Primary Respiratory Syncytial Virus Research Articles

Association of disease severity and genetic variation during primary Respiratory Syncytial Virus infections

BackgroundRespiratory Syncytial Virus (RSV) disease in young children ranges from mild cold symptoms to severe symptoms that require hospitalization and sometimes result in death. Studies have shown a statistical association between RSV subtype or phylogenic lineage and RSV disease severity, although these results have been inconsistent. Associations between variation within RSV gene coding regions or residues and RSV disease severity has been largely unexplored.MethodsNasal swabs from children (< 8 months-old) infected with RSV in Rochester, NY between 1977–1998 clinically presenting with either mild or severe disease during their first cold-season were used. Whole-genome RSV sequences were obtained using overlapping PCR and next-generation sequencing. Both whole-genome phylogenetic and non-phylogenetic statistical approaches were performed to associate RSV genotype with disease severity.ResultsThe RSVB subtype was statistically associated with disease severity. A significant association between phylogenetic clustering of mild/severe traits and disease severity was also found. GA1 clade sequences were associated with severe disease while GB1 was significantly associated with mild disease. Both G and M2-2 gene variation was significantly associated with disease severity. We identified 16 residues in the G gene and 3 in the M2-2 RSV gene associated with disease severity.ConclusionThese results suggest that phylogenetic lineage and the genetic variability in G or M2-2 genes of RSV may contribute to disease severity in young children undergoing their first infection.

Post-immunobiotics increase resistance to primary respiratory syncytial virus infection and secondary pneumococcal pneumonia.

Previously, we demonstrated that post-immunobiotics derived from Lactobacillus gasseri TMT36, TMT39, and TMT40 strains (HK36, HK39 and HK40, respectively) differentially regulated Toll-like receptor 3 (TLR3)-mediated antiviral respiratory immunity in infant mice. In this work, we investigated whether the HK36, HK39 and HK40 nasal treatments were able to improve the resistance against primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. Our results demonstrated that the three treatments increased the resistance to primary viral infection by reducing variations in body weight, RSV titers and lung damage of infected infant mice. Post-immunobiotics significantly enhanced the expressions of interferon (IFN)-λ, IFN-β, IFN-γ, interleukin(IL) - 1β, IL-6, IL-27, Mx1, RNAseL and 2'-5'-oligoadenylate synthetase 1 (OAS1) genes and decreased tumour necrosis factor (TNF)-α in alveolar macrophages of RSV-challenged mice. In addition, the studies in the model of RSV-Streptococcus pneumoniae superinfection showed that the HK39 and HK40 treatments were capable of reducing lung damage, lung bacterial cell counts, and the dissemination of S. pneumoniae into the blood of infant mice. The protective effect was associated with increases in IFN-β, IFN-γ, IL-10, and IL-27 in the respiratory tract. This study demonstrates that the nasal application of the post-immunobiotics HK39 and HK40 stimulates innate respiratory immunity and enhances the defences against primary RSV infection and secondary pneumococcal pneumonia offering an alternative to combat respiratory superinfections in children, which can be fatal.

The Ability of Postimmunobiotics from L. rhamnosus CRL1505 to Protect against Respiratory Syncytial Virus and Pneumococcal Super-Infection Is a Strain-Dependent Characteristic.

Previously, we demonstrated that the non-viable strain Lacticaseibacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulated the respiratory innate antiviral immune response triggered by Toll-like receptor (TLR)-3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In this work, we evaluated the effect of other non-viable L. rhamnosus strains and their peptidoglycans on the respiratory immune response and their impact on primary and secondary respiratory infections. In addition, the duration of the protective effect induced by NV1505 and PG1505 as well as their ability to protect against different Streptococcus pneumoniae serotypes were evaluated. Our results showed that among the five selected L. rhamnosus strains (CRL1505, CRL498, CRL576, UCO25A and IBL027), NV1505 and NVIBL027 improved the protection against viral and pneumococcal infections by modulating the respiratory immune response. Of note, only the PG1505 presented immunomodulatory activities when compared with the other purified peptidoglycans. Studies on alveolar macrophages showed that NV1505 and PG1505 differentially modulated the expression of IL-6, IFN-γ, IFN-β, TNF-α, OAS1, RNAseL and IL-27 genes in response to RSV infection, and IL-6, IFN-γ, IL-1β, TNF-α, CCL2, CXCL2, CXCL10 and IL-27 in response to pneumococcal challenge. Furthermore, we demonstrated that NV1505 and PG1505 treatments protected mice against secondary pneumococcal pneumonia produced by different serotypes of S. pneumoniae until 30 days after stimulation with poly(I:C). This work advances the characterization of the protective effect of NV1505 and PG1505 by demonstrating that they increase resistance against the pneumococcal serotypes 3, 6B, 14 and 19F, with an effect that lasts up to 30 days after the primary viral inflammation. The results also confirm that the immunomodulatory properties of NV1505 and PG1505 are unique and are not shared by other members of this species, and suggest the existence of a capacity to stimulate trained immunity in alveolar macrophages.

