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To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of β-coronaviruses: SARS-CoV-2 S2 apex residues 980–1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

There is growing interest in identifying antibodies that protect against infectious diseases, especially for high-risk individuals and pathogens for which no vaccine is yet available. However, pathogens that manifest as opportunistic or latent infections express complex arrays of virulence-associated proteins and are adept at avoiding immune responses. Some pathogens have developed strategies to selectively destroy antibodies, whereas others create decoy epitopes that trick the host immune system into generating antibodies that are at best nonprotective and at worst enhance pathogenesis. Antibody engineering strategies can thwart these efforts by accessing conserved neutralizing epitopes, generating Fc domains that resist capture or degradation and even accessing pathogens hidden inside cells. Design of pathogen-resistant antibodies can enhance protection and guide development of vaccine immunogens against these complex pathogens. Here, we discuss general strategies for design of antibodies resistant to specific pathogen defense mechanisms. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 14 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain–specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies., Science Immunology, 7 (78), ISSN:2470-9468

Inflammatory back pain is a hallmark feature of axial spondyloarthritis, a heterogeneous group of inflammatory disorders which affects the sacroiliac joints and spine. Imaging plays a key role in diagnosis of this disease and in facilitating appropriate treatment. This document provides evidence-based recommendations on the appropriate use of imaging studies during multiple stages of the clinical evaluation of patients with suspected or known axial spondyloarthritis. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity. As a result, these systems are not easily portable or reusable for on-demand protein production applications. In this study, we employ Bioproduced Proteins On Demand (Bio-POD), a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable, on-demand production and secretion of medium- and high-molecular weight proteins (in this case, SEAP, α-amylase, and anti-HER2). The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration. The hydrogel platform described here is the first hydrogel immobilization using a P. pastoris system to produce recombinant proteins of this breadth. These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production.

Objective To determine whether there is a correlation between pain and the amount of fluid present at the distal insertion of the iliotibial band (ITB) in runners, as measured by USG. Method Our retrospective cross-sectional study evaluated 100 male and female runners prior to the start of a race. A valid and reliable questionnaire collected demographic, pain, and training data. If a runner reported knee pain, a numeric pain rating scale was used to record the degree of pain. Participants then underwent USG on both knees to determine the presence or absence of fluid at the distal insertion of the ITB. Result We found no statistically significant correlations of fluid measurements with pain score, running experience in years, or age. In addition, we found no other differences in fluid measurements between those with and without knee pain or between the sexes. Conclusions Our findings indicate that the presence or absence of fluid at the distal insertion of the ITB does not correlate with knee pain in runners, regardless of age, running experience, or sex.

Diversity, equity, and inclusion have been recognized as important drivers of excellence and innovation in the physician workforce. Given the historical underrepresentation of women in medicine, gender diversity is of interest. In this cross-sectional study, we sought to quantify leadership representation of female physicians in primary care sports medicine settings, including primary care sports medicine fellowship programs, select sports medicine societies, and select sports medicine-related scientific journals. Data were collected by querying the corresponding web site for each fellowship program, society, and journal and analyzed in a descriptive manner. Results showed that fewer female physicians hold primary care sports medicine leadership roles than men do. This work establishes a baseline for female representation in primary care sports medicine leadership; efforts should continue to increase the presence of women in leadership positions.

It is unknown whether ultrasound findings and symptoms of Achilles tendinopathy in runners correlate with foot strike patterns. We aimed to examine the relationships among Achilles tendon ultrasound findings in runners with or without Achilles tendinopathy, their foot strike patterns, and their training regimens. We recruited marathon runners 18 years of age or older with no history of Achilles tendon pain or surgery participating in the 2018 DONNA Marathon. Participants completed surveys and underwent Achilles tendon sonographic evaluations and were categorized by foot strike patterns. Seventy-nine runners were included; 22 (28%) with forefoot, 30 (38%) midfoot, and 27 (34%) hindfoot strike patterns. Foot strike pattern was not associated with tendon hyperaemia (

