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

[摘要] ENGLISH ABSTRACT:Since the advent of recombinant DNA technology in the 1970s, variousmicrobial hosts have been employed for the efficient high-level heterologousproduction of a variety of proteins, ranging from enzymes and reagents totherapeutics and vaccines. More recent microbial hosts to be employed forthese purposes are filamentous fungi, and particularly the genus Aspergillus.Aspergilli have been associated with industrial processes for many years andare used in the production of antibiotics, enzymes, citric acid and Oriental foodsand beverages, and thus strains such as Aspergillus niger and Aspergillusoryzae have been afforded GRAS (Generally Regarded 8s ~afe) status. Theyalso secrete copious amounts of homologous and heterologous proteins andare able to perform post-translational modifications effectively. Various proteinsof pharmaceutical interest have been successfully expressed in Aspergillus, butthe potential of these fungi to produce heterologous viral proteins has not beenexplored extensively.In this study, we evaluated the potential of the filamentous fungi A. niger andAspergillus awamori as alternative hosts for the heterologous production ofhepatitis B viral proteins. Hepatitis B is a serious, potentially lethal liver diseasethat affects 2000 million people world-wide and has a high endemicity inSouthern Africa. Currently there is no effective treatment for viral hepatitis andthus a mass vaccination strategy is the only solution to curb the spread of thedisease. This kind of vaccination strategy requires a cheap, safe and effectivevaccine and these objectives have been achieved in the development ofrecombinant 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 threeproteins, 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 thepreS1preS2S gene). When produced in the above-mentioned yeasts, the Sprotein was shown to spontaneously assemble into pseudoviral particles devoidof viral DNA, which were then purified and used as vaccine.In the present study the Sand preS1preS2S genes from a local isolate ofhepatitis B subtype adw2 were placed under transcriptional control of theconstitutive Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase(gpdA) promoter and the inducible A. niger glucoamylase (glaA) promoter. Therespective viral genes were also fused to the region encoding the catalyticdomain of the highly expressed and secreted A. niger glucoamylase, whichserved as a carrier moiety to possibly facilitate viral protein secretion. Thevarious gene constructs were subsequently transformed to laboratory strains ofA. niger and A. awamori and numerous transformants were obtained. OneA. niger transformant carrying the S gene under control of the gpdA promotercontained approximately 7 integrated copies of the expression cassette andproduced hepatitis B pseudoviral particles intracellularly at levels of 0.4 mg/Iculture. These levels are approximately ten-fold higher than those initiallyobtained from the yeast S.cerevisiae, which showed yields of 0.01 to0.025 mg/I. None of the other transformants could be shown to producerecombinant S or L protein and no secretion of viral protein could bedemonstrated. This could be attributed to numerous factors, including vectorcopy number, site of integration or proteolytic activity. The most importantinsight emerging from this work regarding secretion of heterologous viral proteinwas that the addition of a carrier protein hampered, rather than enhancedsecretion of the viral envelope protein, due to the inherent properties of viralprotein assembly.This work also serves as a proof of principle, showing that Aspergillus isindeed a viable alternative host for the production of hepatitis B pseudoviralparticles, and could be investigated further for its potential as host for theheterologous expression of other viral proteins.

[发布机构] Stellenbosch University