Browsing by Author "Douglass, Nicola"

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    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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    Assessment of an LSDV-Vectored Vaccine for Heterologous Prime-Boost Immunizations against HIV
    (2021-11-05) Chapman, Ros; van Diepen, Michiel; Douglass, Nicola; Galant, Shireen; Jaffer, Mohamed; Margolin, Emmanuel; Ximba, Phindile; Hermanus, Tandile; Moore, Penny L; Williamson, Anna-Lise
    The modest protective effects of the RV144 HIV-1 vaccine trial have prompted the further exploration of improved poxvirus vector systems that can yield better immune responses and protection. In this study, a recombinant lumpy skin disease virus (LSDV) expressing HIV-1 CAP256.SU gp150 (Env) and a subtype C mosaic Gag was constructed (LSDVGC5) and compared to the equivalent recombinant modified vaccinia Ankara (MVAGC5). In vitro characterization confirmed that cells infected with recombinant LSDV produced Gag virus-like particles containing Env, and that Env expressed on the surface of the cells infected with LSDV was in a native-like conformation. This candidate HIV-1 vaccine (L) was tested in a rabbit model using different heterologous vaccination regimens, in combination with DNA (D) and MVA (M) vectors expressing the equivalent HIV-1 antigens. The four different vaccination regimens (DDMMLL, DDMLML, DDLMLM, and DDLLMM) all elicited high titers of binding and Tier 1A neutralizing antibodies (NAbs), and some regimens induced Tier 1B NAbs. Furthermore, two rabbits in the DDLMLM group developed low levels of autologous Tier 2 NAbs. The humoral immune responses elicited against HIV-1 Env by the recombinant LSDVGC5 were comparable to those induced by MVAGC5.
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    Comparative analysis of avian poxvirus genomes, including a novel poxvirus from lesser flamingos (Phoenicopterus minor), highlights the lack of conservation of the central region
    (BioMed Central, 2017-12-06) Carulei, Olivia; Douglass, Nicola; Williamson, Anna-Lise
    Background: Avian poxviruses are important pathogens of both wild and domestic birds. To date, seven isolates from subclades A and B and one from proposed subclade E, have had their genomes completely sequenced. The genomes of these isolates have been shown to exhibit typical poxvirus genome characteristics with conserved central regions and more variable terminal regions. Infection with avian poxviruses (APVs) has been reported in three species of captive flamingo, as well as a free-living, lesser flamingo at Kamfers dam, near Kimberley, South Africa. This study was undertaken to further characterise this virus which may have long term effects on this important and vulnerable, breeding population. Results: Gene content and synteny as well as percentage identities between conserved orthologues was compared between Flamingopox virus (FGPV) and the other sequenced APV genomes. Dotplot comparisons revealed major differences in central regions that have been thought to be conserved. Further analysis revealed five regions of difference, of differing lengths, spread across the central, conserved regions of the various genomes. Although individual gene identities at the nucleotide level did not vary greatly, gene content and synteny between isolates/species at these identified regions were more divergent than expected. Conclusion: Basic comparative genomics revealed the expected similarities in genome architecture but an in depth, comparative, analysis showed all avian poxvirus genomes to differ from other poxvirus genomes in fundamental and unexpected ways. The reasons for these large genomic rearrangements in regions of the genome that were thought to be relatively conserved are yet to be elucidated. Sequencing and analysis of further avian poxvirus genomes will help characterise this complex genus of poxviruses.
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    A comparative analysis of cowpox virus (CPV WT) and a deletion mutant lacking the gene encoding the inflammation modulatory protein (CPV IMP)
    (2007) Paulsen, Janis; Douglass, Nicola; Williamson, Anna-Lise
    Cowpox virus has been found to encode the inflammation modulatory protein (IMP) (Miller, C.G., 1997), a homologue vaccinia virus complement control protein (VCP). VCP belongs to regulation of complement activation (RCA) protein superfamily. It has been shown to inhibit both the alternative and classical pathways of complement activation by binding to the proteins C3 and C4, thereby preventing complementmediated opsonisation of virus, antibody-mediated lysis of infected cells and migration of inflammatory cells into the site of infection. VCP also possesses heparin binding sites.
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    Comparison of the two lumpy skin disease virus vaccines, Neethling and Herbivac, and construction of a recombinant Herbivac-Rift Valley fever virus vaccine
    (2015) Omar, Ruzaiq; Williamson, Anna-Lise; Douglass, Nicola
    There are two broad aims to this project. The first aim is to compare and characterise two lumpy skin disease virus (LSDV) vaccines namely the vaccine based on attenuated Neethling LSDV (nLSDV) and Herbivac®LS (Herbivac). The second aim is to construct a recombinant LSDV expressing Rift Valley fever virus (RVFV) genes. An LSDV vaccine is critical for sustainable control of lumpy skin disease (LSD). There are four commercially available live attenuated vaccines for LSDV, nLSDV, Herbivac, Lumpyvax and the Kenyan strain sheeppox virus (KS-1). In this study Herbivac was characterised by comparing it to its parent, nLSDV. Growth curves of the two viral strains were conducted in cell culture as well as in embryonated hens’ eggs. No notable difference in the growth rate of the two strains could be detected when the viruses were grown in cell culture, however a notable difference was detected when the viruses were grown on the chick allantoic membranes (CAMs) of embryonated hens’ eggs. When grown on CAMs a faster growth rate was observed for nLSDV compared to Herbivac. nLSDV also killed the embryos at 4 d.p.i where Herbivac did not. The two strains were then further characterised through histological analysis of CAMs after infection with each of the viruses. Overall, higher levels of hyperplasia and hypertrophy were observed in CAMs infected with either nLSDV or Herbivac compared to uninfected CAMs. Herbivac-infected CAMs resulted in thicker chorionic membranes and larger pocks compared to nLSDV. RVFV and LSDV both contribute to the disease burden among cattle in Africa and the Arabian Peninsula. The main aim of this study was to construct a recombinant Herbivac which expresses immunogenic proteins of Rift Valley fever virus (Herbivac-RVFV). Herbivac-RVFV was designed to express specific RVFV genes selected for their antigenic properties. The genes selected are also representative of the genes from recent viral outbreaks in the horn of Africa. The selection of outbreak relevant RVFV genes involved phylogenetic analysis of all full length M-segment and NC gene sequences available on Genbank. Phylogenetic trees were constructed for M-segments and NC genes and groups identified which were highly representative of sequences from recent outbreaks of the virus. Consensus sequences were derived from these groups and included in the transfer vector. The phylogenetic analysis also revealed that the sequences of current RVFV vaccines are phylogenetically distant from viruses isolated from current outbreaks, although high levels of sequence conservation was maintained across all viral strains. This is the first study in which the RVFV genes coding for proteins that will induce a protective immune response (Gn and Gc, as well as the nucleocapsid (NC) gene) were selected so as to be representative of current outbreak strains of the virus. These genes were inserted between LSDV ORFs 49 and 50, a novel insertion site. The transfer vector also contained an eGFP marker gene and an ECO-GPT selection gene, located outside of the LSDV flanking sequences. This meant a two-step isolation procedure, first to isolate the recombinant containing the entire transfer vector with eGFP and ECO-GPT, and then to isolate a recombinant with only the RVFV genes and not eGFP and ECO-GPT. Transient expression of RVFV proteins in cells infected with Herbivac and then transfected with the transfer vector was confirmed via western blotting and immunofluorescence. Here the proteins Gn, Gc and NC were shown to be expressed. In the present study, a single crossover Herbivac-RVFV recombinant was isolated through multiple passaging of cell lysates, originally obtained from Herbivac-infected FBT cells transfected with the transfer vector, in the presence of mycophenolic-acid selection medium.
