Synergism and Antagonism of Bacterial-Viral Coinfection in the Upper Respiratory Tract.

Streptococcus pneumoniae (the pneumococcus) is a leading cause of pneumonia in children under 5 years of age. Coinfection by pneumococci and respiratory viruses enhances disease severity. Little is known about pneumococcal coinfections with respiratory syncytial virus (RSV). Here, we developed a novel infant mouse model of coinfection using pneumonia virus of mice (PVM), a murine analogue of RSV, to examine the dynamics of coinfection in the upper respiratory tract, an anatomical niche that is essential for host-to-host transmission and progression to disease. Coinfection increased damage to the nasal tissue and increased production of the chemokine CCL3. Nasopharyngeal pneumococcal density and shedding in nasal secretions were increased by coinfection. In contrast, coinfection reduced PVM loads in the nasopharynx, an effect that was independent of pneumococcal strain and the order of infection. We showed that this "antagonistic" effect was absent using either ethanol-killed pneumococci or a pneumococcal mutant deficient in capsule production and incapable of nasopharyngeal carriage. Colonization with a pneumococcal strain naturally unable to produce capsule also reduced viral loads. The pneumococcus-mediated reduction in PVM loads was caused by accelerated viral clearance from the nasopharynx. Although these synergistic and antagonistic effects occurred with both wild-type pneumococcal strains used in this study, the magnitude of the effects was strain dependent. Lastly, we showed that pneumococci can also antagonize influenza virus. Taken together, our study has uncovered multiple novel facets of bacterial-viral coinfection. Our findings have important public health implications, including for bacterial and viral vaccination strategies in young children. IMPORTANCE Respiratory bacterial-viral coinfections (such as pneumococci and influenza virus) are often synergistic, resulting in enhanced disease severity. Although colonization of the nasopharynx is the precursor to disease and transmission, little is known about bacterial-viral interactions that occur within this niche. In this study, we developed a novel mouse model to examine pneumococcal-viral interactions in the nasopharynx with pneumonia virus of mice (PVM) and influenza. We found that PVM infection benefits pneumococci by increasing their numbers in the nasopharynx and shedding of these bacteria in respiratory secretions. In contrast, we discovered that pneumococci decrease PVM numbers by accelerating viral clearance. We also report a similar effect of pneumococci on influenza. By showing that coinfections lead to both synergistic and antagonistic outcomes, our findings challenge the existing dogma in the field. Our work has important applications and implications for bacterial and viral vaccines that target these microbes.

Rhinovirus Reduces the Severity of Subsequent Respiratory Viral Infections by Interferon-Dependent and -Independent Mechanisms.

Coinfection by heterologous viruses in the respiratory tract is common and can alter disease severity compared to infection by individual virus strains. We previously found that inoculation of mice with rhinovirus (RV) 2 days before inoculation with a lethal dose of influenza A virus [A/Puerto Rico/8/34 (H1N1) (PR8)] provides complete protection against mortality. Here, we extended that finding to a second lethal respiratory virus, pneumonia virus of mice (PVM), and analyzed potential mechanisms of RV-induced protection. RV completely prevented mortality and weight loss associated with PVM infection. Major changes in host gene expression upon PVM infection were delayed compared to PR8. RV induced earlier recruitment of inflammatory cells, which were reduced at later times in RV-inoculated mice. Findings common to both virus pairs included the upregulated expression of mucin-associated genes and dampening of inflammation-related genes in mice that were inoculated with RV before lethal virus infection. However, type I interferon (IFN) signaling was required for RV-mediated protection against PR8 but not PVM. IFN signaling had minor effects on PR8 replication and contributed to controlling neutrophilic inflammation and hemorrhagic lung pathology in RV/PR8-infected mice. These findings, combined with differences in virus replication levels and disease severity, suggest that the suppression of inflammation in RV/PVM-infected mice may be due to early, IFN-independent suppression of viral replication, while that in RV/PR8-infected mice may be due to IFN-dependent modulation of immune responses. Thus, a mild upper respiratory viral infection can reduce the severity of a subsequent severe viral infection in the lungs through virus-dependent mechanisms. IMPORTANCE Respiratory viruses from diverse families cocirculate in human populations and are frequently detected within the same host. Although clinical studies suggest that infection by multiple different respiratory viruses may alter disease severity, animal models in which we can control the doses, timing, and strains of coinfecting viruses are critical to understanding how coinfection affects disease severity. Here, we compared gene expression and immune cell recruitment between two pairs of viruses (RV/PR8 and RV/PVM) inoculated sequentially in mice, both of which result in reduced severity compared to lethal infection by PR8 or PVM alone. Reduced disease severity was associated with suppression of inflammatory responses in the lungs. However, differences in disease kinetics and host and viral gene expression suggest that protection by coinfection with RV may be due to distinct molecular mechanisms. Indeed, we found that antiviral cytokine signaling was required for RV-mediated protection against lethal infection by PR8 but not PVM.

