PR8 Influenza Virus Research Articles

Characterization of recombinant influenza A virus as a vector expressing respiratory syncytial virus fusion protein epitopes.

Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and the elderly, and no vaccine against this virus has yet been licensed. Here, we report a recombinant PR8 influenza virus with the RSV fusion (F) protein epitopes of the subgroup A gene inserted into the influenza virus non-structural (NS) gene (rFlu/RSV/F) that was generated as an RSV vaccine candidate. The rescued viruses were assessed by microscopy and Western blotting. The proper expression of NS1, the NS gene product, and the nuclear export protein (NEP) of rFlu/RSV/F was also investigated using an immunofluorescent assay. The rescued virus replicated well in the MDCK kidney cell line, A549 lung adenocarcinoma cell line and CNE-2Z nasopharyngeal carcinoma cell line. BALB/c mice immunized intranasally with rFlu/RSV/F had specific haemagglutination inhibition antibody responses against the PR8 influenza virus and RSV neutralization test proteins. Furthermore, intranasal immunization with rFlu/RSV/F elicited T helper type 1-dominant cytokine profiles against the RSV strain A2 virus. Taken together, our findings suggested that rFlu/RSV/F was immunogenic in vivo and warrants further development as a promising candidate vaccine.

Phosphoinositide-3-kinase p110δ isoform promotes influenza pathogenicity (MUC5P.858)

Abstract We have found that mice deficient in a tissue specific isoform of the signaling enzyme phosphoinositide-3 kinase (PI3K) p110δ are protected from PR8 influenza virus infection and have reduced weight loss and lung viral loads post-infection. Lung inflammation and immune cell infiltration is also decreased after viral challenge. In addition, p110δ KO mice had fewer influenza virus infected alveolar epithelial cells when infected with a GFP-flu virus and GFP expression was measured in lung epithelial cells. Cytokine measurements in bronchoalveolar lavages suggest p110δ KO mice exhibit increased IFN-gamma and MIG post PR8 infection. Slightly increased numbers of NK cells are detected in the lungs of p110δ KO mice as early as day 3 post-infection however activation was decreased within this population as compared to controls, suggesting they are not the primary source of resistance to influenza. In order to determine which cell type was causing the decreased pathogenicity we used two different tissue specific knockout mouse lines. Mice with a myeloid specific KO of p110δ showed similar weight loss and viral loads to C57BL/6 controls, suggesting alveolar macrophages are not primarily responsible for the observed protection in p110δ KO mice. However, mice with alveolar epithelial cell (AEC) specific deletion of p110δ were protected from influenza virus challenge. These data suggest PI3K signaling cascade is important for influenza virus protection within AEC.

Inclusion of flagellin increases antibody responses in nonhuman primate neonates following influenza vaccination (VAC8P.1002)

Abstract Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response to vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for use in this vulnerable population. To address this issue we developed an African Green Monkey model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a TLR5 agonist, to serve as an effective adjuvant for vaccination of neonates. Animals were primed and boosted with inactivated PR8 influenza virus in the presence of either flagellin or an inactive form of the protein. Inclusion of flagellin significantly increased the level of influenza-specific serum IgG present following boosting as well as at early times after virus challenge. Animals receiving flagellin adjuvanted vaccination also exhibited higher levels of respiratory influenza-specific IgG at early times post challenge. Interestingly, there was no flagellin mediated increase in IgM. The presence of flagellin also resulted in an increase in neutralizing antibody. This study supports further exploration of flagellin as an effective adjuvant for inducing vaccine responses against influenza in neonates.

Transient ablation of alveolar macrophages leads to massive pathology of influenza infection without affecting cellular adaptive immunity

Alveolar macrophages (AMs), localized at the pulmonary air-tissue interface, are one of the first lines of defense that interact with inhaled airborne pathogens such as influenza viruses. By using a new CD169-DTR transgenic mouse strain we demonstrate that specific and highly controlled in vivo ablation of this myeloid cell subset leads to severe impairment of the innate, but not adaptive, immune responses and critically affects the progression of the disease. In fact, AM-ablated mice, infected with a normally sublethal dose of PR8 influenza virus, showed dramatically increased virus load in the lungs, severe airway inflammation, pulmonary edema and vascular leakage, which caused the death of the infected animals. Our data highlight the possibilities for new therapeutic strategies focusing on modulation of AMs, which may efficiently boost innate responses to influenza infections.

