Journal of Microbiological Methods 117 (2015) 22–27
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The epitope analysis of an antibody speciﬁcally against Vibrio cholerae O1 Ogawa by phage library study Shiliang Cheng a, Zhen Lin b, Xinfeng Liu a, Wen Zheng a, Gang Lu c, Zhiguang Tu d, Jun Zhang c, Jian Zheng b, Xiaolin Yu d,⁎ a
Clinical Laboratory, Shandong Jiaotong Hospital, Jinan, Shandong, China Key Laboratory of Molecular Biology on Infectious Diseases, Chongqing Medical University, Chongqing, China Artron BioResearch Inc., Burnaby, British Columbia, Canada d Key Laboratory of Diagnostic Medicine, Chongqing Medical University, Chongqing, China. b c
a r t i c l e
i n f o
Article history: Received 8 May 2015 Received in revised form 3 July 2015 Accepted 5 July 2015 Available online 12 July 2015 Keywords: Cholera Ogawa Monoclonal antibody Phage display library Mimic peptide Lipopolysaccharide
a b s t r a c t To prevent epidemic and pandemic cholera disease, an indispensible approach is to develop cholera vaccines based on comprehensive epitope information of this pathogen. This study aimed to utilize our previously raised monoclonal antibody IXiao3G6, which can recognize an epitope in lipopolysaccharide (LPS) sites of Ogawa, to identify mimetic peptides, which may represent Ogawa LPS's epitope information. A phage display library screening using IXiao3G6 antibody resulted in identiﬁcation of a mimic peptide (MP) with high avidity. A recombinant protein, containing one cholera toxin subunit B (CTB) and two MP repeats (CTB-(MP)2), was subsequently constructed and investigated for its immunological characteristics. The ﬁndings collectively demonstrated that the MP presenting phages and CTB-(MP)2 recombinant protein were both capable of inhibiting the interaction between IXiao3G6 and Ogawa/Ogawa LPS speciﬁcally in a dose-dependent manner. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Cholera, caused by the Gram-negative bacterium Vibrio cholerae, is an acute malignant infectious dehydrating diarrheal disease, which occurs rapidly and spreads widely, leading to severe dehydrating diarrhea and vomiting, even high rates of mortality in some cases. There is annually more than 5 million people affected leading to at least 3% mortality rate. Epidemics as well as pandemics of cholera remain a major health problem in many parts of the world, especially in developing countries (Cholera, 2011, 2012). In addition to development of new rapid diagnosis tools for accurate and prompt diagnosis in time, it is imperative to expedite vaccine research and development as an additional tool for cholera control. Currently, available vaccines for cholera are based on killed, whole-cell, or live-attenuated formulations, which mostly provide only partial short term protection. An alternative approach which may be used to improve vaccination is to develop deﬁned component formulations. LPS, which is composed of three distinct domains: lipid A, core polysaccharide (inner and outer) and O-speciﬁc polysaccharide (O-SP), can induce protective humoral immune responses in humans and animal models during ⁎ Corresponding author at: The Key Laboratory of Diagnostic Medicine, Chongqing Medical University, No.1 Yixueyuan Road, Yuzhong District, Chongqing 400016, China. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.mimet.2015.07.006 0167-7012/© 2015 Elsevier B.V. All rights reserved.
