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Infect Immun, February 1998, p. 870-873, Vol. 66, No. 2
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Anti-Lipid A Monoclonal Antibody Centoxin (HA-1A)
Binds to a Wide Variety of Hydrophobic Ligands
E. J.
Helmerhorst,1
J. J.
Maaskant,2 and
B.
J.
Appelmelk2,*
Department of Oral
Biochemistry1 and
Department of Medical
Microbiology,2 Vrije Universiteit, 1081 BT
Amsterdam, The Netherlands
Received 19 September 1997/Returned for modification 27 October
1997/Accepted 26 November 1997
 |
ABSTRACT |
This note describes the binding specificities of four lipid A
monoclonal antibodies (MAbs) including Centoxin
(HA-1A); these MAbs display similar binding properties. MAbs reacted
with lipid A and heat-killed smooth bacteria, whereas no
reactivity was observed with smooth lipopolysaccharide (LPS).
Immunoblotting of bacterial extracts separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis showed that the MAbs
bound to many polypeptide bands including the molecular weight
markers. Denaturation of bovine serum albumin (BSA)
by boiling or dithiothreitol treatment unmasked antibody epitopes. In
addition, binding both to a hydrophobic aliphatic
C12 chain covalently coupled to BSA and to single-stranded DNA was
observed. The polyreactivity of these clones is most likely mediated by
a preferential reactivity with hydrophobic molecular patches.
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TEXT |
Severe infections with gram-negative
organisms are still an important cause of death. One of the outer
membrane components of such bacteria, endotoxin (LPS) plays a pivotal
role in the pathogenesis of GNB. There is substantial evidence that LPS
initiates a cascade of events leading to the onset of the sepsis
syndrome (13). Since lipid A is the toxic moiety of LPS,
many attempts to prepare protective agents against LPS have focused on
the preparation of ligands to the lipid A moiety. It has been
postulated that antibodies directed against the conserved core or lipid
A region of LPS may cross-react with LPS produced by phylogenetically
diverse gram-negative bacteria involved in GNB (5). Centoxin
(also called HA-1A) is a human monoclonal antibody raised against the rough LPS of Escherichia coli J5 (Rc chemotype) and selected
on binding to lipid A. Both animal studies (16, 18) and
phase III human clinical trials gave discrepant results as to the
protective efficacy of Centoxin against GNB (14, 19). Since
the publication of these reports, questions about the epitope
specificity of this MAb have arisen. While it has been described by
some authors as a lipid A-specific MAb (8, 10), others
defined the antibody as polyreactive since cross-reaction was observed
with i antigen present on cord erythrocytes, a ligand on human B
lymphocytes, and several anionic polymers such as ssDNA, chondroitin
sulfate, and cardiolipin (6, 7). In the present study we
demonstrate that there might be a unifying principle to explain the
cross-reactivity with several apparently different antigens. The
epitope specificities of a number of anti-lipid A MAbs developed in our
laboratory that showed a binding profile comparable to that of HA-1A
are described. By ELISA, SDS-PAGE, and dot spot techniques it has been
made plausible that these MAbs recognize hydrophobic molecular patches
present in lipid A, denatured proteins and in aliphatic chains.
Abbreviations used.
BSA, bovine serum albumin; DTT,
dithiothreitol; CHAPS,
3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate; ELISA,
enzyme-linked immunosorbent assay; GNB, gram-negative bacteremia; LPS,
lipopolysaccharide; MAb, monoclonal antibody; OD, optical density;
OMPs, outer membrane proteins; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; ssDNA, single-stranded
DNA; Ig, immunoglobulin.
The antibodies used in this study were murine MAbs of the IgM class
(clones 28, 37, 38, 40, and 43) or of the IgG class (clone 3). Centoxin
(HA-1A) is a human MAb of the IgM class and was obtained upon
immunization with E. coli J5 (18). MAbs 28 and 40 were raised against alkaline- and acid-treated Salmonella
minnesota R595 cells (Re chemotype), respectively (2).
MAbs 37 and 38 were raised against heat-killed S. minnesota
R4 cells (Rd chemotype). The epitope specificity of anti-lipid A MAb 43 has been described before (3, 11, 12); this MAb recognizes
the hydrophillic part of lipid A (8a). MAb 3 was raised
against heat-killed E. coli J5 bacteria (3).
ELISA was carried out as described previously (
1), and the
antibody titers, defined as the lowest MAb concentrations at
which
significant binding occurred, i.e., with an OD at 492 nm
above control
values by more than 0.200 without a first antibody,
were determined. At
10 ng/ml MAbs 28, 37, and 40 still reacted
(data not shown) with
heat-killed bacterial cells of
E. coli O111
and other
species and with isolated lipid A; in contrast, even
at 2,000 ng/ml no
reaction with smooth LPS was observed. Centoxin
reacted in a similar
pattern, i.e., a good reaction with bacterial
cells at 100 ng/ml and no
reaction with smooth LPS at 32,000 ng/ml.
Anti-lipid A MAb 43 reacted
with lipid A but not with LPS or heat-killed
bacteria.
It has been proposed that HA-1A binds to bacteria by the lipid A
epitope. The binding of HA-1A to smooth bacteria is enhanced
by
antibiotic treatment, which would unmask the lipid A epitope
(
17). To investigate the epitopes of MAbs 28, 37, 40, and
HA-1A
on bacteria, SDS-PAGE and immunoblotting were performed on
heat-killed
S. minnesota R5 (Rc chemotype) bacteria (OD = 3) and on an extract
of the OMPs. The OMPs of
S. minnesota
R5 bacteria were extracted
as follows. A concentrated bacterial
suspension was washed three
times in 50 mM Tris-HCl (pH 7.3) and
incubated under agitation
in the same buffer supplemented with 0.5%
CHAPS for 1 h at 4°C.
Proteolytic digests were prepared by
incubation of a suspension
of heat-killed bacteria (OD = 3) or
OMPs with proteinase K (final
concentration, 500 µg/ml) for 1 h
at 37°C. SDS-PAGE was performed,
and the gels were blotted on
Immobilon-P blotting paper (Boehringer
GmbH, Mannheim, Germany),
blocked for 1 h with blocking buffer
(Boehringer), washed in
phosphate-buffered saline supplemented
with 0.1% Tween 80 (Sigma), and
incubated with MAb 40 (1.8 µg/ml),
Centoxin (20 µg/ml), or
-Rc
MAb 3 (3 µg/ml). Figure
1a shows
that
MAb 40 binds to a wide variety of OMPs (lane 2). No binding
to
proteinase K-treated OMPs was observed (lane 1). A wide range
of
protein bands was also recognized in heat-killed R5 bacteria
(lane 3);
the banding pattern was comparable to that produced
by total protein
staining with Congo red (data not shown). For
proteinase K-treated R5
bacteria, only one predominant band of
36 kDa recognized by the
antibody was left (lane 4). This band
could represent either a
proteinase K-resistant protein or a nonprotein
component. Remarkably,
all protein molecular weight markers (phosphorylase
B, BSA, ovalbumin,
carbonic anhydrase, soybean trypsin inhibitor,
and lysozyme; Pharmacia)
were also recognized by MAb 40 (lane
5). Just like MAb 40, Centoxin
recognized many proteins in heat-killed
S. minnesota R5
(Fig.
1b, left panel, lane 3) and one band in
the proteolytic digest
(lane 2). In accordance with the binding
studies performed by ELISA, no
binding of Centoxin to R5 LPS (lane
1), which was near the front of the
gel and which was visualized
with
-Rc LPS antibody, was seen (Fig.
1b, right panel, lanes
1 and 3). In contrast to what was seen with MAb
40 and Centoxin,
proteinase K treatment of whole bacteria did not
abolish the epitope
of MAb 3 (lane 2). The binding of Centoxin to
coextracted porin
proteins in LPS samples has previously been described
by Mascelli
et al. (
15). Our binding studies further
emphasize the great
affinity of Centoxin for proteins separated by
SDS-PAGE. These
proteins have undergone a series of denaturing
treatments, such
as breaking of cysteine bridges in DTT, dissolution in
SDS, and
heating. To study whether the unfolding of proteins could
unmask
antibody epitopes, a well-defined system was chosen for this
subject
of investigation. BSA (Sigma) was exposed to a number of
conditions
that affect its conformational characteristics: incubation
in
2, 5, and 8 M urea overnight at room temperature, incubation for
1 h in 200 mM DTT at room temperature, boiling for 10 min, and
a
combination of these conditions. In a dot spot experiment (Fig.
2), no binding of clone 40 to native BSA
(1 mg/ml; spot 10) or
to BSA incubated with urea alone (spots 2, 3, and
4) was observed.
Clear immunoreactivity was observed with BSA treated
with 200
mM DTT (spot 8) and with boiled BSA (spot 11). By these
treatments
the protein is expected to unfold to expose the inner parts
of
the molecule to the outside. It was concluded that partial
denaturation
of the protein, achieved by either boiling or DTT
treatment, uncovers
protein patches that are hidden in native BSA.
These inner parts,
that are known to have a hydrophobic character, are
recognized
by MAb 40.
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FIG. 1.
(a) Western blot analysis of S. minnesota R5
bacteria, R5 OMPs, and proteolytic digests with MAb 40. Blots were
incubated for 2 h at 37°C with MAb 40 (1.8 µg/ml) in
phosphate-buffered saline supplemented with 0.1% Tween 80. Lane 1, S. minnesota R5 OMPs (7.5 µl) incubated with proteinase K
(500 µg/ml); lane 2, S. minnesota R5 OMPs (7.5 µl); lane
3, S. minnesota R5 bacteria (OD = 3; 7.5 µl); lane 4, S. minnesota R5 bacteria (OD = 3; 7.5 µl) treated
with proteinase K (500 µg/ml); lane 5, molecular weight markers; lane
6, proteinase K (12.5 µg). kD, kilodaltons. (b) Western blot analysis
of S. minnesota R5 bacteria, R5 LPS, and proteolytic digests
with anti-lipid A and anti-Rc MAb. Left panel: blot incubated with MAb
HA-1A (20 µg/ml); right panel: blot incubated with -Rc MAb 3 (3 µg/ml). Lanes 1, R5 LPS (7.5 µg); lanes 2, S. minnesota
R5 bacteria (OD = 3; 7.5 µl) treated with proteinase K (500 µg/ml); lanes 3, S. minnesota R5 bacteria (OD = 3;
7.5 µl); lanes 4, proteinase K (12.5 µg).
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FIG. 2.
Dot spot analysis of BSA (1 mg/ml) preincubated at
various denaturing conditions (DTT, urea, boiling) with anti-lipid A
MAb 43 (1.8 µg/ml) (A), without a first antibody (B), and with MAb 40 (1.8 µg/ml) (C). Spot 1, 8 M urea plus 200 mM DTT; spots 2, 3, and 4, BSA incubated for 24 h in 2, 5, and 8 M urea, respectively; spots
5, 6, and 7, BSA incubated in 2, 5, and 8 M urea, respectively, plus
200 mM DTT; spot 8, BSA incubated in 200 mM DTT; spot 9, 200 mM DTT;
spot 10, native BSA; spot 11, boiled BSA.
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The binding of MAbs 40 and HA-1A to all molecular weight markers and to
bacterial proteins in SDS-PAGE and to denatured BSA
but not to native
BSA in a dot spot assay suggests that hydrophobic
protein patches are
recognized. In additional ELISA experiments
it was observed that MAbs
28, 37, 40, and HA-1A also have great
affinity for non-protein-derived
hydrophobic ligands, such as
a polymeric aliphatic chain (C12, dodecyl)
covalently coupled
to BSA (
9) (Fig.
3). No binding to native BSA was observed
(data not shown). In addition to observing the binding to dodecyl-BSA,
we confirmed the finding of Bhat et al. (
6) that Centoxin
binds
to ssDNA (origin: calf thymus type I; Sigma) and found that MAbs
28, 37, and 40 also bind to ssDNA (data not shown); DNase treatment
of
the DNA sample completely abolished the binding (data not shown).
The
binding of MAb 40 and Centoxin to solid-phase bound ssDNA
could be
inhibited by fluid-phase dodecyl-BSA and heat-killed
bacteria (data not
shown); this suggests that the same antigen-binding
site is involved in
the interaction with the various ligands.
Bhat et al. (
6)
provided convincing evidence for a highly specific
binding of Centoxin
to i antigen, a carbohydrate structure present
on cord erythrocytes. In
agreement with this was the observation
that the MAb was coded for by
VH4.21, a gene often utilized by
anti-i antibodies (
7). They
explained the cross-reactivity
with lipid A by assuming that an
acyl-substituted disaccharide
structure, present both in i antigen and
lipid A, was the common
epitope (
4). Alternatively, the data
provided by us suggest
that the hydrophobicity of the antigen-binding
site is responsible
for its polyreactivity. The specificity of MAb 28 differs from
that of Centoxin, i.e., MAb 28 reacts specifically and
strongly
with LPS of
S. minnesota R595 (
2) and
does not bind to LPS
of other chemotypes. Yet MAb 28 displays a
polyreactivity similar
to that of Centoxin; in Centoxin and MAb 28 hydrophobicity is
a common property.
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FIG. 3.
Binding of anti-lipid A MAbs to immobilized dodecyl-BSA
by ELISA. Ninety-six-well microtiter plates were coated with
dodecyl-BSA (1 µg/ml). Data for twofold dilution series for MAbs 28 (+), 37 ( ), 40 ( ), and HA-1A (×) and control MAb 38 ( ) are
shown. The OD was measured at 492 nm and expressed as a function of
antibody concentration. Data are representative for two independent
experiments.
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|
Our study emphasizes the preferential reactivity of Centoxin and MAbs
28, 37, and 40 with hydrophobic structures. These are
present in lipid
A (acyl chains), in unfolded proteins (hydrophobic
amino acid side
chains), and in dodecyl-BSA (polymeric aliphatic
chain). The binding of
Centoxin to ssDNA can be explained by the
hydrophobic nature of the DNA
bases, which are only exposed in
the single-stranded conformation.
Indeed Centoxin does not bind
to double-stranded DNA (
7,
8).
Moreover it was found that
MAbs 37 and Centoxin had a greater affinity
for monophosphoryl
lipid A than for diphosphoryl lipid A (data not
shown), which
is less hydrophobic than monophosphoryl lipid A.
In summary, this binding study shows that a number of MAbs raised
against rough mutants of bacteria involved in GNB and selected
on
binding to lipid A are polyreactive. The multispecific binding
pattern
can be explained by a strong interaction with hydrophobic
molecular
patches, and in addition, these Mabs might react more
specifically with
carbohydrate epitopes such as those present
in the i antigen or in R595
LPS (
2). As it has now been demonstrated
that Centoxin has a
great affinity for denatured proteins and
likely also for hydrophic
patches present in native proteins such
as bacterial porins,
contamination of LPS samples with coextracted
proteins may have led to
misinterpretations in studies of Centoxin
binding to LPS performed in
the past.
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ACKNOWLEDGMENTS |
We thank H. Brade (Borstel, Germany) for providing lipid A, LPS,
and bacterial strains.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Phone: 31 20 4448297. Fax: 31 20 4448318. E-mail: BJ.Appelmelk.mm{at}med.vu.nl.
Editor: R. E. McCallum
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Infect Immun, February 1998, p. 870-873, Vol. 66, No. 2
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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