Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective human immunodeficiency virus (HIV) vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost following eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

mRNA-LNP prime boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Human immunoglobulin gene allelic variation impacts germline-targeting vaccine priming.

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials.

The effect of restrictive fluid management on outcomes among geriatric hip fractures: a retrospective cohort study at five level I trauma centers.

Restrictive fluid management (RFM) for hemodynamically unstable trauma patients has reduced mortality rates. The objective was to determine whether RFM benefits geriatric hip fracture patients, who are usually hemodynamically stable. Design: Retrospective propensity-matched study. Setting: Five Level I trauma centers (January 1, 2018-December 12, 2018). Patients: Geriatric patients (65 years or older) with hip fractures were included in this study. Patients with multiple injuries, nonoperative management, and preoperative blood products were excluded. Intervention: Patients were grouped by fluid volume (normal saline, lactated Ringer, dextrose, electrolytes, and medications) received preoperatively or ≤24 hours of arrival; patients with standard fluid management (SFM) received ≥150 mL and RFM <150 mL of fluids. Main Outcome Measurements: The primary outcomes were length of stay (LOS), delayed ambulation (>2 days postoperatively), and mortality. Paired Student t-tests, Wilcoxon paired rank sum tests, and McNemar tests were used; an α value of < 0.05 was considered statistically significant. Results: There were 523 patients (40% RFM, 60% SFM); after matching, there were 95 patients per arm. The matched patients were well-balanced, including no difference in time from arrival to surgery. RFM and SFM patients received a median of 80 mL and 1250 mL of preoperative fluids, respectively (P < 0.001). Postoperative fluid volumes were 1550 versus 2000 mL, respectively, (P = 0.73), and LOSs were similar between the two groups (5 versus 5 days, P = 0.83). Mortality and complications, including acute kidney injuries, were similar. Delayed ambulation rates were similar overall. When stratified by preinjury ambulation status, SFM was associated with delayed ambulation for patients not walking independently before injury (P = 0.01), but RFM was not (P = 0.09). Conclusions: RFM seems to be safe in terms of laboratory results, complications, and disposition. SFM may lead to delayed ambulation for patients who are not walking independently before injury.

Human immunoglobulin gene allelic variation impacts germline-targeting vaccine priming.

Vaccine priming immunogens that activate germline precursors for broadly neutralizing antibodies (bnAbs) have promise for development of precision vaccines against major human pathogens. In a clinical trial of the eOD-GT8 60mer germline-targeting immunogen, higher frequencies of vaccine-induced VRC01-class bnAb-precursor B cells were observed in the high dose compared to the low dose group. Through immunoglobulin heavy chain variable (IGHV) genotyping, statistical modeling, quantification of IGHV1-2 allele usage and B cell frequencies in the naive repertoire for each trial participant, and antibody affinity analyses, we found that the difference between dose groups in VRC01-class response frequency was best explained by IGHV1-2 genotype rather than dose and was most likely due to differences in IGHV1-2 B cell frequencies for different genotypes. The results demonstrate the need to define population-level immunoglobulin allelic variations when designing germline-targeting immunogens and evaluating them in clinical trials. One-Sentence Summary: Human genetic variation can modulate the strength of vaccine-induced broadly neutralizing antibody precursor B cell responses.

Improving Medical Student Mentorship in Orthopaedic Surgery.

Introduction: Owing to limited clinical clerkships and travel restrictions related to COVID-19, recent medical student mentorship in orthopaedic surgery has been impacted negatively. The purpose of this quality improvement (QI) project was to determine if medical student awareness of orthopaedics as a possible career field may be improved through a mentoring program designed and delivered by orthopaedic residents. Methods: A five-resident QI team developed four educational sessions aimed at a medical student audience. Forum topics included: (1) orthopaedics as a career, (2) fracture conference, (3) splinting workshop, and (4) residency application process. Pre- and post-forum surveys were administered to student participants to assess changes in their perceptions regarding orthopaedic surgery. Data derived from the questionnaires were analyzed with nonparametric statistical tests. Results: Of 18 forum participants, 14 were men and 4 were women. A total of 40 survey pairs were collected, averaging 10 per session. In the all-participant encounter analysis, there were statistically significant improvements in all outcome measures including interest in, exposure to, and knowledge of orthopaedics; exposure to our training program; and ability to interact with our residents. Those undecided regarding their specialty demonstrated larger increases in post-forum responses, suggesting that the learning experience was more impactful for that subgroup. Conclusions: This QI initiative was a successful demonstration of orthopaedic resident mentorship of medical students, wherein perceptions of orthopaedics were influenced favorably by the educational experience. For some students with limited access to orthopaedic clerkships or formal one-on-one mentoring, forums like these may be an acceptable alternative.

