Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Since the advent of recombinant DNA technology in the 1970s, various microbial hosts have been employed for the efficient high-level heterologous production of a variety of proteins, ranging from enzymes and reagents to therapeutics and vaccines. More recent microbial hosts to be employed for these purposes are filamentous fungi, and particularly the genus Aspergillus. Aspergilli have been associated with industrial processes for many years and are used in the production of antibiotics, enzymes, citric acid and Oriental foods and beverages, and thus strains such as Aspergillus niger and Aspergillus oryzae have been afforded GRAS (Generally Regarded 8s ~afe) status. They also secrete copious amounts of homologous and heterologous proteins and are able to perform post-translational modifications effectively. Various proteins of pharmaceutical interest have been successfully expressed in Aspergillus, but the potential of these fungi to produce heterologous viral proteins has not been explored extensively. In this study, we evaluated the potential of the filamentous fungi A. niger and Aspergillus awamori as alternative hosts for the heterologous production of hepatitis B viral proteins. Hepatitis B is a serious, potentially lethal liver disease that affects 2000 million people world-wide and has a high endemicity in Southern Africa. Currently there is no effective treatment for viral hepatitis and thus a mass vaccination strategy is the only solution to curb the spread of the disease. This kind of vaccination strategy requires a cheap, safe and effective vaccine and these objectives have been achieved in the development of recombinant subunit vaccines from yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris that are commercially available. The hepatitis B virus envelope consists of a membrane fraction and three proteins, namely the major (S) protein (encoded by the S gene), the middle (M) protein (encoded by the preS2S gene) and the large (L) protein (encoded by the preS1preS2S gene). When produced in the above-mentioned yeasts, the S protein was shown to spontaneously assemble into pseudoviral particles devoid of viral DNA, which were then purified and used as vaccine. In the present study the Sand preS1preS2S genes from a local isolate of hepatitis B subtype adw2 were placed under transcriptional control of the constitutive Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter and the inducible A. niger glucoamylase (glaA) promoter. The respective viral genes were also fused to the region encoding the catalytic domain of the highly expressed and secreted A. niger glucoamylase, which served as a carrier moiety to possibly facilitate viral protein secretion. The various gene constructs were subsequently transformed to laboratory strains of A. niger and A. awamori and numerous transformants were obtained. One A. niger transformant carrying the S gene under control of the gpdA promoter contained approximately 7 integrated copies of the expression cassette and produced hepatitis B pseudoviral particles intracellularly at levels of 0.4 mg/I culture. These levels are approximately ten-fold higher than those initially obtained from the yeast S.cerevisiae, which showed yields of 0.01 to 0.025 mg/I. None of the other transformants could be shown to produce recombinant S or L protein and no secretion of viral protein could be demonstrated. This could be attributed to numerous factors, including vector copy number, site of integration or proteolytic activity. The most important insight emerging from this work regarding secretion of heterologous viral protein was that the addition of a carrier protein hampered, rather than enhanced secretion of the viral envelope protein, due to the inherent properties of viral protein assembly. This work also serves as a "proof of principle", showing that Aspergillus is indeed a viable alternative host for the production of hepatitis B pseudoviral particles, and could be investigated further for its potential as host for the heterologous expression of other viral proteins. / AFRIKAANSE OPSOMMING: Sedert die ontwikkeling van rekombinante DNA tegnologie in die sewentigerjare is verskeie mikroorganismes reeds gebruik vir die