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    Development and Characterization of a Modified Vaccinia Ankara Vaccine Candidate Expressing the SARS-CoV-2 Spike Glycoprotein
    (2022) Khumalo, Fezokuhle Ncedile; Margolin, Emmanuel; Williamson, Anna-Lise; Douglass, Nicola
    Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2, SARS-CoV-2. Given the ongoing COVID-19 pandemic and the continued evolution of the virus to escape host immunity, new vaccines and refinement of first-generation vaccines to improve protection against SARS-CoV-2 variants of concern is vital. In Africa, the cost of vaccine manufacturing as well as the scarcity in resources for storage and distribution have all contributed to the inequitable access to vaccines and heavy reliance on donations. Modified Vaccinia Virus Ankara (MVA) is a low-cost production vector platform which is suitable in this context. This project falls into a bigger study where our group compared different vector platforms, including MVA. The project serves as a proof of concept that this platform can be used to produce vaccines encompassing different variants of SARS-CoV-2 as they emerge. The most recent variant, Omicron, has proven to be highly immune evasive and demonstrates this need well. As the virus mutates, the variants of concern each present with differing characteristics and subsequently differ in immunogenicity and pathogenicity. Sub-Saharan Africa has been ransacked by the pandemic, resulting in loss of lives and livelihoods; the effects of which will undoubtedly be felt for decades to come. This study had two aims: 1. The development of a candidate vaccine, MVA-SARS-CoV2-S∆TM by using the widely used MVA platform and poxvirus recombinant vaccine strategies used in our research group, 2. The testing of this vaccine's immunogenicity in mice. The MVA-based vaccine was constructed by infection of BHK21 cells with wildtype MVA, and transfection with transfer vector pMVA-FNK2. The transfer vector contains a truncated form of the SARS-CoV-2 spike glycoprotein gene, the vaccinia virus host-range gene K1L and reporter gene eGFP, flanked by gene sequences to allow homologous recombination into MVA. The presence of the K1L gene in the recombinant virus allowed for selection by passaging in RK13 cells, which were not permissive for the parent MVA. Following the potentially successful isolation, the recombinant MVA-SARS-CoV2-S∆TM was validated and characterized by PCR, Sanger sequencing, Western blot analysis and immunofluorescence, which all confirmed the presence and expression of the spike protein. Large scale propagation of the vaccine was done in RK13 cells, and the stock was titrated to yield a titre of 3.9 x 106 ffu/ml. Balb/c mice were inoculated three times with MVA-SARS-CoV2-S∆TM at a dose of 3 x 105 to assess immunogenicity of the vaccine. Results from the immunogenicity assessments demonstrated that the vaccine induced T-cell responses shown by an enzyme-linked immunosorbent spot (ELISpot) assay. An enzyme-linked immunosorbent assay (ELISA) confirmed the ability of MVA-SARSCoV2-S∆TM to induce increasing titres of binding antibodies in mice over a period of 56 days. However, the vaccine did not induce neutralizing antibodies against a matched SARS-CoV-2 pseudovirus, highlighting the need for further refinement of the vaccine. In conclusion, recombinant MVA expressing a truncated SARS-CoV-2 spike protein was successfully constructed and tested for immunogenicity in mice. The candidate vaccine induced good T-cell responses and binding antibodies, but not neutralizing antibodies. This study provides additional evidence that MVA can be used as a platform for a SARS-CoV-2 vaccine, as has been previously demonstrated by others, and this could potentially be adapted for emerging variants of concern. This work also allows for the direct comparison of the MVA platform to other platforms employed by our group (DNA and plant-based subunit) as SARS-CoV-2 vaccines.
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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.
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    The development of penguinpox virus (PEPV) as a vaccine vector : transfer vector construction and rescue of virus growth in rabbit kidney cells (RK-13) by vaccinia virus K1
    (2010) Hu, Nai-Chung; Williamson, Anna-Lise; Douglass, Nicola
    Of the many vaccine trials which have taken place, the most promising results have been obtained from the recent phase 3 clinical trial which tested the ability of a dual protein and Canarypox virus recombinant to protect humans against HIV-1 infections. Because poxviruses are being developed as vaccine vectors against a number of diseases, it is important to continue the search for novel poxvirus vectors, in particular, those that do not cross-neutralize one another. This thesis describes the preliminary work performed on the development of Penguinpox virus (PEPV) as a vaccine vector.
