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Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.

Significance Despite the high antigenic diversity of the HIV envelope trimer (Env), broadly neutralizing antibodies (bnAbs) have identified conserved regions that serve as targets for vaccine design. One of these regions is located at the apex of Env and is expressed fully only in the context of the correctly folded trimer. This work describes the isolation of bnAbs that target this region using a recombinant native-like Env trimer as an affinity reagent to sort specific antibody-producing cells. Characterization of these antibodies reveals a highly diverse antibody response against the trimer apex and provides molecular information that will be useful in the design of immunogens to elicit bnAbs to this region of Env. Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC ₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.

Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012–2013. Here we describe an antibody, F045–092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045–092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045–092 extends its recognition to divergent subtypes, including H1, H2 and H13, using the enhanced avidity of its IgG to overcome lower-affinity Fab binding, as observed with other antibodies that target the receptor-binding site. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small-molecule therapeutics. Influenza A H3 viruses circulate in humans and bind host cells using the haemagglutinin (HA) glycoprotein. Here, Lee et al. perform a structural analysis of antibody F045–092 with broadly neutralizing activity against the H3 subtype and reveal its interaction with the HA receptor binding site.

Background Total joint replacements are an established treatment for patients suffering from reduced mobility and pain due to severe joint damage. Aseptic loosening due to stress shielding is currently one of the main reasons for revision surgery. As this phenomenon is related to a mismatch in mechanical properties between implant and bone, stiffness reduction of implants has been of major interest in new implant designs. Facilitated by modern additive manufacturing technologies, the introduction of porosity into implant materials has been shown to enable significant stiffness reduction; however, whether these devices mitigate stress-shielding associated complications or device failure remains poorly understood. Methods In this systematic review, a broad literature search was conducted in six databases (Scopus, Web of Science, Medline, Embase, Compendex, and Inspec) aiming to identify current design approaches to target stress shielding through controlled porous structures. The search keywords included ‘lattice,’ ‘implant,’ ‘additive manufacturing,’ and ‘stress shielding.’ Results After the screening of 2530 articles, a total of 46 studies were included in this review. Studies focusing on hip, knee, and shoulder replacements were found. Three porous design strategies were identified, specifically uniform, graded, and optimized designs. The latter included personalized design approaches targeting stress shielding based on patient-specific data. All studies reported a reduction of stress shielding achieved by the presented design. Conclusion Not all studies used quantitative measures to describe the improvements, and the main stress shielding measures chosen varied between studies. However, due to the nature of the optimization approaches, optimized designs were found to be the most promising. Besides the stiffness reduction, other factors such as mechanical strength can be considered in the design on a patient-specific level. While it was found that controlled porous designs are overall promising to reduce stress shielding, further research and clinical evidence are needed to determine the most superior design approach for total joint replacement implants.

Bone-anchored percutaneous implants, commonly referred to as osseointegrated implants, are gaining popularity as an alternative to sockets for attaching a prosthetic limb to an amputated femur. While these implants have several advantages, femoral fractures are relatively common in these patients, occurring in 6.3% of femurs within 4 years. This study aimed to develop a novel method to quantify femoral strength under all possible external loads acting on the distal implant. Finite element analysis was used to explore combinations of forces and moments that, when applied on the bone-anchored implant, were just sufficient to fracture the femur. The 6-dimensional envelope generated in the current study provided a quantitative description of the strength of the femur-implant construct. All the femoral fractures were predicted in the trochanteric region, corresponding to where most in vivo fractures have been reported. The 6-dimensional envelopes developed in the current study may be useful in the design of fail-safe devices that could reduce femoral fractures in patients with bone-anchored percutaneous femoral implants. The framework presented may also be used for determining patient suitability for bone-anchored percutaneous implants, and for evaluating the functional performance of implant designs.

Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes.

Background Total hip replacement surgery is a well-established intervention that restores joint function and provides excellent outcomes for patients. In some cases, bone resorption caused by stress shielding leads to implant loosening. Stress shielding occurs because implants are much stiffer than bone and transmit a significant proportion of the load, leaving the surrounding bone to carry less load compared to an intact femur. Methods To address stress shielding, we aimed to design patient-specific additively manufactured porous femoral stems with reduced stiffness. Diamond lattice structure test specimens of varying porosities were manufactured and tested to measure elastic moduli and yield strengths. These properties were used in subsequent implant optimisation based on finite element analysis. Four implant templates were created based on the region of the implant that was assigned porous material. These templates were referred to as fully porous (FP), proximally porous with a solid distal end (PP), solid distal shell (DS), and fully solid shell (SS). In addition, the elastic modulus within the porous region was assigned either a linear or a radial distribution, resulting in eight possible implant designs. Results Optimisation yielded six distinct solutions, which were evaluated based on the reduction in stress shielding and micromotion at the bone implant interface. While all implant designs reduced stress shielding compared to a solid implant, only the PP and SS stems were predicted to pass the standard fatigue test for femoral stems (ISO 7206-4). Implant-bone micromotion was conducive for bone implant integration for all designs, with the potential exception of the fully porous stem. Of the implants that were predicted to pass the fatigue test, the linear proximal porous stem resulted in the largest reduction in stress shielding (9.5% for walking, 8.1% for stair climbing). Conclusions Based on patient-specific computational models, the porous region of the stems influenced the reduction of stress shielding compared to fully solid stems. Considering implant fatigue failure and bone-implant micromotion, the PP and SS templates were found to be the most suitable design approaches to address femoral bone stress shielding after total hip replacement. Further investigation is required to fully comprehend the implications of porous femoral stems on long-term implant stability.

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.

Background Bone-anchored prostheses (BAPs) address many of the shortcomings of socket prostheses, driving the adoption of the technology for lower limb amputees. However, higher femoral fracture rates have been observed in these patients compared to socket users. It is believed that this increased fracture rate is due to the direct transmission of loads to bone through the bone-anchored implant. While low-impact activities, such as walking, have been extensively studied using finite element (FE) models, other more demanding low-impact activities such as ambulating on stairs and inclines remain poorly understood. Most previously reported subject-specific FE models of femurs fitted with osseointegrated implants were not validated against experimental data. The current study aimed to validate an FE modelling methodology using implanted cadaveric specimens and to calculate the fracture risk of implanted femurs during various activities of daily living. Method Strain responses of cadaveric femurs fitted with press-fit osseointegrated implants under load were recorded and compared to strain predicted by specimen-specific FE models created from CT data. Five different published relationships between Hounsfield units and Young’s Moduli were investigated to determine which relationship gave the most accurate model predictions. The validated FE models of the implanted femurs were later used to simulate in-vivo force measurements during five routine activities of daily living. Lastly, the fracture risk of the implanted femurs during these activities was quantified using factor of safety (FOS), where FOS = 1 denoted fracture was predicted at the given load. Results The validated FE model predicted the experimental strain responses with a coefficient of determination (R 2 ) of 0.91 and a root mean square error (RMSE) of 81.27 με. The FOS for the activities of daily living of individual specimens ranged from 1.8 to 5.0, with walking up an incline having the lowest average FOS (1.9) across all specimens. Compared across different environments, the average FOS for level walking (4.3) and ambulating on stairs (4.0) were similar but almost double that of ambulating on an incline (2.2). Conclusion Male BAP FE models predicted the experimental bone strains and suggested that peak loads during ambulation on level ground, stairs, and inclines are unlikely to cause bone fracture.

Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza.