The male genital tract is a major site of HIV acquisition and transmission. It is an obvious site for inducing immune responses to candidate HIV vaccines, to prevent infection or halt the spread of the virus. There are relatively few published studies characterising T cells in the male genital tract. A challenge that hampers studies at this mucosal surface is obtaining samples with sufficient immune cells. Therefore, the first aim of this study was to establish an optimised method to isolate immune cells from the male genital tract. Cellular activation and inflammation in the genital tract have important implications for both transmission and acquisition of HIV, since they provide target cells for viral replication. Thus, the second and third aim of this study was to investigate mucosal T cell activation and inflammatory cytokine profiles in semen in HIV?infected and uninfected men, and compare the immune milieu of the genital tract with the systemic compartment.

The aim of this study is to utilise the tetracycline dependent gene regulation system to down-regulate recombinant antigen expression in BCG during the expansion of seed stocks and up-regulate expression prior to or post vaccination.

Vaccination continues to represent the most effective means of providing immunological protection against infectious disease in human populations. With the World Health Organisation (WHO) reporting over 1.2 million AIDS related deaths in 2014, an efficacious HIV1 vaccine is urgently needed. The UCT Human Immunodeficiency Virus (HIV) Vaccine Development Group has focussed on understanding novel HIV-1 vaccine vectors, such as modified BCG, combined with modified vaccinia Ankara in the context of heterologous prime boost regimes. The tuberculosis vaccine Bacillus Calmette–Guérin (BCG) has a wellestablished safety profile as well as notable adjuvant activity with an estimated 3 billion doses administered globally since 1921. The development of modern molecular biology techniques in recent times has led to the creation of modified strains of BCG which have been shown in many cases to be safer and/or more immunogenic than the wild type strains in terms of delivering heterologous antigen. This study focuses on exploiting and combining two particular strategies of rBCG modification. The first is the development of auxotrophic strains that contain deletions geared towards preventing the bacteria from synthesising essential growth compounds or amino acids. An example of this is the ΔpanCD auxotroph of rBCG which does not synthesise pantothenic acid and thus has limited intracellular replication leading to less pathology. The UCT group has previously reported that HIV vaccines vectored by the Pasteur strain of rBCG ΔpanCD induced less pathology but improved immunogenicity as compared to Pasteur WT in the murine model (when used a prime in conjunction with an MVA boost). A second notable BCG modification strategy is the inclusion of exogenous genes to improve the immunogenicity of rBCG as a vaccine vector. An example of this is the insertion of the detoxified Clostridium perfringens toxin, perfringolysin O (pfo), into the rBCG genome. Upon expression, pfo forms pores in the endosome which facilitates translocation of vaccine derived antigen into the cytoplasm. This modification has been shown to lead to cross priming of T cells and improve the induction of vaccine specific CD8+ T cell as compared to controls.

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 &amp; 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.

Includes bibliographical references (leaves 114-147). / The increasing diversity of HIV-1 in different geographic regions presents a challenge for the development of an HIV/AIDS vaccine. Even if proven effective, the ability of a vaccine to have cross-subtype and intra-subtype protection remains an important question. The aim of the study was to investigate cross- and intra-clade T cell immune responses in HIV-1 subtype C-infected individuals. The objectives were to genetically characterise full length gag gene sequences from individuals chronically infected with HIV-1 and then assess the degree of cross-reactive immune responses in an ELlS pot assay using peptide reagents based on vaccine candidates.

Includes bibliographical references (leaves 96-114). / 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.

Includes abstract. Includes bibliographical references.

Includes bibliographical references. / 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.

Includes bibliographical references (leaves 89-95). / This study aimed to investigate whether the mycobacterial replicon may also play a role. The common mycobacterial replicon in episomal E. coli-mycobacterial shuttle vectors contains the truncated rap gene, encoding an auxiliary replication factor, and the repA and repB genes which code for essential replication proteins. Specific aspects of this typical mycobacterial replicon were identified as potential targets to improve stability and increase recombinant protein expression levels, and were modified accordingly.

This study characterised the humoral and cellular immune responses to MVA from a candidate MVA-vectored HIV vaccine in non-human primates, and examined the effect of anti-vector immunity on the response to the HIV immunogens.