There is currently no protective vaccine against HIV. It is known that a high mutation rate and the existence of many subspecies or clades generated by point mutations or recombination events, are at least partly responsible for the ability of the virus to escape immune responses elicited by classical vaccines. Protein subunit vaccines may not be effective due to this pronounced viral mutability. An immune evasion mechanism has been postulated in which variable domains occlude conserved epitopes crucial for infectivity. The use of DNA vaccines appeared as a favorable approach. Here, a DNA vaccine approach is presented in which the DNA constructs have been engineered to circumvent the aforementioned problems by 1) introducing elements to enhance expression, such as a heterologous promoter, a heterologous signal sequence and intron sequences, 2) by optimizing codon usage, and 3) by vaccinating with antigens that have a modified glycosylation pattern which will make them more immunogenic. The results indicated that deglycosylation of different clades of gp120 did not affect the protein conformation, and 'in vitro' expression levels were good. Antigen codon optimization dramatically increased antibody production. In the animals vaccinated with non-codon-optimized constructs, the presence of an intron and a heterologous signal sequence was required to achieve a good antibody response. Therefore, antigen engineering is required to obtain a powerful immune response against HIV-1 gp120.

Abstract not available

Since its discovery 15 years ago there has been an explosion of research in the field of DNA immunisation. Unfortunately despite early promises that DNA immunisation had the potential to cure almost any infectious disease, autoimmune disease or even cancer, progress towards clinical trials has been slow. This has been due in part to the huge range of permutations possible in delivering the DNA. One approach is to deliver the DNA by gene gun. Gene gun delivery is a very efficient way of transfecting cells however also has a number of possible disadvantages. These drawbacks include a weak immunogenicity in larger animals as well as the tendency to bias towards the development of a strong type 2 response. In an effort to enhance antigen-specific immune responses and counter the type 2 polarisation of gene gun delivery, a series of DNA vaccines were created where the extracellular portion of the hemagglutinin (HA) gene from influenza A/PR8/34 virus was genetically fused the type 1 cytokines IFNγ, IL-12 and IL-23. Interleukin-23 has been recently discovered and even though both IL-12 and IL-23 contain the p40 subunit they seem to have dissimilar functions. The vaccine constructs were first tested in cellular assays in vitro to ensure correct production and biological activity of the attached cytokines. They were then delivered in various combinations to groups of BALB/c mice to test development of immune responses and the effect of different delivery regimes. Finally mice were immunised then challenged with live influenza virus to determine the different DNA vaccines� protective efficacy. DNA vaccines containing the HA gene alone (pHA) or fused to IFNγ (pIFNγHA), IL-12 (pIL-12HA) or IL-23 (pIL-23HA) were successfully constructed. The fusion of the HA gene to the genes for IFNγ, IL-12 or IL-23 did not significantly disturb the structure of the antigen or prevent the biological actions of the cytokines. Mice immunised three times with pHA had high titres of serum IgG1 antibody and their splenocytes produced approximately equal amounts of IFNγ and IL-5. Co-delivery of IFNγ was unable to alter immune responses regardless of whether it was delivered at the first, last or during all immunisations. Surprisingly co-delivery of IL-12 acted to suppress both antibody and cellular immune responses, possibly through an IFNγ/nitric oxide feedback loop. On the other hand co-delivery of IL-23 tended to enhanced immune responses and, while it did not significantly alter the type 1 to type 2 balance, it was able to increase the ability of mice to clear live influenza virus from their lungs when they were challenged 26 weeks after immunisation. This protection was associated with increased levels of neutralising antibody in the serum of pIL-23HA immunised mice. This research has illuminated several of the pitfalls in the development of DNA vaccines and the use of cytokine as adjuvants. However it has also broadened our understanding of IL-23 and implies that IL-23 could be effectively used to increase the development of longterm immunity after immunisation.

