OpenUCT :: Browsing by Author "Shephard, Enid"

Browsing by Author "Shephard, Enid"

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Open Access
Abrogation of contaminating RNA activity in HIV-1 Gag VLPs
(BioMed Central Ltd, 2011) Valley-Omar, Ziyaad; Meyers, Ann; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. METHODS: HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. RESULTS: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/mug Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. CONCLUSIONS: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.
Open Access
Characterisation of fibrinogen and fibrin proteolysis by the neutrophil membrane
(1999) Kirsch, Richard; Shephard, Enid
Recent studies have identified a novel 600 kDa neutrophil membrane associated protease which degrades fibrinogen, fibrin and C-reactive protein (CRP) during incubation of these ligands with phorbol 12-myristate 13-acetate (PMA, 5-10 ng/ml) stimulated neutrophils. This proteolysis is predominantly an extracellular event which occurs through a ligand dependent release of this protease from the neutrophil. Degradation products arising from this proteolysis not only become neutrophil associated but influence a number of important processes occurring in inflammation and coagulation. The aim of the present 'study was to purify and further characterize this protease and investigate the location of the neutrophil associated fibrinogen and fibrin degradation products. Whilst enzyme purification procedures were unsuccessful, several observations made during these attempts suggested that the neutrophil membrane associated proteolytic activity displayed similar characteristics to proteases of the azurophil granule. The proteolytic activity of the membrane was concluded from inhibitor profiles, zymography, and the apparent molecular mass values and hydrophobicity of the fibrinogen degradation products that it generated, to be the composite action of the azurophil granule proteases, human neutrophil elastase, cathepsin G and possibly proteinase 3. Electron microscopy analysis of PMA stimulated neutrophils incorporated within fibrin clots revealed morphological changes suggestive of neutrophil degranulation, and the proteolytic activity released by these cells was shown to be identical to that of azurophil granule proteases with respect to the apparent molecular mass values of the fibrin products that it generated. Immunoelectron microscopy revealed minimal internalization of fibrin like material during this process suggesting that neutrophil mediated fibrinolysis under these conditions is predominantly an extracellular event. Immunoelectron microscopy was used to localise fibrinogen degradation products previously reported to be associated with the neutrophil following incubation with fibrinogen. This revealed neutrophil associated fibrinogen products to be intracellular. Internalisation appears to be the result of pinocytosis which is stimulated in the presence of PMA. Although internalisation may be enhanced by an initial interaction of fibrinogen with the neutrophil membrane, a large proportion of uptake occurs via the fluid phase. Both intact and degraded forms of fibrinogen can associate with the neutrophil. Internalised material is rapidly degraded intracellularly into low molecular weight products which are partially released into the surrounding medium. This intracellular degradation, however, contributes minimally to the overall degradation of fibrinogen by neutrophils; the major pathway is extracellular. The demonstration in this· study, that the previously identified fibrinogen- fibrin- and CRP-degrading activity of the neutrophil membrane is due to azurophil granule proteases co-incides with numerous recent reports suggesting that membrane bound forms of these proteases, due to their ability to evade naturally occurring protease inhibitors, are the biologically relevant forms of these proteases. The membrane expression of azurophil granule proteases has recently been shown to be under the control of a variety of inflammatory mediators. Thus, neutrophil mediated degradation of fibrinogen, fibrin and CRP in vivo may be tightly controlled by the regulated expression of azurophil granule proteases on the neutrophil membrane.
Restricted
Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles.
(Elsevier, 2008) Halsey, Richard J; Tanzer, Fiona L; Meyers, Ann; Pillay, Sirika; Lynch, Alisson; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward P
HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines.
