OpenUCT :: Browsing by Author "Williamson, Anna-Lise"

Browsing by Author "Williamson, Anna-Lise"

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A deletion and point mutation study of the human papillomavirus type 16 major capsid gene
(2006) Varsani, Arvind; Williamson, Anna-Lise; Jaffer, Mohamed A; Rybicki, Edward P
Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A→T mutation at residue 266 (A266T), and of a C→G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428–483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2–9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55 nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428–465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines.
Open Access
A Study of Genital Human Papillomavirus (HPV) and Evaluation of HPV Testing for Cervical Cancer Screening in Women from the Eastern Cape Province, South Africa
(2021) Taku, Ongeziwe; Williamson, Anna-Lise; Meiring,Tracy L; Mbulawa, Zizipho Z A
Introduction: Human papillomavirus (HPV) infection is an important public health problem facing black African women. Persistent infection with high-risk (HR) HPV types is the key factor for the development of cervical cancer. Coinfection of HPV with other sexually transmitted pathogens contributes to the progression of cervical cancer. Preventative measures including screening for and treating pre-cancerous cervical lesions as well as HPV vaccination have been implemented in parts of South Africa. However, in the rural Eastern Cape Province there is limited information on the prevalence of HPV and the HPV types associated with cervical lesions. Two cohorts were chosen to study HPV in the Eastern Cape (South Africa), a community clinic, and a referral hospital for treatment of cervical lesions. This study aimed at determining the prevalence of HPV, risk factors of HPV, coinfection of HPV with sexually transmitted pathogens and evaluate the performance of a number of HPV tests for HPV detection and cervical cancer screening. The objectives of the study were: • To investigate the prevalence of HR-HPV and factors associated with HR-HPV infection among women from rural Eastern Cape, South Africa. • To investigate the distribution of HPV genotypes among women with cervical intraepithelial lesions according to HIV status from Eastern Cape Province, South Africa. • To investigate HR-HPV prevalence and compare agreement between cliniciancollected and self-collected genital specimens as well as two different HPV tests on clinician-collected samples. • To investigate the prevalence of sexually transmitted pathogens and co-infection of with HR-HPV infection among women from rural Eastern Cape Province, South Africa. Methods: A total of 741 participants were recruited from the Mbekweni Community Clinic (N=417) and the Nelson Mandela Hospital Referral Clinic (N=324) located in the OR Tambo municipality of the Eastern Cape Province. Clinician-collected cervical scrapes from women attending the Community Clinic were screened for HR-HPV prevalence and HR-HPV viral load using Hybrid Capture 2 (HC-2, Qiagen Inc., Gaithersburg, MD; USA); Cervical clinician-collected and vaginal self-collected specimens of women with or without abnormal cytology from both study cohorts were also screened for HR-HPV infection using hpVIR real-time PCR. HPV typing of clinician-collected cervical specimens from women with cervical intraepithelial neoplasia grades 2 and 3 (CIN 2 / 3) was done using Direct Flow Chip HPV kit (Master Diagnostica, Spain). Cervical specimens from the Community Clinic (N=205) were also tested for sexually transmitted infections (STIs) namely Chlamydia trachomatis: CT, Haemophilus ducreyi, Herpes Simplex Virus type 2, Neisseria gonorrhoeae: NG, Treponema pallidum, and Trichomonas vaginalis: TV) and pathobionts (Ureaplasma spp: (UP), Mycoplasma genitalium: MG, and Mycoplasma hominis: MH) using the STD Direct Flow Chip kit (Master Diagnostica, Spain). The univariate and multivariate analysis was used to determine the correlation between HPV infection and potential behavioural risk factors using STATA 14.2 (Stata Corp, College Station, Texas). A chi squared test was used determine the difference in estimated HR-HPV prevalence between self-collected and clinician-collected samples. STIs prevalence and association with behavioural risk factors were analysed using GraphPad Prism v6.01 (GraphPad Software, Inc., San Diego, CA). Results: Of the 417 women from the community clinic, HR-HPV prevalence was significantly higher in HIV-positive women compared to HIV-negative women (40.6%, 63/155 vs 21.4%, 56/262, p< 0.0001). Among women referred to Nelson Mandela Hospital with cervical intraepithelial lesions, HPV prevalence was observed to be significantly higher in HIV-positive than HIV-positive women (98.0% vs 89.1%, p=0.012). Similarly, HIV-positive women (65.3%, 96/147) had higher multiple HPV infections than HIV-negative women (47.8%, 22/46; p=0.034). HPV35 (23.9%), HPV58 (23.9%), HPV45 (19.6%), and HPV16 (17.3%) were the most frequently detected HPV types in CIN2, while HPV35 (22.5%), HPV16 (21.8%), HPV33 (15.6%), HPV58 (14.3%) were commonly detected in women with CIN3 regardless of HIV status. HR-HPV prevalence in clinician-collected samples was equivalent to self-collected samples from both study sites, the community clinic (26.4% vs 27.9%, p=0.601) and the referral clinic (83.6% vs 79.9%, p=0.222). HR-HPV positivity between self-collected and clinician-collected samples showed an agreement of 86.9% for community clinic (k=0.669) and 91.4% for referral clinic (k=0.711). The distribution of HR-HPV genotypes was similar between self-collected and clinician-collected samples from both study sites. The agreement of HR-HPV genotypes between self-collected and clinician ranged from moderate to almost perfect (0.571-0.888). A majority of women reported a high positive response of acceptance for self-collection (community-based clinic: 77.2% and referral clinic: 83.0%). HR-HPV detection agreement between hpVIR real-time PCR and HC-2 was almost perfect (87.7%, k=0.754). The prevalence