Persistent Airway Hyperresponsiveness Following Recovery from Infection with Pneumonia Virus of Mice.

Respiratory virus infections can have long-term effects on lung function that persist even after the acute responses have resolved. Numerous studies have linked severe early childhood infection with respiratory syncytial virus (RSV) to the development of wheezing and asthma, although the underlying mechanisms connecting these observations remain unclear. Here, we examine airway hyperresponsiveness (AHR) that develops in wild-type mice after recovery from symptomatic but sublethal infection with the natural rodent pathogen, pneumonia virus of mice (PVM). We found that BALB/c mice respond to a limited inoculum of PVM with significant but reversible weight loss accompanied by virus replication, acute inflammation, and neutrophil recruitment to the airways. At day 21 post-inoculation, virus was no longer detected in the airways and the acute inflammatory response had largely resolved. However, and in contrast to most earlier studies using the PVM infection model, all mice survived the initial infection and all went on to develop serum anti-PVM IgG antibodies. Furthermore, using both invasive plethysmography and precision-cut lung slices, we found that these mice exhibited significant airway hyperresponsiveness at day 21 post-inoculation that persisted through day 45. Taken together, our findings extend an important and versatile respiratory virus infection model that can now be used to explore the role of virions and virion clearance as well as virus-induced inflammatory mediators and their signaling pathways in the development and persistence of post-viral AHR and lung dysfunction.

Innate immune protection from pneumonia virus of mice induced by a novel immunomodulator is prolonged by dual treatment and mediated by macrophages.

Respiratory syncytial virus (RSV) is responsible for a large proportion of acute lower respiratory tract infections, specifically in children. Pneumonia virus of mice (PVM) causes similar lung pathology and clinical disease in rodents, and is therefore an appropriate model of RSV infection. Previously, we demonstrated that a single intranasal dose of P-I-P, a novel immunomodulator composed of the toll-like receptor 3 agonist poly(I:C), an innate defense regulator peptide and a polyphosphazene, confers protection in Balb/c mice for up to 3 days from lethal PVM-15 infection. In the present study a dual intranasal treatment with P-I-P was shown to extend the duration of the protection conferred by P-I-P from PVM-15 challenge. Balb/c mice treated twice with P-I-P showed higher survival rates and milder clinical signs when compared to animals that received a single P-I-P dose. While the mice treated with two consecutive doses of P-I-P experienced some weight loss, they all recovered. The dual P-I-P treatment mediated infiltration of several innate immune cells into the BALF and lung, including alveolar macrophages, neutrophils, and γδ T cells. Partial depletion of alveolar macrophages decreased survival rates and exacerbated clinical signs of mice subjected to the P-I-P dual treatment regime followed by PVM-15 challenge. This suggests that the alveolar macrophage is at least partially responsible for the protection elicited by this novel prophylactic treatment strategy.

Critical Adverse Impact of IL-6 in Acute Pneumovirus Infection.

Severe respiratory virus infections feature robust local host responses that contribute to disease severity. Immunomodulatory strategies that limit virus-induced inflammation may be of critical importance, notably in the absence of antiviral vaccines. In this study, we examined the role of the pleiotropic cytokine IL-6 in acute infection with pneumonia virus of mice (PVM), a natural rodent pathogen that is related to respiratory syncytial virus and that generates local inflammation as a feature of severe infection. In contrast to Influenza A, PVM is substantially less lethal in IL-6 -/- mice than it is in wild-type, a finding associated with diminished neutrophil recruitment and reduced fluid accumulation in lung tissue. Ly6Chi proinflammatory monocytes are recruited in response to PVM via a CCR2-dependent mechanism, but they are not a major source of IL-6 nor do they contribute to lethal sequelae of infection. By contrast, alveolar macrophages are readily infected with PVM in vivo; ablation of alveolar macrophages results in prolonged survival in association with a reduction in virus-induced IL-6. Finally, as shown previously, administration of immunobiotic Lactobacillus plantarum to the respiratory tracts of PVM-infected mice promoted survival in association with diminished levels of IL-6. We demonstrated in this study that IL-6 suppression is a critical feature of the protective mechanism; PVM-infected IL-6 -/- mice responded to low doses of L. plantarum, and administration of IL-6 overcame L. plantarum-mediated protection in PVM-infected wild-type mice. Taken together, these results connect the actions of IL-6 to PVM pathogenesis and suggest cytokine blockade as a potential therapeutic modality in severe infection.

Murine Pneumonia Virus Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus from an Added Gene Is Highly Attenuated and Immunogenic in Rhesus Macaques.

Human respiratory syncytial virus (RSV) continues to be the leading viral cause of severe acute lower respiratory tract disease in infants and children worldwide. A licensed vaccine or antiviral drug suitable for routine use remains unavailable. Like RSV, Murine pneumonia virus (MPV) is a member of the genus Orthopneumovirus, family Pneumoviridae Humans are not normally exposed to MPV, and MPV is not cross-protective with RSV. We evaluated MPV as an RSV vaccine vector expressing the RSV fusion (F) glycoprotein. The RSV F open reading frame (ORF) was codon optimized, and the encoded RSV F protein was made identical to an early passage of RSV strain A2. The RSV F ORF was placed under the control of MPV transcription signals and inserted at the first (rMPV-F1), third (rMPV-F3), or fourth (rMPV-F4) gene position of a version of the MPV genome that contained a codon-pair-optimized polymerase (L) gene. The recovered viruses replicated in vitro as efficiently as the empty vector, with stable expression of RSV F protein. Replication and immunogenicity of rMPV-F1 and rMPV-F3 were evaluated in rhesus macaques following intranasal and intratracheal administration. Both viruses replicated at low levels in the upper and lower respiratory tracts, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high levels similar to those induced by wild-type RSV replicating to a 5- to 25-fold-higher titer. In conclusion, this study demonstrated that rMPV provides a highly attenuated yet immunogenic vector for the expression of RSV F protein, with potential application in RSV-naive and RSV-experienced populations.IMPORTANCE Human respiratory syncytial virus (RSV) is an important human pathogen that lacks a licensed vaccine or antiviral drug suitable for routine use. We describe here the evaluation of recombinant murine pneumonia virus (rMPV) as a live-attenuated vector that expresses the RSV F protein, the major RSV neutralization antigen, as an experimental RSV vaccine. The rMPV-RSV-F vectors expressing RSV F from the first, third, or fourth gene position were genetically stable and were not restricted for replication in vitro In contrast, the vectors exhibited highly attenuated replication in the respiratory tract of rhesus macaques, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high titers similar to those conferred by wild-type RSV. Given the lack of preexisting immunity to MPV in humans and the lack of cross-neutralization and cross-protection between MPV and RSV, an rMPV-vectored RSV vaccine should be immunogenic in both RSV-naive children and RSV-experienced adults.

Chronic IL-33 expression predisposes to virus-induced asthma exacerbations by increasing type 2 inflammation and dampening antiviral immunity.

BACKGROUND: Rhinovirus infection triggers acute asthma exacerbations. IL-33 is an instructive cytokine of type 2 inflammation whose expression is associated with viral load during experimental rhinovirus infection of asthmatic patients. OBJECTIVE: We sought to determine whether anti-IL-33 therapy is effective during disease progression, established disease, or viral exacerbation using a preclinical model of chronic asthma and in vitro human primary airway epithelial cells (AECs). METHODS: Mice were exposed to pneumonia virus of mice and cockroach extract in early and later life and then challenged with rhinovirus to model disease onset, progression, and chronicity. Interventions included anti-IL-33 or dexamethasone at various stages of disease. AECs were obtained from asthmatic patients and healthy subjects and treated with anti-IL-33 after rhinovirus infection. RESULTS: Anti-IL-33 decreased type 2 inflammation in all phases of disease; however, the ability to prevent airway smooth muscle growth was lost after the progression phase. After the chronic phase, IL-33 levels were persistently high, and rhinovirus challenge exacerbated the type 2 inflammatory response. Treatment with anti-IL-33 or dexamethasone diminished exacerbation severity, and anti-IL-33, but not dexamethasone, promoted antiviral interferon expression and decreased viral load. Rhinovirus replication was higher and IFN-λ levels were lower in AECs from asthmatic patients compared with those from healthy subjects. Anti-IL-33 decreased rhinovirus replication and increased IFN-λ levels at the gene and protein levels. CONCLUSION: Anti-IL-33 or dexamethasone suppressed the magnitude of type 2 inflammation during a rhinovirus-induced acute exacerbation; however, only anti-IL-33 boosted antiviral immunity and decreased viral replication. The latter phenotype was replicated in rhinovirus-infected human AECs, suggesting that anti-IL-33 therapy has the additional benefit of enhancing host defense.

Osteoblasts Are Rapidly Ablated by Virus-Induced Systemic Inflammation following Lymphocytic Choriomeningitis Virus or Pneumonia Virus of Mice Infection in Mice.

A link between inflammatory disease and bone loss is now recognized. However, limited data exist on the impact of virus infection on bone loss and regeneration. Bone loss results from an imbalance in remodeling, the physiological process whereby the skeleton undergoes continual cycles of formation and resorption. The specific molecular and cellular mechanisms linking virus-induced inflammation to bone loss remain unclear. In the current study, we provide evidence that infection of mice with either lymphocytic choriomeningitis virus (LCMV) or pneumonia virus of mice (PVM) resulted in rapid and substantial loss of osteoblasts from the bone surface. Osteoblast ablation was associated with elevated levels of circulating inflammatory cytokines, including TNF-α, IFN-γ, IL-6, and CCL2. Both LCMV and PVM infections resulted in reduced osteoblast-specific gene expression in bone, loss of osteoblasts, and reduced serum markers of bone formation, including osteocalcin and procollagen type 1 N propeptide. Infection of Rag-1-deficient mice (which lack adaptive immune cells) or specific depletion of CD8+ T lymphocytes limited osteoblast loss associated with LCMV infection. By contrast, CD8+ T cell depletion had no apparent impact on osteoblast ablation in association with PVM infection. In summary, our data demonstrate dramatic loss of osteoblasts in response to virus infection and associated systemic inflammation. Further, the inflammatory mechanisms mediating viral infection-induced bone loss depend on the specific inflammatory condition.

Epitope mapping and kinetics of CD4 T cell immunity to pneumonia virus of mice in the C57BL/6 strain.

Pneumonia virus of mice (PVM) infection has been widely used as a rodent model to study the closely related human respiratory syncytial virus (hRSV). While T cells are indispensable for viral clearance, they also contribute to immunopathology. To gain more insight into mechanistic details, novel tools are needed that allow to study virus-specific T cells in C57BL/6 mice as the majority of transgenic mice are only available on this background. While PVM-specific CD8 T cell epitopes were recently described, so far no PVM-specific CD4 T cell epitopes have been identified within the C57BL/6 strain. Therefore, we set out to map H2-IAb-restricted epitopes along the PVM proteome. By means of in silico prediction and subsequent functional validation, we were able to identify a MHCII-restricted CD4 T cell epitope, corresponding to amino acids 37-47 in the PVM matrix protein (M37-47). Using this newly identified MHCII-restricted M37-47 epitope and a previously described MHCI-restricted N339-347 epitope, we generated peptide-loaded MHCII and MHCI tetramers and characterized the dynamics of virus-specific CD4 and CD8 T cell responses in vivo. The findings of this study can provide a basis for detailed investigation of T cell-mediated immune responses to PVM in a variety of genetically modified C57BL/6 mice.

RAGE deficiency predisposes mice to virus-induced paucigranulocytic asthma.

Asthma is a chronic inflammatory disease. Although many patients with asthma develop type-2 dominated eosinophilic inflammation, a number of individuals develop paucigranulocytic asthma, which occurs in the absence of eosinophilia or neutrophilia. The aetiology of paucigranulocytic asthma is unknown. However, both respiratory syncytial virus (RSV) infection and mutations in the receptor for advanced glycation endproducts (RAGE) are risk factors for asthma development. Here, we show that RAGE deficiency impairs anti-viral immunity during an early-life infection with pneumonia virus of mice (PVM; a murine analogue of RSV). The elevated viral load was associated with the release of high mobility group box-1 (HMGB1) which triggered airway smooth muscle remodelling in early-life. Re-infection with PVM in later-life induced many of the cardinal features of asthma in the absence of eosinophilic or neutrophilic inflammation. Anti-HMGB1 mitigated both early-life viral disease and asthma-like features, highlighting HMGB1 as a possible novel therapeutic target.

