Steps toward a Universal Influenza Vaccine: Research Models and Comparison of Current Approaches

The ability of influenza virus to adapt to various species and evade natural immunity makes the ubiquitous pathogen particularly difficult to eradicate. Annual reformulation of influenza vaccines is costly and time-consuming and has varying efficacy against influenza virus strains. Therefore, worldwide efforts aim to develop a universal influenza vaccine to prevent potential healthcare emergencies such as pandemic influenza threats, such as the 1918 Spanish Flu and pandemic Swine Flu of 2009. Efficacy of a universal influenza vaccine must overcome current challenges with subtype diversity, antigenic drift, and adequately protect against emerging reassortants from both environmental and agricultural sources. Furthermore, the manufacturing and production of vaccines largely influence the effectiveness of a vaccine and technological advancements may soon rival current vaccine strategies. This review discusses the evolution and diversity of influenza viruses, how viral glycoprotein hemagglutinin plays a dominant role in influenza surveillance and assessment of protection and compares the methodologies of current and upcoming vaccine options. While the obstacles remain daunting, growing knowledge of influenza evolution and immunity may lead to more viable candidates that protect against broader varieties of influenza viruses and help prevent future international health crises

Respiratory Syncytial Virus NS1 Protein Colocalizes with Mitochondrial Antiviral Signaling Protein MAVS following Infection

Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His6-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production

Innate Immune Responses to Respiratory Syncytial Virus: Age-associated Changes

Respiratory syncytial virus (RSV) infection causes ~64 million cases of respiratory disease and 200,000 deaths annually worldwide, yet there is no broadly effective prophylactic or treatment regimen. RSV can produce acute respiratory illness in patients of all ages but strikes the age extremes, infants and the elderly, with highest frequency presumably due to innate immune deficiencies. A higher morbidity and mortality has been reported for the elderly above 65 years of age, which has been attributed to immune senescence. Efforts to generate an effective vaccine have thus far been unsuccessful. The innate immune system provides the first line of defense against viral pathogens with a repertoire of anatomical barriers, phagocytic immune cells, pattern recognition receptors (PRRs) and antiviral cytokines like interferons (IFNs). The precise mechanism of subversion of innate immunity in young and aged is poorly understood. A better understanding of innate immune pathways is expected to aid in the development of appropriate vaccines or prophylactics for these high-risk groups. Previously, the RSV nonstructural protein 1 (NS1) was shown to antagonize IFN responses by disrupting components of the innate immune system, although the mechanism is not well defined. We hypothesized that NS1 targets constituents of the PRR pathways to evade innate immunity and thus ensure viral survival. Using microscopy and co-immunoprecipitation assays, we found that NS1 localizes to the mitochondria and binds to the mitochondrially associated adaptor protein MAVS, thus preventing MAVS interaction with the RNA helicase, RIG-I. Expression of NS1 was also correlated with upstream IFN-response regulator, LGP2, and its expression was inducible in the absence of a viral infection. Tetracycline-inducible expression of recombinant NS1 in a cell model also promoted viral replication and emphasizes the key contribution of NS1 to RSV survival. Through this study, we demonstrated a mechanism for RSV NS1 in the disruption of early innate responses through mitochondrial localization and alteration of the RLH signaling. Whereas the above studies showed the importance RSV-induced innate immune pathways, whether the expression and signaling of innate immune pathways were adversely affected upon RSV infection in the high-risk groups remains unknown. Since elderly individuals are at an increased risk for severe bronchiolitis and RSV-induced pneumonia, often resulting in hospitalization and medical intervention and adaptive immune cell functionality and responsiveness reportedly decline with age, we hypothesized a similar age-related deterioration of the innate antiviral system. In this investigation, we used an aged mouse model to correlate age-associated changes in innate immune gene expression with RSV pathology. Of 84 antiviral genes examined, five genes including RIG-I, IFNAR1, TLR8, IL-1Β, and osteopontin (OPN) were associated with both age and infection. In response to RSV infection, aged mice had delayed induction of antiviral genes and diminished ability to secrete IL-6 in response to TLR7/8 agonist in primary alveolar macrophages. Lungs from aged, RSV-infected mice had increased cellular infiltration and prolonged infection as compared to young mice. In summary, age-related decline in expression and functionality of antiviral defenses were correlated with enhance RSV-induced lung disease in aged mice. In the absence of infection, aged mice chronically overproduced IL-1Β and OPN relative to young mice. Upon infection, aged mice had impaired ability to secrete higher levels of IL-1Β and mucus. In contrast, OPN secretion remained high and prolonged in aged mice throughout infection. The age-related decline in host antiviral gene induction and delayed cytokine production correlated with enhanced disease pathology. Using a transgenic strain of mice deficient in OPN (OPN-KO), we observed greater resistance to RSV and enhanced secretion of mucus, but unaltered cellular infiltration into the lungs. Therefore, OPN overproduction and defective mucus production likely contribute to pathology in aged mice. These findings demonstrate that RSV targets the innate virus recognition and antiviral cytokine activation pathways but also that the antiviral defense system is significantly affected by age. Consequently, efforts to generate vaccines or develop therapies that stimulate IFN induction may prove unsuccessful in the elderly given that RSV virulence factors and age weaken these responses. This study contributes to our understanding of how aging relates to the RSV subversion of the host antiviral response and should help with the development of better antiviral therapies suited to the growing elderly population

