Carcinoma of the cervix is frequently diagnosed in the department of Radiation Oncology in Charlotte Maxeke Johannesburg Academic Hospital(CMJAH). It is therefore is a condition of priority and there is scarce literature in the management of HIV positive patients. OBJECTIVES: The primary objective of the research is to determine the overall survival of 2yrs and more, as well as to determine acute and late toxicity for patients completing prescribed radiation treatment. The secondary objective was to determine the impact of highly active antiretroviral therapy on survival and toxicity. The study is limited to HIV positive women presenting with cervical cancer. DESIGN & METHOD: The study is a retrospective study of patients treated at Charlotte Maxeke Johannesburg Academic Hospital between 2008-2009. Inclusion criteria: Females between the ages of 18 and 70, Stages IB2 – Stage IIIB carcinoma of the cervix who have completed planned radiation therapy with or without chemotherapy. The sample size was 151 patients. RESULTS: The mean age was 42.7yrs. The median CD4 count was 309 and 26.2% had CD4 counts below 200.The majority of patients had either Stage IIB (55.0%) or IIIB (31.8%). The total dose to Point A was a median dose of 74Gy. The majority of patients had either Grade II (38.4%) or III (31.1%) toxicity. Significant association between these adverse events and HAART status was rated as p=0.0008. The most common late complication was cystitis (15.9%). Overall survival at 2 years was 100% for Stage I, 92.8% for Stage II and 96% for Stage III. CONCLUSION: The median age was lower than in the HIV negative patients. The acute complications for those not on HAART, were higher in comparison to patients on HAART. The overall survival at 2 yrs. was above 90% for all stages in this study / GR2018

A research report submitted to the faculty of Health Sciences, University of the Witwatersrand in partial fulfillment of the requirements for the Master of Science Degree in Epidemiology. / Antiretroviral therapy (ART) recommendations among HIV – infected pregnant women have been revised several times by the World Health Organization (WHO). Option B+, which is the latest recommendation continues to be rolled out in several countries across the globe but mostly in sub-Saharan Africa. Retention in care and adherence to antiretroviral (ARV) drugs taken by these women remains unclear in this new program. We assessed ability to stay in care (retention) and adherence to ART among HIV – infected women starting life-long treatment during pregnancy and after, at an HIV care and treatment clinic in Ifakara Tanzania. Our study provided an opportunity to understand the trends in adherence to ART and retention in care for this population. Methods We analyzed data of HIV-infected pregnant women registered and starting ART for the first time in the Kilombero and Ulanga Antiretroviral Cohort in 2009 and 2010 with a follow up period of up to two years to 2011 and 2012 respectively. Adherence was by patient self-report (PSR) and was sufficient (good) if the woman took all the prescribed pills of the issued batch or insufficient (poor) if she missed two or more pills. Women that missed two or more consecutive scheduled visits to the clinic were not retained while those that honored their scheduled visits were retained in care. Two sample t test and Wilcoxon rank sum test were used to test predictor outcome associations for continuous variables while Pearson’s and Fisher’s exact tests were used for categorical ones. Hazard ratios of each predictor variable were calculated using Cox regression. Student No: 737395 Page v Results A total of 1,282 HIV – infected women were registered in KIULARCO between 2009 and 2010. Fifty (50) were pregnant and started life-long ART upon registration in this period. Of these, 25 (50%) were registered in 2009 and the other 25 (50%) in 2010. Slightly more than half, 52.2% had CD4 cell counts above 350cells/mm3. Almost half, 49% of the women were registered in their final (third) pregnancy trimester. About 82% were in WHO stage one and 60% of all the 50 women were initiated on AZT/3TC/EFV regimen. Only 5.7% had secondary education while the rest had primary or no education at all. Of the women that reported their partners HIV state, 54.5% had partners that had never tested for HIV. Adherence for all participants was reported as sufficient (good) for the entire period the women were in care. No one had insufficient (poor) adherence. Retention in care was higher during pregnancy than after delivery. Generally, loss to follow up was 40%. About 30% were lost during pregnancy and the majority, 70% lost after they had delivered their babies. There was no evidence to prove that any of the factors studied independently predicted non retention. The most likely time to non retention was six months after delivery. Conclusions Our study, despite small sample size, shows that among women diagnosed HIV – infected and starting life-long ART during pregnancy (Option B+), adherence to ART is sufficient and retention in care similar during and after pregnancy. Counseling on the importance of staying in care especially around the first few months after delivery should be emphasized at ANC.

