A B-cell epitope on the bovine herpesvirus 1 (BoAHV1) viral protein gC, referred to as PV116, is implicated in the generation of antibodies and the development of an ELISA kit for the detection of the virus antibody

Abstract

BoAHV1 is a significant cattle pathogen, resulting in substantial economic losses worldwide. B-cell epitopes within the viral proteins are not well understood. In this study, we screened a 12-mer phage display peptide library using a commercial BoAHV1 antibody. A total of 16 phage clones displaying individual 12-mer peptides were identified as reactive with this antibody using dot bot analysis. Through sequence alignment, 10 putative BoAHV1 B-cell epitopes, designated as PV109, PV108, PV116, PV59, PV50, PV130, PV113, PV49, PV62, and PV133, located within viral proteins gB, gC, gG, gM, UL36, UL37, and UL49, respectively, were recognized by both commercial BoAHV1 antibody andBoAHV1 IgG positive cattle serum through dot blot assay. Interestingly, immunization of mice with the synthesized peptide PV116 led to the production of antibodies suitable for Western blot analysis. Furthermore, an ELISA kit for the detection of BoAHV1 IgG antibodies in serum was developed, utilizing the identified epitope PV49, PV108, PV109, and PV116 as coating antigen. Collectively, we have identified 10 novel B-cell epitopes ofBoAHV1. Among them, PV116 is capable of inducing the production of antibodies suitable for Western blotting assay, which also shows potential for the development diagnostic tools.

Introduction

Bovine alphaherpesvirus type 1 (BoAHV1) is an enveloped DNA virus that belongs to the genus Varicellovirus in the subfamily Alphaherpesvirinae under the family Herpesviridae [1,2,3]. The virus can infect cattle of all breeds and is capable of causing diseases at any ages. Among the diseases induced by the virus infection are life-threatening pneumonia known as bovine respiratory disease complex (BRDC). In addition, conjunctivitis, vulvovaginitis, meningoencephalitis, and abortion induced by virus infection are also commonly observed [1, 4, 5]. The virus is now prevalent globally and poses a significant economic burden on the cattle industry. For instance, it is estimated to cost the US cattle industry approximately 3 billion dollars each year [6].

An antigenic epitope, also known as an antigenic determinant, is the specific portion of an antigenic molecule recognized by the immune system, including antibodies, B-cell receptors, or T-cell receptors. Virus B-cell epitopes are crucial antigenic elements of viral proteins that are recognized by either B-cell receptors or antibodies. The elucidation of viral epitopes is vital for understanding virus pathogenesis, the development of potential diagnostic tools, and the generation of epitope-based vaccines [7]. Currently, several B-cell epitopes in BoAHV1 have been identified. For example, using synthesized peptides and a panel of monoclonal antibodies, B-cell epitopes including ²³APRVTVYVD³¹ and ³²³GEPKPGPSPDADRPE³³⁷ within the gD protein have been identified [8, 9]. Through the use of recombinant truncated proteins expressed by baculovirus vectors and synthetic peptides, three B-cell epitopes including ³³¹QQIEGYYKRDMATGRRLKEPV³⁵², ⁴⁷⁵YLQELARSNGTLEGLFAAAAP⁴⁹⁶, and ⁴⁸⁷EGLFAAAAPKPGPRRARRAAP⁵⁰⁸ have been mapped to glycoprotein gB [10]. By screening a phage-displayed peptide library using monoclonal antibodies (mAbs) against BoAHV1 gE, one B-cell epitope located at the C-terminus of the gE protein (amino acids 561–569) has been characterized [11]. The viral genome comprises a 106 kb unique long segment (UL) and a 10 kb unique short region (US), which is flanked by inverted repeats of 11 kb [12]. This genome encodes at least 73 open reading frames (ORFs) that have been identified [13]. We speculate that the virus contains more B-cell epitopes than those already revealed as described above.

Random peptide library displayed on the surface of a filamentous bacteriophage, provides a powerful mean to identify a target peptide recognized by an antibody [11, 14]. Although the amino acid compositions of selected peptides are not completely identical to the natural epitopes in the targets [11], they are able to functionally mimic continuous or discontinuous epitopes, thus are called ‘‘mimotopes’’ [11, 15].

