Immobilizing antimicrobial substances onto biocompatible materials is an important approach for the design of novel, functionalized medical devices. By choosing antimicrobial substances from innate immune systems, the risk for development of resistance in pathogenic microbes is lower than if conventional antibiotics are used. Combining natural antimicrobial peptides and bactericidal enzymes with strong and elastic spider silk through recombinant protein technology would enable large-scale production of materials that could serve as functionalized wound dressings. Herein, fusion proteins with the engineered spider silk sequence 4RepCT and five different antimicrobial substances were constructed using two different strategies. In the first, the fusion proteins had a His-tag as well as a solubility-enhancing domain N-terminally to the antimicrobial agent during expression. The tags were cleaved and separated from the target protein during the purification process. The other approach provided a His-tag but no additional solubility domain. The antimicrobial agents included in the work were a charge engineered enzyme and four antimicrobial peptides herein called Peptide A, Peptide B, Peptide C and Peptide D. Four out of five fusion proteins could be expressed in Escherichia coli without exhibiting noticeable toxicity to the host. However, most target proteins were found in the non-soluble fraction. For D-4RepCT, neither soluble nor non-soluble proteins were identified. An operating strategy for expression and purification of antimicrobial spider silk proteins was developed, where the construct system providing the solubility-enhancing domain N-terminally to the antimicrobial sequence, and long time expression at low temperatures is a promising approach. The fusion proteins A-4RepCT and C-4RepCT could be produced in adequate amounts, and they proved to possess the ability to assemble into stable fibers. When incubating solutions of Escherichia coli on the functionalized silk material A-4RepCT, it showed to decrease the number of living bacteria in solution, in contrary to wild-type 4RepCT on which bacteria continued to proliferate. Initial studies of the viability of bacteria adhered to the surface of the functionalized spider silk are so far inconclusive. A larger sample size, complementary experiments and methodology optimization is needed for a proper assessment of antibacterial properties. However, preliminary results for the development of antimicrobial spider silk are positive, and the approach elaborated in this work is believed to be applicable for the construction of functional spider silk with a wide range of natural antimicrobial agents for future wound healing applications.

No description available.

Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections. / Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.

Previously seed coat color in flax has been used as a phenotypic marker for specialty quality traits and currently there is an increasing demand to use seed coat color in flax to market flax for human and animal nutrition uses. Seed coat color was studied to 1) understand the inheritance of seed coat color conditioned by the b1 locus, to 2) understand the relationship of other important flax traits with seed coat color as well as to 3) identify markers that are linked to seed coat color for future marker assisted selection of seed coat color. Spearman’s rank correlation and an allelism test was used to show the inheritance of the alleles at the b1 locus. Bulked segregant analysis (BSA) was used to identify putatively linked markers with the b1 locus, these were then screened on the CDC Bethune x M96006 recombinant inbred line population. Furthermore, the CDC Bethune x M96006 and CDC Bethune x USDA-ARS Crystal recombinant inbred line populations were used to identify any important flax traits that had a significant relationship with seed coat color. It was shown that seed coat color conditioned by the b1 locus was stably inherited and that b1vg and b1 are allelic to one another. The results of the BSA showed that there were 17 candidates for linkage but when these markers were screened on the population only the Lu456 from linkage group (LG) six was identified to have linkage (χ²=3.90; P<0.05) with the b1 locus. Additionally, it was shown that the b1 seed coat color allele of the b1 locus had a pleiotropic effect on flower color and flower shape and that seed coat color was associated with linolenic fatty acid content. None of the traits examined were found to be associated with the b1vg allele of this locus. These results show that the b1 locus is likely present on linkage group six, more marker coverage on linkage group six of markers that are polymorphic between the two seed coat color parents would increase the accuracy of detection. Lastly, this study showed that plant breeders should consider using the b1vg allele that conditions the variegated seed coat color to mark unique lines with important combinations of traits because it sorted independently for seed quality traits. Whereas, the yellow seed coat color conditioned by the b1 allele was found to be associated with higher linolenic fatty acid content and the semi-lethality of this allele would make it not suitable for use in parental lines.