doeltreffende produksie van 'n verskeidenheid proteïne teen hoë vlakke; onder andere ensieme, reagense, terapeutiese middels en vaksiene. Onlangs is filamentagtige swamme, veral van die genus Aspergillus, ontwikkel vir heteroloë proteïenproduksie. Aspergilli word al vir baie jare in nywerheidsprosesse gebruik, onder andere in die vervaardiging van antibiotika, ensieme, sitroensuur en sekere Oosterse voedsel- en drankprodukte. As gevolg van hierdie jarelange gebruik van rasse soos Aspergillus niger en Aspergillus oryzae, word hulle algemeen aanvaar as veilig vir menslike gebruik. Hierdie swamme besit veral die vermoë om hoë vlakke van homoloë en heteroloë proteïene uit te skei en die na-translasiemodifisering van proteïene korrek uit te voer. Verskeie proteïene van farmaseutiese belang is al suksesvol in Aspergillus uitgedruk, maar die potensiaal van hierdie swamme om virale proteïene te vervaardig is nog nie deeglik ondersoek nie. Hierdie studie ondersoek die geskiktheid van die filamentagtige swamme A. niger en Aspergillus awamori om as alternatiewe gashere vir die heteroloë produksie van hepatitis B proteïene te dien. Hepatitis B is 'n ernstige en selfs dodelike lewersiekte. Omtrent 2000 miljoen mense wêreld-wyd is met die virus geïnfekteer en dit is veral endemies in Suiderlike Afrika. Daar is tans geen doeltreffende behandeling vir virale hepatitis en dus is wêreld-wye inentingsprogramme die enigste oplossing om die verspreiding van die siekte te bekamp. Hierdie inentingsstrategie vereis die beskikbaarheid van 'n bekostigbare, veilige en doeltreffende vaksien. Die rekombinante subeenheidvaksiene wat ontwikkel is deur van gashere soos Saccharomyces cerevisiae, Hansenula polymorpha en Pichia pastoris gebruik te maak, voldoen aan hierdie vereistes en is kommersieel beskikbaar. Die omhulsel van die hepatitis B virus bestaan uit 'n membraangedeelte en drie proteïene, naamlik die hoofproteïen (S) (gekodeer deur die S-geen), die middelproteïen (M) (gekodeer deur die preS2S-geen) en die grootproteïen (L) (gekodeer deur die preS1preS2S-geen). Wanneer die S-proteïen in bo-genoemde giste uitgedruk word, vorm dit spontaan pseudovirale partikels wat nie virale DNA bevat nie. Hierdie partikels word dan gesuiwer en as vaksien gebruik. In hierdie studie is die S- en preS1preS2S-gene, vanaf 'n plaaslike isolaat van hepatitis B subtipe adw2, onder transkripsionele beheer van die konstitutiewe Aspergillus nidulans gliseraldehied-3-fosfaat-dehidrogenasepromoter (gpdA) en die induseerbare A. niger glukoamilasepromoter (glaA) geplaas. Die onderskeie virale gene is ook aan die koderende gedeelte vir die katalitiese domein van A. niger glukoamilase gelas om fusieproteïene te vorm. Glukoamilase word teen hoë vlakke deur Aspergillus vervaardig en uitgeskei en kan dus moontlik dien as draerproteïen om sekresie van die proteïne te bevorder. Transformasie van die geenkonstrukte na laboratoriumrasse van A. niger en A. awamori het verskeie transformante gelewer. Een A. niger transformant bevattende die S-geen onder transkripsionele beheer van die gpdA promoter het minstens sewe kopieë van die uitdrukkingskaset in sy genoom geïntegreer en het hepatitis B pseudovirale partikels intrasellulêr teen vlakke van 0.4 mg/I swamkultuur vervaardig. Hierdie vlakke is omtrent tienvoudig hoër as die vlakke van 0.01 - 0.025 mg/I wat S.cerevisiae oorspronklik opgelewer het. Nie een van die ander transformante het rekombinante S of L proteïene vervaardig nie en sekresie van virale proteïen kon nie getoon word nie. Hierdie verskynsel mag te wyte wees aan verskeie faktore insluitende vektor-kopiegetal, setel van integrasie en proteolitiese aktiwiteit. Die belangrikste insig uit hierdie studie aangaande sekresie van heteroloë virale proteïene is dat die koppeling van die virale omhulsel-proteïen aan 'n draerproteïen sekresie benadeel het, eerder as om dit te bevorder. Hierdie verskynsel is te wyte aan die inherente geneigdheid van virale omhulselproteïene om 'n kompleks te vorm. Die studie dien ook as "bewys van beginsel" dat Aspergillus wel 'n werkbare alternatiewe gasheer vir die produksie van hepatitis B pseudovirale partikels is, en dat dit verder ondersoek sou kon word as potensiële gasheer vir die heteroloë uitdrukking van ander virale proteïene.

