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Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates
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Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates Kinda Alshaikhahmed, Polly Roy ∗ Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, United Kingdom
a r t i c l e
i n f o
Article history: Received 18 November 2015 Received in revised form 17 December 2015 Accepted 19 December 2015 Available online xxx Keywords: Vaccine EHDV Baculovirus Orbivirus
a b s t r a c t Epizootic haemorrhagic disease virus (EHDV) is an insect-transmitted pathogen which causes high mortality in deer populations and may also cause high morbidity in cattle. EHDV belongs to the Orbivirus genus and is closely related to the prototype Bluetongue virus (BTV). To date seven distinct serotypes have been recognized. However, a live-attenuated vaccine is commercially available against only one serotype namely EHDV-2, which has been responsible for multiple outbreaks in North America, Canada, Asia and Australia. Here we expressed four major capsid proteins (VP2, VP3, VP5 and VP7) of EHDV-1 using baculovirus multiple gene expression systems and demonstrated that three-layered VLPs were assembled mimicking the authentic EHDV particles but lacking the viral genomic RNA segments and the transcriptase complex (TC). Antibodies generated with VLPs not only neutralized EHDV-1 infection in cell culture but also showed cross neutralizing reactivity against two other serotypes, EHDV-2 and EHDV-6. For proof of concept, we demonstrated that EHDV-2 VLPs could be generated rapidly by expressing the EHDV-2 variable outer capsid proteins (VP2, VP5) together with EHDV-1 VP3 and VP7, the two inner capsid proteins, which are highly conserved among the 7 serotypes. Data presented in this study validate the VLPs as a potential vaccine and demonstrate that a vaccine could be developed rapidly in the event of an outbreak of a new serotype. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Epizootic haemorrhagic disease (EHD) is a vector-borne, noncontagious viral disease of domestic and wild ruminants, primarily white-tailed deer (Odocoileus virginianus) and occasionally in cattle. The virus, with seven distinct serotypes (EHDV-1-EHDV-7), is endemic in many parts of North America, Australia, and certain Asian countries. More recently, it has emerged in the countries surrounding the Mediterranean Basin. Until 2006, it was believed that EHDV does not cause significant clinical disease in cattle with the exception of EHDV-2/Ibaraki virus (IBAV), which emerged in Japan 1959 when the virus infection killed 4000 cattle and caused high morbidity in 39,000 cattle in the affected areas [1,2]. However, the recent outbreaks of EHDV-6 in the US and EHDV-6 and EHDV-7 in the Mediterranean Basin as well as the French Island of Reunion were also highly pathogenic in cattle, demonstrating
∗ Corresponding author at: London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom. Tel.: +44 0207 927 2324; fax: +44 0207 637 4314. E-mail address: [email protected] (P. Roy).
that not only EHDV-2 but other serotypes can also be pathogenic in domestic animals with considerable economic consequences [3–6]. Further, the emergence of these serotypes in Mediterranean Basin also significantly increases the risk of invasion into central and northern Europe. EHDV infection in animals induces a wide range of clinical signs. In white-tailed deer, infection can be severe with high morbidity and mortality [7], while in cattle, the disease is generally subclinical. However, infected cattle could experience a reduction in milk production, infertility and abortion [5,8]. To date, the only commercially available vaccine is a live-attenuated vaccine to control Ibaraki disease caused by IBAV (EHDV-2) [9]. The major problem associated with this vaccine is an inability to differentiate between vaccinated and naturally infected animals (DIVA). Thus, a safe and efficacious vaccine with DIVA compliance is needed. EHDV is an orbivirus, closely related to Bluetongue virus (BTV), the prototype of the Orbivirus genus in the Reoviridae family. BTV and EHDV particles are non-enveloped capsid structures, made up of seven structural proteins that are organized in two concentric shells enclosing 10 dsRNA segments of viral genome [10,11]. At the amino acid level the capsid proteins of EHDV and BTV are highly homologous indicating that these two viruses are structurally similar [12,13].
http://dx.doi.org/10.1016/j.vaccine.2015.12.069 0264-410X/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Alshaikhahmed K, Roy P. Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2015.12.069
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The outer shell of BTV (and most likely also of EHDV) is composed of 180 molecules of VP2 and 360 molecules of VP5, both of which attach onto the VP7 layer, the surface layer of the capsid, termed as ‘core’. The VP7 layer, which is composed of 260 VP7 trimers (780 molecules), encapsidates a further protein layer of 120 VP3 molecules. The interior of the core also includes three viral enzymes (VP1, VP4 and VP6) that are closely associated with the 10 double-stranded (ds) RNA segments of viral genome [14–17]. While VP2 and VP5 are highly variable at the sequence level among serotypes, the core proteins are highly conserved [18]. The two outer capsid proteins are responsible for attachment and penetration of the host membrane; they take no further parts in the generation of new viral genomes in the host cytoplasm as both proteins are detached and degraded during the entry process. However, consistent with their locations, each is responsible for triggering strong neutralizing antibody response. These have been documented extensively for BTV and to a lesser extent for other orbiviruses, including EHDV. The expression of the four structural proteins (VP2, VP3, VP5 and VP7) of BTV in insect cells, utilizing baculovirus multiple expression vectors, leads to the assembly of 3-layered virus-like particles (VLPs), which structurally mimic the authentic virion particles but lack the viral genome and polymerase complex [19,20]. When used for immunization, BTV VLPs have been shown to afford complete protection against virulent strains challenge with no side effects in BTV-susceptible sheep [21–23], indicating that such vaccines could be highly desirable for EHDV serotype. In this study, we have cloned the genes encoding the four major capsid proteins of EHDV-1 and demonstrated their expression as multilayered recombinant VLPs which induced strong neutralizing antibodies in model animals. Further, the possibility of generation of VLPs for other serotypes as a rapid response to the emergent serotypes has also been documented. 2. Materials and methods 2.1. Virus stocks and cell lines BSR cells, a clone derived from the Baby Hamster Kidney cells (BHK), were cultured in Dulbecco’s modified medium (DMEM) supplemented with 5% foetal calf serum (FCS) in the presence of 50 U/ml antibiotic/antimycotic and l-glutamine (Sigma–Aldrich Ltd, UK). EHDV-1 (New Jersey, US), EHDV-2 (Alberta, Canada) and EHDV-6 (The Reunion Island, France) were propagated and titrated in BSR cells both by plaque assay and TCID50 /ml. Insect cells (Spodoptera frugiperda Sf21 and Sf9) were cultured at 28 ◦ C in suspension in TC-100 (Lonza Ltd, UK) or Insect-Xpress medium, respectively (Lonza Ltd, UK) supplemented with 10% FCS (Sigma–Aldrich Ltd, UK). 2.2. Construction of transfer vectors for baculovirus system For single expression of the structural proteins, bacmid BAC10:KO1962 which has a mutation in the orf1629 and pAcYM1 transfer vector were used [24,25]. For the generation of baculovirus multiple gene expression vector, a modified bacmid from bMNO14272 maintained in Escherichia coli strain EL350 was used as described previously [26,27]. Three transfer vectors, pRN306, pRN260 and pRN296 were utilized for cloning the coding regions of EHDV-1 S3, S7 and/or EHDV-2 S5. Each vector is composed of a ph promoter, Cre recombinase gene (Loxp71 and Loxp66), LacZ␣ gene and a baculovirus locus specific sequence for homologous recombination. The transfer vector pAcYM1 was used to subclone S2 of EHDV-1 and EHDV-2 from the available recombinant plasmids pUc4K-EHDV1.S2 and pGEM-T-EHDV2.S2.