We hypothesize that the peptide backbone is crucial and can sometimes substantially contribute to the recognition by antibodies. Consequently, using the amino acid compositions of mimotopes identified from a phage display library, we can deduce putative epitopes that map to a target viral protein with the aid of alignment software. Thereby, the deduced viral epitope may potentially function as a candidate B cell epitope. In this study, we screened a phage-displayed peptide library taking advantage of a commercial available goat anti-BoAHV1 serum (VMDR, IncPullman, WA, USA). Consequently, 10 potential B-cell epitopes, designated as PV109, PV108, PV116, PV59, PV50, PV130, PV113, PV49, PV62, and PV133, were identified through dot blot assays. Notably, PV116 conjugated to the KLH carrier demonstrated the ability to elicit an antibody production in mice. Furthermore, an indirect ELISA kit for the detection of BoAHV1 antibodies in clinical serum was developed, utilizing the identified B-cell epitope including PV49, PV108, PV109, and PV116 as coating antigens.

Materials and methods

Virus and cells

A549 and MDBK cells were purchased from the Chinese Model Culture Preservation Center in Shanghai, China. They were routinely passaged and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT, USA). Cells with fewer than ten passages were used for this study. BoAHV1 strain NJ-16-1 was isolated from commercial bovine semen [16]. To propagate the virus, monolayer MDBK cells in 100 mm dishes were infected with the virus at a multiplicity of infection (MOI) ranging from 0.01 to 0.1. After a 2-hour (h) incubation period, the inoculum was replaced with fresh DMEM containing 2% FBS. Following a 48 h infection, the virus-infected cell cultures were subjected to freeze-thawing twice and then centrifuged at 12,000 rpm for 10 min to pellet the cellular debris. Subsequently, the clarified virus stocks were aliquoted (in a volume of 100 µL) and stored at -80 °C for future use.

Antibodies and peptides

Goat anti-BoAHV1 serum (cat# PAB-IBR) generated by immunization of goat with purified BoAHV1 viral particles was ordered from VMDR Inc (Pullman, WA, USA). HRP- (horseradish peroxidase-) conjugated goat anti-mouse IgG (cat# BF03001) and HRP-conjugated rabbit anti-bovine IgG (ca# BF03018) was purchased from Biodragon Technology (Suzhou, China). HRP- conjugated donkey anti-goat IgG (ca# ab97110) was provided by Abcam (Cambridge, UK).

Synthetic peptides were provided by GL Biochem Ltd (Shanghai, China) with purity greater than 98% determined by HPLC and mass spectrometry. Peptides were conjugated to a carrier protein of keyhole limpet hemocyanin (KLH) (Sigma, St. Louis, MO, USA) using a hetero-bifunctional cross-linker Sulfo-SMCC (Pierce Biotechnology Inc., Rockford, USA) according to the manufactures’ instructions as we described previously [7].

Ph.D.-12 phage display library

The Ph.D.-12 peptide library kit (Cat # E8110) used for this panning was provided by New England Biolabs (Ipswich, MA, USA). This library is constructed by N-terminal fusion of random dodecapeptides to the minor coat pIII protein of M13 phage vector. The titer of the library is 1 × 10¹³ pfu/mL.

Surface panning of Ph.D.-12 phage display library with a BoAHV1 polyclonal antibody

The phage library panning was performed following the manufacture’s protocol with modification as described elsewhere [7]. In brief, 60 mm dishes were coated with commercial goat anti-BoAHV1 serum (1:100 dilution) in 0.1 M NaHCO3 (pH 8.6) overnight at 4^oC. Then the dishes were blocked with 5 mg/mL BSA in 0.1 M NaHCO₃ (pH 8.6) for 2 h at 4^oC. After rapid washing 6 times with TBST (TBS + 0.1% [v/v] Tween-20), the dishes were incubated with 1 × 10¹¹ clones of the phage from Ph.D.-12 phage display library in 1 ml of TBST for 30 minutes at room temperature with gentle rocking. Unbound phages were washed away by 10 times of washing with TBST at room temperature. Bound phages were eluted with 1 ml of 0.2 M Glycine-HCl (pH 2.2) containing 1 mg/mL BSA with gentle rocking for 15 minutes, then neutralized with 150 µL of 1 M Tris-HCl, pH 9.1. Recovered phages were amplified by incubation with 20 ml of E. coli ER2738 (OD600 0.01–0.05) with vigorous shaking for 4.5 h at 37^oC. The amplified phages, were purified by precipitation with 1/6 volume of 20% PEG/2.5 M NaCl overnight at 4 ^oC, and centrifugation for 30 min at 10,000 g. The purified phages were taken over additional binding/amplification/purification cycles to enrich the pool in favor of binding sequences. After 3 rounds of panning, over 100 individual clones are characterized by DNA sequencing with primer 28gIII primer (5’-GTA TGG GAT TTTGCTAAA CAAC-3’).

Sequence analysis

The inserted DNA sequences were analyzed using the Editseq module of DNASTAR software. Sequence alignments were conducted with the free software DNAMAN7.0 and the online BLAST program. Representative amino acid sequences of BoAHV1 viral proteins were sourced from the virus reference strain with the GenBank Accession No. AJ004801.1 for sequence analysis.

Western blot

Cell lysates were transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% non-fat milk in PBST for 1 h at room temperature, the membranes were incubated with our generated antibody overnight at 4^oC, and HRP-conjugated goat anti-mouse IgG (at a dilution of 1:9000) for 1 h at room temperature. After extensive washing with PBST, the reactive protein bands were developed using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA, cat# 1705061).

Dot blot

Purified phages, purified BoAHV1virions as described [17], synthetic peptides-conjugated to KLH, and KLH solution were dropped onto 0.2 μm pore-size nitrocellulose (NC) membrane (Bio-Rad, Hercules, CA, USA, cat # 10600001) within individual cycled markers, respectively. After drying, the membrane was blocked with 5% non-fat milk (Cell Signaling Technology, Danvers, MA, USA, cat# 9999) for 1 h at room temperature, incubated with either goat anti-BoAHV1 specific antibody (VMDR Inc, Pullman, WA, USA, cat# PAB-IBR) or clinical cattle serum overnight at 4^oC, then donkey anti-goat IgG-HRP (dilution 1:8000) or goat anti-bovine IgG-HRP (dilution 1:8000) for 1 h at room temperature. The immune reactive dots were detected using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA, cat# 1705061). The clinical cattle sera were provided by the Center for Animal Disease Control and Prevention of Hebei Province, collected from local farmers for the routine screening of specific diseases.

Immunization of mice

Animals care and study procedure were following the guideline of the Animal Research Ethics Board of Hebei University (Approval number 2022–009102) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the National Research Council. Mice were purchased from Company of Charles River (Beijing, China). Four groups of Balb/c female mice (n = 3) at six-week old were anesthetized with inhaled isoflurane (4% induction and 2% maintenance), then immunized subcutaneously with 30 µg of KLH-conjugated individual peptides (containing approximately 13.6 µg peptides), including PV116-KLH, PV49-KLH, PV108-KLH, and PV109-KLH, emulsified in complete Freund’s adjuvant (dilution volume of 1:1). A dose of 30 µg KLH-conjugated individual peptides in a volume of 100 µL was selected for immunization because this dose is able to induce antibody production in mice, as referenced in other literature [7]. After two weeks, they received a booster immunization subcutaneously with 30 µg of KLH-conjugated peptides emulsified in incomplete Freund’s adjuvant (in a volume of 100 µL) (Sigma-Aldrich, St. Louis, MO, USA). At four weeks post the second booster, the animals were humanely euthanized via CO2 inhalation for the collection of sera for subsequent studies.

Of note, peptides are incomplete antigens of small molecules. When existing alone, they cannot induce an immune response due to their lack of immunogenicity. However, when conjugated or linked (usually through covalent bonds) with large molecular weight proteins or non-antigenic carriers, they can acquire immunogenicity, thereby inducing an immune response. Thus peptides conjugated to KLH carriers were used for this immunization.

Results

Panning of phage-displayed peptide library using commercial available BoAHV1-specific polyclonal antibody

To identify BoAHV1 specific epitopes, a phage-displayed random-dodecapeptide library Ph.D.-12 was screened using a commercial BoAHV1-specific antibody (VMDR Inc, Pullman, WA, USA, cat# PAB-IBR) with a protocol as we described previously [7]. We have previously demonstrated that this antibody can effectively detect virion-associated proteins using either Western blot or immunofluorescence assays [18,19,20]. After three rounds of consecutive biopanning, the number of selected phages was increased from 5.1 × 10⁵ pfu after the first panning to 2.3 × 10⁷ pfu after the third panning, and the recovery rate was increased from 5.1 × 10^− 6% in the first panning to 2.3 × 10^− 4% in the third panning (Table 1), suggesting that the antibody-bound phages are enriched as expected.

Table 1 Enrichment of selected phages by affinity panning. 1 × 10¹¹ pfu (input) of phage library was employed for binding to a coatedBoAHV1 specific antibody in each found of panning. The eluted phages for each round of panning were tittered as output. Recovery rate was calculated using the formula (input/output) %

After the third round of affinity biopanning, approximately 150 individual phage clones were randomly selected for DNA sequencing. Subsequently, the deduced individual peptide sequences were aligned with those of BoAHV1-encoded proteins using either the online tool Protein BLAST or the free software DNAMAN7.0. Among these, 16 phage clones were identified that presumably contain putative peptides with high similarity to known viral proteins. These deduced peptides were denoted by PP46, PP49, PP50, PP59, PP62, PP64, PP65, PP71, PP76, PP104, PP108, PP109, PP113, PP116, PP130, and PP133, respectively (Fig. 1). Correspondingly, these phage clones displaying individual peptides were designated as Phage-P46, Phage-P49, Phage-P50, Phage-P59, Phage-P62, Phage-P64, Phage-P65, Phage-P71, Phage-P76, Phage-P104, Phage-P108, Phage-P109, Phage-P113, Phage-P116, Phage-P130, and Phage-P133, respectively (Fig. 2). Of note, as shown in Fig. 1, we not only illustrate the derivation of viral peptides through sequence alignment but also present the amino acid sequences of individual peptides, especially those synthesized for further studies. To enhance solubility, some peptides with fewer than 12 amino acids were synthesized for subsequent research. This is why they are depicted as being shorter than 12-mers.

Fig. 1

Analysis of the selected peptide sequences displayed on identified phages. The amino acid sequences of individual peptides were aligned with those of viral proteins using the online program Protein BLAST or the free software DNAMAN7.0. Homologous amino acids and consensus sequences were highlighted in red

Fig. 2

The identification of selected peptides displayed on phages with commercial available goat anti-BoAHV1 serum. Nitrocellulose (NC) membranes were coated with purified phages denoted by Phage-P46, Phage-P49, Phage-P50, Phage-P59, Phage-P62, Phage-P64, Phage-P65, Phage-P71, Phage-P76, Phage-P104, Phage-P108, Phage-P109, Phage-P113, Phage-P116, Phage-P130, and Phage-P133, respectively. Purified virions and a phage expressing an irrelevant peptide recognized by BVDV antibody were used as positive and negative controls, respectively. After blocking with 5% non-fat milk in PBS, the membranes were incubated with goat anti-BoAHV1 serum (1:5000), followed by incubation with donkey anti-goat IgG-HRP (1:8000). The immune reaction was developed using Clarity Western ECL substrate. The images shown are representative of two independent experiments

These 16 phage clones were individually purified, coated onto a nitrocellulose (NC) membrane, and then subjected to dot blot assays using a commercially available BoAHV1-specific antibody (VMDR Inc, Pullman, WA, USA, cat# PAB-IBR). The purified virions and an M13 phage clone expressing a peptide P3 that recognized by BVDV(bovine viral diarrhea virus)-specific antibody as described elsewhere [7], were used as a positive and negative control, respectively. As shown in Fig. 2, these 16 phages clones but not the irrelevant phage P3 could be probed by the antibody, suggesting that this antibody specifically interact with these peptides rather than the vector M13 phages. Therefore, the peptides displayed on M13 phages, including PP46, PP49, PP50, PP59, PP62, PP64, PP65, PP71, PP76, PP104, PP108, PP109, PP113, PP116, PP130, and PP133, respectively are potential BoAHV1 mimotopes. The amino acid sequences of these peptides are depicted in Fig. 1. The amino acid sequences of peptides PP46, PP49, PP64, and PP71 show high homology to the viral protein gG, as illustrated in Fig. 1A, B, F, and H, respectively. Peptides PP50, PP62, PP76, and PP133 exhibit homology to the viral protein UL36, as seen in Fig. 1C, E, I, and P, respectively. PP59 is homologous to the viral protein UL37, as depicted in Fig. 1D. PP65 is homologous to the viral protein gH, shown in Fig. 1G. PP108 and PP109 are homologous to the viral protein gB, as demonstrated in Fig. 1K and L, respectively. PP113 is homologous to the viral protein UL49, as indicated in Fig. 1M. PP116 is homologous to the viral protein gC, displayed in Fig. 1N. PP130 is homologous to the viral protein gM, shown in Fig. 1O. Lastly, PP104 is homologous to the viral capsid protein, as demonstrated in Fig. 1J.

The identification of B-cell epitopes in BoAHV1 viral proteins

The identified potential BoAHV1 mimotopes recognized by this BoAHV1-specific antibody are expressed on the surface of M13 phages through fusion to the minor coat protein pIII. We investigated whether the recognition of these potential mimotopes by the antibody is dependent on the fusion context of the minor coat protein pIII. Additionally, we wondered whether these peptides derived from viral proteins have the potential to be B cell epitopes. For this purpose, peptides including PP46, PP49, PP59, PP64, PP109, PP116, PV46, PV50, PV59, PV62, PV64, PV49, PV108, PV109, PV113, PV116, PV130, and PV133 were chemically synthesized, conjugated to the vector KLH) and subjected to dot blot analysis using commercial BoAHV1 serum (VMDR Inc, Pullman, WA, USA). To be notice that the remaining peptides were not characterized due to poor solubility. As a result, among all the synthesized mimotopes, only PP116 was recognized by the antibody and demonstrated a reliable signal (Fig. 3E). The remaining mimotopes, including PP49, PP59, PP109, and PP64, exhibited faint signals (Fig. 3B, C, and F). Clearly, the peptides displayed on the phages are recognizable by the same antibody. However, this recognition is lost when the peptides are conjugated to the KLH vector. This discrepancy arises because the mimotopes, when fused to the phage coat protein, present a different antigenic background compared to when they are conjugated to the KLH vector. Thus, it seems that the recognition of these potential mimotopes by the antibody is dependent on the fusion context of the minor coat protein pIII.

Fig. 3

The identification of synthesized peptides conjugated to KLH by dot blot using commercial available anti-BoAHV1 serum. (A) The peptides of interest, including PP46, PP49, PP59, PP64, PP109, PP116, PV46, PV50, PV59, PV62, PV64, PV49, PV108, PV109, PV113, PV116, PV130, and PV133, were commercially synthesized, conjugated to the carrier KLH, and dot-loaded onto NC membranes. Purified virions and the vector KLH were used as positive and negative controls, respectively. Approximately 3.5 µg of total proteins were loaded onto each blot. After blocking with 5% non-fat milk in PBST, the membranes were incubated with commercially available goat anti-BoAHV1 antibody (1:5000), followed by incubation with donkey anti-goat IgG-HRP (1:8000). The immune reaction was developed using Clarity Western ECL substrate. The images shown are representative of three independent experiments

Peptides PV46 and PV64 showed faint signals relative to the positive controls of purified virions in the dot blot assay (Fig. 3A and F). Of note, ten peptides with amino acid sequences derived from viral proteins, including PV59 (Fig. 3B), PV109 (Fig. 3C), PV50, PV108, PV130, and PV113 (Fig. 3D), PV116 (Fig. 3E), PV49, PV62, and PV133 (Fig. 3F), were readily detected by this antibody and exhibited strong signaling intensity comparable to that of the positive control of purified virions. Therefore, these ten peptide were potential BoAHV1 B cell epitopes and warrant further analysis with BoAHV1 antiserum from cattle.

The identified B-cell epitopes can be recognized by BoAHV1 antiserum from cattle

To ascertain whether these identified putative B-cell epitopes can be recognized by BoAHV1 antibody from cattle, we conducted additional analyses using clinical bovine serum samples. Specifically, we obtained BoAHV1 IgG positive serum from two cattle, designated as 72 and 189,119, and BoAHV1 IgG negative serum from one cattle, designated as 200,178. These sera were sourced from 2 to 3 year-old cattle that had not received BoAHV1 vaccinations. They were provided by a local dairy farmer and collected by the Center for Animal Disease Control and Prevention of Hebei Province for routine screening of specific diseases. The antibody levels were confirmed using a commercial BoAHV1 IgG indirect ELISA kit (BioStone Animal Health, Southlake, TX, USA, with cat# 10074-05) (Fig. 4A). To further validate these serum samples, we used dot blot analysis with BoAHV1 virions as the coating antigen, and mock-infected cell lysates (MICL) as a negative control. Consequently, the virions were recognized by the positive serum samples from cattle 72 and 189,119 (Fig. 4B and C), but not by the negative serum 200,178 (Fig. 4D). In addition, the virions were recognized by the positive control antibody but not by the negative control antibody provided by the ELISA kit (BioStone Animal Health, Southlake, TX, USA, with cat# 10074-05) (Fig. 4E and F). These findings corroborate the results obtained with the ELISA kit. Hence, these serum samples were utilized for subsequent dot blot analysis.

Fig. 4

The detection of BoAHV1 IgG levels in cattle serum. (A) The serum samples, originally labeled as 72, 189,119, and 200,178, provided by a local dairy farmer, were tested using the AsurDx ™ Infectious Bovine Rhinotracheitis (IBR) gB Antibody ELISA Test Kit (BioStone Animal Health, Southlake, TX, USA, cat# 10074-05). The positive and negative antibody controls were included with the ELISA kit. (B-F) Purified virions and mock-infected cell lysates of MDBK cells (MICL) of approximately 3.5 µg of total proteins were dot-loaded onto NC membranes. After blocking with 5% non-fat milk in PBST, the membranes were incubated with sera 72 (1:3000), 189,119 (1:3000), 200,178 (1:3000), as well as the positive (1:1000) and negative (1:1000) antibody controls included with the ELISA kit, respectively, followed by incubation with rabbit anti-bovine IgG-HRP (1:8000). The immune reaction was developed using Clarity Western ECL substrate. The images shown are representative of three independent experiments

