Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6448-x
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli Weikun Guan & Wenxin Liu & Jun Bao & Jinping Li & Chaowen Yuan & Jie Tang & Dongfang Shi
Received: 6 December 2014 / Revised: 27 January 2015 / Accepted: 28 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTBderived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no crossreaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to
Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6448-x) contains supplementary material, which is available to authorized users. W. Guan : W. Liu : J. Li : C. Yuan : J. Tang : D. Shi Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, People’s Republic of China J. Bao College of Animal Science and Technology, Northeast Agricultural University, Harbin, Heilongjiang 150030, People’s Republic of China J. Bao : D. Shi (*) Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural University, Harbin 150030, People’s Republic of China e-mail: [email protected]
GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent. Keywords LTB . Monoclonal antibodies . Epitope analysis . Neutralizing activity
Introduction Diarrhea is the second leading cause of death to young children who live in developing countries (Black et al. 2010) and continues to be a major threat to global health (2006). Enterotoxigenic Escherichia coli (ETEC) strains producing enterotoxins are the most common bacteria that cause diarrhea and are responsible for 300,000–500,000 deaths of young children annually (2006; Zhang et al. 2013). These ETEC strains produce various bacterial adhesions and one or more enterotoxins (Zhang et al. 2013). Bacterial adhesions mediate ETEC initial attachment to host epithelial cells and subsequent colonization at host small intestines, with both heat-labile (LT, LT-II) and heat-stable (STa) enterotoxins stimulating intestinal epithelial cells to secrete electrolytes and fluids, resulting in diarrhea (Allen et al. 2006). Of enterotoxins, LT is the major disease agent of ETEC (Loc et al. 2010). LT consists of an A subunit (LTA) and a pentameric B subunit (LTB) (Spangler 1992). The A and B subunits have different molecular and biological characteristics which are important in host pathogenesis and immunity (Allen et al. 2006). The A subunit is responsible for the toxicity of LT
Appl Microbiol Biotechnol
and is able to increase levels of intracellular cAMP, which leads to activation of the cAMP-dependent protein kinase A, finally causing diarrhea. Although the A subunit is the toxicity center of LT, it cannot cause diarrhea without assistant of the B subunit. The A subunit is linked to the B subunit by the trypsin-sensitive loop and the long α-helix, then B subunit specifically linked to the GM1 receptor (Mudrak and Kuehn 2010). Only when B subunit linked to GM1 receptor can A subunit exert toxic effects. Therefore, B subunit plays a key role in LT-induced diarrhea. However, the specific binding region of B LTB to GM1 receptor is still unclear. In addition, previous studies showed that pentameric LTB contains most of the immunodominant epitopes, and LTB-specific antibody responses were able to provide protection against LTproducing E. coli infection in human (Nashar et al. 2001). However, to date, no studies concerning epitope screening and identification of LTB protein were reported. Moreover, many studies have indicated that LTB is a potent mucosal adjuvant when coadministered with viral proteins and, when given alone, can block the establishment and progression of autoimmune disease (Kang et al. 2004). The adjuvant activities of LTB have been clearly demonstrated with a number of antigens including herpes simplex virus (HSV) glycoprotein and influenza hemagglutinin. The ability of the B subunits to alter the immune response also results from their binding to GM1 receptor (Nashar et al. 2001). It is thought that the interaction between the LTB protein and the GM1 cell receptor activates B and CD4+ T cells and enhances antigen presentation by activating DCs and other APCs by facilitating antigen uptake through receptor-mediated endocytosis mechanisms (Yamamoto et al. 2001). Therefore, this clearly clarifies that the cross-reaction between LTB protein and GM1 receptor was significant. In this study, the linear B cell epitopes within the LTB protein were predicted with bioinformatic tools by using ABCPred online server. In addition, linear B cell epitopes within the LTB protein were also mapped by using LTB polyclonal antisera and then further compared with the predicted epitopes. Moreover, a LTB-neutralized monoclonal antibody (MAb), which was able to interrupt the LTB protein binding to GM1 receptor, was generated. The results will facilitate the development of epitope-based preventive and diagnostic strategies for ETEC infection and provide preliminary information for better understanding of the reaction between LTB protein and host cells.
