Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice (preprint)

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the worlds population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses (preprint)

The majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

Sequences in the cytoplasmic tail of SARS-CoV-2 spike facilitate syncytia formation (preprint)

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds the cell surface protein ACE2 to mediate fusion of the viral membrane with target cells. S comprises a large external domain, a transmembrane domain (TMD) and a short cytoplasmic tail. To elucidate the intracellular trafficking of S protein in host cells we applied proteomics to identify cellular factors that interact with its cytoplasmic tail. We confirm interactions with components of the COPI, COPII and SNX27/retromer vesicle coats, and with FERM domain actin regulators and the WIPI3 autophagy component. The interaction with COPII promotes efficient exit from the endoplasmic reticulum (ER), and although COPI-binding should retain S in the early Golgi system where viral budding occurs, the binding is weakened by a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates as it lacks an endocytosis motif of the type found in many other coronaviruses. It is known that when at the surface S can direct cell:cell fusion leading to the formation of multinucleate syncytia. Thus, the trafficking signals in the cytoplasmic tail of S protein indicate that syncytia formation is not an inadvertent by-product of infection but rather a key aspect of the replicative cycle of SARS-CoV-2 and potential cause of pathological symptoms.

SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier (preprint)

Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.

Covidex: an ultrafast and accurate tool for virus subtyping (preprint)

Covidex is an open-source, alignment-free machine learning subtyping tool for viral species. It is a shiny app that allows a fast and accurate classification in pre-defined clusters for SARS-CoV-2 and FMDV genome sequences. The user can also build its own classification models with the Covidex model generator. AvailabilityCovidex is open-source, cross-platform compatible, and is available under the terms of the GNU General Public License v3 (http://www.gnu.org/licenses/gpl.txt). Covidex is available via SourceForge https://sourceforge.net/projects/covidex or the web application https://cacciabue.shinyapps.io/shiny2/ Contactcacciabue.marco@inta.gob.ar; marcocacciabue@yahoo.com

Targeting pentose phosphate pathway for SARS-CoV-2 therapy (preprint)

It becomes more and more obvious that deregulation of host metabolism play an important role in SARS-CoV-2 pathogenesis with implication for increased risk of severe course of COVID-19. Furthermore, it is expected that COVID-19 patients recovered from severe disease may experience long-term metabolic disorders. Thereby understanding the consequences of SARS-CoV-2 infection on host metabolism can facilitate efforts for effective treatment option. We have previously shown that SARS-CoV-2-infected cells undergo a shift towards glycolysis and that 2-deoxy-D-glucose (2DG) inhibits SARS-CoV-2 replication. Here, we show that also pentose phosphate pathway (PPP) is remarkably deregulated. Since PPP supplies ribonucleotides for SARS-CoV-2 replication, this could represent an attractive target for an intervention. On that account, we employed the transketolase inhibitor benfooxythiamine and showed dose-dependent inhibition of SARS-CoV-2 in non-toxic concentrations. Importantly, the antiviral efficacy of benfooxythiamine was further increased in combination with 2DG.

Temporal landscape of mutation accumulation in SARS-CoV-2 genomes from Bangladesh: possible implications from the ongoing outbreak in Bangladesh (preprint)

Along with intrinsic evolution, adaptation to selective pressure in new environments might have resulted in the circulatory SARS-CoV-2 strains in response to the geoenvironmental conditions of a country and the demographic profile of its population. Thus the analysis of genomic mutations of these circulatory strains may give an insight into the molecular basis of SARS-CoV-2 pathogenesis and evolution favoring the development of effective treatment and containment strategies. With this target, the current study traced the evolutionary route and mutational frequency of 198 Bangladesh originated SARS-CoV-2 genomic sequences available in the GISAID platform over a period of 13 weeks as of 14 July 2020. The analyses were performed using MEGA 7, Swiss Model Repository, Virus Pathogen Resource and Jalview visualization. Our analysis identified that majority of the circulating strains in the country belong to B and/or L type among cluster A to Z and strikingly differ from both the reference genome and the first sequenced genome from Bangladesh. Mutations in Nonspecific protein 2 (NSP2), NSP3, RNA dependent RNA polymerase (RdRp), Helicase, Spike, ORF3a, and Nucleocapsid (N) protein were common in the circulating strains with varying degrees and the most unique mutations(UM) were found in NSP3 (UM-18). But no or limited changes were observed in NSP9, NSP11, E (Envelope), NSP7a, ORF 6, and ORF 7b suggesting the possible conserved functions of those proteins in SARS-CoV-2 propagation. However, along with D614G mutation, more than 20 different mutations in the Spike protein were detected basically in the S2 domain. Besides, mutations in SR-rich region of N protein and P323L in RDRP were also present. However, the mutation accumulation showed an association with sex and age of the COVID-19 positive cases. So, identification of these mutational accumulation patterns may greatly facilitate drug/ vaccine development deciphering the age and the sex dependent differential susceptibility to COVID-19.

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.

A thermostable, closed, SARS-CoV-2 spike protein trimer. (preprint)

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.