Age-specific hospitalization risk of primary and secondary respiratory syncytial virus infection among young children

ObjectivesElucidating the infection dynamics that lead to severe respiratory syncytial virus (RSV) pneumonia and hospitalization among young children are critical. We explored the role of infection parity as well as age in months for RSV-associated hospitalization among young children in Japan. MethodsWe used a sequential transmission catalytic model to capture the transmission mechanisms of RSV among infants in an endemic state. We investigated data on the age-dependent seroprevalence and incidence rate of hospitalization in Japan, and jointly estimated the age-specific risk of hospitalization during primary RSV infection and relative risk of hospitalization during secondary infection in children aged <5 years. ResultsThe estimated risk of hospitalization with primary infection was 0.08 (95% CI: 0.05-0.14) in infants aged 0-2 months. The estimated relative risk of hospitalization owing to secondary infection was 0.18 (95% CI: 0.01-2.04). ConclusionOur simple models successfully captured the infection dynamics of RSV among young children in Japan. The age group of early infancy may be most vulnerable to infection and hospitalization, offering key insights into future vaccinations. The burden of hospitalization from secondary infection may be less important in young children.

Identifying the Target Population for Primary Respiratory Syncytial Virus Two-Step Prevention in Infants: Normative Outcome of Hospitalisation Assessment for Newborns (NOHAN).

Background: Respiratory syncytial virus (RSV) is the leading cause of acute respiratory infection- related hospitalisations in infants (RSVh). Most of these infants are younger than 6 months old with no known risk factors. An efficient RSVh prevention program should address both mothers and infants, relying on Non-Pharmaceutical (NPI) and Pharmaceutical Interventions (PI). This study aimed at identifying the target population for these two interventions. Methods: Laboratory-confirmed RSV-infected infants hospitalised during the first 6 months of life were enrolled from the Hospices Civils de Lyon birth cohort (2014 to 2018). Clinical variables related to pregnancy and birth (sex, month of birth, birth weight, gestational age, parity) were used for descriptive epidemiology, multivariate logistic regression, and predictive score development. Results: Overall, 616 cases of RSVh in 45,648 infants were identified. Being born before the epidemic season, prematurity, and multiparity were independent predictors of RSVh. Infants born in January or June to August with prematurity and multiparity, and those born in September or December with only one other risk factor (prematurity or multiparity) were identified as moderate-risk, identifying the mothers as candidates for a first-level NPI prevention program. Infants born in September or December with prematurity and multiparity, and those born in October or November were identified as high-risk, identifying the mothers and infants as candidates for a second-level (NPI and PI) intervention. Conclusions: It is possible to determine predictors of RSVh at birth, allowing early enrollment of the target population in a two-level RSV prevention intervention.

