Influenza viral matrix 1 protein aggravates viral pathogenicity by inducing TLR4-mediated reactive oxygen species production and apoptotic cell death.

Influenza virus is one of the most challenging viruses threating human health. Since infection with influenza virus triggers inflammatory responses and induces cell death, the molecular and cellular mechanisms by which the virus-infected cells undergo apoptotic and necrotic cell death have been widely studied. However, most of the studies have focused on the molecular events occurring in the cytosol and there is limited information on the physiological correlation between virus-induced cell death and the viral pathogenesis in vivo. In this study, we demonstrate that the influenza virus matrix 1 (M1) protein is released from virus-infected cells and triggers apoptotic cell death of lung epithelial and pulmonary immune cells, through the activation of Toll-like receptor 4 (TLR4) signaling. Treatment with M1 protein led to robust cellular inflammatory responses, such as the production of proinflammatory cytokines and cellular reactive oxygen species (ROS), and induction of cell death. When M1 protein was administered in vivo, it induced the activation of inflammatory responses and cell death in the lungs. Furthermore, the administration of M1 aggravated lung pathology and mortality of the virus-infected mice in a TLR4-dependent manner. These results demonstrate that M1 is an important pathogenic factor contributing to influenza virus pathogenicity by enhancing cell death in the lungs, thereby expanding our understanding of the molecular mechanism of influenza virus-induced cell death through the interaction with an innate immune receptor.

Pseudomonas stutzeri PM101005 inhaled with atmospheric particulate matter induces lung damage through inflammatory responses.

Atmospheric particulate matter (PM) contains a mixture of chemical and biological elements that pose threat to human health by increasing susceptibility to respiratory diseases. Although the identification of the microorganisms composing the PM has been assessed, their immunological impacts are still questionable. Here, we examined the mechanisms responsible for the pathogenicity of Pseudomonas stutzeri PM101005 (PMPS), a bacterium isolated from fine dust, in lung epithelial cells, alveolar cells, and macrophages. Relative to its comparative strain Pseudomonas stutzeri (PS), infections with PMPS induced higher production of inflammatory cytokines and chemokines, mediated by the activation of NF-κB and MAPK signaling pathways. Additionally, with three-dimensional (3D) airway spheroids which mimic the human bronchial epithelium, we confirmed that PMPS infections lead to relatively higher induction of pro-inflammatory cytokines than PM infections. Consistent results were observed in murine models as the infections with PMPS provoked greater inflammatory responses than the infections with PS. These PMPS-induced responses were mediated by the signaling pathways of the Toll-like receptors (TLRs), which regulated PMPS infection and played an important role in the expression of the antibiotic peptide ß-defensin 3 (BD3) that suppressed PMPS proliferation. Moreover, PM pretreatment enhanced inflammatory responses and tissue damage of PMPS, while reducing BD3 expression. Overall, these results indicate that PM-isolated PMPS induce TLR-mediated inflammatory responses in lung tissues, and contributes to the understanding of the etiology of PM-induced respiratory damage.

Extracellular nucleoprotein exacerbates influenza virus pathogenesis by activating Toll-like receptor 4 and the NLRP3 inflammasome.

Host immune responses, such as those initiated by pattern recognition receptor (PRR) activation, are important for viral clearance and pathogenesis. However, little is known about the interactions of viral proteins with surface PRRs or, more importantly, the association of innate immune activation with viral pathogenesis. In this study, we showed that internal influenza virus proteins were released from infected cells. Among these proteins, nucleoprotein (NP) played a critical role in viral pathogenesis by stimulating neighboring cells through toll-like receptor (TLR)2, TLR4, and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Through the activation of these PRRs, NP induced the production of interleukin (IL)-1ß and IL-6, which subsequently led to the induction of trypsin. Trypsin induced by NP increased the infectivity of influenza virus, leading to increases in viral replication and pathology upon subsequent viral infection. These results reveal the role of released NP in influenza pathogenesis and highlight the importance of the interactions of internal viral proteins with PRRs in the extracellular compartment during viral pathogenesis.

Pretreatment of outer membrane vesicle and subsequent infection with influenza virus induces a long-lasting adaptive immune response against broad subtypes of influenza virus.

Influenza is an acute respiratory disease and a global health problem. Although influenza vaccines are commercially available, frequent antigenic changes in hemagglutinin might render them less effective or unavailable. We previously reported that modified outer membrane vesicle (fmOMV) provided immediate and robust protective immunity against various subtypes of influenza virus. However, the effect was transient because it was innate immunity-dependent. In this study, we investigated the effects of consecutive administration of fmOMV and influenza virus on the adaptive immune response and long-term protective immunity against influenza virus. When the mice were pretreated with fmOMV and subsequently infected with influenza virus, strong influenza-specific antibody and T cell responses were induced in both systemic and lung mucosal compartments without pathogenic symptoms. Upon the secondary viral challenge at week 4, the mice given fmOMV and influenza virus exhibited almost complete protection against homologous and heterologous viral challenge. More importantly, this strong protective immunity lasted up to 18 weeks after the first infection. These results show that pretreatment with fmOMV and subsequent infection with influenza virus efficiently induces broad and long-lasting protective immunity against various virus subtypes, suggesting a novel antiviral strategy against newly-emerging viral diseases without suitable vaccines or therapeutics.

Current Status of COVID-19 Vaccine Development: Focusing on Antigen Design and Clinical Trials on Later Stages.

The global outbreak of coronavirus disease 2019 (COVID-19) is still threatening human health, economy, and social life worldwide. As a counteraction for this devastating disease, a number of vaccines are being developed with unprecedented speed combined with new technologies. As COVID-19 vaccines are being developed in the absence of a licensed human coronavirus vaccine, there remain further questions regarding the long-term efficacy and safety of the vaccines, as well as immunological mechanisms in depth. This review article discusses the current status of COVID-19 vaccine development, mainly focusing on antigen design, clinical trials in later stages, and immunological considerations for further study.

Exosomes released from Shiga toxin 2a-treated human macrophages modulate inflammatory responses and induce cell death in toxin receptor expressing human cells.

Shiga toxins (Stxs) produced by Stx-producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx-mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte-derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage-like differentiated THP-1 cells treated with Stxs secreted Stx-associated exosomes (Stx-Exo) of 90-130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb3 )-dependent manner. Stx2-Exo engulfed by Gb3 -positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK-2. Stx2-Exo contained pro-inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2-Exo-mediated human renal cell death. Stx2-Exo isolated from human primary monocyte-derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx-containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin-sensitive cells. Therapeutic interventions targeting Stx-containing exosomes may prevent or ameliorate Stx-mediated acute vascular dysfunction.

Outer membrane vesicle increases the efficacy of an influenza vaccine in a diet-induced obese mouse model.

Obesity has been associated with increased symptoms and mortality in influenza patients and impaired immune responses to the influenza vaccine. To date, however, there is no effective adjuvant to improve vaccine efficacy for the obese population. To address this issue, we generated a modified outer membrane vesicle with attenuated endotoxicity (fmOMV) and tested its adjuvant effect on the influenza vaccine in comparison with a squalene-based oil-in-water adjuvant (AddaVax) using a diet-induced obese (DIO) mouse model. Although coadministration of fmOMV did not affect neutralizing antibody (Ab) response, it preferentially induced IgG2c antibody response and significantly increased the vaccine-induced T cell response. More importantly, fmOMV conferred significant protection against homologous and heterologous influenza virus challenge, whereas AddaVax showed marginal protection irrespective of the strongest Ab and T cell responses in DIO mice. These results indicate that fmOMV improves the antigen-specific T cell response and the efficacy of an influenza vaccine, suggesting a potential influenza vaccine adjuvant for the obese population.

Bacterial Outer Membrane Vesicles Provide Broad-Spectrum Protection against Influenza Virus Infection via Recruitment and Activation of Macrophages.

Influenza A virus (IAV) poses a constant worldwide threat to human health. Although conventional vaccines are available, their protective efficacy is type or strain specific, and their production is time-consuming. For the control of an influenza pandemic in particular, agents that are immediately effective against a wide range of virus variants should be developed. Although pretreatment of various Toll-like receptor (TLR) ligands have already been reported to be effective in the defense against subsequent IAV infection, the efficacy was limited to specific subtypes, and safety concerns were also raised. In this study, we investigated the protective effect of an attenuated bacterial outer membrane vesicle -harboring modified lipid A moiety of lipopolysaccharide (fmOMV) against IAV infection and the underlying mechanisms. Administration of fmOMV conferred significant protection against a lethal dose of pandemic H1N1, PR8, H5N2, and highly pathogenic H5N1 viruses; this broad antiviral activity was dependent on macrophages but independent of neutrophils. fmOMV induced recruitment and activation of macrophages and elicited type I IFNs. Intriguingly, fmOMV showed a more significant protective effect than other TLR ligands tested in previous reports, without exhibiting any adverse effect. These results show the potential of fmOMV as a prophylactic agent for the defense against influenza virus infection.

Attachment of flagellin enhances the immunostimulatory activity of a hemagglutinin-ferritin nano-cage.

Hemagglutinin (HA) displayed on a ferritin nano-cage has been shown to be effective in generating a potent immune response against a broad range of influenza infections. Here, we showed that conjugation of flagellin together with HA to the exterior surface of the ferritin cage greatly enhanced not only the humoral immune response in mice but also antigen-specific T cell responses that include Th1 cytokine secretion. The effect of flagellin remained essentially unchanged when the molar ratio of flagellin to HA was reduced from 1:1 to 1:3. Injection of the ferritin-HA-flagellin cage provided protection against lethal virus challenge in mice. We used a small immunoglobulin fragment VL12.3 as a convenient method for attaching HA and flagellin to the ferritin cage. This attachment method can be used for rapid screening of a variety of protein cages and nano-assemblies to identify the most suitable carrier and adjuvant proteins for the target antigen.

Outer membrane vesicles harboring modified lipid A moiety augment the efficacy of an influenza vaccine exhibiting reduced endotoxicity in a mouse model.

Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103+ dendritic cells in the mediastinal lymph nodes was significantly increased after fmOMV co-administration. Notably, the mice co-immunized with fmOMV showed a significantly higher protection rate against challenge with a lethal dose of homologous or heterologous influenza viruses without adverse effects. These results show the potential of fmOMV as an effective mucosal adjuvant for intranasal vaccines.

Virulence of a novel reassortant canine H3N2 influenza virus in ferret, dog and mouse models.

An outbreak of a canine influenza virus (CIV) H3N2 reassortant derived from pandemic (pdm) H1N1 and CIV H3N2 in companion animals has underscored the urgent need to monitor CIV infections for potential zoonotic transmission of influenza viruses to humans. In this study, we assessed the virulence of a novel CIV H3N2 reassortant, VC378, which was obtained from a dog that was coinfected with pdm H1N1 and CIV H3N2, in ferrets, dogs, and mice. Significantly enhanced virulence of VC378 was demonstrated in mice, although the transmissibility and pathogenicity of VC378 were similar to those of classical H3N2 in ferrets and dogs. This is notable because mice inoculated with an equivalent dose of classical CIV H3N2 showed no clinical signs and no lethality. We found that the PA and NS gene segments of VC378 were introduced from pdmH1N1, and these genes included the amino acid substitutions PA-P224S and NS-I123V, which were previously found to be associated with increased virulence in mice. Thus, we speculate that the natural reassortment between pdm H1N1 and CIV H3N2 can confer virulence and that continuous surveillance is needed to monitor the evolution of CIV in companion animals.