The outer membrane protein, LamB (maltoporin), is a versatile

Vaccine 32 (2014) 809–815
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Vaccine
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The outer membrane protein, LamB (maltoporin), is a versatile
vaccine candidate among the Vibrio species
Jingsheng Lun 1 , Changyan Xia 1 , Chuanfei Yuan, Yueling Zhang, Mingqi Zhong,
Tongwang Huang, Zhong Hu ∗
Department of Biology, School of Science, Shantou University, Shantou, Guangdong 515063, China
a b s t r a c t
i n f o
Article history:
Received 9 September 2013
Received in revised form 7 December 2013
Accepted 12 December 2013
Available online 29 December 2013
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Maltoporin (LamB) is a family of outer membrane proteins. There has been no report of immunological
characteristics of LamB in the Vibrio species so far. In this study, lamB genes from eight Vibrio strains were
cloned and sequenced. The bioinformatics analysis indicated that sequence similarities of LamB proteins
were ranged from 46.7% to 81.1%. Further, the result showed that their antigenic epitopes were highly
conserved implying that LamB might be a shared antigen among Vibrios. The Western blot of rabbit sera
against recombinant LamB from V. alginolyticus ATCC 33787 with cell lysate of 18 Vibrio strains showed
cross-recognition. Bands observed on cell lysate of Vibrio strains immunoblotted with the anti-LamB
sera ranged between 40 and 49 kDa. The Whole-cell ELISA assay further conﬁrmed that the antisera of
recombinant LamB recognized the tested Vibrio strains indicating the surface-exposed of LamB. Finally,
the cross-protective property of recombinant LamB was evaluated through vaccination and subsequent
challenge with heterogeneous virulent Vibrio strains in zebraﬁsh. Recorded relative percent survival (RPS)
of the vaccinated group varied from 54.1% to 77.8%, showing that zebraﬁsh were protected from Vibrio
infection after immunization with LamB protein. The cumulative evidences in this study suggested that
LamB was a conserved antigen among tested Vibrio species and might be a potentially versatile vaccine
candidate for the prevention of Vibriosis.
© 2013 Elsevier Ltd. All rights reserved.
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1. Introduction
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Keywords:
Vibrio
Outer membrane protein
LamB (maltoporin)
Versatile vaccine
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Vibriosis is one of the most serious infectious diseases in ﬁsh
and shellﬁsh [1–4]. However, due to the diversity of pathogens and
their complicated serotypes, the progress in vaccine development
against Vibriosis has been slow [5–8]. Therefore, development
of versatile Vibriosis vaccines that can ﬁght against as many
pathogens as possible is in an urgent need. Outer membrane proteins (OMPs) as potential vaccine candidates had been reported
recently, including OmpK of Vibrio harveyi [9], OmpA of Edwardsiella tarda [10] and four OMPs (OmpW, OmpV, OmpU and OmpK)
of V. parahaemolyticus [11]. Further studies showed that OmpK was
a conserved protective antigen and might be a potentially versatile vaccine candidate for the prevention of infections caused
by V. harveyi, V. alginolyticus and V. parahaemolyticus [12]. LamB
proteins (or maltoporins) are a family of OMPs. It forms a betabarrel composed of three monomers and ensures the transport
of maltose and maltodextrin in Gram-negative bacteria [13,14].
∗ Corresponding author. Tel.: +86 754 8650 2081; fax: +86 754 8290 2767.
E-mail addresses: [email protected], [email protected] (Z. Hu).
1
These authors contributed equally to this study.
Khushiramani et al. [15] reported that recombinant Aeromonas
hydrophila outer membrane protein 48, which belongs to the maltoporin group of porins, induces a protective immune response
against A. hydrophila and E. tarda. There has been no other report
of immunological characteristics of LamB so far. Interestingly, the
polyclonal antibody raised against the recombinant LamB from V.
alginolyticus ATCC33787 recognized LamB homologues from other
Vibrio species in the Western blot and Whole cell ELISA analysis. In order to evaluate the immunogenicity and cross-protective
property of recombinant LamB, zebraﬁsh were vaccinated with the
puriﬁed recombinant LamB and then challenged with heterogeneous virulent Vibrio strains.
