Note: Descriptions are shown in the official language in which they were submitted.
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CLOSTR:DIUM DIFFICILE VACCINE
FIELD
[0001] The present disclosure provides compositions and methods for
prevention or
treatment of Clostridium difficile infection. More specifically, the
disclosure relates to a
Clostridium difficile spore glycoprotein as well as glycopeptides and glycans
thereof, and their
use as a vaccine against Clostridium difficile.
BACKGROUND
[0002] Clostridium difficile (C. difficile) is a Gram positive, spore
forming anaerobe that is a
major cause of antibiotic-associated diarrhoea. The incidence of C. difficile
infection (CDI) has
been rapidly increasing in North America and Europe in recent years and this
increase in
infections has been associated with higher rates of morbidity and mortality.
Recent estimates of
the incidence of C. difficile associated diarrhoea (CDAD) in the U.S. indicate
as many as
500,000 cases per year with up to 20,000 deaths (Rupnik, M. et al.,
Microbiology 7:526-536,
2009; Ananthakrishnan, A.N. Gastroenterology & Hepatology 8:17-26, 2011).
[0003] Much C. difficile research has focused on two toxins, TcdA and TcdB,
that are
produced by C. difficile and that cause tissue damage and a severe
inflammatory response,
leading in serious cases to potentially lethal pseudomembranous colitis. While
toxin activity is
recognised as the major virulence factor associated with CDAD, other aspects
of C. difficile
virulence are less well understood.
[0004] Spore production in C. difficile is an integral part of the
infectious process. This
recalcitrant, dormant form of C. difficile can survive indefinitely outside
the host and is known to
persist in the hospital environment. It has been demonstrated in mice that
antibiotic treatment
suppresses the diversity of the gut microbiome and promotes the production of
these highly
infectious spores, which are then disseminated into the environment
("Supershedder state")
(Lawley, T.D. et al., Infection and immunity 77: 3661-3669, 2009). As such,
more recently there
has been increased attention on the process of spore formation in C. difficile
as well as studies
of spore structure and biochemical composition. To date, the focus of studies
on spore structure
has been to identify spore coat proteins and demonstrate enzymatic activity.
Spores are
typically pretreated either by enzymatic digestion or sonication to remove the
exosporangial
layer prior to analysis. However, although considerable progress has been made
recently in the
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analysis of spore coat proteins from C. difficile, the identification and
characterisation of both the
exosporangial and glycan-containing components is less well advanced, and the
surface
polysaccharides remain relatively poorly understood.
[0005]
Current therapies for treatment of CDI target the vegetative phase of the
organism's
life cycle. Among these treatments are antibiotics such as vancomycin and
metronidazole.
However, antibiotics are not always effective, and the use of fluoroquinolone
antibiotics, such as
ciprofloxacin and levofloxacin, has unfortunately led to the emergence of new,
highly virulent,
and antibiotic resistant strains of C. difficile. Few effective treatments
exist for patients with
multiple recurrences of C. difficile infection. Fecal bacteriotherapy, or
"stool transplant" has
been used to re-establish normal intestinal bacterial flora in a patient by
transplanting stool from
a healthy donor into the patient's intestine (Bakken, J.S., Anaerobe 2009,
15:285-9; Rohlke, F.
et al., J. Clin. Gastroenterol. 2010, 44(8):567-70). However, stool
transplants can contain
pathogenic bacteria and viruses, are not reproducible and controllable, and
often carry a
psychological stigma for the patient.
[0006]
Vaccine approaches to date have focused on the A and B toxins and vegetative
cell
surface proteins (SLPAs) that are produced by metabolically active bacteria.
International PCT
Application Publication No. WO 2013/084071 describes recombinant fragments of
C. difficile
TcdA and TcdB and their use in the development of vaccines against C.
difficile associated
disease, particularly combinations of a ToxB-GT antigen and a TcdA antigen or
a ToxA-GT
antigen and a TcdB antigen. However, the toxins are produced by vegetative
cells rather than
spores, and anti-toxin strategies only neutralize the toxin without killing
the bacteria.
[0007]
Carbohydrate-based C.difficile vaccines have been described (Oberli, M.A. et
al.,
Chemistry and Biology 18: 580-588, 2011; Monteiro, M.A. et al., Expert Rev.
Vaccines 12: 421-
31, 2013; Martin, C.E. et al., J. Am. Chem. Soc. 135: 9713-22, 2013). These
studies describe
the design and immunogenicity of vaccines composed of raw polysaccharides and
conjugates
thereof containing the PS-I or PS-II surface glycans from C. difficile.
However, such
approaches address primary infection by vegetative stage bacteria, but do not
target the
recalcitrant, dormant, but still infectious, spores.
[0008]
International PCT Application Publication No. WO 2013/071409 describes a novel
lipoteichoic acid (LTA) isolated from C. difficile and its use as a vaccine
against CDI and as a
diagnostic antigen. Use of LTA-based glycoconjugates as vaccines to combat CDI
has also
been explored by Cox, A.D. et al. (Glycoconj. 30: 843-55, 2013). However, LTA
is found
primarily on vegetative cells and cannot be used to target spores
specifically.
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[0009]
International PCT Application Publication No. WO 2012/092469 describes
compositions and methods for the treatment or prevention of CDI in a
vertebrate subject.
Compositions containing an antibody or fragment that binds to a C. difficile
spore polypeptide or
fragment are described, where the spore polypeptide or fragment can be BcIA1,
BcIA2, BcIA3,
Alr, SIpA paralogue, SIpA HMW, CD1021, lunH, Fe-Mn-SOD, or FliD. Methods of
reducing or
preventing CU in a subject are also described, comprising administering to the
subject a C.
difficile spore polypeptide or fragment or variant, which can be BcIA1, BcIA2,
BcIA3, Alr, SIpA
paralogue, SIpA HMW, CD1021, lunH, Fe-Mn-SOD, or FliD. However, BcIA3
glycosylation is
not described, and antisera from mice immunized with BcIA3 polypeptide show no
reactivity with
C. difficile spores, suggesting that BcIA3 polypeptide is not an effective
immunogen.
[0010] There
is a need for a safe and effective vaccine composition for preventing or
treating CDI based on targeting C. difficile spores.
SUMMARY
[0011] It is
an object of the present invention to ameliorate at least some of the
deficiencies
present in the prior art. Embodiments of the present technology have been
developed based on
the inventors' appreciation that there is a need for improved compositions and
methods for
prevention and/or treatment of CDI.
[0012] The
present disclosure relates to a C. difficile spore glycoprotein and uses
thereof.
More specifically, there is provided herein a C. difficile BcIA3 spore
glycoprotein, as well as
glycopeptides and glycans thereof, and their use as a vaccine to prevent or
treat CDI.
[0013]
According to a first aspect of the invention, there is provided an isolated C.
difficile
spore BcIA3 glycoprotein or a glycopeptide thereof. The C. difficile BcIA3
glycoprotein may
comprise the full-length protein or an immunogenic glycopeptide thereof.
Functionally
equivalent or biologically active homologs, fragments, analogs and/or variants
thereof are also
encompassed.
[0014] In an
embodiment, an isolated BcIA3 glycoprotein or glycopeptide comprises the
amino acid sequence set forth in any one of SEQ ID NOs: 1-32, or a homolog,
fragment,
analog, or variant thereof. In
another embodiment, an isolated BcIA3 glycoprotein or
glycopeptide comprises an amino acid sequence at least about 80%, at least
about 85%, at
least about 90%, or at least about 95% identical to the amino acid sequence
set forth in any one
of SEQ ID NOs: 1-32, or a homolog, fragment, analog, or variant thereof.
Isolated BcIA3
glycoproteins and glycopeptides are linked to BcIA3 glycans, as described
herein.
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[0015] In some embodiments, an isolated BcIA3 glycoprotein or glycopeptide
comprises
one or more chain of three or more N-acetyl hexosannine (HexNAc) moieties 0-
linked through a
threonine residue. For example, a BcIA3 glycoprotein or glycopeptide may
comprise three or
more HexNAc moieties, four or more I- 'exNAc moieties, or five or more HexNAc
moieties. In an
embodiment, each HexNAc moiety has a molecular weight of about 203 Da. In
another
embodiment, a BcIA3 glycoprotein or glycopeptide further comprises a glycan
capping moiety at
the end of the HexNAc chain. The glycan capping moiety may have a molecular
weight of about
203 Da, about 215 Da, about 220 Da, 372 Da, about 374 Da, about 429 Da, about
486 Da,
about 462 Da, about 375 Da, about 424 Da, or about 552 Da. In an embodiment,
an isolated
BcIA3 glycoprotein or glycopeptide comprises one or more GIcNAc residue as a
component of
the glycan. In an embodiment, an isolated BcIA3 glycoprotein or glycopeptide
comprises a
single 0-linked N-acetyl glucosamine (GIcNAc) moiety. In yet another
embodiment, a an
isolated BcIA3 glycoprotein or glycopeptide comprises a single HexNAc moiety.
[0016] In yet another embodiment, an isolated BcIA3 glycoprotein or
glycopeptide
comprises a glycopeptide having the nLC-MS/MS spectrum shown in any one of
Figures 3a, 3b,
8a, and 8b. In some embodiments, an isolated BcIA3 glycoprotein or
glycopeptide consists of a
glycopeptide having the nLC-MS/MS spectrum shown in any one of Figures 3a, 3b,
8a, and 8b.
[0017] In another aspect, there is provided an isolated C. difficile spore
BcIA3 glycan. A
BcIA3 glycan may comprise, for example, one or more chain of three or more N-
acetyl
hexosannine (HexNAc) moieties, optionally comprising a glycan capping moiety
at the end of a
HexNAc chain. In an embodiment, a BcIA3 glycan comprises a single HexNAc
moiety.
[0018] In some embodiments, a BcIA3 antigen is conjugated to a carrier
molecule such as,
without limitation, a peptide, a protein, a membrane protein, a carbohydrate
moiety, a linker, or a
combination thereof, or a liposome containing any of the previous carrier
molecules. In some
embodiments, a conjugated BcIA3 antigen comprises a recombinantly synthesized
BcIA3 glycan
conjugated to a carrier protein. In an embodiment, recombinantly synthesized
BcIA3 glycan is
prepared using SgtA glycosyltransferase, e.g., recombinantly expressed SgtA
glycosyltransferase.
[0019] In an embodiment, an isolated BcIA3 glycoprotein or glycopeptide
comprises a
BcIA3 glycan as described herein linked to a protein or peptide consisting of
the amino acid
sequence set forth in any one of SEQ ID NOs: 1-32, or a homolog, fragment,
analog, or variant
thereof. In another embodiment, an isolated BcIA3 glycoprotein or glycopeptide
comprises a
BcIA3 glycan as described herein linked to a protein or peptide consisting of
an amino acid
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sequence at least about 80%, at least about 85%, at least about 90%, or at
least about 95%
identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-32,
or a homolog,
fragment, analog, or variant thereof.
[0020] In yet another aspect, there are provided compositions comprising
the isolated
BcIA3 glycoprotein or glycopeptide, the isolated BcIA3 glycan, or the
conjugated BcIA3 antigen
as described herein, and a pharmaceutically acceptable diluent, carrier, or
excipient.
[0021] In a further aspect, there are provided vaccines for prevention or
treatment of C.
difficile infection comprising the isolated BcIA3 glycoprotein or
glycopeptide, the isolated BcIA3
glycan, or the conjugated BcIA3 antigen described herein.
[0022] In a further aspect, there are provided vaccines for prevention or
treatment of C.
difficile infection comprising the isolated BcIA3 glycoprotein or
glycopeptide, the isolated BcIA3
glycan, or the conjugated BcIA3 antigen described herein, and an adjuvant.
[0023] In a still further aspect, there are provided compositions
comprising an antibody or
fragment thereof that binds to a C. difficile spore glycoprotein or fragment
thereof, wherein the
glycoprotein or fragment thereof comprises BcIA3 glycoprotein or a BcIA3
glycopeptide; and a
pharmaceutically acceptable diluent, carrier, or excipient. The BcIA3
glycoprotein or the
glycopeptide may comprise the amino acid sequence set forth in any one of SEQ
ID NOs: 1-32,
or an amino acid sequence at least about 80%, at least about 85%, at least
about 90%, or at
least about 95% identical to the amino acid sequence set forth in any one of
SEQ ID NOs: 1-32.
In an embodiment, the BcIA3 glycoprotein or the glycopeptide comprises one or
more chain of
three or more N-acetyl hexosamine (HexNAc) moieties 0-linked through a
threonine residue.
The BcIA3 glycoprotein or the glycopeptide may comprise three, four, or five
HexNAc moieties,
each HexNAc moiety optionally having a molecular weight of about 203 Da. In an
embodiment,
the BcIA3 glycoprotein or the glycopeptide comprises a single HexNAc moiety.
In some
embodiments, the BcIA3 glycoprotein or the glycopeptide further comprises a
glycan capping
moiety at the end of the HexNAc chain, the glycan capping moiety optionally
having a molecular
weight of about 203 Da, about 215 Da, about 220 Da, about 372 Da, about 374
Da, about 429
Da, about 486 Da, about 462 Da, about 375 Da, about 424 Da, or about 552 Da.
In an
embodiment, the BcIA3 glycoprotein or glycopeptide comprises one or more
GIcNAc residue as
a component of the glycan. In an embodiment, the BcIA3 glycopeptide has the
nLC-MS/MS
spectrum shown in any one of Figures 3a, 3b, 8a, and 8b.
[0024] In an embodiment, there is provided a composition comprising an
antibody or
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fragment thereof that binds to a C. difficile spore BcIA3 glycan and a
pharmaceutically
acceptable diluent, carrier, or excipient.
[0025] In another aspect, there is provided an isolated antibody or
fragment thereof specific
for C. difficile spores. In an embodiment, the isolated antibody or fragment
thereof is specific for
a C.difficile spore BcIA3 glycoprotein or glycopeptide or glycan thereof. The
isolated antibody or
fragment may bind specifically to one or more of a BcIA3 glycoprotein, a BcIA3
glycopeptide,
and a BcIA3 glycan. In an embodiment, the BcIA3 glycoprotein or glycopeptide
thereof
comprises the amino acid sequence set forth in SEQ ID NOs: 1-32. In another
embodiment, the
BcIA3 glycoprotein or glycopeptide thereof comprises an amino acid sequence at
least about 80
to about 95% identical to any one of the amino acid sequences set forth in SEQ
ID NOs: 1-32.
