Benchmarking B-Cell Epitope Prediction for the Design of Peptide-Based Vaccines: Problems and Prospects

To better support the design of peptide-based vaccines, refinement of methods to predict B-cell epitopes necessitates meaningful benchmarking against empirical data on the cross-reactivity of polyclonal antipeptide antibodies with proteins, such that the positive data reflect functionally relevant cross-reactivity (which is consistent with antibody-mediated change in protein function) and the negative data reflect genuine absence of cross-reactivity (rather than apparent absence of cross-reactivity due to artifactual masking of B-cell epitopes in immunoassays). These data are heterogeneous in view of multiple factors that complicate B-cell epitope prediction, notably physicochemical factors that define key structural differences between immunizing peptides and their cognate proteins (e.g., unmatched electrical charges along the peptide-protein sequence alignments). If the data are partitioned with respect to these factors, iterative parallel benchmarking against the resulting subsets of data provides a basis for systematically identifying and addressing the limitations of methods for B-cell epitope prediction as applied to vaccine design

Characterisation of phosphodiesterase 11 in Drosophila melanogaster

The PDE 11 family of dual specificity phosphodiesterases was first identified in 2000, and has not been well characterised, although mutations in the gene have been linked to multiple disorders, including major depressive disorder, and cancer. DmPDE11 is a dual specificity phosphodiesterase, which shows 96% similarity with the catalytic domain of HsPDE11A, and around 40% similarity along the length of the protein. The focus of this project was to characterise this important enzyme using the model organism Drosophila melanogaster. The resources available to Drosophila researchers are unrivalled, and include a sequenced genome, unparalleled transgenic technology, of which stocks are freely available, and Homophila, a database of human disease genes and their Drosophila orthologues. Drosophila is genetically tractable to an extent not seen in any other multicellular organisms. The genetic dissection of gene function in Drosophila has allowed the identification and characterisation of numerous cell signalling genes. For example, mutations to Dunce were shown to affect olfactory learning. This allowed the identification and cloning of the mammalian dnc homologue PDE4. cAMP (and cGMP) were subsequently shown to modulate learning and memory in mammals. The 5.8 kb expressed sequence tag (EST) SD13096 had previously been shown to contain sequence present in the incomplete PDE11 RA ESTs previously released by Flybase, but also incorporating a 5’ UTR, and an in-frame start codon within two novel 5’ exons. A Northern blot of DmPDE11 RA produced one band of approximately 5.8kb; as this matches the size of the DmPDE11 RA ORF, was accepted that SD13096 encodes the entire PDE11 RA ORF (Day, unpublished). Expression of this EST in S2 cells revealed that the construct produced a protein of the accepted size, and the protein localised to the cytoplasm. However, PDE assays of S2 cell lysate revealed that the enzyme did not appear to encode an enzyme with either cA- or cG-PDE activity. DmPDE11 RA was replaced on Flybase by the new isoforms DmPDE11 RB and DmPDE11 RC, which had two key changes to the RA isoform. Both new isoforms had different N termini, sharing a second exon, with distinct first exons. Furthermore, exon 11 of the RA exon is not present in the newly predicted isoforms. These new isoforms were verified by reverse transcriptase- polymerase chain reaction analysis. In the course of this verification, two further novel isoforms were identified, which shared the novel N termini with the RB and RC isoforms, but include a novel exon/exon boundary within the original exon 19, which results in a truncated isoform. As such the four isoforms were named DmPDE11 RB long, DmPDE11 RB short, DmPDE11 RC long, and DmPDE11 RC short. The open reading frames of these isoforms were cloned from Drosophila cDNA using high-fidelity DNA polymerase and sequenced for fidelity. The open reading frames were tagged with YFP, and this tag was used to verify expression of these isoforms. Each isoform expressed a protein of the predicted size when expressed in Drosophila. DmPDE11 B and C proteins show distinct localisation in the Malpighian tubule, where the long and short isoforms of each isoform display indistinguishable localisations. DmPDE11 B localises to the apical and basolateral membranes, and DmPDE11 C localises to an unknown organelle, or to vesicles. All 4 isoforms were verified as dual specificity cA- and cG- PDEs. The previous finding (Day, unpublished) that DmPDE11 co-immunoprecipitates with cGMP dependent protein kinase activity, and that cGMP dependent protein kinases co-immunoprecipitate with cG-PDE activity, and thus that cG-PDE(s) interact with at least one cGMP dependent protein kinase, directly or indirectly, was investigated. DmPDE11 C long and short were co-transfected in Schneider 2 cells with the cGKs DG1, DG2P1 and DG2P2. Co-immunoprecipitation of these showed that both the long and short isoforms of DmPDE11 C interact with every cGK screened. Time did not permit the application of this protocol to screen DmPDE11 B interaction with the cGKs. Whether this interaction is direct or indirect was screened by peptide array. Peptide arrays were generated representing the sequence of DmPDE11, DG1, and DG2, and proteins were generated fusing fragments of these proteins with HIS6 and Glutathione-S-Transferase tags. These were expressed in E. coli, and verified by western blotting. HIS6 tagged protein expression was shown to be of higher quality, and was thus affinity purified, and used to overlay and probe the peptide arrays for putative direct interactions. When the PDE11 array was overlaid with tagged protein representing the C terminal half of DG1, and the N and C terminal halves of DG2, a putative direct interaction was identified between DG1 and PDE11 on two separate regions of the PDE11 array, which both fell within the sequence of PDE11 represented by the Middle-HIS6 fragment. As such, this was used to probe the PDE11 array. A reciprocal putative interaction was identified on three regions of the DG1 array, representing sequence in both DG1N-HIS6 and DG1C-HIS6 fragments. Unfortunately, although DG1-HIS6 was verified by western blotting at the analytical stage, attempts to affinity purify the protein failed. Time did not permit the probing of the array with DG1N-GST fusion protein, and so further putative interaction sites on PDE11 may remain. The generation of alanine substitution arrays, and subsequent mutagenesis analysis with yeast two hybrid or co-immunoprecipitation would be necessary to confirm this direct protein-protein interaction as bona-fide. The investigation into a putative direct interaction between PDE11 and DG2 did not yield conclusive data, and so further investigation is required. The role of DmPDE11 in immunity was investigated by the use of DmPDE11 RNAi and deletion lines. The DmPDE11 deletion line showed a qualitative reduction in survival in individual survival assays, but when these data were merged a significant decrease in survival compared to controls was seen. However, fly numbers did not permit the inclusion of all of the necessary controls, and so these assays should be repeated with these. However, upon immune challenge, progeny from a DmPDE11 RNAi (line 9) x Act5c (a ubiquitous GAL4 driver line) cross did not show a decrease in survival compared to parental lines. Transgenic Drosophila expressing H. sapiens PDE11A3 were generated. The protein localised to the nucleus at low levels of protein; increased expression led to nuclear exclusion, and localisation to the basolateral and especially apical membranes, with cytosolic localisation also. The work has provided the tools needed to further research PDE11. The implication of this gene as a tumour suppressor gene, and its role in other processes, means that it is of the utmost importance that this enzyme is further characterised

