Intranasal neomycin evokes broad-spectrum antiviral immunity in the upper respiratory tract.

Respiratory virus infections in humans cause a broad-spectrum of diseases that result in substantial morbidity and mortality annually worldwide. To reduce the global burden of respiratory viral diseases, preventative and therapeutic interventions that are accessible and effective are urgently needed, especially in countries that are disproportionately affected. Repurposing generic medicine has the potential to bring new treatments for infectious diseases to patients efficiently and equitably. In this study, we found that intranasal delivery of neomycin, a generic aminoglycoside antibiotic, induces the expression of interferon-stimulated genes (ISGs) in the nasal mucosa that is independent of the commensal microbiota. Prophylactic or therapeutic administration of neomycin provided significant protection against upper respiratory infection and lethal disease in a mouse model of COVID-19. Furthermore, neomycin treatment protected Mx1 congenic mice from upper and lower respiratory infections with a highly virulent strain of influenza A virus. In Syrian hamsters, neomycin treatment potently mitigated contact transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In healthy humans, intranasal application of neomycin-containing Neosporin ointment was well tolerated and effective at inducing ISG expression in the nose in a subset of participants. These findings suggest that neomycin has the potential to be harnessed as a host-directed antiviral strategy for the prevention and treatment of respiratory viral infections.

Enhanced inhibition of MHC-I expression by SARS-CoV-2 Omicron subvariants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that all SARS-CoV-2 VOCs possess the ability to suppress major histocompatibility complex class I (MHC-I) expression. We identified several viral genes that contribute to the suppression of MHC I expression. Notably, MHC-I upregulation was strongly inhibited after SARS-CoV-2 but not influenza virus infection in vivo. While earlier VOCs possess similar capacity as the ancestral strain to suppress MHC-I, the Omicron subvariants exhibited a greater ability to suppress surface MHC-I expression. We identified a common mutation in the E protein of Omicron that further suppressed MHC-I expression. Collectively, our data suggest that in addition to escaping from neutralizing antibodies, the success of Omicron subvariants to cause breakthrough infection and reinfection may in part be due to its optimized evasion from T cell recognition.

Lactate dehydrogenase A inhibition prevents RANKL-induced osteoclastogenesis by reducing enhanced glycolysis.

Osteoclasts are multinucleated, specializes bone-resorbing cells that are derived from the monocyte/macrophage lineage. Excessive resorbing activities of osteoclasts are involved in destructive bone diseases. The detailed mechanism of acidification at the bone adhesion surface during the bone resorption process of osteoclasts remains to be defined. During glycolysis, pyruvate proceeds to the tricarboxylic cycle under aerobic conditions and pyruvate is converted to lactate via lactate dehydrogenase A (LDHA) under anaerobic conditions. However, tumor cells produce ATP during aerobic glycolysis and large amounts of pyruvate are converted to lactate and H+ by LDHA. Lactate and H+ are excreted outside the cell, whereby they are involved in invasion of tumor cells due to the pH drop around the cell. In this study, we focused on aerobic glycolysis and investigated the production of lactate by LDHA in osteoclasts. Expression of LDHA and monocarboxylate transporter 4 (MCT4) was upregulated during osteoclast differentiation. Intracellular and extracellular lactate levels increased with upregulation of LDHA and MCT4, respectively. FX11 (an LDHA inhibitor) inhibited osteoclast differentiation and suppressed the bone-resorbing activity of osteoclasts. We propose that inhibition of LDHA may represent a novel therapeutic strategy for controlling excessive bone resorption in osteoporosis and rheumatoid arthritis.

Inflammasomes and Pyroptosis as Therapeutic Targets for COVID-19.

The inflammatory response to severe acute respiratory syndrome-related coronavirus 2 infection has a direct impact on the clinical outcomes of coronavirus disease 2019 patients. Of the many innate immune pathways that are engaged by severe acute respiratory syndrome-related coronavirus 2, we highlight the importance of the inflammasome pathway. We discuss available pharmaceutical agents that target a critical component of inflammasome activation, signaling leading to cellular pyroptosis, and the downstream cytokines as a promising target for the treatment of severe coronavirus disease 2019-associated diseases.

