Plant-Derived Human Vaccines: Recent Developments.

The idea of producing vaccines in plants originated in the late 1980s. Initially, it was contemplated that this notion could facilitate the concept of edible vaccines, making them more cost effective and easily accessible. Initial studies on edible vaccines focussed on the use of a variety of different transgenic plant host species for the production of vaccine antigens. However, adequate expression levels of antigens, the difficulties predicted with administration of consistent doses, and regulatory rules required for growth of transgenic plants gave way to the development of vaccine candidates that could be purified and administered parenterally. The field has subsequently advanced with improved expression techniques including a shift from using transgenic to transient expression of antigens, refinement of purification protocols, a deeper understanding of the biological processes and a wealth of evidence of immunogenicity and efficacy of plant-produced vaccine candidates, all contributing to the successful practice of what is now known as biopharming or plant molecular farming. The establishment of this technology has resulted in the development of many different types of vaccine candidates including subunit vaccines and various different types of nanoparticle vaccines targeting a wide variety of bacterial and viral diseases. This has brought further acceptance of plants as a suitable platform for vaccine production and in this review, we discuss the most recent advances in the production of vaccines in plants for human use.

Plant expression systems as an economical alternative for the production of iELISA coating antigen AHSV VP7.

African horse sickness (AHS) is a debilitating and highly infectious arthropod-borne disease affecting all species of Equidae. The causative agent of AHS is the non-enveloped dsRNA African horse sickness virus (AHSV), belonging in the genus Orbivirus, family Reoviridae. The identification and surveillance of AHSV by simple and reliable diagnostic tools is essential for managing AHS outbreaks. Indirect ELISAs utilising soluble AHSV antigen or recombinant VP7, an immunodominant and serogroup-specific major core structural protein, are commonly used for serological diagnostic assays. Plant production systems are a significant alternative for recombinant protein production, as they are safe, easily scalable, production rates are rapid and upstream processes are more cost-effective than more traditional expression systems. This pilot study reports the successful production of AHSV-5 VP7 quasi-crystals in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated transient expression using the self-replicating pRIC3.0 plant expression vector. After purification by means of density gradient ultracentrifugation, yields of pure VP7 of 2.66 µg/g fresh leaf mass (FLM) were achieved. Purified plant-produced AHSV-5 VP7 detected AHSV-specific antibodies in horse sera in an indirect ELISA and was able to distinguish between AHSV-positive and negative sera. Additionally, plant-produced AHSV-5 VP7 detected AHSV-specific antibodies to the same degree as E. coli-produced VP7. These results justify further investigation into the diagnostic capability of plant-produced AHSV VP7 quasi-crystals. To the best of our knowledge, this is the first report of AHSV VP7 quasi-crystal production in N. benthamiana and the first time that plant-produced VP7's potential as a diagnostic has been assessed.

A Plant-Produced Virus-Like Particle Displaying Envelope Protein Domain III Elicits an Immune Response Against West Nile Virus in Mice.

West Nile virus (WNV) is a globally disseminated Flavivirus that is associated with encephalitis outbreaks in humans and horses. The continuous global outbreaks of West Nile disease in the bird, human, and horse populations, with no preventative measures for humans, pose a major public health threat. The development of a vaccine that contributes to the "One Health" Initiative could be the answer to prevent the spread of the virus and control human and animal disease. The current commercially available veterinary vaccines are generally costly and most require high levels of biosafety for their manufacture. Consequently, we explored making a particulate vaccine candidate made transiently in plants as a more cost-effective and safer means of production. A WNV virus-like particle-display-based vaccine candidate was generated by the use of the SpyTag/SpyCatcher (ST/SC) conjugation system. The WNV envelope protein domain III (EDIII), which contains WNV-specific epitopes, was fused to and displayed on AP205 phage virus-like particles (VLPs) following the production of both separately in Nicotiana benthamiana. Co-purification of AP205 and EDIII genetically fused to ST and SC, respectively, resulted in the conjugated VLPs displaying EDIII with an average coupling efficiency of 51%. Subcutaneous immunisation of mice with 5 µg of purified AP205: EDIII VLPs elicited a potent IgG response to WNV EDIII. This study presents the potential plants being used as biofactories for making significant pharmaceutical products for the "One Health" Initiative and could be used to address the need for their local production in low- and middle-income countries (LMICs).

