The development of recombinant vaccines against Jembrana disease

Jembrana disease virus (JDV) is a lentivirus causing an acute infection with a 17% case fatality rate in Bali cattle in Indonesia. Control of the disease is currently achieved by identification of infected areas and restriction of cattle movement. A detergent-inactivated whole virus tissue-derived vaccine is sometimes employed in affected areas. This thesis reports initial attempts to produce genetically engineered vaccines to replace the inactivated tissue-derived vaccine, which as it is made from homogenised spleen of infected animals, is expensive to produce and could contain adventitious agents present in the donor animals. 4 potential DNA vaccine constructs were created containing the JDV genes coding for the Tat, capsid (CA), transmembrane (TM) and surface unit (SU) proteins in a commercially available vaccine plasmid. These were assessed for functionality in a range of in vitro and in vivo assays. All proteins were expressed in vitro and administration of 2 of the constructs by a commercial 'gene gun' into the epidermis of mice resulted in antibody production to the appropriate protein. Due to the difficulties of licensing such a DNA vaccine in Indonesia, these vaccines were not progressed further. A mathematical model was developed to describe the progression of the acute phase of Jembrana disease following experimental infection with JDV. The model divided the disease into 6 phases based on the rates of viral replication and clearance calculated from data on sequential plasma viral RNA load detected by quantitative reverse-transcription polymerase chain reaction. This allowed statistical comparison of each phase of the disease and comparison of the severity of the disease process in groups of animals. The use of the model overcame the difficulty of comparing the disease in different animals as a consequence of the animal-to-animal variation in the disease process. The mathematical model was used to identify differences in the pathogenicity of 2 strains of JDV. One strain, JDVTAB caused a more rapid onset of disease in non-vaccinated controls, a significantly higher virus load at the onset of the febrile period and a higher peak viraemia than in animals infected with JDVPUL. This provided the first evidence of variation in pathogenicity of JDV strains. The measurement of virus load also demonstrated that some JDV infected animals developed a clinical disease that was not typical of that which had been reported previously. When infected with less than 1,000 infectious virus particles, up to 20% of infected animals failed to develop a febrile response. Infection of these animals was confirmed, however, by the detection of a high titre of circulating virus particles in plasma. These atypical infections had not been reported previously. Application of the mathematical model describing the progression of the disease in individual animals was used to examine the effect of vaccination with the inactivated tissue-derived vaccine on the progression of the disease. Several effects were noted in vaccinated animals that were subsequently infected with JDV: a reduction in the duration of the febrile response, a reduction in the severity of the febrile response in the early phases of the acute disease, and a reduction in virus load in the early and later phases of the disease process. The effect of vaccination with recombinant Tat, matrix (MA) and CA protein vaccines expressed in a bacterial expression system on subsequent JDV infection was also examined. A vaccine incorporating recombinant Tat and CA vaccine emulsified with Freund's incomplete adjuvant decreased the febrile response particularly in the later stages of the acute disease process, decreased the severity of the leucopenia in the later phases of the acute disease, and decreased the virus load in some but not all phases of the acute disease process. Vaccines administered with Freund's incomplete adjuvant were more efficacious than vaccines administered with QuilA, the latter actually exacerbating the disease process in vaccinated animals

Investigation of the morphological diversity of the potentially zoonotic Trypanosoma copemani in quokkas and Gilbert's potoroos

Trypanosomes are blood-borne parasites that can cause severe disease in both humans and animals, yet little is known of the pathogenicity and life-cycles of trypanosomes in native Australian mammals. Trypanosoma copemani is known to be infective to a variety of Australian marsupials and has recently been shown to be potentially zoonotic as it is resistant to normal human serum. In the present study, in vivo and in vitro examination of blood and cultures from Australian marsupials was conducted using light microscopy, immunofluorescence, scanning electron microscopy and fluorescence in situ hybridization. Promastigote, sphaeromastigote and amastigote life-cycle stages were detected in vivo and in vitro. Novel trypanosome-like stages were also detected both in vivo and in vitro representing an oval stage, an extremely thin stage, an adherent stage and a tiny round stage. The tiny round and adherent stages appeared to adhere to erythrocytes causing potential haematological damage with clinical effects similar to haemolytic anaemia. The present study shows for the first time that trypomastigotes are not the only life-cycle stages circulating within the blood stream of trypanosome infected Australian native marsupials and provides insights into possible pathogenic mechanisms of this potentially zoonotic trypanosome species

Molecular characterization of native Australian trypanosomes in quokka (Setonix brachyurus) populations from Western Australia

The quokka, Setonix brachyurus, is a vulnerable, small marsupial endemic to Western Australia. Blood samples were collected from quokkas from three different geographical locations; Two Peoples Bay, Bald Island and Rottnest Island. The overall prevalence of trypanosomes by nested PCR at the 18S ribosomal RNA gene was 57.3% (63/110) with prevalences of 91.4%, 85.3% and 4.9% respectively for Two Peoples Bay, Bald Island and Rottnest Island. Phylogenetic analysis conducted on 47 18S PCR positives identified two Trypanosoma copemani genotypes, with T. copemani genotype B, the most prevalent genotype infecting quokka populations from the three locations with an overall prevalence of 51.8% (24/47) compared to 34% for T. copemani genotype A (16/47). The overall prevalence of mixed T. copemani genotype A and B infections was 14.9% (7/47). Phylogenetic analysis of 26 quokka isolates at the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locus, largely supported the 18S analysis but identified a mixed infection in one quokka isolate (Q4112-4117 from Two Peoples Bay). T. copemani genotype B has previously only been isolated from quokkas and the Gilbert's potoroo whereas T. copemani genotype A has a wide host range and may be pathogenic. Further work is required to determine the clinical impact of T. copemani on marsupial populations

Characterization of polymorphic microsatellite markers for the Carnaby's cockatoo (Calyptorhynchus latirostris) and related black cockatoo species

Microsatellite loci were isolated from Carnaby's black cockatoo (Calyptorhynchus latirostris: Aves), a highly valued, endangered, and endemic species of bird from Western Australia. This study describes three dinucleotide and one tetranucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with between two and nine alleles and moderate levels of variability. Two additional dinucleotide markers which were monomorphic in the Carnaby's cockatoo were able to amplify and were polymorphic in two other species of black cockatoo, greatly increasing the utility of these markers

Recombinant Jembrana disease virus gag proteins identify several different antigenic domains but do not facilitate serological differentiation of JDV and nonpathogenic bovine lentiviruses

In Indonesia, it is suspected that there are two bovine lentiviruses circulating in the cattle population: a pathogenic Jembrana disease virus (JDV), and a nonpathogenic bovine immunodeficiency-like virus (BIV). Both viruses cross-react antigenically and cannot be differentiated by current serological tests using JDV antigens. To identify possible type-specific epitopes, a series of recombinant protein constructs including the matrix, capsid and nucleocapsid proteins were produced from JDV gag and the expressed proteins were tested by Western blot using JDV and BIV hyperimmune sera. JDV matrix and truncated capsid proteins were recognised by both JDV and BIV hyperimmune sera indicating that there were multiple cross-reactive epitopes present in JDV gag. At least three epitopic regions were identified in these constructs, including the major homology region, by monoclonal antibody binding studies. JDV nucleocapsid recombinant protein was not recognised by either JDV or BIV hyperimmune sera and none of the recombinant gag proteins were able to differentiate between JDV positive sera from Jembrana disease endemic and Jembrana disease-free areas. Additionally, a 40 amino acid recombinant subunit protein encompassing the region recently found to contain an epitope unique to BIV [Zheng, L., Zhang, S., Wood, C., Kapil, S., Wilcox, G.E., Loughin, T.A., Minocha, H.C., 2001. Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity. Clin. Diagn. Lab. Immunol. 8, 283–287] was tested but was not recognised by either JDV positive sera from Jembrana disease-endemic or Jembrana disease-free areas

Data_Sheet_1_A phase I clinical trial assessing the safety, tolerability, and pharmacokinetics of inhaled ethanol in humans as a potential treatment for respiratory tract infections.docx

BackgroundCurrent treatments for respiratory infections are severely limited. Ethanol’s unique properties including antimicrobial, immunomodulatory, and surfactant-like activity make it a promising candidate treatment for respiratory infections if it can be delivered safely to the airway by inhalation. Here, we explore the safety, tolerability, and pharmacokinetics of inhaled ethanol in a phase I clinical trial.MethodsThe study was conducted as a single-centre, open-label clinical trial in 18 healthy adult volunteers, six with no significant medical comorbidities, four with stable asthma, four with stable cystic fibrosis, and four active smokers. A dose-escalating design was used, with participants receiving three dosing cycles of 40, 60%, and then 80% ethanol v/v in water, 2 h apart, in a single visit. Ethanol was nebulised using a standard jet nebuliser, delivered through a novel closed-circuit reservoir system, and inhaled nasally for 10 min, then orally for 30 min. Safety assessments included adverse events and vital sign monitoring, blood alcohol concentrations, clinical examination, spirometry, electrocardiogram, and blood tests.ResultsNo serious adverse events were recorded. The maximum blood alcohol concentration observed was 0.011% immediately following 80% ethanol dosing. Breath alcohol concentrations were high (median 0.26%) following dosing suggesting high tissue levels were achieved. Small transient increases in heart rate, blood pressure, and blood neutrophil levels were observed, with these normalising after dosing, with no other significant safety concerns. Of 18 participants, 15 completed all dosing cycles with three not completing all cycles due to tolerability. The closed-circuit reservoir system significantly reduced fugitive aerosol loss during dosing.ConclusionThese data support the safety of inhaled ethanol at concentrations up to 80%, supporting its further investigation as a treatment for respiratory infections.Clinical trial registration: identifier ACTRN12621000067875.</p

A phase I clinical trial assessing the safety, tolerability, and pharmacokinetics of inhaled ethanol in humans as a potential treatment for respiratory tract infections

BackgroundCurrent treatments for respiratory infections are severely limited. Ethanol’s unique properties including antimicrobial, immunomodulatory, and surfactant-like activity make it a promising candidate treatment for respiratory infections if it can be delivered safely to the airway by inhalation. Here, we explore the safety, tolerability, and pharmacokinetics of inhaled ethanol in a phase I clinical trial.MethodsThe study was conducted as a single-centre, open-label clinical trial in 18 healthy adult volunteers, six with no significant medical comorbidities, four with stable asthma, four with stable cystic fibrosis, and four active smokers. A dose-escalating design was used, with participants receiving three dosing cycles of 40, 60%, and then 80% ethanol v/v in water, 2 h apart, in a single visit. Ethanol was nebulised using a standard jet nebuliser, delivered through a novel closed-circuit reservoir system, and inhaled nasally for 10 min, then orally for 30 min. Safety assessments included adverse events and vital sign monitoring, blood alcohol concentrations, clinical examination, spirometry, electrocardiogram, and blood tests.ResultsNo serious adverse events were recorded. The maximum blood alcohol concentration observed was 0.011% immediately following 80% ethanol dosing. Breath alcohol concentrations were high (median 0.26%) following dosing suggesting high tissue levels were achieved. Small transient increases in heart rate, blood pressure, and blood neutrophil levels were observed, with these normalising after dosing, with no other significant safety concerns. Of 18 participants, 15 completed all dosing cycles with three not completing all cycles due to tolerability. The closed-circuit reservoir system significantly reduced fugitive aerosol loss during dosing.ConclusionThese data support the safety of inhaled ethanol at concentrations up to 80%, supporting its further investigation as a treatment for respiratory infections.Clinical trial registration: identifier ACTRN12621000067875