Download Contribution of defined amino acid residues to the immunogenicity

FEMS Microbiology Letters 192 (2000) 223^229
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Contribution of de¢ned amino acid residues to the immunogenicity
of recombinant Escherichia coli heat-stable enterotoxin
fusion proteins
Isabelle Batisson, Maurice Der Vartanian *
Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix,
63122 Saint-Gene©s-Champanelle, France
Received 28 June 2000; received in revised form 11 September 2000; accepted 19 September 2000
Abstract
We investigated whether the toxicity-associated receptor-binding domain of the non-immunogenic Escherichia coli heat-stable enterotoxin
(STh) as a fusion with a carrier protein and the inclusion of an appropriate spacer are critical factors for eliciting antibody responses against
the native toxin. The immunological properties of three toxic and one non-toxic fusion proteins, consisting of STh N-terminally joined to
the C-terminus of the major subunit ClpG of E. coli CS31A fimbriae, were compared. In contrast to the non-toxic hybrid STh with glycine
and leucine simultaneously substituted for the receptor-interacting Pro13 and Ala14 amino acids, the toxic chimeras responded by producing
high serum levels of anti-STh antibodies in immunized animals. On the other hand, only the toxic ClpG-STh construct with the natural
peptide 47 KSGPESM53 of Pro-STh as spacer stimulated STh-neutralizing responses against both native toxin and enterotoxigenic live
E. coli cells. Altogether, these findings suggest a close relationship between conformational similarity to the native structure of STh and the
ability to elicit specific antibody responses against STh. ß 2000 Federation of European Microbiological Societies. Published by Elsevier
Science B.V. All rights reserved.
Keywords : Fusion protein; Neutralizing antibody ; Serum reactivity ; STa enterotoxin ; Heat-stable enterotoxin ; Escherichia coli
1. Introduction
Escherichia coli heat-stable enterotoxin (STa), produced
by some enterotoxigenic E. coli (ETEC) strains after colonization of the small intestine, is one of the causative
agents of childhood diarrhea in developing countries, in
addition to traveler's diarrhea [1]. The two types of STa,
which vary only slightly, STp and STh, respectively from
porcine and human strains of ETEC, are typical extracellular peptides consisting of 18 (STp) and 19 (STh) amino
acid residues. STh is synthesized in the cytoplasm as a 72
amino acid precursor consisting of Pre, Pro and mature
STh regions [2,3]. After binding to the transmembrane
guanylyl cyclase C receptor [4] on the intestinal brushborder membrane, mature STh activates guanylyl cyclase
* Corresponding author. Tel. : +33 (73) 624-243;
Fax: +33 (73) 624-581; E-mail: [email protected]
C and the resulting rise in mucosal cGMP mediates the
electrolyte-rich, watery diarrhea characteristic of the illness. The receptor-binding and enterotoxic properties of
STh have been mapped to a highly conserved domain including six cysteine residues forming three intramolecular
disul¢de bonds that are absolutely necessary for toxicity of
the molecule [5]. Because STa is non-immunogenic in its
native form, several di¡erent approaches have been explored to obtain non-toxic immunogenic molecules for
safe vaccine designs, one of which is genetic coupling of
STa to appropriate carrier proteins [6^9]. In general, these
constructions either failed to elicit neutralizing antibodies
or retained some degree of STa-associated toxicity, suggesting that the immunogenic properties of STa are in£uenced by conformation and amino acid residues associated
with toxicity. To investigate this possibility, we constructed toxic and non-toxic fusion proteins in which the
N-terminus of STh was variously fused to the C-terminus
of the major protein subunit ClpG of E. coli CS31A ¢mbriae [10], which was used as a carrier and a provider of a
signal peptide. We compared the immunological properties
of these hybrids after parenteral immunizations of mice
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 3 9 - 0
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and rabbit via di¡erent routes with preparations containing either ClpG-STh proteins or whole recombinant E. coli
cells.