Open Access
Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus) by ESAT-6 and CFP-10 IFN-gamma ELISPOT tests following a tuberculosis outbreak
(BioMed Central Ltd, 2008) Chege, Gerald; Warren, Robin; van Pittius, Nico; Burgers, Wendy; Wilkinson, Robert; Shephard, Enid; Williamson, Anna-Lise
BACKGROUND:Both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates (NHP), indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. Screening of mycobacterial infections in NHP is traditionally done by tuberculin skin test (TST), which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. In this study, we investigated the use of ESAT-6 and CFP-10 for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis (TB). METHODS: Peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay (IFN-gamma ELISPOT) assay were obtained from TST positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture. RESULTS: Both ESAT-6 and CFP-10 IFN-gamma ELISPOT assays were able to detect early M. tuberculosis but also M. intracellulare infection. Although this indicates potential cross-reactivity with M. intracellulare antigens, the method was able to distinguish M. bovis BCG vaccination from M. tuberculosis infection. This assay performed better than the TST, which failed to detect one M. tuberculosis and two early M. intracellulare infections. CONCLUSION: These results suggest that the IFN-gamma ELISPOT assay could improve the detection of M tuberculosis infections when screening NHP. There is some doubt, however, concerning specificity, as the assay scored positive three animals infected with M. intracellulare.
Open Access
The effect of two novel C-type lectins, Ba100 and Ba25, isolated from the venom of the puff adder, Bitis arietans on T lymphocyte proliferative responses
(2008) Spearman, Catherine Wendy Nest; Shephard, Enid
Includes abstract. Includes bibliographical references (p. 233-318).
Open Access
Expression of HIV-1 antigens in plants as potential subunit vaccines
(BioMed Central Ltd, 2008) Meyers, Ann; Chakauya, Ereck; Shephard, Enid; Tanzer, Fiona; Maclean, James; Lynch, Alisson; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. RESULTS: Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. CONCLUSION: Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.
Open Access
The fumonisin B₁-fed rat as a model for liver injury, oval ('progenitor') cell proliferation, and carcinogenesis
(1999) Lemmer, Eric Richard; Hall, Pauline; Shephard, Enid; Cruse, Peter
Fumonisin B₁ (FB₁‚ ) is a carcinogenic mycotoxin produced by the fimgus Fusarium moniliforme in maize, and is hepatotoxic and hepatocarcinogenic in rats. The goal of this dissertation was to characterise the FB₁-fed rat as a model for liver injury and carcinogenesis, and to examine the role of oval ('progenitor') cells during these processes. Male Fischer 344 rats were fed FB₁ 250 mg/kg diet for five weeks, and this basic feeding regimen was modified in individual experiments. Short-term feeding of FB₁ caused a severe 'toxic' hepatitis, apoptosis and regeneration of hepatocytes, fibrosis, proliferation of OV-6 positive oval cells, and formation of GST pi positive hepatic foci and nodules. Oval cells were noted inside some of the hepatic nodules. There were marked increases in the expression of mRNA transcripts for mature TGF-β1 and c-myc in livers of FB₁-fed animals. The overexpression of TGF-β1 by hepatocytes may be responsible for the prominent apoptosis and fibrosis seen with liver injury due to FB₁. Increased expression of c-myc and TGF-β1 may cooperate during FB₁-induced promotion of liver tumours, possibly by providing an environment that selects for the growth of TGFβ1-resistant transformed liver cells. In rats given FB₁ in the presence of dietary iron overload, FB₁ augmented iron-induced lipid peroxidation in the liver. However, dietary iron loading appeared to protect against the cancer-promoting properties of FB₁, possibly due to a stimulatory effect on hepatocyte regeneration. Long-term feeding of FB₁ caused fibrosis and regenerative nodules, dysplastic hepatic nodules, cholangiofibrotic lesions, intraductal cholangiocarcinomas, and a hepatocellular carcinoma. 2-Acetylaminofluorene enhanced the effects of FB₁ in the liver, presumably by blocking hepatocyte regeneration in response to FB₁ toxicity. Proliferating oval cells were found inside/adjacent to GST pi positive lesions, dysplastic nodules, and cholangiofibrotic lesions, suggesting that oval cells may be involved in FBI-induced hepato- and cholangiocarcinogenesis in the liver. Furthermore, the OV-6 antigen was expressed by proliferating oval cells and bile ductules, hepatic nodules, cholangiofibrotic lesions, and cystic lesions, indicating that all of these cells may have a common ('stem') cell of origin. In conclusion, the FB₁-fed rat is a promising model for the study of liver injury, oval ('progenitor') cell proliferation, and carcinogenesis.