High ambient temperature dampens adaptive immune responses to influenza A virus infection.

Although climate change may expand the geographical distribution of several vector-borne diseases, the effects of environmental temperature in host defense to viral infection in vivo are unknown. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C impaired adaptive immune responses against infection with viral pathogens, influenza, Zika, and severe fever with thrombocytopenia syndrome phlebovirus. Following influenza virus infection, the high heat-exposed mice failed to stimulate inflammasome-dependent cytokine secretion and respiratory dendritic cell migration to lymph nodes. Although commensal microbiota composition remained intact, the high heat-exposed mice decreased their food intake and increased autophagy in lung tissue. Induction of autophagy in room temperature-exposed mice severely impaired virus-specific CD8 T cells and antibody responses following respiratory influenza virus infection. In addition, we found that administration of glucose or dietary short-chain fatty acids restored influenza virus-specific adaptive immune responses in high heat-exposed mice. These findings uncover an unexpected mechanism by which ambient temperature and nutritional status control virus-specific adaptive immune responses.

Two Conserved Amino Acids within the NSs of Severe Fever with Thrombocytopenia Syndrome Phlebovirus Are Essential for Anti-interferon Activity.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-ß) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-ß responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-ß mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, an association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signaling protein (MAVS)-mediated activation of IFN-ß promoter activity. Although SFTSV NSs did not prevent the ubiquitination of TBK1, it associates with TBK1 through its N-terminal kinase domain (residues 1 to 307) to block the autophosphorylation of TBK1. Furthermore, we found that both wild-type NSs and the 21/23A mutant (NSs in which residues at positions 21 and 23 were replaced with alanine) of SFTSV suppressed NLRP3 inflammasome-dependent interleukin-1ß (IL-1ß) secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signaling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.IMPORTANCE Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection but also in the initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294-amino-acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-ß) production and enhance viral replication. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-ß production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

The RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1ß Secretion.

UNLABELLED: Inflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1ß (IL-1ß) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1ß secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1ß secretion. However, the precise mechanism by which NS1 inhibits IL-1ß secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1ß secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1ß secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1ß secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1ß secretion. IMPORTANCE: Innate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1ß (IL-1ß) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1ß secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1ß secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1ß secretion. These results will facilitate the development of new anti-inflammatory drugs.

SARS-CoV-2-related bat viruses evade human intrinsic immunity but lack efficient transmission capacity.

Circulating bat coronaviruses represent a pandemic threat. However, our understanding of bat coronavirus pathogenesis and transmission potential is limited by the lack of phenotypically characterized strains. We created molecular clones for the two closest known relatives of SARS-CoV-2, BANAL-52 and BANAL-236. We demonstrated that BANAL-CoVs and SARS-CoV-2 have similar replication kinetics in human bronchial epithelial cells. However, BANAL-CoVs have impaired replication in human nasal epithelial cells and in the upper airway of mice. We also observed reduced pathogenesis in mice and diminished transmission in hamsters. Further, we observed that diverse bat coronaviruses evade interferon and downregulate major histocompatibility complex class I. Collectively, our study demonstrates that despite high genetic similarity across bat coronaviruses, prediction of pandemic potential of a virus necessitates functional characterization. Finally, the restriction of bat coronavirus replication in the upper airway highlights that transmission potential and innate immune restriction can be uncoupled in this high-risk family of emerging viruses.

High body temperature increases gut microbiota-dependent host resistance to influenza A virus and SARS-CoV-2 infection.

Fever is a common symptom of influenza and coronavirus disease 2019 (COVID-19), yet its physiological role in host resistance to viral infection remains less clear. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increases host resistance to viral pathogens including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High heat-exposed mice increase basal body temperature over 38 °C to enable more bile acids production in a gut microbiota-dependent manner. The gut microbiota-derived deoxycholic acid (DCA) and its plasma membrane-bound receptor Takeda G-protein-coupled receptor 5 (TGR5) signaling increase host resistance to influenza virus infection by suppressing virus replication and neutrophil-dependent tissue damage. Furthermore, the DCA and its nuclear farnesoid X receptor (FXR) agonist protect Syrian hamsters from lethal SARS-CoV-2 infection. Moreover, we demonstrate that certain bile acids are reduced in the plasma of COVID-19 patients who develop moderate I/II disease compared with the minor severity of illness group. These findings implicate a mechanism by which virus-induced high fever increases host resistance to influenza virus and SARS-CoV-2 in a gut microbiota-dependent manner.

SARS-CoV-2 Omicron subvariants evolved to promote further escape from MHC-I recognition.

SARS-CoV-2 variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that all SARS-CoV-2 VOCs possess the ability to suppress MHC I expression. We identified several viral genes that contribute to the suppression of MHC I expression. Notably, MHC-I upregulation was strongly inhibited after SARS-CoV-2 infection in vivo. While earlier VOCs possess similar capacity as the ancestral strain to suppress MHC I, Omicron subvariants exhibit a greater ability to suppress surface MHC-I expressions. Collectively, our data suggest that, in addition to escape from neutralizing antibodies, the success of Omicron subvariants to cause breakthrough infection and reinfection may in part be due to its optimized evasion from T cell recognition.

Oral Bacteria Combined with an Intranasal Vaccine Protect from Influenza A Virus and SARS-CoV-2 Infection.

The gut microbiota plays a critical role in the induction of adaptive immune responses to influenza virus infection. However, the role of nasal bacteria in the induction of the virus-specific adaptive immunity is less clear. Here, we found that disruption of nasal bacteria by intranasal application of antibiotics before influenza virus infection enhanced the virus-specific antibody response in a MyD88-dependent manner. Similarly, disruption of nasal bacteria by lysozyme enhanced antibody responses to intranasally administered influenza virus hemagglutinin (HA) vaccine in a MyD88-dependent manner, suggesting that intranasal application of antibiotics or lysozyme could release bacterial pathogen-associated molecular patterns (PAMPs) from disrupted nasal bacteria that act as mucosal adjuvants by activating the MyD88 signaling pathway. Since commensal bacteria in the nasal mucosal surface were significantly lower than those in the oral cavity, intranasal administration of HA vaccine alone was insufficient to induce the vaccine-specific antibody response. However, intranasal supplementation of cultured oral bacteria from a healthy human volunteer enhanced antibody responses to an intranasally administered HA vaccine. Finally, we demonstrated that oral bacteria combined with an intranasal vaccine protect from influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our results reveal the role of nasal bacteria in the induction of the virus-specific adaptive immunity and provide clues for developing better intranasal vaccines. IMPORTANCE Intranasal vaccination induces the nasal IgA antibody which is protective against respiratory viruses, such as influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, understanding how mucosal immune responses are elicited following viral infection is important for developing better vaccines. Here, we focused on the role of nasal commensal bacteria in the induction of immune responses following influenza virus infection. To deplete nasal bacteria, we intranasally administered antibiotics to mice before influenza virus infection and found that antibiotic-induced disruption of nasal bacteria could release bacterial components which stimulate the virus-specific antibody responses. Since commensal bacteria in nasal mucosa were significantly lower than those in the oral cavity, intranasal administration of split virus vaccine alone was insufficient to induce the vaccine-specific antibody response. However, intranasal supplementation of cultured oral bacteria from a healthy human volunteer enhanced antibody responses to the intranasally administered vaccine. Therefore, both integrity and amounts of nasal bacteria may be critical for an effective intranasal vaccine.

Intranasal priming induces local lung-resident B cell populations that secrete protective mucosal antiviral IgA.

Antibodies secreted at the mucosal surface play an integral role in immune defense by serving to neutralize the pathogen and promote its elimination at the site of entry. Secretory immunoglobulin A (IgA) is a predominant Ig isotype at mucosal surfaces whose epithelial cells express polymeric Ig receptor capable of transporting dimeric IgA to the lumen. Although the role of IgA in intestinal mucosa has been extensively studied, the cell types responsible for secreting the IgA that protects the host against pathogens in the lower respiratory tract are less clear. Here, using a mouse model of influenza virus infection, we demonstrate that intranasal, but not systemic, immunization induces local IgA secretion in the bronchoalveolar space. Using single-cell RNA sequencing, we found a heterogeneous population of IgA-expressing cells within the respiratory mucosa, including tissue-resident memory B cells, plasmablasts, and plasma cells. IgA-secreting cell establishment within the lung required CXCR3. An intranasally administered protein-based vaccine also led to the establishment of IgA-secreting cells in the lung, but not when given intramuscularly or intraperitoneally. Last, local IgA secretion correlated with superior protection against secondary challenge with homologous and heterologous virus infection than circulating antibodies alone. These results provide key insights into establishment of protective immunity in the lung based on tissue-resident IgA-secreting B cells and inform vaccine strategies designed to elicit highly effective immune protection against respiratory virus infections.

Seasonality of Respiratory Viral Infections.

The seasonal cycle of respiratory viral diseases has been widely recognized for thousands of years, as annual epidemics of the common cold and influenza disease hit the human population like clockwork in the winter season in temperate regions. Moreover, epidemics caused by viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) and the newly emerging SARS-CoV-2 occur during the winter months. The mechanisms underlying the seasonal nature of respiratory viral infections have been examined and debated for many years. The two major contributing factors are the changes in environmental parameters and human behavior. Studies have revealed the effect of temperature and humidity on respiratory virus stability and transmission rates. More recent research highlights the importance of the environmental factors, especially temperature and humidity, in modulating host intrinsic, innate, and adaptive immune responses to viral infections in the respiratory tract. Here we review evidence of how outdoor and indoor climates are linked to the seasonality of viral respiratory infections. We further discuss determinants of host response in the seasonality of respiratory viruses by highlighting recent studies in the field.

Influenza Virus-Induced Oxidized DNA Activates Inflammasomes.

Influenza virus M2 and PB1-F2 proteins have been proposed to activate the Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome in macrophages by altering intracellular ionic balance or mitochondrial reactive oxygen species (ROS) production. However, the precise mechanism by which these viral proteins trigger the NLRP3 inflammasome activation remains unclear. Here we show that influenza virus stimulates oxidized DNA release from macrophages. Ion channel activity of the M2 protein or mitochondrial localization of the PB1-F2 protein was required for oxidized DNA release. The oxidized DNA enhanced influenza virus-induced IL-1ß secretion, whereas inhibition of mitochondrial ROS production by antioxidant Mito-TEMPO decreased the virus-induced IL-1ß secretion. In addition, we show that influenza virus stimulates IL-1ß secretion from macrophages in an AIM2-dependent manner. These results provide a missing link between influenza viral proteins and the NLRP3 inflammasome activation and reveal the importance of influenza virus-induced oxidized DNA in inflammasomes activation.

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.

Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses.

Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.

Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome.

Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1ß) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1ß secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1ß secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K+ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

Consecutive inoculations of influenza virus vaccine and poly(I:C) protects mice against homologous and heterologous virus challenge.

Mucosal immunity induced through natural infection by influenza virus has potent cross-protective activity, compared to subcutaneous vaccination-induced systemic immunity. Compared to natural infection with influenza virus, however, a single intranasal vaccination with an inactivated influenza virus vaccine and poly(I:C) is not sufficient to induce primary immune response in naïve animals. The reasons for this moderate effect are not fully understood. Here, we demonstrated that intranasal vaccination with formalin-inactivated influenza virus vaccine and poly(I:C) for five consecutive days elicits high levels of virus-specific nasal IgA and serum IgG responses, while vaccination without poly(I:C) induced little response. Mice immunized with influenza virus vaccine and poly(I:C) for five consecutive days sustained high levels of virus-specific IgA in nasal wash and IgG in serum until at least 6months after vaccination. Furthermore, intranasal vaccination with influenza virus vaccine and poly(I:C) protected mice against homologous and heterologous influenza virus challenge. These results suggest that consecutive inoculations of influenza virus vaccine and poly(I:C) is an alternative method to induce primary immune responses in naïve subjects.