infection and vaccination, suggesting that LPS can be accepted as a protective immunogen for cholera vaccine development (Taylor et al., 2004). However, LPS application may be problematic, not only because of its toxicity due to lipid A component, which may lead to inﬂammation, multiple organ failure, shock, and potentially death (Chatterjee and Chaudhuri, 2006), but also because LPS is a type of T-lymphocyte independent (TI) antigens, from which immune response generated can be lack of T-lymphocyte memory. One alternative to circumvent these obstacles is to select peptides mimicking the immune-dominant structures, which can potentially stimulate T-lymphocyte dependent (TD) immunity (De Bolle et al., 1999). Smith and Parmley established the phage display technique in 1985, which can express exogenous peptides and present them on surface of the phage (Parmley and Smith, 1988). Devlin and his group further developed phage display library technology using antipolysaccharide monoclonal antibodies (Devlin et al., 1990). A phage library can be screened by using a highly-speciﬁc anti-polysaccharide monoclonal antibody directly against surface carbohydrate ligand structures of pathogenic bacteria. After consecutive cycles of selection and ampliﬁcation, the phage expressing peptides that bind to the antibody can be selected, which can help mimic the ligand in both a structural and functional manner with respect to antibody recognition and elicit a robust immune response directed against the mimicked antigen. If the interaction
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between the selected phage and the target antibody can be inhibited by the polysaccharide, the peptide may mimic the polysaccharide antigen. The phage display libraries has been used to identify peptides capable of inducing antibodies against a variety of carbohydrate epitopes present on bacteria (Weintraub, 2003). Many studies have reported that peptide mimics strategy which may be valuable for ﬁnding potential surrogate antigens of carbohydrates for vaccine development against microorganisms (Hou and Gu, 2003; Lesinski et al., 2001; Maitta et al., 2004; Pincus et al., 1998) and tumors (Hardy and Raiter, 2005; Monzavi-Karbassi et al., 2007). With their high productivity and intrinsic immunogenic properties, peptide mimotopes have advantages over incorporating complex carbohydrate haptens issued from bacterial cell cultures or low yielding syntheses. The present study intended to employ a monoclonal antibody we previously generated, IXiao3G6 (Chen et al., 2014), which is directed against LPS of V. cholerae O1 Ogawa with competitive sensitivity and speciﬁcity, to identify the speciﬁc mimic peptides with high avidity through biopanning using phage random 7 peptide library. 2. Materials and methods 2.1. Bacteria strains V. cholerae O1 serotype Ogawa and Inaba, and V. cholerae O139 were kindly donated by the Sichuan Provincial Center for Disease Control and Prevention. The following bacteria including the standard strains (shown by ATCC number), e.g. Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 13076), Shigella sonnei (ATCC 25931), and clinicalseparated stains Vibrio ﬂuvialis and Aeromonas caviae were kindly provided by the clinical laboratory of the Second Afﬁliated Hospital to Chongqing University of Medical Sciences. The bacteria were ﬁrstly grown on blood agar plates for 24 h at 37 °C, and the colony was removed to Luria–Bertani broth for expansion (started with 3–5 mL mini-scale culture, then 50–100 mL large-scale culture). After bacteria were inactivated at 70 °C for 30 min, the bacteria were centrifuged and the pellets were stored at 4 °C. The inactivated bacteria were quantiﬁed as protein in this study, and the protein content of all preparations was determined by standard BCA assay. After each bacterial strain was biochemically characterized and evaluated as previously described (Chen et al., 2014). 2.2. Biopanning and identiﬁcation of epitope mimic peptide (MP) through Ph.D.-7 Phage Library The Ph.D.™-7 Phage Display Peptide Library was purchased from New England Biolabs (New England Biolabs (Beijing) Ltd., Beijing, China). The 96-well Microtiter™ plates (polystyrene, ﬂat, Thermo Scientiﬁc, China) were ﬁrstly coated with 100 μL McAb IXiao3G6 (100 μg/mL) (Chen et al., 2014), diluted in 0.1 M NaHCO3, and incubated overnight at 4 °C. Plates were then incubated for 2 h with 200 μL blocking buffer 0.1 M NaHCO3/1% BSA at room temperature and washed three times in Tris-Buffered Saline plus 0.05% Tween 20 (TBST). One hundred microliter of phage solution (2 × 1011 pfu) was incubated for 60 min at room temperature, followed by eight times TBST wash. The bound phages were subsequently eluted by incubation with 100 μL 0.2 M Glycine–HCl (pH 2.2) per well for 10 min at room temperature, and neutralized with 30 μL 1 M Tris–HCl (pH 9.0) and transferred to 1.5 mL centrifuge tubes. One microliter of each eluate was used for determining the titer and the rest were transferred to E. coli ER2738 culture (New England Biolabs (Beijing) Ltd., Beijing, China) and incubated for 4–5 h at 37 °C for ampliﬁcation. Ampliﬁed phages were puriﬁed by precipitating the phage with PEG/NaCl (20% polyethelene glycol, 2.5 M NaCl). The recovery rate was calculated as follows: (phage eluted) / (phage input) × 100%. The second and third rounds of panning were carried out using the same procedure except that the amounts of IXiao3G6 coated in the plates were gradually reduced to 10 μg/mL.
