Made available in DSpace on 2016-08-29T15:34:47Z (GMT). No. of bitstreams: 1 tese_4464_.pdf: 3204606 bytes, checksum: a23da35b5188c96d619b059f83d1577a (MD5) Previous issue date: 2010-12-15 / Os HPV são vírus epiteliotrópicos que infectam tecido cutâneo ou mucoso e estão relacionados com desenvolvimento de lesões que, no trato genital, variam de verrugas ao câncer cervical invasivo. As lesões são causadas por diferentes tipos de HPV, que são classificados em baixo e alto risco conforme sua associação com câncer cervical. Sabe-se que mulheres positivas para HIV são mais acometidas por infecções por HPV e estão mais propensas ao desenvolvimento de câncer cervical. O objetivo desse estudo foi avaliar a frequência de tipos de HPV em mulheres soropositivas e soronegativas para HIV. Para isso foram analisadas amostras de escovado cervical, mantidas congeladas, de mulheres conhecidamente positivas para HPV (n=87), atendidas no Centro de Referência DST/AIDS, em Vitória-ES, no período de março a dezembro de 2006. O DNA das amostras foi extraído utilizando kit comercial QIAamp® DNA Mini Kit ou através do método de isotiocinanato de guanidina e sílica. DNA do HPV foi amplificado por PCR utilizando os iniciadores degenerados MY09/MY11 e a genotipagem foi realizada por Restriction Fragment Length Polymorphism (RFLP) e por Reverse Line Blot (RLB). Do total de amostras, 97,7% foram genotipadas e 31 tipos distintos detectados: 6, 11, 13, 16, 18, 26, 31, 31b, 32, 33, 34, 35, 42, 44, 51, 52, 53, 55, 56, 58, 59, 61, 62, 64, 66, 68, 71, 81, 82, 83 e 84. O tipo mais prevalente foi o HPV16, tanto nas mulheres soropositivas quanto nas soronegativas para HIV, seguido pelos tipos 6, 53 e 11. O tipo 13, incomum em amostras cervicais, foi observado nesse estudo, porém a quantidade de amostras não foi suficiente para a realização de seqüenciamento para a confirmação deste tipo viral. Os tipos oncogênicos foram mais comuns nas amostras de mulheres soropositivas para HIV, porém com número semelhante e o número de infecções múltiplas foi maior entre as mulheres HIV positivas. Este estudo revelou uma grande diversidade de tipos de HPV na região. PALAVRAS CHAVES: Human papillomavirus (HPV), Human Immunodeficiency virus (HIV), Restriction Fragment Length Polymorphism (RFLP), Reverse Line Blot (RLB).

High-risk mucosal human papillomaviruses (HPVs) are major aetiological agents for the development of cervical cancer. Thus, the current goal of cervical cancer treatment is to develop vaccines against HPV s. Such vaccines would either prevent cervical cancer by eliminating HPV infection or be useful for treating established lesions by the destruction of cells displaying HPV proteins. The aim of this thesis was to characterise immune responses to the E5 protein of HPV -16, one of several antigens with possible use in vaccination. To determine whether immune responses to HPV -16 E5 existed and whether they could be correlated with disease severity or with the presence of HPV -16 DNA, both cell mediated (Chapter Two) and humoral (Chapter Three) immunity was investigated in women with and without cervical disease. Cellular responses in a minority of women were inversely correlated with disease severity. However, E5 specific antibodies were negatively correlated with the absence of HPV -16 DNA. Thus, although some immune responses were evident, these were generally limited to a small number of subjects and were not associated with the detection of HPV-16 E5 mRNA or DNA sequence variants. Due to the immune responses in women, E5 was further investigated to determine if the absence of HPV -16 E5 specific immune responses was due to the poor antigenicity of HPV -16. Mice were immunised with synthetic peptides corresponding to full length HPV -16 E5 (Chapter Four). As with the human data, cellular responses and weak antibody responses were detected in mice. Some mice also exhibited cytotoxic T -lymphocyte responses and when E5/major histocompatibility class I (MHC-I) interactions were investigated, a number of peptides showed a high percentage of binding. The E5/MHC-I interactions were further investigated (Chapter Five). The surface expression of MHC-I on cells containing HPV-16 or -18 DNA was found to be lower than on HPV DNA negative cell lines even after stimulation with interferon-gamma. Stimulation with E5 synthetic peptides increased expression of cell surface MHC-I molecules on cell lines negative for HPV DNA. Furthermore, the presence of the E5 gene reduced the expression of the ovalbumin gene in normal human keratinocytes. In conclusion, the data contained within this thesis indicate that HPV-16 E5 CMI is inversely correlated with disease status. It is possible to induce cell mediated responses to HPV -16 E5 and low-titre antibody responses. The presence of HPV16 E5 DNA may impair normal cellular function.

