypeptide suitable for use as a vaccine composition in
accordance
with the invention is a polypeptide which is able to generate a protective
immune
response against a streptococcal infection. The polypeptides of the present
invention
can typically be selected by the ability to interfere with bacterial
aggregation or
adhesion to epithelial surfaces. In the context of aggregation and adhesion,
the
sequence (a) may be used as an antiaggregative agent. The corresponding
sequence (b}
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may be used to inhibit aggregation of the streptococcal strain from which the
sequence
derives. In the alternative, the sequences may interfere generally with
streptococcal
aggregation and/or adhesion of the bacteria to epithelial cells. In this
context,
aggregation of the bacteria means that the aggregation seen in streptococci as
exemplified in Example l and measured in absorbence studies. Preferably, the
peptide
will reduce aggregation by 30% more preferably by at least 50%. In addition or
in the
alternative, the polypeptide may interfere with adhesion of the bacteria to
epithelial
cells as exemplified in Example 4.
Fragments of sequences (a) and (b) may also be selected on the basis of their
ability to interfere with bacterial aggregation or adhesion. Modifications,
substitutions,
deletions or rearrangements as outlined above may also be selected to maintain
the
ability of the polypeptide to interfere with bacterial aggregation.
Thus, in an alternative aspect of the invention, we provide a polypeptide of
up to
50 amino acids in length comprising:
(a) the amino acids 150-168 of protein H of S. pyogenes having the
sequence QKQQQLETEKQISEASRKS;
(b) a corresponding sequence to sequence (a) of an outer membrane protein of a
streptococcus strain, said sequence having the ability to interfere with
aggregation or
adhesion of said streptococcal strain;
(c) a fragment of sequence (a) or (b) of 6 or more amino acids, which retains
the
ability to interfere with streptococcal aggregation or adhesion; and
(d) a sequence comprising sequences {a), (b) or (c) modified by deletion,
insertion, substitution or rearrangement, the sequence retaining the ability
to interfere
with streptococcal aggregation or adherence.
The polypeptides of the present application are thus also proposed for use to
inhibit bacterial aggregation or adherence to epithelial cells and thus may be
used to
reduce virulence of the bacteria.
Polypeptides of the invention may be in a substantially isolated form. It will
be
understood that the polypeptide may be mixed with carriers or diluents which
will not
interfere with the intended purpose of the polypeptide and still be regarded
as
substantially isolated. A polypeptide of the invention may also be in a
substantially
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purified form, in which case the polypeptide of the invention will generally
comprise
90%, e.g. more than 95% or more than 99% by weight of the polypeptide in the
preparation produced by the isolation or purification procedure.
Polypeptides of the invention may be made synthetically or recombinantly using
techniques which are widely available in the art. Synthetic production
generally
involves stepwise addition of individual amino acid residues to a reaction
vessel in
which a polypeptide of a desired sequence is being made. Examples of
recombinant
techniques are described below.
The polypeptides of the present invention are up to 50 amino acids in length.
More preferably, the polypeptide is of no more than 40 to 45 amino acids in
length, and
most preferably up to 30, 20 or 15 amino acids in length.
The invention also relates to chimeric proteins comprising a first polypeptide
in
accordance with the invention and a second polypeptide which is not naturally
contiguous to said first polypeptide. Thus, the polypeptide of the invention
may
comprise repeats of the sequences (a), (b), (c) or (d) or combinations
thereof. The
polypeptide may be cyclized. Alternatively the polypeptide may comprise
separate
repeat regions linked through a linker sequence such as a poiylysine bridge.
The
peptides may also be formulated as fusion proteins with a Garner, particularly
when
used as a vaccine with carriers such as keyhole limpet hemacyanin, diptheria
toxoid,
tetanus toxoid.
The polypeptides of the invention may be formulated into pharmaceutical
compositions. The compositions comprise the polypeptides together with a
pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable
carriers or
diluents include those used in formulations suitable for oral, topical or
parenteral (e.g.
intramuscular or intravenous) administration. The formulations may
conveniently be
presented in unit dosage form and may be prepared by any of the methods well-
known
in the art of pharmacy.
For example, formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may contain anti-
oxidants,
buffers, bacteriostats and solutes which render the formulation isotonic with
the blood
of the intended recipient; and aqueous and non-aqueous sterile suspensions
which rnay
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include suspending agents and thickening agents, and liposomes or other micro-
particulate systems which are designed to target the polypeptide within the
body.
The peptides of the invention may also be used to generate an immune response
to provide protection against later infections by Streptococcus.
Means of presentation of the peptide immunogen(s} include, but are not
restricted to: free peptides, as peptides conjugated to suitable carrier
proteins such as
keyhole limpet haemocyanin, bovine serum albumin, ovalbumin, inactived
bacterial
toxins such as tetanus and diptheria toxoids, bacterial or mammalian heat
shock
proteins; as fusion or chimeric proteins, expressed by recombinant bacteria or
viruses
comprising the peptide of choice and one or more peptides or proteins
containing T- or
B-cell epitopes, or specific binding domains for molecules present on the
lymphocyte
surface; as peptides or fusion proteins expressed on the surface of, or
secreted by, live
bacterial or viral vectors.
