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    Design and engineering of self-assembling antigens towards particulate vaccines : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Chen, Shuxiong
    Natural and synthetic self-assembling polymers and proteins could be bioengineered to display and/or encapsulate antigens to serve as innovative antigen carrier systems for the induction of desirable immunities. Polyhydroxyalkanoates (PHAs) are naturally occurring polyesters synthesized as cytoplasmic polyester inclusions (polyester particles) by various bacteria. The particles have been used as an antigen delivery platform by translationally fusing antigens to the particle surface-associated protein, PHA synthase. Furthermore, it has been found that protein inclusion bodies contain a large amount of correctly folded and biologically active proteins and could be engineered to perform as an antigen carrier system. Tuberculosis (TB) is a global health issue for both humans and animals. Inaccurate diagnosis and inefficacious vaccination make TB control problematic. The Mantoux tuberculin skin test gives false positive results if humans or animals are vaccinated with the Bacille Calmette-Guérin (BCG) strain or exposed to environmental mycobacteria. BCG cannot provide effective protection against TB. Subunit vaccines have great promise to protect against infectious diseases, but they are often weak immunogenically. A strategy to circumvent this problem is the use of self-assembly particulate vaccines, which could present multiple copies of antigens and serve as a depot for prolonged multivalent antigen display to induce enhanced immunogenicity. In this thesis, four specific TB diagnostic antigens — CFP10, Rv3615c, ESAT6, and Rv3020c — were displayed on polyester particles. The results showed that polyester particles displaying TB antigens specifically distinguished TB-infected from non-infected cattle. Antigen immunogenicity was dramatically enhanced after the display on polyester particles, which lowered the antigen concentration (0.1 to 3 μg dose/inoculum) required for skin tests. Mycobacterial vaccines H4 (Ag85B-TB10.4) or H28 (Ag85B-TB10.4-Rv2660c) were bioengineered to display H4/H28 on polyester particles and/or self-assemble H4/H28 into protein inclusion bodies. The results demonstrated that polyester particle-/protein inclusion body-based particulate TB vaccines increased overall immunogenicity by enhancing humoral (for example, IgG1 and IgG2c) and cellular (for example, IFNγ and IL17A) immune responses when compared to respective soluble antigens.
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    Novel polyhydroxyalkanoate bead-based vaccines against Pseudomonas aeruginosa infection : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Gonzaga, Zennia Jean
    Pseudomonas aeruginosa infections are increasingly problematic due to their multiple antibiotic resistances. To date, there is no commercial vaccine against P. aeruginosa infection. This study used polyhydroxyalkanoate (PHA) beads as a delivery platform for selected P. aeruginosa antigens to produce novel particulate vaccines against P. aeruginosa. Genetic engineering was used to modify the PHA synthase (PhaC), the enzyme that catalyses PHA bead formation, to produce functionalised PHA beads displaying the antigens. The highly conserved PopB antigen, recently revealed as an effective stimulator of Th17 immunity was displayed on the bead surface together with and without the previously selected antigens Ag (epitopes derived from outer membrane proteins OprI, OprF and AlgE). The PHA beads were produced in two production strains: (1) the pathogen P. aeruginosa itself, with the benefit of co-purifying host cell proteins (HCPs) expanding the antigen repertoire that could boost the immune response, and (2) E. coli strain ClearColiTM, a defined mutant incapable of lipopolysaccharide synthesis enabling production of endotoxin free PHA beads. Vaccination of mice with antigen-coated PHA beads (only PopB or with additional Ag) showed increased production of IL-17, a reflection of induction of a Th17 immunity. Furthermore, Th1 and Th2 mediated immunity were detected from the IgG analysis of the immunised mice with the PHA bead vaccines. Significant enhancement of Th1 immune response using the PHA bead platform was observed compared to the antigen-only counterparts. Challenge of immunised mice with pathogenic P. aeruginosa showed that PopB-displaying PHA beads from P. aeruginosa and Ag-displaying PHA beads from E. coli induced partially protective immunity. The promising PHA bead candidate vaccines can be conjugated with other antigens such as the exopolysaccharide Psl for induction of improved immune response towards protective immunity. Exopolysaccharide Psl is a mannose rich polymer produced by P. aeruginosa in both mucoid and nonmucoid phenotypes. Psl has been revealed to play an essential role in P. aeruginosa pathogenicity such as biofilm formation. Previous studies had provided evidence that CdrA binds directly to Psl, functioning as a Psl cross-linker and possibly tethering Psl to the cell surface. Psl is commercially not available, hence the aim of this study was to develop a Psl production strain. This study created a cdrA knockout mutant that produces free Psl which may be a potential vaccine antigen against P. aeruginosa infections. An isogenic knockout of cdrA was obtained by homologous recombination and this mutant overproduced Psl released from the cell surface. In future studies, this strain will be used to produce Psl to serve as antigen in vaccine formulations.