Identifying the Target Population for Primary Respiratory Syncytial Virus Two-Step Prevention in Infants: Normative Outcome of Hospitalisation Assessment for Newborns (NOHAN)

Identifying the Target Population for Primary Respiratory Syncytial Virus Two-Step Prevention in Infants: Normative Outcome of Hospitalisation Assessment for Newborns (NOHAN)

133. validation of a Global Respiratory Severity Score in Infants with Primary RSV Infection

BackgroundWe recently developed a global respiratory severity score (GRSS) as a research tool from (n=139) infants with primary respiratory syncytial virus (RSV) infection enrolled prospectively in the Assessing Predictors of Infant RSV Effects and Severity (AsPIRES) study. The objective of the present study was to validate our original findings that the GRSS correlates well with clinical outcomes including hospitalization and length of stay (LOS) utilizing an independent cohort.MethodsClinical and demographic data on infants with primary RSV infection were abstracted from the electronic medical record. The GRSS was calculated by applying the original training data formula to the new data set. We compared the mean GRSS between the hospitalized and non-hospitalized group with Welch two sample t-test, and correlated it with hospitalization and LOS using Pearson’s correlation test.ResultsA total of 184 (98 hospitalized and 86 non-hospitalized) subjects were enrolled. The hospitalized and non-hospitalized infants were different in general appearance, the percentage with rales, retractions, lethargy, respiratory rate and oxygen saturation. The hospitalized group had a significantly (t=9.334, p< 0.0001) higher GRSS (4.20±2.10) than the non-hospitalized group (1.76±1.41). Using GRSS ≤3.5 as the classification criterion, we correctly predicted the hospitalization status of 131 (71.2%) subjects. The area under the ROC curve of the GRSS as a classifier of hospitalization is AUC=0.8265 (p< 0.0001). Pearson correlation between the GRSS and LOS is (p< 0.0001).Area under the ROC curve of the GRSS Boxplot demonstrating the difference in GRSS between hospitalized and non hospitalized infants ConclusionInfants hospitalized with RSV have a significantly higher GRSS than non-hospitalized subjects, and the GRSS is strongly associated with LOS. GRSS can also serve as an acceptable predictor of hospitalization. We are currently re-training the GRSS model with both datasets and believe it will lead to an improved predictive power of hospitalization.Disclosures All Authors: No reported disclosures

The Role of Alveolar Macrophages in the Improved Protection against Respiratory Syncytial Virus and Pneumococcal Superinfection Induced by the Peptidoglycan of Lactobacillus rhamnosus CRL1505.

The nasal priming with nonviable Lactobacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulates the respiratory innate immune response in infant mice, improving their resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, it was found that NV1505 or PG1505 significantly enhance the numbers of CD11c+SiglecF+ alveolar macrophages (AMs) producing interferon (IFN)-β. In this work, we aimed to further advance in the characterization of the beneficial effects of NV1505 and PG1505 in the context of a respiratory superinfection by evaluating whether their immunomodulatory properties are dependent on AM functions. Macrophage depletion experiments and a detailed study of their production of cytokines and antiviral factors clearly demonstrated the key role of this immune cell population in the improvement of both the reduction of pathogens loads and the protection against lung tissue damage induced by the immunobiotic CRL1505 strain. Studies at basal conditions during primary RSV or S. pneumoniae infections, as well as during secondary pneumococcal pneumonia, brought the following five notable findings regarding the immunomodulatory effects of NV1505 and PG1505: (a) AMs play a key role in the beneficial modulation of the respiratory innate immune response and protection against RSV infection, (b) AMs are necessary for improved protection against primary and secondary pneumococcal pneumonia, (c) the generation of activated/trained AMs would be essential for the enhanced protection against respiratory pathogens, (d) other immune and nonimmune cell populations in the respiratory tract may contribute to the protection against bacterial and viral infections, and (e) the immunomodulatory properties of NV1505 and PG1505 are strain-specific. These findings significantly improve our knowledge about the immunological mechanisms involved in the modulation of respiratory immunity induced by beneficial microbes.

Immune response to respiratory syncytial virus infection (&lt;i&gt;Orthopneumovirus&lt;/i&gt;)

Respiratory syncytial virus (RSV) infects children as well as elderly and immunocompromised subjects. In 2016, RSV was renamed to Orthopneumovirus owing to virus taxonomy latinization and was also included into the Pneumoviridae family. However, in this review we will use the old and more common RSV name. RSV infection may occur throughout human lifetime as it does not induce sterilizing immunity. During RSV infection, diverse immune cells such as dendritic cells, macrophages, T cells, B cells and eosinophils are involved in the antiviral response. Some of them play an important role in eliminating RSV, while the others can provoke tissue damage. An interaction between these cells occurs through the induced cytokines and chemokines, some of which emerge at early disease stages, whereas the others — at later stages. In addition, the mentioned cells can affect the course of both primary and secondary RSV infection. A prolonged or persistent RSV infection is observed in children with T-cell immunodeficiency, emphasizing the importance of T cells in resolution of acute infection as well as for virus-specific immunological memory development. Almost all the adults and children bear RSV-specific antibodies, but that doesn't protect against the repeated infection. It was shown that high mucosal rather than serum IgG level correlated better with reduced RSV load. A growing body of RSV vaccine candidates has emerged: live-attenuated, protein-based, whole-inactivated, particle-based, subunit antigens, and nucleic acid-based vaccines. While developing vaccines, there should be taken into consideration features of anti-RSV immune response as well as age of subjects to be vaccinated. In particular, to avoid vaccine-associated aggravation of RSV infection it is justified to use live attenuated vaccines in children, whereas middle-aged and the elderly subjects might be applied with subunit vaccines. Currently, no licensed vaccine for RSV infection is available. In this review, we will detail an interaction of the RSV with diverse immune cells as well as our contemporary understanding regarding preventive vaccines in RSV infection.

Aims, Study Design, and Enrollment Results From the Assessing Predictors of Infant Respiratory Syncytial Virus Effects and Severity Study.

BackgroundThe majority of infants hospitalized with primary respiratory syncytial virus (RSV) infection have no obvious risk factors for severe disease.ObjectiveThe aim of this study (Assessing Predictors of Infant RSV Effects and Severity, AsPIRES) was to identify factors associated with severe disease in full-term healthy infants younger than 10 months with primary RSV infection.MethodsRSV infected infants were enrolled from 3 cohorts during consecutive winters from August 2012 to April 2016 in Rochester, New York. A birth cohort was prospectively enrolled and followed through their first winter for development of RSV infection. An outpatient supplemental cohort was enrolled in the emergency department or pediatric offices, and a hospital cohort was enrolled on admission with RSV infection. RSV was diagnosed by reverse transcriptase-polymerase chain reaction. Demographic and clinical data were recorded and samples collected for assays: buccal swab (cytomegalovirus polymerase chain reaction, PCR), nasal swab (RSV qualitative PCR, complete viral gene sequence, 16S ribosomal ribonucleic acid [RNA] amplicon microbiota analysis), nasal wash (chemokine and cytokine assays), nasal brush (nasal respiratory epithelial cell gene expression using RNA sequencing [RNAseq]), and 2 to 3 ml of heparinized blood (flow cytometry, RNAseq analysis of purified cluster of differentiation [CD]4+, CD8+, B cells and natural killer cells, and RSV-specific antibody). Cord blood (RSV-specific antibody) was also collected for the birth cohort. Univariate and multivariate logistic regression will be used for analysis of data using a continuous Global Respiratory Severity Score (GRSS) as the outcome variable. Novel statistical methods will be developed for integration of the large complex datasets.ResultsA total of 453 infants were enrolled into the 3 cohorts; 226 in the birth cohort, 60 in the supplemental cohort, and 78 in the hospital cohort. A total of 126 birth cohort infants remained in the study and were evaluated for 150 respiratory illnesses. Of the 60 RSV positive infants in the supplemental cohort, 42 completed the study, whereas all 78 of the RSV positive hospital cohort infants completed the study. A GRSS was calculated for each RSV-infected infant and is being used to analyze each of the complex datasets by correlation with disease severity in univariate and multivariate methods.ConclusionsThe AsPIRES study will provide insights into the complex pathogenesis of RSV infection in healthy full-term infants with primary RSV infection. The analysis will allow assessment of multiple factors potentially influencing the severity of RSV infection including the level of RSV specific antibodies, the innate immune response of nasal epithelial cells, the adaptive response by various lymphocyte subsets, the resident airway microbiota, and viral factors. Results of this study will inform disease interventions such as vaccines and antiviral therapies.

Virus-Specific Antibody, Viral Load, and Disease Severity in Respiratory Syncytial Virus Infection.