Sulforaphane improves MARCO expression, bacterial clearance and survival in post‐influenza bacterial pneumonia (145.1)

Mechanisms that increase susceptibility for post‐influenza bacterial pneumonia include down‐regulation of the alveolar macrophage (AM) scavenger receptor MARCO by interferon‐γ (IFN‐γ), leading to diminished bacterial clearance. Since MARCO expression can be increased by NRF2 signaling, we tested whether the NRF2‐inducer sulforaphane (SFN) can up‐regulate MARCO expression and improve bacterial clearance after influenza. Human AM‐like monocyte‐derived macrophages (HMDMs) were treated with IFN‐γ (10‐50 IU/ml, 24h) to simulate influenza infection, followed by sulforaphane (SFN, 10µM) for 24h before quantitation of MARCO expression or challenge with green fluorescence protein (GFP)‐expressing S. aureus. Scanning cytometry was used to quantitate MARCO surface labeling and uptake of bacteria. C57/BL6 mice were inoculated i.n. on day 0 with 3500PFU of PR8 influenza virus, and on day 7 with 500 CFU of S. pneumoniae serotype 3. Mice were injected with SFN 25 or 50 mg/kg, or PBS daily from day 6 to 9 after influenza infection. Bacterial clearance was measured on day 1 after S. pneumoniae infection using lung lavage samples. IFN‐γ down‐regulated MARCO expression (p<0.001) of HMDMs and impaired phagocytosis of S. aureus (p<0.001, n=4). SFN added after IFN‐γ significantly improved MARCO expression (p=0.003) and S. aureus phagocytosis (p=0.05), an effect that was inhibited by scavenger receptor blockers poly(I) (p=0.001) or MARCO blocking peptide (P<0.001). In vivo, sulforaphane‐treated mice showed improved bacterial clearance (e.g. 84%, 95% reduction in SFN‐treated mice in 2 trials) and a significantly higher survival rate compared to PBS‐treated mice (75.0% vs. 12.5%, n = 8/grp, p=0.022). Therapeutic targeting of the NRF2 pathway may be beneficial to reduce risk or severity of secondary bacterial pneumonia after influenza.Grant Funding Source: Supported by ES00002

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Hepcidin Induction by Pathogens and Pathogen-Derived Molecules Is Strongly Dependent on Interleukin-6

Hepcidin, the iron-regulatory hormone, is increased during infection or inflammation, causing hypoferremia. This response is thought to be a host defense mechanism that restricts iron availability to invading pathogens. It is not known if hepcidin is differentially induced by bacterial versus viral infections, whether the stimulation of pattern recognition receptors directly regulates hepcidin transcription, or which of the proposed signaling pathways are essential for hepcidin increase during infection. We analyzed hepcidin induction and its dependence on interleukin-6 (IL-6) in response to common bacterial or viral infections in mice or in response to a panel of pathogen-derived molecules (PAMPs) in mice and human primary hepatocytes. In wild-type (WT) mice, hepcidin mRNA was induced several hundred-fold both by a bacterial (Streptococcus pneumoniae) and a viral infection (influenza virus PR8) within 2 to 5 days. Treatment of mice and human primary hepatocytes with most Toll-like receptor ligands increased hepcidin mRNA within 6 h. Hepcidin induction by microbial stimuli was IL-6 dependent. IL-6 knockout mice failed to increase hepcidin in response to S. pneumoniae or influenza infection and had greatly diminished hepcidin response to PAMPs. In vitro, hepcidin induction by PAMPs in primary human hepatocytes was abolished by the addition of neutralizing IL-6 antibodies. Our results support the key role of IL-6 in hepcidin regulation in response to a variety of infectious and inflammatory stimuli.