Background: Multiple factors contribute to mother-to-child transmission (MTCT) of HIV-1, including virological, obstetric and biological factors. Other possible contributory determinants for high MTCT rates include immunological factors such as host genetics and viral genetic variations. Despite several therapeutic, prophylactic and obstetric interventions to reduce the proportion of infants infected during labour and delivery, mechanisms for intrapartum HIV-1 transmission remain elusive and current interventions, could, therefore remain sub-optimal. Much controversy has surrounded the correlation of HIV-1 RNA (viral load) in the systemic and genital compartments of women. The influence of short-term antiretroviral (ARV) drugs on genital tract HIV-1 is also unclear. At the time the present study was initiated, a regimen of maternal intrapartum and neonatal postpartum single-dose Nevirapine (sdNVP) was the standard of care for the prevention of mother-to-child transmission (PMTCT). In most low and middle-income countries, including South Africa, sdNVP has been documented as effective intrapartum HIV-1 prevention based on plasma pharmacokinetic levels, decreased viral loads (HIV-1 RNA) and reduced rates of intrapartum transmission, yet operational studies continue to report high intrapartum transmission rates despite the administration of sdNVP. As a result perinatal HIV-1 transmission remains a significant public health concern in several African countries. Aim: The primary aim of this study was to describe the pharmacological dynamics of Nevirapine in association with virological and immunological risk factors for intrapartum HIV-1 transmission in a South African PMTCT programme where sdNVP was the standard of care. Methods: Following regulatory approval from the Biomedical Research Ethics Committee at the University of KwaZulu-Natal (UKZN), one hundred and twenty pregnant HIV-infected women who received the sdNVP regimen for prevention of mother-to-child HIV-1 transmission were enrolled between April-December 2006 at King Edward VIII Hospital (KEH) in Durban. Blood and cervicovaginal lavage (CVL) samples were collected from women at pre-NVP (during pregnancy) and post-NVP dosing (during labour/delivery). In addition to infant blood sampling at birth (post-NVP), postnatal infants were assessed at four and six weeks postnatally. Pharmacological laboratory investigations involved measurement of NVP drug concentration by Tandem Mass spectrophotometry. Virological investigations comprised HIV-1 RNA (viral load) quantitation, HIV-1 drug resistance testing (HIV-1 transmitting women only) and HIV-1 DNA PCR testing (infants only). Immunological investigations were only undertaken in a selected case-control subset of HIV-1 transmitting women and their infants. In this component, laboratory investigations included the determination of CCL3 and CCL3-L1 gene copy numbers, identification of single nucleotide polymorphisms (SNP’s) and haplotype characterisation of the CCL3 gene. All women were also screened for the presence of sexually transmitted infections (STI’s) during pregnancy. Results: One hundred and twenty women were enrolled onto this study. Of these, 110 women delivered 117 live infants (103 singletons and 7 twin pairs). Twelve (10.9%) women transmitted HIV-1 to their infants, while 95 (86.0%) were classified as non-transmitters. As a result of seven twin deliveries, the infant cohort comprised of 117 infants in total. Following two separate DNA PCR tests, HIV-1 infection was identified in 14 (11.9%) of study infants while the remaining 90 (76.9%) were exposed-uninfected. HIV infection status remained unknown for 13 infants due to infant demise (1.7%), lost to follow-up (7.7%) or study withdrawal (1.7%). During active labour (sampling that was best representative of the intrapartum phase) and within 20 hours of dosing, the median NVP concentration of 1070 ng/ml in the maternal systemic compartment was almost 44 times higher than the NVP levels detected in the genital compartment [24.5 ng/ml] (p < 0.001). NVP drug levels were below the 100 ng/ml therapeutic target in seven (13.7%) of 51 plasma and in all 39 CVL samples. While no significant association was found between NVP concentration in the systemic compartment and HIV-1 transmission (p = 0.4), this association was statistically significant in the genital compartment(p = 0.02). The median plasma NVP level detected among infants at birth was 83 times above the IC50 WT (10 ng/ml) and eight times higher than the 100 ng/ml therapeutic target for NVP. More than 71.0% of the infants achieved NVP drug levels above the therapeutic target. In general, higher levels of HIV-1 RNA (viral load) were observed in maternal plasma when compared to CVL. Following intrapartum sdNVP dosing, reduction in HIV-1 RNA levels did occur, however R80.0% of the women experienced no change to their HIV-1 RNA levels in both systemic and genital compartments during active labour. These findings were further supported by the strong correlation observed when comparing pre and post-NVP HIV-1 RNA levels in both maternal systemic [r = 0.81, p < 0.0001] and genital compartments [r = 0.80, p < 0.0001] during