Effectiveness of monoclonal antibody therapy for COVID-19 patients using a risk scoring system

IntroductionMonoclonal antibody therapy has been reported to be highly effective for preventing hospitalisation and severe cases in patients with Coronavirus Disease 2019 (COVID-19). However, since the drug is not readily available, it is important to rapidly and appropriately identify high-risk patients who can benefit most from therapy. Therefore, we designed a risk scoring system to identify at-risk COVID-19 patients in our region during the largest surge of COVID-19, from July to September 2021.MethodsAccording to the risk scores, confirmed COVID-19 patients were introduced to receive REGN-CoV-2 to our hospital by regional health centre from 18th August (Term 3). The primary outcome was the comparison of the number of hospitalisation and severe condition with other periods, the 4th wave (Term 1) and the early part of the 5th wave (Term 2) in Japan.ResultsDuring Term 3, 115 patients were stratified with the scoring system and administered REGN-COV-2. The number of hospitalisation vs severe cases were 60 (5.2%) vs 14 (1.2%), 8 (1.5%) vs 3 (0.6%) and 21 (1.2%) vs 2 (0.1%), in term 1, 2 and 3, respectively. Among those aged <60 years, compared with term 1, the relative risk of hospitalisation and severe condition were 0.25 (95% CI: 0.12–0.53) and 0.10 (95% CI: 0.01–0.80), respectively, in term 3. Drug adverse events were fever (3: 2.6%), headache (1: 0.9%) and neck rash (1: 0.9%), all events were resolved within 24 h wth no serious adverse event.ConclusionsThe administration of monoclonal antibody therapy using a risk scoring system significantly reduced the number of hospitalisation and disease severity of COVID-19 without any serious adverse events and avoided regional medical collapse.     

Hypocholesterolaemic mechanism of bitter melon aqueous extracts via inhibition of pancreatic cholesterol esterase and reduction of cholesterol micellar solubility

This study investigated the hypocholesterolaemic effects of bitter melon aqueous extracts (BMAE) in vitro, the inhibitory effects of BMAE on pancreatic cholesterol esterase (CEase) and incorporation of cholesterol into micelles were investigated. BMAE decreased the in vitro micellar solubility of cholesterol in a dose-dependent manner. The conformation of CEase was investigated by means of circular dichroism (CD) and fluorescence. The result revealed the decrease of α-helix contents, increase of β-sheet and exposure of aromatic amino acid residuals. The incorporation of cholesterol into micelles was inhibited by BMAE. A complex was observed by transmission electron microscopy (TEM), which indicated interaction between cholesterol and BMAE. The result revealed that BMAE can play a role in decreased intestinal cholesterol absorption via inhibition of CEase, and of micelle formation.     

Collecting and evaluating convalescent plasma for COVID-19 treatment: why and how?

Plasma provided by COVID-19 convalescent patients may provide therapeutic relief as the number of COVID-19 cases escalates steeply worldwide. Prior findings in various viral respiratory diseases including SARS-CoV-related pneumonia suggest that convalescent plasma can reduce mortality, although formal proof of efficacy is still lacking. By reducing viral spread early on, such an approach may possibly downplay subsequent immunopathology. Identifying, collecting, qualifying and preparing plasma from convalescent patients with adequate SARS-CoV-2-neutralizing Ab titres in an acute crisis setting may be challenging, although well within the remit of most blood establishments. Careful clinical evaluation should allow to quickly establish whether such passive immunotherapy, administered at early phases of the disease in patients at high risk of deleterious evolution, may reduce the frequency of patient deterioration, and thereby COVID-19 mortality.     

The respiratory syncytial virus fusion protein-specific B cell receptor repertoire reshaped by post-fusion subunit vaccination

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory illness in children of less than 5 years of age which usually results in hospitalization or even in death. Vaccine development is hampered in consequence of a failed vaccine trial with fatalities in the 1960s. Even though research has been more focused on the RSV fusion protein in its pre-fusion conformation, maternal vaccination with post-fusion protein (post F) was considered as a promising vaccine strategy for passive immunization of babies, because post F preserves very potent neutralizing epitopes. We extensively analyzed post F-binding B cell receptor (BCR) repertoires of three vaccinees who received a post F-subunit vaccine in the context of a first-in-human, Phase 1, randomized, observer-blind, placebo-controlled clinical trial (ClinicalTrials.gov Identifier: NCT02298179). In order to compare the vaccine-induced BCR repertoires with BCR repertoires induced by natural infection, we also analyzed pre F- and post F-binding BCRs isolated from a healthy blood donor with relatively high F-binding memory B cell (MBC) frequencies. Analysis of the vaccine-induced repertoires revealed that preferentially V_H4-encoded BCRs were expanded in response to vaccination. Estimation of antigen-driven selection further demonstrated that expanded BCRs accumulated positively selected replacement mutations which substantiated the hypothesis that post F-vaccination induces diversification of V_H4-encoded BCRs in germinal centers. Comparison of the vaccine-induced BCR repertoires with clonally related pre and post F-binding BCRs of the healthy blood donor suggested that the vaccine expanded pre/post F cross-reactive MBCs. Interestingly, several vaccine-induced BCRs shared stereotypic VDJ gene junctions with known neutralizing Abs. Once expressed for functional characterization, the selected monoclonal Abs demonstrated the predicted neutralization activities in plaque reduction neutralization assays indicating that the post F-vaccine induced expansion of neutralizing BCRs.     

Recent advances in preclinical in vitro approaches towards quantitative prediction of hepatic clearance and drug-drug interactions involving organic anion transporting polypeptide (OATP) 1B transporters

Hepatic uptake mediated by organic anion transporting polypeptide (OATP) 1B1 and 1B3 can serve as a major elimination pathway for various anionic drugs and as a site of drug-drug interactions (DDIs). This article provides an overview of the in vitro approaches used to predict human hepatic clearance (CL_h) and the risk of DDIs involving OATP1Bs. On the basis of the so-called extended clearance concept, in vitro–in vivo extrapolation methods using human hepatocytes as in vitro systems have been used to predict the CL_h involving OATP1B-mediated hepatic uptake. CL_h can be quantitatively predicted using human donor lots possessing adequate OATP1B activities. The contribution of OATP1Bs to hepatic uptake can be estimated by the relative activity factor, the relative expression factor, or selective inhibitor approaches, which offer generally consistent outcomes. In OATP1B1 inhibition assays, substantial substrate dependency was observed. The time-dependent inhibition of OATP1B1 was also noted and may be a mechanism underlying the in vitro–in vivo differences in the inhibition constant of cyclosporine A. Although it is still challenging to quantitatively predict CL_h and DDIs involving OATP1Bs from only preclinical data, understanding the utility and limitation of the current in vitro methods will pave the way for better prediction.     

Vaccination inhibits the human adenoviral transduction in a mouse keratoconjunctivitis model

Adenovirus infections are a major cause of epidemic keratoconjunctivitis (EKC), which can lead to corneal subepithelial infiltrates and multifocal corneal opacity. In the current study, we investigated the use of an E1/E3-deleted adenovirus serotype 5 (Ad5) vector as a vaccine administered intramuscularly (IM) or intranasally (IN) against subsequent challenges with a luciferase-expressing Ad5 (Ad5-Luci) vector via eyedrop. We evaluated the adaptive immune response to Ad5 vector vaccination and confirmed a robust polyfunctional CD8 T cell response in splenic cells. Neutralizing Ad5 antibodies were also measured in the sera of vaccinated mice as well as Ad5 antibody in the eye wash solutions. Upon challenge with Ad5-Luci vector 8 weeks post the primary immunization, transduction was significantly reduced by > 70% in the vaccinated mice, which was slightly better in IM- vs. that in IN-vaccinated animals. Resistance to subsequent challenge was observed 10 months post primary IM vaccination, with sustained reduction up to 60% in the Ad5-Luci vector transduction. Passive immunization of naive mice with antisera from IM to vaccinated mice subsequently challenged with the Ad5-Luci vector resulted in approximately 40% loss in transduction efficiency. Furthermore, the mice that received IM immunization with or without CD8 T cell depletion showed > 40% and 70% reductions, respectively, in Ad8 genomic copies after Ad8 topical challenge. We conclude that Ad-vector vaccination successfully induced an adaptive immune response that prevented subsequent Ad transduction in the cornea and conjunctiva-associated tissues in a mouse model of adenovirus keratoconjunctivitis, and that both cellular and humoral immunity play an important role in preventing Ad transduction.     

Rabies virus glycoprotein serology ELISA for measurement of neutralizing antibodies in sera of vaccinated human subjects

BackgroundVaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects.MethodsUsing a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy.ResultsRABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02–0.06 IU/mL and the linear range was 0.2–10.0 IU/ mL. The inter-assay CVs were in the range of 6.60–10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively.ConclusionsThe validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.     

单抗N糖分析系统适用性对照品的建立

目的建立单抗N糖分析方法的系统适用性对照品,并设定相应的系统适用性要求。方法利用液质联用(LC-MS)仪对N糖系统适用性对照品进行N糖型的表征鉴别,并对对照品进行稳定性评价。结合方法特点和验证数据,对系统适用性要求进行设定。结果建立的系统适用性对照品具有良好的稳定性,其糖型涵盖了单抗主要的N糖型种类。针对3种药典拟收录的单抗N糖分析方法,设定了以下系统适用性要求,包括:图谱与典型图谱相似、G1F(1,6)和G1F(1,3)的分离度应满足具体要求、G0F%应在规定的范围内、G0F保留时间的RSD应≤4%。结论建立了单抗N糖系统适用性对照品,可配合3种2020年版《中国药典》拟收录的N糖分析方法使用。     

Influence of posture on blink reflex prepulse inhibition induced by somatosensory inputs from upper and lower limbs

BackgroundPrepulse inhibition (PPI) is a neurophysiological phenomenon whereby a weak stimulus modulates the reflex response to a subsequent strong stimulus. Its physiological purpose is to avoid interruption of sensory processing by subsequent disturbing stimuli at the subcortical level, thereby preventing undesired motor reactions. An important hub in the PPI circuit is the pedunculopontine nucleus, which is also involved in the control of posture and sleep/wakefulness.ObjectiveTo study the effect of posture (supine versus standing) on PPI, induced by somatosensory prepulses to either upper or lower limb. PPI was measured as the percentage inhibition of the blink reflex response to electrical supraorbital nerve (SON) stimulation.MethodsSixteen healthy volunteers underwent bilateral blink reflex recordings following SON stimulation either alone (baseline) or preceded by an electrical prepulse to the median nerve (MN) or sural nerve (SN), both in supine and standing. Stimulus intensity was 8 times sensory threshold for SON, and 2 times sensory threshold for MN and SN, respectively. Eight stimuli were applied in each condition.ResultsBaseline blink reflex parameters did not differ significantly between the two postures. Prepulse stimulation to MN and SN caused significant inhibition of R2. In supine but not in standing, R2 was significantly more inhibited by MN than by SN prepulses. In standing, SN stimulation caused significantly more inhibition of R2 than in supine, while the inhibition caused by MN prepulses did not differ significantly between postures.SignificancePPI induced by lower limb afferent input may contribute to postural control while standing.