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    Study of an exported protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2004) Copland, Susan Maree
    Johne's disease is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (M. ptb) from the MAIS complex (M. avium, M. ptb, M. intracellulare and M. scrofulaceum). The lack of specific and sensitive diagnostic tests often leads to M. ptb infected animals being diagnosed with bovine tuberculosis, a member of the MTB complex (M. tb, M. bovis, M. bovis BCG, M africanum, M. microti and M. canetti). Secreted proteins from pathogenic mycobacteria have been found to be important for the development of protective immunity, namely a cell mediated immune response (CMI). The development of reliable differential diagnostic tests will require the use of species-specific secreted protein antigens and the CMI response. Due to the taxonomic distance between the MAIS and MTB complexes our hypothesis was that the M. ptb genome may encode for secreted proteins that are absent from members of the MTB complex. If such proteins can stimulate an immune response they may be suitable for use as antigens in a differential diagnostic test for Johne's disease. To this end, the secreted protein library clone pJEMIl-M ptb281 was examined and its insert found to contain the 5' region of the hypothetical M.ptb281 ORF fused in frame with phoA. The entire ORF was determined using M. avium and M. ptb database sequences then cloned into E. coli and mycobacterial expression systems. These systems incorporate 6x histidine (His6) affinity tags into recombinant proteins allowing them to be semi-purified by Ni-NTA affinity chromatography. Semi-purified recombinant proteins tested positive by western blot analysis to highly specific anti-His6-tag antibodies. Amino acid sequencing to confirm the identity of these recombinant proteins and screening for their ability to stimulate an immune response were prevented by time constraints. Homologs to M. ptb281 were absent from M. tb, M. bovis and M. bovis BCG but present in the MAIS complex, making this protein unsuitable for use as an antigen to differentiate between MAIS complex species in a diagnostic test. M. ptb281 homologs found in the genomes of two members of the Acetomycetes order corresponded to hypothetical proteins predicted by computer software programs trained to identify genes, which may indicate that the hypothetical M. ptb281 ORF may encode a functional protein.