Chinese Academy of Agricultural Sciences). SP2/0 myeloma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) in a humidified incubator with 5 % CO2 at 37 °C. All culture media were supplemented with 10 % heated-inactivated fetal bovine serum (FBS, GIBCO, Invitrogen) and antibiotics (0.1 mg/ml of streptomycin and 100 IU/ml of penicillin). E. coli C83903 (LT+) was deposited in the Chinese Veterinary Culture Collection Center (CVCC: C83903). Mouse adrenocortical (Y1) cells were purchased from SZST.CN (Shanghai,Chinese Academy of Sciences), and rabbit polyclonal antibody (PAb) was stored in our laboratory. Sequence analysis and the B cell epitope prediction of LTB protein A primary structure analysis of the LTB protein was conducted using the ProtParam online tool by computing various physical and chemical parameters, including the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (GRAVY) (Sun et al. 2014). The secondary structure and disorder were predicted by means of the PHYRE 2 online server (Kelley and Sternberg 2009). The 3-D structure of LTB protein was gained from PDB online service. The B cell epitopes in the LTB protein were predicted by using ABCPred online server using a threshold setting 0.8 (information taken from website http://www.imtech.res.in/raghava/abcpred/). Recombinant LTB protein preparation The LTB gene (GenBank: CP002732.1) was PCR amplified using genome of E. coli C83903 as the template and the designed primers P1 (5′-GGGATCCATGTTTACGGCGTTA CTATCCT-3′, BamH I) and P2 (5′-GCTCGAGTCAGATT GCCGCAATTGAATTGG-3′, Xho I) for cloning in pET-30a vector. The recombinant LTB protein was prepared as previously described (Feng et al. 2013). Further, the recombinant LTB protein was also analyzed by sodium dodecyl sulfate (SDS)-PAGE and Western blot (WB) as previously described (Zhao et al. 2012). Following characterization of recombinant proteins, rP-ETEC-LTB served as the source antigen for the immunization of mice. Preparation and characterization of MAbs against LTB
Materials and methods Animals, cells, and strains Six-week-old BALB/c mice were supplied by the Centre of Experimental Animals (Harbin Veterinary Research Institute,
Splenocytes harvested from immunized mice were fused with SP2/0 myeloma cells at a ratio of 1:4 using polyethylene glycol (PEG 4000; Sigma). Hybridoma cells were plated into 96well plates in hypoxanthine-aminopterin-thymidine (HAT) selection media (DMEM containing 20 % FBS, 100 mg streptomycin/ml, 100 IU penicillin/ml, 100 mM hypoxanthine,
Appl Microbiol Biotechnol
16 mM thymidine, and 400 mM aminopterin) in a humidified incubator with 5 % CO2 at 37 °C. After 5 days, the HAT media were replaced with hypoxanthine-thymidine (HT) media lacking aminopterin. After HAT/HT selection, three rounds of subcloning by limiting dilution were performed. Hybridoma clone culture supernatants were screened for reactivity against ETEC-LTB protein by indirect ELISA and WB as previously described (Liu et al. 2014). The titer of each MAbs was determined by indirect ELISA as described above. Antibody subtype was determined using mouse MAb isotyping kit (Invitrogen) according to the manufacturer’s instructions.
Comparison and location distribution analysis of the predicted and identified epitopes in the 3-D structure of LTB protein
Cross-reactivity studies with competitive ELISA and WB
Analysis of the specificity and conservation of the identified linear peptide epitopes among similar toxins
A competitive ELISA protocol was developed to check the antibody for cross-reactivity with other toxins of E. coli. Briefly, plates were coated with rP-ETEC-LTB protein overnight. One hundred microliter of diluted hybridoma supernatants was incubated with an equal volume of PBS containing various amounts of STa (heat-stable enterotoxin I), STb (heatstable enterotoxin II), or LT toxins. After 1 h at 37 °C, 50 μl of the incubated samples was added into the plates for 1 h at 37 °C, and the plate was then processed as in the ELISA protocol described above. LT, STa, STb, Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), and Stx2e (Shiga toxin II variant) were separated using 12 % SDS-PAGE; then, the culture supernatants were used as primary antibodies with an horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody as previously described (Liu et al. 2014).
We next compared the experimentally identified LTB antibody epitopes with computationally predicted epitopes to assess the accuracy of B cell epitope prediction using the artificial neural network. The spatial distribution of the predicted and identified epitopes on the LTB protein was analyzed by mapping epitope locations onto a 3-D model of the ETEC LTB protein using PyMOL software based on the results of the PHYRE 2 online server.
