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

Platelet-Rich Plasma Promotes the Proliferation of Human Muscle Derived Progenitor Cells and Maintains Their Stemness

Human muscle-derived progenitor cells (hMDPCs) offer great promise for muscle cell-based regenerative medicine; however, prolonged ex-vivo expansion using animal sera is necessary to acquire sufficient cells for transplantation. Due to the risks associated with the use of animal sera, the development of a strategy for the ex vivo expansion of hMDPCs is required. The purpose of this study was to investigate the efficacy of using platelet-rich plasma (PRP) for the ex-vivo expansion of hMDPCs. Pre-plated MDPCs, myoendothelial cells, and pericytes are three populations of hMDPCs that we isolated by the modified pre-plate technique and Fluorescence Activated Cell Sorting (FACS), respectively. Pooled allogeneic human PRP was obtained from a local blood bank, and the effect that thrombin-activated PRP-releasate supplemented media had on the ex-vivo expansion of the hMDPCs was tested against FBS supplemented media, both in vitro and in vivo. PRP significantly enhanced short and long-term cell proliferation, with or without FBS supplementation. Antibody-neutralization of PDGF significantly blocked the mitogenic/proliferative effects that PRP had on the hMDPCs. A more stable and sustained expression of markers associated with stemness, and a decreased expression of lineage specific markers was observed in the PRP-expanded cells when compared with the FBS-expanded cells. The in vitro osteogenic, chondrogenic, and myogenic differentiation capacities of the hMDPCs were not altered when expanded in media supplemented with PRP. All populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers in vivo. Our data demonstrated that PRP promoted the proliferation and maintained the multi-differentiation capacities of the hMDPCs during ex-vivo expansion by maintaining the cells in an undifferentiated state. Moreover, PDGF appears to be a key contributing factor to the beneficial effect that PRP has on the proliferation of hMDPCs. © 2013 Li et al

RT-PCR analysis.

<p>hMDPCs were expanded with FBS or PRP for 3 weeks. The controls were hMDPCs cultured at day0. Data shown are means ± SD.</p><p>n = 3,</p>*<p><i>P</i><0.05 compare to control,</p>#<p><i>P</i><0.05 compare to FBS group.</p><p>N/A means the expression level was not detectable.</p

Differentiation abilities of hMDPCs were maintained after expansion with PRP.

<p>hMDPCs expanded either with PRP or FBS were tested for their multi-lineage differentiation abilities. <b>A.</b> For osteogenesis, data showed the mineralized bone volume of the pellets at different time points; for chondrogenesis, data showed GAG content of pellets; for myogenesis, data showed the percentage of f-MHC expressing nuclei per total nuclei. <b>B.</b> representative data of pericytes. For osteogenesis, pictures of 3D mineralization within the pellets; for chondrogenesis, alcian blue staining of the pellets; for myogenesis, immuno-staining of f-MHC (red) and nuclei (blue). Cells expanded in both culture conditions consistently differentiated into osteogenic, chondrogenic, and myogenic lineages. There were no significant differences in regard to the multi-lineage differentiation ability between the PRP-expanded and FBS-expanded hMDPCs (n = 3).</p

Flow cytometry analysis.

<p>hMDPCs were expanded with FBS or PRP for 3 weeks. The controls were hMDPCs cultured at day0; FBS group was hMDPCs cultured in FBS supplemented media; PRP group was hMDPCs expanded in PRP supplemented media. The percentage of positively stained cells was quantified by flow cytometry. Data shown are mean ± SD. n = 3,</p>*<p><i>P</i><0.05 compare to control,</p>#<p><i>P</i><0.05 compare to FBS group.</p

Influence of PRP on the long-term proliferation of hMDPCs.

<p>hMDPCs were cultured in 20% PRP (PRP group). The mean cumulative population doubling rate (CPD) was compared among the cells cultured in 20% FBS (FBS group) until passage 8. n = 3, ^*<i>P</i><0.05 compared to FBS group. Data showed that CPD was significantly higher when hMDPCs were cultured in PRP rather than FBS.</p

List of primer sequences for the tested genes.

<p>List of primer sequences for the tested genes.</p

The role of growth factors in PRP on the proliferation of hMDPCs.

<p>Neutralizing antibodies against PDGF, TGF-β1, and VEGF were added to the media to determine the role that these growth factors played in the beneficial effect that PRP had on hMDPCs proliferation. n = 4, ^*<i>P</i><0.05. Data showed that PDGF within the PRP played a role in stimulating the proliferation of the hMDPCs.</p

<i>In vivo</i> myogenic potential of PRP-expanded hMDPCs.

<p>PRP- and FBS-expanded hMDPCs were injected into mdx-SCID gastrocnemius muscles damaged with cardiotoxin. Cryo-sections were prepared 4 weeks after cell injection. <b>A.</b> HE staining and immuno-fluorescence staining of human major histocompatibility complex (MHC) class-I (red), human mitochondria (green), and DAPI (blue) were performed on the slides that contained the injection sites. Human MHC-I-positive and human mitochondria-positive myofibers were detected in the injured area. <b>B.</b> Quantification of human MHC-I-positive fibers per 1×10⁵ injected cells. Data showed that all populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers, and no significant differences were found between FBS and PRP expanded cells (n = 4).</p

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