Intranasal inoculation of female BALB/c mice with replication-deficient human adenovirus type 5 expressing SARS-CoV-2 nucleocapsid protein aggravates lung pathology upon re-encountering the antigen.

Preclinical studies indicate that SARS-CoV-2 nucleocapsid (N)-based vaccines, along with other viral protein(s), confer protection in various animal models against infection by SARS-CoV-2 ancestral virus and variants of concern. However, the optimal vaccination procedure and the role of N-specific host adaptive immune responses remain elusive. Here, we report that intranasal inoculation with replication-deficient human adenovirus type 5 expressing SARS-CoV-2 N protein (Ad5-N) conferred no protection in the lung of female BALB/c mice upon re-encountering the antigen, either by 10-fold Ad5-N re-exposure or sublethal infection of mouse-adapted SARS-CoV-2. By contrast, this procedure led to aggravated lung pathology with more necroptotic CD3+ T cells and Ly6G+ granulocytes, which was associated with the accumulation of IFN-γ-expressing antigen-experienced CD4+ and CD8+ T cells. These findings pre-caution the clinical application of this vaccination procedure. Furthermore, our data suggest that excessive host adaptive immune responses against N protein contributes to COVID-19 pathogenesis.

Mechanical isolation of neonatal and adult mouse dura leukocytes for flow cytometry analysis.

The meninges, consisting of the pia, arachnoid, and dura layers, provide immunosurveillance of the central nervous system with both innate and adaptive immune cells. Here we present an optimized protocol for isolating dura leukocytes from neonatal and adult mice. We describe steps for harvesting the skull cap, extracting the dura mater, mechanical isolation of dura leukocytes, and flow cytometry analysis. Unlike the time-consuming enzymatic digestion isolation which makes dura hematopoietic stem cells (HSCs) undetectable, this rapid and simplified technique permits dura HSC identification. For complete details on the use and execution of this protocol, please refer to Niu et al. (2022).1.

Identification of hematopoietic stem cells residing in the meninges of adult mice at steady state.

Steady-state extramedullary hematopoiesis during adulthood is an emerging field of great interest. The meninges contain both innate and adaptive immune cells, which provide immunosurveillance of the central nervous system (CNS). Hematopoietic progenitors that give rise to meningeal immune cells remain elusive. Here, we report that steady-state meninges of adult mice host hematopoietic stem cells (HSCs), as defined by long-term, efficient, multi-lineage reconstitution and self-renewal capacity in the meninges, blood, spleen, and bone marrow of sublethally irradiated adult recipients. HSCs lodge in the meninges after birth with local expression of pro-hematopoietic niche factors. Meningeal HSCs are locally maintained in homeostasis and get replenished from the blood only when the resident pool is reduced. With a tissue-specific expression profile, meningeal HSCs can provide the CNS with a constant supply of leukocytes more adapted to local microenvironment.