The prevalence of class 3 obesity (body mass index ≥40 kg/m2) is 7.7% of the United States adult population; thus, more than 25 million people may be medically appropriate for consideration of bariatric surgery as therapy for severe obesity. Although bariatric surgery is the most effective therapy for patients with severe obesity, the surgery is performed in less than 1% of patients annually for whom it may be appropriate. Patients' and medical professionals' misperceptions about obesity and bariatric surgery create barriers to accessing bariatric surgery that are not given adequate attention and clinical consideration. Commonly cited patient barriers are lack of knowledge about the severity of obesity, the perception that obesity is a lifestyle problem rather than a chronic disease, and fear that bariatric surgery is dangerous. Medical professional barriers include failing to recognize causes of obesity and weight gain, providing recommendations that are inconsistent with current obesity treatment guidelines, and being uncomfortable counseling patients about treatment options for severe obesity. Previous research has revealed that medical professional counseling and accurate perception of the health risks associated with severe obesity are strong predictors of patients' willingness to consider bariatric surgery. This article reviews patient and medical professional barriers to acceptance of bariatric surgery as a treatment of medical necessity and offers practical advice for medical professionals to rethink perspectives about bariatric surgery when it is medically and psychologically appropriate.

Antibody-based therapeutics enjoy considerable clinical and commercial successes as cancer treatments. However, they can also cause serious toxicities due to recognition of tumor-associated antigens in noncancerous tissues, which can prevent antibody use in certain patient populations and therapeutic modalities. Here, we discuss recent efforts to develop advanced antibody therapeutics with activities restricted to the solid tumor microenvironment. With the intent of decreasing toxicities and expanding therapeutic windows, protein engineering strategies can render ligand binding sensitive to multiple tumor-specific characteristics. These triggers can be intrinsic to solid tumor microenvironments, such as low pH, high extracellular ATP, and the presence of specific proteases. Emerging strategies rely instead on exogenous triggers such as light and ultrasound to provide spatial and temporal control over antibody activation. These multilayered approaches to targeting diseased tissues are expected to usher in a new generation of precision therapeutics.

RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMβ2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMβ2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I–III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMβ2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines., Author summary Diverse bacterial pathogens use the type 1 secretion system (T1SS) to secrete RTX leukotoxins, which target host leukocytes during infection. T1SS substrates all contain a repetitive C-terminal ‘RTX’ domain that adopts a characteristic β-roll fold and is involved in secretion. Notably, The RTX domain of Bordetella pertussis adenylate cyclase toxin (ACT) mediates leukocyte targeting via binding to the αMβ2 integrin receptor, and antibodies that block receptor binding neutralize toxin activity. However, ACT also contains multiple non-neutralizing epitopes, and precise knowledge of the sites targeted by neutralizing antibodies is desirable for vaccine design. Here we determine the crystal structure of an ACT fragment in complex with two neutralizing antibodies and define the key neutralization-sensitive sites within the RTX domain. This first structure of a heterotypic protein–protein interaction formed by an RTX domain suggests the linker regions between β-roll segments engage binding partners.

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin's role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin's role in infection and understand the impact of antipertactin antibodies on bacterial fitness.

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen deuterium exchange mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-EM, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation—an open trimer— contains easily accessible RBDs. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.One Sentence SummaryAn alternative conformation of SARS-CoV-2 spike ectodomain modulated by temperature, binding, and sequence variants.

T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV-specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nm affinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications.

Integrins are ubiquitous cell-surface heterodimers that are exploited by pathogens and toxins, including leukotoxins that target β

Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

Despite the exquisite specificity and high affinity of antibody-based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor FcγRIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc-FcγRIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced FcγRIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ∼2-fold pH-selectivity for FcγRIIIa binding based on the ratio of equilibrium dissociation constants K

Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity. As a result, these systems are not easily portable or reusable for on-demand protein production applications. In this study, we employ Bioproduced Proteins On Demand (Bio-POD), a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable, on-demand production and secretion of medium- and high-molecular weight proteins (in this case, SEAP, α-amylase, and anti-HER2). The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration. The hydrogel platform described here is the first hydrogel immobilization using a P. pastoris system to produce recombinant proteins of this breadth. These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production., Graphical abstract Image 1, Highlights • Hydrogel immobilization used for first time for Pichia pastoris protein production. • Diffusion of large protein from hydrogels is not a hindrance to protein production. • Yeast-laden hydrogel samples lost minimal productivity following lyophilization. • Encapsulation-based production outperformed suspension cultures. • Bio-POD enabled portable, reusable, preservable, on-demand production of proteins.

Stabilizing the prefusion SARS-CoV-2 spike The development of therapeutic antibodies and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on the spike (S) protein that decorates the viral surface. A version of the spike ectodomain that includes two proline substitutions (S-2P) and stabilizes the prefusion conformation has been used to determine high-resolution structures. However, even S-2P is unstable and difficult to produce in mammalian cells. Hsieh et al. characterized many individual and combined structure-guided substitutions and identified a variant, named HexaPro, that retains the prefusion conformation but shows higher expression than S-2P and can also withstand heating and freezing. This version of the protein is likely to be useful in the development of vaccines and diagnostics. Science , this issue p. 1501

The most recent International Consensus Statement on Concussion in Sport reports that full recovery of symptoms following sport-related concussions (SRC) typically occurs approximately 10–14 days after injury. Delayed recovery occurs when symptoms persist beyond the expected time frames (i.e., >10–14 days in adults and > 4 weeks in children). Demographic characteristics such as younger age ( 5 days has negative correlation with recovery, early progression of physical activity within the first week (guided by the symptom exacerbation threshold) shows an inverse relationship. Screening for the presence of prognostic risk factors is recommended to initiate earlier intervention and/or appropriate referrals. Return-to-activity/play programs need to be individualized for each patient in order to achieve better outcomes.

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

Understanding protein stability is central to combatting protein aggregation diseases and developing new protein therapeutics. At the high concentrations often present in biological systems, purified proteins can exhibit undesirable high solution viscosities and poor solubilities mediated by short-range electrostatic and hydrophobic protein-protein interactions. The interplay between protein amino acid sequence, protein structure, and solvent conditions to minimize protein-protein interactions is key to designing well-behaved pharmaceutical proteins. However, theoretical approaches have yet to yield a general framework to address these problems. Here, we analyzed the high concentration behavior of superfolder GFP (sfGFP) and two supercharged sfGFP variants engineered to have formal charges of -18 or +15. Under low cosolute conditions, sfGFP and the -18 variant formed a gel or phase separated at ∼10 mg/mL. Under conditions that screen surface charges, including formulations with high histidine or high NaCl concentrations, all three variants attained concentrations up to 250 mg/mL with moderate viscosities. Moreover, all three variants exhibited very similar viscosity-concentration profiles over this range. This effect was not mimicked by high sugar concentrations that exert excluded-volume effects without shielding charge. Collectively, these data demonstrate that charge shielding neutralizes not only long-range electrostatic interactions but also, surprisingly, short-range electrostatic effects due to surface charge anisotropy. This work shows that supercharged sfGFP behavior under high ionic strength is largely determined by particle geometry, a conclusion that is supported by colloid models and may be applicable to pharmaceutically relevant proteins.

Computational antibody engineering efforts to date have focused on improving binding affinities or biophysical characteristics. De novo design of antibodies binding specific epitopes could greatly accelerate discovery of therapeutics as compared to conventional immunization or synthetic library selection strategies. Here, we employed de novo complementarity determining region (CDR) design to engineer targeted antibody–antigen interactions using previously described in silico methods. CDRs predicted to bind the minimal FLAG peptide (Asp–Tyr–Lys–Asp) were grafted onto a single-chain variable fragment (scFv) acceptor framework. Fifty scFvs comprised of designed heavy and light or just heavy chain CDRs were synthesized and screened for peptide binding by phage ELISA. Roughly half of the designs resulted in detectable scFv expression. Four antibodies, designed entirely in silico, bound the minimal FLAG sequence with high specificity and sensitivity. When reformatted as soluble antigen-binding fragments (Fab), these clones expressed well, were predominantly monomeric and retained peptide specificity. In both formats, the antibodies bind the peptide only when present at the amino-terminus of a carrier protein and even conservative peptide amino acid substitutions resulted in a complete loss of binding. These results support in silico CDR design of antibody specificity as an emerging antibody engineering strategy.

