JAK inhibition during the early phase of SARS-CoV-2 infection worsens kidney injury by suppressing endogenous antiviral activity in mice.

Coronavirus disease 2019 (COVID-19) induces respiratory dysfunction as well as kidney injury. Although the kidney is considered a target organ of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and affected by the COVID-19-induced cytokine storm, the mechanisms of renal reaction in SARS-CoV-2 infection are unknown. In this study, a murine COVID-19 model was induced by nasal infection with mouse-adapted SARS-CoV-2 (MA10). MA10 infection induced body weight loss along with lung inflammation in mice 4 days after infection. Serum creatinine levels and the urinary albumin/creatinine ratio increased on day 4 after MA10 infection. Measurement of the urinary neutrophil gelatinase-associated lipocalin/creatinine ratio and hematoxylin and eosin staining revealed tubular damage in MA10-infected murine kidneys, indicating kidney injury in the murine COVID-19 model. Interferon (IFN)-γ and interleukin-6 upregulation in the sera of MA10-infected mice, along with the absence of MA10 in the kidneys, implied that the kidneys were affected by the MA10 infection-induced cytokine storm rather than by direct MA10 infection of the kidneys. RNA-sequencing analysis revealed that antiviral genes, such as the IFN/Janus kinase (JAK) pathway, were upregulated in MA10-infected kidneys. Upon administration of the JAK inhibitor baricitinib on days 1-3 after MA10 infection, an antiviral pathway was suppressed, and MA10 was detected more frequently in the kidneys. Notably, JAK inhibition upregulated the hypoxia response and exaggerated kidney injury. These results suggest that endogenous antiviral activity protects against SARS-CoV-2-induced kidney injury in the early phase of infection, providing valuable insights into the pathogenesis of COVID-19-associated nephropathy.NEW & NOTEWORTHY Patients frequently present with acute kidney injury or abnormal urinary findings after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we investigated how the kidneys respond during SARS-CoV-2 infection using a murine coronavirus disease 2019 (COVID-19) model and showed that Janus kinase-mediated endogenous antiviral activity protects against kidney injury in the early phase of SARS-CoV-2 infection. These findings provide valuable insights into the renal pathophysiology of COVID-19.

Vaccine using community-acquired respiratory distress syndrome toxin as an antigen against Mycoplasma pneumoniae in mice.

Mycoplasma pneumoniae (Mp) is one of the most common causes of bacterial community-acquired pneumonia in humans. Because of the frequent epidemics and the emergence of antibiotic-resistant Mp, vaccines for Mp are urgently needed to ameliorate the pneumonia and secondary complications. The community-acquired respiratory distress syndrome (CARDS) toxin produced by Mp is a pathogenic factor that induces severe inflammatory responses in lung. Although blocking CARDS toxin is expected to mitigate the severity of Mp pneumonia, the potential of CARDS toxin as a vaccine antigen has not been assessed. Here, we examined the effectiveness of vaccine using recombinant CARDS toxin (rCARDS toxin) as an antigen in mice. Immunization with rCARDS toxin induced both rCARDS toxin- and Mp-specific antibody responses, indicating that CARDS toxin is located on the surface of Mp. In addition, immunization with rCARDS toxin decreased not only lung injury, neutrophil infiltration, and the production of inflammatory cytokines but also the persistence of Mp in lung after Mp challenge. Furthermore, we elucidated that the CARDS toxin on the surface of Mp facilitates the adherence of Mp to epithelial cells. In conclusion, we have demonstrated the potential of rCARDS toxin as a vaccine antigen to ameliorate Mp pneumonia by suppressing the inflammatory responses induced by Mp and the persistence of Mp in lung. These data support the development of novel vaccines for Mp pneumonia.

Nasal-subcutaneous prime-boost regimen for inactivated whole-virus influenza vaccine efficiently protects mice against both upper and lower respiratory tract infections.