All challenges associated with influenza A viruses including antigenic variation in hemagglutinin (HA) and neuraminidase (NA), the evolving drug resistance and the drawbacks of current vaccines hinder our ability to control this constant threat. Furthermore, gene reassortment as well as the direct transmission of highly pathogenic avian viruses to humans can result in an occasional emergence of novel influenza strains with devastating pandemic potential. Therefore, it is crucial to investigate alternative approaches to better control these viruses and to develop new prophylactic and treatment options. Targeting highly conserved epitopes or antigens among the different subtypes of influenza A virus could offer protection against broad range of influenza viruses, including emerging strains. In my research, I have investigated the potential of broadly neutralizing antibodies against HA and conducted mechanistic study of a prototype vaccine based on the highly conserved nucleoprotein (NP). We recently found that the 14 amino acids of the amino-terminus of the fusion peptide of influenza HA2 subunit is the only universally conserved sequence in all HA subtypes of influenza A and the two lineages of influenza B viruses. Here, I show that universal antibodies targeting this linear sequence in the viral HA (Uni-1 antibodies) can cross-neutralize multiple subtypes of influenza A virus by inhibiting the pH-dependant fusion of viral and cellular membranes. It is noted that the influenza NP is a highly conserved antigen and has the potential to induce heterosubtypic immunity against divergent subtypes of influenza A virus. However, NP-based vaccination only affords weak protective immunity compared to HA. This is mostly due to the non-sterilizing immunity induced by NP. Using CD40 ligand (CD40L), a key regulator of the immune system, as both a targeting ligand and a molecular adjuvant, I show that single immunization with recombinant adenovirus carrying a fused gene encoding the secreted NP-CD40L fusion protein provided robust and long-lasting protection against influenza in normal mice. It enhanced both B-cell and T-cell responses and augmented the role of both NP-specific antibodies and CTLs in protection. Importantly, it afforded effective protection in CD40L and CD4 deficient mice, confirming that the induced protection is CD40L-mediated and CD4+ T cell-independent. The rapid evolution of the influenza A viruses necessitates the development of new alternatives to contain this medically important pathogen. The results of these studies could significantly contribute to future vaccine development and avert the necessity of yearly vaccine updates.

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.

Aeromonas hydrophila is an opportunistic pathogen that causes a wide range of symptoms and diseases in fish. Development of a commercial vaccine has been problematic due to the heterogenicity between isolates of A. hydrophila. A new approach using immunoproteomics was used in this study to try to develop a vaccine that would protect against a wide range of A. hydrophila strains. The virulence of 14 isolates of A. hydrophila from different geographical regions was determined in common carp (Cyprinus carpio) indicating that 6 isolates were virulent, while 8 isolates were avirulent. Expression of cellular and extracellular products (ECP) of six of these isolates (4 virulent and 2 avirulent isolates) were examined following culture of the bacterium in vitro, in tryptic soy broth, and in vivo, in dialysis tubing placed within the peritoneal cavity of carp. Two types of molecular weight cut off tubes (25 and 100 kDa) were used for the implants. Whole cell (WC), outer membrane protein (OMP) and ECPs of the bacteria grown in vitro and in vivo were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations of A. hydrophila grown in vitro and in vivo. The production of unique proteins and up and down-regulation of protein expression were observed in all the preparations of bacteria grown in vitro and in vivo. Unique bands were seen in the 1D SDS-PAGE at 58 and 55 kDa for WC and OMP preparations, respectively, for all the isolates cultured in vivo. Bands of increased intensity were observed at 70, 55, 50 and 25 kDa with WC preparations for the virulent isolates cultured in vivo. Analysis of WC preparations by 2D SDS-PAGE indicated differences in the expression of spots between bacteria cultured in vitro and in vivo. A number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0-6.0 were observed in the bacteria grown in vivo. The protein profiles of different preparations (WC, OMP, ECP) of bacteria cultured in vitro and in vivo were screened by 1D Western blot using antibodies from carp artificially infected with different isolates of A. hydrophila to identify potential vaccine candidates. The WC preparations of A. hydrophila (T4 isolate) grown in vitro were also analysed by 2D Western blot. A 50 kDa protein of A. hydrophila was found to be the most immunogenic molecule in both WC and OMP of bacteria grown both in vitro and in vivo. The protection efficacy of this protein was determined in goldfish by vaccinating fish with electro-eluted 50 kDa protein then challenging the fish with A. hydrophila. Fish were also passively immunised with fish sera raised to the 50 kDa protein and then challenged. The relative percentage survival (RPS) was 67 % in the vaccination trial, while the results were inconclusive for the passive immunisation trial. The 50 kDa protein was confirmed to be the S-layer protein of A. hydrophila following identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Recombinant S-layer protein was then produced and the cross-protection efficacy of this protein against six virulent isolates of A. hydrophila was confirmed in a large scale vaccination trial using carp. The RPS value for the 6 isolates of A. hydrophila ranged from between 56 and 87 %. The results of this project suggest that the immunogenic S-layer protein of A. hydrophila could be used as a common antigen to protect fish against infection by different isolates of this pathogenic bacterium.

The highly pathogenic Avian Influenza subtype H5N1 can lead to 100 % mortality in chickens. The main issue in prevention of H5N1 is the development of efficient poultry vaccines. Influenza haemagglutinin (HA) derived recombinant polypeptides would not elicit an immune response against internal viral proteins. Thus HA polypeptide use facilitates differentiation between infected and vaccinated animals (DIVA). Serological tests using recombinant immune-dominant proteins devoid of non-specific moieties present in whole cell preparations might have higher sensitivity and specificity. In the present study, four non-overlapping sequences of different functional domains of influenza A virus subtype H5 virus (A / Thailand / 1 (Kan-1) / 2004) designated P1, P2, P5 and rHA1 were cloned and expressed in Pichia pastoris for vaccination and diagnosis purposes. The four polypeptides were expressed successfully in P. pastoris using peptone methanol (1 % (w/v) yeast extract, 2 % (w/v) peptone, 2 % (v/v) methanol). P1, P2 and rHA1 polypeptides were purified using nickel affinity chromatography, whereas, P5 was purified using lectin affinity chromatography. Correct expression was analysed by SDS-PAGE and western blot, glycosylation analysis and MALDI-TOF.The immune responses of P1, P2 and rHA1 polypeptides were assessed in BALB/C mice. To enhance antibody response, recombinant polypeptides were mixed with the Gerbu adjuvant and injected subcutaneously. Vaccination of mice induced high subtype specific antibody titres in mice as analysed by Elisa (using recombinant antigens or whole H5N1 antigen) and Immunofluorescence assay (IFA) performed on Vero cells infected with H5 (A / Thailand / 1 (Kan-1) / 2004). The immunogenicity of P1, P2, P5 and rHA1 polypeptides was determined in commercial layer chickens. Results showed that P1, P2 and rHA1 polypeptides induced high subtype specific antibody titres in chickens as analysed by Elisa (using recombinant antigens or whole H5N1 antigen), IFA (performed on Vero cells infected with H5N1 A / Thailand / 1 (Kan-1) / 2004) and microneutralization test (µNT). However, P5 polypeptide was not immunogenic in chickens. Neutralizing antibodies could be detected in chicken sera immunized with P1, P2 and rHA1 polypeptides as analyzed with microneutralization test. IgY was analysed in egg yolk of chickens immunized with recombinant polypeptides. The IgY of chicken immunized with P1 and rHA1, transferred to the egg yolk was proportional to maternal serum IgY. However, IgY could not be detected in egg yolk of chickens immunized with P2 and P5 recombinant polypeptides. The more immunogenic polypeptides P1 and rHA1 were used in an recombinant Elisa (rElisa) for detection of influenza A subtype H5 in chickens and duck sera.The optimal antigen for the concentrations of rHA1, P1 was 50 ng / well, 50 ng / well. Analysis of 25 positive sera and 25 negative sera to H5 antibodies revealed that, the sensitivity of Western blot, whole H5N1 Elisa, agar gel immunodiffusion test (AGID), P1-Elisa and rHA1-Elisa was 100 %, 100 %, 52 %, 80 % and 100 %, respectively, while the specificity was 100 %, 100 %, 100 %, 72 %, and 100 %, respectively. Moreover, duck sera, with haemagglutination inhibiting titer ranged from 4 - 8 log2, were tested positive by rHA1 Elisa compared with negative duck sera. Further analysis of 179 serum samples with rHA1-Elisa in comparison with haemagglutination inhibition (HI) and commercial Elisa proved to be highly sensitive and specific. The agreement ratio between rElisa and HI was 84.9 % and between commercial Elisa (Flock check) and HI was 76.5 %. In conclusion, P. pastoris may allow development of an effective recombinant influenza vaccine based on truncated sequences of HA that might provide broader protection against H5 influenza viruses. The possibilities to use rHA1, P1 and P5 recombinant polypeptides as a vaccine against H5 influenza should be further studied. Also our study demonstrates the potential utility of recombinant Elisa as a tool for improvement of serological diagnosis of influenza A subtype H5 in chickens and ducks. / Die hochpathogene aviäre Influenza des Subtyps H5N1 erreicht beim Ausbruch von Infektionen in Nutzgeflügelbeständen Mortalitätsraten von bis zu 100 %. Effektive und kostengünstige Impfstoffe werden benötigt, die möglichst auch eine Differenzierung zwischen geimpften Tieren und mit Wild-Virus infizierten Tieren zulassen. In diesem Zusammenhang könnten Peptid-Vakzine eine mögliche Alternative zu den herkömmlichen Impfstoffen darstellen, bei denen unter Verwendung des Vollvirus Antikörper gegen mehrere Virusproteine induziert werden. Außerdem, könnten rekombinante Antigene in serologischen Tests zur Diagnose von H5 Virus in Nutzgeflügel eingesetzt werden. Von dem Einsatz spezifischer rekombinanter Antigene ist eine Verbesserung der Serodiagnostik zu erwarten. In dieser Arbeit, wurden vier verkürzte Sequenzen des Hämagglutinins (P1, P2, P5 und rHA1) von Subtyp H5 (A / Thailand / 1 (Kan-1) / 2004) rekombinant in Pichia Pastoris exprimiert. Dazu erfolgten zunächst eine Klonierung in der Expressionsvektor pAOX und die Transformation von Pichia Pastoris. Die Expression wurde durch Methanol induziert. Der Nachweis der rekombinanten Fusionspeptiden mit C-terminalen Histidin-Tag erfolgte durch SDS-PAGE, Western Blot, Glycolysierungsanalyse, und MALDI-TOF. Der Histidin-Tag ermöglichte die Reinigung von P1, P2 und rHA1 mit Metall-Affinitätschromatographie. Polypeptid P5 hingegen wurde mittels Lectin-Affinitäts- chromatographie gereinigt. Balb/c Mäuse wurden mit Polypeptid P1, P2 bzw. rHA1, versetzt mit Gerbu Adjuvans, immunisiert. Zur Untersuchung der Immunantwort wurden die murinen Seren mittels Elisa (unter Verwendung rekombinanter Antigene oder Voll-H5N1 Antigen) sowie IFA (durchgeführt in Vero- Zellen infiziert mit A / Thailand / 1 (Kan-1) / 2004) analysiert. Dabei wurde die präferentielle Induktion von H5-spezifischen Antikörpern detektiert. Die Immunogenität der P1, P2, P5 und rHA1-Polypeptide wurde in kommerziellen Legehennen bestimmt. Seren wurden mit ELISA, IFA, und Mikroneutralizationstest (μNT) analysiert. Die ELISA-Ergebnisse zeigten, dass die Polypeptide P1, P2 und rHA1 hohe Subtyp-spezifische Antikörpertiter in Hühnern induzierten. Im µNT konnte nur ein niedriger neutralisierender Antikörpertiter nachgewiesen werden. Das P5- Polypeptid ist bei Hühnern nicht immunogen. Im Eigelb von Hühnern, die mit den rekombinanten Polypeptiden P1 und rHA1 immunisiert wurden, konnten H5-spezifische IgY Antikörper detektiert werden. Hühner, die mit P2 und P5 immunisiert wurden, zeigten keine IgY im Eigelb. Die rekombinanten Antigene P1 und rHA1 wurden im ELISA auf ihre potenzielle Eignung für die Serodiagnostik untersucht. Die optimale Antigenkonzentration war 50 ng / well. Die serologische Analyse von 25 positiven und 25 negativen Seren auf Antikörper gegen H5 zeigte, dass Sensitivität und Spezifität von Western Blot, Voll-H5N1 ELISA und rHA1-ELISA bei jeweils 100 % lagen. Bei Agargel- Immunodiffusiontest (AGID) lagen Sensitivität und Spezifität bei 52 % und 100 %, während im P1-Elisa lediglich eine Sensitivität von 80 % und eine Spezifität von 72 % erreicht wurden. Somit eignet sich rHA1 für die Anwendung in der Serodiagnostik. Bei der serologischen Untersuchung von 175 Hühnerseren wurde eine Überbestimmung zwischen rHA1-ELISA und Hämagglutinationshemmungstest (HAI) 84.9 % festgestellt, während diese zwischen dem kommerziellen ELISA (Flock Check) und HAI 76.5 % betrug. Die Ergebnisse zeigten, dass das Expressionssystem P. pastoris als Produktionssystem rekombinanter Antigene für die Serodiagnostik von H5 Influenza geeignet ist. Challenge-Versuche sind nötig, um die Eignung von rekombinanten Antigenen als möglichen Impfstoff gegen H5 Influenza zu untersuchen.

No description available.