Biotechnol Lett (2014) 36:2109–2116 DOI 10.1007/s10529-014-1586-2
ORIGINAL RESEARCH PAPER
Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry Li-Ming Xu • Miao Liu • Jing-Zhuang Zhao • Yong-Sheng cao • Jia-Sheng Yin • Hong-Bai Liu Tongyan Lu
•
Received: 26 March 2014 / Accepted: 4 June 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments,
except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.
L.-M. Xu M. Liu J.-Z. Zhao Y.-S. cao J.-S. Yin H.-B. Liu T. Lu (&) Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences, Harbin 150070, People’s Republic of China e-mail: [email protected]
Introduction
L.-M. Xu e-mail: [email protected] M. Liu e-mail: [email protected] J.-Z. Zhao e-mail: [email protected] Y.-S. cao e-mail: [email protected] J.-S. Yin e-mail: [email protected] H.-B. Liu e-mail: [email protected]
Keywords Bacterial display technology Epitope mapping Fluorescence-activated cell sorting Glycoprotein Infectious hematopoietic necrosis virus Linear B cell Epitopes
Research into epitope mapping has led to the development of a variety of immunological assays that are used to detect the presence of antibodies. These have played crucial roles in diagnosing diseases, monitoring humoral immune responses and identifying molecules of biological or medical significance. Highthroughput, rapid and easily visualized screening of epitopes is therefore significant for the understanding of immunological events, the development of epitopebased marker vaccines, and as diagnostic tools for various diseases (Peng et al. 2008). Common methods of identifying B cell epitopes include the identification of antigens, synthetic peptide scan (PEPSCAN), and phage display random peptide libraries. The use of PEPSCAN is limited by the length
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of synthetic peptides and the number of overlapping amino acids that cause the omission of certain epitopes. Compared with a bacterial display, phage display depends on random peptide libraries and has some drawbacks: monoclonal antibodies (MAbs) are required as ligands; phage displays are not able to be monitored in real time; and greater than three or four bio-pannings are necessary to identify specific binding. In a previous study, an expression system was devised that enables target protein anchoring on the periplasmic face of the Escherichia coli inner membrane via a lipoprotein-targeting motif (Miroux and Walker 1996). The new lipoprotein (NlpA) is a non-essential E. coli lipoprotein that exclusively localizes to the inner membrane. NlpA is secreted across the membrane via the Sec pathway and, once in the periplasm, a diacylglycerol group is attached through a thioether bond to a cysteine residue on the C-terminal side of the signal sequence. The signal peptide is cleaved by signal peptidase II and the protein is acylated at the modified cysteine residue. The lipophilic fatty acid then inserts itself into the membrane to anchor the protein. The NlpA leader peptide with the first six amino acids of the mature NlpA, containing the putative fatty acylation and inner membrane targeting sites, were used for generating this bacterial display technology. The bacterial display technology, generated as our previous studies (Li et al. 2013), relies on the anchored expression of antigens on the periplasmic side of the inner membrane (Harvey et al. 2004, 2006). Upon removal of the outer membrane by spheroplasting, fluorescent antibodies can enter into the periplasmic space where it is recognized by the membrane-tethered antigens, and the antigen–antibody binding ability can be analyzed by fluorescence-activated cell sorting (FACS) (Fig. 1). Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes acute, systemic disease in salmonid fish and also occurs in asymptomatic fish hosts. As one of the most important viral diseases in aquaculture facilities, outbreaks of IHN result in losses approaching 100 % depending on the species and size of the fish, the virus strain and environmental conditions (Breyta et al. 2013; Enzmann et al. 2005; Kim et al. 2007; Kolodziejek et al. 2008; Nichol et al. 1995; Nishizawa et al. 2006; Rudakova et al. 2007). Due to the extensive
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Fig. 1 Schematic of truncated glycoprotein displayed by bacteria-display technology. a Truncated glycoprotein antigens were expressed and anchored to the inner membrane of E. coli in the periplasm. b Binding of antigens with pAb and fluorescence labeled anti-rabbit antibody. c Analysis of antigen-binding ability using FCM. pAb: rabbit anti-glycoprotein polyclonal antibody; CDQSSS New lipoprotein A amino acids 1–6. FCM flow cyotmetry
economic losses caused by IHNV in fish culture facilities, the immunological characterization of the virus should be deeply analyzed. In this study, B cell epitopes of the glycoprotein of IHNV prevalent in China was mapped by FCM without the need for protein expression and purification steps.
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Fig. 2 Schematic illustration of epitope prediction and bacteria-display plasmid construction. The long black bar represents the full length glycoprotein, and the gray bars represent the
glycoprotein fragments that were cloned into the bacteriadisplay vector pBFD plasmid. The short black bars represent the most hydrophilic regions
Materials and methods
proteins. Amplicons were ligated into pMD18-Tsimple vector; following sequence verification of the recombinant plasmids, the ten truncated fragments of the glycoprotein gene were inserted downstream of the NlpA leader peptide and the first six amino acids (CDQSSS) of the mature NlpA protein in the pBFD vector (Xu et al. 2011).
