Bluetongue virus (BTV) is a non-enveloped dsRNA virus belonging to the family Reoviridae, genus Orbivirus, and typically infects ruminants and causes severe haemorrhagic disease in sheep [1]. The eradication and control of BTV is a challenge due to the existence of 29 different serotypes and their limited serological cross-reactivity [2]. Serotypes are determined by the variability of segment 2 of the genome encoding the VP2 structural protein which elicits a neutralising antibody response. During infection, the tip domain of VP2 points outwards from the virion, and binding of VP2-specific antibodies can induce serotype-specific neutralisation [3]. South Africa has an extensive diversity of BTV, with at least 21 serotypes in circulation [4]. Currently, vaccination using a modified live virus (MLV) vaccine, or the inactivated vaccine is the most effective method of controlling BTV spread and preventing severe disease. The MLV regime consists of three doses containing five serotypes each, which can be onerous to administer. The inactivated vaccine is a safer alternative, as there are no risks of reassortment of vaccine and wild isolates or reversion to virulence like the MLV. However, the short-lived immunogenicity of inactivated vaccines has encouraged the development of alternative candidates which are safer, more cost-effective, are efficacious for a longer period, and protective against multiple serotypes.
A potentially very suitable vaccine regime would be the use of virus-like particles (VLPs): these mimic the protein shell of the native virion but lack any genetic material, making them non-infectious and unable to undergo replication or cause viraemia [5]. BTV VLPs are assembled by co-expression of the 4 major structural viral proteins - VP2, VP3, VP5 and VP7 - and several research groups have produced BTV VLPs representing different serotypes using the baculovirus / insect cell expression system [6]. Despite their immunogenicity and efficacy, however, this production system is insufficiently cost effective to compete with the commercially available inactivated vaccines [6]. The use of plants as a production platform, however, has the appeal of low complexity of the upstream process, far greater ease of scale-up and lower operating costs with no need for upstream sterility. Moreover, there are numerous examples published of plant-made vaccines developed for veterinary use [7]. We have previously shown that plant-produced BTV8 VLPs elicited serotype-specific antibodies in sheep which protected them against BTV8 challenge [8]; furthermore, Mokoena et al. showed that plant-produced double chimaeric BTV VLPs targeting serotype 3 and 4 induced seroconversion in sheep [9].
The requirement for co-expression of full length serotype specific BTV structural proteins to make BTV VLPs targeting all or even most of the 29 serotypes is very cumbersome. Accordingly, we investigated a streamlined approach to produce chimaeric VLPs which have the potential to provide protection against multiple serotypes either through cross protection by a single chimaeric VLP, or through use of a cocktail of chimaeric VLPs targeting different serotypes. In this work we explored the substitution of the immunogenic “tip” domain of BTV8 VP2 with that of the corresponding domain of BTV1 to generate a chimaeric VP2 which, when co-expressed in plants with the remaining BTV8 VP3, VP5 and VP7, resulted in chimaeric BTV1/8 VLPs. These were used to immunise guinea pigs, and their immunogenicity and capability to elicit neutralising antibodies were evaluated.
The BTV1/8 chimaeric VP2 gene was synthesised by GenScript (USA). A consensus sequence for the BTV1 VP2 tip domain (base pairs 579–1241), created by aligning BTV1 VP2 genes from 37 different BTV1 strains available at the time on GenBank, was human codon-optimised and substituted in the corresponding region of BTV8 VP2 (BTV1/8VP2) (Fig. 1A). AgeI and XhoI restriction enzyme sites on the N and C termini of BTV1/8VP2, respectively, facilitated cloning into the pEAQ-HT vector to generate chimaeric pEAQ-HT-BTV1/8 VP2. One hundred ng of pEAQ-HT-BTV1/8 VP2 was electroporated into Rhizobium radiobacter AGL-1 cells (ATCC BAA-101). Transformed colonies from cells plated on LB agar supplemented with carbenicillin (25ug/mL) and kanamycin (30ug/mL) and incubated at 27°C for 2 days were screened by PCR to confirm the presence of pEAQ-HT-BTV1/8 VP2.
For infiltration, cells harbouring constructs encoding BTV8 proteins (pEAQ-HT-BTV8 VP2, VP3, VP5 and VP7) described previously [8] (Fig. 1A) and R. radiobacter harbouring pEAQ-HT-BTV1/8 VP2 were cultured in LB broth supplemented with appropriate antibiotics overnight at 27°C overnight with agitation. For co-infiltration, cell cultures containing each construct were adjusted to an OD600 of 0.5 in infiltration medium as previously described [10]. Combined cultures harbouring constructs encoding either all four BTV8 VPs or chimaeric BTV1/8 VP2 and BTV8 VP3, VP5 and VP7 were vacuum-infiltrated into whole Nicotiana benthamiana plants and incubated as described previously [11]. Plants similarly infiltrated with a culture harbouring pEAQ-HT lacking any gene of interest served as negative controls.