An analysis to assess the conservation of identified epitopes among similar toxins was conducted. Amino acid sequences corresponding to identified epitope regions of LTB protein and several structural similar toxins were aligned using MegAlign bioinformatics software (Lasergene, DNASTAR Inc., Madison, WI, USA). GM1-ganglioside-binding assay for LT The binding of LT to GM1 ganglioside receptor was determined by GM1-ELISA (mlbio biotech, Shanghai, China) following the manufacturer’s protocol with slight modification. Briefly, 50 μl of cell supernatants from the prepared MAb was mixed with an equal volume of LT enterotoxin for 2 h at 37 °C.
Polypeptide design and preparation
In vitro neutralization ability of the MAb against LT toxin
A panel of ten partially overlapping peptides, spanning the entirety of the LTB protein, was designed and expressed as maltose-binding protein (MBP)-fused polypeptides (named MBP-LTB-1 through MBP-LTB-10) (Sun et al. 2012). The recombinant plasmids were transformed into Rossetta (DE3) E. coli cells for expression as described above (Feng et al. 2014). SDS-PAGE and WB analysis were performed to confirm the presence of each MBP-fused recombinant polypeptide. Briefly, anti-MBP MAbs were used as primary antibodies with an HRP-conjugated goat anti-mouse secondary antibody as previously described (Liu et al. 2014).
To determine the neutralizing ability of the prepared anti-LTB MAb, the cell supernatant of the MAb and SP2/0 was evaluated via Y1 cell neutralization assay. Briefly, 50 μl of antibody samples in twofold serial dilutions (from 1:2 to 1:512) was mixed with an equal volume of LT toxin (50 CD50). Then, the antibody-toxin mixture was added to Y1 cells and incubated at 37 °C under 5 % CO2 for 24 h. Cell viability was stained with crystal violet, and the numbers of infected cells and normal cells were counted as previously described (Gentry and Dalrymple 1980; Rocha et al. 2012).
B cell linear epitope mapping using PAb and MAb
In vivo neutralization ability of the MAb against LT toxin in mice model
The reactivity of the rabbit PAb and murine MAb with the overlapping MBP-fused LTB polypeptide series was screened by WB. Briefly, each of the ten MBP-fused LTB polypeptides was performed by SDS-PAGE and used as target antigen for the WB to identify linear epitopes recognized by MAb and the rabbit polyclonal antiserum as previously described (Liu et al. 2014).
A total of 20 6-week-old female BALB/c mice (Liaoning Changsheng Biotechnology Co., LTD, China) were divided into two groups (experimental and control) of ten mice per group. The LT toxin (50 median lethal dose LD50) was neutralized with antibody samples as described above. Then, the mice of experimental group received an intraperitoneal (IP) injection of the antibody-toxin mixture of 0.2 ml. Mice of
Appl Microbiol Biotechnol
control group received an IP injection of the equal volume of SP2/0-toxin mixture as the negative control. The outcome was Bdead^ or Balive^ after a 2-h interval. Ligated intestinal loop assay in rabbit model Ligated intestinal loop assay in rabbit model was performed as previously described (Whipp 1991). Briefly, a total of six 10week-old rabbits (Liaoning Changsheng Biotechnology Co., LTD, China) were divided into two groups (experimental and control) of three rabbits per group. The LT toxin (50 LD50 in mice model) was neutralized with antibody samples as described above. For experimental group, rabbit loops were injected with 2 ml of the antibody-toxin mixture, and the adjacent loops were injected with 2 ml of the 0.9 % saline. For control group, rabbit loops were injected with 2 ml of the SP2/ 0-toxin mixture and the adjacent loops were injected with 2 ml of the 0.9 % saline. The concentrations of cAMP in fluid were quantified using a cAMP enzyme immunoassay kit according t o t h e m a n u f a c t u r e r ’s i n s t r u c t i o n s ( E l a b s c i e n c e Biotechnology, Wuhan, China). Intestinal segments were fixed with formalin solution and were prepared using standard paraffin-embedding procedures by sectioning at 5-μm thickness and staining with hematoxylin and eosin. A total of 15 intact, well-oriented crypt-villus units were measured from each broiler. Villus height and crypt depth were determined using scale plate of the microscope and were expressed as micrometers.
experimental group were administered enema with 10 ml of the MAb supernatant, and the piglets in control group were administered enema with 10 ml of the SP2/0 supernatant. Then, the anuses of the piglets were closed by a clamp. After 8 h, clinical symptoms were observed. Then, villus height and crypt depth were determined as mentioned above.
Statistical analysis Statistical analysis (ANOVA) was performed using SPSS version 19.0 and GraphPad Prism 5.0. P values of
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli Weikun Guan & Wenxin Liu & Jun Bao & Jinping Li & Chaowen Yuan & Jie Tang & Dongfang Shi
Received: 6 December 2014 / Revised: 27 January 2015 / Accepted: 28 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTBderived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no crossreaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to
Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6448-x) contains supplementary material, which is available to authorized users. W. Guan : W. Liu : J. Li : C. Yuan : J. Tang : D. Shi Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, People’s Republic of China J. Bao College of Animal Science and Technology, Northeast Agricultural University, Harbin, Heilongjiang 150030, People’s Republic of China J. Bao : D. Shi (*) Synergetic Innovation Center of Food Safety and Nutrition, Northeast Agricultural University, Harbin 150030, People’s Republic of China e-mail: [email protected]
GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent. Keywords LTB . Monoclonal antibodies . Epitope analysis . Neutralizing activity
Introduction Diarrhea is the second leading cause of death to young children who live in developing countries (Black et al. 2010) and continues to be a major threat to global health (2006). Enterotoxigenic Escherichia coli (ETEC) strains producing enterotoxins are the most common bacteria that cause diarrhea and are responsible for 300,000–500,000 deaths of young children annually (2006; Zhang et al. 2013). These ETEC strains produce various bacterial adhesions and one or more enterotoxins (Zhang et al. 2013). Bacterial adhesions mediate ETEC initial attachment to host epithelial cells and subsequent colonization at host small intestines, with both heat-labile (LT, LT-II) and heat-stable (STa) enterotoxins stimulating intestinal epithelial cells to secrete electrolytes and fluids, resulting in diarrhea (Allen et al. 2006). Of enterotoxins, LT is the major disease agent of ETEC (Loc et al. 2010). LT consists of an A subunit (LTA) and a pentameric B subunit (LTB) (Spangler 1992). The A and B subunits have different molecular and biological characteristics which are important in host pathogenesis and immunity (Allen et al. 2006). The A subunit is responsible for the toxicity of LT
Appl Microbiol Biotechnol
and is able to increase levels of intracellular cAMP, which leads to activation of the cAMP-dependent protein kinase A, finally causing diarrhea. Although the A subunit is the toxicity center of LT, it cannot cause diarrhea without assistant of the B subunit. The A subunit is linked to the B subunit by the trypsin-sensitive loop and the long α-helix, then B subunit specifically linked to the GM1 receptor (Mudrak and Kuehn 2010). Only when B subunit linked to GM1 receptor can A subunit exert toxic effects. Therefore, B subunit plays a key role in LT-induced diarrhea. However, the specific binding region of B LTB to GM1 receptor is still unclear. In addition, previous studies showed that pentameric LTB contains most of the immunodominant epitopes, and LTB-specific antibody responses were able to provide protection against LTproducing E. coli infection in human (Nashar et al. 2001). However, to date, no studies concerning epitope screening and identification of LTB protein were reported. Moreover, many studies have indicated that LTB is a potent mucosal adjuvant when coadministered with viral proteins and, when given alone, can block the establishment and progression of autoimmune disease (Kang et al. 2004). The adjuvant activities of LTB have been clearly demonstrated with a number of antigens including herpes simplex virus (HSV) glycoprotein and influenza hemagglutinin. The ability of the B subunits to alter the immune response also results from their binding to GM1 receptor (Nashar et al. 2001). It is thought that the interaction between the LTB protein and the GM1 cell receptor activates B and CD4+ T cells and enhances antigen presentation by activating DCs and other APCs by facilitating antigen uptake through receptor-mediated endocytosis mechanisms (Yamamoto et al. 2001). Therefore, this clearly clarifies that the cross-reaction between LTB protein and GM1 receptor was significant. In this study, the linear B cell epitopes within the LTB protein were predicted with bioinformatic tools by using ABCPred online server. In addition, linear B cell epitopes within the LTB protein were also mapped by using LTB polyclonal antisera and then further compared with the predicted epitopes. Moreover, a LTB-neutralized monoclonal antibody (MAb), which was able to interrupt the LTB protein binding to GM1 receptor, was generated. The results will facilitate the development of epitope-based preventive and diagnostic strategies for ETEC infection and provide preliminary information for better understanding of the reaction between LTB protein and host cells.
