O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Contribute to the Mutational Trajectory in Variants of Concern

The emergence of a polybasic cleavage motif for the protease furin in the SARS-CoV-2 spike protein has been established as a major factor for enhanced viral transmission in humans. The peptide region N-terminal to that motif is extensively mutated in major variants of concern including Alpha, Delta and Omicron. Besides furin, spike proteins from these variants appear to rely on other proteases for maturation, including TMPRSS2 that may share the same cleavage motif. Glycans found near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, with a suite of chemical tools, we establish O-linked glycosylation as a major determinant of SARS-CoV-2 spike cleavage by the host proteases furin and TMPRSS2, and as a likely driving force for the emergence of common mutations in variants of concern. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, and this glycosylation event is suppressed by many, but not all variant mutations. A novel strategy for rapid bioorthogonal modification of Thr678-containing glycopeptides revealed that introduction of a negative charge completely abrogates furin activity. In a panel of synthetic glycopeptides containing elaborated O-glycans, we found that the sole incorporation of N-acetylgalactosamine did not substantially impact furin activity, but the presence of sialic acid in elaborated O-glycans reduced furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on spike cleavage by TMPRSS2. With a chemistry-centered approach, we firmly establish O-glycosylation as a major determinant of spike maturation and propose that a disruption of O-GalNAc glycosylation is a substantial driving force for the evolution of variants of concern. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/508093v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@71b54eorg.highwire.dtl.DTLVardef@1361b4aorg.highwire.dtl.DTLVardef@139bdd9org.highwire.dtl.DTLVardef@1df192b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Broad human and animal coronavirus neutralisation by SARS-CoV-2 S2-targeted vaccination

Several common-cold coronaviruses (HCoVs) are endemic in humans and several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current Coronavirus disease 2019 (COVID-19) pandemic. Whilst antibody cross-reactivity with the Spike glycoproteins (S) of diverse coronaviruses has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to or mediate protection, when induced naturally or through vaccination. Using a mouse model, we show that prior HCoV-OC43 S immunity primes neutralising antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, mouse vaccination with SARS-CoV-2 S2 elicits antibodies that neutralise diverse animal and human alphacoronaviruses and betacoronaviruses in vitro, and protects against SARS-CoV-2 challenge in vivo. Lastly, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induces stronger and broader neutralising antibody response than booster Wuhan S vaccination, suggesting it may prevent repertoire focusing caused by repeated homologous vaccination. The data presented here establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern (VOCs), as well as to unpredictable, yet inevitable future coronavirus zoonoses.

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp13 Helicase

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterised pharmaceuticals for nsp13 inhibitors using a FRET-based high-throughput screening (HTS) approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains

We examined the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 that arose in the United Kingdom and spread globally. Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa B.1.351 variant, than of the infecting variant. The drop in cross-reactivity was more pronounced following B.1.1.7 than parental strain infection, indicating asymmetric heterotypic immunity induced by SARS-CoV-2 variants.

SARS-CoV-2 recruits a haem metabolite to evade antibody immunity

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans

Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections1. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions2,3. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Scalable and Resilient SARS-CoV2 testing in an Academic Centre

The emergence of the novel coronavirus SARS-CoV-2 has led to a pandemic infecting more than two million people worldwide in less than four months, posing a major threat to healthcare systems. This is compounded by the shortage of available tests causing numerous healthcare workers to unnecessarily self-isolate. We provide a roadmap instructing how a research institute can be repurposed in the midst of this crisis, in collaboration with partner hospitals and an established diagnostic laboratory, harnessing existing expertise in virus handling, robotics, PCR, and data science to derive a rapid, high throughput diagnostic testing pipeline for detecting SARS-CoV-2 in patients with suspected COVID-19. The pipeline is used to detect SARS-CoV-2 from combined nose-throat swabs and endotracheal secretions/ bronchoalveolar lavage fluid. Notably, it relies on a series of in-house buffers for virus inactivation and the extraction of viral RNA, thereby reducing the dependency on commercial suppliers at times of global shortage. We use a commercial RT-PCR assay, from BGI, and results are reported with a bespoke online web application that integrates with the healthcare digital system. This strategy facilitates the remote reporting of thousands of samples a day with a turnaround time of under 24 hours, universally applicable to laboratories worldwide.