The ability of a novel fish oil emulsion antigen-delivery system administered orally and by immersion, to stimulate antibody responses in the dab, was measured in a further experiment. On this occasion, oral intubation of HGG (25 mg) in saline induced no detectable responses. Immersion in a bath containing HGG in lipid emulsion induced a transient ASC response in the blood only. Anal intubation of HGG (25 mg) in saline induced a slight ASC response in the gut and blood. Oral intubation of HGG (25 mg) in lipid emulsion induced ASC responses in the gut and transiently in the gill but no response (above background) in the head kidney. None of the above methods of immunisation induced serum antibody titres. Intraperitoneal injection of HGG (1 mg) in saline induced high numbers of ASC in the head, kidney, gut and gill as well as serum antibody. The ASC response in the head kidney was detected from week 5 to 10, peaking at week 5. The response in the gill was from week 3 to 10, peaking at week 6, and the response in the gut was from week 5 to 10, peaking at week 8. The results indicate that systemic stimulation induced ASC responses in both systemic and mucosal compartments. The orally protected HGG in lipid emulsion was more effective than oral HGG in saline and anal HGG in saline in inducing ASC responses in the gut and the gill without including serum antibody, suggesting that oral immunisation can induce a common mucosal response independently of the head kidney. Leucocytes were isolated from the perfused gill of rainbow trout (<I>Oncorhynchus mykiss</I>) and fractioned on a 40-70% Percoll gradient into two subpopulations, top and bottom cell fractions. On stimulation with calcium ionophore, the isolated gill cells, following nylon wool filtration, were shown to be capable of producing chemoattractants for head kidney leucocytes at a dilution of 1:8. Only the bottom cell fraction exhibited migration toward a 2% dilution of trout serum while dilutions of 0.25% and 0.5% rainbow trout serum were not chemoattractive for either head kidney or gill leucocyte populations. The highest migration index was achieved after 1.5 h and the optimal cell number for migration was 4.65x10⁷ cells/ml. Respiratory burst activity was undetectable with isolated gill cells. Mitogenic responses of isolated and fractionated gill cells to LPS and PHA suggested the presence of few B-cells and a preponderance of T-cells.

No description available.

Crimean-Congo hemorrhagic fever (CCHF) is an inflammatory disease caused by the tick-borne pathogen CCHF virus (CCHFV). CCHFV is widely distributed with a endemic area including central and western Asia, the Middle East, southeastern Europe and the African continent, and can be transmitted to humans directly by tick bite or by contact with body fluids of infected animals or people. Most animals, while susceptible to CCHFV infection, do not display disease signs following infection, therefore, the development of CCHF disease models has been severely hampered. The lack of disease models has resulted in a lack of characterization of disease progression and lack of evaluated clinical countermeasures. The aims of this study were to further characterize a recently reported small animal model, the interferon alpha/beta receptor knockout (IFNAR-/-) mouse model of CCHF and to utilize this model to evaluate the protective efficacy of Adenovirus (Ad) vaccines as well as mechanisms of protection. To achieve the goals of the study hematological, coagulation, virological, pathological and cytokine/chemokine parameters of IFNAR-/- mice were assessed chronologically following CCHFV challenge. Vaccines were developed by construction of Ad expressing CCHFV nucleocapsid protein (Ad-NP) and glycoproteins (complete glycoprotein precursor Ad-GPC, and mature glycoproteins Ad-GN and Ad-GC). IFNAR-/- mice were then evaluated for adaptive immune responses following Ad vaccination and then challenged with a lethal dose of CCHFV and monitored for survival. To determine the roles of humoral and cell mediated adaptive immune responses in protection against CCHFV, experiments using adoptive transfers of Ad vaccinated IFNAR-/- mice to naïve IFNAR-/- mice and depletion of B- and/or T-cells were undertaken. The results demonstrate that IFNAR-/- mice develop an inflammatory, lethal disease following CCHFV challenge that resembles human CCHF; Ad-NP and Ad-NP/Ad-GPC vaccinated IFNAR-/- mice are protected from CCHFV iii challenge; and that humoral responses are essential for protection from CCHFV while T-cell responses are dispensable, at least in this vaccine platform, in this animal model. These studies provide the basis for more detailed work in vivo and suggest which mechanisms of protection may be important for subsequent advances in CCHFV vaccinology.

The protein monomer of bacterial flagella, FliC, is known to stimulate human innate immunity through activation of Toll-like receptor five. Linking native Salmonella FliC with various antigens has demonstrated an increased immune response as compared to single antigen presentation. To drastically reduce production time and allow for a more cost effective recombinant vaccine adjuvant, a synthetic construct was created that enables genetic linkage of FliC to other known antigens. The construct contains the necessary components for immune system stimulation while the non-essential regions were replaced with commonly used restriction enzyme recognition sites to aid in ligation with other antigens and cloning into various expression vectors and hosts. After synthesis in the inducible expression vector pJ404, the construct was transformed into competent BL21 E. coli and expression was confirmed through SDS-PAGE, Western blot, and MALDI MS/MS. The cells were adapted to fermentation media and re-screened for expression, and upon confirmation a 20-liter fermentation was conducted. The resulting samples were analyzed for expression within the insoluble and soluble cellular fractions to further optimize fermentation conditions. Once purified, this synthetic FliC will serve as a platform technology for the standardized co-expression of the TRL5 activator with a variety of antigens in both prokaryotic and eukaryotic systems.

Adjuvants are compounds that enhance immune responses to antigens present in a vaccine. They are particularly important in subunit vaccines; without adjuvants, these vaccines are often poorly immunogenic. A novel adjuvant platform developed at VIDO-InterVac is comprised of CpG-ODN or poly I:C, innate defense regulator peptides, and a new class of adjuvant called polyphosphazene. The polyphosphazenes have demonstrated a great potential as a safe and effective adjuvant. In particular, the polysphosphazenes poly[di(carboxylatophenoxy)-phosphazene] PCPP and poly[di(sodium carboxylatoethylpehnoxy)-phosphazene](PCEP) have been used in numerous animal studies where they not only have been shown to enhance the quality and quantity of the adaptive immune response, but also were shown to induce parenteral and mucosal immune responses with many different antigens, demonstrating their versatility. However the mechanisms by which the polyphosphazenes stimulate the innate immune response are only partially understood. Antigen presenting cells (APCs) are capable of facilitating the uptake of antigen and directing the immune response. Based upon the proposed mechanism of action of another adjuvant, we chose to investigate whether porcine monocytes could be induced to secrete pro-inflammatory cytokines IL-1 and IL-18 in response to stimulation with polyphosphazenes PCEP and PCPP. The release of these cytokines is thought to be mediated by the Nod-Like Receptors (NLRs), which are cytosolic pattern recognition receptors expressed in APCs. It is suggested that these receptors act in conjunction with TLR transcription pathways to control caspase-1 and release associated pro-inflammatory cytokines IL-1and IL-18 (Kawai and Akira, 2011). We first investigated the relative gene expression of three Nod-like receptor genes: nod1, nod2 and nlrp3 in various populations of porcine peripheral blood mononuclear cells (PBMCs) and found that monocytes, dendritic cells and B cells express increased relative levels of these receptors as compared to T cells. Subsequently, we evaluated the relative NLR expression in several porcine mucosal and lymphoid tissues and observed genes to be most significantly expressed in nasal mucosa, bronchial mucosa, and lung while limited in tissues associated with Peyer’s patches, jejunal wall. Both the mesenteric lymph node and bronchial lymph node exhibited similar patterns and levels of expression of nod1, nod2 and nlrp3. To characterize the activation of NOD1, NOD2 and NLRP3 receptors in response to stimulation with polyphosphazenes, porcine monocytes were stimulated with PCEP or PCPP in both the presence and absence of a second signal (poly I:C and CpG-ODN, TLR-7 and -9 agonists respectively). We found that PCEP and PCPP alone did not significantly upregulate nod1, nod2 and nlrp3, nor genes for cell activation markers such as CD80 and CD86. However monocytes cultured with the combination of CpG-ODN, Poly I:C and PCPP appeared to moderately express IL-18, CD80 and CD86. The secretion of pro-inflammatory cytokines from cultured monocytes was determined with Enzyme Linked Immunosorbent Assays (ELISA). It was found that IL-1was secreted in significantly higher quantities in the supernatant of cells stimulated with both polyphosphazene and TLR ligands, as opposed to those cultured with polyphosphazene alone. Assays for IL-18, IL-6, IL-10 and IL-12 did not detect a significant presence of these proteins in the supernatant. Furthermore, we found that a soluble caspase inhibitor did not significantly reduce the production of IL-1by monocytes, and was likely attributable to cell death at high concentrations. Taken together, these results suggest that porcine monocytes, B cells and dendritic cells express elevated levels of the NLRs as compared to T cells. Additionally, areas of the respiratory tract appear to express increased levels of these receptors relative to mucosal and lymphoid tissues of the gastrointestinal tract. Neither PCPP or PCEP alone were capable of inducing significant production of IL-1 or IL-18 by cultured monocytes, however stimulation of these cells with a combination of CpG-ODN, poly I:C and polyphosphazene resulted in the secretion of IL-1.

