Browsing by Department "Department of Integrative Biomedical Sciences (IBMS)"
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- ItemOpen AccessA clinico-pathological study of schistosomiasis in South Central Africa.(1948) Gelfand, MichaelA study of bilharziasis as a whole, based largely on pathological and clinical experience, has not been attempted in Africa outside of Egypt. The most impressive work in Egypt in this connection is that Fairley on the experimental infection of monkeys, which he published in 1920. Elsewhere in Africa, a number of smaller publications dealing with a particular or restricted clinical branch of schistosomiasis has been contributed by various authors, notably from South Africa. Th most important of these is Cawston. Valuable contributions from the Congo were made by such workers as Fisher and Chesterman. From West Africa particular mention must be made of Blacklock, and from Nyasaland of Dye and Gopsill. Interesting clincial papers have also been published on bilharziasis from East Africa.
- ItemOpen AccessA post-gene silencing bioinformatics protocol for plant-defence gene validation and underlying process identification: case study of the Arabidopsis thaliana NPR1(2017) Yocgo, Rosita E; Geza, Ephifania; Chimusa, Emile R; Mazandu, Gaston KAdvances in forward and reverse genetic techniques have enabled the discovery and identification of several plant defence genes based on quantifiable disease phenotypes in mutant populations. Existing models for testing the effect of gene inactivation or genes causing these phenotypes do not take into account eventual uncertainty of these datasets and potential noise inherent in the biological experiment used, which may mask downstream analysis and limit the use of these datasets. Moreover, elucidating biological mechanisms driving the induced disease resistance and influencing these observable disease phenotypes has never been systematically tackled, eliciting the need for an efficient model to characterize completely the gene target under consideration.
- ItemOpen AccessA study of the biochemical changes which occur in experimental cadmium poisoning(1965) Gain, Adrian ConalCertain of these isotopes appear suitable for activation analysis by neutron or other type of bombardment, which, in the future, could provide a much more sensitive technique for determination of trace quantities of the element than the spectrographic or oolorimetrio methods currently employed. The metal tarnishes in air and burns when heated forming the oxide. It occurs naturally in small quantities associated with sine, and was discovered by Strongmeyer in 1817 as an impurity in zinc carbonate. Cadmium volatilizes before zinc during the course of preparation of the metal, and condenses as a brown oxide, which is then reduced with carbon. It forms a number of salts, the chloride and sulfate being readily available in high degree of purity.
- ItemOpen AccessAn African Genome Variation Database and its applications in human diversity and health(2021) Todt, Davis; Mulder, NicolaAfrican genomes exhibit the highest levels of sequence and haplotype diversity of all extant human populations. A combination of historical as well as geographical factors have contributed toward the high level of genetic diversity in Ancestral populations in Africa. Additionally, a series of concomitant migration events out of Africa, with founder populations harbouring only a subset of this genetic variation, have contributed to the relatively lower genetic diversity observed in non-Africans. Population genetic studies have refined our understanding of human evolutionary history and clinical genomic studies have resulted in improved patient outcomes. However, despite the increased throughput and decreased cost afforded from next-generation sequencing (NGS) and despite the relatively higher genetic variation in Africans, relatively little of the genomic data currently available is representative of diverse African populations. This may result in adverse outcomes in the context of minority populations with little representation in clinical databases. Given the under-representation of African genetic variation and the importance of highlighting and further characterizing it, the objectives of this project were to design, develop and deploy a proof of concept database and web application for the storage, analysis and visualization of African genetic variant data – the African Genome Variation Database (AGVD). The AGVD was developed according to software industry design standards. The project also explored available genomic tools and databases in order to leverage existing software solutions where suitable. Additionally, relevant data sets were identified for use during testing and validation of the pilot phase of the project. To this end, the open access 1000 Genomes Project phase 3 dataset was selected and the genotypes for several chromosomes were loaded into the AGVD. The AGVD leverages the scalable, performant, and open source genomics engine OpenCGA for data storage and analysis. A custom front-end web application was developed by applying a novel approach to render and serve static Vue JS assets from the Python Flask microframework. The web application supports rich data search and filtering operations of loaded variants and allows end-users to visualize annotations of genomic loci and allele change, variant type, associated gene and transcript consequences, clinical significance, and allele frequency information for all annotated cohorts in a highly interactive manner. A bespoke REST API also supports future analytical functionality. The AGVD has demonstrated proof of concept in the secure and scalable storage and visualization of African genomic data, providing a viable solution for H3ABioNet to further extend in future iterations of the project and a valuable resource for researchers to explore African genetic variation.
- ItemOpen AccessAn immunoproteomic approach to identifying cancer-associated autoantibody biomarkers(2018) Smith, Muneerah; Blackburn, JonathanCancer is a heterogenous disease capable of forming and spreading in most tissues of the human body. Cancer screening and diagnosis can be performed through medical procedures, which are highly invasive, requiring an intensive infrastructure. It is therefore important to create cost-effective, non-invasive cancer diagnostic tools that also gives an indication of disease prognosis. With this in mind, the Blackburn lab previously created a cancer-testis antigen microarray (CT100plus) functionalised with tumour-associated and tumour-specific antigens, capable of detecting plasma- or serum-derived autoantibodies in the picogram per millilitre (pg/ml) range. In this thesis, a newly established statistical pipeline was used to analyse colorectal cancer (CRC) patient-derived CT100plus data. Using the pipeline, the 10 antigens with the highest receiver operator characteristic (ROC)-derived area under the ROC curve (AUC)-values were identified as potential autoantibody-based biomarkers. The top 10 antigen biomarker candidates include CEACAM 1, COL6A1, GRWD1, MAGEA1, MAGEA5, MAGEA10, NY-CO-1, SGY-1, SPANXB1 and THEG. Using these biomarker candidates, distinct clusters of healthy controls (HCs) and CRC patients were observed using both unsupervised hierarchical clustering and principle component analysis (PCA) analysis. Combinatorial ROC analysis indicates that CEACAM1 and GRWD1 as the top autoantigen combination for CRC diagnosis, together producing sensitivity-, and specificity-, and AUC-values of 1.00, 0.77 and 0.94, respectively. Furthermore, other top autoantigens, including COL6A1, THEG and CEACAM7, a homologue of CEACAM1, were also identified in this thesis by affinity purification-mass spectrometry (AP-MS) for patients from the same cohort, providing supporting evidence that these antigens are associated with CRC. The CT100plus microarray content was enzymatically modified to include citrullinated proteins, with the subsequent assessment of CRC patient autoantibody response. Significantly (p-value ≤ 0.05; adjusted p-value ≤ 0.05) higher signal intensities were detected in CRC patients versus HCs for citrullinated CDK7, MAGEB1, MAGEB5, MAGEB6 and SYCP1, whereas no significant (adjusted p-value > 0.05) difference in autoantibody signal was detected for these autoantigens on the noncitrullinated microarray for the same patient cohort. ROC analyses of these antigens resulted in 2 an AUC-, sensitivity- and specificity-values of 0.91, 0.87 and 0.89, respectively. Together, this thesisshowsfor the first time that cancer patients elicit an autoantibody response to citrullinated proteins, resulting in potential novel CRC biomarkers. A novel AP-MS assay was developed to detect autoantibody responses to autologous native CRC tissue proteins. Using the optimised methodology, proteins or homologues of proteins that were significantly (> cut-off value) detected on the CT100plus microarray for the same 5 patients were re-identified by the orthogonal AP-MS method. Using the methodology, PAD2, an enzyme that catalyses the conversion of arginine to citrulline was also identified. In addition, citrullinated antigens associated with cancer were identified, including homologues of CDK7 and MAGEB supporting the conclusion that citrullinated homologues of these proteins induce an autoantibody response in CRC patients. Finally, serum and/or plasma samples of a cohort melanoma patients were analysed using the CT100plus microarray, and autoantibody signals were compared to those of healthy control (HC) samples. Using the established statistical pipeline, the 10 antigens with the highest ROC-derived AUC-values were identified as potential biomarkers. The top 10 biomarker autoantigen candidates for melanoma included CEACAM 1, DPPA2, FGFR2, ITGB1, MAGEA10, NANOG, PIM1, SPANXB1, THEG and XAGE1B. Using these biomarker candidates, distinct clusters of HCs and melanoma patients were identified in both unsupervised hierarchical clustering and PCA analysis. Combinatorial ROC analysis indicates that CEACAM1 and FGFR2 were identified as the top antigens for melanoma diagnosis, together producing sensitivity-, and specificity-, and AUCvalues of 0.96, 0.94 and 0.93, respectively. In conclusion, a statistical pipeline was established for microarray data, enabling the identification of potential antigens associated with cancer diagnosis, and the potential to determine disease prognosis. Using the established pipeline, cancer antigens associated with CRC and melanoma were identified. In addition, an AP-MS assay was developed for the identification of known and novel cancer antigen that can be added to the customisable CT100plus microarray.
- ItemOpen AccessAn in-depth analysis of comorbidities in the context of HIV burden, in a cohort of patients seeking healthcare at Khayelitsha facilities in 2016-2017(2023) Osei-Yeboah, Richard; Tiffin, NicolaIntroduction: Improvements in early detection of human immunodeficiency virus (HIV), linkage to treatment, and availability of antiretroviral therapy (ART) have contributed to increasing life expectancy for people living with HIV (PLHIV) in South Africa. These improvements have resulted in the decline of HIV cause-specific mortalities. In addition to existing tuberculosis burden in PLHIV, cases of chronic non-communicable diseases (NCDs) are increasing in the general population. Considering the ageing population of PLHIV in South Africa, it is important to understand their health needs, as well as identify potential drivers of comorbidities that may provide avenues for future interventions. This study aimed at exploring HIV and comorbidity profiles in a virtual cohort of a population of healthcare clients accessing care in public facilities in Khayelitsha, Cape Town. Methods: Routinely collected data for healthcare clients accessing care in public facilities in 2016/17 were obtained from the Western Cape Provincial Health Data Centre, and analysed to describe ascertained comorbidities, comparing the profiles of PLHIV and HIV-negative individuals. The risks of comorbidity occurrence in PLHIV, in the context of other comorbidities and HIV metrics such as ART duration, viral load and CD4 cell counts, including the contribution of comorbidities to unsuppressed viral load levels in PLHIV were explored. Findings: The findings show that accessing HIV care may lead to earlier ascertainment of common chronic NCDs – hypertension, diabetes, chronic kidney disease (CKD), cervical cancer in PLHIV, compared to HIV-negative clients. Analysis of routine health data suggests that ascertainment of comorbidities differs for healthcare clients due to sub-population differences including age, sex, HIV status and reasons for accessing care. Routine laboratory testing results for renal function reflect distinct healthcare experiences by age for healthcare clients with and without HIV. Analysis of routine data shows that presence of an existing comorbidity may contribute to the incidence of other comorbidities and unsuppressed viral load levels in PLHIV. Conclusion: From real life routine health data, this study has explored comorbidities profiles of PLHIV and HIV-negative clients and observed that routine health data could provide a better understanding of disease profiles, healthcare access and requirements for both PLHIV and HIV-negative clients.
- ItemOpen AccessAnalysis of the impact on phylogenetic inference of non-reversible nucleotide substitution models(2023) Sianga, Rita; Martin, DarrinMost phylogenetic trees are inferred using time-reversible evolutionary models that assume that the relative rates of substitution for any given pair of nucleotides are the same regardless of the direction of the substitutions. However, there is no reason to assume that the underlying biochemical mutational processes that cause substitutions are similarly symmetrical. Here, we evaluate the effect on phylogenetic inference in empirical viral and simulated data of incorporating non-reversibility into models of nucleotide substitution processes. I consider two non-reversible nucleotide substitution models: (1) a 6-rate nonreversible model (NREV6) that is applicable to analyzing mutational processes in double-stranded genomes in that complementary substitutions occur at identical rates; and (2) a 12-rate non-reversible model (NREV12) that is applicable to analyzing mutational processes in single-stranded (ss) genomes in that all substitution types are free to occur at different rates. Using likelihood ratio and Akaike Information Criterion-based model tests, we show that, surprisingly, NREV12 provided a significantly better fit than the General Time Reversible (GTR) and NREV6 models to 21/31 dsRNA and 20/30 dsDNA datasets. As expected, however, NREV12 provided a significantly better fit to 24/33 ssDNA and 40/47 ssRNA datasets. I tested how non-reversibility impacts the accuracy with which phylogenetic trees are inferred. As simulated degrees of non-reversibility (DNR) increased, the tree topology inferences using both NREV12 and GTR became more accurate, whereas inferred tree branch lengths became less accurate. I conclude that while non-reversible models should be helpful in the analysis of mutational processes in most virus species, there is no pressing need to use these models for routine phylogenetic inference. Finally, I introduce a web application, RpNRM, that roots phylogenetic trees using a non-reversible nucleotide substitution model. The phylogenetic tree is rooted on every branch and the likelihoods of each rooting are determined and compared with the highest likelihood tree being identified as that with the most plausible rooting. The rooting accuracy of RpNRM was compared to that of the outgroup rooting method, the midpoint rooting method and another non-reversible model-based rooting method implemented in the program IQTREE. I find that although the RpNRM and IQTREE reversible model-based methods are not as accurate on their own as outgroup or midpoint rooting methods, they nevertheless provide an independent means of verifying the root locations that are inferred by these other methods.