CHARACTERIZATION OF THE ASSEMBLY OF HUMAN CYTOMEGALOVIRUS gH/gL/gO and gH/gL/UL128-131 COMPLEXES AND STUDIES ON THEIR FUNCTIONS DURING VIRUS ENTRY AND TROPISM

Human cytomegalovirus (HCMV) is a human pathogen that can cause severe diseases in immunocompromised individuals, and also is a leading cause for congenital infection, making it a major public health concern. Currently, there is no effective vaccine available and antiviral treatment is often associated with problems, like drug toxicity, and drug resistance. Intervention in the virus entry process during the replication cycle could serve as a useful therapeutic strategy. The overall aims of the research in this dissertation are to characterize the roles of HCMV two gH/gL glycoprotein complexes during virus entry and tropism, and to study the molecular basis for the regulation of the assembly of those two complexes. The work has revealed that gH/gL/gO complex promotes virus fusion into all cell types whereas gH/gL/UL128-­‐131 complex provides a non-­‐fusion but necessary function for virus entry into select cell types. Importantly, the work also demonstrated that different HCMV strains vary dramatically in the relative abundance of those two gH/gL complexes on the virion envelope, and that could have a fundamental impact on virus efficiency of entry. The regulation of the assembly of those two complexes is likely influenced by multiple viral factors. This work will help us better understand the molecular biology of how HCMV initiates infection of different cell types, and will aid in the development of antiviral strategies in the future

Characterisation and functional analysis of the developmentally regulated expression site associated gene 9 family in trypanosoma brucei