Peptides may also be entrapped within, or presented on the surface of,
liposomes; be incorporated into biodegradable microspheres formulated from
poly
(D,L) lactic co-glycolic acid or other polymers, or presented on the surface
of micelles
formulated from saponins such as Quil A, cholesterol and phospholipid suitable
detergent.
Multiple copies of the peptide may be administered to enhance the immune
response as synthetic polypeptides containing between 2 and 20 copies of the
peptide
vaccinogen and/or single or multiple copies of additional T- or B-cell
epitopes.
Alternatively, between 2 and 20 copies of the peptide vaccinogen andlor
multiple copies
of additional T- and B-cell epitopes may be presented as chemically
synthesised
branched oligomers formed around a core matrix of lysine or another amino
acid.
Peptides used for vaccination may be chemically modified to facilitate
conjugation to protein carriers and/or to increase their immunogenicity.
Suitable
modifications include, but are not confined to, the addition of cysteine
residues at each
terminus to permit polymerisation via disulphide bond formation. Peptides may
be
modified to increase their immunogenicity by conjugation to lipids such as a-
aminohexadecanoic acid at the N-terminus.
The preparation of vaccines which contain an immunogenic polypeptide(s) as
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active ingredient(s), is known to one skilled in the art. Typically, such
vaccines are
prepared as injectables, either as liquid solutions or suspensions; solid
forms suitable for
solution in, or suspension in, liquid prior to injection may also be prepared.
The
preparation may also be emulsified, or the protein encapsulated in liposomes.
The
active immunogenic ingredients are often mixed with excipients which are
pharmaceutically acceptable and compatible with the active ingredient.
Suitable
excipients are, for example, water, saline, dextrose, glycerol, ethanol, or
the like in
combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering agents and/or
adjuvants
which enhance the effectiveness of the vaccines. Examples of adjuvants which
may be
effective include but are not limited to: alum or other aluminum salts,
calcium salts;
water-in-oil emulsions containing mineral oil, squalene or squalane; oil-in-
water
emulsions of squaiene, squalene or oils in combination with surfactants such
as Tween
80 or Span 85. These emulsions may also include components such as N-acetyl-
muramyl-L-alanyl-D-isoglutamine (MDP), or derivatives of synthetic
sulpholippolysaccharides, or non-ionic block copolymers; compositions
containing
saponins such as Quil A and/or monophosphoryl lipid A (MPL); carbohydrate
polymers
such as mannan or beta 1-3 glucose; natural or recombinant bacterial toxins
such as
cholera toxin or Escherichia coli labile toxin; natural or recombinant
cytokines such as
human interleukin-1 (IL-1), IL-2, IL-4 or IL-12. The effectiveness of an
adjuvant may
be determined by measuring the amount of antibodies directed against an
immunogenic
peptide resulting from administration of this peptide in vaccines which are
also
comprised of the various adjuvants.
The vaccines are conventionally administered parenterally by injection, for
example, either subcutaneously or intramuscularly. Additional formulations
which are
suitable for other modes of administration include suppositories and, in some
cases, oral
formulations or topical application to the nasal, rectal or vaginal mucosa, or
through
inhalation of a liquid or powder formulation. For suppositories, traditional
binders and
carriers may include for example, polyalkylene glycol triglycerides. Oral
formulations
include such normally employed excipients as, for example, pharmaceutical
grades of
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mannitol, lactose, starch, magnesium stearate, sodium saccarine, cellulose,
magnesium
carbonate and the like. These compositions take the form of solutions,
suspensions,
tablets, pills, capsules, sustained release formulations or powders. Where the
vaccine
composition is lyophilised, the lyophilised material may be reconstituted
prior to
administration, e.g. as a suspension.
Capsules, tablets and pills for oral administration to a patient may be
provided
with an enteric coating comprising, for example, Eudragit "S", Eudragit "L",
cellulose
acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
The polypeptides of the invention may be formulated into the vaccines as
natural or salt form. Pharmaceutically acceptable salts include the acid
addition salts
(formed with free amino acid groups of the peptide) and which are formed with
inorganic acids such as, for example, hydrochloric or phosphoric acids, or
such organic
acids such as acetic, oxalic, tartaric and malefic. Salt forms for the free
carboxyl groups
may also be derived from inorganic bases such as, for example, sodium,
potassium,
ammonium, calcium, or ferric hydroxide and such organic bases as
isopropylamine and
trimethylamine, 2-ethylamino ethanol, histidine and procaine.
Peptide vaccinogens may be given as single or repeat doses, with each does
consisting of between 2 and 5000 micrograms, preferably between 10 and 1000
micrograms of the peptide. Preferably two or more repeat doses will be given.
When formulating vaccine compositions, additional antigenic components
derived from streptococcal proteins may be incorporated. For example,
streptococcal
extracellular cysteine proteinase (SCP) may be incorporated in the vaccine
formulation
or an antigenic fragment thereof. SCP is thought to play an important role in
the
virulence of S. pyogenes S.pyogenes which are deficient in active SCP
production have
been shown to lose virtually all their virulence. Synthetic peptides such as
those
described in W096/08569 representing conserved immuno-dominant epitopes within
SCP may readily be incorporated in a suitable vaccine composition.
