Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

COVID-19 vaccine hesitancy: a survey in a population highly compliant to common vaccinations.

Vaccination is a key protective factor against COVID-19. Some vaccines have already received emergency authorization from Health Agencies, but growing skepticism and vaccine hesitancy will probably affect COVID-19 vaccination campaigns. In the attempt to shed light on this issue, we conducted an online survey in a population of parents referring to 4 pediatric practices in Naples, Italy in whom we evaluated potential vaccine acceptability in relation to socio-demographic characteristics, perception of personal health and of the impact of COVID-19, and attitudes toward general vaccination practices. Vaccination rates were analyzed also in the corresponding pediatric population.Almost 27% of participants declared they were in favor of vaccinations, and in fact real life vaccination rates in children exceeded the national mean. Only 26.5% of respondents declared they would receive COVID-19 vaccine. Vaccine refusal was attributed to safety concerns in 76% of parents. Specific vaccine attributes further reduced the acceptance rate. Female gender, younger age and lower education level were associated with non-adherence to vaccination. Among extrinsic factors of COVID-19 vaccination, only information from National Health Authorities was significantly associated to vaccine acceptance.The rate of potential COVID-19 vaccine acceptability was very poor in our population of parents. Vaccine hesitancy was mainly due to safety concerns. Demographic and educational factors were correlated to vaccine acceptability. Health education and communication strategies are needed to achieve large-scale vaccine acceptability and finally herd immunity.

Development of a new, simple, rapid ultra-high-performance liquid chromatography (UHPLC) method for the quantification of 2-phenoxyethanol in vaccines for human use.

A new, simple and rapid method for the quantitative determination of the antimicrobial preservative 2-phenoxyethanol, based on reverse phase ultra-high-performance liquid chromatography has been developed. The validation was performed according the ICH Q2 guideline "Validation of Analytical Procedures". The desired chromatographic separation was achieved on a Waters Symmetry C18 (150 × 4.6 mm, 5 µm) column using an isocratic elution, with detection at 270 nm wavelength. The mobile phase consisted of acetonitrile/water (55:45, v/v), pumped at a flow rate of 1 mL/min. The calibration curve and the analytical procedure are linear (r2 = 0.999) from the concentration of 0.07 mg/mL to 1.1 mg/mL. The percent relative standard deviation for intra- and inter-day precision was <1%. The recovery of 2-phenoxyethanol in vaccines ranged between 96.5 and 100.60%. The limits of detection and quantitation were 1.3 × 10-4 and 2.7 × 10-4 mg/mL, respectively. The method was found to be robust by changing the column working temperature, the percentage of acetonitrile of the mobile phase and the flow rate. The validated method can be successfully and reliably used to quantify as well as to exclude presence of 2-phenoxyethanol preservative in marketed vaccines.

Effects of a Video-Based Preoperative Educational Intervention Plus Nurse-Led Reinforcement Discussion on Knowledge, Self-Efficacy, and Resilience in Patients Undergoing Major Surgery.

This study aimed to verify the feasibility of a video-based preoperative educational intervention plus one-to-one, nurse-led reinforcement discussion in patients undergoing elective major surgery and to assess the impact of this combined intervention on patient- and nurse-perceived patient knowledge, self-efficacy, and resilience. Patients received written material at pre-admission and were offered the intervention at admission. Patients reported their knowledge and self-efficacy at pre-admission and after the intervention, and resilience at pre-admission and discharge. Nurses assessed patients' knowledge and self-efficacy after the intervention. In all, 88/97 (90.7%) patients completed the intervention. The 80 patients with complete data reported a significant increase in their knowledge (p < .001) and self-efficacy (p < .001), but no difference in resilience (p = .72). Nurse-perceived patient knowledge agreed with patients' perceptions (p = .57) but nurses scored patients' self-efficacy lower (p < .001). The combined intervention was feasible, and patients perceived an improvement in their knowledge and self-efficacy. Nurses' assessment partially agreed with patients' perceptions.

Teaching and Learning Computational Drug Design: Student Investigations of 3D Quantitative Structure-Activity Relationships through Web Applications.

