Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

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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.

We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.

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The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8 + T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response, but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

139During B cell development, the near random nature of the VDJ recombination process leads to the unavoidable production of self-reactive antibodies. In fact, studies in humans have demonstrated that many if not most newly generated B cells are autoreactive ( 1 ). Extensive studies of self-tolerant B cells in transgenic mouse models have revealed the complicated systems of B cell selection used to avoid autoimmunity. Current models suggest that B cells expressing a transgenic surface Ig that binds DNA or protein autoantigens fi rst attempt to alter the B cell receptor (BCR) by further variable gene rearrangement using " receptor editing " ( 2, 3 ). If receptor editing is unsuccessful, then the offending B cell may be eliminated by clonal deletion ( 4, 5 ) or it may enter maturity but with reduced or altered function so that it no longer reacts to the self-antigens, which is referred to as clonal anergy ( 6 -8 ). In this paper, we describe a human B cell population that is anergic. Clonal anergy was fi rst conceived by Nossal and Pike in 1980 ( 6 ) to explain why injection of neonatal mice with high dosages of an antigen induced deletion of the specifi c B cells, whereas lesser dosages allowed retention of the specifi c B cells, but the cells were incapable of becoming antibody-secreting cells. In 1988, Goodnow et al. ( 8 )

The metabolic effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on human blood plasma were characterized using multiplatform metabolic phenotyping with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry (LC-MS). Quantitative measurements of lipoprotein subfractions, α-1-acid glycoprotein, glucose, and biogenic amines were made on samples from symptomatic coronavirus disease 19 (COVID-19) patients who had tested positive for the SARS-CoV-2 virus ( n = 17) and from age- and gender-matched controls ( n = 25). Data were analyzed using an orthogonal-projections to latent structures (OPLS) method and used to construct an exceptionally strong (AUROC = 1) hybrid NMR-MS model that enabled detailed metabolic discrimination between the groups and their biochemical relationships. Key discriminant metabolites included markers of inflammation including elevated α-1-acid glycoprotein and an increased kynurenine/tryptophan ratio. There was also an abnormal lipoprotein, glucose, and amino acid signature consistent with diabetes and coronary artery disease (low total and HDL Apolipoprotein A1, low HDL triglycerides, high LDL and VLDL triglycerides), plus multiple highly significant amino acid markers of liver dysfunction (including the elevated glutamine/glutamate and Fischer’s ratios) that present themselves as part of a distinct SARS-CoV-2 infection pattern. A multivariate training-test set model was validated using independent samples from additional SARS-CoV-2 positive patients and controls. The predictive model showed a sensitivity of 100% for SARS-CoV-2 positivity. The breadth of the disturbed pathways indicates a systemic signature of SARS-CoV-2 positivity that includes elements of liver dysfunction, dyslipidemia, diabetes, and coronary heart disease risk that are consistent with recent reports that COVID-19 is a systemic disease affecting multiple organs and systems. Metabolights study reference: MTBLS2014.