Fate and plasticity of SARS-CoV-2-specific B cells during memory and recall response in humans (preprint)

B cell responses to different pathogens recruit tailored effector mechanisms, resulting in functionally specialized subsets. For human memory B cells (MBCs), these include CD21+ resting, CD21-CD27+ activated, and CD21-CD27- atypical cells. Whether these subsets follow deterministic or interconnected fates is unknown. We demonstrate in COVID-19 patients that single clones of SARS-CoV-2-specific MBCs followed multiple fates with distinctive phenotypic and functional characteristics. 6-12 months after infection, most circulating MBCs were CD21+ resting cells, which also accumulated in peripheral lymphoid organs where they acquired markers of tissue residency. Conversely, at acute infection and following SARS-CoV-2-specific immunization, CD21- MBCs became the predominant subsets, with atypical MBCs expressing high T-bet, inhibitory molecules, and distinct chemokine receptors. B cell receptor sequencing allowed tracking of individual MBC clones differentiating into CD21+, CD21-CD27+, and CD21-CD27- cell fates. Collectively, single MBC clones can adopt functionally different trajectories, thus contributing to immunity to infection.

Exposure to avian coronavirus vaccines is associated with increased levels of SARS-CoV-2-cross-reactive antibodies (preprint)

Background: Although avian coronavirus infectious bronchitis virus (IBV) and SARS-CoV-2 belong to different genera of the Coronaviridae family, exposure to IBV may result in the development of cross-reactive antibodies to SARS-CoV-2 due to homologous epitopes. We aimed to investigate whether antibody responses to IBV cross-react with SARS-CoV-2 in poultry farm personnel who are occupationally exposed to aerosolized IBV vaccines. Methods: : We analyzed sera from poultry farm personnel, COVID-19 patients, and pre-pandemic controls. IgG levels against the SARS-CoV-2 antigens S1, RBD, S2, and N and peptides corresponding to the SARS-CoV-2 ORF3a, N, and S proteins as well as whole virus antigens of the four major S1-genotypes 4/91, IS/1494/06, M41, and D274 of IBV were investigated by in-house ELISAs. Moreover, live-virus neutralization test (VNT) was performed. Results: : A subgroup of poultry farm personnel showed elevated levels of specific IgG for all tested SARS-CoV-2 antigens compared to pre-pandemic controls. Moreover, poultry farm personnel, COVID-19 patients, and pre-pandemic controls showed specific IgG antibodies against IBV strains. These antibody titers were higher in long-term vaccine implementers. We observed a strong correlation between IBV-specific IgG and SARS-CoV-2 S1-, RBD-, S2-, and N-specific IgG in poultry farm personnel compared to pre-pandemic controls and COVID-19 patients. However, no neutralization was observed for these cross-reactive antibodies from poultry farm personnel using the VNT. Conclusion: We report here for the first time the detection of cross-reactive IgG antibodies against SARS-CoV-2 antigens in humans exposed to IBV vaccines. These findings have implications for future vaccination strategies and possibly cross-reactive T cell immunity.

Autoantibodies in COVID-19 correlate with anti-viral humoral responses and distinct immune signatures (preprint)

Background: Several autoimmune features occur during coronavirus disease 2019 (COVID-19), with possible implications for disease course, immunity, and autoimmune pathology. Objective: We longitudinally screened for clinically relevant systemic autoantibodies to assess their prevalence, temporal trajectory, and association with immunity, comorbidities, and severity of COVID-19. Methods: We performed highly sensitive indirect immunofluorescence assays to detect anti-nuclear antibodies (ANA) and anti-neutrophil cytoplasmic antibodies (ANCA), along with serum proteomics and virome-wide serological profiling in a multicentric cohort of 175 COVID-19 patients followed-up to one year after infection, eleven vaccinated individuals, and 41 unexposed controls. Results: Compared to healthy controls, similar prevalence and patterns of ANA were present in patients during acute COVID-19 and recovery. However, paired analysis revealed a subgroup of patients with transient presence of certain ANA patterns during acute COVID-19. Furthermore, patients with severe COVID-19 exhibited a high prevalence of ANCA during acute disease. These autoantibodies were quantitatively associated with higher SARS-CoV-2-specific antibody titers in COVID-19 patients and in vaccinated individuals, thus linking autoantibody production to increased antigen-specific humoral responses. Notably, the qualitative breadth of antibodies cross-reactive with other coronaviruses was comparable in ANA-positive and ANA-negative individuals during acute COVID-19. In autoantibody-positive patients, multiparametric characterization demonstrated an inflammatory signature during acute COVID-19 and alterations of the B cell compartment after recovery. Conclusion: Highly sensitive indirect immunofluorescence assays revealed transient autoantibody production during acute SARS-CoV-2 infection, while the presence of autoantibodies in COVID-19 patients correlated with increased anti-viral humoral immune responses and inflammatory immune signatures.

CD8+ T cell signature in acute SARS-CoV-2 infection identifies memory precursors (preprint)

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1, 2. Since the outbreak of the ongoing coronavirus disease 19 (COVID-19) pandemic, a key question has focused on whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor (TCR) sequencing we longitudinally characterize individual SARS-CoV-2-specific CD8+ T cells of COVID-19 patients from acute infection to one year into recovery and find a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting one year after acute infection re-express CD45RA and interleukin-7 receptor (CD127), upregulate T cell factor-1 (TCF1), and maintain low CCR7, thus resembling CD45RA+ effector-memory T (TEMRA) cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones giving rise to long-lived cells, whereas prolonged proliferation and mammalian target of rapamycin (mTOR) signaling are associated with clone contraction and disappearance. Collectively, we identify a transcriptional signature differentiating short-from long-lived memory CD8+ T cells following an acute virus infection in humans.

