A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters.

Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparator mammalian antigen. Animals vaccinated with the plant-derived antigen exhibited reduced viral loads following challenge, as well as significant protection from SARS-CoV-2 disease as evidenced by reduced lung pathology, lower viral loads and protection from weight loss. Nonetheless, animals immunized with the mammalian cell-culture-derived protein were better protected in this challenge model suggesting that more faithfully reproducing the native glycoprotein structure and associated glycosylation of the antigen may be desirable.

Optimal size of DNA encapsidated by plant produced human papillomavirus pseudovirions.

Human papillomaviruses (HPVs) are known to be the cause of anogenital and oropharyngeal cancers as well as genital and common warts. HPV pseudovirions (PsVs) are synthetic viral particles that are made up of the L1 major and L2 minor HPV capsid proteins and up to 8 Kb of encapsidated pseudogenome dsDNA. HPV PsVs are used to test novel neutralising antibodies elicited by vaccines, for studying the virus life cycle, and potentially for the delivery of therapeutic DNA vaccines. HPV PsVs are typically produced in mammalian cells, however, it has recently been shown that Papillomavirus PsVs can be produced in plants, a potentially safer, cheaper and more easily scalable means of production. We analysed the encapsidation frequencies of pseudogenomes expressing EGFP, ranging in size from 4.8 Kb to 7.8 Kb, by plant-made HPV-35 L1/L2 particles. The smaller pseudogenomes were found to be packaged more efficiently into PsVs as higher concentrations of encapsidated DNA and higher levels of EGFP expression were obtained with the 4.8 Kb pseudogenome, compared to the larger 5.8-7.8 Kb pseudogenomes. Thus, smaller pseudogenomes, of 4.8 Kb, should be used for efficient plant production of HPV-35 PsVs.

Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants.

Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and ß1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

Characterization of a dynamic self-replicating mammalian expression vector based on the circular ssDNA genome of beak and feather disease virus.

In vivo nucleic expression technologies using DNA or mRNA offer several advantages for recombinant gene expression. Their inherent ability to generate natively expressed recombinant proteins and antigens allows these technologies to mimic foreign gene expression without infection. Furthermore, foreign nucleic acid fragments have an inherent ability to act as natural immune adjuvants and stimulate innate pathogen- and DNA damage-associated receptors that are responsible for activating pathogen-associated molecular pattern (PAMP) and DNA damage-associated molecular pattern (DAMP) signalling pathways. This makes nucleic-acid-based expression technologies attractive for a wide range of vaccine and oncolytic immunotherapeutic uses. Recently, RNA vaccines have demonstrated their efficacy in generating strong humoral and cellular immune responses for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). DNA vaccines, which are more stable and easier to manufacture, generate similar immune responses to RNA, but typically exhibit lower immunogenicity. Here we report on a novel method of constructing self-amplifying DNA expression vectors that have the potential to amplify and enhance gene/antigen expression at a cellular level by increasing per cell gene copy numbers, boost genomic adjuvating effects and mitigate through replication many of the problems faced by non-replicating vectors such as degradation, methylation and gene silencing. These vectors employ a viral origin rolling circle replication cycle in mammalian host cells that amplifies the vector and gene of interest (GOI) copy number, maintaining themselves as nuclear episomes. We show that these vectors maintain persistently elevated GOI expression levels at the cellular level and induce morphological cellular alterations synonymous with increased cellular stress.

Characterization of a Novel Chimeric Theileria parva p67 Antigen Which Incorporates into Virus-like Particles and Is Highly Immunogenic in Mice.

The current method to protect cattle against East Coast Fever (ECF) involves the use of live Theileria parva sporozoites. Although this provides immunity, using live parasites has many disadvantages, such as contributing to the spread of ECF. Subunit vaccines based on the sporozoite surface protein p67 have been investigated as a replacement for the current method. In this study, two DNA vaccines expressing recombinant forms of p67 designed to display on retrovirus-like particles were constructed with the aim of improving immunogenicity. The native leader sequence was replaced with the human tissue plasminogen activator leader in both vaccines. The full-length p67 gene was included in the first DNA vaccine (p67); in the second, the transmembrane domain and cytoplasmic tail were replaced with those of an influenza A virus hemagglutinin 5 (p67HA). Immunofluorescent staining of fixed and live transfected mammalian cells showed that both p67 and p67HA were successfully expressed, and p67HA localised on the cell surface. Furthermore, p67HA was displayed on the surface of both bovine leukaemia virus (BLV) Gag and HIV-1 Gag virus-like particles (VLPs) made in the same cells. Mice vaccinated with DNA vaccines expressing p67 and p67HA alone, or p67HA with BLV or HIV-1 Gag, developed high titres of p67 and BLV Gag-binding antibodies. Here we show that it is possible to integrate a form of p67 containing all known antigenic domains into VLPs. This p67HA-VLP combination has the potential to be incorporated into a vaccine against ECF, as a DNA vaccine or as other vaccine platforms.

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.

Immunogenicity of Plant-Produced Human Papillomavirus (HPV) Virus-Like Particles (VLPs).

Cervical cancer is ranked fourth among the top cancers in women and is the second most common cancer in low- and middle-income regions, with ~570,000 new cases reported in 2018, which attributed to 84% of worldwide cervical cancer cases. Three commercially available prophylactic Human papillomavirus (HPV) vaccines are effective at preventing HPV infections. However, these vaccines are expensive due to their complex production systems, therefore limiting their use in developing countries. Recently, the use of plants to produce vaccines has emerged as a cost-effective alternative to conventionally used expression systems. Here, L1 proteins of eight high-risk (HPV 16, 18, 31, 33, 35, 45, 52, and 58) and two low risk (HPV 6 and 34) HPV types were successfully expressed in Nicotiana benthamiana, and transmission electron microscopy (TEM) analysis showed the presence of VLPs and/or capsomeres. Immunogenicity studies were conducted in mice utilizing HPV 35, 52, and 58 and showed that type-specific L1-specific antibodies were produced which were able to successfully neutralize homologous HPV pseudovirions in pseudovirion-based neutralization assays (PBNAs). This work demonstrated the potential for using plant-based transient expression systems to produce affordable and immunogenic HPV vaccines, particularly for developing countries.

Extended Set of GoldenBraid Compatible Vectors for Fast Assembly of Multigenic Constructs and Their Use to Create Geminiviral Expression Vectors.

Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more and more popular in plant synthetic and molecular biology. These methods enable routine construction of large and complex multigene DNA structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set of novel α-level plasmids compatible with the GB convention that extend the ability of GB to rapidly assemble more complex genetic constructs, while maintaining compatibility with all existing GB parts as well as most MoClo parts and GB modules. With the use of our new plasmids, standard GB parts can be assembled into complex assemblies containing 1, 5, 10 and up to theoretically 50 units in each successive level of infinite loop assembly. Assembled DNA constructs can be also combined with conventional binary GB-assemblies (1, 2, 4, 8… units). We demonstrate the usefulness of our framework on single tube assembly of replicating plant expression constructs based on the geminivirus Bean yellow dwarf virus (BeYDV).

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors.

KEY MESSAGE: This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.

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.

Plant molecular farming of virus-like nanoparticles as vaccines and reagents.

The use of plants for the production of virus-like nanoparticles (VNPs) dates back to separating natural empty capsids of plant viruses from whole virions nearly 70 years ago, through to the present use of transgenic plants or recombinant Agrobacterium tumefaciens and/or plant virus-derived vectors for the transient expression of engineered viral or other structural proteins in plants-a production system also known as molecular farming. Plant production of heterologous proteins has major advantages in terms of convenience-whole plants are generally used, and processes do not need to be sterile-and cost, as bulk biomass production is significantly cheaper than by any other method. Plant-made VNPs in current use for nanotechnology include whole virions and naturally occurring empty capsids of plant viruses, and particles made by reassembly of coat protein (CP) purified from virions or by recombinant expression. Engineered VNP-forming animal or human virus CPs expressed in plants include L1 protein from human papillomaviruses, human norovirus CP, hepatitis B surface and core antigens, influenza virus HA protein and HIV Gag polyprotein forming large enveloped particles by budding, orbi- and rotavirus particles that require assembly of four co-expressed proteins, and polio- and foot and mouth disease viruses which require proteolytic processing of a polyprotein precursor to form 4-component VNPs. Both plant and animal virus-derived plant-made VNPs can be used for surface and internal display of heterologous peptides or even whole proteins. A significant recent development has been the production of pseudovirions in plants, comprising plant or animal virus CPs and RNA or DNA pseudogenomes that can be used to deliver nucleic acid payloads into cultured cells or specific tissues or tumors in whole animals. This article is characterized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > in vivo Nanodiagnostics and Imaging.

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.

Substitution of Human Papillomavirus Type 16 L2 Neutralizing Epitopes Into L1 Surface Loops: The Effect on Virus-Like Particle Assembly and Immunogenicity.

Cervical cancer caused by infection with human papillomaviruses (HPVs) is the fourth most common cancer in women globally, with the burden mainly in developing countries due to limited healthcare resources. Current vaccines based on virus-like particles (VLPs) assembled from recombinant expression of the immunodominant L1 protein are highly effective in the prevention of cervical infection; however, these vaccines are expensive and type-specific. Therefore, there is a need for more broadly protective and affordable vaccines. The HPV-16 L2 peptide sequences 108-120, 65-81, 56-81, and 17-36 are highly conserved across several HPV types and have been shown to elicit cross-neutralizing antibodies. To increase L2 immunogenicity, L1:L2 chimeric VLPs (cVLP) vaccine candidates were developed. The four L2 peptides mentioned above were substituted into the DE loop of HPV-16 L1 at position 131 (SAC) or in the C-terminal region at position 431 (SAE) to generate HPV-16-derived L1:L2 chimeras. All eight chimeras were transiently expressed in Nicotiana benthamiana via Agrobacterium tumefaciens-mediated DNA transfer. SAC chimeras predominantly assembled into higher order structures (T = 1 and T = 7 VLPs), whereas SAE chimeras assembled into capsomeres or formed aggregates. Four SAC and one SAE chimeras were used in vaccination studies in mice, and their ability to generate cross-neutralizing antibodies was analyzed in HPV pseudovirion-based neutralization assays. Of the seven heterologous HPVs tested, cross-neutralization with antisera specific to chimeras was observed for HPV-11 (SAE 65-18), HPV-18 (SAC 108-120, SAC 65-81, SAC 56-81, SAE 65-81), and HPV-58 (SAC 108-120). Interestingly, only anti-SAE 65-81 antiserum showed neutralization of homologous HPV-16, suggesting that the position of the L2 epitope display is critical for maintaining L1-specific neutralizing epitopes.

Immunogenicity of plant-produced porcine circovirus-like particles in mice.

Porcine circovirus type 2 (PCV-2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus-associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV-2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV-2 capsid protein (CP) from plants is an essential first step towards the goal of a plant-produced PCV-2 vaccine candidate. In this study, the PCV-2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV-2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self-assembled into virus-like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant-produced PCV-2 VLPs elicited specific antibody responses to PCV-2 CP. This is the first report describing the expression of PCV-2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.

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.