Maternally derived serum antibody and viral load are thought to influence disease severity in primary respiratory syncytial virus (RSV) infection. As part of the AsPIRES study of RSV pathogenesis, we correlated various serum antibody concentrations and viral load with disease severity. Serum neutralizing antibody titers and levels of immunoglobulin G (IgG) to RSV fusion protein (F), attachment proteins of RSV group A and B, the CX3C region of G, and nasal viral load were measured in 139 full-term previously healthy infants with primary RSV infection and correlated with illness severity. Univariate analysis showed no relationship between measures of serum antibody and severity. However, a multivariate model adjusting for age at the time of infection found a significant 0.56 decrease in severity score for each 2-fold increase in antibody concentration to RSV F. The benefit of antibody was greatest in infants ≤ 2 months of age. Additionally, estimated antibody titer at birth was correlated with age at infection, suggesting that higher antibody titers delay infection. Viral load did not differ by illness severity. Our data support the concept of maternal immunization with an RSV vaccine during pregnancy as a strategy for reducing the burden of RSV infection in full-term healthy infants exposed to RSV during their first winter.

Association of Dynamic Changes in the CD4 T-Cell Transcriptome With Disease Severity During Primary Respiratory Syncytial Virus Infection in Young Infants.

Nearly all children are infected with respiratory syncytial virus (RSV) within the first 2 years of life, with a minority developing severe disease (1%-3% hospitalized). We hypothesized that an assessment of the adaptive immune system, using CD4+ T-lymphocyte transcriptomics, would identify gene expression correlates of disease severity. Infants infected with RSV representing extremes of clinical severity were studied. Mild illness (n = 23) was defined as a respiratory rate (RR) < 55 and room air oxygen saturation (SaO2) ≥ 97%, and severe illness (n = 23) was defined as RR ≥ 65 and SaO2 ≤ 92%. RNA from fresh, sort-purified CD4+ T cells was assessed by RNA sequencing. Gestational age, age at illness onset, exposure to environmental tobacco smoke, bacterial colonization, and breastfeeding were associated (adjusted P < .05) with disease severity. RNA sequencing analysis reliably measured approximately 60% of the genome. Severity of RSV illness had the greatest effect size upon CD4 T-cell gene expression. Pathway analysis identified correlates of severity, including JAK/STAT, prolactin, and interleukin 9 signaling. We also identified genes and pathways associated with timing of symptoms and RSV group (A/B). These data suggest fundamental changes in adaptive immune cell phenotypes may be associated with RSV clinical severity.

Peptidoglycan from Immunobiotic Lactobacillus rhamnosus Improves Resistance of Infant Mice to Respiratory Syncytial Viral Infection and Secondary Pneumococcal Pneumonia.

Several research works have demonstrated that beneficial microbes with the capacity to modulate the mucosal immune system (immunobiotics) are an interesting alternative to improve the outcome of bacterial and viral respiratory infections. Among the immunobiotic strains with the capacity to beneficially modulate respiratory immunity, Lactobacillus rhamnosus CRL1505 has outstanding properties. Although we have significantly advanced in demonstrating the capacity of L. rhamnosus CRL1505 to improve resistance against respiratory infections as well as in the cellular and molecular mechanisms involved in its beneficial activities, the potential protective ability of this strain or its immunomodulatory cellular fractions in the context of a secondary bacterial pneumonia has not been addressed before. In this work, we demonstrated that the nasal priming with non-viable L. rhamnosus CRL1505 or its purified peptidoglycan differentially modulated the respiratory innate antiviral immune response triggered by toll-like receptor 3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that peptidoglycan from L. rhamnosus CRL1505 significantly improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-β+ alveolar macrophages with the consequent increases of IFN-γ, IL-10, and IFN-β in the respiratory tract. Our results also showed that the increase of these three cytokines is necessary to achieve protection against respiratory superinfection since each of them are involved in different aspect of the secondary pneumococcal pneumonia that have to be controlled in order to reduce the severity of the infectious disease: lung pneumococcal colonization, bacteremia, and inflammatory-mediated lung tissue injury.

Development of a Global Respiratory Severity Score for Respiratory Syncytial Virus Infection in Infants.