of the six traditional STIs (CT, TV, NG, HSV-2, TP, and Haemophilus ducreyi) was high (22.9%, 47/205). TV was the most frequently detected STI (15.6%, 32/205). UP (70.2%, 144/205) and MH (36.6%, 47/205) were the most frequently identified pathobionts. Multiple infections/coinfections with more than two STIs/pathobionts was found in 52.7% (108/205) of women with UP/MH (26.9%) and UP/HPV (21.3%) the frequently identified coinfections. HR-HPV infection was significantly associated with HIV infection (p=0.017) and HSV-2 (p=0.026). Conclusion: This study shows that HIV infection and sexual behaviour increased the risk of HPV infection among women from the community clinic. HIV-positive women had significantly higher HPV viral load and multiple HPV type infections compared with HIV-negative women with or without cervical lesions. Since HIV positive women are at higher risk of HPV infection they need to continue to be screened more regularly for cervical lesions and treated when appropriate. In addition, the high prevalence of HPV in the community of HIV negative women indicates that a robust cervical screening programme is needed to implement the cervical screening policy of South Africa. Thus, the women get the allocated three cervical smears in a life time. Distribution of HPV types was similar among women with CIN2 & 3 with HPV35 being the most frequently detected HPV type regardless of HIV status. This highlights the importance for the inclusion of HPV-35 in the next generation of HPV prophylactic vaccines. The findings of this study add to the limited information on genital HPV infection in women from this province. Moreover, our data now acts as a baseline/reference data for future investigations. The data will also contribute to discussions on HPV testing as the primary screening strategy for cervical cancer and HPV vaccination in South Africa. The hpVIR real-time PCR test between self-collected and clinician-collected specimens showed comparable agreement for the detection of HPV infection. The type-specific concordance between self-collected and clinician-collected showed moderate to an excellent agreement, indicating that self-collection can be utilised as the alternative screening tool for cervical cancer. The participants showed a high positive response for the self-collection method, indicating that introducing this method can positively impact the cervical cancer screening program. However, hpVIR real-time PCR is an in-house test which is not practical to introduce on a large scale in South Africa. Therefore, future research should be done to determine what other HPV tests could be done on these types of specimens. Presently, syndromic management is used to treat STI at clinics in South Africa. The high prevalence of sexually transmitted pathogens necessitates the need to enhance the current screening methods for these pathogens.
Open Access
A study of the genital microbiotas of black South African women and men: associations with human papillomavirus and HIV infections
(2018) Onywera, David Harris; Meiring, Tracy L; Williamson, Anna-Lise
Persistent genital infection with oncogenic or high-risk human papillomavirus (HPV) is causally associated with cervical cancer in women and some penile cancers in men. The role of the complex genital microbiota in HPV infection has not been extensively addressed. This study characterised the genital microbiotas of heterosexually-active Black South African women and men, predominantly of the Xhosa ethnicity, recruited from a community in Cape Town, South Africa. The association of the genital microbiotas with prevalent HPV, HIV, demographic, behavioural, and clinical characteristics of the participants was examined. In Chapter 2 the bacterial communities in cervicovaginal samples from 62 HIVseronegative South African women were profiled by Ion Torrent PGM sequencing of the V4 hypervariable region of the bacterial 16S rRNA gene (IT-V4 method). The cervicovaginal microbiotas (CVMs) were found to cluster into three distinct community state types (CSTs): Lactobacillus iners-dominated CVMs (CST I (38.7%, 24/62)), unclassified Lactobacillusdominated CVMs (CST II (4.8%, 3/62)), and diverse CVMs (CST III (56.5%, 35/62)) with an array of heterogeneous bacteria, predominantly the bacterial vaginosis (BV)-associated Gardnerella, Prevotella, Sneathia, and Shuttleworthia. The majority of the women had nonLactobacillus-dominated CVMs. Lactobacilli are recognised as protective against sexually transmitted infections. Among the Lactobacillus species detected in the women, L. iners was the most prevalent and abundant. This species is recognised as the least protective amongst the vaginal lactobacilli. Women in CST I were more likely to be on hormonal contraception compared to women in CST III (relative risk (RR): 2.6 [95% CI 1.3-5.3]; p=0.005). Further research is required to confirm this association and to determine the biological mechanism. Microbiome research methodologies are constantly improving and in Chapter 3 the performance of two bacterial 16S rRNA gene amplicon-based methodologies were compared. The CVMs of 19 women were characterised using the IT-V4 method (Chapter 2) and using the Illumina 16S rRNA metagenomics method (IM-V3/V4 method). The latter method involves sequencing the V3 and V4 hypervariable regions of the 16S rRNA gene on the Illumina MiSeq platform. The two methods showed a high degree of correlation (r=0.89, p< 0.0001) in the average relative abundance of shared bacterial taxa. Procrustes analyses of the weighted UniFrac distances further showed a statistically consistent clustering between the two methods (M 2 =0.3, p< 0.0001). The IM-V3/V4 method proved to have a greater throughput, longer read-length, and lower error rates than the IT-V4 method and was therefore used in the subsequent chapters (4 and 5). In Chapter 4, the CVMs of 87 HIV-seronegative women from the same cohort were examined using the IM-V3/V4 method. The CVMs clustered into eight CSTs. Only 23 women (26.4%) had CVMs dominated by a single Lactobacillus species, this included two women (2.3%) with