Trypanosoma brucei is a protozoan parasite that is the causative agent of sleeping sickness in sub-Saharan Africa. T. brucei has a complex life cycle involving passage between a mammalian host and the tsetse fly. The parasite evades the mammalian immune system via expression of Variant Surface Glycoprotein (VSG) on the cell surface. VSG genes are expressed at telomeric expression sites and at these sites are a number of Expression Site Associated Genes (ESAGs). One unusual ESAG, ESAG9, is developmentally regulated: RNA for these genes accumulates during the transition from slender to stumpy cells in the mammalian bloodstream and cellassociated protein is only detected transiently in stumpy and differentiating cells. Transgenic cell lines were generated which ectopically express one or more members of the ESAG9 gene family. Biochemical and cytological analyses using these cell lines indicated that some members of this family are glycosylated and GPI-anchored, and also that one gene, ESAG9-K69, is secreted. ESAG9-K69 is also secreted by wild-type stumpy parasites. In vivo experiments with tsetse flies did not conclusively show whether ESAG9 proteins play a role in the establishment of a tsetse fly mid-gut infection by transgenic trypanosomes. However, In vivo and ex vivo experiments using the mouse model of trypanosomiasis indicated that expression of ESAG9 proteins may alter parasitaemia in the mouse and results in a significant decrease in the proportion of CD4+ T cells in the mouse spleen

The role of ATP binding cassette A3 (ABCA3) in health and disease using pluripotent stem cell-derived type II alveolar epithelial cells

The most common causes of childhood interstitial lung disease (chILD) are autosomal recessive mutations in the gene encoding ATP Binding Cassette A3 (ABCA3) protein, a lamellar body (LB) associated lipid transporter exclusively expressed within the alveolar epithelial type II cells (AEC2s) in the lung. Instability of primary AEC2s in culture has prevented studies of ABCA3 mutations, resulting in limited understanding of disease pathogenesis. To overcome this challenge, we developed AEC2-like cells from human pluripotent stem cells (PSCs) in vitro, allowing study of normal ABCA3 function and perturbations that result from ABCA3 mutations. To develop an AEC2 model that would recapitulate ABCA3 biology, we targeted human PSC lines with a knock-in GFP fusion reporter (ABCA3:GFP). Differentiations of PSCs into AEC2s (iAEC2s) resulted in exclusive expression of ABCA3:GFP in iAEC2s and intracellular localization to LAMP3+ vesicles, reminiscent of endogenous ABCA3. Moreover, we find these ABCA3:GFP+ iAEC2s express LBs, process surfactant proteins, and secret surfactant lipids, indicative of preserved ABCA3 function. To study the effects of ABCA3 mutations using our model, we generated two sets of PSC reporter lines: 1) two patient-derived iPSC lines carrying rare homozygous E690K and W308R ABCA3 mutations predicted to affect ABCA3 function or trafficking, respectively, and their two syngeneic gene-corrected lines each targeted with the AEC2-specific knock-in fluorescent reporter SFTPCtdTomato; and 2) three syngeneic ABCA3:GFP knock-in iPSC lines encoding wildtype, E690K, or W308R proteins. Directed differentiation of patient iPSCs into iAEC2s revealed attenuated secretion of surfactant-specific lipids, recapitulating clinical findings of surfactant deficiency. Examination of ABCA3 protein trafficking using the ABCA3:GFP fusion reporter revealed retained E690K and W308R mutant ABCA3 protein processing and trafficking compared to the wildtype protein by confocal microscopy and western blot analyses, however mutant iAEC2s exhibited smaller LBs, indicative of defective ABCA3-dependent lipid transport. Bulk RNA sequencing of mutant and gene-corrected SFTPCtdTomato- or ABCA3:GFP-expressing iAEC2s revealed enrichment of the TNF-NFB pathway in both W308R and E690K mutant iAEC2s, validated by lentiviral reporter assays and secretion of NFB-driven cytokines. Thus, we provide insights into how ABCA3 mutations alter AEC2 physiology and developed a platform to study other genetic AEC2 diseases through our ABCA3:GFP reporter system.2021-05-26T00:00:00