Respiratory syncytial virus (RSV) infection in infants has recognizable clinical signs and symptoms. However, quantification of disease severity is difficult, and published scores remain problematic. Thus, as part of a RSV pathogenesis study, we developed a global respiratory severity score (GRSS) as a research tool for evaluating infants with primary RSV infection. Previously healthy infants <10 months of age with RSV infections representing the spectrum of disease severity were prospectively evaluated. Clinical signs and symptoms were collected at 3 time points from hospitalized infants and those seen in ambulatory settings. Data were also extracted from office, emergency department, and hospital records. An unbiased data-driven approach using factor analysis was used to develop a GRSS. A total of 139 infants (84 hospitalized and 55 nonhospitalized) were enrolled. Using hospitalization status as the output variable, 9 clinical variables were identified and weighted to produce a composite GRSS. The GRSS had an area under the receiver operator curve of 0.961. Construct validity was demonstrated via a significant correlation with length of stay (r = 0.586, P < .0001). Using routine clinical variables, we developed a severity score for infants with RSV infection that should be useful as an end point for investigation of disease pathogenesis and as an outcome measure for therapeutic interventions.

Cytokine responses in primary and secondary respiratory syncytial virus infections.

Primary respiratory syncytial virus (RSV) infections are characterized by high levels of IL-8 and an intense neutrophilia. Little is known about the cytokine responses in secondary infections. Preschool children experiencing RSV secondary infections were recruited from the siblings of infants admitted to hospital with RSV acute bronchiolitis. Fifty-one infants with acute bronchiolitis (39 RSV positive, 12 RSV negative) and 20 age-matched control infants were recruited. In addition, seven older siblings of infants from the RSV-positive cohort and confirmed RSV infection were recruited. Samples of nasal secretions were obtained using a flocked swab, and secretions extracted using centrifugation. Cytokine bead array was used to obtain levels of interleukin (IL)-17A, IL-8, IL-6, IL-21, and tumor necrosis factor-α. Levels of IL-8 and IL-6 were significantly lower in the RSV-positive siblings compared with the RSV-positive infants. There were no significant differences between levels of the other cytokines in the primary and secondary infections. The very high levels of IL-8 and IL-6 response characteristic of the primary RSV infection was not observed in secondary RSV-positive infections and this did not appear to be due to a global reduction in cytokine production.

STAT4 deficiency fails to induce lung Th2 or Th17 immunity following primary or secondary respiratory syncytial virus (RSV) challenge but enhances the lung RSV-specific CD8+ T cell immune response to secondary challenge.

Immune-mediated lung injury is a hallmark of lower respiratory tract illness caused by respiratory syncytial virus (RSV). STAT4 plays a critical role in CD4+ Th1 lineage differentiation and gamma interferon (IFN-γ) protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4-/- mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to wild-type (WT) mice. We performed primary and secondary RSV challenges in WT and STAT4-/- mice and used STAT1-/- mice as a positive control for the development of RSV-specific lung Th2 and Th17 inflammation during primary challenge. Primary RSV challenge of STAT4-/- mice resulted in decreased T-bet and IFN-γ expression levels in CD4+ T cells compared to those of WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4-/- mice. Decreased IFN-γ expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4-/- mice compared to WT mice. Following secondary challenge, WT and STAT4-/- mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4-/- mice resulted in enhanced weight loss, an increased lung IFN-γ expression level, and an increased lung RSV-specific CD8+ T cell response compared to those of WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge. STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infections.

Axl Receptor Blockade Ameliorates Pulmonary Pathology Resulting from Primary Viral Infection and Viral Exacerbation of Asthma