- ItemOpen AccessAnalysis of urinary lipid biomarker candidates from tuberculosis patients by multiple reaction monitoring(2019) Waldron, Elizabeth Louise; Blackburn, JonathanBackground: Tuberculosis (TB) is an aggressive disease and is the leading cause of death by infectious disease in South Africa. With early diagnosis and correct treatment, almost all TB cases can be cured. The main diagnostic tests in South Africa are limited for people living in rural areas, require sputum which cannot be produced by very ill patients, and have low sensitivity in immune compromised individuals. There is an urgent need for a non-invasive and robust diagnostic test which uses an easily accessible biofluid and can be performed at the point-of-care. Urine has shown promise as a diagnostic biofluid for biomarker investigation. Full scan mass spectrometry is the gold standard for the unbiased discovery of biomarker candidates, and targeted multiple reaction monitoring (MRM) is the method of choice for subsequent validation of biomarker candidates. A list of candidate urinary biomarkers has previously been generated which can discriminate between latent and active TB infection using MS1 mass spectrometry, but these biomarkers have not yet been verified by targeted mass spectrometry. Aims and Objectives: The aim of this project is to verify a list of biomarker candidates using MRM assays by: 1) developing MRM assays for known fatty acid standards, and 2) developing MRM assays for unidentified urinary lipid biomarker candidates de novo, which can be applied to clinical cohorts for future validation. Methods: Fatty acid standards were initially assessed using direct infusion full-scan MS1 mass spectrometry on an orbitrap mass analyser. They were then optimised for fragmentation by compound optimisation on a triple-quadrupole mass analyser, the data from which was used to build MRM assays. Liquid chromatography was optimised for these lipids and the MRMs were validated by spiking the lipid standards into a complex mixture. For the second part of the project, lipid extract (containing unidentified biomarker candidates) from patient derived urine samples were analysed by data-dependent acquisition with inclusion lists on an orbitrap mass analyser. From this experiment MS/MS data was acquired for biomarker candidates which were then compiled into MRM assays and verified using a triple-quadrupole mass analyser. Results: From six fatty acid standards, reliable MRM assays were generated for five of them. The biomarker candidates formed a list of 70 molecules which were further refined to 10 molecules which were reproducibly measured by MRM assay. Discussion and Conclusions: From this work the fatty acid standards can be used as internal retention time predictors for future lipidomic work and quality checks, as they eluted across a wide retention time range. The biomarker candidates have been verified using MRM assays and can be validated in larger clinical cohorts in the future. The end-goal is to use these biomarker candidates as part of a panel which represents a unique biosignature according to the disease state of the patient.
- ItemOpen AccessBiochemical studies in drug-induced porphyrias in the rat : with a review of the literature on experimental porphyria and an investigation of thirteen human cases(1964) Ginsburg, Aubrey DavidThe group of disorders characterized by disturbances of porphyrin metabolism pose .many fascinating problems. The drug-induced porphyrias in animals provide a model whereby these problems may be investigated by techniques which cannot be applied to man. The consumption of alcoholic beverages or other chemical substances has been associated with the development of porphyrinuria and porphyria, but it is controversial whether these agents are of primary aetiological importance. or 'Whether they precipitate the· disease in genetically predisposed individuals. The Turkish epidemic of porphyrla has shown conclusively·that this disease may occur as an acquired condition in man. and a variety of compounds has been shown to be capable of inducing porphyria in animals.
- ItemOpen AccessCell-free DNA and tumor exosome cargo as diagnostic and prognostic marker for prostate cancer(2023) Temilola, Dada; zerbini, luizAccording to the Global Cancer Statistics 2020, prostate cancer (PCa) is the second most commonly diagnosed male cancer and second leading cause of cancer death among men globally. Prostate cancer is known to be more aggressive among men of African origin with reasons not fully known. Previous studies have revealed PCa to be of a serious disease burden among African populations with PCa being the major cause of male cancer mortality. Prostate specific antigen (PSA) has long been introduced as a biomarker for screening in PCa diagnosis. However, serum PSA has low sensitivity for PCa diagnosis which has led to serious harm such as overdiagnosis and other complications of treatment for indolent disease. This makes it imperative to search for other novel biomarkers with high sensitivity and specificity for early diagnosis and management of prostate cancer. This study was aimed to characterize plasma and urinary cfDNA and tumour exosome cargo as diagnostic biomarker for PCa in South African populations with the goal of discovery of reliable, non-invasive, and novel biomarkers of PCa. We performed miRNA sequencing of exosomal RNA extracted from high and low Gleason's score PCa plasma samples. We performed differential expression (DE) of TCGA data and exosomal miRNA data and which we identified 185 miRNA and 65 miRNAs respectively. A comparison of the differential expressed TCGA miRNA and exosomal miRNA showed 13 miRNAs common between the two data with 7 of the 13 miRNAs expressed in the same direction. We further validated the expression of the 7 miRNAs using real time PCR in exosomal miRNA of high and low Gleason's score PCa samples and benign prostatic hyperplasia (BPH). We also performed whole exome sequencing of urinary cell free DNA and we identified 31 mutated genes. We reported for the first time an association between 27 of these genes and PCa in African populations. Four of the genes have earlier been identified as promising biomarker for prostate cancer diagnosis among African men. We also performed real time PCR quantification of cell free DNA to determine the concentration and DNA integrity of cfDNA in PCa and BPH of plasma and urine samples and which we were able to identify significantly higher plasma cfDNA level in PCa than BPH samples. We identified herein putative diagnostic biomarkers in plasma and urinary cfDNA and exosomes cargo for diagnosis of PCa in South African populations.
- ItemOpen AccessCharacterisation of dysregulated proteins in macrophages infected with Mycobacterium smegmatis focusing on matrix metalloproteases and their effectors(2019) Seele, Palesa Pamela; Sturrock, Edward; Blackburn, JonathanCavitation is a key facilitator in transmission of Mycobacterium tuberculosis (M. tb). Upregulation of matrix metalloproteases (MMPs) has been documented in patients with tuberculosis (TB), while their tissue inhibitor (TIMPs) levels remained the same. Animals which can develop cavities have well-conserved MMP-1 orthologs suggesting a pivotal role of MMP-1 in cavitation. The migration of immune cells to the site of infection and maturation of the granuloma is associated with MMP-9 expression. Our understanding of the phenotypic changes induced by Mycobacterium smegmatis (M. smeg) in THP-1 macrophages is central to understanding its avirulent nature especially its effects on MMPs. The aim of this study was to evaluate the role of MMP-1 and MMP-9, and their effectors in macrophages infected with M. smeg. Differentiated THP-1 monocytes were incubated in serum-free media with or without bacilli. Thereafter, the secretome and lysate were harvested at different time points. The activity of MMPs was analysed by zymography. The activity of MMP-1 and MMP-9 were specifically determined using an MMP-1 fluorogenic assay and a non-fluorogenic MMP-9 substrate monitored using the HPLC. Discovery proteomics was performed for the 18-hour time point with the use of mass spectrometry. The generated data was used to evaluate dysregulated proteins and those that act as upstream and downstream effectors of MMPs. The phenotypic changes induced by M. smeg were also analysed. In addition to that, the hosts’ response to lipoarabinomannan H37Rv (LAM) treatment was assessed by discovery proteomics and zymography. There was an increase in gelatinase activity of secreted MMP-9 which was maintained between the 1 and 18-hour time points. The fold difference in activity between uninfected and infected declined at 24 hours, and at the 72-hour time point the uninfected was slightly higher versus the infected. The data also suggests a switch in the proteolytic repertoire of the macrophages between the 6- and 18-hour time points to one that potentially generates the same degradation products as the uninfected macrophages. The intracellular gelatinase activity of MMP-9 (82 kDa) was not significantly altered by the M. smeg infection, in fact the activity was slightly higher in the uninfected in the 18 and 24-hour time points. In contrast to MMP-9, MMP-1 was secreted in the later time points and was significantly decreased by the infection. This supports the postulation that upregulation of MMP-1 is specific for M. tb infection. The proteomics data depict significant upregulation of MMP-9 in the lysate and secretome, while TIMP-1 was exclusively expressed and secreted by infected macrophages, validating the non-destructive ECM phenotype induced by M. smeg. The dysregulation of IL-1β and COX pathways were implicated in the overexpression of MMP-9, as well as tRNA aminoacylation in alternative splicing of MMPs. The GO enrichment of exosomes is postulated to play a role in the recycling of MMP-9. Intercellular communication is hypothesised to be delivered to neighbouring cells through exosomes carrying DNA, RNA, proteins and DNA/RNA binding proteins, and via signalling scaffolds formed by the 14-3-3 proteins amongst others. LAM treatment did not induce dysregulation in the activity of expressed and secreted MMP-9, however, TIMP-1 was upregulated explaining the lack of differential gelatinase activity between treated and non-treated macrophages. The C-type mannose receptor 2 (MRC2) and C-type lectin domain family 11 (CLEC11A) were only expressed by the LAM-treated macrophages and may partake in the recognition and uptake of M. tb. Interestingly, the data indicates the presence of chromatin in the secretome which may be responsible for the formation of extracellular traps (NETs) and facilitating the transport of LAM across the glomerular basal membrane (GBM) through exosomes. Inhibition of MMP activity by TIMPs could result in decreased aggregation of NETS (aggNETS) that trap the LAM from being transported by binding to the chromatin. This decreases the concentration of LAM in urine and means MMP inhibitors that chelate the active-site zinc could decrease the sensitivity of urine-LAM detection kits.
- ItemOpen AccessCharacterisation of HIV-1 subtype C envelope functional determinants of dual infected individuals(2018) Omar, Shatha Sultan Ahmed; Woodman, ZendaIdentification 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.
- ItemOpen AccessCharacterisation of human surfactant protein A and recombinant human vimentin in their modulation of HPV16 pseudovirus infection(2019) Carse, Sinead; Schafer, Georgia; Katz, AriehInfection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, where low-and middle-income countries (LMIC) have the highest incidence. Prophylactic HPV vaccines exist but LMIC have limited access. Therefore, alternative preventative measures against HPV infection and cervical cancer progression are needed. Two human proteins have been identified in our laboratory that modulate HPV16 pseudovirus (HPV16-PsVs) infection in vitro, namely surfactant protein A (SP-A) and recombinant human vimentin (rhVim). Previous work suggested SP-A mediated immune recognition of HPV since SP-A-coated HPV16- PsVs enhanced viral uptake by RAW264.7 murine macrophages. These initial observations were confirmed using a murine C57BL/6 cervicovaginal challenge model: pre-incubation of HPV16- PsVs with purified human SP-A significantly reduced the level of HPV16-PsV infection in vivo. Moreover, when isolated cells from female reproductive tracts of naïve C57BL/6 mice were incubated with HPV16-PsVs and stained for selected innate immune cell populations by flow cytometry, significant increases in viral uptake by eosinophils, neutrophils, monocytes and macrophages were observed over time using SP-A-pre-coated virions compared to control particles. Compared to SP-A mediated modulation of HPV infection through activation of innate immune responses, rhVim was suggested to directly interfere with HPV entry into host cells. Indeed, supplementation with non-filamentous rhVim resulted in decreased viral uptake by NIKS cells which was confirmed in vivo using the murine C57BL/6 cervicovaginal HPV16-PsVs challenge model. Co-localisation analysis employing confocal imaging, revealed that rhVim-coated HPV16- PsVs co-localised, to a lesser degree, with surface-expressed heparan sulphate proteoglycans (HSPGs) than control particles. Removal of surface HSPGs on NIKS cells decreased HPV16-PsVs cell surface binding and internalisation, while pre-incubation of HPV16-PsVs with rhVim decreased viral particle binding and internalisation to a greater extent. This indicates that rhVim may modulate HPV16 infection by interfering with its attachment to HSPGs as well as viral engagement with the yet unknown entry receptor(s). In summary, both SP-A and vimentin modulate HPV16-PsVs infection by different mechanisms. These in vivo studies strongly confirm previous in vitro observations, rendering both proteins potentially suitable for further development into possible candidates for use in topical microbicides, which may provide protection against new HPV infections.
- ItemOpen AccessCharacterisation of Kaposi's sarcoma-associated herpesvirus (KSHV)-driven pathology and disease outcome in HIV infected South African patients(2020) Blumenthal, Melissa; Schafer, Georgia; Katz, AriehKaposi'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.
- ItemOpen AccessCharacterising the mechanism of DCUN1D1 activity in prostate cancer and identifying DCUN1D1 inhibitors for prostate cancer treatment(2020) Vava, Akhona; Zerbini, Luiz FernandoDCUN1D1 is an E3 ligase of the neddylation pathway. It mediates the posttranslational modification of majority of the cullin family of proteins with NEDD8. This activity is known to enhance ubiquitination of the cullin RING E3 ligases, however, the extent of the impact of DCUN1D1's activity is underexplored. Studies performed previously in our lab demonstrated the role of DCUN1D1 in prostate cancer in vitro and in vivo. We also identified potential inhibitors of DCUN1D1 which inhibited the proliferation of prostate cancer cells in a DCUN1D1-specific manner. This study seeks to determine the mechanism of action of DCUN1D1 in prostate cancer and to identify DCUN1D1 inhibitors using a proteomics approach. Immunoprecipitation-coupled mass spectrometry was performed to identify DCUN1D1 binding partners and we identified some known substrates of DCUN1D1 in the form of cullin 3, cullin 4B and cullin 5. We also observed that the DCUN1D1 pulldown products implicated the ubiquitin proteasome pathway, transcription, lipid metabolism and inflammatory pathways. SILAC quantitative proteomics analysis was also performed to determine the proteins that were differentially expressed in DU145 DCUN1D1 knockdown cells relative to DU145 control cells. Interestingly, we did not identify the cullin proteins or classical components of the neddylation pathway but identified the ubiquitin activating enzyme, UBA1. We also found that dysregulation of DCUN1D1 in prostate cancer led to a dysregulation in translation-related and protein processing activities such as dysregulation of eukaryotic protein translation, and protein processing in the endoplasmic reticulum. We also observed the recurrence of the WNT signalling pathway across the proteomics approaches. This culminated in the exploration of the mechanism of action of DCUN1D1 in prostate cancer using changes in protein expression as measured by western blot analysis. Significantly, we determined that DCUN1D1 mediates its mechanism of action in prostate cancer, through the neddylation pathway and preferential neddylation of cullin proteins. We also observed that knockdown of DCUN1D1 in prostate cancer led to the dysregulation of the ubiquitination and WNT/β-catenin pathways. Furthermore, advanced connectivity map analysis was performed to identify potential inhibitors of DCUN1D1 based on a proteomics approach. The drugs found to strongly connect with the DCUN1D1 knockdown signature included kinase inhibitors and anti-inflammatory agents. The above observations could lead to improved understanding of DCUN1D1 and its potential for molecular target based treatment of prostate cancer.