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    B lymphocyte activities in the opossum, Trichosurus vulpecula : a thesis presented in partial fulfilment of the requirement for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1980) Ramadass, Pachaikani
    The evolution of vertebrate immunity from the level of the protochordates to that of the metatherians is reviewed. Using standard methods IgG, IgM and IgA were isolated from the serum or intestinal fluid of the Australian brush-tailed opossum, Trichosurus vulpecuia. These were characterized in terms of their molecular weights, amino acid and carbohydrate compositions and values for their concentrations in serum were calculated. Two forms of IgG were seen which differed in their abilities to bind to insoluble matrices and also in their molecular weights. No antigenic differences were seen between them on analysis by agar diffusion . The molecular weight of the IgA seen in intestinal fluid and results from its analysis by agar diffusion suggest that the molecule may lack secretory component. B lymphocytes were identified by their surface immunoglobulin and their complement and Fc receptors. The number of these cells in blood and various lymphoid tissues of T.vulpecula was found to be similar to the values reported for mice and humans. Lymphocyte fractionation on nylon wool columns confirmed that the markers employed were associated with an adherent cell population. Blood lymphocytes were stimulated in vitro with a range of mitogens and the degree of transformation achieved with each was assessed by the cells uptake of tritiated thymidine. Insoluble concanavalin A, pokeweed mitogen and lipopolysaccharide, in that order, were the most effective of the mitogens used on unfractionated blood lymphocytes. These three mitogens were further used in studies in which nylon wool fractionation of blood lymphocytes was used to prepare B cell- and T cell-enriched cultures. Lipopolysaccharide was the only mitogen to stimulate B cells more than T cells. Insoluble concanavalin A consistently stimulated T cells to a greater extent than B cells as did pokeweed mitogen. The ultrastructure of mitogen-stimulated cells was studied by electron microscopy and it was shown that lipopolysaccharide induced the formation of plasmablasts which resembled those of eutherians. Mitogen-stimulated cells were also analysed for their production of immunoglobulins, the levels of de novo synthesised materials being measured by their incorporation of isotope-labelled leucine provided in the culture medium. Both secreted and intracellular proteins were measured in this way. Lipopolysaccharide, pokeweed mitogen and insoluble concanavalin A all induced significantly increased levels of 19S and 7S secreted proteins, these proteins being separated by gel filtration. Pokeweed mitogen induced the synthesis of significantly increased levels of both 19S and 7S intracellular proteins, while lipopolysaccharide and insoluble concanavalin A significantly increased the levels of 19S protein only. The presence of IgM and IgG in the 19S and 7S fractions was shown by their precipitation with class-specific antisera. The immune responses of T.vulpecula to a particulate and a soluble antigen were compared with those of rabbits to the same antigens. Sheep erythrocytes, at two dose levels, were injected intravenously. The responses of opossums to 5x109 erythrocytes were appreciably more rapid than those of the rabbits. The responses of the two species to 25x109 erythrocytes were similar in the titres attained and the time taken to do so. The distribution of haemagglutinating activity between IgM and IgG was studied and found to be essentially the same for both species for both levels of antigen. The responses of opossums to bovine serum albumin injected intramuscularly with Freund's adjuvants were similar to those of rabbits. It is concluded that the B cell-dependent immune functions of T.vulpecula are as efficient as those of other metatherians and compare favourably with those of eutherians.
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    Development and use of polyhydroxybutyrate biopolyester as particulate vaccine beads : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand
    (Massey University, 2012) Parlane, Natalie Anne
    3-hydroxybutyric acid) (PHB) is the most commonly produced polyhydroxyalkanoate formed naturally inside many genera of bacteria and archaea when nutrients are limited and a carbon-source is available in excess. These water-insoluble biopolyester spherical beads in the size range of 20-800 nm can be recombinantly produced by insertion of the required PHB biosynthesis genes into alternative bacterial hosts and then culturing the organisms under suitable conditions. A gene fusion can also be made to enable production of PHB beads which display the selected proteins abundantly at the surface of the bead. Vaccines are needed which stimulate cell-mediated immunity and are effective at reducing intracellular infections such as tuberculosis, neosporosis and many viral infections. These diseases are responsible for a huge burden to human and animal health. Particulate vaccines target antigen presenting cells and cellular immune responses to protein antigens are enhanced when particulate vaccines are used. This thesis describes the development of a novel vaccine delivery system in which PHB beads were engineered to display vaccine antigen on the surface of the beads. Investigations were made into the process of vaccine bead design, production and validation to enable their use in vaccine trials. PHB synthesis genes from Cupriavidus necator were inserted into production strains to enable production of PHB. Escherichia coli was initially used as a bacterial production host and then Lactococcus lactis was introduced as an alternative, due to its lack of lipopolysaccharide, previous use as a production host for recombinant proteins and history of safe use for a range of human foods and products. To expand the repertoire of PHB vaccine beads, different vaccine antigens were used: hepatitis C core antigen and mycobacterial antigens (antigen-85A and 6 kDA early secretory antigenic target). Antigen specific cellular immune responses were produced in mice vaccinated with PHB vaccine beads and protection against tuberculosis was observed in mice immunized with these vaccines. Preliminary studies into the mechanism of uptake of PHB beads by dendritic cells (DCs) showed PHB beads were taken up readily by DCs, with maturation of DCs and subsequent secretion of interleukin-12.
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    A genetic approach to identify Mycobacterium bovis exported protein antigens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology, Massey University
    (Massey University, 1997) Borich, Suzanne Marie
    A novel approach, combining phoA-fusion technology with T cell screening of a recombinant cosmid library, was used to detect Mycobacterium bovis exported T cell antigens. An M. bovis BCG library of phoA-fusions was constructed in Escherichia coli and Mycobacterium smegmatis using the plasmid vector pJEM11. The M. bovis BCG DNA inserts from ten PhoA+ clones were partially sequenced and used to search databases for similarities to known genes. These revealed similarities to a family of genes coding for high temperature-requirement serine proteases and a Mycobacterium leprae putative exported lipoprotein gene (pel). The DNA inserts from PhoA+ clones were used to probe an M. bovis cosmid library expressed in M. smegmatis 10 identify cosmids containing the full-length genes coding for these exported proteins. Culture filtrates (CFs) prepared from selected M. smegmatis recombinants (cosmids) were assayed for their ability to induce proliferation and IFN-γ-production from peripheral blood mononuclear cells (PBMCs) taken from M. bovis BCG-immunised and non-immunised control cattle. Culture filtrates from two recombinant M. smegmatis (cosmids 44 and 56) induced significant IFN-γ-production and proliferation by PBMCs from immunised animals. An exported protein gene, identified using the phoA-fusion technology, was subcloned from cosmid 56 and its sequence determined and analysed. Database searches using the deduced amino acid sequence of this gene revealed similarities to an M. leprae putative exported lipoprotein (Pel) and a family of MalE maltose-binding proteins. The M. bovis pel gene was shown to be expressed by recombinant M. smegmatis. Preliminary evidence from this study indicates that the M. bovis Pel protein is recognised by antigen-specific lymphocytes from M. bovis BCG-immunised animals. The PBMCs taken from M. bovis challenged and M. bovis BCG vaccinated / challenged cattle also recognised CF from recombinant M. smegmatis expressing the pel gene in in vitro immunoassays. The combined strategy of using phoA-gene fusions and T cell screening of CFs from a recombinant M. bovis cosmid library proved a sensitive and rapid method for the detection of potential M. bovis T cell antigens.
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    Identification and characterisation of an exported immunogenic protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Dupont, Christine
    Exported proteins of mycobacteria are available to interact with the immune system at an early stage of infection and are potent inducers of immune responses. Potentially exported proteins of Mycobacterium avium subspecies paratuberculosis were identified using alkaline phosphatase gene fusion technology. A library of partial gene fusions from a New Zealand clinical isolate of M. a. paratuberculosis was constructed in the shuttle vector pJEM11 and expressed in the surrogate hosts E. coli and M. smegmatis. The DNA inserts from a portion of the resulting clones expressing alkaline phosphatase-positive fusion proteins were partially sequenced to identify the proteins. Eleven proteins not previously described for M. a. paratuberculosis were identified as containing signal sequences for export. One of these, a putative lipoprotein named P22 was selected for further study. The full nucleic acid sequence of the p22 gene was determined and the open reading frame was cloned into die mycobacterial expression vector pMIP12. This enabled P22 to be produced as a polyhistidine-tagged protein in M. smegmatis and facilitated purification by chromatography. N-terminal sequencing of the recombinant protein confirmed cleavage of an N-terminal signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. a. paratuberculosis strain 316F using rabbit antibody raised to P22. Investigation of the presence of genes similar to p22 in other mycobacterial species, revealed p22 was present in Mycobacterium avium subspecies avium and similar genes existed in M. intracellularae (88.5% identity) and M. scrofulaceum (87.7% identity). Database searches showed P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in M. leprae and in members of the M. tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit significantly increased interferon-gamma secretion in blood from a group of eight sheep vaccinated with a live, attenuated strain of M. a. paratuberculosis (strain 316F) compared to a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.