active labour. HIV-1 transmitting women had significantly higher viral loads than their non-transmitting counterparts in systemic and genital compartments, before and after intrapartum sdNVP administration. In terms of perinatal transmission this observation was only statistically significant for plasma (p = 0.02) and not CVL (p = 0.7). Maternal viral load was inversely correlated with maternal CD4 cell counts in both systemic and genital compartments. Almost 40.0% of women in this study had at least one type of STI detected during pregnancy. Maternal STI’s were detected in four (66.6%) intrapartum transmitting women and in 38 (38.8%) of non-transmitting women. No significant association was observed between the presence of maternal STI’s and the risk for intrapartum MTCT (p = 0.2,RR: 2.90, 95% CI: 0.60-15.40). The presence of maternal STI’s was associated with higher median viral loads in both systemic and genital compartments of all women, independent of intrapartum HIV-1 transmission. Despite trial-like conditions and optimal sdNVP dosing, the overall MTCT rate in this exclusively formula-fed cohort was 11.9%, of which 50.0% were in utero and 50.0% were intrapartum HIV-1 transmissions. In utero and intrapartum MTCT rates were 5.9% and 5.9% respectively. Discussion/Conclusion: Detectable CVL HIV-1 RNA that correlated well with plasma HIV-1 RNA, in conjunction with sub-optimal NVP drug concentration in maternal CVL during active labour, suggests that intrapartum HIV-1 infected women continue to act as reservoirs for both vertical and horizontal HIV-1 transmission throughout the duration of pregnancy. These findings confirm that the role of sdNVP in PMTCT was primarily one of infant prophylaxis. This was further supported by relatively unchanged maternal HIV-1 RNA (viral load) during active labour, in both systemic and genital compartments. Early identification of women who need highly active antiretroviral therapy (HAART), and initiation of such therapy as early as possible during pregnancy, not only benefits maternal health but remains the best prophylaxis against mother-to-child HIV-1 transmission. Universal access to HAART and improving strategies to optimize coverage of the current dual ARV regimen sdNVP and Zidovudine for PMTCT remain urgent research priorities in several resource-limited settings. Ongoing STI counseling, intensive screening/testing of women and their partners together promotion of condom usage, safer sex practices and aggressive STI treatment are simple interventions with tremendous impact for PMTCT in resource-limited settings. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2009.

In response to the urgent call for investigations into antiretroviral (ARV)-African traditional medicine (ATM) interactions, this research was undertaken to ascertain whether chronic administration of the ATM, Sutherlandia frutescens (SF) may alter the bioavailability of the protease inhibitor (PI), atazanavir (ATV), which may impact on the safety or efficacy of the ARV. Prior to investigating a potential interaction between ATV and SF in vitro and in vivo, a high performance liquid chromatography method with ultraviolet detection (HPLC-UV) was developed and validated for the bioanalysis of ATV in human plasma and liver microsomes. An improved and efficient analytical method with minimal use of solvents and short run time was achieved in comparison to methods published in the literature. In addition, the method was selective, linear, accurate and precise for quantitative analysis of ATV in these studies. Molecular docking studies were conducted to compare the binding modes and affinities of ATV and two major SF constituents, Sutherlandioside B and Sutherlandin C, with the efflux transporter, P-glycoprotein (P-gp) and the CYP450 isoenzyme, CYP3A4 to determine the potential for these phytochemicals to competitively inhibit the binding of ATV to these two proteins, which are mediators of absorption and metabolism. These studies revealed that modulation of P-gp transport of ATV by Sutherlandioside B and Sutherlandin C was not likely to occur via competitive inhibition. The results further indicated that weak competitive inhibition of CYP3A4 may possibly occur in the presence of either of these two SF constituents. The Caco-2 cell line was used as an in vitro model of human intestinal absorption. Accumulation studies in these cells were conducted to ascertain whether extracts and constituents of SF have the ability to alter the absorption of ATV. The results showed that the aqueous extract of SF significantly reduced ATV accumulation, suggesting decreased ATV absorption, whilst a triterpenoid glycoside fraction isolated from SF exhibited an opposing effect. Analogous responses were elicited by the aqueous extract and a triterpenoid glycoside fraction in similar accumulation studies in P-gp overexpressing Madin–Darby Canine Kidney Strain II cells (MDCKII-MDR1), which signified that the effects of this extract and component on ATV transport in the Caco-2 cells were P-gp-mediated. The quantitative analysis of ATV in human liver microsomes after co-incubation with extracts and components of SF was conducted to