Binding of SARS-CoV-2 nucleocapsid protein to uninfected epithelial cells induces antibody-mediated complement deposition (preprint)

SARS-CoV-2 infection triggers strong antibody response toward Nucleocapsid-Protein (NP), suggesting extracellular presence beyond its intra-virion RNA binding. Interestingly, NP was found to decorate infected and proximal uninfected cell-surfaces. Here, we propose a new mechanism through which extracellular NP on uninfected cells contributes to COVID-19 pathogenicity. We show that NP binds to cell-surface sulfated linear-glycosaminoglycans by spatial rearrangement of its RNA-binding sites facilitated by the flexible, positively charged, linker. Coating of uninfected lung-derived cells with purified NP attracted anti-NP-IgG from lung fluids and sera collected from COVID-19 patients. The magnitude of this immune recognition was significantly elevated in moderate compared to mild COVID-19 cases. Importantly, binding of anti-NP-IgG present in sera generated clusters that triggered C3b deposition by the classical complement pathway. Heparin analog enoxaparin outcompeted NP-binding, rescuing cells from anti-NP IgG-mediated complement deposition. Our findings unveil how extracellular NP may exacerbate COVID-19 tissue damage, and suggest leads for preventative therapy.

Human nasal and lung tissues infected ex vivo with SARS-CoV-2 provide insights into differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tract (preprint)

The nasal-mucosa constitutes the primary entry site for respiratory viruses including SARS-CoV-2. While the imbalanced innate immune response of end-stage COVID-19 has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local-mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon stimulated genes, cytokines and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tract, that are distinct to SARS-CoV-2. The studies shed light on the role of the nasal-mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCEIn order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal-mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues, infected in parallel with SARS-CoV-2 and influenza virus, we have revealed distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal-mucosal infection model can be employed to assess the impact of viral evolutionary changes, and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2 (preprint)

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

Lessons from applied large-scale pooling of 133,816 SARS-CoV-2 RT-PCR tests (preprint)

Pooling multiple swab samples prior to RNA extraction and RT-PCR analysis was proposed as a strategy to reduce costs and increase throughput of SARS-CoV-2 tests. However, reports on practical large-scale group testing for SARS-CoV-2 have been scant. Key open questions concern reduced sensitivity due to sample dilution; the rate of false positives; the actual efficiency (number of tests saved by pooling) and the impact of infection rate in the population on assay performance. Here we report analysis of 133,816 samples collected at April-September 2020, tested by pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the reality of frequently changing prevalence rate (0.5%-6%). Surprisingly, we observed pooling efficiency and sensitivity that exceed theoretical predictions, which resulted from non-random distribution of positive samples in pools. Overall, the findings strongly support the use of pooling for efficient large high throughput SARS-CoV-2 testing.

Clinical Correlates of Early IgG Response in Patients with Mild COVID-19 (preprint)

Background: We determined the temporal pattern of early SARS-CoV-2 IgG response in patients with mild COVID-19, and sought to identify predictive clinical and laboratory features. Methods:  Serum samples were prospectively obtained from 111 convalescent COVID-19 patients, staying in dedicated Isolation–hotels, and tested for the presence of SARS-CoV-2 IgG by anti-S1 protein ELISA.Results: SARS-CoV-2 IgG was detected in 78 (70.3%) patients tested within the first month from diagnosis. While highly variable between patients, the rate of antibody detection generally increased with time, from 47.1% to 93.8% at the first and fourth weeks from diagnosis, respectively, with the largest shift observed between the second and third week. Notably, the presence of more profound symptoms at presentation, namely, fever and chills, positively and independently correlated with early antibody response. IgG-positive patients had higher ferritin levels (p=0.039). Older age (p<0.001) and increased CRP levels (p=0.001) were associated with higher SARS-CoV-2 IgG levels. Conclusions: The identified temporal pattern along with the correlation between inflammation-related clinical and laboratory parameters and early IgG response in patients with mild COVID-19, could provide a basis for better prediction and understating of the immune response to SARS-CoV-2, and inform therapeutic donor-plasma selection.  

Pooled RNA extraction and PCR assay for efficient SARS-CoV-2 detection (preprint)

Testing for active SARS-CoV-2 infection is a fundamental tool in the public health measures taken to control the COVID-19 pandemic. Due to the overwhelming use of SARS-CoV-2 RT-PCR tests worldwide, availability of test kits has become a major bottleneck. Here we demonstrate pooling strategies to perform RNA extraction and RT-PCR in pools, significantly increasing throughput while maintaining clinical sensitivity. We implemented the method in a routine clinical diagnosis setting of asymptomatic populations, and already tested 5,464 individuals for SARS-CoV-2 using 731 RNA extraction and RT-PCR kits. We identified six SARS-CoV-2 positive patients corresponding to 0.11% of the tested population.