ABSTRACT
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (Covid-19), which is most frequently mild yet can be severe and life-threatening. Virus-neutralizing monoclonal antibodies are predicted to reduce viral load, ameliorate symptoms, and prevent hospitalization. METHODS: In this ongoing phase 2 trial involving outpatients with recently diagnosed mild or moderate Covid-19, we randomly assigned 452 patients to receive a single intravenous infusion of neutralizing antibody LY-CoV555 in one of three doses (700 mg, 2800 mg, or 7000 mg) or placebo and evaluated the quantitative virologic end points and clinical outcomes. The primary outcome was the change from baseline in the viral load at day 11. The results of a preplanned interim analysis as of September 5, 2020, are reported here. RESULTS: At the time of the interim analysis, the observed mean decrease from baseline in the log viral load for the entire population was -3.81, for an elimination of more than 99.97% of viral RNA. For patients who received the 2800-mg dose of LY-CoV555, the difference from placebo in the decrease from baseline was -0.53 (95% confidence interval [CI], -0.98 to -0.08; P = 0.02), for a viral load that was lower by a factor of 3.4. Smaller differences from placebo in the change from baseline were observed among the patients who received the 700-mg dose (-0.20; 95% CI, -0.66 to 0.25; P = 0.38) or the 7000-mg dose (0.09; 95% CI, -0.37 to 0.55; P = 0.70). On days 2 to 6, the patients who received LY-CoV555 had a slightly lower severity of symptoms than those who received placebo. The percentage of patients who had a Covid-19-related hospitalization or visit to an emergency department was 1.6% in the LY-CoV555 group and 6.3% in the placebo group. CONCLUSIONS: In this interim analysis of a phase 2 trial, one of three doses of neutralizing antibody LY-CoV555 appeared to accelerate the natural decline in viral load over time, whereas the other doses had not by day 11. (Funded by Eli Lilly; BLAZE-1 ClinicalTrials.gov number, NCT04427501.).
Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Neutralizing/administration & dosage , COVID-19 Drug Treatment , Immunologic Factors/administration & dosage , SARS-CoV-2/isolation & purification , Viral Load/drug effects , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Neutralizing/adverse effects , COVID-19/virology , Dose-Response Relationship, Drug , Double-Blind Method , Female , Hospitalization/statistics & numerical data , Humans , Immunologic Factors/adverse effects , Male , Middle Aged , Outpatients , RNA, Viral/blood , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Severity of Illness Index , Young AdultABSTRACT
BACKGROUND: Patients with underlying medical conditions are at increased risk for severe coronavirus disease 2019 (Covid-19). Whereas vaccine-derived immunity develops over time, neutralizing monoclonal-antibody treatment provides immediate, passive immunity and may limit disease progression and complications. METHODS: In this phase 3 trial, we randomly assigned, in a 1:1 ratio, a cohort of ambulatory patients with mild or moderate Covid-19 who were at high risk for progression to severe disease to receive a single intravenous infusion of either a neutralizing monoclonal-antibody combination agent (2800 mg of bamlanivimab and 2800 mg of etesevimab, administered together) or placebo within 3 days after a laboratory diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The primary outcome was the overall clinical status of the patients, defined as Covid-19-related hospitalization or death from any cause by day 29. RESULTS: A total of 1035 patients underwent randomization and received an infusion of bamlanivimab-etesevimab or placebo. The mean (±SD) age of the patients was 53.8±16.8 years, and 52.0% were adolescent girls or women. By day 29, a total of 11 of 518 patients (2.1%) in the bamlanivimab-etesevimab group had a Covid-19-related hospitalization or death from any cause, as compared with 36 of 517 patients (7.0%) in the placebo group (absolute risk difference, -4.8 percentage points; 95% confidence interval [CI], -7.4 to -2.3; relative risk difference, 70%; P<0.001). No deaths occurred in the bamlanivimab-etesevimab group; in the placebo group, 10 deaths occurred, 9 of which were designated by the trial investigators as Covid-19-related. At day 7, a greater reduction from baseline in the log viral load was observed among patients who received bamlanivimab plus etesevimab than among those who received placebo (difference from placebo in the change from baseline, -1.20; 95% CI, -1.46 to -0.94; P<0.001). CONCLUSIONS: Among high-risk ambulatory patients, bamlanivimab plus etesevimab led to a lower incidence of Covid-19-related hospitalization and death than did placebo and accelerated the decline in the SARS-CoV-2 viral load. (Funded by Eli Lilly; BLAZE-1 ClinicalTrials.gov number, NCT04427501.).
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , COVID-19 Drug Treatment , Adolescent , Adult , Aged , Antibodies, Monoclonal, Humanized/adverse effects , COVID-19/ethnology , COVID-19/virology , Child , Double-Blind Method , Drug Therapy, Combination , Female , Hospitalization/statistics & numerical data , Humans , Infusions, Intravenous , Kaplan-Meier Estimate , Male , Middle Aged , Patient Acuity , Risk Factors , SARS-CoV-2/isolation & purification , Viral Load/drug effects , Young AdultABSTRACT
BACKGROUND: Based on interim analyses and modeling data, lower doses of bamlanivimab and etesevimab together (700/1400 mg) were investigated to determine optimal dose and expand availability of treatment. METHODS: This Phase 3 portion of the BLAZE-1 trial characterized the effect of bamlanivimab with etesevimab on overall patient clinical status and virologic outcomes in ambulatory patients ≥12 years old, with mild-to-moderate coronavirus disease 2019 (COVID-19), and ≥1 risk factor for progressing to severe COVID-19 and/or hospitalization. Bamlanivimab and etesevimab together (700/1400 mg) or placebo were infused intravenously within 3 days of patients' first positive COVID-19 test. RESULTS: In total, 769 patients were infused (median age [range]; 56.0 years [12, 93], 30.3% of patients ≥65 years of age and median duration of symptoms; 4 days). By day 29, 4/511 patients (0.8%) in the antibody treatment group had a COVID-19-related hospitalization or any-cause death, as compared with 15/258 patients (5.8%) in the placebo group (Δ[95% confidence interval {CI}]â =â -5.0 [-8.0, -2.1], Pâ <â .001). No deaths occurred in the bamlanivimab and etesevimab group compared with 4 deaths (all COVID-19-related) in the placebo group. Patients receiving antibody treatment had a greater mean reduction in viral load from baseline to Day 7 (Δ[95% CI]â =â -0.99 [-1.33, -.66], Pâ <â .0001) compared with those receiving placebo. Persistently high viral load at Day 7 correlated with COVID-19-related hospitalization or any-cause death by Day 29 in all BLAZE-1 cohorts investigated. CONCLUSIONS: These data support the use of bamlanivimab and etesevimab (700/1400 mg) for ambulatory patients at high risk for severe COVID-19. Evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants will require continued monitoring to determine the applicability of this treatment. CLINICAL TRIALS REGISTRATION: NCT04427501.
Subject(s)
COVID-19 Drug Treatment , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing , Child , Humans , Middle Aged , Prognosis , SARS-CoV-2 , Viral LoadABSTRACT
Importance: Coronavirus disease 2019 (COVID-19) continues to spread rapidly worldwide. Neutralizing antibodies are a potential treatment for COVID-19. Objective: To determine the effect of bamlanivimab monotherapy and combination therapy with bamlanivimab and etesevimab on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in mild to moderate COVID-19. Design, Setting, and Participants: The BLAZE-1 study is a randomized phase 2/3 trial at 49 US centers including ambulatory patients (N = 613) who tested positive for SARS-CoV-2 infection and had 1 or more mild to moderate symptoms. Patients who received bamlanivimab monotherapy or placebo were enrolled first (June 17-August 21, 2020) followed by patients who received bamlanivimab and etesevimab or placebo (August 22-September 3). These are the final analyses and represent findings through October 6, 2020. Interventions: Patients were randomized to receive a single infusion of bamlanivimab (700 mg [n = 101], 2800 mg [n = 107], or 7000 mg [n = 101]), the combination treatment (2800 mg of bamlanivimab and 2800 mg of etesevimab [n = 112]), or placebo (n = 156). Main Outcomes and Measures: The primary end point was change in SARS-CoV-2 log viral load at day 11 (±4 days). Nine prespecified secondary outcome measures were evaluated with comparisons between each treatment group and placebo, and included 3 other measures of viral load, 5 on symptoms, and 1 measure of clinical outcome (the proportion of patients with a COVID-19-related hospitalization, an emergency department [ED] visit, or death at day 29). Results: Among the 577 patients who were randomized and received an infusion (mean age, 44.7 [SD, 15.7] years; 315 [54.6%] women), 533 (92.4%) completed the efficacy evaluation period (day 29). The change in log viral load from baseline at day 11 was -3.72 for 700 mg, -4.08 for 2800 mg, -3.49 for 7000 mg, -4.37 for combination treatment, and -3.80 for placebo. Compared with placebo, the differences in the change in log viral load at day 11 were 0.09 (95% CI, -0.35 to 0.52; P = .69) for 700 mg, -0.27 (95% CI, -0.71 to 0.16; P = .21) for 2800 mg, 0.31 (95% CI, -0.13 to 0.76; P = .16) for 7000 mg, and -0.57 (95% CI, -1.00 to -0.14; P = .01) for combination treatment. Among the secondary outcome measures, differences between each treatment group vs the placebo group were statistically significant for 10 of 84 end points. The proportion of patients with COVID-19-related hospitalizations or ED visits was 5.8% (9 events) for placebo, 1.0% (1 event) for 700 mg, 1.9% (2 events) for 2800 mg, 2.0% (2 events) for 7000 mg, and 0.9% (1 event) for combination treatment. Immediate hypersensitivity reactions were reported in 9 patients (6 bamlanivimab, 2 combination treatment, and 1 placebo). No deaths occurred during the study treatment. Conclusions and Relevance: Among nonhospitalized patients with mild to moderate COVID-19 illness, treatment with bamlanivimab and etesevimab, compared with placebo, was associated with a statistically significant reduction in SARS-CoV-2 viral load at day 11; no significant difference in viral load reduction was observed for bamlanivimab monotherapy. Further ongoing clinical trials will focus on assessing the clinical benefit of antispike neutralizing antibodies in patients with COVID-19 as a primary end point. Trial Registration: ClinicalTrials.gov Identifier: NCT04427501.
Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Neutralizing/administration & dosage , Antiviral Agents/administration & dosage , COVID-19 Drug Treatment , SARS-CoV-2/isolation & purification , Viral Load/drug effects , Adult , Aged , Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Neutralizing/adverse effects , Antiviral Agents/adverse effects , COVID-19/mortality , COVID-19/virology , Dose-Response Relationship, Drug , Double-Blind Method , Drug Therapy, Combination , Female , Hospitalization/statistics & numerical data , Humans , Infusions, Intravenous , Male , Middle Aged , SARS-CoV-2/drug effects , Severity of Illness IndexABSTRACT