2. Materials and methods
2.1. Bacterial strains
Several Vibrio species, commonly considered as pathogens, were
used in this study. V. parahaemolyticus ATCC 17802, V. alginolyticus ATCC 33787, V. mimicus ATCC 33653, V. vulniﬁcus ATCC 27562,
V. ﬂurialis ATCC 33810, V. furnissii ATCC 33813, V. proteolyticus
ATCC 15338, V. natriegens ATCC 14048 and V. pelagius ATCC 25916
were purchased from American Type Culture Collection (ATCC,
0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.12.035
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J. Lun et al. / Vaccine 32 (2014) 809–815
Manassas, VA, USA). V. anguillarum NBRC 13266, V. harveyi NBRC
15634, V. damsel NBRC 15633, V. ﬁscheri NBRC 101058, V. splendidus NBRC 101061, V. metschnikovii NBRC 103153 and V. campbellii
NBRC 15631 were purchased from NITE Biological Resource Center
(NBRC, Japan). Another two testing and quality control strains, V.
cholera non-01 Vb0 and V. parahaemolyticus VPL4-90, were purchased from Huankai Microbial (HKM, Guangzhou, China). The
Vibrio strains were cultured in Luria-Bertani broth (LB) at 28 ◦ C.
Another 4 non Vibrio strains including A. hydrophila NBRC 12658,
Escherichia coli DH5␣, E. coli BL21 (DE3), Staphyloccocus aureus and
Bacillus subtilis were cultured in LB at 37 ◦ C.
the protocol. Then the puriﬁed recombinant proteins were determined by the mass spectrometric analysis (MALDI-TOF/TOF). All
protein samples were characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) which contained
5% stacking gel and 12% separating gel. Antisera against the puriﬁed
recombinant LamB was raised by immunizing a New Zealand rabbit. Brieﬂy, 500 ␮g puriﬁed LamB protein emulsiﬁed with Freund’s
complete adjuvant was injected subcutaneously into the rabbit. The
ﬁrst injection was followed by two other injections with the LamB
emulsiﬁed Freund’s incomplete adjuvant at intervals of one week.
The antisera were collected and stored at −80 ◦ C until used.
2.2. Cloning and sequence analysis of lamB genes of the Vibrio
species
2.5. Western blot analysis
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Bacteria cells of the tested strains were harvested by centrifugation at 7000 × g for 10 min. After washing with PBS, the
cells were suspended with PBS, and lysed by sonication on ice.
The concentration of total proteins was determined using the
Bradford method. Protein samples of the tested strains (100 ␮g
total proteins) were electrophoresed by SDS-PAGE (5% stacking
gel and 12% separating gel), and then transferred onto 0.45 ␮m
polyvinylidene ﬂuoride (PVDF) membranes (Merck, Germany). The
membranes were washed with PBS containing 0.1% Tween-20
(PBS-T). The remaining binding sites were blocked with PBS-T
containing 5% skim milk (PBS-TS) at 37 ◦ C for 2 h. After three
washes with PBS-T, the membranes were incubated for 1 h at 37 ◦ C
with the anti-LamB sera (1:2000 diluted in PBS-TS). After washing for three times with PBS-T, the membranes were incubated
with horseradish perosidase (HRP) conjugated goat-anti-rabbit IgG
(Beyotime, Jiangsu, China) (1:2000 diluted in PBS-TS) at 37 ◦ C
for 1 h. After another three washing steps, the membranes were
visualized with 3,3 -diaminobenzidine (DAB) substrate and the
reaction was stopped by washing the membrane with distilled
water.
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The LamB protein sequence of V. parahaemolyticus RIMD
2210633 was utilized as the template for the BLAST search. Then the
nucleotide sequences, from the LamB homologues found in other
Vibrio species, were used for multiple alignment analysis and to
generate the degenerated LamB primers. The primers, lamB-f: 5 ATG AAA AAA GTA AGT SNY ATT GCA G-3 and lamB-r: 5 -TTA CCA
CCA AGC TTC NRC TTG -3 , were designed to amplify the complete
coding sequence of lamB genes from the genomic DNA of the Vibrio
species. The PCR ampliﬁcation was carried out in a thermal cycler
as follows: 5 min at 95 ◦ C; 30 cycles of 30 s at 95 ◦ C, 45 s at 58 ◦ C,
1 min at 72 ◦ C; then 10 min at 72 ◦ C for further extension. PCR products were ligated to pMD19-T vector (TaKaRa, Dalian, China) and
transformed into E. coli DH5␣. The recombinant plasmids pMD19T-lamB were conﬁrmed by PCR and sequenced at least twice by
Beijing Genomics Institute (BGI, Beijing, China).
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2.3. Database searching and bioinformatics analysis
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Multiple alignment of deduced protein sequences of lamB genes
were analyzed using ClustalX, and the phylogenic tree was constructed using Neighbor-Joining of MEGA (bootstrap = 1000 times).
T cell and B cell epitope prediction were performed by the online
software CTLpred (http://www.imtech.res.in/raghava/ctlpred) and
Antigenic Index of Protean (DNAStar 5.0), respectively. Homology searches of lamB genes were performed using BLASTn
and BLASTp in National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). The cleavage site for
the signal peptide was predicted by the SignalP 3.0 Server
(http://www.cbs.dtu.dk/services/SignalP).
2.4. Expression, puriﬁcation and antisera preparation of
recombinant LamB
The primers, LamBeF: 5 -CGCGGATCCGCAGTGGATTTTAAC3 with Bam HI restriction digest site and LamBeR: 5 CCGCTCGAGATTCGGCTTTTACCAC-3 with Xho I restriction digest
site, were designed to amplify the lamB gene of V. alginolyticus ATCC
33787 and to remove its signal peptide coding sequence. The PCR
products were cloned into the expression vector pET-32a (Novagen,
Germany) which was available for producing cleavable 6×His-Tag
fusion proteins for detection and puriﬁcation. The recombinant
plasmids, conﬁrmed by PCR, restriction enzyme digestion and DNA
sequencing, were transformed into E. coli BL21 (DE3) (Novagen,
Germany). The E. coli BL21 (DE3) transformed with pET-32a-lamB
was cultured in LB (with 100 mg/mL ampicillin) at 37 ◦ C. Protein expressions were induced with 1 mmol/L IPTG at cell density
(OD600 ) of 0.6. Bacteria cells were harvested by centrifugation at
7000 × g for 10 min. The cells were suspended with phosphatebuffered saline (PBS, pH 7.4), and lysed by sonication on ice. The
6 × His-Tag fusion proteins were puriﬁed with a Ni2+ -nitriloacetate
(NTA) super ﬂow resin column (QIAGEN, Germany) according to
2.6. Whole-cell enzyme-linked immunosorbent assay (ELISA)
Bacteria were cultured in LB to OD600 of 0.6, washed in PBS,
resuspended in PBS (with 0.5% formalin) and incubated overnight
at 4 ◦ C. After that, bacteria cells were washed three times and resuspended with PBS. The 96-well plates (Corning costar, USA) were
coated overnight at 4 ◦ C with 100 ␮L (105 CFU) bacterial suspension per well. After wash the plate three times with PBS-T, the
remaining binding sites on the plates were blocked 1 h at 37 ◦ C with
PBS-TS. Equal volumes of anti-LamB sera (1:1000 diluted in PBS-TS)
were added in triplicate to the wells and incubated 2 h at 37 ◦ C. The
rabbit sera collected before immunization was used as negative
control. After washing three times with PBS-T, the goat-anti-rabbit
IgG conjugated with HRP (1:1000 diluted in PBS-TS) was added
and incubated for another 1 h at 37 ◦ C. After another three washing
steps, the color was developed with 3,3 ,5,5 -tetramethylbenzidine
(TMB) for 15 min at 37 ◦ C and the reaction was stopped by adding
50 ␮L 2 mol/L H2 SO4 . The absorbance of each well was read at
450 nm by a microplate reader (Thermo Scientiﬁc Multiskan MK3,
USA). The result was considered as positive when the absorbance
of the tested group was at least twice as high as compared to the
negative control group.
2.7. Fish vaccination
The puriﬁed recombinant LamB was resuspended in PBS to
a concentration of 500 ␮g/mL and used as antigen to vaccinate
zebraﬁsh (Danio reiro, ∼5 cm). The ﬁsh in the vaccination group
were each injected i.p. with 10 ␮L of puriﬁed LamB, while the ﬁsh
in the control group were each injected i.p. with equal volume of
aseptic PBS. Two weeks later, the ﬁsh in the vaccination group
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V. harveyi NBRC 15634 (JF747211)
100
V. parahaemolyticus RIMD 2210633 (NP_801154)
V. parahaemolyticus VPL4-90 (FJ904926)
85
V. alginolyticus 12G01 (EAS78178)
99
95
Vibrio sp. Ex25 (ZP_04920785)
V. alginolyticus ATCC 33787 (JF747203)
92
V. harveyi HY01 (ZP_01986109)
V. parahaemolyticus ATCC 17802 (JF747207)
63
95
100 V. mimicus VM573 (ZP_05717266)
100
100
811
V. mimicus ATCC 33653 (JF747206)
V. cholerae RC385 (ZP_06941856)
49
V. cholera Vb0 (JF747210)
V. furnissii NCTC 11218 (ADT89237)
100
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V. furnissii ATCC 33813 (JF747208)
100
V. flurialis ATCC 33810 (JF747209)
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V. splendidus 12B01 (ZP_00993172)
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Fig. 1. A phylogenetic tree with bootstrap values based on the deduced amino acid sequences of LamB. The tree was constructed using a neighbor-joining method. LamB
cloned in this study is shown in bold.
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were boosted with 5 ␮g of puriﬁed LamB, while the ﬁsh in the
control group were boosted with PBS. Sera were collected from
vaccinated and unvaccinated ﬁsh (ﬁve at each time point) at different times post-vaccination. Antibody titer was determined from
the immunized ﬁsh by sandwich ELISA. Brieﬂy, the sera were serially diluted in carbonate/bicarbonate buffer, pH7.4 (1:1, v/v) and
added in triplicate to wells of the plates. The plates were incubated overnight at 4 ◦ C. The remaining binding sites were blocked
1 h at 37 ◦ C with PBS-TS. Then 100 ␮L puriﬁed LamB (0.5 ␮g/well)
was added to the plates. After incubation at 37 ◦ C for 2 h and
washing three times with PBS-T, rabbit anti-LamB sera (1:1000
diluted in PBS-TS) were added to the plates. The plates were
incubated and washed as above. HRP conjugated with goat-antirabbit IgG (1:1000 diluted in PBS-TS) was added and incubated
for another 1 h at 37 ◦ C. Finally reaction was achieved as above.
The positive readings were deﬁned as above and antibody titer
was presented as the highest dilution that gave rise to a positive
reading.
2.8. Experimental challenge and calculation of relative percent
survival (RPS)
At the 15th day post-boost, thirty ﬁsh from the vaccination
group or control group were challenged using ten times their lethal
dose (LD50 ) of ﬁve Vibrio strains including V. alginolyticus ATCC
33787 (5.6 × 107 CFU), V. parahaemolyticus VPL4-90 (5.6 × 108 CFU),
V. parahaemolyticus ATCC 17802 (3.2 × 108 CFU), V. harveyi NBRC
15634 (3.2 × 108 CFU) and V. mimicus ATCC 33653 (3.2 × 108 CFU),
respectively. The Vibrio strains were previously cultured in LB at
28 ◦ C to OD600 of 0.6. The cultures were collected, washed and resuspended in aseptic PBS then counted with a blood counting chamber.
Each ﬁsh was injected i.p. with 10 ␮L bacterial suspension. After
the injection, each group of ﬁsh was then maintained in a separate
tank at 28 ◦ C for 5 days. Dead ﬁsh were collected daily and livers
were streaked on thiosulfate citrate bile salts sucrose (TCBS) agar
plate (HKM, Guangzhou, China) to conﬁrm the presence of Vibrios.
Relative percentage survival (RPS) was calculated from the cumulative mortalities using the following equation: RPS = 1 − (mortality
of vaccinated ﬁsh/mortality of unvaccinated control ﬁsh) × 100%.
Statistical analysis was performed by Student’s t-test.
3. Results
3.1. Clone and bioinformatic analysis of LamB
To investigate the LamB’s characters, lamB genes of Vibrios were
cloned and then subjected to bioinformatic analysis. From eight of
the eighteen Vibrio strains, the lamB genes were successfully cloned
by the use of the degenerated primers, and their sequences were
submitted to the GenBank. Sequence analysis showed that lamB
genes contained an open reading frame (ORF) of 1185–1278 bp
corresponding to a polypeptide of 394–425 amino acids with a
calculated molecular weight of 42.8–46.4 kDa (data not shown).
Comparison of the deduced amino acid sequences of LamB revealed
that their similarities were 46.7–81.1% between V. alginolyticus
ATCC 33787 and other Vibrio species. The phylogenetic tree, based
upon their similarities, showed the inferred evolutionary relationships among tested Vibrio species (Fig. 1). Furthermore, the epitopes
of LamB of V. alginolyticus ATCC 33787 were predicted and utilized as primers for BLAST analysis in other tested Vibrios. As a
result, homologous epitopes were found in LamB and shared by
different species of Vibrios (Fig. 2). Six T cell epitopes FAVDFNGYM,
DIHITDFYF, GFNQTVFQY, GFRLINWGV, WEMGHQLAY and AMGDSFWAR were recognized by T cell. Two epitopes MYKWNDTMRTV
and DSFWARPELRVY were recognized by B cell. The epitope WAGKTYYQRKDIHI was recognized by both T cell and B cell. These
results indicated that the lamB gene not only had high degree of
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3.2. Expression, puriﬁcation and antisera preparation of
recombinant LamB
the fusion protein, including 17.7 kDa His-Tag in N-terminal and
43.7 kDa LamB protein, was consistent with the predicted result.
The recombinant LamB proteins puriﬁed by Ni-NTA afﬁnity
chromatography were characterized by SDS-PAGE (Fig. 3B) and
conﬁrmed by the mass-spectrography (data not shown). The antisera against puriﬁed recombinant LamB, which were raised by
immunizing rabbit and antibody titers of the antisera were more
than 1:40000 (data not shown). This result demonstrated that the
antisera were qualiﬁed for subsequently use in Western bolt and
Whole-cell ELISA.
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similarities in deduced amino acid sequences among tested Vibrio
strains, but also shared homologous epitopes.
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Fig. 2. Homologous epitopes of LamB found in eight different Vibrio strains. (A) The predicted T cell epitopes. (B) The predicted B cell epitopes. (C) Epitope recognized by
both B and T cells. The epitopes of V. alginolyticus ATCC 33787 LamB were predicted. Then the predicted epitopes were utilized as templates for multiple alignment analysis
in other tested Vibrio strains. Identical amino acid residues were boxed in black, amino acid residues with similar side chains were boxed in gray, and amino acid residues
with different side chains were boxed in white.
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To investigate the immunological properties of LamB, lamB gene
was cloned and expressed in E. coli BL21 (DE3). The lamB gene of
V. alginolyticus ATCC 33787 contains a 1221 bp ORF with the ﬁrst
20 amino acid residues functioning as the signal peptide. The signal peptide truncated lamB gene was cloned and ligated to the
expression plasmid pET-32a. A prominent protein band in SDSPAGE gel corresponding to 60 kDa was detected in E. coli harboring
pET-32a-lamB induced with IPTG, which was not found when IPTG
was not added into the media (Fig. 3A). The molecular weight of
Fig. 3. Expression and puriﬁcation of recombinant LamB. Proﬁle of all protein samples were analyzed by a 12% SDS-PAGE gel. M indicated protein molecular weight
marker. (A) Total proteins of BL21 transformed with pET-32a-lamB. Lane 1, BL21
transformed with pET-32a-lamB induced with 1 mmol/L IPTG; lane 2, BL21 transformed with pET-32a-lamB without IPTG induction. (B) The recombinant protein
LamB puriﬁed with Ni-NTA. Lane 1, puriﬁed recombinant LamB.
3.3. Western blot analysis
Antisera of recombinant LamB were used as the primary antibodies on the Western blot. The recombinant LamB and its antisera
were derived from V. alginolyticus ATCC 33787. The lane of V. alginolyticus ATCC 33787, which was known to produce a 43.7 kDa
LamB was taken as a positive control in Western bolt. As shown
Fig. 4. Western blot for investigation of cross-reaction properties of anti-LamB sera
against different bacteria. Total protein of tested strains were used as antigens and
antisera of recombinant LamB as the primary antibodies. For the antisera of recombinant LamB was V. alginolyticus-derived, the lane of V. alginolyticus ATCC 33787
which known as producing a 43.7 kDa LamB was taken as positive control in this
study. Assays were performed in duplicate.
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Table 1
The Whole-cell ELISA analysis of recombinant protein anti-LamB sera with Vibrios.
Tested strains
Test results
V. alginolyticus ATCC 33787
V. parahaemolyticus VPL4-90
V. parahaemolyticus ATCC 17802
V. harveyi NBRC 15634
V. mimicus ATCC 33653
V. cholerae Vb0
V. furnissii ATCC 33813
V. ﬂurialis ATCC 33810
+++
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++
++
+
++
3.5. Cross-protective analysis of LamB recombinant protein
To examine the cross-protective potential of LamB, the recombinant protein was used to immunize zebraﬁsh. Antibody response of
the ﬁsh was determined by sandwich ELISA, and the result showed
that the antibody titer was over 1:2000 at two weeks after the
second booster vaccination (data not shown). Table 2 showed the
cumulative mortalities of zebraﬁsh in the challenge test with ﬁve
important Vibrio pathogens of aquatic animals. The results showed
that cumulative mortalities of zebraﬁsh in LamB vaccinated group
were signiﬁcantly lower than that of control group (P < 0.05). The
recorded RPSs ranged from 54.1% to 77.8%. The result indicated that
LamB was a cross-protective antigen among tested Vibrio species
and was a potentially versatile vaccine candidate for the prevention of Vibriosis, including V. alginolyticus, V. parahaemolyticus, V.
harveyi and V. mimicus. Interestingly, the cross-protective capacity
of recombinant LamB was correlative with the degree of identity
of deduced amino acid sequences of LamB between challenging
strain and V. alginolyticus ATCC 33787. In addition, it was reported
that zebraﬁsh is sensitive to Vibrios infection, although it is a kind of
freshwater ﬁsh [16–18]. In challenge trials, the dead ﬁshes showed
typically clinical symptom of Vibriosis, and no other pathogen but
Vibrio species was isolated from dead ﬁshes.
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+++ indicated positive (P)/negative control (N) ≥ 5.0; ++ indicated P/N ≥ 3.0; + indicated P/N ≥ 2.1; indicated P/N < 2.1.
4. Discussion
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Due to the diversity of pathogens and their complicated
serotypes, the development of a versatile vaccine that provides
heterologous protection for Vibriosis has been a concern. Outer
membrane proteins (OMPs) are highly immunogenic bacterial
components due to their exposed epitopes on the cell surface.
Recently, much attention has been focused on the study of immunogenic cross-reaction among OMPs. Li et al. [19,20] developed a
new approach combining heterogeneous antisera-based immunoproteomics with a bacterial challenge test after immunization to
identify broadly cross-protective antigens among different species,
genera and families of bacteria. Antigenic cross-reactivity of OMPs
has been investigated in several species of bacterial pathogens.
LamB proteins (or maltoporins) are a family of OMPs, and there has
been no report of immunological characteristics of LamB in Vibrio
species so far. In this study, the lamB genes of eight Vibrio strains
were cloned and sequenced. The similarities of deduced amino acid
sequences of LamB ranged from 46.7% to 81.1% (Fig. 1). Furthermore, highly conserved epitopes for both B and T cells were found
in the tested Vibrios (Fig. 2). The result implies that LamB may be a
shared antigen among Vibrio species.
To conﬁrm this hypothesis, immunological techniques based
on rabbit sera against the recombinant LamB from V. alginolyticus ATCC 33787 were applied. The Western blot analysis showed
that the antisera of recombinant LamB recognized predominantly a
polypeptide(s) between 40 and 49 kDa in the tested strains (Fig. 3).
This result indicated that LamB homologue is distributed widely in
the Vibrio species included in this study. Moreover, three obvious
bands were observed in Western blot with V. ﬂurialis ATCC 33810. It
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in Fig. 4, bands observed on tested strains ranged between 40
and 49 kDa, consistent with predicated molecular weight of LamB.
It showed that the antibodies derived from V. alginolyticus ATCC
33787 recognized homologous LamB of the tested strains including
V. alginolyticus ATCC 33787, V. proteolyticus ATCC 15338, V. parahaemolyticus VPL4-90, V. parahaemolyticus ATCC 17802, V. cholera
Vb0, V. vulniﬁcus ATCC 27562, V. mimicus ATCC 33653, V. ﬂurialis
ATCC 33810, V. furnissii ATCC 33813, V. natriegens ATCC 14048, V.
pelagius ATCC 25916, V. damsel NBRC 15633, V. harveyi NBRC 15634,
V. campbellii NBRC 15631, V. splendidus NBRC 101061, V. anguillarum NBRC 13266 and A. hydrophila NBRC 12658. Three non Vibrio
species were used for the purpose of negative control, whereas S.
aureus and E. coli were shown to be cross-reactivity against the
anti-LamB sera. There were no obvious reaction bands on two Vibrio
species (V. metschnikovii NBRC 103153 and V. ﬁscheri NBRC 101058)
and on B. subtilis. This is further evidence that the LamB is widely
distributed and shows cross-reactions antigenicity in the tested
Vibrio strains.
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3.4. Investigation of cross-reaction properties of anti-LamB sera
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Whole-cell ELISA was performed to investigate the crossreaction properties of the anti-LamB antibodies to react with the
whole cells of the tested strains. We selected eight Vibrio strains
which had obvious reaction bands in Western blot and their lamB
genes which had been cloned in this study. The result showed
that the strongest immune response was from V. alginolyticus
ATCC 33787, from which the anti-LamB sera derived. The second
highest reactions came from V. parahaemolyticus VPL4-90, V. parahaemolyticus ATCC 17802, V. harveyi NBRC 15634 and V. cholerae
Vb0, and a lower cross-reaction came from V. mimicus ATCC 33653.
However, anti-LamB sera showed the cross-reaction ability against
V. furnissii ATCC 33813 and V. ﬂurialis ATCC 33810 in Western blot
but not in Whole-cell ELISA assay (Table 1). The result indicated that
the anti-LamB antibodies recognized surface-localized epitopes of
this antigen. It further conﬁrmed that LamB is exposed on the cell
surface.
Table 2
Cumulative percent mortality and relative percent survival (RPS) of vaccinated zebraﬁsh, following intraperitoneal challenge with pathogen Vibrios.
Challenge strain
Accumulating death
dates in control group
Accumulating death dates in
immunized groupa
RPS
V. alginolyticus ATCC 33787
V. parahaemolyticus VPL4-90
V. parahaemolyticus ATCC 17802
V. harveyi NBRC 15634
V. mimicus ATCC 33653
90.0% (27/30)
80.0% (24/30)
93.3% (28/30)
83.3% (25/30)
80.0% (24/30)
20.0% (6/30)
30.0% (9/30)
40.0% (12/30)
33.3% (10/30)
36.7% (11/30)
77.8%
62.5%
57.1%
60.0%
54.1%
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b
Results obtained in this study were analyzed using Student’s t-test, and differences between immunized group and control group were signiﬁcant (P < 0.05).
Identity of amino acid sequence of LamB between challenge strain and homogenous V. alginolyticus ATCC 33787.
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Identityb
100%
48.3%
75.1%
48.8%
75.4%
J. Lun et al. / Vaccine 32 (2014) 809–815
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with the degree of the identity of deduced amino acid sequences
of LamB (Table 2). On the other hand, highly conserved epitopes
of LamB were found in our bioinformatics analysis. Thus, homologous epitopes of LamB may contribute to broad cross-protective
activity. However, the antigenic epitopes include not only the linear
epitopes (composed of continuous amino acid residues) but also the
conformational epitopes (composed of folding discontinuous peptide segments). In protein antigen, different afﬁnities could occur
with small changes in primary sequence (e.g., the conservative substitution of threonine for serine), or with changes in conformation,
such as the cleavage of the protein into fragments [28]. Therefore,
what is the role of the predicted LamB epitopes (mainly linear epitopes) in the cross-protective activity needs further analysis.
In summary, the present study found for the ﬁrst time that the
LamB shared a high degree of similarity, and distributed widely
among tested Vibrio species. The anti-LamB sera of V. alginolyticus ATCC 33787 had a signiﬁcant cross-reaction capacity against
several pathogenic Vibrios. Furthermore, although further work is
needed for the development of a vaccine, our results suggest that
LamB is a broad cross-protective vaccine candidate for Vibrios.
or
C
Acknowledgements
This work was sponsored by grants from The Agricultural Science and Technology Achievements Transformation Fund of China
(2013GB2E000368) and Science and Technology Department of
Guangdong province, P.R.China (2012B020308008). We would like
to thank Dr. Chiju Wei and Dr. Dan Trotter for their effort in revising
the English writing in the manuscript.
ad
implied that there might be common epitopes between LamB protein and some other proteins presented in a cell lysate which could
be recognized by the LamB antibodies.