Viruses use Tyro3, Axl, and Mertk (TAM) receptor tyrosine kinases to infect and modulate the immune properties of various cell types, which led us to investigate whether TAM receptor activation affected primary viral infection and viral exacerbation of asthma in experimental models. In these lung-specific models, we observed that Axl was the most abundantly induced TAM receptor protein. During primary respiratory syncytial virus (RSV) infection, anti-Axl mAb treatment significantly increased the number of IFN-γ-producing T cells and NK cells and significantly suppressed RSV replication and whole lung levels of IL-4 and IL-13. Intrapulmonary H1N1 infection induced lethal pulmonary inflammation, but anti-Axl mAb treatment of infected mice significantly increased the number of IFN-β-producing macrophages and dendritic cells and significantly suppressed neutrophil infiltration. Consequently, the lethal effect of H1N1 infection in this model was significantly reduced in the mAb-treated group compared with the IgG control-treated group. Targeting Axl also inhibited airway hyperresponsiveness, IL-4 and IL-13 production, and goblet cell metaplasia in an Aspergillus fumigatus-induced asthma model. Finally, infection of mice with RSV during fungal asthma significantly exacerbated airway inflammation, goblet cell metaplasia, and airway remodeling, but all of these features in this viral exacerbation model were ameliorated by anti-Axl mAb treatment. Taken together, these results demonstrate that Axl modulates the pulmonary immune response during viral and/or allergic pathology, and they also suggest that targeting this TAM receptor might provide a novel therapeutic approach in these infectious diseases.

Opposing Roles of Membrane and Soluble Forms of the Receptor for Advanced Glycation End Products in Primary Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV), a common respiratory pathogen in infants and the older population, causes pulmonary inflammation and airway occlusion that leads to impairment of lung function. Here, we have established a role for receptor for advanced glycation end products (RAGE) in RSV infection. RAGE-deficient (ager−/−) mice were protected from RSV-induced weight loss and inflammation. This protection correlated with an early increase in type I interferons, later decreases in proinflammatory cytokines, and a reduction in viral load. To assess the contribution of soluble RAGE (sRAGE) to RSV-induced disease, wild-type and ager−/− mice were given doses of sRAGE following RSV infection. Of interest, sRAGE treatment prevented RSV-induced weight loss and neutrophilic inflammation to a degree similar to that observed in ager−/− mice. Our work further elucidates the roles of RAGE in the pathogenesis of respiratory infections and highlights the opposing roles of membrane and sRAGE in modulating the host response to RSV infection.

The Role of Notch-Notch Ligand Signaling in Primary and Secondary Respiratory Syncytial Virus (RSV) Infection

The Role of Notch-Notch Ligand Signaling in Primary and Secondary Respiratory Syncytial Virus (RSV) Infection

S25 IL 17 production in primary and secondary Respiratory Syncytial Virus (RSV) infection and neutrophil transmigration

<h3>Introduction and Objectives</h3> RSV causes winter epidemics of respiratory disease particularly in infants and the immunocompromised. Infected ciliated airway epithelial cells, local macrophages and dendritic cells secrete cytokines including interleukins (IL) 6 and 8, promoting a strong neutrophilic response that is important in disease clearance and airway inflammation. The IL 17 inflammatory pathway, which is important in surface immunity, has been little studied in RSV infection to date. The following hypothesis was explored: <i>The IL17 pathway is important in neutrophil chemotaxis and restriction of RSV replication</i>. <h3>Methods</h3> A transwell model of the airway was devised, co-culturing A549s (an immortalised bronchial epithelial cell line) and neutrophils to study the effect of RSV and IL17 on the transmigration of neutrophils. Neutrophil transmigration was assessed using light microscopic immunocytochemistry. Nasal swabs were taken from infants with RSV positive Bronchiolitis (n=49), RSV negative Bronchiolitis (n=12), the symptomatic older siblings of RSV positive infants (n=15) and uninfected, asymptomatic children (n=20). Cytokines released (IL6/IL8/IL17a/IL21/TNF) were analysed by cytokine bead array. <h3>Results</h3> IL 17 and RSV caused significantly increased number of neutrophils to transmigrate compared to either IL 17 or RSV alone. Primary RSV infections which caused hospitalisation, are characterised by high levels of nasal epithelial derived cytokines (IL 6, IL 8). Less severe secondary RSV infections are characterised by relatively low levels of IL 6 and IL 8 and relatively high levels of IL 17. <h3>Conclusions</h3> There is evidence that IL 17 is synergistic with RSV induced IL 8 in promoting neutrophil transmigration <i>in vitro</i>. There is evidence to support IL 17 as an important cytokine restricting RSV production in secondary infection <i>in vivo</i>. This study suggests that the IL 17 pathway is important in the pathogenesis of RSV bronchiolitis and could be potentially important in the development of novel therapies for this ubiquitous and important cause of respiratory disease.