Understanding the structural and functional basis of cell polarity regulation in Trichoplax adhaerens

Submitted in total fulfillment of the requirements for the degree of Doctor of Philosophy to the School of Agriculture, Biomedicine and Environment, La Trobe University, Victoria, Australia.</p

Characterisation of an Influenza B virus-derived peptide presented by HLA-B*18:01

The influenza B virus (IBV) can pose a significant threat to global health. Despite this, IBV is understudied compared with influenza A virus (IAV). CD8+ T cells have proven highly effective in reducing influenza disease severity. In addition, pre-existing immune responses towards IAV epitopes may provide protection against homologous IBV-derived peptides due to T cell cross-reactivity. To exploit the advantages of T cells for future vaccine developments, a better understanding of the IBV-derived peptide presentation by the highly polymorphic human leukocyte antigen (HLA) is required. We previously determined that the IAV-derived PB1177-A peptide was presented by the HLA-B*18:01 molecule and induced CD8+ T cell responses. Here, we assessed the PB1177-A IBV homologue (PB1177-B). Intracellular cytokine staining assays showed that PB1177-B was unable to activate CD8+ T cells from several HLA-B*18:01+ samples tested. We determined that the IAVand IBV-derived PB1177 adopted different stability and conformation in the cleft of HLA-B*18:01. Molecular dynamics analysis on the crystal structure showed that PB1177-B had a central flexible region with a large hydrophobic patch formed by two phenylalanine residues, not present in PB1177-A. The flexibility and the lower stability of the HLA-B*18:01-PB1177-B complex may hinder CD8+ T cell receptor binding, underpinning the lack of CD8+ T cell activation observed. This provides additional insights into the differences between IAVand IBV-specific CD8+ T cell responses.</p

Structural insight into the Scribble PDZ domains interaction with the oncogenic Human T-cell lymphotrophic virus-1 (HTLV-1) Tax1 PBM

Scribble (Scrib) is a highly conserved cell polarity regulator that harbours potent tumour suppressor activity and plays an important role in cell migration. Dysregulation of polarity is associated with poor prognosis during viral infections. Human T-cell lymphotrophic virus-1 (HTLV-1) encodes for the oncogenic Tax1 protein, a modulator of the transcription of viral and human proteins that can cause cell cycle dysregulation as well as a loss of genomic integrity. Previous studies established that Scribble interacts with Tax1 via its C-terminal PDZ-binding motif (PBM), leading to aggregation of polarity regulators and subsequent perturbation of host cell adhesion, proliferation, and signalling. Using isothermal titration calorimetry, we now show that all four PDZ domains of Scribble bind to Tax1 PBM. We then determined crystal structures of Scribble PDZ1, PDZ2 and PDZ3 domains bound to Tax1 PBM. Our findings establish a structural basis for Tax1-mediated subversion of Scribble-mediated cell polarity signalling and provide the platform for mechanistic studies to examine Tax1 induced mislocalization of Scribble and the associated changes in cellular architecture and subsequent tumorigenesis

Beyond acute infection: mechanisms underlying post-acute sequelae of COVID-19 (PASC)

Immune dysregulation is a key aspect of post-acute sequelae of coronavirus disease 2019 (PASC), also known as long COVID, with sustained activation of immune cells, T cell exhaustion, skewed B cell profiles, and disrupted immune communication thereby resulting in autoimmune-related complications.  The gut is emerging as a critical link between microbiota, metabolism and overall dysfunction, potentially sharing similarities with other chronic fatigue conditions and PASC.  Immunothrombosis and neurological signalling dysfunction emphasise the complex interplay between the immune system, blood clotting, and the central nervous system in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.  Clear research gaps in the design of PASC studies, especially in the context of longitudinal research, stand out as significant areas of concern.</p

Characterisation of novel influenza‐derived HLA‐B*18:01‐restricted epitopes

Objectives: Seasonal influenza viruses cause roughly 650 000 deaths annually despite available vaccines. CD8+ T cells typically recognise influenza-derived peptides from internal structural and non-structural influenza proteins and are an attractive avenue for future vaccine design as they could reduce the severity of disease following infection with diverse influenza strains. CD8+ T cells recognise peptides presented by the highly polymorphic Human Leukocyte Antigens class I molecules (HLA-I). Each HLA-I variant has distinct peptide binding preferences, representing a significant obstacle for designing vaccines that elicit CD8+ T cell responses across broad populations. Consequently, the rational design of a CD8+ T cell-mediated vaccine would require the identification of highly immunogenic peptides restricted to a range of different HLA molecules. Methods: Here, we assessed the immunogenicity of six recently published novel influenza-derived peptides identified by mass-spectrometry and predicted to bind to the prevalent HLA-B*18:01 molecule. Results: Using CD8+ T cell activation assays and protein biochemistry, we showed that 3/6 of the novel peptides were immunogenic in several HLA-B*18:01+ individuals and confirmed their HLA-B*18:01 restriction. We subsequently compared CD8+ T cell responses towards the previously identified highly immunogenic HLA-B*18:01-restricted NP219 peptide. Using X-ray crystallography, we solved the first crystal structures of HLA-B*18:01 presenting immunogenic influenza-derived peptides. Finally, we dissected the first TCR repertoires specific for HLA-B*18:01 restricted pathogen-derived peptides, identifying private and restricted repertoires against each of the four peptides. Conclusion: Overall the characterisation of these novel immunogenic peptides provides additional HLA-B*18:01-restricted vaccine targets derived from the Matrix protein 1 and potentially the non-structural protein and the RNA polymerase catalytic subunit of influenza viruses.</p

The impact of SARS-CoV-2 spike mutation on peptide presentation is HLA allomorph-specific

CD8+ T cells are crucial for viral elimination and recovery from viral infection. Nonetheless, the current understanding of the T cell response to SARS-CoV-2 at the antigen level remains limited. The Spike protein is an external structural protein that is prone to mutations, threatening the efficacy of current vaccines. Therefore, we have characterised the immune response towards the immunogenic Spike-derived peptide (S976-984, VLNDILSRL), restricted to the HLA-A*02:01 molecule, which is mutated in both Alpha (S982A) and Omicron BA.1 (L981F) variants of concern. We determined that the mutation in the Alpha variant (S982A) impacted both the stability and conformation of the peptide, bound to HLA-A*02:01, in comparison to the original S976-984. We identified a longer and overlapping immunogenic peptide (S975-984, SVLNDILSRL) that could be presented by HLA-A*02:01, HLA-A*11:01 and HLA-B*13:01 allomorphs. We showed that S975-specific CD8+ T cells were weakly cross-reactive to the mutant peptides despite their similar conformations when presented by HLA-A*11:01. Altogether, our results show that the impact of SARS-CoV-2 mutations on peptide presentation is HLA allomorph-specific, and that post vaccination there are T cells able to react and cross-react towards the variant of concern peptides

A broadly cross-reactive i-body to AMA1 potently inhibits blood and liver stages of Plasmodium parasites

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.</p