Humoral and cell-mediated immune responses to plant-produced African horse sickness virus VP7 quasi-crystals.

African horse sickness (AHS) is a devastating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS, and commercially used live-attenuated vaccines have various undesirable side effects. Previous studies have shown that mice inoculated with insoluble African horse sickness virus (AHSV) VP7 crystals are protected from live challenge with a lethal dose of AHSV. This study investigates the humoral and cell-mediated immune responses in guinea-pigs to a safer monovalent vaccine alternative based on AHSV-5 VP7 quasi-crystals produced in plants. Guinea-pigs received prime- and boost-inoculations of between 10 and 50 µg of purified plant-produced AHSV VP7. Western immunoblot analysis of the humoral response showed stimulation of high titres of anti-VP7 antibodies 28 days after the boost-inoculation in sera from three of the five experimental animals. In addition, RNA-seq transcriptome profiling of guinea-pig spleen-derived RNA highlighted thirty significantly (q ≤ 0.05) differentially expressed genes involved in innate and adaptive immunity. Differential expression of genes involved in Th1, Th2 and Th17 cell differentiation suggest a cell-mediated immune response to AHSV-5 VP7. Upregulation of several important cytokines and cytokine receptors were noted, including TNFSF14, CX3CR1, IFNLR1 and IL17RA. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. While further investigation is needed to validate these findings, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic and induce both humoral and cell-mediated responses.

Engineering the Plant Secretory Pathway for the Production of Next-Generation Pharmaceuticals.

Production of biologics in plants, or plant molecular pharming, is a promising protein expression technology that is receiving increasing attention from the pharmaceutical industry. Previously, low expression yields of recombinant proteins and the realization that certain post-translational modifications (PTMs) may not occur optimally limited the widespread acceptance of the technology. However, molecular engineering of the plant secretory pathway is now enabling the production of increasingly complex biomolecules using tailored protein-specific approaches to ensure their maturation. These involve the elimination of undesired processing events, and the introduction of heterologous biosynthetic machinery to support the production of specific target proteins. Here, we discuss recent advances in the production of pharmaceutical proteins in plants, which leverage the unique advantages of the technology.

Characterization and Immunogenicity of HIV Envelope gp140 Zera® Tagged Antigens.

HIV-1 envelope glycoprotein (Env) remains the most relevant target for the elicitation of functional antibodies to HIV by vaccination. However, soluble Env antigens often do not elicit the desired immune responses. Delivering subunit antigens on particulate nanoparticles is an established approach to improve their immunogenicity. In this study the sequence encoding Zera®, a proline-rich domain derived from the γ-zein storage protein, was fused to either the C- or N-terminus of the superinfecting HIV-1 CAP256 gp140 envelope: Zera® generally induces the formation of protein bodies (PBs), which can significantly improve both the immunogenicity and yields of the partner protein. The expression of gp140-Zera® and Zera®-gp140 (N- and C-terminal fusions respectively) in mammalian cells was confirmed by western blot analysis and immunostaining. However, isopycnic ultracentrifugation showed that neither gp140-Zera® nor Zera®-gp140 accumulated in characteristic electron-dense PBs. gp140-Zera® elicited higher binding antibody titers in rabbits to autologous gp140 and V1V2 scaffold than Zera®-gp140. Rabbit anti-gp140-Zera® sera also had significantly higher Tier 1A neutralizing antibody titers than anti-Zera®-gp140 sera. Neither gp140-Zera® nor Zera®-gp140-specific sera neutralized Tier 1B or autologous Tier 2 viruses. These results showed that HIV-1 gp140 tagged with Zera® at either the N- or C-termini elicited high titers of gp140 and V1V2 binding antibodies, and low levels of Tier 1 neutralizing antibodies in rabbits.

Co-expression of human calreticulin significantly improves the production of HIV gp140 and other viral glycoproteins in plants.