Thesis (MScIng)--University of Stellenbosch, 2005. / Accompanied by CD in end pocket of book. Bibliography. Also available via the Internet.

The objectives of this study are: (1) To compare the effectiveness of two dosing schedules of hepatitis B vaccine in achieving compliance within the vaccines; (2) To determine the immunization requirements in U.S. pharmacy schools both at admission and before the students begin clinical clerkships; and, (3) To design an immunization program for pharmacy students at the University of the Pacific in an attempt to enhance compliance.

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2005. / The development of non-replicating vaccines is an emerging option for safe, effective vaccines, several of which contain virus-like particles (VLPs). Many recombinant expression systems have been evaluated as hosts for VLP production for the prevention of infectious diseases. The filamentous fungi Aspergillus niger has emerged as a potential alternative expression system for cost effective VLP vaccine production. Hepatitis B surface antigen (HBsAg) was used as a model VLP product to benchmark A. niger’s production capacity with those of Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha. Bioprocessing strategies were used to optimise VLP production by recombinant A. niger in batch culture. In particular, the effect of the parameters culture temperature, inoculum concentration, agitation intensity, dissolved oxygen (dO2) concentration and culture pH on biomass formation, morphology and VLP (HBsAg) production concentration was quantified. At an optimum agitation of 100 rpm and optimum dO2 concentration of 50 %, HBsAg production levels were increased 9-fold compared to yields obtained in shakeflask cultivation. Highest HBsAg production levels of 3.6 mg.ℓculture -1 and 350 μg.gDW -1 were recorded, at a biomass concentration of 10.5 gDW.ℓculture -1. These production levels compare favourable with those obtained by other production systems under similar conditions. HBsAg VLPs mostly accumulated intracellularly, although under optimum bioreactor conditions significant HBsAg accumulation in the cytoplasm and culture supernatant was also observed. The impact of these process parameters on VLP production and cell morphology was attributed to environmental stress conditions. Volumetric biomass and HBsAg production levels were maximised under conditions of lowest environmental stress, resulting in the most optimal small-pelleted morphology. These results indicate a substantial potential for further engineering of the A. niger production system for the high level of intracellular and extracellular VLP production.

No description available.

Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production which in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection.

Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production which in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-02-04T09:24:45Z No. of bitstreams: 2 Dissertação - Leandro Nascimento da Silva - 2014.pdf: 6086146 bytes, checksum: 75f020e70d97640ac095871c4913b275 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-05T14:16:04Z (GMT) No. of bitstreams: 2 Dissertação - Leandro Nascimento da Silva - 2014.pdf: 6086146 bytes, checksum: 75f020e70d97640ac095871c4913b275 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-05T14:16:04Z (GMT). No. of bitstreams: 2 Dissertação - Leandro Nascimento da Silva - 2014.pdf: 6086146 bytes, checksum: 75f020e70d97640ac095871c4913b275 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-09 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Crack is considered a public health problem in Brazil and in the world because of its impact on social relationships, physical and mental integrity of the user, and the risk associated with infections, such as those caused by the hepatitis B virus (HBV). This study investigated the epidemiology of infection with the hepatitis B virus and immunization status among users of crack institutionalized in Goiania, Brazil. During August 2012 to April 2013, a total of 600 individuals were interviewed, and blood samples collected for the detection of serological markers of HBV (HBsAg, total anti HBc and anti-HBs) by enzyme-linked immune sorbent assay (ELISA). Subsequently a cohort of individuals susceptible to hepatitis B was formed to assess compliance, completion of the vaccination series, and vaccine response against hepatitis B, using an accelerated scheme. Prior exposure to HBV (anti-HBc) was 7.0% (95% CI: 5.22 to 9.32), and 17.7% (95% CI: 14.8 to 20.9) were anti -HBs isolated, suggesting previous vaccination against hepatitis B. The use of crack cocaine through improvised pipes, history of sexually transmitted disease, and exchanging sex for drugs or money were significantly associated with exposure to HBV (p < 0.05). Of the total of individuals who received the first dose of hepatitis B vaccine and eligible to complete the full vaccine scheme (n = 406), 229 (56.4%) and 96 (26.6%) received the second and third doses, respectively. It was possible to evaluate the vaccine response in only 23/96 subjects, and 78% responded with protective titers. The high frequency of risk behaviors, the low frequency of vaccinations, and improper compliance with the vaccination schedule, even using the accelerated scheme, highlights the need for strategies for health education and prevention to reach this population so vulnerable to sexually transmitted infections and parenteral transmission of hepatitis B. / O crack é considerado um problema de saúde pública no Brasil e no mundo devido ao seu impacto nas relações sociais, na integridade física e mental do usuário e no risco associado às infecções, como a causada pelo vírus da hepatite B (HBV). Este estudo investigou a epidemiologia da infecção pelo vírus da Hepatite B e situação vacinal em usuários de crack institucionalizados em Goiânia – Goiás. Durante agosto de 2012 a abril de 2013 um total de 600 indivíduos foram entrevistados e amostras sanguíneas coletadas para detecção dos marcadores sorológicos do HBV (HBsAg, anti-HBc total e anti-HBs) pelo ensaio imunoenzimático (ELISA). Posteriormente foi formada uma coorte de indivíduos suscetíveis a hepatite B para avaliação da adesão, completude do esquema e resposta vacinal contra hepatite B, utilizando-se um esquema super acelerado. A exposição prévia ao HBV (anti-HBc) foi de 7,0% (IC 95%: 5,22-9,32), e 17,7% (IC 95%: 14,8-20,9) apresentaram positividade isolada para o anti-HBs, sugerindo vacinação prévia contra hepatite B. O consumo de crack por meio de lata improvisada como cachimbo, história de doença sexualmente transmissível e troca de sexo por droga ou dinheiro foram significativamente associados à exposição ao HBV (p< 0,05). Do total de indivíduos que recebeu a primeira dose da vacina contra hepatite B e elegíveis para completar o esquema (n=406), 229 (56,4%) e 96 (26,6%) receberam a segunda e terceira doses, respectivamente. Em somente 23/96 indivíduos foi possível avaliar a resposta vacinal, sendo que 78% responderam com títulos protetores. A frequência elevada de comportamentos de risco, a baixa frequência de indivíduos vacinados e adesão ao esquema vacinal, mesmo com esquema super acelerado, evidencia a necessidade de estratégias de educação em saúde e prevenção que alcancem essa população vulnerável as doenças de transmissão sexual e parenteral como a hepatite B.