Another streptococcal protein which may be useful in a vaccine formulation is
the streptococcal inhibitor of complement-mediated lysis, namely protein SIC.
Again,
this protein plays a role in S. pyogenes pathogenicity and virulence and is
described in
W097/13 786.
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As outlined above, the peptides of the invention may be made by recombinant
techniques. Thus, the invention also provides nucleic acids encoding
polypeptides of
the invention. Particularly preferred are the polynucleotides set out in
Figure I 0 for the
33-mer sequence or fragments thereof encoding the 19-mer APP sequence (a).
A polynucleotide of the invention is capable of hybridising selectively with
the
coding sequence of sequence (a) for example the sequence set out in Figure 10
for the
33-mer sequence or the fragment thereof encoding the 19-mer APP sequence (a),
or to a
sequence complementary to that coding sequence. Polynucleotides of the
invention
include variants of the sequence shown in Figure 10 which encode the amino
acid
sequences of APP or the 33-mer and variants thereof which encode the
polypeptide
having the sequence (b), (c) or (d) of the invention. Typically, a
polynucleotide of the
invention is a contiguous sequence of nucleotides which is capable of
selectively
hybridising to the polynucleotide sequence of the 33-mer or 19-mer as set out
in Figure
10 or to the complement of that sequence.
A polynucleotide of the invention hybridising to the sequence of Figure 10
encoding the 19-mer APP sequence (a) can hybridise at a level significantly
above
background. Background hybridisation may occur, for example, because of other
DNAs present in a DNA library. The signal level generated by the interaction
between
a polynucleotide of the invention and the polynucleotide sequence of Figure 10
24 encoding the 19-mer sequence (a) is typically at least 10 fold, preferably
at least 100
fold, as intense as interactions between other polynucleotides and the
polynucleotide
sequence of Figure 10 encoding sequence (a). The intensity of interaction may
be
measured, for example by radiolabelling the probe e.g. with'ZP. Selective
hybridisation
is typically achieved using conditions of medium to high stringency, for
example, 0.03
M sodium chloride and 0.03 M sodium citrate at from about 50 ° C to
about 60 ° C.
A nucleotide sequence capable of selectively hybridising to the DNA coding
sequence of sequence (a) or to the sequence complementary to that coding
sequence
will be generally at least 50%, preferably at Least 70 or 80% and more
preferably at least
90 or 95% homologous to the polynucleotide of Figure I 0 or its complement
over a
region of at least 30, preferably at least 40, for instance, at least 60, 80
or 90 contiguous
nucleotides over the entire length of the polynucleotide sequence set out in
Figure 10
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for the 33-mer sequence. Methods of measuring polynucleotide homology are well
known in the art. The UWGCG package, which provides the BESTFIT program can be
used to calculate homology, e.g. on its default settings (Deveraux et al,
Nucl. Acids.
Res. 12. 387-395, 1984).
Any combination of the above mentioned degrees of homology and minimum
size may be used to define polynucleotides of the invention, with the more
stringent
combinations, i.e. higher homology over longer lengths being preferred.
Polynucleotides of the invention can be incorporated into a recombinant
replicable vector. The vector may be used to replicate the nucleic acid in a
compatible
host cell. Thus, in a further embodiment, the invention provides a method of
making
polynucleotides of the invention by introducing a polynucleotide of the
invention into a
replicable vector, introducing the vector into a compatibie host cell, and
growing the
host cell under conditions which bring about replication of the vector. The
vector may
be recovered from the host cell. Suitable host cells include bacteria such as
E. coli,
yeast, mammalian cell lines and other eukaryotic cell lines, for example,
insect SF9
cells.
Preferably, the polynucleotide of the invention in a vector is operably linked
to a
regulatory sequence which is capable of providing for the expression of the
coding
sequence of the host cell, i.e. the vector is an expression vector. The term
operably
linked refers to a juxtaposition where the components described are in a
relationship
permitting them to function in their intended manner. A regulatory system
operably
linked to a coding sequence is located in such a way that expression of the
coding
sequence is achieved under conditions compatible with the control sequences.
Such vectors may be transformed or transfected into a suitable host cell as
described above to provide for expression of a polypeptide of the invention.
This
process may comprise culturing a host cell transformed with an expression
vector as
described above under conditions to provide expression by the vector of a
coding
sequence encoding the polypeptide, and optionally recovering the expressed
polypeptide.
The vector may be for example, plasmid or virus vectors provided with an
origin
of replication, optionally a promoter for the expression of the said
polynucieotide and
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optionally a regulator of the promoter. The vectors may contain one or more
selectable
marker genes, for example an ampicillin resistance gene in the case of a
bacterial
plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be
used
in vitro, for example for the production of RNA or used to transfect or
transform a host
cell.
Promoters/enhancers and other expression regulation signals may be selected to
be compatible with the host cell for which the expression vector is designed.
For
example, yeast promoters include S. cerevisiae GAL4 and ADH promoters. Viral
promoters include the SV40 large T antigen promoter, retroviral LTR promoters
and
adenovirus promoters. All these promoters are readily available in the art.
