U.S. patent application number 11/730659 was filed with the patent office on 2007-10-11 for ides, an igg-degrading enzyme of streptococcus pyogenes.
This patent application is currently assigned to HANSA MEDICAL RESEARCH AB. Invention is credited to Lars Bjorck, Bjorn Johansson, Ulrich Von Pawel-Rammingen.
Application Number | 20070237784 11/730659 |
Document ID | / |
Family ID | 9927839 |
Filed Date | 2007-10-11 |
United States Patent
Application |
20070237784 |
Kind Code |
A1 |
Von Pawel-Rammingen; Ulrich ;
et al. |
October 11, 2007 |
IdeS, an IgG-degrading enzyme of Streptococcus pyogenes
Abstract
A polypeptide isolated from S. pyogenes is described, having IgG
cysteine protease activity. The protease is designated IdeS,
Immunoglobulin G-degrading enzyme of S. pyogenes. A polypeptide
comprises SEQ ID NO: 1 and variants and fragments thereof having
IgG cysteine protease activity or the ability to generate an immune
response against S. pyogenes in an individual. Polynucleotides
encoding these polypeptides and the polypeptides may be used in
generating an immune response in an individual. IdeS protease
inhibitors may be used in the treatment of S. pyogenes
infection.
Inventors: |
Von Pawel-Rammingen; Ulrich;
(Lund, SE) ; Johansson; Bjorn; (Lund, SE) ;
Bjorck; Lars; (Lund, SE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
HANSA MEDICAL RESEARCH AB
Lund
SE
|
Family ID: |
9927839 |
Appl. No.: |
11/730659 |
Filed: |
April 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10499143 |
Nov 30, 2004 |
|
|
|
PCT/EP02/14427 |
Dec 17, 2002 |
|
|
|
11730659 |
Apr 3, 2007 |
|
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|
Current U.S.
Class: |
424/190.1 ;
514/25; 530/350 |
Current CPC
Class: |
A61K 2039/505 20130101;
C12N 9/52 20130101; A61K 39/00 20130101; A61K 38/00 20130101; C07K
14/36 20130101; A61K 2039/53 20130101; C12N 9/641 20130101 |
Class at
Publication: |
424/190.1 ;
514/025; 530/350 |
International
Class: |
A61K 39/09 20060101
A61K039/09; A61K 38/16 20060101 A61K038/16; C07K 14/315 20060101
C07K014/315 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 18, 2001 |
GB |
0130228.0 |
Claims
1. A polypeptide comprising: (a) the amino acid sequence of SEQ ID
NO: 1; (b) a variant thereof having at least 50% identity to the
amino acid sequence of SEQ ID NO: 1 and having IgG cysteine
protease activity; (c) a fragment of either thereof having IgG
cysteine protease activity; or (d) a fragment of (a) or (b) which
is capable of generating an immune response to S. pyogenes in an
individual.
2. A polypeptide according to claim 1, wherein said variant has at
least 80% identity to SEQ ID NO: 1.
3. A polypeptide according to claim 2, wherein said variant has at
least 95% identity to SEQ ID NO: 1.
4. A polypeptide according to claim 3, wherein said variant has at
least 97% identity to SEQ ID NO: 1.
5. A polypeptide according to claim 4, wherein said variant has at
least 99% identity to SEQ ID NO: 1.
6. A polypeptide according to claim 1, wherein said fragment is a
fragment of at least 50 amino acids in length.
7. A polypeptide according to claim 6, wherein said fragment is a
fragment of at least 100 amino acids in length.
8. A polypeptide according to claim 7, wherein said fragment is a
fragment of at least 200 amino acids in length.
9. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and/or diluent and a polypeptide according to
claim 1.
10. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a polypeptide according to claim 1, wherein
said composition is a vaccine composition.
Description
[0001] This application is a divisional of application Ser. No.
10/499,143, filed Nov. 30, 2004, which is the U.S. national phase
of international application PCT/EP02/14427 filed Dec. 17, 2002,
which designated the U.S. PCT/EP02/14427 claims priority to GB
Application No. 0130228.0 filed Dec. 18, 2001. The entire contents
of these applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a new Streptococcus pyogenes
protein which displays IgG cysteine protease activity. The
invention further relates to the treatment, vaccination and
diagnosis of S. progenes infection and to the development of new
tools for biotechnology.
BACKGROUND OF THE INVENTION
[0003] S. pyogenes (Group A streptococcus) is an important human
bacterial pathogen best known as the cause of skin and throat
infections. Streptococcal infections vary in severity from
relatively mild diseases, like impetigo and pharyngitis, to serious
life threatening conditions such as septicemia, necrotizing
fascitis, and streptococcal toxic-shock syndrome (Bisno and
Stevens, 1996; Cunningham, 2000). Sequelae to S. pyogenes skin and
throat infections include serious conditions such as acute
rheumatic fever and post-streptococcal glomerulonephritis.
[0004] S. pyogenes expresses cell wall-anchored surface proteins
with the ability to interact with abundant extracellular human
proteins such as albumin, IgG, IgA, fibrinogen, fibronectin, and
.alpha..sub.2-macroglobulin (for references see Navarre and
Schneewind, 1999). Many of these protein-protein interactions are
mediated by members of the M-protein family.
SUMMARY OF THE INVENTION
[0005] The present inventors have identified, purified and
characterised a new extracellular cysteine protease produced by S.
pyogenes. The protease, designated IdeS (Immunoglobulin G-degrading
enzyme of S. pyogenes) displays a high specificity for IgG,
cleaving in the hinge region of the immunoglobulin. The protease
cleaves not only IgG bound to the bacterial surface by
IgGFc-binding proteins, but also opsonising IgG, and so appears to
have a role in helping S. pyogenes to evade the host immune system.
The inventors have shown that IdeS is expressed in both the
logarithmic and stationary phases of bacterial growth, and in a
number of clinically relevant S. pyogenes strains, including those
of the M1, M12 and M55 serotypes. Antibodies to IdeS were found in
individuals suffering from S. pyogenes infection, with those found
in convalescent sera capable of blocking IdeS enzymatic activity.
IdeS is therefore of use in the treatment and diagnosis of
conditions associated with S. pyogenes infection. The protease is
also useful for developing new biotechnological tools.
[0006] Accordingly the invention provides a polypeptide
comprising:
[0007] (a) the amino acid sequence of SEQ ID NO: 1;
[0008] (b) a variant thereof having at least 50% identity to the
amino acid sequence of SEQ ID NO: 1 and having IgG cysteine
protease activity; or
[0009] (c) a fragment of either thereof having IgG cysteine
protease activity.
[0010] The invention also provides a polypeptide for use in
generating an immune response in an individual comprising:
[0011] (a) the amino acid sequence of SEQ ID NO: 1;
[0012] (b) a variant thereof having at least 50% identity to the
amino acid sequence of SEQ ID NO: 1 and having IgG cysteine
protease activity; or
[0013] (c) a fragment of either thereof which is capable of
generating an immune response to S. pyogenes in an individual.
[0014] In another aspect the invention provides a polynucleotide
which comprises:
[0015] (a) SEQ ID NO: 3 or a complementary sequence thereto;
[0016] (b) a sequence which hybridises under stringent conditions
to the sequence defined in (a);
[0017] (c) a sequence which is degenerate as a result of the
genetic code to a sequence as defined in (a) or (b);
[0018] (d) a sequence having at least 60% identity to a sequence as
defined in (a), (b) or (c); or
[0019] (e) a fragments of any of the sequences (a), (b), (c) or
(d), and which encodes a polypeptide having IgG cysteine protease
activity or capable of generating an immune response against S.
pyogenes in an individual.
[0020] The invention also relates to expression vectors comprising
a polynucleotide of the invention and host cells transformed with
such expression vectors.
[0021] In another aspect, the invention relates to a method for
identifying an agent that modulates IgG cysteine protease activity
of a polypeptide having the amino acid sequence of SEQ ID NO: 1
comprising:
[0022] (i) contacting a polypeptide as defined above and IgG with a
test substance under conditions that would permit IgG cysteine
protease activity in the absence of the test substance; and
[0023] (ii) determining thereby whether the test substance
modulates the said activity.
[0024] Inhibitors, of the cysteine protease of the invention, for
example identifiable by the other method are provided for use in
the treatment of S. pyogenes infection.
[0025] The polypeptides of the invention may be used in a method of
generating Fc or Fab fragments of IgG comprising contacting IgG
with the polypeptide.
[0026] The invention also relates to a method of generating an
immune response in an individual comprising administering a
polypeptide, polynucleotide or expression vector of the invention.
Preferably, the polypeptide or polynucleotide is used to generate a
protective immune response. Methods of treating S. pyogenes
infection are also described, comprising administering an antibody
or an IdeS protease inhibitor to an individual.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 shows the amino acid sequence found in the hinge
region of human IgG including the cleavage site for IdeS.
[0028] FIG. 2 shows a schematic representation of an open reading
frame (ORF) encoding IdeS isolated from S. pyogenes AP1, including
a putative signal sequence and RGD motif.
[0029] FIG. 3 shows survival factors for S. pyogenes bacteria in
macrophage like cells, after
[0030] incubation of the bacteria in immune or non-immune plasma,
and with or without IdeS.
[0031] FIG. 4 shows IdeS cleavage of IgG bound to S. pyogenes
bacterial surface.
BRIEF DESCRIPTION OF THE SEQUENCES
[0032] SEQ ID NO:1 is an amino acid sequence encoding IdeS isolated
from S. pyogenes AP1. [0033] SEQ ID NO:2 is an amino acid sequence
encoding IdeS isolated from S. pyogenes AP1, including a putative
signal sequence. [0034] SEQ ID NO:3 is nucleic acid sequence
encoding IdeS, isolated from S. pyogenes AP1 (including a signal
sequence). [0035] SEQ ID NO:4 is PCR primer Ide1. [0036] SEQ ID
NO:5 is PCR primer Ide2. [0037] SEQ ID NO:6 is PCR primer
Ide5.times. [0038] SEQ ID NO:7 is PCR primer Ide3.times. [0039] SEQ
ID NO:8 is N terminal amino acid sequence of an IdeS human IgG
cleavage product. [0040] SEQ ID NO:9 is N terminal amino acid
sequence of IdeS isolated from S. pyogenes AP1. [0041] SEQ ID NO:10
is a cell wall attachment signal found in a number of bacterial
proteins.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention provides certain polypeptides. In particular,
in accordance with the invention these polypeptides may be used in
the prophylaxis and diagnosis of infection by S. pyogenes
strains.
[0043] Polypeptides in accordance with the invention are those
which comprise the amino acid sequence of SEQ ED NO:1 and display
IgG cysteine protease activity, together with functional variants,
derivatives and fragments thereof. The invention also relates to
variants and fragments of SEQ ID NO:1 which have the ability to
generate an immune response in an individual and in particular
those which generate antibodies having the ability to block the
enzymatic activity of IdeS, or to generate a protective immune
response. Preferably, the polypeptide comprises the sequence of SEQ
ID NO:1. The polypeptide may additionally include a signal sequence
as in SEQ ID NO:2.
