Evaluation of Aspergillus as a host for the production of viral proteins using hepatitis B as a model

Pluddemann, Annette, 1972- (2002-12)

Dissertation (PhD)--University of Stellenbosch, 2002.

Thesis

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.

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