Lethal Influenza Infection Research Articles

FimH, a TLR4 ligand, induces innate antiviral responses in the lung leading to protection against lethal influenza infection in mice

Fimbriae H protein (FimH) is a novel TLR4 ligand that has been shown to stimulate the innate immune system and elicits protective responses against bacterial and viral infections. Here, we evaluated the protective role of local delivery of FimH against influenza A infection in a mouse model. We show that intranasal delivery of FimH prior to lethal challenge with influenza A virus, resulted in decreased morbidity and mortality in wild-type, but not TLR4 −/−, mice. Importantly, FimH was able to reduce the early viral burden in the lung leading to minimal cell infiltration into the airway lumen and reduced pulmonary pathology following infection in wild type mice compared to TLR4 −/− mice. Local delivery of FimH to C57BL/6, not TLR4 −/−, mice in a prophylactic manner increased the IL-12 and RANTES responses as well as neutrophil recruitment into the airway lumen. These effects correlate to the course of influenza infection. The FimH-mediated antiviral response against influenza virus appears to be partially dependent on alveolar macrophages. The antiviral effects are likely mediated by the innate mediators (TNF-α, IL-12 or RANTES) and/or by activation of a feedback inhibition loop to curtail the pulmonary inflammation possibly be the potential mechanisms involved in FimH-mediated protection. FimH thus holds promise to be a possible prophylactic mean of control against influenza viral infection.

Immunogenicity and Efficacy of Flagellin-Fused Vaccine Candidates Targeting 2009 Pandemic H1N1 Influenza in Mice

We have previously demonstrated that the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2, a TLR5 ligand) elicits protective immunity to H1N1 and H5N1 lethal influenza infections in mice (Song et al., 2008, PLoS ONE 3, e2257; Song et al., 2009, Vaccine 27, 5875–5888). These fusion proteins can be efficiently and economically manufactured in E. coli fermentation systems as next generation pandemic and seasonal influenza vaccines. Here we report immunogenicity and efficacy results of three vaccine candidates in which the HA globular head of A/California/07/2009 (H1N1) was fused to STF2 at the C-terminus (STF2.HA1), in replace of domain 3 (STF2R3.HA1), or in both positions (STF2R3.2xHA1). For all three vaccines, two subcutaneous immunizations of BALB/c mice with doses of either 0.3 or 3 µg elicit robust neutralizing (HAI) antibodies, that lead to > = 2 Log10 unit reduction in day 4 lung virus titer and full protection against a lethal A/California/04/2009 challenge. Vaccination with doses as low as 0.03 µg results in partial to full protection. Each candidate, particularly the STF2R3.HA1 and STF2R3.2xHA1 candidates, elicits robust neutralizing antibody responses that last for at least 8 months. The STF2R3.HA1 candidate, which was intermediately protective in the challenge models, is more immunogenic than the H1N1 components of two commercially available trivalent inactivated influenza vaccines (TIVs) in mice. Taken together, the results demonstrate that all three vaccine candidates are highly immunogenic and efficacious in mice, and that the STF2R3.2xHA1 format is the most effective candidate vaccine format.

GM-CSF in the Lung Protects against Lethal Influenza Infection

Alveolar macrophages contribute to host defenses against influenza in animal models. Enhancing alveolar macrophage function may contribute to protection against influenza. To determine if increased expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the lung increases resistance to influenza. Wild-type mice and transgenic mice that expressed GM-CSF in the lung were infected with influenza virus, and lung pathology, weight loss, and mortality were measured. We also administered GM-CSF to the lungs of wild-type mice that were infected with influenza virus. Wild-type mice all died after infection with different strains of influenza virus, but all transgenic mice expressing GM-CSF in the lungs survived. The latter also had greatly reduced weight loss and lung injury, and showed histologic evidence of a rapid host inflammatory response that controlled infection. The resistance of transgenic mice to influenza was abrogated by elimination of alveolar phagocytes, but not by depletion of T cells, B cells, or neutrophils. Transgenic mice had far more alveolar macrophages than did wild-type mice, and they were more resistant to influenza-induced apoptosis. Delivery of intranasal GM-CSF to wild-type mice also conferred resistance to influenza. GM-CSF confers resistance to influenza by enhancing innate immune mechanisms that depend on alveolar macrophages. Pulmonary delivery of this cytokine has the potential to reduce the morbidity and mortality due to influenza virus.