O câncer de cervical é causado por uma infecção persistente por algum dos tipos oncogênicos de Papilomavírus Humano (HPV) e continua a ser um problema de saúde pública, especialmente nos países em desenvolvimento. Por Peptide Phage Display, foram selecionadas sequências de peptídeos com afinidade de ligação a linhagens celulares ou a tumores associados ao HPV. Dentre elas, foi possível identificar sequências contidas em moléculas importantes para a progressão de tumores, como a -manosidase e triptase, enzimas que desempenham um papel importante na progressão tumoral. Para caracterizar o efeito da inibição de -manosidase II na terapia de tumores cervicais em camundongos, utilizamos a droga Swainsonina (SW), previamente descrita como inibidor desta enzima. Nós testamos o efeito desta droga tratando animais inoculados com células tumorais que expressam os oncogenes E6 e E7 de HPV16. Observamos que os animais tratados com Swainsonina apresentaram crescimento tumoral significativamente mais rápido do que os animais controle. Investigando os mecanismos por trás desse efeito, descobrimos que embora SW module parcialmente os macrófagos associados aos tumores, o tratamento induz o acúmulo de células com fenótipo mieloderivado supressor no baço dos animais, potencializando o efeito tolerogênico dos tumores sobre o sistema imune. Sendo assim, sugerimos cautela no uso deste fármaco para a terapia de pacientes com tumores HPV&#43. Outro braço do trabalho foi avaliar o papel da triptase, que é produzida por mastócitos, no infiltrado inflamatório. Para isto padronizamos um modelo de co-cultura de esferóides tumorais da linhagem TC-1 com a linhagem de mastócitos murinos PT18. Através deste modelo pudemos observar que a linhagem tumoral consegue induzir a desgranulação dos mastócitos independentemente de anticorpos. Além disso, quando em co-cultura, a linhagem tumoral parece estar aumentando a meia-vida dos mastócitos e estimulando a proliferação destes. Em experimentos in vivo observamos que tumores induzidos com as células PT18 e TC-1 cresceram mais rapidamente do que tumores induzidos apenas com TC-1. Através da imunofenotipagem dos tumores ficou evidenciado um aumento de células CD31+ e no infiltrado inflamatório total de tumores induzidos com o co-cultivo. Justificando o fato destes crescerem mais rapidamente, sugerindo que os mastócitos podem ter efeitos tanto proliferativos quanto no processo de angiogênese tumoral. / Cervical cancer is caused by a persistent infection by some of the oncogenic types of Human Papillomavirus (HPV) and continues to be a public health problem, especially in developing countries. By Peptide Phage Display peptide sequences were selected with binding affinity to cell lines or to HPV-associated tumors. Among them, it was possible to identify sequences contained in molecules important for the progression of tumors, such as -mannosidase and tryptase, enzymes that play an important role in tumor progression. To characterize the effect of -mannosidase II inhibition on cervical tumor therapy in mice, we used the drug Swainsonina (SW), previously described as an inhibitor of this enzyme. We tested the effect of this drug by treating animals inoculated with tumor cells expressing HPV16 E6 and E7 oncogenes. We observed that Swainsonine treated animals had significantly faster tumor growth than control animals. Investigating the mechanisms behind this effect, we found that although SW partially modulates tumor-associated macrophages, the treatment induces the accumulation of cells with suppressive myeloderivative phenotype in the animals spleens, enhancing the tolerogenic effect of tumors on the immune system. Therefore, we suggest caution in the use of this drug for the therapy of patients with HPV&#43 tumors. Another arm of the study was to evaluate the role of tryptase, which is produced by mast cells, in the inflammatory infiltrate. For this we standardized a co-culture model of tumor spheroids of the TC-1 lineage with the murine mast cell line PT18. Through this model we could observe that the tumoral lineage can induce mast cell degranulation independently of antibodies. In addition, when co-cultured, the tumoral lineage appears to be increasing the half-life of mast cells and stimulating the proliferation of these. In in vivo experiments we observed that tumors induced with PT18 and TC-1 cells grew faster than tumors induced only with TC-1. Tumor immunophenotyping revealed an increase in CD31&#43 cells and in the total inflammatory infiltrate of tumors induced with co-culture. Justifying the fact that they grow faster, suggesting that mast cells can have both proliferative effects and the process of tumor angiogenesis.