[0026] In some embodiments, the antibody or fragment thereof is a
polyclonal antibody. In
alternative embodiments, the antibody or fragment thereof is a monoclonal
antibody. The
antibody or fragment thereof may be humanized, human, or chimeric. In some
embodiments,
the antibody or fragment thereof comprises a whole immunoglobulin molecule; a
single-chain
antibody; a single-chain variable fragment (scFv); a single domain antibody;
an Fab fragment;
an F(ab')2 fragment; or a disulfide-linked Fv (di-scFv). The antibody or
fragment thereof may
comprise a heavy chain immunoglobulin constant domain selected from human IgM,
human
IgG1, human IgG2, human IgG3, human IgG4, and human IgA1/2. Further, the
antibody or
fragment thereof may comprise a light chain immunoglobulin constant domain
selected from
human Ig kappa and human Ig lambda. In some embodiments, the antibody or
fragment binds
to an antigen with an affinity constant of at least about 109 M or at least
about 1010 M.
[0027] In some embodiments, compositions provided herein further comprise a
second
agent for preventing or treating C. difficile infection. In some embodiments,
the second agent
comprises, without limitation, one or more of: an antibody that binds to toxin
A; an antibody that
binds to toxin B; an antibody that binds to LTA; an antibody that binds to PS-
I; an antibody that
binds to PS-II; an antibody that binds to a C. difficile vegetative cell
surface protein; and, an
antibody that binds to a C. difficile spore cell surface protein selected from
BcIA1, BcIA2, Alr,
SIpA paralogue, SIpA HMW, CD1021, lunH, Fe-Mn-SOD, and FliD. In another
embodiment, the
second agent comprises an antibiotic such as, without limitation,
metronidazole or vancomycin.
[0028] In another aspect, there are provided methods for preventing or
treating C. difficile
infection comprising administering to a subject the BcIA3 antigens, conjugated
antigens,
compositions, vaccines, or antibodies or fragments thereof described herein,
such that C.
difficile infection is prevented or treated in the subject. Methods of
inducing immunity against C.
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difficile infection in a subject, such that C. difficile infection is
prevented or treated in the subject,
are also provided.
[0029] A
composition, vaccine, antibody or fragment thereof may be administered
intravenously, subcutaneously, intramuscularly, or orally. In some
embodiments, a composition,
vaccine, antibody or fragment thereof is administered in combination with a
second agent for
preventing or treating C. difficile infection. The
second agent may be administered
concomitantly with the composition, accine, antibody or fragment thereof, or
they may be
administered sequentially, i.e., one before the other.
[0030] Use
of a C. difficile spore BcIA3 glycoprotein, glycopeptide, glycan or conjugated
BcIA3 antigen in the manufacture of a vaccine for prevention or treatment of
C. difficile infection
is also provided.
[0031] In
another aspect, there is provided an isolated C. difficile SgtA
glycosyltransferase
and use thereof for preparation of a C. difficile spore antigen, e.g., a C.
difficile spore BcIA3
glycan. In an embodiment, SgtA glycosyltransferase is recombinantly expressed
and used for
recombinant BcIA3 glycan production.
[0032] In
yet another aspect, there are provided kits for preventing or treating CDI,
comprising one or more C. difficile BcIA3 spore antigen, antibody,
composition, and/or vaccine
as described herein. Instructions for use or for carrying out the methods
described herein may
also be provided in a kit. A kit may further include additional reagents,
solvents, buffers,
adjuvants, etc., required for carrying out the methods described herein. Kits
for diagnosing CDI
in a subject or for determining that a subject is at risk of recurrence of CD!
comprising reagents
for detecting the presence of one or more of a BcIA3 glycoprotein,
glycopeptide, and glycan in a
subject are also provided.
[0033] In an
aspect, there are provided diagnostic methods for detecting the presence of
C. difficile in a subject. In an embodiment, diagnostic methods for detecting
the presence of C.
difficile spores in a subject are provided. In some embodiments, there are
provided methods of
detecting the presence of C. difficile in a subject comprising obtaining a
stool sample from the
subject and assaying the stool sample for the presence of a BcIA3
glycoprotein, glycopeptide,
and/or glycan thereof, wherein the presence of the BcIA3 glycoprotein,
glycopeptide, and/or
glycan thereof in the stool sample indicates the presence of C. difficile
and/or C. difficile spores
in the subject. In some embodiments, the assay comprises an immunoassay. There
are further
provided uses of the BcIA3 antigens and uses of the antibodies or fragments
described herein
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for detecting the presence of C. difficile and/or C. difficile spores in a
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] For a better understanding of the invention and to show more clearly
how it may be
carried into effect, reference will now be made by way of example to the
accompanying
drawings, which illustrate aspects and features according to preferred
embodiments of the
present invention, and in which:
[0035] Figure 1 is a schematic diagram showing the genetic organization of
the putative C.
difficile exosporiunn glycoprotein genes and related glycosyltransferase gene.
(a) Bacillus
anthracis Sterne strain has been shown to possess two exosporium glycoprotein
genes, BAS
1130 and 2281 (coloured black in the figure) denoted bcIA and bcIB. A
glycosyltransferase has
also been identified lying adjacent to bcIA (BAS 1131, denoted white). Genetic
organisation of
the bcl homologs (black arrows) and putative glycosyl transferase sgtA (white
arrows) in C.
difficile 630 (b), R20291 (c) and QCD-32g58 (d) are shown.
[0036] Figure 2 shows NuPAGE gel analysis of C. difficile endospore surface
protein
extracts. (a) Silver stained 3-8% NuPage, (b) Pro-Emerald Q glycostain 3-8%
NuPage. Lane 1:
HiMark Prestained molecular weight marker; Lane 2: 630Aerm spore surface
extract; Lane 3:
R20291 spore surface extract; Lane 4: QCD-32g58 spore surface extract. Arrows
indicate
regions of the gel that were excised, enzymatically digested, and analysed by
nLC-MS/MS.
[0037] Figure 3 shows mass spectrometry analysis of peptides from
proteinase K digestion
of C. difficile R20291 endospore surface extracts. (a) nLC-MS/MS spectrum of
the doubly
protonated glycopeptide ion at m/z 811.8 is shown. Peptide type y and b ions
were visible and
gave the peptide sequence AGLIGPTGATGV, a peptide from the BcIA3 protein. The
spectrum
was dominated in the high m/z region by sequential neutral losses of 203 Da,
with the
unmodified peptide ion observed at m/z 1013.5. Combined with the observed
intense glycan
oxonium ion at m/z 204, with neutral losses of water to give glycan related
ions at m/z 186 and
168, this spectrum suggested the peptilie to be modified with a chain of 3
HexNAc moieties. (b)
nLC-MS/Ms spectrum of a doubly protonated glycopeptides ion at m/z 1129 is
shown. Peptide
type y and b ions corresponded to a sequence of TGPTGATGADGITGP, corresponding
to the
BcIA3 protein. The high m/z region of the spectrum was dominated by sequential
neutral losses
of 374-203-203-203. An intense oxonium ion was observed at m/z 375 and a very
weak
oxonium ion at m/z 204 (not indicated). Glycan related fragment ions were
observed at m/z 300
and 272. (c) peptide sequence coverage map of BcIA3 protein homolog from spore
surface
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protein extraction, band 1 is shown. Boldface and underlining indicates
peptides modified with
glycan moieties. Two of the peptides shown to be modified with glycan are
shown in bold grey
text to indicate that the amino acid sequences appear in the BcIA3 protein
more than once. A
dotted underline indicates a glycopeptide sequence that is common to both
BcIA3 and BcIA2
proteins.
[0038] Figure 4 shows immunofluorescence of anti-r3-0-GIcNAc binding to
spores. (A) Wild
type spores of strains of C. difficile, from a range of ribotypes and
geographical locations are
shown. 630Aerm, GIcNAc binding at poles is marked with arrows. (B) sgtA mutant
spores of
strains R20291 and 630Aerm are shoivn. (A) and (B), top row of column, merged
image of
phase contrast, DAPI and FITC; 2nd row DAPI channel only; 3rd row FITC channel
only; 4th row
phase contrast only. GIcNAc was visualised with mouse anti-13-0-GIcNAc and
anti-mouse IgM-
FITC conjugate. (C) shows percentage of spores reacting with anti-0-0-GIcNAc
after 7 days of
growth. At least 100 spores were counted in triplicate on three independent
occasions for anti-13-
0-GIcNAc binding to surface, analysed by immunofluorescence microscopy.
[0039] Figure 5 shows restoration of anti-GIcNAc reactivity through
complementation. A
western blot of 72 hour plate grown cultures run on 3-8% Nu-PAGE gel is shown.
Complemented strains were induced with 500 ng anhydrotetracycline. Lane 1:
HiMark
(Invitrogen); Lane 2: R20291; Lane 3: R20291ACDR3194; Lane 4:
R20291A3194p3350; Lane
5: 630Aerm; Lane 6: 630A3350; Lane 7: 630A3350p3350.
[0040] Figure 6 shows resistance of R20291 wild type and AsgtA spores to 80
C for 20
minutes. Spores were incubated for 20 minutes in a water bath at 80 C and then
cfu/ml was
determined. Percentage survival was calculated by comparing inocula to post
heat treatment.
Statistical analysis is t-test with Welch's correction, p = <0.0001.
[0041] Figure 7 shows adherence and invasion of J774A.1 macrophage cells.
Percentage
of spores adhering to or internalised into J774A.1 macrophages after 30
minutes incubation at
37 C 5% CO2 is shown. Percentage wase calculated based on known MOI and final
adhered
spores. Fifty J774A.1 cells were counted in triplicate on three independent
occasions. Statistical
analysis is t-test with Welch's correctior, (* p=<0.05; *** p = <0.0001).
[0042] Figure 8 shows gel electrophoresis and mass spectrometry analyses of
C. difficile
QCD-32g58 endospore cell surface protein extract. (a) shows strain QCD-32g58;
the MSMS
spectrum of the doubly charged precursor ion at m/z 927.4. The y and b ion
sequence
corresponded to the peptide sequence 39GPTGATGVTGADGA323 from putative
exosporium
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protein (CdifQ_040500019311) with modification with three putative HexNAc
residues. (b)
shows the MSMS spectrum of the doubly charged peptide precursor on at m.z
1088.5 gave a
series of peptide y and b ions, corresponding to the putative exosporial
peptide
304AGLIGPTGATGV317 . Neutral losses corresponding to three HexNac moieties and
an
unknown glycan of 552 Da were observed in the high m/z region of the spectrum.
This gave a
total mass excess of 1162 Da. De novo sequencing of the resulting MSMS spectra
showed
peptides corresponding to a putative exosporium glycoprotein
(CdifQ_040500019311). (c) A
total of nine glycopeptides were identified, corresponding to 17-21 % sequence
coverage.
[0043] Figure 9 shows immunofluorescence of anti-13-0-GIcNAc binding to
vegetative cells.
(a) 630Aerm; (b) R20291, comparing fµfild type to respective mutant strains
(a) ACD3350 (b)
ACDR3194. Left hand column shows FITC labelling only; right hand column shows
merged
images of FITC, DAPI and transmitted light channels. GIcNAc was visualised
with mouse anti-f3-
0-GIcNAc and anti-mouse IgM-FITC conjugate.
[0044] Figure 10 shows RT-PCR analysis demonstrating co-transcription of
CD3350 and
CD3349. Upper panel shows expected size of each product with primer pairs P1
(CD3350), P2
(intergenic region) and P3(CD3349); lower panel shows agarose gel analysis of
products. RT
lanes: RT-PCR was performed using total RNA from C. difficile 630 cells. RNA
lanes: standard
PCR reaction was performed with same primers using total RNA to demonstrate no
contaminating DNA in RNA samples. M: 500bp DNA marker.
[0045] Figure 11 shows restoration of anti-GIcNAc reactivity through
complementation. 72
hour plate grown cultures of (a) 630Aerm, (b) R20291, comparing wild type to
ACD3350/ACDR3194 and ACDR3194p3350; complements were induced with 500 ng
anyhdrotetracycline. Merged images of FITC, DAPI and transmitted light
channels are shown.
GIcNAc was visualised with mouse anti-(3-0-GIcNAc and anti-mouse IgM-FITC
conjugate.
[0046] Figure 12 shows glycostaining of C. difficile spore surface
extracts. Surface extracts
were run on 3-8% Tris-Acetate NuPAGE prior to glycostaining with Pro-Emerald
Q. Lane 1:
630Aerm; Lane 2: 630AsgtA; Lane 3: R20291; Lane 4: R20291AsgtA; Lane 5: QCD-
32g58.
[0047] Figure 13 shows resistance assays with (a) lysozyme and (b) ethanol.
(a) R20291
1/VT and AsgtA spores were incubated with 250 pgiml lysozyme for 1 hour at 37
C and then
percentage survival was calculated. (b) R20291 WT and AsgtA spores were
incubated in 70%
ethanol for 20 minutes at room temperature and then percentage survival was
calculated.
Assays were performed in triplicate on three independent occasions.
Statistical analysis is t-test
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with Welch's correction (* p = <0.05).
[0048] Figure 14 shows a graph illustrating results from an ELISA assay in
which CD5
rabbit polyclonal serum (A) and preimmune serum (=) were tested against viable
R20291
spores.
[0049] Figure 15 shows a Western blot of spore surface extracts. Lane 1:
R20291 spore
extract, CD5 preimmune serum; Lane 2: R20291 sgtA spore extract, CD5 preimmune
serum;
Lane 3: R20291 spore extract, CD5 immune serum; Lane 4: R20291 sgtA spore
extract, CD5
immune serum.
DETAILED DESCRIPTION
[0050] The present disclosure relates to the identification and
characterization of the BcIA3
homolog from C. difficile as a major component of the C. difficile spore
surface. We report
herein that C. difficile BcIA3 is a surface associated glycoprotein modified
with a novel
oligosaccharide, specifically an 0-linked glycan structure. Further, we have
demonstrated that
antibodies raised against the spore BcIA3 glycoprotein recognize C. difficile
spores and spore
surface extracts. In addition, a glycosyltransferase gene involved in the
biosynthesis of surface-
associated glycan components was identified, and immunoreactivity of
antibodies raised against
the spore BcIA3 glycoprotein was abrogated in glycosyltransferase mutants.
Reactivity of a 13-0
GIcNAc specific antibody with glycan structures on the C. difficile spore
surface confirmed the
presence of GIcNAc residue(s) as a component of spore glycan. We have thus
demonstrated
the immunogenicity and significance of the BcIA3 glycan structure and
identified the BcIA3
glycoprotein as a key immunogen for C. difficile spores.
