Lethal Influenza Infection Research Articles

Notch4 regulatory T cells and SARS-CoV-2 viremia shape COVID19 survival outcome.

Immune dysregulation and SARS-CoV-2 plasma viremia have been implicated in fatal COVID-19 disease. However, how these two factors interact to shape disease outcomes is unclear. We carried out viral and immunological phenotyping on a prospective cohort of 280 patients with COVID-19 presenting to acute care hospitals in Boston, Massachusetts and Genoa, Italy between June 1, 2020 and February 8, 2022. Disease severity, mortality, plasma viremia, and immune dysregulation were assessed. A mouse model of lethal H1N1 influenza infection was used to analyze the therapeutic potential of Notch4 and pyroptosis inhibition in disease outcome. Stratifying patients based on %Notch4+ Treg cells and/or the presence of plasma viremia identified four subgroups with different clinical trajectories and immune phenotypes. Patients with both high %Notch4+ Treg cells and viremia suffered the most disease severity and 90-day mortality compared to the other groups even after adjusting for baseline comorbidities. Increased Notch4 and plasma viremia impacted different arms of the immune response in SARS-CoV-2 infection. Increased Notch4 was associated with decreased Treg cell amphiregulin expression and suppressive function whereas plasma viremia was associated with increased monocyte cell pyroptosis. Combinatorial therapies using Notch4 blockade and pyroptosis inhibition induced stepwise protection against mortality in a mouse model of lethal H1N1 influenza infection. The clinical trajectory and survival outcome in hospitalized patients with COVID-19 is predicated on two cardinal factors in disease pathogenesis: viremia and Notch4+ Treg cells. Intervention strategies aimed at resetting the immune dysregulation in COVID-19 by antagonizing Notch4 and pyroptosis may be effective in severe cases of viral lung infection.

High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

A protective and broadly binding antibody class engages the influenza virus hemagglutinin head at its stem interface.

Influenza infection and vaccination impart strain-specific immunity that protects against neither seasonal antigenic variants nor the next pandemic. However, antibodies directed to conserved sites can confer broad protection. Here we identify and characterize a class of human antibodies that engage a previously undescribed, conserved epitope on the influenza hemagglutinin (HA) protein. Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal influenza infection in mice. Antibody isotypes that direct clearance of infected cells enhance this protection. Head-stem interface antibodies bind to most influenza A serotypes and seasonal human variants, and are present at low frequencies in the memory B cell populations of multiple human donors. Vaccines designed to elicit these antibodies might contribute to "universal" influenza immunity.

Development of a Recombinant Live Attenuated Influenza Vaccine Virus Expressing Pneumococcal Surface Antigen A as a Strategy for Combined Protection Against Influenza and Bacterial Coinfection Caused by Streptococcus pneumoniae

Introduction Bacterial superinfection with Streptococcus pneumoniae following the influenza virus infection complicates the course of the disease and is a major cause of mortality during influenza virus epidemics. The effectiveness of licensed polysaccharide vaccines is limited by the serotypes included in the vaccine and possible immune tolerance during revaccination. Pneumococcal surface protein A (PspA), which includes conserved regions and has at least two functions in pathogen virulence, is considered a promising target for the development of new-generation vaccines, including gene-engineering constructions. Therefore, the development of an influenza virus vector-based vaccine expressing conserved bacterial proteins seems to be a promising strategy for designing combined vaccines against influenza and bacterial pathogens. Methods A recombinant live attenuated influenza virus (LAIV) expressing PspA fragment in a modified hemagglutinin was rescued on the A/Leningrad/134/17/57 (H2N2) backbone. This recombinant virus was assessed for its growth characteristics in vitro, as well as for its immunogenicity and protective capacity, using a mouse model of influenza-bacterial coinfection. Results The rescued recombinant LAIV/HA+PspA virus was genetically stable after sequential passaging in embryonated chicken eggs and possessed an attenuated phenotype similar to the classical LAIV strain. The LAIV/HA+PspA bivalent vaccine-induced IgG antibodies specific to both influenza virus and S. pneumoniae and provided complete protection of vaccinated mice against lethal influenza infection, as well as a 40% survival rate for lethal homologous and heterologous influenza infection complicated by concomitant bacterial infection with S. pneumoniae. Conclusion The presented design of a recombinant influenza virus carrying immunogenic fragments of a bacterial pathogen can be considered a promising strategy for the combined protection of vaccinated individuals against influenza and its bacterial complications, and further in-depth studies of such recombinant viruses in preclinical studies are warranted.

The effect of triterpene glycoside on survival and changes in the cytokine profile in the spleen of BALB/c mice under conditions of lethal influenza infection A/WSN/1/33(H1N1)

BACKGROUND: Currently, the search and development of compounds, including herbal ones, with anti-influenza activity are relevant.
 AIM: To study the effect of oral administration of the triterpene glycoside saponin tauroside Sx1 on survival and changes in the cytokine profile of the spleen of mice under conditions of lethal influenza infection.
 MATERIAL AND METHODS: 78 male BALB/c mice aged 4–6 weeks, weighing 16–18 g, were used. They were divided into groups: first — control group; the second was a group of animals infected with influenza virus A/WSN/1/33(H1N1); the third was a group of infected animals that received saponin tauroside Sx1, isolated from the Crimean ivy Hedera taurica (Hibberd) Carrière at a dose of 11.8 mg/kg per day against the background of a viral infection for 3 days. The survival and expression of interleukins-1β and -10 in the spleen of mice on the 4th and 14th days of the experiment were studied. Spleen fragments were stained with hematoxylin and eosin. Microscopic analysis was performed using a video microscopy system. Immunohistochemical study was performed according to a standard protocol. Intergroup differences were assessed using the Kruskal–Wallis test and the Mann–Whitney U test using MS Office (Excel 2010) and Statistica 10.0.
 RESULTS: Administration of saponin tauroside Sx1 significantly increased the average life expectancy of animals by 4.6 days compared to the group without correction; survival rate was 30.0% (p=0.0233). Against the background of influenza infection, an increase in the number of cells secreting interleukin-1β was detected in the spleen of mice. In the mantle zone of the second group, the level of cytokine expression exceeded the control values by 3.41 times (p=0.0001), and the values of the third group — by 3.38 times (p=0.0001). By the 14th day of the experiment in the mantle zone, expression in the group with saponin administration increased, exceeding the indicators of the group without correction by 34.78% (p=0.0482). By the 4th day of the experiment, in both experimental groups, the number of cells positive for interleukin-10 increased in the mantle zone and decreased compensatoryly in the marginal zone. By the 14th day, in the group without correction, a decrease in expression by 24.86% (p=0.0487) was recorded; after the administration of saponin, the number of immunopositive cells was practically no different from the control (p=0.0443).
 CONCLUSION: Therapeutic administration of saponin tauroside Sx1 stabilizes the cytokine profile of the spleen of mice and promotes their better survival in conditions of lethal influenza infection.

Antiviral Activity of Double-Stranded Ribonucleic Acid and Interferon Alpha Composition in the Model of Experimental Influenza Infection of Mice

High variability of influenza viruses requires the development of agents for nonspecific resistance stimulation, along with the development of new drugs for prevention and treatment. Among the antiviral drugs, interferons and their inducers are known to exhibit a universally wide spectrum of action. The Institute of Medical Biotechnology, a branch of SRC VB «Vector», Rospotrebnadzor, has developed the technology and obtained pharmaceutical compositions containing an interferon inducer — double stranded ribonucleic acid (dsRNA) from the killer strain of Saccharomyces cerevisiae yeast and recombinant human interferon-alpha-2b (IFN- alpha-2b). The specific antiviral activity of the preparations and synergistic effect of the components within the compositions were shown in L-68 and L-929 cell cultures. The aim of this work was to study antiviral activity of intranasal forms of the pharmaceutical compositions containing yeast dsRNA and recombinant human interferon-alpha-2b in a model of lethal influenza infection in mice. The outbred ICR/CD1 mice were intranasally infected with influenza A/Aichi/2/68 (H3N2) virus. The study compositions were intranasally administered 3 hours before infection with influenza virus, as well as 1 and 3 days post infection. The doses of active components in the administered compositions were as follows: for dsRNA — 2.5 mg/kg, for IFN-alpha-2b — 500 IU/kg, 2500 IU/kg, or 5000 IU/kg. The antiviral activity of the drugs was assessed based on the mortality rate and the average life expectancy of mice. It was shown that a three-time intranasal administration of the composition of dsRNA (2.5 mg/kg) and IFN-alpha-2b (2500 IU) into the infected mice according to therapeutic-prophylactic regimen led to an increase in the rates of survival and average life expectancy of animals, which were comparable to the effect of Tamiflu. The comparison preparations — dsRNA and IFN-alpha-2b — administered intranasally at the same doses and regimen exerted no antiviral effect in this mouse model of viral infection. The data obtained confirm the prospects for further development of new dosage forms of dsRNA and interferons for intranasal application as agents for prevention and treatment of influenza.

IL-4/IL-4 Ab complex enhances the accumulation of both antigen-specific and bystander CD8 T cells in mouse lungs infected with influenza A virus

BackgroundUnlike conventional T cells, innate and virtual-memory CD8 T cells in naïve mice acquire their memory phenotypes and functions in the absence of antigenic encounters in a cytokine-dependent manner. The relevant cytokines include interleukin-4 (IL-4), type I interferon, and interleukin-15 (IL-15). Moreover, exogenous IL-4 can also induce de novo generation and/or expansion of the virtual-memory CD8 T cell population. In this study, we investigated whether exogenous IL-4 could enhance the immune response to a viral infection.ResultsIn vivo administration of IL-4 and an anti-IL-4 antibody complex (IL-4C) increased CXCR3 expression in both memory and naïve phenotype CD8 T cells in the absence of antigenic stimulation, and protected mice from lethal influenza infection. Flow cytometric analysis of lung-infiltrating immune cells on day 5 after virus infection revealed higher numbers of antigen-specific and bystander CD8 T cells in IL-4C-treated mice than in control mice. In particular, the bystander CD8 T cells were a naïve or evident memory phenotypes. Crucially, an anti-CXCR3 blocking antibody abrogated this IL-4C effect, reflecting that the increased accumulation of CD8 T cells in the lungs after IL-4C treatment is dependent on CXCR3.ConclusionsThese data demonstrate that exogenous IL-4C plays a protective role by enhancing CXCR3-dependent migration of CD8 T cells into influenza-infected lungs.

Prevention of severe lung immunopathology associated with influenza infection through adeno-associated virus vector administration

BackgroundInfluenza A viruses (IAVs) have long posed a threat to humans, occasionally causing significant morbidity and mortality. The initial immune response is triggered by infected epithelial cells, alveolar macrophages and dendritic cells. However, an exaggerated innate immune response can result in severe lung injury and even host mortality. One notable pathology observed in hosts succumbing to severe influenza is the excessive influx of neutrophils and monocytes into the lung. In this study, we investigated a strategy for controlling lung immunopathology following severe influenza infection.ResultsTo evaluate the impact of innate immunity on influenza-associated lung injury, we employed CB17.SCID and NOD.SCID mice. NOD.SCID mice exhibited slower weight loss and longer survival than CB17.SCID mice following influenza infection. Lung inflammation was reduced in NOD.SCID mice compared to CB17.SCID mice. Bulk RNA sequencing analysis of lung tissue showed significant downregulation of 827 genes, and differentially expressed gene analysis indicated that the cytokine-cytokine receptor interaction pathway was predominantly downregulated in NOD.SCID mice. Interestingly, the expression of the Cxcl14 gene was higher in the lungs of influenza-infected NOD.SCID mice than in CB17.SCID mice. Therefore, we induced overexpression of the Cxcl14 gene in the lung using the adeno-associated virus 9 (AAV9)-vector system for target gene delivery. However, when we administered the AAV9 vector carrying the Cxcl14 gene or a control AAV9 vector to BALB/c mice from both groups, the morbidity and mortality rates remained similar. Both groups exhibited lower morbidity and mortality than the naive group that did not receive the AAV9 vector prior to IAV infection, suggesting that the pre-administration of the AAV9 vector conferred protection against lethal influenza infection, irrespective of Cxcl14 overexpression. Furthermore, we found that pre-inoculation of BALB/c mice with AAV9 attenuated the infiltration of trans-macrophages, neutrophils and monocytes in the lungs following IAV infection. Although there was no difference in lung viral titers between the naive group and the AAV9 pre-inoculated group, pre-inoculation with AAV9 conferred lung injury protection against lethal influenza infection in mice.ConclusionsOur study demonstrated that pre-inoculation with AAV9 prior to IAV infection protected mouse lungs from immunopathology by reducing the recruitment of inflammatory cells.

An Advax-CpG adjuvanted recombinant H5 hemagglutinin vaccine protects mice against lethal influenza infection

There is a major unmet need for strategies to improve the immunogenicity of vaccines to protect against highly pathogenic avian influenza strains with pandemic potential. This study tested the ability of adjuvants based on delta inulin (Advax™) alone or combined with a TLR9 agonist (Advax-CpG™) to enhance the immunogenicity of recombinant H5 hemagglutinin antigen expressed in insect cells (rH5HA) to protect mice against lethal influenza infection. The Advax-adjuvanted rH5HA induced high serum hemagglutination inhibition activity, as well as Th1 and Th2 cytokine secreting CD4 and CD8 T cells. Immunization protected mice against a lethal heterosubtypic H5N1 virus challenge. Mice immunized with an Advax-adjuvanted rHA2 stem antigen prepared by enzymatic cleavage of rH5HA produced serum antibodies devoid of hemagglutination inhibition activity, but these anti-HA2 antibodies were nevertheless able to transfer protection against lethal H1N1 or H3N2 infections to naïve mice. We hypothesize that the enhanced protection afforded by Advax-adjuvanted rH5HA may be mediated by the combination of neutralizing antibodies directed at the HA head, anti-HA2 stem antibodies plus memory CD4 + and CD8 + T cells. This outcome supports further development of the Advax-adjuvanted rH5 pandemic influenza vaccine platform.

Combination adjuvants and antigens provide heterosubtypic protection against lethal influenza infection.

Abstract Influenza A virus (IAV) is a negative strand RNA virus that undergoes antigenic drift and shift, leading to serologically distinct strains that can cause tens of thousands of deaths annually in the US. The wide variety of hemagglutinin (HA) and neuraminidase (NA) proteins necessitates yearly reformulation of the vaccine to protect against novel seasonal and pandemic strains. Current IAV vaccines do not contain adjuvants, however, immune modulators such as pattern recognition receptor (PRR) agonists may enhance antigen presentation to T cells and provide broader protection against IAV strains that differ in HA and NA outer coat proteins. We set out to investigate whether TLR4 (lipopolysaccharide) and TLR3 (poly IC) agonists in combination with HA (H3N2) and nucleoprotein (NP) or NP and matrix (M1) proteins could provide protection against lethal challenge with mismatched IAV strain H1N1. Mice given H3 and NP with LPS + pIC in a prime/boost strategy demonstrated 80% survival after heterosubtypic H1N1 challenge while H3 + NP in PBS showed 20% survival. Similar results were seen using LPS + pIC in combination with NP and M1 protein with 90% survival. High endpoint titers of anti-NP antibodies (that also bound H1N1 virus) correlated with heterosubtypic protection suggesting a role for non-neutralizing antibodies in promoting survival. In addition, LPS and pIC enhanced dendritic cell (DC) function in vitro and increased migratory monocyte DC in the lymph node in vivo, coupled with early upregulation of T cell marker CD69. This strategy may provide increased efficacy over current IAV vaccines by inducing non-neutralizing antibodies and enhanced T cell responses to internal proteins. Supported by Trudeau Institute and Clarkson University.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity invivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

STING agonist-containing microparticles improve seasonal influenza vaccine efficacy and durability in ferrets over standard adjuvant.

The current pandemic highlights the need for effective vaccines against respiratory viruses. An ideal vaccine should induce robust and long-lasting responses with high manufacturing scalability. We use an adjuvant comprised of a Stimulator of Interferon Genes (STING) agonist incorporated in a scalable microparticle platform to achieve durable protection against the influenza virus. This formulation overcomes the challenges presented by the cytosolic localization of STING and the hydrophilicity of its agonists. We evaluated a monoaxial formulation of polymeric acetalated dextran microparticles (MPs) to deliver the STING agonist cyclic GMP-AMP (cGAMP) which achieved >10× dose-sparing effects compared to other published work. Efficacy was evaluated in ferrets, a larger animal model of choice for influenza vaccines. cGAMP MPs with recombinant hemagglutinin reduced viral shedding and improved vaccine outcomes compared to a seasonal influenza vaccine. Importantly, sustained protection against a lethal influenza infection was detected a year after a single dose of the vaccine-adjuvant.

C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus

SummaryThe development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro. Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection.

RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice.

Seasonal influenza epidemics represent a significant global health threat. The exacerbated immune response triggered by respiratory influenza virus infection causes severe pulmonary damage and contributes to substantial morbidity and mortality. Regulator of G-protein signaling 10 (RGS10) belongs to the RGS protein family that act as GTPase activating proteins for heterotrimeric G proteins to terminate signaling pathways downstream of G protein-coupled receptors. While RGS10 is highly expressed in immune cells, in particular monocytes and macrophages, where it has strong anti-inflammatory effects, its physiological role in the respiratory immune system has not been explored yet. Here, we show that Rgs10 negatively modulates lung immune and inflammatory responses associated with severe influenza H1N1 virus respiratory infection in a mouse model. In response to influenza A virus challenge, mice lacking RGS10 experience enhanced weight loss and lung viral titers, higher mortality and significantly faster disease onset. Deficiency of Rgs10 upregulates the levels of several proinflammatory cytokines and chemokines and increases myeloid leukocyte accumulation in the infected lung, markedly neutrophils, monocytes, and inflammatory monocytes, which is associated with more pronounced lung damage. Consistent with this, influenza-infected Rgs10-deficent lungs contain more neutrophil extracellular traps and exhibit higher neutrophil elastase activities than wild-type lungs. Overall, these findings propose a novel, in vivo role for RGS10 in the respiratory immune system controlling myeloid leukocyte infiltration, viral clearance and associated clinical symptoms following lethal influenza challenge. RGS10 also holds promise as a new, potential therapeutic target for respiratory infections.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Influenza Vaccine-Induced CD4 Effectors Require Antigen Recognition at an Effector Checkpoint to Generate CD4 Lung Memory and Antibody Production.

Previously, we discovered that influenza-generated CD4 effectors must recognize cognate Ag at a defined effector checkpoint to become memory cells. Ag recognition was also required for efficient protection against lethal influenza infection. To extend these findings, we investigated if vaccine-generated effectors would have the same requirement. We compared live infection with influenza to an inactivated whole influenza vaccine. Live infection provided strong, long-lasting Ag presentation that persisted through the effector phase. It stimulated effector generation, long-lived CD4 memory generation, and robust generation of Ab-producing B cells. In contrast, immunization with an inactivated virus vaccine, even when enhanced by additional Ag-pulsed APC, presented Ag for 3 d or less and generated few CD4 memory cells or long-lived Ab-producing B cells. To test if checkpoint Ag addition would enhance this vaccine response, we immunized mice with inactivated vaccine and injected Ag-pulsed activated APC at the predicted effector checkpoint to provide Ag presentation to the effector CD4 T cells. This enhanced generation of CD4 memory, especially tissue-resident memory in the lung, long-lived bone marrow Ab-secreting cells, and influenza-specific IgG Ab. All responses increased as we increased the density of peptide Ag on the APC to high levels. This suggests that CD4 effectors induced by inactivated vaccine require high levels of cognate Ag recognition at the effector checkpoint to most efficiently become memory cells. Thus, we suggest that nonlive vaccines will need to provide high levels of Ag recognition throughout the effector checkpoint to optimize CD4 memory generation.

Intranasal Bifidobacterium longum protects against viral-induced lung inflammation and injury in a murine model of lethal influenza infection.

BackgroundProphylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections.MethodsWe examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified.FindingsIntranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain.InterpretationExposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung.FundingThis work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne – Center for Allergy Research and Education (CK-CARE).

Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.

LAIV vaccination prior to pregnancy protects against lethal influenza infection and prevents infection-associated outcomes in offspring

Abstract Influenza is a highly contagious respiratory infection of public health concern. Pregnant women are among those at greater risk of suffering from serious influenza A virus (IAV) infection. Compromised cell-mediated immune responses, low inactivated vaccine efficacy, and poor vaccine uptake all increase the likelihood that exposure to the virus during pregnancy will result in infection-associated maternal and gestational complications such as stillbirths, preterm delivery, and low birthweights. The protective potential of IAV-specific immunological T cell memory induced by vaccination with a cold adapted live attenuated influenza vaccine (LAIV) during pregnancy has not been described; this immunity has the potential to mediate universal protection against influenza. Here, we investigated how LAIV priming before pregnancy and subsequent heterosubtypic IAV challenge during pregnancy impacts maternal and fetal outcomes in mice. LAIV priming prior to pregnancy completely protects pregnant dams against the morbidity and mortality of infection that is lethal to unprimed mice and primed dams go on to drop average sized litters. Pups born to unprimed dams are significantly smaller in terms of weight on days 3, 7, 14, and 21 when compared to pups born to LAIV primed dams. In contrast, morphological development determined by crown-rump length and skeletal measurements, while initially hindered, does not significantly differ between pups born to unprimed and primed dams after the first week following birth. LAIV priming prior to pregnancy thus appears to prevent adverse maternal and fetal IAV-associated outcomes. These data support that LAIV has potential for use as a universal vaccine to protect pregnant females against pandemic IAV.

Influenza infection elicits an expansion of gut population of endogenous Bifidobacterium animalis which protects mice against infection

BackgroundInfluenza is a severe respiratory illness that continually threatens global health. It has been widely known that gut microbiota modulates the host response to protect against influenza infection, but mechanistic details remain largely unknown. Here, we took advantage of the phenomenon of lethal dose 50 (LD50) and metagenomic sequencing analysis to identify specific anti-influenza gut microbes and analyze the underlying mechanism.ResultsTransferring fecal microbes from mice that survive virulent influenza H7N9 infection into antibiotic-treated mice confers resistance to infection. Some gut microbes exhibit differential features to lethal influenza infection depending on the infection outcome. Bifidobacterium pseudolongum and Bifidobacterium animalis levels are significantly elevated in surviving mice when compared to dead or mock-infected mice. Oral administration of B. animalis alone or the combination of both significantly reduces the severity of H7N9 infection in both antibiotic-treated and germ-free mice. Functional metagenomic analysis suggests that B. animalis mediates the anti-influenza effect via several specific metabolic molecules. In vivo tests confirm valine and coenzyme A produce an anti-influenza effect.ConclusionsThese findings show that the severity of influenza infection is closely related to the heterogeneous responses of the gut microbiota. We demonstrate the anti-influenza effect of B. animalis, and also find that the gut population of endogenous B. animalis can expand to enhance host influenza resistance when lethal influenza infection occurs, representing a novel interaction between host and gut microbiota. Further, our data suggest the potential utility of Bifidobacterium in the prevention and as a prognostic predictor of influenza.