Open Access
HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice
(BioMed Central Ltd, 2010) Pillay, Sirika; Shephard, Enid; Meyers, Ann; Williamson, Anna-Lise; Rybicki, Edward P
Several approaches have been explored to eradicate HIV; however, a multigene vaccine appears to be the best option, given their proven potential to elicit broad, effective responses in animal models. The Pr55Gag protein is an excellent vaccine candidate in its own right, given that it can assemble into large, enveloped, virus-like particles (VLPs) which are highly immunogenic, and can moreover be used as a scaffold for the presentation of other large non-structural HIV antigens. In this study, we evaluated the potential of two novel chimaeric HIV-1 Pr55Gag-based VLP constructs - C-terminal fusions with reverse transcriptase and a Tat::Nef fusion protein, designated GagRT and GagTN respectively - to enhance a cellular response in mice when used as boost components in two types of heterologous prime-boost vaccine strategies. A vaccine regimen consisting of a DNA prime and chimaeric HIV-1 VLP boosts in mice induced strong, broad cellular immune responses at an optimum dose of 100 ng VLPs. The enhanced cellular responses induced by the DNA prime-VLP boost were two- to three-fold greater than two DNA vaccinations. Moreover, a mixture of GagRT and GagTN VLPs also boosted antigen-specific CD8+ and CD4+ T-cell responses, while VLP vaccinations only induced predominantly robust Gag CD4+ T-cell responses. The results demonstrate the promising potential of these chimaeric VLPs as vaccine candidates against HIV-1.
Open Access
A neutral protease of the neutrophil surface : role in the proteolysis of C-reactive protein and fibrinogen
(1995) Kelly, Sharon Lesley; Shephard, Enid
Both of the acute phase reactants, C-reactive protein and fibrinogen, as well as neutrophils have been shown to accumulate at sites of tissue injury or inflammation. The association of C-reactive protein with neutrophils and the concomitant degradation of this ligand by a phorbol 12-myristate 13 acetateactivatable membrane-associated neutral protease has been shown in previous studies. Degradation of C-reactive protein by the neutrophil protease was shown to result in peptides with an ability to modulate various immune functions of the neutrophil. The aim of this study has been to investigate specific characteristics of the protease, with respect to cellular distribution and molecular size. The ability of this neutrophil membrane-associated protease to degrade the acute phase protein, fibrinogen was investigated. The mechanism of degradation of both C-reactive protein and fibrinogen during their association with the neutrophil was also examined. The neutrophil protease, capable of degrading C-reactive protein, was also associated with the cytoskeleton and was proposed to be a submembrane protease localised at sites of attachment of the membrane with the cytoskeleton. The protease was found to have a molecular mass of approximately 600 kDa which, on sodium dodecyl sulphate polyacrylamide gel electrophoresis, separated into four bands which migrated to molecular mass values of 209 kDa, 316 kDa, 398 kDa and 501 kDa. This protease also possessed fibrinogenolytic activity. The fibrinogen degradation products generated by this neutrophil membrane-associated protease were distinct from the products generated by the fibrinogenolytic systems of plasmin, human neutrophil elastase and neutrophil lysosomal enzymes and were unclottable through cleavage of the Aα chain from the N-terminus and the Bβ and γ chains from the C-terminus. N-terminal cleavage of the Aα chain by the neutrophil membrane-associated protease generated the Aα1-21 peptide, previously regarded as a unique consequence of elastase activity. Degradation of C-reactive protein and fibrinogen occurred as a result of their interaction with the neutrophil near to the CD11c integrin receptor. This interaction resulted in the egress of proteolytic activity into the extracellular medium. The fibrinogen products generated outside the cell associated with the neutrophil via the β₂ integrin receptors and the IgG Fc receptor. The interaction of the Creactive protein degradation products with the neutrophil could not be determined. Both C-reactive protein and fibrinogen are degraded by non-stimulated neutrophils but activation with phorbol 12- myristate 13 acetate resulted in maximum degradation This upregulation of activity was achieved through activation of H7 and trifluoperazine inhibitable cellular kinases and changes in microfilament assembly. The generation of non-clottable fibrinogen together with possible modulation of neutrophil receptormediated functions by the fibringen degradation products as well as the knowledge that the neutrophil protease generates C-reactive protein peptides with immunomodulatory activity implicates this neutrophil membrane-associated protease in the modulation of various inflammatory processes.