MicroRNA Expression Profile of Mouse Lung Infected with 2009 Pandemic H1N1 Influenza Virus

MicroRNAs have been implicated in the regulation of gene expression of various biological processes in a post-transcriptional manner under physiological and pathological conditions including host responses to viral infections. The 2009 pandemic H1N1 influenza virus is an emerging reassortant strain of swine, human and bird influenza virus that can cause mild to severe illness and even death. To further understand the molecular pathogenesis of the 2009 pandemic H1N1 influenza virus, we profiled cellular microRNAs of lungs from BALB/c mice infected with wild-type 2009 pandemic influenza virus A/Beijing/501/2009 (H1N1) (hereafter referred to as BJ501) and mouse-adapted influenza virus A/Puerto Rico/8/1934 (H1N1) (hereafter referred to as PR8) for comparison. Microarray analysis showed both the influenza virus BJ501 and PR8 infection induced strain- and temporal-specific microRNA expression patterns and that their infection caused a group of common and distinct differentially expressed microRNAs. Characteristically, more differentially expressed microRNAs were aroused on day 5 post infection than on day 2 and more up-regulated differentially expressed microRNAs were provoked than the down-regulated for both strains of influenza virus. Finally, 47 differentially expressed microRNAs were obtained for the infection of both strains of H1N1 influenza virus with 29 for influenza virus BJ501 and 43 for PR8. Among them, 15 microRNAs had no reported function, while 32 including miR-155 and miR-233 are known to play important roles in cancer, immunity and antiviral activity. Pathway enrichment analyses of the predicted targets revealed that the transforming growth factor-β (TGF-β) signaling pathway was the key cellular pathway associated with the differentially expressed miRNAs during influenza virus PR8 or BJ501 infection. To our knowledge, this is the first report of microRNA expression profiles of the 2009 pandemic H1N1 influenza virus in a mouse model, and our findings might offer novel therapy targets for influenza virus infection.

Induction of human Beta-Defensin-1 gene expression is dependent on IRF-7 and PU.1. (P1379)

Abstract Beta-defensins have been gaining recognition as immunomodulators and regulators of the inflammatory response. Human beta-defensin (hBD) peptides 1, 2 and 3 are differentially modulated in primary gingival and tracheal epithelial cells (EC) by viruses. We showed that both HSV-1, Sendai and PR8 influenza viruses increase the gene expression of hBD-1 in monocytes and plasmacytoid dendritic cells (PDC) and suppress gene expression of hBD-1 in target EC. Deleting the hBD-1 homologue in mice also increased influenza-induced morbidity, mortality and lung inflammation compared with wild type C57BL/6 mice, highlighting its importance in viral innate immunity. In this study, we investigated how hBD-1 gene expression is regulated. In PDC, no increased hBD-1 gene expression occurred when cells were incubated with the TLR9 agonist, CpG-A, indicating that hBD-1 is not induced via TLR9. When PDC were incubated with anti-interferon (IFN)-alpha-receptor-1 antibody, no change in viral induction by HSV-1 occurred. In addition, in a luciferase reporter system in A549 cells co-transfected with expression vectors containing IRF3, IRF5, IRF7 and PU.1, IRF7 and PU.1 were the only transcription factors that increased activation of the hBD-1 promoter. These results suggest that HSV-1 induction of hBD-1 occurs independently of IFN-alpha at the receptor level but that transcription factors common to IFN-alpha are involved in the regulation of hBD-1 gene expression.

Prime-boost vaccination with a live attenuated influenza vaccine elicits T cell-mediated cross-strain protective immunity (P4287)

Abstract Antigen drift and shift of influenza viruses requires frequent reformulation of influenza vaccines. This stresses the need for a universal influenza vaccine. In this study, mice were vaccinated with the trivalent live attenuated (LAIV, nasal spray FluMist) and inactivated (TIV, flu shot FluZone) influenza vaccines by 1-3 dose prime-boost vaccination regimens. While vaccination with both LAIV and TIV elicited strong humoral responses, only LAIV induced significant protection against challenge with the non-vaccine strain H1N1 PR8 influenza virus. Furthermore, protective immunity against PR8 was dose-dependent. Of note, IL-2 and IFN-γ cytokine secretion in the lung alveolar fluid were significantly elevated in mice immunized with LAIV. Moreover, T cell depletion of LAIV immunized mice compromised protection, indicating that T cell-mediated immunities were required. In contrast, passive transfer of sera from mice immunized with LAIV into naïve mice failed to protect against PR8 challenge. To verify these results, we performed virus neutralization assays in vitro and confirmed that LAIV did not induce cross-strain neutralizing antibodies against PR8 virus. Finally, we showed that 3 doses of LAIV also provided protection against challenge with additional two heterologous viruses FM/47 (H1N1) and HK/68 (H3N2). These results support the potential use of the LAIV as a universal influenza vaccine under prime-boost vaccination regimen.