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    Genetic and phenotypic analysis of novel South African Avian poxviruses
    (2018) Carulei, Olivia; Williamson, Anna-Lise; Douglass, Nicola
    Avian poxviruses are important pathogens of both wild and domestic birds and exhibit a large degree of intragenus diversity at a genomic level. These viruses are known to differ in growth characteristics (in vitro and in vivo), virulence, and cross-protection, with little known about the genomic contributions to these differences. Only six isolates from subclades A and B and one from proposed subclade E have had their genomes completely sequenced. These genomes have been shown to exhibit typical poxvirus genome characteristics with conserved central regions and more variable terminal regions, however all isolates exhibit major differences in defined central regions. This study aimed to analyze and characterize novel isolates from South Africa in terms of growth characteristics and phylogenetic relationships. It also added to the pool of genome sequences available for comparative genomic analyses to further investigate genome architecture. Poxvirus isolates from lesser flamingo (Phoenicopterus minor) and African penguin (Spheniscus demersus) were chosen for analysis from a larger pool of donated isolates by comparison of macroscopic growth characteristics on chorioallantoic membranes, membrane histology and phylogenetic analyses based on nucleotide alignment of partial P4b sequences. Flamingopox virus was shown to group in subclade A3, induce membrane thickening and mesodermal hyperplasia while Penguinpox virus grouped in subclade A2, and did not induce membrane thickening or hyperplasia. The genomes of the above isolates were sequenced and compared to other available avipoxvirus genomes. Dotplot comparisons revealed major differences in central regions that have traditionally been thought to be conserved. Further analysis revealed five regions of difference, of varying lengths, spread across the central regions of the various genomes. Although individual gene identities at the nucleotide level did not vary greatly, gene content and synteny between isolates/species at these identified regions were far more divergent than expected. The reasons for these large genomic rearrangements are yet to be elucidated and will need to be considered in future phylogenetic studies and vaccine vector design. Sequencing and analysis of further avian poxvirus genomes will help characterize this complex genus of poxviruses.
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    HIV-1 subtype C mosaic Gag expressed by BCG and MVA elicits persistent effector t cell responses in a prime-boost regimen in mice
    (Public Library of Science, 2016) Jongwe, Tsungai Ivai; Chapman, Ros; Douglass, Nicola; Chetty, Shivan; Chege, Gerald; Williamson, Anna-Lise
    Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C) viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG Δ panCD auxotroph and modified vaccinia Ankara (MVA) vaccines expressing HIV-1C mosaic Gag (Gag M ) were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-Gag M vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-Gag M and boosting with MVA-Gag M elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-Gag M only and MVA-Gag M only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4 + and CD8 + T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (10 4 pfu) can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C).
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    Improvement of the Capripoxvirus, lumpy skin disease virus for use as a vaccine vector
    (2018) Munyanduki, Henry Munyaradzi; Williamson, Anna-Lise; Douglass, Nicola
    Lumpy skin disease (LSD) is a notifiable viral infection due both to its morbidity in cattle and its severe economic burden. The disease was confined to Sub-Saharan Africa but has in recent years spread to the Middle East and Europe. Vaccination is the only way of preventing LSD. Live attenuated lumpy skin disease virus (LSDV) has been used as a vaccine against LSD. The most successful LSD vaccine is the Neethling vaccine strain (nLSDV) from South Africa. There are however, reports of nLSDV being too attenuated or too virulent in different breeds of cattle. A South African produced vaccine strain of LSDV, Herbivac, was said to be more immunogenic than nLSDV (personal communication, Deltamune). Whole genomic sequence comparison of Herbivac with nLSDV revealed a single potentially significant change in open reading frame (ORF) 131. This ORF encodes a superoxide dismutase (SOD) homologue. The mutation identified in Herbivac is a 2bp deletion which causes a frameshift mutation that restores the SOD homologue to resemble the full-length SOD homolog encoded by the virulent field strain. This SOD homologue gene is truncated in nLSDV. Protein structural alignment of SOD homologues from LSDV and other characterised SOD homologues was done. Both the truncated and full-length SOD homologues lacked the catalytic arginine at position 142 which is involved in SOD activity. Some similarities with known SOD homologues which act as SOD decoys, such as the Leporipoxviruses myxoma and Shope Fibroma virus, were observed. Like Leporipoxvirus’ SOD homologues, the full-length Herbivac SOD homologue contained regions of homology with the copper chaperone for SOD (CCS). The putative SOD protein from Herbivac, and not nLSDV, contained 6 out of 8 metal binding residues. Unlike the SOD decoy from myxoma virus, the full-length SOD homolog from Herbivac did not include a cysteine molecule at position 56 that stabilizes the SOD-CCS heterodimer. The alignment suggests that all SOD homologs from LSDV are inactive as enzymes. Transcriptome analysis of messenger RNA from spleens of mice infected with nLSDV or Herbivac for 24hrs was carried out to determine the effect of Herbivac or nLSDV infection on host gene expression. Compared to the PBS control, nLSDV and Herbivac induced the differential expression of 98 genes in common, largely related to response to viral infection. nLSDV differed in the unique expression of 6 genes and Herbivac differed in the unique expression of 36 genes, including granzyme A and Poly (ADP-ribose) polymerase (Parp 9). Herbivac upregulated genes associated with pathogen pattern recognition, interferon response, immune response and cell death. More Gene ontology (GO) processes were enriched after Herbivac infection than nLSDV infection. Amongst these processes were immune response processes, the interferon and cell death related responses. To characterise the SOD homolog with respect to SOD activity, cell death and whether it plays a role in growth of viruses expressing it, recombinant viruses were constructed. The first set of recombinants included a SOD knock-out virus where the SOD gene from nLSDV was replaced with reporter gene GFP. A SOD knock-in virus was constructed with the SOD homologue gene altered to improve the stability of the gene. A reporter gene mCherry was also inserted. Histological examination of CAMs infected with nLSDVdSOD-M showed vacuolation and oedema to a greater degree than nLSDV, Herbivac and nLSDVSODis-M. Herbivac showed greater immune cell infiltration. SOD knock-in, nLSDVSODis-M showed increased epithelial hyperplasia and fibroplasia. Another set of recombinants without marker genes was made, nLSDVdSOD-UCT (SOD knock-out), and nLSDVSODis-UCT (SOD knock-in). Deleting the SOD homolog reduced virus yield by approximately ten-fold in MDBK cells. Histologically, the presence of the SOD homolog in nLSDVSODis-UCT caused greater inflammatory changes in the mesoderm after 5 days of infection compared to nLSDVdSOD-UCT. In vitro functional studies were done in MDBK cells which are permissible to LSDV infection. No difference in SOD activity could be detected amongst the different viruses. Differences could, however, be detected in induction and inhibition of apoptosis. There was increased induction of apoptosis following infection by all viruses containing full-length SOD compared to infection with truncated or SOD knock-out virus. Viruses expressing a full-length SOD showed greater inhibition of camptothecin induced apoptosis than nLSDV or the SOD knockout. Similarly, full-length SOD induced cell death by necrosis to a greater extent than the SOD knock-out or nLSDV; and all viruses inhibited camptothecin induced necrosis. These results suggest that the presence of a SOD homolog in a vaccine could be advantageous with respect to growth of the vaccine to higher titres and to improved immunogenicity of the vaccine. Further work is required to test this hypothesis in a bovine animal model.
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    Influence of the Viral Superoxide Dismutase (SOD) Homologue on Lumpy Skin Disease Virus (LSDV) Growth, Histopathology and Pathogenicity
    (2020-11-07) Douglass, Nicola; Munyanduki, Henry; Omar, Ruzaiq; Gers, Sophette; Mutowembwa, Paidamwoyo; Heath, Livio; Williamson, Anna-Lise
    Lumpy skin disease is an important economic disease of cattle that is controlled by vaccination. This paper presents an investigation into the role of the lumpy skin disease virus (LSDV) superoxide dismutase (SOD) homologue on growth and histopathology of the virus both in vitro and in vivo. SOD homologue knock-out and knock-in recombinants (nLSDV∆SOD-UCT and nLSDVSODis-UCT, respectively) were constructed and compared to the Neethling vaccine (nLSDV) for growth in a permissive bovine cell line as well as on fertilized chick chorioallantoic membranes (CAMs). The infected CAMs were scored for histological changes. Deletion of the SOD homologue from LSDV reduced virus growth both in Madin-Darby bovine kidney (MDBK) cells as well as on CAMs. Furthermore, the knockout virus showed reduced inflammation in CAMs and more ballooning degeneration. A pilot experiment was performed in cattle to compare the lesions produced by the different LSDV constructs in the same animal. One animal developed a larger lesion to nLSDV∆SOD-UCT compared to both nLSDVSODis-UCT and nLSDV. Histological analysis of biopsies of these lesions shows less inflammation and necrosis associated with nLSDVSODis-UCT compared to nLSDV and nLSDV∆SOD-UCT. None of the vaccinated animals showed disseminated LSDV disease, indicating that the candidate vaccines are safe for further testing. Our results suggest that the SOD homologue may improve immunogenicity and reduce virulence.