Open Access
Nonalcoholic steatohepatitis (NASH) : animal studies of interactive hepatoxicity
(2004) Clarkson, Vivian; Hall, Pauline de la Motte; Marais, David; Shephard, Enid
The term "Nonalcoholic steatohepatitis" (NASH) describes a form of liver disease indistinguishable from alcoholic liver disease, but present in persons who consume insigniﬁcant amounts of alcohol. The spectrum of non-alcoholic liver disease ranges from non-progressive steatosis, through to NASH and sometimes to cirrhosis. Risk factors predisposing to NASH include obesity, diabetes mellitus, hypertriglyceridermia and procedures leading to rapid and profound weight loss such as gastric bypass and bulimia. Given the high incidence of type II diabetes, obesity and various types of hyperlipidaemia in the Western Cape, NASH has become one of the most commonly encountered liver diseases in the Liver Clinic, Groote Schuur Hospital. Statistics also suggest nonalcoholic fatty liver disease underlies most cases of elevated liver enzymes in many other parts of the world. The exact pathogenic mechanisms involved are not well understood, and no effective treatment options are currently available. However, animal models of NASH now provide unique opportunities for study of this disease in the laboratory setting. This thesis employs a dietary animal model, which restricts methionine and choline intake, to replicate aspects of the injurious process in human NASH. Two lines of inquiry are pursued. The ﬁrst involves an assessment of the effect of alcohol consumption on fatty liver of non- alcoholic aetiology. Elevated liver enzymes, altered levels of lipid peroxides, and increased cytochrome P450 activity recorded in this study, suggest alcohol may exacerbate nonalcoholic fatty liver disease. The second line of inquiry is an exploration of the role of Tumor Necrosis Factor alpha (TNFα, a cytokine implicated in pathogenesis of alcoholic liver disease and thought to be pivotal in NASH pathogenesis. The role of TNFα in the dietary model of NASH is investigated using mice lacking functional TNFα genes, as well as wild type mice in which expression of this cytokine is induced by endotoxin challenge. Endotoxin challenge does elicit a marked inflammatory response and enhanced generation of lipid peroxides in the fatty liver, both of which may contribute to injury. However, as knockout mice still develop experimental NASH, TNFα cannot be considered the sole driving force required for the development of NASH. This strength of the project lies in the use of a variety of techniques (in the ﬁelds of Pathology, Immunology and Lipid Biochemistry) to investigate a range of aspects of NASH. The results of the alcohol study may provide the first empirical evidence supporting the need to restrict alcohol intake in patients with NASH, while the data on TNFα is vital for illuminating the complex, (potentially) counter-intuitive role of this cytokine in NASH. Where deﬁnitive conclusions cannot be drawn from the data, the questions posited may constitute the basis for future research.
Open Access
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.
Open Access
An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice
(BioMed Central Ltd, 2009) Chin'ombe, Nyasha; Bourn, William; Williamson, Anna-Lise; Shephard, Enid
BACKGROUND:The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli beta-galactosidase alpha-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. RESULTS: High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-gamma and IL-4) immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-gamma and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-gamma produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05). The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05). CONCLUSION: These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.
Open Access
The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine
(BioMed Central Ltd, 2011) Tanzer, Fiona; Shephard, Enid; Palmer, Kenneth; Burger, Marieta; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND:One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1) and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1), an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. RESULTS: The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap) alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. CONCLUSIONS: We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.
Open Access
Pre-clinical assessment of novel candidate HIV-1 vaccines using the Chacma baboon
(2006) Chege, Gerald Kimani; Williamson, Anna-Lise; Shephard, Enid
Includes bibliographical references (leaves 176-221).
Open Access
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.
Open Access
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.