Thesis (Dr. P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / "May 2008." Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0911. Adviser: Lu-Yu Hwang. Includes bibliographical references (leaves 54-62).

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-09-05T13:10:46Z No. of bitstreams: 2 Dissertação - Paulie Marcelly Ribeiro dos Santos Carvalho - 2016.pdf: 5626599 bytes, checksum: 9af96621473e8d99237c7811c44ef4fc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-05T13:19:07Z (GMT) No. of bitstreams: 2 Dissertação - Paulie Marcelly Ribeiro dos Santos Carvalho - 2016.pdf: 5626599 bytes, checksum: 9af96621473e8d99237c7811c44ef4fc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-05T13:19:07Z (GMT). No. of bitstreams: 2 Dissertação - Paulie Marcelly Ribeiro dos Santos Carvalho - 2016.pdf: 5626599 bytes, checksum: 9af96621473e8d99237c7811c44ef4fc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-02-18 / Infection caused by the hepatitis B virus (HBV) continues to have a major impact on global public health, even while being vaccine-preventable. People living on the streets, homeless, are at high risk for sexually transmitted infections (STIs), including hepatitis B. To investigate the epidemiology of HBV infection among homeless people in Goiania, Goias, from August 2014 to June 2015, 353 individuals served by a public shelter at the capital were interviewed using a structured questionnaire containing questions about socio-demographic characteristics, clinical and risk factors for HBV infection. Next, all were tested for serologic markers of HBV. It was observed the predominance of males (81.3%), mixed race selfdeclared (61%), single (59.8%), low income (70%) and low level of education (53,3%). The global prevalence of HBV was estimated at 21.81% (95% CI 17.82 to 26.41): two individuals were HBsAg / anti-HBc positive, 61 were anti-HBc / anti-HBs, and 16 showed reactivity to only the anti-HBc marker. Additionally, 19.55% (CI: 95%: from 15.75 to 24.00) tested positive for isolated anti-HBs, suggesting immunity to HBV. Analyzing potential risk factors to HBV showed that: age over 50 years, being gay or bisexual, and being mixed race/blackselfdeclared were independently associated of exposure to HBV. The results confirm the vulnerability of this populational subgroup and a high prevalence of exposure to HBV. Still. the low frequency of serological evidence of immunization against HBV, specially among older subjects, makes evident the need of drawing up strategies of vaccination at the support places to homeless. / A infecção causada pelo vírus da hepatite B (HBV) continua apresentando grande impacto na saúde pública mundial, mesmo sendo imunoprevenível. Populações em situação de rua (PSR) apresentam um risco elevado para infecções sexualmente transmissíveis (IST), incluindo a hepatite B. Para investigar a epidemiologia da infecção pelo HBV em indivíduos em situação de rua em Goiânia, Goiás, durante agosto de 2014 a junho de 2015, 353 indivíduos atendidos em um albergue público da capital foram entrevistados utilizando-se um roteiro estruturado, contendo questões sobre características sociodemograficas, clínicas e fatores de risco para a infecção pelo HBV. A seguir, todos foram testados para os marcadores sorológicos do HBV. Observou-se predomínio de indivíduos do sexo masculino (81,3%), de cor/raça parda autodeclarada (61%), solteiros (59,8%), de baixa renda (70%) e baixo nível de escolaridade (53,3%). Estimou-se uma prevalência global para o HBV de 21,81% (IC 95%: 17,82 - 26,41): dois indivíduos foram HBsAg/anti-HBc positivos, 61 foram anti-HBc/anti-HBs e 16 apresentaram reatividade do marcador anti-HBc isolado. Ainda, 19,55% (IC: 95%: 15,75 - 24,00) apresentaram positividade isolada para o anti-HBs, sugerindo imunidade para o HBV. A análise de potenciais fatores de risco para HBV mostrou que: idade > 50 anos, ser homossexual ou bissexual e ser de cor preta/negra autodeclarada foram independentemente associados a exposição ao HBV. Os resultados ratificam a vulnerabilidade desse subgrupo populacional e uma elevada prevalência de exposição ao HBV. Ainda, a baixa freqüência de evidência sorológica de imunização contra o HBV, especialmente nos indivíduos mais velhos, evidencia a necessidade de elaboração de estratégias de vacinação nos locais de apoio a PSR.