3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The role of HIV-1 Rev during allosteric inhibition of integrase(2018) Abrahams, ShaakiraThe HIV-1 integration step is a pertinent process in the HIV-1 replication cycle that integrates the viral genome into the host chromosome. HIV-1 integrase is crucial in facilitating the integration step. Integration is dependent on many co-factors and viral- as well as host accessory proteins. Lens epithelium-derived growth factor (LEDGF/p75) is a host protein that binds to the hydrophobic pocket of integrase formed by a dimer of integrase. The function of LEDGF/p75 is to tether integrase to the host chromosome for successful integration, and consequently provides a drug target for a novel class of HIV-1 inhibitors. The latest studies on the HIV-1 integration process revealed that the HIV-1 regulatory factor, Rev, plays a cardinal role in the regulation of the integration step. Previous studies have demonstrated that Rev and LEDGF/p75 compete for the hydrophobic pocket located on integrase where Rev is able to dissociate the integrase-LEDGF/p75 complex and subsequently form integrase-Rev complexes and LEDGF/p75-Rev complexes. Interestingly, the latest class of HIV-1 inhibitors known as allosteric integrase inhibitors (ALLINIs), compete with LEDGF/p75 and Rev for the same hydrophobic site located on integrase. Further to this, ALLINIs and Rev inhibit the integration step in the early phase of the replication cycle by inducing an integrase oligomerization shift. Consequently, inactive multimers of integrase are formed that are unable to bind to the host chromosome thus preventing integration. Due to the similarities between Rev and ALLINIs in the mechanism of inhibiting integration and the shared binding site on integrase, this study postulated that ALLINIs interfere with the interaction between Rev and integrase. The first objective was to determine whether ALLINIs, Mut 101 and CX05168, had an effect on HIV-1 subtype C Rev and integrase through in vitro selection resistant studies followed by genotypic- as well as phenotypic analysis. This study reports the first mutations against Mut 101 within the HIV-1 subtype C IN gene. The integrase mutations identified include: H171L for the 05ZAFV3 (FV 3) isolate and Q164L, L172I, and L172I/H171Q for the 05ZAFV26 (FV 26) isolate. In vitro phenotypic inhibition studies demonstrated the phenotypic significance of the mutations through a shift in EC50 values (0.53 ± 0.01µM for drug naive; 0.21 ± 0.11µM for Q164L mutated isolate; 2.67 ± 0.95µM for L172I mutated isolate) and the infectious ability of the isolates (FV 3 H171L and FV 26 L172I/H171Q). Molecular docking studies further confirmed the effects of the mutations by predicting the interaction between integrase harbouring the mutations and Mut 101. An increase in the binding energies of the predicted models suggests that the presence of mutations are responsible for weaker interactions between integrase harbouring the identified mutations and Mut 101. Data for in vitro resistance studies selected by CX05168 could not be generated as CX05168 failed to inhibit viral replication of the FV 3 and FV 26 isolates at its reported EC50 for HIV-1 subtype B isolates. This prompted in vitro phenotypic inhibition studies to determine the EC50 values of CX05168 against subtype C isolates (FV 3 = 4.3 ± 0.4µM and FV 26 = 40.1 ± 0.8µM) which was significantly higher than the reported EC50 of CX05168 against HIV-1 subtype B (2.35 ± 0.28µM). Furthermore, genotypic analysis of the rev gene was not achieved as the amplification thereof faced continuous challenges. The second objective was to delineate how Rev affect the oligomerization of integrase in the presence of Mut 101 and CX05168. AlphaScreen dimerization assays, BS3 crosslinking studies and dynamic light scattering experiments confirmed the multimerization of integrase in the presence of Rev, Mut 101 and CX05168. However, the aberrant multimerization of integrase induced by the ALLINIs was significantly reduced in the presence of Rev. This finding suggests that Rev and the ALLINIs have distinct pathways to induce integrase multimerization and that Rev indeed modulates the multimerization mechanism of integrase induced by ALLINIs. The third objective aimed to investigate the integrase-Rev interaction in the presence of Mut 101 and CX05168. Through an ELISA method, the dissociation constant (Kd) of integrase-Rev was calculated at 461 ± 5.65nM. Order of addition ELISA experiments demonstrated that ALLINIs do not interfere with the integrase-Rev interaction once Rev is already bound to integrase. However, when ALLINIs are added to integrase before the addition of Rev, a significant shift in Kd was observed. The Kd increased to 850 ± 70.71nM in the presence of Mut 101 and 1010 ± 127.27nM in the presence of CX05168. The integrase-Rev interaction was confirmed through isothermal titration calorimetry where the Kd (419nM) obtained significantly correlated with the Kd calculated through ELISA studies. This study is the first to report the thermodynamic properties of the integrase-Rev interaction. The interaction elicited an exothermic reaction with an enthalphy change (∆H) = -5350J/mol, a temperature entropy change (T∆S) = 103.9J/mol.K, stoichiometry of 1 and free energy (∆G) = -5453.9J/mol which indicated that the reaction was enthalpically driven. Thermodynamics of the integrase-Rev interaction could not be established in the presence of Mut 101 and CX05168 suggesting that multimerization of integrase could have intercepted the reaction. Overall, this study reports novel data and provides insight on the hypothesis of the study which establishes that ALLINIs distort the interaction between Rev and integrase and has downstream effects on integrase multimerization. The data presented in this study may lead to a better understanding of ALLINIs in the replication process, especially in HIV-1 subtype C isolates, and may add value to the development process of future ALLINIs.Item Sequential adeno-associated virus deliverd HIV-1 subtype C ENV glycoproteins as novel vaccine immunogens(2018) Moodie, MelanieAn efficacious HIV-1 vaccine that can elicit broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) is likely to provide protection from infection. To date, no vaccine immunogen has elicited bNAbs against HIV-1. Recent evidence from infected individuals suggests that bNAbs develop over time in response to constant antigenic challenge provided by an evolving Env that drives the somatic hypermutation of these antibodies and is necessary for their development. Env sequences required to engage the unmutated germline Bcells and drive the somatic hypermutation required to develop a bNAb response are available. Recombinant adeno-associated viral (rAAV) vectors present an attractive novel strategy for in vivo delivery and long-lived expression of sequential HIV-1 Env immunogens. Thus, this study aimed to provide proof-of-concept that rAAV can be used for the delivery and expression of sequentially-derived HIV-1 Env sequences in mammalian cells. Five sequential Env sequences (from a total of 292), over an infection period of 78 weeks, replicating in vivo viral evolution from patient CH505 who was infected with HIV-1 subtype C, were selected, codon optimized, and cloned into pcDNA3.3 for mammalian expression of Env. Recombinant Env glycoproteins were transiently expressed in HEK293 T cells, purified, and biochemically characterized by SDS-PAGE, Western Blotting and ELISA. The matched env sequences were also sub-cloned into the rAAV-Luc vector, allowing for the production of rAAV following a polyethylenimine (PEI)-based triple transfection protocol of HEK293 T cells. rAAVs were purified via polyethylene glycol (PEG) precipitation, followed by iodixanol gradient centrifugation, and quantified via droplet-digital PCR (ddPCR). These rAAVs were used to transduce HEK293 T cells and characterize the expression of Env in vitro by ELISA. Consensus Env sequences were generated from all time points, and following bioinformatics analyses, sequences from weeks 4, 20, 30, 53, and 78 post infection were selected. All pcDNA3.3-CH505 Env constructs were successfully expressed and purified, apart from the week 4 Env. Binding of the recombinant Envs to a panel of bNAbs, as determined by ELISA, confirmed they were functional and conformationally intact. Interestingly, differences in antibody binding were noted between weeks 20 – 78, highlighting potential changes in epitope exposure. All 5 envs were successfully sub-cloned into the pAAV backbone vector with intact ITRs, as confirmed by restriction enzyme digest analysis. The env transgenes were then packaged into serotype 2 rAAV vectors, and rAAVs were successfully