Chinese Academy of Agricultural Sciences). SP2/0 myeloma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) in a humidified incubator with 5 % CO2 at 37 °C. All culture media were supplemented with 10 % heated-inactivated fetal bovine serum (FBS, GIBCO, Invitrogen) and antibiotics (0.1 mg/ml of streptomycin and 100 IU/ml of penicillin). E. coli C83903 (LT+) was deposited in the Chinese Veterinary Culture Collection Center (CVCC: C83903). Mouse adrenocortical (Y1) cells were purchased from SZST.CN (Shanghai,Chinese Academy of Sciences), and rabbit polyclonal antibody (PAb) was stored in our laboratory. Sequence analysis and the B cell epitope prediction of LTB protein A primary structure analysis of the LTB protein was conducted using the ProtParam online tool by computing various physical and chemical parameters, including the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (GRAVY) (Sun et al. 2014). The secondary structure and disorder were predicted by means of the PHYRE 2 online server (Kelley and Sternberg 2009). The 3-D structure of LTB protein was gained from PDB online service. The B cell epitopes in the LTB protein were predicted by using ABCPred online server using a threshold setting 0.8 (information taken from website http://www.imtech.res.in/raghava/abcpred/). Recombinant LTB protein preparation The LTB gene (GenBank: CP002732.1) was PCR amplified using genome of E. coli C83903 as the template and the designed primers P1 (5′-GGGATCCATGTTTACGGCGTTA CTATCCT-3′, BamH I) and P2 (5′-GCTCGAGTCAGATT GCCGCAATTGAATTGG-3′, Xho I) for cloning in pET-30a vector. The recombinant LTB protein was prepared as previously described (Feng et al. 2013). Further, the recombinant LTB protein was also analyzed by sodium dodecyl sulfate (SDS)-PAGE and Western blot (WB) as previously described (Zhao et al. 2012). Following characterization of recombinant proteins, rP-ETEC-LTB served as the source antigen for the immunization of mice. Preparation and characterization of MAbs against LTB
Materials and methods Animals, cells, and strains Six-week-old BALB/c mice were supplied by the Centre of Experimental Animals (Harbin Veterinary Research Institute,
Splenocytes harvested from immunized mice were fused with SP2/0 myeloma cells at a ratio of 1:4 using polyethylene glycol (PEG 4000; Sigma). Hybridoma cells were plated into 96well plates in hypoxanthine-aminopterin-thymidine (HAT) selection media (DMEM containing 20 % FBS, 100 mg streptomycin/ml, 100 IU penicillin/ml, 100 mM hypoxanthine,
Appl Microbiol Biotechnol
16 mM thymidine, and 400 mM aminopterin) in a humidified incubator with 5 % CO2 at 37 °C. After 5 days, the HAT media were replaced with hypoxanthine-thymidine (HT) media lacking aminopterin. After HAT/HT selection, three rounds of subcloning by limiting dilution were performed. Hybridoma clone culture supernatants were screened for reactivity against ETEC-LTB protein by indirect ELISA and WB as previously described (Liu et al. 2014). The titer of each MAbs was determined by indirect ELISA as described above. Antibody subtype was determined using mouse MAb isotyping kit (Invitrogen) according to the manufacturer’s instructions.
Comparison and location distribution analysis of the predicted and identified epitopes in the 3-D structure of LTB protein
Cross-reactivity studies with competitive ELISA and WB
Analysis of the specificity and conservation of the identified linear peptide epitopes among similar toxins
A competitive ELISA protocol was developed to check the antibody for cross-reactivity with other toxins of E. coli. Briefly, plates were coated with rP-ETEC-LTB protein overnight. One hundred microliter of diluted hybridoma supernatants was incubated with an equal volume of PBS containing various amounts of STa (heat-stable enterotoxin I), STb (heatstable enterotoxin II), or LT toxins. After 1 h at 37 °C, 50 μl of the incubated samples was added into the plates for 1 h at 37 °C, and the plate was then processed as in the ELISA protocol described above. LT, STa, STb, Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), and Stx2e (Shiga toxin II variant) were separated using 12 % SDS-PAGE; then, the culture supernatants were used as primary antibodies with an horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody as previously described (Liu et al. 2014).
We next compared the experimentally identified LTB antibody epitopes with computationally predicted epitopes to assess the accuracy of B cell epitope prediction using the artificial neural network. The spatial distribution of the predicted and identified epitopes on the LTB protein was analyzed by mapping epitope locations onto a 3-D model of the ETEC LTB protein using PyMOL software based on the results of the PHYRE 2 online server.