Open Access
Proteases of the neutrophil membrane represent an alternative fibrinolytic pathway to that mediated by plasmin
(1996) Adams, Susan Ann; Shephard, Enid
The cellular components of the blood, which become associated with fibrin through specific cellular adhesive processes, play a significant role in the breakdown of fibrin. Fibrinolysis by elastase and cathepsin G, enzymes present within the azurophilic granules of the neutrophil, has previously been shown. Recent studies have demonstrated neutrophil-mediated fibrinogenolysis by a membrane-associated protease which suggests that proteases connected with the neutrophil membrane might also be capable of clot dissolution. Investigations showed that neutrophil-mediated clot lysis was effected by a membrane-associated serine protease that can be dissociated by SDS-PAGE to bands that migrate to apparent molecular weights of 501 kDa, 398 kDa, 316 kDa, 245 kDa and 209 kDa. This degradation was distinct from that produced by plasmin, neutrophil lysosomal enzymes and purified human neutrophil elastase and enhanced the action of plasmin in clot solubilization. Preincubation of neutrophils with monoclonal antibodies directed against the CD 11 c/CD 18 integrin was able to significantly inhibit neutrophil membrane-dependent fibrinolytic activity. Upregulation of enzyme activity occurred following association of fibrin substrate with the cell membrane and was dependent on the activation of cellular kinases, in particular protein kinase C. Fibrin products generated by neutrophil membrane proteolytic activity were found to possess biological activity. The low molecular weight peptides effected substantial inhibition of thrombin-induced platelet aggregation while the presence of the higher molecular weight material could partially overcome platelet-induced resistance to plasmic lysis. No modulation of platelet-mediated fibrin clot retraction was observed using these same fibrin products. Neutrophil lysosomal enzyme activity was shown to further degrade the end products of plasmic fibrin degradation into low molecular weight material, followed by reassembly of higher molecular weight products in a process dependent on calcium and factor XIII. The reformed products have a similar molecular weight to those produced by plasmic lysis of fibrin, as well as a putative crosslinked site. However, the isoelectric point of these reformed products indicates they are distinctly different from plasmin-derived fibrin products. These reassembled products were recognized by a monoclonal antibody raised against D-dimer. Processing by neutrophils of the end products of plasmic fibrin degradation may have the potential for modulating the immune response as well as compromising the predictive value of tests measuring D-dimer, used as a laboratory marker of a number of thromboembolic disorders encountered in clinical practice.
Open Access
RNA transmission and expression from inert HIV candidate vaccine virus-like-particles
(2008) Valley-Omar, Ziyaad; Rybicki, Ed; Meyers, Ann; Shephard, Enid
HIV-1 Gag virus-like-particles (VLPs) produced in various expression systems are potent stimulators of both cellular and humoral immune responses in animal models. The encapsidation of large concentrations of random cellular RNA species is known to accompany the assembly of HIV virus particles. This RNA plays a crucial role by serving as a molecular scaffold for the assembly of Gag structural proteins into particles. Non-pseudotyped VLPs that do not present any HIV envelope glycoproteins are regarded as inert particles as they contain no replicative nucleic acid and are presumed to be unable to deliver encapsidated RNA for expression in inoculated individuals. Live virus cellular entry studies have shown that non-pseudotyped Gag particles are destined for degradation in acidified vesicles subsequent to receptor independent cellular entry. In addition to host cell RNA incorporation, Gag VLPs produced in insect cell-based, baculovirus expression systems have been observed to incorporate the baculovirus-derived Gp64 envelope glycoprotein. Gp64 has been shown to be efficient at enabling the delivery and expression of genes from recombinant baculoviruses and other Gp64 pseudotyped live viruses in mammalian cell lines both in vivo and in vitro. This study, therefore, set out to establish for the first time whether inert, baculovirus-derived (Gp64 pseudotyped) Gag VLPs could mediate delivery and expression of randomly encapsidated RNAs in mammalian cell lines.
Open Access
The role of the cytolytic mediators, granulysin and perforin, in tuberculosis
(2008) Semple, Patricia Lynn; Ress, Stanley; Shephard, Enid
Protective immunity against mycobacterial infection requires an effective cytolytic response, in addition to an intact Type l (Th1) cytokine pathway. Natural killer (NK) cells and cytolytic T-cells (CTL) are essential components of protective immunity against tuberculosis (TB) and mediate granule-dependent killing of infected cells. Granulysin, an antimicrobial protein, and perforin, a pore-forming molecule, have been found to co-localise in the granules of these two cell types. Granulysin has been shown to be directly cytotoxic to extracellular Mycobacterium tuberculosis (M.tb) and, together with perforin, is cytolytic against intracellular mycobacteria. This project evaluated the role of these two cytolytic mediators in TB.
Open Access
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.