Early cytokine dysregulation contributes to mortality during lethal influenza infection. (49.18)

Abstract Recent studies have suggested that pandemic influenza viral strains induce high pro-inflammatory cytokine levels in the lung that may contribute to increased mortality. However, the mechanisms linking highly virulent virus infection and mortality have not been fully elucidated. Here we compare a sublethal dose of highly pathogenic H1N1 virus, A/Puerto Rico/34/8 (PR8) with a lethal dose to determine the extent by which viral load affects inflammatory responses in the lung. We observed a 5-10 fold increase in viral load that correlated with a 10 fold increase in TNF-α, IL-1β and MIP-1α secretion in lethally infected mice compared to sublethally infected mice at day 2 post infection (p. i.). Surprisingly, this difference was not apparent at later time points after infection indicating early cytokine dysregulation may contribute to mortality. IL-6, MCP-1 and MIP-1β were also upregulated at day 2 p. i. and peaked at day 6 suggesting sustained levels of certain cytokines may also contribute to pathology. Furthermore, we find that RIG-I and TLR-3 are upregulated in CD45- epithelial cells leading to high expression of IFN-β and IL-6 while TLR-7 and TLR-9 are highly upregulated in CD45+ immune cells correlating with expression of TNF-α. Thus, differential PRR usage and cytokine secretion may be a contributing factor in the balance between protective immunity and pathogenicity.

Parasite induced IFN-γ treats severe H5N1 influenza infection (49.11)

Abstract Outbreaks of influenza A viruses are a continued burden to society in terms of both human health and economic costs. Given the continuous evolution of the virus away from our limited antiviral therapies, new treatments are desperately needed. An alternative approach is through the identification of immune enhancement treatments. We show that mice chronically infected with the immune modulating protistian parasite Toxoplasma gondii survive lethal influenza infection. Because a non-infectious fraction of parasite extract elicits an immunologic response similar to live parasites, it was tested as a post-infection treatment for severe influenza virus. Extract administration two days post- lethal influenza infection enhanced survival, lowered viral titers, and reduced clinical disease. Examination of treated, influenza virus infected mice lacking lymphocytes showed that while the adaptive immune response is not absolutely required for enhanced survival, it is necessary for extract-mediated viral clearance. Mechanistic analysis of the extract induced-innate immune responses showed that interferon gamma (IFNγ) production is both necessary and sufficient for survival. These studies demonstrate that T. gondii may have beneficial properties for its warm-blooded host and a non-infectious component from one microbe can be used to enhance the immune response to a second microbe.

Evaluation of anti-influenza efficiency of polyclonal IgG antibodies specific to the ectodomain of M2 protein of influenza A virus by passive immunization of mice

We attempted to quantify the protective potential of polyclonal IgG antibodies specific to the ectodomain of M2 protein (eM2) of influenza A virus (IAV) against lethal influenza infection of mice. For this purpose, eM2 conjugated with keyhole limpet hemocyanin (KLH) or KLH alone were administered with Freund's adjuvant intraperitoneally (i.p.) to BALB/c mice. IgG antibodies specific to the KLH-eM2 conjugate (anti-KLH-eM2 IgGs) and KLH (anti-KLH IgGs), respectively, were purified from ascitic fluids. Analysis of the preparation of anti-KLH-eM2 IgGs by ELISA revealed that it contained about 25% of anti-eM2 IgGs and 75% of anti-KLH IgGs. Taking into account this finding mice were passively immunized by intravenous route with 320, 160, 80, and 40 µg of anti-eM2 IgGs per mouse, respectively, while 320 µg of anti-KLH IgGs were used in control. Following subsequent infection with 3 LD50 IAV the survival of mice was determined. An absolute protection (100% survival) was obtained with 320 µg of anti-eM2 IgGs, and a relatively strong significant protection (~80% survival, p = 0.024) with 160 µg. The amount 160 µg of IgGs represents approx. 100 µg IgGs per 1 ml of blood.