An analysis to assess the conservation of identified epitopes among similar toxins was conducted. Amino acid sequences corresponding to identified epitope regions of LTB protein and several structural similar toxins were aligned using MegAlign bioinformatics software (Lasergene, DNASTAR Inc., Madison, WI, USA). GM1-ganglioside-binding assay for LT The binding of LT to GM1 ganglioside receptor was determined by GM1-ELISA (mlbio biotech, Shanghai, China) following the manufacturer’s protocol with slight modification. Briefly, 50 μl of cell supernatants from the prepared MAb was mixed with an equal volume of LT enterotoxin for 2 h at 37 °C.
Polypeptide design and preparation
In vitro neutralization ability of the MAb against LT toxin
A panel of ten partially overlapping peptides, spanning the entirety of the LTB protein, was designed and expressed as maltose-binding protein (MBP)-fused polypeptides (named MBP-LTB-1 through MBP-LTB-10) (Sun et al. 2012). The recombinant plasmids were transformed into Rossetta (DE3) E. coli cells for expression as described above (Feng et al. 2014). SDS-PAGE and WB analysis were performed to confirm the presence of each MBP-fused recombinant polypeptide. Briefly, anti-MBP MAbs were used as primary antibodies with an HRP-conjugated goat anti-mouse secondary antibody as previously described (Liu et al. 2014).
To determine the neutralizing ability of the prepared anti-LTB MAb, the cell supernatant of the MAb and SP2/0 was evaluated via Y1 cell neutralization assay. Briefly, 50 μl of antibody samples in twofold serial dilutions (from 1:2 to 1:512) was mixed with an equal volume of LT toxin (50 CD50). Then, the antibody-toxin mixture was added to Y1 cells and incubated at 37 °C under 5 % CO2 for 24 h. Cell viability was stained with crystal violet, and the numbers of infected cells and normal cells were counted as previously described (Gentry and Dalrymple 1980; Rocha et al. 2012).
B cell linear epitope mapping using PAb and MAb
In vivo neutralization ability of the MAb against LT toxin in mice model
The reactivity of the rabbit PAb and murine MAb with the overlapping MBP-fused LTB polypeptide series was screened by WB. Briefly, each of the ten MBP-fused LTB polypeptides was performed by SDS-PAGE and used as target antigen for the WB to identify linear epitopes recognized by MAb and the rabbit polyclonal antiserum as previously described (Liu et al. 2014).
A total of 20 6-week-old female BALB/c mice (Liaoning Changsheng Biotechnology Co., LTD, China) were divided into two groups (experimental and control) of ten mice per group. The LT toxin (50 median lethal dose LD50) was neutralized with antibody samples as described above. Then, the mice of experimental group received an intraperitoneal (IP) injection of the antibody-toxin mixture of 0.2 ml. Mice of
Appl Microbiol Biotechnol
control group received an IP injection of the equal volume of SP2/0-toxin mixture as the negative control. The outcome was Bdead^ or Balive^ after a 2-h interval. Ligated intestinal loop assay in rabbit model Ligated intestinal loop assay in rabbit model was performed as previously described (Whipp 1991). Briefly, a total of six 10week-old rabbits (Liaoning Changsheng Biotechnology Co., LTD, China) were divided into two groups (experimental and control) of three rabbits per group. The LT toxin (50 LD50 in mice model) was neutralized with antibody samples as described above. For experimental group, rabbit loops were injected with 2 ml of the antibody-toxin mixture, and the adjacent loops were injected with 2 ml of the 0.9 % saline. For control group, rabbit loops were injected with 2 ml of the SP2/ 0-toxin mixture and the adjacent loops were injected with 2 ml of the 0.9 % saline. The concentrations of cAMP in fluid were quantified using a cAMP enzyme immunoassay kit according t o t h e m a n u f a c t u r e r ’s i n s t r u c t i o n s ( E l a b s c i e n c e Biotechnology, Wuhan, China). Intestinal segments were fixed with formalin solution and were prepared using standard paraffin-embedding procedures by sectioning at 5-μm thickness and staining with hematoxylin and eosin. A total of 15 intact, well-oriented crypt-villus units were measured from each broiler. Villus height and crypt depth were determined using scale plate of the microscope and were expressed as micrometers.
experimental group were administered enema with 10 ml of the MAb supernatant, and the piglets in control group were administered enema with 10 ml of the SP2/0 supernatant. Then, the anuses of the piglets were closed by a clamp. After 8 h, clinical symptoms were observed. Then, villus height and crypt depth were determined as mentioned above.
Statistical analysis Statistical analysis (ANOVA) was performed using SPSS version 19.0 and GraphPad Prism 5.0. P values of