Philosophiae Doctor - PhD / Purpose: High risk human papillomavirus (HR-HPV) is well established as a causative agent of squamous cell carcinoma (SCC) of the orophaynx. HR-HPV has also been reported in periocular cancers and precancers, but controversy exists about its overall incidence and clinicopathologic profile. The purpose of this study is to evaluate the role of HR-HPV infection in periocular cancers and precancers, using multiple methods of detection. Design: Retrospective observational case series with laboratory investigations. Methods: Sequential surgical samples of 87 carcinomas (invasive SCC, SCC in situ and sebaceous carcinoma) from three different periocular sites (conjunctiva, lacrimal sac and the eyelid) diagnosed over a 15-year period (2000-2015) were selected for evaluation. Unstained paraffin sections of 87 cases of periocular carcinomas were analyzed with immunohistochemistry (IHC) for p16 as a screening test. p16 positive conjunctival- and lacrimal sac SCC were further evaluated for HR-HPV using DNA in situ hybridization (DNA ISH), and a subset was also analyzed by DNA Polymerase Chain Reaction (DNA PCR). p16 positive periocular sebaceous carcinomas (SC) were analyzed with PCR, and a subset of 18cases was further studied with a novel method of mRNA ISH, an advanced technique with an enhanced sensitivity and specificity. Relevant patient clinical information was obtained from review of the electronic medical records.

Philosophiae Doctor - PhD / PURPOSE: High risk human papillomavirus (HR-HPV) is well established as a causative agent of squamous cell carcinoma (SCC) of the orophaynx. HR-HPV has also been reported in periocular cancers and precancers, but controversy exists about its overall incidence and clinicopathologic profile. The purpose of this study is to evaluate the role of HR-HPV infection in periocular cancers and precancers, using multiple methods of detection. DESIGN: Retrospective observational case series with laboratory investigations. METHODS: Sequential surgical samples of 87 carcinomas (invasive SCC, SCC in situ and sebaceous carcinoma) from three different periocular sites (conjunctiva, lacrimal sac and the eyelid) diagnosed over a 15-year period (2000-2015) were selected for evaluation. Unstained paraffin sections of 87 cases of periocular carcinomas were analyzed with immunohistochemistry (IHC) for p16 as a screening test. p16 positive conjunctival- and lacrimal sac SCC were further evaluated for HR-HPV using DNA in situ hybridization (DNA ISH), and a subset was also analyzed by DNA Polymerase Chain Reaction (DNA PCR). p16 positive periocular sebaceous carcinomas (SC) were analyzed with PCR, and a subset of 18cases was further studied with a novel method of mRNA ISH, an advanced technique with an enhanced sensitivity and specificity. Relevant patient clinical information was obtained from review of the electronic medical records.

In Canada, parental acceptance and uptake of the HPV vaccine has been low. There is a need for more effective HPV vaccination health messages for parents. Whether a message is framed in terms of the benefits of engaging in the behaviour (gain frame), the costs of failing to engage in the behaviour (loss frame) or both the benefits and the costs (mixed frame) has potential to impact parents’ decision making. The appropriate frame of a message may depend on the recipient’s sex and involvement with the health issue. The purpose of this study was to investigate the persuasiveness of gain-, loss- and mixed-framed messages on mothers’ and fathers’ intentions to have their young son or daughter vaccinated against HPV. The study used a 3 Frame x 2 Sex of Parent x 2 Sex of Child design. We randomly assigned participants (n=367) to read a framed message and then complete a 29-item questionnaire assessing theoretical determinants of parental consent for vaccination. ANCOVAs revealed a three-way interaction for intentions to speak to a doctor about the HPV vaccine, F(2, 342)=3.66, p =.03, perceived severity of HPV, F(2, 347) = 3.10, p = .05, and for anxiety about their child contracting HPV, F(2, 342)=3.58, p=.02. Effect size comparisons revealed that gain-framed messages seem to persuade parents who are the opposite sex to the child for whom they are considering the vaccine. In turn, loss- and mixed-framed messages may persuade parents who are the same sex as the child for whom they are considering the vaccine. Perceived severity of HPV and anxiety about HPV mediated the relationship between message frame and intentions for some parent-child dyads. Findings have implications for constructing effective messages encouraging parents to consider having their child vaccinated against HPV. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2010-07-07 23:06:25.757