Protection from H1N1 Influenza Virus Infections in Mice by Supplementation with Selenium: A Comparison with Selenium-Deficient Mice

The present paper describes protective effects of supplemental selenium in mice infected with influenza virus. The effects of supplemental selenium on serum selenium levels, mortality, lung virus titers, and cytokine titers were investigated in mice inoculated intranasally with suspensions of influenza virus. Whereas the mortality of the virus-infected Se-deficient mice was 75%, along with a marked reduction in body weight, lower levels of TNF-α and IFN-γ and lower serum selenium concentrations, the mortality of mice maintained on feed containing 0.5mg Se/kg in the form of sodium selenite was 25%.There were no significantly differences, however, in viral titer between the Se-adequate and the selenium-supplemented groups. The data indicate that selenium supplementation may provide a feasible approach to improving the immune response to viral infections, such as lethal influenza infection.

Plasmacytoid Dendritic Cells Enhance Mortality during Lethal Influenza Infections by Eliminating Virus-Specific CD8 T Cells

Previous studies have shown that the reduction in CD8 T cell immunity observed during high-dose influenza A virus (IAV) infection is mediated via lymph node (LN) dendritic cells (DCs) that express Fas ligand (FasL) and drive FasL-Fas (DC-T)-induced apoptosis. However, the specific DC subset(s) within the LN and the additional factors required for DC-mediated elimination of IAV-specific CD8 T cells remain unknown. In this paper, we demonstrate that plasmacytoid DCs (pDCs), which downregulate FasL during sublethal, but not lethal, IAV infection, accumulate to greater numbers within the LNs of lethal dose-infected mice. Further our findings show that pDCs from lethal, but not sublethal, dose IAV infections drive elimination of Fas(+) CD8 T cells and that this elimination occurs only in the absence of TCR recognition of IAV peptide-MHC class I complexes. Together, these results suggest that pDCs play a heretofore unknown deleterious role during lethal dose IAV infections by limiting the CD8 T cell response.

Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

BackgroundRecombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.Methodology/Principal FindingsPeptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.Conclusions/SignificanceAP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.

The mechanism of protection against influenza by transfer of Tc17, an IL-17 secreting CD8 T cell subset. (46.5)

Abstract Interleukin (IL) -17 secreting CD4 T (Th)17 and CD8 T (Tc)17 effector cells are found in the lung following primary challenge with influenza A and blocking antibody to IL-17 increases weight loss and reduces survival. Tc17 effectors can be generated in vitro using naïve CD8 T cells from OT-1 T cell receptor (TcR) transgenic mice. T cell numbers expand 20 fold and a majority secretes IL-17, but little IFN-γ. Tc17 effectors are negative for granzyme B, for perforin message and lack cytolytic activity in vitro, in contrast to Tc1 effectors. Many of the Tc17 effectors become IFN-γ/IL-17 double producers after adoptive transfer and kill targets by a Fas dependent, perforin independent, pathway in an in vivo CTL assay. Adoptively transferred Tc17 cells expand greatly after transfer to naïve recipients following challenge and can protect them against lethal influenza infection. Transfer of Tc17 effector cell enhances the infiltration of neutrophils and monocytes to lung after influenza infection and protects against the development of respiratory distress much more effectively than transfer of equivalent numbers of Tc1 effectors. Additional mechansims of protection are being explored. This work was supported by NIH AI046530 and the Trudeau Institute.

Tc17, a Unique Subset of CD8 T Cells That Can Protect against Lethal Influenza Challenge

We show here that IL-17-secreting CD4 T (Th)17 and CD8 T (Tc)17 effector cells are found in the lung following primary challenge with influenza A and that blocking Ab to IL-17 increases weight loss and reduces survival. Tc17 effectors can be generated in vitro using naive CD8 T cells from OT-I TCR-transgenic mice. T cell numbers expand 20-fold and a majority secretes IL-17, but little IFN-gamma. Many of the IL-17-secreting cells also secrete TNF and some secrete IL-2. Tc17 are negative for granzyme B, perforin message, and cytolytic activity, in contrast to Tc1 effectors. Tc17 populations express message for orphan nuclear receptor gammat and FoxP3, but are negative for T-bet and GATA-3 transcription factors. The FoxP3-positive, IL-17-secreting and IFN-gamma-secreting cells represent three separate populations. The IFN-gamma-, granzyme B-, FoxP3-positive cells and cells positive for IL-22 come mainly from memory cells and decrease in number when generated from CD44(low) rather than unselected CD8 T cells. Cells of this unique subset of CD8 effector T cells expand greatly after transfer to naive recipients following challenge and can protect them against lethal influenza infection. Tc17 protection is accompanied by greater neutrophil influx into the lung than in Tc1-injected mice, and the protection afforded by Tc17 effectors is less perforin but more IFN-gamma dependent, implying that different mechanisms are involved.

A Signaling Polypeptide Derived from an Innate Immune Adaptor Molecule Can Be Harnessed as a New Class of Vaccine Adjuvant

Modulation of intracellular signaling using cell-permeable polypeptides is a promising technology for future clinical applications. To develop a novel approach to activate innate immune signaling by synthetic polypeptides, we characterized several different polypeptides derived from the caspase recruitment domain (CARD) of IFN-beta promoter stimulator 1, each of which localizes to a different subcellular compartment. Of particular interest was, N'-CARD, which consisted of the nuclear localization signal of histone H2B and the IFN-beta promoter stimulator 1CARD and which localized to the nucleus. This polypeptide led to a strong production of type I IFNs and molecular and genetic analyses showed that nuclear DNA helicase II is critically involved in this response. N'-CARD polypeptide fused to a protein transduction domain (N'-CARD-PTD) readily transmigrated from the outside to the inside of the cell and triggered innate immune signaling. Administration of N'-CARD-PTD polypeptide elicited production of type I IFNs, maturation of bone marrow-derived dendritic cells, and promotion of vaccine immunogenicity by enhancing Ag-specific Th1-type immune responses, thereby protecting mice from lethal influenza infection and from outgrowth of transplanted tumors in vivo. Thus, our results indicate that the N'-CARD-PTD polypeptide belongs to a new class of vaccine adjuvant that directly triggers intracellular signal transduction by a distinct mechanism from those engaged by conventional vaccine adjuvants, such as TLR ligands.

Augmented Lung Inflammation Protects against Influenza A Pneumonia

BackgroundInfluenza pneumonia causes high mortality every year, and pandemic episodes kill millions of people. Influenza-related mortality has been variously ascribed to an ineffective host response that fails to limit viral replication, an excessive host inflammatory response that results in lung injury and impairment of gas exchange, or to bacterial superinfection. We sought to determine whether lung inflammation promoted or impaired host survival in influenza pneumonia.Methods and FindingsTo distinguish among these possible causes of influenza-related death, we induced robust lung inflammation by exposing mice to an aerosolized bacterial lysate prior to challenge with live virus. The treatment induced expression of the inflammatory cytokines IL-6 and TNF in bronchoalveolar lavage fluid 8- and 40-fold greater, respectively, than that caused by lethal influenza infection. Yet, this augmented inflammation was associated with striking resistance to host mortality (0% vs 90% survival, p = 0.0001) and reduced viral titers (p = 0.004). Bacterial superinfection of virus infected lungs was not observed. When mice were repeatedly exposed to the bacterial lysate, as would be clinically desirable during an influenza epidemic, there was no tachyphylaxis of the induced viral resistance. When the bacterial lysate was administered after the viral challenge, there was still some mortality benefit, and when ribavirin was added to the aerosolized bacterial lysate, host survival was synergistically improved (0% vs 93.3% survival, p<0.0001).ConclusionsTogether, these data indicate that innate immune resistance to influenza can be effectively stimulated, and suggest that ineffective rather than excessive inflammation is the major cause of mortality in influenza pneumonia.

Distinctly different expression of cytokines and chemokines in the lungs of two H5N1 avian influenza patients

The pathogenesis of human H5N1 influenza remains poorly understood and controversial. 'Cytokine storm' has been hypothesized to be the main cause of the severity of this disease. However, the significance of this hypothesis has been called into question by a recent report, which demonstrates that inhibition of the cytokine response did not protect against lethal H5N1 influenza infection in mice. Here we showed discrepant findings in two adult H5N1 autopsies and a fetus obtained at autopsy which also raise doubt about this hypothesis. Antigens of 10 cytokines/chemokines which were found to be significantly elevated in previous H5N1-infected patients and in vitro experiments, and mRNA of eight of these, were absent from the lungs of a pregnant woman and her fetus. In contrast, antigens of seven cytokines/chemokines and mRNA of six of these were found to be increased in the lungs of a male autopsy. The cells expressing these cytokines and chemokines were identified as type II pneumocytes, bronchial epithelial cells, macrophages and vascular endothelial cells. Levels of cytokines and chemokines in the serum of the male case were also significantly higher than those of infectious (infection other than by H5N1) and non-infectious controls. In comparison with results from our previous study, it appeared that the male case had increased cytokine/chemokine expression but reduced viral load, while the pregnant female had diminished cytokine/chemokine expression but a significantly increased viral load in the lungs. These disparate findings in these two cases suggest that 'cytokine storm' alone could not be a sufficient explanation for the severe lung injury of this newly emerging disease.

Inhibition of the cytokine response does not protect against lethal H5N1 influenza infection

Because proinflammatory cytokines are markedly elevated during H5N1 influenza virus infection, the "cytokine storm" is hypothesized to be the main cause of mortality. Here, we demonstrate that mice deficient in the hallmark inflammatory cytokines TNF-alpha, IL-6, or CC chemokine ligand 2 succumb to infection with A/Vietnam/1203/04 (H5N1) virus, as do wild-type mice treated with glucocorticoids for suppression of cytokines. Because cytokine inhibition does not protect against death, therapies that target the virus rather than cytokines may be preferable.

IL-17 receptor-deficient mice have increased resistance to otherwise lethal influenza infection (96.14)

Abstract IL-17 is a CD4+ T cell-derived cytokine that has been previously been shown to play a role in the immune response to several different lung infections. Specifically, it has been shown to induce neutrophil recruitment and inflammation in response to a variety of pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycoplasma pneumoniae. Additionally, it has been shown that IL-17 is upregulated in respiratory syncitial virus (RSV) infection, but its role in other viral pulmonary infections remains poorly described. In order to investigate the role of IL-17 in influenza infections, BALB/c mice lacking the IL-17 receptor were challenged intranasally with a lethal dose of influenza A/Puerto Rico/8/34 (H1N1). The mice were monitored for weight loss and signs of clinical illness on a daily basis. When compared to BALB/c controls, the IL-17 receptor knockout mice exhibited delayed weight loss and had higher survival rates. Additionally, the control mice exhibited higher levels of protein and lactate dehydrogenase in the BAL fluid, markers of acute lung injury, during the course of infection. These data suggest that the effects of IL-17R signaling contributes to lung injury and the pathology of a lethal influenza infection in a murine model.

N-Acetylcysteine Synergizes with Oseltamivir in Protecting Mice from Lethal Influenza Infection

Many studies have shown that oxidative stress is important in the pathogenesis of pulmonary damage during influenza virus infections. Antioxidant molecules are therefore potentially useful against viral infection. Our previous studies show that N-acetylcysteine (NAC) has a protective effect in a model of lethal influenza infection in mice. NAC administration significantly decreased the mortality in infected mice. Further studies have demonstrated that NAC enhanced survival in combination with the antiviral agent ribavirin. In the present study, we report the effect of combined treatment with NAC and Oseltamivir, clinically used in the treatment and prevention of influenza virus infection, in a murine model of lethal influenza infection. NAC was given as a single daily dose of 1000 mg/kg starting from 4 h before infection and until day 4 after infection; Oseltamivir was given twice daily at dose of 1 mg/kg/die for 5 days, starting from 4 h before infection. End-point evaluation was 21-days survival. NAC alone was slightly effective (20%), since a suboptimal treatment was used. Survival increased to 60% with Oseltamivir and to 100% with Oseltamivir and NAC used in combination. Since NAC alone does not show any antiviral action, the present findings suggest that antioxidant therapy increase survival by an improvement in host defense mechanisms, and/or by a direct antioxidant effect against oxidative stress associated with viral infection. Our studies demonstrate the effectiveness of combining agents acting through different mechanisms, such as antiviral drugs oseltamivir and the antioxidant NAC, indicating a possible advantage of combining the two treatments.

Immunogenicity of novel consensus-based DNA vaccines against avian influenza

The frequency of H5N1 avian influenza outbreaks in China and Eastern Europe has raised concern in the world health community regarding the potential for an influenza pandemic. Efforts to monitor the disease will only provide minimal warning in a global society, and steps must be taken to prevent the morbidity and mortality associated with past pandemics. The current stockpiling of antibody-inducing “bird flu” vaccines assumes the strain that emerges will be the same as strains currently circulating. We propose a novel consensus-based approach to vaccine development, employing a DNA vaccine strategy that can provide more highly cross-reactive cellular immunity against lethal influenza infection. We show such constructs can induce strong cellular immunity against H5 influenza antigens.

SAP Is Required for Th Cell Function and for Immunity to Influenza

Ab is a crucial component of protective immunity to infection, but Ab responses do not proceed normally when defects occur in a protein called signaling lymphocytic activation molecule-associated protein (SAP). To explain this Ab defect, we analyzed B cell and plasma cell responses under conditions of SAP deficiency. Our results demonstrate that SAP-deficient (SAP knockout (KO)) mice have a profound CD4 T cell-intrinsic defect in generating Ag-specific plasma cells following challenge with model Ags or influenza virus, resulting in low Ag-specific Ab titers. We also show that SAP is required in CD4 T cells for normal division and expansion of B cells. These B cell and plasma cell defects were observed during the expansion phase of the primary immune response, indicating early defects in Th cell activity. In fact, additional experiments revealed a nearly complete lack of T cell help for B cells in SAP KO mice. Our work suggests that the ability of SAP to promote T-dependent humoral immune responses is important for antiviral immunity because mice lacking SAP are unable to prevent high dose secondary influenza infection, and because passive transfer of IgG in immune serum from wild-type, but not SAP KO mice can protect mice from an otherwise lethal influenza infection. Overall, our results demonstrate that SAP is required in CD4 T cells for their ability to help B cell responses and promote influenza-specific immunity.

De Kruif's Boast: Vaccine Trials and the Construction of a Virus

This article describes the sustained effort by American researchers between the mid-1930s and the mid-1950s to develop an effective influenza vaccine. From almost the beginning of this project, researchers succeeded in protecting laboratory animals from lethal influenza infection, and they believed that success with humans would follow quickly. Yet although they succeeded in producing a vaccine that proved effective in field trials in 1943 and 1945, that same vaccine failed to offer any protection in 1947. This vaccine failure forced researchers to reconsider the growing evidence of antigenic variation and challenged the model of the virus that had been taken for granted.