Browsing by Author "Chapman, Rosamund"

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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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    An Investigation of the SHIV reservoirs in the lymphoid organs of HIV-vaccinated rhesus monkeys after SHIV challenge
    (2022) Benn, Kealan Timothy; Chapman, Rosamund; Hurdayal, Ramona
    The development of an effective vaccine against HIV-1 is thought to be a key component of combating the current HIV epidemic. This study aimed to investigate viral reservoirs in the blood and lymphoid tissues after HIV vaccination and virus challenge using the rhesus macaque model. In addition, the study investigated the envelope sequences of the virus located in the potential reservoir sites in an attempt to understand viral variability. Cryopreserved peripheral blood mononuclear cells (PBMC) and cells isolated from lymphoid tissues obtained at termination of vaccine group (P4, P11, P25, P47, P52) and control group (P67, P70, P71, P72) macaques were processed for determination of viral RNA and proviral DNA loads, and HIV-1 Env amino acid sequences using single genome amplification (SGA) sequencing method. In addition, cryopreserved plasma obtained at key time points pre- and post- vaccination and SHIV challenge were used to measure the levels of HIV Env and SIV Gag antibodies by Western blotting, and HIV-1 Env gp140 ELISA. RNA viral loads were low for all animals with the median of the averages of the viral load for PBMCs being 3.82 copies/107 cells and a range of < 1 copies/107 cells to 61.15 copies/107 cells. For the control group, only P67 had a detectable viral load in the PBMC. The median of the averages for proviral DNA load for PBMCs was 1903.98 copies/107 cells and a range of 696.93 copies/107 cells to 28 663.88 copies/107 cells. Proviral DNA levels were higher than the RNA viral loads indicating a larger amount of integrated provirus and potentially the presence of reservoirs within the macaques. While the low viral load values could mean there are few actively replicating viruses present in the PBMCs. The median of the averages for the RNA viral loads in the inguinal tissue cells was 58.08 copies/107 cells with a range of 8.85 copies/107 cells to 911.61 copies/107 cells. The median of the average for proviral DNA load in the inguinal tissue cells was 3160.36 copies/107 cells with a range of 604.67 copies/107 cells to 73 140.68 copies/107 cells. Proviral DNA levels in the inguinal tissues were higher than the RNA viral load levels which indicates a larger amount of integrated provirus present in the cells of the inguinal tissues and the presence of a potential reservoir. This also indicates that there is less circulating virus. The Western blots showed that the macaques developed binding antibodies to both HIV Env and SIV Gag, however, non-specific binding of antibodies to proteins other than HIV Env and SIV Gag was also seen. The ELISA effectively showed that both the vaccine group and control group macaques were able to produce binding antibodies. Macaques P4, P47, and P52 had the highest endpoint titre for the vaccine group of 5120. Macaque P71 had the highest overall endpoint titre of 20 480. Macaques P11 and P25 of the vaccine group had an endpoint titre of 1280 which was the same endpoint titre for control group macaques P67 and P70, while macaque P72 had an endpoint titre of 5120. Single genome amplification and sequencing showed that there were very few amino acid changes between sequences found in macaques. There was minimal variation within the vaccine group and between the vaccine and control groups with amino acid changes in the variable region 1 (V1), glycan N276 in the conserved region 2 (C2) and variable region 4 (V4) resulting in potential N-glycosylation sites (PNGS) being shifted as well as new glycosylation sites being formed while other lost. This study demonstrates that RNA viral loads and proviral DNA loads were detectable in both macaque groups with no significant difference being present in the viral RNA and proviral DNA loads between the two macaque groups. The lack of changes seen in the sequences of the inguinal and PBMC tissues are due to the early virus seeding the reservoir. Once the reservoir is seeded active replication does not occur in the reservoir resulting in the viruses having similar sequences.
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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 novel HIV-1 vaccines using modified recombinant BCG
    (2016) Chetty, Shivan; Williamson, Anna-Lise; Chapman, Rosamund
    Vaccination continues to represent the most effective means of providing immunological protection against infectious disease in human populations. With the World Health Organisation (WHO) reporting over 1.2 million AIDS related deaths in 2014, an efficacious HIV1 vaccine is urgently needed. The UCT Human Immunodeficiency Virus (HIV) Vaccine Development Group has focussed on understanding novel HIV-1 vaccine vectors, such as modified BCG, combined with modified vaccinia Ankara in the context of heterologous prime boost regimes. The tuberculosis vaccine Bacillus Calmette–Guérin (BCG) has a wellestablished safety profile as well as notable adjuvant activity with an estimated 3 billion doses administered globally since 1921. The development of modern molecular biology techniques in recent times has led to the creation of modified strains of BCG which have been shown in many cases to be safer and/or more immunogenic than the wild type strains in terms of delivering heterologous antigen. This study focuses on exploiting and combining two particular strategies of rBCG modification. The first is the development of auxotrophic strains that contain deletions geared towards preventing the bacteria from synthesising essential growth compounds or amino acids. An example of this is the ΔpanCD auxotroph of rBCG which does not synthesise pantothenic acid and thus has limited intracellular replication leading to less pathology. The UCT group has previously reported that HIV vaccines vectored by the Pasteur strain of rBCG ΔpanCD induced less pathology but improved immunogenicity as compared to Pasteur WT in the murine model (when used a prime in conjunction with an MVA boost). A second notable BCG modification strategy is the inclusion of exogenous genes to improve the immunogenicity of rBCG as a vaccine vector. An example of this is the insertion of the detoxified Clostridium perfringens toxin, perfringolysin O (pfo), into the rBCG genome. Upon expression, pfo forms pores in the endosome which facilitates translocation of vaccine derived antigen into the cytoplasm. This modification has been shown to lead to cross priming of T cells and improve the induction of vaccine specific CD8+ T cell as compared to controls.
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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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    Lumpy Skin Disease — An Emerging Cattle Disease in Europe and Asia
    (Multidisciplinary Digital Publishing Institute, 2023-03-02) Whittle, Leah; Chapman, Rosamund; Williamson, Anna-Lise
    Lumpy skin disease virus (LSDV) is a member of the Capripoxvirus genus, mainly infecting cattle and buffalo, which until relatively recently was only endemic in parts of Africa and then spread to the Middle East and lately Europe and Asia. Lumpy skin disease (LSD) is a notifiable disease with a serious impact on the beef industry as it causes mortality of up to 10% and has impacts on milk and meat production, as well as fertility. The close serological relationship between LSDV, goat poxvirus (GTPV) and sheep poxvirus (SPPV) has led to live attenuated GTPV and SPPV vaccines being used to protect against LSD in some countries. There is evidence that the SPPV vaccine does not protect from LSD as well as the GTPV and LSDV vaccines. One of the LSD vaccines used in Eastern Europe was found to be a combination of different Capripoxviruses, and a series of recombination events in the manufacturing process resulted in cattle being vaccinated with a range of recombinant LSDVs resulting in virulent LSDV which spread throughout Asia. It is likely that LSD will become endemic throughout Asia as it will be very challenging to control the spread of the virus without widespread vaccination.
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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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    Optimisation of the mycobacterial replicon of an E. coli-mycobacterial shuttle vector
    (2007) Griffin, Sarah; Chapman, Rosamund
    This study aimed to investigate whether the mycobacterial replicon may also play a role. The common mycobacterial replicon in episomal E. coli-mycobacterial shuttle vectors contains the truncated rap gene, encoding an auxiliary replication factor, and the repA and repB genes which code for essential replication proteins. Specific aspects of this typical mycobacterial replicon were identified as potential targets to improve stability and increase recombinant protein expression levels, and were modified accordingly.
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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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    Priming with recombinant auxotrophic BCG expressing HIV-1 Gag, RT and Gp120 and boosting with recombinant MVA induces a robust T cell response in mice
    (Public Library of Science, 2013) Chapman, Rosamund; Stutz, Helen; Jr, William Jacobs; Shephard, Enid; Williamson, Anna-Lise
    In previous studies we have shown that a pantothenate auxotroph of Myocbacterium bovis BCG (BCGΔ panCD ) expressing HIV-1 subtype C Gag induced Gag-specific immune responses in mice and Chacma baboons after prime-boost immunization in combination with matched rMVA and VLP vaccines respectively. In this study recombinant BCG (rBCG) expressing HIV-1 subtype C reverse transcriptase and a truncated envelope were constructed using both the wild type BCG Pasteur strain as a vector and the pantothenate auxotroph. Mice were primed with rBCG expressing Gag and RT and boosted with a recombinant MVA, expressing a polyprotein of Gag, RT, Tat and Nef (SAAVI MVA-C). Priming with rBCGΔ panCD expressing Gag or RT rather than the wild type rBCG expressing Gag or RT resulted in higher frequencies of total HIV-specific CD8 + T cells and increased numbers of T cells specific to the subdominant Gag and RT epitopes. Increasing the dose of rBCG from 10 5 cfu to 10 7 cfu also led to an increase in the frequency of responses to subdominant HIV epitopes. A mix of the individual rBCGΔ panCD vaccines expressing either Gag, RT or the truncated Env primed the immune system for a boost with SAAVI MVA-C and generated five-fold higher numbers of HIV-specific IFN-γ-spot forming cells than mice primed with rBCGΔ panCD containing an empty vector control. Priming with the individual rBCGΔ panCD vaccines or the mix and boosting with SAAVI MVA-C also resulted in the generation of HIV-specific CD4 + and CD8 + T cells producing IFN-γ and TNF-α and CD4 + cells producing IL-2. The rBCG vaccines tested in this study were able to prime the immune system for a boost with rMVA expressing matching antigens, inducing robust, HIV-specific T cell responses to both dominant and subdominant epitopes in the individual proteins when used as individual vaccines or in a mix.
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    The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen
    (Public Library of Science, 2014) Chapman, Rosamund; Bourn, William R; Shephard, Enid; Stutz, Helen; Douglass, Nicola; Mgwebi, Thandi; Meyers, Ann; Chin'ombe, Nyasha; Williamson, Anna-Lise
    Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10 7 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10 6 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.