Analysis of SARS-CoV-2 Emergent Variants Following AZD7442 (Tixagevimab/Cilgavimab) for Early Outpatient Treatment of COVID-19 (TACKLE Trial)

Introduction: AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). // Methods: Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. // Results: Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. // Conclusion: These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment

Pollutant-Induced Changes in Ca2+ Channels Alter Dream-Mediated Gene Transcription

Two proteins involved in many aspects of Ca2+ signaling throughout the body include the ryanodine receptor (RyR) and the voltage gated L-type Ca2+ channel (CaV1). These Ca2+ channels are essential to physiological processes, including excitation-contraction coupling in striated muscle, neuronal network development, endocrine health, and neurodegenerative disorders. Several environmental pollutants, such as polychlorinated biphenyl (PCB) congeners and triclosan, are capable of causing Ca2+ signal disruption (CSD) by altering the activity of RyR or CaV1 channels, respectively. The aim of this study was to determine if CSD, caused by cellular exposure to PCBs and triclosan, can lead to changes in gene transcription as regulated by the Ca2+ sensitive transcriptional repressor, downstream regulatory element antagonistic modulator (DREAM). The goal of this work was to connect CSD to DREAM-mediated gene transcription. Specifically, I investigated whether levels of gonadotropin releasing hormone (GnRH), neuronal PAS domain protein 4 (NPAS4), and brain-derived neurotrophic factor (BDNF), genes known to be regulated by DREAM, were altered when the GT1-7 hypothalamic cell line was exposed to CSD chemicals. Cells were exposed to varying concentrations of CSD chemicals for 3, 12, and 24h. Following exposure, RNA was extracted and qPCR used to analyze changes in gene transcription. Cellular exposure to the non-coplanar PCB and RyR activator, PCB 95, did not cause significant changes in GnRH, NPAS4, or BDNF transcription, consistent with a low RyR mRNA expression in GT1-7 cells. Conversely, exposure to triclosan, a known CaV1 inhibitor, decreased GnRH and increased NPAS4 transcript levels in GT1-7 cells in a dose and time dependent manner. Interestingly, BDNF transcript levels varied across dose and time; however, these were not significant relative to mRNA levels in the vehicle control. The second aim of this study was to determine if these changes in GnRH transcription were mediated by DREAM. The GT1-7 cells were transfected with small interfering RNA (siRNA) targeting the DREAM mRNA. Transfected cells were then exposed to control treatments or triclosan for 3h and GnRH, NPAS4, and BDNF mRNA levels were compared using qPCR. DREAM knockdown in cells caused a significant decrease in GnRH as compared to the non-targeting vehicle control. In the presence of CSD chemicals, siRNA transfected cells did not experience additional changes in GnRH transcription as compared to the control knockdown cells. DREAM’s direct connection with intracellular Ca2+ levels and altered gene transcription could eventually be developed as a cellular screening assay for other CSD compounds that lead to DREAM, or related proteins, mediated gene transcription. DREAM is located throughout the body and is known to be important in the digestive system, central nervous system, and skeletal and cardiac muscle. Altering its regulation of gene transcription may contribute to dysfunction in these systems and has been tied to alterations in pain reception, learning and memory, thyroid-gland health, and pituitary gene transcription

Analysis of SARS-CoV-2 Emergent Variants Following AZD7442 (Tixagevimab/Cilgavimab) for Early Outpatient Treatment of COVID-19 (TACKLE Trial)

Abstract Introduction AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). Methods Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. Results Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. Conclusion These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment