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Infection and Immunity, July 2000, p. 3949-3955, Vol. 68, No. 7
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Selection of Recombinant Antibodies Specific for
Pathogenic Streptococcus suis by Subtractive Phage
Display
Astrid
de
Greeff,1,2,*
Loek
van Alphen,1,3 and
Hilde E.
Smith2
Department of Medical Microbiology,
University of Amsterdam, Academical Medical Center,
Amsterdam,1 Department of
Bacteriology, Institute of Animal Science and Health, 8200 AB
Lelystad,2 and Laboratory of Vaccine
Development and Immune Mechanisms, RIVM, National Institute of
Public Health and the Environment, 3720 BA
Bilthoven,3 The Netherlands
Received 6 January 2000/Returned for modification 14 February
2000/Accepted 23 April 2000
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ABSTRACT |
A semisynthetic antibody phage display library was used to select
recombinant antibodies directed against surface components of a
pathogenic strain of Streptococcus suis serotype 2 and
against extracellular factor (EF), a protein known to be exclusively
associated with pathogenic S. suis serotype 2 strains.
Three distinct monoclonal phage antibodies directed against
conformational epitopes of surface protein components of S. suis were selected. In addition, three different monoclonal phage
antibodies were isolated that recognized EF. To isolate antibody
fragments that recognize epitopes specific for a pathogenic S. suis serotype 2 strain, compared to a nonpathogenic serotype 2 strain, we applied a subtractive selection procedure. With this
procedure, only one distinct phage antibody was found, and it was shown
to be directed against EF. This demonstrates the selectivity of the
applied procedure and confirms that EF is indeed differentially
expressed by pathogenic and nonpathogenic strains. It also shows that
EF is a very dominant antigen in phage antibody selections.
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INTRODUCTION |
Antibody phage display is a very
powerful technique for selecting recombinant antibodies from a large
library (15, 18, 30). An antibody phage library consists of
the variable regions of heavy (VH) and light
(VL) chains of human antibodies, which are randomly
combined and linked together by a polypeptide linker to form a
single-chain fragment (scFv). These scFvs are fused to a minor coat
protein of bacteriophage M13, pIII, resulting in phages displaying
antibody fragments. The display of scFvs on a filamentous phage offers
the possibility to select phage antibodies without using hybridoma
technology. Phage antibodies are selected by panning the library for
several rounds on an immobilized antigen. At present, large synthetic
libraries are available, which are created from unrearranged V gene
segments from nonimmunized healthy human donors. These libraries can be
used to select antibodies against any given antigen, including foreign
antigens, self antigens, nonimmunogenic antigens, and toxic
antigens (30). In addition, subtractive selection strategies
to select for phage antibodies against differentially expressed
structures on the surface of different cell types, like thymic cells
(25) and human blood cells (16), as well as
against proteins differentially expressed on two types of strains of
the gram-negative bacterium Moraxella catarrhalis
(2), have been described.
Streptococcus suis is a gram-positive bacterium that can
cause severe infections in pigs. Young pigs can suffer from meningitis, septicemia, and arthritis and often do not survive an S. suis infection (3, 27). Occasionally, S. suis can also cause meningitis in humans (1). Until
now, no effective vaccines have been available. Besides, very little is
known of S. suis in general and its pathogenesis in
particular. This makes it difficult to control the disease. So far, 35 capsular serotypes of S. suis have been described (6, 7, 12, 19). Worldwide, S. suis serotype 2 is the most
frequently isolated serotype. Strains of serotype 2 can differ in their
virulence: pathogenic, weak-pathogenic, and
nonpathogenic strains are recognized (26, 28).
Previously, we showed that the expression of two proteins,
muramidase-released protein (MRP) and extracellular factor (EF) is
strongly associated with pathogenic strains of serotype 2 (28,
29). Therefore, these proteins are considered virulence
markers for S. suis serotype 2. However, besides MRP and EF,
other proteins may be important in the pathogenesis of an S. suis infection (5, 8, 9, 13, 14, 28, 29). The use of a
phage display library may be of great help in identifying these
proteins and in determining the difference between pathogenic and
nonpathogenic S. suis strains.
Since a considerable number of virulence factors of pathogenic bacteria
are either secreted or located on the cell surface, we first tried to
select phage antibodies against whole cells of a virulent strain of
S. suis serotype 2. In addition, phage antibodies were
selected against EF. Finally, phage antibodies were selected against
cell-associated structures of a pathogenic strain of S. suis
after subtraction with a nonpathogenic strain. Three distinct anti-EF
phage antibodies, as well as three distinct anti-S. suis
phage antibodies, were selected. After subtraction, one phage antibody
remained, which recognized EF. These data clearly show the successful
selection of a phage antibody directed against a protein exclusively
expressed by a pathogenic strain of S. suis serotype 2 and
that EF is a very dominant protein in phage antibody selections.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Two
Escherichia coli strains were used in this study as hosts
for bacteriophages: TG1 [K-12 
(lac-pro) supE thi
hsdD5/F' traD36 proA+ B+
lacIq lacZM15] and HB2151 [K-12
ara (lac-pro) thi/F'
proA+ B+ lacIq
ZM15]. Both strains were grown on tryptone yeast extract
(TYE) plates (17) containing 1% glucose and antibiotics
when required. Cultures were grown in 2× TYE broth (2× TY)
(17). S. suis strain 10 expresses EF and MRP,
while S. suis strain T15 does not (23, 29).
Strain 10 was proven to be pathogenic, while strain T15 was proven to
be nonpathogenic in an experimental pig model (23, 29).
S. suis strain 10cpsEF is an isogenic mutant of S. suis strain 10 that is deficient in capsular polysaccharide
production (21). S. suis strains were grown on
Columbia agar blood base plates (code CM331; Oxoid, Ltd., London,
United Kingdom), containing 6% (vol/vol) horse blood. Cultures were
grown in Todd-Hewitt broth (code CM 189; Oxoid, Ltd.).
Preparation of antigens.
Stationary-phase S. suis
cells (100 ml) were centrifuged for 20 min at 2,500 × g and washed twice with 100 ml of phosphate-buffered saline (PBS)
(0.1 M NaCl, 33 mM Na2HPO4, 17 mM
NaH2PO4 · 2H2O; pH 7.4). The
cells were resuspended in 50 ml of PBS. This suspension was used for
coatings, both for the selection procedure and for the enzyme-linked
immunosorbent assay (ELISA). The supernatant was collected for use on
Western blots. To prepare protoplast supernatant, exponential-phase
S. suis cells (100 ml) were centrifuged for 30 min at
2,500 × g and washed in 100 ml of PBS. The pellet was
resuspended in 10 ml of protoplast buffer (30 mM Tris, 3 mM MgCl2, 25% sucrose, 30 µg of lysozyme ml
1
[Boehringer GmbH, Mannheim, Germany]), incubated for 1 h at
37°C, and centrifuged for 10 min at 11,600 × g. The
supernatant was collected to coat immunotubes and microtiter plates. It
was also used as an antigen on Western blots.
Phage display library.
The Griffin.1 library (a generous
gift from Greg Winter, Centre for Protein Engineering, Cambridge,
United Kingdom) was used to select phage antibodies. Griffin.1 is a
semisynthetic phage library containing more than 109
clones. The library was constructed by recloning the VH and
VL variable regions from the lox library vectors into the
phagemid vector pHEN2 (11).
Rescue of the phage library.
Rescue of the library was
essentially done as described previously (11). Briefly, over
1010 CFU of the Griffin.1 library in strain TG1 were
inoculated in 500 ml of 2× TY broth containing 100 µg of ampicillin
(AMP) ml1 (Boehringer) and 1% glucose (GLU) (2×
TY-AMP-GLU) and incubated at 37°C (with shaking at 200 rpm) until the
optical density at 600 nm (OD600) was approximately 0.5. Twenty-five milliliters of this culture was infected with
1010 PFU of VCS-M13 helper phage (Stratagene, La Jolla,
Calif.) and incubated for 30 min at 37°C. The infected cells were
collected by centrifugation (10 min at 2,500 × g) and
resuspended in 300 ml of 2× TY broth containing 100 µg of AMP
ml1 and 25 µg of kanamycin ml1
(Boehringer) (2× TY-AMP-kanamycin). Subsequently, the cells were incubated overnight at 37°C and with shaking at 200 rpm. Bacterial cells were removed by centrifugation (30 min at 2,500 × g), and phages present in the supernatant were precipitated with
polyethylene glycol-NaCl (20% polyethylene glycol 6000, 2.5 M NaCl)
twice and resuspended in PBS to a final concentration of about
1013 PFU ml1.
Selection procedure.
The selection procedure was performed
with immunotubes and was carried out as described previously
(18). Briefly, Maxisorp immunotubes (5.0-ml; Nunc, Roskilde,
Denmark) were either incubated with 4 ml of purified EF protein
(H. J. Wisselink et al., submitted for publication) at a
concentration of 10 µg ml1 or with 4 ml of intact
S. suis cells. The tubes were incubated for 1 h at
37°C, followed by an incubation for 16 h at 4°C. Subsequently, the immunotubes were blocked with PBS containing 2% skimmed milk (MPBS) for 2 h at 37°C and washed three times with PBS. The
phage library (about 1013 PFU in 2% MPBS) was added, and
the tubes were incubated on a turntable for 30 min at room temperature,
followed by a standing incubation for 90 min at room temperature.
Unbound phages were removed by washing the tubes 20 times with PBS
containing 0.1% Tween 20 and 20 times with PBS. Bound phages were
eluted in 1 ml of 100 mM triethylamine (for 10 min, while being rotated
on a turntable at room temperature). Eluted phages were neutralized with 0.5 ml of 1 M Tris-HCl (pH 7.4) and used to infect E. coli strain TG1. To determine the number of eluted phages,
infected E. coli cells were plated in a serial dilution on
TYE-AMP-GLU plates. For the subtractive selection, the immunotubes were
incubated for 1 h at 37°C either with 4 ml of protoplast
supernatant of S. suis strain 10, diluted 1:100 in PBS, or
with 4 ml of intact S. suis cells, followed by an incubation
for 16 h at 4°C. The phage library (about 1013 PFU
of library cells) was preincubated in 2% MPBS for 15 min at room
temperature either with 1 ml of undiluted protoplast supernatant or
with 1 ml of intact cells of S. suis strain T15, and
subsequently the mixture was added to the immunotube. The selection
procedure was then performed as described above.
PCR and restriction enzyme analysis of phagemid DNA.
Individual colonies of phage-infected TG1 cells were transferred into a
PCR mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM
MgCl2, 10 pmol of each deoxynucleoside triphosphate, 10 pmol of the forward primer fdSeq1 (5'-GAATTTTCTGTATGAGG-3'), 10 pmol of the backward primer LMB3
(5'-CAGGAAACAGCTATGAC-3'), and 2 U of Taq
polymerase (Perkin-Elmer, Foster City, Calif.). DNA amplification was
carried out with a Perkin-Elmer 9600 thermal cycler, and the program
consisted of an incubation for 10 min at 94°C and 30 cycles of 1 min
at 94°C, 1 min at 55°C, and 1.5 min at 72°C, followed and 5 min
at 72°C. PCR products were analyzed on a 1.5% agarose gel containing
0.5 µg of ethidium bromide ml1. PCR products were
digested with BstNI (New England Biolabs, Beverly, Mass.) or
AvaII (Promega, Madison, Wis.). The restriction mixture,
containing 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM dithiothreitol, and 4 U of BstNI or
AvaII, was added to the PCR mixture in a 1:1 ratio. The
mixture was incubated for 2 h at 60°C for BstNI and
for 2 h at 37°C for AvaII. The restriction products
were analyzed on a 4% agarose gel containing 50% multipurpose agarose
and 50% Metaphor agarose (FMC Bioproducts, Rockland, Maine) and 0.5 µg of ethidium bromide ml1.
Phage ELISA.
Microtiter plates (Greiner Labortechnik,
Frickenhausen, Germany) were coated with purified EF protein or with
intact S. suis cells as described above for the coating of
the immunotubes. Before being used, the plates were washed three times
with PBS and blocked with 2% MPBS for 2 h at 37°C. Polyethylene
glycol-precipitated polyclonal phage antibodies (PoPhAbs) or a culture
supernatant of superinfected TG1 cells containing monoclonal phage
antibodies (MoPhAbs) was used in serial dilutions in 2% MPBS and
incubated for 90 min at 37°C. Plates were washed three times with PBS
containing 0.05% Tween 20 and three times with PBS. Phages were
detected by using the Detection Module Recombinant Phage Antibody
system (Pharmacia, Uppsala, Sweden) according to the manufacturer's recommendations.
Expression of soluble antibody fragments.
The E. coli nonsuppressor strain, HB2151, was infected with phages as
described for TG1. Individual HB2151 colonies were transferred into 150 µl of 2× TY-AMP-GLU. Plates were incubated at 37°C (with shaking
at 200 rpm) until the OD600 of the cells was about 0.9. Isopropyl-
-D-thiogalactopyranoside (IPTG) (Eurogentec,
Seraing, Belgium) was added to a final concentration of 1 mM. Plates
were incubated for 16 to 24 h at 30°C (with shaking at 200 rpm).
Bacteria were centrifuged at 1,800 × g for 10 min.
Supernatants containing scFvs were collected for further use.
Western blot analysis.
Proteins were separated by sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and
transferred to a nitrocellulose membrane by standard procedures
(20). The membrane was blocked in Tris-buffered saline (TBS)
(50 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing 4% skimmed milk
and 0.05% Tween 20 (Blotto) for 1 h. To detect specific antigens,
the membranes were incubated with a 1:1 dilution of scFvs in Blotto for
at least 90 min. Bound scFvs were visualized with a 1:1,000 dilution of
anti-c-myc monoclonal antibody (MAb) (Boehringer) in Blotto-TBS (1:1),
followed by an incubation with a 1:1,000 dilution of alkaline
phosphatase-conjugated anti-mouse antibody. As a substrate, we used
Nitro Blue Tetrazolium (Merck, Darmstadt, Germany)-bromochloroindolyl
phosphate (Sigma, St. Louis, Mo.). All washing steps were performed in
Blotto-TBS (1:1). A hybridoma-derived MAb directed against EF and
convalescent serum raised against S. suis strain 10 in swine
were used as positive controls (28).
Dotblot analysis.
Proteins were spotted onto nitrocellulose
by using a Bio Dot Apparatus (Bio-Rad Laboratories, Richmond, Calif.)
according to the manufacturer's recommendations. Three different
protein samples were used: native protoplast supernatant diluted 1:1 in TBS, protoplast supernatant diluted 1:1 in SDS loading buffer (100 mM
Tris-HCl [pH 6.8], 4% SDS, 0.2% bromophenol blue, 20% glycerol),
and protoplast supernatant diluted 1:1 in native loading buffer (100 mM
Tris-HCl [pH 6.8], 10% glycerol, and 0.25
bromophenol blue). The
blots were blocked in Blotto for 1 h. To detect specific proteins,
the membrane was incubated for at least 90 min with a 1:1 dilution of
MoPhAbs in Blotto. Phages were detected with the Detection Module
Recombinant Phage Antibody System (Pharmacia), according to the
manufacturer's recommendations. The signal was visualized by using
ECL+ (Amersham Pharmacia Biotech, N.J.) according to the
manufacturer's recommendations. Signals were detected on the Storm
(Molecular Dynamics, Sunnyvale, Calif.). All washing steps were
performed in Blotto-TBS (1:1).
Nucleotide sequence analysis.
Nucleotide sequences were
determined with a 373A DNA Sequencing System (Applied Biosystems,
Warrington, United Kingdom). Samples were prepared with an ABI/PRISM
Dye Terminator Cycle Sequencing Ready Reaction kit (Applied
Biosystems). Custom-made sequencing primers were purchased from Life
Technologies. Primers used were as follows: Boli 189 (5'-GCCTACGGCAGCCGCTCGAT-3'), FOR_LinkSeq (5'-GCCACCTCCGCCTGAACC-3'), Boli 101 (5'-GGTGGAGGCGGTTCACGCGCAGGTGGCTCT-3'), and fdSeq1
(5'-GAATTTTCTGTATGAGG-3'). Sequencing data were assembled and analyzed with the Lasergene program (DNASTAR). The V-BASE sequence
directory described by Tomlinson et al. (24) was used to
assign germ line VH and VL segments.
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RESULTS |
Selection of phage antibodies.
The Griffin.1 phage library was
used to select phage antibodies against purified EF protein. Six rounds
of selection were performed on EF. Input phage titers were very
uniform, between 5.5 × 1012 and 4.7 × 1013. After each round of selection, the number of eluted
phages was determined. The results (Table
1) show that phage titers decreased in
the first two rounds of selection and rose again in the third round.
This indicates antigen-specific phage antibodies were selected and
enriched.
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TABLE 1.
Phage titers obtained after biopanning on purified
EF protein and intact S. suis cells and after a
subtractive selection procedure
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PoPhAbs derived from the library and the successive selection rounds
were subsequently tested for their capacity to bind to
purified EF
protein in an ELISA (Fig.
1). An increase
in the absorption
signal was observed for the phage antibodies obtained
after the
second selection round. This indicates that antigen-specific
PoPhAbs
had been selected and enriched after round 2. To confirm this
data further, 96 individual, randomly chosen colonies from each
selection round were used to prepare MoPhAbs. These MoPhAbs were
subsequently tested with an ELISA for their capacity to bind to
EF. As
shown in Table
2, the number of MoPhAbs
which showed specific
binding to EF increased after the successive
rounds of selection.
The MoPhAbs did not bind to 3% bovine serum
albumin, 2% MPBS,
Todd-Hewitt medium, or uncoated plates
(OD
600 < 0.150).
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FIG. 1.
Enrichment for phages recognizing purified EF protein
(A) or S. suis cells (B) as determined by ELISA. The
absorbance of the positive control present in the phage detection kit
was set at 100%. The data were expressed as percentages of absorbance
relative to that of the control. Every column represents three
individual experiments; error bars indicate the standard error of the
mean.
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Subsequently, the Griffin.1 library was used to select phage antibodies
against intact
S. suis strain 10 cells. Four rounds
of
selection were performed on intact bacterial cells. Input phage
titers
were between 5.5 × 10
12 and 4.7 × 10
13. After each selection round, the eluted phage titers
were determined.
The titers decreased in the first two rounds but
increased again
in the third round (Table
1), indicating that
enrichment of antigen-specific
phages had taken
place.
PoPhAbs derived from the library and the four rounds of selection were
tested for their capacity to bind to intact
S. suis cells.
Figure
1 shows an increase in the absorption signal for
PoPhAbs derived
from the third selection round, confirming the
selection of
antigen-specific phage antibodies. Ninety-six individual,
randomly
chosen colonies from each selection round were induced
to produce
MoPhAbs. These MoPhAbs were tested with an ELISA on
intact
S. suis cells. The number of MoPhAbs that specifically
recognized
S. suis increased after the successive rounds of selection.
The selected MoPhAbs did not bind to 3% bovine serum albumin,
2%
MPBS, Todd-Hewitt medium, or uncoated plates (OD
600 < 0.150).
In conclusion, these data indicate that MoPhAbs specific for
purified
EF protein and intact
S. suis strain 10 cells have
been selected
and
enriched.
Diversity of isolated phage antibodies.
To determine the
diversity among the selected phage antibodies, 96 individual clones
obtained after the fifth round of selection against purified EF protein
and after the third round of selection against S. suis
cells were subjected to PCR. The PCR products were analyzed by
restriction enzyme analysis. As a control, 10 randomly chosen clones of
the original library were analyzed in the same way. Amplification of a
complete scFv fragment will result in a PCR product of about 1 kb. Of
96 clones obtained after selection against EF, 36 showed a PCR product
of 1 kb and 11 showed a PCR product of 0.7 to 0.8 kb; for 51 clones, no
PCR product was obtained. Of the 96 clones obtained after selection
against S. suis cells, 52 showed a PCR product of 1 kb and
24 showed a product of 0.7 to 0.8 kb; for 20 clones, no PCR product was
obtained. Moreover, nearly all clones which yielded a PCR product of 1 kb were positive by ELISA. In contrast, clones which did not result in
a PCR product were never positive by ELISA. From clones which yielded a
PCR product of 0.7 to 0.8 kb, the ELISA signal varied between negative and weakly positive (data not shown). From all 10 clones randomly selected from the library, a 1-kb PCR product was obtained. As expected, these 10 clones showed 10 different BstNI
restriction patterns. Among the 36 anti-EF clones, three distinct
BstNI restriction patterns were found (E-H1, E-D9, and E-H3)
(Fig. 2). Of the anti-EF clones, 92%
were of the E-H1 type, 6% of the E-H3 type, and 3% of the E-D9 type.
Surprisingly, after PCR and restriction analysis of the sixth selection
round, only one clone was found, E-H1 (data not shown). This indicates
that the increase of phage titers observed after round 6 was the result
of enrichment of a subpopulation of the EF-binding clones. Among the 50 anti-S. suis clones, three other unique BstNI
restriction patterns were found (S-A7, S-B1, and S-F7) (Fig. 2). Of the
anti-S. suis clones, 63% were of the S-B1 type, 12% of the
S-F9 type, and 4% of the S-A7 type.
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FIG. 2.
BstNI fingerprints of the inserts of phages
selected against purified EF protein and S. suis cells.
Individual clones were subjected to PCR and restricted with
BstNI. Lane 1, EF-specific clone E-H1; lane 2, EF-specific
clone E-D9; lane 3, EF-specific clone E-H3; lane 4, S. suis-specific clone S-A7; lane 5, S. suis-specific
clone S-B1; lane 6, S. suis-specific clone S-F9. The size of
products is indicated in base pairs.
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Immunoblot analysis.
The binding specificity of MoPhAb E-H1
was analyzed as representative for anti-EF selected MoPhAbs with a
Western blot of culture supernatant of S. suis strain 10. A
hybridoma-derived MAb raised against EF and convalescent serum raised
against S. suis strain 10 were included as controls. Figure
3 shows that EF, a 110-kDa protein
(28), was clearly detected with the MAb as well as with the
convalescent serum. A protein band of the same size was detected
by using the scFv preparation of the selected MoPhAb, E-H1. These
results show that E-H1 was indeed specifically directed against EF.
Therefore, the antibody phage display is a fast and efficient method to
select MoPhAbs against purified EF.
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FIG. 3.
Western blot analysis of the culture supernatant of
S. suis strain 10, probed with a classical MAb raised
against EF (lane 1); convalescent serum raised against S. suis (lane 2); and scFvs of clone E-H1 (lane 3). Arrowhead,
110-kDa EF protein.
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To characterize the MoPhAbs selected against intact
S. suis
cells, Western blots containing either culture supernatant or
protoplast supernatant of
S. suis strain 10 were incubated
with
MoPhAbs or scFvs of the three
S. suis-specific clones.
No specific
band was detected. One possible explanation for this is
that the
selected MoPhAbs are directed against cytoplasmic components
and
that these components were not present on the blots used. However,
because intact
S. suis cells were used in the selection
procedure,
it is not very likely that the antibodies react with
cytoplasmic
proteins. Since strain 10 of
S. suis serotype 2 is highly encapsulated,
phage antibodies selected against intact
S. suis cells may be
directed against capsular
polysaccharides. To test this possibility,
we determined the capacity
of the three anti-
S. suis MoPhAbs to
bind to strain
10cps
EF, an isogenic mutant of strain 10 deficient
in capsular
polysaccharide production (
20). All three phage
antibodies
bound to the capsular mutant strain as efficiently
as to the wild-type
strain (OD
600 > 0.350), thereby excluding
the
possibility that the phage antibodies are directed against
capsular
polysaccharide components. A further possibility is that
the selected
MoPhAbs recognize conformational epitopes and therefore
do not
recognize the denatured and reduced antigens on the Western
blot.
Protoplast supernatant was spotted on a blot under denaturing
and
nondenaturing conditions, and the dot blot was subsequently
incubated
with MoPhAbs prepared from clones S-A7, S-B1, and S-F9.
Figure
4 shows that nondenatured proteins
present in protoplast
supernatant were recognized by the three selected
MoPhAbs, whereas
the denatured proteins were not recognized, except for
clone S-F9,
which showed a weak reaction with denatured proteins. This
result
shows that the MoPhAbs were directed against cell surface
proteins
of
S. suis and that they recognize conformational
epitopes.
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FIG. 4.
Dot blot analysis of the protoplast supernatant of
strain 10, probed with three anti-S. suis MoPhAbs selected
against S. suis cells. (A) protoplast supernatant in TBS;
(B) protoplast supernatant in SDS loading buffer; (C) native loading
buffer. Proteins were either incubated with S-A7 (lane 1), S-B1 (lane
2), or S-F9 (lane 3). Convalescent serum raised against S. suis strain 10 was used as a positive control (lane 4).