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    An investigation into the Use of Lumpy Skin Disease Virus as a Vaccine Vector for a Potential HIV-1 vaccine
    (2010) Shen, Yen-Ju; Williamson, Anna-Lise; Shephard, Enid G; Douglass, Nicola
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    Investigation of local South African avipoxviruses as potential vaccine vectors
    (2014) Offerman, Kristy-Maree; Williamson, Anna-Lise; Douglass, Nicola
    Avipoxviruses are large, genetically diverse DNA viruses which are particularly desirable for use as vaccine vectors as a result of their excellent safety profile and host range restriction. In this study, 8 novel South African (SA) avipoxvirus isolates were characterized. They could be divided into five groups, according to gross pathology and pock appearance on CAMs. Histopathology revealed distinct differences in epidermal and mesodermal cell proliferation, as well as immune cell infiltration, caused by the different avipoxviruses. Phylogenetic analysis was performed based on several conserved poxvirus genetic regions, corresponding to vaccinia virus (VACV) A3L (fpv167 locus, VACV P4b), G8R (fpv126 locus, VLTF-1), H3L (fpv140 locus, VACV H3L) and A11R–A12L (fpv175–176 locus). The SA isolates all grouped in clade A, either in subclade A2 or A3 of the genus Avipoxvirus, with branching patterns which differed according to the locus analysed.
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    LSDV-Vectored SARS-CoV-2 S and N Vaccine Protects against Severe Clinical Disease in Hamsters
    (2023-06-21) de Moor, Warren R. J.; Williamson, Anna-Lise; Schäfer, Georgia; Douglass, Nicola; Gers, Sophette; Sutherland, Andrew D.; Blumenthal, Melissa J.; Margolin, Emmanuel; Shaw, Megan L.; Preiser, Wolfgang; Chapman, Rosamund
    The SARS-CoV-2 pandemic demonstrated the need for potent and broad-spectrum vaccines. This study reports the development and testing of a lumpy skin disease virus (LSDV)-vectored vaccine against SARS-CoV-2, utilizing stabilized spike and conserved nucleocapsid proteins as antigens to develop robust immunogenicity. Construction of the vaccine (LSDV-SARS2-S,N) was confirmed by polymerase chain reaction (PCR) amplification and sequencing. In vitro characterization confirmed that cells infected with LSDV-SARS2-S,N expressed SARS-CoV-2 spike and nucleocapsid protein. In BALB/c mice, the vaccine elicited high magnitude IFN-γ ELISpot responses (spike: 2808 SFU/106 splenocytes) and neutralizing antibodies (ID50 = 6552). Testing in hamsters, which emulate human COVID-19 disease progression, showed the development of high titers of neutralizing antibodies against the Wuhan and Delta SARS-CoV-2 variants (Wuhan ID50 = 2905; Delta ID50 = 4648). Additionally, hamsters vaccinated with LSDV-SARS2-S,N displayed significantly less weight loss, lung damage, and reduced viral RNA copies following SARS-CoV-2 infection with the Delta variant as compared to controls, demonstrating protection against disease. These data demonstrate that LSDV-vectored vaccines display promise as an effective SARS-CoV-2 vaccine and as a potential vaccine platform for communicable diseases in humans and animals. Further efficacy testing and immune response analysis, particularly in non-human primates, are warranted.
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    Needle-Free Devices and CpG-Adjuvanted DNA Improve Anti-HIV Antibody Responses of Both DNA and Modified Vaccinia Ankara-Vectored Candidate Vaccines
    (2023-02-07) Chapman, Rosamund; van Diepen, Michiel; Douglass, Nicola; Hermanus, Tandile; Moore, Penny L.; Williamson, Anna-Lise
    The combination of mosaic Gag and CAP256 envelope in an HIV vaccine regimen comprising DNA prime and modified vaccinia Ankara (MVA) boost followed by protein boost has previously been shown to generate robust autologous Tier 2 neutralizing antibodies (nAbs) in rabbits. Further refinements of this strategy have been investigated to improve antibody responses. The delivery of both DNA and recombinant MVA vaccines with a needle-free device was compared to delivery by injection, and the effect of formulating the DNA vaccine with adjuvant CpG ODN 1826 was determined. The Pharmajet Stratis® needle-free injection device (PharmaJet, Golden, CO, USA) improved binding antibody responses to the DNA vaccine as well as both binding and neutralizing antibody responses to the MVA vaccines. Formulation of the DNA vaccines with CpG adjuvant further improved the antibody responses. A shortened vaccination regimen of a single DNA inoculation followed by a single MVA inoculation did not elicit Tier 1B nor Tier 2 neutralization responses as produced by the two DNA, followed by two MVA vaccination regimen. This study showed the immunogenicity of HIV DNA and MVA vaccines administered in a DDMM regimen could be improved using the PharmaJet Stratis needle-free injection device and formulation of the DNA vaccines with CpG adjuvant.
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    A novel candidate HIV vaccine vector based on the replication deficient Capripoxvirus, Lumpy skin disease virus (LSDV)
    (BioMed Central Ltd, 2011) Shen, Yen-Ju; Shephard, Enid; Douglass, Nicola; Johnston, Nicolette; Adams, Craig; Williamson, Carolyn; Williamson, Anna-Lise
    BACKGROUND: The Capripoxvirus, Lumpy skin disease virus (LSDV) has a restricted host-range and is being investigated as a novel HIV-1 vaccine vector. LSDV does not complete its replication cycle in non-ruminant hosts. METHODS: The safety of LSDV was tested at doses of 104 and 106 plaque forming units in two strains of immunocompromised mice, namely RAG mice and CD4 T cell knockout mice. LSDV expressing HIV-1 subtype C Gag, reverse transcriptase (RT), Tat and Nef as a polyprotein (Grttn), (rLSDV-grttn), was constructed. The immunogenicity of rLSDV-grttn was tested in homologous prime-boost regimens as well as heterologous prime-boost regimes in combination with a DNA vaccine (pVRC-grttn) or modified vaccinia Ankara vaccine (rMVA-grttn) both expressing Grttn. RESULTS: Safety was demonstrated in two strains of immunocompromised mice.In the immunogenicity experiments mice developed high magnitudes of HIV-specific cells producing IFN-gamma and IL-2. A comparison of rLSDV-grttn and rMVA-grttn to boost a DNA vaccine (pVRC-grttn) indicated a DNA prime and rLSDV-grttn boost induced a 2 fold (p < 0.01) lower cumulative frequency of Gag- and RT-specific IFN-gamma CD8 and CD4 cells than a boost with rMVA-grttn. However, the HIV-specific cells induced by the DNA vaccine prime rLSDV-grttn boost produced greater than 3 fold (p < 0.01) more IFN- gamma than the HIV-specific cells induced by the DNA vaccine prime rMVA-grttn boost. A boost of HIV-specific CD4 cells producing IL-2 was only achieved with the DNA vaccine prime and rLSDV-grttn boost. Heterologous prime-boost combinations of rLSDV-grttn and rMVA-grttn induced similar cumulative frequencies of IFN- gamma producing Gag- and RT-specific CD8 and CD4 cells. A significant difference (p < 0.01) between the regimens was the higher capacity (2.1 fold) of Gag-and RT-specific CD4 cells to produce IFN-gamma with a rMVA-grttn prime - rLSDV-grttn boost. This regimen also induced a 1.5 fold higher (p < 0.05) frequency of Gag- and RT-specific CD4 cells producing IL-2. CONCLUSIONS: LSDV was demonstrated to be non-pathogenic in immunocompromised mice. The rLSDV-grttn vaccine was immunogenic in mice particularly in prime-boost regimens. The data suggests that this novel vaccine may be useful for enhancing, in particular, HIV-specific CD4 IFN- gamma and IL-2 responses induced by a priming vaccine.
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    Phylogenetic analysis of three genes of Penguinpox virus corresponding to Vaccinia virus G8R (VLTF-1), A3L (P4b) and H3L reveals that it is most closely related to Turkeypox virus, Ostrichpox virus and Pigeonpox virus
    (BioMed Central Ltd, 2009) Carulei, Olivia; Douglass, Nicola; Williamson, Anna-Lise
    Phylogenetic analysis of three genes of Penguinpox virus, a novel Avipoxvirus isolated from African penguins, reveals its relationship to other poxviruses. The genes corresponding to Vaccinia virus G8R (VLTF-1), A3L (P4b) and H3L were sequenced and phylogenetic trees (Neighbour-Joining and UPGMA) constructed from MUSCLE nucleotide and amino acid alignments of the equivalent sequences from several different poxviruses. Based on this analysis, PEPV was confirmed to belong to the genus Avipoxvirus, specifically, clade A, subclade A2 and to be most closely related to Turkeypox virus (TKPV), Ostrichpox virus (OSPV)and Pigeonpox virus (PGPV).
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    Priming with a recombinant pantothenate auxotroph of Mycobacterium bovis BCG and boosting with MVA elicits HIV-1 Gag specific CD8+ T cells
    (Public Library of Science, 2012) Chapman, Rosamund; Shephard, Enid; Stutz, Helen; Douglass, Nicola; Sambandamurthy, Vasan; Garcia, Irene; Ryffel, Bernhard; Jacobs, William; Williamson, Anna-Lise
    A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔ panCD ) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔ panCD [pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8 + T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4 + T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge.
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    Six host-range restricted poxviruses from three genera induce distinct gene expression profiles in an in vivo mouse model
    (BioMed Central Ltd, 2015) Offerman, Kristy; Deffur, Armin; Carulei, Olivia; Wilkinson, Robert; Douglass, Nicola; Williamson, Anna Lise
    BACKGROUND: Host-range restricted poxviruses make promising vaccine vectors due to their safety profile and immunogenicity. An understanding of the host innate immune responses produced by different poxvirus vectors would aid in the assessment, selection and rational design of improved vaccines for human and veterinary applications. Novel avipoxviruses are being assessed to determine if they are different from other poxvirus vectors. Analysis of the transcriptome induced in a mouse model would aid in determining if there were significant differences between different poxvirus vectors which may reflect different adjuvant potential as well as establish if they should be further evaluated as vaccine vectors. RESULTS: We compared host transcript abundance in the spleens of BALB/c mice twenty four hours after intravenous infection (10 5 pfu/mouse) with six host-restricted poxvirus species from three genera, namely Lumpy Skin Disease virus (LSDV), Canarypox virus (CNPV), Fowlpox virus (FWPV), modified vaccinia Ankara (MVA) and two novel South African avipoxviruses, Feral Pigeonpox virus (FeP2) and Penguinpox virus (PEPV). These six viruses produced qualitatively and quantitatively distinct host responses with LSDV, followed by MVA, inducing the greatest interferon (IFN) response. FeP2 and PEPV caused very little change to host transcript abundance compared to the other 4 viruses tested. CNPV and FWPV induced the up regulation of two immunoglobulin genes (Ighg and Ighg3 (IgG3)) with CNPV inducing a third, Ighm (IgM). HIV-1-specific IgG3 antibodies have been correlated with decreased risk of HIV-1 infection in the RV144 trial, which included a CNPV-based vector (Yates et al. (Sci Transl Med, 6(228) p228, 2014). Up regulation of IgG3 by CNPV and FWPV but not the other poxviruses tested in vivo, implies that these two avipoxvirus-vector backbones may be involved in stimulation of the clinically important IgG3 antibody subclass. Differential transcript abundance associated with the different poxviruses is further discussed with particular emphasis on responses related to immune responses. CONCLUSION: Six, genetically diverse host-restricted poxviruses produce different responses in a mouse model early after infection. These differences may affect the immune response induced to vaccine antigen in vectors based on these viruses. The two novel avipoxviruses were clearly distinguishable from the other viruses.