The liver flukes Fasciola hepatica and F. gigantica cause infectious disease in ruminants and humans. The geographical range of these two parasite species (temperate and tropical respectively) ensures that infection can occur worldwide. Although anthelmintic treatment is effective against disease, emerging drug resistant strains leads to the development of a vaccine. However, despite several decades of research, there is no commercial vaccine available. The main challenge at present is to produce recombinant proteins in an immunologically active form using recombinant DNA technology. This is an essential step in Fasciola vaccine production. Cysteine proteases are probably the most important facilitators of virulence in flukes and are produced by all stages of the fluke life-cycle. Two classes of cysteine protease are found in the excretory and secretory material of liver flukes- these are cathepsin L and cathepsin B. As such, the major aims of this thesis were to investigate the expression and purification of Fasciola recombinant cysteine proteins, and characterisation by SDS-PAGE and immunoblotting using monoclonal and polyclonal antibodies. These studies demonstrate the production of functionally active cathepsin proteins in S. cerevisiae BJ3505 cells which will lead to vaccine candidate analysis. The second aim of this thesis was to determine the protective efficacy of stage specific target antigens against experimental infection. In addressing this issue, the protective efficacy of single and multivalent recombinant protein vaccinations of adult stage F. hepatica cathepsin L5, immature F. gigantica cathepsin L1g and juvenile F. hepatica cathepsin B were analysed in Sprague Dawley rats against F. hepatica infection. This study demonstrates that juvenile fluke target antigen-cathepsin B induces better immune protection than adult fluke antigen-cathepsin L5. Cocktails of juvenile and adult stage fluke recombinant proteins (cathepsin B and L5) elicited the highest protective immunity against experimental infection and this combination showed not only reduction in fluke recovery and size of flukes, but also marked diminution in the intensity of liver lesions in vaccinated rats. In order to assess the immunogenic property of an early infective stage fluke secreting cysteine protease as a vaccine candidate, DNA vaccination vectors encoding cathepsin B were analysed in BALB/c mice. In this study, the ability of four DNA vaccination strategies such as secretory, chemokine-activating, lymph node targeting vectors encoding cathepsin B were assessed by antibody titre, antibody avidity, western blotting and ELIPSOT assay. The results have further validated the immunoprophylactic potential of a cathepsin B vaccine against F. hepatica. In this study, we have expressed and attained high yields of F. gigantica cathepsin L1g from E. coli BL21, and compared this to a yeast-expressed system. This protease was over-expressed and formed insoluble inclusion bodies that were subsequently solubilised with urea or guanidine hydrochloride. In order to purify the urea-solubilised protein, step-wise urea gradient chromatography was used. For refolding of solubilised protein, a dilution and dialysis procedure was utilised. Proteolytic activity was confirmed by gelatin SDS-PAGE analysis. In conclusion, the determination of the immune potential of recombinant stage specific antigens allows the development of effective vaccines against Fasciola infection.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Bovine herpesvirus-1 (BoHV-1) causes recurrent respiratory and genital infections in cattle; and predisposes them to lethal secondary bacterial infections. Vaccination is a primary strategy to prevent and reduce the severity of disease associated with BoHV-1, and to reduce virus transmission. While modified live (MLV) or killed (KV) BoHV-1 vaccines exist, these are expensive to produce, can cause disease (MLV) or may be ineffective (KV). Development of a DNA vaccine for BoHV-1 has the potential to address these shortcomings, but the very small amount of antigen expressed after DNA immunization presents a barrier to successful immunization of large animals. Engineering the vaccine to target this limited quantity of antigen to dendritic cells (DCs), the cells that prime immune responses, by attracting immature DCs (iDCs) to the vaccination site, is one way that DNA vaccine efficacy might be improved. Beta (β)-defensins are chemotactic peptides that, in studies with mice, improve induction of immune responses to DNA vaccines and this is due, at least in part, to their ability to attract iDCs to the site of vaccination. Accordingly, the objective of the studies described in this thesis was to determine whether using a bovine β-defensin in a DNA vaccine would enhance immune responses to the vaccine and subsequently protect cattle upon challenge with BoHV-1. First I characterized the bovine iDC and then used these cells to screen a panel of synthesized bovine β-defensins for chemotactic activity. The results showed that bovine neutrophil β-defensin (BNBD) 3, BNBD9 and enteric β-defensin (EBD) were equally the most chemotactic of the fourteen synthesized peptides for bovine iDCs. Because BNBD3 is the most abundant of the thirteen BNBDs and was able to attract CD1+ DCs when injected into the skin, I chose BNBD3 as the peptide I would use for the rest of the project. Next I constructed plasmids that expressed BNBD3; either alone or as a fusion construct with the BoHV-1 antigen truncated glycoprotein D (tgD), and then tested the effects of the plasmids as vaccines in both mice and cattle. In cattle, the addition of BNBD3 as a fusion strengthened the Th1 bias and increased cell-mediated immune responses to the DNA vaccine but not antibody response or protection from BoHV-1 infection. Given that inefficient humoral immune responses have been implicated in a lack of protection from BoHV-1 challenge, these results suggested that the successful BoHV-1 DNA vaccine would need to induce a much stronger humoral response. Lastly I assessed the ability of BNBD3 to improve humoral responses to pMASIA-tgD when complexed with the DNA vaccine and found that the vaccine complexed at a nanomolar peptide to DNA ratio of 125:1 increased humoral responses of mice. In vitro, treatment of mouse bone-marrow DCs with BNBD3 induced phenotypic and functional maturation/activation. This is an important aspect for vaccination in the skin, since after uptake, the DC must “mature” in order to traffic from the site of vaccination to the draining lymph node where induction of antigen-specific responses, by activated DCs, takes place. The findings in this thesis show that bovine β-defensins are chemotactic for bovine iDCs. I also show that using a bovine β-defensin as a fusion construct in a DNA vaccine enhances cell mediated but not humoral responses of cattle and yet this vaccine is protective against BoHV-1 challenge. I demonstrate that a bovine β-defensin, when used as a peptide to complex an antigen-encoding plasmid, can increase humoral responses. My work shows a multifunctional ability of bovine β-defensins to modulate and increase immune responses and suggests that bovine β-defensins likely have further untapped potential to enhance efficacy of DNA vaccines for large animals.