Inflammation regulates the differentiation of CD4 T cells with cytolytic potential (P6088)

Abstract Cytolytic CD4 T cells have been described during viral infections; however, the factors necessary for driving the cytolytic phenotype have not been identified. Our data indicate IL-2 regulates granzyme B (GrB) expression in CD4 T cells but IL-12 and IL-6 enhance GrB expression and promote perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of inflammation in CD4 CTL generation in vivo, we used an adoptive transfer system and infection with highly pathogenic influenza virus PR8, compared to x31 with lower pathogenicity. At relatively low doses (1000 EID50), Ova specific CD4 cells expressing GrB differentiated in PR8/Ova infected mice but not x31/Ova infected mice. This pattern was observed at 10-fold higher dose of x31/Ova compared to PR8/Ova. In addition, CD4 T cell influx to the lung and IFN-γ expression was increased in mice infected with PR8/Ova compared to x31/Ova. Importantly, these differences were seen in the endogenous, polyclonal population of CD4 T cells. Inflammatory mediators including IL-6, CXCL-9, CXCL-10 and MIP1β were 3-5 fold higher in PR8/Ova infected lungs at day 4 post infection compared to x31/Ova infected lungs. Whether these inflammatory cytokines are responsible for inducing CD4 CTL is being investigated. These data suggest that CD4 CTL are induced in vivo by strong inflammatory responses and may provide an extra layer of protection against highly pathogenic IAV infection.

Mutations in haemagglutinin that affect receptor binding and pH stability increase replication of a PR8 influenza virus with H5 HA in the upper respiratory tract of ferrets and may contribute to transmissibility

The H5N1 influenza A viruses have circulated widely in the avian population for 10 years with only sporadic infection of humans observed and no sustained human to human transmission. Vaccination against potential pandemic strains is one strategy in planning for future influenza pandemics; however, the success of live attenuated vaccines for H5N1 has been limited, due to poor replication in the human upper respiratory tract (URT). Mutations that increase the ability of H5N1 viruses to replicate in the URT will aid immunogenicity of these vaccines and provide information about humanizing adaptations in H5N1 strains that may signal transmissibility. As well as mediating receptor interactions, the haemagglutinin (HA) protein of influenza facilitates fusion of the viral membrane and genome entry into the host cell; this process is pH dependent. We have shown in this study that the pH at which a panel of avian influenza HA proteins, including H5, mediate fusion is higher than that for human influenza HA proteins, and that mutations in the H5 HA can reduce the pH of fusion. Coupled with receptor switching mutations, increasing the pH stability of the H5 HA resulted in increased viral shedding of H5N1 from the nasal cavity of ferrets and contact transmission to a co-housed animal. Ferret serum antibodies induced by infection with any of the mutated H5 HA viruses neutralized HA pseudotyped lentiviruses bearing homologous or heterologous H5 HAs, suggesting that this strategy to increase nasal replication of a vaccine virus would not compromise vaccine efficacy.

The Lipid Mediator Protectin D1 Inhibits Influenza Virus Replication and Improves Severe Influenza

Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.

Enhanced immune response induced by a potential influenza A vaccine based on branched M2e polypeptides linked to tuftsin

Vaccination is the most effective means for preventing influenza-associated morbidity and mortality. Since the influenza virus mutates frequently, the virus strains for new vaccine production should be changed according to predicted epidemic strains. The extracellular domain of matrix protein 2 (M2e) is 24 amino acids long, which is highly conserved and therefore a good target for the development of a universal vaccine which may protect against a much wider range of influenza A virus strains. However its low antigenicity and immunogenicity, which are related to its small size, poses a big challenge for vaccine development. Multiple antigen peptide system (MAP) is based on an inert core molecule of radially branching lysine dendrites onto which a number of peptide antigens are anchored. Tuftsin is an immuno-stimulant molecule peptide. Here we developed a novel peptide vaccine by connecting a tuftsin to a branched, four-copy M2e. Not only did this increase the molecular mass, but also potentiate the immunogenicity. Two branched peptides, (M2e)4-tuftsin and (M2e)4-G4(tuftsin was replaced with four glycines), and a M2e monomer were synthesized using standard solid-phase methods. In vitro and in vivo studies were performed to compare their antigenicity and immunogenicity. Experiments in BALB/c mice demonstrated that the branched M2e could induce stronger humoral and cellular immune responses than the M2e monomer, and (M2e)4-tuftsin induced stronger humoral and cellular immune response than (M2e)4-G4. After lethal challenge with influenza virus PR8 strain, up to 80% of the animals in the (M2e)4-tuftsin vaccinated group still survived, in contrast to 44% in the (M2e)4-G4 group and 30% in the M2e monomer group. The combination of branched polypeptides and tuftsin in vaccine design is presented here for the first time, and the results show that the new construct is a promising candidate for a universal vaccine against the influenza A virus.

