IL-11, a novel target for drug development, has been associated with several fibroinflammatory diseases including thyroid eye disease (TED), where it plays an important role in signaling to stromal cells activating multiple intracellular pathways. In TED patient tissue, IL-11 is elevated and stimulates multiple effects important in disease progression, including the production of proinflammatory cytokines, hyaluronic acid (HA) and fibrotic markers. LASN01, a potent antibody to IL-11 receptor, inhibits these effects and is a potential therapeutic agent for TED. Teprotumumab, an antibody to IGF-1 receptor, inhibits HA production and adipogenesis and is effective in reduction of proptosis. Activation of the IGF-1 and IL-11 pathways in TED tissue induces the expression of fibroinflammatory genes regulated by LASN01 and lipid biosynthetic genes regulated by Teprotumumab. Clinical studies show that LASN01 is well tolerated and in a placebo-controlled phase II trial in TED, LASN01 resulted in a statistically significant resolution of clinical activity score (CAS) in 88% of treated patients (p = 0.028), but had lesser effects on proptosis. The data supports the importance of IL-11 biology in fibroinflammatory disease and that IL-11 receptor is a pharmacologically active target for drug development.

Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Although recombinant mIL18 has demonstrated efficacy in mouse tumor models, recombinant human IL18 has not been efficacious in clinical studies, likely due to upregulation of IL18 binding protein (IL18BP), a negative regulator of the IL18 signaling axis. IL18BP is induced 10 - 100 fold after IL18 administration, binds to IL18 with high affinity, and prevents IL18 binding to the IL18 receptor. Analysis of expression data from the TCGA database as well as archived patient samples from select indications revealed that both IL18 and IL18BP are co-expressed in a number of tumor types, suggesting there may be a preexisting pool of IL18/IL18BP complex at the site of the tumor in some patients. Inhibition of IL18/IL18BP binding using a monoclonal antibody may therefore allow release of IL18 in situ, leading to IL18-mediated proinflammatory effects and enhanced anti-tumor immunity. Although the interaction of IL18 with IL18BP is a high affinity interaction, we have been able to identify very high affinity antibodies which can both inhibit complex formation and disrupt existing mouse IL18/IL18BP complexes. These antibodies are active in a number of cell-based functional assays, resulting in potent stimulation of interferon gamma production from cell lines or splenocytes. In vivo, we observed efficacy in a mouse syngeneic tumor model using an anti-mIL18BP antibody. RNA and protein analysis demonstrated expression of both IL18BP and IL18 within the tumors. Combined, these data suggest that inhibition of IL18/IL18BP binding may prove efficacious in the treatment of cancers where existing pools of IL18/IL18BP complexes prevent an IL18-mediated proinflammatory response. Experiments exploring anti-IL18BP therapy in combination with other checkpoint inhibitors such as anti-PD-1 are under current investigation. Citation Format: H. Toni Jun, Deborah A. Witherden, Eric K. Elliot, Christine M. Chidester, Maggie Willen, Bryan R. Peguero, Merrick Chai, Robert A. Horlick, David J. King. Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1824.

