Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2 (preprint)

Why individuals with Down Syndrome (DS, trisomy 21) are particularly susceptible to SARS-CoV-2 induced neuropathology remains largely unclear. Since the choroid plexus (CP) performs important barrier and immune-interface functions, secretes the cerebrospinal fluid and strongly expresses the ACE2 receptor and the chromosome 21 encoded TMPRSS2 protease, we hypothesized that the CP could play a role in establishing SARS-CoV-2 infection in the brain. To investigate the role of the choroid plexus in SARS-CoV-2 central nervous system infection in DS, we established a new type of brain organoid from DS and isogenic euploid control iPSC that consists of a core of appropriately patterned functional cortical neuronal cell types that is surrounded by a patent and functional choroid plexus (CPCOs). Remarkably, DS-CPCOs not only recapitulated abnormal features of DS cortical development but also revealed defects in ciliogenesis and epithelial cell polarity of the developing choroid plexus. We next demonstrate that the choroid plexus layer facilitates SARS-CoV-2 replication and infection of cortical neuronal cells, and that this is increased in DS-CPCOs. We further show that inhibition of TMPRSS2 and Furin activity inhibits SARS-CoV-2 replication in DS-CPCOs to the level observed in euploid organoids. We conclude that CPCOs are a useful model for dissecting the role of the choroid plexus in euploid and DS forebrain development and enables screening for therapeutics that can inhibit SARS-CoV-2 induced neuro-pathogenesis.

An alpaca-derived nanobody neutralizes the SARS-CoV-2 omicron variant (preprint)

The SARS-CoV2 Omicron variant sub-lineages spread rapidly through the world, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for anti-SARS-CoV-2 agents that are effective against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production and potential for delivery via inhalation. Here, we characterize the RBD-specific nanobody W25, which we previously isolated from an alpaca, and show superior neutralization activity towards Omicron lineage BA.1 in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike surface glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. Furthermore, we show that W25 also binds the spike protein from the emerging, more infectious Omicron BA.2 lineage with picomolar affinity. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse prioritization of W25 for further clinical development.

Omicron BA.5 infects human brain organoids and is neuroinvasive and lethal in K18-hACE2 mice (preprint)

A frequently repeated premise is that viruses evolve to become less pathogenic. This appears also to be true for SARS-CoV-2, although the increased level of immunity in human populations makes it difficult to distinguish between reduced intrinsic pathogenicity and increasing protective immunity. The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described and appears to be due to reduced utilization of TMPRRS2. That this reduced pathogenicity remains true for omicron BA.5 was recently reported. In sharp contrast, we show that a BA.5 isolate was significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, with BA.5 infection showing increased neurovirulence, encephalitis and mortality, similar to that seen for an original strain isolate. BA.5 also infected human cortical brain organoids to a greater extent than a BA.1 and original strain isolate. Neurons were the target of infection, with increasing evidence of neuron infection in COVID-19 patients. These results argue that while omicron virus may be associated with reduced respiratory symptoms, BA.5 shows increased neurovirulence compared to earlier omicron sub-variants.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike-ACE2 receptor interaction (preprint)

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation utilising a model of human monocyte-derived microglia. We identified that SARS-CoV-2 isolates can bind and enter microglia, triggering inflammasome activation in the absence of viral replication. Mechanistically, microglial NLRP3 could be both primed and activated with SARS-CoV-2 spike glycoprotein in a NF{kappa}B and ACE2-dependent manner. Notably, virus- and spike protein-mediated inflammasome activation in microglia was significantly enhanced in the presence of -synuclein fibrils, which was entirely ablated by NLRP3-inhibition. These results support a possible mechanism of microglia activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in certain COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

SARS-CoV-2 triggers complement activation through interactions with heparan sulfate (preprint)

The complement system has been heavily implicated in severe COVID-19 with clinical studies revealing widespread gene induction, deposition, and activation. However, the mechanism by which complement is activated in this disease remains incompletely understood. Herein we examined the relationship between SARS-CoV-2 and complement by inoculating the virus in lepirudin-anticoagulated human blood. This caused progressive C5a production after 30 minutes and 24 hours, which was blocked entirely by inhibitors for factor B, C3, C5, and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface deposition of complement and interactions with heparan sulfate. Additional functional analysis revealed that complement-dependent granulocyte and monocyte activation was delayed. Indeed, C5aR1 internalisation and CD11b upregulation on these cells only occurred after 24 hours. Thus, SARS-CoV-2 is a non-canonical complement activator that triggers the alternative pathway through interactions with heparan sulfate.