Plant molecular farming (PMF) is rapidly gaining traction as a viable alternative to the currently accepted paradigm of producing biologics. While the platform is potentially cheaper and more scalable than conventional manufacturing systems, expression yields and appropriate post-translational modifications along the plant secretory pathway remain a challenge for certain proteins. Viral fusion glycoproteins in particular are often expressed at low yields in plants and, in some cases, may not be appropriately processed. Recently, however, transiently or stably engineering the host plant has shown promise as a strategy for producing heterologous proteins with more complex maturation requirements. In this study we investigated the co-expression of a suite of human chaperones to improve the production of a human immunodeficiency virus (HIV) type 1 soluble gp140 vaccine candidate in Nicotiana benthamiana plants. The co-expression of calreticulin (CRT) resulted in a dramatic increase in Env expression and ameliorated the endoplasmic reticulum (ER) stress response - as evidenced by lower transcript abundance of representative stress-responsive genes. The co-expression of CRT similarly improved accumulation of glycoproteins from Epstein-Barr virus (EBV), Rift Valley fever virus (RVFV) and chikungunya virus (CHIKV), suggesting that the endogenous chaperone machinery may impose a bottleneck for their production. We subsequently successfully combined the co-expression of human CRT with the transient expression of human furin, to enable the production of an appropriately cleaved HIV gp140 antigen. These transient plant host engineering strategies are a promising approach for the production of high yields of appropriately processed and cleaved viral glycoproteins.

Production and Immunogenicity of Soluble Plant-Produced HIV-1 Subtype C Envelope gp140 Immunogens.

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.

African Horse Sickness: A Review of Current Understanding and Vaccine Development.

African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines that utilize viral vectors or are based on reverse genetics or virus-like particle technologies. This review summarizes the current understanding of AHSV structure and the viral replication cycle and also evaluates existing and potential vaccine strategies that may be applied to prevent or control the disease.

Chimaeric Rift Valley Fever Virus-Like Particle Vaccine Candidate Production in Nicotiana benthamiana.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live-attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus-like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail-encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg-1 fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne-HA particles of 49-60 nm which are immunogenic, eliciting Gn-specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne-HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen.

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Minimally processed crude leaf extracts of Nicotiana benthamiana containing recombinant foot and mouth disease virus-like particles are immunogenic in mice.

Foot-and-mouth disease (FMD) remains one of the most feared viral diseases affecting cloven-hoofed animals, and results in severe economic losses. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of recombinant FMDV-like particles (VLPs) as subunit vaccines has gained importance because of their immunogenic properties and safety. We evaluated the production of FMD VLPs, via Agrobacterium-mediated transient expression, and the immunogenicity of these structures in mice. Leaves were infiltrated with pEAQ-HT and pRIC 3.0 vectors encoding the capsid precursor P1-2A and the protease 3C. The recombinant protein yield was 3-4 mg/kg of fresh leaf tissue. Both groups of mice immunized with purified VLPs and mice immunized with the crude leaf extract elicited a specific humoral response with similar antibody titers. Thus, minimally processed plant material containing transiently expressed FMD VLPs could be a scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses.

African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.

Transient expression of heat- and acid-resistant foot-and-mouth disease virus P1-2A mutants in Nicotiana benthamiana.

Recombinant foot-and-mouth disease virus-like particles (VLPs) can be expressed in a number of expression systems including plants. However, yields in plants have formerly been shown to be low, possibly due to their acid and/or heat lability, previously shown to affect VLP yields produced in other systems. This work describes the introduction of mutations into the FMDV structural protein-encoding gene (P1-2A) which have been previously shown to increase acid and thermostability. VLPs expressed in plants using the mutant constructs had negative rather than positive effects on yield and temperature and acid stability compared to the control.

Production of complex viral glycoproteins in plants as vaccine immunogens.

Plant molecular farming offers a cost-effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant-derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post-translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana.

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

Therapeutic vaccines for high-risk HPV-associated diseases.

Cancer is the second leading cause of death worldwide, and it is estimated that Human papillomavirus (HPV) related cancers account for 5% of all human cancers. Current HPV vaccines are extremely effective at preventing infection and neoplastic disease; however, they are prophylactic and do not clear established infections. Therapeutic vaccines which trigger cell-mediated immune responses for the treatment of established infections and malignancies are therefore required. The E6 and E7 early genes are ideal targets for vaccine therapy due to their role in disruption of the cell cycle and their constitutive expression in premalignant and malignant tissues. Several strategies have been investigated for the development of therapeutic vaccines, including live-vector, nucleic acid, peptide, protein-based and cell-based vaccines as well as combinatorial approaches, with several vaccine candidates progressing to clinical trials. With the current understanding of the HPV life cycle, molecular mechanisms of infection, carcinogenesis, tumour biology, the tumour microenvironment and immune response mechanisms, an approved HPV therapeutic vaccine seems to be a goal not far from being achieved. In this article, the status of therapeutic HPV vaccines in clinical trials are reviewed, and the potential for plant-based vaccine production platforms described.

Novel expression of immunogenic foot-and-mouth disease virus-like particles in Nicotiana benthamiana.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals and is endemic in Africa, parts of South America and southern Asia. The causative agent, FMD virus (FMDV) is a member of the genus Aphthovirus, family Picornaviridae. Vaccines currently used against FMDV are chemically inactivated virus strains which are produced under high-level biocontainment facilities, thus raising their cost. The development of recombinant FMDV vaccines has focused predominantly on FMDV virus-like particle (VLP) subunit vaccines for which promising results have been achieved. These VLPs are attractive candidates because they avoid the use of live virus in production facilities, but conserve the complete repertoire of conformational epitopes of the virus. Recombinant FMDV VLPs are formed by the expression and assembly of the three structural proteins VP0, VP1 and VP3. This can be attained by co-expression of the three individual structural capsid proteins or by co-expression of the viral capsid precursor P1-2A together with the viral protease 3C. The latter proteolytically cleaves P1-2A into the respective structural proteins. These VLPS are produced in mammalian or insect cell culture systems, which are expensive and can be easily contaminated. Plants, such as Nicotiana benthamiana, potentially provide a more cost-effective and very highly scalable platform for recombinant protein and VLP production. In this study, P1-2A was transiently expressed in N. benthamiana alone, without the 3C protease. Surprisingly, there was efficient processing of the P1-2A polyprotein into its component structural proteins, and subsequent assembly into VLPs. The yield was ∼0.030µg per gram of fresh leaf material. Partially purified VLPs were preliminarily tested for immunogenicity in mice and shown to stimulate the production of FMDV-specific antibodies. This study, has important implications for simplifying the production and expression of potential vaccine candidates against FMDV in plants, in the absence of 3C expression.

Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine.

African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live-attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost-effective recombinant vaccine. Here, we report the plant-based production of a virus-like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens-mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell-based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.

Development of plant-produced protein body vaccine candidates for bluetongue virus.

BACKGROUND: Bluetongue is a disease of domestic and wild ruminants caused by bluetongue virus serotypes (BTV), which have caused serious outbreaks worldwide. Commercially available vaccines are live-attenuated or inactivated virus strains: these are effective, but there is the risk of reversion to virulence or reassortment with circulating strains for live virus, and residual live virus for the inactivated vaccines. The live-attenuated virus vaccines are not able to distinguish naturally infected animals from vaccinated animals (DIVA compliant). Recombinant vaccines are preferable to minimize the risks associated with these vaccines, and would also enable the development of candidate vaccines that are DIVA-compliant. RESULTS: In this study, two novel protein body (PB) plant-produced vaccines were developed, Zera®-VP2ep and Zera®-VP2. Zera®-VP2ep contained B-cell epitope sequences of multiple BTV serotypes and Zera®-VP2 contained the full-length BTV-8 VP2 codon-optimised sequence. In addition to fulfilling the DIVA requirement, Zera®-VP2ep was aimed at being multivalent with the ability to stimulate an immune response to several BTV serotypes. Both these candidate vaccines were successfully made in N. benthamiana via transient Agrobacterium-mediated expression, and in situ TEM analysis showed that the expressed proteins accumulated within the cytoplasm of plant cells in dense membrane-defined PBs. The peptide sequences included in Zera®-VP2ep contained epitopes that bound antibodies produced against native VP2. Preliminary murine immunogenicity studies showed that the PB vaccine candidates elicited anti-VP2 immune responses in mice without the use of adjuvant. CONCLUSIONS: These proof of concept results demonstrate that Zera®-VP2ep and Zera®-VP2 have potential as BTV vaccines and their development should be further investigated.