Importance: Preventive interventions are needed to protect residents and staff of skilled nursing and assisted living facilities from COVID-19 during outbreaks in their facilities. Bamlanivimab, a neutralizing monoclonal antibody against SARS-CoV-2, may confer rapid protection from SARS-CoV-2 infection and COVID-19. Objective: To determine the effect of bamlanivimab on the incidence of COVID-19 among residents and staff of skilled nursing and assisted living facilities. Design, Setting, and Participants: Randomized, double-blind, single-dose, phase 3 trial that enrolled residents and staff of 74 skilled nursing and assisted living facilities in the United States with at least 1 confirmed SARS-CoV-2 index case. A total of 1175 participants enrolled in the study from August 2 to November 20, 2020. Database lock was triggered on January 13, 2021, when all participants reached study day 57. Interventions: Participants were randomized to receive a single intravenous infusion of bamlanivimab, 4200 mg (n = 588), or placebo (n = 587). Main Outcomes and Measures: The primary outcome was incidence of COVID-19, defined as the detection of SARS-CoV-2 by reverse transcriptase-polymerase chain reaction and mild or worse disease severity within 21 days of detection, within 8 weeks of randomization. Key secondary outcomes included incidence of moderate or worse COVID-19 severity and incidence of SARS-CoV-2 infection. Results: The prevention population comprised a total of 966 participants (666 staff and 300 residents) who were negative at baseline for SARS-CoV-2 infection and serology (mean age, 53.0 [range, 18-104] years; 722 [74.7%] women). Bamlanivimab significantly reduced the incidence of COVID-19 in the prevention population compared with placebo (8.5% vs 15.2%; odds ratio, 0.43 [95% CI, 0.28-0.68]; P < .001; absolute risk difference, -6.6 [95% CI, -10.7 to -2.6] percentage points). Five deaths attributed to COVID-19 were reported by day 57; all occurred in the placebo group. Among 1175 participants who received study product (safety population), the rate of participants with adverse events was 20.1% in the bamlanivimab group and 18.9% in the placebo group. The most common adverse events were urinary tract infection (reported by 12 participants [2%] who received bamlanivimab and 14 [2.4%] who received placebo) and hypertension (reported by 7 participants [1.2%] who received bamlanivimab and 10 [1.7%] who received placebo). Conclusions and Relevance: Among residents and staff in skilled nursing and assisted living facilities, treatment during August-November 2020 with bamlanivimab monotherapy reduced the incidence of COVID-19 infection. Further research is needed to assess preventive efficacy with current patterns of viral strains with combination monoclonal antibody therapy. Trial Registration: ClinicalTrials.gov Identifier: NCT04497987.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Neutralizing/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/prevention & control , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/adverse effects , Antibodies, Monoclonal, Humanized/immunology , Antiviral Agents/adverse effects , Antiviral Agents/immunology , Assisted Living Facilities , COVID-19/epidemiology , Double-Blind Method , Drug Approval , Female , Health Personnel , Humans , Immunization, Passive , Incidence , Infusions, Intravenous , Male , Middle Aged , SARS-CoV-2/isolation & purification , Severity of Illness Index , Skilled Nursing Facilities , Young AdultABSTRACT
Among the proteins that mediate calcium-stimulated transmitter release, the synaptic vesicle protein 2 (SV2) stands out as a unique modulator specific to the neurons and endocrine cells of vertebrates. In synapses, SV2 regulates the expression and trafficking of the calcium sensor protein synaptotagmin, an action consistent with the reduced calcium-mediated exocytosis observed in neurons lacking SV2. Yet SV2 contains amino acid motifs consistent with it performing other actions that could regulate presynaptic functioning and that might underlie the mechanism of drug action. To test the role of these functional motifs, we performed a mutagenic analysis of SV2A and assessed the ability of mutant SV2A proteins to restore normal synaptic transmission in neurons from SV2A/B knockout mice. We report that SV2A-R231Q, harboring a mutation in a canonical transporter motif, restored normal synaptic depression (a measure of release probability and signature deficit of neurons lacking SV2). In contrast, normal synaptic depression was not restored by SV2A-W300A and SV2A-W666A, harboring mutations of conserved tryptophans in the 5th and 10th transmembrane domains. Although they did not rescue normal neurotransmission, SV2A-W300A and SV2A-W666A did restore normal levels of synaptotagmin expression and internalization. This indicates that tryptophans 300 and 666 support an essential action of SV2 that is unrelated to its role in synaptotagmin expression or trafficking. These results indicate that SV2 performs at least two actions at the synapse that contribute to neurotransmitter release.
Subject(s)
Exocytosis/physiology , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Neurotransmitter Agents/metabolism , Protein Isoforms/metabolism , Synaptic Transmission/physiology , Animals , Cells, Cultured , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Mutation , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/metabolism , Patch-Clamp Techniques , Protein Isoforms/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synapses/metabolismABSTRACT
We report a thorough analysis of neurotransmission in cultured hippocampal neurons lacking synaptic vesicle protein 2 (SV2), a membrane glycoprotein present in all vesicles that undergo regulated secretion. We found that SV2 selectively enhances low-frequency neurotransmission by priming morphologically docked vesicles. Loss of SV2 reduced initial release probability during a train of action potentials but had no effect on steady-state responses. The amount and decay rate of asynchronous release, two measures sensitive to presynaptic calcium concentrations, are not altered in SV2 knock-outs, suggesting that SV2 does not act by modulating presynaptic calcium. Normal neurotransmission could be temporarily recovered by delivering an exhaustive stimulus train. Our results indicate that SV2 primes vesicles in quiescent neurons and that SV2 function can be bypassed by an activity-dependent priming mechanism. We propose that SV2 action modulates synaptic networks by ensuring that low-frequency neurotransmission is faithfully conveyed.
Subject(s)
Hippocampus/cytology , Membrane Glycoproteins/physiology , Nerve Tissue Proteins/physiology , Neurons/metabolism , Neurotransmitter Agents/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Action Potentials , Animals , Astrocytes/physiology , Calcium/pharmacology , Cells, Cultured , Evoked Potentials/physiology , Genotype , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Patch-Clamp Techniques , Synaptic Vesicles/ultrastructureABSTRACT
Generating microislands of culture substrate on coverslips by spray application of poly-d lysine is a commonly used method for culturing isolated neurons that form self (autaptic) synapses. This preparation has multiple advantages for studying synaptic transmission in isolation; however, generating microislands by spraying produces islands of non-uniform size and thus cultures vary widely in the number of islands containing single neurons. To address these problems, we developed a high-throughput method for reliably generating uniformly shaped microislands of culture substrate. Stamp molds formed of poly(dimethylsiloxane) (PDMS) were fabricated with arrays of circles and used to generate stamps made of 9.2% agarose. The agarose stamps were capable of loading sufficient poly D-lysine and collagen dissolved in acetic acid to rapidly generate coverslips containing at least 64 microislands per coverslip. When hippocampal neurons were cultured on these coverslips, there were significantly more single-neuron islands per coverslip. We noted that single neurons tended to form one of three distinct neurite-arbor morphologies, which varied with island size and the location of the cell body on the island. To our surprise, the number of synapses per autaptic neuron did not correlate with arbor shape or island size, suggesting that other factors regulate the number of synapses formed by isolated neurons. The stamping method we report can be used to increase the number of single-neuron islands per culture and aid in the rapid visualization of microislands.
Subject(s)
Cell Culture Techniques/methods , Neurons/cytology , Animals , Cell Communication/physiology , Cells, Cultured , Dendrites/physiology , Hippocampus/cytology , Hippocampus/physiology , Mice , Neurons/physiology , Synapses/physiology , Synaptic Transmission/physiologyABSTRACT
SV2 (Synaptic Vesicle Protein 2) is expressed in neurons and endocrine cells where it is required for normal calcium-evoked neurosecretion. In mammals, there are three SV2 genes, denoted SV2A, B and C. SV2A interacts with synaptotagmin, the prime candidate for the calcium sensor in exocytosis. Here, we report that all isoforms of native SV2 bind synaptotagmin and that binding is inhibited by calcium, indicating that all isoforms contain a common calcium-inhibited synaptotagmin-binding site. The isolated amino termini of SV2A and SV2C supported an additional interaction with synaptotagmin, and binding at this site was stimulated by calcium. The amino-terminal binding site was mapped to the first 57 amino acids of SV2A, and removal of this domain decreased calcium-mediated inhibition of binding to synaptotagmin, suggesting that it modulates calcium's effect on the SV2-synaptotagmin interaction. Introduction of the amino terminus of SV2A or SV2C into cultured superior cervical ganglion neurons inhibited neurotransmission, whereas the amino terminus of SV2B did not. These observations implicate the SV2-synaptotagmin interaction in regulated exocytosis and suggest that SV2A and SV2C, via their additional synaptotagmin binding site, function differently than SV2B.