BcIA3 Spore Antigens
[0051] There is provided herein a C. difficile spore antigen comprising a
BcIA3 glycoprotein,
or an immunogenic glycopeptide or glycan thereof. The terms "BcIA3 spore
antigen", "spore
BcIA3 antigen", "BcIA3 antigen", "C. difficile spore antigen", and "C.
difficile BcIA3 spore antigen"
are used interchangeably herein to refer to immunogenic molecules comprising a
C. difficile
BcIA3 glycoprotein, a glycopeptide thereof, a glycan thereof, and/or a
conjugate thereof, as well
as homologs, analogs, variants, and/Or fragments thereof. It should be
understood that any
immunogenic BcIA3 glycoprotein, glycopeptide, glycan, conjugate, or homolog,
analog, variant,
or fragment or portion thereof, is encompassed by the present invention, and
may be used in
compositions and methods provided herein.
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[0052] The term "glycoprotein" is used herein to refer to a protein that is
post-translationally
modified to include glycosylation, i.e., linkage to one or more carbohydrates.
The term "glycan"
is used to refer to the carbohydrate moiety of a glycoprotein, i.e., the
carbohydrates attached to
the protein in a glycoprotein. The terms "glycopolypeptide" and "glycopeptide"
are used
interchangeably herein to refer to a polymer of amino acids attached to one or
more
carbohydrates post-translationally. As used herein, the term "glycoprotein"
generally refers to a
full-length protein, e.g., the full-length BcIA3 glycoprotein, and the term
"glycopeptide" is used to
refer to a shorter glycosylated fragment or portion thereof.
[0053] As used herein, the term "BcIA3 glycoprotein" refers to a full-
length glycosylated
BcIA3 protein from C. difficile such as, without limitation, a glycoprotein
having the amino acid
sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 24. Many different strains of
C. difficile are
known and the glycoproteins expressed by different strains may vary slightly
in their amino acid
sequences and/or their glycan structures. However, the BcIA3 glycoprotein
provided herein is
not meant to be limited to the glycoprotein expressed by any particular
strain. It is intended that
homologs, variants, fragments, and analogs are encompassed by the present
technology. In an
embodiment, a BcIA3 glycoprotein comprises the BcIA3 homolog in a C.difficile
strain, such as
but not limited to the strains listed in Table 5.
[0054] As used herein, the term "BcIA3 glycopeptide" refers to an
immunogenic and
glycosylated peptide fragment or portion of a BcIA3 glycoprotein. In some
embodiments, a
BcIA3 glycopeptide has the amino acid sequence set forth in any one of SEQ ID
NOs: 2-23 and
25-32. In an embodment, a BcIA3 glycopeptide comprises the amino acid sequence
set forth in
any one of SEQ ID NOs: 2-23 and 25-32. In another embodiment, a BcIA3
glycopeptide
consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2-23
and 25-32, the
amino acid sequence being 0-linked to a glycan, e.g., a chain of three or more
N-acetyl
hexosamine (HexNAc) moieties.
[0055] In some embodiments, a BcIA3 spore antigen comprises a BcIA3
glycoprotein or
glycopeptide having the amino acid sequence set forth in any one of SEQ ID
Nos: 1 - 32 and
one or more BcIA3 glycan, the BcIA3 glycan comprising a chain of three or more
N-acetyl
hexosamine (HexNAc) moieties. In an embodiment, the BcIA3 glycan is 0-linked,
e.g., 0-linked
through a threonine residue to the BcIA3 protein or peptide. In one
embodiment, a BcIA3 glycan
comprises three HexNAc moieties. In another embodiment, a BcIA3 glycan
comprises five
HexNAc moieties. In an embodiment, each HexNAc moiety has a molecular weight
of about 203
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Da. In some embodiments, a BcIA3 glycan further comprises a glycan capping
moiety at the
end of a HexNAc chain. The glycan capping moiety may have a molecular weight
of, for
example, about 203 Da, about 215 Da, about 220 Da, about 372 Da, about 374 Da,
about 429
Da, about 486 Da, about 462 Da, about 375 Da, about 424 Da, or about 552 Da.
In an
embodiment, a BcIA3 glycan comprises one or more GIcNAc residue. In a
particular
embodiment, a BcIA3 glycopeptide comprises or consists of a glycopeptide
having the nLC-
MS/MS spectrum shown in any one of Figures 3a, 3b, 8a, and 8b.
[0056] In some embodiments, a BcIA3 spore antigen comprises at least one
carbohydrate
chain comprising an 0-linked N-acetyl glucosamine (GIcNAc) moiety. In an
embodiment, a
BcIA3 spore antigen comprises a single 0-linked N-acetyl glucosamine (GIcNAc)
moiety.
[0057] The amino acid sequences of the protein/peptide portion of exemplary
BcIA3
glycoproteins and glycopeptides are given in Table 1.
Table 1. Amino acid sequences of exemplary BcIA3 glycoproteins and
glycopeptides.
SEQ Amino acid sequence Source
ID
NO.
1 MSRNKYFGPFDDNDYNNGYDKYDDCNNGRDDYNSCDCHHCCPPSCVGPTGPMGPRGRTGPTGPT BcIA3
GPTGPGVGGT GPTGPT GPTGPT GNTGNTGATGLRGPTGATGGTGPTGATGAI GFGVTGPTGPTG
glycoprotein
PTGATGATGADGVT GPTGPTGATGADGI TGPTGATGATGFGVTGPTGPTGATGVGVTGAT GL IG (full-
length),
PTGATGTPGATGPTGAI GAT GIGI TGPTGATGATGADGATGVTGPTGPT GATGADGVTGPTGAT
GATGIGITGPTGATGATGIGITGATGL I GPTGAT GATGATGPTGVTGAT GAAGL I GPTGATGVT C.
difficile
GADGATGATGATGATGPTGADGLVGPTGATGATGADGLVGPTGPTGATGVGITGATGATGATGP R20291 strain
TGADGLVGPT GAT GATGADGVAGPTGAT GATGNTGADGATGPTGATGPTGADGLVGPTGATGAT
GLAGATGAT GP I GAT GPTGADGAT GATGAT GPTGADGLVGPTGATGAT GATGPTGPTGASAI I P
FASGI PLSLTTIAGGLVGTPGFVGFGSSAPGLS IVGGVI DLTNAAGTLTNFAFSMPRDGT IT S
SAYFSTTAAL SLVGSTITITATLYQSTAPNNSFTAVPGATVTLAPPLTGILSVGS I S SGIVTGL
NIAATAETRFLLVFTATASGLSLVNTVAGYASAGIAIN
2 AGLIGPTGATGV C. difficile
R20291 and
QCD-32g58
strains
3 TGPTGATGADGITGP C. difficile
R20291 strain
4 VGPTGATGA C. difficile
R20291 strain
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C. difficile
GLIGPTGATGTPGA
R20291 strain
C. difficile
6 TGATGLIGPTGATGA
R20291 strain
C. difficile
7 TGIGITGPTGATGA
R20291 strain
C. difficile
8 TGIGITGPTGA
R20291 strain
C. difficile
9 GLIGPTGATGVTGA
R20291 strain
C. difficile
TGVTGATGAAGLIGP R20291
strain
C. difficile
11 TGATGLIGPTGATGA
R20291 strain
C. difficile
12 IGPTGATGTPGATGPTGA
R20291 strain
C. difficile
13 TGPTGATGPTGADGL
R20291 and
QCD-32g58
strains
C. difficile
14 GVTGPTGPTGPTGATGA
R20291 strain
C. difficile
GVTGPTGPTGATGV
R20291 strain
C. difficile
16 VGPTGATGATGADGL
R20291 strain
C. difficile
17 VGPTGPTGATGV
R20291 strain
C. difficile
18 IGPTGATGTPGATGPTGA
R20291 strain
C. difficile
19 IGPTGATGVTGADGA
R20291 strain
C. difficile
VGPTGATGATGL
R20291 and
QCD-32g58
strains
C. difficile
21 VGPTGATGATGADGV
R20291 strain
C. difficile
22 VGPTGPTGATGV
R20291 strain
C. difficile
23 ATASGLSLVNTVA
R20291 strain
24 MSRNKYFGPFDDNDYNNGYDKYDDCNNGRDDYNSCDCHHCCPPSCVGPTGPMGPRGRTGPTGPT BcIA3
GPTGPGVGGTGPTGPTGPTGPTGNTGNTGATGLRGPTGATGGTGPTGATGAIGFGVTGPTGPTG glycoprotein
ATGATGADGVTGPTGPTGATGADGITGPTGATGATGFGVTGPTGPTGATGVGVTGATGLIGPTG (full-
length), C.
ATGTPGATGPTGAIGATGIGITGPTGATGATGADGATGVTGPTGPTGATGADGVTGPTGATGAT
difficile
GIGITGPTGATGATGIGITGATGLIG2TGATGATGATGPTGVTGATGAAGLIGPTGATGVTGAD
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GATGAT GAT GATGPTGADGLVGPTGATGAT GADGLVGPTGPTGATGVGI T GATGATGATGPTGA QCD-3208
DGLVGPTGATGATGADGVAGPTGATGATGNTGADGATGPTGATGPTGADGLVGPTGATGATGLA strain
GAT GATGP I GATGPTGADGAT GATGATGPT GADGLVGPTGATGATGATGPTGPTGASAI I P FAS
GI PLSLTTIAGGLVGT PGFVGFGSSAPGLS IVGGVI DLTNAAGTLTNFAFSMPRDGT IT S I SAY
FS TTAAL SLVGST IT ITATLYQSTAPNNS FTAVPGATVTLAP PLTGILSVGS I SSGIVTGLN IA
ATAETRFLLVFTATASGLSLVNTVAGYASAGIAIN
C. difficile
25 TGPTGVTGATGA
QCD-32g58
strain
C. difficile
26 GVTGPTGPTGATGA
QCD-32g58
strain
C. difficile
27 GVTGPTGPTGATGV
QCD-32g58
strain
C. difficile
28 TGPTGADGL
QCD-32g58
strain
C. difficile
29 GLVGPTGPTGATGV
QCD-32g58
strain
C. difficile
30 AGPTGATGATGNTGADGA
QCD-32g58
strain
31 TGPTGATGPTGADGLVGPTGATGATGLA C.
difficile
QCD-32g58
strain
C. difficile
32 IGPTGATGVTGADGA
QCD-32g58
strain
[0058] Variants, analogs, and fragments of BcIA3 glycoproteins and
glycopeptides are also
encompassed. As used herein, a "variant" refers to an amino acid sequence of
the naturally
occurring protein or peptide in which a small number of amino acids have been
substituted,
inserted, or deleted, and which retains the relevant biological activity or
function of the starting
protein. For example, in the case of an antigen for use in a vaccine, a
variant may retain the
immunogenic characteristics of the starting protein, sufficient for its
intended use in inducing
immunity. In the case of an antibody, a variant may retain the antigen-binding
properties of the
starting protein, sufficient for its intended use in binding specifically to
antigen.
[0059] In some embodiments, a variant includes one or more conservative
amino acid
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substitutions, one or more non-conservative amino acid substitutions, one or
more deletions,
and/or one or more insertions. A conservative substitution is one in which an
amino acid residue
is substituted by another amino acid residue having similar characteristics
(e.g., charge or
hydrophobicity). In general, a conservative amino acid substitution will not
substantially change
the functional properties of a protein. Examples of groups of amino acids that
have side chains
with similar chemical properties include: 1) aliphatic side chains: glycine,
alanine, valine,
leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and
threonine; 3) amide-
containing side chains: asparagine and glutamine; 4) aromatic side chains:
phenylalanine,
tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and
histidine; 6) acidic side
chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains:
cysteine and
methionine. Exemplary conservative amino acids substitution groups are: valine-
leucine-
isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine -valine,
glutamate-aspartate, and
asparagine-glutamine. Other conservative amino acid substitutions are known in
the art and are
included herein. Non-conservative substitutions, such as replacing a basic
amino acid with a
hydrophobic one, are also well-known in the art.
[0060] As used herein, an "analog" refers to an amino acid sequence of the
naturally
occurring protein in which one or more amino acids have been replaced by amino
acid analogs.
Non-limiting examples of amino acid analogs include non-naturally occurring
amino acids,
synthetic amino acids, amino acids which only occur naturally in an unrelated
biological system,
modified amino acids from mammalian systems, polypeptides with substituted
linkages, as well
as other modifications known in the art, both naturally occurring and non-
naturally occurring. In
some embodiments, analogs include modifications which increase glycoprotein or
glycopeptide
stability. In one embodiment, an analog includes a beta amino acid, a gamma
amino acid, or a
D-amino acid.
[0061] A "fragment" refers to a portion of the starting molecule which
retains the relevant
biological activity or function (e.g, antigenicity, antigen-binding,
immunogenicity) of the starting
molecule.
[0062] A "biologically active" or "functionally equivalent" fragment,
variant, or analog
generally retains biological activity or function of the starting molecule,
sufficient for use in the
present compositions and methods. Thus, a "biologically active" or
"functionally equivalent"
fragment, variant, or analog may retain the binding specificity, the
antigenicity, or the
immunogenicity of the starting molecule. In some embodiments, a fragment,
variant or analog
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has at least about 70%, at least about 80%, at least about 85%, at least about
90%, at least
about 95%, or at least about 98% sequence identity to the starting molecule
(e.g., protein).
When referring to an antibody, "functionally equivalent" generally refers to a
fragment,
derivative, variant, analog, or fusion protein of the antibody that maintains
sufficient antigen-
binding affinity, specificity and/or selectivity for use in the present
compositions and methods.
The antigen-binding properties of a functionally equivalent antibody or
fragment need not be
identical to those of the reference antibody so long as they are sufficient
for use in the present
compositions and methods for preventing or treating CDI.
[0063] Variants, fragments, or analogs may also be modified at the N-
and/or C-terminal
ends to allow the polypeptide or fragment to be conformationally constrained
and/or to allow
coupling to an immunogenic carrier.
[0064] There are further provided conjugated BcIA3 spore antigens
comprising a BcIA3
antigen conjugated to a carrier molecule. A carrier molecule may be any
suitable molecule such
as, without limitation, a peptide, a protein, a membrane protein, a
carbohydrate moiety, or one
or more liposomes loaded with any of the previously recited types of carrier
molecules or loaded
with a BcIA3 antigen itself. Many such carrier molecules are known in the art
and may be used
in the conjugated BcIA3 antigens provided herein. In one embodiment, a
conjugated BcIA3
antigen comprises a BcIA3 antigen conjugated to a carrier protein. In an
embodiment, a
conjugated BcIA3 antigen comprises a BcIA3 glycan. In another embodiment, a
BcIA3 antigen is
conjugated to a linker molecule or a protein carrier.
[0065] Further, a carrier molecule may be linked to a BcIA3 antigen, e.g.,
a BcIA3 glycan,
using any suitable method known in the art. For example, a carrier molecule
may be linked to a
BcIA3 antigen by a covalent bond or an ionic interaction, either directly or
using a linker. Linkage
may be achieved by chemical cross-linking, e.g., a thiol linkage. A carrier
protein or peptide
may be linked to a BcIA3 glycan through, for example, 0-linkage of the glycan
to a threonine
residue in the peptide. Methods for linking glycans to carrier molecules are
well-known in the
art, as are methods for preparing glycoconjugate vaccines. In an embodiment, a
conjugated
glycan antigen is prepared by conjugating a recombinantly-synthesized glycan
to a carrier
protein.
[0066] In another embodiment, a spore antigen is produced as a fusion
protein or a
conjugate that contains other distinct amino acid sequences that are not part
of the C. difficile
spore antigen sequences disclosed herein, such as amino acid linkers or signal
sequences or
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immunogenic carriers, as well as ligands useful in protein purification, such
as glutathione-S-
transferase, histidine tag, and staphylococcal protein A. A heterologous
polypeptide can be
fused, for example, to the N- terminus or C-terminus of a BcIA3 peptide or
protein. Further,
more than one BcIA3 peptide can be present in a fusion protein
[0067] As used herein, the term "isolated" refers to a molecule that by
virtue of its origin or
source of derivation (1) is not associated with naturally associated
components that accompany
it in its native state, (2) is free of other macromolecules (e.g., proteins,
glycans) from the same
species, (3) is expressed by a cell from a different species, or (4) does not
occur in nature.
Thus, a glycoprotein, glycopeptide or glycan that is chemically synthesized or
synthesized in a
cellular system different from the cell from which it naturally originates
will be "isolated" from its
naturally associated components. A glycoprotein, glycopeptide or glycan may
also be rendered
substantially free of naturally associated components by isolation, using
purification or
separation techniques well-known in the art. BcIA3 spore antigens used in
compositions and
methods described herein are generally provided in purified or substantially
purified form, i.e.,
substantially free from other glycopeptides and polypeptides, particularly
from other C. difficile
or host cell glycopeptides or polypeptides. In some embodiments, BcIA3 spore
antigens are at
least about 50% pure, at least about 60% pure, at least about 70% pure, at
least about 80%, at
least about 90% pure, or at least about 95% pure (by weight).
[0068] BcIA3 glycoproteins, glycopeptides and glycans thereof can be
prepared by various
means (e.g., recombinant expression, purification from cell culture, chemical
synthesis, etc.). In
some embodiments, a BcIA3 spore antigen is chemically synthesized. For
example, a glycan
may be chemically synthesized and then coupled to a protein or peptide, which
protein or
peptide may also be chemically synthesized (e.g., using solid phase peptide
synthesis) or
purified after recombinant expression in a cell line. Althernatively, a BcIA3
spore antigen may
be purified after recombinant expression in a cell line. For example, a
polynucleotide encoding
a BcIA3 protein or peptide can be introduced into an expression vector that
can be expressed in
a suitable expression system using techniques well-known in the art, followed
by isolation or
purification of the expressed protein or peptide. A variety of bacterial,
yeast, plant, mammalian,
and insect expression systems are available in the art and any such suitable
expression system
can be used. A glycoprotein or glycopeptide can be expressed in systems, e.g.
cultured cells,
cell lines, etc., which produce substantially the same postranslational
modifications present as
when the protein or peptide is expressed natively. Alternatively, a
polynucleotide encoding a
BcIA3 protein or peptide can be translated in a cell-free translation system.
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[0069] In cases where the expression system or cell line is competent to
glycosylate the
expressed BcIA3 protein or peptide appropriately, then the glycosylated BcIA3
spore antigen
may be purified from the expression system or cell line. Alternatively, an
unglycosylated BcIA3
protein or peptide may be linked to a glycan subsequently, after expressing
and purifying the
protein or peptide from a host cell. For example, a glycan can be chemically
synthesized and
then coupled to a protein or peptide in vitro. In an embodiment, an
unglycosylated BcIA3
protein or peptide is incubated with a glycosylation enzyme, e.g., SgtA
glycosyltransferase, in
appropriate conditions to allow glycosylation of the BcIA3 protein or peptide.
In an embodiment,
reconnbinantly expressed SgtA glycosyltransferase is used for production of a
BcIA3 spore
antigen, or for production of a recombinant glycan. BcIA3 antigen conjugates,
e.g, BcIA3 glycan
conjugates, can be prepared using similar techniques, including without
limitation chemical
synthesis, recombinant expression, use of recombinantly expressed SgtA
glycosyltransferase,
and/or a combination thereof, as well as other techniques known in the art.
[0070] A spore antigen can also be produced as a fusion protein or a
conjugate that
contains other distinct amino acid sequences that are not part of the C.
difficile spore antigen
sequences disclosed herein, such as amino acid linkers or signal sequences or
immunogenic
carriers, as well as ligands useful in protein purification, such as
glutathione-S-transferase,
histidine tag, and staphylococcal protein A. A heterologous polypeptide can be
fused, for
example, to the N- terminus or C-termit ius of a BcIA3 peptide or protein.
Further, more than one
BcIA3 peptide can be present in a fusion protein.
[0071] Many variations of techniques described herein are known in the art
and may be
used to prepare BcIA3 spore antigens.
Pharmaceutical Compositions and Methods
[0072] There are provided herein compositions and methods for the
prevention or
treatment of C. difficile infection (CDI) in a subject comprising immunogenic
BcIA3 spore
antigens. Compositions and methods for inducing an immune response to C.
difficile are also
provided. Methods provided herein comprise administration of a C. difficile
BcIA3 spore antigen
(e.g., BcIA3 glycoprotein, glycopeptide or glycan thereof), to a subject in an
amount effective to
induce an immune response against C. difficile spores, thereby reducing,
eliminating,
preventing, or treating CD'. Compositions and methods are also provided for
the generation of
antibodies for use in passive immunization against CDI.
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[0073] C. difficile infection (CDI) is a bacterial infectious disease of
the gastrointestinal tract
caused by Clostridium difficile (C. difficile), a toxin-producing Gram-
positive anaerobic, spore-
forming bacillus. As used herein, CDI includes recurrent CD!, which is defined
as complete
resolution of COI while on appropriate therapy, followed by recurrence of CD!
after treatment
has been stopped. CDI is often associated with disorders of the
gastrointestinal tract such as
dysbiosis, Crohn's disease, ulcerative colitis, enteritis, irritable bowel
syndrome, inflammatory
bowel disease, diarrhea, antibiotic-associated diarrhea, and diverticular
disease. In some
embodiments, there are provided compositions and methods for prevention or
treatment of
disorders of the gastrointestinal tract associated with CDI such as, without
limitation, dysbiosis,
Crohn's disease, ulcerative colitis, enteritis, irritable bowel syndrome,
inflammatory bowel
disease, diarrhea, antibiotic-associated diarrhea, and diverticular disease.
[0074] The terms "subject" and "patient" are used interchangeably herein to
refer to a
subject in need of prevention or treatment for CD! or for a disorder of the
gastrointestinal tract
associated with CD, including those at risk of contracting CD! for the first
time and those at risk
of recurrence of CDI. A subject may be a vertebrate, such as a mammal, e.g., a
human, a non-
human primate, a rabbit, a rat, a mouse, a cow, a horse, a goat, or another
animal. Animals
include all vertebrates, e.g., mammals and non-mammals, such as mice, sheep,
dogs, cows,
avian species, ducks, geese, pigs, chickens, amphibians, and reptiles. In an
embodiment, a
subject is a human.
[0075] "Treating" or "treatment" refers to either (i) the prevention of
infection or reinfection,
e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of the
disease of interest, e.g.,
therapy. "Treating" or "treatment" can refer to the administration of a
composition comprising a
BcIA3 glycoprotein, glycopeptide, or glycan of interest, e.g., C. difficile
BcIA3 spore antigens, or
to the administration of antibodies raised against these antigens. Treating a
subject with the
composition can prevent or reduce the risk of infection and/or recurrence
and/or induce an
immune response to C. difficile spores. In some embodiments, spore germination
is inhibited or
delayed; pathogen burden is reduced; spore colonization is inhibited; and/or
spore adherence to
the GI tract is blocked in a subject.
[0076] Treatment can be prophylactic (e.g., to prevent or delay the onset
of the disease, to
prevent the manifestation of clinical or subclinical symptoms thereof, or to
prevent recurrence of
the disease) or therapeutic (e.g., suppression or alleviation of symptoms
after the manifestation
of the disease). "Preventing" or "prevention" refers to prophylactic
administration or vaccination
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with BcIA3 spore antigens or antigen compositions in a subject who has not
been infected or
who is symptom-free after CDI and at risk of recurrence of CDI.
[0077] As used herein, the term "immune response" refers to the response of
immune
system cells to external or internal stimuli (e.g., antigens, cell surface
receptors, cytokines,
chemokines, and other cells) producing biochemical changes in the immune cells
that result in
immune cell migration, killing of target cells, phagocytosis, production of
antibodies, production
of soluble effectors of the immune response, and the like. An "immunogenic"
molecule is one
that is capable of producing an immune response in a subject after
administration.
[0078] "Active immunization" refers to the process of administering an
antigen (e.g., an
immunogenic molecule) to a subject in order to induce an immune response. In
contrast,
"passive immunization" refers to the administration of active humoral
immunity, usually in the
form of pre-made antibodies, to a subject. Passive immunization is a form of
short-term
immunization that can be achieved by the administration of an antibody or an
antigen-binding
fragment thereof. Antibodies can be administered in several possible forms,
for example as
human or animal blood plasma or serum, as pooled animal or human
immunoglobulin, as high-
titer animal or human antibodies from immunized subjects or from donors
recovering from a
disease, as polyclonal antibodies, or as monoclonal antibodies. Typically,
immunity derived from
passive immunization provides immediate protection or treatment but may last
for only a short
period of time.
[0079] In some embodiments, there are provided compositions and methods for
active
immunization against C. difficile. Compositions and methods are provided for
inducing an
immune response to C. difficile bacteria in a subject, comprising
administering to the subject a
C. difficile BcIA3 spore antigen and an adjuvant in an amount effective to
induce an immune
response in the subject. In one embodiment, there is provided a composition
comprising an
effective immunizing amount of an isolated C. difficile BcIA3 spore antigen
and an adjuvant,
wherein the composition is effective to prevent or treat CD! in a subject in
need thereof. In an
embodiment, the BcIA3 spore antigen comprises one or more BcIA3 glycoprotein,
glycopeptide,
glycan, or conjugated BcIA3 antigen (e.g., conjugated BcIA3 glycan antigen),
as described
herein. In an embodiment, an adjuvant is not required, i.e., compositions and
methods are
provided for inducing an immune response to C. difficile bacteria in a
subject, comprising
administering to the subject a C. difficile BcIA3 spore antigen and a
pharmaceutically acceptable
carrier, excipient, or diluent, in an amount effective to induce an immune
response in the
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subject.
[0080]
Adjuvants generally increa._ie the specificity and/or the level of immune
response.
An adjuvant may thus reduce the quantity of antigen necessary to induce an
immune response,
and/or the frequency of injection necessary in order to generate a sufficient
immune response to
benefit the subject. Any compound or compounds that act to increase an immune
response to
an antigen and are suitable for use in a subject (e.g., pharmaceutically-
acceptable) may be
used as an adjuvant in compositions, vaccines, and methods of the invention.
In some
embodiments, the adjuvant may be the carrier molecule (for example, but not
limited to, cholera
toxin B subunit, liposome, etc.) in a conjugated or recombinant antigen. In
alternative
embodiments, the adjuvant may be an unrelated molecule known to increase the
response of
the immune system (for example, but not limited to attenuated bacterial or
viral vectors,
AMVAD, etc.). In one embodiment, the adjuvant may be one that generates a
strong mucosal
immune response such as an attenuated virus or bacteria, or aluminum salts.
Other suitable
adjuvants are well-known to those of skill in the art.
[0081]
Compositions, formulations and vaccines including one or more BcIA3 spore
antigen
described herein can be prepared by uniformly and intimately bringing into
association the
antigen and the adjuvant using techniques well-known to those skilled in the
art including, but
not limited to, mixing, sonication and microfluidation. An adjuvant will
typically comprise about
to 50% (v/v), about 20 to 40% (v/v), or about 20 to 30% or 35% (v/v) of the
composition.
[0082] In
other embodiments, there are provided compositions and methods for passive
immunization comprising an antibody or an antigen-binding fragment thereof
specific for a C.
difficile spore BcIA3 antigen. As used herein, the term "antibody" refers to
any immunoglobulin
or intact molecule as well as to fragments thereof that bind to a specific
antigen or epitope. Such
antibodies include, but are not limited to polyclonal, monoclonal, chimeric,
humanized, single
chain, Fab, Fab', F(ab')2, F(ab)' fragments, and/or F(v) portions of the whole
antibody and
variants thereof. All isotypes are emcompassed by this term, including IgA,
IgD, IgE, IgG, and
IgM.
[0083] As
used herein, the term "antibody fragment" refers to a functionally equivalent
fragment or portion of antiody, i.e., to an incomplete or isolated portion of
the full sequence of an
antibody which retains the antigen binding capacity (e.g., specificity,
affinity, and/or selectivity)
of the parent antibody. Non-limiting examples of antigen-binding portions
include: (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains;
(ii) a F(alp')2
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fragment, a bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a
Fv fragment
consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment, which
consists of a VH domain; (vi) an isolated complementarity determining region
(CDR); and (vii) a
single chain Fv (scFv), which consists of the two domains of the Fv fragment,
VL and VH. Other
non-limiting examples of antibody fragments are Fab fragments; diabodies;
linear antibodies;
single-chain antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0084] An intact "antibody" comprises at least two heavy (H) chains and two
light (L) chains
inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy
chain variable
region (VH) and a heavy chain constant region. The heavy chain constant region
is comprised of
three domains, CHi, CH2 and CH3. Each light chain is comprised of a light
chain variable region
(VL) and a light chain constant region. The light chain constant region is
comprised of one
domain, CL. The VH and VL regions can be further subdivided into regions of
hypervariability,
termed complementarity determining regions (CDR), interspersed with regions
that are more
conserved, termed framework regions (FR). Each VH and VL is composed of three
CDRs and
four FRs, arranged from amino-terminus to carboxyl-terminus in the following
order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light
chains
contain a binding domain that interacts with an antigen. The constant regions
of the antibodies
can mediate the binding of the immunoglobulin to host tissues or factors,
including various cells
of the immune system (e.g., effector cells) and the first component (Clq) of
the classical
complement system.