The nucleotide sequences of the invention and expression vectors can also be
used as vaccine formulations as outlined above. The vaccines may comprise
naked
nucleotide sequences or in combination with cationic lipids, polymers or
targeting
systems.
The immunogenic polypeptides prepared as described above can be used to
produce antibodies, both polyclonai and monoclonal. If polyclonal antibodies
are
desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is
immunised with an
immunogenic polypeptide of the invention. Serum from the immunised animal is
collected and treated according to known procedures. If serum containing
polyclonal
antibodies to the poiypeptide contains antibodies to other antigens, the
polyclonal
antibodies can be purif ed by immunoaffinity chromatography. Techniques for
producing and processing polyclonal antisera are known in the art.
Monoclonal antibodies directed against Streptococcal epitopes in the
polypeptides of the invention can also be readily produced by one skilled in
the art. The
general methodology for making monoclonal antibodies by hybridomas is well
known.
Immortal antibody-producing cell lines can be created by cell fusion, and also
by other
techniques such as direct transformation of B lymphocytes with oncogenic DNA,
or
transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced
against
polypeptides of the invention can be screened for various properties; i.e.,
for isotype and
epitope affinity.
Antibodies, both monoclonal and polyclonal, which are directed against
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polypeptides of the invention are particularly useful in diagnosis, and those
which are
neutralising are useful in passive immunotherapy. Monoclonal antibodies, in
particular,
may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are
immunoglobulins which carry an "internal image" of the antigen of the
infectious agent
against which protection is desired.
Techniques for raising anti-idiotype antibodies are known in the art. These
anti-idiotype antibodies may also be useful for treatment of Streptococci, as
well as for
an elucidation of the immunogenic regions of polypeptides of the invention.
It is also possible to use fragments of the antibodies described above, for
example, Fab fragments.
EXAMPLES
Example 1 - Surface proteins promote aggregation ofAPl bacteria
S. pyogenes bacteria of the AP1 strain (40/58) from the WHO Collaborating
Centre for Reference and Research on Streptococci, Prague, Czech Republic,
were
found to aggregate following growth overnight at 37°C in Todd Hewitt
broth (TH)
(Difco, Detroit, MI). These visible aggregates rapidly fall to the bottom of
the testtube.
By measuring optical density at 620 nm at various time intervals, the degree
of
aggregation was determined. Human plasma (10% solution) or human IgG (1.4
mg/ml)
(Sigma Chemical Co., St. Louis, MO) present during growth led to no
aggregation
being seen and much slower settling of cultures (Fig. 1 ). Microscopic
analysis of the
cultures revealed large aggregates of bacteria except in those containing
plasma or IgG,
where short chains and almost no aggregates could be seen {not shown).
At the bacterial surface protein H and M1 protein are responsible for IgG-
binding, and a possible role for these molecules in the formation of
aggregates was
proposed.
Papain and a cysteine proteinase produced by S. pyogenes can remove proteins
H and M 1 from the surface of AP 1 bacteria AP 1 bacteria were suspended in
0.01 M
Tris-HCI, pH 8.0, to I% (v/v) {2x10' cellslml). The bacteria were incubated
with
papain (Sigma) and L-cystein (100 p,g papain and 28 ~1 1M L-cystein/ml cell
solution)
at 37°C for 1 h. Iodoacetic acid (Sigma) was added to a final
concentration of 10 mM to
terminate the digestion. Bacteria were collected by centrifugation at 3000 x
g, washed
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twice with PBS and submitted to sedimentation analysis. Binding of's'I-
labelled IgG to
the cells was also performed. For digestion with streptococcal cysteine
proteinase, 0.5
ml AP1 bacteria (2x10'° cells/ml PBS) were incubated with 5 p,g of the
activated
enzyme for 3 h at 37°C. The enzyme was inactivated by the addition of
iodoacetic acid
to 6 mM and cells were washed twice with PBS and analyzed for sedimentation
and
binding of'ZSI-labelled IgG.
Following treatment with these enzymes or cyanogen bromide (CNBr), the
bacterial suspensions settled slowly and no longer showed IgG-binding activity
(Fig.
2A, B). Peptides solubilized with CNBr were separated by SDS-PAGE and gave
rise to
bands with apparent molecular masses of 54, 49 and 44 kDa, respectively. These
bands,
denoted I, II and III in Figure 2C, were subjected to NHZ-terminal amino acid
sequencing. The sequences of bands I and II were determined to Asn-Gly-Asp-Gly-
Asn
and Glu-Val-Ala-Gly-Arg, sequences that start at positions 42 and 82,
respectively, in
M 1 protein, whereas the NHZ-terminal sequence of band III (Glu-Gly-Ala-Lys-
Ile)
corresponds to a protein H fragment starting at position 42. The size of the
fragments
generated with CNBr correspond well with the positions of methionine residues
in the
M 1 protein and protein H sequences. These data, together with the
observations
concerning IgG-binding and aggregation, indicate that protein H and/or M1
participate
in cell-cell interactions resulting in the formation of large bacterial
aggregates.
Example 2 Protein H binds to itself
Radiolabelled protein H or M 1 protein, labelled with '~sI using the Bolton
and
Hunter reagent (Amersham, UK}, was incubated with AP l bacteria in TH as
before.
Protein H was found to bind to the bacterial cells (Fig. 3A}. The AP6 strain
of the M6
serotype is a non-protein H-expressing strain and neither protein H nor M 1
protein
showed affinity for these bacteria. Included in Figure 3A, as a positive
control, are also
the binding curves obtained with'zsI-labelled IgG. The binding of protein H to
AP1
bacteria could be inhibited with protein H efficiently (see below, Fig. 4B).
Pmtein H
and M1 protein were also applied in slots to PVDF membranes and probed
with'ZSI-
labelled protein H or M 1 protein. An interaction could be seen between the
protein H
molecules, whereas M1 protein bound neither protein H nor M1 protein itself
(Fig. 3B).
Furthermore, protein H immobilized on Sepharose was also found to specifically
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interact with'ZSI-labelled protein H (see below). These observations suggest
that
protein H, through homophilic interactions, contributes to AP I aggregation.
Example 3 Identification of a self interacting region in protein H
By using various protein H fragments, the binding sites on the protein H
molecule for IgG, albumin, and FNIII domains have been determined. For IgG and
FNIII the binding sites are located within the NH,-terminal AB and A region
respectively, whereas the binding of albumin resides in the C repeats of
protein H.
Figure 4C shows a schematic representation of protein H, where additionally
the
fragments used in the experiments below are indicated.
Radiolabelled protein H and fragment AB, but not fragment A, showed affinity
for AP 1 bacteria (Fig. 4A). The binding of radiolabelled protein H to AP 1
bacteria
could also be blocked with fragment AB but not with fragment A, suggesting
that the
binding is located in the B region (Fig. 4B). Nevertheless, compared to
protein H a
larger amount of AB was required for inhibition. Thus, to obtain 50%
inhibition, about
300-fold more of the AB fragment was needed. Moreover, fibronectin and albumin
could not block the binding of protein H to AP 1 bacteria (data not shown),
which also
suggests that B represents the self associating region.
IgG-binding to protein H has been mapped to a region covering the NHZ
terminal part of B and the COOH-terminal part of A. The fact that IgG
interferes with
bacterial aggregation indicated that the self associating region could overlap
the IgG-
binding site. However, when radiolabelled protein H was applied to a column of
protein H-Sepharose, the bound radioactivity could not be eluted with an
excess of
unlabelled IgG (Fig. 5). In contrast, the radioactivity was readily eluted
with 3 M
KSCN. The protein H fragments were also tested for binding to protein H-
Sepharose,
and again only the AB fragment had affinity (not shown). This fragment
includes also
the 10 NHZ-terminal amino acid residues from the C1 repeat (see Fig. 4C),
which raised
the possibility that this sequence and/or the COOH-terminal part of B
represents the
self associating region. A peptide covering this region was therefore
synthesized (Fig.
4C) and designated APP; gggregative protein H peptide I50-168.
At the bacterial surface protein H and other members of the M protein family
form a-helical coiled-coil dimers. The homobifunctional cross-linker
disuccinimidyl
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suberate (DSS) from Pierce in Me~SO,, was added to a final concentration of 1
mM to
protein solutions in PBS for 30 min. at 4°C. 1M Tris-HCl pH7.5 and 1M
NaCI were
added to terminate the reaction. DSS was found to dimerize protein H in
solution (Fig.
6, stain, lanes 1 and 2). To test if APP could interact with the dimers,
protein H was
S incubated with'ZSI-labelled APP in the absence or presence of a 1000-fold
molar excess
of unlabelled APP, followed by cross-linking with DSS and SDS-PAGE. One gel
was
stained (Fig. 6, stain) and one was dried and autoradiographed (Fig. 6,
autoradiogram).
The ,ZSI-labelled APP preferentially bound to protein H dimers (Fig. 6,
autoradiogram,
lane 3) and the binding was inhibited by unlabelled APP (Fig. 6,
autoradiogram, lane 4).
The incomplete dimerization of protein H in the presence of an excess of APP
(Fig. 6,
stain, lane 4) is probably due to binding of the peptide to protein H
monomers, whereby
dimerization and subsequent covalent cross-linking is disturbed. Whereas the
experiments with APP demonstrate a physical interaction with protein H,
unrelated
peptide was not cross-linked with protein H (Fig. 6 autoradiogram, lane 5).
This peptide
I S corresponds to one of the Ig light chain-binding domains of protein L,
another bacterial
surface protein. These results show that the APP sequence is part of the self
associating
region of protein H and that the APP peptide can interact with this region.
Example 4 The APP .sequence is related to bacterial aggregation, adherence
and resistance to phagocytosis
To investigate whether the APP sequence also influences bacterial aggregation,
the APP peptide was added to AP 1 cultures. APP, and proteins H and M 1,
reduced the
sedimentation rate (Table below), whereas a peptide from the COOH-terminal
part of
protein H and protein L-derived peptides were without effect.
30
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Table 1
Inhibition of the aggregation ofAPl (% of OD drop after Ih)~
Protein/pentide nmol/ml % Inhibition
Protein H 1.7 99.5
M 1 protein 1.3 98.6
APP 82.5 51.5
Protein H peptide 351-376 64.6 0
Protein L, B 1-B4 5,0 0
Protein L peptide 1 150.8 p
Protein L peptide 2 82.4 0
' AP 1 was grown in TH over night in the presence of various
proteins/peptides.