- ItemRestrictedComparison of the therapeutic efficacy of SNAP-tag fusion protein and the synergistic actions of chemotherapy and photodynamic therapy in killing resistant melanoma(2019) Biteghe, Fleury Augustin Nsole; Barth, StefanCutaneous melanoma is the deadliest form of skin cancer, which arises from epidermal pigment-producing cells called melanocytes. In melanoma, surgical excision of primary nonmetastatic tumor remains the gold standard of therapy worldwide. Upon metastases, melanoma becomes highly resistant to conventional radio-and chemotherapy. Chemotherapy, using dacarbazine (DTIC), remains the standard treatment option. In melanoma, chemotherapy failure has partly been attributed to a resistant population which is endowed with higher clonogenic potential, and aberrant expression of membrane proteins known as ABC transporters (ABCB5 and ABCG2), which mediate cellular resistance by extruding cytotoxic molecules from cells. To palliate these adverse effects, this study primarily aimed to investigate the efficacy of the synergistic actions of chemotherapy (Dacarbazine:DTIC) and hypericin-activated photodynamic therapy (HYP-PDT) in reducing chemoresistance in DTIC resistant (UCT Mel1DTICR2) and non-resistant melanoma cells (UCT Mel-1). To achieve these goals, the therapeutic efficacy of conventional therapies (DTIC, HYP-PDT and DTIC+HYP-PDT) was evaluated based on their ability to reduce cell viability; therapeutic resistant subpopulations; clonogenicity and ABC transporters (ABCB5 and ABCG2) in both melanoma cells. Additionally, the ability of the therapeutic treatments to efficiently halt cell division and activating cell death mechanisms, was assessed using cell cycle analysis and an Annexin-V assay. The results obtained showed that combination therapy was the most efficient therapy which was associated with a reduction in main populations (therapeutic resistant sub-population not expressing ABC transporters: MP), and clonogenic capacity in both melanoma cell types. Similarly, DTIC displayed a therapeutic efficacy which significantly reduced side populations (therapeutic resistant sub-population which were expressing ABC transporters: SP), and clonogenicity in UCT Mel-1 only. Interestingly, both ABCG2 and ABCB5 expressions were significantly increased in both melanoma cells, post combination therapy. Lastly, combination therapy and PDT were equally shown to induce a G1 cell cycle arrest, as opposed to DTIC which induced an S phase arrest. These cell cycle arrests were associated with efficient activation of apoptosis and necrosis, depending on the melanoma cell type, post HYP-PDT and combination therapy. Nevertheless, the efficacy of DTIC and HYP-PDT might respectively be limited by off target effects harming normal cells and low dosage in tumour cells which limiting their clinical utility. To address these challenges, this study aimed to develop a targeted tumor therapy, using the self-labelling activity of SNAP-tag fusion protein to conjugate synthetic small molecule lead substance toxin such as monomethyl Auristatin F (MMAF or AURIF). This cytotoxic payload was delivered to targeted cells, through genetic fusion of SNAP-tag to three different single chain fragments of an antibody (scFv1711, LsFv49 and scFv#34), which specifically treated tumor cells expressing epidermal growth factor (EGFR), melanotransferrin (p97) orfibroblast activation protein alpha (FAP-α) receptors, respectively. Achievement of this targeted therapy was performed through construction of three scFv-SNAP fusion proteins, which were expressed in HEK 293T cells. Thereafter, the purified scFv-SNAP fusion proteins were conjugated to BG-substrates to investigate their efficacy in specifically killing tumor cells. Binding and cytotoxic activities of the scFv-SNAP fusion proteins were performed using flow cytometry, and the XTT cell viability assay. All scFv-SNAP-fusion proteins were specifically bound to their target cells, indicating that AURIF conjugation did not compromise the binding activity of scFv-SNAP fusions. Finally, the cytotoxic assay confirmed that all scFv-SNAPAURIF conjugates induced a 50 percent reduction in cell viability at nanomolar concentrations in targeted cells which expressed their cognate antigens. To conclude, combination therapy was shown to be more efficient than monotherapies in killing chemoresistant melanoma cells, while SNAP-tag technology provided a superior, targeted therapeutic efficacy by sparing normal cells from unwanted toxic effects, and reducing the therapeutic requirement for high concentration of cytotoxic payloads.
- ItemOpen AccessCreating and analysing an African pan-genome(2022) Bourn, Jessica Jean; Mulder, NicolaThe human reference genome is currently a core resource for understanding the role of genetics in human health, disease, and variation, and has been invaluable in the development of clinical and computational tools for these purposes. However, the limited number of individual genomes used to create the reference has resulted in an underrepresentation of the extensive genetic diversity present in different human populations. Since an important use of the reference genome is to identify genetic variants that may be implicated in disease, this lack of diversity could limit the scientific utility of the reference for ethnic groups that are poorly represented in it. As a result, adaptations to the reference genome structure have been proposed. One such proposal has been the use of multiple reference genomes, each of which represent different human populations. A logical and highly practical method of achieving this is through the use of a pan-genome, which is a curated collection of all the DNA sequences that are found within a population under study. Despite the fact that African populations exhibit the greatest genetic diversity and variation in the world, the many and sometimes ancient ethnolinguistic groups from Africa are among those least represented within the reference genome. Consequently, this study aimed to explore the feasibility of creating and analysing an African pangenome, and to begin developing tools to achieve this. Several distinct African regional ancestral groups – namely east African Nilo-Saharan, east African Afro-Asiatic, far west Niger-Congo, central west Niger-Congo, Bantu-speaking Niger-Congo, central African rainforest hunter-gatherer, and the Khoe and San – have previously been identified, and this study included and analysed samples from each group in order to assemble a more inclusive and representative pan-genome. A software pipeline developed by Duan et al. (2019), termed the HUman Pan-genome ANalysis (HUPAN) pipeline, was used here to assemble the African pan-genome. As the HUPAN pipeline was originally designed to analyse only single populations, the inclusion of multiple populations required modifications and improvements, which were implemented following the testing and analysis of the pipeline using a smaller dataset of whole genome sequences. Subsequently, a final dataset of 168 African high- and medium-coverage whole genome sequences representing the seven separate regional ancestral groups was submitted to the adapted HUPAN pipeline. For each group, nucleotide sequences that were absent from the human reference genome were assembled and extracted, which resulted in the identification of 43.37 Mbp of non-redundant non-reference genomic sequence and 31 novel predicted protein-coding genes from African individuals. Alignment to other pan-genome sequences, whole genomes from different human populations, and the complete telomere-to-telomere human genome validated a large portion of the sequences as nonreference and confirmed that the dataset contained sequences specific to African populations. However, the gene presence-absence variation analysis of the pan-genome within all 168 samples revealed patterns of gene presence and absence that were strongly correlated to the sample dataset of origin, rather than to the ancestral group of origin. This hindered the identification of genuine genetic variation specific to the groups analysed. Further, it appears that previous pan-genomic research has not investigated the degree to which the genetic variation identified is dataset-specific or truly population-specific. Consequently, the failure to acknowledge and account for the effects of spurious inter-dataset variation in previous pan-genomic research indicates that those analyses may be incomplete or ambiguous. This, therefore, calls into question the methods currently used for pangenomic research, and highlights that robust, standardised methods for human pan-genome research must be agreed on to ensure that comprehensive population-specific pan-genomes are produced in the future. Despite this inherent weakness of pan-genomic research, this study successfully enabled the creation and analysis of a comprehensive and inclusive African pan-genome. Unique sets of non-reference sequences specific to African regional ancestral groups were identified and obtained, enabling the assembly of a non-redundant set of pan-African non-reference sequences. Furthermore, certain complex but previously unconsidered aspects of pan-genome research were identified and explored, and these observations may play a role in the advancement of pan-genome research in future.
- ItemOpen AccessDevelopment of potential immunodiagnostic & therapeutic techniques using SNAP-fusion proteins as tools for the validation of Triple-negative Breast Cancer(2020) Magugu, Freddy-Junior Siybaulela; Barth, Stefan; Naran, KrupaGlobally, breast cancer is the leading cause of death in the female population aged 45 and below with a breast cancer incidence reaching 18.1 million in the year 2018. Triple negative breast cancer (TNBC) is part of a group of cancers that lack the expression of Progesterone receptor (PR), Estrogen receptor (ER) and Human epidermal growth factor receptor 2 (HER2). TNBC is commonly associated with early stage metastasis with low survival rates as well as a high frequency of recurrence and proves to be problematic in both the young and elderly female populations. Conventional diagnostic methods for TNBCs include mammography, magnetic resonance imaging (MRI) and ultrasound while therapeutic methods include mastectomy and breast conserving surgery (coupled with radiation therapy). The lack of effective therapeutic options, poor prognostic value and high rates of metastasis, has made treatment of TNBC difficult. The major focus of this work was on the following tumour associated antigens (TAAs): CSPG4 (a transmembrane protein found in 50% of TNBC cases), EGFR (which is overexpressed in 13-76% of TNBCs), and MSLN (which is overexpressed in 67% of TNBCs) as potential targets for monospecific therapy. The evolution of antibody-based immunotherapy strategies has led to applications of single chain variable fragment (scFv) & single domain/nanobody (VHH) antibody formats for diagnostic and therapeutic purposes. In this work, these recombinant antibody fragments have been combined with SNAP-tag, a modified version of the human DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT), which autocatalytically binds benzyl-guanine modified substrates such as fluorophores or small molecule toxins covalently in a 1:1 stoichiometry. In this study, the primary aim was the comparison of different antibody formats fused to SNAPtag and the potential of these biopharmaceuticals towards immunodiagnosis and therapy of TNBCs. First functionalities of two scFv SNAP fusion proteins and one VHH SNAP fusion protein previously not having been described are provided through binding analyses on receptor positive tumour cell lines. This was achieved by in-silico design and molecular cloning of genetically fused antiCSPG4(scFv), -MSLN(scFv), -MSLN(VHH), -EGFR(scFv) & -EGFR(VHH) to SNAP-tag. The final constructs were confirmed by Sanger sequencing and subsequently transfected into a mammalian vector system (HEK293T) for transient expression of the engineered fusion proteins. Full length protein purified from cell culture supernatant was analysed for diagnostic/therapeutic activities dependant on the substrate attached in the form of a fluorophore or small molecule toxin resulting in recombinant antibody-drug conjugates (ADCs). The study shows promise in providing new immunodiagnostic and therapeutic agents that are specific and less harmful than the current state of the art procedure
- ItemOpen AccessDevelopment of SNAP-tag based fusion proteins as novel auristatin F-containing immunoconjugates and photoimmunotheranostics in the detection and treatment of triple-negative breast cancer(2022) Mungra, Neelakshi; Barth, StefanBreast cancer represents one of the most common forms of female malignancy of the 21st century. Among the various breast cancer subtypes, triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). As an idiosyncratic disease, TNBC displays a conspicuously aggressive and invasive clinical course, with an unexplained partiality towards women of African ancestry. Its acute heterogeneity and complexity behave as mutually reinforcing negative factors, which further complicate prognosis, thereby increasing the burden of breast cancer-related mortality. With the absence of well-defined molecular targets in TNBC, there is a heightened reliance on tri-modality therapy (surgery, radiotherapy and chemotherapy), albeit with an increasing incidence of adverse effects and disease relapse. To this end, there is an urgent need to develop an arsenal of targeted diagnostics and therapeutics, which can be synergized to cover the vast majority of triple-negative breast tumors, paving the way towards the development of personalized regimens suitable for the particular needs and disease of each patient. As such, achieving selective cytotoxicity, with minimal or no collateral damage to healthy tissues, embodies the holy grail of targeted anti-cancer therapies. For instance, the high affinity and specificity of monoclonal antibodies (mAbs) and derivatives thereof, have cemented their application as revolutionary tools in the selective delivery of drugs to malignant cells. These therapeutic proteins, also known as antibody-drug conjugates (ADCs), might exhibit several advantages compared to their small-molecule counterparts, but their widespread clinical use is hampered by various developmental considerations. Traditional conjugation strategies employed to arm mAbs with cytotoxic warheads, usually give rise to heterogeneous mixtures of ADC species, bearing non-uniform drug-to-antibody ratios (DARs), pharmacologic characteristics and safety profiles. Fortunately, the implementation of self-labeling tags (such as SNAP-tag, CLIP-tag and Halo-tag) are providing renewed impetus to homogeneous recombinant immunotherapeutics development. More precisely, SNAP-tag is an engineered mutant of the human O(6)-alkylguanine-DNA alkyltransferase, endowed with the ability to specifically and irreversibly react with benzylguanine (BG) derivatives, forming a stable product. Based on the above premises, this research aims to use SNAP-tag technology as a cutting-edge site-specific conjugation method to: (1) develop a comprehensive antibody platform, consisting of single-chain antibody fragments (scFvs) genetically fused to SNAP-tag, to specifically screen and evaluate their predictive potential for chondroitin sulfate proteoglycan 4 (CSPG4), CD44 and aspartate (aspartyl/asparaginyl) β-hydroxylase (ASPH)-positive TNBC cells, (2) generate functional recombinant ADC formulations as robust delivery systems carrying the antimitotic drug monomethyl auristatin F (MMAF/AURIF), concurrently overcoming production constraints to yield therapeutically viable and homogeneous combination products, and (3) provide a fail-safe system that overcomes the lack of specificity of photodynamic therapy (PDT), by coupling scFv-SNAP-tag to the potent near-infrared (NIR) photosensitizer (PS) IRDye700DX (IR700) and to demonstrate its selective dose-dependent cytotoxic activities in vitro. Following in silico design of the open reading frames (ORFs) coding for each construct, standardized molecular cloning techniques were implemented to generate recombinant mammalian expression plasmids, encompassing Ig-Kappa leader as an efficient protein secretory system. After confirmation of the DNA integrity, protein expression was achieved through transient transfection into HEK293T cells. Thereafter, the resulting histidine-tagged fusion proteins (αCSPG4(scFv)-SNAP, αCD44(scFv)-SNAPf and αASPH(scFv)-SNAP) were harvested from the cell culture supernatant and subjected to immobilized metal affinity chromatography (IMAC). In order to evaluate the outcome of this protein expression and purification step, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were used to confirm the presence of full-length recombinant SNAP-tag based fusion proteins based on their molecular weights. Integration of the fluorescent dye Alexa Fluor 488 into the fusion proteins was carried out to investigate the self-labeling activity of the SNAP-tag moiety, as well as to provide qualitative and quantitative insights into the binding potential of the antibody fragments towards their cognate antigens. Subsequently, the AURIF and IR700-based immunoconjugates were generated by conjugating scFv-SNAP with their respective BG-modified substrates, in a defined 1:1 stoichiometric reaction. The specific and dose-dependent biological activities of the resulting bifunctional therapeutic proteins were then assessed on TNBC cells. In this study, pCB-αCD44(scFv)-SNAPf was successfully cloned and all 3 fusion proteins were effectively expressed, although with low yields and purity, yet adequate for downstream in vitro characterization. After showcasing the self-labeling potential of the SNAP-tag component, surface binding of the fluorescently labeled product was demonstrated on antigen-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell killing ability of the novel AURIF-based recombinant ADCs and IR700 photoimmunoconjugates, was illustrated by the induction of a 50% reduction in cell viability (IC50 value) at nanomolar to micromolar concentrations on target cell lines. This observable selective cytotoxicity revealed that conjugation of BG derivatives to SNAP-tag, did not affect the binding potential of the antibody fragment, nor abrogated the cytocidal activity of the payload. As a proof of concept, this research builds on existing work that promulgates the use of SNAP-tag as a state-of-the-art conjugation strategy that can circumvent the challenges associated with the use of antibodies as effective delivery systems for therapeutic molecules. By harnessing the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically acceptable immunoconjugates, the results herein presented, also highlight the prospects of such agents in disease-specific tumor suppression. While various architectural modifications could further improve cytotoxic activities of future combination products, this research underscores the duality of SNAP-tag in the development of immunodiagnostics and therapeutics, that could potentially be instrumental in instilling a shift towards a personalized medicine stratagem. In conclusion, the combination of such immunoconjugates with a robust companion diagnostic panel provided by SNAP-tag, represents a first step towards the effective management of TNBC, with potential impact on the economic, social and clinical settings.
- ItemOpen AccessDifferential proteomic profiling towards elucidation of TB-IRIS pathogenesis(2022) Peyper, Janique Michelle; Blackburn, Jonathan; Meintjes, GraemeBackground Up to 59% of tuberculosis (TB)/human immunodeficiency virus (HIV) co-treated patients develop paradoxical TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) after addition of antiretroviral (ARV) therapy to anti-tuberculous therapy (ATT). The course can be prolonged and the average mortality rate is 2% (75% for TB-IRIS involving the central nervous system (CNS)). Immune elements – including neutrophils - involved in the anti-Mycobacterium tuberculosis (Mtb) response are implicated in pathogenesis, which remains incompletely understood. Diagnosis is one of exclusion, no reliable laboratory markers exist, corticosteroid-mediated prophylaxis and therapy are only partially effective, and no treatment targets tissue damage. Disentangling cause and effect in complex disorders such as TB-IRIS requires techniques capable of interrogating complex biological systems. Neutrophils are the major circulating leukocyte population, the earliest innate system responders, and exhibit various unusual immunometabolic functional specialisations. Proteins represent the most functionally-proximal and commonly pharmacologically-targeted cellular biomolecules. Label-free high-performance liquid chromatography-coupled tandem mass spectrometry (HPLCMS/MS) is well-suited to differentially profiling the ex vivo neutrophil proteome in an unbiased manner, in order to investigate TB-IRIS predisposition and pathogenesis. Methods Applying first principles to existing human literature, the most parsimonious holistic hypothetical model regarding paradoxical TB-IRIS predisposition and pathogenesis was inferred. A clinical cohort of control (CTRL) and matched TB-IRIS case (IRIS) study participants was assembled. Demographic, clinical, and biochemical characteristics were analysed for statistically-significant differences and to identify potential risk/protective factors (relative risk (RR) with 95% confidence interval (CI)). A small group (n = 9) of TB-HIV- healthy volunteers (HVs) was also assembled. Phlebotomy occurred at two timepoints: just prior to ARV initiation (week 0) and at the typical time of IRIS manifestation (week 2). Neutrophils were isolated and lysed, proteins underwent on-filter protein trypsinisation, peptide salts and detergents were removed, and neutrophil-optimised HPLC-MS/MS was conducted. Spectra were submitted to MaxQuant for parent protein identification and quantitation. Comparisons of (a) CTRL0 and IRIS0 to HV (and resultant differences) identified class-differential impacts of partial ATT-treated coinfection (IRIS predisposition) and of (b) CTRL2 to CTRL0 and IRIS2 to IRIS0 (and resultant differences) identified class-differential impacts of ARV therapy (IRIS pathogenesis). Class-discriminating proteomic differences were visualised using principal components analysis (PCA), protein differential expression analysis was performed (including for detectable/undetectable and significantly differentiallyexpressed (SDE) proteins), and results informed differential functional profiling via gene ontology overrepresentation analysis (GO-ORA) and pathway activation state prediction. To address shortcomings of current knowledgebases and automated tools, a novel deep manual analysis approach focused on key inference-friendly proteins, convergent findings, and neutrophil-specific functional modules. Integrated findings extended the literature-derived TB-IRIS model, generating testable novel hypotheses, one of which was partially validated using live-cell fluorescence microscopy. Proteomic data were additionally analysed to detect Mtb proteins, preliminarily analyse variable post-translational modifications (PTMs) of interest, and identify candidate prognostic and diagnostic biomarkers. Finally, mechanistic hypotheses facilitated identification of novel potential prophylactic and therapeutic targets. Results (1) Literature suggests advanced TB/HIV-coinfection (including a higher Mtb/antigen load) as the major TB-IRIS risk factor. Attendant significant immunometabolic state perturbations include myeloid overactivation, metabolic stress (possibly including adaptogen depletion), a lack of regulatory receptors, impaired pro-inflammatory signal transduction, and impaired antigen clearance. These likely predispose to lytic cell death - including release of host- and pathogen-derived inflammatory/cytotoxic molecules and proteolytic enzymes - and less restrained/more abnormal inflammation as well as tissue damage, on restoration of HIV-suppressed inflammatory signalling pathways by ARV therapy. (2) Regarding the clinical cohort, a sample size of > 42 participants per characteristic-matched comparison class provides > 95% power to detect a two-fold change with 99% confidence. Cases exhibited known TB-IRIS risk factors, and largely expected white cell count (WCC), body mass index (BMI), and C-reactive protein (CRP) level changes in response to ARV therapy (e.g. WCC increase and BMI decrease). None of the few participants on alternate (efavirenz (EFV)- or tenofovir (TDF)-lacking) regimens developed TB-IRIS. Pre-ARV prednisone (or incidental antihistamine/anti-fungal) use was associated with a non-significantly decreased TB-IRIS risk. Interestingly, smoking is associated with a significant decrease in TB-IRIS risk by 60%. (3) Regarding sample processing and analysis, the average sample collection to neutrophil isolation interval was within recommended limits. Isolation yield exceeded 15 x 106 and 25 x 106 per sample in the CTRL and IRIS groups, respectively. Isolated neutrophil purity exceeded 80% in both groups; the few low-purity samples were excluded from subsequent proteomic analysis. Lysates from 5 x 106 neutrophils routinely yielded over 100μg total protein, tryptic digestion was efficient (on average < 96% missed cleavages), and equivalent peptide injection volumes yielded comparable total ion chromatogram (TIC) profiles and intensities. An average 23% spectral identification rate resulted in a total of 2532 protein group identifications, the deepest neutrophil proteome coverage achieved to date without pre-fractionation, representing ~12% of human protein coding genes, and ~25% of the detectable human proteome. Samples were analysed in two (randomised) batches, producing independent datasets A (N = 37) and B (N =74). Withindataset technical replicates exhibit excellent agreement in protein identities and quantities; betweendataset protein identities and functional inferences also exhibit excellent agreement. We identify a number of proteins apparently not known to be expressed by human neutrophils, as well as one predicted human protein never before observed empirically. Overall, parent pathways of level-altered proteins suggest perturbation of nine major neutrophil function modules at both time-points: (a) signal transduction, (b) pattern recognition receptor (PRR) and cytokine signalling, (c) the eicosanoid cascade, (d) neutrophil antimicrobial functions, (e) carbon-energy metabolism, (f) protein homeostasis, (g) integrated nitrogen-sulfur-B-vitamin and redox/xenobiotic/glyoxal metabolism, (h) gene expression, and (i) cytoskeletal dynamics. (4) Regarding impacts of partially ATT-treated co-infection (week 0), neutrophil proteomic profiles successfully distinguish between HV and IRIS0 or CTRL0. Many differences from HV are shared between IRIS0 and CTRL0 (i.e. driven by partially ATT-treated co-infection), but some are class-unique (i.e. driven by factors predisposing to or protecting from TB-IRIS). Findings are supported by a head-to-head comparison of the CTRL0 and IRIS0 proteomes, including changes suggesting: more prevalent type I IFN, TGFβ, and Th2-type cytokine signalling; poorer capacity for restraint of alternate complement activation; mitochondrial and oxidative stress (including proneness to necrosis); impaired function (e.g. microbicidality, TLR/IL-1R-MyD88-NFκB signalling, and caspase 1- mediated IL-1β and IL-18 maturation) of activated neutrophils; and enhanced lipid and upstream (but inhibited downstream) isoprenoid synthesis (including decreased steroidogenesis). Candidate biomarkers distinguish CTRL- and IRIS-class partially ATT-treated neutrophils from HV neutrophils and from each other. (5) Regarding impacts of ARV therapy (week 2), both IRIS and CTRL neutrophil proteomes exhibit significant changes in response to ARV therapy. Many changes are shared between IRIS and CTRL (i.e. driven by ARV therapy and declining viral load (VL)), but some are class-unique (i.e. driven by factors preventing/contributing to TB-IRIS pathogenesis). Findings are supported by a head-to-head comparison of the CTRL2 and IRIS2 proteomes, including changes suggesting: a slower decline in type I IFN signalling; increased inflammatory cytokine (e.g. IL-6, TNFα, and IFNγ) signalling and protease (e.g. MMP-8) activity; decreased sensitivity to immunoregulatory glucocorticoids and vitamin A; and increased mitochondrial, endoplasmic reticulum (ER), and oxidative stress. Candidate biomarkers distinguish CTRL- and IRIS-group ARV-exposed neutrophils from baseline and from each other. (6) Livecell fluorescence microscopy of HV neutrophils suggests that in vivo-equivalent levels of EFV rapidly alter mitochondrial, lysosomal, and aggresomal architecture in a manner consistent with organelle and protein folding stress, and suggesting cell death commitment. (7) Integrated neutrophil immunometabolic changes suggested by proteomic findings support and extend the biologically compelling literature-derived model. Model highlights include more advanced baseline TB/HIV (including higher type I IFN, TGFβ, and possibly Th2-type cytokine levels), with consequent impaired myeloid-mediated Mtb antigen clearance and depletion of cellular adaptogens. The resultant abnormal immunometabolic state produces myeloid cells less able to counteract metabolic stress and primed for less-restrained inflammation. Introduction of mitotoxic ARV drugs and rapid lifting of HIVmediated immune embargoes escalates myeloid metabolic (including oxidative) stress and overactivation (including via NLRC4, CASP4/5, TLR/IL-1R-MyD88-NFκB, and MAPK-AP1 signalling), producing - instead of Mtb clearance - inflammatory cell death with release of immune-activating and tissue-damaging host- and Mtb-derived molecules. Reactivation of Mtb lymphocyte memory responses likely only produces clinically-apparent inflammation (TB-IRIS) when multiple simultaneous but incompatible immune programmes (e.g. overzealous myeloid activity, Th1, Th2, Th17, and Treg) coexist. Based on this model, existing compounds with the potential for rational, safe, effective TB-IRIS prophylaxis/therapy are identified (e.g. glutathione, vitamins B-complex and A/D/E, rapamycin, and metformin) which may assist in restoring system homeostasis.
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