Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The link between infection with Human Papillomavirus (HPV) and the development of cervical
cancer has been established by several epidemiology studies. Cervical cancer is the second most
common cancer among women and it occurs at a rate of 22.8 cases per 100 000 women in South
Africa. Approximately 86% of newly reported cases of cervical cancer occur in developing
countries where limited access to medical facilities hampers efforts to prevent and screen for
HPV infection. Two commercial virus-like particle (VLP) vaccines consisting of HPV major
structural protein L1, which protect against the most common high-risk HPV-types, are currently
available. The high cost and type specificity of these commercially available vaccines have
necessitated the development of a low cost, broad-spectrum HPV vaccine. Inclusion of the minor
structural protein L2 has been shown to induce broadly cross-neutralizing antibodies and
therefore a chimera was constructed that contains an epitope of L2 inserted within the L1
sequence. This construct, renamed SAF, was shown to be highly immunogenic and thus has the
potential to be used as a prophylactic cervical cancer vaccine. Methylotrophic yeasts are known
to be excellent producers of recombinant proteins due to their strongly inducible promoters that
allow culturing of these yeasts to very high cell densities. Pichia pastoris and Hansenula
polymorpha have been employed in several studies for heterologous protein production and
levels of protein higher than 1 g/L have been reported. These yeasts also have GRAS status and
can therefore be used to manufacture products for use in humans.
In this study, the potential of H. polymorpha and P. pastoris to produce SAF intracellularly was
evaluated. The effect of increased gene dosage and peroxisomal targeting on SAF production
was examined as possible strategies to increase the yield of SAF. Peroxisomal targeting was
achieved by fusing the SAF gene at the C-terminal end with the Peroxisomal Targeting
Sequence 1 (PTS1) which consists of a short tri-peptide: –SKL. The functionality of PTS1 was
confirmed using green fluorescent protein (GFP), fluorescence microscopy and peroxisome
isolation. Peroxisomal targeting was shown to have a negative effect on SAF production levels
in both H. polymorpha and P. pastoris. An increase in gene dosage had no discernable effect on
SAF yield in H. polymorpha which is in contrast to previous research. The highest production
levels were achieved by P. pastoris KM71 (24.86 mg/L) which compares well to levels of L1 achieved by other research groups. The most significant insight emerging from this work was
that all the strains that produced SAF at detectable levels were equally efficient at the production
of SAF. Increased biomass was therefore the biggest contributor to high SAF levels (mg/L) in
the P. pastoris strains as significantly higher cell densities were achieved during culturing of
these strains. With the necessary optimisation, the methylotrophic yeasts have the potential to be
used as hosts for the production of a broad-spectrum HPV vaccine. / AFRIKAANSE OPSOMMING: Die skakel tussen infeksie met Mens Papilloomvirus (HPV) en die ontwikkeling van servikale
kanker is deur verskeie epidemiologiese studies bevestig. Servikale kanker is die tweede mees
algemene kanker onder vroue en dit kom voor teen ‘n tempo van 22.8 gevalle per 100 000 vroue
in Suid Afrika. Ongeveer 86% van alle nuwe gevalle kom voor in ontwikkelende lande waar
beperkte toegang tot mediese fasiliteite pogings om HPV infeksie te voorkom en te behandel,
belemmer. Twee pseudovirale-partikel (VLP) entstowwe teen HPV is tans op die mark
beskikbaar en hierdie entstowwe verleen immuniteit teen die mees algemene hoë-risiko HPV
tipes. Die hoë koste en nou spektrum van hierdie entstowwe het dit nodig gemaak om ‘n
goedkoop, wye-spektrum HPV entstof te ontwikkel. Navorsing het bewys dat die insluiting van
die strukturele L2 proteïen in die VLP entstof, lei tot die indusering van neutraliserende
teenliggame, wat wye spektrum antigenisiteit tot gevolg het. ‘n Chimeriese proteïen wat ‘n
epitoop van L2 binne die L1 volgorde bevat is gekonstrueer, en hierdie proteïen is benoem SAF.
SAF het hoë immunogenisiteit en kan dus potensieel as ‘n voorkomende servikale kanker entstof
gebruik word. Metielotrofiese giste is bekend vir hulle vermoë om hoë vlakke rekombinante
proteïene te produseer as gevolg van hulle induseerbare promotors wat groei tot baie hoë sel
digthede toelaat. Pichia pastoris en Hansenula polymorpha is in menigte studies gebruik om
heteroloë proteïene te produseer tot vlakke bo 1 g/L. Hierdie giste en die proteïen produkte wat
hulle vorm word algemeen aanvaar as veilig vir menslike gebruik.
In hierdie studie het ons die potensiaal van H. polymorpha en P. pastoris om SAF intrasellulêr te
produseer, geevalueer. Die effek op SAF produksie van verhoogde geen dosering asook die
teiken van SAF na die peroksisoom was ondersoek as moontlike strategieë om die opbrengs van
SAF te verhoog. Die teiken van SAF na die peroksisoom is behaal deur die Peroksisomale
Teiken Volgorde 1 (PTS1) aan die C-terminaal van SAF te heg. Die funksionaliteit van PTS1
was bevestig deur gebruik te maak van groen fluoroserende proteïen (GFP), fluoressensie
mikroskopie en isolering van peroksisome. Teiken van SAF na die peroksisoom het ‘n negatiewe
uitwerking gehad op SAF uitdrukking in beide H. polymorpha en P. pastoris. ‘n Verhoging in
geen dosering het geen onderskeibare effek gehad op SAF opbrengs in H. polymorpha nie wat in
teenstelling is met vorige navorsing. Die hoogste produksie vlakke is opgelewer deur P. pastoris KM71 (24.86 mg/L) wat goed vergelyk met vlakke van L1 wat deur ander navorsings groepe
behaal is. Die belangrikste gevolgtrekking wat gemaak kan word uit hierdie studie is dat al die
rasse wat SAF geproduseer het in meetbare hoeveelhede ewe effektief was. Verhoogde biomassa
was dus die grootste bydraende faktor tot hoë SAF vlakke (mg/L) in die P. pastoris rasse as
gevolg van die hoë sel digthede wat hierdie rasse kan bereik. Dit is duidelik dat metielotrofiese
giste, met die nodige optimisering, oor die potensiaal beskik om as gasheer sisteme te dien vir die
produksie van ‘n wye spektrum HPV entstof. / The NRF and the Department of Microbiology for financial support
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6908 |
| Date | 03 1900 |
| Creators | Burke, Arista |
| Contributors | Van Zyl, W. H., Gorgens, J. F., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xviii, 149 p. : ill. |
| Rights | Stellenbosch University |
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