In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin–mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2–induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2–induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

Based on its ability to express high levels of protein, baculovirus has been widely used for recombinant protein production in insect cells for more than thirty years with continued technical improvements. In addition, baculovirus has been successfully applied for foreign gene delivery into mammalian cells without any viral replication. However, several CpG motifs are present throughout baculoviral DNA and induce an antiviral response in mammalian cells, resulting in the production of pro-inflammatory cytokines and type I interferon through a Toll-like receptor (TLR)-dependent or -independent signaling pathway, and ultimately limiting the efficiency of transgene expression. On the other hand, by taking advantage of this strong adjuvant activity, recombinant baculoviruses encoding neutralization epitopes can elicit protective immunity in mice. Moreover, immunodeficient cells, such as hepatitis C virus (HCV)- or human immunodeficiency virus (HIV)-infected cells, are more susceptible to baculovirus infection than normal cells and are selectively eliminated by the apoptosis-inducible recombinant baculovirus. Here, we summarize the application of baculovirus as a gene expression vector and the mechanism of the host innate immune response induced by baculovirus in mammalian cells. We also discuss the future prospects of baculovirus vectors.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)–specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Broadly protective vaccines against SARS-related coronaviruses that may cause future outbreaks are urgently needed. The SARS-CoV-2 spike receptor-binding domain (RBD) comprises two regions, the core-RBD and the receptor-binding motif (RBM); the former is structurally conserved between SARS-CoV-2 and SARS-CoV. Here, in order to elicit humoral responses to the more conserved core-RBD, we introduced N-linked glycans onto RBM surfaces of the SARS-CoV-2 RBD and used them as immunogens in a mouse model. We found that glycan addition elicited higher proportions of the core-RBD–specific germinal center (GC) B cells and antibody responses, thereby manifesting significant neutralizing activity for SARS-CoV, SARS-CoV-2, and the bat WIV1-CoV. These results have implications for the design of SARS-like virus vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), employs host-cell angiotensin-converting enzyme 2 (ACE2) for cell entry. Genetic analyses of ACE2 have identified several single-nucleotide polymorphisms (SNPs) specific to different human populations. Molecular dynamics simulations have indicated that several of these SNPs could affect interactions between SARS-CoV-2 and ACE2, thereby providing a partial explanation for the regional differences observed in SARS-CoV-2 infectivity and severity. However, the significance of population-specific ACE2 SNPs in SARS-CoV-2 infectivity is unknown, as no in vitro validation studies have been performed. Here, we analyzed the impact of eight SNPs found in specific populations on receptor binding and cell entry in vitro. Except for a SNP causing a nonsense mutation that reduced ACE2 expression, none of the selected SNPs markedly altered the interaction between ACE2 and the SARS-CoV-2 spike protein (SARS-2-S), which is responsible for receptor recognition and cell entry, or the efficiency of viral cell entry mediated by SARS-2-S. Our findings indicate that ACE2 polymorphisms have limited impact on the ACE2-dependent cell entry of SARS-CoV-2 and underscore the importance of future studies on the involvement of population-specific SNPs of other host genes in susceptibility toward SARS-CoV-2 infection.

Intranasal vaccines are anticipated to be powerful tools for combating many infectious diseases, including SARS-CoV-2, because they induce not only systemic immunity but also mucosal immunity at the site of initial infection. However, they are generally inefficient in inducing an antigen-specific immune response without adjuvants. Here, we developed an adjuvant-free intranasal vaccine platform that utilizes the preexisting immunity induced by previous infection or vaccination to enhance vaccine effectiveness. We made RBD-HA, a fusion of the receptor-binding domain (RBD) of spike derived from SARS-CoV-2 as a vaccine target with HA derived from influenza A virus (IAV) as a carrier protein. Intranasal immunization of previously IAV-infected mice with RBD-HA without an adjuvant elicited robust production of RBD-specific systemic IgG and mucosal IgA by utilizing both HA-specific preexisting IgG and CD4⁺ T cells. Consequently, the mice were efficiently protected from SARS-CoV-2 infection. Additionally, we demonstrated the high versatility of this intranasal vaccine platform by assessing various vaccine antigens and preexisting immunity associated with a variety of infectious diseases. The results of this study suggest the promising potential of this intranasal vaccine platform to address problems associated with intranasal vaccines.