Leaking in Intimate Partner Homicide: A Systematic Review.

Intimate partner homicides (IPH) are serious offenses by a heterogeneous group of offenders with diverse risk factors that are too unspecific for the successful prediction of an offense. Recent research suggested several warning signs that may precede IPH and enhance its prevention, but little is still known about "leaking." Leaking comprises all offense-related statements, behaviors, or actions that express the perpetrator's thoughts, fantasies, ideas, interests, feelings, intentions, plans, or positive evaluations of an own violent act or previous similar offenses prior to the own attack. This review aims to identify the forms, recipients, and media of leaking as well as potential subgroup differences in cases of IPH. We identified 47 relevant publications via a systematic search of eight databases and additional methods. We included publications that did not explicitly use the term, but described behaviors that could be interpreted as leaking. Up to now, leaking has not been systematically researched in cases of IPH. Nevertheless, publications described several behaviors that are in line with our definition of leaking and were categorized into five broader categories: (a) homicide announcements, (b) previous severe acts of violence, (c) suicidal behavior, (d) planning activities, and (e) interest in similar offenses/offenders. Information on recipients and media as well as subgroup differences was sparse. Leaking is relevant in IPH, but more systematic research is needed to understand its potential role in future risk analyses procedures and prevention of IPH.

Intermediate conformations of CD4-bound HIV-1 Env heterotrimers.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3-9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

Delineating clinical and developmental outcomes in STXBP1-related disorders.

STXBP1-related disorders are among the most common genetic epilepsies and neurodevelopmental disorders. However, the longitudinal epilepsy course and developmental end points, have not yet been described in detail, which is a critical prerequisite for clinical trial readiness. Here, we assessed 1281 cumulative patient-years of seizure and developmental histories in 162 individuals with STXBP1-related disorders and established a natural history framework. STXBP1-related disorders are characterized by a dynamic pattern of seizures in the first year of life and high variability in neurodevelopmental trajectories in early childhood. Epilepsy onset differed across seizure types, with 90% cumulative onset for infantile spasms by 6 months and focal-onset seizures by 27 months of life. Epilepsy histories diverged between variant subgroups in the first 2 years of life, when individuals with protein-truncating variants and deletions in STXBP1 (n = 39) were more likely to have infantile spasms between 5 and 6 months followed by seizure remission, while individuals with missense variants (n = 30) had an increased risk for focal seizures and ongoing seizures after the first year. Developmental outcomes were mapped using milestone acquisition data in addition to standardized assessments including the Gross Motor Function Measure-66 Item Set and the Grasping and Visual-Motor Integration subsets of the Peabody Developmental Motor Scales. Quantification of end points revealed high variability during the first 5 years of life, with emerging stratification between clinical subgroups. An earlier epilepsy onset was associated with lower developmental abilities, most prominently when assessing gross motor development and expressive communication. We found that individuals with neonatal seizures or early infantile seizures followed by seizure offset by 12 months of life had more predictable seizure trajectories in early to late childhood compared to individuals with more severe seizure presentations, including individuals with refractory epilepsy throughout the first year. Characterization of anti-seizure medication response revealed age-dependent response over time, with phenobarbital, levetiracetam, topiramate and adrenocorticotropic hormone effective in reducing seizures in the first year of life, while clobazam and the ketogenic diet were effective in long-term seizure management. Virtual clinical trials using seizure frequency as the primary outcome resulted in wide range of trial success probabilities across the age span, with the highest probability in early childhood between 1 year and 3.5 years. In summary, we delineated epilepsy and developmental trajectories in STXBP1-related disorders using standardized measures, providing a foundation to interpret future therapeutic strategies and inform rational trial design.

Calorie restriction modulates the transcription of genes related to stress response and longevity in human muscle: The CALERIE study.

The lifespan extension induced by 40% caloric restriction (CR) in rodents is accompanied by postponement of disease, preservation of function, and increased stress resistance. Whether CR elicits the same physiological and molecular responses in humans remains mostly unexplored. In the CALERIE study, 12% CR for 2 years in healthy humans induced minor losses of muscle mass (leg lean mass) without changes of muscle strength, but mechanisms for muscle quality preservation remained unclear. We performed high-depth RNA-Seq (387-618 million paired reads) on human vastus lateralis muscle biopsies collected from the CALERIE participants at baseline, 12- and 24-month follow-up from the 90 CALERIE participants randomized to CR and "ad libitum" control. Using linear mixed effect model, we identified protein-coding genes and splicing variants whose expression was significantly changed in the CR group compared to controls, including genes related to proteostasis, circadian rhythm regulation, DNA repair, mitochondrial biogenesis, mRNA processing/splicing, FOXO3 metabolism, apoptosis, and inflammation. Changes in some of these biological pathways mediated part of the positive effect of CR on muscle quality. Differentially expressed splicing variants were associated with change in pathways shown to be affected by CR in model organisms. Two years of sustained CR in humans positively affected skeletal muscle quality, and impacted gene expression and splicing profiles of biological pathways affected by CR in model organisms, suggesting that attainable levels of CR in a lifestyle intervention can benefit muscle health in humans.

Physicochemical and Biochemical Characterization of Collagen from Stichopus cf. horrens Tissues for Use as Stimuli-Responsive Thin Films.

The mutable collagenous tissue (MCT) of sea cucumber, with its ability to rapidly change its stiffness and extensibility in response to different environmental stress conditions, serves as inspiration for the design of new smart functional biomaterials. Collagen, extracted from the body wall of Stichopus cf. horrens, a species commonly found in the Philippines, was characterized for its suitability as stimuli-responsive films. Protein BLAST search showed the presence of sequences commonly found in type VII and IX collagen, suggesting that Stichopus horrens collagen is heterotypic. The maximum transition temperature recorded was 56.0 ± 2 °C, which is higher than those of other known sources of marine collagen. This suggests that S. horrens collagen has better thermal stability and durability. Collagen-based thin films were then prepared, and atomic force microscopy (AFM) imaging showed the visible collagen network comprising the films. The thin films were subjected to thermomechanical analysis with degradation starting at >175 °C. At 100-150 °C, the collagen-based films apparently lose their translucency due to the removal of moisture. Upon exposure to ambient temperature, instead of degrading, the films were able to revert to the original state due to the readsorption of moisture. This study is a demonstration of a smart biomaterial developed from S. cf. horrens collagen with potential applications in food, pharmaceutical, biomedical, and other collagen-based research.

Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 Å resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. Double electron-electron resonance spectroscopy provided further evidence for enriched occupancy of more open Env conformations. Consistent with conformational flexibility, 1059 Envs were associated with resistance to most bnAbs that exhibit reduced potency against functional Env intermediates. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal, typically occluded, epitopes. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that poorly recognize functional Env intermediates. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.

Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

Delineating clinical and developmental outcomes in STXBP1-related disorders.

STXBP1-related disorders are among the most common genetic epilepsies and neurodevelopmental disorders. However, the longitudinal epilepsy course and developmental endpoints have not yet been described in detail, which is a critical prerequisite for clinical trial readiness. Here, we assessed 1,281 cumulative patient-years of seizure and developmental histories in 162 individuals with STXBP1-related disorders and established a natural history framework. STXBP1-related disorders are characterized by a dynamic pattern of seizures in the first year of life and high variability in neurodevelopmental trajectories in early childhood. Epilepsy onset differed across seizure types, with 90% cumulative onset for infantile spasms by 6 months and focal-onset seizures by 27 months of life. Epilepsy histories diverged between variant subgroups in the first 2 years of life, when individuals with protein-truncating variants and deletions in STXBP1 (n=39) were more likely to have infantile spasms between 5 and 6 months followed by seizure remission, while individuals with missense variants (n=30) had an increased risk for focal seizures and ongoing seizures after the first year. Developmental outcomes were mapped using milestone acquisition data in addition to standardized assessments including the Gross Motor Function Measure-66 Item Set and the Grasping and Visual-Motor Integration subsets of the Peabody Developmental Motor Scales. Quantification of endpoints revealed high variability during the first five years of life, with emerging stratification between clinical subgroups, most prominently between individuals with and without infantile spasms. We found that individuals with neonatal seizures or early infantile seizures followed by seizure offset by 12 months of life had more predictable seizure trajectories in early to late childhood than compared to individuals with more severe seizure presentations, including individuals with refractory epilepsy throughout the first year. Characterization of anti-seizure medication response revealed age-dependent response over time, with phenobarbital, levetiracetam, topiramate, and adrenocorticotropic hormone effective in reducing seizures in the first year of life, while clobazam and the ketogenic diet were effective in long-term seizure management. Virtual clinical trials using seizure frequency as the primary outcome resulted in wide range of trial success probabilities across the age span, with the highest probability in early childhood between 1 year and 3.5 years. In summary, we delineated epilepsy and developmental trajectories in STXBP1-related disorders using standardized measures, providing a foundation to interpret future therapeutic strategies and inform rational trial design.

Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk with altered NTD dynamics.

SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

Structural characterization of HIV-1 Env heterotrimers bound to one or two CD4 receptors reveals intermediate Env conformations.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120/gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops, and in fully-saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops. To investigate changes resulting from sub-stoichiometric CD4 binding, we solved 3.4Å and 3.9Å single-particle cryo-EM structures of soluble, native-like Envs bound to one or two CD4 molecules. Env trimer bound to one CD4 adopted the closed, prefusion Env state. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state that included gp120 outward rotation but maintained the prefusion, three-stranded gp120 bridging sheet and showed only partial V1V2 displacement and V3 exposure. We conclude that engagement of one CD4 molecule was insufficient to stimulate CD4-induced conformational changes, while binding two CD4 molecules led to Env opening in CD4-bound protomers only. Together, these results illuminate HIV-1 Env intermediate conformations and illustrate the structural plasticity of HIV-1 Env.

The Enhancing Life Research Laboratory: Tools for Addressing Orientational Distress in the Medical Profession.

PURPOSE: To explore distress in the medical profession and how it was highlighted by the ongoing COVID-19 pandemic. The term "orientational distress" was developed to name the experience of a breakdown in the patterns of moral self-understanding and one's capacity to navigate professional responsibilities. METHOD: The Enhancing Life Research Laboratory at the University of Chicago convened a 5-session online workshop (total 10 hours, May-June 2021) to explore orientational distress and to promote collaboration between academics and physicians. Sixteen participants from Canada, Germany, Israel, and the United States engaged in discussions of the conceptual framework and toolkit to address orientational distress within institutional settings. The tools included 5 dimensions of life, 12 dynamics of life, and the role of counterworlds. Follow-up narrative interviews were transcribed and coded using a consensus-based iterative process. RESULTS: Participants reported that the concept of orientational distress helped explain their professional experiences better than burnout or moral distress. Moreover, participants strongly endorsed the project's supporting thesis that collaborative work on orientational distress and the tools provided in the research laboratory had a specific intrinsic value and provided benefits not found in other support instruments. CONCLUSIONS: Orientational distress compromises medical professionals and threatens the medical system. Next steps include the dissemination of materials from the Enhancing Life Research Laboratory to more medical professionals and medical schools. In contrast to burnout and moral injury, the concept of orientational distress may better enable clinicians to understand and more fruitfully navigate the challenges of their professional situations.

CD4 binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wild-type animals.

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3. As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

Combining staged laparoscopic colectomy with robotic completion proctectomy and ileal pouch-anal anastomosis (IPAA) in ulcerative colitis for improved clinical and cosmetic outcomes: a single-center feasibility study and technical description.

Robotic proctectomy has been shown to lead to better functional outcomes compared to laparoscopic surgery in rectal cancer. However, in ulcerative colitis (UC), the potential value of robotic proctectomy has not yet been investigated, and in this indication, the operation needs to be adjusted to the total colectomy typically performed in the preceding 6 months. In this study, we describe the technique and analyze outcomes of a staged laparoscopic and robotic three-stage restorative proctocolectomy and compare the clinical outcome with the classical laparoscopic procedure. Between December 2016 and May 2021, 17 patients underwent robotic completion proctectomy (CP) with ileal pouch-anal anastomosis (IPAA) for UC. These patients were compared to 10 patients who underwent laparoscopic CP and IPAA, following laparoscopic total colectomy with end ileostomy 6 months prior by the same surgical team at our tertiary referral center. 27 patients underwent a 3-stage procedure for refractory UC (10 in the lap. group vs. 17 in the robot group). Return to normal bowel function and morbidity were comparable between the two groups. Median length of hospital stay was the same for the robotic proctectomy/IPAA group with 7 days [median; IQR (6-10)], compared to the laparoscopic stage II with 7.5 days [median; IQR (6.25-8)]. Median time to soft diet was 2 days [IQR (1-3)] vs. 3 days in the lap group [IQR 3 (3-4)]. Two patients suffered from a major complication (Clavien-Dindo ≥ 3a) in the first 90 postoperative days in the robotic group vs. one in the laparoscopic group. Perception of cosmetic results were favorable with 100% of patients reporting to be highly satisfied or satisfied in the robotic group. This report demonstrates the feasibility of a combined laparoscopic and robotic staged restorative proctocolectomy for UC, when compared with the traditional approach. Robotic pelvic dissection and a revised trocar placement in staged proctocolectomy with synergistic use of both surgical techniques with their individual advantages will likely improve overall long-term functional results, including an improved cosmetic outcome.

Anti-BCMA/CD19 CAR T Cells with Early Immunomodulatory Maintenance for Multiple Myeloma Responding to Initial or Later-Line Therapy.

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)-negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment. SIGNIFICANCE: CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy. This article is highlighted in the In This Issue feature, p. 101.

Antibody Recognition of CD4-Induced Open HIV-1 Env Trimers.

