Spreading Standardized Documentation of Pediatric Occupational and Physical Therapy Dose: A Quality Improvement Initiative.

IMPORTANCE: Clinicians and researchers can leverage clinical documentation of therapy services for quality improvement and research purposes. However, documentation is often institution specific and may not adequately capture the dose of therapy delivered, thus limiting collaboration. OBJECTIVE: To implement documentation of pediatric occupational and physical therapy dose from one institution to another institution. Dose documentation includes the frequency, intensity, time, and type of interventions delivered (FITT Epic® Flowsheet) at each session. DESIGN: Prospective time-series quality improvement study. SETTING: Two large urban pediatric hospitals. PARTICIPANTS: Occupational and physical therapy staff members. INTERVENTION: Staff training and feedback loops utilizing existing groups. OUTCOMES AND MEASURES: The process measure (number of available staff trained and using the FITT Epic Flowsheet over time) and the outcome measure (percentage of FITT Epic Flowsheets used for treatment visits each month) were analyzed using a statistical process control chart. The balancing measure (percentage of notes closed before 7 p.m. on the same day as the encounter) was analyzed using mean per month across three time periods. RESULTS: Fifty-seven staff members (68%) attended formal training. On average, clinicians documented 90% of sessions using the FITT Epic Flowsheet after implementation. There was no change observed in the balancing measure. CONCLUSIONS AND RELEVANCE: Documentation of dose was spread from one institution to another. Shared documentation will facilitate future collaboration for quality improvement and research purposes. Occupational therapy practitioners and leaders should consider implementing documentation with common dose elements. Plain-Language Summary: Occupational therapy documentation is often institution specific and may not adequately capture the dose of therapy (frequency, intensity, time) or types of interventions that were delivered, thus limiting opportunities for collaboration between institutions. This article adds to the literature on administrative supports for clinical and quality improvement research by illustrating a specific example of how documentation of dose can be shared from one institution to another. The data show that clinicians at one institution started using a new style of documentation using the FITT Epic® Flowsheet and shared discrete dose elements with another institution, creating new opportunities for collaboration. Shared documentation can facilitate future collaboration for quality improvement and research purposes.

Immune response to SARS-CoV-2 variants after immunization with different vaccines in Mexico.

There is limited information on the antibody responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in subjects from developing countries with populations having a high incidence of co-morbidities. Here, we analysed the immunogenicity of homologous schemes using the ChAdOx1-S, Sputnik V, or BNT162b2 vaccines and the effect of a booster dose with ChAdOx1-S in middle-aged adults who were seropositive or seronegative to the SARS-CoV-2 spike protein before vaccination. The study was conducted post-vaccination with a follow-up of 4 months for antibody titre using enzyme-linked immunosorbent assay (ELISA) and pseudovirus (PV) neutralization assays (PNAs). All three vaccines elicited a superior IgG anti-receptor-binding domain (RBD) and neutralization response against the Alpha and Delta variants when administered to individuals with a previous infection by SARS-CoV-2. The booster dose spiked the neutralization activity among individuals with and without a prior SARS-CoV-2 infection. The ChAdOx1-S vaccine induced weaker antibody responses in infection-naive subjects. A follow-up of 4 months post-vaccination showed a drop in antibody titre, with about 20% of the infection-naive and 100% of SARS-CoV-2 pre-exposed participants with detectable neutralization capacity against Alpha pseudovirus (Alpha-PV) and Delta PV (Delta-PV). Our observations support the use of different vaccines in a country with high seroprevalence at the vaccination time.

Implementing Parent Coaching in Hospital-Based Pediatric Occupational Therapy: A Multisite Quality Improvement Project.