Materials The pMD18-T-glycoprotein and pBFD-IL-1b vectors were laboratory stocks, and the glycoprotein gene sequence was submitted to the GenBank database (Accession No. KC660147). We used E. coli DH5a for displaying the truncated glycoprotein, while E. coli Rosetta and pET27b (?) were used for expression of the truncated glycoprotein proteins. The pBFD used for glycoprotein display was laboratory stock. Rabbit anti-IHNV glycoprotein polyclonal antibody (pAb) and full length IHNV glycoprotein were prepared by our previous study. HRP-goat anti-rabbit IgG and mouse anti-His IgG were purchased from R&D (Minneapolis, MO). A fluorescein iso-thiocyanate (FITC)-labeled goat anti-rabbit antibody (Santa Cruz Biotechnology, USA) was used for epitopes FCM analysis. Mouse anti-Flag IgG and FITC-goat antimouse IgG (Santa Cruz Biotechnology) were used for expression analysis of truncated proteins. Rabbit antiinfectious bursal disease virus pAb was donated by Harbin Veterinary Research Institute, Harbin, China. Generation of pBFD-glycoprotein The glycoprotein gene was truncated to ten fragments as depicted in Fig. 2. Ten truncated fragments of the glycoprotein gene were cloned from pMD18-T-glycoprotein. The truncated gene fragments were amplified with specific primers and designed to have a Flag tag at the N-terminus of truncated glycoprotein
Software prediction of epitopes The nucleotide sequences of glycoprotein were compiled and edited with Lasergene v7.1, and multiple sequence alignments were assembled with Protean. Based on the truncated glycoprotein amino acid sequence, glycoprotein epitopes were predicted using antigenic index. Preparation of spheroplasts and epitope FACS analysis All colonies from the pBFD- truncated glycoproteintransformed DH5a were collected and cultured in lysogeny broth (LB). When the culture OD600 was between 0.4 and 0.6, synthesis of glycoprotein fragments was induced by the addition of 0.25 mM IPTG, and the cells were transferred to 25 °C for 4 h. Spheroplasts were prepared as previously described (Jeong et al. 2007). The prepared spheroplasts were incubated with the anti-glycoprotein pAb and the FITC-goat anti-rabbit IgG. Spheroplasts bearing pBFD-IL-1b were used as a negative control. FACS analysis was conducted using a FACSAria Cell Sorter (BD Biosciences, USA).
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Fig. 3 DNAStar analysis of potential epitopes in glycoprotein. The antigenic index represents the strength of antigen-binding. The frames indicate potential antigenic epitopes
FACS analysis of truncated glycoprotein-Flag proteins displayed in periplasm Expression levels of truncated glycoprotein proteins in the periplasm were analyzed by FCM. Spheroplasts were prepared and incubated with a mouse anti-Flag IgG (1:2,000 dilution in 1 % BSA/PBS) for 1 h at room temperature. After washed with 0.1 % (v/v) Tween 20/PBS five times, the spheroplasts were incubated with a FITC- goat anti-mouse IgG for 1 h at room temperature. After washing, the fluorescence intensity of the spheroplasts was detected by FACS. Three negative controls were included: control 1 (spheroplasm without any plasmid), control 2 (spheroplasm bearing pBFD-full length glycoprotein without any tag), and control 3 (spheroplasm bearing pBFDfull length glycoprotein with Flag tag, incubated with FITC-mouse anti-His IgG).
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Expression of truncated glycoprotein The ten truncated fragments of glycoprotein were cloned from pMD18-T-glycoprotein. PCR products were ligated into the prokaryotic expression vector pET-27b between the NcoI and BamHI sites. After sequence verification, the plasmids were transformed into E. coli Rosetta to generate the recombinant proteins in vitro. Western blotting of purified truncated glycoprotein proteins Purified glycoprotein fragments were detected by western blotting, with ovalbumin used as a negative control, and rabbit anti- glycoprotein pAb used as the prey. The binding activity of pAb to the antigen was visualized using ECL techniques.
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ELISA analysis For ELISA, 96-well microtiter plates were coated with 200 nM purified truncated fragments or full length glycoprotein (positive control) in 100 ll NaHCO3/ Na2CO3 buffer (pH 8.7) overnight at 4 °C. Triplicate samples were washed three times with PBS-T (0.5 %, v/v), and 5 % (v/v) skimmed milk in PBS was used to block the remaining non-specific binding sites at 37 °C for 2 h. After washing, each well was incubated with 100 ll rabbit anti-IHNV glycoprotein pAb (200fold dilution) at 37 °C for 1 h, followed by HRP-goat anti-rabbit antibody (7500-fold dilution). Full length IHNV glycoprotein incubated with negative rabbit serum was used as a negative control. The assay was developed using TMB solution and the development of color product was terminated by the addition of 50 ll 2 M H2SO4. The absorbance of each well was measured by an ELISA reader at 450 nm. The rabbit anti-IHNV glycoprotein pAb and full length IHNV glycoprotein were prepared as our previous studies (Xu et al. 2013).
Results
Fig. 4 Epitope mapping of glycoprotein by flow cytometry. M mean fluorescence intensity. Count number of spheroplasm
between M values of G23 and G3. The results indicated that there was no linear epitope in fragment G2. The M values were ranked as G14 [ G45 [ G13 [ G4 [ G23 [ G3 [ G12 [ G5 [ G1 [ G2 (Fig. 4).
Epitope prediction The amino acid sequences of the truncated glycoprotein were analyzed using DNAstar (Fig. 3). The antigenic index represents the strength of an antigenbinding. The regions containing peaks indicate potential antigenic epitopes. G1, G3, G4 and G5 contain many regions with high antigenic index, but there is only one region containing a single peak within the G2 fragment. FACS epitope analysis of glycoprotein fragments After subsequent incubation with the rabbit antiglycoprotein pAb and the FITC-conjugated goat antirabbit antibody, the spheroplasts bearing truncated glycoprotein fragments were detected by FACS. Spheroplasts bearing pBFD-IL-1b were used as a negative control. Compared with the negative control, there was an obvious increase in the mean fluorescence intensity (M value) for all fragments except G2. The M value of G12 was approximately the same with that of G1, and there were also negligible differences
Detection of the truncated glycoprotein fragments displayed in the periplasm To exclude the influence of antigen expression levels in the periplasm on FACS fluorescence intensity, a Flag tag was fused to each truncated glycoprotein fragment and cloned into pBFD display vector. Expression levels were measured by FACS. No obvious differences of mean fluorescent intensity (M) were observed from each spheraplasts containing truncated glycoprotein-Flag fusions (Fig. 5). The expression level of the truncated glycoprotein proteins in the periplasm was strong enough to be captured by corresponding antibodies. Western blotting of purified truncated glycoprotein fragments The ten truncated glycoprotein proteins were expressed as inclusion bodies in E. coli Rosetta by pET-27b (?), and were purified by denaturation and renaturation. Western blotting was conducted to
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Fig. 5 Detection of truncated glycoprotein-Flag fragments displayed in the periplasm of E. coli by flow cytometry. Blank spheroplasm without any plasmid; NC1 spheroplasm bearing
pBFD-full length glycoprotein without any tag; NC2 spheroplasm bearing pBFD-full length glycoprotein with Flag tag incubated with FITC-mouse anti-His IgG
evaluate if purified truncated proteins could bind with the rabbit anti- glycoprotein pAb. The fragments containing epitopes reacted with the rabbit antiglycoprotein pAb. The distribution of epitopes was identified by western blotting. Except for fragment G2, all fragments strongly reacted with the rabbit antiglycoprotein pAb and bands with expected size were observed (Fig. 6a). The G2 protein and the ovalbumin (negative control) failed to react with the rabbit antiglycoprotein pAb.
fragments could bind with pAb and showed much higher OD450 than that of negative control, but a little lower than that of positive control (Fig. 6b). The OD450 was ranked as PC [ G14 [ G45 [ G13 [ G4 [ G23 [ G3 [ G12 [ G5 [ G1 [ G2 [ NC. The results were accordant with FCM analysis and indicated that there were epitopes located in all fragments but G2.
Discussion ELISA analysis Binding ability of fragments to pAb was determined by ELISA assay. Except the fragment G2, all
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In our current study, bacterial display technology combined with FCM was used for mapping linear B cell epitopes of the IHNV glycoprotein. The IHNV
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Fig. 6 Immunological characterization of the purified truncated protein. a Western blotting of the purified truncated glycoprotein fragments. NC negative control; M standard protein marker. b ELISA analysis of the purified truncated glycoprotein fragments. The data represent the mean ± SD. of quadruple samples. **p \ 0.01 versus NC. NC negative control; PC positive control
glycoprotein was truncated into ten overlapped fragments, and linear B cell epitopes of these fragments were determined by FCM according to M value. Our findings clearly indicate that the fragments G1, G3, G4, and G5 react with the pAbs, suggesting that these fragments contain linear B cell epitopes. The antigenic intensity of these fragments was ranked as G4 [ G3 [ G1 [ G5. The G2 fragment contains no antigenic epitope. To validate the results we obtained from FCM, the amino acid sequences of fragments were analyzed by DNAStar. There are multiple long hydrophilic regions with high antigenic index in the G1, G3, G4 and G5 fragments, but only few short hydrophilic regions were detected in the G2 fragment. These results are in agreement with the results obtained from FCM. To validate the results further, traditional methods were employed to analyze the antigenic epitopes of these fragments. The truncated fragments were expressed in E. coli and the purified fragments were examined by western blotting and ELISA analysis. Only the fragment G2 did not react with the pAb. The results are in accordance with those obtained by the bacterial display method we established.