Approximately 25-30g of leaf material was harvested from infiltrated plants at 5 days post infiltration (dpi) and homogenised using an IKA® T25 digital ULTRA-TURRAX in 2 volumes of bicine buffer (50mM bicine, 400mM NaCl, pH9) containing 1X Roche® EDTA-free complete protease inhibitor. Homogenates were incubated at 4°C with gentle agitation for 1 h, then centrifuged (Beckman Coulter Avanti® J25-I centrifuge) at 25931 x g for 30 min, filtered through one layer of Miracloth™ (Merck Millipore) and finally re-centrifuged at 25931 x g for 20 min. The pH of the clarified supernatants was adjusted to 8.4 with 1M NaOH, and they were incubated at 4°C with gentle agitation for approximately 48 h. The clarified plant extracts were centrifuged (Beckman Coulter Avanti® J25-I centrifuge) at 25931 x g for 20 min and the supernatants loaded onto discontinuous Optiprep™ (Sigma Aldrich) iodixanol gradients (2mL 50%, 2mL 40%, 2mL 30%, 2mL 20%) diluted in bicine buffer. The gradients were re-centrifuged (Beckman Coulter Optima™ L-100 XP Ultracentrifuge) at 60973 x g (22400 rpm) for 3 h at 10°C using a Beckman SW 32 Ti rotor. Fractions of 500uL were collected from the bottom of the centrifuge tubes and analysed by SDS-PAGE, western blotting and transmission electron microscopy (TEM) as described previously [8]. The purified VLP samples were quantitated by gel densitometry of Coomassie-stained polyacrylamide gels using GeneTools software (Syngene) and a BSA standard curve.
Immunogenicity of the BTV8 and chimaeric purified VLPs was tested in female guinea pigs. This study was approved by the Animal Ethics Committee at UCT (AEC020-023). Ten to 12-week old animals were randomly separated into 3 groups of five guinea pigs each (n = 5). Group 1 was inoculated with mock antigen comprising the equivalent fraction extracted from negative control leaves and purified in a similar manner to the VLPs. Groups 2 and 3 were inoculated subcutaneously with 15ug BTV8 or chimaeric BTV1/8 VLPs, respectively and mixed with 5% Montanide™ ISA 50 V2 adjuvant (Seppic). Pre-bleed serum was harvested prior to immunisation and animals were boosted at 13 days post-immunisation. Final bleeds were collected on day 41 post-immunisation by cardiac puncture.
Indirect ELISAs were conducted on sera to evaluate the vaccine immune responses using methods described by Stander et al. with some modifications [11]: ELISA plates were coated with 100ng plant-produced BTV8 or chimaeric BTV1/8 VLPs; pooled sera were diluted to 1:10 000 in TBS prior to loading into the wells containing the respective antigens against which they were raised; and, goat anti-guinea pig IgG (whole molecule) alkaline phosphatase conjugated secondary antibody was diluted to 1:30 000 in 1 x TBS. Statistical analysis was conducted using GraphPad Prism version 9.3.1 (GraphPad, CA, USA). Error bars on the graphs represent the mean ± SEM (standard error of the mean) from three independent experiments with technical triplicates for each group. The comparison of the response between groups was calculated using the Student’s two-tailed t-test. The significance threshold (p-value) was set at 5% (p = 0.05) with p < 0.05 representing a statistically significant result.
Sera from animals in Groups 2 and 3 and a pooled sample from Group 1 were assayed for virus neutralisation capability against BTV serotypes 1 and 8 using the microneutralisation method. Briefly, two-fold serial dilutions of test sera at 25 µl volumes were made in tissue culture medium in wells of flat-bottomed microtitre plates. Approximately 100–300 TCID50 of reference BTV serotype 1 and 8 were added in equal volumes to each well containing the test sera and gently mixed. The plates were incubated for 1 hour at 370C and 5% CO2, following which nearly 104 cells were added per well in volumes of 100 µl, and the plates incubated for up to 7 days. Positive and negative antisera and cell controls and virus back-titration plates were set up per serotype accordingly, and the plates were observed for development of cytopathic effect (CPE) daily using an inverted microscope. Final test results were read when there was 75–100% CPE in the wells containing test virus, and virus and negative control antisera, and no CPE was observed in the cell control, and virus and positive control antisera containing wells. Sera were considered to be serologically identical to reference BTV serotypes if they neutralised the virus in the test [12].
