West Nile virus (WNV) is a flavivirus that has disseminated globally as a significant cause of viral encephalitis in humans. MircoRNA-155 (miR-155) regulates various aspects of innate and adaptive immune responses. We previously reported that WNV infection induces upregulation of miR-155 in mice brains. In the current study, we demonstrate the critical role of miR-155 in restricting the pathogenesis of WNV infection in mice. Compared to wild-type (WT) mice, miR-155 knockout mice exhibited significantly higher morbidity and mortality after infection with either a lethal strain, WNV NY99, or a non-lethal strain, WNV Eg101. Increased mortality in miR-155^−/− mice was associated with significantly high WNV burden in the serum and brains. Protein levels of interferon (IFN)-α in the serum and brains were higher in miR-155^−/− mice. However, miR-155^−/− mice exhibited significantly lower protein levels of anti-viral interleukin (IL)-1β, IL-12, IL-6, IL-15, and GM-CSF despite the high viral load. Primary mouse cells lacking miR-155 were more susceptible to infection with WNV compared to cells derived from WT mice. Besides, overexpression of miR-155 in human neuronal cells modulated anti-viral cytokine response and resulted in significantly lower WNV replication. These data collectively indicate that miR-155 restricts WNV production in mouse and human cells and protects against lethal WNV infection in mice.

Transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) have been extensively used to investigate the pathogenesis and tissue tropism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Neuroinvasion and the replication of SARS-CoV-2 within the central nervous system (CNS) of K18-hACE2 mice is associated with increased mortality; although, the mechanisms by which this occurs remain unclear. In this study, we generated primary neuronal cultures from K18-hACE2 mice to investigate the effects of a SARS-CoV-2 infection. We also evaluated the immunological response to SARS-CoV-2 infection in the CNS of K18-hACE2 mice and mouse neuronal cultures. Our data show that neuronal cultures obtained from K18-hACE2 mice are permissive to SARS-CoV-2 infection and support productive virus replication. Furthermore, SARS-CoV-2 infection upregulated the expression of genes involved in innate immunity and inflammation, including IFN-α, ISG-15, CXCL10, CCL2, IL-6 and TNF-α, in the neurons and mouse brains. In addition, we found that SARS-CoV-2 infection of neurons and mouse brains activates the ZBP1/pMLKL-regulated necroptosis pathway. Together, our data provide insights into the neuropathogenesis of SARS-CoV-2 infection in K18-hACE2 mice.

Neurological manifestations are a significant complication of coronavirus disease 2019 (COVID-19), but the underlying mechanisms are yet to be understood. Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced neuroinvasion and encephalitis were observed in K18-hACE2 mice, leading to mortality. Our goal in this study was to gain insights into the molecular pathogenesis of neurological manifestations in this mouse model. To analyze differentially expressed genes (DEGs) in the brains of mice following SARS-CoV-2 infection, we performed NanoString gene expression analysis using three individual animal samples at 1, 3, and 6 days post-infection. We identified the DEGs by comparing them to animals that were not infected with the virus. We found that genes upregulated at day 6 post-infection were mainly associated with Toll-like receptor (TLR) signaling, RIG-I-like receptor (RLR) signaling, and cell death pathways. However, downregulated genes were associated with neurodegeneration and synaptic signaling pathways. In correlation with gene expression profiles, a multiplexed immunoassay showed the upregulation of multiple cytokines and chemokines involved in inflammation and cell death in SARS-CoV-2-infected brains. Furthermore, the pathway analysis of DEGs indicated a possible link between TLR2-mediated signaling pathways and neuroinflammation, as well as pyroptosis and necroptosis in the brain. In conclusion, our work demonstrates neuroinflammation-associated gene expression profiles, which can provide key insight into the severe disease observed in COVID-19 patients.

The emergence of new SARS-CoV-2 variants continues to cause challenging problems for the effective control of COVID-19. In this study, we tested the hypothesis of whether a strategy of multivalent and sequential heterologous spike protein vaccinations would induce a broader range and higher levels of neutralizing antibodies against SARS-CoV-2 variants and more effective protection than homologous spike protein vaccination in a mouse model. We determined spike-specific IgG, receptor-binding inhibition titers, and protective efficacy in the groups of mice that were vaccinated with multivalent recombinant spike proteins (Wuhan, Delta, Omicron), sequentially with heterologous spike protein variants, or with homologous spike proteins. Trivalent (Wuhan + Delta + Omicron) and sequential heterologous spike protein vaccinations were more effective in inducing serum inhibition activities of receptor binding to spike variants and virus neutralizing antibody titers than homologous spike protein vaccination. The higher efficacy of protection was observed in mice with trivalent and sequential heterologous spike protein vaccination after a challenge with a mouse-adapted SARS-CoV-2 MA10 strain compared to homologous spike protein vaccination. This study provides evidence that a strategy of multivalent and sequential heterologous variant spike vaccination might provide more effective protection against emerging SARS-CoV-2 variants than homologous spike vaccination and significantly alleviate severe inflammation due to COVID-19.

Widespread and rapidly evolving SARS-CoV-2 posed an unprecedented challenge to vaccine developers. GeoVax has designed a multiantigen SARS-CoV-2 vaccine, designated GEO-CM02 based on a Modified Vaccinia Virus (MVA) vector that expresses spike (S), membrane (M), and envelope (E) antigens. This experimental vaccine was tested in the hACE2 transgenic mouse model to assess immunogenicity and efficacy. Administration of the vaccine in a two-dose regimen elicited high levels of neutralizing antibodies and provided complete protection, effectively reducing lung, olfactory bulb, and brain viral load and reducing lung inflammation following infection with original B.1 virus and the B.1.1.529 variant. In addition, GEO-CM02 conferred 80% protection against a lethal infection with the B.1.351 variant. GEO-CM02 vaccine efficacy studies also demonstrated a complete level of vaccine-induced protection with a single dose against the original B.1 virus and B.1.1.529 variant. GEO-CM02 effectively elicited functional T-cell responses in both prime and prime–boost groups. These data indicate that vaccination with the GEO-CM02 vaccine can induce immune responses that protect against severe disease induced by SARS-CoV-2 and its variants in a highly relevant pre-clinical model.

Despite current vaccines and therapeutics targeting SARS-CoV-2, the causative agent of the COVID-19 pandemic, cases remain high causing a burden on health care systems. Spike-protein mediated membrane fusion of SARS-CoV-2 is a critical step in viral entry. Herein, we describe entry inhibitors identified by first screening a library of about 160 compounds and then analogue synthesis. Specifically, compound 261 was found to inhibit SARS-CoV-2 infection in a tissue model with IC₅₀ of 0.3 µM. Using NMR, we found that 261 interacts with key residues in the aromatic-rich region of the spike protein directly next to the transmembrane domain. Molecular dynamic simulations of the 261 binding pocket in the spike protein was also mapped to the transmembrane domain, consistent with NMR findings. The amino acids in the binding site are conserved among different coronaviruses known to infect humans; therefore, inhibitors targeting this conserved binding site could be a useful addition to current therapeutics and may have pan-coronavirus antiviral activities.