IHNV belongs to the genus Novirhabdovirus. Its glycoprotein is a membrane protein and contains antigenic epitopes that are responsible for eliciting neutralizing antibodies (Huang et al. 1996). The antigenic epitopes of IHNV isolated in America have been studied and mapped (Huang et al. 1996; Xu et al. 1991). The results suggests that there are two epitopes within G4, one in G5 and one in G23 (G2 fused with G3). In this study, the mean fluorescence intensity of G4 was far higher than other fragments, and these results are in accordance with previous studies. Additionally, G23 was truncated into G2 and G3 in this study. The mean fluorescence intensity of G3 was almost the same as that of fragment G23, and far higher than that of negative control and fragment G2. It indicated that there was no epitope in G2 but in G3. These data indicate that the method is valid for systematic mapping of linear B cell epitopes for a given antigen. The new strategy established in this study offers several advantages over previous epitope mapping approaches. First, the bacterial display system can be used to detect a broad spectrum of antigens, as long as the polypeptides can be expressed in E. coli. Second,
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because the bacterial display system is based on an expression plasmid, it provides an easy way to create an epitope library with a large capacity to systematically analyze a large number of antigens in a short period of time. In this study, the glycoprotein was truncated into five fragments to construct a mini library. The epitope libraries cannot be analyzed by traditional methods because of the large amount of work involved in protein expression and purification. Third, because of the larger size of the bacterial cells, the incorporation of the bacteria-display system with FCM detection and screening technology dramatically enhances the feasibility of the technology. The combination of the bacterial display system with FACS, which confers real-time and high-throughput detection and screening capacities, makes this technology an attractive method for rapid and high-throughput mapping of linear B cell epitopes for any given antigen. Epitope clusters of glycoprotein of Chinese IHNV isolate were located in this study. According to the results, truncated glycoprotein fragment with high antigenicity will be chosen to be expressed and utilized to prepare antibody for the detection of IHNV isolate prevalent in China. As with a previous study, neutralization antigenic epitopes were located in the fragment, G4, which has the strongest antigenicity as revealed in this study. Thus, fragment G4 will not only be used for antibody preparation but also a subunit vaccine for Chinese IHNV isolate. Acknowledgments This study was supported by grants from sub-project of National 12th 5-year Support Key Projects (2012BAD25B02) and the Central-Level Non-profit Scientific Research Institutes Special Funds (HSY201204).
References Breyta R, Jones A, Stewart B, Brunson R, Thomas J, Kerwin J, Bertolini J, Mumford S, Patterson C, Kurath G (2013) Emergence of MD type infectious hematopoietic necrosis virus in Washington State coastal steelhead trout. Dis Aquat Organ 104:179–195 Enzmann PJ, Kurath G, Fichtner D, Bergmann SM (2005) Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American genogroup M. Dis Aquat Organ 66:187–195 Harvey BR, Georgiou G, Hayhurst A, Jeong KJ, Iverson BL, Rogers GK (2004) Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc Nat Acad Sci USA 101(25):9193–9198
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Biotechnol Lett (2014) 36:2109–2116 Harvey BR, Shanafelt AB, Baburina I, Hui R, Vitone S, Iverson BL, Georgiou G (2006) Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression. J Immunol Methods 308:43–52. doi:10.1016/j.jim.2005.09.017 Huang C, Chien MS, Landolt M, Batts W, Winton J (1996) Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus. J Gen Virol 77:3033–3040 Jeong KJ, Seo MJ, Iverson BL, Georgiou G (2007) APEx 2-hybrid, a quantitative protein–protein interaction assay for antibody discovery and engineering. Proc Nat Acad Sci USA 104:8247–8252 Kim WS, Oh MJ, Nishizawa T, Park JW, Kurath G, Yoshimizu M (2007) Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene. Arch Virol 152:2119–2124 Kolodziejek J, Schachner O, Durrwald R, Latif M, Nowotny N (2008) ‘‘Mid-G’’ region sequences of the glycoprotein gene of Austrian infectious hematopoietic necrosis virus isolates form two lineages within European isolates and are distinct from American and Asian lineages. J Clin Microbiol 46:22–30 Li T, Xu L, Ren G, Yin C, Zhou B, Ye X, Li Q, Li N, Li D (2013) A neutralization scFv antibody against IL-1beta isolated from a NIPA-based bacterial display library. Curr Pharm Biotechnol 14:571–581 Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298 Nichol ST, Rowe JE, Winton JR (1995) Molecular epizootiology and evolution of the glycoprotein and non-virion protein genes of infectious hematopoietic necrosis virus, a fish rhabdovirus. Virus Res 38:159–173 Nishizawa T, Kinoshita S, Kim WS, Higashi S, Yoshimizu M (2006) Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene. Dis Aquat Organ 71:267–272 Peng WP, Hou Q, Xia ZH, Chen D, Li N, Sun Y, Qiu HJ (2008) Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of Classical swine fever virus by phage-displayed random peptide library. Virus Res 135:267–272 Rudakova SL, Kurath G, Bochkova EV (2007) Occurrence and genetic typing of infectious hematopoietic necrosis virus in Kamchatka, Russia. Dis Aquat Organ 75:1–11 Xu L, Mourich DV, Engelking HM, Ristow S, Arnzen J, Leong JC (1991) Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli. J Virol 65:1611–1615 Xu LM, Yin CK, Ren GP, Tian H, Wang XQ, Ding LJ, Li DS (2011) Establishment of bacteria-display technology for Fab antibody library screening (in Chinese). Chinese J Cell Mol Immunol 27:1090–1093 Xu LM, Liu HB, Yin JS, Lu TY (2013) Prokaryotic expression and immunogenicity analysis of glycoprotein from infectious hematopoietic necrosis virus (in Chinese). Chinese J Virol 29:529–534
ORIGINAL RESEARCH PAPER
Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry Li-Ming Xu • Miao Liu • Jing-Zhuang Zhao • Yong-Sheng cao • Jia-Sheng Yin • Hong-Bai Liu Tongyan Lu
•
Received: 26 March 2014 / Accepted: 4 June 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments,
except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.