2.3. Generation and amplification of recombinant baculoviruses To purify high-copy bacmid DNA from the bacterial culture, Invitrogen PureLinkTM HiPure Plasmid DNA Purification Kit (InvitrogenTM, UK) was used. For single and multiple gene expression, 2 g of Bacmid DNA together with 500 ng of specific recombinant transfer vectors were used for transfection. The confluent Sf21 insect cells in the presence of 10 l Insect GeneJuice (Novagen) and 100 l TC-100 medium according to the manufacturer’s instructions. For baculovirus amplification, the supernatant containing the recombinant baculovirus progeny was harvested 72 h post cotransfection and subjected to 4 rounds of amplification in fresh Sf9 insect cells. The virus infection was analyzed daily by light microscopy and confirmed further by plaque assay. 2.4. Protein expression Suspensions of Sf21 cells at density of 1.2 × 106 cells/ml were infected at multiplicity of infection (MOI) of 2–5 and incubated for 72 h at 28◦ C. Samples were harvested and analyzed by SDS-PAGE followed by coomassie blue staining or western blot analysis using specific EHDV-1 polyclonal antibodies. 2.5. Particles purification For the expression and purification of the recombinant corelike particles (CLPs, composed of VP3 and VP7) of EHDV-1, Sf9 insect cells were infected with the recombinant dual baculovirus Bac1629 :EHDV1.VP7.VP3 at a MOI of 3–5, incubated at 28 ◦ C and harvested 72 hpi. CLPs were purified as previously described [18]. For EHDV-1 VLPs, the recombinant proteins were generated by infection of Sf9 insect cells with the quadruple recombinant baculovirus consisting of S2, S3, S5 and S7 of EHDV-1. For the generation of heterologous VLPs of EHDV-2, Sf9 insect monolayers were coinfected with the dual baculoviruses; Bac1629 :EHDV1.S3.S7 and Bac1629 :EHDV2.S2.S5. Homologous and chimeric VLPs were purified according to the methods described by Stewart et al. [28]. As positive controls, EHDV-1 cores and virions were purified according to the modified methods described previously [29,30]. 2.6. Antibodies production For polyclonal antibody production, 2.5 mg/ml of the CLPs quantified by Bradford reagent (Sigma–Aldrich Ltd, UK) and 2.0 mg/ml of VLPs were supplied to a commercial company. Immunization and work with vertebrate animals (rabbits) was performed at ThermoFisher Scientific in accordance with the UK Animals (Scientific Procedures) Act 1986 (UK Government 1986) using the principles outlined in the Home Office guidance ‘Antibody Production: Principles for Protocols of Minimal Severity’ (Home Office 2000). The work adhered to the principles of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (see https://www.nc3rs.org.uk/arrive-guidelines). Animals were immunized first with 500 g of antigen in 10 sites followed by boosting twice with 250 g antigen at 14 and 28 respectively in the presence of Incomplete Fruend’s adjuvant. 2.7. Electron microscopy Aliquots (2 l) of purified EHDV-1 cores, CLPs and VLPs were adsorbed onto copper 400-mesh Formvar carbon-coated grids (TAAB Laboratories Equipment Ltd, UK) for 2 min and stained with filtered 2% (w/v) phosphotungstic acid (PTA), pH 6.8. Grids were examined under JEOL 1200 EX transmission microscope. 2.8. Neutralizing and cross neutralizing antibody response Antibodies raised against purified EHDV-1 VLPs were collected at (0, 35 and 56–58 days) and analyzed for their neutralizing activity
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against EHDV-1 virus. In addition, cross neutralizing activities were assessed against EHDV-2 and EHDV-6. Serum neutralization of 50 TCID50 and plaque reduction based standard neutralizing assay were carried out. All the dilutions were performed in triplicate and assays were repeated three times. 3. Results 3.1. Cloning and expression of individual EHDV major structural proteins Initially, we sought to assess if each of the four major structural proteins of the virion particle could be synthesized as recombinant proteins in Sf9 insect cells. Therefore, cloning and expression of the individual outer and inner capsid proteins VP2, VP3, VP5 and VP7 of EHDV-1 in insect cells were assessed using baculovirus single expression vector. The plasmid pAcYM1, containing AcMNPV ph promoter, was utilized to generate recombinant viruses that expressed high levels of the targeted genes. To generate recombinant pAcYM1 transfer vectors containing each EHDV-1 gene, the open reading frames (ORFs) of S2 (aa971), S5 (aa527) and S7 (aa349) were amplified using specific sense and antisense primers. The amplified ORFs were then sub-cloned into the pAcYM1 vector and the correct orientation of the inserts was confirmed by sequencing. The recombinant pAcYM1-EHDV1.S3 (aa899) was generated previously [31]. Recombinant baculoviruses expressing each protein singly were then generated and lysates from cells infected with each recombinant virus were subsequently analyzed by SDS-PAGE. Each recombinant virus showed high level expression of each capsid protein (Fig. 1, left panels). The authenticity of each protein was subsequently demonstrated by western analysis using EHDV1 polyclonal antibody with reactive bands corresponding to the estimated sizes of EHDV-1 structural proteins (Fig. 1, right panels). 3.2. Simultaneous expression of EHDV structural proteins by a single recombinant baculovirus
3
encoding for the inner core proteins (VP3 and VP7) of EHDV-1 were sub-cloned into transfer vectors targeting two specific genetic loci in the baculovirus genome, the 39K and egt loci, as described by Noad et al. [27]. The expression of each of the inner capsid proteins was examined in Sf21 insect cells and their assembly into particles was assessed by fractionation on a 25–50% discontinuous sucrose gradient. The gradient band corresponding to putative CLPs was isolated and analyzed by SDS-PAGE. Two protein bands corresponding to the estimated sizes of VP3 (103 kDa) and VP7 (39 kDa) were present in the middle of the gradient (Fig. 2A) and peak fractions were further purified using a second sucrose gradient, resulting in only VP3 and VP7 by SDS-PAGE analysis (Fig. 2B, lane 2). The VP3 material showed a doublet consistent with N-terminal trimming analogues to that observed for BTV CLPs [32]. Micrographs of the negatively stained samples showed particulate structures with a core-like morphology (Fig. 2C, right panel) similar in size and appearance to authentic EHDV-1purified cores (Fig. 2C, left panel). To generate VLPs, the genes encoding for the outer shell proteins VP2 and VP5 were sub-cloned individually and each recombined into AcMNPV genome encoding of VP3 and VP7, using two alternative loci, ph and odv-e56, respectively. To demonstrate that all four proteins of EHDV were expressed from one single recombinant baculovirus, recombinant virus-infected cell lysate was analyzed by SDS-PAGE. Coomassie blue staining of the gel exhibited the presence of all four EHDV proteins with sizes corresponding to those of the virion derived proteins (Fig. 3A, lane 3). As before, EHDV-1 VLPs were purified using discontinuous sucrose gradients and the composition of the purified VLPs was analyzed by SDS-PAGE. All four EHDV-1 proteins, VP2, VP5, VP3, and VP7 were identified (Fig. 3B, lane 3) with sizes corresponding to the recombinant individual EHDV-1 proteins expressed previously (Fig. 3B, lanes 4–7). The yield of purified VLPs from 100 ml culture was estimated approximately 1 mg. By electron microscopy the negatively stained samples of the peak fractions (Fig. 3C, left panel) of VLPs exhibited similar in size and morphology to EHDV-1 virion particles (Fig. 3C, right panel).
To synthesize VLPs efficiently, all four major structural proteins were expressed by a single recombinant baculovirus. As outer capsid formation requires the inner capsid as scaffold, the synthesis of EHDV-1 core-like particles (CLPs) was first assessed. The genes
Fig. 1. Individual expression of EHDV-1 structural capsid proteins. Sf9 insect cells were infected with each recombinant baculovirus at MOI of 3 and harvested at 72 hpi. The expression of each recombinant protein (VP2, VP3, VP5 and VP7) was analyzed by SDS-PAGE stained by coomassie blue (left panel) and immunoblotting with polyclonal antibodies against EHDV-1 (right panel). As controls, uninfected Sf9 cells (C) and Sf9 cells infected with a baculovirus expressing GFP protein (negative control, −ve) were included. Position of EHDV-1 protein bands and the molecular mass of standard proteins (M) are indicated.
Fig. 2. Assembly of EHDV-1 VP3 and VP7 into CLPs. EHDV-1 CLPs were purified by two consecutive sucrose gradient centrifugations. Aliquots of the first (A) and the second (B) centrifugations were analyzed by SDS-PAGE to confirm the expression of VP3 (103 kDa) and VP7 (39 kDa) (lane 2). Positions of the proteins and the molecular mass of standard (M) are indicated. (C) Electron micrographs of EHDV-1 purified cores (left panel) and CLPs (right panel). Bars indicate 100 nm.
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Fig. 3. Simultaneous expression of four capsid proteins of EHDV and their assembly into homologous VLPs. (A) Cell lysate infected with the recombinant baculovirus expressing VP2 (112 kDa), VP3 (103 kDa), VP5 (59 kDa) and VP7 (39 kDa) was analyzed by SDS-PAGE (lane 3). (B) VLPs were purified by sucrose gradient centrifugation and analyzed by SDS-PAGE. Note that all proteins were detected in the same fraction (lane 3, arrows). Proteins expressed singly were used as controls (lanes 4–7, arrowheads). As control, uninfected Sf9 cells (C) were used. Positions of the proteins and the molecular mass of standard (M) are indicated. (C) Electron micrographs of virion particles (left panel) and VLPs (right panel). Bars indicate 100 nm.
3.3. Generation of EHDV-2 VLPs using EHDV-1 CLPs as scaffolding Since VP3 and VP7 of CLPs are highly conserved proteins across the different serotypes, EHDV-1 CLPs could acquire VP2 and VP5 of a heterologous EHDV serotype to generate chimeric VLPs. To assess this, a dual expressing baculovirus was constructed with the coding region of EHDV-2 VP5 (aa527) and VP2 (aa982) inserted at the odve56 and ph loci respectively. The expression of the two structural proteins in the insect cells was analyzed by SDS-PAGE (Fig. 4A, Lane 3) and were shown to co-migrate with the individual recombinant EHDV-2 VP2 and EHDV-2 VP5 (Fig. 4A, lanes 4 and 5) described
Fig. 4. Simultaneous expression of EHDV-2 outer capsid proteins and their assembly into chimeric VLPs. (A) SDS-PAGE analysis of cell lysates infected either with a dual baculovirus expressing EHDV-2 VP2 and VP5 (lane 3), or with a single recombinant virus expressing each protein, VP2 (lane 4) and VP5 (lane 5). Lane 2, uninfected Sf9 cells (C) and lane 1, protein marker (M). (B) Assembly of EHDV-2 chimeric VLPs. An aliquot of purified chimeric VLPs was analyzed by SDS-PAGE (lane 4). As controls, partially purified EHDV-1 VLP sample (lane 3) and uninfected Sf9 cells (C) (lane 2) were used. Position of the proteins and the molecular marker (M) are indicated.
above. The production of chimeric VLPs was achieved by coinfection of insect cells with the recombinant baculovirus expressing EHDV-2 VP2 and VP5 with the inner capsid proteins VP3 and VP7 of EHDV1. The presence of chimeric VLPs was assessed by fractionation of infected cell lysates on sucrose gradients followed by SDS-PAGE analysis. All four proteins required for the formation of VLPs were present in the peak fraction (Fig. 4B, lane 4) with molecular mass of each protein similar to those of partially purified EHDV-1 VLPs used as a positive control (Fig. 4B, lane 3). 3.4. Immunogenic properties of EHDV CLPs and VLPs Antisera raised against purified EHDV-1 CLPs or VLPs were produced in rabbits and specificity determined by western blot of EHDV-1 infected BSR cells. Two specific protein bands were visualized with sizes consistent with EHDV-1 VP3 and VP7 with the CLP antisera (Fig. 5A, lane 3), while four bands corresponding to each of the EHDV-1 major structural proteins were clearly detectable by VLP antisera (Fig. 5B, lane 3). To test whether VLP antiserum would neutralize EHDV-1, serum neutralization assays by TCID50 and the plaque reduction assays were done. Both assays yielded similar results with 128 neutralization titres as shown in Table 1. Furthermore, to determine if VLP antiserum against EHDV-1 could neutralize infection by other EHDV serotypes, two additional serotypes, EHDV-2 and EHDV6 were assessed. Lower, but significant, cross-neutralization was apparent for both viruses in comparison to the control serum (Table 1). A live-attenuated vaccine, the only currently available EHDV vaccine, was developed following the outbreaks of the Ibaraki disease in Japan in 1980s. This vaccine was effective at least until 1997 as neither any evidence of Ibaraki disease nor any seroconversion was documented in cattle in that region prior to 1997.
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Fig. 5. Detection of EHDV-1 proteins from purified cores or virions by anti CLP or anti VLP antisera. (A) Western blot analysis of core proteins VP3 and VP7 using the anti CLP antiserum (lane 3). (B) Western blot analysis of four structural proteins VP2, VP3, VP5 and VP7 of virion using the anti VLP antiserum (lane 3). As control, uninfected cells were used (C). Position of the molecular mass (M) and recombinant proteins are indicated.
However, during 1997 outbreaks, new clinical signs that were never observed in the previous outbreaks were recorded due to the emergence of a more virulent strain of Ibaraki virus [9]. Live attenuated virus vaccine is however, neither DIVA compliant nor without risk. Furthermore, there are at least six additional serotypes circulating and controlling infection by each requires type-specific vaccine. The development of DIVA compliant efficacious vaccines against all EHDV serotypes is therefore desirable and economically important. In particular, recent epidemics by different serotypes in various European bordering countries, in which cattle farms were mainly affected, have emphasized the urgency for stock-piling multi-serotype vaccines. VLP vaccines are generally DIVA compliant due to the absence of non-structural and enzymatic proteins and have been proven to be highly efficacious for a number of animal viruses including orbiviruses [22,23]. VLPs for non-enveloped (HEV and HPV) and enveloped (HBV, Influenza A) viruses have been produced using different expression systems and shown to be highly immunogenic [33–37]. However, the production of VLPs for the viruses of the family Reoviridae is more challenging as VLPs consist of complex multiple interacting viral capsid proteins organized in several concentric layers. In this study, we investigated the possibility of generating EHDV VLPs by simultaneous expression of four major structural proteins using baculovirus multiple gene expression vectors. Electron microscopy analysis of VLPs revealed the particulate structures analogous to the authentic EHDV particles. However, the VLPs lacked the central density due to the absence of the viral genomic RNA. Antibodies raised to EHDV-1 VLPs particles had a strong neutralizing titre against EHDV-1 and a higher than typical neutralization level in natural convalescent sera from infected animals [38]. These data indicate that the epitopes of the outer capsids proteins of VLPs are presented in correct immunogenic
Table 1 EHDV-1 VLPs antisera neutralization titre determined by TCID50 in BSR cells infected with EHDV-1, -2 and 6. EHDV-1 VLPs serum
EHDV-1 EHDV-2 EHDV-6
FCS
0 dpi
35 dpi
56–58 dpi
0 0 0
32 6 6
128 8 8
2 2 2
5
conformations. These VLPs are likely to afford complete protection in animals against EHDV-1 infection. The sequence analysis of different orbiviruses has shown that VP2 and VP5 are the most variable proteins. Nevertheless, depending on the serotype, both proteins exhibit overlapping conserved sequences to other serotypes and in some cases, cross neutralization activities have also been demonstrated [39]. Consistent with these, low level cross neutralization of EHDV-2 and EHDV6 was achieved by EHDV-1 anti-VLP antiserum, emphasizing that these three serotypes are genetically related and certain immunogenic epitopes are shared among these serotypes. The data also suggests that VLP vaccines of less than seven different serotypes would be capable of affording protective immunity against all seven serotypes. Comparative analysis of the inner core protein sequence of the seven EHDV serotypes showed a high conservation among these proteins, with identity up to 90% for VP3 and 80% for VP7 [6]. Here we showed that it was indeed possible to assemble EHDV-2 outer capsids proteins onto the CLPs of EHDV-1 and to generate EHDV-2 VLPs. This is highly encouraging as using a common CLP scaffold, VLPs could be produced rapidly in response to emerging serotypes similarly. 4. Conclusions Overall, data presented in this study demonstrated that immunogenic VLPs of different EHDV serotypes could be generated by simultaneous expression of all major structural proteins using baculovirus multiple expression system, as opposed to coinfections of insect cells with four different recombinant viruses, which is not ideal for vaccine purposes. These VLPs are potential vaccine candidates against EHDV infection in animals and are also DIVA compliant as none of the NS proteins, which could serve as diagnostic reagents, are present in these protein-based VLPs. Acknowledgements We are grateful to Dr. J. Richt (Kansas State University, USA) for providing us with EHDV-2 plasmid constructs. This work was funded by the FP7 Framework of European Commission. References [1] Omori T, Inaba Y, Morimoto T, Tanaka Y, Ishitani R. Ibaraki virus, an agent of epizootic disease of cattle resembling bluetongue. I. Epidemiologic, clinical and pathologic observations and experimental transmission to calves. Jpn J Microbiol 1969;13(2):139–57. [2] Inaba U. Ibaraki disease and its relationship to bluetongue. Aust Vet J 1975;51(4):178–85. [3] Breard E, Sailleau C, Hamblin C, Graham SD, Gourreau JM, Zientara S. Outbreak of epizootic haemorrhagic disease on the island of Reunion. Vet Rec 2004;155(14):422–3. [4] Gibbs EP, Lawman MJ. Infection of British deer and farm animals with epizootic haemorrhagic disease of deer virus. J Comp Pathol 1977;87(3):335–43. [5] Temizel EM, Yesilbag K, Batten C, Senturk S, Maan NS, Clement-Mertens PP, et al. Epizootic hemorrhagic disease in cattle Western Turkey. Emerg Infect Dis 2009;15(2):317–9. [6] Allison AB, Goekjian VH, Potgieter AC, Wilson WC, Johnson DJ, Mertens PP, et al. Detection of a novel reassortant epizootic hemorrhagic disease virus (EHDV) in the USA containing RNA segments derived from both exotic (EHDV-6) and endemic (EHDV-2) serotypes. J Gen Virol 2010;91(Pt 2):430–9. [7] Gaydos JK, Crum JM, Davidson WR, Cross SS, Owen SF, Stallknecht DE. Epizootiology of an epizootic hemorrhagic disease outbreak in West Virginia. J Wildl Dis 2004;40(3):383–93. [8] Yadin H, Brenner J, Bumbrov V, Oved Z, Stram Y, Klement E, et al. Epizootic haemorrhagic disease virus type 7 infection in cattle in Israel. Vet Rec 2008;162(2):53–6. [9] Ohashi S, Yoshida K, Watanabe Y, Tsuda T. Identification and PCR-restriction fragment length polymorphism analysis of a variant of the Ibaraki virus from naturally infected cattle and aborted fetuses in Japan. J Clin Microbiol 1999;37(12):3800–3.
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[10] Huismans H, Van Dijk AA. Bluetongue virus structural components. Curr Top Microbiol Immunol 1990;162:21–41. [11] Verwoerd DW, Els HJ, De Villiers EM, Huismans H. Structure of the bluetongue virus capsid. J Virol 1972;10(4):783–94. [12] Iwata H, Hirasawa T, Roy P. Complete nucleotide sequence of segment 5 of epizootic haemorrhagic disease virus; the outer capsid protein VP5 is homologous to the VP5 protein of bluetongue virus. Virus Res 1991;20(3):273–81. [13] Iwata H, Yamagawa M, Roy P. Evolutionary relationships among the gnattransmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences. Virology 1992;191(1):251–61. [14] Prasad BV, Yamaguchi S, Roy P. Three-dimensional structure of single-shelled bluetongue virus. J Virol 1992;66(4):2135–42. [15] Hewat EA, Booth TF, Roy P. Structure of bluetongue virus particles by cryoelectron microscopy. J Struct Biol 1992;109(1):61–9. [16] Nason EL, Rothagel R, Mukherjee SK, Kar AK, Forzan M, Prasad BV, et al. Interactions between the inner and outer capsids of bluetongue virus. J Virol 2004;78(15):8059–67. [17] Zhang X, Boyce M, Bhattacharya B, Schein S, Roy P, Zhou ZH. Bluetongue virus coat protein VP2 contains sialic acid-binding domains, and VP5 resembles enveloped virus fusion proteins. Proc Natl Acad Sci U S A 2010;107(14): 6292–7. [18] French TJ, Roy P. Synthesis of bluetongue virus (BTV) core like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. J Virol 1990;64(4):1530–6. [19] Hewat EA, Booth TF, Roy P. Structure of correctly self-assembled bluetongue virus-like particles. J Struct Biol 1994;112(3):183–91. [20] French TJ, Marshall JJ, Roy P. Assembly of double-shelled, virus like particles of bluetongue virus by the simultaneous expression of four structural proteins. J Virol 1990;64(12):5695–700. [21] Roy P, French T, Erasmus BJ. Protective efficacy of virus-like particles for bluetongue disease. Vaccine 1992;10(1):28–32. [22] Roy P, Bishop DH, LeBlois H, Erasmus BJ. Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection. Vaccine 1994;12(9):805–11. [23] Stewart M, Dovas CI, Chatzinasiou E, Athmaram TN, Papanastassopoulou M, Papadopoulos O, et al. Protective efficacy of Bluetongue virus-like and subviruslike particles in sheep: presence of the serotype-specific VP2, independent of its geographic lineage, is essential for protection. Vaccine 2012;30(12): 2131–9. [24] Zhao Y, Chapman DA, Jones IM. Improving baculovirus recombination. Nucleic Acids Res 2003;31(2):E6. [25] Matsuura Y, Possee RD, Overton HA, Bishop DH. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol 1987;68(Pt 5):1233–50.
[26] Luckow VA, Lee SC, Barry GF, Olins PO. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 1993;67(8):4566–79. [27] Noad RJ, Stewart M, Boyce M, Celma CC, Willison KR, Roy P. Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA. BMC Mol Biol 2009;10:87. [28] Stewart M, Bhatia Y, Athmaran TN, Noad R, Gastaldi C, Dubois E, et al. Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus. Vaccine 2010;28(17):3047–54. [29] Mertens PP, Burroughs JN, Anderson J. Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4. Virology 1987;157(2):375–86. [30] Van Dijk AA, Huismans H. The in vitro activation and further characterization of the bluetongue virus-associated transcriptase. Virology 1980;104(2):347–56. [31] Le Blois H, Fayard B, Urakawa T, Roy P. Synthesis and characterization of chimeric particles between epizootic hemorrhagic disease virus and bluetongue virus: functional domains are conserved on the VP3 protein. J Virol 1991;65(9):4821–31. [32] Limn CK, Staeuber N, Monastyrskaya K, Gouet P, Roy P. Functional dissection of the major structural protein of bluetongue virus: identification of key residues within VP7 essential for capsid assembly. J Virol 2000;74(18):8658–69. [33] McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ, Hilleman MR. Human hepatitis B vaccine from recombinant yeast. Nature 1984;307(5947):178–80. [34] Zhu FC, Zhang J, Zhang XF, Zhou C, Wang ZZ, Huang SJ, et al. Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a largescale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet 2010;376(9744):895–902. [35] Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E, Alvarez FB, et al. A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 2002;347(21):1645–51. [36] Harper DM, Franco EL, Wheeler CM, Moscicki AB, Romanowski B, RoteliMartins CM, et al. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet 2006;367(9518):1247–55. [37] Anggraeni MR, Connors NK, Wu Y, Chuan YP, Lua LH, Middelberg AP. Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle. Vaccine 2013;31(40):4428–35. [38] Dubay SA, deVos Jr JC, Noon TH, Boe S. Epizootiology of hemorrhagic disease in mule deer in central Arizona. J Wildl Dis 2004;40(1):119–24. [39] Anthony SJ, Maan S, Maan N, Kgosana L, Bachanek-Bankowska K, Batten C, et al. Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup. Virus Res 2009;145(2):200–10.
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Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates Kinda Alshaikhahmed, Polly Roy ∗ Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, United Kingdom
a r t i c l e
i n f o
Article history: Received 18 November 2015 Received in revised form 17 December 2015 Accepted 19 December 2015 Available online xxx Keywords: Vaccine EHDV Baculovirus Orbivirus
a b s t r a c t Epizootic haemorrhagic disease virus (EHDV) is an insect-transmitted pathogen which causes high mortality in deer populations and may also cause high morbidity in cattle. EHDV belongs to the Orbivirus genus and is closely related to the prototype Bluetongue virus (BTV). To date seven distinct serotypes have been recognized. However, a live-attenuated vaccine is commercially available against only one serotype namely EHDV-2, which has been responsible for multiple outbreaks in North America, Canada, Asia and Australia. Here we expressed four major capsid proteins (VP2, VP3, VP5 and VP7) of EHDV-1 using baculovirus multiple gene expression systems and demonstrated that three-layered VLPs were assembled mimicking the authentic EHDV particles but lacking the viral genomic RNA segments and the transcriptase complex (TC). Antibodies generated with VLPs not only neutralized EHDV-1 infection in cell culture but also showed cross neutralizing reactivity against two other serotypes, EHDV-2 and EHDV-6. For proof of concept, we demonstrated that EHDV-2 VLPs could be generated rapidly by expressing the EHDV-2 variable outer capsid proteins (VP2, VP5) together with EHDV-1 VP3 and VP7, the two inner capsid proteins, which are highly conserved among the 7 serotypes. Data presented in this study validate the VLPs as a potential vaccine and demonstrate that a vaccine could be developed rapidly in the event of an outbreak of a new serotype. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Epizootic haemorrhagic disease (EHD) is a vector-borne, noncontagious viral disease of domestic and wild ruminants, primarily white-tailed deer (Odocoileus virginianus) and occasionally in cattle. The virus, with seven distinct serotypes (EHDV-1-EHDV-7), is endemic in many parts of North America, Australia, and certain Asian countries. More recently, it has emerged in the countries surrounding the Mediterranean Basin. Until 2006, it was believed that EHDV does not cause significant clinical disease in cattle with the exception of EHDV-2/Ibaraki virus (IBAV), which emerged in Japan 1959 when the virus infection killed 4000 cattle and caused high morbidity in 39,000 cattle in the affected areas [1,2]. However, the recent outbreaks of EHDV-6 in the US and EHDV-6 and EHDV-7 in the Mediterranean Basin as well as the French Island of Reunion were also highly pathogenic in cattle, demonstrating
∗ Corresponding author at: London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom. Tel.: +44 0207 927 2324; fax: +44 0207 637 4314. E-mail address: [email protected] (P. Roy).
that not only EHDV-2 but other serotypes can also be pathogenic in domestic animals with considerable economic consequences [3–6]. Further, the emergence of these serotypes in Mediterranean Basin also significantly increases the risk of invasion into central and northern Europe. EHDV infection in animals induces a wide range of clinical signs. In white-tailed deer, infection can be severe with high morbidity and mortality [7], while in cattle, the disease is generally subclinical. However, infected cattle could experience a reduction in milk production, infertility and abortion [5,8]. To date, the only commercially available vaccine is a live-attenuated vaccine to control Ibaraki disease caused by IBAV (EHDV-2) [9]. The major problem associated with this vaccine is an inability to differentiate between vaccinated and naturally infected animals (DIVA). Thus, a safe and efficacious vaccine with DIVA compliance is needed. EHDV is an orbivirus, closely related to Bluetongue virus (BTV), the prototype of the Orbivirus genus in the Reoviridae family. BTV and EHDV particles are non-enveloped capsid structures, made up of seven structural proteins that are organized in two concentric shells enclosing 10 dsRNA segments of viral genome [10,11]. At the amino acid level the capsid proteins of EHDV and BTV are highly homologous indicating that these two viruses are structurally similar [12,13].
http://dx.doi.org/10.1016/j.vaccine.2015.12.069 0264-410X/© 2016 Elsevier Ltd. All rights reserved.
