Induction of broad multifunctional CD8+ and CD4+ T cells by hepatitis B virus antigen-based synthetic long peptides ex vivo.

Introduction: Therapeutic vaccination based on synthetic long peptides (SLP®) containing both CD4+ and CD8+ T cell epitopes is a promising treatment strategy for chronic hepatitis B infection (cHBV). Methods: We designed SLPs for three HBV proteins, HBcAg and the non-secreted proteins polymerase and X, and investigated their ability to induce T cell responses ex vivo. A set of 17 SLPs was constructed based on viral protein conservation, functionality, predicted and validated binders for prevalent human leukocyte antigen (HLA) supertypes, validated HLA I epitopes, and chemical producibility. Results: All 17 SLPs were capable of inducing interferon gamma (IFNÉ£) production in samples from four or more donors that had resolved an HBV infection in the past (resolver). Further analysis of the best performing SLPs demonstrated activation of both CD8+ and CD4+ multi-functional T cells in one or more resolver and patient sample(s). When investigating which SLP could activate HBV-specific T cells, the responses could be traced back to different peptides for each patient or resolver. Discussion: This indicates that a large population of subjects with different HLA types can be covered by selecting a suitable mix of SLPs for therapeutic vaccine design. In conclusion, we designed a set of SLPs capable of inducing multifunctional CD8+ and CD4+ T cells ex vivo that create important components for a novel therapeutic vaccine to cure cHBV.

Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.

BACKGROUND AND AIMS: HEV infection is the most common cause of liver inflammation, but the pathogenic mechanisms remain largely unclear. We aim to explore whether HEV infection activates inflammasomes, crosstalk with antiviral interferon response, and the potential of therapeutic targeting. APPROACH AND RESULTS: We measured IL-1ß secretion, the hallmark of inflammasome activation, in serum of HEV-infected patients and rabbits, and in cultured macrophage cell lines and primary monocyte-derived macrophages. We found that genotypes 3 and 4 HEV infection in rabbits elevated IL-1ß production. A profound increase of IL-1ß secretion was further observed in HEV-infected patients (1,733 ± 1,234 pg/mL; n = 70) compared to healthy persons (731 ± 701 pg/mL; n = 70). Given that macrophages are the drivers of inflammatory response, we found that inoculation with infectious HEV particles robustly triggered NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in primary macrophages and macrophage cell lines. We further revealed that the ORF2 capsid protein and the formed integral viral particles are responsible for activating inflammasome response. We also identified NF-κB signaling activation as a key upstream event of HEV-induced NLRP3 inflammasome response. Interestingly, inflammasome activation antagonizes interferon response to facilitate viral replication in macrophages. Pharmacological inhibitors and clinically used steroids can effectively target inflammasome activation. Combining steroids with ribavirin simultaneously inhibits HEV and inflammasome response without cross-interference. CONCLUSIONS: HEV infection strongly activates NLRP3 inflammasome activation in macrophages, which regulates host innate defense and pathogenesis. Therapeutic targeting of NLRP3, in particular when combined with antiviral agents, represents a viable option for treating severe HEV infection.

Empirical Evaluation of the Use of Computational HLA Binding as an Early Filter to the Mass Spectrometry-Based Epitope Discovery Workflow.

Immunopeptidomics is used to identify novel epitopes for (therapeutic) vaccination strategies in cancer and infectious disease. Various false discovery rates (FDRs) are applied in the field when converting liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra to peptides. Subsequently, large efforts have recently been made to rescue peptides of lower confidence. However, it remains unclear what the overall relation is between the FDR threshold and the percentage of obtained HLA-binders. We here directly evaluated the effect of varying FDR thresholds on the resulting immunopeptidomes of HLA-eluates from human cancer cell lines and primary hepatocyte isolates using HLA-binding algorithms. Additional peptides obtained using less stringent FDR-thresholds, although generally derived from poorer spectra, still contained a high amount of HLA-binders and confirmed recently developed tools that tap into this pool of otherwise ignored peptides. Most of these peptides were identified with improved confidence when cell input was increased, supporting the validity and potential of these identifications. Altogether, our data suggest that increasing the FDR threshold for peptide identification in conjunction with data filtering by HLA-binding prediction, is a valid and highly potent method to more efficient exhaustion of immunopeptidome datasets for epitope discovery and reveals the extent of peptides to be rescued by recently developed algorithms.

The Potential of Soluble Human Leukocyte Antigen Molecules for Early Cancer Detection and Therapeutic Vaccine Design.

Human leukocyte antigen (HLA) molecules are essential for anti-tumor immunity, as they display tumor-derived peptides to drive tumor eradication by cytotoxic T lymphocytes. HLA molecules are primarily studied as peptide-loaded complexes on cell membranes (mHLA) and much less attention is given to their secretion as soluble HLA-peptide complexes (sHLA) into bodily fluids. Yet sHLA levels are altered in various pathologies including cancer, and are thus of high interest as biomarkers. Disconcordance in results across studies, however, hampers interpretation and generalization of the relationship between sHLA levels and cancer presence, thereby impairing its use as a biomarker. Furthermore, the question remains to what extent sHLA complexes exert immunomodulatory effects and whether shifts in sHLA levels contribute to disease or are only a consequence of disease. sHLA complexes can also bear tumor-derived peptides and recent advancements in mass spectrometry now permit closer sHLA peptide cargo analysis. sHLA peptide cargo may represent a "liquid biopsy" that could facilitate the use of sHLA for cancer diagnosis and target identification for therapeutic vaccination. This review aims to outline the contradictory and unexplored aspects of sHLA and to provide direction on how the full potential of sHLA as a quantitative and qualitative biomarker can be exploited.

The relation of brain ouabain-like compounds and idiopathic intracranial hypertension.

OBJECTIVES: To determine the relationship between levels of ouabain-like compounds (OLC) in the cerebrospinal fluid (CSF) and the occurence of idiopathic intracranial hypertension (IIH). BACKGROUND: OLC are naturally occurring inhibitors of the sodium-potassium ATPase that are found in the CSF of mammals. Since the production of CSF is dependent upon sodium-potassium ATPase activity, and since there is evidence that the increased intracranial pressure found in the condition of IIH may be the result of increased CSF production, we hypothesized that the level of endogenous OLC would be reduced in the CSF of patients with IIH. METHODS: CSF samples were obtained from n = 7 patients with IIH and n = 31 patients with neurological disorders other than IIH ("control" patients) who had lumbar puncture as part of their routine evaluation or treatment. The concentration of OLC in the CSF samples was measured using an established ELISA. RESULTS: Patients with IIH exhibited a concentration of OLC in the CSF of 0.11 +/- 0.03 ng/mL. In comparison, the concentration of OLC in CSF samples from non-IIH control patients was 0.12 +/- 0.01 ng/mL. These values were not statistically different when compared with a t-test (P= 0.31). However, the concentration of OLC did negatively correlate to the opening pressure on lumbar puncture, but only in the IIH group (r=-0.80, P= .03). Furthermore, IIH patients who were newly diagnosed or who were unsuccessfully treated (n = 5 of 7 IIH patients) exhibited OLC concentrations of 0.06 +/- 0.1 ng/mL, which is nearly lower than that of the control group (P= .06). CONCLUSIONS: The average concentration of OLC in IIH patients (treated and untreated) is unlikely to be distinguishable from that in non-IIH control patients with other neurological conditions. However, the concentration of OLC may be inversely related to the intracranial pressure in patients with IIH, and it may prove to be lower in the subgroup of untreated and unsuccessfully treated IIH patients.

Primary care physicians' attitudes about obesity and its treatment.

OBJECTIVE: This study was designed to assess physicians' attitudes toward obese patients and the causes and treatment of obesity. RESEARCH METHODS AND PROCEDURES: A questionnaire assessed attitudes in 2 geographically representative national random samples of 5000 primary care physicians. In one sample (N = 2500), obesity was defined as a BMI of 30 to 40 kg/m(2), and in the other (N = 2500), obesity was defined as a BMI > 40. RESULTS: Six hundred twenty physicians responded. They rated physical inactivity as significantly more important than any other cause of obesity (p < 0.0009). Two other behavioral factors-overeating and a high-fat diet-received the next highest mean ratings. More than 50% of physicians viewed obese patients as awkward, unattractive, ugly, and noncompliant. The treatment of obesity was rated as significantly less effective (p < 0.001) than therapies for 9 of 10 chronic conditions. Most respondents (75%), however, agreed with the consensus recommendations that a 10% reduction in weight is sufficient to improve obesity-related health complications and viewed a 14% weight loss (i.e., 78 +/- 5 kg from an initial weight of 91 kg) as an acceptable treatment outcome. More than one-half (54%) would spend more time working on weight management issues if their time was reimbursed appropriately. DISCUSSION: Primary care physicians view obesity as largely a behavioral problem and share our broader society's negative stereotypes about the personal attributes of obese persons. Practitioners are realistic about treatment outcomes but view obesity treatment as less effective than treatment of most other chronic conditions.