The baboon model of Bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date. However, among the 15 experimentally infected baboons in this study, a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count. Moreover, cultures of nasopharyngeal wash samples from several baboons suggested B. bronchiseptica coinfection. Analysis of serum antibodies recognizing filamentous hemagglutinin, pertussis toxin and B. pertussis lipo-oligosaccharide indicated that several baboons had likely been previously exposed to Bordetella species and that prior exposure correlated with partial protection from B. pertussis infection. Notably, all animals with a baseline Fha titer of 5 IU/ml or below exhibited symptoms typical of the model, suggesting this value can be used as inclusion criteria for animals prior to study enrollment. While B. pertussis infection is endemic to human populations and B. bronchiseptica is common in wild small mammals, this study illustrates that baboons can readily harbor both organisms. Awareness of Bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis.

While antibody libraries are traditionally screened in phage, bacterial, or yeast display formats, they are produced in large scale for pharmaceutical and commercial use in mammalian cell lines. The simpler organisms used for screening have significantly different folding and glycosylation machinery than mammalian cells; consequently, clones resulting from these libraries may require further optimization for mammalian cell expression. To streamline the antibody discovery process, we developed a Chinese hamster ovary (CHO) cell-based selection system that allows for long-term display of antibody Fab fragments. This system is facilitated by a semi-stable Epi-CHO episomal platform to maintain antibody expression for up to 2 months and is compatible with standard PCR-based mutagenesis strategies. This protocol describes the simple and accessible use of CHO display coupled with flow cytometry to enrich for antibody variants with increased ligand-binding affinity from large libraries of ~10

While antibody libraries are traditionally screened in phage, bacterial, or yeast display formats, they are produced in large scale for pharmaceutical and commercial use in mammalian cell lines. The simpler organisms used for screening have significantly different folding and glycosylation machinery than mammalian cells; consequently, clones resulting from these libraries may require further optimization for mammalian cell expression. To streamline the antibody discovery process, we developed a Chinese hamster ovary (CHO) cell-based selection system that allows for long-term display of antibody Fab fragments. This system is facilitated by a semi-stable Epi-CHO episomal platform to maintain antibody expression for up to 2 months and is compatible with standard PCR-based mutagenesis strategies. This protocol describes the simple and accessible use of CHO display coupled with flow cytometry to enrich for antibody variants with increased ligand-binding affinity from large libraries of ~106 variants, using HER2-binding antibodies as an example.

An antibody prevents the symptoms of whooping cough in newborn baboons when administered 5 weeks before infection., Pertussis continues to cause considerable infant mortality world-wide, which could be addressed in part by passive immunization strategies. Antibody hu1B7 is a candidate therapeutic that potently neutralizes pertussis toxin in vitro, prevents leukocytosis in mice and treats established disease in weanling baboons as part of an antibody cocktail. Here, we evaluated the potential for hu1B7 and an extended half-life hu1B7 variant to prevent death, leukocytosis and other clinical symptoms in a newborn baboon model that mimics many aspects of human disease. We administered a single antibody dose to newborn baboons five weeks prior to experimental infection. While all animals were heavily colonized with Bordetella pertussis, prophylaxed animals showed significantly greater survival (P < 0.005), delayed and suppressed leukocytosis (P < 0.01) and enhanced clinical outcomes, including coughing (P < 0.01), as compared to controls. Together, this work demonstrates that a single neutralizing anti-PTx antibody is sufficient to prevent clinical pertussis symptoms.

Bordetella pertussis(B. pertussis) is the causative agent of pertussis (whooping cough). Since the 1990s, pertussis has re-emerged in the United States despite an estimated 95% vaccine coverage. Our goal was to characterize neutrophil responses and gene expression profiles of murine lungs in the context of vaccination andB. pertussischallenge. We utilized a bioluminescent neutrophil mouse model (NECre luc) to track neutrophil recruitment. NECre luc mice were immunized with whole cell vaccine (WCV), acellular vaccine (ACV), or a truncated adenylate cyclase toxoid (RTX) vaccine. Neutrophil recruitment was measured in live mice across time and corroborated by flow cytometry and other data. WCV immunized mice showed signs of neutrophilia in response toB. pertussischallenge. Mice immunized with either ACV or WCV cleared the challenge infection; however immunization with RTX alone was not protective. RNA sequencing revealed distinctive gene expression profiles for each immunization group. We observed an increase in expression of genes associated with responses to infection, and changes in expression of distinct genes in each vaccine group, providing a complex view of the immune response toB. pertussisinfection in mice. This study suggests that combination of immunological analysis with transcriptomic profiling can facilitate discovery of pre-clinical correlates of protection for vaccine development.

The use of animal models and in vitro assays has allowed the identification and functional characterization of an iconic set of Bordetella pertussis factors that contribute to its pathogenesis. Much research on B. pertussis has been focused on the effects of pertussis toxin and adenylate cyclase toxin on pathogenesis and disease progression, and on the function of adherence factors. However, much larger sets of factors have been identified and proposed to be involved in host–pathogen interactions, including B. pertussis manipulating the host’s metabolism to its advantage. The identification of a third player, the resident microbiota, reveals a complex picture that has yet to be investigated. The ongoing studies of the molecular components produced by B. pertussis and their effect on the host during infection, commonly referred to as ‘pathogenesis’ research, are crucial to generate understanding of the host–pathogen interactions necessary to design new approaches to prevent and cure disease.

Understanding the molecular interactions between biological cells and engineered nanoparticles is a key to evaluating potential toxicities to humans and the environment. This study developed a method to determine the mechanisms by which fullerene aggregates are distributed into a representative cell line, human intestinal Caco-2 cells. First, we determined that the presence of fetal bovine serum (FBS) in the cell culture media changes the particle characteristics and inhibits particle adsorptions onto cell surfaces. Second, significantly lower amounts of fullerene were internalized at 4°C, a temperature at which active transport mechanisms are effectively impeded, than at 37°C. Third, metabolic inhibitors of active transport and a microtubule transport inhibitor decreased fullerene uptake at 37°C. Fourth, cellular uptake of fullerene increased with increasing fullerene concentration, suggesting that passive diffusion into lipid membranes contributed to uptake over the broad concentration range used in this study. Together, these results indicate fullerene transport into cells occurs via two mechanisms: passive diffusion across the lipid bilayer and active transport including microtubule involved endocytosis. The results also suggest that simple physical-chemical partitioning models do not fully describe fullerene uptake, and instead, active transport models are also required to estimate the cellular uptake and toxicity of fullerene.

Bispecific antibodies are a rapidly growing class of therapeutic molecules, originally developed for the treatment of cancer but recently explored for the treatment of autoimmune and infectious diseases. Bordetella pertussis is a reemerging pathogen, and several of the key symptoms of infection are caused by the pertussis toxin (PTx). Two humanized antibodies, hu1B7 and hu11E6, bind distinct epitopes on PTx and, when coadministered, mitigate disease severity in murine and baboon models of infection. Here we describe the generation of a bispecific human IgG1 molecule combining the hu1B7 and hu11E6 binding sites via a knobs-in-holes design. The bispecific antibody showed binding activity equivalent to that of the antibody mixture in a competition enzyme-linked immunosorbent assay (ELISA). A CHO cell neutralization assay provided preliminary evidence for synergy between the two antibodies, while a murine model of PTx-induced leukocytosis definitively showed synergistic neutralization. Notably, the bispecific antibody retained the synergy observed for the antibody mixture, supporting the conclusion that synergy is due to simultaneous blockade of both the catalytic and receptor binding activities of pertussis toxin. These data suggest that a hu1B7/hu11E6 bispecific antibody is a viable alternative to an antibody mixture for pertussis treatment.

