Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

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Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening disease occurring several weeks after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Deep immune profiling showed acute MIS-C patients had highly activated neutrophils, classical monocytes and memory CD8+ T-cells; increased frequencies of B-cell plasmablasts and double-negative B-cells. Post treatment samples from the same patients, taken during symptom resolution, identified recovery-associated immune features including increased monocyte CD163 levels, emergence of a new population of immature neutrophils and, in some patients, transiently increased plasma arginase. Plasma profiling identified multiple features shared by MIS-C, Kawasaki Disease and COVID-19 and that therapeutic inhibition of IL6 may be preferable to IL1 or TNF-α . We identified several potential mechanisms of action for IVIG, the most commonly used drug to treat MIS-C. Finally, we showed systemic complement activation with high plasma C5b-9 levels is common in MIS-C suggesting complement inhibitors could be used to treat the disease.

Reasons for performing study: Damage to the flexor tendons, particularly the superficial digital flexor tendon (SDFT), is one of the most common musculoskeletal injuries sustained by horses competing in all disciplines. Our previous work has shown that SDFTs from different individuals show a wide variation in mechanical strengths; this is important clinically as it may relate to predisposition to injury. The high mechanical strength of tendon relies on the correct orientation of collagen molecules within fibrils and stabilisation by the formation of chemical cross-links between collagen molecules. It is not known whether the variation in SDFT mechanical properties between individuals relates to differences in collagen cross-link levels. Hypothesis: Enzyme-derived, intermolecular cross-linking of tendon collagen correlates with mechanical properties of the SDFT. Methods: SDFTs were collected from 38 horses and mechanically tested to failure. Structural and material properties were calculated from the load/deformation plot and cross-sectional area for each tendon. Following mechanical testing, pyrrolic cross-link levels were measured using a spectrophotometric assay for Ehrlich's reactivity and pyridinoline levels were quantified by HPLC. Cross-link levels were correlated with mechanical properties and statistical significance tested using a Pearson's correlation test. Results: Pyrrole cross-link levels showed a significant positive correlation with ultimate stress (P = 0.004), yield stress (P = 0.003) and elastic modulus (P = 0.018) of the tendons, despite being a minor cross-link in these tendons. There was no significant correlation of mechanical properties with either hydroxylysyl-or lysyl-pyridinoline levels. Conclusions: Given the low absolute levels of pyrrole, we suggest that the correlation with high mechanical strength is through an indirect mechanism. Understanding the nature of the relationships between pyrrole cross-links, other matrix characteristics and tendon material properties may allow development of strategies to identify horses at risk from tendon injury and be of value in informing training practices.