The sedimentation was measured and the decrease in optical density (OD drop)
after 1 h
was calculated and compared to the sedimentation of AP 1 in TH alone. Mean
values
from at least two experiments are given.
APP-related sequences were identified in several M and M-like proteins,
including the M1 protein (Fig. 7). Among M serotypes in which APP-related
sequences
were identified, three additional strains of M serotypes 4, 12 and 49 were
tested and
found to aggregate, an aggregation that could be blocked with protein H (data
not
shown). Although many M and M-like proteins remain to be sequenced, the
proteins
containing the APP-related sequences listed in Figure 7, are found in some of
the most
frequent and important Streptococcal strains and in particular S. Pyogenes
serotypes.
The related serotypes set out in Fig. 7 represent 46 percent of clinical
isolates of S.
pyogenes in the UK during the period 1980-1990 Colman et al J. Med Microbiol
1993
Vol. 39 165-178. Thus, APP-related sequences are very common in clinical
isolates
implicating that aggregation contributes to the pathogenicity of S. Pyogenes,
and other
Streptococci.
Adherence to mucosal surfaces is an important and early step in S. Pyogenes
infections. We therefore analyzed the significance of APP-related sequences at
the
streptococcal surface for aggregation and adherence, using mutants of the API
strain
and the M6 protein-expressing strain JRS4 which is protein H deficient.
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Detroit 562 human (carcinoma) pharynx epithelial cells (ATCC CCL 138) were
cultured in Minimal essential medium with Earle's salt (MEM) from ICN,
supplemented
with 0.1 mM glutamine (ICN), I O% fetal calf serum (FCS) from Life
Technologies and
penicillin/streptomycin {5000 units/ml; 5000 pg/ml, PEST; ICN) at 37°C
in an
atmosphere containing 5% COz. For adhesion assays cells were grown in 24-well
tissue
culture plates to almost confluence. Cells were washed three times with
antibiotic free
medium before use. Bacteria were grown in TH at 37°C over night,
collected by
centrifugation, washed once in PBS and resuspended in MEM supplemented with 10
FCS. 2 x 10' bacteria were added per well and plates were incubated for 2h at
37°C.
The wells were washed three times with PBS to remove non-adherent bacteria 0.1
ml
trypsin (2.5 mg/ml in PBS) and 0.5 ml Triton X-100 (0.025% in PBS) were added
to
each well for lysis of the epithelial cells. To determine the number of viable
bacteria
appropriate dilutions were plated, on TH plates, in triplicate from each well.
As shown in Figure 8 both wild-type strains are highly aggregative and they
also
adhere well to epithelial cells. The BM27.6 is a mutant of AP 1 which does not
express
protein H, whereas BJM7I lacks both protein H and M1 protein. The data of
Figure 8
demonstrate that bacterial aggregation and adherence are significantly reduced
when
protein H is missing at the bacterial surface, and that the removal of also M
1 protein
further decreases aggregation. The results with the M6 protein-negative mutant
JRS 145
also suggest that the presence of surface proteins containing APP-related
sequences
promotes aggregation and adherence.
Like most M protein-expressing strains of S pyogenes, the AP 1 strain studied
here survives and multiplies in human whole blood. This is in contrast to the
non-
aggregating BMJ71 mutant which is rapidly killed. As demonstrated above,
soluble
2S protein H and APP both interfere with AP1 aggregation. If aggregation
contributes to
the anti-phagocytic effect, these peptides should inhibit growth of AP1
bacteria in
human blood.
Bacteria were grown to early midlog-phase (ODbzo 0.15) and serially diluted in
TH. 100 p,l of the bacterial solution were incubated with proteins or peptides
for 30
min. on ice, mixed with 1 ml heparin-treated blood from different donors, and
rotated
end over end at 3?°C. Samples of 100 pl were drawn at different times,
added to 2.5 ml
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TH with O.S% agar, spread on TH-agar plates, and incubated at 37°C
overnight
As shown in Table 2 below the addition of protein H or APP, but not an
unrelated peptide, reduced the multiplication of AP1 bacteria in human whole
blood,
suggesting that APP-mediated aggregation protects S. pyogenes against
phagocytosis.
S Table 2 Survival of API in human blood
Time of incubation (h)
0 S
CFU/ml blood' % Inhibition
AP1 + pgS 88 t 67 126476 f 9U500
AP 1 + protein H 72 t 86 52632 + 60627 57.429.4
( 1.2S nmol)
API + App 88 + 82 43938 t 39205 6S.9t2S.1
( 1000 nmol)
AP1 + protein L 173 t 118 256333 t 102372 0
1 S ( 1.67 nmol)
Values are mean t SD from five experiments using blood from three different
donors.
Example 6 Immunisation studies
Subsequently, studies were carried out to investigate the ability of APP to
generate antibodies, indicative of its potential as a vaccine candidate.
