Implementation and Analysis of a 5-Year Online Esophageal Motility Curriculum for Gastroenterology Fellows.

BACKGROUND: Motility disorders are frequently encountered in gastroenterology (GI) practice, yet a national structured training curriculum for GI fellows in motility disorders is lacking. Since GI fellowships vary considerably in opportunities for specialized esophageal motility (EM) training, novel educational technology may be leveraged to provide standardized EM curriculum to train GI fellows in esophageal manometry. METHODS: GI fellows participated in an online EM learning program at a single academic center from 2017 to 2022. Fellows answered case-based questions and were provided with evidence-based, corrective feedback related to core EM learning objectives. The primary outcome was change in knowledge and comfort in interpretation and clinical application of EM studies. RESULTS: Sixty-nine fellows actively participated in the online EM curriculum. 65 fellows completed a pre-curriculum test, and 54 fellows completed a post-curriculum test. There was a cumulative improvement between pre-curriculum test and post-curriculum test scores from 70 to 87%, respectively (p < 0.001). Fellows had a mean improvement of 19% in questions as they progressed through the curriculum. Prior to enrolling in the EM course, 26% of fellows felt comfortable in interpreting EM studies compared to 54% of fellows after completion of the program (p < 0.001). CONCLUSION: An online, technology-based curriculum was effective in educating GI fellows on core competencies of EM. Fellows demonstrated improvement in proficiency of clinically important EM studies and increased comfort in interpreting EM studies. Further studies are needed to evaluate the use of technology-based learning to widely disseminate a structured training curriculum in EM, particularly in training programs without a motility presence.

Prevalence and Factors Associated With Eyeglass Wear Compliance Among Preschoolers From Low-Income Families in San Francisco, California.

IMPORTANCE: Despite growing support for early school-based vision screening and eyeglass provision, few studies have rigorously monitored the compliance of eyeglass wear among preschool-aged children who receive eyeglasses through such programs. OBJECTIVE: To assess the prevalence and factors associated with eyeglass wear compliance among preschoolers from low-income families who receive eyeglasses through the See Well to Learn program. DESIGN, SETTING, AND PARTICIPANTS: Longitudinal cross-sectional study of eyeglass wear compliance patterns among 188 children 3 to 5 years of age from 51 Bay Area Head Start preschools in San Francisco, California. The study conducted during the 2017 to 2018 school year included students with a failed vision screening who met predetermined refractive criteria following cycloplegic refraction and received eyeglasses through the See Well to Learn program. EXPOSURES: Eyeglass distribution. MAIN OUTCOMES AND MEASURES: Eyeglass wear compliance, measured by a school-year's worth of weekly teacher reports, was a longitudinal measure of consistent eyeglass wear, defined by eyeglass wear for more than 50% of every school day (compliance score of 4). RESULTS: Of 188 students (91 boys [49%]; 94 girls [51%]; mean [SD] age, 3.89 [0.5] years), 133 (71%; 95% CI, 64%-77%) maintained a mean compliance score throughout the school year of 4 or higher. Compliance prevalence was relatively stable throughout the school year, ranging from 139 students (74%) to 164 students (87%). Baseline uncorrected visual acuity in both the better-seeing and worse-seeing eyes was the only assessed factor that was associated with compliance. In the better-seeing eye, the mean uncorrected visual acuity of students with eyeglass wear compliance was 0.473 logMAR (95% CI, 0.433-0.514) (Snellen equivalent, 20/60) compared with 0.394 logMAR (95% CI, 0.334-0.454) (Snellen equivalent, 20/50) for students with noncompliance (P = .03). In the worse-seeing eye, the mean uncorrected visual acuity of students with compliance was 0.576 logMAR (95% CI, 0.530-0.623) (Snellen equivalent, 20/75) compared with 0.492 logMAR (95% CI, 0.433-0.551) (Snellen equivalent, 20/62) for students with noncompliance (P = .03). In the better-seeing eye, the difference between students with compliance vs noncompliance was 0.079 logMAR (95% CI, 0.009-0.150) (5 Snellen letter difference) compared with 0.084 logMAR (95% CI, 0.007-0.160) (5 Snellen letter difference) in the worse-seeing eye. CONCLUSIONS AND RELEVANCE: This study found that nearly 3 of 4 preschool students consistently wore their glasses at school during their first year of use, supporting the continued implementation of preschool-based vision screening programs. These findings suggest that programs involving school-based screening and eyeglass delivery may lessen disparities in accessing pediatric vision care. Consistent with previous studies, students with poorer uncorrected baseline visual acuity were found to be more likely to wear eyeglasses compliantly.

Soluble Prefusion Closed DS-SOSIP.664-Env Trimers of Diverse HIV-1 Strains.

The elicitation of autologous neutralizing responses by immunization with HIV-1 envelope (Env) trimers conformationally stabilized in a prefusion closed state has generated considerable interest in the HIV-1 vaccine field. However, soluble prefusion closed Env trimers have been produced from only a handful of HIV-1 strains, limiting their utility as vaccine antigens and B cell probes. Here, we report the engineering from 81 HIV-1 strains of soluble, fully cleaved, prefusion Env trimers with appropriate antigenicity. We used a 96-well expression-screening format to assess the ability of artificial disulfides and Ile559Pro substitution (DS-SOSIP) to produce soluble cleaved-Env trimers; from 180 Env strains, 20 yielded prefusion closed trimers. We also created chimeras, by utilizing structure-based design to incorporate select regions from the well-behaved BG505 strain; from 180 Env strains, 78 DS-SOSIP-stabilized chimeras, including 61 additional strains, yielded prefusion closed trimers. Structure-based design thus enables the production of prefusion closed HIV-1-Env trimers from dozens of diverse strains.

Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV.

Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.

Evaluation of candidate vaccine approaches for MERS-CoV.

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.

Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env.

UNLABELLED: Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. IMPORTANCE: The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions.

Transplanting supersites of HIV-1 vulnerability.

One strategy for isolating or eliciting antibodies against a specific target region on the envelope glycoprotein trimer (Env) of the human immunodeficiency virus type 1 (HIV-1) involves the creation of site transplants, which present the target region on a heterologous protein scaffold with preserved antibody-binding properties. If the target region is a supersite of HIV-1 vulnerability, recognized by a collection of broadly neutralizing antibodies, this strategy affords the creation of "supersite transplants", capable of binding (and potentially eliciting) antibodies similar to the template collection of effective antibodies. Here we transplant three supersites of HIV-1 vulnerability, each targeted by effective neutralizing antibodies from multiple donors. To implement our strategy, we chose a single representative antibody against each of the target supersites: antibody 10E8, which recognizes the membrane-proximal external region (MPER) on the HIV-1 gp41 glycoprotein; antibody PG9, which recognizes variable regions one and two (V1V2) on the HIV-1 gp120 glycoprotein; and antibody PGT128 which recognizes a glycopeptide supersite in variable region 3 (glycan V3) on gp120. We used a structural alignment algorithm to identify suitable acceptor proteins, and then designed, expressed, and tested antigenically over 100-supersite transplants in a 96-well microtiter-plate format. The majority of the supersite transplants failed to maintain the antigenic properties of their respective template supersite. However, seven of the glycan V3-supersite transplants exhibited nanomolar affinity to effective neutralizing antibodies from at least three donors and recapitulated the mannose9-N-linked glycan requirement of the template supersite. The binding of these transplants could be further enhanced by placement into self-assembling nanoparticles. Essential elements of the glycan V3 supersite, embodied by as few as 3 N-linked glycans and ∼ 25 Env residues, can be segregated into acceptor scaffolds away from the immune-evading capabilities of the rest of HIV-1 Env, thereby providing a means to focus the immune response on the scaffolded supersite.