[0085] As used herein, the term "monoclonal antibody" or "mAb" refers to a
preparation of
antibody molecules of single molecular composition. A monoclonal antibody
composition
displays a single binding specificity and affinity for a particular epitope. A
"human monoclonal
antibody" refers to antibodies displaying a single binding specificity which
have variable and
constant regions (if present) derived from human germline imnnunoglobulin
sequences. In one
aspect, human monoclonal antibodies are produced by a hybridoma which includes
a B cell
obtained from a transgenic non-human animal, e.g., a transgenic mouse, having
a genome
comprising a human heavy chain transgene and a light chain transgene fused to
an
immortalized cell. A "humanized antibody" refers to at least one antibody
molecule in which the
amino acid sequence in the non-antigen binding regions and/or the antigen-
binding regions has
been altered so that the antibody more closely resembles a human antibody, and
still retains its
original binding properties. Humanized antibodies are typically antibody
molecules from non-
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human species having one or more CDRs from the non-human species and a
framework region
from a human immunoglobulin molecule. The term "chimeric antibody" refers to
an antibody in
which different portions are derived from different animal species, e.g., an
antibody having a
variable region derived from a murine mAb and a human immunoglobulin constant
region.
[0086] As used herein, the term "antigen" refers to a substance that
prompts the generation
of antibodies and can cause an immune response. The terms "antigen" and
"immunogen" are
used interchangeably herein, although, in the strict sense, immunogens are
substances that
elicit a response from the immune system, whereas antigens are defined as
substances that
bind to specific antibodies. An antigen or fragment thereof can be a molecule
(i.e., an epitope)
that makes contact with a particular antibody. When a glycoprotein or a
fragment thereof is used
to immunize a host animal, numerous regions of the glycoprotein can induce the
production of
antibodies (i.e., elicit the immune response), which bind specifically to the
antigen (given
regions or three-dimensional structures on the glycoprotein).
[0087] The terms "specific for" or "specifically binding" are used
interchangeably to refer to
the interaction between an antibody and its corresponding antigen. The
interaction is dependent
upon the presence of a particular structure of the protein recognized by the
binding molecule
(i.e., the antigen or epitope). In order for binding to be specific, it should
involve antibody binding
of the epitope(s) of interest and not background antigens, i.e., no more than
a small amount of
cross reactivity with other antigens (such as other proteins or glycan
structures, host cell
proteins, etc.). Antibodies, or antigen-binding fragments, variants or
derivatives thereof of the
present disclosure can also be described or specified in terms of their
binding affinity to an
antigen. The affinity of an antibody for an antigen can be determined
experimentally using
methods known in the art. The term "high affinity" for an antibody typically
refers to an
equilibrium association constant (Kaff) of at least about 1 x 107 liters/mole,
or at least about 1 x
108 liters/mole, or at least about 1 x 109 liters/mole, or at least about 1 x
1010 liters/mole, or at
least about 1 x 1011 liters/mole, or at least about 1 x 1012 liters/mole, or
at least about 1 x 1013
liters/mole, or at least about 1 x 1014 liters/mole or greater. KD, the
equilibrium dissociation
constant, can also be used to describe antibody affinity and is the inverse of
Kaff.
[0088] BcIA3 spore antigens and antibodies described herein are typically
combined with a
pharmaceutically acceptable carrier or excipient to form a pharmaceutical
composition.
Pharmaceutically acceptable carriers can include a physiologically acceptable
compound that
acts to, e.g., stabilize, or increase or decrease the absorption or clearance
rate of a
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pharmaceutical composition. Physiologically acceptable compounds can include,
e.g.,
carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as
ascorbic acid or
glutathione, chelating agents, low molecular weight proteins, compositions
that reduce the
clearance or hydrolysis of glycopeptides, or excipients or other stabilizers
and/or buffers. Other
physiologically acceptable compounds include wetting agents, emulsifying
agents, dispersing
agents or preservatives which are particularly useful for preventing the
growth or action of
microorganisms. Various preservatives are well known and include, e.g., phenol
and ascorbic
acid. Detergents can also be used to stabilize or to increase or decrease the
absorption of the
pharmaceutical composition, including liposomal carriers. Pharmaceutically
acceptable carriers
and formulations are known to the skilled artisan and are described in detail
in the scientific and
patent literature, see e.g., the latest edition of Remington's Pharmaceutical
Science, Mack
Publishing Company, Easton, Pa. ("Remington's"). One skilled in the art would
appreciate that
the choice of a pharmaceutically acceptable carrier including a
physiologically acceptable
compound depends, for example, on the route of administration of the
composition, antigen, or
antibody of the invention, and on its particular physio-chemical
characteristics.
[0089] Compositions and vaccines of the present invention may be
administered by any
suitable means, for example, orally, such as in the form of pills, tablets,
capsules, granules or
powders; sublingually; buccally; parenterally, such as by subcutaneous,
intravenous,
intramuscular, intraperitoneal or intrastemal injection or using infusion
techniques (e.g., as
sterile injectable aqueous or non-aqueous solutions or suspensions); nasally,
such as by
inhalation spray, aerosol, mist, or nebulizer; topically, such as in the form
of a cream, ointment,
salve, powder, or gel; transdermally, such as in the form of a patch;
transmucosally; or rectally,
such as in the form of suppositories. The present compositions may also be
administered in a
form suitable for immediate release or extended release. Immediate release or
extended
release may be achieved by the use o suitable pharmaceutical compositions, or,
particularly in
the case of extended release, by the use of devices such as subcutaneous
implants or osmotic
pumps.
[0090] It is often advantageous to formulate oral or parenteral
compositions in dosage unit
form for ease of administration and uniformity of dosage. Dosage unit form
refers to physically
discrete units suited as unitary dosages for the subject to be treated; each
unit containing a
predetermined quantity of active compound calculated to produce the desired
therapeutic or
immunogenic effect in association with the required pharmaceutical carrier.
Compositions of
peptides, glycans or antibodies, when administered orally, can be protected
from digestion,
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using methods known in the art (see, e.g., Fix, Pharm Res. 13: 1760-1764,
1996; Samanen, J.
Pharnn. Pharmacol. 48: 119-135, 1996).
[0091] In an embodiment, a composition or vaccine is prepared as an
injectable, either as a
liquid solution or suspension, or as a solid form which is suitable for
solution or suspension in a
liquid vehicle prior to injection. In another embodiment, a composition or
vaccine is prepared in
solid form, emulsified or encapsulated ,n a liposome vehicle or other
particulate carrier used for
sustained delivery. For example, a vaccine can be in the form of an oil
emulsion, a water in oil
emulsion, a water-in-oil-in-water emulsion, a site-specific emulsion, a long-
residence emulsion,
a sticky emulsion, a microemulsion, a nanoemulsion, a liposome, a
microparticle, a
microsphere, a nanosphere, or a nanoparticle. A vaccine may include a
swellable polymer such
as a hydrogel, a resorbable polymer such as collagen, or certain polyacids or
polyesters such
as those used to make resorbable sutures, that allow for sustained release of
a vaccine.
[0092] In some embodiments, compositions provided herein include one or
more additional
therapeutic or prophylactic agents for CDI. For example, a composition may
contain a second
agent for preventing or treating C. difficile infection. Examples of such
second agents include,
without limitation, antibiotics (such as metronidazole and vancomycin) and
antibodies (such as
antibodies that bind to toxin A, toxin B, lipoteichoic acid (LTA), PS-I, PS-
II, a C. difficile
vegetative cell surface protein, or a C. difficile spore cell surface protein
such as BcIA1, BcIA2,
Alr, SIpA paralogue, SIpA HMW, CD1021, lunH, Fe-Mn-SOD, and FliD).
[0093] In alternative embodiments, compositions of the present invention
may be employed
alone, or in combination with other suitable agents useful in the prevention
or treatment of CD.
In some embodiments compositions of the present invention are administered
concomitantly
with a second composition comprising a second therapeutic or prophylactic
agent for CDI.
[0094] As used herein, a "therapeutically effective amount" or "an
effective amount" refers
to an amount of a composition, vaccine, antigen, or antibody that is
sufficient to prevent or treat
CU, to alleviate (e.g., mitigate, decrease, reduce) at least one of the
symptoms associated with
CDI, and/or to induce an immune response to C. difficile, such that benefit to
the subject is
provided. The effective amount of a composition, vaccine, antigen, or antibody
may be
determined by one of ordinary skill in the art. Exemplary dosage amounts for
an adult human
include, without limitation,from about 0.1 to 500 mg/kg of body weight of
antigen or antibody per
day, which may be administered in a single dose or in the form of individual
divided doses, such
as from 1 to 5 times per day, or weekly, or bi-weekly.
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[0095] In some embodiments, an effective amount of a composition comprising
a protein
contains about 0.05 to about 1500 pg protein, about 10 to about 1000 pg
protein, about 30 to
about 500 pg, or about 40 to about 300 pg protein, or any integer between
those values. For
example, a protein may be administered to a subject at a dose of about 0.1 pg
to about 200 mg,
e.g., from about 0.1 pg to about 5 pg, from about 5 pg to about 10 pg, from
about 10 pg to about
25 pg, from about 25 pg to about 50 pg, from about 50 pg to about 100 pg, from
about 100 pg to
about 500 pg, from about 500 pg to about 1 mg, or from about 1 mg to about 2
mg, with optional
boosters given at, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, two months,
three months,
6 months and/or a year later.
[0096] In some embodiments, an effective amount of an antibody composition
for passive
immunization ranges from about about 0.001 to about 30 mg/kg body weight, for
example,
about 0.01 to about 25 mg/kg body weight, about 0.1 to about 20 mg/kg body
weight, about 1 to
about 10 mg/kg, or about 10 mg/kg to about 20 mg/kg.
[0097] A composition, vaccine, antigen or antibody may also be administered
once per
month, twice per month, three times per month, every other week (qow), once
per week (qw),
twice per week (biw), three times per week (tiw), four times per week, five
times per week, six
times per week, every other day (qod), daily (qd), twice a day (qid), or three
times a day (tid).
For prophylactic purposes, the amount of peptide in each dose is selected as
an amount which
induces an immunoprotective response without significant adverse side effects
in a typical
vaccine. Following an initial vaccination, subjects may receive one or several
booster
immunisations adequately spaced.
[0098] It will be understood that the specific dose level and frequency of
dosage for any
particular subject may be varied and will depend upon a variety of factors
including the activity
of the specific compound employed, the metabolic stability and length of
action of that
compound, the species, age, body weight, general health, sex and diet of the
subject, the mode
and time of administration, rate of excretion and clearance, drug
combinations, and severity of
the particular condition.
SgtA glycosyltransferase
[0099] There is also reported herein the identification of a SgtA
glycosyltransferase.
Insertional inactivation of the glycosyltransferase gene, sgtA
(CD3350/CDR3194), provided
direct evidence for a role of the glycosyltransferase enzyme in spore surface
13-0 linked GIcNAc
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reactivity, as well as in the production of glycosylated BcIA3. The sgtA gene
is thus linked to
production of C. difficile spore glycans and BcIA3 spore antigens.
[00100] There is provided herein a SgtA glycosyltransferase for use in
preparing C. difficile
spore antigens as described herein. In an embodiment, SgtA glycosyltransferase
is used for
production of a BcIA3 glycoprotein, glycopeptide or glycan, e.g., in vitro or
in a recombinant
expression system. In an embodiment, SgtA glycosyltransferase is used for
recombinant glycan
production. SgtA glycosyltransferase may be prepared using known techniques,
e.g., it may be
expressed recombinantly and then isolated or purified, or used in an extract
from an expression
system or cell line.
Spore diagnostics and detection
[00101] There are provided methods of diagnosing CDI based on detecting the
presence of
C. difficile in a subject using a BcIA3 spore antigen as described herein.
Known methods of
detecting bacterial antigens in a sample from a subject may be used to detect
the presence of a
BcIA3 glycoprotein, glycopeptide, or glycan, the presence of the BcIA3
glycoprotein,
glycopeptide, or glycan being indicative of the presence of C. difficile in
the subject. In an
embodiment, the presence of the BcIA3 glycoprotein, glycopeptide, or glycan in
a sample from a
subject is indicative of the presence of C. difficile spores in the subject.
[00102] A sample may be, for example, a stool sample, inlcuding without
limitation a liquid
stool sample, a solid stool sample, a rectal swab, etc.. Antigens may be
detected using a variety
of common techniques in the art, such as without limitation detection using an
antibody reagent
specific for a BcIA3 antigen, e.g., using an enzyme immunoassay.
Alternatively, a nucleic acid
amplification test such as PCR may be used to detect the C. difficile BcIA3
gene in a sample
from a subject.
[00103] In one embodiment, there is provided use of the isolated BcIA3
glycoprotein or
glycopeptide according to any one of rlaims 1 to 10 or the isolated BcIA3
glycan according to
any one of claims 11 to 16 for detecting the presence of C. difficile in a
subject. In another
embodiment, there is provided use of the antibody or fragment of any one of
claims 39 to 51 for
detecting the presence of C. difficile in a subject. In yet another
embodiment, there is provided
a method of detecting the presence of C. difficile in a subject comprising:
obtaining a stool
sample from the subject, and assaying the stool sample for the presence of a
BcIA3
glycoprotein, glycopeptide, and/or glycan thereof, wherein the presence of the
BcIA3
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glycoprotein, glycopeptide, and/or glycan thereof in the stool sample
indicates the presence of
C. difficile in the subject.
[00104] In some embodiments, methods provided herein are used to detect the
presence of
C. difficile spores in a subject. Diagnostic methods provided herein may thus,
in some
embodiments, provide an advantage over current diagnostic methods for CD! that
rely on the
detection of C. difficile toxin; as C. difficile toxin is produced by
vegetative cells and not spores,
such methods are not highly effective at detecting the presence of C.
difficile spores in patients,
e.g., in patients at risk of recurrence of Ca In an embodiment, methods
provided herein are
used to diagnose patients at risk of recurrence of CDI based on detecting the
presence of C.
difficile spores in a subject using a BcIA3 spore antigen as described herein
Kits
[00105] Kits are provided for preventing or treating CD', comprising one or
more BcIA3
spore antigen, antibody, composition, and/or vaccine as described herein.