A. 19-mer APP-peptide conjugated to KLH (Pierce) was used for raising of
antibodies in rabbits. 0.4 mg of 19-mer APP-peptide and 0.1 mg KLH, in a
volume of
O.S ml, were mixed with O.S ml Freunds adjuvant (Sigma, St. Louis, MO) (1 part
complete and 2 parts incomplete) and 10 x 0.1 mI were injected subcutaneously
in the
back of a rabbit. Immunisation was done on day 1, and a booster of the same
amount of
protein was injected in the same way as above at day 30 and day 60.
Each well in a 96-microwell plate (Nunc, Maxisorp) was coated overnight at
+4°C with 200 pl of a 1 pg/ml solution of respectively protein, protein
H, APP-peptide
and protein L-peptide (Actigen, Cambridge UK) diluted in coupling buffer
(0.016 M
na2CO3, 0.035 M NaHC03 pH 9.6). After washing, in PBS, the wells were
incubated
with 200 pl preimmunserum or antiserum for 1 h at 37°C. The sera were
serially
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diluted, 1:1000; 1:2000; 1:4(?00; 1:8000; 1:16000; 1:32000; 1:64000 and
1:128000, in
PBS + 0.05% Tween ""-20, 2% bovine serum albumin (BSA).
A secondary antibody, goat anti-rabbit HRP-labelled (BioRad, Hercules, CA)
diluted 1:3000 in PBS + 0.05% Tween'M-20, 2% BSA was added for detection of
activity. 200 mg of ABTS (2, 2'-Azino-di (3-ethylbensthiazolinsulfonat (NH4)2-
salt was
dissolved in 10 water. A colour reagent mixture consisting of 40 ml substrate
buffer
(0.1 M citric acid, 0.1 M NazHP04 x 2H20, pH 4.5}, 2m1 ABTS solution, and 0.8
ml of
30% HZOZ Was prepared.
The plate was developed by adding 200 pl of colour reagent mixture to each
well followed by incubation for 30 minutes at 37°C. The plates were
read in an ELISA
reader at 405 nm.
As shown in Figure 9, APP bound antibody confirming generation of anti-APP
antibodies.
B. Sheep were primed with SOOItg Spy-PH YQE33
(YQWQLQKQQQLETEKQISEASRKSLSRDLEASRC-COOH) conjugated to
Keyhole Limpet Haemacyanin (KLH) administered subcutaneously in Freunds
complete adjuvant at 6 individual sites. On days 28, 56 and 82 following
primary
immunisation animals were boosted with 350~g Spy-PH YQE33-KLH conjugate
administered subcutaneously in Freunds complete adjuvant at 6 individual
sites.
Fourteen days after the final booster animals were bled and sera prepared for
determination of anti-Spy-PH-YQE33 antibodies by ELISA.
Microtitre plates (96 well) were coated with Spy-PH-YQE33 peptide by
addition of S pg/ml peptide solution in 0.05 M bicarbonate buffer (pH 9.6) and
incubation (60 min, 37°C). Plates were blocked (60 min, 37°C)
with 1% bovine serum
albumin (BSA) in PBS supplemented with 0.05% Tween-20 (PBS-Tween) to minimise
background binding. Sheep immune serum prepared as described above was diluted
1:10000 in PBS-Tween and pre-incubated (60 min, 37°C) with 100 pg/ml
Spy-PH-
QKQ19 (QKQQQLETEKQLSEASRKSC-COON} relevant peptide or 100~g/ml Spy-
LP-KEY17 {CKEY TDKLDKLDKESKDK-COOH) irrelevant peptide. Control serum
was incubated in the absence of peptide. Pre-incubated sera was added to the
plates and
incubated for 60 min at 37°C. Donkey anti-sheep IgG conjugated to horse
radish
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peroxidase (1:1000 dilution in PBS-Tween) was added to the plate (60 min,
37°C)
followed by enzyme substrate solution containing 3,3',5,5'-tetramethyl-
bemzidine in
phosphate citrate buffer at pH 5.0 ( 10 min, RT). The colorimetric reaction
was stopped
by addition of 2 M HCl and optical density determined at 450 nm. Plates were
washed
between each stage of the ELISA for a minimum of 3 times with PBS-Tween.
The results are shown in Figure 12.
C. Recombinant protein H was obtained by expression in E. coli as
described in Berge et al., ( 1997) J. Biol. Chem. 272, 20774-20781. Protein H
was
purified from E. coli cell lysates in a single affinity chromatography step
using a human
IgG-Sepharose column. Bound protein H was eluted with 3 M KSCN and dialysed
against PBS.
Protein extracts were prepared from S. pyogenes by partial digestion of the
cell
wall with lysozyme and rnutanolysin. Approximately 2 ml of cells grown to
ODD"",
of 1.0 were pelleted by low speed centrifugation and resuspended in 100 ~1 of
10 mM
Tris-HCl containing Smg/ml of freshly added lysozyme (Sigma) and 100 U/ml of
mutanolysin (Sigma). After 10 min incubation at 37 °C the cells were
recovered by
centrifugation and washed twice with 1 ml of 10 mM Tris-HCl (pH 8.0) and then
lysed
by the addition of 150 pl of NuPage sample buffer (Novex) containing 50 mM
DTT.