After the enrichment, phage plaques were randomly picked up from LB/IPTG/Xgal plates and tested for their ability to bind to the IXiao3G6 by phage ELISA. 2.3. Screening of phage eluate for positive clones by phage ELISA The 96-well Microtiter™ plates (polystyrene, ﬂat, Thermo Scientiﬁc, China) were ﬁrstly coated with 100 μL previously puriﬁed McAb IXiao3G6, diluted in 0.1 M NaHCO3 (10 μg/mL), and incubated overnight at 4 °C. Plates were then incubated for 2 h with 200 μL blocking buffer (0.1 M NaHCO3/1% BSA) at room temperature and washed ﬁve times in TBST. Fifty microliter of each ampliﬁed phage supernatant (1.0 × 106 pfu/μL) plus 150 μL of TBST, or 2 μL original phage (2.0 × 1010 pfu/μL) from Ph.D.™-7 Phage Display Peptide Library plus 200 μL of TBST (negative control) were placed in each of the wells and incubated for 60 min at room temperature. After being washed in TBST for eight times, plates were incubated with 100 μL anti-M13horseradish peroxidase (HRP) (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China) diluted in blocking buffer (1:5000) for 1 h at room temperature. Finally, plates were washed 5 times with TBST and developed with 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system for ELISA (Sigma-Aldrich, Beijing, China) in the dark for 15 min at room temperature. The reaction was terminated by supplementing 50 μL of 1 M H2SO4 and absorbance values were determined at 450 nm with BioTek™ ELx808™ Absorbance Microplate Readers (ThermoFisher Scientiﬁc Inc., Beijing, China) against the blank. Clones were scored as positive when the mean OD450 value of triplicate wells was at least twice the signal of negative controls. 2.4. Determine DNA sequences of displayed peptides from positive phage clones The positive phage clones were ﬁrst expanded by propagating them in 2 mL cultures of logarithmically growing E. coli K12 ER2738 culture. The bacteria were removed from the culture suspension by a brief centrifugation to recover the phage. Ampliﬁed phage were precipitated by PEG/NaCl, and the pellet containing the phage was resuspended 100 μL of iodide buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 4 M NaI) and 250 μL of absolute ethanol. After centrifugation for 10 min, the phage DNA was washed with 70% ethanol, dried, and resuspended in 30 μL of TE buffer. One hundred nanograms of phage DNA was subjected to dideoxyoligonucleotide termination reactions using a DNA sequencing kit (PerkinElmer, Beijing, China) and the M13-96gIII sequencing primer (5′-CCCTCATAGTTAGCGTAACG-3′). The sequence of the positive clones was obtained by running the above reaction products through an automated DNA sequencer from Applied Biosystems (Life Technologies, Beijing, China). Based on the revealed DNA sequence, the translated peptide sequence was subjected to search database Non-redundant protein sequences (nr) using Blastp (protein–protein BLAST, available on NCBI website). 2.5. The speciﬁcity and afﬁnity determination of mimic peptide to IXiao3G6 The 96-well plates were coated with 10 μg/mL McAb IXiao3G6 and blocked as described above. After being washed in TBST for 5 times, plates were then incubated with 50 μL of IXiao3G6, Dao2H8 (for Inaba) and O4D7 (for O139) McAbs (both generated from the same laboratory) at different concentrations (100, 50, 25, 12.5, 6.25, 3.125 μg/mL) for 10 min at 37 °C. Control wells were incubated with 50 μL 0.1 M NaHCO3 and 50 μL P12 phage clone (1.0 × 107 pfu) were subsequently incubated for 50 min at 37 °C. The rest of the procedures, e.g. antiM13-HRP incubation and color development, were the same as described in phage ELISA. The inhibition rate was calculated as follows: (ODcontrol − ODAb) / ODcontrol × 100%. The experiment was repeated 3 times and the mean values were used for plotting.