[0044] Variant polypeptides are those for which the amino acid
sequence varies from that in SEQ ID NO:1, but which retain the same
essential character or basic functionality as IdeS. The variant
polypeptides may therefore display IgG cysteine protease activity
or the ability to generate an immune response in an individual. In
particular such variants include those which are able to generate
antibodies having the ability to block the enzymatic activity of
IdeS, or to generate a protective immune response. Typically,
polypeptides with more than about 50%, 55% or 65% identity
preferably at least 80% or at least 90% and particularly preferably
at least 95%, at least 97% or at least 99% identity, with the amino
acid sequence of SEQ ID NO:1 are considered variants of the
protein. Such variants may include allelic variants and the
deletion, modification or addition of single amino acids or groups
of amino acids within the protein sequence, as long as the peptide
maintains a basic functionality of IdeS.
[0045] The inventors have also found that it is possible to provide
mutants of IdeS, in which mutation in the catalytic domain removes
the cysteine protease activity of the protein. Such a mutant may
comprise replacement or deletion of the catalytic cysteine residue
at position 94 (C94) of IdeS. For example, cysteine may be replaced
with glycine. The utility of such variants is described in more
detail below. The invention also relates to variants of fragments
of such a mutated IdeS, but which maintain other functions of IdeS,
such as the ability to generate an immune response or bind to
IgG.
[0046] Amino acid substitutions may be made, for example from 1, 2
or 3 to 10, 20 or 30 substitutions. The modified polypeptide
generally retains activity as an IgG-specific cysteine protease.
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. TABLE-US-00001 ALIPHATIC Non-polar G A
P I L V Polar-uncharged C S T M N Q Polar-charged D E K R AROMATIC
H F W Y
[0047] Preferably the polypeptides comprise a cysteine residue and
a histidine residue at a spacing typically found in cysteine
proteases. For example, in SEQ ID NO:1, these residues are found at
a spacing of about 130 aa, as is typically found in cysteine
proteases.
[0048] Shorter polypeptide sequences or fragments are within the
scope of the invention. For example, a peptide of at least 20 amino
acids or up to 50, 60, 70, 80, 100, 150 or 200 amino acids in
length is considered to fall within the scope of the invention as
long as it demonstrates a basic functionality of IdeS. In
particular, but not exclusively, this aspect of the invention
encompasses the situation when the protein is a fragment of the
complete protein sequence and may represent an IgG-binding region
or an epitope. Such fragments may not retain IgG cysteine protease
activity.
[0049] Polypeptides of the invention may also be chemically
modified, e.g. post-translationally modified. For example, they may
be glycosylated or comprise modified amino acid residues. They may
be modified by the addition of histidine, residues to assist their
purification or by the addition of a signal sequence to promote
insertion into the cell membrane. It may be desirable to provide
the peptides or proteins in a form suitable for attachment to a
solid support. The proteins or peptides may thus be modified to
enhance their binding to a solid support for example by the
addition of a cystine residue. Such modified polypeptides fall
within the scope of the term "polypeptide" of the invention.
[0050] Typically, polypeptides for use in accordance with the
invention display immunoglobulin cysteine protease activity, and in
particular IgG cysteine protease activity. Preferably, the
polypeptide cleaves IgG in the hinge region and more particularly
in the hinge region of the heavy chain. Preferably, cleavage
results in production of Fc and Fab fragments of IgG. Preferably
the activity is specific for IgG. The cysteine protease activity
may be determined by means of a suitable assay. For example, a test
polypeptide may be incubated with IgG at a suitable temperature,
such as 37.degree. C. The starting materials and the reaction
products may then be analysed by SDS PAGE to determine whether the
desired IgG cleavage product is present. Typically this cleavage
product is a 31 kDa fragment. Typically there is no further
degradation of IgG after this first cleavage. The cleavage product
may be subjected to N-terminal sequencing to verify that cleavage
has occurred in the hinge region of IgG. Preferably the N-terminal
sequence comprises the sequence in SEQ ID NO:8.
[0051] The cysteine protease activity of the polypeptides can be
further characterised by inhibition studies. Preferably, the
activity is inhibited by the peptide derivateZ-LVG-CHN.sub.2 and/or
by iodoacetic acid both of which are protease inhibitors. However,
the activity is generally not inhibited by E64.
[0052] The cysteine protease activity of the polypeptides is
generally IgG-specific in that the polypeptides may not degrade the
other classes of Ig, namely IgM, IgA, IgD and IgE, when incubated
with these immunoglobulins under conditions that permit cleavage of
IgG. In preferred embodiments the polypeptide has the ability to
cleave human, rabbit or goat IgG, and preferably does not have the
ability to cleave murine IgG.
[0053] The invention also relates to mutant IdeS, in which the
catalytic cysteine protease activity has been reduced or lost. The
absence of cysteine protease activity may be assayed as described
for non-mutant IdeS. Such mutants may retain the ability to bind
IgG. Binding of IgG can be assayed by binding studies, for example
immobilising IdeS, and contacting said immobilised IdeS with IgG,
and monitoring for the presence of any bound IgG. Such a mutant may
display no cysteine protease activity or reduced cysteine protease
activity compared to a polypeptide not so modified.
[0054] According to one aspect of the invention, the polypeptides
provided are capable of generating an immune response, preferably a
protective immune response to S. pyogenes in an individual. These
polypeptides are useful for inclusion in vaccines targeting S.
pyogenes infection. In preferred embodiments, the polypeptide
generates antibodies which have the ability to block the enzymatic
activity of IdeS. This activity may be monitored, for example as
described for IdeS activity, in which IdeS or a variant thereof
retaining IgG cysteine protease activity is incubated with IgG in
the presence of the generated antibody. Cleavage of IgG by IdeS can
be monitored as before. The polypeptides can also be used to
generate antibodies which can be used in the diagnosis or treatment
by immunotherapy of S. pyogenes infection. Such polypeptides may
comprise an epitope of the IdeS polypeptide and may not otherwise
demonstrate the IgG cysteine protease activity. Preferably the
polypeptides are fragments. For example, the fragments may be at
least 6 amino acids in length, preferably at least 10, such as at
least 12 or 15 or up to 20, 30 or 40 amino acids. Longer fragments
such as up to 60 or 150 aa in length may also be used.
[0055] A peptide for generating an immune response may be
identified by immunisation studies. For example, a candidate
peptide may be administered to an animal and subsequently the
antibody or T-cell response generated which is specific for the
peptide may be determined. Antiserum generated following
administration of a peptide to an animal may be evaluated for the
ability to bind the peptide or to bind IdeS. Subsequently the
animal may be challenged with S. pyogenes to evaluate whether a
protective immune response has been generated.
[0056] 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 purified form, in which case it will generally
comprise the polypeptide in a preparation in which more than 50%,
e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide
in the preparation is a polypeptide of the invention.
[0057] A protein or peptide of the invention may be labelled with a
revealing label. The revealing label may be any suitable label
which allows the protein or peptide to be detected. Suitable labels
include radioisotopes such as .sup.125I, .sup.35S or enzymes,
antibodies, polynucleotides and linkers such as biotin. Labelled
polypeptides of the invention may be used in diagnostic procedures
such as immunoassays. In such assays it may be preferred to provide
the peptides attached to a solid support, for example, the surface
of an immunoassay well or dipstick. The present invention also
relates to such labelled and/or immobilized polypeptides packaged
in the form of a kit in a container. The kit may optionally contain
other suitable reagent(s), control(s) or instructions and the
like.
[0058] Polypeptides for use in the present invention may be
isolated from suitable IdeS expressing strains of S. pyogenes.
Suitable strains may be identified by a number of techniques. For
example, S. pyogenes strains may initially be tested for the
presence an ideS gene. Polynucleotide primers or probes may be
designed based on for example, SEQ ID Nos 1, 2 or 3. Suitable
primers are set out in SEQ ID NOs 4, 5, 6 and 7. The presence of
the ides gene can then be verified by PCR using the primers or by
hybridisation of the probes to genomic DNA of the S. pyogenes
strain.
[0059] S. pyogenes strains expressing active IdeS can be identified
by assaying for IgG cysteine protease activity in the culture
supernatant. Preferably inhibitor E64 is added to the supernatant
to inhibit any SpeB cysteine protease activity. The present
inventors have shown that at least five strains tested express
active IdeS: strains AP1, AP12, AP55, KTL3 and SF370. Preferably
the expressing strain is selected from AP1, AP12 and AP55.
[0060] Isolation and purification of IdeS from an expressing S.
pyogenes culture is typically on the basis of IgG cysteine protease
activity. Preferably the purification method involves an ammonium
sulphate precipitation step and an ion exchange chromatography
step. According to one method, the culture medium is fractionated
by adding increasing amounts of ammonium sulphate. The amounts of
ammonium sulphate may be 10 to 80%. Preferably the culture medium
is fractionated with 50% animonium sulphate, and the resulting
supernatant is fuirther precipitated with 70% ammonium sulphate.
Pelleted proteins may then be subjected to ion exchange
chromatography, for example by FPLC on a Mono Q column. Eluted
fractions may be assayed for IgG cysteine protease activity and
peak activity factions may be pooled. Fractions may be analysed by
SDS PAGE. For example, an N-terminal sequence can be obtained from
the SDS PAGE protein band. Fractions may be stored at -20.degree.
C.
[0061] Polypeptides for use in the invention may also be prepared
as fragments of such isolated proteins. Further, the proteins and
peptides of the invention may also be made synthetically or by
recombinant means as discussed below.
[0062] The amino acid sequence of proteins and polypeptides of the
invention may be modified to include non-naturally occurring amino
acids or to increase the stability of the compound. When the
proteins or peptides are produced by synthetic means, such amino
acids may be introduced during production. The proteins or peptides
may also be modified following either synthetic or recombinant
production.
[0063] The proteins or peptides of the invention may also be
produced using D-amino acids. In such cases the amino acids will be
linked in reverse sequence in the C to N orientation. This is
conventional in the art for producing such proteins or
peptides.
[0064] A number of side chain modifications are known in the art
and may be made to the side chains of the proteins-or peptides of
the present invention. Such modifications include, for example,
modifications of amino acids by reductive alkylation by reaction
with an aldehyde followed by reduction with NaBH.sub.4, amidination
with methylacetimidate or acylation with acetic anhydride.
[0065] The invention also relates to polynucleotides encoding the
above polypeptides, and their use in medicine. In particular the
invention relates to polynucleotides comprising or consisting of
(a) the coding sequence of SEQ ID NO:3 or a complementary sequence
thereto; (b) sequence which hybridises under stringent conditions
to the sequences defined in (a); (c) sequence which is degenerate
as a result of the genetic code to sequence as defined in (a) or
(b); (d) sequence having at least 60% identity to sequences defined
in (a) (b) or (c); and (e) fragments of the above sequences.
