Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

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.

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.

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.

Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization.

Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1-naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1-naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 10(7)unique HCDR3 amino acid regions from 70 different HIV-1-naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1-naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts.

Application of Suzuki-Miyaura and Buchwald-Hartwig Cross-coupling Reactions to the Preparation of Substituted 1,2,4-Benzotriazine 1-Oxides Related to the Antitumor Agent Tirapazamine.

Many 1,2,4-benzotriazine 1,4-dioxides display the ability to selectively kill the oxygen-poor cells found in solid tumors. As a result, there is a desire for synthetic routes that afford access to substituted 1,2,4-benzotriazine 1-oxides that can be used as direct precursors in the synthesis of 1,2,4-benzotriazine 1,4-dioxides. Here we describe the use of Suzuki-Miyaura and Buchwald-Hartwig cross-coupling reactions for the construction of various 1,2,4-benzotriazine 1-oxide analogs bearing substituents at the 3-, 6-, and 7-positions.

Human germline antibody gene segments encode polyspecific antibodies.

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

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.

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

A protective 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 after 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.

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.

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.

Maturation-induced cloaking of neutralization epitopes on HIV-1 particles.

To become infectious, HIV-1 particles undergo a maturation process involving proteolytic cleavage of the Gag and Gag-Pol polyproteins. Immature particles contain a highly stable spherical Gag lattice and are impaired for fusion with target cells. The fusion impairment is relieved by truncation of the gp41 cytoplasmic tail (CT), indicating that an interaction between the immature viral core and gp41 within the particle represses HIV-1 fusion by an unknown mechanism. We hypothesized that the conformation of Env on the viral surface is regulated allosterically by interactions with the HIV-1 core during particle maturation. To test this, we quantified the binding of a panel of monoclonal antibodies to mature and immature HIV-1 particles by immunofluorescence imaging. Surprisingly, immature particles exhibited markedly enhanced binding of several gp41-specific antibodies, including two that recognize the membrane proximal external region (MPER) and neutralize diverse HIV-1 strains. Several of the differences in epitope exposure on mature and immature particles were abolished by truncation of the gp41 CT, thus linking the immature HIV-1 fusion defect with altered Env conformation. Our results suggest that perturbation of fusion-dependent Env conformational changes contributes to the impaired fusion of immature particles. Masking of neutralization-sensitive epitopes during particle maturation may contribute to HIV-1 immune evasion and has practical implications for vaccine strategies targeting the gp41 MPER.

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.

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.

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.

Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus.

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.

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.

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.