Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology. (preprint)

Aging is the primary risk factor for most neurodegenerative diseases, and recently coronavirus disease 2019 (COVID-19) has been associated with severe neurological manifestations that can eventually impact neurodegenerative conditions in the long-term. The progressive accumulation of senescent cells in vivo strongly contributes to brain aging and neurodegenerative co-morbidities but the impact of virus-induced senescence in the aetiology of neuropathologies is unknown. Here, we show that senescent cells accumulate in physiologically aged brain organoids of human origin and that senolytic treatment reduces inflammation and cellular senescence; for which we found that combined treatment with the senolytic drugs dasatinib and quercetin rejuvenates transcriptomic human brain aging clocks. We further interrogated brain frontal cortex regions in postmortem patients who succumbed to severe COVID-19 and observed increased accumulation of senescent cells as compared to age-matched control brains from non-COVID-affected individuals. Moreover, we show that exposure of human brain organoids to SARS-CoV-2 evoked cellular senescence, and that spatial transcriptomic sequencing of virus-induced senescent cells identified a unique SARS-CoV-2 variant-specific inflammatory signature that is different from endogenous naturally-emerging senescent cells. Importantly, following SARS-CoV-2 infection of human brain organoids, treatment with senolytics blocked viral retention and prevented the emergence of senescent corticothalamic and GABAergic neurons. Furthermore, we demonstrate in human ACE2 overexpressing mice that senolytic treatment ameliorates COVID-19 brain pathology following infection with SARS-CoV-2. In vivo treatment with senolytics improved SARS-CoV-2 clinical phenotype and survival, alleviated brain senescence and reactive astrogliosis, promoted survival of dopaminergic neurons, and reduced viral and senescence-associated secretory phenotype gene expression in the brain. Collectively, our findings demonstrate SARS-CoV-2 can trigger cellular senescence in the brain, and that senolytic therapy mitigates senescence-driven brain aging and multiple neuropathological sequelae caused by neurotropic viruses, including SARS-CoV-2.

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.

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.

Serological Profile Of Specific Antibodies Against Dominant Antigens Of SARS-CoV-2 In Chilean COVID-19 Patients. (preprint)

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has been a pandemic since March 2020. Currently, the virus has infected more than 50 million people worldwide and more than half a million in Chile. For many coronaviruses, Spike (S) and Nucleocapsid (N) proteins are described as major antigenic molecules, inducing seroconversion and production of neutralizing antibodies. In this work, we evaluated the presence in serum of IgM, IgA and IgG antibodies against N and S proteins of SARS-CoV-2 using western blot, and developed an ELISA test for the qualitative characterization of COVID-19 patients. Patients with an active infection or who have recovered from COVID-19 showed specific immunoblotting patterns for the recombinants S protein and its domains S1 and S2, as well as for the N protein of SARS-CoV-2. Anti-N antibodies were more frequently detected than anti-S or anti-S1-RBD antibodies. People who were never exposed to SARS-CoV-2 did not show reactivity. Finally, indirect ELISA assays using N and S1-RBD proteins, alone or in combination, were established with variable sensitivity and specificity depending on the antigen bound to the solid phase. Overall, Spike showed higher specificity than the nucleocapsid, and comparable sensitivity for both antigens. Both approaches confirmed the seroconversion after infection and allowed us to implement the analysis of antibodies in blood for research purposes in a local facility.

Fast isolation of sub-nanomolar affinity alpaca nanobody against the Spike RBD of SARS-CoV-2 by combining bacterial display and a simple single-step density gradient selection (preprint)

Despite the worldwide efforts to avoid disease progression of COVID-19 into a severe acute respiratory syndrome and avoid its severe impact on health systems; the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are required to meet the worldwide demand: recombinant antibodies such as alpaca Nanobodies fulfill these requirements. Here, we develop a fast track for nanobody isolation against the receptor-binding-domain (RBD) SARS-CoV-2 Spike protein following an optimized immunization, efficient construction of the VHH library for E. coli surface display, and single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. Following this procedure, we isolate and characterize an alpaca Nanobody against Spike RBD of SARS-CoV-2 in the sub-nanomolar range.