As investigated by using commercial available BoAHV1 serum, peptides PP46 and PV46 were not well recognized by sera from cattle 72 and 189,119 (Fig. 5A and G). The mimotope PP59 was recognized by serum 72 (Fig. 5B), but not by serum 189,119 (Fig. 5H). Notably, the titers of BoAHV1 IgG are much higher in serum sample 72 compared to 189,119 (Fig. 4A), which further suggests that the mimotope PP59 is not well recognized by the serum and corroborates the findings as performed using commercial available BoAHV1 antibody (Fig. 3B). It is an interesting issue to explore whether No. 189,119 partially recognizes non-conserved amino acids between PP59 and PV59, which warrants extensive studies in the future. Epitopes derived from viral proteins and conjugated to the vector KLH, such as PV59 (Fig. 5B and H), PV109 (Fig. 3C and I), PV50, PV108, PV130, and PV113 (Fig. 3D and J), PV116 (Fig. 3E and K), PV49, PV62, and PV133 (Fig. 3F and L), were unanimously recognized by both sera from cattle 72 and 189,119. Peptides PV46 and PV64 showed faint signals, when probed with cattle serum 72 relative to the positive controls of purified virions in the dot blot assay (Fig. 5A and F). As expected, all these peptides conjugated to the vector KLH were not readily recognized by the negative serum 200,178 (Fig. 5M-R). The virions, but not the KLH vector along were readily recognized by the positive serum from cattle of both 72 and 189,119 (Fig. 5). Thus, these identified B-cell epitopes including PV59, PV109, PV50, PV108, PV130, PV113, PV116, PV49, PV62, and PV133 are capable of being recognized by BoAHV1 antibody positive cattle serum regardless of the fusion contexts, albeit with varying efficacy. These findings are consistent with observations conducted using the commercial antibody as depicted in Fig. 3. However, without conjugation to the KLH carrier, these peptides alone could not be recognized by either commercially available anti-BoAHV-1 or BoAHV-1 antibody positive serum from cattle (data not shown).

Fig. 5

The identification of synthesized peptides conjugated to KLH by dot blot using BoAHV1 IgG positive cattle serum. (A) The peptides including PP46, PP49, PP59, PP64, PP109, PP116, PV46, PV50, PV59, PV62, PV64, PV49, PV108, PV109, PV113, PV116, PV130, and PV133, were commercially synthesized, conjugated to the carrier KLH, and dot-loaded onto NC membranes. Purified virions and the vector KLH were used as positive and negative controls, respectively. Approximately 3.5 µg of total proteins were loaded onto each blot. After blocking with 5% non-fat milk in PBST, the membranes were incubated with sera from cattle 72, 189,119, and 200,178 (1:3000), followed by incubation with goat anti-bovine IgG-HRP (1:8000). The immune reaction was developed using Clarity Western ECL substrate. The images shown are representative of three independent experiments

The identified B-cell epitopes were mapped to distinct BoAHV1 viral proteins

Based on the dot blot analysis using sera from BoAHV1 IgG positive cattle and commercial available BoAHV1antibody (VMDR Inc, Pullman, WA, USA) (Figs. 3 and 5), we identified ten B-cell epitopes of BoAHV1, including PV49 (³¹⁷VLTVPLNLPPG³²⁷), PV50 (⁷³⁶YFLRGAQYSARA⁷⁴⁷), PV59 (¹⁶⁹GSAAVIRPLLQ¹⁷⁹), PV62 (³⁰RAVPPSTPVEQ⁴⁰), PV108 (¹³⁸RLAPARPCPEY¹⁴⁸), PV109 (⁵⁸⁴AASAALDRRAA⁵⁹⁴), PV116 (³²SPSPPPSPSPT⁴²), PV113 (⁶⁹AARGRDRA⁷⁶), PV130 (¹⁹⁸ERSPRAHR²⁰⁵), and PV133 (²⁰⁶AGDAVYLFDPHGSGDV²²¹).

Based on the alignment of amino acid sequences, PV49 was mapped to the viral protein US4 (gG), PV59 to UL37 (a virion protein), PV62 and PV133 to UL36 (a very large virion protein), PV50 also to UL36, PV113 to UL49 (a virion protein), PV116 to UL44 (gC), PV108 and PV109 to UL27 (gB), and PV130 to UL10 (gM) (Fig. 6).