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Subtractive selection on S. suis strain 10.
To
identify proteins exclusively expressed by a pathogenic S. suis serotype 2 strain, phage antibodies were selected against intact cells and against protoplast supernatant of the pathogenic S. suis strain 10, after subtraction with the nonpathogenic
S. suis strain T15, lacking protein EF. Five rounds of
selection were performed on both antigens. Input phage titers for both
selections were fairly constant, between 5 × 1012 and
3.8 × 1013.
For selection on intact cells of
S. suis strain 10, phage
titers decreased in the first four rounds of selection and increased
slightly after the fifth round (Table
1). To test the specificity
of
the selected phage antibodies, 96 randomly chosen colonies
from the
library and from selection rounds four and five were
induced to produce
MoPhAbs. The binding of these MoPhAbs to proteins
present on intact
cells of
S. suis strain 10 was determined by
an ELISA. After
five rounds of selection, three positive clones
were found (Table
2).
For selection on protoplast supernatant of
S. suis strain
10, phage titers decreased in the first two rounds of selection
and
increased again after the fourth round of selection (Table
1),
indicating enrichment of antigen-binding phages. To test
the
specificity of the selected phage antibodies, 96 randomly
chosen
colonies from each selection round were induced to produce
MoPhAbs. The
binding of the MoPhAbs to proteins present in the
protoplast
supernatant of
S. suis strain 10 was determined. The
number
of MoPhAbs which showed specific binding to the protoplast
supernatant
of
S. suis strain 10 in ELISA increased in the successive
selection rounds (Table
2), indicating enrichment of
S. suis-specific
clones.
Clones that were positive in the monoclonal phage ELISAs on intact
cells or on protoplast supernatant of
S. suis strain 10
were
subsequently tested by PCR and fingerprint analysis. Ninety-seven
of
these clones, including the three clones selected on intact
S. suis cells, showed a PCR product of 1 kb. Moreover, all 97
clones
showed an identical
BstNI restriction pattern. Surprisingly,
this pattern was identical to the pattern of clone E-H1, indicating
that clone E-H1 and the subtractive clone (Sub-B3) are identical.
Clones E-H1 and Sub-B3 were further characterized by
AvaII
fingerprints.
Figure
5 shows that the
AvaII restriction patterns of E-H1 and
Sub-B3 were
identical. These data strongly indicate that the phage
antibodies
selected by the subtractive procedure are similar to
those of E-H1 and
therefore directed against EF. Since strains
10 and T15 are known to
differ in the expression of EF (
28,
29), these data suggest
that the subtractive selection procedure
has succeeded. This was
further confirmed by Western blots of
culture supernatants of
S. suis strains 10 and T15 that were incubated
with scFvs derived
from Sub-B3. As control, a MAb raised against
EF was used. The 110-kDa
EF protein was recognized by Sub-B3 and
the MAb against EF in culture
supernatant of strain 10 but not
in culture supernatant of strain T15
(Fig.
6).
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FIG. 5.
BstNI and AvaII fingerprints of
the inserts of EF-specific clone E-H1 and S. suis-specific
clone Sub-B3. Individual clones were subjected to PCR and restricted
with BstNI and AvaII. Lane 1, EF specific-clone
E-H1 restricted with BstNI; lane 2, S. suis-specific clone Sub-B3 restricted with BstNI; lane
3, EF-specific clone E-H1 restricted with AvaII; lane 4, S. suis-specific clone Sub-B3 restricted with
AvaII. The size of products is indicated in base pairs.
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FIG. 6.
Western blot analysis of the culture supernatant of
S. suis strain 10 (lanes 1 and 3) and strain T15 (lanes 2 and 4) with scFvs of clone Sub-B3 (lanes 1 and 2) and with a classical
MAb raised against EF (lanes 3 and 4). Arrowhead, 110-kDa EF protein.
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|
Nucleotide sequence analysis of V regions of selected
MoPhAbs.