Pneumovirus-Induced Lung Disease in Mice Is Independent of Neutrophil-Driven Inflammation.

The human pneumovirus respiratory syncytial virus (RSV) is the most common pathogen causing lower respiratory tract disease in young children worldwide. A hallmark of severe human RSV infection is the strong neutrophil recruitment to the airways and lungs. Massive neutrophil activation has been proven detrimental in numerous diseases, yet in RSV the contribution of neutrophils to disease severity, and thereby, the relevance of targeting them, is largely unknown. To determine the relevance of potential neutrophil targeting therapies, we implemented antibody-mediated neutrophil depletion in a mouse pneumonia virus of mice (PVM) model. PVM is a host specific murine pneumovirus closely related to human RSV, which reproduces many of the features of RSV infection, such as high viral replication and neutrophil recruitment. Clinical disease and markers of lung inflammation and injury were studied in PVM-infected mice treated with either depleting or isotype control antibodies. To confirm our results we performed all experiments in two mice strains: C57Bl6 and BALBc mice. Neutrophil depletion in blood and lungs was efficient throughout the disease. Remarkably, in both mouse strains we found no difference in clinical disease severity between neutrophil-depleted and control arms. In line with this observation, we found no differences between groups in histopathological lung injury and lung viral loads. In conclusion, our study shows that in mice neutrophil recruitment to the lungs does not affect disease outcome or viral clearance during severe PVM infection. As such, this model does not support the notion that neutrophils play a key role in mouse pneumovirus disease.

Intranasal treatment with a novel immunomodulator mediates innate immune protection against lethal pneumonia virus of mice.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and young children. There are no licensed RSV vaccines available, and the few treatment options for high-risk individuals are either extremely costly or cause severe side effects and toxicity. Immunomodulation mediated by a novel formulation consisting of the toll-like receptor 3 agonist poly(I:C), an innate defense regulator peptide and a polyphosphazene (P-I-P) was evaluated in the context of lethal infection with pneumonia virus of mice (PVM). Intranasal delivery of a single dose of P-I-P protected adult mice against PVM when given 24 h prior to challenge. These animals experienced minimal weight loss, no clinical disease, 100% survival, and reduced lung pathology. Similar clinical outcomes were observed in mice treated up to 3 days prior to infection. P-I-P pre-treatment induced early mRNA and protein expression of key chemokine and cytokine genes, reduced the recruitment of neutrophils and eosinophils, decreased virus titers in the lungs, and modulated the delayed exacerbated nature of PVM disease without any short-term side effects. On day 14 post-infection, P-I-P-treated mice were confirmed to be PVM-free. These results demonstrate the capacity of this formulation to prevent PVM and possibly other viral respiratory infections.

IL-12p40 gene-deficient BALB/c mice exhibit lower weight loss, reduced lung pathology and decreased sensitization to allergen in response to infection with pneumonia virus of mice.

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and pneumonia in infants and pneumonia virus of mice (PVM) causes similar disease. BALB/c mice are highly susceptible, while C57BL/6 mice are more resistant to PVM. IL-12 was significantly more up-regulated in response to PVM infection in BALB/c than in C57BL/6 mice. IL-12p40-deficient neonatal and adult BALB/c mice showed significantly less weight loss than wild-type mice after PVM challenge. The percentage of regulatory T cells, as well as IFN-ß and IL-18 expression, was higher in the lungs of both neonatal and adult IL-12p40-/- mice. Adult IL-12p40-/- mice also showed enhanced TGF-ß and IL-10 expression and reduced inflammatory responses. Furthermore, IL-12p40-/- mice showed decreased sensitization to inhaled cockroach antigen after PVM infection when compared to wild-type mice. In conclusion, these data suggest that a depressed regulatory capacity in BALB/c mice to PVM infection results in enhanced immunopathology and sensitization to allergen.

Signaling via pattern recognition receptors NOD2 and TLR2 contributes to immunomodulatory control of lethal pneumovirus infection.