Characterisation of the Vaccinia Virus Protein, C16, and its Role in Infection

Characterisation and manipulation of Vaccinia virus (VACV) immunomodulators is important for engineering recombinant VACV vaccines with enhanced immunogenicity. To this end, the goal of this study was to characterise the putative immunomodulator, C16, a protein of interest due to proposed similarity to the interleukin 1 receptor antagonist protein. C16 is a highly conserved, early protein of approximately 37 kDa that actively shuttles between the cytoplasm and nucleus in transfected and infected cells. In order to study the function of C16, a VACV mutant lacking the C16L gene (vΔC16) and a revertant virus wherein the gene was reinserted into the deletion mutant (vC16Rev) were constructed. In cell culture, vΔC16 had similar replication kinetics to wild type and revertant control viruses, but formed a smaller plaque. The loss of C16 led to a significantly attenuated phenotype in a murine intranasal model of infection that was characterised by reduced weight-loss and signs of illness. While virus titres between the virus sets were comparable in the initial days of infection, virus titres of vΔC16 dropped more rapidly over the course of the infection. In vΔC16-infected mice there was also an accelerated recruitment and activation of lymphocytes. In contrast, there was no significant difference in the murine intradermal model of infection. Furthermore, the omission of C16 did not decrease the efficacy of protective vaccination. The attenuation of vΔC16 in vivo may be due to the effect of C16 on IL-6 production which has been demonstrated in vitro and in vivo. This is reminiscent of one of the functions of IL-1Ra though the precise mechanism of this downregulation of IL-6 by C16 remains to be determined. C16 also downregulates ISRE-dependent gene expression, via a mechanism independent of STAT1 but which may involve p38 MAPK

The elaborate interplay of natural killer cells and vaccinia virus

Vaccinia virus (VACV) is a poxvirus and is the vaccine used to eradicate smallpox. It is also an expression vector for heterologous antigens and an oncolytic virus for cancer therapy. VACV encodes multiple proteins that aid evasion of the host immune response. There is, however, an incomplete understanding of how this immune suppression by VACV is consistent with such a strong immune response and subsequent memory. There is especially little known about the relationship between natural killer (NK) cells and VACV. Previous studies showed that during VACV infection, NK cells proliferate, are activated, limit viral replication, kill VACV-infected cells and display memory-like qualities. However, how NK cells recognise and interact with VACV-infected cells, which ligand(s) trigger NK cell activation, which NK receptors (NKR) are involved, and whether VACV uses strategies to interfere with the NK cell response remains elusive. This thesis aimed to address such questions using screening methods in parallel to candidate-based approach to tackle the challenges caused by the high diversity of NKRs and NK ligands. First, the responsiveness of murine NK cells to systemic VACV infection was studied ex vivo. Transcriptomic analysis, along with validation at the protein level, indicated NK cell activation and preparedness to mediate effector functions. Analysis of NK cell transcriptomic signature indicated that the stimuli triggering NK cell activation in the context of VACV infection correspond primarily with direct cell recognition but also cytokines such as Il-12 and -18, and IFNs. NKRs expression level in response to VACV was investigated, both at the transcript and protein level, and candidate NKRs involved specifically in the response to VACV were defined. Using a published dataset, the human and murine NK cell response to VACV was compared, revealing strong similarities. Second, the modulation of the plasma membrane (PM) proteome after VACV infection was studied using a proteomic screen that allowed to i) determine how VACV affects NK ligands expression; ii) give insights into VACV host surface protein modulation mechanism; iii) highlight previously unrecognised VACV strategies to evade NK cell response, iv) establish for the first time, a comprehensive analysis of VACV proteins expressed at the host PM and, v) suggest VACV surface proteins that potentially engage with NK cells. Third, the impact of the absence of VACV A56, an NK ligand candidate, and the murine natural cytotoxicity receptor (NCR) NKp46, were studied, in vitro and in vivo, in the context of VACV infection. This confirmed that VACV A56 prevents cell fusion, anchors VACV K2 and VCP (virus complement control protein) at the cell surface and enhances the binding of human and murine NCRs to VACV-infected cells. Further, it revealed that A56 deletion did not affect plaque size or EEV (extracellular enveloped virus) release, did not alter NK ligands surface expression, but led to decreased VACV-infected cells killing by murine NK cells. Lastly, the impact of VACV A56 and NKp46 deletion, on VACV infection outcome were assessed in vivo, in the acute and the memory stage and did not reveal substantial differences. Collectively, these data constitute a valuable resource concerning the interaction of VACV with NK cells and the factors influencing their interplay. These data can contribute to improve the development of VACV-based vaccines vectors and oncolytic viruses, and further our understanding of host-pathogen interactions