Although influenza vaccines are effective for reducing viral transmission and the severity of clinical symptoms, influenza viruses still induce considerable morbidity and mortality worldwide. Seasonal influenza viruses infect the upper respiratory tract initially but then often induce severe pulmonary complications in the lower respiratory tract. Therefore, influenza vaccines that prevent viral infection at both the upper and lower respiratory tracts are highly anticipated. Here, we examined whether using different vaccination routes for priming and boosting achieved protection in both regions of the respiratory tract. To this end, we used inactivated whole-virion influenza vaccines to immunize mice either subcutaneously or intranasally for both priming and boosting. Regardless of the route used for boosting, the levels of virus-specific IgG in plasma were higher in mice primed subcutaneously than those in control mice, which received PBS only. In addition, intranasal priming followed by subcutaneous boosting induced higher levels of virus-specific IgG in plasma than those in control mice. The levels of virus-specific nasal IgA were higher in mice that were primed intranasally than in control mice or in mice primed subcutaneously. Furthermore, intranasal priming but not subcutaneous priming provided protection against viral challenge in the upper respiratory tract. In addition, when coupled with subcutaneous boosting, both subcutaneous and intranasal priming protected against viral challenge in the lower respiratory tract. These results indicate that intranasal priming followed by subcutaneous boosting induces both virus-specific IgG in plasma and IgA in nasal washes and protects against virus challenge in both the upper and lower respiratory tracts. Our results will help to develop novel vaccines against influenza viruses and other respiratory viruses.

Peptides with the multibasic cleavage site of the hemagglutinin from highly pathogenic influenza viruses act as cell-penetrating via binding to heparan sulfate and neuropilins.

Cell-penetrating peptides (CPPs) show promise as an attractive delivery vehicle for therapeutic molecules-including nucleic acids, peptides, proteins, and even particulates-into several cell types. It is important to identify new CPPs and select the optimal CPP for each application, because CPPs differ in their internalized efficiency and internalization mechanisms. Here, we identified new CPPs derived from the peptides with the hemagglutinin cleavage site (pHACS) of highly pathogenic influenza viruses. We compared the potential of peptides from the pHACS of four subtypes of influenza A virus (H1, H3, H5, and H7) and an influenza B virus (H1-pHACS, H3-pHACS, H5-pHACS, H7-pHACS, and B-pHACS, respectively) to serve as CPPs. H5-pHACS and H7-pHACS, but not the other peptides, bound to mouse dendritic cells and human epithelial cells and were internalized efficiently into these cells. H5-pHACS and H7-pHACS required glycosaminoglycans, especially heparan sulfate and neuropilins, to bind to the cells. In addition, we designed a mutant H7-pHACS with superior cell-binding capability by changing a single amino acid. Furthermore, when conjugated with antigen, H5-pHACS and H7-pHACS induced antigen-specific antibody responses, demonstrating the usefulness of this antigen-delivery vehicle. Our results will improve our understanding of the mechanisms of CPPs and facilitate the development of novel drug-delivery vehicles designed to improve therapeutic efficacy.

A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice.

Introduction: Vaccinations are ideal for reducing the severity of clinical manifestations and secondary complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, SARS-CoV-2 continues to cause morbidity and mortality worldwide. In contrast to parenteral vaccines such as messenger RNA vaccines, nasal vaccines are expected to be more effective in preventing viral infections in the upper respiratory tract, the primary locus for viral infection and transmission. In this study, we examined the prospects of an inactivated whole-virion (WV) vaccine administered intranasally against SARS-CoV-2. Methods: Mice were immunized subcutaneously (subcutaneous vaccine) or intranasally (nasal vaccine) with the inactivated WV of SARS-CoV-2 as the antigen. Results: The spike protein (S)-specific IgA level was found to be higher upon nasal vaccination than after subcutaneous vaccination. The level of S-specific IgG in the serum was also increased by the nasal vaccine, although it was lower than that induced by the subcutaneous vaccine. The nasal vaccine exhibited a stronger defense against viral invasion in the upper respiratory tract than the subcutaneous vaccine and unimmunized control; however, both subcutaneous and nasal vaccines provided protection in the lower respiratory tract. Furthermore, we found that intranasally administered inactivated WV elicited robust production of S-specific IgA in the nasal mucosa and IgG in the blood of mice previously vaccinated with messenger RNA encoding the S protein. Discussion: Overall, these results suggest that a nasal vaccine containing inactivated WV can be a highly effective means of protection against SARS-CoV-2 infection.

Replication of Human Sapovirus in Human-Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Cells.

Sapoviruses, like noroviruses, are single-stranded positive-sense RNA viruses classified in the family Caliciviridae and are recognized as a causative pathogen of diarrhea in infants and the elderly. Like human norovirus, human sapovirus (HuSaV) has long been difficult to replicate in vitro. Recently, it has been reported that HuSaV can be replicated in vitro by using intestinal epithelial cells (IECs) derived from human tissues and cell lines derived from testicular and duodenal cancers. In this study, we report that multiple genotypes of HuSaV can sufficiently infect and replicate in human-induced pluripotent stem cell-derived IECs. We also show that this HuSaV replication system can be used to investigate the conditions for inactivation of HuSaV by heat and alcohol, and the effects of virus neutralization of antisera obtained by immunization with vaccine antigens, under conditions closer to the living environment. The results of this study confirm that HuSaV can also infect and replicate in human normal IECs regardless of their origin and are expected to contribute to future virological studies.

Effective SARS-CoV-2 replication of monolayers of intestinal epithelial cells differentiated from human induced pluripotent stem cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe acute respiratory symptoms in humans. Controlling the coronavirus disease pandemic is a worldwide priority. The number of SARS-CoV-2 studies has dramatically increased, and the requirement for analytical tools is higher than ever. Here, we propose monolayered-intestinal epithelial cells (IECs) derived from human induced pluripotent stem cells (iPSCs) instead of three-dimensional cultured intestinal organoids as a suitable tool to study SARS-CoV-2 infection. Differentiated IEC monolayers express high levels of angiotensin-converting enzyme 2 and transmembrane protease serine 2 (TMPRSS2), host factors essential for SARS-CoV-2 infection. SARS-CoV-2 efficiently grows in IEC monolayers. Using this propagation system, we confirm that TMPRSS2 inhibition blocked SARS-CoV-2 infection in IECs. Hence, our iPSC-derived IEC monolayers are suitable for SARS-CoV-2 research under physiologically relevant conditions.

Intranasal immunization with an RBD-hemagglutinin fusion protein harnesses preexisting immunity to enhance antigen-specific responses.

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.

Efficient antigen delivery by dendritic cell-targeting peptide via nucleolin confers superior vaccine effects in mice.

Efficient delivery of subunit vaccines to dendritic cells (DCs) is necessary to improve vaccine efficacy, because the vaccine antigen alone cannot induce sufficient protective immunity. Here, we identified DC-targeting peptides using a phage display system and demonstrated the potential of these peptides as antigen-delivery carriers to improve subunit vaccine effectiveness in mice. The fusion of antigen proteins and peptides with DC-targeting peptides induced strong antigen-specific IgG responses, even in the absence of adjuvants. In addition, the DC-targeting peptide improved the distribution of antigens to DCs and antigen presentation by DCs. The combined use of an adjuvant with a DC-targeting peptide improved the effectiveness of the vaccine. Furthermore, nucleolin, located on the DC surface, was identified as the receptor for DC-targeting peptide, and nucleolin was indispensable for the vaccine effect of the DC-targeting peptide. Overall, the findings of this study could be useful for developing subunit vaccines against infectious diseases.

SARS-CoV-2 disrupts respiratory vascular barriers by suppressing Claudin-5 expression.

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.

Neutrophil-Mediated Lung Injury Both via TLR2-Dependent Production of IL-1α and IL-12 p40, and TLR2-Independent CARDS Toxin after Mycoplasma pneumoniae Infection in Mice.

Mycoplasma pneumoniae (Mp) residing extracellularly in the respiratory tract is the primary cause of bacterial community-acquired pneumonia in humans. However, the detailed pathological mechanism of Mp infection, especially inflammation in the lung, remains unclear. This study examined the role of the neutrophils in the inflammation of Mp-induced pneumonia in mice and the mechanism of neutrophil infiltration into the lungs in the Mp-induced pneumonia. We observed massive infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) and lung injury after the Mp challenge. The neutrophils were shown to contribute to lung injury in Mp pneumonia but were not involved in eliminating Mp, suggesting that neutrophils are detrimental to the host in Mp pneumonia. Mp also induced the production of inflammatory cytokines and chemokines in the BALF in a toll-like receptor 2 (TLR2)-dependent manner. Particularly, both interleukin (IL)-1α and IL-12 p40 played a crucial role in neutrophil infiltration into the BALF in a coordinated manner. Both IL-1α and IL-12 p40 were released from the alveolar macrophages depending on the TLR2 and reactive oxygen species. In addition, the community-acquired respiratory distress syndrome (CARDS) toxin from Mp were found to induce neutrophil infiltration into BALF in a TLR2-independent and IL-1α-dependent manner. Collectively, the TLR2-dependent production of both IL-1α and IL-12 p40, and CARDS toxin have been elucidated to play an important role in neutrophil infiltration into the lungs subsequently leading to the lung injury upon Mp infection in mice. These data will aid in the development of therapeutics and vaccines for Mp pneumonia. IMPORTANCE Although Mp-induced pneumonia is usually a self-limiting disease, refractory life-threatening pneumonia is often induced. In addition, the development of alternative therapeutic strategies for Mp is expected because of the emergence of antibiotic-resistant Mp. However, the lack of knowledge regarding the pathogenesis of Mp-induced pneumonia, especially inflammation upon the Mp infection, makes it tedious to design novel therapeutics and vaccines. For example, although neutrophil infiltration is widely recognized as one of the characteristics of Mp-induced pneumonia, the precise role of neutrophils in the aggravation of Mp pneumonia remains unclear. This study showed that the infiltration of neutrophils in the lungs is detrimental to the host in Mp-induced pneumonia in mice. Furthermore, the TLR2-dependent IL-1α and IL-12 p40 production, and CARDS toxin play important roles in neutrophil infiltration into the lung, following lung injury. Our findings apply to the rational design of novel therapeutics and vaccines against Mp.