L.-M. Xu M. Liu J.-Z. Zhao Y.-S. cao J.-S. Yin H.-B. Liu T. Lu (&) Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences, Harbin 150070, People’s Republic of China e-mail: [email protected]
Introduction
L.-M. Xu e-mail: [email protected] M. Liu e-mail: [email protected] J.-Z. Zhao e-mail: [email protected] Y.-S. cao e-mail: [email protected] J.-S. Yin e-mail: [email protected] H.-B. Liu e-mail: [email protected]
Keywords Bacterial display technology Epitope mapping Fluorescence-activated cell sorting Glycoprotein Infectious hematopoietic necrosis virus Linear B cell Epitopes
Research into epitope mapping has led to the development of a variety of immunological assays that are used to detect the presence of antibodies. These have played crucial roles in diagnosing diseases, monitoring humoral immune responses and identifying molecules of biological or medical significance. Highthroughput, rapid and easily visualized screening of epitopes is therefore significant for the understanding of immunological events, the development of epitopebased marker vaccines, and as diagnostic tools for various diseases (Peng et al. 2008). Common methods of identifying B cell epitopes include the identification of antigens, synthetic peptide scan (PEPSCAN), and phage display random peptide libraries. The use of PEPSCAN is limited by the length
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of synthetic peptides and the number of overlapping amino acids that cause the omission of certain epitopes. Compared with a bacterial display, phage display depends on random peptide libraries and has some drawbacks: monoclonal antibodies (MAbs) are required as ligands; phage displays are not able to be monitored in real time; and greater than three or four bio-pannings are necessary to identify specific binding. In a previous study, an expression system was devised that enables target protein anchoring on the periplasmic face of the Escherichia coli inner membrane via a lipoprotein-targeting motif (Miroux and Walker 1996). The new lipoprotein (NlpA) is a non-essential E. coli lipoprotein that exclusively localizes to the inner membrane. NlpA is secreted across the membrane via the Sec pathway and, once in the periplasm, a diacylglycerol group is attached through a thioether bond to a cysteine residue on the C-terminal side of the signal sequence. The signal peptide is cleaved by signal peptidase II and the protein is acylated at the modified cysteine residue. The lipophilic fatty acid then inserts itself into the membrane to anchor the protein. The NlpA leader peptide with the first six amino acids of the mature NlpA, containing the putative fatty acylation and inner membrane targeting sites, were used for generating this bacterial display technology. The bacterial display technology, generated as our previous studies (Li et al. 2013), relies on the anchored expression of antigens on the periplasmic side of the inner membrane (Harvey et al. 2004, 2006). Upon removal of the outer membrane by spheroplasting, fluorescent antibodies can enter into the periplasmic space where it is recognized by the membrane-tethered antigens, and the antigen–antibody binding ability can be analyzed by fluorescence-activated cell sorting (FACS) (Fig. 1). Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes acute, systemic disease in salmonid fish and also occurs in asymptomatic fish hosts. As one of the most important viral diseases in aquaculture facilities, outbreaks of IHN result in losses approaching 100 % depending on the species and size of the fish, the virus strain and environmental conditions (Breyta et al. 2013; Enzmann et al. 2005; Kim et al. 2007; Kolodziejek et al. 2008; Nichol et al. 1995; Nishizawa et al. 2006; Rudakova et al. 2007). Due to the extensive
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Fig. 1 Schematic of truncated glycoprotein displayed by bacteria-display technology. a Truncated glycoprotein antigens were expressed and anchored to the inner membrane of E. coli in the periplasm. b Binding of antigens with pAb and fluorescence labeled anti-rabbit antibody. c Analysis of antigen-binding ability using FCM. pAb: rabbit anti-glycoprotein polyclonal antibody; CDQSSS New lipoprotein A amino acids 1–6. FCM flow cyotmetry
economic losses caused by IHNV in fish culture facilities, the immunological characterization of the virus should be deeply analyzed. In this study, B cell epitopes of the glycoprotein of IHNV prevalent in China was mapped by FCM without the need for protein expression and purification steps.
Biotechnol Lett (2014) 36:2109–2116
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Fig. 2 Schematic illustration of epitope prediction and bacteria-display plasmid construction. The long black bar represents the full length glycoprotein, and the gray bars represent the
glycoprotein fragments that were cloned into the bacteriadisplay vector pBFD plasmid. The short black bars represent the most hydrophilic regions
Materials and methods
proteins. Amplicons were ligated into pMD18-Tsimple vector; following sequence verification of the recombinant plasmids, the ten truncated fragments of the glycoprotein gene were inserted downstream of the NlpA leader peptide and the first six amino acids (CDQSSS) of the mature NlpA protein in the pBFD vector (Xu et al. 2011).
