Browsing by Author "Suzuki, Akiko"

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    Open Access
    Advancements in the Growth and Construction of Recombinant Lumpy Skin Disease Virus (LSDV) for Use as a Vaccine Vector
    (2021-10-04) van Diepen, Michiel; Chapman, Rosamund; Douglass, Nicola; Whittle, Leah; Chineka, Nicole; Galant, Shireen; Cotchobos, Christian; Suzuki, Akiko; Williamson, Anna-Lise
    Attenuated vaccine strains of lumpy skin disease virus (LSDV) have become increasingly popular as recombinant vaccine vectors, to target both LSDV, as well as other pathogens, including human infectious agents. Historically, these vaccine strains and recombinants were generated in primary (lamb) testis (LT) cells, Madin–Darby bovine kidney (MDBK) cells or in eggs. Growth in eggs is a laborious process, the use of primary cells has the potential to introduce pathogens and MDBK cells are known to harbor bovine viral diarrhea virus (BVDV). In this study, data is presented to show the growth of an attenuated LSDV strain in baby hamster kidney (BHK-21) cells. Subsequently, a recombinant LSDV vaccine was generated in BHK-21 cells. Partial growth was also observed in rabbit kidney cells (RK13), but only when the vaccinia virus host range gene K1L was expressed. Despite the limited growth, the expression of K1L was enough to serve as a positive selection marker for the generation of recombinant LSDV vaccines in RK13 cells. The simplification of generating (recombinant) LSDV vaccines shown here should increase the interest for this platform for future livestock vaccine development and, with BHK-21 cells approved for current good manufacturing practice, this can be expanded to human vaccines as well.
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    Development and characterisation of recombinant LSDV-vectored dual vaccines against bovine leukaemia virus and lumpy skin disease virus
    (2019) Suzuki, Akiko; Williamson, Anna-Lise; Chapman, Rosamund
    Bovine leukaemia virus (BLV) and lumpy skin disease virus (LSDV) are endemic to Africa and cause significant economic losses to the beef and dairy industries. Vaccines are the most cost-effective and efficient way to prevent infection and outbreaks. Currently, there is no commercially available vaccine against BLV. In contrast, there are several live attenuated vaccines against LSDV. A recombinant LSDV which could protect cattle against both LSDV and BLV would be of great benefit to the African continent. This Master’s degree project involved three objectives. Firstly, the genetic variabilities and phylogenetic relationships of eight South African BLV isolates with other BLV strains from different geographical regions worldwide with known genotypes were determined. The BLV full-length envelope (env) and gag genes were successfully sequenced from total DNA extracted from the blood of BLV-infected cattle from a single herd. The analyses indicated that the seven of the South African isolates characterised in this study belonged to genotype 4 and the eighth to genotype 1. Furthermore, amino acid substitutions in the BLV Env and Gag sequences unique to the South African isolates were identified. Secondly, the activity of five selected poxvirus promoters in cells infected with LSDV was assessed by the detection of transient expression of an enhanced green fluorescent protein (eGFP) reporter gene driven by the poxvirus promoters. The promoters tested were a modified early fowlpox virus promoter (pmFP), an early-late promoter of a 7.5 kilodalton polypeptide gene of vaccinia virus (VACV) (p7.5), a synthetic early-late promoter of VACV (pS), a modified early-late promoter of the H5 gene of VACV (pmH5) and a synthetic early-late optimised promoter of VACV (pLEO). The results showed that all the poxvirus promoters were functional in the LSDV-infected cells and the eGFP expression was stable over the 72-hour study period. Lastly, two LSDV-vectored dual vaccines containing BLV immunogen(s) were developed and characterised. The first recombinant LSDV-vectored vaccine contained the BLV Env and Gag immunogens and the second recombinant LSDV-vectored vaccine contained the BLV Env immunogen alone. The presence of the BLV env gene in the recombinant LSDV vaccine was confirmed by polymerase chain reaction (PCR) and the BLV env sequence was confirmed by Sanger sequencing. Furthermore, BLV Env and Gag protein expression were confirmed by immunofluorescent staining and Western blotting, respectively. Future work will involve further purification of the recombinant viruses, confirmation of the production of BLV Gag virus-like particles and the preparation of high titre stocks of the vaccines to test in cattle.
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    The Development of Dual Vaccines against Lumpy Skin Disease (LSD) and Bovine Ephemeral Fever (BEF)
    (2021-10-20) Douglass, Nicola; Omar, Ruzaiq; Munyanduki, Henry; Suzuki, Akiko; de Moor, Warren; Mutowembwa, Paidamwoyo; Pretorius, Alri; Nefefe, Tshifhiwa; Schalkwyk, Antoinette van; Kara, Pravesh; Heath, Livio; Williamson, Anna-Lise
    Dual vaccines (n = 6) against both lumpy skin disease (LSD) and bovine ephemeral fever (BEF) were constructed, based on the BEFV glycoprotein (G) gene, with or without the BEFV matrix (M) protein gene, inserted into one of two different LSDV backbones, nLSDV∆SOD-UCT or nLSDVSODis-UCT. The inserted gene cassettes were confirmed by PCR; and BEFV protein was shown to be expressed by immunofluorescence. The candidate dual vaccines were initially tested in a rabbit model; neutralization assays using the South African BEFV vaccine (B-Phemeral) strain showed an African consensus G protein gene (Gb) to give superior neutralization compared to the Australian (Ga) gene. The two LSDV backbones expressing both Gb and M BEFV genes were tested in cattle and shown to elicit neutralizing responses to LSDV as well as BEFV after two inoculations 4 weeks apart. The vaccines were safe in cattle and all vaccinated animals were protected against virulent LSDV challenge, unlike a group of control naïve animals, which developed clinical LSD. Both neutralizing and T cell responses to LSDV were stimulated upon challenge. After two inoculations, all vaccinated animals produced BEFV neutralizing antibodies ≥ 1/20, which is considered protective for BEF.