L. crispatus (CST-1), two (2.3%) with L. jensenii (CST-2), and 19 (21.8%) with L. iners (CST-3). The majority of the women (64.4% (56/87)), however, had diverse and heterogeneous CVMs (CST-8) that were associated with BV (p2, p<0.05). In the final experimental chapter, the penile microbiotas of 238 Black South African men were characterised. This is the first large-scale study of the penile microbiota of South African men. Corynebacteriaceae (47.2%) and Prevotellaceae (6.6%) were found to be the most abundant bacterial families. The penile bacterial communities clustered into six CSTs (designated 1-6). A majority of the men (53.4% (127/238)) had Corynebacterium-dominated microbiotas (CST-1). The remaining CSTs (2-6) had lower relative abundances of Corynebacterium than CST-1 and were colonised with several vaginal bacteria. The prevalences of these CSTs (2-6) in men together with their respective most abundant genera (besides Corynebacterium) were as follows: CST-2 (9.2%; unclassified Clostridiales and Porphyromonas), CST-3 (8.8%; Gardnerella), CST-4 (7.6%; Chryseobacterium and Acinetobacter), CST-5 (18.5%; unclassified Clostridiales and Porphyromonas), and CST-6 (2.5%; Lactobacillus). One hundred and thirty (54.6%) and 102 (42.9%) of the men were positive for HPV and high-risk HPV, respectively, as detected by the Roche Linear Array HPV Genotyping assay. Of the 130 HPV-positive men, 37 (28.5%) and 93 (71.5%) had single and multiple HPV types, respectively. Men in CST-1 were less likely to have high-risk HPV and multiple HPV infections relative to men in CSTs 2-6 (RR: high-risk HPV: 0.8 [95% CI 0.6-1.0]; p=0.027 and multiple HPV: 0.8 [95% CI 0.6-1.0]; p=0.042). LefSe revealed that prevalent HPV infection was strongly associated with higher relative abundances of Sneathia, Porphyromonas, Prevotella, Dialister, and Campylobacter (LDA score >3, p3, p< 0.05). The relative abundances of the latter three bacteria together with Peptoniphilus were strongly associated with high-risk HPV infection (LDA score >3, p <0.05). In our cohort, 88 men (37.0%) were positive for HIV. Of these, 71.6% and 60.2% were positive for HPV and highrisk HPV infection, respectively. Among the HIV-negative men (n=150), 44.7% and 32.7% were positive for HPV and high-risk HPV infection, respectively. Although HIV status did not impact the overall composition of the penile microbiotas, HIV-infected men had higher relative abundances of Staphylococcus, Faecalibacterium, Strenotrophominas, Jonquetella, Ruminococcus, Roseburia, and Lamia (LDA score >2, p<0.05) Men with BV-negative female sexual partners (66.5% (157/236)) had higher relative abundances of Lactobacillus in their penile microbiotas than men with BV-positive female partners (p=0.007). Atopobium, Sneathia, and Saccharofermentans were significantly more prevalent in men with BV-positive female partners than men with BV-negative partners (p<0.020). The main limitations of our study include relatively small sample size of women, insufficient participant information such as host genetics, other STIs (e.g., herpes simplex virus) and abnormal vaginal flora (e.g., aerobic vaginitis), using a less sensitive method to diagnose BV in women, and inherent biases evident in any retrospective study. Moreover, we did not adjust for confounding factors in our analysis due to the small sample size. Despite the underscored limitations, our findings provide insight into the baseline genital microbiotas of the Black South African women and men. The associations identified in this cross-sectional study between specific microbiota members and HPV infection, particularly the association between Sneathia and HPV/high-risk HPV infection, identified in both women and men, are hypothesis-generating and warrant further investigation. The study forms a critical starting point for future longitudinal confirmatory association studies and studies examining these bacteria as potential biomarkers or risk factors for HPV infection.
Open Access
Abrogation of contaminating RNA activity in HIV-1 Gag VLPs
(BioMed Central Ltd, 2011) Valley-Omar, Ziyaad; Meyers, Ann; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward
BACKGROUND: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. METHODS: HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. RESULTS: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/mug Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. CONCLUSIONS: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.
Open Access
Advancements in the Growth and Construction of Recombinant Lumpy Skin Disease Virus (LSDV) for Use as a Vaccine Vector
(2021-10-04) van Diepen, Michiel; Chapman, Rosamund; Douglass, Nicola; Whittle, Leah; Chineka, Nicole; Galant, Shireen; Cotchobos, Christian; Suzuki, Akiko; Williamson, Anna-Lise
Attenuated vaccine strains of lumpy skin disease virus (LSDV) have become increasingly popular as recombinant vaccine vectors, to target both LSDV, as well as other pathogens, including human infectious agents. Historically, these vaccine strains and recombinants were generated in primary (lamb) testis (LT) cells, Madin–Darby bovine kidney (MDBK) cells or in eggs. Growth in eggs is a laborious process, the use of primary cells has the potential to introduce pathogens and MDBK cells are known to harbor bovine viral diarrhea virus (BVDV). In this study, data is presented to show the growth of an attenuated LSDV strain in baby hamster kidney (BHK-21) cells. Subsequently, a recombinant LSDV vaccine was generated in BHK-21 cells. Partial growth was also observed in rabbit kidney cells (RK13), but only when the vaccinia virus host range gene K1L was expressed. Despite the limited growth, the expression of K1L was enough to serve as a positive selection marker for the generation of recombinant LSDV vaccines in RK13 cells. The simplification of generating (recombinant) LSDV vaccines shown here should increase the interest for this platform for future livestock vaccine development and, with BHK-21 cells approved for current good manufacturing practice, this can be expanded to human vaccines as well.
Open Access
The allelic distribution of -308 Tumor Necrosis Factor-alpha gene polymorphism in South African women with cervical cancer and control women
(BioMed Central Ltd, 2006) Govan, Vandana; Constant, Debbie; Hoffman, Margaret; Williamson, Anna-Lise
BACKGROUND:Cervical cancer is due to infection with specific high-risk types of human papillomavirus (HPV). Although the incidence of genital HPV infection in various population groups is high, most of these regress without intervention. Investigating genetic host factors and cellular immune responses, particularly cytokines, could help to understand the association between genital HPV infection and carcinogenesis. The tumor necrosis factor alpha (TNF-alpha) cytokine plays an important role in all stages of cervical cancer and has the ability to induce the regression of human tumors. Therefore the aim of the study was to investigate the allelic distribution of -308 TNF-alpha gene polymorphism in South African women with cervical cancer compared to control women. METHODS: Included in our study were women with histologically proven cancer of the cervix (n = 244) and hospital-based controls (n = 228). All patients and controls were from mixed race and black population groups in South Africa. The detection of a bi-allelic -308 (A/G) polymorphism in the promoter region of TNF-alpha was investigated using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. The distributions of the allelic frequencies were stratified in both patients and controls into two South African ethnic population groups. RESULTS: In this study we observed no association between the distribution of -308 TNF-alpha polymorphism and the risk of developing cervical cancer even after combining the data from the two ethnic populations (X2 = 2.26). In addition, using the chi-squared test we found no significant association between the known risk factors for cervical cancer and the allele distribution of -308 TNF-alpha. However, the frequency of the rare high-producing allele -308A of TNF-alpha was significantly lower in the South African population when compared to Caucasians and Chinese population groups. CONCLUSION: We demonstrated no association between -308 TNF-alpha polymorphism and the risk of cervical cancer among two South African ethnic population groups. However, as the distribution of the -308A TNF-alpha was notably different between the control groups of South Africa and other population groups this result suggests that ethnic disparity may influence the levels of TNF-alpha produced.
Open Access
Assessment of an LSDV-Vectored Vaccine for Heterologous Prime-Boost Immunizations against HIV
(2021-11-05) Chapman, Ros; van Diepen, Michiel; Douglass, Nicola; Galant, Shireen; Jaffer, Mohamed; Margolin, Emmanuel; Ximba, Phindile; Hermanus, Tandile; Moore, Penny L; Williamson, Anna-Lise
The modest protective effects of the RV144 HIV-1 vaccine trial have prompted the further exploration of improved poxvirus vector systems that can yield better immune responses and protection. In this study, a recombinant lumpy skin disease virus (LSDV) expressing HIV-1 CAP256.SU gp150 (Env) and a subtype C mosaic Gag was constructed (LSDVGC5) and compared to the equivalent recombinant modified vaccinia Ankara (MVAGC5). In vitro characterization confirmed that cells infected with recombinant LSDV produced Gag virus-like particles containing Env, and that Env expressed on the surface of the cells infected with LSDV was in a native-like conformation. This candidate HIV-1 vaccine (L) was tested in a rabbit model using different heterologous vaccination regimens, in combination with DNA (D) and MVA (M) vectors expressing the equivalent HIV-1 antigens. The four different vaccination regimens (DDMMLL, DDMLML, DDLMLM, and DDLLMM) all elicited high titers of binding and Tier 1A neutralizing antibodies (NAbs), and some regimens induced Tier 1B NAbs. Furthermore, two rabbits in the DDLMLM group developed low levels of autologous Tier 2 NAbs. The humoral immune responses elicited against HIV-1 Env by the recombinant LSDVGC5 were comparable to those induced by MVAGC5.
Open Access
CCR2-V64I polymorphism is associated with increased risk of cervical cancer but not with HPV infection or pre-cancerous lesions in African women
(BioMed Central Ltd, 2010) Chatterjee, Koushik; Dandara, Collet; Hoffman, Margaret; Williamson, Anna-Lise
BACKGROUND: Cervical cancer, caused by specific oncogenic types of human papillomavirus (HPV), is the second most common cancer in women worldwide. A large number of young sexually active women get infected by HPV but only a small fraction of them have persistent infection and develop cervical cancer pointing to co- factors including host genetics that might play a role in outcome of the HPV infection. This study investigated the role of CCR2-V64I polymorphism in cervical cancer, pre-cancers and HPV infection in South African women resident in Western Cape. CCR2-V64I polymorphism has been previously reported to influence the progression to cervical cancer in some populations and has also been associated with decreased progression from HIV infection to AIDS. METHODS: Genotyping for CCR2-V64I was done by PCR-SSP in a case-control study of 446 women (106 black African and 340 mixed-ancestry) with histologically confirmed invasive cervical cancer and 1432 controls (322 black African and 1110 mixed-ancestry) group-matched (1:3) by age, ethnicity and domicile status. In the control women HPV was detected using the Digene Hybrid Capture II test and cervical disease was detected by cervical cytology. RESULTS: The CCR2-64I variant was significantly associated with cervical cancer when cases were compared to the control group (P = 0.001). Further analysis comparing selected groups within the controls showed that individuals with abnormal cytology and high grade squamous intraepitleial neoplasia (HSIL) did not have this association when compared to women with normal cytology. HPV infection also showed no association with CCR2-64I variant. Comparing SIL positive controls with the cases showed a significant association of CCR2-64I variant (P = 0.001) with cervical cancer. CONCLUSIONS: This is the first study of the role of CCR2-V64I polymorphism in cervical cancer in an African population. Our results show that CCR2-64I variant is associated with the risk of cervical cancer but does not affect the susceptibility to HPV infection or HSIL in South African women of black and mixed-ancestry origin. This result implies that the role of CCR2 is important in invasive cancer of the cervix but not in HPV infection or in the development of pre-cancers.
Open Access
Cellular immune responses to human papillomavirus (HPV) type 16 at the cervix of women with HPV-associated squamous intraepithelial neoplasia
(2005) Milner, Michelle; Passmore, Jo-Ann; Williamson, Anna-Lise
Cervical cancer is the most common cause of cancer-related death in black South African women. Human papillomavirus (HPV) has been found to be a necessary causative agent of cervical cancer and has been reported to be associated with 84% of cervical intraepithelial neoplasia (CIN). HPV type 16 (HPV-16) is the most prevalent HPV type associated CIN and cervical cancer with ±56% of women with cervical disease being infected with HPV 16. Yet studies have shown that 47-85% of CIN regressed, suggesting that perhaps an effective immune response could result in HPV clearance and lesion regression. Since HPV infection does not disseminate and there is no systemic phase of infection, it is hypothesized that local cervical immune responses are important in lesion regression and clearance of HPV infection. There are, however, very few studies of mucosal immune responses to HPV infection. The aim of this study was to determine the type of mucosal immune response elicited by the CD4 and CD8 T cell subsets to HPV infection at the cervix of women diagnosed with varying grades of CIN and to compare these to systemic responses.
Open Access
Characterisation and evolutionary dynamics of ten novel Gammapapillomavirus types from South African penile swabs
(2019) Murahwa, Alltalents Tutsirayi; Williamson, Anna-Lise; Meiring,Tracy L
Human papillomaviruses (HPVs) are genetically diverse, belonging to five distinct genera: Alpha, Beta, Gamma, Mu and Nu. We discovered ten novel Gammapapillomaviruses (Gamma-HPVs). Genomic characterisation and phylogenetic evaluation of the ten novel Gamma-HPV types were done: HPV211, HPV212, HPV213, HPV214, HPV215, HPV216, HPV219, HPV220, HPV221 and HPV222. These HPVs were previously identified in a study that was done on 218 penile samples (104 HIV negative and 114 HIV positive) using high throughput sequencing (Roche 454) of amplimers obtained using FAP59/64 primers which were designed to detect “cutaneous” or Beta- and Gamma-HPVs. Fifteen putative novel HPV types were identified from the short HPV L1 FAP fragments HPV211 (CT02, KY063000), HPV212 (CT03, KY063001), HPV213 (CT04, KY063002), HPV214 (CT06, KY063004), HPV215 (CT07, KY063005), HPV216 (CT12, KY063010), HPV219 (CT01, KY062999), HPV220 (CT08, KY063006), HPV221 (CT09, KY063007) and HPV222 (CT155, AY009886) with prevalences varying from 0.5% to 4.1% of men sampled. Multiple full genome clones for each novel type were generated through whole genome amplification, cloning and next generation sequencing. Complete genome sizes were: HPV211 (7253 bp), HPV212 (7208 bp), HPV213 (7096 bp), HPV214 (7357 bp), HPV215 (7186 bp), HPV216 (7233 bp), HPV219 (7108 bp), HPV220 (7381 bp), HPV221 (7326 bp) and HPV222 (7275 bp). Phylogenetically the novel Papillomaviruses (PVs) all clustered with Gamma-HPVs: HPV211 is most closely related to HPV168 (72% identity in the L1 nucleotide sequence) of the Gamma-8 species, HPV212 is most closely related to HPV144 (82.9%) of the Gamma-17 species, HPV213 is most closely related to HPV153 (71.8%) of the Gamma-13 species, HPV214 is most closely related to HPV103 (75.3%) of the Gamma-6 species, HPV215 and HPV216 are most closely related to HPV129 (76.8% and 79.2% respectively) of the Gamma-9 species. HPV219 is phylogenetically most closely related to HPV213 (87% identity in L1 gene) of the Gamma-13 species, HPV220 to HPV212 (72%) of Gamma-17, HPV221 to HPV142 (80%) of Gamma-10, HPV222 to HPV162 (73%) of Gamma-19. The novel HPV types demonstrated the classical genomic organisation of Gamma-HPVs, with seven open reading frames (ORFs) encoding five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) proteins. Typical of Gamma-HPVs, the novel types all lacked the E5 ORF and HPV214 also lacked the E6 ORF. We further examined variation of the novel types in clinical specimens from which they were identified. All the clones of HPV211, HPV214, HPV216, HPV219 and HPV221 were identical and showed 100% pairwise identity. The clones of HPV213, HPV215, HPV212, HPV220 and HPV222 had several differences. Analysis of mismatches between the nine genomic clones of HPV212 showed a total of 67 mismatch positions that varied along the 7208 bp genome and all the clones were unique. Analysis of mismatches between the 10 genomic clones of HPV213 showed a total of 51 mismatch positions that varied along the 7096 bp genome and it had 5 unique clones. The 6 genomic clones of HPV215 showed a total of 50 mismatch positions along a 7186 bp genome and it had 3 identical and 3 different clones. HPV220 had 4 different genomic clones that showed 17 mismatch positions along a 7381 bp genome. The 5 different clones of HPV222 showed a total of 24 mismatch positions along the 7275 bp genome. Conserved domains observed among the novel types were the Zinc finger binding Domain and PDZ domains. A retinoblastoma binding protein (pRB) binding domain in the E7 protein was additionally identified in HPV214 and HPV222. PVs are thought to evolve slowly because they co-opt high-fidelity host cellular DNA polymerases for their replication. Despite extensive efforts to catalogue all the HPV species that infect humans, it is likely that many still remain undiscovered. We used the genome sequences of the ten novel viruses and related HPVs to analyse the evolutionary dynamics of these viruses at the whole genome and individual gene scales. We found statistically significant incongruences between the phylogenetic trees of different genes which imply gene-to-gene variation in the evolutionary processes underlying the diversification of Gamma-PVs. We were, however, only able to detect convincing evidence of a single recombination event which, on its own, cannot explain the observed incongruences between gene phylogenies. The divergence times of the last common ancestor (LCA) of the Alpha, Beta, Mu, Nu and Gamma genera was predicted to have existed between 49.7-58.5 million years ago before splitting into the five main lineages. The LCA of the presently sampled Gamma-PVs was predicted to have existed between 45.3 and 67.5 million years ago: approximately at the time when the simian and tarsier lineages of the primates diverged. The discovery, characterisation and classification of HPV211, HPV212, HPV213, HPV214, HPV215 HPV216, HPV219, HPV220, HPV221 and HPV222 add these novel types to the repertoire of the ever expanding Gamma-HPVs genus hence expanding our knowledge of these viruses.