Influenza has been one of the most infectious diseases for roughly 2400 years. The most effective way to prevent influenza outbreaks and eliminate their seasonal effects is vaccination. The distribution of influenza vaccine to various groups in the population becomes an important decision determining the effectiveness of vaccination for the entire population. We developed a simulation model using the Epifire C++ application program [2] to simulate influenza transmission under a given vaccination strategy. Our model can generate a network that can be configured with different degree distributions, transmission rates, number of nodes and edges, infection periods, and perform chain-binomial based simulation of SIR (Susceptible-Infectious-Recovered) disease transmission. Furthermore, we integrated NOMAD (Nonlinear Optimization by Mesh Adaptive Direct Search) for optimizing vaccine allocation to various age groups. We calibrate our model according to age specific attack rates from the 1918 pandemic. In our simulation model, we evaluate three different vaccine policies for 36 different scenarios and 1000 individuals: The policy of the Advisory Committee on Immunization Practices (ACIP), former recommendations of the Centers for Disease Control and Prevention (CDC), and new suggestions of the CDC. We derive the number of infected people at the end of each run and calculated the corresponding cost and years of life lost. As a result, we observe that optimized vaccine distribution ensures less infected people and years of life lost compared to the fore-mentioned policies in almost all cases. On the other hand, total costs for the policies are close to each other. Former CDC policy ensures slightly lower cost than other policies and our proposed in some cases.

First in 1997, and later re-emerging in 2003, highly pathogenic avian influenza A virus, subtype H5N1, has spread from wild bird reservoirs to domestic bird flocks. As a result, cross-transmission has been confirmed in people living or working in close contact with infected birds. H5N1 virus infection is associated with a high mortality rate (>60%) in humans and the rapid evolution of the virus suggests that it could potentially develop into a new, and possibly severe, pandemic influenza virus. To-date, conventional inactivated and live-attenuated vaccine strategies offers the best protection against influenza virus infection; however, poor immunogenicity and weaker efficacy have been observed against H5N1 viruses. It was hypothesized that experimental adenovirus-based vaccines based on human adenovirus serotype 5 (AdHu5) or porcine adenovirus serotype 3 (PAV3) can offer protection against a broad range of avian influenza, subtype H5N1, viruses. Ad vaccine vectors are highly immunogenic and have demonstrated protective efficacy against several disease models. However, natural immunity against AdHu5 can interfere with vector efficacy. The nonhuman PAV3 was not neutralized by pooled human serum from 10,000-60,000 individuals and offers a promising alternative to AdHu5-based vectors. Systematic antigen screening using DNA vaccines identified the hemagglutinin (HA) glycoprotein as the most immunogenic H5N1 antigen. HA was then inserted directly into PAV3 or AdHu5. Comparable immune responses were observed between both vectors but, interestingly, the PAV3-based vaccine generated stronger T-cell responses and better rapid protection 8 days following immunization. Additionally, better long-term protection 1 year following vaccination was observed with the PAV3-HA vaccine. The co-administration of multiple H5N1 antigens was also screened to improve protection against divergent H5N1 challenge. Combinations of DNA vaccines expressing (HA+NA) and (HA+NP) offered the best promise for enhancing protection against homologous and heterologous H5N1 challenges, respectively. However, addition of three or more antigens reduced overall protection possibly by antigen dilution, competition, or interference. Co-administration of PAV3 or AdHu5 vectors expressing both the HA and NP antigens reduced protection against homologous and heterologous H5N1 virus challenges. For all combination vaccines, T-cell responses were strong against HA but significantly decreased against additional antigens in each combination vaccine. Overall, the experimental porcine-based Ad-based vaccine offered better protection than the H5N1 conventional vaccine against a broad range of different H5N1 viruses. Understanding of the relationship between immune parameters and protection will be critical in future improvement of adenovirus-based and other vaccines against avian influenza H5N1.

As the number of drug-resistant malaria parasites continues to grow, pressure is increasing to find an effective, cross-protective, multi-valent malaria vaccine (32). Expression library immunisation is an un-biased screening technique that leads to the identification of novel, protective antigens that can be administered as components of a multivalent DNA vaccine (9, 50, 75, 86, 92). Here, a P. c. adami DS expression library has been evaluated as a malaria vaccine in mice, and several subpools of cross-protective plasmids have been identified. Upon vaccination with these plasmid subpools, mice demonstrate significantly lower mean cumulative parasitemia values than control vaccinated mice, when challenged with avirulent heterologous P. c. adami DK parasites. These cross-protective responses correlate with the induction of opsonizing antibodies against infected red blood cells and the production of IFN-gamma by T-cells. The determination of P. c. adami antigens capable of inducing strain-transcending immunity implies the identification of orthologues in the P. falciparum genome that may be applied as components of a human malaria vaccine.

No description available.

xii, 205, xxviii leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--Dept. of Microbiology, University of Adelaide, 1980