Complete protection by a single dose skin patch delivered SARS-CoV-2 spike vaccine (preprint)

SARS-CoV-2 has infected over 160 million people and resulted in more than 3.3 million deaths, and we still face many challenges in the rollout of vaccines. Here, we use the high-density microarray patch to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show the vaccine, dry-coated on the patch is thermostable, and delivery of spike via HD-MAP induced greater cellular and antibody immune responses, with serum able to potently neutralize clinically relevant isolates including those from the B.1.1.7 and B.1.351 lineages. Finally, a single dose of HD-MAP-delivered spike provided complete protection from a lethal virus challenge, demonstrating that HD-MAP delivery of a SARS-CoV-2 vaccine is superior to traditional needle-and-syringe vaccination and has the potential to greatly impact the ongoing COVID-19 pandemic.

Human genome integration of SARS-CoV-2 contradicted by long-read sequencing (preprint)

A recent study proposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we applied deep (>50x) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2, and did not find any evidence of the virus existing as DNA. By examining ONT data from separate HEK293T cultivars, we resolved the complete sequences of 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV) positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions via ONT sequencing. That we found no evidence of SARS-CoV-2 integration suggests such events in vivo are highly unlikely to drive later oncogenesis or explain post-recovery detection of the virus.

Pediatric nasal epithelial cells are less permissive to SARS-CoV-2 replication compared to adult cells (preprint)

Rationale: Young children (typically those <10 years old) are less susceptible to SARS-CoV-2 infection and symptoms compared to adults. However, the mechanisms that underlie these age-dependent differences remain to be determined and could inform future therapeutics for adults. Objective: To contrast the infection dynamics of SARS-CoV-2 in primary nasal epithelial cells from adults and children. Methods: Viral replication was quantified by plaque assay. The cellular transcriptome of infected and uninfected cells was assessed by RNA-seq. ACE2 and TMPRSS2 protein expression were quantified by Western Blot Measurements and Main Results: We report significantly higher SARS-CoV-2 replication in adult compared to pediatric nasal epithelial cells. This was restricted to SARS-CoV-2 infection, as the same phenomenon was not observed with influenza virus infection. The differentiational SARS-CoV-2 replication dynamics were associated with an elevated type I and III interferon response, and a more pronounced inflammatory response in pediatric cells. No significant difference between the two age groups was observed in the protein levels of ACE2 and TMPRSS2. Conclusions: Our data suggest that the innate immune response of pediatric nasal epithelial cells, and not differential receptor expression, may contribute to the reported reduced SARS-COV-2 infection and symptoms reported amongst children.

Pixatimod (PG545), a clinical-stage heparan sulfate mimetic, is a potent inhibitor of the SARS-CoV-2 virus (preprint)

A major global effort is currently ongoing to search for therapeutics and vaccines to treat or prevent infection by the SARS-CoV-2 virus. Repurposing existing entities is one attractive approach. The heparan sulfate mimetic pixatimod is a clinical-stage synthetic sulfated compound that is a potent inhibitor of the glycosidase heparanase, and has known anti-cancer, anti-inflammatory and also antiviral properties. Here we show that pixatimod binds directly to the SARS-CoV-2 spike protein S1 receptor binding domain (RBD) and alters its conformation. Notably, this site overlaps with the known ACE2 binding site in the S1 RBD. We find that pixatimod inhibits binding of recombinant S1 RBD to Vero cells which express the ACE2 receptor. Moreover, in assays with three different isolates of live SARS-CoV-2 virus we show that pixatimod effectively inhibits viral infection of Vero cells. Importantly, its potency is well within its safe therapeutic dose range. These data provide evidence that pixatimod is a potent antiviral agent against SARS-CoV-2. Together with its other known activities this provides a strong rationale for its clinical investigation as a new multimodal therapeutic for the current COVID-19 pandemic.