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To test whether the identiﬁed mimic peptide can inhibit the interaction between McAb IXiao3G6 and Ogawa, the 10 μg/mL McAb IXiao3G6 coated plates were incubated with 50 μL inactivated bacteria (V. cholerae O1 Ogawa and Inaba, V. cholerae O139, E. coli, S. typhi, S. sonnei, V. ﬂuvialis, and A. caviae) (refer to Section 2.1 for details), or alternatively their corresponding LPS extracts (prepared as previously described (Chen et al., 2014)) at different concentrations (10, 5, 2.5, 1.25, 0.625, 0.31 μg/mL) diluted in PBS for 10 min at 37 °C. After being washed in TBST for 5 times, plates were incubated with either 50 μL P16 phage clone (1.0 × 107 pfu) or P17 phage clone (1.0 × 107 pfu) respectively, followed by anti-M13-HRP incubation and color development. 50 μL 0.1 M NaHCO3 was included in both conditions and served as a positive control. The inhibition rate was calculated as follows: (ODcontrol − ODAb) / ODcontrol × 100%. Each experiment was repeated 3 times and the mean values were used for plotting. The above experiments were repeated using either 50 μL original phage (2.0 × 1010 pfu) from Ph.D.™-7 Phage Display Peptide Library or 50 μL P9 phage clone (1.0 × 107 pfu) to replace P12, P16 and P17 to examine the level of background binding. 2.6. Recombinant protein construction and puriﬁcation Cholera toxin subunit B (CTB) was ﬁrstly ampliﬁed by primers 5′CGCGGTACCATGATTAAATTAAAATTTGGTG-3′ and 5′-GCCGGATCCATT TGCC ATACTAATTGCG-3′, which has a KpnI restriction site and a BamHI site respectively. The ampliﬁed DNA fragment was then cloned into pET-32a (+) vector (Merck Millipore, Beijing, China) to construct pET-32a (+)–CTB plasmid. Secondly, the fragment (GPGAPAIP ASGPGAPAIPASGPG) containing the duplicated mimic peptides separated by spacer glycine–proline–glycine (GPG) was ampliﬁed by primers 5′-GCGGGATCCGGTCCAGGTGCACCTGCTATTCCGGCTTCTGGTCCAG GTG-3′ and 5′-GCAAAGCTTACCTGGACCAGAAGCCGGAATAGCAGGTG CACCTGGACCA-3′, which has a BamHI site and a HindIII site respectively. The ﬁnal construct was assembled as pET-32a (+)–CTB(MP)2. All the PCR reactions were performed as previously described (Chen et al., 2014). The constructed plasmids pET-32a (+)–CTB and pET-32a (+)–CTB-(MP)2 were then transformed into E. coli strain JM109 for expression. The overexpressed CTB or CTB-(MP) 2 peptides, containing a C-terminal His-tag, were puriﬁed by afﬁnity chromatography using Ni2 +-NTA column according to the established methods (Park et al., 2001). 2.7. Antigenicity analysis of the recombinant CTB-(MP)2 The antigenicity of CTB-(MP)2 was analyzed by both indirect ELISA and western-blotting approaches. For indirect ELISA approach, the 96well microplates were coated with 100 μL 10 μg/mL recombinant proteins CTB-(MP)2, CTB, and pET-32a (+) vector expression, diluted in 0.1 M NaHCO3, and incubated overnight at 4 °C. Plates were blocked for 2 h with 200 μL blocking buffer (TBS/1% BSA) at room temperature and washed three times in TBST (TBS/0.5% Tween-20). Plates were then incubated with different concentrations (100, 50, 25, 12.5, 6.25 μg/mL) of primary antibody IXiao3G6 at 37 °C for 1 h. After being washed with PBST ﬁve times, plates were incubated with 100 μL secondary antibody goat-anti mouse IgG-HRP (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China) in blocking buffer (1:5000) for 30 min at 37 °C. Finally, plates were washed ﬁve times with TBST and developed with 100 μL TMB in the dark for 15 min at room temperature. The reaction was terminated by supplementing 50 μL of 1 M H2SO4 and absorbance values were determined at 450 nm. The experiment was repeated 3 times and the mean values were used for plotting. The western blotting analysis was performed as previously described and the blot was detected by McAb IXiao3G6 (Chen et al., 2014). The inhibition effect of the recombinant protein CTB-(MP)2 on binding between Ogawa or its LPS and IXiao3G6 was examined as follows. The 96-well plates were coated with 100 μL 10 μg/mL inactivated
Ogawa bacteria culture or 10 μg/mL LPS extracted from Ogawa overnight at 4 °C. After being washed in TBST for 5 times, different concentrations of CTB-(MP)2 , CTB, and pET-32a (+) expression products (500, 250, 125, 62.5, 31.3 μg/mL) were co-incubated with 10 μg/mL IXiao3G6 for 1 h at 37 °C. After TBST wash, the plates were incubated with goat-anti mouse IgG-HRP and subsequently TMB for color development. The inhibition rate was calculated as follows: (ODcontrol − ODAb) / ODcontrol × 100%. Each experiment was repeated 3 times and the mean values were used for plotting. 3. Results 3.1. Biopanning and identiﬁcation of MP The phage library was effectively enriched after three rounds of biopanning selection and the recovery rate was enhanced from 1.6 × 10−4 to 1.9 × 10−2 (~118 times). The randomly selected 25 clones were conﬁrmed by phage ELISA that 22 of them were positive clones. The following DNA sequencing of these 22 clones illustrated that 20 of them had the identical amino acid sequence (APAIPAS). The other two clone sequences were respectively (APALPAS) and (APSIPAS). The online BLAST search of peptide (APAIPAS) did not reveal any of the known sequence. 3.2. The speciﬁcity characterization of MP Three positive clones were randomly selected (P12, P16, P17) from those 20 identical clones described above, and they bound to IXiao3G6 at the same level. The phage ELISA experiment results showed that the ratio of their mean OD450 values to negative control were 27.3 (P12), 28.1 (P16) and 26.4 (P17) respectively (data not shown). These random selections were subjected to study the interaction between mimic peptide and IXiao3G6. Firstly, only IXiao3G6 could inhibit P12 clone to bind immobilized IXiao3G6 but not the McAbs Dao2H8 and O4D7, and the inhibition was dose-dependent (Fig. 1A). Secondly, the competition experiment demonstrated that only V. cholerae O1 Ogawa could inhibit the binding between P16 clone and IXiao3G6 (Fig. 1B), but not other tested bacteria, e.g. V. cholerae O1 Inaba, V. cholerae O139, E. coli, S. typhi, S. sonnei, V. ﬂuvialis, and A. caviae, and the inhibition was positively correlated to the concentration of Ogawa culture. Similarly, only LPS extracted from Ogawa, but not from other bacteria, could inhibit the binding between P17 clone and IXiao3G6 (Fig. 1C), and the inhibition exhibited a positive correlation manner. It was conﬁrmed that neither the original phage from Ph.D.™-7 Phage Display Peptide Library nor the irrelevant phage clone, P9, which does not bind to IXiao3G6, contributed to the inhibition effect (data not shown). The inhibition levels for P12, P16, and P17 to either Ogawa or its LPS were very similar. The inhibition rates of 10 μg/mL Ogawa bacteria for different clones were 89.1% (P16), 91.3% (P17), and 88.2% (P12) respectively (data not shown). The inhibition rates of 10 μg/mL Ogawa LPS for different clones were 90.3% (P16), 90.5% (P17), and 89.3% (P12) respectively (data not shown). 3.3. Recombinant protein CTB-(MP)2 overexpression and antigenicity analysis The overexpressed recombinant proteins, pET-32a (+) vector protein, CTB and CTB-(MP)2 (Fig. 2A left panel, lanes 1, 2, 3 respectively) were puriﬁed by Ni–NTA afﬁnity column successfully with more than 90% purity. The corresponding western blotting analysis of these recombinant proteins illustrated that McAb IXiao3G6 could speciﬁcally detect recombinant CTB-(MP)2, but not CTB or pET-32a (+) vector protein (Fig. 2A right panel, lanes 3, 2, 1 respectively). Fig. 2B presented phage ELISA result demonstrated that McAb IXiao3G6 only reacted with recombinant CTB-(MP)2 in a dose-dependent manner, but not with CTB
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Fig. 1. Characterization of the mimic peptide. A: The inhibition effect of IXiao3G6 and other McAbs, Dao2H8 (against Inaba) and O4D7 (against O139), on a positive phage clone (P12) binding to coated IXiao3G6. X-axis: McAb concentration, Y-axis: inhibition rate. B: The inhibition effect of Ogawa and other bacteria on a phage clone (P16) binding to coated IXiao3G6. X-axis: bacteria concentration, Y-axis: inhibition rate. C: The inhibition effect of extracted LPS from Ogawa and other bacteria on a phage clone (P17) binding to coated IXiao3G6. X-axis: LPS concentration, Y-axis: inhibition rate.