Human immunodeficiency virus type 1 (HIV-1) envelope (Env), a heterotrimer of gp120-gp41 subunits, mediates fusion of the viral and host cell membranes after interactions with the host receptor CD4 and a coreceptor. CD4 binding induces rearrangements in Env trimer, resulting in a CD4-induced (CD4i) open Env conformation. Structural studies of antibodies isolated from infected donors have defined antibody-Env interactions, with one class of antibodies specifically recognizing the CD4i open Env conformation. In this study, we characterized a group of monoclonal antibodies isolated from HIV-1 infected donors (V2i MAbs) that displayed characteristics of CD4i antibodies. Binding experiments demonstrated that the V2i MAbs preferentially recognize CD4-bound open Env trimers. Structural characterizations of V2i MAb-Env-CD4 trimer complexes using single-particle cryo-electron microscopy showed recognition by V2i MAbs using different angles of approach to the gp120 V1V2 domain and the ß2/ß3 strands on a CD4i open conformation Env with no direct interactions of the MAbs with CD4. We also characterized CG10, a CD4i antibody that was raised in mice immunized with a gp120-CD4 complex, bound to an Env trimer plus CD4. CG10 exhibited characteristics similar to those of the V2i antibodies, i.e., recognition of the open Env conformation, but showed direct contacts to both CD4 and gp120. Structural comparisons of these and previously characterized CD4i antibody interactions with Env provide a suggested mechanism for how these antibodies are elicited during HIV-1 infection. IMPORTANCE The RV144 HIV-1 clinical vaccination trial showed modest protection against viral infection. Antibody responses to the V1V2 region of HIV-1 Env gp120 were correlated inversely with the risk of infection, and data from three other clinical vaccine trials suggested a similar signal. In addition, antibodies targeting V1V2 have been correlated with protections from simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) infections in nonhuman primates. We structurally characterized V2i antibodies directed against V1V2 isolated from HIV-1 infected humans in complex with open Env trimers bound to the host receptor CD4. We also characterized a CD4i antibody that interacts with CD4 as well as the gp120 subunit of an open Env trimer. Our study suggests how V2i and CD4i antibodies were elicited during HIV-1 infection.

HIV-1 CD4-binding site germline antibody-Env structures inform vaccine design.

BG24, a VRC01-class broadly neutralizing antibody (bNAb) against HIV-1 Env with relatively few somatic hypermutations (SHMs), represents a promising target for vaccine strategies to elicit CD4-binding site (CD4bs) bNAbs. To understand how SHMs correlate with BG24 neutralization of HIV-1, we report 4.1 Å and 3.4 Å single-particle cryo-EM structures of two inferred germline (iGL) BG24 precursors complexed with engineered Env-based immunogens lacking CD4bs N-glycans. Structures reveal critical Env contacts by BG24iGL and identify antibody light chain structural features that impede Env recognition. In addition, biochemical data and cryo-EM structures of BG24iGL variants bound to Envs with CD4bs glycans present provide insights into N-glycan accommodation, including structural modes of light chain adaptations in the presence of the N276gp120 glycan. Together, these findings reveal Env regions critical for germline antibody recognition and potential sites to alter in immunogen design.

Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk and enhanced NTD dynamics

SARS-CoV-2 emergent variants are characterized by increased transmissibility and each show multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track correlative changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in Omicron BA.1 S traced through Alpha S and Delta S variants. Stabilization preceded progressive enhancement in dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from specific S mutations detail the evolutionary trajectory of S protein in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

Comparing methods for immobilizing HIV-1 SOSIPs in ELISAs that evaluate antibody binding.

Enzyme-linked immunosorbent assays (ELISAs) are used to evaluate binding of broadly neutralizing antibodies (bNAbs) and polyclonal sera to native-like HIV-1 Env SOSIPs. Methods for immobilizing SOSIPs on plates differ, which can lead to variable or, in some cases, misleading results. Three methods used to immobilize SOSIPs were compared to determine how antigen immobilization methods affect Env conformation and ELISA results. HIV-1 SOSIPs were directly coated on polystyrene plates, captured by a monoclonal antibody against a C-terminal affinity tag, or randomly biotinylated and coated on a streptavidin plate. Binding of bNAbs with known epitopes were compared for each immobilization method. Binding of bNAbs targeting the V1V2, V3, CD4 binding site, and gp120/gp41 interface was comparable for all antigen immobilization methods. However, directly coated HIV-1 SOSIP ELISAs showed detectable binding of 17b, a CD4-induced antibody that binds a V3 epitope that is concealed on closed prefusion Env trimers in the absence of added CD4, whereas antibody-immobilized and randomly biotinylated Env-coated ELISAs did not show detectable binding of 17b in the absence of CD4. We conclude direct coating of HIV-1 SOSIPs on ELISA plates can result in exposure of CD4-induced antibody epitopes, suggesting disruption of Env structure and exposure of epitopes that are hidden in the closed, prefusion trimer.