Veterinary Microbiology 174 (2014) 565–569

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Short communication

Linear epitope recognition antibodies strongly respond to the C-terminal domain of HP-PRRSV GP5 Xinglong Wang a,*, Li Qui a, Yu Dang b, Sha Xiao a, Shuxia Zhang a, Zengqi Yang a a b

College of Veterinary Medicine, Northwest A&F University, No. 22 Xinong road, Yangling 712100, China Shaanxi University of Technology, Hanzhong 723001, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 April 2014 Received in revised form 17 August 2014 Accepted 8 September 2014

A total of 155 peptides derived from the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) glycoprotein 5 (GP5) were printed on a chip to reveal the antigen reaction characteristics of the protein. The reactions of these peptides to HP-PRRSV-specific pig serum were scanned and quantified using fluorescence intensity via the PepSlide1 Analyzer software. The intensity plots showed different reactions in the different sectors of GP5. The highest reaction intensity value reached 3894.5, with a peptide sequence of IVEKGGKVEVEGHLI. Seventeen peptides that showed relatively high reaction levels with HP-PRRSV-specific pig serum were selected as epitope candidates. Furthermore, the antigenic character was predicted using a software and was compared with the peptide scan results. In contrast to the software prediction, the HP-PRRSV-specific antibodies strongly responded to the C-terminal domain of GP5. The acquired data may be useful for understanding the antigenic characteristics of HP-PRRSV GP5. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Antigenic regions Peptide scan HP-PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, is endemic in most swine-producing countries. It causes numerous problems, ranging from subclinical infections to severe reproductive failure in sows and respiratory disease in piglets. PRRSV isolates show different pathogenicities. Some exceptionally severe PRRS outbreaks with high abortion frequencies and sow mortalities have been reported in the US around 1996 (Halbur and Bush, 1997; Hurd et al., 2001). More recently, China and its neighboring countries have suffered from an extremely severe epidemic that was attributed to highly pathogenic (HP) PRRSV isolates (Li et al., 2007; Ni et al., 2012; Tian et al., 2007).

* Corresponding author. Tel.: +86 29 87091106; fax: +86 29 87091032. E-mail addresses: [email protected] (X. Wang), [email protected] (Z. Yang). http://dx.doi.org/10.1016/j.vetmic.2014.09.004 0378-1135/ß 2014 Elsevier B.V. All rights reserved.

PRRSV is a spherical, enveloped, single-stranded, positive-sense RNA virus (Benfield et al., 1992; Wensvoort et al., 1991). The genome of the virus is approximately 15 kb in length and is subdivided into nine open reading frames (ORFs). ORF1a and ORF1b, which encode vital replicase polyproteins, comprise 80% of the genome, whereas ORF2a, ORF2b, and ORFs 3–7 encode the viral structural proteins GP2, E, GP3, GP4, GP5, M, and N, respectively (Stadejek et al., 2008). GP5 is a major viral envelope protein and is the most variable of the six structure proteins. Its amino acid (aa) sequence identity between the European and North American types is only 51–55% (Indik et al., 2000; Mardassi et al., 1995). GP5 contains some epitopes involved in virus neutralization in the N-terminal ectodomain (Pirzadeh and Dea, 1997); it also contains some nonneutralizing epitopes. An immunodominant epitope (170–201 aa or 178–199 aa) was found in the C terminus of the GP5 of the European isolates (Ostrowski et al., 2002; Rodriguez et al., 2001);

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however, it was not precisely identified. Several epitopes (1–15 aa, 31–45 aa, and 187–200 aa) were identified in GP5 of the North American isolates (de Lima et al., 2006). An increasing number of studies have provided useful data for understanding PRRSV antigen recognition, but the research based on monoclonal antibodies is random and incomplete. Monoclonal antibody recognition of all the epitopes in one protein cannot be performed because of the limitations of the current technology in preparing monoclonal antibodies. Therefore, a peptide microarray using 14 overlapping peptides covering the full length of the mature GP5 was used in this study. The reaction characteristics of these peptides that reflect the antigen recognition of the liner B response of the protein were recorded.

1. Materials and methods 1.1. Virus and serum The GP5 amino sequence of the HP-PRRS virus strain SY0608 (Li et al., 2007) was used as a template. The hyperimmune SY0608 specific serum was provided by Dr P. Jiang (Nanjing Agricultural University, China). The serum was collected from pigs vaccinated with killed SY0608 virus (2 times with 2 weeks interval, 5  106 TCID 50/ml, 1:1 mixed with Montanide ISA 206) and then challenged with the same virus (5  106 TCID 50/ml). The serum was collected at 14 days post challenge. Before the immunization, the infection of the piglets by PRRSV was excluded by PCR and antibody detection. A secondary antibody from Goat anti-swine IgG (H + L) DyLight680 was provided by PEPperPRINT GmbH. 1.2. GP5 peptide chip and peptide scan A total of 155 peptides with a 14-aa length overlap covering the full length of GP5 (without a signal peptide, the first 15 peptides (CKPCFSSSLSDIKTN) were printed on the chip with 14 duplicated aa with the second, and by such analogy, these 155 peptides were designed and printed on the protein microarray chip. Therefore, each peptide had two replicates on the chip. Surrounding the target peptide points were the interval FLAG peptides DYKDDDDKGG and HA Peptide YPYDVPDYAG as controls. The peptide chip was blocked with Rockland blocking buffer MB-070 for 60 min prior to the first assay. Prestaining of the peptide array was performed using the secondary goat anti-swine IgG (H + L) DyLight680 antibody at a dilution of 1:5000 to investigate any background interactions that could interfere with the subsequent main assays. Subsequent incubation of the peptide array with HP-PRRSV SY0608 specific serum at a dilution of 1:1000 in an incubation buffer (PBS, pH 7.4 with 0.05% Tween 20 and 10% Rockland blocking buffer) was followed by staining with the secondary goat anti-swine IgG (H + L) DyLight680 antibody, which was then read using the scanning intensities of 5 and 7 (red). Quantification of spot intensities and peptide annotations were performed using the PepSlide1 Analyzer.

A software algorithm was used to break down the fluorescence intensities of each spot into raw, foreground, and background signals, and to calculate the standard deviation of the foreground median intensities. The foreground intensities were the differences of the raw intensities and the background intensities, except these minus values and which were marked as zeros. Averaged spot intensities for prestaining with the secondary antibody and the main assay with the pig serum against the GP5 sequence from the N to the C terminus were plotted to better visualize overall spot intensities and signal to noise ratios. Fluorescence Intensity plots was prepared using Origin.Lab.Origin.V8.0 software. 1.3. Antigenic analysis using software The antigenic character of SY0608 GP5 was analyzed using the method by Kolaskar and Tongaonkar (Kolaskar and Tongaonkar, 1990) given by bioinformatics serve of the Immunomedicine Group of the Universidad Complutense de Madrid (http://imed.med.ucm.es/Tools/antigenic.pl).

2. Results 2.1. Peptide scan The peptide array was prestained using the secondary goat anti-swine IgG (H + L) DyLight680 antibody at 1:5000 dilution to investigate any background interactions that could interfere with the subsequent main assays. The subsequent incubation of the peptide array with pig serum at a dilution of 1:1000 in the incubation buffer was followed by staining with the secondary goat anti-swine IgG (H + L) DyLight680 antibody. The results were determined at scanning intensities of 5 and 7 (red). Spot fluorescence intensity was quantified, and peptide annotation was conducted using the PepSlide1 Analyzer and listed in an Excel file named ‘‘MicroarrayData_GP5_PigSerum.xlsx.’’ The values are provided in the Supplementary data. A software algorithm was used to break down the fluorescence intensity of each spot into raw, foreground, and background signals (Supplementary data ‘‘Mapping Raw Data’’ tabs) and to calculate the standard deviation of the foreground median intensities (Supplementary data ‘‘Mapping Summary’’ tab). The average spot fluorescence intensity of the prestaining with the secondary antibody was plotted to visualize the overall spot fluorescence intensities (Background Value, BV, Fig. 1). The main assay using pig HP-PPRSV-specific serum against the GP5 sequence from the N to the C terminus was also plotted (Main Assay Valued, MV, Fig. 1). Three reaction active regions were observed on the fluorescence intensity plot covering 32–55 aas, 73–93 aas, and 140–200 aas of GP5. These sites were overlapped with these peptides 1– 24, 42–62 and 109–155 (Fig. 1). HA and FLAG control peptides framing the peptide microarray were subsequently stained and scanned at a high intensity. Their homogeneous staining of the controls shows that the chip was evenly dyed (data not shown).

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antigenic characters. The antigenic plot of HP-PRRSV GP5 was obtained from their website. As shown in Fig. 2, the antigenic determinant was relatively concentrated in the region covering 29–98 aa. The average antigenic propensity of the N-terminal endodomain of GP5 was lower than that of the C-terminal endodomain. 3. Discussion

Fig. 1. Data quantification was followed by generation of intensity plots for prestaining (Red line) and main assay (Grey line). In accordance with microarray scan and intensity map, strong and polyclonal response of pig serum was observed with overlapping peptides in the C-terminal endodomain of GP5. Relatively low intensities were found in prestaining, although several sites show low affinity reaction with secondary antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.2. Epitope candidates in GP5 Based on the scanning results, these peptides with fluorescence intensity higher than 1000 were picked out and used as epitope candidates. The results of the chosen peptides, their fluorescence intensity values, and the BVs are shown in Table 1. Even though some epitopes overlapping with those reported ones, some new epitopes may be harbored in these candidates, as no epitope has been reported in the sites overlapping with peptides ‘‘78SYGALTTSHFLDTV92G’’,‘‘154RWRSPVIVEKGGKV169E’’and ‘‘LDGSAATPLTRVSAE’’, which showed relative higher fluorescence intensities in our experiment. 2.3. Antigenic analysis using software The full length of the SY0608 GP5 aa sequence was submitted to the Immunomedicine Group of the Universidad Complutense de Madrid for them to analyze its

The major envelope protein GP5 (ORF5) contains 200 aas and its molecular weight is approximately 25 kDa (Meulenberg et al., 1995). It contains a 32-aa signal peptide, and this signal peptide is probably cleaved off during translocation. After the signal peptide, a short putative ectodomain of 30 aa is present. Two, three, or four putative N-glycosylation sites occupied by complex-type glycans were found in this region. Viruses tend to evolve more glycosylation sites in their ectodomains (Costers et al., 2010). An increasing number of PRRSV isolates with three or four N-glycosylation sites have been collected from clinical samples. Following the putative GP5 ectodomain is a long stretch of hydrophobic residue (63–127 aa). This region can be subdivided into one, two, or three membrane-spanning helices based on structural analysis. The long C terminus is the GP5 endodomain when the protein spans the membrane once or thrice, whereas this sequence determines a second ectodomain, when the protein spans the membrane twice. GP5 and Matrix Protein form disulfide-linked heterodimers in the viral envelope (Mardassi et al., 1996). These heterodimers are important in both virus infection and immune protection. Glycosylation and complex structure probably compromise antibody accessibility to the fully processed protein (Ansari et al., 2006), which is not surprising for the low level of reactivity of the protein. The antigenic characters of different PRRSV isolates vary from each other probably because of the numbers and locations of N-glycosylation sites. NA- and EU-type viruses have different GP5-specific antibody responses; for example, NA-type PRRSV antibodies are produced against both the predicted C-terminal endodomain and the N-terminal

Table 1 Epitope candidates in GP5. N-aa sites

Peptides

C-aa sites

MV

BV

Noise radio

35 51 53 78 148 154 156 157 158 160 161 162 165 166 181 182 183

NSSHIQLIYNLTLCE NGTDWLAQKFDWAVE TDWLAQKFDWAVETF SYGALTTSHFLDTVG TKGRLYRWRSPVIVE RWRSPVIVEKGGKVE RSPVIVEKGGKVEVE SPVIVEKGGKVEVEG PVIVEKGGKVEVEGH IVEKGGKVEVEGHLI VEKGGKVEVEGHLID EKGGKVEVEGHLIDL GKVEVEGHLIDLKRV KVEVEGHLIDLKRVV LDGSAATPLTRVSAE DGSAATPLTRVSAEQ GSAATPLTRVSAEQW

39 65 67 92 162 169 170 171 172 174 175 176 179 180 195 196 197

1405.0 1631.5 1480.0 1082.5 2065.0 1014.0 2595.0 1516.0 1243.0 3894.5 3736.5 2865.0 1703.0 2729.5 1586.0 1475.0 1013.0

122.0 107.5 23.0 160.5 331.0 67.0 124.0 176.0 65.0 328.5 370.5 150.0 4.0 348.5 64.0 112.0 20.0

8.7% 6.6% 1.6% 14.8% 16.0% 6.6% 4.8% 11.6% 5.2% 8.4% 9.9% 5.2% 0.2% 12.8% 4.0% 7.6% 2.0%

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Fig. 2. Antigenic plot of HP-PRRSV GP5 obtained by bioinformatics analysis. The antigenic determent was relatively concentrated in the region between 29 and 98 aa.

ectodomain. Studies on the antibody response against EUtype viruses show that only antibodies against the GP5 C terminus are produced (de Lima et al., 2006; Mulupuri et al., 2008; Rodriguez et al., 2001). In the present study, peptides in both the C-terminal endodomain and the Nterminal ectodomain reacted with the HP-PRRSV-specific pig serum. This finding is consistent with previously reported results using Escherichia coli-expressing GP5 from NA-type PRRSV (Zhou et al., 2009). Some new characteristics of HP-PRRSV GP5 were observed: the C terminus of GP5 more strongly responds to the pig serum because high levels of fluorescence intensity and antigenic determinant density compared with the N terminus were observed. In previous research, a high level of antigenic response was reported in the C terminus of GP5 of the EU-type PRRSV. Three linear antigenic regions (ARs) were identified. Two of these ARs were refractory to antibody neutralization, although they were expected to be nonneutralizing epitopes (Vanhee et al., 2011). In this study, seventeen peptides located from 35–197 aa sites were identified in this research as epitope candidates. These epitope candidates may be used in further research for epitope identification or diagnosis. Previous epitope studies of PRRSV mainly focused on the N-terminal ectodomain of GP5. Some neutralizing MAbs against the North American type PRRSV were generated and one neutralizing epitope (37–45 aa) was identified in the middle of the GP5 ectodomain (Ostrowski et al., 2002; Plagemann et al., 2002). In a more recent research; three minimal epitopes (R152LYRWR156 E169GHLIDLKRV178 and Q196WGRL200) were identified in the C terminus of the North American type PRRSV isolated in China, using these MAbs and overlapping peptide series. The lengths of the epitopes were mostly 7–9 aas long (range: 4–12) as reported in a previous study using peptide scan (Buus et al., 2012). This was also consisted with previous epitope study on GP5, as eptiope A contains 4 core aas (27A/(V)L28V29N30), eptiope B contains 5 aas (H38 and I43Y44N45), and the other epitope were 6, 10, or 5 aas in length. Three in 5 of the epitopes identified in previous reports were overlapped with the peptides giving in Table 1. One of the other two sits in signal peptide and another is at the C terminal end of the GP5. Comparing the fluorescence

intensities of these peptides harboring the identified epitope with the others, their fluorescence intensities were not the highest. By this analogy, some other important epitopes may be contained in those peptides. Peptides ‘‘NGTDWLAQKFDWAVE’’, ‘‘SYGALTTSHFLDTVG’’, ‘‘RSPVIVEKGGKVEVE’’, and ‘‘LDGSAATPLTRVSAE’’ have no overlapped reported epitopes and they show higher fluorescence intensities. We are sure that new epitopes can be indentified in these peptides in future. Although further research is needed before we can obtain the exact sites of the new predicted epitopes, we believe this is one of the largest collections of fully substituted antibody epitope mappings reported on American type PRRSV GP5. Protein structure prediction (Moult et al., 2011) and antigen region analysis (Lancaster et al., 2007) have become basic technologies in protein studies with the development of bioinformatics in recent years. An increasing number of biology software have been developed for antigen character analysis (Burland, 1999; Lancaster et al., 2007). In addition, several websites provide free antigen forecasting services. The antigenic propensity of HP-PRRSV GP5 was predicted using the service provided by the Immunomedicine Group (http://imed.med.ucm.es/ Tools/antigenic.pl). Three antigenic determinant groups are shown on the antigenic propensity plot (Fig. 2). These antigenic determinants are located in the C-terminal endodomain or in the middle. However, the average antigenic propensity of the N-terminal ectodomain in GP5 is relatively low. These results contradict those from the peptide scan. These differences may be caused by protein glycosylation or the specified structure of GP5. Protein glycosylation (Faaberg et al., 2006; Jiang et al., 2007) and immune decoy epitopes (Lopez and Osorio, 2004; Ostrowski et al., 2002) are identified as immune evasion strategies used by the GP5 of PRRSV (Costers, 2008). The N-glycans in the GP5 of NA-type PRRSV were involved in shielding neutralizing epitopes. This finding is consistent with the fact that NA-type GP5 is targeted by virus-neutralizing antibodies (Pirzadeh and Dea, 1997). The GP5 of SY0608 contains four predicted N-glycans at 30, 35, 44, and 51 aa sites (http://www.cbs.dtu.dk/services/ NetNGlyc/). The peptides starting from the 35 and 51 aa sites respond to the PRRSV serum and the first peptide ‘‘NSSHIQLIYNLTLCE’’ containing one identified epitope

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(SSHIQLIYNLT). N-terminal glycosylation may be the reason for the low antigenic propensity of the GP5 Nterminal. This phenomenon may also be the reason for the different results between the software prediction and the actual response. 4. Conclusion The GP5 antigen reaction panorama of HP-PRRSV created using the peptide scan method demonstrated strongly B cells respond happen to the C-terminal domain of HP-PRRSV GP5. These conflicted with software prediction results. This difference may arise from the complex structure or protein glycosylation of GP5. In addition, seventeen epitope candidates arose in GP5, which can be used for further epitope identification. Acknowledgments Project supported by the National Natural Science Foundation of China (Grant No. 31302122) and Basic Research Universities Special Fund Operations (QN2011106). We thank Dr. Jiang for providing the SY0608 specific pig serum.

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