IMPORTANCE: Parent coaching (PC) is a best practice for young children with, or at high risk for, cerebral palsy (CP). Occupational therapy practitioners in outpatient settings encounter barriers to implementing PC. OBJECTIVE: To increase the documented use of PC in outpatient occupational therapy visits for children younger than age 2 yr with, or at high risk for, CP from 0% to 80%. DESIGN: Multicenter quality improvement (QI) initiative with a time-series design. SETTING: Three pediatric tertiary-care institutions, each with multiple outpatient occupational therapy clinics. PARTICIPANTS: Practitioners in the outpatient clinics and patients <2 yr old with, or at high risk for, cerebral palsy. INTERVENTION: Plan-do-study-act cycles included interventions packaged as a toolkit: education sessions, quick references, electronic medical record (EMR) supports, and site-specific strategies. OUTCOMES AND MEASURES: The primary outcome measure was the use of PC in outpatient sessions. Process measures included pre- and posteducation practitioner knowledge scores and an EMR checklist. Balancing measures (ensuring that changes do not cause problems in other areas) of parent satisfaction/experience and practitioner productivity were measured pre- and postintervention. RESULTS: The primary outcome measure goal (80% documented use of PC in sessions) was attained in the seventh month of the study, sustained for 4 mo, and settled at 79.1% for the remaining 6 mo. Practitioner knowledge scores increased from 83.1% to 87.9% after initial education sessions, t[56] = 3.289, p = .001. Parent satisfaction/experience and practitioner productivity scores did not change. CONCLUSIONS AND RELEVANCE: QI methodology can support PC implementation in pediatric outpatient practice. What This Article Adds: This multisite QI initiative shows that outpatient occupational therapy practitioners can implement PC as a best practice with the use of a toolkit. Results suggest that education alone does not result in changes to practitioner behavior and that QI methods can help when implementing best practices in a clinical setting.

Trauma radiology teaching for foundation doctors working within the Scottish Trauma Network improves radiology requests and patient safety: a multidepartmental quality improvement project.

The importance of radiology in trauma management is particularly relevant today as Scotland develops its Scottish Trauma Network. Trauma and radiology are scarcely covered in the 2016 and 2021 Foundation Programme Curriculum. Trauma is a significant and endemic public health challenge, while radiology is ever growing in use as a diagnostic and interventional tool. Currently, the majority of radiological investigation requests in trauma are made by foundation doctors. Accordingly, there is an urgent need to ensure foundation doctors are adequately trained in trauma radiology. This prospective, multidepartmental quality improvement project at a single major trauma centre primarily analysed the impact of trauma radiology teaching on the quality of foundation doctors' radiology requests according to Ionising Radiation Medical Exposure Regulations (IRMER) criteria. As a secondary outcome, the impact of teaching on patient safety was also evaluated. 50 foundation doctors across three departments dealing with trauma patients had their trauma radiology requests analysed before and after the intervention of trauma-focused radiology teaching. Results showed cancelled and altered radiology requests decreased from 20% to 5% and from 25% to 10%, respectively, with a p value of 0.01. This translated to fewer delays in trauma patients receiving radiological investigations. The foundation curriculum would benefit from the introduction of trauma radiology teaching for its foundation doctors, in parallel to the demands of a growing national trauma network. Education globally improves radiology request quality by raising awareness and respect of IRMER criteria and ultimately leads to positive changes for patient safety.

Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine.

Influenza virus poses an ongoing human health threat with pandemic potential. Due to mutations in circulating strains, formulating effective vaccines remains a challenge. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) proteins is a promising vaccine strategy to protect against a wide range of current and future influenza viruses. Though effective in preclinical studies, the mechanistic basis driving the broad reactivity of COBRA proteins remains to be elucidated. Here, we report the crystal structure of the COBRA HA termed P1 and identify antigenic and glycosylation properties that contribute to its immunogenicity. We further report the cryo-EM structure of the P1-elicited broadly neutralizing antibody 1F8 bound to COBRA P1, revealing 1F8 to recognize an atypical receptor binding site epitope via an unexpected mode of binding.

Pediatric Outpatient Occupational Therapy: Transitioning From In-Person Services to Telehealth.

The COVID-19 pandemic necessitated rapid adoption of telehealth for outpatient pediatric occupational therapy practice. The dose of therapy may have varied across diagnostic and geographical groups despite efforts to ensure access for all patients. The objective of the study was to describe the visit length of outpatient pediatric occupational therapy practice for three diagnostic groups at one institution both during and prior to the COVID-19 pandemic. Retrospective review of electronic health records for two time periods using both practitioner-entered and telecommunications data. Data were analyzed using descriptive statistics and generalized linear mixed model. Prior to the pandemic, average treatment length did not vary by primary diagnosis. During the pandemic, average visit length varied by primary diagnosis, with feeding disorder (FD) visits significantly shorter than cerebral palsy (CP) and autism spectrum disorder (ASD) visits. During the pandemic, visit length was associated with rurality for the whole sample and for patients with ASD and CP, but not FD. Patients with FD may have been seen for shorter durations during telehealth visits. The technology gap may affect services for patients living in rural communities.

The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes.

Clinical Focus: Findings and Clinical Implications for Thickening Formula With Infant Cereal Using the International Dysphagia Diet Standardisation Initiative Flow Test.

PURPOSE: The International Dysphagia Diet Standardisation Initiative (IDDSI) framework was established to provide standardized terminology and objective measures to assess foods and liquids for persons with swallowing difficulties. This clinical focus article reports the findings and clinical implications of the flow testing of infant formulas thickened with infant cereal completed as part of the transition process to IDDSI for one large pediatric quaternary care hospital. METHOD: To determine a common recipe that could be used to thicken formulas with infant cereal to the appropriate IDDSI levels, three clinicians completed flow testing on 94 infant formulas. To examine intra- and interclinician variability in the process, they repeated flow testing with three commonly used formulas and infant cereal. RESULTS: Clinicians were unable to identify a standard recipe (infant formula + infant cereal combination) that consistently thickened different formula brands to a desired IDDSI thickness level, as there was pronounced variability across and within infant formulas. Reliability testing revealed that, overall, clinician mixers were consistent in replicating similar results to themselves and to each other and that, instead, greater variability lies within the formula (and infant formula + infant cereal combination). CONCLUSIONS: Based on findings of pronounced variability within and across infant formulas, our institution determined that the creation of a standard recipe for achieving IDDSI thickness levels of formula mixed with infant cereal was not feasible or clinically appropriate. We offer recommendations for similar institutions for advancing clinical management of infant dysphagia using the IDDSI flow test and directions for future research.

Loss to patient-reported outcome measure follow-up after hip arthroplasty and knee arthroplasty : patient satisfaction, associations with non-response, and maximizing returns.

AIMS: The aim of this study was to determine satisfaction rates after hip and knee arthroplasty in patients who did not respond to postoperative patient-reported outcome measures (PROMs), characteristics of non-responders, and contact preferences to maximize response rates. METHODS: A prospective cohort study of patients planned to undergo hip arthroplasty (n = 713) and knee arthroplasty (n = 737) at a UK university teaching hospital who had completed preoperative PROMs questionnaires, including the EuroQol five-dimension health-related quality of life score, and Oxford Hip Score (OHS) and Oxford Knee Score (OKS). Follow-up questionnaires were sent by post at one year, including satisfaction scoring. Attempts were made to contact patients who did not initially respond. Univariate, logistic regression, and receiver operator curve analysis was performed. RESULTS: At one year, 667 hip patients (93.5%) and 685 knee patients (92.9%) had undergone surgery and were alive. No response was received from 151/667 hip patients (22.6%), 83 (55.0%) of whom were ultimately contacted); or from 108/685 knee patients (15.8%), 91 (84.3%) of whom were ultimately contacted. There was no difference in satisfaction after arthroplasty between initial non-responders and responders for hips (74/81 satisfied vs 476/516 satisfied; p = 0.847) or knees (81/93 satisfied vs 470/561 satisfied; p = 0.480). Initial non-response and persistent non-response was associated with younger age, higher BMIs, and worse preoperative PROMs for both hip and knee patients (p < 0.050). Being in employment was associated with persistent non-response for hip patients (p = 0.047). Multivariate analysis demonstrated that younger age (p < 0.038), higher BMI (p = 0.018), and poorer preoperative OHS (p = 0.031) were independently associated with persistent non-response to hip PROMs. No independent associations were identified for knees. Using a threshold of > 66.4 years predicted a preference for contact by post (area under the curve 0.723 (95% confidence interval (CI) 0.647 to 0.799; p < 0.001, though this CI crosses the 0.7 limit considered reliable). CONCLUSION: The majority of initial non-responders were ultimately contactable with effort. Satisfaction rates were not inferior in patients who did not initially respond to PROMs. Cite this article: Bone Jt Open 2022;3(4):275-283.