[0066] Typically the polynucleotide is DNA. However, the invention
may comprise RNA polynucleotides. The polynucleotides may be single
or double stranded, and may include within them synthetic or
modified nucleotides.
[0067] A polynucleotide of the invention can hybridize to the
coding sequence or the complement of the coding sequence of SEQ ID
NO: 3 at a level significantly above background. Background
hybridization 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 coding, sequence
or complement of the coding sequence of SEQ ID NO: 3 is typically
at least 10 fold, preferably at least 100 fold, as intense as
interactions between other polynucleotides and the coding sequence
of SEQ ID NO: 3. The intensity of interaction may be measured, for
example, by radiolabelling the probe, e.g. with .sup.32P. Selective
hybridisation may typically be achieved using conditions of medium
to high stringency. However,.such hybridisation may be carried out
under any suitable conditions known in the art (see Sambrook et al,
1989. For example, if high stringency is required suitable
conditions include from 0.1 to 0.2.times.SSC at 60.degree. C. up to
65.degree. C. If lower stringency is required suitable conditions
include 2.times.SSC at 60.degree. C.
[0068] The coding sequence of SEQ ID NO: 3 may be modified by
nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50
or 100 substitutions. The polynucleotide of SEQ ID NO: 3 may
alternatively or additionally be modified by one or more insertions
and/or deletions and/or by an extension at either or both ends.
Additional sequences such as signal sequences may also be included.
The modified polynucleotide generally encodes a polypeptide which
has IgG specific cysteine protease activity. Alternatively, a
polynucleotide encodes an epitope portion of an IdeS polypeptide.
Degenerate substitutions may be made and/or substitutions may be
made which would result in a conservative amino acid substitution
when the modified sequence is translated, for example as shown in
the Table above.
[0069] A nucleotide sequence which is capable of selectively
hybridizing to the complement of the DNA coding sequence of SEQ ID
NO: 3 will generally have at least 60%, at least 70%, at least 80%,
at least 90%, at least 95%, at least 98% or at least 99% sequence
identity to the coding sequence of SEQ ID NO: 3 over a region of at
least 20, preferably at least 30, for instance at least 40, at
least 60, more preferably at least 100 contiguous nucleotides or
most preferably over the full length of SEQ ID NO: 3.
[0070] For example the UWGCG Package provides the BESTFIT program
which can be used to calculate homology (for example used on its
default settings) (Devereux et al (1984) Nucleic Acids Research 12,
p 387-395). The PILEUP and BLAST algorithms can be used to
calculate homology or line up sequences (typically on their default
settings), for example as described in Altschul (1993) J. Mol.
Evol. 36:290-300; Altschul et al (1990) J. Mol. Biol.
215:403-10.
[0071] Software for performing BLAST analyses is publicly available
through the National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al, 1990). These
initial neighborhood word hits act as seeds for initiating searches
to find HSPs containing them. The word hits are extended in both
directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The BLAST
program uses as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of
10, M=5, N=4, and a comparison of both strands.
[0072] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g., Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a sequence is considered
similar to another sequence if the smallest sum probability in
comparison of the first sequence to the second sequence is less
than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0073] Any combination of the above mentioned degrees of sequence
identity and minimum sizes may be used to define polynucleotides of
the invention, with the more stringent combinations (i.e. higher
sequence identity over longer lengths) being preferred. Thus, for
example a polynucleotide which has at least 90% sequence identity
over 25, preferably over 30 nucleotides forms one aspect of the
invention, as does a polynucleotide which has at least 95% sequence
identity over 40 nucleotides.
[0074] Polynucleotide fragments, such as those suitable for use as
probes or primers will preferably be at least 10, preferably at
least 15 or at least 20, for example at least 25, at least 30 or at
least 40 nucleotides in length. They will typically be up to 40,
50, 60, 70, 100 or 150 nucleotides in length. Probes and fragments
can be longer than 150 nucleotides in length, for example up to
200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a
few nucleotides, such as five or ten nucleotides, short of the
coding sequence of SEQ ID NO: 3.
[0075] Polynucleotides according to the invention may be produced
recombinantly, synthetically, or by any means available to those of
skill in the art. They may also be cloned by standard techniques.
The polynucleotides are typically provided in isolated and/or
purified form.
[0076] In general, primers will be produced by synthetic means,
involving a step wise manufacture of the desired nucleic acid
sequence one nucleotide at a time. Techniques for accomplishing
this using automated techniques are readily available in the
art.
[0077] Longer polynucleotides will generally be produced using
recombinant means, for example using PCR (polymerase chain
reaction) cloning techniques. This will involve making a pair of
primers (e.g. of about 15-30 nucleotides) to a region of the ideS
gene which it is desired to clone, bringing the primers into
contact with DNA obtained from a bacterial cell, performing a
polymerase chain reaction under conditions which bring about
amplification of the desired region, isolating the amplified
fragment (e.g. by purifying the reaction mixture on an agarose gel)
and recovering the amplified DNA. The primers may be designed to
contain suitable restriction enzyme recognition sites so that the
amplified DNA can be cloned into a suitable cloning vector.
Suitable primers are for example, those in SEQ ID Nos 4, 5, 6 or
7.
[0078] Such techniques may be used to obtain all or part of the
ideS gene sequence described herein. Although in general the
techniques mentioned herein are well known in the art, reference
may be made in particular to Sambrook et al, Molecular Cloning: A
Laboratory Manual, 1989.
[0079] The polynucleotides according to the invention have utility
in production of the polypeptides according to the invention, which
may take place in vitro, in vivo or ex vivo. The polynucleotides
may be used as therapeutic or immunisation agents in their own
right or may be involved in recombinant protein synthesis.
[0080] Polynucleotides of the invention may be used as a primer,
e.g. a PCR primer, a primer for an alternative amplification
reaction, a probe e.g. labelled with a revealing label by
conventional means using radioactive or non-radioactive labels, or
the polynucleotides may be cloned into vectors.
[0081] Polynucleotides or primers of the invention may carry a
revealing label. Suitable labels include radioisotopes such as
.sup.32P or .sup.35S, enzyme labels, or other protein labels such
as biotin. Such labels may be added to polynucleotides or primers
of the invention and may be detected using techniques known per
se.
[0082] Polynucleotides or primers of the invention or fragments
thereof, labelled or unlabelled, may be used by a person skilled in
the art in nucleic acid-based tests for detecting or sequencing
ideS in a sample.
[0083] Such tests for detecting generally comprise bringing a
sample containing DNA or RNA into contact with a probe comprising a
polynucleotide or primer of the invention under hybridizing
conditions and detecting any duplex formed between the probe and
nucleic acid in the sample. Such detection may be achieved using
techniques such as PCR or by immobilizing the probe on a solid
support, removing nucleic acid in the sample which is not
hybridized to the probe, and then detecting nucleic acid which has
hybridized to the probe. Alternatively, the sample nucleic acid may
be immobilized on a solid support, and the amount of probe bound to
such a support can be detected.
[0084] The probes of the invention may conveniently be packaged in
the form of a test kit in a suitable container. In such kits the
probe may be bound to a solid support where the assay formats for
which the kit is designed requires such binding. The kit may also
contain suitable reagents for treating the sample to be probed,
hybridizing the probe to nucleic acid in the sample, control
reagents, instructions, and the like.
[0085] The polynucleotides of the invention may be incorporated
into a recombinant replicable vector. The vector may be used to
replicate the nucleic acid in a compatible host cell. Therefore,
polynucleotides of the invention may be made by introducing a
polynucleotide of the invention into a replicable vector,
introducing the vector into a compatible host cell and growing the
host cell under conditions which bring about replication of the
vector.
[0086] Preferably the vector is an expression vector comprising a
nucleic acid sequence that encodes a polypeptide of the invention.
Such expression vectors are routinely constructed in the art of
molecular biology and may for example involve the use of plasmid
DNA and appropriate initiators, promoters, enhancers and other
elements, which may be necessary, and which are positioned in the
correct orientation, in order to allow for protein expression.
Other suitable vectors would be apparent to persons skilled in the
art. By way of further example in this regard we refer to Sambrook
et al. 1989.
[0087] Polynucleotides according to the invention may also be
inserted into the vectors described above in an antisense
orientation in order to provide for the production of antisense
RNA. Antisense RNA or other antisense polynucleotides or
interfering RNA, iRNA may also be produced by synthetic means. Such
antisense polynucleotides or iRNA may be used as test compounds in
the assays of the invention or may be useful in a method of
treatment of the human or animal body by therapy.
[0088] Preferably, a polynucleotide of the invention or for use in
the invention in a vector is operably linked to a control sequence
which is capable of providing for the expression of the coding
sequence by the host cell, i.e. the vector is an expression vector.
The term "operably linked" refers to a juxtaposition wherein the
components described are in a relationship permitting them to
function in their intended manner. A regulatory sequence, such as a
promoter, "operably linked" to a coding sequence is positioned in
such a way that expression of the coding sequence is achieved under
conditions compatible with the regulatory sequence.
[0089] The vectors may be for example, plasmid, virus or phage
vectors provided with a origin of replication, optionally a
promoter for the expression of the said polynucleotide and
optionally a regulator of the promoter. The vectors may contain one
or more selectable marker genes, for example an ampicillin
resistence gene in the case of a bacterial plasmid or a resistance
gene for a fungal vector.
[0090] Promoters and other expression regulation signals may be
selected to be compatible with the host cell for which expression
is designed. For example, yeast promoters include S. cerevisiae
GAL4 and ADH promoters, S. pombe nmt1 and adh promoter. Mammalian
promoters include the metallothionein promoter which can be induced
in response to heavy metals such as cadmium. Viral promoters such
as the SV40 large T antigen promoter or adenovirus promoters may
also be used. All these promoters are readily available in the
art.
[0091] Mammalian promoters, such as .beta.-actin promoters, may be
used. Tissue-specific promoters are especially preferred. Viral
promoters may also be used, for example the Moloney murine
leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma
virus (RSV) LTR promoter, the SV40 promoter, the human
cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such
as the HSV IE promoters), or HPV promoters, particularly the HPV
upstream regulatory region (URR). Viral promoters are readily
available in the art.
[0092] The vector may further include sequences flanking the
polynucleotide giving rise to polynucleotides which comprise
sequences homologous to eukaryotic genomic sequences, preferably
mammalian genomic sequences, or viral genomic sequences. This will
allow the introduction of the polynucleotides of the invention into
the genome of eukaryotic cells or viruses by homologous
recombination. In particular, a plasmid vector comprising the
expression cassette flanked by viral sequences can be used to
prepare a viral vector suitable for delivering the polynucleotides
of the invention to a mammalian cell. Other examples of suitable
viral vectors include herpes simplex viral vectors and
retroviruses, including lentiviruses, adenoviruses,
adeno-associated viruses and HPV viruses. Gene transfer techniques
using these viruses are known to those skilled in the art.
Retrovirus vectors for example may be used to stably integrate the
polynucleotide giving rise to the polynucleotide into the host
genome. Replication-defective adenovirus vectors by contrast remain
episomal and therefore allow transient expression.
[0093] Vectors may be used in vitro, for example for the production
of DNA or RNA or used to transfect or transform a host cell, for
example, a mammalian host cell. The vectors may also be adapted to
be used in vivo, for example in a method of gene therapy or nucleic
acid immunisation.
[0094] Expression vectors may be transformed into a suitable host
cell to provide for expression of a polypeptide or polypeptide
fragment of the invention. The host cell, transformed or
transfected with an expression vector as described above, is
cultivated under conditions to allow for expression of the
polypeptide or fragment, and the expressed polypeptide or fragment
is recovered. Isolation and purification may be carried out as
described above. Host cells will be chosen to be compatible with
the vector and will preferably be bacterial. Host cells may also be
cells of a non-human animal, or a plant transformed with a
polynucleotide of the invention.
[0095] According to another aspect, the present invention also
relates to antibodies capable of specific binding to a polypeptide
of the invention. Such antibodies are for example useful in
purification, isolation or screening methods or indeed as
therapeutic agents in their own right.
[0096] Antibodies may be raised against specific epitopes of the
polypeptides according to the invention. An antibody, or other
compound, "specifically binds" to a protein when it binds with
preferential or high affinity to the protein for which it is
specific but does substantially bind not bind or binds with only
low affinity to other proteins. A variety of protocols for
competitive binding or immunoradiometric assays to determine the
specific binding capability of an antibody are well known in the
art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226,
1993). Such immunoassays typically involve the formation of
complexes between the specific protein and its antibody and the
measurement of complex formation.
[0097] For the purposes of this invention, the term "antibody",
unless specified to the contrary, includes fragments which bind a
polypeptide of the invention. Such fragments include Fv, F(ab') and
F(ab').sub.2 fragments, as well as single chain antibodies.
Furthermore, the antibodies and fragment thereof may be chimeric
antibodies, CDR-grafted antibodies or humanised antibodies.
[0098] Antibodies of the invention can be produced by any suitable
method. Means for preparing and characterising antibodies are well
known in the art, see for example Harlow and Lane (1988)
"Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. For example, an antibody may be
produced by raising antibody in a host animal against the whole
polypeptide or a fragment thereof, for example an antigenic epitope
thereof, herein after the "immunogen".
[0099] A method for producing a polyclonal antibody comprises
immunising a suitable host animal, for example an experimental
animal, with the immunogen and isolating immunoglobulins from the
animal's serum. The animal may therefore be inoculated with the
immunogen, blood subsequently removed from the animal and the IgG
fraction purified.
[0100] A method for producing a monoclonal antibody comprises
immortalising cells which produce the desired antibody. Hybridoma
cells may be produced by fusing spleen cells from an inoculated
experimental animal with tumour cells (Kohler and Milstein (1975)
Nature 256, 495-497). An immortalized cell producing the desired
antibody may be selected by a conventional procedure. The
hybridomas may be grown in culture or injected intraperitoneally
for formation of ascites fluid or into the blood stream of an
allogenic host or immunocompromised host. Human antibody may be
prepared by in vitro immunisation of human lymphocytes, followed by
transformation of the lymphocytes with Epstein-Barr virus.
[0101] For the production of both monoclonal and polyclonal
antibodies, the experimental animal is suitably a goat, rabbit, rat
or mouse. If desired, the immunogen may be administered as a
conjugate in which the immunogen is coupled, for example via a side
chain of one of the amino acid residues, to a suitable carrier. The
carrier molecule is typically a physiologically acceptable carrier.
The antibody obtained may be isolated and, if desired,
purified.
[0102] Antibodies, both monoclonal and polyclonal, which are
directed against polypeptides of the invention are particularly
useful in diagnosis.
[0103] Antibodies may be used in a method for detecting
polypeptides of the invention in a biological sample. Generally
such a method comprises (a) incubating a biological sample with the
antibody under conditions which allow for the formation of an
antibody-antigen complex; and (b) determining whether
antibody-antigen complex comprising the antibody is formed. A
sample may be for example a tissue extract, blood, serum and
saliva. Similarly, a polypeptide of the invention may be used to
detect the presence of anti-IdeS antibodies in a sample, for
example to provide an indicator of S. pyogenes infection.
Preferably, a polypeptide of the invention for use in accordance
with this aspect of the invention comprises a mutant polypeptide
which does not have cysteine protease activity or has reduced
cysteine protease activity, but maintains the ability to bind
IgG.
[0104] Antibodies or polypeptides of the invention may be bound to
a solid support and/or packaged into kits in a suitable container
along with suitable reagents, controls, instructions, etc.
Antibodies or polypeptides may be linked to a revealing label and
thus may be suitable for use in methods of in vivo imaging.
[0105] Antibodies, including antibody fragments are also 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 well known in
the art. These anti-idiotype antibodies may also be useful for
treatment of S. pyogenes, as well as for an elucidation of the
immunogenic regions of polypeptides of the invention.
[0106] The invention is also concerned with modulatory agents which
modulate the IgG cysteine protease activity and/or expression of
the present polypeptides, in particular, agents which inhibit the
activity. The agents may bind to the polypeptides. The agents may
modulate IgG binding of the polypeptides and/or cysteine protease
activity. The present inventors have shown that inhibitors of IdeS
include iodoacetic acid and Z-LVG-CHN.sub.2 and also antibodies to
IdeS.
[0107] Modulatory agents may be identified in screening methods
using the present polypeptides. In general such screening methods
comprise: [0108] (i) contacting a polynucleotide of the invention,
a vector of the invention, a polypeptide of the invention or a cell
of the invention and a test substance under conditions that would
permit IgG cysteine protease activity in the absence of the test
substance; and [0109] (ii) determining thereby whether the said
substance modulates the activity and/or expression of the
polypeptide. Any suitable assay format may be used. Assay formats
which allow high through put screening are preferred.
[0110] The assay may be carried out on a cell harbouring the
polynucleotide or vector or on a cell extract comprising the
polynucleotide or vector. The cells may express the polypeptide
naturally or the polypeptide may be recombinantly expressed. The
cell or cell extract will typically allow transcription and
translation of the polynucleotide or vector in the absence of a
test substance.
[0111] The assay may also be carried out using a polypeptide of the
invention. The polypeptide may be in a purified preparation or for
example in a culture supernatant. Most preferably such an assay
would be carried out in a single well of a plastics microtitre
plate so that high through-put screening may be carried out.
Typically the polypeptide is incubated with a test substance-in the
dark at a temperature of 25 to 42.degree. C. The enzyme reaction is
commenced by addition of IgG. Reaction products may then be
analysed by SDS PAGE.
[0112] In addition to the polypeptide, test substance and IgG
substrate, the reaction mixture may contain a suitable buffer. A
suitable buffer includes any suitable biological buffer that can
provide buffering capability at a pH conducive to the reaction
requirements of the enzyme. The assay of the invention may be
carried out at any temperature at which the polypeptide, in the
absence of inhibitor, is active. Typically the assay will be
carried out in the range of from 25 to 42.degree. C., in particular
at 37.degree. C.
[0113] Typically control assays are carried out in the absence of
the test substance. The substance tested may be tested with any
other polypeptide/enzyme to exclude the possibility that the
substance is a general inhibitor of gene expression or enzyme
activity. Control experiments may be carried out on cells which do
not express the polypeptide of the invention to establish whether
the desired responses are the result of inhibition or activation of
the polypeptide. Preferably the assay is carried out in the
presence of E64, an inhibitor of the SpeB cysteine protease,
particularly where S. pyogenes cells are used in the assay.
[0114] Assays can also be carried out using constructs comprising
an IdeS gene promoter operably linked to a heterologous coding
sequence, to identify compounds which modulate expression of IdeS
at the transcriptional level.
[0115] A promoter means a transcriptional promoter. IdeS promoters
can be isolated via methods known to those skilled in the art and
as described above. The term "heterologous" indicates that the
coding sequence is not operably linked to the promoter in nature;
the coding sequence is generally from a different organism to the
promoter. The promoter may be fused directly to a coding sequence
or via a linker. The linker sequence may comprise a sequence having
enhancer characteristics, to boost expression levels.
[0116] Preferably the promoter is operably linked to the coding
sequence of a reporter polypeptide. The reporter polypeptide may
be, for example, the bacterial polypeptide .beta.-glucuronidase
(GUS), green fluoresent protein (GFP), luciferase (luc),
chloramphenicol transferase (CAT) or .beta.-galactosidase
(lacZ).
[0117] Promoter:reporter gene constructs such as those described
above can be incorporated into a recombinant replicable vector. The
vector may be used to replicate the nucleic acid construct in a
compatible host cell. The vectors may be, for example, plasmid,
virus or phage vectors provided with an origin of replication. Any
host cell may be used in which the promoter is functional, but
typically the host cell will be a cell of the species from which
the promoter derives. The promoter:reporter gene constructs of the
invention may be introduced into host cells using conventional
techniques.
[0118] Thus the invention provides a method for identifying a
modulator of IdeS expression. Typically a promoter:reporter
polypeptide construct or a cell harbouring that construct will be
contacted with a test substance under conditions that would permit
the expression of the reporter polypeptide in the absence of the
test substance.
[0119] Any reporter polypeptide may be used, but typically GUS or
GFP are used. GUS is assayed by measuring the hydrolysis of a
suitable substrate, for example
5-bromo-4-chloro-3-indolyl-.beta.-D-glucoronic acid (X-gluc)or 4
-methylumbelliferyl-.beta.-glucuronide (MUG). The hydrolysis of MUG
yields a product which can be measured fluorometrically. GFP is
quantified by measuring fluorescence at 590 nm after excitation at
494 nm. These methods are well known to those skilled in the
art.
[0120] Test substances may also be assayed directly for binding to
a polypeptide of the invention. For example, a radiolabelled test
substance can be incubated with a polypeptide of the invention and
binding of the test substance to the polypeptide monitored.
Non-specific binding of the test substance may also be determined
by carrying out a competitive binding assay. Substance that inhibit
the interaction of a polypeptide of the invention with IgG may also
be identified using a protein interaction assay such as
immunoprecipitation or an ELISA based technique.
[0121] A test substance which modulates the expression or activity
of a polypeptide of the invention may do so by binding directly to
the relevant gene promoter, thus inhibiting or activating
transcription of the gene. Inhibition may occur by preventing the
initiation or completion of transcription. Activation may occur,
for example by increasing the affinity of the transcription complex
for the promoter. Alternatively a modulator may bind-to a protein
which is associated with the promoter and is required for
transcription.
[0122] A substance which modulates the activity of the polypeptide
may do so by binding to the enzyme. Such binding may result in
activation or inhibition of the protein. Inhibition may occur, for
example, if the modulator resembles,the substrate and binds at the
active site of the enzyme. The IgG is thus prevented from binding
to the same active site and the rate of catalysis is reduced by
reducing the proportion of enzyme molecules bound to substrate. A
modulator which inhibits activity may do so by binding to the
substrate.
[0123] Suitable test substances which can be tested in the above
assays include combinatorial libraries, defined chemical entities
and compounds, peptide and peptide mimetics, oligonucleotides and
natural product libraries, such as display (e.g. phage display
libraries) and antibody products.
[0124] Typically, organic molecules will be screened, preferably
small organic molecules which have a molecular weight of from 50 to
2500 daltons. Candidate products can be biomolecules including,
saccharides, fatty acids, steroids, purines, pyrimidines,
derivatives, structural analogs or combinations thereof. Candidate
agents are obtained from a wide variety of sources including
libraries of synthetic or natural compounds. Known pharmacological
agents may be subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification, etc. to produce structural analogs.
[0125] Test substances may be used in an initial screen of, for
example, 10 substances per reaction, and the substances of these
batches which show inhibition or activation tested individually.
Test substances may be used at a concentration of from 1 nM to 1000
.mu.M, preferably from 1 .mu.M to 100 .mu.M, more preferably from 1
.mu.M to 10 .mu.M. Preferably, the activity of a test substance is
compared to the activity shown by a known activator or
inhibitor.
[0126] A modulator of expression and/or activity of the present
polypeptide is one which produces a measurable reduction or
increase in expression and/or activity in assays such as those
described above. Thus, modulators may be inhibitors or activators
of expression and/or activity.
[0127] Preferred inhibitors are those which inhibit expression
and/or activity by at least 10%, at least 20%, at least 30%, at
least 40% at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 95% or at least 99% at a concentration of
the inhibitor of 1 .mu.g ml.sup.-1, 10 .mu.g ml.sup.-1, 100 .mu.g
ml.sup.-1, 500 .mu.g ml.sup.-1, 1 mg ml.sup.-1, 10 mg ml.sup.-1,
100 mg ml.sup.-1.
[0128] Preferred activitors are those which activate expression
and/or activity by at least 10%, at least 25%, at least 50%, at
least 100%, at least, 200%, at least 500% or at least 1000% at a
concentration of the activator 1 .mu.g ml.sup.-1, 10 .mu.g
ml.sup.-1, 100 .mu.g ml.sup.-1, 500 .mu.g ml.sup.-1, 1 mg
ml.sup.-1, 10 mg ml.sup.-1, 100 mg ml.sup.-1.
[0129] The percentage inhibition or activation represents the
percentage decrease or increase in expression/activity in a
comparison of assays in the presence and absence of the test
substance. Any combination of the above mentioned degrees of
percentage inhibition or activation and concentration of inhibitor
or activator may be used to define an inhibitor or activator of the
invention, with greater inhibition or activation at lower
concentrations being preferred.
[0130] The present invention provides the polypeptides,
polynucleotides, antibodies and agents described above for use in
therapy or prophylexis. In particular, the polypeptides,
polynucleotides, antibodies and agents are useful for the treatment
of S. pyogenes infection of a human or animal. Treatment may be
therapeutic or prophylactic.
[0131] Preferably, the infecting S. pyogenes strain is an
IdeS-expressing strain. Such strains may be identified as described
above. Typically the strain is of the M1, M12 or M55 serotype.
Examples of suitable strains include AP1, AP12, AP55, KTL3 and
SF370. In a preferred embodiment, the strain is of M1 serotype,
such as AP1.
[0132] Conditions which may be usefully targeted include those
associated with acute infection and also sequelae following acute
infection. Examples include but are not limited to impetigo,
pharyngitis, septicaemia, necrotizing fascitis, streptococcal
toxic-shock syndrome, acute rheumatic fever and post-streptoccal
glomerulonephritis.
[0133] Preferably the individual to be treated is human.
[0134] The invention additionally provides pharmaceutical
compositions comprising the polypeptides, polynucleotides,
antibodies or agents of the invention and a pharmaceutically
acceptable carrier or diluent.
[0135] Formulation with standard pharmaceutically acceptable
carriers and/or excipients may be carried out using routine methods
in the pharmaceutical art. For example, an active substance may be
dissolved in physiological saline or water for injections. The
exact nature of a formulation will depend upon several factors
including the particular substance to be administered and the
desired route of administration. Suitable types of formulation are
fully described in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Eastern Pennsylvania, 17.sup.th Ed. 1985, the
disclosure of which is included herein of its entirety by way of
reference.
[0136] The substances may be administered by enteral or parenteral
routes such as via oral, buccal, anal, pulmonary, intravenous,
intra-arterial, intramuscular, intraperitoneal, topical or other
appropriate administration routes.
[0137] The polypeptides and polynucleotides of the invention are
useful for prophylactic treatment of individuals. Typically the
polypeptide or polynucleotide used represents or encodes an epitope
of IdeS. In general, the polypeptide or polynucleotide is capable
of generating an immune, in particular a protective immune response
in the individual to be treated. Preferably antibodies that have
the ability to block the IgG enzymatic activity of IdeS are
generated. Such polypeptides and polynucleotides may be identified
by the methods described above.
[0138] Generally for such uses, the polypeptides, polynucleotides
are incorporated in vaccine compositions.
[0139] Vaccines may be prepared from one or more of the proteins or
peptides of the invention and a physiologically acceptable carrier
or diluent. 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 a liposome. The active immunogenic ingredient may
be mixed with an excipient which is pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol, of the like
and combinations thereof.
[0140] 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 vaccine. Examples of adjuvants which may be
effective include but are not limited to: aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred
to as MTP-PE), and RIBI, which contains three components extracted
from bacteria, monophosphoryl lipid A, trehalose dimycolate and
cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80
emulsion. The effectiveness of an adjuvant may be determined by
measuring the amount of antibodies directed against an immunogenic
polypeptide containing an IdeS antigenic sequence resulting from
administration of this polypeptide in vaccines which are also
comprised of the various adjuvants.
[0141] The vaccines are conventionally administered parentally, by
injection, for example, either subcutaneously or intramuscularly.
Additional formulations which are suitable for other modes of
administration include suppositories, oral formulations and
formulations for transdermal administration. For suppositories,
traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such
normally employed excipients as, for example, pharmaceutical grades
of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, and the like. These
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain 10% to 95% of active ingredient, preferably 25% to 70%.
Where the vaccine composition is lyophilised, the lyophilised
material may be reconstituted prior to administration, e.g. a
suspension. Reconstitution is preferably effected in buffer.
[0142] 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.
[0143] Vaccine compositions suitable for delivery by needleless
injection, for example, transdermally, may also be used.
[0144] The proteins or peptides of the invention may be formulated
into the vaccine as neutral or salt forms. Pharmaceutically
acceptable salts include the acid addition salt (formed with free
amino 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 maleic.
Salts formed with the free carboxyl groups may also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylantino ethanol, histidine
and procaine.
[0145] The vaccines are administered in a manner compatible with
the dosage formulation and in such amount will be prophylactically
and/or therapeutically effective. The quantity to be administered,
which is generally in the range of 5 .mu.g to 100 mg, preferably
250 .mu.g to 10 mg of antigen per dose, depends on the subject to
be treated, capacity of the subject's immune system to synthesize
antibodies, and the degree of protection desired. Precise amounts
of active ingredient required to be administered may depend on the
judgement of the practitioner and may be peculiar to each
subject.
[0146] The vaccine may be given in a single dose schedule, or
preferably in a multiple dose schedule. A multiple does schedule is
one in which a primary course of vaccination may be 1-10 separate
doses, followed by other doses given at subsequent time intervals
required to maintain and or reinforce the immune response, for
example at 1 to 4 months for a second dose, and if needed, a
subsequent dose(s) after several months. The dosage regimen will
also, at least in part, be determined by the need of the individual
and be dependent upon the judgement of the practitioner.
[0147] The nucleotide sequences of the invention and expression
vectors can also be used as vaccine formulations as outlined above.
Preferably, the nucleic acid, such as RNA or DNA, in particular
DNA, is provided in the form of an expression vector, which may be
expressed in the cells of the individual to be treated. The
vaccines may comprise naked nucleotide sequences or be in
combination with cationic lipids, polymers or targeting systems.
The vaccines may be delivered by any available technique. For
example, the nucleic acid may be introduced by needle injection,
preferably intradermally, subcutaneously or intramuscularly.
Alternatively, the nucleic acid may be delivered directly across
the skin using a nucleic acid delivery device such as
particle-mediated gene delivery. The nucleic acid may be
administered topically to the skin, or to mucosal surfaces for
example by intranasal, oral, intravaginal or intrarectal
administration.
[0148] Uptake of nucleic acid constructs may be enhanced by several
known transfection techniques, for example those including the use
of transfection agents. Examples of these agents includes cationic
agents, for example, calcium phosphate and DEAE-Dextran and
lipofectants, for example, lipofectam and transfectam. The dosage
of the nucleic acid to be administered can be altered. Typically
the nucleic acid is administered in the range of 1 pg to 1 mg,
preferably to 1 pg to 10 .mu.g nucleic acid for particle mediated
gene delivery and 10 .mu.g to 1 mg for other routes.
[0149] Inhibitory agents, for example, identified according to the
above screening methods may also be useful in preventing or
treating infection-associated conditions. These agents may be
formulated with standard pharmaceutically acceptable carriers
and/or excipients using routine methods.
[0150] The antibodies and agents of the invention may be useful for
therapeutic treatment of S. pyogenes infections.
[0151] Antibodies of the invention, both polyclonal and monoclonal,
which are neutralising, are useful in passive immunotherapy.
Monoclonal antibodies in particular, may be used to raise
anti-idiotype antibodies as above. These anti-idiotype antibodies
may also be useful for treatment of S. pyogenes, as well as for an
elucidation of the immunogenic regions of polypeptides of the
invention. Antibody fragments, for example, Fab fragments, may also
be useful in immunotherapy of S. pyogenes infection.
[0152] The antibodies of the invention may be formulated with a
pharmaceutically acceptable carrier and delivered in the same way
as set out for the vaccine compositions. Preferably the antibody is
administered in an amount effective to ameliorate S. pyogenes
infection in the individual.
[0153] Inhibitors of IdeS activity, for example those identified by
the above screening methods may be useful for therapeutic treatment
of S. pyogenes infections. These agents may be formulated with
standard pharmaceutically acceptable carriers and/or excipients
using routine methods and delivered in the same way as set out for
the vaccine compositions. The inhibitor is administered to an
individual in a therapeutically effective amount. The dose of an
inhibitor may be determined according to various parameters,
especially according to the substance used; the age, weight and
condition of the patient to be treated; the route of
administration; and the required regimen. A physician will be able
to determine the required route of administration and dosage for
any particular patient.
[0154] In one aspect, an S. pyogenes infection may be treated by
administration of both an antibody and an inhibitory agent. The
antibody and the agent may be administered simultaneously,
separately, or sequentially. Accordingly the invention also relates
to products in which both antibody and agent are supplied for use
in such a treatment regimen.
[0155] The invention is also concerned with the diagnosis of S.
pyogenes infection in an individual, preferably a human. The
polypeptides and antibodies of the invention may be used for such
diagnosis. The polypeptides may be used to detect antibodies
specific to the polypeptides in the individual or vice versa, thus
determining infection. Polypeptides suitable for use in diagnosis
are those which retain specific antibody binding capability. For
example, such polypeptides typically comprise an epitope of IdeS.
Suitable polypeptides can be identified by the methods described
above. Antibodies for use in diagnosis may be identified and
produced by the methods described above.
[0156] The diagnostic method may be practised in vitro or in vivo.
Preferably the method is carried out in Vitro using a sample taken
from the individual to be tested for S. pyogenes infection. A
sample may be for example a tissue extract, blood, serum or saliva.
Generally, the method may involve (i) contacting a biological
sample taken from the individual with a polypeptide or antibody of
the invention under conditions that allow for the formation of an
antibody-polypeptide complex; and (ii) determining whether
antibody-polypeptide complex is formed.
[0157] Typically the polypeptides or antibodies for use in testing
are suitably labelled. Suitable label and detection systems are
known in the art. The polypeptides or antibodies may be bound to a
solid support and/or packaged into kits in a suitable container
along with suitable reagents, controls, instructions, etc.
Antibodies may also be linked to a revealing label and thus may be
suitable for use in methods of in vivo imaging.
[0158] In a further aspect, the present polypeptides may provide
useful tools for biotechnology. For example, the polypeptides may
be used for specific in vitro cleavage of IgG, in particular human
IgG. In such a method, the polypeptide may be incubated with the
sample containing IgG under conditions which permit the specific
cysteine protease activity to occur. Specific cleavage can be
verified, and the cleavage products isolated using the methods
described above. The method of the invention can be used in
particular to generate Fc and Fab fragments. The polypeptides may
be used to cleave IgG in a sample, for example in a method to
remove IgG from a sample. Such methods may be used to assist in
removing immunoglobulin from a sample, or to remove IgG in the
purification of other immunoglobulin.
[0159] Modified polypeptides of the invention which no longer have
cysteine protease activity may be used to bind and isolate or
purify IgG. For example, such polypeptide may be bound to a solid
support and a sample containing IgG contacted with the support
under conditions which allow IgG to bind to the polypeptide. Such a
method may also be used to remove IgG from a sample, the remainder
of the sample, free of IgG being collected for subsequent use or
analysis. IgG may subsequently be desorbed from the solid support
if required.
[0160] The polypeptides may also be used to detect IgG in a sample.
In general such a detection method involves incubating the
polypeptide with the sample under conditions which permit
IgG-specific binding and cleavage. The presence of IgG can be
verified by detection of the specific IgG cleavage products as
above.
EXAMPLES
Materials and Methods
[0161] Unless indicated otherwise, the methods used are standard
biochemistry and molecular biology techniques. Examples of suitable
methodology textbooks include Sambrook et al., Molecular Cloning, A
Laboratory Manual (1989) and Ausubel et al., Current Protocols in
Molecular Biology (1995), John Wiley and Sons, Inc.
Bacterial Strains and Growth Conditions
[0162] S. pyogenes strains used in this study are listed in Table
I. TABLE-US-00002 IgG cleavage (in the PCR Strain M-serotype
Reference or source presence of E64) product SF370 1 (Suvorov and
Feretti, 1996; (+/-) + Ferretti et al., 2001) AP1 1 WHO Prague
collection.sup.a + + AL1 1 speB mutant of AP1 (Collin and +
nd.sup.d Olsen, 2001a) BMJ71 1 mga. mutant of AP1 (Kihlberg et + nd
al., 1995) KTL3 1 Finnish institute for health (+) + AP4 4 WHO
Prague collection - + M5 5 Sequencing in progess.sup.b - + AP6 6
WHO Prague collection - + AP12 12 WHO Prague collection + + AP49 49
WHO Prague collection - + AP53 53 WHO Prague collection - + AP55 55
WHO Prague collection + + AP57 57 WHO Prague collection - +
[0163] Streptococci were routinely grown in Todd Hewitt broth (TH)
(Difco) at 37.degree. C. in 5% CO.sub.2. Strains BMJ71 and AL1 were
grown in the presence of 10 .mu.g/ml tetracycline or 150 .mu.g/ml
kanamycin, respectively. In some cases bacteria were grown in the
presence of the cysteine proteinase inhibitor
trans-epoxysuccinyl-L-leucylamido-(4-Guanidino) butane (E64)
(Sigma).
SDS-PAGE Analysis and N-Terminal Sequence Determination
[0164] Proteins of S. pyogenes growth media were precipitated with
trichloroacetic acid (final concentration 5%), washed twice with 1
ml acetone, and resuspended in sample buffer. Proteins were
separated by 12% SDS-PAGE (all SDS-PAGEs in this work were
performed under reducing conditions) and stained with Coomassie
Blue. For N-terminal amino acid sequence analysis proteins were
separated by 10% SDS-PAGE electrophoresis and blotted onto a PVDF
membrane using 10 mM CAPS buffer, 10% methanol. Proteins were
visualized in 0.1% Coomassie Blue R-250, 50% methanol.
[0165] After destaining in 50% methanol, membranes were dried,
protein bands cut out with a scalpel and stored at -20.degree. C.
until sequencing. Sequencing was performed at Eurosequence Company
(Groningen, The Netherlands).
Purification of IdeS
[0166] IdeS was purified by growing bacteria to an OD.sub.620 of
.about.0.4 and fractionating the culture supernatant with 50%
ammonium sulfate. The resulting precipitate was discarded and
ammonium sulfate was added to the remaining supernatant to a final
concentration of 70%. The second precipitate was resuspended in
1/100 of the starting volume with 20 mM Tris-HCl, pH 8.0, and
dialyzed against the same buffer. The material was further
fractionated by FPLC on a Mono Q column (Pharmacia). Proteins were
eluted by a linear NaCl gradient, and a peak eluted at 0.1M NaCl
was found to contain the IdeS activity. Corresponding fractions
were collected, analyzed by SDS-PAGE, and saved at -20.degree. C.
until use.
IdeS Activity Assays
[0167] For standard IdeS activity assays, bacteria cultures were
grown to OD.sub.620=0.4. Bacteria were pelleted by centrifugation
and supernatants were sterile-filtered through a 0.22 .mu.m
membrane (Millipore) prior to use. For activity assays, 25 .mu.l of
supernatant were mixed with 5 .mu.l of IgG (10 mg/ml, Sigma) and
the volume was adjusted with PBS to 100 .mu.l. For screening of
IdeS activity in different S. progenes strains, E64 was added to a
final concentration of 40 .mu.M. The mixtures were incubated at
37.degree. C. for 30 min and samples were analyzed by 12% SDS-PAGE.
For cleavage assays of different classes of Ig, purified IdeS (0.3
.mu.g/ml) was incubated with 3 .mu.g 1 g for 2 h at 37.degree. C.,
and analyzed by 12% SDS-PAGE analysis.
PCR Analysis of Genomic DNA for Identification of Ides
[0168] To analyze the presence of the ideS gene in different
streptococcal isolates, PCR template DNA was prepared by boiling S.
pyogenes bacteria for 5 min in sterile water.
[0169] Cell debris was removed by centrifugation and 1 .mu.l of the
boiled lysate was used with PCR primers Ide1 (5'-CGT TAC TTC CGT
TTG GAT CCA AGG-3') and Ide2 (5'-GAA ATA GCT ACT TCT CGA GCG GAA
TT-3'). PCR products were analyzed by agarose (1%) gel
electrophoresis.
Recombinant Expression of IdeS in E. Coli
[0170] For PCR amplification of ideS, template DNA was prepared by
boiling S. pyogenes bacteria (strain AP1) in sterile water. The
cell debris was removed by centrifugation and 5 .mu.l of the boiled
lysate was used with PCR primers Ide5X (5'-TCG GTA GAT CGT GGG ATC
CTA GCA GAT AGT-3') creating a BanmHI restriction site, and Ide3X
(5'-CGG AAT TCT TAA TTG GTC TGA TTC CAA C-3'), creating an EcoRI
restriction site. A PCR fragment covering bp 79-1020 of the intact
ideS gene was generated, cleaved with restriction enzymes, and
cloned into the corresponding sites of plasmid pGEX-5X-3 (Amersham
Pharmacia Biotech). The resulting plasmid was transformed into
Escherichia coli strain BL21(DE3) pLysS, according to standard
protocols (Sambrook et al., 1989). Protein expression was induced
by addition of 0.1 mM isopropyl-.beta.-D-thiogalactopyranoside
(IPTG) at an OD.sub.620 of .about.0.2. Growth was continued for 3
h, and lysates were prepared by freezing bacterial pellets at
-70.degree. C., followed by resuspension in PBS. Cell debris was
removed by centrifagation and 2 .mu.L of supernatant was incubated
with 5 .mu.l of IgG (10 mg/ml) in PBS, and separated by 12%
SDS-PAGE for analysis of recombinantly expressed IdeS.
Proteinase Inhibition Assays
[0171] Partially purified IdeS (0.3 .mu.g/ml) was incubated with
either 20 mM iodoacetic acid, Z-LVG-CHN.sub.2 (Bjorck et al., 1989)
at 0.4 mg/ml in 1% DMSO, or E64 (40 .mu.M). The tubes were kept in
the dark and incubated for 30 min at room temperature. As controls,
IdeS was also kept in phosphate buffered saline (PBS) or in 1%
DMSO, the solvent for Z-LVG-CIN.sub.2. After 30 min, 5 .mu.l of 10
mg/ml IgG (Sigma) were added, and the volume was adjusted to 100
.mu.l with PBS. Incubation was continued for 60 min at 37.degree.
C. The reaction was stopped by the addition of SDS-PAGE sample
buffer and samples were analyzed by 12% SDS-PAGE.
Cell Culture and Infection of Eukaryotic Cells
[0172] The murine macrophage-like cell line RAW 264.7 was cultured
in RPMI 1640 medium (Life Technologies), supplemented with 10% FCS,
and antibiotics (100 units/ml.sup.-1 penicillin; 100 .mu.g
ml.sup.-1 streptomycin), at 5% CO.sub.2 with 100% relative
humidity.
[0173] To study phagocytic killing, S. pyogenes strain AP1 was
grown overnight at 37.degree. C. AP1 bacteria were incubated with
either immune or non-immune plasma, washed and treated with IdeS or
a buffer control, for 2 h at 37.degree. C. Subsequently, bacteria
were washed and diluted in antibiotic-free cell culture medium
prior to infection. Cell lines were carefully washed in
antibiotic-free cell culture medium and bacteria were added (0.1-1
bacteria/cell) to confluent RAW264.7 cells. Infections were
synchronized by gentle centrifugation at 400 g for 3 min by
incubation at 37.degree. C. Ten minutes after infection, the cell
cultures were carefully washed in antibiotic-free medium to remove
non-adherent bacteria (time 0 h). Control cells were lysed in
ice-cold lysis buffer (0.1% Tween), diluted, and spread onto TH
plates. Parallel cell cultures were incubated at 37.degree. C. for
1 h. Subsequently, growth media was removed and cells were lysed
and treated as described above. For analysis of bacterial survival
the number of surviving bacteria after 1 h was divided by the
number of adherent bacteria at time 0 h.
Results
S. pyogenes Secretes an IgG-Cleaving Enzyme Distinct from SpeB, the
Classical Streptococcal Cysteine Proteinase
[0174] The proteolytic activity of extracellular enzymes of S.
pyogenes strain AP1, was analyzed by growing AP1 bacteria in Todd
Hewitt (TH) medium supplemented with 10% human plasma. Following
growth to stationary phase, bacteria were removed by centrifugation
and the supernatant was subjected to SDS-PAGE (all SDS-PAGE's in
this work were performed under reducing conditions). The band
pattern was compared to the pattern of human plasma proteins that
had not been in contact with bacteria (data not shown). The
bacterial supernatant contained a protein band of approximately 31
kDa, which was absent in the plasma control. The N-terminal
sequence of this protein was determined to GPSVFLFP, which
corresponds to amino acids 237-244 of the hinge region of human
IgG.sub.1 (FIG. 1). Recent work has shown that the streptococcal
cysteine proteinase, SpeB, cleaves IgG at this site. However, most
strains of S. pyogenes, including AP1, do not express the speB gene
in TH medium. Moreover, the proteolytic activity of SpeB is
efficiently blocked by the specific cysteine proteinase inhibitor
E64, but E64 did not inhibit the cleavage of IgG when added to the
AP1 growth medium, as evidenced by the continued generation of the
31 kDa IgG cleavage product (data not shown). In addition, growth
medium from the isogenic SpeB deficient mutant strain AL1, also
contained IgG-cleaving activity. Taken together, these data
demonstrate that SpeB is not responsible for the cleavage of IgG in
TH medium.
[0175] The streptococcal strain AP1 studied here, expresses a
surface-associated C5a peptidase, and the IgGFc binding proteins, H
and M1. The genes encoding these surface proteins are controlled by
the transcriptional activator Mga, and BMJ71 is an isogenic mutant
of AP1, carrying a Tn916 insertion within the mga gene. The 31 kDa
IgG cleavage product is also generated in growth medium of BMJ71,
showing that the proteolytic activity is not under Mga control.
Purification and Sequence Characteristics of IdeS, a Novel
Proteinase of S. pyogenes
[0176] As the IgG proteolytic activity was found in the growth
medium of strain AP1 we fractionated culture medium proteins after
bacterial growth by adding increasing amounts of ammonium sulfate
(10 to 80%). These initial experiments revealed that precipitates
of 60 to 70% ammonium sulfate contained most of the IgG-cleaving
activity. For purification, the growth medium was fractionated with
50% ammonium sulfate, the resulting pellet was discarded and the
ammonium sulfate concentration in the supernatant was adjusted to
70%. Proteins pelleted by this second precipitation were subjected
to ion-exchange chromatography and peak fractions were tested for
enzymatic activity. Maximum IgG-cleaving activity was eluted at
0.1M NaCl and the corresponding fractions contained a major band of
approximately 34 kDa as judged by SDS-PAGE. This protein band was
excised and subjected to N-terminal sequence analysis. The sequence
obtained, DSFSANQEIRY, was used to search the Streptococcal Genome
Sequencing Project (SGSP) database. A perfect match was found in an
open reading frame of 339 amino acids designated SPy0861. The
obtained N-terminal sequence corresponds to amino acids 30-40 (FIG.
2) and was preceded by a potential signal sequence of 29 amino
acids as predicted by the SignalP algorithm. The protein does not
contain a cell wall attachment signal (LPXTGX), a common feature of
cell wall-anchored proteins of S. pyogenes, and the predicted size
of the protein, without the potential signal sequence, is 34.9 kDa,
which is in accordance with the size of the purified protein
estimated by SDS-PAGE. Apart from the putative signal sequence, the
protein has an RGD motif at amino acids 214-216 (FIG. 2). This
motif is important for ligand recognition by integrins, and a
variety of bacterial and viral pathogens have been shown to bind to
host cell integrins. The full-length putative protein sequence was
used in a similarity search against the DDBJ/EMBL/GenBank database
using a BLASTp algorithm. This search revealed no similarities to
any prokaryotic protein and a weak similarity (24% identity in a
region of 204 amino acid residues) to human MAC-1 integrin alpha M
precursor (Arnaout et al., 1988). Due to the absence of any
previously reported function, and based on the enzymatic activity
against human IgG, the protein was denoted IdeS, for Immunoglobulin
degrading enzyme of Streptococcus pyogenes.
[0177] To further confirm that the identified IdeS protein has IgG
cleaving activity, the ideS gene was cloned in plasmid pGEX-5X-3
(Amersham Pharmacia Biotech) and expressed in Escherichia coli.
Partially purified lysates were incubated with IgG and analyzed by
SDS-PAGE. Lysates from E. coli carrying the ideS gene generated the
31 kDa IgG-derived band, whereas extracts from cells carrying only
a plasmid control did not cleave IgG (data not shown).
IdeS is a Novel Cysteine Proteinase Highly Specific for IgG
[0178] We noticed that the sequence of the IdeS protein contains a
single cysteine residue at position 94 (FIG. 2). Despite the lack
of sequence homology to other cysteine proteinases, IdeS also has a
histidine residue at a distance (His 224) from the cysteine, which
is often found in other cysteine proteinases, although the
enzymatic activity was not inhibited by the cysteine proteinase
inhibitor E64. The peptide derivate Z-LVG-CHN.sub.2, structurally
based on the inhibitory reactive site of cystatin C and carrying a
diazomethyl ketone group to inactivate the sulfhydryl group of the
catalytic cysteine, has previously been shown to irreversibly
inhibit papain and SpeB. Moreover, cysteine proteinases are also
inactivated by iodoacetic acid through an irreversible modification
of the catalytic sulfhydryl group. We therefore investigated
whether treatment with these specific inhibitors would affect the
enzymatic activity of IdeS. Analysis of IgG incubated with IdeS
alone or with IdeS preincubated with inhibitors, revealed that
Z-LVG-CHN.sub.2 and iodoacetic acid efficiently inhibited the
activity of IdeS whereas E64 had no effect on the enzyme (data not
shown). The activity of IdeS, its sequence characteristics, and
inhibition profile, establish IdeS as a new member of the cysteine
proteinase family.
[0179] Recently the streptococcal cysteine proteinase SpeB was
shown to cleave the heavy chains of all classes of human
immunoglobulins; IgG, IgM, IgA, IgD, and IgE. In contrast, when
human IgG, IgM, IgA, IgD, or IgE, were incubated with purified IdeS
for 2 h at 37.degree. C., only IgG was degraded (data not shown).
We also analyzed the activity of IdeS against the different
subclasses of IgG, and found that all were susceptible for IdeS
digestion, although, when compared to the other subclasses, IgG2
was less efficiently digested (not shown). The high specificity of
IdeS is further emphasized by the observation that only the 31 kDa
IgG-derived band and no additional degradation products could be
identified following incubation of human plasma with purified IdeS.
Although it cannot be excluded that the enzyme has other
substrates, these data show that IdeS has a higher degree of
specificity for IgG than any previously described proteinase.
Distribution and expression of the ideS Gene in S. Pyogenes
Strains
[0180] The distribution of ideS among S. pyogenes strains was
investigated by PCR analysis using primers designed to amplify the
internal coding region of ideS. We analyzed chromosomal DNA
preparations from 11 S. pyogenes strains of 9 different M
serotypes, and were able-to amplify identical PCR fragments of the
expected size from all strains (Table I, data not shown). However,
when analyzing the cleavage of IgG during bacterial growth in TH
medium, only five of the tested strains expressed the IgG-degrading
activity (AP1, KTL3, SF370, AP12, and AP55), and among these
strains KTL3 and SF370 showed weak activity. Thus, although the
IdeS gene seems to be present in all S. pyogenes isolates,
expressed enzyme activity under the conditions used here, is
restricted to some strains and varies even within the same M
serotype (Table I). The secretion pattern of the IgG-cleaving
activity during growth of strain AP1 in TH medium was also
investigated. Samples were taken from the growth medium at
different time points during bacterial growth, and tested for
enzymatic activity against IgG. IgG-degrading activity started to
appear in samples taken during early logarithmic growth phase, and
the activity increased during logarithmic growth as determined from
the degree of IgG cleavage. The enzymatic activity did not further
increase in stationary phase supernatants but appeared to be
persistent at a constant level (FIG. 3).
Fc-Mediated Phagocytosis and Killing of S. Pyogenes is Inhibited by
IdeS
[0181] Opsonizing IgG antibodies bound to surface antigens of S.
pyogenes will expose their Fc regions to complement factor C1q and
Fc.gamma.-receptors of phagocytic cells, and thereby facilitate
phagocytosis and killing of the bacteria. To test the hypothesis
that IdeS by proteolytic cleavage of IgG, could interfere with this
defense mechanisms, AP1 bacteria were incubated with either human
immune or non-immune plasma. After incubation bacteria were washed
and incubated with IdeS, or with a buffer control, followed by
another washing step to remove IdeS and degradation products.
Confluent RAW264.7 macrophage-like cells were then infected with
these bacteria at .about.0.1-1 bacteria/cell. Infections were
synchronized by gentle centrifugation and cells were lysed
immediately to determine the number of cfu's at time zero. In
parallel infections, cell cultures were carefully washed to remove
non-adherent bacteria and incubations were continued for 1 h, after
which cells were lysed and the number of cfu's was determined. The
ratios of cfu's at time 1 h, divided by the number of cfu's at time
zero were determined as survival factors and are shown in FIG. 3.
The relatively short incubation time was chosen to minimize IgG
independent phagocytosis. While bacteria incubated in non-immune
plasma survived contact with macrophage-like cells, the number of
bacteria, which had been exposed to opsonizing immunoglobulins in
immune plasma was significantly reduced (p<0.03) in the presence
of macrophages. However, this effect was abolished when bacteria
carrying opsonizing IgG were treated with IdeS prior to incubation
with phagocytes (FIG. 3).
[0182] AP1 bacteria express surface proteins that bind several
abundant human plasma proteins. As previously reported, following
plasma absorptions, the major protein bands eluted from AP1
bacteria represent albumin, fibrinogen, and IgG heavy and light
chains. The same protein pattern was obtained following absorption
of non-immune or immune plasma, and in both cases IgG was cleaved
by IdeS, generating IgGFc fragments, which under the reducing
conditions used, give rise to the 31 kDa band (data not shown).
These Fc fragments are associated with IgGFc-binding proteins,
interactions that efficiently block their capacity to bind
complementation factor C1q. However, as shown in FIG. 3, IdeS
protects bacteria preincubated with plasma containing specific IgG
antibodies. These antibodies are bound to the streptococcal surface
via their antigen-binding Fab regions, suggesting that cleavage of
this IgG population by IdeS will result in the removal of Fc
fragments from the bacterial surface. These data demonstrate that
cleavage of IgG by IdeS can occur at the bacterial surface and that
IgG cleavage by IdeS increases the capacity of S. pyogenes to evade
phagocytic cells.
[0183] The streptococcal cysteine proteinase SpeB, is
well-established as a virulence determinant, and SpeB was recently
shown to cleave the hinge region of IgG and to degrade the heavy
chains of all human immunoglobulin classes. Therefore, the
discovery of an additional extracellular cysteine proteinase in S.
pyogenes was unexpected. However, at least under laboratory
conditions, SpeB is not expressed until S. pyogenes reaches
stationary growth phase, which makes a possible function of SpeB as
an enzyme cleaving opsonizing IgG questionable. Thus, it should be
important for such a proteinase to be present continuously during
infection. IdeS production starts already during early logarithmic
growth and continues into late stationary growth phase, which makes
the enzyme more suitable to remove opsonizing IgG from
the-bacterial surface. -Still, the actions of IdeS and SpeB could
well be complementary. In fact, the identification and
characterization of IdeS might explain some previous and puzzling
observations. IdeS is not affected by the cysteine proteinase
inhibitor E64, but is inhibited by a synthetic peptide derivative
(Z-LVG-CHN.sub.2), structurally based on the proteinase-binding
center of cystatin C, a human cysteine proteinase inhibitor.
Z-LVG-CHN.sub.2 and E64 both irreversibly block the proteolytic
activity of SpeB, but only Z-LVG-CHN.sub.2 inhibited streptococcal
growth in vitro and in vivo. However, the observation that IdeS is
inhibited by Z-LVG-CHN.sub.2, but not by E64, suggests that the
previously observed effect of Z-LVG-CHN.sub.2 on S. pyogenes growth
and virulence, could be due to interference with both SpeB and
IdeS.
[0184] In severe invasive S. pyogenes infections, strains of the M1
serotype are the most common, and the AP1 strain studied here and
expressing IdeS, is of this serotype. Strains of serotypes M12 and
M55, also producing proteolytically active IdeS under the growth
conditions used, are phylogenetically closely related, and
represent clinically relevant strains often connected with
post-streptococcal glomerulonephritis. This correlation suggests a
role for IdeS both during acute infections and in aseptic sequelae
following acute S. pyogenes infections.
[0185] IgG is the dominant Ig class and IgGFc has important
functions in complement activation and recruitment of phagocytic
cells. Moreover, Fc.gamma. receptors are expressed by all
immunologically active cells. It seems that S. pyogenes has evolved
a specific IgG-cleaving enzyme, and its specificity underlines a
potential role for IdeS in preventing contact between S. pyogenes
and phagocytes,by cleaving opsonizing IgG in the hinge region.
Opsonizing IgG antibodies bind to various S. pyogenes surface
structures via the Fab regions. However, most S. pyogenes strains
express surface proteins of the M protein family with affinity for
IgGFc. The AP1 strain studied here has two such proteins, proteins
H and M1, which are structurally closed related. Large amounts of
these IgGFc-binding proteins are present at the bacterial surface,
and bind IgG with high affinity. As a result, AP1 bacteria
surrounded by plasma or inflammatory exudate, are covered with IgG
bound to these proteins through the IgGFc-binding proteins. This
IgG population will be present in vast amounts compared to antigen
specific IgG bound to the bacterial surface via Fab. However, the
data reported here demonstrate that IdeS not only cleaves
opsonizing antibodies, but also IgG bound to the surface via
Fc.
Results of Further Studies
1) Substrate Specificity
[0186] IdeS exhibits high substrate specificity and has so far been
found to cleave only IgG. We examined whether IdeS could have
additional substrates e.g. eukaryotic plasma proteins. An extensive
search for additional substrates and natural occurring inhibitors
was performed.
[0187] i) Species Restrictions
[0188] IdeS was found to cleave human, rabbit and goat IgG, but not
murine IgG, although the primary amino acid sequences at the
cleavage site are conserved between the species.
[0189] ii) Cleavage or Other Human Proteins
[0190] No cleavage products could be detected when IdeS was
incubated with whole human plasma or with fibrinogen, fibronectin,
albumin, transferrin, lactoferrin, laminin, H-kininogen,
.alpha.1-antitrypsin, aprotihin, cystatin D or cystatin C.
[0191] No cleavage products could be observed when cystatin C from
either chicken, rat or mouse were incubated with IdeS.
[0192] iii) Activity Towards Natural and Synthetic Peptides
[0193] Incubation of IdeS with antimicrobial peptide LL-37 or a
synthetic peptide homologous to amino acids 234 to 241 of human IgG
did not reveal any degradation of the peptides. Neither was the
synthetic peptide Z-LVG-CHN.sub.2, which inhibits IdeS activity,
cleaved by excess IdeS.
[0194] Thus, to date the only known substrate for IdeS is IgG.
However, we found that a Fc-fragment generated by papain cleavage,
including 12 amino acids of the hinge region is cleaved by IdeS,
suggesting that the recognition site of IdeS on human IgG is
located in the C.gamma.2 region of the molecule. The
C.gamma.2-C.gamma.3 interphase region of IgG has been ruled out as
binding site for IdeS, as streptococcal proteins H and G, both of
which bind to this region, cannot inhibit IdeS activity.
2) Neutralizing Antibodies Against the Enzymatic Activity of
IdeS
[0195] An extensive analysis of antibody titers against IdeS in 80
healthy blood donors and 70 patients suffering from invasive S.
pyogenes infections was performed.
[0196] All blood donors showed detectable antibody levels against
IdeS, emphasizing that IdeS is expressed iii vivo during the course
of streptococcal infections. Patients with invasive S. pyogenes
infections showed significantly higher acute phase antibody levels
against IdeS (mean ELISA index increase from 0.64 in blood donors
to 1.24 in patient sera; p<0.05). Several serum samples from
patients recovering from streptococcal infections (pharyngitis and
sepsis) show increased antibody titers against IdeS compared to
acute phase titers. Antibodies in the majority of the convalescent
sera analyzed, but not in all, had the ability to block the
enzymatic activity of IdeS. This finding is a very strong
indication for the importance of the enzymatic activity in vivo.
Sequence CWU 1
1
10 1 310 PRT S. Pyogenes 1 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg
Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr
Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp
Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp
Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly
Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80
Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85
90 95 Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala
Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu
Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His
Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn
Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro
Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp
Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu 180 185 190 Thr Ser
Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205
Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His 210
215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly
Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr
Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys
Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser
Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu
Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln
Thr Asn 305 310 2 339 PRT S. Pyogenes 2 Met Arg Lys Arg Cys Tyr Ser
Thr Ser Ala Ala Val Leu Ala Ala Val 1 5 10 15 Thr Leu Phe Val Leu
Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25 30 Ser Ala Asn
Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 35 40 45 Thr
Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50 55
60 Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr
65 70 75 80 Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys
Gly Ala 85 90 95 Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp
Gln Asn Lys Asp 100 105 110 Gln Ile Lys Arg Tyr Leu Glu Glu His Pro
Glu Lys Gln Lys Ile Asn 115 120 125 Phe Asn Gly Glu Gln Met Phe Asp
Val Lys Glu Ala Ile Asp Thr Lys 130 135 140 Asn His Gln Leu Asp Ser
Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala 145 150 155 160 Phe Pro Tyr
Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val 165 170 175 Ile
Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185
190 Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe
195 200 205 Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr
Ser Arg 210 215 220 His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser
Asp Leu Ile Lys 225 230 235 240 Lys Glu Leu Thr Glu Gly Lys Ala Leu
Gly Leu Ser His Thr Tyr Ala 245 250 255 Asn Val Arg Ile Asn His Val
Ile Asn Leu Trp Gly Ala Asp Phe Asp 260 265 270 Ser Asn Gly Asn Leu
Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275 280 285 Ala Ser Ile
Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290 295 300 Lys
Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala 305 310
315 320 Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp
Asn 325 330 335 Gln Thr Asn 3 1020 DNA S. Pyogenes 3 atgagaaaaa
gatgctattc aacttcagct gcagtattgg cagcagtgac tttatttgtt 60
ctatcggtag atcgtggtgt tatagcagat agtttttctg ctaatcaaga gattagatat
120 tcggaagtaa caccttatca cgttacttcc gtttggacca aaggagttac
tcctccagca 180 aacttcactc aaggtgaaga tgtttttcac gctccttatg
ttgctaacca aggatggtat 240 gatattacca aaacattcaa tggaaaagac
gatcttcttt gcggggctgc cacagcaggg 300 aatatgcttc actggtggtt
cgatcaaaac aaagaccaaa ttaaacgtta tttggaagag 360 catccagaaa
agcaaaaaat aaacttcaat ggcgaacaga tgtttgacgt aaaagaagct 420
atcgacacta aaaaccacca gctagatagt aaattatttg aatattttaa agaaaaagct
480 ttcccttatc tatctactaa acacctagga gttttccctg atcatgtaat
tgatatgttc 540 attaacggct accgccttag tctaactaac cacggtccaa
cgccagtaaa agaaggtagt 600 aaagatcccc gaggtggtat ttttgacgcc
gtatttacaa gaggtgatca aagtaagcta 660 ttgacaagtc gtcatgattt
taaagaaaaa aatctcaaag aaatcagtga tctcattaag 720 aaagagttaa
ccgaaggcaa ggctctaggc ctatcacaca cctacgctaa cgtacgcatc 780
aaccatgtta taaacctgtg gggagctgac tttgattcta acgggaacct taaagctatt
840 tatgtaacag actctgatag taatgcatct attggtatga agaaatactt
tgttggtgtt 900 aattccgctg gaaaagtagc tatttctgct aaagaaataa
aagaagataa tattggtgct 960 caagtactag ggttatttac actttcaaca
gggcaagata gttggaatca gaccaattaa 1020 4 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 4 cgttacttcc gtttggatcc
aagg 24 5 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 5 gaaatagcta cttctcgagc ggaatt 26 6 30 DNA
Artificial Sequence Description of Artificial Sequence Primer 6
tcggtagatc gtgggatcct agcagatagt 30 7 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 7 cggaattctt aattggtctg
attccaac 28 8 8 PRT Homo sapiens 8 Gly Pro Ser Val Phe Leu Phe Pro
1 5 9 11 PRT S.Pyogenes 9 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg
Tyr 1 5 10 10 6 PRT Bacteria Xaa (1)..(6) ANY AMINO ACID 10 Leu Pro
Xaa Thr Gly Xaa 1 5
* * * * *
References