A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/ gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fctagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

Central nervous system (CNS) tumors in children are a devastating diagnosis and delay in diagnosis is well documented in the literature. The aim of this study was to document and characterize time to diagnosis of CNS tumors among children 0 to 17 years of age in a pediatric center. A retrospective chart review was conducted of medical records of children with CNS tumors from 2000 to 2016 in British Columbia, Canada and 148 reports were available for review. Average age at diagnosis was 87.8 months (SD=59.7; median=72). One third (30%) were diagnosed after a single visit to a health care provider and 11 (7.7%) after more than 4 visits. Median time to diagnosis (prediagnostic symptomatic interval [PSI]) was 62 days (average 197±341 d; range, 0 to 2047 d). Longest period was time from first symptom to first health care provider visit (PSI1, median 37 d). Tumors in the posterior fossa and symptoms of ataxia or paresis were associated with a significantly shorter PSI. CNS tumors in children continue to pose a diagnostic challenge with variability in time to diagnosis. Our population-based study suggests variability in time to diagnosis with a need for education of families to identify symptoms associated with CNS tumors.

Timely care for the cleft/craniofacial patient populations represents a challenge for the CPP. Although half of patients may meet the general ACPA guidelines, only 32% of patients are meeting the CPP patient-specific recommendations. To provide better patient care, future adjustments are needed, which may include improved resource allotment and program support.

Expansion and death of effector CD8 T cells are regulated to limit immunopathology and cells that escape contraction go on to generate immunological memory. CD44, a receptor for the extracellular matrix component hyaluronan, is a marker of activated and memory T cells. Here, we show with a murine model that the increase in CD44 expression and hyaluronan binding induced upon CD8 T cell activation was proportional to the strength of TCR engagement, thereby identifying the most strongly activated T cells. When CD44 and CD44 OT-I CD8 T cells were adoptively transferred into mice challenged with Listeria-OVA, there was a slight increase in the percentage of CD44 cells at the effector site. However, CD44 cells were out-competed by CD44 cells after the contraction phase in the lymphoid tissues, and the CD44 cells preferentially formed more memory cells. The hyaluronan-binding CD44 CD8 effector T cells showed increased pAkt expression, higher glucose uptake, and were more susceptible to cell death during the contraction phase compared to non-binding CD44 and CD44 OT-I CD8 T cells, suggesting that CD44 and its engagement with hyaluronan skews CD8 T cells toward a terminal effector differentiation state that reduces their ability to form memory cells.

Background: Incremental hemodialysis, a strategy to individualize dialysis prescription based on residual kidney function, may be associated with enhanced quality of life and decreased health care costs compared with conventional hemodialysis. Objective: We surveyed practicing Canadian nephrologists to assess knowledge, perceptions, and practice pattern on the use of incremental hemodialysis. Design/Setting: We distributed a cross-sectional, web-based survey. We asked about incremental hemodialysis prescribing practices, including frequency of prescription, clinical factors used to determine suitability for treatment, and barriers to implementation. The survey was conducted from September 21 to October 30, 2020. Participants: We distributed the survey to practicing Canadian nephrologists identified from a private membership list of the Canadian Society of Nephrology (CSN), as well as to nephrologists named on a publicly available national list of practicing Canadian nephrologists created from provincial College of Physician registries. These were samples of convenience. Methods: We conducted descriptive analysis of categorical data including frequencies for nominal variables and measures of central tendency (mean) and dispersion (standard deviation) for ordinal variables. We used chi-square analysis to identify association between participant and practice characteristics and their opinions and attitudes toward incremental dialysis. We used simple thematic analysis on free-text responses on questions regarding the prescription of incremental hemodialysis, focusing on age and baseline management of cardiac and noncardiac comorbidities. Results: The response rate was 35% (243/691). Most (138/211, 65%) of the participants prescribed incremental hemodialysis using an individualized approach at the nephrologist’s discretion. Most participants (200/203, 98%) did not report any policy for implementation. Residual urine output was identified as the most important factor for eligibility (112/172, 65%), followed by electrolyte stability (76/172, 44%) and patient goals of care (69/117, 40%). Most participants agreed that dialysis prescriptions should take residual kidney function into consideration; however, 74% of the participants disagreed with a statement that there was strong evidence supporting incremental hemodialysis. Barriers identified included patient safety, patient acceptance of dose escalation, and logistics of scheduling. Despite these barriers, 82% of participants felt that that incremental hemodialysis is feasible with their current resources and 78% agreed that with specific criteria, it is a safe option. Limitations: The generalizability of our study is limited by its response rate of 35%; however, this is comparable with typical response rates seen in electronic surveys. Most participants practice in an academic setting, which may have introduced bias to the results. Conclusions: Despite the perception of limited evidence and a lack of guidance on implementation, incremental hemodialysis is frequently practiced by Canadian nephrologists. Barriers to implementation were identified, highlighting the need for research to guide practice.

PURPOSE Delay in diagnosis of central nervous system (CNS) tumors in children is well documented. The aims of this study were to characterize the symptomatology of CNS tumors and the time to diagnosis in a large pediatric hospital in Canada. METHODS Retrospective chart review of children diagnosed with a CNS tumor between 2000 and 2016 in Vancouver, British Columbia, Canada was performed. Data collected included demographics, symptomatology, tumor type, age at diagnosis, known visits to healthcare professionals, neuroimaging, therapy and post treatment relapse or progression. RESULTS 148 children with complete medical records were reviewed. The average age at diagnosis was 87.8 months (standard deviation (SD) = 59.7; median = 72). 50.7% of patients had posterior fossa tumors and 49.3% had supratentorial tumors. 30% of patients were diagnosed after a single visit to a health care provider. 7.7% of children needed more than 4 visits. Median total time to diagnosis (PSI) was 62 days (range = 0-2047 days). The longest prediagnostic interval was first symptom onset to first healthcare provider visit (PSI1, median 37 days). Patients with posterior fossa tumors, presence of metastases, and symptoms of ataxia and paresis were associated with shorter PSI. CONCLUSIONS CNS tumors in children continue to pose a diagnostic challenge with significant variability in time to diagnosis. Our population-based study found that median time from symptoms to seeking medical advice by parents was over a month. It is essential to uncover the reasons for delay and address them where possible.

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