Proteins were electrophoretically separated using 10% NuPage Bis-Tris-HCl
buffered acrylamide gels and the NuPage MES SDS running buffer to optimise
separation proteins in molecular weight range 39 to 60 kDa. Details of the
NuPage
electrophoresis system can be obtained from Novex Electrophoresis GmbH,
Brueningstrasse 50, Building C 584, D-65929 Frankfurt/M. The proteins were
transferred to PVDF membrane filters using the Novex XCell II blotting
apparatus and
the conditions recommended by the manufacturer. After transfer the PVDF
filters were
blocked using a 1:1 mixture of UHT virtually fat free milk and phosphate
buffered
saline containing 0.05% v/v Tween 20 (PBST) for 30 min at room temperature.
The
filters were incubated with the primary antibody serum ( 1: i 000) in blocking
buffer ( 1
part fat free milk to 3 parts PBST) for 1 h at room temperature and then
washed for 3 x
5 min in blocking buffer. The filters were incubated with the anti-sheep IgG
alkaline
phosphate conjugate diluted 1:5000 in blocking buffer for 30 min at room
temperature.
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The filters were finally washed for 2 x 10 min with PBST and 2 x S min with 10
mM
Tris-HCl (pH 8.0), 150 mM NaCI before addition of bromo-chloro-indolyl
phosphate
nitro-blue tetrazolium (BCIP/NBT substrate kit, Sigma) a chromogenic substrate
for
alkaline phosphatase.
Immunoblotting was carried out with recombinant protein H (800 ng) and
protein extracts from M6 type (non-protein H expressor) and AP1 (M1 type
protein H
expressor) strains of S, pyogenes. A major band was detected in the tracks
loaded with
purified recombinant protein H and a slightly higher molecular weight band was
detected in the track loaded with AP 1 extracts that may represent protein H
enzymaticaily released in small quantities from the cell wall of strain AP 1.
These results demonstrate that immunisation with APP peptide conjugated to
carrier protein ICLH elicits IgG serum antibodies that bind to purified
recombinant
protein H and wild type protein H from S. pyogenes. Differences in the
molecular
weight of purified recombinant protein H and wild type protein H from S.
pyogenes can
be accounted for by the fact that recombinant protein H is 30 amino acids
shorter in
length.
Example 7 Bactericidal Analysis
Streptococcus pyogenes group A (AP 1 ) bacteria were grown to early midlog-
phase (OD6ZO = 0.15) in Todd Hewitt broth (TH) (Difco, Difco Laboratories,
Detroit,
MN) at 37°C in S% CO2. The bacteria were serially diluted five times in
TH-broth, 0.4
ml bacteria + 4.6 ml TH. 100 pl of the fourth or fifth dilution was incubated,
30 min.
on ice, with 100 Itl of undiluted preimmunserum (pre-APP) or 100 ~1 of
undiluted
antiserum (anti-APP), 100 pl of 1 mg/ml Ig-fractions of preimmunserum (pre-
IgG) or
antiserum (anti-IgG). 1 ml heparin-treated human blood was added to the 200 ~1
mixture and rotated end over end at 37°C.
At various time points samples of 100 ~1 were drawn, added to 2.5 ml TH with
0.5% agar, spread on TH-agar plates and incubated at 37°C overnight.
Colony forming
units were counted. IgG-fractions from preimmunserum and antiserum
respectively
were purified on protein LG-Sepharose.
The results are shown in Table 3 below, demonstrating the ability of anti-APP
serum to inhibit the survival of AP1 in human blood. APP itself also has an
inhibiting
activity, outlining the potcntial of APP not only as a vaccine candidate but
also as an
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anti-aggregative agent.
Table 3
Inhibition of the survival of AP1 in human blood
I hi 'tor % Inhibition
pre-APP serum' 30.9 ~ 24.8
anti-APP serum' 35.5 f 15.9
IgGz affinity purified from pre-APP serum 50.3 f 28.9
IgG2 affinity purified from anti-APP serum 80.2 + 11.8
~ Mean values from five experiments using blood from three different donors
Z Mean values from three experiments using blood from two different donors
Example 8 Challenge study with APP peptide and S. pyogenes bacteria
The usefulness of APP in increasing survival through its anti-aggregative
properties was investigated.
1 S A S. pyogenes strain AP 1 of M 1 serotype were grown in Todd Hewitt
culture
medium overnight at 37°C in 5% COZ atmosphere. Bacteria were harvested
by
centrifugation at 3,800 x g for 10 minutes. The resulting bacterial pellet was
washed
twice and resuspended in 1 x phosphate buffered saline, PBS. A total of 2 mg
19-mer
APP-peptide, dissolved in PBS, and 106 colony forming units of AP1 bacteria
were
mixed in a volume of 0.2 ml. This mixture was then injected subcutaneously
into mice,
using the so-called air sac model. In this method, a syringe is filled and
mixed with 0.8
ml air and the 0.2 ml bacteria/peptide mixture. This creates the so-called air-
sac model.
The mice were left in their cages and survival was monitored over time. The
Results
are set out in Table 4 below.
Table 4
Bacteria (cfu/ml) Bacteria + App-peptide Survival
106 - all died
106 2 mg all survived
The mice that received only bacteria died within 24 hours, while mice that
were
given bacteria plus APP-peptide were still alive 4 weeks later.