The increasing use of information technology in the discovery of new molecular entities encourages the use of modern molecular-modeling tools to help teach important concepts of drug design to chemistry and pharmacy undergraduate students. In particular, statistical models such as quantitative structure-activity relationships (QSAR)-often as its 3D QSAR variant-are commonly used in the development and optimization of a leading compound. We describe how these drug discovery methods can be taught and learned by means of free and open-source web applications, specifically the online platform www.3d-qsar.com. This new suite of web applications has been integrated into a drug design teaching course, one that provides both theoretical and practical perspectives. We include the teaching protocol by which pharmaceutical biotechnology master students at Pharmacy Faculty of Sapienza Rome University are introduced to drug design. Starting with a choice among recent articles describing the potencies of a series of molecules tested against a biological target, each student is expected to build a 3D QSAR ligand-based model from their chosen publication, proceeding as follows: creating the initial data set (Py-MolEdit); generating the global minimum conformations (Py-ConfSearch); proposing a promising mutual alignment (Py-Align); and finally, building, and optimizing a robust 3D QSAR models (Py-CoMFA). These student activities also help validate these new molecular modeling tools, especially for their usability by inexperienced hands. To more fully demonstrate the effectiveness of this protocol and its tools, we include the work performed by four of these students (four of the coauthors), detailing the satisfactory 3D QSAR models they obtained. Such scientifically complete experiences by undergraduates, made possible by the efficiency of the 3D QSAR methodology, provide exposure to computational tools in the same spirit as traditional laboratory exercises. With the obsolescence of the classic Comparative Molecular Field Analysis Sybyl host, the 3dqsar web portal offers one of the few available means of performing this well-established 3D QSAR method.

Ratiometric sweat secretion optical test in cystic fibrosis, carriers and healthy subjects.

We have simplified the published procedure (5) for measuring sweat rates in individual human sweat glands. Sweat secretion rates were obtained from sweat drops secreted on the forearm by multiple individual glands. We computed a ratio between CFTR-dependent (by intradermal microinjection of a ß adrenergic cocktail) and CFTR-independent (by methacoline as cholinergic stimulus) sweat secretion rates. We obtained a reproducible, approximately linear readout of CFTR function with measurements performed by two different independent teams. We considered three groups (CF subjects, CF carriers and non-CF controls, n=22 in each group); their mean ratios was respectively 0.000, 0.104 and 0.205 The average ratio of CF subjects was consistent with diagnosis in 3 additional cases clinically resembling CF. All groups were clearly discriminated, with sensibility and specificity ranging from 82% to 100%. A software was developed for detecting sweat droplets. This bioassay is suitabile for multicentre studies focusing on CFTR targeted therapies, controversial diagnosis and functional relevance of rare CFTR mutations.

Dornase alfa as postoperative therapy in cystic fibrosis sinonasal disease.

OBJECTIVE: To determine the benefit of nasally inhaled dornase alfa in patients with cystic fibrosis and nasal symptoms. DESIGN: Double-blind placebo-controlled trial. SETTING: Cystic Fibrosis Regional Center of Campania at the University of Naples "Federico II." PATIENTS: A total of 24 patients with cystic fibrosis and chronic sinusitis. INTERVENTIONS: Patients underwent sinonasal surgery during a 3-year period and received once-daily doses of either dornase alfa (2.5 mg) or hypotonic saline solution (5 mL) beginning 1 month after surgery and for a 12-month period. MAIN OUTCOME MEASURES: Primary outcomes were nasal-related symptoms and nasal endoscopic appearance; secondary outcomes were forced expiratory volume in 1 second, nasal computed tomography findings, and saccharine clearance test results. Patients were evaluated before and after treatment. RESULTS: After surgery, all outcomes were significantly improved for each treatment at 1 month (P<.05); primary outcomes were improved at 24 and 48 weeks in the group receiving dornase alfa (P<.05), and at 12 weeks in the group receiving placebo. Secondary outcomes were better in the dornase alfa group (P<.01) than in the placebo group at 12 months except for the saccharine clearance test results. In particular, median relative difference in forced expiratory volume in 1 second between dornase alfa and placebo was significantly improved in the dornase alfa group (P<.01). CONCLUSIONS: Nasally inhaled dornase alfa can be effective in patients with cystic fibrosis and sinonasal disease who do not respond to conventional therapy after surgical treatment. Further studies should be carried out to determine the long-term effect on sinus disease, recurrence of polyps, and quality of life.