U.S. patent application number 15/692543 was filed with the patent office on 2018-01-11 for enterococcus faecalis and/or enterococcus faecium antigen.
The applicant listed for this patent is Universitatsklinikum Freiburg. Invention is credited to Otto HOLST, Johannes HUBNER, Zbigniew KACZYNSKI, Christian THEILACKER.
Application Number | 20180009834 15/692543 |
Document ID | / |
Family ID | 39277653 |
Filed Date | 2018-01-11 |
United States Patent
Application |
20180009834 |
Kind Code |
A1 |
HUBNER; Johannes ; et
al. |
January 11, 2018 |
ENTEROCOCCUS FAECALIS AND/OR ENTEROCOCCUS FAECIUM ANTIGEN
Abstract
The present invention generally relates to the field of
detecting and preventing infectious diseases caused by Enterococcus
faecalis and/or Enterococcus faecium. More specifically, the
invention relates to an Enterococcus faecalis and/or Enterococcus
faecium antigen which comprises at least one unit having the
following general formula: ##STR00001##
Inventors: |
HUBNER; Johannes; (Freiburg,
DE) ; HOLST; Otto; (Bad Oldesloe, DE) ;
THEILACKER; Christian; (Zurich, CH) ; KACZYNSKI;
Zbigniew; (Gdansk, PL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Universitatsklinikum Freiburg |
Freiburg |
|
DE |
|
|
Family ID: |
39277653 |
Appl. No.: |
15/692543 |
Filed: |
August 31, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12514653 |
Jun 17, 2009 |
9796744 |
|
|
PCT/EP2007/009813 |
Nov 13, 2007 |
|
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15692543 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/315 20130101;
C07H 15/04 20130101; A61P 37/04 20180101; A61K 39/07 20130101; G01N
33/56944 20130101; A61P 31/04 20180101; A61P 37/00 20180101; C08B
37/006 20130101 |
International
Class: |
C07H 15/04 20060101
C07H015/04; A61K 39/07 20060101 A61K039/07; C08B 37/00 20060101
C08B037/00; G01N 33/569 20060101 G01N033/569 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 13, 2006 |
DE |
102006053385.2 |
Claims
1. An isolated antibody, or an antibody fragment, to an
Enterococcus faecalis and/or Enterococcus faecium antigen, wherein
the antigen comprises at least one unit having the following
general formula: ##STR00004## wherein R.sub.1 are independently of
one another selected from the group consisting of H, OH, OCH.sub.3,
OAc, OC.sub.nH.sub.m, OFo, OAcyl, SH, SCH.sub.3, SC.sub.nH.sub.m,
SFo, SAc, SAcyl, NH.sub.2, NHCH.sub.3, NHC.sub.nH.sub.m, NHFo,
NHAc, NHAcyl, PO.sub.2(OR.sub.2).sub.2, F, Cl, Br and I, wherein Fo
is formyl, R.sub.2 is independently selected from the group
consisting of H, C.sub.nH.sub.m, C.sub.nH.sub.m-1NHR.sub.3 and
C.sub.nH.sub.m-1N(CH.sub.3).sub.3, wherein R.sub.3 is independently
selected from the group consisting of H, Fo, Ac and acyl, X are
independently of one another selected from the group consisting of
O, S, CH.sub.2, NH and POOR.sub.4, wherein R.sub.4 is H,
C.sub.nH.sub.m, C.sub.nH.sub.m-1NHR.sub.3 and
C.sub.nH.sub.m-1N(CH.sub.3).sub.3, Y are independently of one
another selected from the group consisting of O, S, CH.sub.2 and
HPO.sub.4, CA is independently of one another selected from the
group consisting of C.sub.1-C.sub.6-acyl radicals, in particular
hydroxyacyl radicals, preferably lactyl, H, OH, OCH.sub.3, OAc,
OC.sub.nH.sub.m, OFo, OAcyl, SH, SCH.sub.3, SC.sub.nH.sub.m, SFo,
SAc, SAcyl, NH.sub.2, NHCH.sub.3, NHC.sub.nH.sub.m, NHFo, NHAc,
NHAcyl, PO.sub.2OR.sub.2, F, Cl, Br and I, wherein Fo is formyl,
R.sub.2 is independently selected from the group consisting of H,
OC.sub.nH.sub.m, OC.sub.nH.sub.m-1NHR.sub.3 and OC.sub.nH.sub.m-1N
(CH.sub.3).sub.3, wherein R.sub.3 is independently selected from
the group consisting of H, Fo, Ac and acyl, wherein m=2n+1 and n is
selected from the set of natural numbers from 1 to 10.
2. The antibody, according to claim 1, wherein the antigen has two
sugars that are in the D configuration.
3. The antibody, according to claim 1, wherein the antigen
comprises a disaccharide consisting of a furanose Gal and a
pyranose Glc has a structure that is selected from the following:
.fwdarw.-v)-D-Galf-(1.fwdarw.-z)-D-Glcp-(1.fwdarw.-,
.fwdarw.-v)-D-Galf-(1.fwdarw.-z)-D-Glcf-(1.fwdarw.-,
.fwdarw.-v)-D-Galp-(1.fwdarw.-z)-D-Glcp-(1.fwdarw.- or
.fwdarw.-v)-D-Galp-(1.fwdarw.-z)-D-Glcf-(1.fwdarw.-, wherein v and
z are in each case 1, 2, 3, 4, 5 or 6.
4. The antibody, according to claim 1, wherein the antigen has the
following formula: ##STR00005##
5. The antibody, according to claim 1, wherein, for the antigen,
R.sub.1 is OH, X is O, Y is O and CA is lactyl.
6. The antibody, according to claim 1, wherein the antigen has a
molecular weight of approximately 50,000 to 150,000 Da.
7. The antibody, according to claim 1, said unit of the antigen
occurs at least 100 times.
8. The antibody, according to claim 1, that is a recombinant
antibody, or a recombinant fragment thereof.
9. A composition comprising an antibody according to claim 1.
10. The composition as claimed in claim 9, comprising a
pharmaceutically acceptable carrier.
11. The composition as claimed in claim 9, characterized in that it
is a vaccine against Enterococcus faecalis and/or Enterococcus
faecium.
12. A kit for detecting Enterococcus faecalis and/or Enterococcus
faecium in a sample, comprising an antibody according to claim 1,
optionally bound to a support.
13. The kit according to claim 12, characterized in that the
antigen or the antibody is labeled with a marker.
14. A method for immunizing a subject against Enterococcus faecalis
and/or Enterococcus faecium, wherein said method comprises
administering to the subject an antibody of claim 1.
15. An isolated antibody, or an antibody fragment, to an
Enterococcus faecalis and/or Enterococcus faecium antigen, wherein
the antigen comprises at least one unit having the following
general formula: ##STR00006## wherein R.sub.1 is OH, X is O, Y is O
and CA is lactyl.
16. A method for immunizing a subject against Enterococcus faecalis
and/or Enterococcus faecium, wherein said method comprises
administering to the subject an antibody of claim 15.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/514,653, filed Jun. 17, 2009; which is the national stage
application of International Application No. PCT/EP2007/009813,
filed Nov. 13, 2007; which claims the benefit of German Patent
Application No. 10 2006 053 385.2, filed Nov. 13, 2006; all of
which are incorporated herein by reference in their entirety.
[0002] The present invention generally relates to the field of
detecting and preventing infectious diseases caused by Enterococcus
faecalis and/or Enterococcus faecium.
[0003] More specifically, the present invention relates to an
antigen, antibodies, compositions, methods, a kit, and to a
use.
[0004] Nosocomial infections (hospital-acquired infections) are
infections which are caused by microorganisms and which are
causally linked to hospitalization.
[0005] The proportion of nosocomial infections increased by 36% in
the USA from 1975-1995 so that, in 1995, about 10 of 1000 patients
were affected by such an infection.
[0006] Thus, nosocomial infections alone are responsible for
44000-98000 deaths per annum in the USA.
[0007] Hospital-acquired infections are responsible for a large
part of all hospital-based complications, and avoiding them is
essential for the quality of medical care and nursing of the
patients.
[0008] They are therefore a serious problem for any hospital, since
the supreme aim of any quality-oriented actions in hospital care is
of course a healthy patient.
[0009] Nosocomial infections not only put a strain on the patient
himself due to additional pain, but usually also prolong
hospitalization--on average by about 4 days--and therefore
contribute to hospitals and the health-care system being
overburdened. Nosocomial infections produce costs of from 17 to 29
billion US dollars per year in the USA.
[0010] Despite indisputable medical progress, the frequency of
nosocomial infections is expected rather to increase in the
future.
[0011] The following factors are inter alia held responsible for
this development: [0012] the number of elderly patients increases,
[0013] an increasing number of patients whose endogenous infection
defenses are weak are treated in the hospitals, [0014] more
complicated and difficult operations are carried out owing to the
progress in surgical technologies, [0015] it is increasingly
possible to carry out invasive measures involving complicated
apparatus and having an increased risk of infection, [0016]
therapeutic measures which lower the defenses are increasingly
carried out, and [0017] antibiotics, in particular broad-spectrum
antibiotics, are increasingly employed, resulting in an increase in
multiresistant microorganisms.
[0018] More recent studies carried out in this connection (Am. J.
Infect. Control., 32:470-485, 2004) have demonstrated that up to
25% of Enterococcus faecium isolates from hospitals are resistant
to glycopeptidic antibiotics.
[0019] Glycopeptidic antibiotics attack the cell wall and insert
directly into the structure of the cell wall, thereby producing
holes and enabling water to enter. An example of glycopeptidic
antibiotics is vancomycin.
[0020] DiazGranados, C. A., et al. recently demonstrated that
vancomycin-resistant enterococci cause considerable morbidity and
mortality, in particular in patients whose immune system has
already been weakened (Clin. Infect. Dis., 41:327-333, 2005).
[0021] There is therefore continuing interest in developing
immunotherapeutic approaches which allow such infections to be
prevented or at least controlled.
[0022] Although the use of carbohydrate antigens in the development
of vaccines is known in the prior art in principle (Ada, G., et
al., Clin. Microbiol. Infect., 9: 79-85, 2003), little is known up
to now about cell wall-associated and capsular polysaccharides in
Enterococcus faecalis.
[0023] Four carbohydrate antigens have been described up to now
(Hancock, et al., Proc. Natl. Acad. Sci USA, 99:1574-1579, 2002;
Hsu et al., BMC Microbiol., 6:62, 2006; Pazur et al., J. Biol.
Chem., 248: 279-284, 1973; Wang et al., Carbohydr. Res.
[0024] 316: 155-160, 1999; Xu et al., Infect. Immun., 65:4207-4215,
1997; Xu et al., Infect. Immun. 66:4313-4323, 1998; Xu et al.,
Infect. Immun., 68:815-823, 2000), however, a complete structural
analysis has been carried out only for one of them. A carbohydrate
antigen which is exposed on the cell surface of the Enterococcus
faecalis strain FA2-2 and which consists of glucose, galactose and
glycerol phosphate has also been described.
[0025] In order to solve the problems caused by the appearance of
multiresistant Enterococcus faecalis strains and the closely
related Enterococcus faecium strains, there is a considerable need
in the prior art for characterizing antigens exposed on the surface
of Enterococcus faecalis and/or Enterococcus faecium, in order to
use said antigens for overcoming the problems described above.
[0026] It is therefore the object of the present invention to
provide previously unknown antigens of Enterococcus faecalis and/or
Enterococcus faecium.
[0027] Another object of the present invention is to provide
antibodies to these previously unknown antigens of Enterococcus
faecalis and/or Enterococcus faecium.
[0028] Another object of the present invention is to provide a
composition which comprises at least one antigen of Enterococcus
faecalis and/or Enterococcus faecium or an antibody thereto, which
composition is preferably suitable for active or passive
immunization against Enterococcus faecalis and/or Enterococcus
faecium infections.
[0029] Another object of the present invention is to provide a
method which enables the previously unknown antigen to be detected
in a sample.
[0030] Another object of the present invention is to provide a
method which enables antibodies to the previously unknown antigen
to be detected in a sample.
[0031] Another object of the present invention is to provide a kit
which enables the previously unknown antigen or antibodies to this
antigen to be detected.
[0032] Another object of the present invention is to provide a
possible use of the previously unknown antigen for detecting or
producing corresponding antibodies or antibody fragments and to
provide a corresponding method.
[0033] These objects of the present invention are achieved by the
antigen, by the antibodies, by the compositions, by the methods, by
a kit, and by a use.
[0034] More specifically, the present invention provides an
Enterococcus faecalis and/or Enterococcus faecium antigen which is
characterized in that it comprises at least one unit having the
following general formula:
##STR00002##
[0035] wherein R.sub.1 are independently of one another selected
from the group consisting of H, OH, OCH.sub.3, OAc,
OC.sub.nH.sub.m, OFo, OAcyl, SH, SCH.sub.3, SC.sub.nH.sub.m, SFo,
SAc, SAcyl, NH.sub.2, NHCH.sub.3, NHC.sub.nH.sub.m, NHFo, NHAc,
NHAcyl, PO.sub.2(OR.sub.2).sub.2, F, Cl, Br and I,
[0036] wherein Fo is formyl, R.sub.2 is independently selected from
the group consisting of H, C.sub.nH.sub.m,
C.sub.nH.sub.m-1NHR.sup.3 and C.sub.nH.sub.m-1N
(CH.sub.3).sub.3,
[0037] wherein R.sub.3 is independently selected from the group
consisting of H, Fo, Ac and acyl,
[0038] X are independently of one another selected from the group
consisting of O, S, CH.sub.2, NH and POOR.sub.4,
[0039] wherein R.sub.4 is H, C.sub.nH.sub.m,
C.sub.nH.sub.m-1NHR.sup.3 and
C.sub.nH.sub.m-1N(CH.sub.3).sub.3,
[0040] Y are independently of one another selected from the group
consisting of O, S, CH.sub.2 and HPO.sub.4,
[0041] CA is independently of one another selected from the group
consisting of C.sub.1-C.sub.6-acyl radicals, in particular
hydroxyacyl radicals, preferably lactyl, H, OH, OCH.sub.3, OAc,
OC.sub.nH.sub.m, OFo, OAcyl, SH, SCH.sub.3, SC.sub.nH.sub.m, SFo,
SAc, SAcyl, NH.sub.2, NHCH.sub.3, NHC.sub.nH.sub.m, NHFo, NHAc,
NHAcyl, PO.sub.2OR.sup.2, F, Cl, Br and I,
[0042] wherein Fo is formyl, R.sub.2 is independently selected from
the group consisting of H, OC.sub.nH.sub.m,
OC.sub.nH.sub.m-1NHR.sup.3 and OC.sub.nH.sub.m-1N
(CH.sub.3).sub.3,
[0043] wherein R.sub.3 is independently selected from the group
consisting of H, Fo, Ac and acyl,
[0044] wherein m=2n+1 and n is selected from the set of natural
numbers from 1 to 10.
[0045] The two sugars in this unit are preferably in the D
configuration.
[0046] Particular preference is given to the antigen of the present
invention, characterized in that the disaccharide consisting of a
furanose Gal and a pyranose Glc has a structure which is selected
from the following: [0047]
.fwdarw.-v)-D-Galf-(1.fwdarw.-z)-D-Glcp-(1.fwdarw.-, [0048]
.fwdarw.-v)-D-Galf-(1.fwdarw.-z)-D-Glcf-(1.fwdarw.-, [0049]
.fwdarw.-v)-D-Galp-(1.fwdarw.-z)-D-Glcp-(1.fwdarw.- or [0050]
.fwdarw.-v)-D-Galp-(1.fwdarw.-z)-D-Glcf-(1.fwdarw.-,
[0051] wherein v and z are in each case 1, 2, 3, 4, 5 or 6.
[0052] An antigen particularly preferred in accordance with the
present invention has the following formula:
##STR00003##
[0053] Further preference is given to R.sub.1 being OH, X being O,
Y being O and CA being lactyl.
[0054] The molecular weight of the antigens of the present
invention is noncritical in principle and is therefore not
restricted any further. Typically, however, the antigens of the
present invention have molecular weights of approx. 1000-200000 Da,
more preferably of approx. 50000-150000 Da, particularly preferably
of approx. 100000 Da. Molecular weights which are too small may
possibly result in the antigen not being able to form any
structurally stable epitopes, due to the few intramolecular
interactions, while molecular weights which are too high result in
such antigens frequently being difficult to synthesize and their
lifetime being limited.
[0055] It is sufficient in principle for the antigen of the present
invention to include the unit of claim 1 at least once. It is
however possible to increase the antigenicity of the antigen of the
present invention by said unit occurring at least 5 times,
preferably at least 10 times, more preferably at least 100 times,
particularly preferably at least 1000 times, per antigen.
[0056] For various analytical applications of the antigen of the
present invention, preference may further be given to said antigen
being immobilized. This may restrict the presence of the antigen
locally, so that many different samples are contacted with the
antibody and analyzed simultaneously but nevertheless separately,
for example within the framework of an array. Immobilized antigens
are also useful in affinity-based purification methods, for example
for corresponding antibodies.
[0057] Therefore, in a preferred embodiment of the invention, the
antigen is bound to a support, preferably to an immunosupport.
[0058] Immobilization may be carried out in principle by any type
of interactions, such as, for example, van der Waals forces, ionic
interactions, dipole-dipole interactions, hydrogen bonding or
hydrophobic interactions. Preference is given, however, to the
binding to the support being of the covalent type.
[0059] Preference is given to the antigen of the present invention
being provided in the form of a composition. According to a
preferred embodiment, such a composition comprises at least one
pharmaceutically acceptable support. A support substance here is
any substance to which the antigen can be added on and/or
physically bound. Thus it is possible, for example, for the antigen
whose dose can usually be administered only with difficulty to be
bound to a support whose dose can be administered more easily.
Examples of such supports are starch and maltodextrine.
Pharmaceutically acceptable here means that the support used is
nontoxic and does not interfere with the action of the antigen.
[0060] The antigen of the present invention may be administered as
a preventive measure to a patient within the framework of an active
vaccination against infectious diseases caused by Enterococcus
faecalis and/or Enterococcus faecium. The aim of such a vaccination
is to stimulate the endogenous immune system itself to form
specific antibodies, thereby producing a specific immunity to
Enterococcus faecalis and/or Enterococcus faecium. A patient may
therefore be any living being that has an immune system. Of
particular importance, however, are mammals such as, for example,
humans, nonhuman primates, dogs, cats, pigs, cows, horses, goats
and sheep, and birds such as, for example, chickens, ducks, geese,
turkeys or ostriches.
[0061] In this respect, the antigen of the present invention, in a
preferred embodiment, is a vaccine against Enterococcus faecalis
and/or Enterococcus faecium.
[0062] The present invention likewise relates to an antibody to the
above-described antigen.
[0063] This antibody may be a monoclonal or a polyclonal
antibody.
[0064] Antibodies consist of two identical heavy chains (H) and two
identical light chains (L), which are linked to one another by
covalent disulfide bonds to give a Y-shaped structure. Depending on
the organism and immunoglobulin subclass, the two light chains are
either of the kappa type or of the lambda type and, together with
the heavy chain portion located upstream of the hinge region, form
the antigen-binding fragment, Fab, which can be removed
enzymatically from the downstream crystalline fragment, Fc.
[0065] The marked variability of the antibody bonding sites
(abbreviated CDR, Complementarity Determining Region) is achieved
by the organism via V(D)J recombination.
[0066] The light chains consist in each case of a variable and a
constant domain. These are referred to as V.sub.L and C.sub.L. The
heavy chains, in contrast, have in each case one variable and three
constant domains. These are referred to analogously as V.sub.E and
C.sub.H1, C.sub.H2, C.sub.H3.
[0067] Antibodies in accordance with the present invention are
whole antibodies or fragments thereof, for example Fab or scFv,
provided that the latter are capable of binding to the antigen, at
least to a limited extent.
[0068] The present invention likewise comprises humanized and
chimeric antibodies.
[0069] An antibody of this kind and/or the antigen of the present
invention may be employed, for example, within the framework of
[0070] an ELISA method of quantifying antigens or antibodies, for
example in the serum, in cell culture supernatant, etc., by means
of enzyme-coupled antibodies, [0071] an ELISPOT method of detecting
antibody- or antigen-secreting cells (plasma cells) by means of
enzyme-coupled antibodies, [0072] an FACS method of quantifying
cells by means of fluorescently coupled antibodies to antigens on
the cell surface, in the zytoplasm or in the nucleus, [0073] a
Western blot, [0074] a super gel shift experiment (also EMSA),
[0075] a phage display, [0076] a drugwipe assay, [0077] an abzyme
method, or [0078] a method of purifying the antigens or antibodies
of the present invention by appropriate affinity methods.
[0079] The antibody of the present invention, like the antigen, is
preferably provided within the framework of a composition which in
turn--like the antigen--preferably comprises a pharmaceutically
acceptable support.
[0080] The antibody of the present invention is used within the
framework of a particularly preferred embodiment of the present
invention for passive vaccination against Enterococcus faecalis
and/or Enterococcus faecium and thus represents a vaccine against
Enterococcus faecalis and/or Enterococcus faecium.
[0081] Passive vaccination involves vaccination with a vaccine
serum which includes, preferably high concentrations of, the
specific antibody of the present invention to Enterococcus faecalis
and/or Enterococcus faecium.
[0082] Preference is given in principle to the compositions of the
present invention being pharmaceutical compositions. These are
distinguished in that they are suitable for administration within
the framework of a therapy or prophylaxis.
[0083] The present invention further relates to a method of
detecting Enterococcus faecalis and/or Enterococcus faecium
antibodies, said method comprising the following steps: [0084]
contacting antigens according to the present invention with a
sample to be tested for Enterococcus faecalis and/or Enterococcus
faecium antibodies, and [0085] detecting antibody-antigen or
antibody-antigen conjugate complexes,
[0086] wherein the presence of such complexes indicates the
presence of Enterococcus faecalis and/or Enterococcus faecium
antibodies in the sample.
[0087] The present invention likewise comprises a method of
detecting Enterococcus faecalis and/or Enterococcus faecium
antigens, having at least the following steps: [0088] contacting
antibodies according to the present invention, optionally
immobilized on a support, with a sample to be tested for
Enterococcus faecalis and/or Enterococcus faecium antigens, and
[0089] detecting antigen-antibody or antibody conjugate-antigen
complexes,
[0090] wherein the presence of such complexes indicates the
presence of Enterococcus faecalis and/or Enterococcus faecium
antigens in the sample.
[0091] In the two methods above, preference is given to the
antigens or antibodies being immobilized on a matrix, in particular
on a solid matrix, preferably on a microtiter plate.
[0092] As an alternative, it is also conceivable for the sample to
be examined to be immobilized to a matrix.
[0093] In each case, detection of antigen-antibody conjugates
formed is particularly facilitated, since unbound sample components
can optionally be removed from the matrix, for example by washing,
prior to detecting the complexes. This reduces noise and increases
measurement accuracy, and the use of a solid matrix also
facilitates handling of the samples by machine, and, as a result,
the method of the present invention has, inter alia, excellent
suitability for automation and thus, for example, for high
throughput screening.
[0094] Detection of the antigen-antibody conjugates may further be
facilitated by providing the antigen or the antibodies of the
present invention with a detectable marker.
[0095] If, for example, the sample to be examined, which may
contain anti-Enterococcus faecalis and/or Enterococcus faecium
antibodies, has been immobilized on a matrix and if this
immobilized sample is then contacted with labeled antigens of the
invention, then samples containing anti-Enterococcus faecalis
and/or Enterococcus faecium antibodies, after washing, may already
be recognized by way of the signal released by the marker on the
antigen.
[0096] A suitable compound which can be used as marker is any
compound that can be contacted with the antibodies or antigens of
the present invention, without said contact totally interrupting
the antigen-antibody interactions, and which generate a detectable
signal of any kind directly or indirectly, where appropriate after
appropriate activation or introduction of substrate.
[0097] Preferred markers in accordance with the present invention
are radioactive markers, colored markers, enzymatic markers and
magnetic markers.
[0098] The detection method of the present invention may be
accelerated further if the marker displays a property change after
binding to an antibody or to an antigen. This would enable unbound
labeled antigens, or antibodies, to be distinguished from bound
ones without the need for a washing step, for example.
[0099] The present invention likewise provides for a kit for
detecting Enterococcus faecalis and/or Enterococcus faecium
antibodies and/or Enterococcus faecalis and/or Enterococcus faecium
antigens, which kit comprises the antigen and/or the antibody of
the present invention in any of the embodiments described
above.
[0100] More specifically, the antigen of the invention and/or the
antibody of the invention may be bound to a support, preferably to
a solid matrix, and/or labeled with a marker, preferably a
radioactive marker, a colored marker, an enzymatic marker or a
magnetic marker.
[0101] The present invention furthermore comprises the use of the
antigen of the invention according to claims 1 to 10 for detecting
or producing Enterococcus faecalis and/or Enterococcus faecium
antibodies and/or antibody fragments, in particular Fab or
scFv.
[0102] The Enterococcus faecalis and/or Enterococcus faecium
antibodies and/or antibody fragments produced in this way are
designed in particular for passive immunotherapy or for prophylaxis
against Enterococcus antigens.
[0103] The antibodies or antibody fragments of the invention, in
particular Fab or scFv, can be prepared, for example, by a method
which comprises administering the antigen of the invention to an
animal, in particular a mammal, in an amount sufficient for
producing antibodies or antibody fragments.
[0104] Polyclonal antibodies of the present invention may be
prepared by firstly selecting and producing the antigen of the
invention to which the antibody is intended to be directed. This
may be achieved in various ways, for example by isolating an
antigen from Enterococcus faecalis and/or Enterococcus faecium
synthesizing said antigen in vitro or producing it recombinantly,
for example in bacteria. The antigen is then administered to an
animal whose immune system then forms antibodies to the antigen.
Suitable antibody producers are in particular mice, rats and
rabbits but also goats, sheep and horses. Preference is given to
repeating the immunization several times. After a few weeks,
polyclonal antibodies are taken from the blood or the serum of the
antibody producer.
[0105] Monoclonal antibodies may be produced as described for
production of polyclonal antibodies--by immunizing animals,
preferably mice and/or rats, and then obtaining their plasma cells
(from spleen or lymph nodes). These plasma cells may be fused with
tumor cell lines, thereby enabling "hybridoma cell lines" to be
generated which, in theory, live forever but secrete a single type
of monoclonal antibodies which in turn may be isolated from the
cell culture supernatant.
[0106] Another embodiment of the present invention is a method of
producing an antibody of the invention or a fragment thereof, in
particular Fab or scFv, in recombinant form, which method comprises
cloning a DNA sequence which codes for said antibody or its
fragment into an expression vector, transforming a cell, in
particular an E. coli cell or a yeast cell or a eukaryotic cell
(such as, for example, a CHO cell), with this construct and
expressing the recombinant antibody or a fragment thereof.
[0107] A recombinant antibody obtainable by such a method is
characterized in that it corresponds structurally to the antibodies
of the present invention. In the same way, recombinant fragments of
the antibody of the invention, in particular Fab or scFv,
correspond to the natively purifiable antibody fragments of the
present invention.
[0108] The recombinant antibodies and antibody fragments of the
present invention have the particular advantage that they can be
produced in large quantities without complicated technology and
without animals having to be used as antibody producers. It is
furthermore possible to achieve a considerably higher purity of the
antibody preparations, and direct contact with blood and the risk
of infection associated therewith are avoided.
[0109] It is obvious to the skilled worker that he can combine all
embodiments of the present invention which are listed here by way
of example in any way without departing from the scope of the
disclosure of the present invention.
[0110] Furthermore, reference is made to the entire contents of any
citations herein.
[0111] Further advantages and embodiments of the invention will be
evident from the description of individual exemplary embodiments
and from the drawing.
[0112] In the figures,
[0113] FIG. 1 depicts the .sup.1H NMR spectrum of the capsular
polysaccharide from E. faecalis strain type 5. The spectrum was
recorded at 600 MHz and 27.degree. C. The letters refer to the
carbohydrate radicals as depicted in FIG. 3, and the Arabic
numerals refer to the protons on the corresponding radicals; LA,
lactic acid.
[0114] FIG. 2 depicts sections of the ROESY spectrum of the
capsular polysaccharide from E. faecalis strain type 5. The
spectrum was recorded at 600 MHz and at 27.degree. C. The letters
refer to the carbohydrate radicals as depicted in FIG. 3, and the
Arabic numerals refer to the protons on the corresponding radicals.
The NOE contacts between the radicals are underlined and depicted
in italics.
[0115] FIG. 3 depicts the chemical structure of the repetitious
unit of the capsular polysaccharide of E. faecalis strain type 5.
LA, lactic acid. The Galf radical is assumed to have the D
configuration.
[0116] FIG. 4 depicts binding of a rabbit antiserum to the
bacterial cells of E. faecalis strain type 5. The antigens used are
indicated in the legend. The values indicated represent in each
case the average of at least two measurements.
[0117] FIG. 5 depicts the inhibition of opsonophagocytosis of E.
faecalis strain type 5 bacterial cells by rabbit antiserum. All
antisera were used as 1:200 dilutions. The inhibitors are indicated
in the legend. The data indicated represent an average of at least
4 measured values, and the error bars represent the SEM. The
opsonophagocytotic activity without inhibitor was >70% for all
antisera.
[0118] FIG. 6 depicts the binding of antibodies generated in
rabbits to capsular polysaccharide of E. faecalis type 2 and type 5
to the corresponding purified antigen.
[0119] FIG. 7 depicts the determination of curing by
opsonophagocytosis of E. faecalis type 2, which were produced
against the purified antiserum. The values clearly demonstrate that
protective antibodies were produced in rabbits, which resulted in
the in vitro assay to destroy the bacteria.
EXAMPLE 1
[0120] Elimination of Enterococcus faecalis by opsonophagocytosis
is accomplished inter alia by antibodies which are directed to
carbohydrate antigens of the cell wall and of the bacterial
capsule. Recently, lipoteichoic acid (LTA) has been identified as
target of the opsonizing antibodies in E. faecalis strain 12030.
However, serum raised against purified LTA does not kill any
bacterial strains having the CPS-C and -D serotypes.
[0121] The present example comprises isolating a novel capsular
polysaccharide from the E. faecalis type 5 strain, a CPS-D strain,
by enzymatic digestion of the cell wall and by gel permeation and
anion exchange chromatography.
[0122] The isolated polysaccharide is analyzed by sugar analysis,
one-dimensional and two-dimensional homonuclear and heteronuclear
.sup.1H and .sup.13C NMR spectroscopy.
[0123] A novel E. faecalis capsular polysaccharide is identified
which includes an unusual
.fwdarw.6)-3-O-[1-carboxyethyl]-.beta.-Galf-(1.fwdarw. unit in the
repetitious unit.
[0124] A rabbit antiserum induced by means of immunization of
heat-inactivated E. faecalis type 5 bacteria cells contained both
antibodies specific to the novel capsular polysaccharide and
antibodies to LTA of said strain.
[0125] However, opsonophagocytosis of E. faecalis type 5 by this
antiserum was inhibited only by the purified polysaccharide but not
by LTA.
[0126] This example therefore illustrates a possibility of how to
identify a novel capsular polysaccharide as antigen in E. faecalis
type 5 that is immunogenic and can serve as target for opsonizing
antibodies.
[0127] This example further illustrates how to assay the
immunogenicity of antigens derived from E. faecalis.
EXAMPLE 2
[0128] 2a) Bacteria Strains and Cultures
[0129] Capsular polysaccharides were isolated from E. faecalis type
5 (Maekawa, S., et al., Microbiol. Immunol., 36:671-681, 1992) a
CPS-D strain, using a recently described serotyping system
(Hufnagel, M., et al., J. Clin. Microbial. 42:2548-2557, 2004).
Bacteria cells were cultured from the starter cultures in a
Columbia nutrient solution (Becton Dickinson, Sparks, MD, USA)
enriched with 1% glucose, without agitation at 37.degree. C. for 2
hours.
[0130] 2b) Antisera
[0131] Antisera to whole bacteria cells of E. faecalis type 5 have
been described in the past (Hufnagel, M., et al., J. Clin.
Microbiol. 42:2548-2557, 2004). The antiserum to LTA was produced
with LTA purified from the strain 12030, as described previously
(Theilacker, C., et al., Infect. Immun. 74, 2006). A female white
New Zealand rabbit was immunized subcutaneously with 100 .mu.g of
LTA suspended in complete Freund's adjuvant, and thereafter with
the same LTA dose suspended in incomplete Freund's adjuvant seven
days later, and then with 10 .mu.g booster doses every three days
in the following week.
[0132] 2c) Preparation and Characterization of the Capsular
Colysaccharide.
[0133] E. faecalis LTA was isolated as described previously
(Huebner, J., et al., Infect. Immun. 67:1213-1219, 1999;
Theilacker, C., et al., Infect. Immun. 74, 2006). The antigen
described herein was prepared as follows: briefly, bacterial cells
were harvested by centrifugation and digested by adding mutanolysin
and lysozyme (in each case 100 pg/ml, Sigma Chemicals, St. Louis,
Mo., USA in PBS enriched with 5 mM MgCl.sub.2, 1 mM CaCl.sub.2 and
0.05% NaN.sub.3) at 37.degree. C. for 18 hours. Insoluble material
was removed by centrifugation, and the supernatant was treated with
nucleases (DNase I and RNase A, 100 pg/ml) at 37.degree. C. for 4
hours, followed by 18 hours of adding proteinase K (100 pg/ml, all
available from Sigma Chemicals) at 56.degree. C. The supernatant
was precipitated by adding ethanol (final volume 80%) and then
collected by centrifugation. After dialysis against deionized
H.sub.2O, the material was lyophilized. For gel permeation
chromatography, the material was dissolved in 0.01 M ammonium
bicarbonate buffer solution and applied to a Sephacryl S-400 column
(1.6.times.90 cm) (GE Healthcare, Uppsala, Sweden). Fractions
eluting at a K.sub.av of about 0.45 were combined, dialyzed and
lyophilized. The material was resuspended in 20 mM of NaHCO.sub.3,
pH 8.4 and applied to an anion exchange column (Sepharose Q FF, GE
Healthcare). Bound antigen was eluted from the column by way of a
linear NaCl gradient, and fractions comprising polysaccharides were
identified by a Dubois assay (Dubios, M., et al., Anal. Chem.
28:350-356, 1956.) and immunoblotting using a rabbit anti-type 5
immune serum. Immunoreactive material eluting at 450 mM NaCl was
combined, dialyzed and lyophilized. The final purification step
performed was a gel permeation chromatography on a 1.5.times.75 cm
Toyopearl HW-40 (Tosoh Corporation, Tokyo, Japan) column. The
purity of the isolated material was confirmed by means of SDS PAGE
using a 10% Bis-Tris gel and an MOPS running buffer (Invitrogen,
Karlsruhe, Germany) and by Coomassie (Invitrogen) and PAS (Sigma)
staining according to the manufacturer's instruction.
[0134] Furthermore, a Western blot of material removed by SDS PAGE
was stained using an anti-type 5 rabbit antiserum.
[0135] 2d) Result of the Purification of the Capsular
Polysaccharide
[0136] Capsular polysaccharide of the E. faecalis strain type 5 was
mobilized by enzymatic digestion of peptidoglycan from the
bacterial cells. The extracted material eluted in the form of two
carbohydrate-containing fractions in Sephacryl S-400 gel
chromatography. One fraction eluting in the void volume comprised
LTA as determined by .sup.1H NMR analysis (data not shown). A
large, second fraction at a K.sub.av of around 0.45 was further
purified by anion exchange chromatography using Q Sepharose. Small
amounts of immunoreactive material eluted at 450 mM NaCl, which
material comprised only glucose and galactose, and which was
subjected to further analysis after gel permeation chromatography
on Toyopearl HW-40S. The SDS PAGE of this purified material showed
a single broad band at 100 kDa which was stained by PAS but not by
Commassie blue. A Western blot stained with anti-type 5 antiserum
likewise showed a single broad band at 100 kDa and no further bands
(data not shown).
EXAMPLE 3
[0137] Provision of LTA
[0138] LTA was prepared by butanol extraction and hydrophobic
interaction chromatography as described previously (Theilacker, C.,
et al., Infect. Immun. 74, 2006). The purity of the LTA
preparations was evaluated by SDS PAGE and Western blot analysis
using the corresponding antiserum to whole bacterial cells (cf.
above). The structural identity of LTA was confirmed by NMR
spectroscopy as recently described (Theilacker, C., et al., Infect.
Immun. 74, 2006).
[0139] General and Analytical Methods
[0140] Hydrolysis was carried out using 2 M trifluoroacetic acid
(120.degree. C., 3 h). Monosaccharides were converted to alditol
acetates and analyzed by GC in a Hewlett-Packard 5890 chromatograph
with an SPB-5 column (30 m.times.0.25 mm.times.0.25 pm, Supelco,
Munich, Germany), using a temperature program of 150.degree. C. for
3 minutes, then 3.degree. C. min.sup.-1 up to 300.degree. C. The
absolute arrangement of the sugar radicals was determined as
described previously (Haseley, S. R., et al., Eur. J. Biochem.
244:761-766, 1997; Leontein, K., et al., Carb. Res.
62:359-362,1978).
[0141] NMR Spectroscopy
[0142] The sample was substituted three times with 99.0%
.sup.2H.sub.2O, lyophilized and redispersed in 99.9%
.sup.2H.sub.2O. All one- and two-dimensional spectra were recorded
on a Bruker DRX Avance 600 MHz spectrometer (working frequencies of
600.31 MHz for .sup.1H NMR and 150.96 MHz for .sup.13C NMR) using a
conventional Bruker Software (Bruker, Rheinstetten, Germany) at
27.degree. C. The chemical shifts are indicated in relation to
acetone (.delta.H 2.225; .delta.C 31.45). Correlation spectroscopy
(COSY), and total correlation spectroscopy (TOCSY), and ROESY were
recorded using datasheets (t1.times.t2) containing 4096.times.512
points, and 32 scans were carried out.
[0143] TOCSY and ROESY were carried out in a phase-sensitive manner
according to the method of States et al., with a mixing time of 100
ms being used for TOCSY (States, D. J., et al., J. Magn. Reson.
48:286-292, 1982). The .sup.1H, .sup.13C correlations were measured
in .sup.1H detection mode by means of a multiple quantum coherence
(HMQC) with proton decoupling in the .sup.13C domain using datasets
containing 2048.times.256 points, with 128 scans being recorded for
each t.sub.1 value (Bax, A., et al., J. Am. Chem. Soc.
109:2093-2094, 1986; Summers, M. F., et al., J. Am. Chem. Soc.
108:4285-4294, 1986).
[0144] Chemical Analysis and NMR Spectroscopy
[0145] In the low field region, the .sup.1H NMR spectrum of the
purified polysaccharide (FIG. 1) showed two anomeric signals at
.delta. 5.315 (radical A, [.sup.3J.sub.H1,H2<2 Hz]), and at
.delta. 4.542 (radical B, [.sup.3J.sub.H1,H2=7.8 Hz]), which were
identified as .beta.-Galf and .beta.-D-Glcp. Furthermore, the
Dublett at .delta. 1.361 was identified as a methyl group which
belongs to a lactic acid radical (LA) (Knirel, Y. A., et al.,
Carbohydr. Res. 259, 1994; , Knirel, Y. A., et al., Carbohydr. Res.
235:C19-23, 1992). The presence of a D-Glcp radical was confirmed
by chemical analysis, assigning of the absolute configuration, and
by NMR spectroscopy data. The Galf radical substituted with lactic
acid in position C-3 was identified only by NMR data (Beynon, L.
M., et al., Eur. J. Biochem. 250:163-167, 1997; Knirel, Y. A., et
al., Carbohydr. Res. 259, 1994; , Knirel, Y. A., et al., Carbohydr.
Res. 235:C19-23, 1992). All .sup.1H and .sup.13C chemical shifts of
the capsular polysaccharide of E. faecalis type 5 (Table 1) were
determined from .sup.1H,.sup.1H COSY and TOCSY, and from
.sup.1H,.sup.13C HMQC spectra.
TABLE-US-00001 TABLE 1 Chemical shift .sup.1H and .sup.13C
[.delta.] H1 H2 H3 H4 H5 H6.sup.a Radical C1 C2 C3 C4 C5 C6
H6.sup.b A 5.315 4.346 3.932 4.225 4.040 3.768 4.019
.fwdarw.6)-.beta.- 109.26 80.26 84.96 82.41 70.55 71.90 Galf- B
4.542 3.460 3.663 3.465 3.500 3.742 3.930 .fwdarw.3)-.beta.- 103.22
74.05 82.44 68.80 76.26 61.26 D-Glcp- LA 4.038 1.361 Lactic 181.29
77.72 19.21 acid
[0146] Carbon atom signals shifted to low field indicated
substitutions on C-6 and C-3 of .beta.-Galf (radical A, .delta.
71.90 and .delta. 84.96) and substitutions on C-3 of .beta.-D-Glcp
(radical B, .delta. 82.44). The sequence of the radicals in the
repetitious unit was determined by ROESY experiments. Strong NOE
contacts between the radicals were found between the protons A1
(.delta. 5.315) and B3 (.delta. 3.663), and B1(.delta. 4.542) and
A6a (.delta. 3.768) (FIG. 2). Thus the structure of the repetitious
unit of the isolated polysaccharide was as depicted in FIG. 3.
EXAMPLE 4
[0147] ELISA Studies
[0148] ELISA experiments were carried out by customary methods as
described previously (Theilacker, C., et al., Infect. Immun. 74,
2006). Briefly, microtiter plates were coated with various
carbohydrate antigens derived from E. faecalis (10 pg/ml in 0.04 M
phosphate buffer, pH 7.0), and left at 4.degree. C. for 18 hours.
Washing steps were carried out using PBS containing 0.05% Tween 20.
The plates were blocked with 3% skimmed milk in PBS-0.02% sodium
azide at 37.degree. C. for 2 hours. The secondary antibody used was
a goat anti-rabbit IgG alkalinephosphatase conjugate (Sigma),
diluted to 1:1000, with p-nitrophenyl phosphate being used as
substrate (Sigma). After incubation at 37.degree. C. for 60
minutes, absorption was measured at 405 nm.
EXAMPLE 5
[0149] Opsonophagocytosis Assay
[0150] An opsonophagocytosis assay was performed as described
previously (Theilacker, C., et al., Infect. Immun. 74, 2006). Baby
rabbit serum (Cedarlane Laboratories, Hornby, Ontario, Canada)
absorbed with the target bacterial strain was used as complement
source. The opsonic activity of the immune sera was compared to
that of the controls containing normal rabbit serum. The immune
serum was heat-inactivated at 56.degree. C. for 30 minutes before
use. Negative controls comprised sample tubes which either did not
contain any polymorphonuclear leucocytes or any complement or any
serum. The opsonic activity of the serum was calculated as follows:
[1-(CFU immune serum at 90 min/CFU preimmune serum at 90
min)].times.100.
[0151] Opsonophagocytosis inhibition was studied by incubating
antiserum in a concentration of 1:200 with from 0.08 to 100
.mu.g/ml at 4.degree. C. for 60 minutes. After incubation, the
absorbed serum was added and the opsonophagocytosis assay was
continued as described above. Without inhibition, all sera had a
minimum opsonophagocytosis activity of >70% of the inoculum.
EXAMPLE 6
[0152] Immunochemical Characterization
[0153] Serum against bacteria cells of E. faecalis type 5 (Maekawa,
S., et al., Microbiol. Immunol. 36:671-681, 1992) was reactive to
the purified polysaccharide (FIG. 4). The antiserum also contained
high levels of IgG antibodies to E. faecalis LTA (FIG. 4). Since
anti-LTA antibodies mediate opsonophagocytosis of the E. faecalis
strain 12030, we intended to determine the specificity of
opsonizing antibodies for the E. faecalis type 5 strain. Purified
LTA did not inhibit the opsonophagocytosis activity of anti-type 5
serum (FIG. 5), in agreement with the observation that there is
little cross-reactivity of opsonizing antibodies between E.
faecalis type 5 and E. faecalis 12030 (Hufnagel, M., et al., J.
Clin. Microbiol. 42:2548-2557, 2004). However, the purified
capsular polysaccharide was a potent inhibitor of opsonizing
antibodies to type 5 (FIG. 5). Rabbit serum against purified LTA,
which promotes opsonophagocytosis of strain 12030 in the
opsonophagocytosis assay, was non-opsonizing against the type 5
strain, confirming the results from our inhibition studies (data
not shown).
EXAMPLE 7
[0154] Antibodies to the two polysaccharides of type 2 and of type
5 were raised in rabbits: a female white New Zealand rabbit was
immunized subcutaneously with 100 .mu.g of purified polysaccharide
of type 2 and type 5 which had been suspended in complete Freund's
adjuvant, and was then immunized with the same dose of
polysaccharide suspended in incomplete Freund's adjuvant seven days
later, and thereafter with 10 .mu.g booster doses every three days
for the following week.
[0155] The polysaccharides were obtained by the method described
under 2d). In each case two rabbits were used for immunization and
the antiserum was subsequently obtained from the rabbits. The
antisera obtained from the rabbits were studied in an ELISA. To
this end, both the polysaccharide of type 2 and the polysaccharide
of type 5 were bound to the microtiter plates. Surprisingly,
antibodies were found to be produced both against purified
polysaccharide of type 5 and against polysaccharide of type 2. This
demonstrated that the antigen is immunogenic, a fact that was not
necessarily expected in view of the fact that it is a "T
cell-independent" antigen. Production of the antibodies is depicted
in FIG. 6.
EXAMPLE 8
[0156] Opsonophagocytic Assay
[0157] This in vitro assay involved combining granulocytes,
antiserum and bacteria and testing whether it was possible to kill
the Enterococcus faecalis bacteria by the antibodies generated. The
exact conditions of the assay mixture were as follows: fresh whole
blood was obtained from healthy donors and admixed with a heparin
dextran buffer. The white blood cells were then purified and
adjusted to a defined number (5.times.10.sup.6 cells/ml). The
bacteria to be studied were removed by centrifugation from a
culture at average logarithmic growth and adjusted
spectrophotometrically likewise to 5.times.10.sup.6 cells/ml. The
complement source used was lyophilized baby rabbit serum diluted
1:15 with cell culture medium and absorbed with the target bacteria
strain in order to remove existing antibodies to the Enterococcus
strain used. The rabbit serum was likewise diluted with cell
culture medium in accordance with the experimental set up. For the
experiment, in each case 100 .mu.l of the bacterial suspension, 100
.mu.l of leucocytes (bacteria: leucocytes ratio of 1:1), 100 .mu.l
of the complement source and 100 .mu.l of the corresponding
antibody dilution were mixed. The initial bacterial count was
determined by dilution and plating out, and the experimental
mixture was then incubated in an end over end rotator at 37.degree.
C. for 90 minutes. At the end of the experiment, the bacteria in
the experimental mixture were likewise diluted again and plated
out, and the number of colonies was counted on the next day. The
reduction of the bacterial count between inoculum and the bacterial
count at the end of the experiment was expressed as
opsonophagocytosis-mediated killing in percent. This value
represents the best surrogate marker for a protective immuno
response to bacterial infectious pathogens.
[0158] The results of the experiment are depicted in FIG. 7. They
demonstrate that serum dilutions of from 1:50 to 1:200 result in a
marked killing of approx. 80%, while this effect is lost with
further dilutions of the serum. This indicates that higher antibody
titers to this antigen represent a protection of the organism from
infection with E. faecalis.
* * * * *