Pattern recognition receptors (PRRs) engage microbial components in the lung, although their role in providing primary host defense against respiratory virus infection is not fully understood. We have previously shown that Gram-positive Lactobacillus plantarum (Lp) administered to the respiratory tract promotes full and sustained protection in response to an otherwise lethal mouse pneumovirus (PVM) infection, a robust example of heterologous immunity. While Lp engages PRRs TLR2 and NOD2 in ex vivo signaling assays, we found that Lp-mediated protection was unimpaired in single gene-deleted TLR2(-/-) and NOD2(-/-) mice. Here we demonstrate substantial loss of Lp-mediated protection in a double gene-deleted NOD2(-/-)TLR2(-/-) strain. Furthermore, we demonstrate protection against PVM infection by administration of the bi-functional NOD2-TLR2 agonist, CL-429. The bi-functional NOD2-TLR2 ligand CL-429 not only suppresses virus-induced inflammation, it is significantly more effective at preventing lethal infection than equivalent amounts of mono-molecular TLR2 and NOD2 agonists. Interestingly, and in contrast to biochemical NOD2 and/or TLR2 agonists, Lp remained capable of eliciting primary proinflammatory responses from NOD2(-/-)TLR2(-/-) mice in vivo and from alveolar macrophages challenged ex vivo. Taken together, we conclude that coordinate engagement of NOD2 and TLR2 constitutes a key step in the genesis of Lp-mediated protection from a lethal respiratory virus infection, and represents a critical target for modulation of virus-induced inflammatory pathology.

Priming of the Respiratory Tract with Immunobiotic Lactobacillus plantarum Limits Infection of Alveolar Macrophages with Recombinant Pneumonia Virus of Mice (rK2-PVM).

UNLABELLED: Pneumonia virus of mice (PVM) is a natural rodent pathogen that replicates in bronchial epithelial cells and reproduces many clinical and pathological features of the more severe forms of disease associated with human respiratory syncytial virus. In order to track virus-target cell interactions during acute infection in vivo, we developed rK2-PVM, bacterial artificial chromosome-based recombinant PVM strain J3666 that incorporates the fluorescent tag monomeric Katushka 2 (mKATE2). The rK2-PVM pathogen promotes lethal infection in BALB/c mice and elicits characteristic cytokine production and leukocyte recruitment to the lung parenchyma. Using recombinant virus, we demonstrate for the first time PVM infection of both dendritic cells (DCs; CD11c(+) major histocompatibility complex class II(+)) and alveolar macrophages (AMs; CD11c(+) sialic acid-binding immunoglobulin-like lectin F(+)) in vivo and likewise detect mKATE2(+) DCs in mediastinal lymph nodes from infected mice. AMs support both active virus replication and production of infectious virions. Furthermore, we report that priming of the respiratory tract with immunobiotic Lactobacillus plantarum, a regimen that results in protection against the lethal inflammatory sequelae of acute respiratory virus infection, resulted in differential recruitment of neutrophils, DCs, and lymphocytes to the lungs in response to rK2-PVM and a reduction from ∼ 40% to <10% mKATE2(+) AMs in association with a 2-log drop in the release of infectious virions. In contrast, AMs from L. plantarum-primed mice challenged with virus ex vivo exhibited no differential susceptibility to rK2-PVM. Although the mechanisms underlying Lactobacillus-mediated viral suppression remain to be fully elucidated, this study provides insight into the cellular basis of this response. IMPORTANCE: Pneumonia virus of mice (PVM) is a natural mouse pathogen that serves as a model for severe human respiratory syncytial virus disease. We have developed a fully functional recombinant PVM strain with a fluorescent reporter protein (rK2-PVM) that permits us to track infection of target cells in vivo. With rK2-PVM, we demonstrate infection of leukocytes in the lung, notably, dendritic cells and alveolar macrophages. Alveolar macrophages undergo productive infection and release infectious virions. We have shown previously that administration of immunobiotic Lactobacillus directly to the respiratory mucosa protects mice from the lethal sequelae of PVM infection in association with profound suppression of the virus-induced inflammatory response. We show here that Lactobacillus administration also limits infection of leukocytes in vivo and results in diminished release of infectious virions from alveolar macrophages. This is the first study to provide insight into the cellular basis of the antiviral impact of immunobiotic L. plantarum.

Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal challenge with pneumonia virus of mice.

Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replication-deficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response in such animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8(+) T cell response and, to a lesser extent, a CD4(+) T cell response. These findings suggest that T-cell responses may be more important than serum IgG in the observed protection induced by rAd5N.

Blunted inflammatory and mucosal IgA responses to pneumonia virus of mice in C57BL/6 neonates are correlated to reduced protective immunity upon re-infection as elderly mice.