Synergistic effect of non-neutralizing antibodies and interferon-γ for cross-protection against influenza.

Current influenza vaccines do not typically confer cross-protection against antigenically mismatched strains. To develop vaccines conferring broader cross-protection, recent evidence indicates the crucial role of both cross-reactive antibodies and viral-specific CD4+ T cells; however, the precise mechanism of cross-protection is unclear. Furthermore, adjuvants that can efficiently induce cross-protective CD4+ T cells have not been identified. Here we show that CpG oligodeoxynucleotides combined with aluminum salts work as adjuvants for influenza vaccine and confer strong cross-protection in mice. Both cross-reactive antibodies and viral-specific CD4+ T cells contributed to cross-protection synergistically, with each individually ineffective. Furthermore, we found that downregulated expression of Fcγ receptor IIb on alveolar macrophages due to IFN-γ secreted by viral-specific CD4+ T cells improves the activity of cross-reactive antibodies. Our findings inform the development of optimal adjuvants for vaccines and how influenza vaccines confer broader cross-protection.

Susceptibility Analysis in Several Mouse Strains Reveals Robust T-Cell Responses After Mycoplasma pneumoniae Infection in DBA/2 Mice.

Mycoplasma pneumoniae (Mp) is a highly contagious respiratory pathogen responsible for human community-acquired pneumonia. The number of antibiotic-resistant Mp strains is increasing; therefore, to develop novel therapeutics, it is crucial to precisely understand the pathogenesis of mycoplasma pneumonia. Herein, we examined the susceptibility and response to Mp among eight inbred mouse strains. Following infection, the bacterial load in the bronchoalveolar lavage fluid (BALF) from DBA/2 mice was higher than that in the other tested strains such as BALB/c mice, which are frequently used in Mp research. In contrast, the numbers of CD45+ immune cells and neutrophils in BALF were comparable between BALB/c and DBA/2 mice, with lower numbers observed in C57BL/6J and CBA/N mice than in BALB/c mice. Among the tested strains, the BALF level of interleukin 12 subunit p40 was highest in DBA/2 mice; however, significant differences in other cytokines levels were not observed between BALB/c and DBA/2 mice. After Mp infection, Mp-specific Th1 and Th17 responses were significantly enhanced in DBA/2 mice when compared with BALB/c mice. Furthermore, prior infection with Mp increased the number of neutrophils in BALF after the reinfection of DBA/2 mice through an Mp-specific CD4+ T cell-dependent mechanism. Thus, DBA/2 may be an appropriate strain for evaluating Mp infection. Moreover, a comparison of responses revealed by various inbred mouse strains could be useful for elucidating the pathogenesis of Mycoplasma pneumonia.

Vaccination using inactivated Mycoplasma pneumoniae induces detrimental infiltration of neutrophils after subsequent infection in mice.