or vector proteins. On the other hand, the competition assay showed that only CTB-(MP)2 was capable of suppressing the binding between IXiao3G6 and V. cholerae O1 Ogawa (Fig. 2C) or LPS extracted from Ogawa (Fig. 2D), but not the CTB or vector protein, and this inhibition was positively correlated to Ogawa or Ogawa LPS concentration. 4. Discussion At the present study, we utilized previously generated McAb IXiao3G6, which targets LPS sites of Ogawa, to decrease the nonspeciﬁc phage absorption and assist high avidity selection. The phage ELISA results demonstrated that the MP presenting phage clones only
reacted with McAb IXiao3G6 but not other related species, e.g. Dao2H8 and O4D7. Additionally, as expected, the competition assay demonstrated that only V. cholerae O1 Ogawa or its LPS extract but not other bacteria or their corresponding LPS extracts could inhibit the reaction between the selected phage clones and McAb IXiao3G6. The inhibition effect was speciﬁc and dose dependent. After the successfully sequencing this mimetic peptide (APAIPAS), we constructed a fusion protein CTB-(MP)2 and investigated its immunological properties. The duplicated MPs and the CTB component were also segregated via a few glycine–proline–glycine linkers, which is beneﬁcial to preserve the desired spatial orientation, and in turn not only guarantees each epitope resides relatively independently, but also
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Fig. 2. Characterization of the recombinant protein CTB-(MP)2. A: SDS-PAGE (left panel) and western blot analysis (right panel) of the recombinant proteins (1: pET32a(+) protein; 2: CTB fusion protein; 3. CTB-(MP)2 fusion protein). The western blot was detected against IXiao3G6. B: Three different recombinant proteins were detected against different concentrations of IXiao3G6. C: Competitive inhibition of binding between IXiao3G6 and Ogawa by different recombinant proteins at various concentrations (X-axis: recombinant protein concentration; Y-axis: inhibition rate). D: Competitive inhibition of binding between IXiao3G6 and Ogawa LPS by different concentrations recombinant proteins (X-axis: recombinant protein concentration; Y-axis: inhibition rate).
minimizes the possible spatial impact of CTB on mimic peptides. The rationale for constructing this complex is that several problems could be associated with short peptide antigens, such as their poor chemical stability (rapid degradation) and subsequent lower antigenicity in vivo (Partidos et al., 2001). It has been reported that vaccination at mucosal surfaces and combinatorial vaccination strategies that linking immunostimulatory molecules to antigens have been developed to enhance vaccine efﬁcacy (Langridge et al., 2010). Because the non-toxic CTB subunit displays both carrier and immunostimulatory properties when linked to pathogen antigens as a potent mucosal adjuvant, and this unique characteristic has been applied to the development of vaccine either for cholera (Price et al., 2013) or other diseases (Olivera et al., 2014), we believe that it is a suitable partner candidate for our identiﬁed MP. The examination of immunogenicity of recombinant proteins demonstrated promisingly that only the fusion protein CTB-(MP)2, but not CTB alone or vector protein, could speciﬁcally react with McAb IXiao3G6, and this result was conﬁrmed by both indirect ELISA and western blotting approaches. The competition experiments further showed that only CTB-(MP)2 could inhibit the interaction between IXiao3G6 and Ogawa or its LPS extract in a dose-dependent manner. Collectively, this study successfully conﬁrmed that the phage library strategy can be employed for epitope mimicking of Ogawa LPS, and it is a useful technique which has importance in developing vaccines. However, due to the limited characterization of monoclonal antibody IXiao3G6, it is inefﬁcient to conclude that the antibody has potential protective efﬁcacy. Therefore, it is not deﬁnite to presume that this identiﬁed MP or corresponding recombinant protein CTB-(MP)2 is capable of developing LPS-speciﬁc protective responses in this report. Therefore, further studies especially in vivo administration investigation should be carried out for deep understanding of the epitope information of this
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