To further advance a subcutaneous injection of monoclonal antibodies (mAbs) at elevated concentrations, novel concepts are needed to lower the viscosity. The addition of high concentrations of cosolutes, namely, arginine glutamate (Arg·Glu) or Arg·HCl, reduced the viscosity of a ∼250 mg/mL mAb solution up to 6-fold. With Arg·Glu, the viscosity of the mAb solution was reduced to 30 cP and for a polyclonal sheep IgG solution to 17 cP both at ∼250 mg/mL. Viscosities went through a maximum at the mAb isoelectric point for solutions with Arg·Glu or Arg·HCl. In contrast the viscosity was only weakly affected by NaCl or the preferentially excluded molecule trehalose. The large viscosity reduction from Arg may be attributed to direct binding to the mAb, resulting in suppression of both hydrophobic and local anisotropic electrostatic attraction. Aggregate formation was negligible for high cosolute mAb solutions as demonstrated by SEC even after 8 weeks of 25 °C storage.

Invasin is a key adhesin displayed on the outer membrane of Yersinia enterocolitica and Y. pseudotuberculosis that mediates the initial stages of infection. Invasin specifically targets microfold (M) cells in the small intestine by binding β1 integrins and is sufficient to trigger eukaryotic uptake of invasin-coated particles, including Yersinia, Escherichia coli, and latex beads. As a result, invasin has generated interest to mediate oral delivery of vaccines and other biologics. Integrin binding affinity has been shown to correlate with particle uptake; thus we hypothesized that invasin variants with higher affinity would confer enhanced internalization. We first performed alanine scanning of surface-exposed tyrosine residues to identify those contributing to integrin binding. We identified two residues, which, when substituted with alanine, reduced binding to soluble α5β1 integrin. Next, we constructed four targeted mutagenesis libraries spanning these and other residues known to contribute to binding, followed by enrichment of variants able to mediate Caco-2 cellular invasion and to bind soluble α5β1 integrin. We identified three amino acid substitutions that increased α5β1 integrin binding affinity as measured by flow cytometry and ELISA assays, two of which created a novel RGD motif surrounding the D911 residue critical for binding. This variant confers enhanced internalization into CHO cells but not Caco-2 cells when expressed on the E. coli surface. Further analysis showed that inclusion of an RGD expands invasin-integrin specificity, thereby impacting cellular selectivity. This work provides a molecular explanation for the lack of an RGD motif in invasin that is present in many other adhesins.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis. RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice.

Sports-related concussions continue to generate widespread interest. A concussion is a complex pathophysiologic process, with or without loss of consciousness, that results in a disturbance of brain function. Risk factors include age

Despite high vaccination rates, the incidence of whooping cough has steadily been increasing in developing countries for several decades. The current acellular pertussis (aP) vaccines all include the major protective antigen pertussis toxin (PTx) and are safer, but they appear to be less protective than infection or older, whole-cell vaccines. To better understand the attributes of individual antibodies stimulated by aP, we isolated plasmablast clones recognizing PTx after booster immunization of two donors. Five unique antibody sequences recognizing native PTx were recovered and expressed as recombinant human IgG1 antibodies. The antibodies all bind different epitopes on the PTx S1 subunit, B oligomer, or S1-B subunit interface, and just one clone neutralized PTx in an in vitro assay. To better understand the epitopes bound by the nonneutralizing S1-subunit antibodies, comprehensive mutagenesis with yeast display provided a detailed map of the epitope recognized by antibodies A8 and E12. Residue R76 is required for antibody A8 binding and is present on the S1 surface but is only partially exposed in the holotoxin, providing a structural explanation for A8's inability to neutralize holotoxin. The B-subunit-specific antibody D8 inhibited PTx binding to a model receptor and neutralized PTx in vitro as well as in an in vivo leukocytosis assay. This is the first study, to our knowledge, to identify individual human antibodies stimulated by the acellular pertussis vaccine and demonstrates the feasibility of using these approaches to address outstanding issues in pertussis vaccinology, including mechanisms of accelerated waning of protective immunity despite repeated aP immunization.