Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DR hi CD11c hi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR lo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

No abstract

Highlights d A standardized, ultra-high-throughput clinical platform for serum and plasma proteomics d Platform enables high precision quantification of 180 human proteomes per day at low cost d 27 biomarkers are differentially expressed between WHO severity grades for COVID-19 d Biomarkers include proteins not previously associated with COVID-19 infection

Two elementary parameters for quantifying viral infection and shedding are viral load and whether samples yield a replicating virus isolate in cell culture. We examined 25,381 German SARS-CoV-2 cases, including 6110 from test centres attended by pre-symptomatic, asymptomatic, and mildly-symptomatic (PAMS) subjects, 9519 who were hospitalised, and 1533 B.1.1.7 lineage infections. The youngest had mean log10 viral load 0.5 (or less) lower than older subjects and an estimated ~78% of the peak cell culture replication probability, due in part to smaller swab sizes and unlikely to be clinically relevant. Viral loads above 109 copies per swab were found in 8% of subjects, one-third of whom were PAMS, with mean age 37.6. We estimate 4.3 days from onset of shedding to peak viral load (8.1) and cell culture isolation probability (0.75). B.1.1.7 subjects had mean log10 viral load 1.05 higher than non-B.1.1.7, with estimated cell culture replication probability 2.6 times higher.

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC 50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.

42 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a rapidly 43 unfolding pandemic, overwhelming health care systems worldwide 1 . Clinical manifestations of 44 Coronavirus-disease 2019 (COVID-19) vary broadly, ranging from asymptomatic infection to 45 acute respiratory failure and death 2 , yet the underlying mechanisms for this high variability are 46 still unknown. Similarly, the role of host immune responses in viral clearance of COVID-19 47 remains unresolved. For SARS-CoV (2002/03), however, it has been reported that CD4 + T cell 48responses correlated with positive outcomes 3,4 , whereas T cell immune responses to SARS-49CoV-2 have not yet been characterized. Here, we describe an assay that allows direct detection 50and characterization of SARS-CoV-2 spike glycoprotein (S)-reactive CD4 + T cells in peripheral 51blood. We demonstrate the presence of S-reactive CD4 + T cells in 83% of COVID-19 patients, 52as well as in 34% of SARS-CoV-2 seronegative healthy donors (HD), albeit at lower 53 frequencies. Strikingly, S-reactive CD4 + T cells in COVID-19 patients equally targeted N-54terminal and C-terminal epitopes of S whereas in HD S-reactive CD4 + T cells reacted almost 55exclusively to the C-terminal epitopes that are a) characterized by higher homology with spike 56 glycoprotein of human endemic "common cold" coronaviruses (hCoVs), and b) contains the S2 57 subunit of S with the cytoplasmic peptide (CP), the fusion peptide (FP), and the transmembrane 58 domain (TM) but not the receptor-binding domain (RBD). In contrast to S-reactive CD4 + T 59 cells in HD, S-reactive CD4 + T cells from COVID-19 patients co-expressed CD38 and HLA-60DR, indivative of their recent in vivo activation. Our study is the first to directly measure SARS-61CoV-2-reactive T cell responses providing critical tools for large scale testing and 62 characterization of potential cross-reactive cellular immunity to SARS-CoV-2. The presence of 63 pre-existing SARS-CoV-2-reactive T cells in a subset of SARS-CoV-2 naïve HD is of high 64interest but larger scale prospective cohort studies are needed to assess whether their presence 65 is a correlate of protection or pathology for COVID-19. Results of such studies will be key for 66 a mechanistic understanding of the SARS-CoV-2 pandemic, adaptation of containment 67 methods and to support vaccine development.

Background Heterologous vaccine regimens have been widely discussed as a way to mitigate intermittent supply shortages and to improve immunogenicity and safety of COVID-19 vaccines. We aimed to assess the reactogenicity and immunogenicity of heterologous immunisations with ChAdOx1 nCov-19 (AstraZeneca, Cambridge, UK) and BNT162b2 (Pfizer-BioNtech, Mainz, Germany) compared with homologous BNT162b2 and ChAdOx1 nCov-19 immunisation. Methods This is an interim analysis of a prospective observational cohort study enrolling health-care workers in Berlin (Germany) who received either homologous ChAdOx1 nCov-19 or heterologous ChAdOx1 nCov-19-BNT162b2 vaccination with a 10-12-week vaccine interval or homologous BNT162b2 vaccination with a 3-week vaccine interval. We assessed reactogenicity after the first and second vaccination by use of electronic questionnaires on days 1, 3, 5, and 7. Immunogenicity was measured by the presence of SARS-CoV-2-specific antibodies (full spike-IgG, S1-IgG, and RBD-IgG), by an RBD-ACE2 binding inhibition assay (surrogate SARS-CoV-2 virus neutralisation test), a pseudovirus neutralisation assay against two variants of concerns (alpha [B.1.1.7] and beta [B.1.351]), and anti-S1-IgG avidity. T-cell reactivity was measured by IFN-γ release assay.