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.

A candidate dual vaccine against influenza and noroviruses

The extracellular domain of the matrix protein 2 (M2e) of influenza viruses is highly conserved among all influenza A subtypes, making it a suitable target for a universal influenza vaccine. In this study, we demonstrated an enhanced immune response and protection of a chimeric M2e vaccine against influenza A viruses using our newly developed vaccine platform, the norovirus P particle, to present the M2e peptide. The 23-amino acid peptide was inserted into one of the surface loops of the P protein, resulting in 24 copies of M2e presented on each P particle. Significantly (P<0.001) increased antibody responses to M2e were observed in mice immunized with the P particle-M2e chimera compared with those immunized with the free M2e peptides. Mice immunized with the P particle-M2e vaccine were fully protected (100% survived) against lethal challenge of a mouse adapted human influenza virus PR8 (H1N1), while only low survival rates (<12.5%) were found in mice immunized with the free M2e peptides or wild type P particle. In addition, the mouse sera collected after immunization with the P particle-M2e vaccine were able to block the binding of norovirus virus-like particle and P particle to histo-blood group antigen receptors. These results suggest that the P particle-M2e chimera can be used as dual vaccine against both noroviruses and influenza viruses.

Influenza virus pathophysiology and brain invasion in mice with functional and dysfunctional Mx1 genes

Mice with a dysfunctional myxovirus resistance-1 (dMx1) gene transport intranasally-instilled PR8 influenza virus to the olfactory bulb (OB) within 4 h post-infection. To determine if the presence of a functional Mx1 (fMx1) gene would influence this brain viral localization and/or disease, we infected mature C57BL/6 dMx1 and fMx1 mice under the same conditions and observed sickness behaviors, viral nucleoprotein (NP) RNA expression and innate immune mediator (IIM) mRNA expression in selected tissues at 15 and 96 h post-infection. Virus invaded the OB and lungs comparably in both sub-strains at 15 and 96 h as determined by nested PCR. In contrast, virus was present in blood and somatosensory cortex of dMx1, but not fMx1 mice at 96 h. At 15 h, sickness behaviors were comparable in both sub-strains. By 96 h dMx1, but not fMx1, were moribund. In both 15 and 96 h lungs, viral NP was significantly elevated in the dMx1 mice compared to the fMx1 mice, as determined by quantitative PCR. OB expression of most IIM mRNAs was similar at both time periods in both sub-strains. In contrast, lung IIM mRNAs were elevated in fMx1 at 15 h, but by 96 h were consistently reduced compared to dMx1 mice. In conclusion, functional Mx1 did not alter OB invasion by virus but attenuated illness compared to dMx1 mice. Inflammation was similar in OBs and lungs of both strains at 15 h but by 96 h it was suppressed in lungs, but not in OBs, of fMx1 mice.

Protective essential oil attenuates influenza virus infection: An in vitro study in MDCK cells

BackgroundInfluenza is a significant cause of morbidity and mortality. The recent pandemic of a novel H1N1 influenza virus has stressed the importance of the search for effective treatments for this disease. Essential oils from aromatic plants have been used for a wide variety of applications, such as personal hygiene, therapeutic massage and even medical practice. In this paper, we investigate the potential role of an essential oil in antiviral activity.MethodsWe studied a commercial essential oil blend, On Guard™, and evaluated its ability in modulating influenza virus, A/PR8/34 (PR8), infection in Madin-Darby canine kidney (MDCK) cells. Influenza virus was first incubated with the essential oil and infectivity in MDCK cells was quantified by fluorescent focus assay (FFA). In order to determine the mechanism of effects of essential oil in viral infection inhibition, we measured hemagglutination (HA) activity, binding and internalization of untreated and oil-treated virus in MDCK cells by flow cytometry and immunofluorescence microscopy. In addition, the effect of oil treatment on viral transcription and translation were assayed by relative end-point RT-PCR and western blot analysis.ResultsInfluenza virus infectivity was suppressed by essential oil treatment in a dose-dependent manner; the number of nascent viral particles released from MDCK cells was reduced by 90% and by 40% when virus was treated with 1:4,000 and 1:6,000 dilutions of the oil, respectively. Oil treatment of the virus also decreased direct infection of the cells as the number of infected MDCK cells decreased by 90% and 45% when virus was treated with 1:2,000 and 1:3,000 dilutions of the oil, respectively. This was not due to a decrease in HA activity, as HA was preserved despite oil treatment. In addition, oil treatment did not affect virus binding or internalization in MDCK cells. These effects did not appear to be due to cytotoxicity of the oil as MDCK cell viability was only seen with concentrations of oil that were 2 to 6 times greater than the doses that inhibited viral infectivity. RT-PCR and western blotting demonstrated that oil treatment of the virus inhibited viral NP and NS1 protein, but not mRNA expression.ConclusionsAn essential oil blend significantly attenuates influenza virus PR8 infectivity in vitro without affecting viral binding or cellular internalization in MDCK cells. Oil treated virus continued to express viral mRNAs but had minimal expression of viral proteins, suggesting that the antiviral effect may be due to inhibition of viral protein translation.

Effect of intranasal administration of Lactobacillus pentosus S-PT84 on influenza virus infection in mice

Lactobacillus pentosus strain S-PT84 isolated from Kyoto pickles enhances splenic natural killer (NK) cell activity and exhibit anti-allergic effects by modulating the Th1/Th2 (T-helper1/T-helper2) balance. In the present study, we investigated whether the immune response could be activated by intranasal administration of S-PT84 in the respiratory immune system and protected against influenza virus infection in mice. When BALB/c mice received intranasal administration of S-PT84 once daily for 3 consecutive days, S-PT84 strongly induced interleukin-12 (IL-12) and gamma interferon (IFN-γ) production in mediastinal lymph node (MLN) cells. At intranasal infection with influenza virus PR8 (a mouse-adapted H1N1 strain) after S-PT84 treatment, the survival rates of mice improved in a dose-dependent manner, and the titer of influenza virus in bronchoalveolar lavage fluids (BALF) was significantly decreased by S-PT84 administration. Production of IL-12 and alpha-interferon (IFN-α) in BALF were significantly higher in mice treated with S-PT84 compared to the control mice. Lung NK activity was also significantly augmented in S-PT84-treated mice. These results suggested that the L. pentosus strain S-PT84 showed inhibitory activity against influenza virus infection.

Efficacy of poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) as mucosal adjuvant to induce protective immunity against respiratory pathogens

Polyphosphazene polyelectrolyte, a potent new mucosal adjuvant candidate, was tested for its ability to elicit protective immunity against several respiratory diseases. Groups of mice were intranasally (i.n.) vaccinated with poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) together with several vaccine antigens such as pertussis toxoid, pneumococcal surface protein A, and formalin-inactivated PR8 influenza virus. Results showed predominant levels of antigen-specific IgG and IgA antibodies in serum and bronchial alveolar lavage fluids after vaccination with PCPP plus antigen when compared to antigen alone. In addition, there were significantly higher levels of the secretory form of IgA antibody in the mucosal secretions (i.e., nasal wash, saliva, vaginal wash, and fecal extracts). Moreover, i.n. vaccination with PCPP resulted in brisk numbers of IgG and IgA antibody-forming cells in the nasal passage, lung, and sub-mandibular glands of vaccinated mice. Of note, PCPP administration resulted in mixed Th1 and Th2 type responses (i.e., high levels of IgG2a and IgG1 as well as IFN-γ and IL-4). Most interestingly, i.n. challenge with vaccine antigens together with PCPP elicited strong protective efficacy against respiratory infection with Bordetella pertussis, Streptococcus pneumoniae, and influenza virus. Taken together, these results suggest that PCPP may be a promising candidate for mucosal adjuvant to elicit protective immunity against respiratory infectious diseases.