Nucleocapsid Specific Diagnostics for the Detection of Divergent SARS-CoV-2 Variants

Isaacs, Ariel; Amarilla, Alberto A.; Aguado, Julio; Modhiran, Naphak; Albornoz, Eduardo A; Baradar, Alireza A.; McMillan, Christopher L. D.; Choo, Jovin J. Y.; Idris, Adi; Supramaniam, Aroon; McMillan, Nigel A.J.; Muller, David A.; Young, Paul R.; Woodruff, Trent M.; Wolvetang, Ernst J.; Chappell, Keith J.; Watterson, Daniel

doi:10.3389/fimmu.2022.926262

Cited by 12 publications

(10 citation statements)

References 31 publications

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“…Six-week-old female mice carrying the human ACE2 gene under the control of the keratin 18 promoter (K18-hACE2) on C57BL/6J background were purchased from Animal Resource Centre (ARC) in Australia and randomly assigned to cages. Group sizes were determined based on our previous studies [ 4 , 29 ], and contained n = 6 for clinical scoring and n = 4 for tissue collection. Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by intranasal inoculation of 20 µL of the ancestral SARS-CoV-2 (Wu strain) at 5 x 10 3 FFU per mouse, or RPMI additive free medium for the uninfected control group.…”

Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein

et al. 2022

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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. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson’s disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

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Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein

et al. 2022

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“…These test systems allowed detection of as low as 8 pg of a purified protein or 625 TCID 50 /mL of a virus, and they cross-reacted with the P.1 and B.1.617.2 variants [ 52 ]. The use of N-specific detection by highly sensitive nanobodies C2 and E2, which are specific to B.1, P.1 and B.1.617.2, was demonstrated by Isaacs et al [ 53 ]. Molecular modeling of the interaction of anti-N mAbs specific to 501Y.V1-V3, obtained by Yamaoka et al [ 54 ], revealed binding with exterior protein surface epitopes, and immunochromatographic test systems, combined with silver amplification technology, were developed.…”

Section: Discussionmentioning

confidence: 99%

Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model

Rak

Горбунов

Kostevich

et al. 2023

Viruses

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COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.

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“…019-19741); anti-SARS-CoV-2 Nucleocapsid C2, 1:1,000 (ref. 62 ); anti-SARS-CoV-2 Spike protein, 1:1,000 (ref. 63 ); anti-γH2AX (Millipore, 1:1,000, no.…”

Section: Methodsmentioning

confidence: 99%

Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology

Aguado,

Amarilla,

Taherian Fard

et al. 2023

Nat Aging

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Aging is a major risk factor for neurodegenerative diseases, and coronavirus disease 2019 (COVID-19) is linked to severe neurological manifestations. Senescent cells contribute to brain aging, but the impact of virus-induced senescence on neuropathologies is unknown. Here we show that senescent cells accumulate in aged human brain organoids and that senolytics reduce age-related inflammation and rejuvenate transcriptomic aging clocks. In postmortem brains of patients with severe COVID-19 we observed increased senescent cell accumulation compared with age-matched controls. Exposure of human brain organoids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced cellular senescence, and transcriptomic analysis revealed a unique SARS-CoV-2 inflammatory signature. Senolytic treatment of infected brain organoids blocked viral replication and prevented senescence in distinct neuronal populations. In human-ACE2-overexpressing mice, senolytics improved COVID-19 clinical outcomes, promoted dopaminergic neuron survival and alleviated viral and proinflammatory gene expression. Collectively our results demonstrate an important role for cellular senescence in driving brain aging and SARS-CoV-2-induced neuropathology, and a therapeutic benefit of senolytic treatments.

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Nucleocapsid Specific Diagnostics for the Detection of Divergent SARS-CoV-2 Variants

Cited by 12 publications

References 31 publications

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein

Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model

Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology

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