Spider venoms constitute incredibly diverse libraries of compounds, many of which are involved in prey capture and defence. Polyamines are often prevalent in the venom and target ionotropic glutamate receptors. Here we show that a novel spider polyamine, PA366, containing a hydroxyphenyl-based structure is present in the venom of several species of tarantula, and has selective toxicity against MCF-7 breast cancer cells. By contrast, a polyamine from an Australian funnel-web spider venom, which contains an identical polyamine tail to PA366 but an indole-based head-group, is only cytotoxic at high concentrations. Our results suggest that the ring structure plays a role in the cytotoxicity and that modification to the polyamine head group might lead to more potent and selective compounds with potential as novel cancer treatments.
The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG1 and IgG3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.
In the pursuit of improved diagnostic tests for infectious diseases, several classes of molecules have been scrutinized as prospective biomarkers. Small (18-22 nucleotide), non-coding RNA transcripts called microRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential, due to their role in numerous diseases, previously established methods for quantitation and their stability within biofluids. Despite efforts to identify, characterize and apply miRNA signatures as diagnostic markers in a range of non-infectious diseases, their application in infectious disease has advanced relatively slowly. Here, we outline the benefits that miRNA biomarkers offer to the diagnosis, management, and treatment of infectious diseases. Investigation of these novel biomarkers could advance the use of personalized medicine in infectious disease treatment, which raises important considerations for validating their use as diagnostic or prognostic markers. Finally, we discuss new and emerging miRNA detection platforms, with a focus on rapid, point-of-care testing, to evaluate the benefits and obstacles of miRNA biomarkers for infectious disease.
Necator americanus is one of several hookworms that infect humans. The other notable species are Ancylostoma duodenale and Ancylostoma ceylanicum. Hookworms infect approximately 540-730 million people worldwide with more than a billion at risk however there are currently no vaccines to limit the global burden of disease. Activation associated secreted protein 2 (ASP-2) is one of the most highly up-regulated genes upon the transition of hookworm larvae from the free-living to the infective stage. Vaccine trials in numerous animal models of hookworm disease showed that ASP-2 was efficacious and worthy of development as a human hookworm vaccine. Na-ASP-2 was safe and immunogenic in a phase 1a human clinical trial in hookwormnaive volunteers in the U.S. However in a phase 1b trial in a hookworm-endemic area of Brazil, a subset of the trial patients exhibited immediate systemic hypersensitivity reactions, due to circulating IgE antibodies against the vaccine as a result of prior hookworm exposure, hence the vaccine was shelved. These studies highlighted the fact that there was insufficient knowledge about the biology of ASP-2 and its interactions with the human host.
My hypothesis was twofold: 1) De-allergenisation (reduce IgE reactivity) of Na-ASP-2 to get a better vaccine; 2) ASP-2 has specific molecular and/or cellular binding partners within the human host to exert its effector functions.
In this thesis, I mapped the IgG and IgE epitopes of Na-ASP-2 to both recombinant fragments of the protein as well as overlapping synthetic 13mer peptides. I then used this information to design and express site-directed mutants where single amino acids in major IgE epitopes were substituted to Alanine in an attempt to de-allergenise the protein. Four distinct IgE mutants were expressed in recombinant form and screened for reduced IgE reactivity using sera from phase 1b trial patients. Single point mutations resulted in reduced IgE binding, albeit non-significant and future efforts should focus on a single protein that incorporates all four mutations.
The crystal structure of Na-ASP-2 from N. americanus reveals a putative equatorial binding groove containing a conserved tandem Histidine motif located in the centre of the groove. This putative binding groove was thought to provide a yet to be determined catalytic activity, prompting speculation that it interacts with proteins from its human host. In this thesis I employed two distinct approaches to elucidate putative host-derived binding partners for ASP-2. Firstly, by panning a random 12-mer peptide phage library, 16 peptides were identified after three successive rounds of panning. Most of the identified peptides were enriched for Glutamine and Histidine residues, and 3 peptides possessed a HXXQH motif, and a fourth peptide contained the highly similar HXXXH motif. BLASTp searches of public databases with an emphasis on the human proteome identified many distinct proteins containing varying degrees of similarity to different regions within the peptides; however, no peptide had a perfect match across its entire sequence length (12 residues) to known proteins, implying that binding of Na-ASP-2 to its native ligand is dependent on shorter peptides, possibly those containing an HXXQH motif. One such protein was the SK3 small conductance calcium-activated potassium channel, a transmembrane protein that contained a HNHQH motif within its extracellular domain. Binding of the synthetic SK3 peptide to recombinant Na-ASP-2 was confirmed by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.
To further explore potential binding partners of Na-ASP-2, the recombinant hookworm protein was used to probe a human proteome microarray, whereupon it bound selectively to the B cell Igα receptor CD79a. This is the first description of a host-pathogen protein:protein interaction identified using proteome microarrays. Using flow cytometry I confirmed the association that Na-ASP-2 bound to human B lymphocytes ex vivo. To investigate the biological effects of ASP-2 on human cells, B cell RNA was extracted and submitted to Next Generation RNA Sequencing, and revealed downregulated transcription of approximately 1000 B cell mRNAs while only approximately 100 mRNAs were upregulated, compared with control-treated cells. The expression of a range of molecules was affected by Na-ASP-2, including factors involved in leukocyte transendothelial migration pathways and the B cell signaling receptor pathway. Of note was the downregulated transcription of lyn and pi3k, molecules that are known to interact with CD79A and control B cell receptor signaling processes. Together, these results highlight a previously unknown interaction between a hookworm-secreted protein and B cells, which has implications for helminth-driven immunomodulation and vaccine development. Further, the novel use of human protein microarrays to identify host–pathogen interactions, coupled with ex vivo binding studies and subsequent analyses of global gene expression in human host cells, demonstrates a new pipeline by which to explore the molecular basis of infectious diseases.
ABSTRACT The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domain, previously C6orf106) is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we have utilised RNA-seq to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection. We find that inhibition of ILRUN expression increases cellular expression of several members of the renin-angiotensin aldosterone system (RAAS), including the SARS-CoV-2 entry receptor angiotensin converting enzyme 2 (ACE2). Furthermore, inhibition of ILRUN results in increased SARS-CoV-2 replication. These data identify ILRUN as a novel inhibitor of SARS-CoV-2 replication and represents, to our knowledge, the first report of ILRUN as a regulator of the RAAS. SIGNIFICANCE STATEMENT There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions and the development of innovative and exciting therapeutic strategies and new knowledge and tools to better protect against the impacts of disease. The human protein-coding gene ILRUN is a recently-characterised inhibitor of the transcription regulators p300 and CREB-binding protein (CBP). Here we present the first evidence that ILRUN modulation has implications for SARS-CoV-2 infections. Virus infectivity assays confirmed that gene silencing of ILRUN had a proviral effect and increased SARS-CoV-2 replication, whilst over-expression of ILRUN inhibited SARS-CoV-2 production. Additionally, we observed that ILRUN also regulates the expression of key elements of the RAAS. These data have important implications for the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.
Pigs play an essential role in the epidemiology of FMD in endemic countries. In disease-free countries, the virus amplifier role of pigs is a concern should an incursion occur. Although vaccination is an option, improved tools are required to measure immune responses and predict correlates of protection. Intradermal Application of Liquids (IDAL) technology has shown that intradermal (ID) vaccine delivery is comparable to intramuscular (IM) vaccination without causing local granulomatous reactions at the site of vaccination. We compared the immunological outcome of the different vaccine delivery routes using a Systems Immunology (SI) based approach to analyse the transcriptomic data by studying the differentially expressed genes in peripheral blood mononuclear cells (PBMCs). Groups of pigs received a prime-only IM or ID monovalent vaccination, and blood was collected on −3 and 3 days post-vaccination (dpv). PBMCs were isolated, and messenger RNA was sequenced. Transcribed genes were grouped according to biological function in the context of known Blood Transcriptome Modules (BTM). Although there were no detectable FMDV-specific antibody responses in either of the two vaccine groups at 3 dpv, there was a notable difference in the extent to which the two vaccination routes modulated innate and adaptive immune responses. IM and ID vaccination promoted BTM families involved in natural killer and T-cell differentiation, activation, signalling, co-stimulation, and proliferation. ID vaccination positively modulated dendritic cell antigen processing, presentation, and activation; type I interferon response; and inflammatory responses and complement activation. B-cell development and differentiation, B-cell receptor signalling, and immunoglobulins were negatively modulated in both groups when comparing post- and pre-vaccination gene expression. The novel SI approach provides a tool to measure the early immune responses in vaccinated pigs before antibodies are detected. Expansion of this work is required to correlate the detection of these early responses to booster vaccination and downstream protection from challenge.
The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains; previously C6orf106) was identified as a proviral factor for Hendra virus infection and was recently characterized to function as an inhibitor of type I interferon expression. Here, we have utilized transcriptome sequencing (RNA-seq) to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of Caco-2 cells. We find that inhibition of ILRUN expression by RNA interference alters transcription profiles of numerous cellular pathways, including upregulation of the SARS-CoV-2 entry receptor ACE2 and several other members of the renin-angiotensin aldosterone system. In addition, transcripts of the SARS-CoV-2 coreceptors TMPRSS2 and CTSL were also upregulated. Inhibition of ILRUN also resulted in increased SARS-CoV-2 replication, while overexpression of ILRUN had the opposite effect, identifying ILRUN as a novel antiviral factor for SARS-CoV-2 replication. This represents, to our knowledge, the first report of ILRUN as a regulator of the renin-angiotensin-aldosterone system (RAAS). IMPORTANCE There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions to assist with the development of innovative and exciting therapeutic strategies. Here, we present the first evidence that modulation of the human protein-coding gene ILRUN functions as an antiviral factor for SARS-CoV-2 infection, likely through its newly identified role in regulating the expression of SARS-CoV-2 entry receptors ACE2, TMPRSS2, and CTSL. These data improve our understanding of biological pathways that regulate host factors critical to SARS-CoV-2 infection, contributing to the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.
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