Outer membrane proteins, being localized at the exteriors of
bacterial cells, are ideal targets for bactericidal and protective antibodies as they may be retained on the bacterial surface. Whole-cell
ELISA was used to investigate the subcellular location of the target proteins [21,22]. LamB proteins (or maltoporins) are a family of
outer membrane proteins. To investigate the subcellular location of
LamB in the native background of the Vibrios, Whole-cell ELISA was
performed in this study. The observation that the antisera raised
against recombinant LamB from V. alginolyticus ATCC 33787 recognized the tested strains including V. alginolyticus ATCC 33787,
V. parahaemolyticus VPL4-90, V. parahaemolyticus ATCC 17802, V.
harveyi NBRC 15634, V. cholerae Vb0 and V. mimicus ATCC 33653
suggests that LamB is exposed on the surface of the cell, at least
partially surface-presented. However, the anti-LamB sera failed to
recognize V. furnissii ATCC 33813 and V. ﬂurialis ATCC 33810 in the
Whole-cell ELISA assay. On the one hand, comparing with other
tested Vibrio strains, these two strains were in a different cluster
indicating the lower homology in the nucleotide sequence (Fig. 1).
On the other hand, it is well-known that sodium dodecyl sulfate
(SDS) is commonly used in preparing proteins for electrophoresis
in the SDS-PAGE technique. This compound works by disrupting
non-covalent bonds in the proteins, and causing proteins to unfold
into a rod-like shape. For this reason, the LamB antibodies had more
chances to interact with the LamB epitopes in Western blot based
on the effects of SDS, whereas these antibodies interacted only
with the LamB epitopes exposed on the cell surface in Whole-cell
ELISA. This may explain why the anti-LamB sera showed the crossreaction ability against V. furnissii ATCC 33813 and V. ﬂurialis ATCC
33810 in the Western blot but not in the Whole-cell ELISA assay.
In addition, antigenic cross-reactivity of OMPs has been reported
among Gram-negative bacteria [23]. Interestingly, in our study,
signiﬁcant antibody response also appeared on S. aureus, indicating that LamB might have homologue between the Gram-positive
and -negative bacteria. Although the immunological characteristic
of LamB is yet to be further investigated, the results shown here
strongly suggest that LamB has a broad cross-reaction property.
The present study demonstrated that native or recombinant
OMPs could induce a cross-protective immune response. Inoue
et al. [24,25] ﬁrstly reported that the 26 kDa OmpK of V. parahaemolyticus could serve as the receptor for the vibriophage
KVP40. Immunoblotting analyses using anti-OmpK sera revealed
that OmpK and its homologs were distributed widely among Vibrio and Photobacterium strains. Recent research has shown that
OmpK was an effective vaccine candidate and could provide crossprotection challenge with heterogeneous virulent Vibrio strains
[9,11,12,26,27]. To evaluate the cross-protection ability of LamB,
challenge tests with pathogens were applied. The result showed
that the protective capacity of recombinant LamB against V.
parahaemolyticus ATCC 17802, V. parahaemolyticus VPL4-90, V. alginolyticus ATCC 33787, V. mimicus ATCC 33653 and V. harveyi NBRC
15634 was signiﬁcantly higher (RPS > 50%) than that of control
saline solution (Table 2). These results suggested that LamB was
a potentially versatile vaccine candidate for the prevention of Vibriosis.
In addition, consistent with the results from the crossprotection experiment, the phylogenetic analysis of the deduced
amino acid sequences of LamB showed that the top three Vibrio strains with the highest RPS (V. alginolyticus, RPS = 77.8%; V.
parahaemolyticus, RPS = 62.5%; V. harveyi, RPS = 60.0%) had close
distance in the phylogenic tree (Fig. 1). Xu et al. [23] indicated
that the less distance of phenogram the higher possibility of sharing cross-protective OMPs. This result is similar to the ﬁndings
that cross-protective capacity of recombinant LamB was correlative
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