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The outer shell of BTV (and most likely also of EHDV) is composed of 180 molecules of VP2 and 360 molecules of VP5, both of which attach onto the VP7 layer, the surface layer of the capsid, termed as ‘core’. The VP7 layer, which is composed of 260 VP7 trimers (780 molecules), encapsidates a further protein layer of 120 VP3 molecules. The interior of the core also includes three viral enzymes (VP1, VP4 and VP6) that are closely associated with the 10 double-stranded (ds) RNA segments of viral genome [14–17]. While VP2 and VP5 are highly variable at the sequence level among serotypes, the core proteins are highly conserved [18]. The two outer capsid proteins are responsible for attachment and penetration of the host membrane; they take no further parts in the generation of new viral genomes in the host cytoplasm as both proteins are detached and degraded during the entry process. However, consistent with their locations, each is responsible for triggering strong neutralizing antibody response. These have been documented extensively for BTV and to a lesser extent for other orbiviruses, including EHDV. The expression of the four structural proteins (VP2, VP3, VP5 and VP7) of BTV in insect cells, utilizing baculovirus multiple expression vectors, leads to the assembly of 3-layered virus-like particles (VLPs), which structurally mimic the authentic virion particles but lack the viral genome and polymerase complex [19,20]. When used for immunization, BTV VLPs have been shown to afford complete protection against virulent strains challenge with no side effects in BTV-susceptible sheep [21–23], indicating that such vaccines could be highly desirable for EHDV serotype. In this study, we have cloned the genes encoding the four major capsid proteins of EHDV-1 and demonstrated their expression as multilayered recombinant VLPs which induced strong neutralizing antibodies in model animals. Further, the possibility of generation of VLPs for other serotypes as a rapid response to the emergent serotypes has also been documented. 2. Materials and methods 2.1. Virus stocks and cell lines BSR cells, a clone derived from the Baby Hamster Kidney cells (BHK), were cultured in Dulbecco’s modified medium (DMEM) supplemented with 5% foetal calf serum (FCS) in the presence of 50 U/ml antibiotic/antimycotic and l-glutamine (Sigma–Aldrich Ltd, UK). EHDV-1 (New Jersey, US), EHDV-2 (Alberta, Canada) and EHDV-6 (The Reunion Island, France) were propagated and titrated in BSR cells both by plaque assay and TCID50 /ml. Insect cells (Spodoptera frugiperda Sf21 and Sf9) were cultured at 28 ◦ C in suspension in TC-100 (Lonza Ltd, UK) or Insect-Xpress medium, respectively (Lonza Ltd, UK) supplemented with 10% FCS (Sigma–Aldrich Ltd, UK). 2.2. Construction of transfer vectors for baculovirus system For single expression of the structural proteins, bacmid BAC10:KO1962 which has a mutation in the orf1629 and pAcYM1 transfer vector were used [24,25]. For the generation of baculovirus multiple gene expression vector, a modified bacmid from bMNO14272 maintained in Escherichia coli strain EL350 was used as described previously [26,27]. Three transfer vectors, pRN306, pRN260 and pRN296 were utilized for cloning the coding regions of EHDV-1 S3, S7 and/or EHDV-2 S5. Each vector is composed of a ph promoter, Cre recombinase gene (Loxp71 and Loxp66), LacZ␣ gene and a baculovirus locus specific sequence for homologous recombination. The transfer vector pAcYM1 was used to subclone S2 of EHDV-1 and EHDV-2 from the available recombinant plasmids pUc4K-EHDV1.S2 and pGEM-T-EHDV2.S2.
2.3. Generation and amplification of recombinant baculoviruses To purify high-copy bacmid DNA from the bacterial culture, Invitrogen PureLinkTM HiPure Plasmid DNA Purification Kit (InvitrogenTM, UK) was used. For single and multiple gene expression, 2 g of Bacmid DNA together with 500 ng of specific recombinant transfer vectors were used for transfection. The confluent Sf21 insect cells in the presence of 10 l Insect GeneJuice (Novagen) and 100 l TC-100 medium according to the manufacturer’s instructions. For baculovirus amplification, the supernatant containing the recombinant baculovirus progeny was harvested 72 h post cotransfection and subjected to 4 rounds of amplification in fresh Sf9 insect cells. The virus infection was analyzed daily by light microscopy and confirmed further by plaque assay. 2.4. Protein expression Suspensions of Sf21 cells at density of 1.2 × 106 cells/ml were infected at multiplicity of infection (MOI) of 2–5 and incubated for 72 h at 28◦ C. Samples were harvested and analyzed by SDS-PAGE followed by coomassie blue staining or western blot analysis using specific EHDV-1 polyclonal antibodies. 2.5. Particles purification For the expression and purification of the recombinant corelike particles (CLPs, composed of VP3 and VP7) of EHDV-1, Sf9 insect cells were infected with the recombinant dual baculovirus Bac1629 :EHDV1.VP7.VP3 at a MOI of 3–5, incubated at 28 ◦ C and harvested 72 hpi. CLPs were purified as previously described [18]. For EHDV-1 VLPs, the recombinant proteins were generated by infection of Sf9 insect cells with the quadruple recombinant baculovirus consisting of S2, S3, S5 and S7 of EHDV-1. For the generation of heterologous VLPs of EHDV-2, Sf9 insect monolayers were coinfected with the dual baculoviruses; Bac1629 :EHDV1.S3.S7 and Bac1629 :EHDV2.S2.S5. Homologous and chimeric VLPs were purified according to the methods described by Stewart et al. [28]. As positive controls, EHDV-1 cores and virions were purified according to the modified methods described previously [29,30]. 2.6. Antibodies production For polyclonal antibody production, 2.5 mg/ml of the CLPs quantified by Bradford reagent (Sigma–Aldrich Ltd, UK) and 2.0 mg/ml of VLPs were supplied to a commercial company. Immunization and work with vertebrate animals (rabbits) was performed at ThermoFisher Scientific in accordance with the UK Animals (Scientific Procedures) Act 1986 (UK Government 1986) using the principles outlined in the Home Office guidance ‘Antibody Production: Principles for Protocols of Minimal Severity’ (Home Office 2000). The work adhered to the principles of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (see https://www.nc3rs.org.uk/arrive-guidelines). Animals were immunized first with 500 g of antigen in 10 sites followed by boosting twice with 250 g antigen at 14 and 28 respectively in the presence of Incomplete Fruend’s adjuvant. 2.7. Electron microscopy Aliquots (2 l) of purified EHDV-1 cores, CLPs and VLPs were adsorbed onto copper 400-mesh Formvar carbon-coated grids (TAAB Laboratories Equipment Ltd, UK) for 2 min and stained with filtered 2% (w/v) phosphotungstic acid (PTA), pH 6.8. Grids were examined under JEOL 1200 EX transmission microscope. 2.8. Neutralizing and cross neutralizing antibody response Antibodies raised against purified EHDV-1 VLPs were collected at (0, 35 and 56–58 days) and analyzed for their neutralizing activity
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against EHDV-1 virus. In addition, cross neutralizing activities were assessed against EHDV-2 and EHDV-6. Serum neutralization of 50 TCID50 and plaque reduction based standard neutralizing assay were carried out. All the dilutions were performed in triplicate and assays were repeated three times. 3. Results 3.1. Cloning and expression of individual EHDV major structural proteins Initially, we sought to assess if each of the four major structural proteins of the virion particle could be synthesized as recombinant proteins in Sf9 insect cells. Therefore, cloning and expression of the individual outer and inner capsid proteins VP2, VP3, VP5 and VP7 of EHDV-1 in insect cells were assessed using baculovirus single expression vector. The plasmid pAcYM1, containing AcMNPV ph promoter, was utilized to generate recombinant viruses that expressed high levels of the targeted genes. To generate recombinant pAcYM1 transfer vectors containing each EHDV-1 gene, the open reading frames (ORFs) of S2 (aa971), S5 (aa527) and S7 (aa349) were amplified using specific sense and antisense primers. The amplified ORFs were then sub-cloned into the pAcYM1 vector and the correct orientation of the inserts was confirmed by sequencing. The recombinant pAcYM1-EHDV1.S3 (aa899) was generated previously [31]. Recombinant baculoviruses expressing each protein singly were then generated and lysates from cells infected with each recombinant virus were subsequently analyzed by SDS-PAGE. Each recombinant virus showed high level expression of each capsid protein (Fig. 1, left panels). The authenticity of each protein was subsequently demonstrated by western analysis using EHDV1 polyclonal antibody with reactive bands corresponding to the estimated sizes of EHDV-1 structural proteins (Fig. 1, right panels). 3.2. Simultaneous expression of EHDV structural proteins by a single recombinant baculovirus
3
encoding for the inner core proteins (VP3 and VP7) of EHDV-1 were sub-cloned into transfer vectors targeting two specific genetic loci in the baculovirus genome, the 39K and egt loci, as described by Noad et al. [27]. The expression of each of the inner capsid proteins was examined in Sf21 insect cells and their assembly into particles was assessed by fractionation on a 25–50% discontinuous sucrose gradient. The gradient band corresponding to putative CLPs was isolated and analyzed by SDS-PAGE. Two protein bands corresponding to the estimated sizes of VP3 (103 kDa) and VP7 (39 kDa) were present in the middle of the gradient (Fig. 2A) and peak fractions were further purified using a second sucrose gradient, resulting in only VP3 and VP7 by SDS-PAGE analysis (Fig. 2B, lane 2). The VP3 material showed a doublet consistent with N-terminal trimming analogues to that observed for BTV CLPs [32]. Micrographs of the negatively stained samples showed particulate structures with a core-like morphology (Fig. 2C, right panel) similar in size and appearance to authentic EHDV-1purified cores (Fig. 2C, left panel). To generate VLPs, the genes encoding for the outer shell proteins VP2 and VP5 were sub-cloned individually and each recombined into AcMNPV genome encoding of VP3 and VP7, using two alternative loci, ph and odv-e56, respectively. To demonstrate that all four proteins of EHDV were expressed from one single recombinant baculovirus, recombinant virus-infected cell lysate was analyzed by SDS-PAGE. Coomassie blue staining of the gel exhibited the presence of all four EHDV proteins with sizes corresponding to those of the virion derived proteins (Fig. 3A, lane 3). As before, EHDV-1 VLPs were purified using discontinuous sucrose gradients and the composition of the purified VLPs was analyzed by SDS-PAGE. All four EHDV-1 proteins, VP2, VP5, VP3, and VP7 were identified (Fig. 3B, lane 3) with sizes corresponding to the recombinant individual EHDV-1 proteins expressed previously (Fig. 3B, lanes 4–7). The yield of purified VLPs from 100 ml culture was estimated approximately 1 mg. By electron microscopy the negatively stained samples of the peak fractions (Fig. 3C, left panel) of VLPs exhibited similar in size and morphology to EHDV-1 virion particles (Fig. 3C, right panel).
To synthesize VLPs efficiently, all four major structural proteins were expressed by a single recombinant baculovirus. As outer capsid formation requires the inner capsid as scaffold, the synthesis of EHDV-1 core-like particles (CLPs) was first assessed. The genes
Fig. 1. Individual expression of EHDV-1 structural capsid proteins. Sf9 insect cells were infected with each recombinant baculovirus at MOI of 3 and harvested at 72 hpi. The expression of each recombinant protein (VP2, VP3, VP5 and VP7) was analyzed by SDS-PAGE stained by coomassie blue (left panel) and immunoblotting with polyclonal antibodies against EHDV-1 (right panel). As controls, uninfected Sf9 cells (C) and Sf9 cells infected with a baculovirus expressing GFP protein (negative control, −ve) were included. Position of EHDV-1 protein bands and the molecular mass of standard proteins (M) are indicated.
Fig. 2. Assembly of EHDV-1 VP3 and VP7 into CLPs. EHDV-1 CLPs were purified by two consecutive sucrose gradient centrifugations. Aliquots of the first (A) and the second (B) centrifugations were analyzed by SDS-PAGE to confirm the expression of VP3 (103 kDa) and VP7 (39 kDa) (lane 2). Positions of the proteins and the molecular mass of standard (M) are indicated. (C) Electron micrographs of EHDV-1 purified cores (left panel) and CLPs (right panel). Bars indicate 100 nm.
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Fig. 3. Simultaneous expression of four capsid proteins of EHDV and their assembly into homologous VLPs. (A) Cell lysate infected with the recombinant baculovirus expressing VP2 (112 kDa), VP3 (103 kDa), VP5 (59 kDa) and VP7 (39 kDa) was analyzed by SDS-PAGE (lane 3). (B) VLPs were purified by sucrose gradient centrifugation and analyzed by SDS-PAGE. Note that all proteins were detected in the same fraction (lane 3, arrows). Proteins expressed singly were used as controls (lanes 4–7, arrowheads). As control, uninfected Sf9 cells (C) were used. Positions of the proteins and the molecular mass of standard (M) are indicated. (C) Electron micrographs of virion particles (left panel) and VLPs (right panel). Bars indicate 100 nm.
3.3. Generation of EHDV-2 VLPs using EHDV-1 CLPs as scaffolding Since VP3 and VP7 of CLPs are highly conserved proteins across the different serotypes, EHDV-1 CLPs could acquire VP2 and VP5 of a heterologous EHDV serotype to generate chimeric VLPs. To assess this, a dual expressing baculovirus was constructed with the coding region of EHDV-2 VP5 (aa527) and VP2 (aa982) inserted at the odve56 and ph loci respectively. The expression of the two structural proteins in the insect cells was analyzed by SDS-PAGE (Fig. 4A, Lane 3) and were shown to co-migrate with the individual recombinant EHDV-2 VP2 and EHDV-2 VP5 (Fig. 4A, lanes 4 and 5) described
Fig. 4. Simultaneous expression of EHDV-2 outer capsid proteins and their assembly into chimeric VLPs. (A) SDS-PAGE analysis of cell lysates infected either with a dual baculovirus expressing EHDV-2 VP2 and VP5 (lane 3), or with a single recombinant virus expressing each protein, VP2 (lane 4) and VP5 (lane 5). Lane 2, uninfected Sf9 cells (C) and lane 1, protein marker (M). (B) Assembly of EHDV-2 chimeric VLPs. An aliquot of purified chimeric VLPs was analyzed by SDS-PAGE (lane 4). As controls, partially purified EHDV-1 VLP sample (lane 3) and uninfected Sf9 cells (C) (lane 2) were used. Position of the proteins and the molecular marker (M) are indicated.
above. The production of chimeric VLPs was achieved by coinfection of insect cells with the recombinant baculovirus expressing EHDV-2 VP2 and VP5 with the inner capsid proteins VP3 and VP7 of EHDV1. The presence of chimeric VLPs was assessed by fractionation of infected cell lysates on sucrose gradients followed by SDS-PAGE analysis. All four proteins required for the formation of VLPs were present in the peak fraction (Fig. 4B, lane 4) with molecular mass of each protein similar to those of partially purified EHDV-1 VLPs used as a positive control (Fig. 4B, lane 3). 3.4. Immunogenic properties of EHDV CLPs and VLPs Antisera raised against purified EHDV-1 CLPs or VLPs were produced in rabbits and specificity determined by western blot of EHDV-1 infected BSR cells. Two specific protein bands were visualized with sizes consistent with EHDV-1 VP3 and VP7 with the CLP antisera (Fig. 5A, lane 3), while four bands corresponding to each of the EHDV-1 major structural proteins were clearly detectable by VLP antisera (Fig. 5B, lane 3). To test whether VLP antiserum would neutralize EHDV-1, serum neutralization assays by TCID50 and the plaque reduction assays were done. Both assays yielded similar results with 128 neutralization titres as shown in Table 1. Furthermore, to determine if VLP antiserum against EHDV-1 could neutralize infection by other EHDV serotypes, two additional serotypes, EHDV-2 and EHDV6 were assessed. Lower, but significant, cross-neutralization was apparent for both viruses in comparison to the control serum (Table 1). A live-attenuated vaccine, the only currently available EHDV vaccine, was developed following the outbreaks of the Ibaraki disease in Japan in 1980s. This vaccine was effective at least until 1997 as neither any evidence of Ibaraki disease nor any seroconversion was documented in cattle in that region prior to 1997.