determine the effects of SF on the metabolism of ATV. The aqueous and methanolic extracts of SF inhibited ATV metabolism, whilst the triterpenoid glycoside fraction had a converse effect. Analogous effects by the extracts were demonstrated in experiments conducted in CYP3A4-transfected microsomes, suggesting that the inhibition of ATV metabolism in the liver microsomes by these SF extracts was CYP3A4-mediated. A combination of Sutherlandiosides C and D also inhibited CYP3A4-mediated ATV metabolism, which was in contrast to the response elicited by the triterpenoid fraction in the liver microsomes, where other unidentified compounds, shown to be present therein, may have contributed to the activation of ATV metabolism. The in vitro studies revealed the potential for SF to alter the bioavailability of ATV, therefore a clinical study in which the effect of a multiple dose regimen of SF on the pharmacokinetics (PK) of a single dose of ATV was conducted in healthy male volunteers. The statistical analysis showed that the 90 % confidence intervals around the geometric mean ratios (ATV + SF/ATV alone) for both Cmax and AUC0-24 hours, fell well below the lower limit of the "no-effect" boundary of 0.8 – 1.25, implying that the bioavailability of ATV was significantly reduced in this cohort of subjects. It may thus be concluded that if the reduction in bioavailability observed in this clinical study is found to be clinically relevant, co-administration of SF commercial dosage forms and ATV in HIV/AIDS patients may potentially result in subtherapeutic ATV levels, which may in turn contribute to ATV resistance and/or treatment failure. This research has therefore highlighted the potential risk for toxicity or lack of efficacy of ARV regimens which may result when ATMs and PIs are used concurrently and that patients and health care practitioners alike should be aware of these perils.

In order for a medicinal product to produce a consistent and reliable therapeutic response, it is essential that the final composition of the product is invariable and that the active ingredient/s is/are present in appropriate, non-toxic amounts. However, due to the complexity involved in the standardization of natural products, quality control (QC) criteria and procedures for the registration and market approval of such products are conspicuously absent in most countries around the world. African Potato (AP) is of great medical interest and this particular plant has gained tremendous popularity following the endorsement by the South African Minister of Health as a remedy for HIV/ AIDS patients. Very little information has appeared in the literature to describe methods for the quantitative analysis of hypoxoside, an important component in AP. It has also been claimed that sterols and sterolins present in AP are responsible for its medicinal property but is yet to be proven scientifically. To-date, no QC methods have been reported for the simultaneous quantitative analysis of the combination, β- sitosterol (BSS)/ stigmasterol (STG)/ stigmastanol (STN), purported to be present in preparations containing AP. The effect of concomitant administration of AP and other herbal medicines on the safety and efficacy of conventional medicines has not yet been fully determined. Amongst the objectives of this study was to develop and validate quantitative analytical methods that are suitable for the assay and quality control of plant material, extracts and commercial formulations containing AP. Hypoxoside was isolated from AP and characterized for use as a reference standard for the quality control of AP products and a stability-indicating HPLC/ UV assay method for the quantitative determination of hypoxoside was developed. In addition, a quantitative capillary zone electrophoretic (CZE) method was developed to determine hypoxoside, specifically for its advantages over HPLC. A HPLC method was also developed and validated for the quantitative analysis of BSS, STG and STN in commercially available oral dosage forms containing AP material or extracts thereof. The antioxidant activity of an aqueous extract of lyophilized corms of AP along with hypoxoside and rooperol were investigated. In comparison with the AP extracts and also with hypoxoside, rooperol showed significant antioxidant activity. The capacity of AP, (extracts, formulations, hypoxoside and rooperol as well as sterols to inhibit in vitro metabolism of drug substrates by human cytochrome P450 (CYP) enzymes such as CYP 3A4, 3A5 and CYP19 were investigated. Samples were also assessed for their effect on drug transport proteins such as P-glycoprotein (P-gp). Various extracts of AP, AP formulations, stigmasterol and the norlignans, in particular the aglycone rooperol, exhibited inhibitory effects on CYP 3A4, 3A5 and CYP19 mediated metabolism.These results suggest that concurrent therapy with AP and other medicines, in particular antiretroviral drugs, can have important implications for safety and efficacy. Large discrepancies in marker content between AP products were found. Dissolution testing of AP products was investigated as a QC tool and the results also revealed inconsistencies between different AP products.

Original published work submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctorate of Philosophy in Virology. Johannesburg 2017. / Introduction: HIV-infected and HIV-exposed-uninfected children have a heightened susceptibility to some vaccine preventable disease. There is a paucity of data on immunogenicity of vaccines in these children, including HIV-infected children who are initiated on early antiretroviral therapy (ART). We evaluated the effect of maternal HIV-exposure and timing of ART in HIV-infected children on antibody responses to combined diphtheria-toxoid (DT) -tetanus-toxoid (TT)-whole cell pertussis (wP) and Haemophilus influenzae type b conjugate vaccine (HibCV); monovalent hepatitis B vaccine (HepB) and live-attenuated measles vaccine (MV). Methods: Samples obtained from children aged 6–12 weeks who had been enrolled into the CIPRA-SA study were analysed. Briefly, HIV-uninfected children born to HIV-uninfected (HIV-unexposed) and HIV-infected mothers (HEU). Additionally, we enrolled perinatally HIV-infected children with CD4+%≥25% randomized to deferred-ART (i.e. initiated when clinically or immunologically indicated per the then WHO recommended treatment criteria; ART-Def) or immediate-ART initiation (i.e. initiated on ART immediately upon confirmation of HIV-infection status at 4-10 weeks of age; ART-Immed). Children enrolled in the ART-Immed arm were further randomized to interrupt ART at one-year (ART/12m) or two-years of age (ART/24m). Additionally, a convenience sample of HIV-infected children with CD4+<25% initiated on immediate-ART was enrolled (ART-CD4+<25%). Children received a primary series of DTwP-HibCV/HepB at 6, 10 and 14 weeks of age; and MV at 40 weeks of age. Booster dose of DTwP and MV was given at 15-18 months of age. Sampling time-points were: prior to the first dose of vaccine, four weeks after the third dose (18 weeks age), 24 weeks after the third dose (39.3 weeks of age), at the time of the booster dose (15- 18 months age), two to four weeks after the booster dose and at 24 months of age. Samples were analysed for antibodies for DT, TT, PT, FHA, HepB measured by Luminex microbead-immunoassay; and MV antibodies were quantified by an indirect enzyme immunoassay. Results: Antibody kinetics and response to primary series of DTwP-HibCV/HepB: Pre-vaccination GMCs were higher in HIV-unexposed than HEU children for TT, but lower for HepB, DT and FHA. Post-vaccination, sero-conversion, sero-protection and GMCs were similar in HEU and HIV-unexposed children for all vaccines. Furthermore, GMCs were higher in HIV-unexposed for TT, DT, HepB and FHA than in ART-Immed children; and for TT, HepB and PT than in ART-Def children. Nevertheless, there was no difference in proportion of HIV-unexposed and HIV-infected children who developed sero-protective vaccine-specific antibody levels post-vaccination. The timing of ART initiation generally did not affect immune responses to vaccines between HIV-infected groups. Antibody kinetics and booster responses to DTwP-HibCV/HepB vaccines: Pre-booster GMCs were generally higher in HIV-unexposed than HIV-infected children for all vaccine epitopes. Post-booster and at 24 months of age the ART-Def group had lower GMCs (except to FHA), and were less likely to have sero-protective antibody levels compared to HIV-unexposed group. Also, post-booster and at 24 months of age, GMC were generally higher in HIV-unexposed than ART-Immed children, and a higher percentage of HIV-unexposed than ART-Immed children maintained antibody levels ≥1IU/ml to TT and DT at 24 months of age. The GMCs and percentage of children with sero-protective thresholds were similar pre-booster and at 24 months of age between HIV-unexposed and HEU children. Antibody kinetics and response to measles virus vaccine: At 7.3 weeks of age, the proportion with sero-protective titers was higher in HIV-unexposed (65.2%) compared to any HIV-infected group (range: 16.7% to 41.8%); but dropped to <17% in all Groups at age 19.6 weeks. Twenty-eight weeks following the first measles-vaccine, ART/12m were less likely to have sero-protective titers (79.3%) compared to HIV-unexposed (94.8%; p<0.001), ART-Def (95.7%; p=0.003) or ART/24m (92.1%; p=0.02). Although the proportion with sero-protective levels were similar between groups immediately post-booster dose, this was lower in HEU (79.6%; p=0.002) and ART/12m (80.3%; p=0.01) compared to HIV-unexposed (94.3%) 41-weeks later. Conclusion: Primary vaccination with DTwP-HibCV/HBV of HIV-infected children initiated on early-ART confers similar immunity compared to HIV-unexposed children. HIV-infected children had poor anamnestic responses, if ART was not initiated prior to primary vaccination. In contrast, the memory response and persistence of antibody to most vaccine epitopes were similar between HIV-unexposed and HEU children. Increased waning of vaccine induced immunity over a 24 month period in ART-Def, ART/12m and HEU children following MV booster-dose; indicating the need for further booster doses after two-years of age in these children. I recommend close monitoring of HEU children, as this group makes up most children born to HIV-infected mothers and what facets of the immune system have been impacted by maternal exposure to HIV. / MT2017

The HIV/AIDS pandemic has implications at every level of social functioning. It affects individuals, families, communities and organisations. The burden of caring for those exposed, affected and infected is vast, but one of the most significant developments which have the potential to reduce disease burden is antiretroviral therapy. Antiretroviral therapy (ART) is complex and difficult to administer, and requires a learning process which is mediated through a number of means. Vygotskian theory was utilised to better understand the process of adherence through mediated learning, and as a framework for explaining compliance. In this study, mediated learning occurs both in the context of the clinic staff and the clinic attendees, and the caregivers and the child. Therefore Vygotsky‟s theory offers useful insight into this process. This qualitative study aimed to research the factors which contribute to ART adherence in a small sample of HIV positive children who are attending a local clinic. Eight child-caregiver dyads were interviewed, and drawings utilised to better understand child and caregiver factors which contribute to compliance. There were a number of psychosocial factors identified which contribute to compliance, or lack thereof, including social support, stigma, medication fatigue, disclosure, access difficulties, psychoeducation, and motivation. A number of qualitative differences were also identified between children who knew their HIV status and those who did not. These differences emerged primarily through the analysis of the child participants‟ drawings and there appeared to be a number of inter- and intrapersonal benefits to disclosure. The factors identified in this study, if better understood, can inform interventions to improve compliance on ART. / Thesis (M.Soc.Sci.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

There are currently an estimated 33.2 million people living with human immunodeficiency virus (HIV) worldwide. For these individuals, long-term use of combination antiretroviral therapy (cART) is not feasible for a variety of reasons including major adverse complications, multi-drug resistance, poor adherence, and high cost. Hence, development of novel therapeutic strategies that can reduce the life-long dependency on cART is highly desired. In order to develop effective therapeutic strategies such as a therapeutic vaccine, we need to have a greater understanding of the immune correlates of viral control in chronic HIV infection. In this thesis, we used treatment interruption (TI) as a tool to test the efficacy of several therapeutic approaches and immune parameters for their association with effective control of viral replication. / In Chapter 2 we showed that cART intensification and Remune vaccination resulted in reduced viral load (VL) plateau during sequential TIs. Although HIV-specific immune responses measured by interferon-gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) increased in the same time frame, neither their breadth nor magnitude correlated with the decrease in VL plateau. In Chapter 3 the effect of ALVAC-vCP1425 plus Remune vaccination on HIV proteome-wide HIV-specific responses was monitored using a dual color IFN-gamma/interleukin-2 (IL-2) ELISPOT assay. We observed an increase in the magnitude of HIV-specific IFN-gamma/IL-2 responses, as well as in the breadth of Gag-specific IFN-gamma responses in the vaccinated groups compared to placebo groups. A shift towards an increased contribution of Gag-specific responses to total HIV-specific vaccine induced immune response was associated with longer delay to viral rebound during TI. In Chapters 4 and 5, we examined baseline pre-TI immune parameters and their association with viral rebound and CD4 count change during TI in HIV-infected individuals in the chronic phase of infection experiencing virologic failure before TI (Chapter 4) or with different levels of VL control while on therapy prior to TI (Chapter 5). We saw that chronic antigen stimulation from persistent viremia as well as co-infections such as with cytomegalovirus are associated with T-cell senescence, which may result in less favourable clinical outcomes during TI. / Consequently, results from this thesis contribute to further understanding of immune correlates of viral control in chronic HIV infection. New therapeutic vaccines and interventions should induce polyfunctional HIV-specific immune responses, broad Gag-specific immune responses, as well as reducing chronic antigen stimulation to prevent irreversible T-cell exhaustion. Taken together, these insights could potentially lead to the development of novel treatment interventions that could effectively control viral replication off cART.

No description available.

引發獲得性免疫缺陷綜合癥（AIDS）的人類免疫缺陷病毒（HIV）是一種逆轉錄病毒。過去的十餘年間，高效抗逆轉錄病毒治療療法（HARRT），在抗病毒感染方面取得了很大的成功。高效抗逆轉錄病毒治療療法是一種將多種抗逆轉錄病毒藥物複合的藥物聯用療法。然而，因為病毒的逆轉錄過程極易突變，導致HIV已經可以對大多數使用的抑製藥物產生抗藥性。因此，有越來越多的需要去尋找新型的抗病毒複製機理，例如將人體細胞蛋白作為載體，來達到克服病毒抗藥性的目的。 / HIV-1的複製離不開宿主細胞的剪接因子，例如SR蛋白。選擇性剪接因子ASF/SF2，一個典型的調控pre-RNA剪接的SR蛋白，在HIV-1的pre-mRNA剪接和複製中起到了很重要的調控作用。ASF/SF2和其他SR蛋白一樣，都被丝氨酸/苏氨酸蛋白激酶（SRPK）磷酸化，磷酸化位點位於C端的丝氨酸/苏氨酸結構域（RS domain）。SRPK通過磷酸化來調節ASF/SF2在細胞中的分佈。對於SRPK 和ASF/SF2複合物的結構學和功能學研究指出，ASF/SF2的docking motif和SRPK1的遠離活性位點的docking groove存在很強的相互作用。而這種相互作用是調節磷酸化過程關鍵。所以，在我們的研究過程中，我們希望通過阻斷2個蛋白的相互作用來干擾ASF/SF2的磷酸化，進而抑制其在HIV-1 pre-mRNA剪接過程中的活性。 / 我們採用以結構為基礎的藥物模擬篩選，來選擇潛在的抑制物，達到通過抑制物與docking groove的相互作用來阻斷ASF/SF2和SRPK1的相互作用，以達到抑制磷酸化的目的。我們使用的數據庫來自于ZINC數據庫（UCSF），包括天然產物數據庫和SPECS。我們採用AutoDock Vina 和AutoDock 4.2 二個模擬軟件來栓選數據庫中351473个化合物。并從中選出50個潛在的化合物用作之後的化學生物學測試。體外的激酶活性試驗顯示，6個化合物對ASF/SF2的磷酸化有抑製作用。 / 體外的HIV-1 pre-mRNA剪接實驗顯示，5個化合物在逆轉錄PCR（RT-PCR）中有一定得抑制效果。和DMSO對照組相比，在抑製劑作用下剪接產物的生成被抑制。HIV-1病毒合胞體感染實驗顯示，有一個化合物對病毒的感染起到了一定的抑制作用。 / 其他的測試實驗還在進行中，包括對SRPK1和抑制物複合物的結構研究，從而更好的研究抑制物的作用機理。以及，採用表面等離子共振波譜來進行動力學研究和其他關於化合物在病毒複製過程中的實驗測試。 / Human immunodeficient virus (HIV) is a retrovirus that cause acquired immunodeficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) is a treatment of HIV infection that uses combinations of antiretroviral drugs and has achieved great success in the past two decades. However, since the reverse transcription process of viral RNA is notoriously prone to error, HIV-1 can acquire resistance to nearly all known inhibitors and has started to develop resistance to HAART. Therefore, there is an ongoing search for new drugs with novel inhibitory mechanism such as targeting cellular proteins essential for HIV-1 replication to overcome drug resistance of the virus. / HIV-1 mRNA undergoes complex splicing and the expression of the integrated HIV-1 provirus is largely dependent on the host’s splicing machinery which assembly requires splicing factors such as serine-arginine rich proteins (SR proteins). Alternative splicing factor/splicing factor 2 (ASF/SF2), a prototypic SR protein that is essential for pre-mRNA splicing, has been shown to play critical roles during HIV-1 pre-mRNA splicing and replication. ASF/SF2, like other SR proteins, is phosphorylated by SR protein-specific kinases (SRPKs) at its C-terminal arginine/serine (RS) domain, which governs its localization and metabolism. Structural and functional studies of SRPK1 in complex with ASF/SF2 has revealed that a docking groove on SRPK1 that is distal to the active site interacts strongly with a docking motif and the RS domain of ASF/SF2, leading to high affinity binding as well as regulating the mechanism of phosphorylation. In this study, we propose that by blocking this interaction, we might interfere the phosphorylation of ASF/SF2 and inhibit its activity during splicing of HIV-1 pre-mRNA. / Structure-based in silico screening method is adopted to identify potential inhibitors that bind to the docking groove of SRPK1 to block the binding and phosphorylation of ASF/SF2. The compound libraries being used include the Natual Products Database and SPECS database from ZINC (UCSF). 351,473 compounds have been screened using the program Autodock Vina as well as Autodock 4.0. Until now 50 potential candidates of inhibitor have been selected for biochemical analyses. In vitro kinase assays showed that six compounds exhibit inhibitory activity against the phosphorylation of ASF/SF2. / To test the effect of the selected inhibitors on the splicing of HIV-1 mRNA, ex vivo splicing assay has been performed. Current results showed that the synthesis of splicing products extracted from drug-treated cells was less efficient when compared to untreated cells. Biological assays testing the inhibitory effects of the compounds on viral infection are currently underway. Our preliminary result suggested that one of the compounds could indeed inhibit HIV-1 viral infection. / Other biochemical and biological analyses including structural study of kinase-inhibitor complexes to understand the mode of inhibition; measurement of binding kinetics using surface plasmon resonance spectroscopy (SPR); and biological assays testing the inhibitory effects of the compounds on replication are underway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yu, Xiyao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 95-107). / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / TABLE OF CONTENTS --- p.VI / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.XI / Chapter Chapter I --- : Introduction --- p.1 / Chapter 1.1 --- HIV, HAART and HIV Drug Resistance --- p.2 / Chapter 1.2 --- HIV-1 alternative splicing mechanism --- p.9 / Chapter 1.3 --- SR Protein Family --- p.13 / Chapter 1.4 --- Functional roles of SR protein in HIV pre-mRNA splicing --- p.16 / Chapter 1.5 --- Phosphorylation States of SR Proteins --- p.18 / Chapter 1.6 --- SR protein Kinase --- p.20 / Chapter 1.7 --- Interaction between SRPK1 and ASF/SF2 --- p.23 / Chapter 1.8 --- IDC16 and SPRIN340 --- p.26 / Chapter 1.9 --- Structure-based drug screening --- p.27 / Chapter 1.10 --- AutoDock Suite --- p.29 / Chapter 1.11 --- Kinase-substrate interaction inhibitors --- p.30 / Chapter 1.12 --- Focus of study --- p.34 / Chapter Chapter II --- : Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Bacterial strain --- p.36 / Chapter 2.1.2 --- Antibodies --- p.36 / Chapter 2.1.3 --- Cell line --- p.36 / Chapter 2.1.4 --- Plasmid --- p.36 / Chapter 2.1.5 --- Reagents --- p.38 / Chapter 2.2 --- Expression and purification of Recombinant protein --- p.38 / Chapter 2.3 --- In silico screening of inhibitors --- p.44 / Chapter 2.4 --- Kinase Glo Assay --- p.45 / Chapter 2.5 --- In vitro kinase assay --- p.45 / Chapter 2.6 --- Cell Culture --- p.46 / Chapter 2.7 --- MTT Assay --- p.46 / Chapter 2.8 --- Immunocytochemistry --- p.47 / Chapter 2.9 --- Ex vivo splicing assay --- p.47 / Chapter 2.10 --- Surface plasmon resonance spectroscope --- p.48 / Chapter Chapter III --- : Results --- p.50 / Chapter 3.1 --- In silico screening of inhibitors --- p.51 / Chapter 3.2 --- Selected Compounds Inhibits SRPK1 in Vitro --- p.60 / Chapter 3.2.1 --- Protein purification --- p.60 / Chapter 3.2.2 --- Inhibits ASF/SF2 Phosphorylation by SRPK --- p.66 / Chapter 3.3 --- Surface Plasmon Resonance Binding Competition Assay --- p.76 / Chapter 3.4 --- Inhibitors Alters HIV-1 Alternative Splicing ex Vivo --- p.79 / Chapter 3.5 --- Cytotoxic effect of candidate compound on HeLa cells --- p.84 / Chapter 3.6 --- Nature compound alters ASF/SF2 localization --- p.86 / Chapter Chapter IV --- : Discussion and Conclusion --- p.89 / References --- p.95