The Human Phenotype Ontology in 2024: phenotypes around the world.

The Human Phenotype Ontology (HPO) is a widely used resource that comprehensively organizes and defines the phenotypic features of human disease, enabling computational inference and supporting genomic and phenotypic analyses through semantic similarity and machine learning algorithms. The HPO has widespread applications in clinical diagnostics and translational research, including genomic diagnostics, gene-disease discovery, and cohort analytics. In recent years, groups around the world have developed translations of the HPO from English to other languages, and the HPO browser has been internationalized, allowing users to view HPO term labels and in many cases synonyms and definitions in ten languages in addition to English. Since our last report, a total of 2239 new HPO terms and 49235 new HPO annotations were developed, many in collaboration with external groups in the fields of psychiatry, arthrogryposis, immunology and cardiology. The Medical Action Ontology (MAxO) is a new effort to model treatments and other measures taken for clinical management. Finally, the HPO consortium is contributing to efforts to integrate the HPO and the GA4GH Phenopacket Schema into electronic health records (EHRs) with the goal of more standardized and computable integration of rare disease data in EHRs

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses

<div><p>Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (K_D) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.</p></div

Phage-displayed scFvs detect GI.1 VLPs.

<p>Phages displaying the novel single-chain antibodies detect GI.1 VLPs directly coated to the ELISA plate <b>(A)</b> and GI.1 VLPs captured by anti-NV polyclonal antibodies <b>(B)</b>. As a negative control, phages were also tested for binding against directly coated BSA <b>(A)</b> or immobilized antibody with 1% MPBS added for capture without VLPs, labeled “Ab-only” <b>(B)</b>. A phage-displayed 12-mer peptide, called NV-N-R5-1 (with sequence LPSWYLAYQKII), known to bind GI.1, is used as a positive control (shown in red) and M13KE phage is used as a negative control for VLP binding. In both ELISAs, 5 × 10¹⁰ cfu of the appropriate phage was added to detect VLPs in each well. Signals produced by each phage are reported as the ratio with the signals produced by M13KE in the same conditions. Detection of VLP by a given phage is defined as positive when the signal ratio is greater than or equal to two (indicated by the dotted line). Significance is also indicated based on t-tests comparing the signal produced by each phage with VLP and the negative control where p < 0.05. Results in part <b>(A)</b> represent the averages and standard deviations from two replicate experiments, and results in part <b>(B)</b> are from four replicate experiments.</p

Detection of natural virus in clinical stool samples.

<p>Phage-displayed scFvs effectively detect GI.1 virus in clinical stool samples from three patient volunteers, numbered 710, 715 and 721. Stool samples were serially diluted in PBS starting with a 10% stool suspension down to 0.02%. Neither phage-displayed scFv exhibits a positive binding signal with PBS that contains no stool, which is indicated as a 0% stool suspension. Negative control phage M13KE showed little variability in signal, so the signal observed at the highest concentration of each stool sample (10% stool suspensions) was used to determine the signal ratios for every other condition.</p

Mapping the binding of phage-displayed scFvs on the major capsid protein.

<p>Phage-displayed scFvs bind to the protruding domain (P-domain) of GI.1. Both NJT-R3-A2 and NJT-R3-A3 phages show positive binding signals against the full GI.1 VLP (black) and purified GI.1 P-domain (blue) based on signal ratios above 2 indicated by the dotted line. These signals are also statistically significant based on Fisher’s t-tests when compared to BSA where p < 0.05. Negative signals were seen for the S-domain (CT303) and negative control BSA. Each well contained the same amount of the appropriate phage at 5 × 10¹⁰ cfu/well.</p

Detection of GI.1 VLPs in dot blot format.

<p>Phage-displayed scFvs effectively detect GI.1 VLPs diluted in 10% norovirus-negative stool to amounts smaller than 0.39 ng of VLP per spot (NJT-R3-A2) and 0.78 ng (NJT-R3-A3). As a positive control, anti-NV (GI.1) polyclonal antibody showed dose-dependent positive binding signals to spots with VLPs, while negative control phage M13KE produced no binding signals for any concentration of VLPs or to stool by itself (0 ng VLP).</p

Dose response to GI.1 or GI.7 in 10% negative stool.

<p>Phage-displayed scFvs detect GI.1 and GI.7 VLPs specifically in 10% norovirus-negative stool captured by 3912 monoclonal antibodies. VLPs were diluted from a maximum of 400 ng/well to a minimum of 0.10 ng/well. The horizontal dotted line at y = 2 indicates the threshold for positive binding signals based on the definition for positive signals described earlier.</p

Binding specificity to norovirus genotypes.

<p>Binding specificity was evaluated for a panel of norovirus genotypes from genogroups GI and GII. Both NJT-R3-A2 and NJT-R3-A3 phages showed positive binding signals with antibody-captured GI.1 and GI.7, which are defined by signal ratios above 2:1 (labeled with the dotted line). These signals are also statistically significant based on t-tests where p < 0.05. No positive binding signals were observed with any of the GII genotypes tested or the BSA negative control. Each ELISA well received 5 × 10¹⁰ cfu of the appropriate phage.</p