Co-infiltration of BTV8 VP2-, VP3-, VP5- and VP7-encoding constructs resulted in expression of all 4 proteins and assembly of VLPs as expected from previous work [8]. To determine whether chimaeric BTV1/8 VLPs could be assembled in plants, the chimaeric BTV1/8 VP2 construct was co-infiltrated with the BTV8 VP3, VP5 and VP7 constructs.
After purification and fractionation, SDS-PAGE confirmed that the majority of the 4 BTV VP proteins for both samples accumulated at the interface of the 30 to 40% Optiprep™ gradient in fractions 7 and 8, further confirmed by western blotting (Fig. S1 - VP2 111 kD, VP3 103 kD, VP5 59 kD and VP7 38 kD). Both fractions from each of the samples were visualised using TEM and confirmed the presence of VLPs (Fig. 1B). Core-like particles (CLPs) – consisting of VP3 and VP7 only – were also visible in both samples, possibly a result of interruption of assembly on harvesting of leaves. BTV8 and BTV1/8 samples had similar densities of particles and comparative ratios of VLPs to CLPs. Purified samples were quantitated for dosing guinea pigs, with concentrations of approximately 35mg/kg of fresh leaf weight (FLW).
BTV8 and BTV1/8 VLPs (15ug total BTV protein) were administered to guinea pigs using a prime-boost regimen to compare their ability to elicit serotype-specific immune responses. Indirect ELISA of pooled pre- and post-immune sera (1:10 000 dilution) for each of the VLP vaccines (groups 2 and 3) using VLPs as coating antigen showed a statistically significant difference (p < 0.01) in the response between pre- and post-immunised sera, whereas there was no difference between the responses in serum from animals immunised with the negative control (group 1) (Fig. 2A). This confirmed that both VLP vaccines were capable of stimulating a specific response. A comparison between the antibody-specific responses of the 2 VLP vaccines using the post-immunisation sera (1:10 000 dilution) from each individual guinea pig in the vaccination groups showed that sera to both BTV8 and BTV1/8 VLPs (groups 2 and 3) produced significantly higher (at least 5-fold) mean OD405 absorbance values, 0.997 and 0.633 respectively, than the negative control group (OD405 0.119) (Fig. 2B). Additionally, the BTV8 VLP vaccine induced a significantly greater (1.5-fold) immune response than the chimaeric VLP group (see p-values Fig. 2B). This suggests that the chimaeric vaccine is not as potent as the BTV8 vaccine. A strong neutralising antibody response requires the VP2 protein and its epitopes to be displayed in the correct conformation. The chimaeric nature of the VP2 protein may have affected the epitope display or the interaction of VP2 with VP5, making it less effective in inducing an antibody response [13].
Serum neutralisation analysis was carried out on individual sera from each guinea pig. Sera from guinea pigs immunised with BTV8 VLPs (samples 1 to 5) neutralised BTV serotype 8 with virus neutralisation titres (vnt) ranging from 1:40 to 1:160 (Table 1). Interestingly, these sera also neutralised BTV serotype 1, albeit with lower titres than those measured for BTV serotype 8. It is possible that this may be due to cross neutralisation between the 2 BTV VP2 domains of serotypes 1 and 8. It has been shown previously that sera from animals infected with BTV8 could neutralise a BTV1 cell-culture-adapted strain [14]. In addition, Martinelle et al [15] have reported partial cross-reactivity between BTV1 and BTV8 in calves pre-vaccinated with BTV8 and subsequently infected with BTV1. Sera from guinea pigs immunised with BTV1/8 chimaeric VLPs (samples 6 to 9) neutralised BTV serotype 1 with vnts ranging from 1:20 to 1:40. The positive titres suggest that the substituted BTV1 VP2 domain may be responsible for conferring BTV1 specificity. The fact that sera from these same guinea pigs did not confer any BTV8 neutralisation capability further confirms this. This also suggests that the domains of BTV8 VP2 which are common to both BTV8 and BTV1/8 VLPs did not elicit a measurable VP2-specific response, and therefore confirms the importance of the tip domain as a major neutralisation epitope.
Having previously shown that BTV serotype 8 VLPs made in plants are immunogenic, are able to elicit serotype-specific neutralising antibodies and are efficacious, this additional investigation showed that it is potentially possible to generate chimaeric VLPs targeting alternative serotypes which are also immunogenic and have neutralising capability. The retention of the serotype 8 backbone comprising VP3, VP5 and VP7 and substitution of the immunogenic tip domain of VP2 with that of a desired BTV serotype simplifies manipulation. The development of a vaccine cocktail of chimaeric VLPs of this nature could potentially be administered to animals to protect against multiple circulating serotypes. In addition, this vaccine design would be more accommodating for combatting outbreaks when the serotypes in circulation cannot be easily predicted prior to an outbreak.