Multiplexed proteomics and imaging of resolving and lethal SARS-CoV-2 infection in the lung (preprint)

Normal tissue physiology and repair depends on communication with the immune system. Understanding this communication at the molecular level in intact tissue requires new methods. The consequences of SARS-CoV-2 infection, which can result in acute respiratory distress, thrombosis and death, has been studied primarily in accessible liquid specimens such as blood, sputum and bronchoalveolar lavage, all of which are peripheral to the primary site of infection in the lung. Here, we describe the combined use of multiplexed deep proteomics with multiplexed imaging to profile infection and its sequelae directly in fixed lung tissue specimens obtained from necropsy of infected animals and autopsy of human decedents. We characterize multiple steps in disease response from cytokine accumulation and protein phosphorylation to activation of receptors, changes in signaling pathways, and crosslinking of fibrin to form clots. Our data reveal significant differences between naturally resolving SARS-CoV-2 infection in rhesus macaques and lethal COVID-19 in humans. The approach we describe is broadly applicable to other tissues and diseases.

Evidence of antigenic imprinting in sequential Sarbecovirus immunization (preprint)

Antigenic imprinting, which describes the bias of antibody response due to previous immune history, can influence vaccine effectiveness and has been reported in different viruses. Give that COVID-19 vaccine development is currently a major focus of the world, there is a lack of understanding of how background immunity influence antibody response to SARS-CoV-2. This study provides evidence for antigenic imprinting in Sarbecovirus, which is the subgenus that SARS-CoV-2 belongs to. Specifically, we sequentially immunized mice with two antigenically distinct Sarbecovirus strains, namely SARS-CoV and SARS-CoV-2. We found that the neutralizing antibodies triggered by the sequentially immunization are dominantly against the one that is used for priming. Given that the impact of the background immunity on COVID-19 is still unclear, our results will provide important insights into the pathogenesis of this disease as well as COVID-19 vaccination strategy.

The Strand-biased Transcription of SARS-CoV-2 and Unbalanced Inhibition by Remdesivir (preprint)

SARS-CoV-2, a positive single-stranded RNA virus, caused the COVID-19 pandemic. Although its sense-mRNA architecture was reported, its anti-sense strand was unexplored. Here, we deeply sequenced both strands of RNA and found SARS-CoV-2 transcription is strongly biased to form the sense strand. During negative strand synthesis, apart from canonical sub-genomic ORFs, numerous non-canonical fusion transcripts are formed, driven by 3-15 nt sequence homology scattered along the genome but more prone to be inhibited by SARS-CoV-2 RNA polymerase inhibitor Remdesivir. The drug also represses more of the negative than the positive strand synthesis as supported by a mathematic simulation model and experimental quantifications. Overall, this study opens new sights into SARS-CoV-2 biogenesis and may facilitate the anti-viral drug design.

Repurposed antiviral drugs for COVID-19; interim WHO SOLIDARITY trial results (preprint)

BACKGROUND WHO expert groups recommended mortality trials in hospitalized COVID-19 of four re-purposed antiviral drugs. METHODS Study drugs were Remdesivir, Hydroxychloroquine, Lopinavir (fixed-dose combination with Ritonavir) and Interferon-{beta}1a (mainly subcutaneous; initially with Lopinavir, later not). COVID-19 inpatients were randomized equally between whichever study drugs were locally available and open control (up to 5 options: 4 active and local standard-of-care). The intent-to-treat primary analyses are of in-hospital mortality in the 4 pairwise comparisons of each study drug vs its controls (concurrently allocated the same management without that drug, despite availability). Kaplan-Meier 28-day risks are unstratified; log-rank death rate ratios (RRs) are stratified for age and ventilation at entry. RESULTS In 405 hospitals in 30 countries 11,266 adults were randomized, with 2750 allocated Remdesivir, 954 Hydroxychloroquine, 1411 Lopinavir, 651 Interferon plus Lopinavir, 1412 only Interferon, and 4088 no study drug. Compliance was 94-96% midway through treatment, with 2-6% crossover. 1253 deaths were reported (at median day 8, IQR 4-14). Kaplan-Meier 28-day mortality was 12% (39% if already ventilated at randomization, 10% otherwise). Death rate ratios (with 95% CIs and numbers dead/randomized, each drug vs its control) were: Remdesivir RR=0.95 (0.81-1.11, p=0.50; 301/2743 active vs 303/2708 control), Hydroxychloroquine RR=1.19 (0.89-1.59, p=0.23; 104/947 vs 84/906), Lopinavir RR=1.00 (0.79-1.25, p=0.97; 148/1399 vs 146/1372) and Interferon RR=1.16 (0.96-1.39, p=0.11; 243/2050 vs 216/2050). No study drug definitely reduced mortality (in unventilated patients or any other subgroup of entry characteristics), initiation of ventilation or hospitalisation duration. CONCLUSIONS These Remdesivir, Hydroxychloroquine, Lopinavir and Interferon regimens appeared to have little or no effect on hospitalized COVID-19, as indicated by overall mortality, initiation of ventilation and duration of hospital stay. The mortality findings contain most of the randomized evidence on Remdesivir and Interferon, and are consistent with meta-analyses of mortality in all major trials. (Funding: WHO. Registration: ISRCTN83971151, NCT04315948)

Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration (preprint)

The Spike protein of SARS-CoV-2, its receptor binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.