Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax

Preoperative Point-of-Care Ultrasound to Identify Frailty and Predict Postoperative Outcomes: A Diagnostic Accuracy Study

BackgroundFrailty is increasingly being recognized as a public health issue, straining healthcare resources and increasing costs to care for these patients. Frailty is the decline in physical and cognitive reserves leading to increased vulnerability to stressors such as surgery or disease states. The goal of this pilot diagnostic accuracy study was to identify whether point-of-care ultrasound measurements of the quadriceps and rectus femoris muscles can be used to discriminate between frail and not-frail patients and predict postoperative outcomes. This study hypothesized that ultrasound could discriminate between frail and not-frail patients before surgery.MethodsPreoperative ultrasound measurements of the quadriceps and rectus femoris were obtained in patients with previous computed tomography scans. Using the computed tomography scans, psoas muscle area was measured in all patients for comparative purposes. Frailty was identified using the Fried phenotype assessment. Postoperative outcomes included unplanned intensive care unit admission, delirium, intensive care unit length of stay, hospital length of stay, unplanned skilled nursing facility admission, rehospitalization, falls within 30 days, and all-cause 30-day and 1-yr mortality.ResultsA total of 32 patients and 20 healthy volunteers were included. Frailty was identified in 18 of the 32 patients. Receiver operating characteristic curve analysis showed that quadriceps depth and psoas muscle area are able to identify frailty (area under the curve-receiver operating characteristic, 0.80 [95% CI, 0.64 to 0.97] and 0.88 [95% CI, 0.76 to 1.00], respectively), whereas the cross-sectional area of the rectus femoris is less promising (area under the curve-receiver operating characteristic, 0.70 [95% CI, 0.49 to 0.91]). Quadriceps depth was also associated with unplanned postoperative skilled nursing facility discharge disposition (area under the curve 0.81 [95% CI, 0.61 to 1.00]) and delirium (area under the curve 0.89 [95% CI, 0.77 to 1.00]).ConclusionsSimilar to computed tomography measurements of psoas muscle area, preoperative ultrasound measurements of quadriceps depth shows promise in discriminating between frail and not-frail patients before surgery. It was also associated with skilled nursing facility admission and postoperative delirium.Editor’s perspectiv

Kinetic analysis of mAb 29 and cAb 29 binding to PA.

<p>SPR sensograms obtained during injection of (A) 100–2000 nM of PA on anti-human captured cAb 29 (∼300 RU), refers to the direct format; or (B) 5–100 nM of cAb 29 on immobilized PA (∼900 RU), refers to the indirect format. Similar sensograms were obtained for the mAb 29 (results not shown).</p

Kinetics of anti-PA and PA-neutralizing antibody development.

<p>Average titer values of total anti-PA ELISA antibodies (filled circles) of eight immunized mice and LeTx neutralizing antibody titers (empty circles) of mice 3, 4, 5 and 7, throughout the immunization process. Arrows indicate PA-immunization time points; Data points are mean±STD.</p

Binding kinetics of the PA-specific antibodies.

a<p>Binding kinetics values derived from global curve-fitting analysis using divalent analysis model.</p>b<p>Binding kinetics values derived from global curve-fitting analysis using 1∶1 Langmuir binding model.</p

<i>In vivo</i> LeTx neutralization.

<p>Rats (n≥6 for each group) were i.m. administered with the indicated doses of (A) cAb 29 or (B) mAb 29, followed by i.v. challenge with LeTx (20 µg PA and 10 µg LF), 17 hours later. Animal survival was monitored for the next 24 hours.</p

<i>In vitro</i> LeTx neutralization.

<p>Toxin complex (5 µg/ml PA and 2 µg/ml LF) was pre-incubated for one hour with increasing concentrations of cAb 29 (triangles) or mAb 29 (squares), and added to J774A.1 mouse macrophage cells for 5 hours. Cell survival was then determined by MTT, and was plotted as percent of untreated control cells. Points are mean±STD of triplicate determinants.</p

Circulatory clearance profiles of cAb 29 and mAb 29.

<p>Guinea pigs (n = 3 for each group) were i.m. administered with 5 mg/kg of either cAb 29 (diamonds) or mAb 29 (squares). Blood samples drawn at various time points were assayed for Ab concentration by ELISA. Mean±STD values are presented as percentages of maximum blood levels (C_max).</p