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Fig. 5. Detection of EHDV-1 proteins from purified cores or virions by anti CLP or anti VLP antisera. (A) Western blot analysis of core proteins VP3 and VP7 using the anti CLP antiserum (lane 3). (B) Western blot analysis of four structural proteins VP2, VP3, VP5 and VP7 of virion using the anti VLP antiserum (lane 3). As control, uninfected cells were used (C). Position of the molecular mass (M) and recombinant proteins are indicated.
However, during 1997 outbreaks, new clinical signs that were never observed in the previous outbreaks were recorded due to the emergence of a more virulent strain of Ibaraki virus [9]. Live attenuated virus vaccine is however, neither DIVA compliant nor without risk. Furthermore, there are at least six additional serotypes circulating and controlling infection by each requires type-specific vaccine. The development of DIVA compliant efficacious vaccines against all EHDV serotypes is therefore desirable and economically important. In particular, recent epidemics by different serotypes in various European bordering countries, in which cattle farms were mainly affected, have emphasized the urgency for stock-piling multi-serotype vaccines. VLP vaccines are generally DIVA compliant due to the absence of non-structural and enzymatic proteins and have been proven to be highly efficacious for a number of animal viruses including orbiviruses [22,23]. VLPs for non-enveloped (HEV and HPV) and enveloped (HBV, Influenza A) viruses have been produced using different expression systems and shown to be highly immunogenic [33–37]. However, the production of VLPs for the viruses of the family Reoviridae is more challenging as VLPs consist of complex multiple interacting viral capsid proteins organized in several concentric layers. In this study, we investigated the possibility of generating EHDV VLPs by simultaneous expression of four major structural proteins using baculovirus multiple gene expression vectors. Electron microscopy analysis of VLPs revealed the particulate structures analogous to the authentic EHDV particles. However, the VLPs lacked the central density due to the absence of the viral genomic RNA. Antibodies raised to EHDV-1 VLPs particles had a strong neutralizing titre against EHDV-1 and a higher than typical neutralization level in natural convalescent sera from infected animals [38]. These data indicate that the epitopes of the outer capsids proteins of VLPs are presented in correct immunogenic
Table 1 EHDV-1 VLPs antisera neutralization titre determined by TCID50 in BSR cells infected with EHDV-1, -2 and 6. EHDV-1 VLPs serum
EHDV-1 EHDV-2 EHDV-6
FCS
0 dpi
35 dpi
56–58 dpi
0 0 0
32 6 6
128 8 8
2 2 2
5
conformations. These VLPs are likely to afford complete protection in animals against EHDV-1 infection. The sequence analysis of different orbiviruses has shown that VP2 and VP5 are the most variable proteins. Nevertheless, depending on the serotype, both proteins exhibit overlapping conserved sequences to other serotypes and in some cases, cross neutralization activities have also been demonstrated [39]. Consistent with these, low level cross neutralization of EHDV-2 and EHDV6 was achieved by EHDV-1 anti-VLP antiserum, emphasizing that these three serotypes are genetically related and certain immunogenic epitopes are shared among these serotypes. The data also suggests that VLP vaccines of less than seven different serotypes would be capable of affording protective immunity against all seven serotypes. Comparative analysis of the inner core protein sequence of the seven EHDV serotypes showed a high conservation among these proteins, with identity up to 90% for VP3 and 80% for VP7 [6]. Here we showed that it was indeed possible to assemble EHDV-2 outer capsids proteins onto the CLPs of EHDV-1 and to generate EHDV-2 VLPs. This is highly encouraging as using a common CLP scaffold, VLPs could be produced rapidly in response to emerging serotypes similarly. 4. Conclusions Overall, data presented in this study demonstrated that immunogenic VLPs of different EHDV serotypes could be generated by simultaneous expression of all major structural proteins using baculovirus multiple expression system, as opposed to coinfections of insect cells with four different recombinant viruses, which is not ideal for vaccine purposes. These VLPs are potential vaccine candidates against EHDV infection in animals and are also DIVA compliant as none of the NS proteins, which could serve as diagnostic reagents, are present in these protein-based VLPs. Acknowledgements We are grateful to Dr. J. Richt (Kansas State University, USA) for providing us with EHDV-2 plasmid constructs. This work was funded by the FP7 Framework of European Commission. References [1] Omori T, Inaba Y, Morimoto T, Tanaka Y, Ishitani R. Ibaraki virus, an agent of epizootic disease of cattle resembling bluetongue. I. Epidemiologic, clinical and pathologic observations and experimental transmission to calves. Jpn J Microbiol 1969;13(2):139–57. [2] Inaba U. Ibaraki disease and its relationship to bluetongue. Aust Vet J 1975;51(4):178–85. [3] Breard E, Sailleau C, Hamblin C, Graham SD, Gourreau JM, Zientara S. Outbreak of epizootic haemorrhagic disease on the island of Reunion. Vet Rec 2004;155(14):422–3. [4] Gibbs EP, Lawman MJ. Infection of British deer and farm animals with epizootic haemorrhagic disease of deer virus. J Comp Pathol 1977;87(3):335–43. [5] Temizel EM, Yesilbag K, Batten C, Senturk S, Maan NS, Clement-Mertens PP, et al. Epizootic hemorrhagic disease in cattle Western Turkey. Emerg Infect Dis 2009;15(2):317–9. [6] Allison AB, Goekjian VH, Potgieter AC, Wilson WC, Johnson DJ, Mertens PP, et al. Detection of a novel reassortant epizootic hemorrhagic disease virus (EHDV) in the USA containing RNA segments derived from both exotic (EHDV-6) and endemic (EHDV-2) serotypes. J Gen Virol 2010;91(Pt 2):430–9. [7] Gaydos JK, Crum JM, Davidson WR, Cross SS, Owen SF, Stallknecht DE. Epizootiology of an epizootic hemorrhagic disease outbreak in West Virginia. J Wildl Dis 2004;40(3):383–93. [8] Yadin H, Brenner J, Bumbrov V, Oved Z, Stram Y, Klement E, et al. Epizootic haemorrhagic disease virus type 7 infection in cattle in Israel. Vet Rec 2008;162(2):53–6. [9] Ohashi S, Yoshida K, Watanabe Y, Tsuda T. Identification and PCR-restriction fragment length polymorphism analysis of a variant of the Ibaraki virus from naturally infected cattle and aborted fetuses in Japan. J Clin Microbiol 1999;37(12):3800–3.
Please cite this article in press as: Alshaikhahmed K, Roy P. Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2015.12.069
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Please cite this article in press as: Alshaikhahmed K, Roy P. Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2015.12.069