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Thesis (M.Sc.Eng.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The conformational epitope(CE) is a spatially grouped cluster of residues belonging to an antigen to which an antibody or B cell immunoglobulins binds (Muller and Jacoby, 2009). Accurate and reliable predictions of CEs are necessary for rational vaccine design and immunodiagnostics (Ponomarenko and Regenmortel, 2009). In this work, we explore the relationship between residues involved in crystal contacts and those belonging to the CE. With this purpose in mind, we present a new computational approach for predicting portions of CEs based on the premise that proteins establish specific crystal contacts to optimize the enthalpic contribution to the free energy during crystallization (Durbin and Feher, 1996) and in doing so mark those residues with high binding affinity - an attribute shared by epitope residues. Furthermore, it has been shown that a disproportionate amount of binding free energy of an antibody-antigen complex is accounted for by a small cluster of residues at the interface, regarded as a "hot spot"(Van Regenmortel, 2002). By identifying the crystal contacts of antigens, we explore the notion that these residues likely belong to the epitope and correspondingly, the hot spot. We hypothesize that crystal contacts are established at specific locations to involve residues that largely contribute to the binding free energy of an antibody-antigen complex. In our approach, we first find the antigen's crystal contacts, before calculating the solvent accessible surface area(SA) and energy of interaction for each crystal contact. Additionally, we calculate the protrusion index of the residues involved in the crystal contact using the computational package ElliPro (Ponomarenko et al., 2008). We measure the viability that the residues of a specific contact are participating in the epitope based on a combined measure of their solvent accessible surface area, interaction energy, and protrusivity. A benchmark dataset of bound antibody-antigen complexes and corresponding unbound antigens is used for analysis. We show that we can with predict a subset of residues belonging to the epitope with high relatively high accuracy. Furthermore, our approach illustrates that the residues driving the establishment of crystal contacts are shared by those used to establish antibody-antigen binding. / 2031-01-01

Human immunodeficiency virus-1 (HIV-1) and tuberculosis (TB) are significant global health issues today and the immunological correlates of protection to both diseases remain poorly defined. HIV-1 is the greatest risk factor for the development of TB and HIV-associated TB contributes significantly to the global TB burden, particularly in sub-Saharan Africa. The host T cell response to Mycobacterium tuberculosis (Mtb) is critical for control and is weakened in HIV-1 disease. However, the extent and precise mechanisms remain incompletely elucidated. Antiretroviral therapy (ART) for HIV-1 reduces TB risk in treated individuals significantly, although not to levels seen in HIV-uninfected individuals. This thesis studies HIV- and Mtb-specific T cell responses in 2 cohorts of individuals from a community with high HIV-1 and TB prevalence in Bloemfontein, South Africa. An analysis of HIV-specific CD8 T cell responses in Chapter 4, confirms the superior role of HIV-1 Gag-specific CD8 responses in controlling HIV-1 viraemia and demonstrates that the loss of such responses contributes to HIV-1 disease progression and likely susceptibility to opportunistic infection. I investigated the impact of HIV-1 infection on the Mtb-specific T cell response through a cross-sectional comparison of T cell responses in HIV-infected and HIV-uninfected individuals (Chapters 5 and 6). HIV-infected individuals had a significant depletion of both Th1 and Th17 CD4 responses to Mtb-specific antigens as well inhibition of Mtb-specific CD8 T cell responses, in comparison to those uninfected with HIV-1. PPD- and Rv2031c-specific responses were particularly reduced in HIV-infected individuals. Over a 12 month period of therapy, ART partially restored the Mtb-specific CD4 T cell response (Chapters 5 and 7). This effect was greater for Th1 than Th17 responses and had no detectable effect on the Mtb-specific CD8 response. However, despite some evident restoration, there remains a significant quantitative deficit in individuals on ART that is likely to contribute to persistent elevated TB risk. Overall, these data contribute to a better understanding of the mechanisms of susceptibility to TB during HIV-1 disease and ART, as well as of the correlates of protective immune responses to both pathogens.

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Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.

There is huge academic interest and clinical need associated with the development of biomarker immunoassays where general aims are the generation of highly specific, convenient and sensitive sensing formats. In this project, a powerful electrochemical technique, electrochemical impedance spectroscopy (EIS), is applied in the establishment of powerful biomarker detecting protocols. Firstly, ultrasensitive, label-free and reusable insulin sensors, based on an antibody-PEGylated thiol self-assembly monolayer (PEG thiol SAM) interface, were produced and characterised via Faradaic EIS, presenting a detection limit (LOD) of 1.2 pM, a linear range across four orders of magnitude, and high sensitivity in even 50 % serum. By applying similar surface chemistry, a label-free biosensor, specific for the detection of α-synuclein antibodies, was fabricated. The α-synuclein interfaces used enabled the reliable detecting of this biomarker in patient sample serum. The concentration levels in the control and a patient group were determined to be significantly different, and, significantly, this difference was consistently across two different cohorts. Strikingly, this could potentially underpin an entirely new means of early Parkinson’s disease (PD) diagnosis. Non-Faradaic EIS methods were additionally applied to label-free insulin assays at both PEG thiol SAM and zwitterionic polymer film interfaces. The latter presented not only an exceptionally non-fouling interface, but also one seemingly both highly biocompatible and facilitating enhanced receptor: target binding. Finally, impedance assays, though potent, generally, operate by sampling only one of a limited number of available experimental variables, typically, Rct for Faradaic EIS, or C or Z for non-Faradaic EIS. Work carried out herein also explores the generation and utility of a portfolio of mathematically derived immittance functions all obtained from the same raw data sets. A particular focus was the examination of whether these were capable of increasing assay sensitivity and efficiency above normal impedance treatments.

<b>Background and objectives:</b> It has been argued that the binary framework frequently adopted to analyse test accuracy does not represent the clinical reality of diagnostic practice, and the recognition of an intermediate category of test result could make the utility of diagnostic tests more transparent. The objective of this thesis is to explore the value of moving away from the binary framework when evaluating and interpreting quantitative diagnostic tests. <b>Methods:</b> This thesis starts with an overview of the key arguments against dichotomising quantitative test results and a summary of some of the alternative methods proposed. Four distinct studies are then reported: 1) a systematic review of the methods currently used to evaluate the accuracy of quantitative cancer biomarkers, 2) a survey of GPs exploring preferences for threshold guidance 3) an evaluation of existing methods for identifying an intermediate range of test values, and 4) an assessment of the feasibility of applying these methods to the results of a meta-analysis. <b>Results:</b> The binary framework remains the most common method for evaluating the accuracy of quantitative tests, despite the survey of GPs indicating that a single threshold interpretation is less helpful than identifying rule-in and rule-out thresholds. Existing methods for identifying an intermediate range of values require some adaptation to incorporate the cost trade-offs relating to different outcomes but, given complete reporting at the primary research, could be applied to the results of a meta-analysis. <b>Conclusion:</b> The 2 x 2 diagnostic framework frequently fails to capture many of the realities and complexities of clinical research questions. Standardised methods that facilitate complete reporting of test accuracy in primary diagnostic accuracy studies are required to allow for greater flexibility when producing threshold recommendations further down the evidence pathway.

It has been demonstrated that retinal photoreception among mammals extends beyond rods and cones to include a small number of intrinsically photosensitive retinal ganglion cells (pRGCs), which are capable of responding to light due to expression of the melanopsin (OPN4) photopigment. OPN4 may have therapeutic potential if ectopically expressed in the degenerate retina in cases where photoreceptors are lost, but the other molecules involved in this light induced transduction cascade are less well characterized. Therefore I sought to probe further the mechanism of OPN4 mediated light sensitivity by siRNA mediated knock down of specific molecules in two mice models in which complete loss of rods and cones renders them almost exclusively dependent on the OPN4 pathway for light sensitivity. I generated siRNA probes against the long transcript variant of murine Opn4 mRNA and first tested these probes on the murine Neuro2A (N2a) cell line, before assessing effects in C3H/HeN rd and rodless/coneless rd/rd cl mice. siRNA was injected intravitreally into one eye and pupillometry was assessed, combined with molecular analyses at various timepoints. Reverse transcription polymerase chain reaction (RT-PCR) analysis in N2a cells confirmed Opn4 knockdown and immunolabelling techniques identified >85% silencing with siRNA. Pupil responses in the rd and rd/rd cl mice were inhibited by the siRNA injections in vivo which confirmed the functional effect of Opn4 silencing detected by molecular analysis. I therefore present a novel reproducible in vivo model in which siRNA induced silencing of the melanopsin pathway can be assessed by pupillometry and compared to levels of mRNA and protein at specific timepoints. Probes against other putative candidate genes, such as TRPC3, may unravel the molecular interactions of this pathway. This may help in future to induce light sensitivity in other retinal neurons in patients who are completely blind from photoreceptor loss.

With the continued need for drug discovery and the quest to understand disease and treatment, there remains a requirement for improved methods to study protein-protein interactions and to identify potential drug leads for protein targets. We sought to develop a new approach to directly link genotype and phenotype to use as a probe for the identification of binding partners of proteins. The method creates millions of water-in-oil emulsions, each of which functions as a micro-environment for the amplification of a library of random peptide-encoding DNA molecules, which covalently bind to a bead. Subsequently the emulsions are broken and the bead-DNA complexes are recovered, which subsequently form another emulsion with in vitro transcription and translation components and incubated under suitable protein synthesis conditions. The synthetic peptide is designed with tags that link to the same bead which it is translated from. In chapter 3, the detailed design and optimisation of the method will be discussed. Cross-clade neutralising antibodies specific to HIV-1 are rare, partly because glycosylation restricts access to conserved backbone residues of gp120. In chapter 4, we hypothesized that peptides may have greater access than relatively large antibody structures, and so used our method to display random peptides on beads using a protein domain scaffold. Using a single round of selection, we identified 22 gp120-binding peptides, 4 of which were able to inhibit HIV-1 replication in vitro. One of the inhibitory peptides was found to bind the CCR5/CXCR4-binding site of gp120 and was able to inhibit clade B and C CCR5-tropic isolates of HIV-1. We have identified HIV-1 cross-clade neutralising peptides using a novel in vitro bead display library. Comprehensive antigenic characterization of a T cell population of unknown specificity is challenging. Existing MHC class I expression systems are limited by the practical difficulty of probing cell populations with an MHC class I peptide library and the cross-reactivity of T cells that are able to recognise many variants of an index peptide. We reasoned that a bead-based display high-throughput approach may overcome these challenges. Using emulsion PCR and emulsion in vitro transcription/translation of a random library of peptides conjugated to β-2-microglobulin on beads, we refolded with exogenous wild-type HLA-A*0201 or CD8-null A*0201 (domains 1 and 2 of HLA-A*0201 and domain 3 of Kb with mutated residues 226A/227L). The HLA bead libraries were used to probe HLA-A*0201-restricted T cells with specificity for influenza, CMV and EBV. High-throughput sequencing was used to rank sequences of identified peptides. Compared to pre-selection sequences, we observed significant enrichment for sequences containing HLA-A2 anchors and correct viral fragments for all T cell populations. HLA bead display provides a novel approach to identify the specificity of T cells. In summary, we combine the convenient handling of beads, the homogeneity of micro-environment in emulsion, and next-generation sequencing to create an attractive alternative to identify ligands of protein targets and antigenic peptides.