Persistent infection of the cervix with high risk (HR) types of Human Papilloma Virus (HPV) (HR-HPV) can result in precancerous lesions and cancers. However, most HPV infections can be cleared naturally by the immune response without causing disease. Although genetic variations have long been considered as the main explanation for individual heterogeneity in cancer susceptibility, the underlying mechanisms remain unclear. In this project, a panel of routinely taken clinical samples was assessed for 32 rationally selected SNPs with allele frequency related to disease outcome using the Taqman® OpenArray® system. The panel incorporated 475 HR-HPV negative, cytologically-normal cervical samples, 413 HR-HPV positive cervical high grade squamous intraepithelial lesion (HSIL) cases and 62 HR-HPV positive cervical cancers. Two SNPs, rs2234671 and rs2623047, were found with significant differences between HR-HPV negative, cytologically-normal samples and HR-HPV positive cervical HSIL cases. In the validation step, these two SNPs were further genotyped in the same set of samples using TaqMan® SNP genotyping assay and/or LightSNiP assay and in additional samples including 83 HR-HPV positive, cytologically-normal cervical samples, 21 HR-HPV positive cervical cancer cases, 129 HR-HPV positive vulval intraepithelial neoplasia cases and 23 HR-HPV positive vulval cancer cases. Statistical analysis was then performed based on pooled and re-grouped genotyping data of the above-mentioned samples under different genetic models so as to evaluate the associations with different stages in the disease process. After validation, SULF1 rs2623047 revealed a strong significant association with the susceptibility to HR-HPV infection but not with the development of high-grade squamous intraepithelial lesion and the progression to cervical cancer. CXCR1 rs2234671, by contrast, was associated with the progression of HR-HPV-related cancers and the minor allele CXCR1 827C was significantly enriched in HPV16 positive cancers. CXCR1 is a receptor for the chemokine CXCL8/IL-8 and CXCR1 rs2234671 leads to a serine to threonine change in an extracellular loop of the receptor. Functionally, the CXCR1 827C allele was shown to enhance cell motility in response to IL-8 stimulation in a chemotaxis assay with transiently transfected fibroblasts (HEK293 cells) and also in a wound healing assay with stably transduced cervical cancer (CaSki) cells. In addition, significantly increased cell proliferation upon IL-8 treatment was observed in two cervical cancer derived cell lines, CaSki and SiHa, transduced with CXCR1-827C allele, but not in their CXCR1 827G transduced counterparts. These findings suggest that SULF1 rs2623047 and CXCR1 rs2234671 may be genetic risk factors for HR-HPV-related cervical disease and CXCR1 rs2234671 might affect HR-HPV-related cancer susceptibility by functionally altering IL-8-CXCR1 signalling. This information has potential for use in the risk stratification of HR-HPV infected women and may also suggest new therapeutic targets to be exploited for treatment of cervical cancer patients.