Lymphopenia-induced T cell proliferation is a hallmark of severe COVID-19 (preprint)

Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a broad clinical presentation ranging from asymptomatic infection to fatal disease. Different features associated with the immune response to SARS-CoV-2, such as hyperinflammation and reduction of peripheral CD8+ T cell counts are strongly associated with severe disease. Here, we confirm the reduction in peripheral CD8+ T cells both in relative and absolute terms and identify T cell apoptosis and migration into inflamed tissues as possible mechanisms driving peripheral T cell lymphopenia. Furthermore, we find evidence of elevated serum interleukin-7, thus indicating systemic T cell paucity and signs of increased T cell proliferation in patients with severe lymphopenia. Following T cell lymphopenia in our pseudo-longitudinal time course, we observed expansion and recovery of poly-specific antiviral T cells, thus arguing for lymphopenia-induced T cell proliferation. In summary, this study suggests that extensive T cell loss and subsequent T cell proliferation are characteristic of severe COVID-19.

A distinct innate immune signature marks progression from mild to severe COVID-19 (preprint)

Coronavirus disease 2019 (COVID-19) manifests with a range of severities, but immune signatures of mild and severe disease are still not fully understood. Excessive inflammation has been postulated to be a major factor in the pathogenesis of severe COVID-19 and innate immune mechanisms are likely to be central in the inflammatory response. We used 40-plex mass cytometry and targeted serum proteomics to profile innate immune cell populations from peripheral blood of patients with mild or severe COVID-19 and healthy controls. Sampling at different stages of COVID-19 allowed us to reconstruct a pseudo-temporal trajectory of the innate immune response. Despite the expected patient heterogeneity, we identified consistent changes during the course of the infection. A rapid and early surge of CD169+ monocytes associated with an IFN{gamma}+MCP-2+ signature quickly followed symptom onset; at symptom onset, patients with mild and severe COVID-19 had a similar signature, but over the course of the disease, the differences between patients with mild and severe disease increased. Later in the disease course, we observed a more pronounced re-appearance of intermediate/non-classical monocytes and mounting systemic CCL3 and CCL4 levels in patients with severe disease. Our data provide new insights into the dynamic nature of the early inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and identifies sustained pathological innate immune responses as a likely key mechanism in severe COVID-19, further supporting investigation of targeted anti-inflammatory interventions in severe COVID-19.

Population-wide evolution of SARS-CoV-2 immunity tracked by a ternary immunoassay (preprint)

We describe the evolution of severe acute respiratory coronavirus 2 (SARS-CoV-2) seroprevalence in the greater area of Zurich, Switzerland, a region that has been only mildly hit by the pandemic in spite of hosting an international airport hub and a highly mobile population. Seroprevalence studies in low-prevalence settings require large sample sizes and high-specificity methodologies. To address this particular challenge, we developed a Tripartite Automated Blood Immunoassay to assess the IgG response against three SARS-CoV-2 proteins on approximately 40'000 samples from university hospital patients and healthy blood donors. The seroprevalence increased in March 2020 (0.3%; CI95%: 0.1% - 0.5%) in the cohort of the hospital patients but rapidly plateaued in April at 1.1-1.3%, with a slight drop in June, then decreased in July to 0.3-0.7%. Seropositive samples were confirmed with Western Blotting and liquid-phase binding assays. Employing a dynamic transmission model that describes SARS-CoV-2 transmission and seroconversion in the general population of the Canton of Zurich, we estimated an infection fatality ratio of 0.6% (CI95%: 0.4%-0.8%). We conclude that a small proportion of the population in the greater area of Zurich has been exposed to SARS-CoV-2, with an IFR that is similar to that of other European areas. The evolution of seroprevalence points to a high effectiveness of containment measures and/or to rapid loss of humoral responses.

Systemic and mucosal antibody secretion specific to SARS-CoV-2 during mild versus severe COVID-19 (preprint)

BackgroundInfection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an acute illness termed coronavirus disease 2019 (COVID-19). Humoral immune responses likely play an important role in containing SARS-CoV-2, however, the determinants of SARS-CoV-2-specific antibody responses are unclear. MethodsUsing immunoassays specific for the SARS-CoV-2 spike protein, we determined SARS-CoV-2-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) in sera and mucosal fluids of two cohorts, including patients with quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR)-confirmed SARS-CoV-2 infection (n = 56; median age 61 years) with mild versus severe COVID-19, and SARS-CoV-2-exposed healthcare workers (n = 109; median age 36 years) with or without symptoms and tested negative or positive by RT-qPCR. FindingsOn average, SARS-CoV-2-specific serum IgA titers in mild COVID-19 cases became positive eight days after symptom onset and were often transient, whereas serum IgG levels remained negative or reached positive values 9-10 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers as a function of duration since symptom onset, independent of patient age and comorbidities. Very high levels of SARS-CoV-2-specific serum IgA correlated with severe acute respiratory distress syndrome (ARDS). Interestingly, some of the SARS-CoV-2-exposed healthcare workers with negative SARS-CoV-2-specific IgA and IgG serum titers had detectable SARS-CoV-2-specific IgA antibodies in their nasal fluids and tears. Moreover, SARS-CoV-2-specific IgA levels in nasal fluids of these healthcare workers were inversely correlated with patient age. InterpretationThese data show that systemic IgA and IgG production against SARS-CoV-2 develops mainly in severe COVID-19, with very high IgA levels seen in patients with severe ARDS, whereas mild disease may be associated with transient serum titers of SARS-CoV-2-specific antibodies but stimulate mucosal SARS-CoV-2-specific IgA secretion. The findings suggest four grades of antibody responses dependent on COVID-19 severity.