The nucleotide sequence of all seven selected MoPhAbs was
determined and analyzed by use of the V-BASE sequence directory described by Tomlinson et al. (24) (Table
3). As expected, based on their identical
restriction patterns, clones E-H1 and Sub-B3, both recognizing EF but
selected on different antigens, used the same V genes. Remarkably,
however, the CDR3 region of both clones was different, both in amino
acid composition and in charge. Both the V genes and the CDR3 sequences
of the other five clones were very variable, as was expected based on
the large differences between the restriction patterns of those clones.
View this table:
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TABLE 3.
Deduced amino acid sequences of heavy-chain CDR3 and
usage of VH and VL genes by seven MoPhAbs
selected against EF or S. suis
|
|
| |
DISCUSSION |
A semisynthetic antibody phage library was used to select
recombinant antibodies directed against surface components of a pathogenic strain of S. suis serotype 2, including EF, a
protein known to be exclusively associated with pathogenic S. suis serotype 2, including EF, a protein known to be exclusively
associated with pathogenic S. suis serotype 2 strains
(28, 29). Using purified EF protein as an antigen, three
unique anti-EF phage antibodies were selected, probably directed
against different linear epitopes of EF. On a Western blot, anti-EF
clone E-H1 recognized EF as efficiently as a hybridoma-derived MAb
raised against EF. This clearly shows that selection on purified EF
protein yielded MoPhAbs specific for EF.
Using intact S. suis serotype 2 cells as an antigen, three
distinct phage antibodies were selected. These MoPhAbs recognized proteins present in the protoplast supernatant fraction of S. suis strain 10, indicating that the MoPhAbs are directed against proteins present on the cell surface of S. suis. The phages
reacted with nondenatured proteins of encapsulated S. suis and a nonencapsulated mutant, indicating that the
MoPhAbs were directed against conformational protein epitopes.
As determined by PCR, only 50% of the selected clones contained a
full-sized insert of 1 kb. Twenty-five percent of the clones showed a
small-sized product of about 0.7 to 0.8 kb, and 25% did not contain an
insert at all. Similar results were described previously by de Bruin et
al. (4). These authors also described the selection of
phages containing small-sized inserts after using the Griffin.1 library. In addition, they showed that these phages were already present in the original library and represented a few phages that did
not obtain a VH region during the construction of the
library. In mixed cultures, phages containing small-sized inserts
tended to overgrow the phages containing full-sized inserts
(4).
With a pathogenic and a nonpathogenic strain in a subtractive selection
procedure, one distinct phage antibody (Sub-B3) was selected that
seemed to be identical to the phage antibody selected on EF-coated
immunotubes (E-H1), as determined by PCR and fingerprint analysis.
Nucleotide sequence analysis confirmed that both clones used the same V
genes. Remarkably, clones E-H1 and Sub-B3 used different CDR3 regions.
Therefore, Sub-B3 may bind to EF with a different affinity or to
another epitope than E-H1. Whether this is true remains to be
determined. Sub-B3 was shown to recognize EF on a Western blot. This
clone was found both after subtractive selection on intact S. suis cells and on protoplast supernatant of S. suis.
Since no EF-recognizing scFvs were selected when the library was panned
on intact S. suis cells without subtraction, it can be
concluded that the subtractive selection procedure was successful.
Clones E-H1 and Sub-B3 seemed to be very dominant in the selection
procedure: after selection on purified EF protein, 92% of the anti-EF
clones were of the E-H1 type; after subtractive selection both on
intact cells and on protoplast supernatant, 100% of the clones were of
the Sub-B3 type. Taken together, this indicates that EF is very capable
of catching specific phage antibodies. This is the first example of the
selection of phage antibodies directed against differentially expressed
proteins on gram-positive bacteria after a subtractive selection
procedure. So far, no clones against MRP or other differentially
expressed proteins were selected, although strain T15 used for
subtraction lacks MRP and EF.
To select phages specific for proteins other than EF exclusively
expressed by the pathogenic S. suis serotype 2 strains, an isogenic mutant of pathogenic strain 10 deficient in the expression of
EF may be helpful.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Bacteriology, Institute of Animal Science and Health, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Phone: 31 320 238403. Fax: 31 320 238153. E-mail: a.degreeff{at}id.wag-ur.nl.
Editor:
W. A. Petri Jr.
| |
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Infection and Immunity, July 2000, p. 3949-3955, Vol. 68, No. 7
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
