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

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

Mechanisms of C-H Bond Activation by Platinum(II)

The rates of C–H bond activation for various alkanes by [(N–N)PtII(CH₃)(TFE-d₃)]⁺ (N–N = Ar–N=C(CH₃)–C(CH₃)=N–Ar; Ar = 3,5-di-tert-butylphenyl; TFE-d₃ = CF₃CD₂OD) were studied. Both linear and cyclic alkanes give the corresponding [(N–N)PtII(H)(alkene)]⁺ cation. Second-order rate constants for cycloalkane activation (CnH2n) are proportional to the size of the ring (k ~ n). For cyclohexane, the deuterium kinetic isotope effect (kH/kD) of 1.28(5) is consistent with the proposed rate determining alkane coordination to form a C–H σ complex. Comparing the relative rates of cyclic and linear alkanes indicates that the platinum center is relatively unselective with respect to different C–H bonds: the rate constants (per C–H bond) for the substrates examined all fall into a narrow range, and there does not appear to be any significant preference for either primary or secondary C–H bonds. The protonolysis of platinum(II) methyl complexes was investigated by both experiment and computation. Experimental results showed that protonolysis of (COD)PtII(CH₃)₂ (COD = 1,5 – cyclooctadiene) by trifluoroacetic acid gave abnormally large (greater than 10) kinetic isotope effects (kH/kD) at room temperature and higher. The temperature dependence of kH/kD for the protonolysis of (COD)PtII(CH₃)₂ gave Arrhenius parameters outside semi-classical limits. On the other hand, protonolysis of (tmeda)PtII(CH₃)Cl (tmeda = N,N,N’,N’–tetramethyl–ethylenediamine) by trifluoroacetic acid gave normal kinetic isotope effects and classical Arrhenius parameters. Density functional theory (DFT) was used to examine the mechanism of protonolysis of these two systems, and the results were found to be consistent with experimental observations. Based on our experimental and computational work, we propose that protonolysis of methylplatinium(II) complexes can occur through either a concerted or stepwise pathway that is highly ligand dependent; more electron-rich ligands will favor the stepwise mechanism, while electron-deficient ligands with stronger trans influence will favor the concerted mechanism. Finally, we propose that the presence of abnormally large KIEs is an indication for a concerted pathway, and that there is a connection between the magnitude and temperature dependence of the KIE and mechanism. In aqueous solution, [PtII(glycinato)Cl₂]⁻ catalyzes oxidation by [PtIVCl₆]²⁻ of the methyl group of p-toluenesulfonate to the corresponding alcohol and aldehyde, with no further oxidation to the carboxylic acid. Both rate and selectivity are improved in comparison to the original Shilov system that employs [PtIICln(H₂O)4-n]2-n as the catalyst.</p

Display of antigens on polyester inclusions lowers the antigen concentration required for a bovine tuberculosis skin test

The tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target (ESAT6), 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c, or a peptide cocktail derived from those proteins, in the skin test greatly enhanced test specificity, with minimal loss of test sensitivity. The proteins are present in members of the pathogenic Mycobacterium tuberculosis complex but are absent in or not expressed by the majority of environmental mycobacteria and the BCG vaccine strain. To produce a low-cost skin test reagent, the proteins were displayed at high density on polyester beads through translational fusion to a polyhydroxyalkanoate synthase that mediates the formation of antigen-displaying inclusions in recombinant Escherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins (0.1 to 3 μg of mycobacterial protein/inoculum) in the skin test. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have high sensitivity for detection of M. bovis-infected cattle and induced minimal responses in animals exposed to environmental mycobacteria or vaccinated with BCG.Full Tex

Functional protein display on the surface of biobeads produced by recombinant Escherichia coli : 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