Materials The pMD18-T-glycoprotein and pBFD-IL-1b vectors were laboratory stocks, and the glycoprotein gene sequence was submitted to the GenBank database (Accession No. KC660147). We used E. coli DH5a for displaying the truncated glycoprotein, while E. coli Rosetta and pET27b (?) were used for expression of the truncated glycoprotein proteins. The pBFD used for glycoprotein display was laboratory stock. Rabbit anti-IHNV glycoprotein polyclonal antibody (pAb) and full length IHNV glycoprotein were prepared by our previous study. HRP-goat anti-rabbit IgG and mouse anti-His IgG were purchased from R&D (Minneapolis, MO). A fluorescein iso-thiocyanate (FITC)-labeled goat anti-rabbit antibody (Santa Cruz Biotechnology, USA) was used for epitopes FCM analysis. Mouse anti-Flag IgG and FITC-goat antimouse IgG (Santa Cruz Biotechnology) were used for expression analysis of truncated proteins. Rabbit antiinfectious bursal disease virus pAb was donated by Harbin Veterinary Research Institute, Harbin, China. Generation of pBFD-glycoprotein The glycoprotein gene was truncated to ten fragments as depicted in Fig. 2. Ten truncated fragments of the glycoprotein gene were cloned from pMD18-T-glycoprotein. The truncated gene fragments were amplified with specific primers and designed to have a Flag tag at the N-terminus of truncated glycoprotein
Software prediction of epitopes The nucleotide sequences of glycoprotein were compiled and edited with Lasergene v7.1, and multiple sequence alignments were assembled with Protean. Based on the truncated glycoprotein amino acid sequence, glycoprotein epitopes were predicted using antigenic index. Preparation of spheroplasts and epitope FACS analysis All colonies from the pBFD- truncated glycoproteintransformed DH5a were collected and cultured in lysogeny broth (LB). When the culture OD600 was between 0.4 and 0.6, synthesis of glycoprotein fragments was induced by the addition of 0.25 mM IPTG, and the cells were transferred to 25 °C for 4 h. Spheroplasts were prepared as previously described (Jeong et al. 2007). The prepared spheroplasts were incubated with the anti-glycoprotein pAb and the FITC-goat anti-rabbit IgG. Spheroplasts bearing pBFD-IL-1b were used as a negative control. FACS analysis was conducted using a FACSAria Cell Sorter (BD Biosciences, USA).
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Fig. 3 DNAStar analysis of potential epitopes in glycoprotein. The antigenic index represents the strength of antigen-binding. The frames indicate potential antigenic epitopes
FACS analysis of truncated glycoprotein-Flag proteins displayed in periplasm Expression levels of truncated glycoprotein proteins in the periplasm were analyzed by FCM. Spheroplasts were prepared and incubated with a mouse anti-Flag IgG (1:2,000 dilution in 1 % BSA/PBS) for 1 h at room temperature. After washed with 0.1 % (v/v) Tween 20/PBS five times, the spheroplasts were incubated with a FITC- goat anti-mouse IgG for 1 h at room temperature. After washing, the fluorescence intensity of the spheroplasts was detected by FACS. Three negative controls were included: control 1 (spheroplasm without any plasmid), control 2 (spheroplasm bearing pBFD-full length glycoprotein without any tag), and control 3 (spheroplasm bearing pBFDfull length glycoprotein with Flag tag, incubated with FITC-mouse anti-His IgG).
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Expression of truncated glycoprotein The ten truncated fragments of glycoprotein were cloned from pMD18-T-glycoprotein. PCR products were ligated into the prokaryotic expression vector pET-27b between the NcoI and BamHI sites. After sequence verification, the plasmids were transformed into E. coli Rosetta to generate the recombinant proteins in vitro. Western blotting of purified truncated glycoprotein proteins Purified glycoprotein fragments were detected by western blotting, with ovalbumin used as a negative control, and rabbit anti- glycoprotein pAb used as the prey. The binding activity of pAb to the antigen was visualized using ECL techniques.
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ELISA analysis For ELISA, 96-well microtiter plates were coated with 200 nM purified truncated fragments or full length glycoprotein (positive control) in 100 ll NaHCO3/ Na2CO3 buffer (pH 8.7) overnight at 4 °C. Triplicate samples were washed three times with PBS-T (0.5 %, v/v), and 5 % (v/v) skimmed milk in PBS was used to block the remaining non-specific binding sites at 37 °C for 2 h. After washing, each well was incubated with 100 ll rabbit anti-IHNV glycoprotein pAb (200fold dilution) at 37 °C for 1 h, followed by HRP-goat anti-rabbit antibody (7500-fold dilution). Full length IHNV glycoprotein incubated with negative rabbit serum was used as a negative control. The assay was developed using TMB solution and the development of color product was terminated by the addition of 50 ll 2 M H2SO4. The absorbance of each well was measured by an ELISA reader at 450 nm. The rabbit anti-IHNV glycoprotein pAb and full length IHNV glycoprotein were prepared as our previous studies (Xu et al. 2013).
Results
Fig. 4 Epitope mapping of glycoprotein by flow cytometry. M mean fluorescence intensity. Count number of spheroplasm
between M values of G23 and G3. The results indicated that there was no linear epitope in fragment G2. The M values were ranked as G14 [ G45 [ G13 [ G4 [ G23 [ G3 [ G12 [ G5 [ G1 [ G2 (Fig. 4).