Since its discovery 15 years ago there has been an explosion of research in the field of DNA immunisation. Unfortunately despite early promises that DNA immunisation had the potential to cure almost any infectious disease, autoimmune disease or even cancer, progress towards clinical trials has been slow. This has been due in part to the huge range of permutations possible in delivering the DNA. One approach is to deliver the DNA by gene gun. Gene gun delivery is a very efficient way of transfecting cells however also has a number of possible disadvantages. These drawbacks include a weak immunogenicity in larger animals as well as the tendency to bias towards the development of a strong type 2 response. In an effort to enhance antigen-specific immune responses and counter the type 2 polarisation of gene gun delivery, a series of DNA vaccines were created where the extracellular portion of the hemagglutinin (HA) gene from influenza A/PR8/34 virus was genetically fused the type 1 cytokines IFNγ, IL-12 and IL-23. Interleukin-23 has been recently discovered and even though both IL-12 and IL-23 contain the p40 subunit they seem to have dissimilar functions. The vaccine constructs were first tested in cellular assays in vitro to ensure correct production and biological activity of the attached cytokines. They were then delivered in various combinations to groups of BALB/c mice to test development of immune responses and the effect of different delivery regimes. Finally mice were immunised then challenged with live influenza virus to determine the different DNA vaccines� protective efficacy. DNA vaccines containing the HA gene alone (pHA) or fused to IFNγ (pIFNγHA), IL-12 (pIL-12HA) or IL-23 (pIL-23HA) were successfully constructed. The fusion of the HA gene to the genes for IFNγ, IL-12 or IL-23 did not significantly disturb the structure of the antigen or prevent the biological actions of the cytokines. Mice immunised three times with pHA had high titres of serum IgG1 antibody and their splenocytes produced approximately equal amounts of IFNγ and IL-5. Co-delivery of IFNγ was unable to alter immune responses regardless of whether it was delivered at the first, last or during all immunisations. Surprisingly co-delivery of IL-12 acted to suppress both antibody and cellular immune responses, possibly through an IFNγ/nitric oxide feedback loop. On the other hand co-delivery of IL-23 tended to enhanced immune responses and, while it did not significantly alter the type 1 to type 2 balance, it was able to increase the ability of mice to clear live influenza virus from their lungs when they were challenged 26 weeks after immunisation. This protection was associated with increased levels of neutralising antibody in the serum of pIL-23HA immunised mice. This research has illuminated several of the pitfalls in the development of DNA vaccines and the use of cytokine as adjuvants. However it has also broadened our understanding of IL-23 and implies that IL-23 could be effectively used to increase the development of longterm immunity after immunisation.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The South African ostrich industry is under enormous threats due to diseases contracted by the ostriches. H5N2 virus (avian influenza) outbreaks the past two years have resulted in thousands of ostriches having to be culled. However, the more silent respiratory infectious agents of ostriches are the three ostrich-specific mycoplasmas. Named Ms01, Ms02, and Ms03, these three mycoplasmas are responsible for dramatic production losses each year, due to their intrusive nature and the fact that no vaccines are currently available to prevent mycoplasma infections in ostriches. The use of antibiotics does not eradicate the disease completely, but only alleviates symptoms. The ostrich industry commissioned investigations into the development of three specific vaccines using the relatively novel approach of DNA vaccination. The concept of DNA vaccine development is based on the availability of complete genome sequences of the pathogen against which the vaccine is to be developed. This is necessary in order to identify vaccine candidate genes through comparative genomic studies. The Ms02 genome has previously been sequenced, resulting in 28 large contiguous sequences. This thesis used the technique of Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) to attempt assembly of these 28 contiguous sequences. The number was reduced to 14 large contiguous sequences, which were then subjected to repetitive sequence analysis and open reading frame analysis. Bioinformatic