produced and purified, with an average yield of 1,4 x 1011 genome copies/ml, as quantified by digital droplet PCR. Transduction assays and characterization revealed that they were functional and produced recombinant Env glycoproteins, with the exception of week 4, with optimal expression between 6-8 days posttransduction. Overall, these results provide the first evidence that HIV-1 Env can be successfully cloned into rAAV, and the associated recombinant proteins can be expressed in HEK293 T cells. Moreover, we provide reagents for future prime-boost immunizations using a panel of sequential Env expressing AAVs with matched recombinant proteins for testing in small animal models.Item Allosteric effects of chicoric acid on human immunodeficiency virus type 1 integrase(2018) Fish, Muhammad QasimHuman immunodeficiency virus (HIV) integrase (IN) is an essential viral protein involved in the integration of the viral DNA into the host genome. Although having a specific catalytic function, it is apparent from mutagenesis studies that IN is pleiotropic and affects the viral life cycle at multiple points other than integration. Compounds that bind to allosteric sites on IN typically disrupt its ordered multimerization and stalls the viral life cycle at various points. Chicoric acid (CA) is a well-known IN inhibitor, however, its mechanism does not follow a conventional active site inhibition, yet it presents with antiviral activity. We thus, hypothesised that CA has an allosteric inhibitory mechanism. To test the hypothesis we aimed to determine alloteric effects of CA on HIV IN. Site directed mutagenesis was used to develop IN mutants resistant to conventional IN inhibitors and these proteins were purified. These were used in an enzyme-linked immunosorbent assay (ELISA) for comparative resistance profiling of CA and raltegravir (RAL). The ELISA also compared magnesium (Mg2+) and manganese (Mn2+) dependent differences on CA inhibition and to determine the importance of order of addition of assay components. An AlphaScreen assay was developed to test for the disruption of the IN/LEDGF interaction. Crosslinking assays and size exclusion chromatography (SEC) was performed to determine the multimeric state of IN in the presence of CA. Surface Plasmon resonance (SPR) was used to confirm binding of CA to IN and determine the kinetics. In silico docking onto the IN catalytic core (CCD) structures was used to identify a possible binding mode of CA at the allosteric binding site. Resistance profiling depicted a clear distinction between CA and RAL. IN resistance mutants: INQ148H, INN155H, ING140S/Q148H and INE92Q/N155H, showed a fold change in IC50 (FCIC50) of 3.42, 1.09, 1.43 and 2.90 for CA respectively. While the same mutants showed an FCIC50 of 947.99, 392.44, 1262.41 and 583.92 for RAL respectively. Additionally, cooperativity trends between the compound’s inhibition profiles were different. It was concluded that CA and RAL have different resistance profiles. Metal dependent differences show that the IN soluble mutant is resistant to CA inhibition only in the presence of Mg2+. This indicates that metal specific structural differences may play a role in resistance. Order of addition indicated that if DNA was present after CA incubation with IN the inhibition was cooperative, while if DNA was added before incubation with CA the inhibition was non-cooperative. This posits that CA may bind to a single site, possibly only the allosteric site, when DNA is already present in the IN active site. When DNA is absent CA binds to multiple sites, possibly both the active site and allosteric sites. The AlphaScreen indicated that the IN/LEDGF interaction is disrupted by CA with IC50 of 237nM (±27nM). Also the disruption is dependent on the order of addition of assay components. Multimerization of IN was increased in the presence of CA as shown by crosslinking assays as well as SEC. Not only did CA induce multimerization of free IN, a nonreducing gel electrophoresis indicated that virus assembled in the presence of CA had increased multimerization of IN, similar to a control. SPR indicated that CA binds to the full length IN with similar kinetics compared to the catalytic core domain (INCCD) indicating that this domain likely contains the CA binding site. The kinetics indicated a slow kon and koff rate for CA binding. These slow binding kinetics are indicative of a high barrier to resistance. Finally, in sillico molecular docking of CA to the active site as well as the allosteric site indicated that it potentially has a dual binding mode on the IN apo-enzyme. The results show that CA has allosteric effects on IN and may provide a good pharmacophore for further development of allosteric IN inhibitors.Item HIV coreceptors: distribution and modulation(2003-07-15) Shalekoff., Sharon.The chemokine receptors, CXCR4 and CCR5, are the major coreceptors for HIV-1 entry into CD4+ cells. In addition, they mediate chemotaxis in response to stromal cell-derived factor (SDF- la) (CXCR4) and macrophage inflammatory protein (MlP)-la, MIP-ip and regulated on activation, normal T cell expressed and secreted (RANTES) (CCR5). Therefore, cell surface expression of these receptors has important implications for the study of the pathogenesis of HIV disease, in terms of viral entry as well as cellular trafficking.Item Expression and characterization of functional HIV-1 subtype C envelope glycoproteins in insect cells.(2002-09-20) Nkosi., Praise-God Sibusiso.The full-length envelope glycoproteins from four South African HIV-1 subtype C isolates exhibiting different phenotypic properties were expressed in insect cells, purified, and their biological characteristics investigated. Isolates 98ZA151Du, 99ZACM9, 98ZA179DU and 99ZASW7 that utilize the CCR5, CCR5/CXCR4, CCR5/CXCR4 and CXCR4 coreceptors for cell entry, respectively were selected for this study. All four full-length env genes were PCR amplified, cloned into a baculovirus expression system and the recombinant envelope glycoproteins were expressed in Spodoptera frugiperda (Sf9) insect cells.Item Characterisation of nef from HIV-1 subtype c-infected individuals(2001-08-31) Mashishi., Tumelo. Nkoenyana.This dissertation examines HIV-1 subtype C nucleotide and amino acid Nef sequences from South African recently-infected (n=12) and chronically-infected individuals (n=9) and from AIDS patients (n=ll). The overall aim is to determine which regions of subtype C Nef are variable and which are conserved and to associate these regions with targeting by the immune system. Phylogenetic analysis and sequence alignments showed that there was no association of Nef variations with stage of disease. Nef amino acid alignments of 32 sequences showed a high degree of conservation in areas of functional and structural importance. The mean intrasubtype variation from these isolates was 15.8%, with sequences from the AIDS cohort being significantly (p<0.05) more variable than those from recent or chronic infectionItem Genetic variation in gp 120 V1?V2 and neutralization sensitivity of HIV-1 subtype C isolates from children with rapid and slow disease progression(2003) Choge, Isaac Ang' Ang' AThis project explore genetic variation and antibody neutralization sensitivity in gp 120 V1/V2 and C2-V5 region amongst HIV-1 subtype C pediatric isolates. Isolates from 25 slow progressing children and 16 rapid progressing infants were used in subtyping and quasispecies analysis performed with a novel V1/V2 heteroduplex mobility assay (HMA)Item HIV-1 Subtype B and C Envelope Glycoprotein based immunogens as preventative vaccines(2017) Grant, MichelleThe ability to induce a potent and broadly neutralizing antibody (bNAb) response following vaccination is critical in developing an effective HIV-1 vaccine. To date, no HIV-1 envelope glycoprotein (Env) immunogens have elicited bNAbs in preclinical or human clinical trials. This study compared the antigenicity and immunogenicity of a panel of HIV-1 subtype B and C Env-based immunogens in various immunization regimens in a small animal model. The Env-based immunogens used included matched monomeric (gp120) and trimeric (gp140GCN4(+)) conformations of 6 subtype C Env’s (IN26191, IN25710, IN25925, ZACAP45, ZACAP210 and ZA706010164; all designed during the course of this study), and subtype B stabilized Env derivatives Cyc4OD gp120 cyclic permutant, gp140 cyclic permutant h-CMP V1cyc 144-142, ODECCOBPICS gp120 fragment, VRC01 engrafted scaffold peptides 1WR2 and 1ORC, and DNA (Wt-JRFL-Env, JRFL Env-570D and JRLF Env-SEKS; all obtained from collaborators). The six HIV-1 subtype C Env sequences (IN26191, IN25710, IN25925, ZACAP45, ZACAP210 and ZA706010164) were selected, matched gp120 and gp140GCN4(+) constructs were designed, codon optimized and cloned into a mammalian expression vector. All 12 env constructs were expressed in HEK293T or 293FS cell lines, and the Env purified by lectin affinity chromatography, followed by size exclusion chromatography. Additionally, two domain soluble CD4 (2dCD4) wildtype, folding defective and an S60C mutant were expressed in a bacterial system and purified by nickel affinity chromatography. 2dCD4S60C-liganded Env was prepared, and purified further, as required. The antigenicity of all 12 Env’s was evaluated against 2dCD4 and a panel of bNAbs using surface plasmon resonance (SPR). The immunogenicity of two of these subtype C Env variants (liganded and unliganded to 2dCD4S60C) were subsequently compared to that of the subtype B immunogens in rabbits, using various prime-boost regimens. Rabbit sera were subsequently tested for anti-Env binding antibody titres by enzyme linked immunosorbent assay (ELISA), and neutralization by an in vitro phenotypic neutralization assay against a panel of HIV-1 pseudoviruses. All 12 recombinant Envs were successfully expressed and purified to homogeneity. Binding of all the gp120/gp140GCN4(+) Env variants to 2dCD4 variants (Wt and S60C) confirmed that they were all functional and conformationally intact with an accessible CD4 binding site (CD4bs). Binding to a panel of CD4bs directed bNAbs (IgG1b12, VRC01, HJ16, VRC-CH31, NIH45-46G54W, VRC03) revealed that overall, the trimeric gp140GCN4(+) variants showed higher binding affinities to these bNAbs compared to the matched monomeric gp120, attributed to their resemblance to the native trimer on the viral surface. With the exception of VRC03 and IgG1b12, the Indian Env variants bound with an approximately 10-fold higher affinity compared to the South African Env’s. Overall, nine different immunization regimens were performed. Immunization of rabbits induced high titres of antibodies (Abs) for all the immunogens tested, as determined by ELISA, however, minimal neutralization breadth (against Tier 1 pseudoviruses) was obtained for the Env-only variants for subtype B and C immunogens. Of these, the VRC01 engrafted scaffold peptide (1ORC) showed improved neutralization of the Tier 1 pseudovirus SF162 compared to the other Env only based immunogens. The only promising neutralization results were obtained from rabbits immunized with the Env/2dCD4S60C liganded immunogens that potently neutralized both subtype B and C, Tier 1, 2 and 3 pseudoviruses. This response was improved for the trimeric Env/2dCD4S60C complexes compared to the monomeric ones and was consistently elicited regardless of the Env sequence used. The neutralization response is likely either due to Abs targeting one or more epitopes on 2dCD4 or Env or both. Therefore, the use of CD4 liganded Env immunogens in vaccine design should be investigated further as they provide a promising “Ibalizumab-like” neutralization response. Overall, based on emerging evidence on how the bNAb responses evolve in HIV-1 infected individuals, the findings in this study are promising and lay the groundwork for further testing of these HIV-1 Env based immunogens in various combinations using sequential prime-boost strategies to optimally drive affinity maturation of the appropriate B cell lineages.Item The prevalence of helminth and malaria infections and the effects of de-worming on disease progression markers in HIV-1 infected pregnant women on antiretroviral therapy in Rwanda(2017) Emil, IvanBackground: Helminth and malaria co-infections have been hypothesized to be factors driving the HIV-1 epidemic in Africa, and the fact that both cause anaemia highlights the importance of addressing the interactions between HIV/AIDS, malaria and intestinal helminthic infections in pregnancy for individuals in resource limited settings. Aims: The aims of this thesis were to determine the prevalence and risk factors for malariahelminthic dual infections among HIV positive pregnant women on antiretroviral therapy in Rwanda. The second aim was to determine the effect of deworming on immune markers of HIV/AIDs disease progression among HIV-infected pregnant women on antiretroviral therapy (ART), and to elucidate the benefits of deworming, specifically in targeted versus untargeted deworming. Methods: A cross-sectional study was carried out in 328 HIV-positive pregnant women receiving ART. We determined the prevalence of helminth and malaria dual infections and the effects of ART on these infections were also examined. This cross sectional study acted as a pilot study for a deworming intervention, which took the form of a longitudinal study of targeted and untargeted deworming in which 980 HIV-infected pregnant women were randomized to ‘targeted’ and ‘untargeted’ arms with albendazole therapy. The effects of deworming on the prevalence of helminth infection and CD4 counts, viral load and haemoglobin levels were measured over time at 4 visits. Measurements were at baseline and every 3 months thereafter. The presence of Plasmodium falciparum was tested at each visit and anti-malarial therapy (Coartem: artemether-lumefantrine) was administered to all subjects who tested positive for P. falciparum. Baseline data was used to determine the risk factors for helminth infection. Helminthic infection was diagnosed using the Kato Katz method, whilst the presence of P. falciparum was identified from blood smears. The CD4 counts and viral load levels were also determined using standard laboratory methods. Results: Within the pilot study of 328 women residing in rural (n=166) and peri-urban (n=162) locations, 38% of those tested harboured helminths, 21% had malaria and 10% were infected with both. The most prevalent helminth species were Ascaris lumbricoides (20.7%), followed by Trichiuris trichiura (9.2%), Ancylostoma duodenale and Necator Americanus (1.2%). Helminth infections were characterized by low haemoglobin levels and low CD4 E. Ivan PhD thesis Page iii counts. Subjects treated with a d4T-3TC-NVP regimen had a reduced risk of Trichuris trichiura infection (OR, 0.27; 95% CIs, 0.10-0.76; p<0.05) and malaria-helminth dual infection (OR, 0.29; 95% CI, 0.11-0.75; p<0.05) compared to those receiving AZT-3TC-NVP therapy. Within the longitudinal study of deworming in 980 pregnant, HIV-infected females, analysis of the baseline data showed that education and employment reduced the risk of all types of infection whilst hand washing protected against helminth infection (0.29 [0.19-0.46]; p<0.0005). Logistic regression analysis, at baseline (odds ratio [95% CIs]), demonstrated that TDF-3TC-NVP (3.47 [2.21-5.45]; p<0.0005), D4T-3TC-NVP (2.47 [1.27-4.80]; p<0.05) and AZT-NVP (2.60 [1.33-5.08]; p<0.05) regimens each yielded higher helminth infection rates than the AZT-3TC-NVP regimen. Anti-retroviral therapy had no effect on the risk of malaria. The prevalence of P. falciparum infection was similar at all-time points for the targeted and non-targeted anti-helminth treatment arms, with a significant fall in helminth prevalence in both arms by visit 2. Albendazole therapy was associated with favourable changes in haemoglobin levels, CD4 counts and viral loads, in those subjects with helminth infections. Haemoglobin levels were similar in both arms at all study visits, rising significantly from visit 1 to visit 2 in both groups and peaking by visit 3. Thereafter, levels fell significantly (p<0.0005 for both comparisons) by visit 4. Conclusions: The prevalence of helminth infection in HIV infected pregnant women on antiretroviral therapy is common in rural and peri-urban settings in Rwanda. This study clearly shows that, albendazole treatment is associated with an increase in CD4 counts, a fall in viral loads and an increase in haemoglobin levels. The effects of albendazole are mediated by the eradication of helminth infection. The study also shows that treatment with albendazole using a targeted or non-targeted regimen is equally effective. The mechanism by which certain ART regimens reduce the risk of helminth infection warrants further study.Item Mannose binding lectin genetic polymorphism: association with HIV-1 infection in adults and children in Zimbabwe(2017) Zinyama-Gutsire, Rutendo Beaunah LynmarryBackground HIV infection has remained a major global health burden since its discovery in 1983 and Sub-Saharan Africa remains the region hardest hit by the HIV/AIDS pandemic. The HIV pandemic continues to ravage most parts of Southern African countries, current prevalence between 10-20%. Individuals worldwide differ in their degree of susceptibility to HIV infection and genetic polymorphisms play a major role. Mannose Binding Lectin (MBL) is one such immunological factor found in serum/plasma, it is a normal liver-derived protein and is a key component of the innate immune defence system. MBL deficiency, due to mutations in the MBL2 gene and promoter region, leading to decreased plasma/serum MBL concentration, characterised by defective opsonisation activities of the innate immune system and increased susceptibility to infections including HIV-1 and schistosomiasis. Rationale While there is a lot of advancement in HIV prevention and treatment in Southern African countries, there is still need to investigate host genetic molecules in adults and mother-baby pairs that could be playing a role in HIV-1 transmission/acquisition, disease progression and survival. It was imperative to carry out this study because of the need to quantify the burden of MBL deficiency in this Zimbabwean adult and PMTCT study populations. Alsoto contribute to the knowledge gap on the role of MBL deficiency in HIV-1 transmission, disease progression and survival in African populations in adults and children. The available literature shows that the majority of studies on the association of MBL deficiency and HIV-1 infection in adults and children have been done on populations outside the African continent. There is dearth of information on the role of MBL in this era when access to ART has greatly improved even in developing countries like Zimbabwe. This will be the second study that will assess MBL2 genes and promoter typing in mother-infant pairs in HIV vertical transmission/acquisition. This study aimed to identify and explore potential biomarkers for susceptibility to HIV infection and disease progression. We assessed role of MBL deficiency in HIV-1 and schistosoma infections in Zimbabwean adults enrolled in the Mupfure Schistosomiasis and HIV Cohort (MUSH Cohort) (Paper 1).We also assessed the role of MBL deficiency on HIV progression and survival in this African adult population. We hypothesized that MBL deficiency has a role to play in HIV infection by increasing HIV disease progression and decreasing survival (Paper 2). We also determined prevalence of MBL deficiency, as estimated by MBL2 haplotypes among Zimbabwean mothers and their children aged 9-18 months old as well as its association with risk of HIV-1 infection and vertical transmission from their HIV positive mothers (Paper 3). Main Aim The broad objective of this study was to determine the relationship between MBL deficiency and HIV infection in an adult population of males and females and among mother-infant pairs in Zimbabwe. Study Specific Objectives 1. To determine the prevalence of MBL deficiency among the Zimbabwean adult population. 2. To determine the relationship of MBL deficiency with HIV infection among the Zimbabwean adult population. 3. To determine the effect of MBL deficiency on disease progression and survival among the Zimbabwean adult population. 4. To determine prevalence of MBL deficiency among mothers and their infants in a Zimbabwean population. 5. To determine the relationship between MBL deficiency and HIV transmission from mother to child in a Zimbabwean population. Methods DNA and plasma samples for MBL and HIV analysis were collected from the 379 adult males and females from the MUSH cohort and stored dried blood samples from 622 mother infant pairs from a national PMTCT survey. HIV-1, S. haematobium and S. mansoni infections were determined at baseline using HIV commercial kits and parasitologically respectively. Plasma MBL concentration was measured by ELISA and MBL2 genotypes determined by PCR. We calculated and compared the proportions of plasma MBL deficiency, MBL2 structural variant alleles B (codon 54A>G), C (codon 57A>G), and D (codon 52T>C) as well as MBL2 promoter variants -550(H/L), -221(X/Y) and +4(P/Q) between HIV-1 and schistosoma co-infection and control groups using Chi Square test (Paper 1). We also assessed the role of MBL deficiency on HIV disease progression and survival inthe adult (MUSH) cohort.We analysed blood samples for MBL levels, MBL2 genotypes, HIV-1 status, viral load and CD4+ T cell counts (Paper 1). Participants were followed up for 3 years wherein the endpoints were measured at baseline, 6 weeks, 3, 6, 12, 24 and 36 months. Disease progression was measured as the rate of decline in CD4+ T cell counts and the rate of increase in HIV viral load (Paper 2). Generalised Estimating Equations (GEE) models were used to compare rates of change of the CD4+ T cell count and viral load measurements over the three-year follow-up period. The role of plasma MBL deficiency and MBL2 genetic variants on survival over the 3-year period were estimated using the Cox proportional hazard models. Regression analysis was used to test for interaction and confounding between MBL deficiency, MBL2 genetic variance, age and sex. We used the Wald Chi-square statistic to choose between full and nested models. We also assessed MBL2 polymorphisms in Zimbabwean HIV positive mothers and their children enrolled in a national PMTCT survey carried out in 2012. MBL deficiency was defined as presence of A/O and O/O genotypes in the mothers and their children. We extracted DNA from two dried blood spots for 622 mothers and infant pairs using the Gene Extract and Amp kit reagents. MBL2 Exon 1 genotypes and promoter region alleles -221(X/Y) and -550(H/L) SNP were detected by pyrosequencing. Differences in distribution frequency between HIV infected and uninfected children, of the MBL2 genotypes, promoter region variants and MBL2 haplotypes, were determined by the Chi square test or Fisher’s exact tests (Paper 3). Key findings For specific objective number 1, we assessed 379 adults, 80% females, median age (IQR) 30 (17-41) years. HIV-1, S. haematobium and S. mansoni prevalence were 26%, 43% and 18% respectively in the MUSH baseline survey. Median (IQR) plasma MBL concentration was 800μg/L (192-1936μg/L). Prevalence of plasma MBL deficiency was 18% with high frequency of the C (codon 57G>A) mutant allele (20%). For specific objective number 2, we found no significant difference in median plasma MBL levels between HIV negative (912μg/L) and HIV positive (688μg/L), p=0.066. However plasma MBL levels at the assay detection limit of 20μg/L were more frequent among the HIV-1 infected (p=0.007). S. haematobium andS. mansoni infected participants had significantly higher MBL levels than uninfected. All MBL2 variants were not associated with HIV-1 infection but promoter variants LY and LL were significantly associated with S. haematobium infection (Paper 1). For specific objective number 3, we assessed 197 HIV positive adults where 83% (164) were women with a median age of 31 years old. Prevalence of plasma MBL deficiency (less than 100μg/L) and MBL2 deficient genetic variants (A/O and O/O genotypes) was 21% (42 out of 197) and 39% (74 out of 190), respectively. We did not observe a significant role to explain individual variation in mortality, change of CD4+ T cell count and viral load by MBL plasma deficiency or MBL2 genetic variants from baseline to 3 years follow up period in this adult population (Paper 2). For specific objective number 4, from the PMTCT study, the median age (IQR) of the mothers was 30(26 - 34) years and the children mean age (IQR) was 12 (11-15) months old at the time of enrolment. All 622 mothers were HIV-1 infected, 574 babies were HIV negative and 48 were HIV-1 positive babies. MBL2 normal structural allele A and variants B (codon 5A>G), C (codon 57 A>G) and promoter region SNPs -550(H/L) and -221(X/Y) were detected. Prevalence of MBL deficiency was 34% among the mothers and 32% among the children. For specific objective number 5, we found no association between maternal MBL2 deficiency and HIV-1 transmission to their children. We found no difference in the distribution of HIV-1 infected and uninfected children between the MBL2 genotypes of the mothers and those of the children (Paper 3). Conclusions The results from our study indicate high prevalence of MBL deficiency but we found no evidence of association between MBL deficiency and HIV-1 infection. However, lower plasma MBL levels were associated with reduced prevalence of both S. haematobium and S. mansoni infections and MBL2 promoter and variants LY and LL were associated with increased susceptibility to S. haematobium infection (Paper 1). Our findings attest to the large between-population variability in a host of factors that can predispose individuals susceptible to HIV progression and mortality. We therefore cannot recommend at this time the use of plasma MBL levels or MBL2 genetic variants as a prognostic marker in HIV infection, disease progression and survival in this adult population in Africa (Paper 2). MBL deficiency was not associated with HIV-1 infection among the children nor was it associated with HIV-1 vertical transmission in this study population (Paper 3).