Structure-Based Design and Antigenic Validation of Respiratory Syncytial Virus G Immunogens.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of children, the elderly, and immunocompromised individuals. Currently, there are no FDA-approved RSV vaccines. The RSV G glycoprotein is used for viral attachment to host cells and impairment of host immunity by interacting with the human chemokine receptor CX3CR1. Antibodies that disrupt this interaction are protective against infection and disease. Nevertheless, development of an RSV G vaccine antigen has been hindered by its low immunogenicity and safety concerns. A previous study described three engineered RSV G proteins containing single-point mutations that induce higher levels of IgG antibodies and have improved safety profiles compared to wild-type RSV G (H. C. Bergeron, J. Murray, A. M. Nuñez Castrejon, et al., Viruses 13:352, 2021, https://doi.org/10.3390/v13020352). However, it is unclear if the mutations affect RSV G protein folding and display of its conformational epitopes. In this study, we show that the RSV G S177Q protein retains high-affinity binding to protective human and mouse monoclonal antibodies and has equal reactivity as wild-type RSV G protein to human reference immunoglobulin to RSV. Additionally, we determined the high-resolution crystal structure of RSV G S177Q protein in complex with the anti-RSV G antibody 3G12, further validating its antigenic structure. These studies show for the first time that an engineered RSV G protein with increased immunogenicity and safety retains conformational epitopes to high-affinity protective antibodies, supporting its further development as an RSV vaccine immunogen. IMPORTANCE Respiratory syncytial virus (RSV) causes severe lower respiratory diseases of children, the elderly, and immunocompromised populations. There currently are no FDA-approved RSV vaccines. Most vaccine development efforts have focused on the RSV F protein, and the field has generally overlooked the receptor-binding antigen RSV G due to its poor immunogenicity and safety concerns. However, single-point mutant RSV G proteins have been previously identified that have increased immunogenicity and safety. In this study, we investigate the antibody reactivities of three known RSV G mutant proteins. We show that one mutant RSV G protein retains high-affinity binding to protective monoclonal antibodies, is equally recognized by anti-RSV antibodies in human sera, and forms the same three-dimensional structure as the wild-type RSV G protein. Our study validates the structure-guided design of the RSV G protein as an RSV vaccine antigen.

Rapid Implementation of Telerehabilitation for Pediatric Patients During Covid-19.

The COVID-19 pandemic necessitated a sudden limitation of in-person outpatient occupational and physical therapy services for most patients at a large, multisite pediatric hospital located in the Midwest, United States. To ensure patient and staff safety, the hospital rapidly shifted to deliver most of these services via telerehabilitation. The purposes of this study were to (1) describe the rapid implementation of telerehabilitation during the COVID-19 pandemic, (2) describe the demographic characteristics of patients who continued in-person services and those who received telerehabilitation, and (3) evaluate the therapists' perceptions of telerehabilitation for physical and occupational therapy. Most of the children (83.4% of n=1352) received telerehabilitation services. A family was more likely to choose to continue in-person visits if their child was <1-year-old, had a diagnosis of torticollis, received serial casting, or was post-surgical. Occupational and physical therapy therapists (n=9) completed surveys to discern their perceptions of the acceptability of telerehabilitation, with most reporting that telerehabilitation was as effective as in-person care.

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.

Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry.

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide semi-quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated "dip-and-read" format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete semi-quantitative results are obtained in less than 20 min. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens.

Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry.

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated "dip-and-read" format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete quantitative results are obtained in less than 20 minutes. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens.

Gene editing in CHO cells to prevent proteolysis and enhance glycosylation: Production of HIV envelope proteins as vaccine immunogens.

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.

Feasibility and Acceptability of Clinical Pediatric Telerehabilitation Services.

OBJECTIVE: Telerehabilitation has long been recognized as a promising means of providing pediatric services; however, significant barriers such as cost, payor reimbursement, and access prevented widespread use. The advent of the COVID-19 pandemic necessitated rapid adoption of telerehabilitation into clinical practice to provide access to care while maintaining social distancing. The purpose of this study is to present clinical data on the feasibility and acceptability of speech-language pathology, developmental occupational and physical therapies, and sports and orthopedic therapies telerehabilitation delivered in a pediatric hospital setting. METHODS: Telerehabilitation services were rapidly implemented in three stages: building the foundation, implementing, and refining this service delivery model. Paper patient satisfaction surveys were administered as part of ongoing quality improvement efforts throughout 2019 and were adapted for online administration in 2020 for telerehabilitation patients. Outpatient visit counts by type (in-person, phone, and video) were extracted from the electronic medical record using data warehousing techniques. RESULTS: Historical patient satisfaction rates from 2019 indicated high patient satisfaction (98.97% positive responses); these results were maintained for telerehabilitation visits (97.73%), indicating that families found telerehabilitation services acceptable. Patient volume returned to 73.5% of pre-pandemic volume after the implementation of telerehabilitation services. CONCLUSIONS: Pediatric telerehabilitation is feasible to provide in clinical settings, and the services are acceptable to patient families. Future work is needed to evaluate the impact of telerehabilitation services on patient care and applications for ongoing use of this delivery model.

Unusual Cysteine Content in V1 Region of gp120 From an Elite Suppressor That Produces Broadly Neutralizing Antibodies.

Although it is now possible to produce recombinant HIV envelope glycoproteins (Envs) with epitopes recognized by the 5-6 major classes of broadly neutralizing antibodies (bNAbs), these have failed to consistently stimulate the formation of bNAbs in immunized animals or humans. In an effort to identify new immunogens better able to elicit bNAbs, we are studying Envs derived from rare individuals who possess bNAbs and are able to control their infection without the need for anti-retroviral drugs (elite supressors or ES), hypothesizing that in at least some people the antibodies may mediate durable virus control. Because virus evolution in people with the ES only phenotype was reported to be limited, we reasoned the Env proteins recovered from these individuals may more closely resemble the Envs that gave rise to bNAbs compared to the highly diverse viruses isolated from normal progressors. Using a phenotypic assay, we screened 25 controllers and identified two for more detailed investigation. In this study, we examined 20 clade B proviral sequences isolated from an African American woman, who had the rare bNAb/ES phenotype. Phylogenetic analysis of proviral envelope sequences demonstrated low genetic diversity. Envelope proteins were unusual in that most possessed two extra cysteines within an elongated V1 region. In this report, we examine the impact of the extra cysteines on the binding to bNAbs, virus infectivity, and sensitivity to neutralization. These data suggest structural motifs in V1 can affect infectivity, and that rare viruses may be prevented from developing escape.

Pediatric Nasal Reconstruction by Washio Procedure.

The use of the Washio retroauricular flap for nasal reconstruction has been infrequently covered in recent literature, particularly concerning the pediatric population. A retrospective study was conducted between 2014 and 2018 and included all pediatric patients who underwent a Washio retroauricular flap procedure for nasal reconstruction operated on by the same surgeon at a referral center for pediatric plastic and maxillofacial surgery. The mean age at the time of the first stage of the Washio procedure was just under 8 years of age (range: 6 years 3 months-8 years 10 months). The Washio retroauricular flap procedure was successfully employed in three patients with three different anatomical defects, including the nasal alae, nasal tip, and columella, without postoperative healing complications. Arguably, the Washio method is sufficiently versatile to be used in various defect types, allows space and planning for subsequent surgical corrections, avoids additional visible scarring of the face, and spares flaps that may be required at the end of the growth, such as the pedicled forehead flap. It is a safe procedure, provided that at least a two-stage procedure is performed, and a progressive postoperative verticalization is prescribed to limit venous drainage complications.

Identification and CRISPR/Cas9 Inactivation of the C1s Protease Responsible for Proteolysis of Recombinant Proteins Produced in CHO Cells.

Proteolysis associated with recombinant protein expression in Chinese Hamster Ovary (CHO) cells has hindered the development of biologics including HIV vaccines. When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1's (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s-/- MGAT1- CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs). The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation. Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases.