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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Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, there is little information about vaccine efficacy against variants of concern (VOC) in individuals above eighty years of age1. Here we analysed immune responses following vaccination with the BNT162b2 mRNA vaccine2 in elderly participants and younger healthcare workers. Serum neutralization and levels of binding IgG or IgA after the first vaccine dose were lower in older individuals, with a marked drop in participants over eighty years old. Sera from participants above eighty showed lower neutralization potency against the B.1.1.7 (Alpha), B.1.351 (Beta) and P.1. (Gamma) VOC than against the wild-type virus and were more likely to lack any neutralization against VOC following the first dose. However, following the second dose, neutralization against VOC was detectable regardless of age. The frequency of SARS-CoV-2 spike-specific memory B cells was higher in elderly responders (whose serum showed neutralization activity) than in non-responders after the first dose. Elderly participants showed a clear reduction in somatic hypermutation of class-switched cells. The production of interferon-γ and interleukin-2 by SARS-CoV-2 spike-specific T cells was lower in older participants, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high-risk population and that specific measures to boost vaccine responses in this population are warranted, particularly where variants of concern are circulating.

NKG2D is an activation receptor that allows natural killer (NK) cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the receptor and reduced function upon subsequent receptor engagement. However, it is not clear whether in addition to modulation the NKG2D receptor complex and/or its signaling capacity is preserved. We show here that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of cell-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can be activated via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive interferon ␥ (IFN␥) production, suggesting sustained signal IntroductionNumerous activation receptors can trigger natural killer (NK) cell-mediated cytotoxic and/or cytokine responses. The NKG2D receptor is specific for endogenous ligands (ie, histocompatibility 60 (H60), retinoic acid early inducible gene-1 (RAE-1), and mouse UL16-binding proteinlike transcript 1 (MULT1) in mice), which are usually not expressed on normal resting cells but which are readily induced upon different forms of cellular distress (for a review see Raulet 1 ). Thus, the de novo expression of NKG2D ligands renders cells susceptible to NK cell-mediated lysis. On many tumor cell lines of distinct tissue origins, NKG2D ligands are constitutively expressed. The ectopic expression of NKG2D ligands induces the rejection of several transplantable tumor cell lines. 2,3 Despite NKG2D's prominent role in NK cell activation and T-cell costimulation, it is noteworthy that many established tumor cell lines constitutively express NKG2D ligands, suggesting that tumors can escape NKG2D recognition. Indeed, T and NK cells, which were infiltrating major histocompatibility class I chain-related protein A (MICA)-positive tumors had reduced NKG2D cell surface levels. 4,5 In vitro, soluble MICA or tumor cells expressing MICA induced the down-modulation and degradation of NKG2D expressed by CD8 ϩ T cells, which led to reduced effector functions. 4 In addition, NK cells derived from nonobese diabetic (NOD) mice (a strain of mice prone to autoimmune diabetes) displayed impaired NKG2D function. 6 NKG2D dysfunction and modulation were explained by an interaction of NKG2D with its ligands, which are aberrantly induced on NOD NK cells upon culture. 6 In general, reduced NKG2D function is thought to be the consequence of and directly related to the lower NKG2D cell surface levels. However, it has not been determined whether sustained NKG2D engagement leaves the NKG2D receptor complex and/or its signal transduction capacity intact. To this end we established a culture system in which NK cells are exposed to tumor cells expressing NKG2D ligand. We show that chronic engagement with tumor cell-bound NKG2D ligand can uncouple NKG2D recognition from the ex...