Approaches to the identification of genetic variants associated with complex brain diseases have evolved in recent decades. This evolution was supported by advancements in medical imaging and genotyping technologies that result in rich data production in the field of imaging genetics and radiogenomics. Studies in these fields have taken different designs and directions from genomewide associations to studying the complex interplay between genetics and structural connectivity of a wide range of brain-related diseases. Nevertheless, such combinations of heterogeneous, high dimensional and inter-related data has introduced new challenges which cannot be handled with traditional statistical methods. In this thesis, we proposed analysis pipelines and methodologies to study the causal relationship between neuroimaging features, including tumour characteristics and connectomics, genetics and clinical factors in brain-related diseases. In doing so, we adopted two longitudinal study designs and modelled the association between Alzheimer's disease progression and genetic factors, utilising local and global brain connectivity networks. In addition to that, we performed a multi-stage radiogenomic analysis in glioblastoma using non-parametric statistical methods. To address some limitations in the methods, we adopted the Structural Equation Model and developed a mathematical model to examine the inter-correlation between neuroimaging and multi-omic characteristics of brain-related diseases. Our findings have successfully identified risk genes that were previously reported in the literature of Alzheimer's and glioblastoma diseases, and discovered potential risk variants which associate with disease progression. More specifically, we found some loci in the genes CDH18, ANTXR2 and IGF1, located in Chromosomes 5, 4 and 12, to have effect on the brain connectivity over time in Alzheimer's disease. We also found that the expression of APP, HFE, PLAU and BLMH have significant effects on the structural connectivity of local areas in the brain, these are the left Heschl gyrus, right anterior cingulate gyrus, left fusiform gyrus and left Heschl gyrus, respectively. These potential association patterns could be useful for early disease diagnosis, treatment and neurodegeneration prediction. More importantly, we identified gaps in the imaging genetics methodologies, we proposed a mathematical model accounting for these limitations and evaluated the model which produced promising results. Our proposed flexible model, BiGen, addresses the gaps in the existing tools by combining neuroimaging, genetics, environmental, and phenotype information to a single complex analysis, accounting for the heterogeneity, inter-correlation, and non-linearity of the variables. Moreover, BiGen adopts an important assumption which is hardly met in the literature of imaging genetics, and that is, all the four variables are assumed to be latent constructs, that means they can not be observed directly from the data, and are measured through observed indicators. This is an important assumption in both neuroimaging, behavioural and genetic studies, and it is one of the reasons why BiGen is flexible and can easily be extended to include more indicators and latent constructs in the context of brain-related diseases.

Identification of HIV-1 Envelope (Env) fitness determinants could provide functionally constrained, accessible regions that could be included in subunit vaccines to induce broadly neutralising antibodies (bnAb). We hypothesised that Env fitness determinants are common to circulating variants but that the plasticity of Env structure limits identification. Rapid evolution; however, could select for sequence changes within the determinants coincident with alterations in function, making identification easier. Dual infection with two phylogenetically distinct HIV-1 variants under the same selective pressures might result in rapid functional evolution, facilitating identification of Env fitness determinants. It has been shown that the Env plays a significant role in viral adaptation to the host environment, which then increases disease progression. Therefore, this study used dual infections as a model system to characterise Env function, its role in in vivo viral outgrowth of variants and disease progression and to identify fitness determinants for future vaccine design. Single-genome amplification (SGA)-derived env sequences of four dual infected individuals sampled at enrolment (0 months), 3, 6, and 12 months post infection (mpi) were analysed using Highlighter plots, RIP, DNA pairwise distance and Neighbourjoining trees to determine the in vivo evolution of infecting viral populations and their relative frequency over time within each participant. Representative amplicons were cloned at each time point and compared using a pseudovirus (PSV) entry efficiency assay. 2 characterised by Affinofile system, T-20 IC50 and Western blotting to identify whether tropism, Env expression/cleavage, incorporation into viral particles and fusogenicity were most likely responsible for the variation in Env entry efficiency. All variants were R5- and T-tropic and only Env fusion capacity correlated significantly with Env entry efficiency data (p = 0.02, r = 0.59), suggesting that variants infecting dual infected participants evolved towards higher fusion capacity. Changes in Env fusogenicity indicated that gp41 might be a fitness determinant of PSV entry efficiency and analysis of SGA sequences indicated that recombination within gp41 was common to 3/4 participants. Env chimeras were generated where gp41 was swapped between clones that either had the same (CAP84) or different (CAP267) PSV entry efficiency. For both participants, and (CAP137) gp41 was identified as a potential determinant of Env fitness. Moreover, two potential N-glycan sites (PNG) at position N332 and N339, previously reported to be involved in neutralising antibody escape, were also identified. While N332 enhanced Env entry efficiency in one participant, N339 attenuated Env entry efficiency in another, potentially due to the escape mutation carrying a fitness cost. However, neither PNG seemed to affect Env expression/cleavage, incorporation into viral particles and fusogenicity. As Env phenotypic characterisation focussed on PSV assays, we wanted to determine whether viral replication was also similarly affected. Infectious molecular clones (IMCs) were generated from two participants using a recombination yeast assay and replication capacity (RC) in peripheral blood mononuclear cells (PBMCs) was assessed using parallel replication. A significant correlation between RC of viruses in PBMCs and Env entry efficiency in TZM-bl and fusion capacity (p = 0.03, r = 0.7; p = 0.04, r = 0.7, respectively) was determined. IMC RC was also associated with in vivo outgrowth of viral populations at 12 mpi although this relationship did not always coincide with the frequency of individual variants. Changes in the RC of the Env chimeras and mutants was not associated with phenotypic changes, suggesting that Env entry efficiency determinants did not play the same role in IMC RC as it did in PSV entry. Lastly, there was a significant negative association (p = 0.046, r = -0.59) between Env entry efficiency and CD4+ T decline, a marker of disease progression, supporting the previous finding that Env entry efficiency could be the driving agent of disease progression. This was also corroborated by the trend in association between RC of IMCs and faster CD4+ T decline. 3 Our findings suggest that despite different host pressures, viral competition in most dual infected individuals selected for rapid recombination within gp41 that enhanced fusion capacity. Enhanced gp41 fusogenicity of the dominant viral population at 12 mpi increased PSV entry efficiency and replicative fitness enabling viral outgrowth. Therefore, vaccines that target gp41 might prevent HIV infection or at least attenuate viral fitness and slow disease progression. On the other hand, we showed that targeting the PNG at position N339 of gp120 might influence viral fitness and increase viral load and/or decrease CD4 T cell count. This is in keeping with the association between CD4 T cell decline and PSV entry efficiency and IMC RC, suggesting that Env fitness plays a role in HIV pathogenicity.

HIV-associated neurocognitive disorders (HAND) affect up to 70% of HIV positive individuals and are the leading cause of dementia in patients under 40 years. Despite this, the molecular mechanisms involved in the onset of HAND are not well understood. Among a number of plausible etiological agents of HAND, HIV-Tat has been shown to be neurotoxic in vitro and in vivo, but the basis of its induced neuronal dysregulation remains relatively poorly characterised, giving rise to various competing theories. This thesis describes differential, quantitative proteomic analyses of HIV-Tat-treated neuronal cells in vitro, the goal being to gain deeper insight into the underlying molecular basis of this HIV-Tat-mediated dysregulation, as well as to potentially inform better patient treatments in the future. To achieve this goal, deep, quantitative proteomic analysis of HIV-Tat treated SILAC-labelled SH-SY5Y neuroblastoma cells was carried out, alongside transcriptomic analysis of the same system in which 3077 proteins were identified and quantified with 407 proteins and 1074 genes being differentially expressed. Subsequently, label-free proteomics analysis was used to study the ability of lithium - a proposed new treatment for HAND - to suppress the HIV-Tat induced dysregulated molecular phenotype in SH-SY5Y cells in which 3757 were identified and quantified with 360 and 531 being significantly differentially expressed in HIV-Tat and HIV-Tat + lithium treated cells, respectively.

Airborne infectious diseases, such as tuberculosis (TB), are spread by airborne infectious particles (viable particles with potential for TB infection) in exhaled air from infectious individuals in enclosed spaces. Exhaled air is the carrier of airborne infectious particles and carbon dioxide is used as a surrogate of this exhaled air. Using carbon dioxide as a surrogate for exhaled air, we modified the Wells-Riley model and the prior modified versions of the model, and obtained a exible but sensitive mathematical model that predicts the risks of airborne infectious diseases, such as TB under steady- state and non-steady-state conditions, without assumptions of well mixed airspace and equilibrium conditions. Applying experimental data from in vivo studies to the mathematical model developed in this study, we explored the probability of exposed guinea pigs acquiring infection in these in vivo stud- ies and quantified the number of surviving airborne infectious particles (infective organisms) required to reach the alveolar to establish infection. Our study shows that the number of infective organisms reported in the in vivo studies might have been markedly underestimated. In this study, we investi- gated TB transmission in congregate settings, such as schools, households, public transport, prisons and health care settings and suggested preventive measures. TB transmission in these locations is attributable to numerous factors, including overpopulation and air pollution, which acts as a carrier of airborne infectious particles. We explored the impact of effective contact rate on TB epidemiology using a mathematical model we developed that consists of five states of susceptible, primary infection, reinfected, active TB and treated individuals. An infectious individual with varying effective contact rate (ranging from 5 to 30 per year) was introduced among 100; 000 fully susceptible individuals and we observed the number of primary infection and reinfected individuals at stability points of a TB epidemic. We found that the number of primary infection individuals decreases at the stability point, while that of reinfected individuals increases with increasing effective contact rate. This implies that a large number of active TB cases might be reinfected individuals. Using an age-structured mathemat- ical model developed in this study that incorporates vaccination, we explored TB disease progression in different age groups (from 0 to ≥ 75 years). We found that TB disease progression is age dependent. High TB notification rate was detected for the age groups [0 - 5); [15 - 25); [45 - 55) and [55 - 65) years, and the lowest TB notification rate was detected in the age group [5 - 15) years. Furthermore, we noted that vaccination decreases active disease progression for the age groups [0 - 5) to [15 - 25) years, while TB notification remains high for the age groups [25-35) to ≥ 75) years. The findings in this study suggest that active disease progression depends on age and average duration of the waning of the vaccine effect.

The purpose of this dissertation was to provide an overview of acute intermittent porphyria, focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD), as well as experimentally demonstrating the use of kinetic, structural and thermodynamic approaches to shed light on the enzyme reaction. The key focus was to investigate the effect of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme. Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme.

Kaposi's sarcoma-associated herpesvirus (KSHV), a gamma-herpesvirus with a particularly high seroprevalence in Sub-Saharan Africa (SSA), is the etiological agent of the endothelial tumour Kaposi's sarcoma (KS), the most common acquired immunodeficiency syndrome (AIDS)-related malignancy worldwide and particularly in SSA. It also causes primary effusion lymphoma (PEL), multicentric Castleman disease (MCD) and KSHV inflammatory cytokine syndrome (KICS). AIDS-related deaths have declined, due to global scale-up of antiretroviral therapy (ART). However, the vast majority of these occurred in SSA, where tuberculosis (TB) is the leading cause of mortality among human immunodeficiency virus (HIV)-infected individuals, accounting for a third of all AIDS-related deaths. The exceptionally high burden of suspected TB in SSA causes misdiagnosis or delayed diagnosis of diseases mimicking TB, such as several pathologies associated with KSHV. KSHV infection is essential but insufficient for the development of KS and other KSHV-associated pathologies; precipitating factors, such as HIV-related immune suppression and potentially genetic predisposition, are required. The erythropoietin-producing hepatocellular carcinoma (Eph) receptor A2 protein (EPHA2) tyrosine kinase receptor is a promising candidate for studies on genetic variants as it potentially acts on two levels: susceptibility to KSHV infection (being one of the key receptors utilised by KSHV for cell entry and intracellular trafficking) and susceptibility to KS development (being implicated in oncogenesis). Despite the high seroprevalence in SSA, the contribution of dysregulated KSHV lytic replication or host KSHV receptor variations to disease outcome in HIV-infected patients is unknown. We hypothesised that KSHV lytic reactivation plays yet unrecognised roles for morbidity and mortality in high HIV settings and to this end, we conducted a cohort study of 682 HIV-positive critically ill patients admitted to Khayelitsha Day Hospital, South Africa, investigated for TB, and followed for 12-weeks to ascertain vital status. We demonstrated that elevated blood KSHV viral load (VL) was a strong predictor of death in hospitalised HIV-infected patients without microbiologically proven TB. Further, we identified and validated variants in the EPHA2 protein tyrosine kinase and sterile alpha motif domains that were significantly associated with susceptibility to infection, KS development and/or KSHV VL in 300 South African HIV-infected patients, by aggregate by-gene analysis. In order to elucidate the functional significance of the identified EPHA2 missense mutations, we knocked out endogenous EPHA2 by CRISPR/Cas9 in the human endothelial cell line, HuARLT2, and reintroduced the wild type and mutant EPHA2 open reading frames by lentiviral transduction. These engineered cells were assessed for baseline EPHA2 phosphorylation levels and susceptibility to KSHV infection utilising recombinant KSHV in binding, internalisation and infection assays. We found that the EPHA2 mutant c.2254T>C (p.Leu700Pro) in the tyrosine kinase domain, associated with KS in our patient cohort, was deficient in tyrosine phosphorylation and less permissive to rKSHV infection when introduced as a single mutation or as a double mutant together with c.2257A>C (p.Asp701Ala) which was found to be in linkage disequilibrium with it. Another tyrosine kinase domain variant, c.2688G>S (p.Ala845Pro), found to be overrepresented among KS patients, had enhanced baseline tyrosine phosphorylation levels. These findings validated the patient-derived data on the molecular level by assigning functional consequences to some mutants which might have implications for the development of future biomarkers predicting KS susceptibility in high-risk populations. In summary, this novel research contributes to the understanding of KSHV-associated pathology and disease outcome. It identified KSHV VL as a potential biomarker to predict KSHV-associated diseases and mortality and assessed the contribution of KSHV entry receptor EPHA2 variations to KSHV-associated pathologies, with potential clinical implications, by facilitating the development of novel diagnostic and surveillance tools.

HIV-1 subtype C is currently the most prevalent in the epidemiology of HIV/AIDS cases in subSaharan Africa. Most clinical protease inhibitors (PIs) were designed against subtype B and are reported to have reduced activity against subtype C proteases. Our initial hypothesis was to create an Escherichia coli based life-or-death selection system for the screening of potential PIs against HIV-1 subtype C protease (PR). This system was engineered by inserting an HIV PR cleavage sequence between the export signal peptide of the commonly used TEM-1 β-lactamase, which upon co-expression of the HIV PR in vivo, would cleave the modified β-lactamase thus preventing its translocation to the periplasmic space. This would result in the host cells' sensitivity to β-lactam antibiotics supplemented in the growth media. The presence of an inhibitor would restore resistance and therefore ‘life'. Despite validation of the E. coliscreening system using the Tobacco Etch Virus (TEV) protease, co-expression of HIV protease subtype C did not inhibit cell growth. Further investigations revealed PR C activity was inhibited by an endogenous E. coli protein. The inhibitor was isolated from E. coli crude cell lysates using ammonium sulphate precipitation, gel filtration and anion exchange chromatography fractionation. It was identified using peptide fingerprinting mass spectrometry (PMF), as alkyl hydroperoxide reductase C22 subunit (AhpC22). Mass-Assisted Laser Desorption-Ionisation-time of flight (MALDI-TOF) analysis of the precursor pre-incubated with AhpC22 revealed reduced autocatalytic cleavage occurring at the N-terminus of PR C. Inhibition kinetics using a recombinant source of AhpC22 characterized the enzyme as a non-competitive inhibitor of PR C activity with an inhibition constant (Ki ) of 0.88 µM. We also describe a protocol to express, purify and refold the HIV-1C protease which is well known for aggregation into inclusion bodies.

Maize streak virus (MSV; Genus: Mastrevirus; Family: Geminiviridae) causes maize streak disease (MSD); a major biotic threat to maize farming especially in sub-Saharan Africa, and it neighbouring Indian and Atlantic Ocean Islands, where its insect vectors in the genus Cicadulina thrive. Of the eleven known MSV strains (called A through K), only MSV-A is economically significant as it is the only one that causes severe disease in maize. MSV is a single stranded DNA (ssDNA) virus which, like RNA viruses, has high mutation and recombination rates. Given that these processes can sometimes promote viral diversity and result in the rapid evolution of new, fitter MSV variants, continuous genomic surveillance of MSV is therefore important. Based on analyses of full genome sequences, MSV-A has been classified into five subtypes (-A1, -A2, -A3, -A4, and -A6) and more than 20 recombinant lineages. Here, I showed using laboratory-based experiments that maize infecting mastreviruses such as Maize streak Reunion virus (MSRV) and MSV-C which have been found maize plants displaying severe streak symptoms do not in fact cause severe streak symptoms in maize when used to infect maize on their own. Although a mixed infection involving MSRV and MSV-B resulted in slight changes in symptom phenotypes it is unlikely that MSRV and MSV-C are responsible for emerging maize diseases. I carried out model-based phylogenetic and phylogeographic analyses of MSV-A movement dynamics in and out of Madagascar, Ethiopia and Rwanda using newly determined MSV-A genome sequences (Madagascar: n = 56; Ethiopia: n = 84) together with other sequences from GenBank. I showed that most movements of MSV-A into Madagascar have been from East Africa between the early 1990s and 2000s. My inferences show that MSV-A1 variants currently found in Ethiopia likely arrived there from Uganda or Kenya between 1985 and 1988. Similarly the MSV-A1 variants found in Rwanda likely also moved there from Ethiopia, Kenya or Uganda between 2007 and 2011. The time periods over which inferred movements of MSV-A1 into Madagascar, Rwanda and Ethiopia occurred all correspond with the period during which trade between these and other East African nations was being liberalized. Although these temporally-scaled phylogeographic analyses indicated that human activities are likely responsible for some of the long-range movements of MSV-A1 variants (such as movements from East Africa to Madagascar), leafhopper-mediated dissemination of these variants also likely played a major role in long and short distance movements of these variants within both Madagascar and between East African countries. Over 90 years of evolution that yielded MSV-A-ZW-MatA_1994 in the MSV-A1 lineage, produced symptoms that have varied in a less concerted ways, or largely remained unchanged. Major harms (intensity of chlorosis, leaf deformation and stunting) have decreased while the amount of colonized cells (chlorotic areas) that determine onward transmission have increased. These data suggest MSV-A has evolved to optimize the number of cells it infects for effective onward transmission, while reducing excessive harm to its hosts. Altogether, these results suggest (1) synergism potentially plays a role in some instances of severe streak disease and (2) the movement of MSV-A1 within the East African region and Madagascar emphasizes the importance of this MSV-A subtype as a major ongoing threat to maize production within these regions; and (3) over the last 90 years, the MSV-A1 subtype has evolved to produce greater chlorotic areas on the leaves of infected maize plants while at the same time either not increasing or reducing the degrees of chloroplast destruction, stunting and deformation caused by infections: characteristics that may have enhanced the transmissibility of this variant and therefore played an important role in the present rise to dominance of this subtype throughout East Africa and Madagascar.

Triple negative breast cancer (TNBC's) is a highly aggressive and invasive subtype of breast cancer, typically characterised by the lack of estrogen receptor (ER), progesterone receptor (PR) and Human epidermal growth factor receptor 2 (HER2) with an inexplicable partiality towards African women. The acute heterogenicity and complexity of TNBC tumours, together with a lack of well-defined molecular targets, complicates prognosis of the diseases resulting in patient reliance on traditional therapies, like chemotherapy, radiotherapy, and surgery, which are associated with elevated incidence of adverse effects and relapse. A major contributor to the heterogenicity of TNBCs is the tumour microenvironment which is composed of tumour infiltrating lymphocytes (TILs), tumour cells, healthy cells, and tumour vasculature. TILs have commonly been used as a prognostic marker and show robust predictive value for TNBC. In-depth analysis of the TIL composition within TNBC tumours may provide greatly beneficial information for the development of newer tumour microenvironment changing therapies and could assist doctors in understanding what therapies a particular patient maybe susceptible to. Thus, the diagnosis and therapy of this disease may greatly benefit from improved molecular profiling and patient stratification. Precision medicine seeks to provide such a solution, by dividing patients into subpopulations based on disease-specific profiles. The identification of new molecular targets would provide the basis for development of novel therapies. To this end, one of the major aims of this thesis was to develop a phage display based screening technique which could be utilised to isolate novel TNBC specific cancer antibodies. Once selected these antibodies could be used to generate TNBC specific therapies. Specific monoclonal antibodies (mAbs) and derivatives thereof, have already been established as a revolutionary tool for drug delivery to cancerous cells. Such antibodies have been conjugated to cytotoxic drugs to form antibody-drug conjugates, which may exhibit multiple advantages over their unconjugated counterparts, but their general use in clinical application has been restricted due to developmental deliberations. Historical conjugation strategies used for the generation of ADCs commonly resulted in heterogeneous mixtures of ADC species, with varying drug-to-antibody ratios resulting in unpredictable pharmacologic characteristics and safety profiles. In more recent time, self-labelling tags such as Snaptag have provided a means of developing homogenised recombinant immunotherapeutics. Snaptag is a modified version of a human DNA repair enzyme, O6 - alkylguanine-DNA-alkyltransferase (AGT) which naturally removes alkyl residues from damaged DNA. The enzyme reacts specifically with benzylguanine (BG) derivatives via irreversible transfer of alkyl groups to cysteine residues forming stable end products. In this thesis, Snaptag technology, together with other antibody discovery and manipulation tools was used to develop a methodology allowing for the generation of disease specific fusion proteins. Specifically, these fusion proteins consist of single-chain antibody fragments genetically fused to snaptag, allowing for the generation of recombinant ADCs that could be used as a drug delivery system carrying any BG-modified drug to a disease specific targets. In addition, snaptag interacts with BG in a 1:1 stoichiometry giving rise to homogenised combination products which when fused to a scFv provides a fail-safe target-specific therapeutic option. In addition to antibody conjugates, one of the most promising of all mAb based therapies currently used, are checkpoint inhibitors. In a balanced immune response, immune activation is counteracted with immunoregulatory pathways such as checkpoint inhibition. These negative regulatory pathways are necessary for maintaining tolerance and preventing hyperactivation, and are governed by cell surface, inhibitory receptors known as ‘'checkpoint inhibitors''. Blocking of checkpoint pathways during chronic infections and cancer has been shown to improve T-cell functions leading to reduced viral load and tumour burden. These findings have been translated into clinical application where checkpoint inhibitors, which are monoclonal antibodies targeting CTLA-4, PD1, PD-L1 or other inhibitory ligands, have been used to block these inhibitory interactions. The main intention of this research was to develop a methodology which could be used to generate snaptag based recombinant fusion proteins with potential diagnostic and therapeutic applications. Several snaptag based fusion proteins were developed using the recommended methodology these included fusion proteins targeting breast cancer specific antigen BCK1, checkpoint inhibitors PDL1, B7.1/CD80 (interacts with CTLA-4),and TIL characterising markers CD3, CD4, CD8, CD19 and CD20. In addition, to demonstrate the versatility and robustness of this methodology we sought to develop a snaptag based fusion protein not targeting breast cancer related antigens. Zika virus, an emerging infectious disease, currently lacking specific therapies was chosen for this purpose. An scFv derived from antibodies targeting the the Zika-DIII envelop protein, which is essential to the viral infection cycle was used in the snap fusion protein. The resulting ZIKA-DII-snap fusion protein demonstrated specific binding to zika virus membrane fractions. This research demonstrates the feasibility of using snaptag technology as a state-of-the-art conjugation strategy capable of bypassing the challenges previously associated with using antibodies as an effective delivery system for therapeutic drugs. By combining the applicability of snaptag technology with other antibody isolation and manipulation tools we were able to generate several functional snaptag based recombinant fusion proteins. Establishment of this methodology represents an important first step in generating medically necessary, pharmaceutically acceptable immunoconjugates that is instrumental in shifting general therapy towards a more personalized precision medicine approach.