PROJECT ONE Introduction HPV is detected in about 99.7% of cervical cancers. However, the HPV type distribution in South African women is unknown. Objectives To determine HPV-type distribution among women with cervical dysplasia in relation to oral contraceptive usage. Methods Prospective cross-sectional study of four groups of patients according to oral contraceptive usage: non-users, users of less than five years duration, users of between five years and ten years and users of more than ten years duration. Swabs of the cervix were analysed for HPV DNA using polymerase chain reaction method. Results A total of 124 women were recruited for the study. There were 75 HIV-infected patients (seroprevalence 61%). Of the 102(82%) HPV-positive patients, 79 patients had high-risk HPV DNA (78%). In terms of the four oral contraceptive groups, high-risk HPV DNA was detected in 70% (n=21), 79% (n=22), 90% (n=21) and 71% (n=15) of patients, respectively. The odds of having HPV DNA was six times higher for the combination of contraceptive users of less than 5 years duration/non-users (OR 5.9, 95% CI: 1.87 - 18.77). There was no change when adjustment was made for age (OR 6.1, 95% CI: 1.9 - 19.4). HPV DNA types 16 and or 18 was present in a total of 21 patients (49%) (non-contraceptive users and users < 5years duration) versus 15 patients (42%) who used oral contraceptives of more than 5 years duration (p=0.524). HPV type 16 was the commonest HPV type detected (20.2%) and HPV type 58 was the next commonest high-risk HPV type (16.1%). HPV types 58 and 33 was detected in a much greater percentage of our population and HPV 16 in a much smaller percentage of our population compared with a non-South African population. Conclusion The findings of this study demonstrate an interesting distribution of HPV types in a South African population. PROJECT TWO Introduction Various risk factors have been implicated in the causation of cervical cancer including human papillomavirus (HPV), the early genes (E6 and E7) of which encode the main transforming proteins. Studies have suggested that steroid hormones may enhance the expression of these genes leading to loss of p53 gene-mediated cell apoptosis. Methods A total of 120 cervical tissue samples were obtained from patients with proven cervical cancer. Patients who used depo-medroxyprogesterone acetate steroid contraception were recruited as part of the study arm. Only HPV DNA type 16 samples were used for the study. Controls included three cell lines (CaSki, SiHa,&C33A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal housekeeping gene. Of 120 patients, there were 111 patients with HPV type 16 identified. Of this number, RNA was present in 63 samples. There were 30 women (30/63) who used steroid contraception. In relation to patients who used contraception, HPV 16 E6 gene expression was present in 79% (n = 23) and 88% (n = 30) of steroid users compared to nonusers, respectively. In total there were 25 patients (40%) with expression of the HPV 16 E6*I gene and 30 patients with expression of the E6*II gene. There were 57% of steroid users (n = 17) who had expression of the E6*I/E6*II gene, compared to 52% (n = 17) of nonusers (P = 0.800). Conclusion From a molecular level, this study reflects almost similar distribution of the HPV 16 E6/E6*1 and E6*11 and does not confirm the role of injectable progesterones in cervical carcinogenesis. Further studies with larger patient numbers are needed. / Thesis (PhD)--University of Pretoria, 2011. / Obstetrics and Gynaecology / unrestricted

Background: Worldwide efforts have been made by some countries to offer HPV vaccination since its introduction in 2006. Population effectiveness of HPV vaccines is presently an active area of research. We review available evidence on the effectiveness of HPV vaccine uptake among female adolescents to prevent HPV infection. Methods: A comprehensive search of published and grey literature was conducted in several electronic databases using a pre-defined search strategy related to HPV prevalence following vaccination. The database searches were complemented by hand-searches of reference lists of eligible studies. Data were extracted onto a purpose-designed data extraction form, pooled in a meta-analysis and stratified by continent considering vaccine type, cross protective and (high/low) risk HPV types as subgroups. Results: Our search yielded 1680 studies, of which thirteen met with our inclusion criteria (8332 vaccinated women aged 12 to 34 years from across the world). The pooled HPV (comprising types 6, 11, 16 and 18) prevalence among vaccinated female adolescents was 7% (95% Confidence Interval (CI): 5% to 9%, 13 studies, n=8,332). The 13 studies were conducted across 3 continents: HPV prevalence for North America was 5% (95% CI: 3% to 7%, 9 studies, n=5781, age range =13 to 34); Europe, 14% (95% CI: 9% to 18%, 3 studies, n=2213, age range =13 to 29) and Australia 5% (95% CI: 3% to 8%, 1 study, n=5781, age range=13 to 34). Of the studies which reported the effect of vaccination on other non-vaccine HPV type prevalence (known as cross protective types) HPV (31, 33, 45, 51 &amp; 58), the overall pooled cross protective HPV prevalence was 9% (95% CI: 6% to 12%, 4 studies, n=3081 age range=13 to 29), by continent North America had 14% (95% CI: 12 to 17%, 1 study, n=753 age range=14 to 24), Europe 7% (95% CI: 6 to 8%, 2 studies, n=1990, age range=13 to 29) and Australia with 8% (95% CI: 5% to 11%, 1 study, n=338 age range=18 to 26). Conclusion: This study showed an HPV prevalence of 7% in women vaccinated against HPV types 6,11,16 and 18, which represents a substantial difference to the 22% HPV prevalence in non-vaccinated women. There was no statistically significant difference between HPV prevalence across the continents. There is however, still a dearth of information on vaccinated women and HPV prevalence, highlighting the need for further studies in this area. Strengths and limitation of this review • The review comprehensively searched multiple databases and bibliographies. We had no language restrictions. • We were stringent in the selection of studies as far as vaccination status was concerned. Studies considering HPV prevalence in unvaccinated women were excluded. • A variety of methods was utilised in collecting data across the studies. However, some of the study participants were not representative of the general population. Caution therefore, needs to be considered when using these results to make inferences or conclusions about prevalence of certain populations.

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