Background Interleukin-18 (IL-18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses and induces high levels of interferon-γ.1 Although recombinant IL-18 has demonstrated efficacy in mouse tumor models,2 recombinant human IL-18 has not been efficacious in clinical studies,3 likely due to upregulation of IL-18 binding protein (IL-18BP), a negative regulator of the IL-18 signaling axis. IL-18BP is induced 10 – 100-fold after IL-18 administration, binds to IL-18 with high affinity, and prevents IL-18 binding to the IL-18 receptor.4 Inhibition of IL-18/IL-18BP binding, and especially the ability to release IL-18 from pre-formed complex, may therefore release IL-18 in situ, leading to IL-18-mediated proinflammatory effects and enhanced anti-tumor immunity. Methods Here we analyze IL-18 and IL-18BP expression in tumors and describe the discovery of a high affinity monoclonal antibody that recognizes IL-18BP. Using in vitro and in vivo assays we analyze the ability of the antibody to disrupt IL-18/IL-18BP complex and activate IL-18-mediated responses. Results Analysis of expression data from the TCGA database and archived patient samples from select indications reveals that both IL-18 and IL-18BP are co-expressed in multiple tumor types, suggesting there is a preexisting pool of IL-18/IL-18BP complex at the site of the tumor in some patients. Although the interaction of IL-18 with IL-18BP is a high affinity interaction (KD 700 pM), we have been able to identify very high affinity antibodies with KD values more than ten-fold lower for both human and mouse IL-18BP. These antibodies can both inhibit complex formation and importantly, disrupt existing IL-18/IL-18BP complexes, liberating endogenous IL-18 at the tumor site. These antibodies are active in cell-based functional assays, resulting in potent stimulation of interferon-γ production from both NK and T cells, including when IL-18/IL-18BP is present as a preexisting complex. In vivo, we observe efficacy in a syngeneic tumor model using an anti-mouse IL-18BP antibody and demonstrate that interferon-γ is upregulated at the tumor site but not in the serum. Combination therapy with anti-PD1 leads to significantly increased efficacy compared to anti PD-1 alone and results in complete responses in more than half of the animals. RNA and protein analysis demonstrates expression of both IL-18BP and IL-18 within the tumors. Additionally, we observe an increase in NK cells and granzyme B expression in response to combination therapy. Conclusions These data suggest that inhibition of IL-18/IL-18BP binding may prove efficacious in the treatment of cancers where IL-18BP prevents an IL-18-mediated proinflammatory response. References Nakamura K, Okamura H, Nagata K, Komatsu T, Tamura T. Purification of a factor which provides a costimulatory signal for gamma interferon production. Infect Immun. 1993;61:64–70. Fabbi M, Carbotti G, Ferrini S. Context-dependent role of IL-18 in cancer biology and counter-regulation by IL-18BP. J Leukoc Biol. 2015;97:665–75. Tarhini AA, Millward M, Mainwaring P, Kefford R, Logan T, Pavlick A, Kathman SJ, Laubscher KH, Dar MM, Kirkwood JM. A phase 2, randomized study of SB-485232, rhIL-18, in patients with previously untreated metastatic melanoma. Cancer. 2009;115:859–68. Dinarello CA, Novick D, Kim S, Kaplanski G. Interleukin-18 and IL-18 binding protein. Front Immunol. 2013;4:289.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

In the original publication [...].

Assays detecting blood transcriptome changes are studied for infectious disease diagnosis. Blood-based RNA alternative splicing (AS) events, which have not been well characterized in pathogen infection, have potential normalization and assay platform stability advantages over gene expression for diagnosis. Here, we present a computational framework for developing AS diagnostic biomarkers. Leveraging a large prospective cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and whole-blood RNA sequencing (RNA-seq) data, we identify a major functional AS program switch upon viral infection. Using an independent cohort, we demonstrate the improved accuracy of AS biomarkers for SARS-CoV-2 diagnosis compared with six reported transcriptome signatures. We then optimize a subset of AS-based biomarkers to develop microfluidic PCR diagnostic assays. This assay achieves nearly perfect test accuracy (61/62 = 98.4%) using a naive principal component classifier, significantly more accurate than a gene expression PCR assay in the same cohort. Therefore, our RNA splicing computational framework enables a promising avenue for host-response diagnosis of infection.

Viruses can evolve to respond to immune pressures conferred by specific antibodies generated after vaccination and/or infection. In this study, an in vitro system was developed to investigate the impact of serum-neutralising antibodies upon the evolution of a foot-and-mouth disease virus (FMDV) isolate. The presence of sub-neutralising dilutions of specific antisera delayed the onset of virus-induced cytopathic effect (CPE) by up to 44 h compared to the untreated control cultures. Continued virus passage with sub-neutralising dilutions of these sera resulted in a decrease in time to complete CPE, suggesting that FMDV in these cultures adapted to escape immune pressure. These phenotypic changes were associated with three separate consensus-level non-synonymous mutations that accrued in the viral RNA-encoding amino acids at positions VP266, VP280 and VP1155, corresponding to known epitope sites. High-throughput sequencing also identified further nucleotide substitutions within the regions encoding the leader (Lpro), VP4, VP2 and VP3 proteins. While association of the later mutations with the adaptation to immune pressure must be further verified, these results highlight the multiple routes by which FMDV populations can escape neutralising antibodies and support the application of a simple in vitro approach to assess the impact of the humoral immune system on the evolution of FMDV and potentially other viruses.

The sequencing of viral genomes provides important data for the prevention and control of foot-and-mouth disease (FMD) outbreaks. Sequence data can be used for strain identification, outbreak tracing, and aiding the selection of the most appropriate vaccine for the circulating strains. At present, sequencing of FMD virus (FMDV) relies upon the time-consuming transport of samples to well-resourced laboratories. The Oxford Nanopore Technologies' MinION portable sequencer has the potential to allow sequencing in remote, decentralised laboratories closer to the outbreak location. In this study, we investigated the utility of the MinION to generate sequence data of sufficient quantity and quality for the characterisation of FMDV serotypes O, A, Asia 1. Prior to sequencing, a universal two-step RT-PCR was used to amplify parts of the 5′UTR, as well as the leader, capsid and parts of the 2A encoding regions of FMDV RNA extracted from three sample matrices: cell culture supernatant, tongue epithelial suspension and oral swabs. The resulting consensus sequences were compared with reference sequences generated on the Illumina MiSeq platform. Consensus sequences with an accuracy of 100% were achieved within 10 and 30 min from the start of the sequencing run when using RNA extracted from cell culture supernatants and tongue epithelial suspensions, respectively. In contrast, sequencing from swabs required up to 2.5 h. Together these results demonstrated that the MinION sequencer can be used to accurately and rapidly characterise serotypes A, O, and Asia 1 of FMDV using amplicons amplified from a variety of different sample matrices.

The autoimmune disease known as Jo-1 positive anti-synthetase syndrome (ASS) is characterized by circulating antibody titers to histidyl-tRNA synthetase (HARS), which may play a role in modulating the non-canonical functions of HARS. Monoclonal antibodies to HARS were isolated by single-cell screening and sequencing from three Jo-1 positive ASS patients and shown to be of high affinity, covering diverse epitope space. The immune response was further characterized by repertoire sequencing from the most productive of the donor samples. In line with previous studies of autoimmune repertoires, these antibodies tended to have long complementarity-determining region H3 sequences with more positive-charged residues than average. Clones of interest were clustered into groups with related sequences, allowing us to observe different somatic mutations in related clones. We postulated that these had found alternate structural solutions for high affinity binding, but that mutations might be transferable between clones to further enhance binding affinity. Transfer of somatic mutations between antibodies within the same clonal group was able to enhance binding affinity in a number of cases, including beneficial transfer of a mutation from a lower affinity clone into one of higher affinity. Affinity enhancement was seen with mutation transfer both between related single-cell clones, and directly from related repertoire sequences. To our knowledge, this is the first demonstration of somatic hypermutation transfer from repertoire sequences to further mature in vivo derived antibodies, and represents an additional tool to aid in affinity maturation for the development of antibodies.

High-throughput sequencing such as those provided by Illumina are an efficient way to understand sequence variation within viral populations. However, challenges exist in distinguishing process-introduced error from biological variance, which significantly impacts our ability to identify sub-consensus single-nucleotide variants (SNVs). Here we have taken a systematic approach to evaluate laboratory and bioinformatic pipelines to accurately identify low-frequency SNVs in viral populations. Artificial DNA and RNA "populations" were created by introducing known SNVs at predetermined frequencies into template nucleic acid before being sequenced on an Illumina MiSeq platform. These were used to assess the effects of abundance and starting input material type, technical replicates, read length and quality, short-read aligner, and percentage frequency thresholds on the ability to accurately call variants. Analyses revealed that the abundance and type of input nucleic acid had the greatest impact on the accuracy of SNV calling as measured by a micro-averaged Matthews correlation coefficient score, with DNA and high RNA inputs (10(7) copies) allowing for variants to be called at a 0.2% frequency. Reduced input RNA (10(5) copies) required more technical replicates to maintain accuracy, while low RNA inputs (10(3) copies) suffered from consensus-level errors. Base errors identified at specific motifs identified in all technical replicates were also identified which can be excluded to further increase SNV calling accuracy. These findings indicate that samples with low RNA inputs should be excluded for SNV calling and reinforce the importance of optimising the technical and bioinformatics steps in pipelines that are used to accurately identify sequence variants.

The uses of artificial intelligence (AI) today seem limitless. It has helped organisations understand their customers more, provide them with better, more tailored services, and helped people with disabilities understand the world they previously could not. There are also many areas of current research for the use of AI. Aiding law-enforcement when they must analyse evidence of an indecent nature is one example where the use of AI, if successful, could enhance detection of indecent images and also reduce the workload and stress on the law enforcement staff employed in such activities. Working with indecent images of minors is particularly stressful. This paper reviews the current stage at which artificial intelligence finds itself when estimating a person’s age. By reviewing its accuracy, it is possible to evaluate the feasibility of its inclusion in an artificial-intelligence-aided evidence analysis tool. With artificial intelligence currently capable of estimating a person’s age to within a few years, its incorporation would most certainly allow photographs to be analysed and flagged if anyone is suspected of being underage.

RNA sequencing (RNA-seq) is the gold standard of expression profiling methods. Our Advanta ™ RNA-Seq NGS Library Prep Kit is an elegant microfluidics-based solution that automates many tedious hands-on steps including poly(A) RNA selection. Advanta RNA-Seq generates up to 48 full-length stranded RNA-seq barcoded libraries from as low as 10 ng of total RNA. Nanoliter reaction volumes significantly reduce reagent consumption to lower overall costs per sample. These barcoded libraries can be pooled prior to bead cleanup steps and subsequent PCR amplifications to further decrease reagent consumption and reduce hands-on time. The results from an internal analytical study and a comparison to results obtained from the Illumina® TruSeq® Stranded mRNA Kit are presented in this poster. Our internal analytical study used a total of 917 samples (Universal Human Reference RNA standard and human brain RNA) at 10 and 100 ng of input. All samples generated genome mapping rates of >80%, with rRNA reads of <10%. Technical replicate correlations were observed to be >98% in all conditions. In addition, we found that a comparable number of genes are detected in UHRR and human brain RNA from libraries prepared with the Advanta RNA-Seq NGS Library Prep Kit and the Illumina TruSeq Stranded mRNA Kit, from both 10 ng and 100 ng starting sample inputs. The Advanta RNA-Seq NGS Library Prep Kit provides an automated RNA-seq library prep solution that substantially minimizes manual pipetting steps and hands-on time and increases walkaway time. Nanoliter-scale reaction volumes significantly decrease reagent consumption to reduce the per-sample costs of RNA-seq library construction, providing an attractive value proposition for high-throughput core laboratories. For Research Use Only. Not for use in diagnostic procedures.

With increases in complexity of graphics in video games, there exists a need to increase the complexity of game world narratives so that players feel they are an active part of an unfolding story, influenced by their actions and behaviours. Drama Management (DM) systems offer an attempt to facilitate this but are an area in need of further exploration for application in real-time narrative games. The aim of the project is to develop a prototype DM system for a real-time game that improves player agency and to analyse the effectiveness of the chosen techniques. An application was developed consisting of a 3D interactive environment, a possibility space of narrative plot points, and an Intelligent Agent that branches the story based on a Player Model, using Heuristic Search Planning. It was determined that the possibility space design has a major role in the application's effectiveness to invoke agency within players. The sense of agency can also be improved by combining the developed framework with additional extensions. This project determined that Drama Management systems arc a viable method of improving the complexity of a narrative's discourse to promote player agency, but also require careful design alongside suitable algorithmic techniques to be fully effective.

His-tRNA synthetase (HARS) is targeted by autoantibodies in chronic and acute inflammatory anti-Jo-1-positive antisynthetase syndrome. The extensive activation and migration of immune cells into lung and muscle are associated with interstitial lung disease, myositis, and morbidity. It is unknown whether the sequestration of HARS is an epiphenomenon or plays a causal role in the disease. Here, we show that HARS circulates in healthy individuals, but it is largely undetectable in the serum of anti-Jo-1-positive antisynthetase syndrome patients. In cultured primary human skeletal muscle myoblasts (HSkMC), HARS is released in increasing amounts during their differentiation into myotubes. We further show that HARS regulates immune cell engagement and inhibits CD4+ and CD8+ T-cell activation. In mouse and rodent models of acute inflammatory diseases, HARS administration downregulates immune activation. In contrast, neutralization of extracellular HARS by high-titer antibody responses during tissue injury increases susceptibility to immune attack, similar to what is seen in humans with anti-Jo-1-positive disease. Collectively, these data suggest that extracellular HARS is homeostatic in normal subjects, and its sequestration contributes to the morbidity of the anti-Jo-1-positive antisynthetase syndrome.