Open Access
Characterization of a Novel Chimeric Theileria parva p67 Antigen Which Incorporates into Virus-like Particles and Is Highly Immunogenic in Mice
(2022-01-28) Whittle, Leah; Chapman, Ros; van Diepen, Michiel; Rybicki, Edward P; Williamson, Anna-Lise
The current method to protect cattle against East Coast Fever (ECF) involves the use of live Theileria parva sporozoites. Although this provides immunity, using live parasites has many disadvantages, such as contributing to the spread of ECF. Subunit vaccines based on the sporozoite surface protein p67 have been investigated as a replacement for the current method. In this study, two DNA vaccines expressing recombinant forms of p67 designed to display on retrovirus-like particles were constructed with the aim of improving immunogenicity. The native leader sequence was replaced with the human tissue plasminogen activator leader in both vaccines. The full-length p67 gene was included in the first DNA vaccine (p67); in the second, the transmembrane domain and cytoplasmic tail were replaced with those of an influenza A virus hemagglutinin 5 (p67HA). Immunofluorescent staining of fixed and live transfected mammalian cells showed that both p67 and p67HA were successfully expressed, and p67HA localised on the cell surface. Furthermore, p67HA was displayed on the surface of both bovine leukaemia virus (BLV) Gag and HIV-1 Gag virus-like particles (VLPs) made in the same cells. Mice vaccinated with DNA vaccines expressing p67 and p67HA alone, or p67HA with BLV or HIV-1 Gag, developed high titres of p67 and BLV Gag-binding antibodies. Here we show that it is possible to integrate a form of p67 containing all known antigenic domains into VLPs. This p67HA–VLP combination has the potential to be incorporated into a vaccine against ECF, as a DNA vaccine or as other vaccine platforms.
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Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles.
(Elsevier, 2008) Halsey, Richard J; Tanzer, Fiona L; Meyers, Ann; Pillay, Sirika; Lynch, Alisson; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward P
HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines.
Open Access
Clinical validation of the HPVIR high-risk HPV test on cervical samples according to the international guidelines for human papillomavirus DNA test requirements for cervical cancer screening
(2019-08-22) Gustavsson, Inger; Aarnio, Riina; Myrnäs, Mattias; Hedlund-Lindberg, Julia; Taku, Ongeziwe; Meiring, Tracy; Wikström, Ingrid; Enroth, Stefan; Williamson, Anna-Lise; Olovsson, Matts; Gyllensten, Ulf
Abstract Background The indicating FTA card is a dry medium used for collection of cervical samples. HPVIR is a multiplex real-time PCR test that detects 12 high-risk human papillomavirus types (hrHPV) and provides single genotype information for HPV16, − 31, − 35, − 39, − 51, − 56, and − 59 and pooled type information for HPV18/45 and HPV33/52/58. The aim of this study was to evaluate whether a strategy with cervical samples collected on the FTA card and subsequently analysed with the HPVIR test complies with the criteria of the international guidelines for a clinically validated method for cervical screening. Methods We performed a non-inferiority test comparing the clinical sensitivity and specificity of the candidate test (FTA card and HPVIR) with a clinically validated reference test (Cobas® HPV test) based on liquid-based cytology (LBC) samples. Two clinical samples (LBC and FTA) were collected from 896 participants in population-based screening. For evaluation of the specificity we used 799 women without ≥ CIN2, and for clinical sensitivity we used 67 women with histologically confirmed ≥ CIN2. The reproducibility was studied by performing inter- and intra-laboratory tests of 558 additional clinical samples. Results The clinical sensitivity and specificity for samples collected on the FTA card and analysed using the HPVIR test were non-inferior to samples analysed with the Cobas® HPV test based on LBC samples (non-inferiority test score, p = 1.0 × 10− 2 and p = 1.89 × 10− 9, respectively). Adequate agreement of > 87% was seen in both the intra- and inter-laboratory comparisons. Conclusions Samples collected on the indicating FTA card and analysed with HPVIR test fulfil the requirements of the international guidelines and can therefore be used in primary cervical cancer screening.