Fig. 6

Diagram showing different locations of the identified B-cell epitopes. PV49 is mapped to the viral protein US4 (gG), PV59 to UL37 (a virion protein), PV50, PV62 and PV133 to UL36(a very large virion protein), PV113 to UL49 (a virion protein), PV116 to UL44 (gC), PV108 and PV109 to UL27 (gB), and PV130 to UL10 (gM)

The identified BoAHV1 B-cell epitope PV116 can be used for generation of antibody against viral proteins

Four epitopes, including PV49 derived from the viral protein US4 (gG), PV108 and PV109 from UL27 (gB), and PV116 from gC, were selected to assess their ability to stimulate B cell responses and induce the production of antibodies against viral proteins. These peptides were chosen for their roles as glycoproteins involved in virus binding, entry, and potential contribution to viral pathogenicity. BALB/c mice were immunized with these individual peptides conjugated to KLH. Sera were collected and utilized to test its reactivity with cell-associated viral proteins by Western blot. For this purpose, MDBK cells were infected with the virus for6, 12, 24, and 36 h. After these time points, cell lysates were prepared and analyzed for viral protein expression using Western blot. A band with a molecular weight of approximately over 130 kDa was clearly detected by sera from mice immunized with PV116-KLH (Fig. 7A). In addition, the cell-associated viral proteins in A549 cells at 48 hpi could also be detected by the antiserum from mice immunized with PV116-KLH, although non-specific faint bands were also observed, particularly in virus-infected cells (Fig. 7B). We recently reported that the host protein 53BP1 plays an important role in viral productive infection [23]. In this study, we found that knockdown of 53BP protein expression by siRNAs, designated as si53BP1-1 and si53BP1-2 [23], significantly reduced the levels of viral protein detected by sera against PV116-KLH (Fig. 7C). Furthermore, the viral lysates could also be recognized by sera against PV116-KLH (Fig. 7D), but not by the sera against KLH (Fig. 7E). Collectively, these findings confirmed the specificity of the serum for vial protein. Notably, sera from mice immunized with PV49-KLH, PV108-KLH, and PV109-KLH could not able to specifically recognize viral proteins (data not shown). Indeed, we could not explain why these three peptides failed to induce antibody production. Perhaps conjugation to KLH alone was insufficient to confer immunogenicity to these peptides. Fortunately, the findings suggest that PV116-KLH is capable of inducing the production of antiserum, which is specific for detecting the viral protein, likely gC.

Fig. 7

The recognization of viral protein gC by mouse serum prepared by immunization of indicated peptides PV116. MDBK (A) and A549 (B) cells were infected with BoAHV1 at an MOI of 1 for the specified time durations. Subsequently, cell lysates (approximately 30 µg of total proteins) were prepared and subjected to detection of viral proteins by Western blot using the mouse serum produced by immunization with peptide PV116. (C) MDBK cells in 6-well plates were transfected with either scrambled siRNA (200 pmol) or 53BP1-specific siRNAs (200 pmol). At 48 h after transfection, the cells were infected with BoAHV1(MOI = 1). After infection for 24 h, cell lysates were prepared and subjected to Western blot to detect viral proteins using the serum against PV116-KLH. (D and E) Viral particles were purified by ultracentrifugation at 20,000 rpm using a Beckman SW28 rotor for 1 h at 4 °C. In parallel, cell debris was also collected for use as a negative control. Both viral particles and cell debris were subjected to detection of viral proteins by Western blot using mouse sera produced by immunization with the peptide PV116-KLH (D) and KLH (E), respectively. The images presented are representative of three independent experiments

The identified BoAHV1 B-cell epitopes are implicated in the generation of an ELISA kit for the detection of BoAHV1 antibody

Given that our findings indicated the identified B-cell epitopes could be specifically recognized by BoAHV1 IgG positive serum from cattle (Fig. 5), we explored the potential of these peptides conjugated to KLH to develop an ELISA kit for the detection of BoAHV1 IgG. The four epitopes, including PV49 from the viral protein US4 (gG), PV108 and PV109 from UL27 (gB), and PV116 from gC were selected for this study. We discovered that when these peptides conjugated to KLH (in a 1:1:1:1 ratio) were coated onto 96-well polystyrene plates (JET BIOFIL, Guangzhou, China) at a dose of 20 µg per well to create an indirect ELISA kit, the detection results matched well with those from a commercially available indirect ELISA kit for 95.5% of the samples (43/44) (Table 2). Thus, these peptides are suggested to be effective in the development of an indirect ELISA kit for the detection of viral antibodies.

Table 2 Detection of BoAHV1 IgG levels using a commercial ELISA kit and our established method with selected cattle sera. Four peptides, including PV49, PV109, PV116, and another PV109 (in a 1:1:1:1 ratio), conjugated to the vector KLH, were coated onto 96-well plates at a dose of 20 µg per well to create the kit. BoAHV1 IgG levels in 44 cattle serum samples provided by a local farmer were detected using both the established kit and the commercial ELISA kit (BioStone Animal Health, Southlake, TX, USA, cat# 10074-05). The results, as determined by OD450 readings from both kits, are listed

Discussion

By screening of a phage-displayed peptide library using a monoclonal antibody (mAb) against BoAHV1 glycoprotein gE, Lehmann et al. have identified one epitope of gE [11]. In order to identify more B-cell epitopes of BoAHV1, we screened a dodecapeptide (Ph.D.-12) phage display library by using a commercial BoAHV1 polyclonal antibody established to recognized more than one viral proteins [20, 24]. Totally, ten B-cell epitopes of BoAHV1 were identified, including PV49(³¹⁷VLTVPLNLPPG³²⁷) mapped to the viral protein US4 (gG), PV59(¹⁶⁹GSAAVIRPLLQ¹⁷⁹) to UL37 (a virion protein), PV50 (⁷³⁶YFLRGAQYSARA⁷⁴⁷), PV62 (³⁰RAVPPSTPVEQ⁴⁰) and PV133(²⁰⁶AGDAVYLFDPHGSGDV²²¹) to UL36(a very large virion protein), PV113(⁶⁹AARGRDRA⁷⁶) to UL49 (a virion protein), PV116(³²SPSPPPSPSPT⁴²) to UL44 (gC), PV108 (¹³⁸RLAPARPCPEY¹⁴⁸) and PV109(⁵⁸⁴AASAALDRRAA⁵⁹⁴) to UL27 (gB), and PV130 (¹⁹⁸ERSPRAHR²⁰⁵) to UL10 (gM) (Figs. 1 and 6). It seems that screening of a library using polyclonal antibody against virions may provide an efficient method to systematically screen viral B-cell epitopes, which may also apply to the other viruses.

At least 12 glycoproteins of the alpha herpesvirus have been identified, including gB, gC, gD, gE, gH, gI, gG, gK, gL, gM, gN, and gJ [6, 25, 26], which are potentially involved in virus infection and pathogenicity. To date, several epitopes on BoAHV1 glycoproteins have been reported, such as those located on viral proteins gD [8, 9], gB [10], and gE [11]. In this study, our identified epitopes are located to viral protein gG, UL36 (a very large virion protein), UL49 (a virion protein), gC, gB, and gM, respectively (Figs. 1 and 6). Notably, these epitopes were also recognized by cattle serum containing BoAHV1 antibody. These findings may extend our understanding on the viral epitopes and their role in viral pathogenicity.

Importantly, immunizing mice with the synthesized B-cell epitope PV116 (³²SPSPPPSPSPT⁴²) successfully induced the production of a polyclonal antibody against viral proteins, as confirmed by Western blot analysis (Fig. 7). This result provides an evidence that identifying B-cell epitopes and using them for animal immunization is an effective method for generating antibodies against viral proteins. Notably, we only tested four epitopes, and further studies are needed to determine whether other conjugated epitopes are antigenic and capable of inducing antibody production. In addition, among the characterized peptides PV49 derived from the viral protein US4 (gG), PV108 and PV109 from UL27 (gB) could not induce antibody production against their respective viral proteins. This may be because that conjugation to KLH alone was insufficient to confer immunogenicity to these peptides, or perhaps maintaining their immunogenicity requires support from additional amino acids flanking the peptides. This warrants extensive future studies. However, conjugation to the carrier KLH was sufficient to confer reactivity, as these peptides can be recognized by anti-BoAHV1 serum derived from various sources. This makes it possible for these four B-cell epitopes to be utilized in the development of an ELISA kit for detecting BoAHV1 antibodies. Therefore, our study demonstrates that the identified B-cell epitopes can be employed to generate reagents for the detection of viral antibodies.

In conclusion, we have identified ten novel B-cell epitopes of BoAHV1. Among them, we discovered the immunogenic B-cell epitope PV116 (³²SPSPPPSPSPT⁴²), which is located on the BoAHV1 gC protein and can be used to generate antibodies against viral proteins. Additionally, we identified four epitopes that have potential for use in the development of an indirect ELISA kit for detecting BoAHV1 antibodies. Therefore, the identification of B-cell epitopes provides an efficient method not only for generating viral antibodies but also for creating reagents necessary for the detection of these antibodies.