Popular media and the bombardment of evolution misconceptions.

Background: Many students enter science classrooms with misconceptions about scientific principles. One of the most perceived controversial scientific principle for students is evolution. Students struggle to learn and accept evolution due to the many misconceptions students have interacted with before they enter a biology class. Evolution misconceptions come from many sources, such as religious beliefs, textbooks, and even unprepared educators. However, with students spending on average over seven hours a day viewing popular media, it is crucial to investigate further the accuracy of the portrayals of evolution in popular media. Results: We gathered data on the sources students saw evolution portrayed in popular media and determined what misconceptions were present in these popular media references. We found that 96% of the popular media references mentioned by students in our study inaccurately depicted evolution. The two most common misconceptions we observed in popular media were that evolution was depicted as a linear process and that individual organisms evolve instead of populations. Conclusion: Popular media does a poor job depicting evolution, which may be why many students are hesitant to learn evolution and overcome misconceptions. We suggest that these incorrect portrayals of evolution may provide an engaging way to teach correct evolutionary principles in the classroom.

Vaccination induces HIV broadly neutralizing antibody precursors in humans.

Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01B had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.

Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors.

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development.

Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice.

Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.

Improving Electronic Patient Handoff in an Orthopaedic Residency using the Listrunner© Application.

Introduction: Miscommunication during shift change and other handoff events is a common source of malpractice claims and patient-care errors. An efficient patient handoff system is imperative to prevent miscommunication. Owning to limitations with our current handoff system and to an ever-increasing reliance on electronic health information, our residency program sought to modernize our handoff method. Methods: To improve handoff communication, the HIPAA-compliant application Listrunner© was adopted. Members of the orthopaedic trauma team were oriented to the new application. Change-of-shift patient handoff was transitioned from the current email system to List-runner©. After three months of using the new application, a web-based questionnaire was administered to all members of the care team to assess their experiences, including perceived benefits and limitations of the Listrunner© application. Results: Seventeen orthopaedic resident physicians and three orthopaedic trauma attending physicians completed the survey. While almost half of the respondents were satisfied using email as a checkout tool, more than half of study participants indicated that it lacked security and several users believed there was a need for improvement. Most indicated that Listrunner© was easy to use, improved clinical efficiency, and improved patient care and safety. Seventeen of 20 respondents reported that they would like to continue using Listrunner© as a check-out tool. Conclusions: The Listrunner© application was adopted quickly by our orthopaedic trauma team, whose members opined that the application increased the efficiency and accuracy of handoff when compared to the previous secure email system.

Significant variations in preoperative fluid resuscitation volumes delivered to elderly hip fracture patients at six level 1 trauma centers: an observational descriptive study.

OBJECTIVE: To describe the variations in administration of preoperative (preop) fluids and in the volumes of fluid administered among geriatric hip fracture patients requiring surgical repair. DESIGN: Observational descriptive. SETTING: Six Level 1 trauma centers. PATIENTS: A total of 595 patients aged ≥65 with ICD-10 codes indicating hip fracture and surgical repair were identified. Of these, 87.9% (n = 525) received preop fluid. The median volume of preop fluid delivered was 1500 mL (IQR: 1000-2250 mL). INTERVENTION: None. MAIN OUTCOME MEASURES: Receipt of preop fluids; median volume of fluid received. RESULTS: Receipt of preop fluid was significantly different by inter-hospital transfer, facility, BMI, hospital length of stay, and postop fluid volume. Age, sex, time to surgery, time to ambulation, and hospital disposition were not associated with preop fluid. There were significant differences in median preop fluid volumes by facility and postop fluid volume. CONCLUSION: This descriptive study of current practices among geriatric trauma patients with isolated hip fractures revealed significant differences in the use of preop fluid resuscitation and the resuscitation volumes administered. Treating facility may be the most substantial source of variation highlighting the need for a guideline on fluid resuscitation. These observed variations may be a result of patient characteristics or provider discretion and should be evaluated further.

Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Creating and Implementing a Protocol for the Management of Patients in Skeletal Traction: A Quality Improvement Project.

INTRODUCTION: Skeletal traction use generally has decreased over generations and is used most often for temporary fracture stabilization. Proper nursing management of patients in skeletal traction is crucial. A hospital protocol was created and implemented to educate and direct registered nurses (RNs) in the care of patients requiring skeletal traction. METHODS: A skeletal traction management protocol was drafted and implemented as hospital policy. Twenty-nine RNs from an orthopaedic unit at a level 1 trauma center attended a financially compensated, 45-minute, in-person, off-shift educational session. An anonymous pre-test utilizing a 5-point Likert scale was completed to assess RN knowledge and comfort regarding the following topics of traction care: pin care, manual traction, frame assembly, weight application and removal, skin evaluation, neurovascular checks, and reporting issues. The RNs were provided with a copy of the new hospital policy and key points were highlighted and demonstrated. After the demonstration, the RNs were given a post-test to assess their perceived knowledge and comfort with traction care. RESULTS: Statistically significant improvements in RN knowledge and comfort were seen in six of the seven evaluated topics. The greatest increase was seen in the manual traction topic. No significant change regarding neurovascular checks was observed with this topic having the highest pre-test scores. CONCLUSIONS: A hospital protocol was created successfully and implemented that significantly improved the level of RN knowledge and comfort with the management of patients requiring skeletal traction. Future studies should assess the effectiveness of annual education regarding the traction policy.

PyRosetta Jupyter Notebooks Teach Biomolecular Structure Prediction and Design.

Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of sixteen modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications like protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in their science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.

PyIR: a scalable wrapper for processing billions of immunoglobulin and T cell receptor sequences using IgBLAST.

BACKGROUND: Recent advances in DNA sequencing technologies have enabled significant leaps in capacity to generate large volumes of DNA sequence data, which has spurred a rapid growth in the use of bioinformatics as a means of interrogating antibody variable gene repertoires. Common tools used for annotation of antibody sequences are often limited in functionality, modularity and usability. RESULTS: We have developed PyIR, a Python wrapper and library for IgBLAST, which offers a minimal setup CLI and API, FASTQ support, file chunking for large sequence files, JSON and Python dictionary output, and built-in sequence filtering. CONCLUSIONS: PyIR offers improved processing speed over multithreaded IgBLAST (version 1.14) when spawning more than 16 processes on a single computer system. Its customizable filtering and data encapsulation allow it to be adapted to a wide range of computing environments. The API allows for IgBLAST to be used in customized bioinformatics workflows.

Correction: Tissue-Specific Expressed Antibody Variable Gene Repertoires.

[This corrects the article DOI: 10.1371/journal.pone.0100839.].

A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses.

Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.

Structure-based design of native-like HIV-1 envelope trimers to silence non-neutralizing epitopes and eliminate CD4 binding.

Elicitation of broadly neutralizing antibodies (bnAbs) is a primary HIV vaccine goal. Native-like trimers mimicking virion-associated spikes present nearly all bnAb epitopes and are therefore promising vaccine antigens. However, first generation native-like trimers expose epitopes for non-neutralizing antibodies (non-nAbs), which may hinder bnAb induction. We here employ computational and structure-guided design to develop improved native-like trimers that reduce exposure of non-nAb epitopes in the V3-loop and trimer base, minimize both CD4 reactivity and CD4-induced non-nAb epitope exposure, and increase thermal stability while maintaining bnAb antigenicity. In rabbit immunizations with native-like trimers of the 327c isolate, improved trimers suppress elicitation of V3-directed and tier-1 neutralizing antibodies and induce robust autologous tier-2 neutralization, unlike a first-generation trimer. The improved native-like trimers from diverse HIV isolates, and the design methods, have promise to assist in the development of a HIV vaccine.