Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Influenza-induced Tpl2 expression within alveolar epithelial cells is dispensable for host viral control and anti-viral immunity.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that regulates the expression of inflammatory mediators in response to Toll-like receptors (TLR) and cytokine receptors. Global ablation of Tpl2 leads to severe disease in response to influenza A virus (IAV) infection, characterized by respiratory distress, and studies in bone marrow chimeric mice implicated Tpl2 in non-hematopoietic cells. Lung epithelial cells are primary targets and replicative niches of influenza viruses; however, the specific regulation of antiviral responses by Tpl2 within lung epithelial cells has not been investigated. Herein, we show that Tpl2 is basally expressed in primary airway epithelial cells and that its expression increases in both type I and type II airway epithelial cells (AECI and AECII) in response to influenza infection. We used Nkx2.1-cre to drive Tpl2 deletion within pulmonary epithelial cells to delineate epithelial cell-specific functions of Tpl2 during influenza infection in mice. Although modest increases in morbidity and mortality were attributed to cre-dependent deletion in lung epithelial cells, no alterations in host cytokine production or lung pathology were observed. In vitro, Tpl2 inhibition within the type I airway epithelial cell line, LET1, as well as genetic ablation in primary airway epithelial cells did not alter cytokine production. Overall, these findings establish that Tpl2-dependent defects in cells other than AECs are primarily responsible for the morbidity and mortality seen in influenza-infected mice with global Tpl2 ablation.

ADAM10 partially protects mice against influenza pneumonia by suppressing specific myeloid cell population.

The influenza virus infection poses a serious health threat worldwide. Myeloid cells play pivotal roles in regulating innate and adaptive immune defense. A disintegrin and metalloproteinase (ADAM) family of proteins contributes to various immune responses; however, the role of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in influenza virus infection remains largely unknown. Herein, we investigated its role, focusing on myeloid cells, during influenza virus infection in mice. ADAM10 gene (Adam10)flox/flox/Lyz2-Cre (Adam10ΔLyz2) and control Adam10flox/flox mice were intranasally infected with 200 plaque-forming units of influenza virus A/H1N1/PR8/34. Adam10ΔLyz2 mice exhibited a significantly higher mortality rate, stronger lung inflammation, and a higher virus titer in the lungs than control mice. Macrophages and inflammatory cytokines, such as TNF-α, IL-1ß, and CCL2, were increased in bronchoalveolar lavage fluid from Adam10ΔLyz2 mice following infection. CD11b+Ly6G-F4/80+ myeloid cells, which had an inflammatory monocyte/macrophage-like phenotype, were significantly increased in the lungs of Adam10ΔLyz2 mice. Adoptive transfer experiments suggested that these cells likely contributed to the poorer prognosis in Adam10ΔLyz2 mice. Seven days after infection, CD11b+Ly6G-F4/80+ lung cells exhibited significantly higher arginase-1 expression levels in Adam10ΔLyz2 mice than in control mice, whereas an arginase-1 inhibitor improved the prognosis of Adam10ΔLyz2 mice. Enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF)/GM-CSF receptor signaling likely contributed to this process. Collectively, these results indicate that myeloid ADAM10 protects against influenza virus pneumonia and may be a promising therapeutic target.

Exacerbation of disease by intranasal liquid administration following influenza virus infection in mice.

Although numerous studies have clarified the synergistic pathogenesis in mouse models of influenza A virus (IAV)-associated dual infections, fewer studies have investigated the influence of intranasal liquid administration on the disease. This study explored the effects of intranasal PBS administration in mouse models of mimic IAV dual infection and the infectious dose of IAV that caused equivalent pathogenesis in different dual infection models. Weights, survival rates, virus loads, lung indexes and lung pathology were compared. We demonstrated that intranasal PBS administration following H1N1 or H3N2 infection increased weight loss, mortality, virus replication and lung damage. No difference was observed if the order was reversed or PBS was given simultaneously with IAV. To induce equivalent virulence, a 20-fold difference in the infectious dose was needed when the H3N2-PBS superinfection and H3N2-PBS coinfection or PBS-H3N2 superinfection groups were compared. Our study demonstrated that the unfavourable effect of intranasal liquid administration should not be neglected and that both the strain and infectious dose of IAV should be considered to avoid an illusion of synergistic pathogenicity when establishing IAV-associated dual infection model. A 20-fold lower dose than that of coinfection may be a better choice for secondary infection following IAV.

Pre-Treatment with Zirconia Nanoparticles Reduces Inflammation Induced by the Pathogenic H5N1 Influenza Virus.

BACKGROUND: New approaches are urgently needed to fight influenza viral infection. Previous research has shown that zirconia nanoparticles can be used as anticancer materials, but their antiviral activity has not been reported. Here, we investigated the antiviral effect of zirconia (ZrO2) nanoparticles (NPs) against a highly pathogenic avian influenza virus. MATERIALS AND METHODS: In this study, the antiviral effects of ZrO2 on H5N1 virus were assessed in vivo, and the molecular mechanism responsible for this protection was investigated. RESULTS: Mice treated with 200 nm positively-charged NPs at a dose of 100 mg/kg showed higher survival rates and smaller reductions in weight. 200 nm ZrO2 activated mature dendritic cells and initially promoted the expression of cytokines associated with the antiviral response and innate immunity. In the lungs of H5N1-infected mice, ZrO2 treatment led to less pathological lung injury, significant reduction in influenza A virus replication, and overexpression of pro-inflammatory cytokines. CONCLUSION: This antiviral study using zirconia NPs shows protection of mice against highly pathogenic avian influenza virus and suggests strong application potential for this method, introducing a new tool against a wide range of microbial infections.

Pneumococcal conjugate vaccine modulates macrophage-mediated innate immunity in pneumonia caused by Streptococcus pneumoniae following influenza.

Pneumococcal conjugate vaccination (PCV) may prevent influenza-related pneumonia, including Streptococcus pneumoniae pneumonia. To investigate PCV efficacy against secondary pneumococcal pneumonia following influenza, PCV was administered intramuscularly 2 and 5 weeks before S. pneumoniae serotype-3 colonization of murine nasopharynges followed by intranasal challenge with a sublethal dose of influenza A virus. Bacterial and viral loads, including innate immune responses were compared across conditions. PCV vaccination improved the survival of mice with secondary pneumococcal pneumonia and significantly reduced the pulmonary bacterial burden. Increased monocyte/macrophage influx into the lungs, alleviated loss of alveolar macrophages and decreased neutrophil influx into the lungs occurred in PCV-treated mice irrespective of pneumococcal colonization. Higher monocyte chemoattractant protein 1 levels and lower levels of CXCL1, interferon-γ, interleukin-17A, and IL-10, were detected in PCV-treated mice. Additionally, PCV treatment activated the macrophage intracellular killing of S. pneumoniae. Collectively, PCV potentially modulates the host's innate immunity and specific antibodies induction. Macrophage-related innate immunity should be further explored to elucidate the efficacy and mechanisms of PCV versus influenza-related life-threatening diseases.

Identification of lncRNA-155 encoded by MIR155HG as a novel regulator of innate immunity against influenza A virus infection.

Long noncoding RNAs (lncRNAs) are single-stranded RNA molecules longer than 200 nt that regulate many cellular processes. MicroRNA 155 host gene (MIR155HG) encodes the microRNA (miR)-155 that regulates various signalling pathways of innate and adaptive immune responses against viral infections. MIR155HG also encodes a lncRNA that we call lncRNA-155. Here, we observed that expression of lncRNA-155 was markedly upregulated during influenza A virus (IAV) infection both in vitro (several cell lines) and in vivo (mouse model). Interestingly, robust expression of lncRNA-155 was also induced by infections with several other viruses. Disruption of lncRNA-155 expression in A549 cells diminished the antiviral innate immunity against IAV. Furthermore, knockout of lncRNA-155 in mice significantly increased IAV replication and virulence in the animals. In contrast, overexpression of lncRNA-155 in human cells suppressed IAV replication, suggesting that lncRNA-155 is involved in host antiviral innate immunity induced by IAV infection. Moreover, we found that lncRNA-155 had a profound effect on expression of protein tyrosine phosphatase 1B (PTP1B) during the infection with IAV. Inhibition of PTP1B by lncRNA-155 resulted in higher production of interferon-beta (IFN-ß) and several critical interferon-stimulated genes (ISGs). Together, these observations reveal that MIR155HG derived lncRNA-155 can be induced by IAV, which modulates host innate immunity during the virus infection via regulation of PTP1B-mediated interferon response.

The parasite-derived rOv-ASP-1 is an effective antigen-sparing CD4+ T cell-dependent adjuvant for the trivalent inactivated influenza vaccine, and functions in the absence of MyD88 pathway.

Vaccination remains the most cost-effective biomedical approach for controlling influenza disease. In times of pandemics, however, these vaccines cannot be produced in sufficient quantities for worldwide use by the current manufacturing capacities and practices. What is needed is the development of adjuvanted vaccines capable of inducing an adequate or better immune response at a decreased antigen dose. Previously we showed that the protein adjuvant rOv-ASP-1 augments influenza-specific antibody titers and survival after virus challenge in both young adult and old-age mice when administered with the trivalent inactivated influenza vaccine (IIV3). In this study we show that a reduced amount of rOv-ASP-1, with 40-times less IIV3 can also induce protection. Apparently the potency of the rOv-ASP-1 adjuvanted IIV3 vaccine is independent of the IIV3-specific Th1/Th2 associated antibody responses, and independent of the presence of HAI antibodies. However, CD4+ T helper cells were indispensable for the protection. Further, rOv-ASP-1 with or without IIV3 elicited the increased level of various chemokines, which are known chemoattractant for immune cells, into the muscle 4 h after immunization, and significantly induced the recruitment of monocytes, macrophages and neutrophils into the muscles. The recruited monocytes had higher expression of the activation marker MHCII on their surface as well as CXCR3 and CCR2; receptors for IP-10 and MCP-1, respectively. These results show that the rOv-ASP-1 adjuvant allows substantial antigen sparing of IIV3 by stimulating at the site of injection the accumulation of chemokines and the recruitment of immune cells that can augment the activation of CD4+ T cell immune responses, essential for the production of antibody responses. Protection elicited by the rOv-ASP-1 adjuvanted IIV3 vaccine also appears to function in the absence of MyD88-signaling. Future studies will attempt to delineate the precise mechanisms by which the rOv-ASP-1 adjuvanted IIV3 vaccine works.

Inflammatory Monocytes Drive Influenza A Virus-Mediated Lung Injury in Juvenile Mice.

Healthy children are more likely to die of influenza A virus (IAV) infection than healthy adults. However, little is known about the mechanisms underlying the impact of young age on the development of life-threatening IAV infection. We report increased mortality in juvenile mice compared with adult mice at each infectious dose of IAV. Juvenile mice had sustained elevation of type I IFNs and persistent NLRP3 inflammasome activation in the lungs, both of which were independent of viral titer. Juvenile mice, but not adult mice, had increased MCP-1 levels that remained high even after viral clearance. Importantly, continued production of MCP-1 was associated with persistent recruitment of monocytes to the lungs and prolonged elevation of inflammatory cytokines. Transcriptional signatures of recruited monocytes to the juvenile and adult IAV-infected lungs were assessed by RNA-seq. Genes associated with a proinflammatory signature were upregulated in the juvenile monocytes compared with adult monocytes. Depletion of monocytes with anti-CCR2 Ab decreased type I IFN secretion, NLRP3 inflammasome activation, and lung injury in juvenile mice. This suggests an exaggerated inflammatory response mediated by increased recruitment of monocytes to the lung, and not an inability to control viral replication, is responsible for severe IAV infection in juvenile mice. This study provides insight into severe IAV infection in juveniles and identifies key inflammatory monocytes that may be central to pediatric acute lung injury secondary to IAV.

GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization.

BACKGROUND: Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. METHODS: Mice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi. RESULTS: Supra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an "M1-like" to a more "M2-like" activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ). CONCLUSIONS: Our data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic "M1-like" macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

Severe Pneumonia Caused by Coinfection With Influenza Virus Followed by Methicillin-Resistant Staphylococcus aureus Induces Higher Mortality in Mice.

Background: Coinfection with influenza virus and bacteria is a major cause of high mortality during flu pandemics. Understanding the mechanisms behind such coinfections is of utmost importance both for the clinical treatment of influenza and the prevention and control of epidemics. Methods: To investigate the cause of high mortality during flu pandemics, we performed coinfection experiments with H1N1 influenza virus and Staphylococcus aureus in which mice were infected with bacteria at time points ranging from 0 to 7 days after infection with influenza virus. Results: The mortality rates of mice infected with bacteria were highest 0-3 days after infection with influenza virus; lung tissues extracted from these co-infected mice showed higher infiltrating cells and thicker lung parenchyma than lung samples from coinfected mice in which influenza virus was introduced at other times and sequences. The levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-6 in the 0-3 day coinfected group were significantly higher than those in the other groups (p < 0.01), as were the mRNA levels of IFN-γ, IL-6, and TNF-α. Coinfection with influenza virus and S. aureus led to high mortality rates that are directly dependent on the sequence and timing of infection by both pathogens. Moreover, coinfection following this particular schedule induced severe pneumonia, leading to increased mortality. Conclusions: Our data suggest that prevention of bacterial co-infection in the early stage of influenza virus infection is critical to reducing the risk of clinical mortality.

Nlrp12 Mediates Adverse Neutrophil Recruitment during Influenza Virus Infection.

Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation, however, neutrophils are necessary for optimal viral control. In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12-/- mice are protected from lethality during IAV infection and show decreased vascular permeability, fewer pulmonary neutrophils, and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12-/- neutrophils and dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp12-/- dendritic cells was not due to a difference in CXCL1 protein stability, but instead to a decrease in Cxcl1 mRNA stability. Together, these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses.

Mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza H5N1 virus infection.

Infection with the highly pathogenic avian influenza H5N1 virus results in a high incidence of mortality in humans. Severe complications from infection are often associated with hypercytokinemia. However, current neuraminidase inhibitors (NAIs) have several limitations including the appearance of oseltamivir-resistant H5N1 virus and the inability to completely ameliorate hyper-immune responses. To overcome these limitations, we evaluated the anti-viral activity of mycophenolic mofetil (MMF) against A/Vietnam/1194/2004 (H5N1) virus infection using MDCK cells and mice. The IC50 of MMF (0.94 µM) was comparable to that of zanamivir (0.87 µM) in H5N1 virus-infected MDCK cells based on ELISA. Time-course assays demonstrated that MMF completely inhibited H5N1 viral mRNA replication and protein expression for approximately 8 h after the initiation of treatment. In addition, MMF treatment protected 100% of mice, and lung viral titers were substantially reduced. The anti-viral mechanism of MMF against H5N1 virus infection was further confirmed to depend on the inhibition of cellular inosine monophosphate dehydrogenase (IMPDH) by exogenous guanosine, which inhibits viral mRNA and protein expression. Moreover, IL-1ß, IFN-ß, IL-6, and IP-10 mRNA expression levels were significantly downregulated in MDCK cells with MMF treatment. These results indicated that MMF could represent a novel inhibitor of viral replication and a potent immunomodulator for the treatment of H5N1 virus infection.

Oral administration of Euglena gracilis Z and its carbohydrate storage substance provides survival protection against influenza virus infection in mice.

Euglena gracilis Z is a micro-algae that is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of Euglena gracilis Z has ß-1, 3-glucan structure. Euglena gracilis Z and paramylon are reported to affect the immune system. In this study, we investigated the protective effects of Euglena gracilis Z and paramylon against influenza virus infection in mice. Euglena gracilis Z and paramylon were administered to mice as a 2% dietary mixture ad libitum. At 2 weeks after initiation of dietary administration, mice were infected intranasally with influenza virus A/PR/8/34 (H1N1). Survival rate was monitored 10 days after infection. In addition, we performed virus titer and cytokine profiles in the lung. High survival rates were observed for Euglena gracilis Z and paramylon-treated groups compared to the control group. Significantly lower virus titer in the lung was observed in the Euglena gracilis Z and paramylon-treated groups compared to the control group from day 1 after infection. Higher amount of IL-1ß, IL-6, IL-12 (p70), IFN-γ, and IL-10 was observed in the paramylon groups compared to the control group. Our data therefore reveals a novel immunoregulatory role of the Euglena gracilis Z and paramylon which provides protection against influenza virus infection.

RIG-I overexpression decreases mortality of cigarette smoke exposed mice during influenza A virus infection.

BACKGROUND: Retinoic acid-inducible gene I (RIG-I) is an important regulator of virus-induced antiviral interferons (IFNs) and proinflammatory cytokines which participate in clearing viral infections. Cigarette smoke (CS) exposure increases the frequency and severity of respiratory tract infections. METHODS: We generated a RIG-I transgenic (TG) mouse strain that expresses the RIG-I gene product under the control of the human lung specific surfactant protein C promoter. We compared the mortality and host immune responses of RIG-I TG mice and their litter-matched wild type (WT) mice following challenge with influenza A virus (IAV). RESULTS: RIG-I overexpression increased survival of IAV-infected mice. CS exposure increased mortality in WT mice infected with IAV. Remarkably, the effect of RIG-I overexpression on survival during IAV infection was enhanced in CS-exposed animals. CS-exposed IAV-infected WT mice had a suppressed innate response profile in the lung compared to sham-exposed IAV-infected WT mice in terms of the protein concentration, total cell count and inflammatory cell composition in the bronchoalveolar lavage fluid. RIG-I overexpression restored the innate immune response in CS-exposed mice to that seen in sham-exposed WT mice during IAV infection, and is likely responsible for enhanced survival in RIG-I TG mice as restoration preceded death of the animals. CONCLUSIONS: Our results demonstrate that RIG-I overexpression in mice is protective for CS enhanced susceptibility of smokers to influenza infection, and that CS mediated RIG-I suppression may be partially responsible for the increased morbidity and mortality of the mice exposed to IAV. Thus, optimizing the RIG-I response may be an important treatment strategy for CS-enhanced lung infections, particularly those due to IAV.

Geldanamycin Reduces Acute Respiratory Distress Syndrome and Promotes the Survival of Mice Infected with the Highly Virulent H5N1 Influenza Virus.

Infections with lethal influenza viruses lead to acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), which may be related to the activation of the host's immune system. Here, in our study, male C57BL/6 mice were infected with 10 LD50 of the H5N1 influenza virus and treated with geldanamycin or oseltamivir 2 h after infection. Lung injury was assessed by histopathology on days 4 and 7. The viral load was quantified by measuring the NP gene expression level on days 2, 4, and 7. Levels of cytokines and chemokines in bronchoalveolar lavage fluids and inflammatory cells were analyzed at different time points. Geldanamycin administration prolonged survival in mice and dramatically reduced lung injury and pulmonary inflammatory compared with other mice. Viral loads in geldanamycin-treated mice also significantly reduced compared with non-treated mice, but not to the extent as the oseltamivir-treated mice. Furthermore, the geldanamycin treatment markedly reduced the production of major proinflammatory cytokines and chemokines and attenuated the infiltration and activation of immune cells, but it did not alter the generation of virus-neutralizing antibodies. In conclusion, geldanamycin plays an important role in attenuating virus infection-induced ALI/ARDS by reducing the host's inflammatory responses and may provide an important reference for clinical treatments.

AIM2 Inflammasome Is Critical for Influenza-Induced Lung Injury and Mortality.

The absent in melanoma 2 (AIM2) inflammasome plays an important role in many viral and bacterial infections, but very little is known about its role in RNA virus infection, including influenza A virus (IAV). In this study, we have designed in vivo and in vitro studies to determine the role of AIM2 in infections with lethal doses of IAVs A/PR8/34 and A/California/07/09. In wild-type mice, IAV infection enhanced AIM2 expression, induced dsDNA release, and stimulated caspase-1 activation and release of cleaved IL-1ß in the lung, which was significantly reduced in AIM2-deficient mice. Interestingly, AIM2 deficiency did not affect the transcription of caspase-1 and IL-1ß. In addition, AIM2-deficient mice exhibited attenuated lung injury and significantly improved survival against IAV challenges, but did not alter viral burden in the lung. However, AIM2 deficiency did not seem to affect adaptive immune response against IAV infections. Furthermore, experiments with AIM2-specific small interfering RNA-treated and AIM2-deficient human and mouse lung alveolar macrophages and type II cells indicated a macrophage-specific function of AIM2 in regulation of IAV-stimulated proinflammatory response. Collectively, our results demonstrate that influenza infection activates the AIM2 inflammasome, which plays a critical role in IAV-induced lung injury and mortality. AIM2 might serve as a therapeutic target for combating influenza-associated morbidity and mortality without compromising the host antiviral responses.

The effect of gamma-irradiation conditions on the immunogenicity of whole-inactivated Influenza A virus vaccine.

Gamma-irradiation, particularly an irradiation dose of 50kGy, has been utilised widely to sterilise highly pathogenic agents such as Ebola, Marburg Virus, and Avian Influenza H5N1. We have reported previously that intranasal vaccination with a gamma-irradiated Influenza A virus vaccine (γ-Flu) results in cross-protective immunity. Considering the possible inclusion of highly pathogenic Influenza strains in future clinical development of γ-Flu, an irradiation dose of 50kGy may be used to enhance vaccine safety beyond the internationally accepted Sterility Assurance Level (SAL). Thus, we investigated the effect of irradiation conditions, including high irradiation doses, on the immunogenicity of γ-Flu. Our data confirm that irradiation at low temperatures (using dry-ice) is associated with reduced damage to viral structure compared with irradiation at room temperature. In addition, a single intranasal vaccination with γ-Flu irradiated on dry-ice with either 25 or 50kGy induced seroconversion and provided complete protection against lethal Influenza A challenge. Considering that low temperature is expected to reduce the protein damage associated with exposure to high irradiation doses, we titrated the vaccine dose to verify the efficacy of 50kGy γ-Flu. Our data demonstrate that exposure to 50kGy on dry-ice is associated with limited effect on vaccine immunogenicity, apparent only when using very low vaccine doses. Overall, our data highlight the immunogenicity of influenza virus irradiated at 50kGy for induction of high titre antibody and cytotoxic T-cell responses. This suggests these conditions are suitable for development of γ-Flu vaccines based on highly pathogenic Influenza A viruses.

DNAzymes Dz13 target the c-jun possess antiviral activity against influenza A viruses.

The emergence of anti-influenza A virus drugs resistant strain highlights the need for more effective therapy. Our earlier study demonstrated that c-jun, a downstream molecule of JNK, might be important in viral infections and inflammatory responses. In the present study, we explored the function of DNAzymes Dz13 that target c-jun in influenza A virus infected mice. Dz13 displayed non-toxic side effects on A549 cells and BALB/c mice. Moreover, Dz13-treated mice had enhanced survival after influenza compared with untreated mice. Simultaneously, the pulmonary inflammatory responses and viral burden were decreased in Dz13 treated mice. Furthermore, proliferation levels of infection-induced CD4+ and CD8+ T cells were impaired. These data demonstrated that Dz13 could reduce viral replication and inflammatory response in vivo, suggesting that Dz13 may potentially be used to treat influenza A viral infection.

Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking.

Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 posttranslational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6A delta (TRAPPC6AΔ), interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6AΔ in the viral life cycle was investigated by the knockdown of endogenous TRAPPC6AΔ with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6AΔ. The results indicated that TRAPPC6AΔ, through its interaction with M2, slows M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice. IMPORTANCE: The influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the posttranslational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6AΔ, as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factor TRAPPC6A/TRAPPC6AΔ. Importantly, we discovered that TRAPPC6AΔ can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.