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Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • The UPLC-MS/MS analyses and quantification of novel 5α-reduced steroids using response factors. • The kinetic characterisation of human steroid 5α-reductase type 1 (SRD5A1), expressed in HEK-293 cells, towards 11OHA4 and 11OHT and their keto derivatives by progress curve analysis. • The subcloning, transformation and functional expression of SRD5A1 in the yeast expression system, P. pastoris. • The conversion of 11OHA4 and 11OHT and their keto derivatives by SRD5A1 expressed in P. pastoris. • The endogenous enzymatic activity in P. pastoris towards the 5α-reduced metabolites in the 11OHA4- and alternate 5α-dione pathways. • The potential application of P. pastoris as a biocatalyst in the production of 5α- reduced C19 steroids. / AFRIKAANSE OPSOMMING: Hierdie ondersoek beskryf: • Die UPLC-MS/MS analise en kwantifisering van nuut-ondekte 5α-gereduseerde steroïede met behulp van responsfaktore. • Die kinetiese karakterisering van menslike steroïed 5α-reduktase tipe 1 (SRD5A1), uitgedruk in HEK-293 selle, vir 11OHA4 en 11OHT en hul ketoderivate deur middel van progressiekurwe-analise. • Die subklonering, transformasie en funksionele uitdrukking van SRD5A1 in die gis P. pastoris. • Die omsetting van 11OHA4 en 11OHT en hul ketoderivate deur SRD5A1 uitgedruk in P. pastoris. • Die omsetting van 5α-gereduseerde steroïede in die 11OHA4 en alternatiewe 5α-dioon paaie deur endogene ensieme in P. pastoris • ‘n Ondersoek na die toepassing van die gisuitdrukkingstelsel as ‘n moontlike OR potensiële biokatalis vir die produksie van 5α-gereduseerde C19 steroïede.

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.

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.

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015