Mycoplasma pneumoniae (Mp) is one of the most common causes of community-acquired pneumonia. Given the emergence and high rates of antibiotic-resistant Mp strains, vaccines that prevent the pneumonia and secondary complications due to Mp infection are urgently needed. Although several studies have shown the protective efficacy of Mp vaccines in human clinical trials, some reports suggest that vaccination against Mp exacerbates disease upon subsequent Mp challenge. Therefore, to develop optimal vaccines against Mp, understanding the immune responses that contribute to post-vaccination exacerbation of inflammation is crucial. Here we examined whether Mp vaccination might exacerbate pneumonia after subsequent Mp infection in mice. We found that vaccination with inactivated Mp plus aluminum salts as an adjuvant induced Mp-specific IgG, Th1 cells, and Th17 cells. Toll-like receptor 2 signaling contributed to the induction of an Mp-specific IgG response and was necessary for Mp-specific Th17-cell-but not Th1-cell-responses in vaccinated mice. In addition, vaccination with inactivated Mp plus aluminum salts suppressed the number of Mp organisms in the bronchoalveolar lavage fluid, indicating that vaccination can reduce Mp infection. However, the numbers of total immune cells and neutrophils in bronchoalveolar lavage fluid after Mp challenge did not differ between vaccinated mice and non-vaccinated control mice. Furthermore, depletion of CD4+ T cells prior to Mp challenge decreased pulmonary neutrophil infiltration in vaccinated mice, suggesting that Th1 or Th17 cells (or both) are responsible for the vaccination-induced neutrophil infiltration. These results suggest that, despite reducing Mp infection, vaccination of mice by using inactivated Mp fails to suppress inflammation, such as neutrophil infiltration into the lung, after subsequent Mp infection.

Lipid Nanoparticles Potentiate CpG-Oligodeoxynucleotide-Based Vaccine for Influenza Virus.

Current influenza vaccines are generally effective against highly similar (homologous) strains, but their effectiveness decreases markedly against antigenically mismatched (heterologous) strains. One way of developing a universal influenza vaccine with a broader spectrum of protection is to use appropriate vaccine adjuvants to improve a vaccine's effectiveness and change its immune properties. Oligodeoxynucleotides (ODNs) with unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG ODNs), which are Toll-like-receptor 9 (TLR9) agonists, are among the most promising adjuvants and are already being used in humans. However, the development of novel delivery vehicles to improve adjuvant effects in vivo is highly desirable. Here, we assessed the potential of lipid nanoparticles (LNPs) as CpG ODN delivery vehicles in mice to augment the vaccine adjuvant effects of CpG ODN and enhance the protective spectrum of conventional influenza split vaccine (SV). In vitro, compared with CpG ODN, LNPs containing CpG ODNs (LNP-CpGs) induced significantly greater production of cytokines such as IL-12 p40 and IFN-α by mouse dendritic cells (DCs) and significantly greater expression of the co-stimulatory molecules CD80 and CD86 on DCs. In addition, after subcutaneous administration in mice, compared with CpG ODN, LNP-CpGs enhanced the expression of CD80 and CD86 on plasmacytoid DCs in draining lymph nodes. LNP-CpGs given with SV from H1N1 influenza A virus improved T-cell responses and gave a stronger not only SV-specific but also heterologous-virus-strain-specific IgG2c response than CpG ODN. Furthermore, immunization with SV plus LNP-CpGs protected against not only homologous strain challenge but also heterologous and heterosubtypic strain challenge, whereas immunization with SV plus CpG ODNs protected against homologous strain challenge only. We therefore demonstrated that LNP-CpGs improved the adjuvant effects of CpG ODN and broadened the protective spectrum of SV against influenza virus. We expect that this strategy will be useful in developing adjuvant delivery vehicles and universal influenza vaccines.

Establishment of a central post-stroke pain model using global cerebral ischaemic mice.

OBJECTIVES: Stroke is the leading cause of disability in the world. Central post-stroke pain (CPSP), an intractable secondary disease, is a serious problem that occurs following cerebral stroke. However, the detailed mechanisms underlying CPSP and standard treatments for it are not well established. Therefore, we examined the nociceptive threshold and alterations in the current stimulus threshold of primary afferent neurons in bilateral carotid artery occlusion (BCAO) mice. METHODS: Male ddY mice were subjected to 30 min of BCAO. The development of mechanical and thermal hyperalgesia and changes in current stimulus threshold in the hind paws were measured after BCAO using the von Frey test, plantar test and a Neurometer, respectively. KEY FINDINGS: The threshold for mechanical and thermal hyperalgesia in both hind paws was significantly decreased on day 3 after BCAO as compared with pre-BCAO treatment. Furthermore, the sensitivity of C and Aß fibres (at stimulation of 5 and 2000 Hz, respectively) was increased on day 3 after BCAO as compared with pre-BCAO treatment, while that of Aδ fibres was not altered. CONCLUSIONS: Our data show the development of bilateral hyperalgesia in this model. Potentially, C and Aß fibre-specific hypersensitization after stroke may have contributed to these symptoms.