ABSTRACT
BACKGROUND: African horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes. RESULTS: In this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species. CONCLUSIONS: We have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.
Subject(s)
African Horse Sickness Virus/immunology , African Horse Sickness/prevention & control , Capsid Proteins/immunology , Nicotiana/metabolism , Viral Vaccines , Animals , Antibodies, Neutralizing/immunology , Capsid Proteins/metabolism , Gene Expression Regulation, Plant , Horses , Plants, Genetically Modified , Recombinant Fusion Proteins , Recombinant Proteins , Vaccines, Attenuated , Vaccines, Virus-Like ParticleABSTRACT
Bluetongue disease in endemic areas is predominantly controlled through vaccination with live-attenuated vaccines. Sequencing of the original master seed viruses used in the production of Onderstepoort Biological Products vaccine was conducted. Nucleotide identities of 82.97%-100% were obtained for all sequences when compared to South African reference strains.
ABSTRACT
Rift Valley Fever virus (RVFV) causes the zoonotic RVF disease, which results in substantial economic losses in livestock industries. Regular vaccination of livestock against RVF is necessary to generate long-term immunity and avoid the loss of livestock. The live attenuated vaccine based on Clone 13 virus strain has been used to reduce the negative impact of RVF disease. The vaccine strain is heat labile and requires stringent conditions for storage and handling. This research evaluated lactose and sucrose-based stabilizers coupled with lyophilisation to enhance stability of the RVF Clone 13 vaccine strain. The glass transition temperature (Tg) of the sucrose-RVF vaccine was 97.0 °C with average residual moisture of below 2 %. The lactose formulation was characterised with Tg of 83.5 °C and residual moisture of above 2 %. The RVF Clone 13 sucrose-based formulation maintained higher antigen titres during lyophilisation compared to the lactose-formulated vaccine. Cellular-mediated and humoral immunity was evaluated and compared for the two newly formulated vaccines. Pheroid® technology was also investigated as a potential adjuvant and its ability to further enhance the immunogenicity conferred by the RVF Clone 13 vaccine formulations in Merino sheep. No adverse reactions were observed following injection of the vaccine formulations in mice, guinea pigs and Merino sheep. Comparable protective humoral immune responses against RVF were obtained for all animals vaccinated with the lactose and sucrose-based stabilisers with and without the Pheroid® adjuvant. No proliferation of CD8+ and CD4+ T-cells as well as expression of IFN-γ was observed for all animals group vaccinated with Pheroid® only. Specific CD8+ IFN-γ+T-cells were expressed at higher levels compared to the CD4+ IFN-γ+T-cells in the RVF Clone 13 vaccines, suggesting that cellular immunity against RVF is through the Class I antigen presentation pathway.
Subject(s)
Rift Valley Fever , Rift Valley fever virus , Viral Vaccines , Animals , Mice , Guinea Pigs , Lactose , Vaccination/veterinary , Vaccines, Attenuated , Adjuvants, Immunologic , Zoonoses , Antibodies, ViralABSTRACT
Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.
Subject(s)
African Horse Sickness Virus , African Horse Sickness , Viral Vaccines , Animals , Antibodies, Neutralizing , Antibodies, Viral , Capsid Proteins , Chromatography, Liquid , Horses , Mice , Serogroup , Tandem Mass Spectrometry , Viral ProteinsABSTRACT
Bovine Ephemeral fever virus (BEFV) is endemic in South Africa and has a negative economic impact on the meat and dairy industries. Bovine ephemeral fever or three-day stiff-sickness is controlled through annual vaccination with a live attenuated virus manufactured by Onderstepoort Biological Products (South Africa). We announce the genome sequences of two South African Bovine Ephemeral Virus strains; the live attenuated vaccine strain (14 876 nucleotides) and a field strain (14 883 nucleotides). A mutation in the alpha 3 open reading frame rendered the gene non-functional in both genomes. Phylogenetic analysis based on the glycoprotein gene showed that the two strains clustered with the South African lineage.
Subject(s)
Cattle Diseases , Ephemeral Fever Virus, Bovine , Ephemeral Fever , Vaccines , Animals , Cattle , Cattle Diseases/epidemiology , Ephemeral Fever/epidemiology , Ephemeral Fever/prevention & control , Ephemeral Fever Virus, Bovine/genetics , Phylogeny , South AfricaABSTRACT
Conventional cell-culture viral quantification methods, namely viral plaque and 50â% tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50â% after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.
ABSTRACT
Lumpy skin disease and Rift Valley fever are two high-priority livestock diseases which have the potential to spread into previously free regions through animal movement and/or vectors, as well as intentional release by bioterrorists. Since the distribution range of both diseases is similar in Africa, it makes sense to use a bivalent vaccine to control them. This may lead to the more consistent and sustainable use of vaccination against Rift Valley fever through a more cost-effective vaccine. In this study, a recombinant lumpy skin disease virus was constructed in which the thymidine kinase gene was used as the insertion site for the Gn and Gc protective glycoprotein genes of Rift Valley fever virus using homologous recombination. Selection markers, the enhanced green fluorescent protein and Escherichia coli guanidine phosphoribosyl transferase (gpt), were used for selection of recombinant virus and in a manner enabling a second recombination event to occur upon removal of the gpt selection-pressure allowing the removal of both marker genes in the final product. This recombinant virus, LSD-RVF.mf, was selected to homogeneity, characterized and evaluated in cattle as a vaccine to show protection against both lumpy skin disease and Rift Valley fever in cattle. The results demonstrate that the LSD-RVF.mf is safe, immunogenic and can protect cattle against both diseases.
ABSTRACT
Bluetongue (BT) is a hemorrhagic non-contagious, biting midge-transmitted disease of wild and domestic ruminants that is caused by bluetongue virus (BTV). Annual vaccination plays a pivotal role in BT disease control in endemic regions. Due to safety concerns of the current BTV multivalent live attenuated vaccine (LAV), a safe efficacious new generation subunit vaccine such as a plant-produced BT virus-like particle (VLP) vaccine is imperative. Previously, homogenous BTV serotype 8 (BTV-8) VLPs were successfully produced in Nicotiana benthamiana plants and provided protective immunity in sheep. In this study, combinations of BTV capsid proteins from more than one serotype were expressed and assembled to form chimaeric BTV-3 and BTV-4 VLPs in N. benthamiana plants. The assembled homogenous BTV-8, as well as chimaeric BTV-3 and chimaeric BTV-4 VLP serotypes, were confirmed by SDS-PAGE, Transmission Electron microscopy (TEM) and protein confirmation using liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing. As VP2 is the major determinant eliciting protective immunity, the percentage coverage and number of unique VP2 peptides detected in assembled chimaeric BT VLPs were used as a guide to assemble the most appropriate chimaeric combinations. Both plant-produced chimaeric BTV-3 and BTV-4 VLPs were able to induce long-lasting serotype-specific neutralizing antibodies equivalent to the monovalent LAV controls. Antibody levels remained high to the end of the trial. Combinations of homogenous and chimaeric BT VLPs have great potential as a safe, effective multivalent vaccine with the ability to distinguish between vaccinated and infected individuals (DIVA) due to the absence of non-structural proteins.