Epitope prediction The amino acid sequences of the truncated glycoprotein were analyzed using DNAstar (Fig. 3). The antigenic index represents the strength of an antigenbinding. The regions containing peaks indicate potential antigenic epitopes. G1, G3, G4 and G5 contain many regions with high antigenic index, but there is only one region containing a single peak within the G2 fragment. FACS epitope analysis of glycoprotein fragments After subsequent incubation with the rabbit antiglycoprotein pAb and the FITC-conjugated goat antirabbit antibody, the spheroplasts bearing truncated glycoprotein fragments were detected by FACS. Spheroplasts bearing pBFD-IL-1b were used as a negative control. Compared with the negative control, there was an obvious increase in the mean fluorescence intensity (M value) for all fragments except G2. The M value of G12 was approximately the same with that of G1, and there were also negligible differences
Detection of the truncated glycoprotein fragments displayed in the periplasm To exclude the influence of antigen expression levels in the periplasm on FACS fluorescence intensity, a Flag tag was fused to each truncated glycoprotein fragment and cloned into pBFD display vector. Expression levels were measured by FACS. No obvious differences of mean fluorescent intensity (M) were observed from each spheraplasts containing truncated glycoprotein-Flag fusions (Fig. 5). The expression level of the truncated glycoprotein proteins in the periplasm was strong enough to be captured by corresponding antibodies. Western blotting of purified truncated glycoprotein fragments The ten truncated glycoprotein proteins were expressed as inclusion bodies in E. coli Rosetta by pET-27b (?), and were purified by denaturation and renaturation. Western blotting was conducted to
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Fig. 5 Detection of truncated glycoprotein-Flag fragments displayed in the periplasm of E. coli by flow cytometry. Blank spheroplasm without any plasmid; NC1 spheroplasm bearing
pBFD-full length glycoprotein without any tag; NC2 spheroplasm bearing pBFD-full length glycoprotein with Flag tag incubated with FITC-mouse anti-His IgG
evaluate if purified truncated proteins could bind with the rabbit anti- glycoprotein pAb. The fragments containing epitopes reacted with the rabbit antiglycoprotein pAb. The distribution of epitopes was identified by western blotting. Except for fragment G2, all fragments strongly reacted with the rabbit antiglycoprotein pAb and bands with expected size were observed (Fig. 6a). The G2 protein and the ovalbumin (negative control) failed to react with the rabbit antiglycoprotein pAb.
fragments could bind with pAb and showed much higher OD450 than that of negative control, but a little lower than that of positive control (Fig. 6b). The OD450 was ranked as PC [ G14 [ G45 [ G13 [ G4 [ G23 [ G3 [ G12 [ G5 [ G1 [ G2 [ NC. The results were accordant with FCM analysis and indicated that there were epitopes located in all fragments but G2.
Discussion ELISA analysis Binding ability of fragments to pAb was determined by ELISA assay. Except the fragment G2, all
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In our current study, bacterial display technology combined with FCM was used for mapping linear B cell epitopes of the IHNV glycoprotein. The IHNV
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Fig. 6 Immunological characterization of the purified truncated protein. a Western blotting of the purified truncated glycoprotein fragments. NC negative control; M standard protein marker. b ELISA analysis of the purified truncated glycoprotein fragments. The data represent the mean ± SD. of quadruple samples. **p \ 0.01 versus NC. NC negative control; PC positive control
glycoprotein was truncated into ten overlapped fragments, and linear B cell epitopes of these fragments were determined by FCM according to M value. Our findings clearly indicate that the fragments G1, G3, G4, and G5 react with the pAbs, suggesting that these fragments contain linear B cell epitopes. The antigenic intensity of these fragments was ranked as G4 [ G3 [ G1 [ G5. The G2 fragment contains no antigenic epitope. To validate the results we obtained from FCM, the amino acid sequences of fragments were analyzed by DNAStar. There are multiple long hydrophilic regions with high antigenic index in the G1, G3, G4 and G5 fragments, but only few short hydrophilic regions were detected in the G2 fragment. These results are in agreement with the results obtained from FCM. To validate the results further, traditional methods were employed to analyze the antigenic epitopes of these fragments. The truncated fragments were expressed in E. coli and the purified fragments were examined by western blotting and ELISA analysis. Only the fragment G2 did not react with the pAb. The results are in accordance with those obtained by the bacterial display method we established.
IHNV belongs to the genus Novirhabdovirus. Its glycoprotein is a membrane protein and contains antigenic epitopes that are responsible for eliciting neutralizing antibodies (Huang et al. 1996). The antigenic epitopes of IHNV isolated in America have been studied and mapped (Huang et al. 1996; Xu et al. 1991). The results suggests that there are two epitopes within G4, one in G5 and one in G23 (G2 fused with G3). In this study, the mean fluorescence intensity of G4 was far higher than other fragments, and these results are in accordance with previous studies. Additionally, G23 was truncated into G2 and G3 in this study. The mean fluorescence intensity of G3 was almost the same as that of fragment G23, and far higher than that of negative control and fragment G2. It indicated that there was no epitope in G2 but in G3. These data indicate that the method is valid for systematic mapping of linear B cell epitopes for a given antigen. The new strategy established in this study offers several advantages over previous epitope mapping approaches. First, the bacterial display system can be used to detect a broad spectrum of antigens, as long as the polypeptides can be expressed in E. coli. Second,
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because the bacterial display system is based on an expression plasmid, it provides an easy way to create an epitope library with a large capacity to systematically analyze a large number of antigens in a short period of time. In this study, the glycoprotein was truncated into five fragments to construct a mini library. The epitope libraries cannot be analyzed by traditional methods because of the large amount of work involved in protein expression and purification. Third, because of the larger size of the bacterial cells, the incorporation of the bacteria-display system with FCM detection and screening technology dramatically enhances the feasibility of the technology. The combination of the bacterial display system with FACS, which confers real-time and high-throughput detection and screening capacities, makes this technology an attractive method for rapid and high-throughput mapping of linear B cell epitopes for any given antigen. Epitope clusters of glycoprotein of Chinese IHNV isolate were located in this study. According to the results, truncated glycoprotein fragment with high antigenicity will be chosen to be expressed and utilized to prepare antibody for the detection of IHNV isolate prevalent in China. As with a previous study, neutralization antigenic epitopes were located in the fragment, G4, which has the strongest antigenicity as revealed in this study. Thus, fragment G4 will not only be used for antibody preparation but also a subunit vaccine for Chinese IHNV isolate. Acknowledgments This study was supported by grants from sub-project of National 12th 5-year Support Key Projects (2012BAD25B02) and the Central-Level Non-profit Scientific Research Institutes Special Funds (HSY201204).
References Breyta R, Jones A, Stewart B, Brunson R, Thomas J, Kerwin J, Bertolini J, Mumford S, Patterson C, Kurath G (2013) Emergence of MD type infectious hematopoietic necrosis virus in Washington State coastal steelhead trout. Dis Aquat Organ 104:179–195 Enzmann PJ, Kurath G, Fichtner D, Bergmann SM (2005) Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American genogroup M. Dis Aquat Organ 66:187–195 Harvey BR, Georgiou G, Hayhurst A, Jeong KJ, Iverson BL, Rogers GK (2004) Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc Nat Acad Sci USA 101(25):9193–9198
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Biotechnol Lett (2014) 36:2109–2116 Harvey BR, Shanafelt AB, Baburina I, Hui R, Vitone S, Iverson BL, Georgiou G (2006) Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression. J Immunol Methods 308:43–52. doi:10.1016/j.jim.2005.09.017 Huang C, Chien MS, Landolt M, Batts W, Winton J (1996) Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus. J Gen Virol 77:3033–3040 Jeong KJ, Seo MJ, Iverson BL, Georgiou G (2007) APEx 2-hybrid, a quantitative protein–protein interaction assay for antibody discovery and engineering. Proc Nat Acad Sci USA 104:8247–8252 Kim WS, Oh MJ, Nishizawa T, Park JW, Kurath G, Yoshimizu M (2007) Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene. Arch Virol 152:2119–2124 Kolodziejek J, Schachner O, Durrwald R, Latif M, Nowotny N (2008) ‘‘Mid-G’’ region sequences of the glycoprotein gene of Austrian infectious hematopoietic necrosis virus isolates form two lineages within European isolates and are distinct from American and Asian lineages. J Clin Microbiol 46:22–30 Li T, Xu L, Ren G, Yin C, Zhou B, Ye X, Li Q, Li N, Li D (2013) A neutralization scFv antibody against IL-1beta isolated from a NIPA-based bacterial display library. Curr Pharm Biotechnol 14:571–581 Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298 Nichol ST, Rowe JE, Winton JR (1995) Molecular epizootiology and evolution of the glycoprotein and non-virion protein genes of infectious hematopoietic necrosis virus, a fish rhabdovirus. Virus Res 38:159–173 Nishizawa T, Kinoshita S, Kim WS, Higashi S, Yoshimizu M (2006) Nucleotide diversity of Japanese isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene. Dis Aquat Organ 71:267–272 Peng WP, Hou Q, Xia ZH, Chen D, Li N, Sun Y, Qiu HJ (2008) Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of Classical swine fever virus by phage-displayed random peptide library. Virus Res 135:267–272 Rudakova SL, Kurath G, Bochkova EV (2007) Occurrence and genetic typing of infectious hematopoietic necrosis virus in Kamchatka, Russia. Dis Aquat Organ 75:1–11 Xu L, Mourich DV, Engelking HM, Ristow S, Arnzen J, Leong JC (1991) Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli. J Virol 65:1611–1615 Xu LM, Yin CK, Ren GP, Tian H, Wang XQ, Ding LJ, Li DS (2011) Establishment of bacteria-display technology for Fab antibody library screening (in Chinese). Chinese J Cell Mol Immunol 27:1090–1093 Xu LM, Liu HB, Yin JS, Lu TY (2013) Prokaryotic expression and immunogenicity analysis of glycoprotein from infectious hematopoietic necrosis virus (in Chinese). Chinese J Virol 29:529–534