Instructions for use
or for carrying out the methods described herein may also be provided in a
kit. A kit may further
include additional reagents, solvents, buffers, adjuvants, etc., required for
carrying out the
methods described herein.
[00106] Also provided are kits for diagnosing CDI in a subject or for
determining that a
subject is at risk of recurrence of CDI comprising reagents for detecting the
presence of one or
more of a BcIA3 glycoprotein, glycopeptide, and glycan in a subject.
Instructions for use or for
carrying out the diagnostic methods described herein may also be provided in a
kit, as well as
additional reagents, solvents, buffers, etc., required for carrying out the
methods described
herein.
[00107] As used herein, the singular forms "a", "an" and "the" include
plural references
unless the content clearly dictates otherwise.
[00108] As used herein, the term "about" refers to a value that is within
the limits of error of
experimental measurement or determination. For example, two values which are
about 5%,
about 10%, about 15%, or about 20% apart from each other, after correcting for
standard error,
may be considered to be "about the same" or "similar". In some embodiments,
"about" refers to
a variation of 20%, 10%, or 5% from the specified value, as appropriate to
perform the
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disclosed methods or to describe the disclosed compositions and methods, as
will be
understood by the person skilled in the art.
[00109] The technology described herein is not meant to be limited to
particular methods,
reagents, compounds, compositions or biological systems, which can, of course,
vary. It should
also be understood that terminology used herein is for the purpose of
describing particular
aspects only, and is not intended to be limiting.
EXAMPLES
[00110] The present invention will be more readily understood by referring
to the following
examples, which are provided to illustic_Ite the invention and are not to be
construed as limiting
the scope thereof in any manner.
[00111] Unless defined otherwise or the context clearly dictates otherwise,
all technical and
scientific terms used herein have the same meaning as commonly understood by
one of
ordinary skill in the art to which this invention belongs. It should be
understood that any
methods and materials similar or equivalent to those described herein can be
used in the
practice or testing of the present technology.
Example 1. Bioinformatic identification of BcIA and BcIB homologs in strains
of C.
difficile.
[00112] In contrast to spores of C. difficile, the spores of another
important toxin producing,
Gram positive pathogen, Bacillus anthracis have been extensively
characterised. These spores
are enclosed by an exosporangial layer which is composed of a number of
different proteins,
and which includes an outermost hair-like nap layer. The filaments of the nap
layer are primarily
composed of a highly immunogenic collagen-like protein BcIA as well as a
second
exosporangial protein, BcIB. Both BcIA and BcIB have been well characterised
and shown to be
glycosylated with an 0-linked pentasaccharide which contains the novel
terminal sugar, 2-0-
methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-D-glucopyranose (also
referred to as
anthrose).
[00113] The Gram positive spore forming bacterium Bacillus anthracis thus
elaborates two
glycosylated spore surface proteins, denoted BcIA (BAS1130, YP_027402 in
strain Sterne) and
BcIB (BAS2281, YP_028542 in strain Sterne) for Bacillus collagen-like protein
(Fig. la).
Homologs of BcIA have also been found within the genome sequences of Bacillus
cereus, and
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Bacillus thuringiensis (Todd, S.J. et al., J. Bacteriology 185:3373-3378,
2003). BLAST searches
of the BcIA and BcIB sequences against genome sequences of three C. difficile
strains revealed
Bc1 protein homologs. C. difficile 630, the first strain to have a completed
genome sequence,
had three ORFs with homology to BcIA and BcIB. These were found in different
regions of the
C. difficile 630 genome, as shown in Fig. lb. Percentage homology and E-values
of significance
are shown in Table 2. The ORF CD3349 was annotated as BcIA3 (YP_001089866),
despite
showing greater homology to BcIB, therefore we will refer to this gene as
BcIA3, to remain
consistant with the genome annotation. Fig. lb also shows C. difficile 630
CD3349 (BcIA3,
YP001089866) to be located 66 bp downstream of a putative glycosyltransferase
(CD3350,
YP_001089867). The glycosyltransferase gene has high homology to a B.
anthracis
glycosyltransferase (BAS 1131, YP_027403) that is likely responsible for
transfer of
carbohydrate components to exosporangial proteins in this species. The
proximity of the genes
and high homology with genes of known function within B. anthracis provided
early suggestions
that C. difficile exosporangial proteins may be glycosylated.
[00114] We next searched the genomes of C. difficile strains QCD-32g58 and
R20291.
Within the strain QCD-32g58 genome, only a single ORF had significant homology
to the Bc1
proteins; CdifQ_040500019311 (WP_009891815), showed 62% homology to BcIA and
50%
homology to BcIB of B. anthracis. A BLAST search of the glycosyltransferase
gene BAS 1131
against the QCD-32g58 genome sequence showed a homolog: a putative
glycosyltransferase
(CdifQ_040500019316, WP_009891817) 78bp upstream of the putative exosporium
glycoprotein gene (CdifQ_040500019311). The genome of strain R20291 contained
two bcl
gene homologs, with the translated product of CDR20291_3090 (BcIA2,
YP_003219565),
showing 64% homology to the B. anthracis BcIB and CDR20291_3193 (BcIA3,
YP_003219669)
showing 56% homology to the B. anthracis BcIA. In addition, CDR20291_3194
(YP_003219670)
which lies 78bp downstream of BcIA3 showed 46% identity with the B. anthracis
glycosyltransferase (Table 2).
[00115] In all three strains, the Bc1 protein homologs have only short,
trypsin susceptible
regions (as predicted by amino acid sequence) at the N and C-termini. The
central region of the
Bc1 protein homologs is comprised of approximately 40 kDa of collagen like
repeats with no
predicted trypsin cleavage site. The, three C. difficile glycosyltransferase
homologs have
identical sequences apart from a single conserved amino acid substitution at
the third amino
acid in the sequence.
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Table 2. BLAST search of Bacillus anthracis exosporangial glycoproteins (BcIA
& BcIB)
translated gene sequences against selected Clostidium difficile translated
genomes. Shown are
the percentage sequence identity and expect values (E-value in brackets).
B.
anthracis C. difficile C. difficile C. difficile
Strain Strain 630 Strain R20291
Strain QCD-32g58
Sterne
Putative
Exosporium Exosporium Exosporium Exosporium Exosporium
exosporium
glycoprotei glycoprotei glycoprotei glycoprotein glycoprotein
n Bc1A1 n Bc1A2 n Bc1A3 Bc1A2 Bc1A3 glycoprotein
Bc1A3
BcIA 60% 57% 49% 56% = 56% 62%
(2e-68) (4e-49) (le-41) (le-52) (7e-32) (7e-41)
BcIB 69% 62% 50% 64% 50% 50%
(le-38) (2e-34) (le-35) (8e-37) = (le-35) (3e-35)
Glycosyl
Glycosyl transferase Glycosyl transferase
transferase
Glycosyl
47% 46% 46%
transferase
(7e-114) (4e-113) (4e-113)
Example 2. Characterisation of C .difficile exosporangial surface extraction.
[00116] It has been demonstrated previously that C. difficile spores possess
an
exosporangial layer which surrounds the spore coat and this layer has been
shown to be
structurally variable amongst isolates. Various treatments of spores have been
utilised to
remove this layer, allowing the characterisation of the underlying spore coat,
however the
structural components of the exosporangial layer have not previously been
characterised. Here
we focused on identifying and characterising spore surface associated protein
components.
Using a detergent based extraction, surface associated components were removed
from spore
preparations which had not been extensively water washed or treated with
enzymes/sonication
to facilitate retention of surface structures. Endospores of strains 630, QCD-
32g58 and R20291
were incubated in detergent solutions to extract the spore surface proteins
and then intact
spores were removed by centrifugation. The protein containing supernatants
were resolved by
NuPAGE 3-8% gradient gel. The high molecular weight region of the gel showed
diffuse
banding patterns reactive with both silver stain (Fig. 2a) and glycostain
(Fig. 2b), suggesting
high molecular weight complexes containing glycoproteins. Proteinase K
digestion of spores
had only a marginal effect on the migration of this material suggesting a more
complex
composition. A distinct pattern of staining was obtained for R20291 and
QCD32g58 spore
extracts when compared to 630 in this region. Glycostaining revealed a
reactive high molecular
weight band in R20291 and QCD32g58 extracts only.
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[00117] Initially, extraction of all gel bands of molecular weight <160 kDa
was performed and
each band was digested with either trypsin or proteinase K and analysed by nLC-
MS/MS. No
BcIA protein identification was made from these analyses. Subsequently, the
high molecular
weight region (> 160 kDa) of each lane was excised in bands, and digested with
trypsin or
proteinase K. The trypsin digests for all three strains did not result in any
protein identifications
by nLC-MS/MS. Analysis of the MS/MS spectra from proteinase K digests,
however, yielded
several spore surface protein identifications, as indicated by the numbered
annotations in Fig.
2a, Lane 3 and summarized in Table 3 for R20291, and Fig. 8 and Table 4 for
QCD-32g58.
[00118] De novo sequencing of the peptide MS/MS spectra from the proteinase
K digests of
gel bands 1-3 from surface extract of R20291 spores revealed a number of
peptides
corresponding to the putative exosporium glycoprotein BcIA3 (CDR20291_3193).
Further
inspection of the MS/MS spectra showed peptides with ions that did not
correspond to peptide y
or b type ions and were characteristic of carbohydrate associated fragment
ions. For example,
from tandem mass spectrometry analyses of band 1 (Fig. 2a) which migrated to a
molecular
mass of greater than 600 kDa, the MS/MS spectrum of the putative glycoprotein
peptide
AGLIGPTGATGV modified with three N-acetyl hexosamine (HexNAc) moieties is
shown in Fig.
3a. The glycan modification was observed as sequential neutral losses of 203
Da from the
glycopeptide precursor ion in the high m/z region of the MS/MS spectrum. In
addition, an
intense glycan oxonium ion was observed at m/z 204 which was common to all of
the identified
glycopeptides. In addition more complex glycosylation patterns were observed
in some cases,
with intense ions observed in glycopeptide spectra that did not correspond to
HexNAc residues
nor peptide type y or b fragment ions (for example, oxonium ions corresponding
to masses of
486Da, 372 Da, 374 Da). Fig. 3b shows an MS/MS spectrum of the BcIA peptide
TGPTGATGADGITGP, modified with three HexNAc moieties and an additional mass of
374 Da.
The sequential neutral losses suggest that HexNAc is the linking sugar. This
glycan neutral
mass was also linked to a putative glycan oxonium ion at m/z 375. Other
intense ions were also
observed in the low m/z region of this MS/MS spectrum, including putative
glycan fragment ions
at m/z 300 and 272. The absence of potential N-linked glycosylation sites
suggested the
glycans to be 0-linked through threonine residues within each peptide.
Observed sequential
neutral losses of 203 Da in the high m/z region of the spectrum and the
presence of an intense
ion corresponding to the unmodified form of the peptide suggest the glycan is
composed of
oligosaccharide chains attached to a single amino acid residue in each
identified glycopeptide.
[00119] Table 3 shows the complete list of surface protein peptides and
glycopeptides
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identified from each of the annotated band numbers indicated in Fig. 2a. The
unknown glycans
varied in observed mass from 281 to 486 Da and it is possible that these are
modified HexNAc
moieties. Tandem mass spectrometry analysis of proteinase K digests of bands 2
and 3 showed
BcIA3 glycopeptides modified with chains of HexNAc moieties, predominantly in
trimers. In
these cases, only peptides modified with HexNAc moieties were observed at
detectable levels.
Table 3. nLC-MS/MS analysis of glyco-reactive peptides. High molecular weight
gel bands of
R20291 spore surface extracts were digested with proteinase K and the
numbering of gel bands
refers to Figure 2. MS/MS spectra were de novo sequenced, and the identified
peptides and
observed glycan moieties are indicated.
Accession Glycan Mass, Da
(Monosaccharide
Band Protein (MW, kDa) Peptide sequence
number neutral
losses, Da)
1 YP 003219669 Exosporium 355AGLIGPTGATGV19 609
(203-203-203)
(CD¨R20291_3193) glycoprotein BcIA3 145TGPTGATGADGITGP159
983 (203-203-203-374)
(59.5 kDa) 344VGPTGATGA352 (also a a 391-399, 406 (203-
203)
aa439+447, aa457-495)
159G L I G PTGATGTPGA2 2 406 (203-203)
275TGATG LI G PTGATGA292 1 186 (203-203-203-203-
374)
275TGATGLIGPTGATGA252 1241 (203-203-203-203-
429)
212TGIGITGPTGATGA225 (also aa259+ 1184 (203-203-203-203-
372)
272)
212TGIGITGPTGA222 (also aa 259-26) 1095 (203-203-203-486)
399G LI G PTGATGVTGA322 1071 (203-203-203-462)
299TGVTGATGAAG L I G P313 609 (203-203-203)
2 YP 003219669 Exosporium 215TGATGLIGPTGATGA2u2
1187 (203-203-203-203-375)
(CD¨R20291_3193) glycoprotein BcIA3 1911 G PTGATGTPGATGPTGA2
8 1661 (203-203-203-222-203-203-424)
(59.5 kDa) 424TGPTGATGPTGADGL435 609 (203-203-203)
3 YP 003219669 Exosporium 119GVTGPTGPTGPTGATGA135
609 (203-203-203)
(CD¨R20291_3193) glycoprotein BcIA3 159GVTGPTGPTGATGV152
609 (203-203-203)
(59.5 kDa) 344VG PTGATGATGADGC55 609 (203-203-203)
359VG PTGPTGATGV79 609 (203-203-203)
1911GPTGATGTPGATGPTGA208 609 (203-203-203)
311IGPTGATGVTGADGA325 609 (203-203-203)
439VG PTGATGATG L45 609 (203-203-203)
391VG PTGATGATGADGV4 5 609 (203-203-203)
359VG PTGPTGATGV37 203
656ATASG LS LVNTVA665
[00120] Table 4 shows a list of surface protein peptides and glycopeptides
identified from
annotated band numbers as indicated in Fig. 8.
