Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.Multiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1–12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13–20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC₅₀ of ∼2–4 µg/mL). Access to infected donors who have high serum titers of bnAbs (21, 22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1–4, 6–8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1–4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG⁺ memory B cells of interest by cell sorting (6, 8, 9, 23, 24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called “GFP-293T^BaL cells,” were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10, 25).The favorable antigenic profile of the soluble BG505 SOSIP.664 gp140 trimer opens the possibility of its use for isolating quaternary-specific antibodies by single-cell sorting (26). To this end, we used BG505 SOSIP.664 gp140 to select for memory B cells from a donor from whom we previously had isolated the trimer-specific bnAbs PGT141–145 (3, 21). (For naming of PGT and PGDM bnAbs, please see SI Materials and Methods, Antibody Nomenclature.) We describe the isolation of previously unidentified somatic variants that are highly divergent from PGT145 and display a range of neutralization breadth and potency, with some being broader and more potent than the previously described PGT145 family members. Overall, the results reveal a mosaic of antibody responses against the trimer-apex site of vulnerability that have important implications for immunogen design in general and for the future optimization of BG505 SOSIP.664 and related native-like trimers as vaccine candidates.     

Subcellular Biochemical Investigation of Purkinje Neurons Using Synchrotron Radiation Fourier Transform Infrared Spectroscopic Imaging with a Focal Plane Array Detector

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“GIOSAT”: a tool to assess CanMEDS competencies during simulated crises

Purpose

Our objective was to develop and evaluate a Generic Integrated Objective Structured Assessment Tool (GIOSAT) to integrate Medical Expert and intrinsic (non-medical expert) CanMEDS competencies with non-technical skills for crisis simulation.

Methods

An assessment tool was designed and piloted using two pediatric anesthesia scenarios (laryngospasm and hyperkalemia). Following revision of the tool, we used previously recorded videos of anesthesia residents (n = 50) who managed one of two intraoperative advanced cardiac life support (ACLS) scenarios (ventricular tachycardia or ventricular fibrillation). Four independent trained raters, blinded to the residents’ level of training, analyzed the video recordings using the GIOSAT scale. Inter-rater reliability was calculated using intraclass correlations (ICCs) for single raters (single measure) and the average of the four raters (average measure), and construct validity was investigated by correlating GIOSAT scores with postgraduate year of residency (PGY).

Results

Total GIOSAT scores for the ACLS scenarios had single measure ICCs of 0.62 and average measure ICCs of 0.85. Inter-rater reliability was substantial for both Medical Expert and intrinsic competencies (single measure ICCs 0.69 and 0.62, respectively; average measure ICCs 0.90 and 0.82, respectively). We found significant correlations between PGY level and total GIOSAT score (r = 0.36; P = 0.011) and between PGY level and Medical Expert competencies (r = 0.42; P = 0.003); however, correlations were not found between PGY level and intrinsic CanMEDS competencies (r = 0.24; P = 0.09).

Conclusion

Inter-rater reliability of the total GIOSAT scores using four trained raters was substantial. Significant correlation between PGY and (i) total GIOSAT score and (ii) Medical Expert competencies supports construct validity. Evidence of validity was not obtained for intrinsic CanMEDS competencies.     

Brief report: Focused transthoracic echocardiography training in a cohort of Canadian anesthesiology residents: a pilot study

Purpose

Bedside transthoracic echocardiography (TTE) is useful for rapid assessment and treatment of hemodynamic disturbances. Transthoracic echocardiography is not standard in Canadian anesthesia training even though undifferentiated hemodynamic disturbances are common in the perioperative setting. The objectives of this pilot study were to determine 1) whether it is feasible to implement a focused bedside TTE curriculum within core anesthesiology training, 2) whether changes could be detected and quantified following the program of study, and 3) whether curriculum implementation might lead to a significant increase in anesthesiology residents’ TTE knowledge-base.

Methods

In this single-centre cohort pilot investigation, anesthesiology residents at Queen’s University received focused bedside TTE training during the winter of 2011. The curriculum consisted of four three-hour sessions with both didactic and practical components. Pre- and post-curriculum examinations were administered, and examination results were compared using non-parametric tests. The primary outcome was the difference in mean pre- and post-curriculum examination scores.

Results

Ten participants completed pre- and post-curriculum examinations. Four residents were unable to participate in the curriculum but served as controls. Mean pretest scores (out of 50) were similar between the two groups (participants 23.9 vs controls 23.5; P = 0.83, Mann-Whitney U). Mean scores improved by 13.0 points following intervention but improved by only 1.3 points for controls, (P = 0.009, Mann-Whitney U).

Conclusion

This pilot investigation suggests that implementation of a focused bedside TTE curriculum within anesthesia training is feasible, quantifiable, and effective for increasing anesthesia residents’ TTE knowledge-base. This pilot study suggests that further investigation is warranted to determine the impact of this perioperative TTE curriculum.     

The impact of surveillance and rapid reduction in immunosuppression to control BK virus‐related graft injury in kidney transplantation

We prospectively screened 609 consecutive kidney (538) and kidney‐pancreas (71) transplant recipients for BK viremia over a 4‐year interval using polymerase chain reaction viral load detection and protocol kidney biopsies. We found that BK viremia is common at our center: total cases 26.7%, cases during first year 21.3% (mean 4 months), and recipients with ≥10 000 copies/ml 12.3%. We found few predictive clinical or demographic risk factors for any BK viremia or viral loads ≥10,000 copies/ml, other than prior treatment of biopsy confirmed acute rejection and/or higher immunosuppressive blood levels of tacrolimus (P = 0.001) or mycophenolate mofetil (P = 0.007). Viral loads at diagnosis (<10 000 copies/ml) demonstrated little impact on graft function or survival. However, rising copy numbers demand early reductions in immunosuppressive drug doses of at least 30–50%. Viral loads >185 000 copies/ml at diagnosis were predictive of BK virus‐associated nephropathy (BKVAN; OR: 113.25, 95% CI: 17.22–744.6, P < 0.001). Surveillance for BK viremia and rapid reduction of immunosuppression limited the incidence of BKVAN to 1.3%. The addition of leflunomide or ciprofloxacin to immunosuppressive dose reduction did not result in greater rates of viral clearance. These data support the role of early surveillance for BK viremia to limit the impact on transplant outcome, although the most effective schedule for screening awaits further investigation.     

Variability of quadriceps femoris motor neuron discharge and muscle force in human aging

The purpose was to determine the contribution of visual feedback and the effect of aging on the variability of knee extensor (KE) muscle force and motor unit (MU) discharge. Single MUs were recorded during two types of isometric trials, (1) visual feedback provided (VIS) and then removed (NOVIS) during the trial (34 MUs from young, 32 from elderly), and (2) only NOVIS (66 MUs from young, 77 from elderly) during the trial. Recruitment threshold (RT) ranged from 0–37% MVC. Standard deviation (SD) and coefficient of variation (CV) of muscle force and MU interspike interval (ISI) was measured during steady contractions at target forces ranging from 0.3 to 54% MVC. Force drift (<0.5 Hz) was removed before analysis. VIS/NOVIS trials: the decrease in the CV of ISI from VIS to NOVIS was greater for MUs from elderly (12.5 ± 4.1 to 9.94 ± 2.6%) than young (10.6 ± 3.3 to 10.3 ± 2.8%, age group × vision interaction, P = 0.006). The change in CV of force from VIS to NOVIS was significantly greater for elderly (1.45 to 1.05%) than young (1.42 to 1.41%). NOVIS only trials: for all MUs, the average RT (6.6 ± 7.7 % MVC), target force above RT (1.20 ± 2.7% MVC), SD of ISI (0.012 ± 0.005 s), and CV of ISI (11.1 ± 3.3%) were similar for young and elderly MUs. The CV of force was similar between age groups for trials between 0 and 3% MVC (1.74 ± 0.74%) and was greater for young subjects from 3 to 10% MVC (1.47 ± 0.5 vs. 1.21 ± 0.4%) and >10% MVC (1.44 ± 0.6 vs. 1.01 ± 0.3%). The CV of ISI was similar between age groups for MUs in 0–3, 3–10, and >10% bins of RT. Thus, the contribution of visuomotor correction to the variability of motor unit discharge and force is greater for elderly adults. The presence of visual feedback appears to be necessary to find greater discharge variability in motor units from the knee extensors of elderly adults.     

Synthesis,evaluation, and molecular docking studies of aryl urea‐triazole‐based derivatives as anti‐urease agents

Considering the importance of urease inhibitors in the treatment of ureolytic bacterial infections, in this work, the synthesis of novel, aryl urea‐triazole‐based derivatives as effective urease inhibitors is described. Dichloro‐substituted derivative 4o , with IC₅₀ = 22.81 ± 0.05 μM, is found to be the most potent urease inhibitor, determined by Berthelot colorimetric assay. Docking studies were also carried out for compound 4o to confirm the effective interactions with the urease active site.