Respiratory syncytial virus is a major cause of bronchiolitis in infants and pneumonia virus of mice (PVM) causes similar disease in mice. The impact of PVM infection in BALB/c and C57BL/6 neonates, and upon re-infection as elderly mice, was compared. As previously shown for adult mice, PVM caused more disease in BALB/c than in C57BL/6 neonates. After PVM-15 infection BALB/c neonates showed higher production of inflammatory mediators, more influx of plasmacytoid dendritic cells and higher IFN-α expression, and more IgA in the lungs than C57BL/6 neonates. After re-infection as elderly, BALB/c mice developed virus neutralizing antibodies in serum and lung, and were protected from clinical disease, whereas C57BL/6 mice did not develop an anamnestic response and were not protected. These results suggest that an effective local innate response, as well as priming of mucosal adaptive responses in neonates after PVM-15 infection is correlated to decreased susceptibility and protection upon re-infection.

Immunobiotic Lactobacillus administered post-exposure averts the lethal sequelae of respiratory virus infection.

We reported previously that priming of the respiratory tract with immunobiotic Lactobacillus prior to virus challenge protects mice against subsequent lethal infection with pneumonia virus of mice (PVM). We present here the results of gene microarray which document differential expression of proinflammatory mediators in response to PVM infection alone and those suppressed in response to Lactobacillus plantarum. We also demonstrate for the first time that intranasal inoculation with live or heat-inactivated L. plantarum or Lactobacillus reuteri promotes full survival from PVM infection when administered within 24h after virus challenge. Survival in response to L. plantarum administered after virus challenge is associated with suppression of proinflammatory cytokines, limited virus recovery, and diminished neutrophil recruitment to lung tissue and airways. Utilizing this post-virus challenge protocol, we found that protective responses elicited by L. plantarum at the respiratory tract were distinct from those at the gastrointestinal mucosa, as mice devoid of the anti-inflammatory cytokine, interleukin (IL)-10, exhibit survival and inflammatory responses that are indistinguishable from those of their wild-type counterparts. Finally, although L. plantarum interacts specifically with pattern recognition receptors TLR2 and NOD2, the respective gene-deleted mice were fully protected against lethal PVM infection by L. plantarum, as are mice devoid of type I interferon receptors. Taken together, L. plantarum is a versatile and flexible agent that is capable of averting the lethal sequelae of severe respiratory infection both prior to and post-virus challenge via complex and potentially redundant mechanisms.

S. mansoni bolsters anti-viral immunity in the murine respiratory tract.

The human intestinal parasite Schistosoma mansoni causes a chronic disease, schistosomiasis or bilharzia. According to the current literature, the parasite induces vigorous immune responses that are controlled by Th2 helper cells at the expense of Th1 helper cells. The latter cell type is, however, indispensable for anti-viral immune responses. Remarkably, there is no reliable literature among 230 million patients worldwide describing defective anti-viral immune responses in the upper respiratory tract, for instance against influenza A virus or against respiratory syncitial virus (RSV). We therefore re-examined the immune response to a human isolate of S. mansoni and challenged mice in the chronic phase of schistosomiasis with influenza A virus, or with pneumonia virus of mice (PVM), a mouse virus to model RSV infections. We found that mice with chronic schistosomiasis had significant, systemic immune responses induced by Th1, Th2, and Th17 helper cells. High serum levels of TNF-α, IFN-γ, IL-5, IL-13, IL-2, IL-17, and GM-CSF were found after mating and oviposition. The lungs of diseased mice showed low-grade inflammation, with goblet cell hyperplasia and excessive mucus secretion, which was alleviated by treatment with an anti-TNF-α agent (Etanercept). Mice with chronic schistosomiasis were to a relative, but significant extent protected from a secondary viral respiratory challenge. The protection correlated with the onset of oviposition and TNF-α-mediated goblet cell hyperplasia and mucus secretion, suggesting that these mechanisms are involved in enhanced immune protection to respiratory viruses during chronic murine schistosomiasis. Indeed, also in a model of allergic airway inflammation mice were protected from a viral respiratory challenge with PVM.

Sustained inflammation and differential expression of interferons type I and III in PVM-infected interferon-gamma (IFNγ) gene-deleted mice.

Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ(-/-) mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNß, IFNλ2/3 and IFN-stimulated 2-5' oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ(-/-) mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include over-production of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ(-/-) mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.