2. Materials and methods
2.1. Bacterial strains and growth conditions
E. coli DH5K (Gibco BRL) was used as the host strain
for all plasmids. The bovine enterotoxigenic E. coli B41
isolate was used as the reference strain producing STp
enterotoxin [11]. Bacteria were grown routinely at 37³C
in Luria^Bertani (LB) broth or on LB agar plates supplemented with appropriate antibiotics.
2.2. Construction of plasmids
Plasmids used in this study are shown in Fig. 1. Plasmids pEH838, pEHSTC22 [12,13] and pEHProSTC28
[12,13] derived from pEH524 [14], a vector carrying the
clp gene cluster that codes for the ClpG export machinery
required for the biogenesis of CS31A ¢mbriae [14] and the
secretion of ClpG-STh fusion proteins by E. coli DH5K
[13]; the wild-type clpG gene from pEH524 was replaced
either by the mutated clpG gene from pHPCO838 [12,13],
giving pEH838, or by the clpG: :sth fusion genes from
pSTC22 and pProSTC28, leading to pEHSTC22 and
pEHProSTC28, respectively. Plasmid pSTCDM1 was
made by inserting a double-stranded oligonucleotide coding for the entire mature STh with glycine and leucine
simultaneously substituted for Pro13 and Ala14 amino
acid residues, between the HpaI and XbaI sites of
pHPCO838. Because of many unsuccessful attempts to
subclone the fusion genes from pSTC17 [12,13] and
pSTCDM1 into pEH524, we trans-complemented
pSTC17 and pSTCDM1 with pDSPH524 [15], which is
pEH524 with clpG deleted, for permitting secretion of fusion proteins. Gene fusions were checked by sequencing.
pended cells on MacConkey lactose agar medium containing the appropriate antibiotic.
2.4. Detection, antigenicity and quanti¢cation of fusion
proteins by ELISA
Detection, antigenicity and quanti¢cation of STh in the
fusions were determined by means of a competitive enzyme-linked immunosorbent assay (ELISA) using the
commercially available assay kit for E. coli STa (COLI
ST EIA) produced by Denka Seiken Co., Ltd., Tokyo,
Japan [16] including microtiter plate-attached STa as solid-phase antigen and peroxidase-conjugated STa-monospeci¢c antibody as secondary antibody [12,13], and by
means of a double antibody sandwich ELISA using mouse
anti-STa monoclonal antibody as coating agent, rabbit
anti-ClpG polyclonal antibody as secondary antibody
and goat anti-rabbit peroxidase-conjugated IgG as tertiary
antibody [13]. Detection and antigenicity were also determined by Western immunoblotting [13].
2.5. Enterotoxicity assay
The suckling mouse enterotoxicity assay [17] was used
to assess any STa-related toxic activity of fusion proteins
and live recombinant bacteria, as described previously
[12,13]. Samples (0.1 ml) were injected intragastrically
into 3 days old Swiss OF1 mice which were killed 3 h after
injection and examined for increased gut-to-carcass weight
ratio as described [17]. A gut/carcass ratio v 0.090 indicated positive toxicity. One mouse unit (MU) was de¢ned
as the enterotoxin activity corresponding to a minimum
e¡ective dose that gave a gut/carcass ratio of 0.090. Determination of minimum e¡ective dose and enterotoxin
titration were performed as described in detail elsewhere
[13]. The minimum e¡ective dose of STa necessary to produce an activity of 1 MU was 8 ng, as determined by using
pure toxin STp (Calbiochem).
2.3. Preparation of cellular and extracellular fractions
2.6. Immunization of animals and antibody response
monitoring
LB broth preculture (1 or 2 ml) containing exponentially growing cells was poured onto LB plates which
were incubated overnight at 37³C in a humid atmosphere
with the agar surface facing up. Bacteria were carefully
harvested by being scraped from the agar surface, and
the ¢nal suspension volume was made up to 2 ml with
phosphate-bu¡ered saline (PBS). After centrifugation at
12 000Ug for 10 min, the resulting supernatants were
stored frozen until used as a source of secreted fusion
proteins. Cell pellets were suspended and washed in PBS,
resuspended in PBS in a ¢nal volume of 2 ml and immediately used as a whole bacterial cell fraction. The bacterial enumeration expressed in CFU (colony-forming units)
ml31 was done by spreading out dilutions of PBS-sus-
One to four New Zealand white rabbits were immunized
intradermally at multiple sites with 15 Wg of fusion protein
and emulsi¢ed in incomplete Freund's adjuvant on days 1,
21, 28, and 35 or intravenously with 5U108 live recombinant bacteria suspended in 0.5 ml PBS on days 1, 8, 15,
and 22. Rabbit antisera were collected 10 days after the
last booster injection, pooled and stored at 320³C until
used.
Five Swiss OF1 mice were intraperitoneally immunized
thrice at 1 week intervals with about 6 Wg of fusion protein
or with 3U108 live recombinant bacteria in PBS. Mouse
antisera were collected by cardiac puncture under general
anesthesia one week after the ¢nal boost, pooled and
stored at 320³C until assayed.
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Anti-STa and anti-ClpG antibody titers in animal antisera were determined using ELISA with microtiter plates
precoated with 1 Wg of puri¢ed synthetic STa [16] or with
1 Wg of puri¢ed ClpG protein [12], respectively. Sera from
immunized rabbits and mice were serially diluted in PBS
(pH 7.2) containing 2% skim milk and 0.5% fetal calf
serum added to the wells and incubated for 2 h at 37³C.
After washing thrice with PBS containing 0.05% Tween 20
then twice with PBS, bound antibody was detected by
adding goat anti-rabbit IgG or goat anti-mouse IgG conjugated to peroxidase (Nordic Immunological Laboratories). After a 20 min enzyme^substrate reaction, the absorbance at 405 nm was read and antibody titers were
expressed as the log10 of the reciprocal dilution. To monitor non-speci¢c reactions, absorbances measured with
sera from animals immunized with the whole bacterial
cell and supernatant fractions from E. coli DH5K
(pDSPH524) strain were subtracted from absorbances obtained with test samples.
2.7. Seroneutralization of toxin activity
For toxin neutralization, the concentration of pure na-
225
tive STa was adjusted with PBS to 25 MU (200 ng) ml31 ,
that of ClpG-STh fusion protein to 40 MU (4 to 18 Wg)
ml31 and that of native STp from E. coli B41 to 40 MU
(320 ng) ml31 , and mixed with an equal volume of sera
pooled from groups of immunized or control animals. For
neutralization of the B41 cell-bound toxicity, density of
bacteria in PBS was adjusted to 1010 CFU ml31 , 2U1010
CFU ml31 , 5U1010 CFU ml31 and 1011 CFU ml31 and
mixed with an equal volume of antiserum. The ¢nal dilution of the sera was 1/2 in all cases. After incubation at
4³C for 18 h, samples (0.1 ml) were administered intragastrically to groups of suckling mice as described above.
3. Results
3.1. Toxicity and antigenicity of fusion proteins
Our recent studies [13] showed that ClpG-STh fusion
proteins from pEHSTC22, pSTC17+pSPH524 and pEHProSTC28 were secreted in the extracellular milieu in a
heat-stable toxic form capable of reacting with antibodies
to either ClpG or STa (Fig. 1). In addition, they desig-
Fig. 1. Structure of fusion proteins. (A) The STh enterotoxin structure. The numbers in boldface above the boxes are the positions of the amino acid
residues relative to the Pre-Pro-STh precursor. Only residues 46^53 of the Pro-STh region are shown. The numbers in boldface below boxes are the positions of the residues relative to the mature STh. The toxic domain in mature STh consists of the sequence from Cys6 to Cys18 which includes the receptor-binding region Cys11 -Asn-Pro-Ala-Cys15 . (B) ClpG-STh fusion proteins. The numbers in normal face above boxes are the positions of the amino
acid residues relative to the signal peptide cleavage site 31/+1 of the ClpG precursor [10]. The additional valine at the C-terminus of ClpG in pEH838
results of the creation of a unique HpaI site in pEH524 allowing in frame insertion of STh. In pEHProSTC28, the inclusion of a glycine residue between the Ser56 and Asn57 residues of the STh precursor minimizes natural cleavage between Pro-STh and mature STh [13]. Data about the secretion,
the enterotoxicity of both ClpG-STh proteins and recombinant whole cells (values represent the speci¢c toxin activity expressed in MU per 1010 CFU)
and the antigenicity of ClpG and STh in the fusion summarize results recently published [13], except for the non-toxic mutant speci¢ed by pSTCDM1
for which data were from this study. Asterisks designate mature STh with glycine and leucine simultaneously substituted for Pro13 and Ala14 residues,
respectively.
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mice with ClpG-STh hybrids (data not shown). Both rabbits and mice immunized with recombinant bacteria developed serum antibodies with an ELISA anti-ClpG titer
close to the serum control in all cases (Fig. 3A), re£ecting
the highly immunogenic nature of ClpG within the fusions. Only the non-toxic strain failed to induce any
anti-STh antibody formation (Fig. 3B), although eliciting
the highest anti-ClpG titer in these animals (Fig. 3A).
Responses to toxic bacteria against STh were identical in
mouse and variable in rabbit, in which maximum titer was
obtained with bacteria carrying pSTC17+pDSPH524 (Fig.
3B). Anti-ClpG titers were always greater than anti-STh
Fig. 2. Immunoblot analysis of the non-toxic fusion protein. Supernatants from agar plate-grown cultures containing 5U109 CFU ml31 of
DH5K (pEH524) (lane 1) and 5U1010 CFU ml31 of DH5K (pSTCDM1+pDSPH524) (lanes 2 and 3) were mixed with an equal volume of 2ULaemmli bu¡er containing L-mercaptoethanol, boiled and
loaded onto 0.1% SDS^15% polyacrylamide gels. After electrophoresis,
proteins were electrotransferred to nitrocellulose membranes and incubated either with ClpG-speci¢c antiserum (lanes 1 and 2) or with the
STa-speci¢c monoclonal antibody 11C (lane 3) as previously described
[13]. Horseradish peroxidase-labeled goat anti-rabbit IgG (lanes 1 and
2) or horseradish peroxidase-labeled goat anti-mouse IgG were used as
secondary antibodies (lane 3). Membranes were then developed with
H2 O2 -K-chloronapthol as substrate. The arrow at left indicates the native ClpG protein.
nated the pEHProSTC28-speci¢ed hybrid as the highest
toxic chimera (Fig. 1). In contrast, the secreted fusion
protein from pSTCDM1+pDSPH524 with glycine and leucine simultaneously substituted for Pro13 and Ala14 residues of STh revealed no toxin activity (Fig. 1). As assessed
by immunoblotting analysis (Fig. 2), this non-toxic chimera reacted with anti-ClpG (lane 2) but not with antiSTa antibodies (lane 3), indicating the lack of STh immunogenicity. In addition, it was detected as a single protein
band migrating more slowly than native ClpG (lane 1),
consistent with the presence of the additional STh sequence. Most likely, this single band represents the fulllength hybrid molecule. Taken together, these results suggest that Pro13 and Ala14 amino acid residues are required
not only for toxicity but also for proper anti-STa antibody
binding and that the lack of toxicity and STh immunogenicity for the fusion protein expressed from pSTCDM1
cannot be attributed to cleavage and loss of the enterotoxin sequence.
3.2. Immunogenicity of fusion proteins
To evaluate the immunogenic potential of the fusion
proteins we compared the anti-ClpG and anti-STh antibody responses following intravenous immunization of
rabbits and intraperitoneal immunization of mice with
live recombinant bacteria (Fig. 3) or after intradermal immunization of rabbits and intraperitoneal immunization of
Fig. 3. ELISA of sera from animals immunized with live bacteria harboring the indicated plasmids for the ability to recognize both ClpG (A)
and STh (B). Rabbits and mice were immunized intravenously (IV) and
intraperitoneally (IP), respectively. E. coli DH5K (pEH524) was used as
negative control and sera from animals immunized with this strain were
used as positive and negative controls for reactivity with ClpG and
STh, respectively. See Section 2 for details. Error bars represent assayto-assay variations.
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227
between immunogenicity and toxicity and that expression
of immunogenicity by the toxic fusion proteins is independent on the nature of the spacer joining ClpG to STh.
3.3. Serum neutralization of STa toxicity
Sera from rabbit and mice variously immunized with
fusion proteins were mixed with either native STa or toxic
ClpG-STh chimeras or native STp from E. coli B41 and
the enterotoxicity of the mixture was determined by the
suckling mouse assay (Fig. 4). Only the antiserum of rabbit intradermally boosted with ClpG-STh protein from
pEHProSTC28 in the presence of incomplete Freund's adjuvant exhibited toxin-neutralizing activity against native
STa, native STp and ClpG-STh proteins (Fig. 4A), and
also against up to 2.5U109 whole E. coli B41 cells (Fig.
4B), explaining why only neutralization results relative to
this antiserum, named SL28, were shown. It is probable
that SL28 recognizes conformational epitopes associated
with toxicity which are common to STa, STp and ClpGSTh from pEHProSTC28. Altogether, these ¢ndings indicate that the route of immunization, the choice of animal,
the origin of antigen (free or cell-associated), the toxicity
state of immunogen, and the inclusion of an appropriate
spacer between the carrier protein and the toxin are important considerations for the development of neutralizing
antibodies.
4. Discussion
Fig. 4. Neutralization of toxin activity in the suckling mouse assay by
the serum SL28 from rabbits immunized intradermally with ClpG-STh
from pEHSTC28 in the presence of incomplete Freund's adjuvant. (A)
A selected dose of native STa toxin, toxic ClpG-STh fusion proteins
and native STp toxin from E. coli B41 was mixed (or not) with SL28.
The ¢nal dose administered per infant mouse was 1.25 MU for STa and
2 MU for the two other toxic samples. Serum from a rabbit immunized
with the culture supernatant of DH5K (pEH524) was used as a negative
control. (B) Various quantities of live E. coli B41 cells were mixed (or
not) with SL28. The suckling mouse assay for STa enterotoxicity and
the signi¢cance for mouse units (MU) are described in Section 2. Gut/
carcass weight ratios of v0.09 (dotted line) are considered positive for
toxicity and, therefore, negative for neutralization. Each bar represents
the mean gut/carcass ratio and the standard errors of a group of four
to 11 mice. *, P 6 0.05; **, P 6 0.01; ***, P 6 0.005.
titers and responses against both ClpG and STh were generally higher in rabbit than in mouse (Fig. 3). Immunizations with fusion proteins led to comparable results, except
that the detoxi¢ed antigen elicited low (in rabbit sera) or
not detectable (in mouse sera) anti-ClpG titers and that
the anti-STh response to toxic samples was, overall, lower
(data not shown). All toxic proteins triggered similar antiSTh responses in mouse, but not in rabbit, in which response to the chimeric protein from pEHProSTC28 was
higher. These results indicate that there is a correlation
Studies on the immunological response to the E. coli
heat-stable enterotoxin STa have been hampered by its
small size, toxicity, poor antigenicity and lack of immunogenicity. Sanchez et al. [8] have reported the construction
of a secretable detoxi¢ed STa fusion protein capable of
eliciting the production of antibodies which could recognize native STa in an ELISA. However, detoxi¢cation
resulted from a single substitution of a disul¢de-linked
cysteine, which drastically disrupted the native three-dimensional structure of the toxin. In addition, whether
these antibodies neutralized the biological activity of STa
was not assessed. This is an important consideration, since
removal of amino acid residues associated with toxicity,
may, in such a small molecule, render the molecule incapable of inducing neutralizing antibodies. On the other
hand, Cardenas and Clements [18] have described a nontoxic, antigenic, and immunogenic recombinant STa capable of inducing antibodies with neutralizing activity when
delivered by a bacterial vector. However, neutralization
occurred in the absence of detectable ELISA reactivity
against STa, which is di¤cult to explain. In addition,
this STa chimera was rendered non-toxic merely by fusion
with the carrier but not by modi¢cation of some amino
acids, making the in£uence of speci¢c residues on the immunogenic properties of STa misunderstood.
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We have previously shown that the three toxic ClpGSTh fusions used in this study were secreted in the extracellular milieu in an antigenic disul¢de-bonded form [13],
indicating that spatial conformation of STh in the fusion
was close to that of the native toxin, which must be very
£exible since the STh moiety in the di¡erent hybrids is
certainly not quite the same. Here we showed, in addition,
that they were highly immunogenic. Indeed, sera from
animals immunized via di¡erent routes with either chimeric molecules or whole recombinant E. coli cells contained
antibodies with high titers that were able to recognize both
the carrier protein ClpG and the native toxin in vitro. In
disagreement with other investigators [6,18], we found that
inclusion of an appropriate linker between the carrier and
STa moieties was not absolutely required for expression of
STa antigenicity and production of antibodies capable of
interacting with native toxin. By contrast, maximum toxicity and ability of the fusion protein to elicit serum-neutralizing antibodies against both native toxin and enterotoxigenic live E. coli cells were obtained only after
inclusion of the peptide 47 KSGPESM53 of Pro-STh as a
spacer between the ClpG and STh domains. Given the
involvement of the Pro region in the maturation pathway
of STa [19], it is conceivable that this natural linker favors
folding of the STh moiety in a more native way. Therefore, it can be assumed that the structure which best resembles the conformation of the native toxin within a
fusion protein would be the most e¡ective for eliciting
neutralizing antibodies.
To investigate whether alteration of some amino acid
residues that interact directly with the toxin receptor guanylyl cyclase C [20,21] in£uenced the immunogenicity of
the chimeric STh, we substituted simultaneously glycine
and leucine for Pro13 and Ala14 residues in STh. Because
single substitution of Pro13 by glycine or Ala14 by leucine
resulted in marked reduction in their ability to bind to
guanylyl cyclase C and in the toxic activity without, however, completely abolishing them [20^22], double mutation
was performed. Unlike the usual disul¢de-linked cysteine
residues [8], replacement of Pro13 and Ala14 by glycine and
leucine residues does not cause structural changes [20], and
thus does not drastically disrupt conformational epitopes
which are common to native toxin. In contrast to toxic
ClpG-STh fusions, and in spite of its ability to be secreted
normally and to trigger high antibody responses against
ClpG, the non-toxic mutant was incapable of eliciting serum antibodies that could either recognize STh antigenically or neutralize the biological activity of STh. Thus, its
lack of toxicity corresponded to its inability to develop
antibodies against the STh moiety, suggesting either that
substitution of Pro13 and Ala14 induced some changes in
toxicity-associated STh conformation, thus a¡ecting immunogenicity or that these amino acid residues belong
to a STh epitope required for the development of antibodies directed against STh.
Acknowledgements
This study was supported by grants from the Conseil
Rëgional Auvergne (France) and the Institut National de
la Recherche Agronomique (France). T. Takeda is gratefully acknowledged for donation of the mouse anti-STa
monoclonal antibody 11C. We thank M. Chavarot, C.
De Martrin, A. Garrivier, B. Ja¡eux and G. Vert for technical assistance and S. Dutilloy and F. Magne for secretarial assistance.
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