Mimotopes selected with a neutralizing antibody against urease B from Helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination.

Abstract

Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the immunogenicity of the selected mimotopes in vivo.
The urease B gene was obtained (GenBank accession No. DQ141576) and the recombinant pGEX-4T-1/UreaseB protein was expressed in Escherichia coli as a 92-kDa recombinant fusion protein with glutathione-S-transferase (GST). Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced immune responses were antigen specific.
The present work demonstrated that phage-displayed mimotopes were accessible to the mouse immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising approach for the development of a vaccine for the diagnosis and treatment of H. pylori infection.

Full text

Page 1

RESEARCH ARTICLEOpen Access
Mimotopes selected with a neutralizing antibody
against urease B from Helicobacter pylori induce
enzyme inhibitory antibodies in mice upon
vaccination
Yan Li1, Yunshan Ning1*, Yundan Wang1, Dandan Peng2, Yaodong Jiang3, Lili Zhang4, Min Long5, Jun Luo5,
Ming Li1*
Abstract
Background: Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric
mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for
the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that
neutralize the enzyme’s activity. We mapped their epitopes by phage display libraries and investigated the
immunogenicity of the selected mimotopes in vivo.
Results: The urease B gene was obtained (GenBank accession No. DQ141576) and the recombinant pGEX-4T-1/
UreaseB protein was expressed in Escherichia coli as a 92-kDa recombinant fusion protein with glutathione-S-
transferase (GST). Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an
immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries
revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.
C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly
recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced
immune responses were antigen specific.
Conclusions: The present work demonstrated that phage-displayed mimotopes were accessible to the mouse
immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising
approach for the development of a vaccine for the diagnosis and treatment of H. pylori infection.
Background
Helicobacter pylori is a helical Gram-negative bacillus
that was originally discovered by Marshall and Warren
in the stomach of patients with gastritis and peptic
ulceration [1]. H. pylori has subsequently been recog-
nised as the major aetiological determinant of various
gastroduodenal diseases. Approximately half of the
world’s population has been estimated to be infected by
H. pylori and harbours the bacterium in their upper
gastrointestinal tract [2]. Even though antibiotic-based
triple therapy is still the most effective treatment for
H. pylori infection, it seems that it is not feasible for
large-scale control of infection, partly because of the
high cost, poor compliance, and emergence of antibio-
tic-resistant strains. Increasing rates of therapeutic treat-
ment failure and high rates of re-infection, together with
low hygiene standards in developing countries have
made it imperative to develop vaccines to control infec-
tion [3].
Currently, most H. pylori vaccines in animal models
have utilised whole-cell preparation of native or recom-
binant proteins from the bacterium, together with
mucosal adjuvant. In general, these vaccines are
designed from a natural form of the pathogen after lysis
or inactivation that differs from natural epitopes [4]. In
response to H. pylori infection, the host triggers
* Correspondence: nys@fimmu.com; mingli2006_2006@126.com
1School of Biotechnology, Southern Medical University, Guangzhou
Dadaobei No.1838, Guangzhou, China, 510515
Full list of author information is available at the end of the article
Li et al. BMC Biotechnology 2010, 10:84
http://www.biomedcentral.com/1472-6750/10/84
© 2010 Li et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

vigorous humoral and cellular immune responses.
Although H. pylori-specific antibodies have been
detected at high titres in inflamed gastric mucosa and in
the serum, the infection can persist and/or never
resolve. This suggests that H. pylori can evade the innate
and adaptive immune responses, and the latter
responses triggered by H. pylori via this natural
approach do not elicit effective immunity [5]. Therefore,
we hypothesise that modified immunity might be
achieved via the use of mimotopes that differ from nat-
ural epitopes. This approach might be able to trigger an
effective immune response that is absent in natural
infections and natural-immunity-based approaches.
Phage display peptide libraries are usually employed to
select epitopes, which mimic the epitopes of natural
proteins recognised by the immune system. Such mimo-
topes are widely used in the development of vaccines
against many diseases [6-8], the design of molecules that
act as agonists or antagonists to many key biomolecules,
and the development of diagnostic reagents [9-12]. It
has been reported that mimotopes induce production of
protective antibodies, and consequently, become candi-
dates for the development of potential vaccines [13,14].
Mimotopes selected from random peptide libraries can
drive an active immune response towards the original
antigen and lead to effective immunity [15-17].
Urease plays a central role in the pathogenesis of
H. pylori infection and promotes colonisation of the sto-
mach and gut. Urease enzymatically hydrolyses urea to
form ammonia and bicarbonate, which neutralise gastro-
intestinal acids and protect the bacteria against the
acidic environment of the stomach. Urease is composed
of two major subunits, urease A and urease B, and the
latter is considered to be an excellent antigen for the
induction of protective immune responses [18,19].
Mucosal vaccination with Lactococcus lactis that
expresses urease B induces the production of IgG in
blood and urease-B-specific faecal IgA against H. pylori
infection [20]. Recently, by transformation of the gene
of urease B into carrot, Zhang et al. have found that
transgenic carrot plants can express the protein of
urease B and effectively induce immune responses in
mice [21]. In addition, immunisation of mice with the
trivalent fusion vaccine that was constructed by geneti-
cally linking heat shock protein A (HspA), H. pylori
adhesion (HpaA) and urease B414 (250-387 aa), has
been shown to protect mice from H. pylori infection
[22]. Thus, urease B appears to be an important target
for the design of a prophylactic vaccine for H. pylori.
Using this technique, we have already obtained the
neutralising mimotopes of Lpp20 and catalase for
H. pylori [23,24]. Whether such mimotopes of urease B
can be exploited to elicit functional antibody responses
against H. pylori has yet to be fully investigated.
In the present study, we applied the phage display
technology to identify the neutralising epitope of
H. pylori urease B with specific monoclonal antibody
(mAb) U001 that has been shown to inhibit significantly
urease activity. Two phage-displayed libraries were
immunoscreened and the selected phage clones were
analysed. The mimotopes were used to immunise
BALB/c mice and the immune responses for the phago-
topes were evaluated.
Results
Expression and purification of recombinant protein
Genomic DNA extracted from H. pylori was used as a
template for PCR. To amplify the DNA that encoded
urease B, which included a major part of the active site,
primers were designed as described in the Methods. An
aliquot of the PCR mixture was analysed by agarose gel
electrophoresis and a DNA fragment of about 1700 bp
was detected, which corresponded to the expected posi-
tion of urease B. The DNA sequence was submitted to
GenBank (accession No. DQ141576). Expression of
recombinant protein urease B-GST was harvested from
Escherichia coli (Top10) and analysed by SDS-PAGE.
We observed a band of 92 kDa, which corresponded to
the predicted molecular size of the fusion protein
(Figure 1). Soluble urease B-GST protein was purified
by glutathione Sepharose (Figure 2) and used as the
immunogen. Western blotting confirmed that sera from
an H. pylori-infected patient specifically recognised the
recombinant urease B protein, whereas the negative sera
could not (Figure 2).
Production and characterisation of anti-urease B mAbs
Five hybridomas, U001-U005, were obtained by cell
fusion. All mAbs were IgG1 (?) and showed high speci-
ficity to urease B. The titre of culture supernatant and
ascites fluid was 1:32-1:64 and 1:32,000-1:128,000,
GST- UreaseBGST- UreaseBGST- UreaseB
200
116
97.4
66.2
45
45
KD
GSTGSTGST
31
21.5
Figure 1 SDS-PAGE analysis of expression of pGEX-4T-1/urease
B in E. coli. 1: pGEX-4T-1 before induction; 2: pGEX-4T-1 after
induction; 3: pGEX-4T-1/urease B before induction; 4: pGEX-4T-1/
urease B after induction; 5: protein marker.
Li et al. BMC Biotechnology 2010, 10:84
http://www.biomedcentral.com/1472-6750/10/84
Page 2 of 12

Page 3

respectively. Triple serial concentrations of the antigen
and each mAb were selected to construct the corre-
sponding curves and to extrapolate the Kaffvalues using
the formula given in the Methods section. The apparent
Kavalues for the five mAbs varied from 1×108/M to
1×1012/M. mAb U001 displayed the highest affinity of
~1012/M (data not shown).
Inhibition and competition studies using increasing
concentrations of inhibitor or competitor mAbs revealed
that four of the five mAbs (U001, U003, U004 and
U005) competed significantly with each other, which
indicated recognition of the same or overlapping epi-
topes. However, U002 did not compete with the other
antibodies and recognised a distinct epitope.
Urease B enzymatic activity was measured and the
inhibitory effects of U001 and U002 were quantitated by
means of an inhibition assay. The inhibition rates were
calculated by comparing mAb-treated and control rates
for Urease B activity (Figure 3). U001 possessed the
strongest inhibitory effect on urease B enzymatic activ-
ity, whereas mAb U002 had a very moderate effect.
Screening of phage display peptide libraries and
characterisation of recombinant phage
To map the neutralising epitopes of urease B that were
recognised by mAb U001, the Ph.D.-12 and Ph.D.-C7C
libraries were screened with this purified mAb. After three
rounds of biopanning, the ratio of output/input increased
(Table 1) and 70 clones (35 each from the Ph.D.-12 and
Ph.D.-C7C libraries) were randomly selected. When tested
by ELISA for their immunoreactivity with mAb U001, 29
dodecapeptide clones and 28 heptapeptide clones gave
positive signals (Figures 4 and 5) with BSA cross-reactivity,
and sequences of 15 clones for each library were tested.
The deduced amino acid sequences of the corresponding
inserts were identified as six different sequences for
the Ph.D.-12 library and five for the Ph.D.-C7C library
(Table 2). The deduced amino acid sequences of selected
phage clones were aligned and analysed. The amino acid
sequences appearing in more than three different selected
clones and matched with urease B protein were grouped
as the consensus residues and +summarised with bold
letters (Table 2). Although these clones did not match the
urease B protein sequence, they shared extensive homol-
ogy with each other and were clustered and classified as
the conformational epitopes of urease B. Peptide
EXXXHDM from the Ph.D.-12 library and EXXXHSM
from the Ph.D.-C7C library showed a good match with the
urease B protein at 347-353 aa. Amino acids E, D, H, M
and S are key residues of the urease B epitope.
Competitive ELISA was performed to confirm the spe-
cificity of the positive clones. The results showed that the
recombinant phage D1 clone (with EHWSHDMFSPGD
sequence) and phage H1 clone (with EKLKHSM
sequence) all inhibited the mAb U001 binding to recom-
binant urease B protein, whereas wild-type M13 phage
had no effect (Figure 6).
Immune response induced by the selected phage clones
To evaluate the immune responses induced by the
selected mimotopes as vaccine candidates, phage clones
ABC
Figure 2 SDS-PAGE analysis of purified urease B and Western
blot analysis of urease B reactivity with patient serum.
(A) Bacteria expressing GST-urease B were lysed by sonication, and
the supernatant was applied to a Glutathione Sepharose 4B column.
Purified urease B was analysed on a 10% SDS-PAGE. 1: purified
urease B; 2: protein marker. (B and C) The gels were transferred to
nitrocellulose membranes and probed with diluted human sera
(1:50) from a normal donor or an H. pylori-infected patient.
Detection was performed with a goat anti-human antibody linked
to HRP (1:10,000), followed by staining with 3-amino-9-
ethylcarbozole. 1: GST; 2: urease B; 3: protein marker.
??
??
??
???
????????????
??????
????????????
??????
????????????
??????
????????????
??????
????
????
???
?
??
????????????????????????????????????????????????????????
?
???????????????????????
?????????????
?????????????
?????????????
Figure 3 Inhibition of urease B enzymatic activity by specific
mAb U001. Urease (25 μl) was incubated with 25 μl mAb U001 or
U002 (equivalent to 0-25 μg) in 96-well microtitre plates overnight
at 4°C, with PBS as a control. On the following day, 50 μl 50 mM
phosphate buffer (pH 6.8) that contained 500 mM urea, 0.02%
phenol red, and 0.1 mM DTT was added to each well. The colour
development was monitored at 550 nm with a microplate reader.
The inhibition ratio was determined by the following equation:
[(activity without Ab - activity with Ab)/(activity without Ab)] × 100.
Li et al. BMC Biotechnology 2010, 10:84
http://www.biomedcentral.com/1472-6750/10/84
Page 3 of 12

Page 4

D1 and H1 were chosen to immunise BALB/c mice by
intraperitoneal injection. Western blot analysis con-
firmed urease B recognition by the antisera (raised by
phage clones and the controls; Figure 7). The sera from
clone D1 (lane 1) and clone H1 (lane 2) reacted specifi-
cally with urease B protein at ~92 kDa, whereas the
pre-immune serum (lane 3), and the serum induced by
wild-type M13 phage (lane 4) or TBS (lane 5) were
negative. Triplicate ELISA measurements demonstrated
significant serum responses against urease B for each
sample and specific antibody responses from clone D1
and H1 were higher than others. The immune response
to wild-type M13 phage control was very low (Figure 8).
Alignment results from MIMOX
Computer-based analysis was performed to define the
consensus amino acid residues of the mimotopes.
Mimox wrapped ClustalW to align a set of mimotope
sequences that were analysed using an embedded ver-
sion of JalView. Figure 9 shows the resultant analysis,
which identified E, H, D and M as the consensus amino
acid residues in the aligned mimotopes.
Discussion
Investigators have demonstrated previously that peptide
mimotopes obtained from phage epitope libraries or by
chemical synthesis can bind antibodies raised against
native structures. In fact, peptide mimotopes have been
found for most, if not all, anti-carbohydrate mAbs [16].
Moreover, peptide epitopes can mimic glycosphingoli-
pids and oligonucleotide structures [15,25]. In the case
of mimotope immunisation, several studies have shown
effective responses in vivo [26,27]. Furthermore, protec-
tive immune responses by mimotope immunisation have
been verified in many infectious diseases [7,28-30].
In the field of vaccine design, the phage-displayed
mimotopes have recently been shown to be possible vac-
cine components that do not necessarily represent the
structural equivalents of the original antigen, but provide
functional images that could replace the original epitopes
Table 1 Enrichment of phage displaying epitope by panning over mAb U001 from ph.D.-12 and Ph.D.-C7C
Library Cyclecoating mAb (mg/L)Washing (TBST)Input phage (pfu)
1.5 × 1011
1.5 × 1011
1.5 × 1011
1.5 × 1011
1.5 × 1011
1.5 × 1011
Output phage (pfu)
8.4 × 103
8.0 × 104
3.0 × 105
7.6 × 103
7.2 × 104
3.5 × 105
Recovery rate (%)
5.6 × 10-6
5.3 × 10-5
2.0 × 10-4
5.4 × 10-6
4.8 × 10-5
2.3 × 10-4
I
II
III
100
10
1
0.1%
0.3%
0.5%
Ph.D.-12
I
II
III
100
10
1
0.1%
0.3%
0.5%
Ph.D.-C7C
?
???
?????? ? ? ?
??????????
?????
????
???
?
???
??????????????????????? ???? ????
??????
????????????
??????
Figure 4 Binding specificity of selected phage from the Ph.D.-12 library. Wells of 96-well microtitre plates were coated with mAb U001 and
BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells and
allowed to bind at 37°C for 1 h. After the unbounded phage was removed, the bound phage was detected by incubation with peroxidase-
labelled murine anti-M13 antibodies. The bound peroxidase was determined by incubation with O-phenylenediamine dihydrochloride and the
reaction was determined in an ELISA reader at OD492. All the assays were carried out in triplicate and the error bars indicate standard deviation.
Li et al. BMC Biotechnology 2010, 10:84
http://www.biomedcentral.com/1472-6750/10/84
Page 4 of 12

Page 5

for vaccine development [31-33]. Application of phage dis-
play technology to elucidate the essential chemical features
of conformational or discontinuous epitopes that are
recognised by some disease-related antibodies can provide
important information about the molecular mechanisms
of these diseases. The selected sets of peptides might also
lead to the future design of more effective reagents for
therapeutic and/or diagnostic purposes. The main advan-
tage of the phage-based approach is that it does not pro-
duce a consensus sequence of a large pool of peptides that
share a common property, but rather yields a list of pep-
tide sequences, each of which is endowed with the same
property. Alignment and comparison of the amino acid
sequences of the selected clones can then determine one
or more consensus sequences. This approach enabled us
to obtain the epitope profile of H. pylori in an efficient
manner. The profiles not only reveal the important roles
of each antigen, but also provide the epitope structures
within the antigenic protein that is involved in the induc-
tion of successful immune responses.
In this study, we first cloned and expressed urease B
of H. pylori, which was then used as an immunogen to
produce mAbs against this recombinant antigen by
hybridoma technology. We obtained two types of mAb
against different epitopes of urease B and observed that
mAb U001 strongly inhibited its enzymatic activity,
whereas mAb U002 did not induce this inhibitory activ-
ity. Therefore, mAb U001 could be beneficial for the
prevention of bacterial growth and attachment to the
gastric mucosa, thus, we identified the neutralising epi-
tope of this mAb via two phage display libraries (Ph.D.-
12 and Ph.D.-C7C). The two mimotopes of urease B
were characterised via affinity screening, binding, com-
petitive inhibition and DNA sequencing. Most clones
from the Ph.D.-12 library share the core EXXXHDM
sequence, therefore, we consider that EXXXHDM might
be a motif of urease B because clone D1 (with this
sequence) competitively inhibited U001 binding to
urease B and induced an immune response in mice.
Clone H1 (EKLKHSM sequence) from the Ph.D.-C7C
library appeared at a high frequency and had the same
function as clone D1. The mimotopes EXXXHDM and
EXXXHSM, which are highly homologous with urease B
residues 347-353 aa are also adjacent to a neutralising
epitope on urease B (321-339 aa), as reported previously
[34]. Li et al. have identified a B-cell-neutralising
?
???
?
???
????? ?????????????????? ????????
??????
????????????
??????
?????
?????
?????
?????
????
????????
????
???
??????
???
Figure 5 Binding specificity of selected phage from the Ph.D.-C7C library. Wells of 96-well microtitre plates were coated with mAb U001
and BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells
and allowed to bind at 37°C for 1 h. After the unbounded phage was removed, the bound phage was detected by incubation with peroxidase-
labelled murine anti-M13 antibodies. The bound peroxidase was determined by incubation with O-phenylenediamine dihydrochloride and the
reaction was determined in an ELISA reader at OD492. All the assays were carried out in triplicate and the error bars indicate standard deviation.
Table 2 The sequences of ph.D.-12 and ph.D.-C7C phage-
displaying peptides from the recombinant phage clones
randomly selected through biopanning
LibraryClone no.Sequence
UreaseB protein
E D T L H D M G I F S I
(347-358)
E H W S H D M F S P G D D1~D3, D5,D6,D8,D9,
D11,D13
D10,D15
D4
D7
D12
D14
E V S L H D M N L A T H
K W L G H D M I M S G T
F N T K H D M Q G D T S
E H N D F P M Y T W R P
T T T H F L A T K F Y K
Ph.D.-12
H1~H2, H4,H6~H9,H11,
H13,H15
H5,H10
H3
H12
H14
E K L K H S M
T K T W Q S M
M S L L G H K
K H G L L S M
M S Q D G H T
Ph.D.-C7C
Li et al. BMC Biotechnology 2010, 10:84
http://www.biomedcentral.com/1472-6750/10/84
Page 5 of 12