Open Access
Comparative analysis of avian poxvirus genomes, including a novel poxvirus from lesser flamingos (Phoenicopterus minor), highlights the lack of conservation of the central region
(BioMed Central, 2017-12-06) Carulei, Olivia; Douglass, Nicola; Williamson, Anna-Lise
Background: Avian poxviruses are important pathogens of both wild and domestic birds. To date, seven isolates from subclades A and B and one from proposed subclade E, have had their genomes completely sequenced. The genomes of these isolates have been shown to exhibit typical poxvirus genome characteristics with conserved central regions and more variable terminal regions. Infection with avian poxviruses (APVs) has been reported in three species of captive flamingo, as well as a free-living, lesser flamingo at Kamfers dam, near Kimberley, South Africa. This study was undertaken to further characterise this virus which may have long term effects on this important and vulnerable, breeding population. Results: Gene content and synteny as well as percentage identities between conserved orthologues was compared between Flamingopox virus (FGPV) and the other sequenced APV genomes. Dotplot comparisons revealed major differences in central regions that have been thought to be conserved. Further analysis revealed five regions of difference, of differing lengths, spread across the central, conserved regions of the various genomes. Although individual gene identities at the nucleotide level did not vary greatly, gene content and synteny between isolates/species at these identified regions were more divergent than expected. Conclusion: Basic comparative genomics revealed the expected similarities in genome architecture but an in depth, comparative, analysis showed all avian poxvirus genomes to differ from other poxvirus genomes in fundamental and unexpected ways. The reasons for these large genomic rearrangements in regions of the genome that were thought to be relatively conserved are yet to be elucidated. Sequencing and analysis of further avian poxvirus genomes will help characterise this complex genus of poxviruses.
Open Access
A comparative analysis of cowpox virus (CPV WT) and a deletion mutant lacking the gene encoding the inflammation modulatory protein (CPV IMP)
(2007) Paulsen, Janis; Douglass, Nicola; Williamson, Anna-Lise
Cowpox virus has been found to encode the inflammation modulatory protein (IMP) (Miller, C.G., 1997), a homologue vaccinia virus complement control protein (VCP). VCP belongs to regulation of complement activation (RCA) protein superfamily. It has been shown to inhibit both the alternative and classical pathways of complement activation by binding to the proteins C3 and C4, thereby preventing complementmediated opsonisation of virus, antibody-mediated lysis of infected cells and migration of inflammatory cells into the site of infection. VCP also possesses heparin binding sites.
Open Access
Comparison of the two lumpy skin disease virus vaccines, Neethling and Herbivac, and construction of a recombinant Herbivac-Rift Valley fever virus vaccine
(2015) Omar, Ruzaiq; Williamson, Anna-Lise; Douglass, Nicola
There are two broad aims to this project. The first aim is to compare and characterise two lumpy skin disease virus (LSDV) vaccines namely the vaccine based on attenuated Neethling LSDV (nLSDV) and HerbivacÂ®LS (Herbivac). The second aim is to construct a recombinant LSDV expressing Rift Valley fever virus (RVFV) genes. An LSDV vaccine is critical for sustainable control of lumpy skin disease (LSD). There are four commercially available live attenuated vaccines for LSDV, nLSDV, Herbivac, Lumpyvax and the Kenyan strain sheeppox virus (KS-1). In this study Herbivac was characterised by comparing it to its parent, nLSDV. Growth curves of the two viral strains were conducted in cell culture as well as in embryonated hensâ€™ eggs. No notable difference in the growth rate of the two strains could be detected when the viruses were grown in cell culture, however a notable difference was detected when the viruses were grown on the chick allantoic membranes (CAMs) of embryonated hensâ€™ eggs. When grown on CAMs a faster growth rate was observed for nLSDV compared to Herbivac. nLSDV also killed the embryos at 4 d.p.i where Herbivac did not. The two strains were then further characterised through histological analysis of CAMs after infection with each of the viruses. Overall, higher levels of hyperplasia and hypertrophy were observed in CAMs infected with either nLSDV or Herbivac compared to uninfected CAMs. Herbivac-infected CAMs resulted in thicker chorionic membranes and larger pocks compared to nLSDV. RVFV and LSDV both contribute to the disease burden among cattle in Africa and the Arabian Peninsula. The main aim of this study was to construct a recombinant Herbivac which expresses immunogenic proteins of Rift Valley fever virus (Herbivac-RVFV). Herbivac-RVFV was designed to express specific RVFV genes selected for their antigenic properties. The genes selected are also representative of the genes from recent viral outbreaks in the horn of Africa. The selection of outbreak relevant RVFV genes involved phylogenetic analysis of all full length M-segment and NC gene sequences available on Genbank. Phylogenetic trees were constructed for M-segments and NC genes and groups identified which were highly representative of sequences from recent outbreaks of the virus. Consensus sequences were derived from these groups and included in the transfer vector. The phylogenetic analysis also revealed that the sequences of current RVFV vaccines are phylogenetically distant from viruses isolated from current outbreaks, although high levels of sequence conservation was maintained across all viral strains. This is the first study in which the RVFV genes coding for proteins that will induce a protective immune response (Gn and Gc, as well as the nucleocapsid (NC) gene) were selected so as to be representative of current outbreak strains of the virus. These genes were inserted between LSDV ORFs 49 and 50, a novel insertion site. The transfer vector also contained an eGFP marker gene and an ECO-GPT selection gene, located outside of the LSDV flanking sequences. This meant a two-step isolation procedure, first to isolate the recombinant containing the entire transfer vector with eGFP and ECO-GPT, and then to isolate a recombinant with only the RVFV genes and not eGFP and ECO-GPT. Transient expression of RVFV proteins in cells infected with Herbivac and then transfected with the transfer vector was confirmed via western blotting and immunofluorescence. Here the proteins Gn, Gc and NC were shown to be expressed. In the present study, a single crossover Herbivac-RVFV recombinant was isolated through multiple passaging of cell lysates, originally obtained from Herbivac-infected FBT cells transfected with the transfer vector, in the presence of mycophenolic-acid selection medium.
Open Access
Construction and evaluation of three candidate vaccines expressing HIV-1 subtype-C mosaic Gag
(2015) Jongwe, Tsungai Ivai; Chapman, Ros; Williamson, Anna-Lise; Douglass, Niki; Chege, Gerald
Of the 35 million people living with HIV-1 globally, approximately 71.4% are in the resource-limited sub-Saharan Africa. The immense sequence diversity of HIV-1, even within subtypes, makes it challenging to develop effective vaccines that target a wide range of HIV subtypes. Mosaic immunogens have been computationally designed to specifically overcome this hurdle by maximizing the inclusion of common T cell epitopes. When compared to consensus immunogens, polyvalent mosaic immunogens of HIV-1 group M have shown increased breadth and depth of antigen-specific T-cell responses. More than 90% of HIV positive individuals in sub-Saharan Africa are infected with HIV-1 subtype C (HIV-1C). We therefore designed, constructed, and evaluated candidate vaccines expressing HIV-1C mosaic Gag (GagM) in a proof of concept study. Gag was chosen as the most appropriate target for a T cell-based vaccine as there are many studies correlating control of HIV viral load with T cell responses to Gag. The immunogen was designed by Fischer et al., 2007 (1). Three different vaccine platforms were chosen based on their different strengths to be used in prime-boost regimens to determine the immunogenicity of HIV-1C GagM in mice. The first was a pantothenic auxotroph of the tuberculosis vaccine Mycobacterium bovis Bacille Calmette Guérin (BCG). The second was a DNA vaccine vector with enhanced expression of transgenes due to a novel enhancer element from porcine circovirus type 1, which has been demonstrated to increase gene expression. The third vaccine vector selected was the well characterised poxvirus modified vaccinia Ankara (MVA).
Open Access
Construction, stability and immunogenicity of recombinant BCG expressing HIV-1 subtype C gag under the control of MtrA promoter, with or without the leader sequences
(2011) Lebeko, Maribanyana R; Chapman, Ros; Williamson, Anna-Lise
This study aimed to compare recombinant mycobacteria expressing HIV-1 gag under the control of different promoters and leader sequences. This was done to determine whether the genetic stability of the recombinant mycobacteria could be improved by modification of these vector features and to gain insight into what types of immune responses may be elicited in mice.
Open Access
Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus) by ESAT-6 and CFP-10 IFN-gamma ELISPOT tests following a tuberculosis outbreak
(BioMed Central Ltd, 2008) Chege, Gerald; Warren, Robin; van Pittius, Nico; Burgers, Wendy; Wilkinson, Robert; Shephard, Enid; Williamson, Anna-Lise
BACKGROUND:Both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates (NHP), indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. Screening of mycobacterial infections in NHP is traditionally done by tuberculin skin test (TST), which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. In this study, we investigated the use of ESAT-6 and CFP-10 for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis (TB). METHODS: Peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay (IFN-gamma ELISPOT) assay were obtained from TST positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture. RESULTS: Both ESAT-6 and CFP-10 IFN-gamma ELISPOT assays were able to detect early M. tuberculosis but also M. intracellulare infection. Although this indicates potential cross-reactivity with M. intracellulare antigens, the method was able to distinguish M. bovis BCG vaccination from M. tuberculosis infection. This assay performed better than the TST, which failed to detect one M. tuberculosis and two early M. intracellulare infections. CONCLUSION: These results suggest that the IFN-gamma ELISPOT assay could improve the detection of M tuberculosis infections when screening NHP. There is some doubt, however, concerning specificity, as the assay scored positive three animals infected with M. intracellulare.
Open Access
Determinants of sexual activity and its relation to cervical cancer risk among South African Women
(BioMed Central Ltd, 2007) Cooper, Diane; Hoffman, Margaret; Carrara, Henri; Rosenberg, Lynn; Kelly, Judy; Stander, Ilse; Denny, Lynnette; Williamson, Anna-Lise; Shapiro, Samuel
BACKGROUND:Invasive cervical cancer is the commonest cause of cancer morbidity and mortality in South African women. This study provides information on adult women's sexual activity and cervical cancer risk in South Africa. METHODS: The data were derived from a case-control study of hormonal contraceptives and cervical cancer risk. Information on age of sexual debut and number of lifetime sexual partners was collected from 524 incident cases and 1541 hospital controls. Prevalence ratios and adjusted prevalence ratios were utilised to estimate risk in exposures considered common. Crude and adjusted relative risks were estimated where the outcome was uncommon, using multiple logistic regression analysis. RESULTS: The median age of sexual debut and number of sexual partners was 17 years and 2 respectively. Early sexual debut was associated with lower education, increased number of life time partners and alcohol use. Having a greater number of sexual partners was associated with younger sexual debut, being black, single, higher educational levels and alcohol use. The adjusted odds ratio for sexual debut < 16 years and [greater than or equal to] 4 life-time sexual partners and cervical cancer risk were 1.6 (95% CI 1.2 - 2.2) and 1.7 (95% CI 1.2 - 2.2), respectively. CONCLUSION: Lower socio-economic status, alcohol intake, and being single or black, appear to be determinants of increased sexual activity in South African women. Education had an ambiguous effect. As expected, cervical cancer risk is associated with increased sexual activity. Initiatives to encourage later commencement of sex, and limiting the number of sexual partners would have a favourable impact on risk of cancer of the cervix and other sexually transmitted infections