Polyhydroxyalkanoic acids (PHAs) are biopolyesters produced by various bacteria. They are deposited as spherical water-insoluble cytoplasmic inclusions (beads) containing an amorphous hydrophobic polyester core and surrounded by a phospholipid monolayer and embedded proteins, including PHA synthase (PhaC), the key enzyme required for PHA bead formation. Although inactive PhaC cannot produce PHA beads, fusing inactive PhaC to green fluorescent protein (GFP) leads to GFP protein bead formation. Both PHA and protein beads could serve as a versatile platform for display of desired proteins suitable for various biotechnological and medical applications. The tuberculin skin test (TST) for diagnosing bovine tuberculosis (TB) in cattle uses the purified protein derivative (PPD) that is prepared from Mycobacterium bovis. However, some antigens in the PPD are also present in environmental mycobacteria. Therefore, the TST lacks specificity if animals are exposed to non-pathogenic environmental mycobacteria. In this study, three specific TB antigens, CFP10, ESAT6, and Rv3615c — which are present in pathogenic but absent in most non-pathogenic mycobacteria — were displayed on the surface of PHA beads. The results demonstrated that these triple antigen-displaying PHA beads can differentiate TB-infected from non-infected cattle, making this an attractive alternative to current skin test diagnostic reagents. IgG binding domains displayed on GFP protein beads have a higher IgG binding ability when compared to their counterpart displayed on PHA beads. However, it is unclear whether an enhancement of IgG binding ability due to GFP protein beads could be achieved by immobilization on other fluorescent protein (FP) beads. The results showed that other FP (including yellow, red and cyan) beads displaying IgG binding domains have an approximately 1.5–2 fold greater IgG binding ability when compared to PHA beads displaying the same binding domains. To investigate whether protein beads displaying iron-binding peptides could be magnetized while maintaining IgG binding function, an iron binding peptide was displayed. The results demonstrated that protein beads displaying both IgG and iron binding peptides can be magnetised by iron oxide and retain a strong IgG binding ability. Finally, this study revealed that different cell disruption techniques could affect the morphology and functionality of FP protein bead

Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

Abstract Background Ovarian retinoid homeostasis plays an important role in the physiological function of the ovary. Retinol-binding protein 4 (RBP4) acts as the mediator for the systemic and intercellular transport of retinol and is heavily involved in cellular retinol influx, efflux, and exchange. However, the expression patterns and regulatory mechanisms of Rbp4 in the ovary remain unclear. Methods The expression pattern of ovarian Rbp4 was examined in immature mice during different developmental stages and in adult mice during different stages of the estrous cycle. The potential regulation and mechanisms of ovarian Rbp4 expression by estrogen and related gonadotropins in mouse ovaries were also investigated. Results The present study demonstrated that the ovarian expression of Rbp4 remained constant before puberty and increased significantly in the peripubertal period. In adult female mice, the expression of Rbp4 increased at proestrus and peaked at estrus at both the mRNA and protein levels. The protein distribution of RBP4 was mainly localized in the granulosa cell and theca cell layer in follicles. In addition, the expression of Rbp4 was significantly induced by follicle-stimulating hormone (FSH) or FSH + luteinizing hormone (LH) in combination in immature mouse (3 weeks old) ovaries in vivo and in granulosa cells cultured in vitro, both at the mRNA and protein levels. In contrast, treatment with LH or 17β-estradiol did not exhibit any observable effects on ovarian Rbp4 expression. Transcription factors high-mobility group AT-hook 1 (HMGA1), steroidogenic factor 1 (SF-1), and liver receptor homolog 1 (LRH-1) (which have been previously shown to be involved in activation of Rbp4 transcription), also responded to FSH stimulation. In addition, H-89, an inhibitor of protein kinase A (PKA), and the depletion of HMGA1, SF-1, and LRH-1 by small interfering RNAs (siRNAs), resulted in a dramatic loss of the induction of Rbp4 expression by FSH at both the mRNA and protein levels. Conclusions These data indicate that the dynamic expression of Rbp4 is mainly regulated by FSH through the cAMP-PKA pathway, involving transcriptional factors HMGA1, SF-1, and LRH-1, in the mouse ovary during different stages of development and the estrous cycle

Enzyme Engineering for In Situ Immobilization

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes

Bioengineering toward direct production of immobilized enzymes: A paradigm shift in biocatalyst design

The need for cost-effectively produced and improved biocatalysts for industrial, pharmaceutical and environmental processes is steadily increasing. While enzyme properties themselves can be improved via protein engineering, immobilization by attachment to carrier materials remains a critical step for stabilization and process implementation. A new emerging immobilization approach, the in situ immobilization, enables simultaneous production of highly active enzymes and carrier materials using bioengineering/synthetic biology of microbial cells. In situ enzyme immobilization holds the promise of cost-effective production of highly functional immobilized biocatalysts for uses such as in bioremediation, drug synthesis, bioenergy and food processing

Meta-analysis of clinical adverse events after CABG vs. PCI in patients with chronic kidney disease and coronary artery disease

Abstract Aim To investigate the efficacy and postoperative clinical adverse events of coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI) for chronic kidney disease (CKD) study participants combined with coronary artery disease (CAD). Methods All randomized controlled trials (RCTs) that focus on the therapeutic effect evaluation of CABG and PCI and their effect on postoperative clinical adverse events as well as main adverse cardiovascular and cerebrovascular events (MACCEs) in CKD study participants with CAD were screened from the following databases, including CNKI, CBM, Wan Fang, VIP, Embase, PubMed, as well as Cochrane library clinical controlled trials. The study was conducted under the PRISMA 2020 criteria. Data were extracted, and quality control was evaluated from the modified Jadad rating scale. Meta-analysis was then undertaken through STATA 16.0 software. Results A total of 5 RCTs were obtained, including 1198 patients. Study participants were subdivided into two groups, including the PCI group (n = 604) and the CABG group (n = 594). Meta-analysis of clinical adverse events results showed that the long-term survival results of CAD patients with CKD who underwent PCI were worsened compared to CABG, such as long-term MACCEs (RR = 1.59, 95%CI: 1.04–2.43) and the long-term repeated revascularization (RR = 2.48, 95%CI: 1.76–3.49). Also, cardiac death (RR = 1.68, 95%CI:1.04–2.71), as well as cerebrovascular accident (RR = 1.74, 95%CI:1.04–2.90) in CABG group was significantly lower than that in PCI group. Conclusion This meta-analysis showed that CABG provided a better therapeutic effect than PCI in CKD patients with CAD when considering long-term prognosis. However, more prospective RCTs are needed to define the proper revascularization strategy for CAD patients with CKD

Removal of phosphate from aqueous solution by sewage sludge-based activated carbon loaded with pyrolusite

Activated carbons were prepared from sewage sludge by chemical activation with pyrolusite (PAC) to develop an efficient adsorbent for phosphate removal from aqueous solution. One percent (wt.) pyrolusite addition was proved to have an important effect on pore formation of the produced carbon. PAC showed 17.06% larger Brunauer–Emmett–Teller (BET) surface area than the sewage sludge-based activated carbon without modification (SAC). The adsorption results showed that the phosphate removal by PAC was 13% higher than SAC's. The adsorption experiments also showed that PAC had very good performance with high phosphate removal rate (ca. 90%) in a wide pH range (pH = 4–8), and could be stable after 30 min reaction. Adsorption isotherm and kinetics studies demonstrated that phosphate adsorption onto the modified adsorbent was well fitted by the Langmuir isotherm and could be described by the pseudo-second-order kinetic model. The modified sewage sludge-based activated carbons were effective and alternative adsorbents for the removal of phosphorus from aqueous solution due to their considerable adsorptive capacities and the low-cost renewable sources

Protective Effects of Astragaloside IV against LPS-Induced Endometritis in Mice through Inhibiting Activation of the NF-κB, p38 and JNK Signaling Pathways

Endometritis, inflammation of the endometrium, is a common reproductive obstacle disease that can lead to infertility in female animals. Astragaloside IV (AS IV), one of the major and active components of the Astragalus membranaceus (Fisch.) Bunge, is known for its anti-inflammatory effects. In the present study, the effects and mechanisms of AS IV on lipopolysaccharide (LPS)-induced endometritis were investigated using a mouse model. Female mice were prepared with AS IV (0.01 mg/g) by gavage for six days before being stimulated with LPS. The results showed that the histopathological changes, levels of inflammatory cytokines (IL-1&#946; and TNF-&#945;), concentration of NO, and myeloperoxidase (MPO) activity in LPS-induced uteri were attenuated significantly by pretreatment with AS IV. Furthermore, LPS-induced activations of NF-&#954;B, p38, and JNK signal pathways were suppressed by pretreatment with AS IV. In conclusion, the data provided new evidence that AS IV effectively attenuates LPS-induced endometritis through inhibition of TLR4-mediated NF-&#954;B, p38, and JNK signaling pathways, implying that AS IV might become a promising potential anti-inflammatory agent for endometritis and other inflammatory diseases