software was also used to predict the origin of replication. The extent of repeats in the Ms02 genome is illustrated, as well as the problems with genome assembly when dealing with repetitive-rich and A+T-rich genomes as those of mycoplasmas. Previous studies determined the mycoplasma oppA gene to be a good vaccine candidate gene, due to its cytadherent properties. This thesis describes the development of three DNA vaccines containing the Ms02 oppA gene, and a preliminary attempt to prove expression of one of these vaccines in a cell culture-based system. The DNA vaccine vectors pCI-neo, VR1012, and VR1020 were chosen for the vaccine development. The Ms02 oppA gene was also cloned into the prokaryotic expression vector pGEX-4T-1 in order to express the OppA protein for purification. The purified protein may be used in future serological tests in ostrich vaccination trials. In this study the protein was used to elicit anti-OppA rabbit antibodies, which were used to attempt detection of the pCI-neo-driven OppA protein expression in an MDA cell line in a transfection study. However, preliminary findings could not detect expression, but did indicate that the currently used colorimetric western blot technique may not be sensitive enough. It is suggested that different cell lines need to be investigated. Further optimisations are also required to decrease the observed non-specific binding. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse volstruisbedryf is onder geweldige druk vanweë siektes wat die volstruise bedreig. Die epidemie van die H5N2 virus (voëlgriep) in die afgelope twee jaar het veroorsaak dat duisende volstruise van kant gemaak moes word. Daar is egter nog ‘n bedreiging wat tot geweldige produksie verliese lei elke jaar: die respiratoriese infeksies wat versoorsaak word deur die drie volstruis mikoplasmas, genoem Ms01, Ms02 en Ms03. Geen entstowwe is tans beskibaar om die infeksies te voorkom nie, en behandeling met behulp van antibiotikas is nie effektief in die genesing van infeksie nie, maar help net om die simptome te verlig. Weens die erns van die saak, het die Suid-Afrikaanse volstruisbedryf ‘n ondersoek geloods na die ontwikkeling van enstowwe teen elkeen van die drie volstruis mikoplasmas. Die relatiewe nuwe benadering van DNA-entstof ontwikkeling was die strategiese keuse. Die beginsel van DNA-entstof ontwikkeling berus op die beskikbaarheid van die genoomvolgordes van die siekte-veroorsakende organisme waarteen die enstof ontwikkel word. Geskikte kandidaat entstof gene word so opgespoor met behulp van vergelykende studies met ander beskikbare genome. Die Ms02 genoomvolgorde is voorheen bepaal en word verteenwoordig deur 28 groot geenvolgorde fragmente. Die tegniek van Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) is gebruik om van die 28 fragmente aan mekaar te las. Die aantal fragmente is verminder na 14 groot geenvolgorde fragmente, wat vervolgens gebruik was om die omvang van herhalende volgordes in die genoom te bepaal, om nuwe leesrame te ondersoek, asook om die oorsprong van DNA replikasie op te spoor met behulp van bioinformatika sagteware. Die omvang van die herhalende aard van die Ms02 genoom word geïllustreer, asook die gepaardgaande probleme met die las van geenvolgorde fragmente wanneer met genome van veelvuldige herhalende volgordes, wat boonop A+T-ryk is, gewerk word, soos die van mikoplasmas. Vorige studies het die mikoplasma oppA geen geïdentifiseer as ‘n geskikte kandidaat entstof geen as gevolg van sy selaanhegting-eienskappe. Hierdie studie behels die invoeging van die Ms02 oppA geen in drie DNA-enstof vektore, naamlik pCI-neo, VR1012, en VR1020, asook die voorlopige poging om bewys van uitdrukking van een van die entstowwe in ‘n selkultuursisteem te bewerkstellig. Die geen is ook gekloneer in die prokariotiese ekspressie vektor pGEX-4T-1, ten einde die Ms02 OppA proteïen te isoleer. Die geïsoleerde proteïen kan in serologiese toetse in toekomstige volstruis enstof proewe gebruik word. In hierdie studie is die proteïen gebruik om konyn teenliggame teen dit op te wek, wat dan gebruik was om vir die pCI-neo-gedrewe ekspressie van die oppA geen te toets in ‘n selkultuur omgewing deur ‘n MDA sellyn te transfekteer. Die voorlopige resultate toon nie ekspressie van die OppA proteïen aan nie, maar het wel uitgelig dat die western blot tegniek wat tans gebruik word, dalk nie sensitief genoeg is nie. Dit kan belowend wees om ander tipes selle te toets. Verdere optimisering is ook nodig om die nie-spesifieke binding wat waargeneem is, te verlaag. / South African Ostrich Business Chamber