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

UNLABELLED: The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5\u27-triphosphate-containing RNA (5\u27pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. IMPORTANCE: The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5\u27pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population

RSV NS1 is present on the mitochondria and co-immunoprecipitates with MAVS in NS1-transfected cells.

<p>(<b>A</b>). A549 cells were transfected with pNS1-Flag (NS1) or pFlag (Vec). Mitochondria were isolated and the proteins were analyzed by western blot for Flag and Cox-IV (<b>B</b>). Total cell extracts were immunoprecipitated with anti-MAVS antibody and analyzed by western blotting with anti-Flag and anti-MAVS antibodies. (<b>C</b>). A549 cells transfected with pNS1-Flag were stained for Flag (green, <i>a</i>), mitochondria (CMXRos, red, <i>b</i>), and MAVS (purple, <i>c</i>) and visualized using the Leica TCS SP2 laser scanning confocal microscope. The squares in<i>d</i>and <i>e</i> show co-localization of NS1 with mitochondria and MAVS respectively, and the box in <i>f</i> shows co-localization of NS1, MAVS and mitochondria. The insets in <i>d,e</i>, and <i>f</i> show the enlargement of the indicated areas.</p

NS1 protein prevents RIG-I from binding to MAVS.

<p>(<b>A</b>) A549 cells were mock-infected or infected with rA2 or rA2ΔNS1 (MOI = 1). Whole-cell lysates were subjected to western blot analysis with anti-RIG-I, anti-MAVS, and anti-NS1antibodies (<i>top panel</i>). The same lysates were immunoprecipitated with anti-RIG-I antibody and the protein complexes were immunoblotted with the indicated antibodies (<i>bottom panel</i>). (<b>B</b>)The bar graph represents the densitometric quantification of the band intensities as the ratio of MAVS to RIG-I. (<b>C</b>) A549 cells were transfected with the indicated plasmids (12 µg of either pVAX or pNS1) and incubated with 0.2 ng/ml poly (I:C). Cytoplasmic extracts from cells 12 and 24 h after transfection were immunoprecipitated with anti-MAVS and immunoblotted with anti-RIG-I antibody. (<b>D</b>) Increasing amounts of pNS1 were co-transfected with constant amounts (6 µg) of pMAVS and pRIG-I, along with 0.2 ng/ml poly (I:C). Competitive inhibition of the interaction of MAVS with RIG-I was tested using IP, as described in (<b>C</b>). Transfection with pVAX was used to keep the total amount of transfected DNA constant.</p

MAVS co-immunoprecipitates with NS1 in epithelial cells infected with rA2- RSV or rA2.

<p>(<b>A</b>) HEp-2 cells were eithermock-infectedor infected with rA2 or rA2ΔNS1or rA2ΔNS2 at an MOI of 1. At 24 h p.i., total cell extracts were immunoprecipitated with anti-MAVS followed by western blot analysis using anti-NS1 or anti-MAVS antibodies. (<b>B</b>) Whole cell lysates from mock or rA2 infected HEp-2 cells were immunoblotted for RSV F protein using anti RSV F antibody (Chemicon MAB8262) (<i>right panel</i>). The lysates from mock or rA2 or rA2ΔNS1 were co-immunoprecipitated with anti-MAVS antibody and probed for MAVS and RSV F protein (<i>left panel</i>).</p

Colocalization of NS1 with MAVS by using immunogold electron microscopy.

<p>A549 cells were mock-infected or infected with 0.5 MOI rA2-His₆-NS1 for 1 h. Cells were harvested at 12 and 24 h post-infection and processed for immunogoldelectron microscopy. Thin sections on nickel grids were treated with rabbit anti-MAVS antibody (1∶100 dilution) for 3 h at room temperature, washed and then stained with 15 nm gold-labeled anti-rabbit secondary antibody. The grids were washed thoroughly and treated with a 1∶20 dilution of 5 nm gold-Ni-NTA (Molecular Probes) that binds to His tags. Cells infected with rA2-His₆-NS1 were stained for His-NS1 with 5 nm gold (red arrow) and for MAVS protein on mitochondria with 15 nm gold (black arrows). (<i>a</i>) Ultrathin sections of uninfected cells showing the 15 nm gold staining of MAVS protein (black arrow). (<i>b</i>) Ultrathin sections of infected cells 12 h p.i. showing His₆ protein stained using 5 nm gold-Ni-NTA. The inset shows His₆-NS1 localized on the mitochondria. (<i>c & d</i>) Sections of cells 12 and 24 h p.i. showing colocalization of NS1 with the MAVS protein. Insets (1 & 2) show colocalization of NS1 (red arrow) and MAVS (black arrow).</p

Co-localization of NS1 with MAVS in mitochondria during RSV infection.

<p>A549 cells were infected with rA2 or rA2ΔNS2 (0.1 MOI).Twelve hours after infection, cells were stained with the mitochondrial stain CMXRos (red). Cells were then fixed and stained with Alexa 488-conjugated anti-NS1 antibody (green) and Alexa 647-conjugated anti-MAVS antibody (purple). Stained cells were viewed under the Leica TCS SP2 laser scanning confocal microscope (<b>A</b>) Images (400× magnification) show individual staining colors, and merged images of NS1-mitochondria and NS1-MAVS are also shown. (<b>B</b>) The images are a 5× enlargement of a 400× image. The top panel shows the cells infected with rA2 and the bottom panel the cells infected with rA2ΔNS2. The areas marked by white circles show the presence of NS1 in mitochondria and colocalization with MAVS. The first pair of images are merges of NS1 and mitochondria. The second pair show NS1 merged with MAVS and the last pair shows all three. (<b>C</b>). Correlation analysis of the co-localization of NS1 with mitochondria in rA2 infected cells. (<b>D</b>). Correlation analysis of the colocalization of NS1 with mitochondria in rA2ΔNS2 infected cells.Pearson's coefficient for MAVS and NS1 is shown along with Manders' coefficients (M1 & M2), which represent the fraction of the mitochondrial red overlapping with NS1 green and the fraction of NS1 green overlapping with the mitochondrial red, respectively.</p

Co-localization of His₆-NS1 with mitochondria in infected cells using Ni²⁺-NTA₂-BM.

<p>(<b>A</b>) Structure of Ni²⁺-NTA₂-BM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029386#pone.0029386-Krishnan1" target="_blank">[26]</a>. (<b>B</b>) rA2-His₆-NS1 or rA2 were used to infect HEp-2 cells at a MOI of 2. At 10h p.i., infected cells were labeled with CMXRos (red), DRAQ5 (blue), and 50 μMNi²⁺-NTA₂-BM. Fluorescence was detected using an Olympus FV1000 MPE multiphoton laser scanning microscope. The cells were maintained at 37°C with 5% CO₂ throughout the imaging (Z min). Shown are micrographs of rA2-His₆-NS1-infected cells collected 10 h p.i (<i>a–c</i>)and rA2 RSV at 10 hp.i. (<i>d–f</i>) demonstrating site-specificity for Ni²⁺-NTA₂-BM. Excitation wavelengths of 405, 563 and 633 nm and differential interference contrast (DIC) were used to collect a series of 0.5 µm Z-stack images for each specimen and experiments were performed in triplicate. Images <i>a</i> and <i>d</i> show the mitochondria (red), <i>b</i> and <i>e</i> represent His₆-NS1 (green) and <i>c</i>and<i>f</i> are the merged images of red and green along with the blue nuclear stain DAPI and DIC. (<b>C</b>). Thresholds for the Z-stack images collected in (<b>B</b>) were acquired using RenyiEntropy AutoThreshold ImageJ plugin (Landini) and Pearson's and Mander's coefficients derived with JACoP plugin (Bolte & Cordelieres). The Mander's coefficient M2 indicates that ∼70% of the His₆-NS1 detected overlaps with CMXRos-stained mitochondria at 10 hrs p.i. in rA2-His₆-NS1-infected cells.</p