Table 4. nLC-MS/MS analysis of glyco-reactive peptides. MS/MS spectra were de
novo
sequenced, and the identified peptides and observed glycan moieties are
indicated.
Unique/repeated m/z Peptide Sequence Glycan
mass, Da
sequence
Unique 1001.512+ '55GVTGPTGPTGATGV 203-203-203-220
(SEQ ID NO: 27)
901.422+ 293TGPTGVTGATGA354 203-203-203-203
(SEQ ID NO: 25)
811.892+ 305AG L I G PTGATG V31 b 203-203-203
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(SEQ ID NO: 2)
1088.52+ JuAG LI G PTGATGVnb 203-203-203-552
(SEQ ID NO: 2)
927.432+ Ju6IGPTGATGVTGADGA'22 203-203-203
(SEQ ID NO: 32)
998.482c 3b4G LVGPTG PTGATGV361 203-203-203-215
(SEQ ID NO: 29)
1130.512' 4u3AGPTGATGATGNTGADGA42u 203-203-203-203
(SEQ ID NO: 30)
1043.492+ 421TGPTGATGPTGADGL435 203-203-203-203
(SEQ ID NO: 13)
1052.032' 421TGPTGATGPTGADGL43b 203-203-203-220
(SEQ ID NO: 13)
806.42' 43bVG PTGATGATG L44( 203-203-203
(SEQ ID NO: 20)
Repeated 597.802+ TGPTGADGL (x4) 203-203
(SEQ ID NO: 28)
978.96+2 GVTGPTGPTGATGA (x3) 203-203-203-203
(SEQ ID NO: 26)
[00121] All of the identified glycopeptides reside within the central
collagen like repeating
domain of the BcIA3 protein. The central collagen-like repeat domains of the
putative exosporial
proteins contained some non-unique regions, which resulted in the
identification of multiple
glycopeptides with amino acid sequerr-,es that repeat within the protein
sequence (for example,
TGIGITGPTGA occurs in BcIA3 at residues 212-222 and 259-269). One of the
identified
peptides, which is repeated in BcIA3 four times, is also common to both BcIA3
and BcIA2
(VGPTGATGA). The non-specific cleavage by proteinase K produced a number of
glycopeptides with overlapping sequences. Furthermore, multiple glycopeptides
were identified
that possessed identical peptide sequences but different glycans.
[00122] As indicated above, strains R20291 and QCD-32g58 showed similar
protein staining
patterns for both silver and glycostains and nLC-MS/MS analysis also showed
that the Bcl
protein of QCD-32g58 was similarily glycosylated predominantly with HexNAC
moieties (Fig. 8).
nLC-MS/MS analysis of the gel digests from strain 630, which showed
significantly different
staining patterns in the high molecular weight region of the gel as compared
to the other two
strains, did not yield any protein or glycoprotein identifications.
Example 3. Anti-p-O-GIcNAc reactivity of C. difficile spores.
[00123] As the most abundant glycan modification observed in the MS
analysis of spore
surface extracted material was shown to have a mass corresponding to an N
acetyl-hexosamine
moiety, we next examined the ability of spores to bind to an 0-linked N-Acetyl
glucosamine (3-
0-GIcNAc) antibody. A monoclonal antibody (MAb) which recognises 0-GIcNAc in a
p-o-
glycosidic linkage to both threonine and serine was utilised in
immunofluorescence experiments
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with intact spores from a number of C. difficile clinical isolates (Fig. 4A).
This antibody had been
used previously to demonstrate presence of 13-0-GIcNAc attached to serine and
threonine
residues of Listeria monocytogenes flagellin (Schirm, M. et al., J.
Bacteriology 186: 6721-6727,
2004).
[00124] When the p-o GIcNAc antibody was used in immunofluorescence
reactions with
spores of R20291 and 630Aerm, both spore preparations reacted strongly with
the antibody.
Interestingly, distinct patterns of reactivity with the spore surface were
observed by this
immunofluorescence method for each strain (Fig. 4A). With R20291 spores anti-p-
O-GIcNAc
was uniformly reactive over the entire spore surface, while with strain
630Aerm, anti-13-0-
GIcNAc reactivity was restricted to the poles of the spores with only limited
labelling of the
central surface (see arrows marking binding at poles; Fig. 4A). Vegetative
cells of both strains
showed no reactivity with anti-p-O-GIcNAc (Fig. 9). To confirm the
conservation of 13-0-GIcNAc
on the surface of multiple C. difficile strains, a range of spores from
different ribotypes and
geographic locations were also tested for anti-p-O-GIcNAc binding. The
reactivity pattern
observed for R20291 spores was found to be conserved in all strains examined,
with the only
exception being spores of 630Aerm (Fig. 4A). Any unstained cells in the images
were either
immature spores or cell debris from the washing process. DAPI binding was
observed only with
vegetative cells and immature phase dark spores. Phase bright spores were
considered mature.
Example 4. Characterization of SgtA glycosyltransferase.
[00125] RT-PCR of CD3350/bc/A3 gene locus. As indicated in Fig. 1, CD3350
(B.
anthracis exosporangial glycosyltransferase gene homolog) and BcIA3 lie
immediately adjacent
to each other and are orientated in the same direction on the chromosome in
both 630 and
R20291 strains. The two genes are separated by only a short intergenic region
suggesting they
may form a single transcriptional unit. Primers which amplified across this
intergenic region
were used to determine if the genes were co-transcribed. RNA samples extracted
from C.
difficile 630 cells were subjected to reverse transcription and an
amplification product of 257bp
linking CD3350 and bcIA3 was obtained confirming cotranscription of these two
genes (Fig. 10).
PCRs using the same primers and total RNA that had not undergone reverse
transcriptase
reaction did not yield any amplification product demonstrating that the RNA
was free of
contaminating DNA.
[00126] Mutagenesis of CD3350/CDR3194 and spore characterisation. We next
generated an insertionally inactivated glycosyltransferase mutant in strains
630Aerm and
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R20291 respectively (ACD3350 and ACDR3194) by using the ClosTron technology as
previously described (Heap, J.T. et al., J. Microbio. methods 80:49-55, 2010).
Insertion of the
TargeTron Erm resistance marker was confirmed by PCR using primers flanking
the gene of
interest and with primers specific to the TargeTron Erm resistance marker
(data not shown).
Vegetative cell growth of both ACD3350 and ACDR3194 were unchanged when
compared to
their respective parent strains and motility was unaffected (data not shown).
Immunofluorescence of spores, with anti-p-O-GIcNAc antibody, revealed a
complete loss of
reactivity for both ACD3350 and ACDR3194 when compared to respective parent
strains (Fig.
4B). The percentage of wild type spores compared to mutant phase bright spores
reacting with
anti-p-O-GIcNAc antibody was quantified microscopically and shown to be 80-95%
compared to
less than 1% for the respective mutants (Fig. 4C).
[00127] Both ACDR3194 and ACD3350 strains were complemented with wild type
copies of
CD3350 using pRPF185 (Fagan, R.P. et al., J. Biol. Chem. 286:27483-27493,
2011) as
evidenced by both western blotting and innmunofluorescence studies (Fig. 5 and
Fig. 11). As
can be seen in Fig. 5 lanes 2 and 5, a positive reaction was observed in
western blot with spore
surface extracts from both R20291 and 630Aernn spores respectively. Spore
extracts of R20291
displayed reactivity with the region of gl corresponding to band 4
(approximately 400 kDa) from
MS analysis. In addition, a second strongly reactive band migrating at
molecular mass of
approximately 170 kDa on 3-8% NuPAGE gel was observed, although we were unable
to
identify peptides from a proteinase K digestion of this region of the gel by
MS analysis. For
strain 630Aerm, no reactivity was observed in the corresponding higher
molecular weight region
of the gel and a series of three distinct reactive bands were observed at
approx. 170 kDa. All
reactivity was lost in CD3350 and CDR3194 mutant strains while the strain
specific pattern of
reactivity was restored upon complementation (Fig. 5 and Fig. 11). On the
basis of these results
this gene was named sgtA for spore glycosyltransferase.
[00128] Characterisation of AsgtA spore surface extract. In parallel with
the spore
surface protein extracts of the wild type strain, spore surface extracts of
the R20291AsgtA
mutant strain were also prepared and analysed by NuPAGE gradient 3-8% Tris
acetate gels to
resolve high molecular weight material. The protein stained gel shows a
diffuse area of staining
at 460 kDa and greater, however, the distinct ¨600 kDa band was not observed.
Similarly,
glycostaining of the same gel showed no detectable reactivity at ¨600 kDa
(Fig. 12). In contrast
to MS studies of gel bands of R20291 spore surface extracts which identified
peptides/glycopeptides in Proteinase K digests, the equivalent region of the
NuPAGE gel of the
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spore surface protein extraction of AsgtA did not yield any peptide or
glycopeptide
identifications. In addition, our analyses of lower molecular weight protein
bands from spore
surface extracts showed no evidence of unglycosylated BcIA3.
[00129] Resistance of R20291AsgtA spores. As the more clinically relevant
strain and as
shown by immunofluorescence to be a more representative strain of C. difficile
spore
morphology, phenotypic assays were undertaken on R20291 wild type spores
compared to
AsgtA spores. Heat resistance of spores was examined as previously described
(Permpoonpattana, P. et al., J. Bacteriology 195:1492-1503, 2013). When
incubated at 80 C for
20 minutes AsgtA spores showed significantly lower survival rates than the
parent R20291
spores (Fig. 6). Susceptibility of spores to 70% ethanol and 250 pg/ml
lysozyme was also
examined, but no significant difference was observed between wild type and
AsgtA spores (Fig.
13).
[00130] Role of sgtA in adherence and internalisation of macrophage cells.
To gain
insight into a possible biological role for the SgtA glycosyltransferase we
next investigated the
ability of spores to adhere to and be internalised by J774A.1 macrophage cell
line (ATCC TIB-
106). Spores were counted based on association with J774A.1 cells, and counted
as adhered if
green/red and internalised if red. Spores not associated with cells were
ignored as were any
remaining vegetative cells based on rod shape. As can be seen in Fig. 7,
adherence and
internalisation of J774A.1 macrophage cells by C. difficile R20291 spores was
affected following
inactivation of sgtA gene, with significantly greater numbers of AsgtA spores
being internalised
compared to wild type.
[00131] In sum, the studies described above present a characterization of
glycoproteins from
C. difficile spores and provide direct evidence demonstrating that BcIA3 is a
glycoprotein which
is glycosylated with chains of 13 0-linked GIcNAc as well as with additional
glycans of novel
mass. Our nLC-MS/MS analysis identified BcIA3 peptides and provides the first
evidence that
this protein is a glycoprotein. The BcIA3 protein of C. difficile is
glycosylated with predominantly
novel tri- or pentasaccharide oligosaccharides, composed of chains of N-Acetyl
hexosamine
sugars which in addition, may be capped with novel glycan moieties.
[00132] It is clear from glycan component neutral masses that the
structural composition of
the C. difficile BcIA3 glycan is quite distinct to that previously reported
for B. anthracis
(Daubenspeck, J.M. et al., J. Biol. Chem. 279:30945-30953, 2004). Gel
migration characteristics
suggested that C. difficile BcIA3 monomers from R20291 and QCD-32g58 form a
stable, higher
38
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molecular weight complex which is resistant to denaturation by heating and
detergents.
[00133] It is noted that p-o linked GIcNAc reactivity of spores from a
number of clinical
isolates has demonstrated for the first time the conserved nature of this
posttranslational
modification on C. difficile spores. Further, insertional inactivation of the
glycosyltransferase
gene, sgtA (CD3350/CDR3194), provided direct evidence for a role of the
glycosyltransferase
enzyme in this spore surface 13-o linked GIcNAc reactivity, as well as in the
production of
glycosylated BcIA3. Our studies thus link the sgtA gene to a specific spore
glycan associated
function.
[00134] The spores of Gram positive bacterial pathogens have gained
considerable attention
in recent years. A role for surface-associated bacterial glycans in host
interactions has been
well documented for many bacterial species. Spores are known to be
recalcitrant to proteolytic
digestion and structural characterization. The studies described herein showed
that spores of a
second important Gram positive pathogen, C. difficile, also carry novel
glycoproteins on surface
associated structures, and that glycans on the spore surface impart resistance
of spores to heat
treatment as well as appear to play a role in macrophage interactions.
MATERIALS AND METHODS
[00135] Bacterial strains and growth conditions. C. difficile strains used
in this study are
listed in Table 5. Initial experiments were carried out using strains 630Aerm
and R20291.
Comparisons with other C. difficile strains from a variety of ribotypes (QCD-
32g58, BI-6, CD20,
CF5, and M68) revealed R20291 to be the more representative strain. R20291 is
also a more
clinically relevant strain, and a better spore former than strain 630. For
these reasons, later
experiments, particularly the biological assays, were focused on R20291
spores. All strains
were routinely grown under anaerobic conditions on brain heart infusion agar
medium (BD,
Sparks, MD) supplemented with 5 g/litre yeast extract, 1.2 g/litre NaCI, 0.5 g
litre cysteine HCI, 5
mg/litre hemin, 1 mg/litre vitamin K, and 1 mg/litre resazurin (BHIS).
Erythromycin (2.5 pg/ml)
and thiamphenicol (15 pg/ml) were added as required for growth of mutant and
complemented
mutant strains.
Table 5. C. difficile strains used in this study.
Strain Characteristics Source
630Aerm Ribotype 012 (*)
Minton, University of
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Nottingham
R20291 Ribotype 027 (**) Wren, LSHTM
630ACD3350 630Aerm CD3350::erm This study
R20291ACDR3194 R20291 CDR3194::erm This study
630ACD3350p3350 630Aerm ACD3350 This study
pRPF185-CD3350
R20291ACDR3194p3350 R20291ACDR3194 This study
pRPF185-CD3350
QCD-32g58 Ribotype 027 (***):
Dascal, Montreal.
BI-6 Ribotype 0176 Wren, LSHTM
CD20 Ribotype 023 Wren, LSHTM
CF5 Riobtype 017 ( )
Wren, LSHTM
M68 Ribotype 017 ( )
Wren, LSHTM
*Hussain, H.A. et al., J. Med. Micro.54:137-141, 2005.
**Stabler, R.A. et al., Genome biology 10:R102, 2009.
***Forgetta, V. et al., J. Clin. Microbio. 49:2230-2238, 2011.
He, M. et al., Proc. Natl. Acad. Sci. 107:7527-7532, 2010.
[00136] Mass Spectrometry (MS) analysis of spores. Spores were harvested
from BHIS
agar plates into PBS, following 7 day incubation under anaerobic conditions,
heat treated at
56 C for 15 minutes, collected by centrifugation (500g, 30 min) and washed
once in PBS. Then
cfu/ml was determined by serial dilution and plating on BHI containing 0.1%
sodium
taurocholate (Sigma-Aldrich, Oakville, Canada) (BHI-ST). Approximately 5 x 109
spores were
resuspended in 200 pl of extraction buffer (2.4 ml 1 M Tris pH 6.8, 0.8 g ASB-
14, 4 ml 100%
glycerol, VA DTT, 3.8 ml ddH20) and were left for 30 minutes at room
temperature. Spores
were removed by centrifugation and soluble material was collected for
analysis.
[00137] Protein containing endospore surface extractions were separated
using 3-8%
NuPage Novex Tris-Acetate minigels following the manufacturer's instructions
(Invitrogen, Life
Technologies). High-molecular-mass Hi-mark' (31 to 500 kDa) were used as
markers. The gel
was stained using Emerald-Q glycostain, as per the manufacturer's instructions
(lnvitrogen, Life
Technologies) and subsquently with non-fixing silver stain (Blum, H. et al.,
Electrophoresis 8:93-
99, 1987). Protein bands were excised, reduced for 1 hour with 10 nnM DTT at
56 C, and
alkylated for 1 hour with 55 mM iodoacetamide in the dark (Gharahdaghi, F. et
al.,
Electrophoresis 20:601-605, 1999) prior to digestion with trypsin as described
previously
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(Fulton, K.M. et al., IJMM 301:591-601, 2011) or with proteinase K. Proteinase
K digests were
carried out using 100 pg/ml of enzym6 in 50 mM ammonium bicarbonate for 15-40
hours. The
resulting peptides were analyzed by nanoliquid chromatography coupled to
tandem MS (nLC-
MS/MS) using electrospray ionization (ESI) as the ion source as recently
described (Fulton,
K.M. et al., IJMM 301:591-601, 2011). Briefly, peptides were analyzed by
nanoflow reversed-
phase liquid chromatography (RPLC) coupled to MS using ESI (nanoRPLC-ESI-MS)
using a
nanoAcquity UltraPerformance LC (UPLC) system coupled to a Q-TOF Ultima hybrid
quadrupole¨TOF mass spectrometer (Waters, Milford, MA). The peptides were
first loaded onto
a 180 pm inner diameter (ID) by 20 mm 5 pm symmetry C18 trap column (Waters,
Milford, MA)
and then eluted to a 100 pm ID by 10 cm 1.7 pm BEH130C18 column (Waters,
Milford, MA)
using a linear gradient from 1% to 45% solvent B (ACN plus 0.1% formic acid)
in 18 min, 45% to
85% solvent B for 3 min, 85% to 1% solvent B over 1 min. Solvent A was 0.1%
formic acid in
HPLC grade water. The peak list files of MS/MS spectra from tryptic digests
were searched
against the NCBI database using the MASCOT search engine (Version 2.3.0 Matrix
Science,
London, United Kingdom). A mass tolerance for precursor ions of 0.8 Da was
used for precursor
and fragment ions. Ion scores of 30 and above indicated identity. In addition,
all spectral
matches were verified manually. Unmatched MS/MS spectra and all MS/MS spectra
from
proteinase K digests were examined manually to determine the sequences of
peptide y and b
type ions.
[00138]
Construction of CD3350/CDR3194 insertional mutants and complemented
mutants. The target site was identified for CD3350 gene from C. difficile 630
using the
Targetron gene knockout system (Sigma Aldrich) and was used to design a 45 bp
retargeting
sequence for the gene. A derivative of plasmid pMTLOO7C-E2 carrying the
retargeting sequence
was obtained from DNA2.0 (Menlo Park, Ca) and used to generate mutants in
strains 630Aerm
and R20291 according to Heap et al. (Heap, J.T. et al., Methods in molecular
biology 646:165-
182, 2010; Heap, J.T. et al., J. Microbio. methods 80:49-55, 2010). A minimum
of two Erm
resistant transconjugants for each strain were checked by PCR using the ErmRAM
primers to
verify splicing of the group I intron following integration and also using
flanking primers for the
CD3350 gene to verify disruption of CD3350/CDR3194 gene by the ernn cassette.
[00139] Each of the CD3350/CDR3194 glycosyltransferase mutant strains were
complemented with a wild type copy of the C. difficile CD3350 gene using
plasmid pRPF185
(Fagan, R.P. et al., J. Biol. Chem. 286:27483-27493, 2011). The entire coding
sequence of the
gene including the Shine Dalgarno sequence was cloned under the control of the
inducible Ptet
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promoter. Plasmids were transferred to ACD3350/ACDR3194 mutant strains via
conjugation
and gene expression induced by plating onto BHIS agar containing anhydrous
tetracycline at
500 ng/nril after growth to mid-late log phase in BHIS broth.
[00140] Western blotting. Spore samples were harvested at 72h and
resuspended to
1x107 spores/100 pl in lx Laemmli loading buffer and heated to 95 C for 5 min.
Spore extracts
were separated on 3-8% NuPage Novex Tris-Acetate minigels and blotted onto
PVDF. The
membrane was probed with 1:5000 dilution of anti-8-0-GIcNAc (Covance,
Montreal, Canada) in
PBS-0.1%Tween 20 (PBS-T). Reactivity was detected with anti-mouse IgM HRP
conjugate
(Sigma Aldrich, Oakville, Canada) secondary antibody at 1:10000 dilution in
PBS-T. Blots were
imaged with ECL Prime western blotting detection kit (GE Healthcare, Baie
D'Urfe, QC,
Canada) according to manufacturer's instructions, followed by exposure to X-
ray film.
[00141] Transcription of CD3350 and BcIA3 genes by RT-PCR. To determine if
the
CD3350 and bcIA3 genes are cotranscribed, reverse transcriptase PCR (RT-PCR)
was
performed using primers designed to amplify across the intergenic region
between the two
genes from C. difficile 630. RNA template was extracted from broth grown cells
(4 h) using a
Trizol extraction procedure (Aubry, A. et al., Infection and immunity 80:3521-
3532, 2012). All
RNA samples were treated with RNase free DNase (Thermo Scientific) to remove
contaminating DNA. RNA was quantified and 30 ng was used for each RT-PCR using
Sensi-
script RT kit (Qiagen) and PCR amplification using TopTaq Master. In addition
PCR
amplifications were performed with the same primers using genomic DNA to
verify amplicon
size and specificity of primer pairs. Control PCR reactions of RNA without
reverse transcriptase
confirmed absence of contaminating DNA in samples.
[00142] Spore production for biological testing. For production of mature
spores, plates
were incubated for 7 days in an anaerobic incubator (Don Whitely Scientific,
UK) on BHI at
37 C. Spores were harvested from agar and heat treated at 60 C for 20 minutes.
To purify
spores, samples were washed x10 in H20 and cfu/ml determined by serial
dilution and plating
on BHI-ST.
[00143] lmmunofluorescence. Spores at 1 x 108/nil were air dried and heat
fixed onto
glass coverslips (VWR). The spores were blocked with 5% milk PBS for 30
minutes at room
temperature, then incubated with 1:100 dilution in PBS of 8-0-GIcNAc
monoclonal antibody
(Covance) for 45 minutes at room temperature. Coverslips were washed with PBS-
T, then
incubated with 1:100 dilution in PBS of anti-mouse IgG + IgM FITC conjugate
(Ca!tag,
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Burlingame, CA) for 45 minutes at room temperature in dark. Coverslips were
washed with
PBS-T then mounted with Vectashield + DAPI (Vector Laboratories, Burlingame,
CA) onto
slides. Slides were examined with Axioplan 200M (Zeiss), with multiple fields
of view observed.
The experiment was performed in duplicate on at least three independent
occasions. For
quantification of GIcNAc reactivity to spores, slides were prepared as stated
above using 7 day
H20 washed spores, then examined by microscopy. Using an Axioplan200 M
microscope
(Zeiss), at least 8 fields of view were examined per slide, with three
replicate slides per sample.
At least 100 spores per slide were counted for anti-13-0-GIcNAc binding to
phase bright spores.
This was performed on at least three occasions to enumerate the percentage of
fully mature
spores that could be bound with anti-13-0-GIcNAc.
[00144] Spore heat resistance assay. Heat resistance of C. difficile spores
was
determined as previously described (Permpoonpattana, P. et al., J.
Bacteriology 195:1492-
1503, 2013). Briefly spores of R20291 and CDR3194 mutant strains were
resuspended in 5 ml
of PBS at 1 x 106/m1 with starting inocula numbers confirmed by serial
dilution and plating on
BHI-ST, incubated anaerobically for 24 hours at 37 C. 1 ml aliquots of spores
were heated to
80 C for 20 minutes in a water bath, then plated on BHI-ST and incubated
anaerobically for 24
hours at 37 C to determine cfu/ml. Percentage survival was determined by
comparing pre and
post heat treatment cfu/ml. The experiment was performed in triplicate on at
least three
independent occasions.
[00145] Spore lysozyme resistance assay. Spores of R20291 and CDR3194
mutant
strains were diluted to 1 x 106/m1 in 5 ml PBS with starting inocula numbers
confirmed by serial
dilution as described above. Lysozyme was added to a final concentration of
250 pg/ml and 1 ml
samples were incubated for 1 hour at 37 C. Cfu/ml was determined by serial
dilution and plating
on BHI-ST. Percentage survival was determined by comparing pre and post
lysozyme treatment
cfu/ml. Experiment performed in triplicate on three independent occasions.
[00146] Spore ethanol resistance assay. Spores of R20291 and CDR3194 mutant
strains
were diluted to 1 x 106/m1 in 5 ml 70 % ethanol. Time point 0 cfu/ml was
confirmed by serial
dilution and plating on BHI-ST as described above. 1 ml aliquots were
incubated at room
temperature for 20 minutes, then plated on BHI-ST and incubated anaerobically
for 24 hours at
37 C to determine cfu/ml. Percentage survival was determined by comparing pre
and post
ethanol treatment cfu/ml. Experiment performed in triplicate on three
independent occasions.
[00147] Macrophage assay. J774A.1 macrophages were cultured at 5 x 105
cells/well on
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coverslips, in 24 well plate in 1 ml RPMI media supplemented with 10% FBS
(R10), at 5% CO2
37 C for 24 hours. Spores were diluted to 5 x 106/m1 (M01 10:1) in R10 and
inocula calculated
by dilution series and plating on BHI-ST. J774A.1 cells were washed with PBS
then 1 ml spores
added to four replicate wells. Plate was incubated for 30 minutes at 37 C 5%
CO2, then wells
were washed with PBS, before cells vvre fixed with 250 pl 4% formaldehyde for
15 minutes at
room temperature. Cells were washed with PBS then incubated with a rabbit anti-
C. difficile
polyclonal antisera (CD3) at 1:100 dilution in PBS for 45 minutes at room
temperature.
Coverslips were washed with PBS, then incubated with anti-rabbit Alexafluor
488 (Invitrogen) at
1:1000 dilution PBS for 45 minutes, at room temperature. Coverslips were
washed with PBS,
then cells were permeabilised with 0.1% Triton-PBS for 15 minutes, at room
temperature.
Coverslips were washed with PBS then incubated for 45 minutes at room
temperature in 1:100
dilution of CD3 antisera in PBS. Coverslips were washed in PBS, then incubated
in 1:1000
dilution of anti-rabbit Alexafluor 594 (Invitrogen) for 45 minutes, at room
temperature. Coverslips
were washed with PBS, then mounted onto slides with Vectashield+DAPI (Vector
Laboratories)
and sealed with nail polish. Slides were examined with Axioplan 200M
microscope (Zeiss).
Three coverslips per strain with 50 J774A.1 cells per coverslip were counted
utilising z stack
images to gain a 3D representation of the cell. Adhesion and internalisation
were quantified by
counting adhered (red/green) and internalised (red) spores and calculating
percentage adhesion
or internalised per J774A.1 cell based on known MOI. Assay was performed in
triplicate on
three independent occasions.
[00148] Statistical analysis. Student's t-test with Welch's correction was
used for pairwise
comparisons.
Example 6. Polyclonal immune serum production to spore surface.
[00149] Further work was completed to investigate the imnnunogenicity of
spore BcIA3
glycoprotein on the spore surface. Formalin killed spores from C. difficile
strain R20291 were
used to immunise New Zealand white rabbit to produce a high titre polyclonal
antiserum (CD5).
Immunization included sub-cutaneous immunisation with 1 x108spores, and two
boosts all in
incomplete Freund's adjuvant (FA). This immune serum was tested against viable
R20291
spores by ELISA. The results showed that rabbit polyclonal serum produced
after immunization
with formalin killed cells clearly recognised R20291 spores when coated on an
ELISA plate (Fig.
14). Spores were immunogenic upon subcutaneous immunisation with incomplete
Freund's
adjuvant.
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Spore surface antigens. Next, the reactivity of this polyclonal immune serum
with spore
surface extracts was examined by western blotting. Spores (5x107) of either
the R20291 parent
strain or R20291::sgtA strain were resuspended in 100 pl of SDS-PAGE
solubilisation buffer
and heated to 100 C for 20 minutes. Insoluble material was removed by
centrifugation in
Eppendorf centrifuge for 15 min and spore surface extract analysed by SDS-
PAGE. Western
blotting with CD5 antiserum revealed a number of reactive bands in the extract
from R20291
parent strain (Fig. 15). In contrast, spore surface extracts from R20291::sgtA
had only limited
reactivity with the CD5 serum indicating loss of immune reactive material as a
consequence of
sgtA inactivation. BcIA3 glycoprotein was previously shown to migrate to
regions of the gel
corresponding to the top two reactive bands in the immunoblot. In conclusion,
immunoblotting
using the CD5 antiserum identified immunoreactive bands in spore extracts by
Western blotting,
and identified BcIA3 as a key immunogen. Further, our results indicate that
insertional
inactivation of glycosyltransferase resulted in almost complete loss of
immunoreactivity,
demonstrating immunogenicity and significance of glycan structure.
[00150] Although this invention is described in detail with reference to
preferred
embodiments thereof, these embodiments are offered to illustrate but not to
limit the invention.
It is possible to make other embodiments that employ the principles of the
invention and that fall
within its spirit and scope as defined by the claims appended hereto.
[00151] The
contents of all documents and references cited herein are hereby incorporated
by reference in their entirety.
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