[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) lower respiratory tract infection (LRI) during infancy has been consistently associated with an increased risk of childhood asthma. In addition, evidence supports that this relationship is causal. However, the mechanisms through which RSV contributes to asthma development are not understood. The INSPIRE (Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure) study objectives are to: 1) characterize the host phenotypic response to RSV infection in infancy and the risk of recurrent wheeze and asthma, 2) identify the immune response and lung injury patterns of RSV infection that are associated with the development of early childhood wheezing illness and asthma, and 3) determine the contribution of specific RSV strains to early childhood wheezing and asthma development. This article describes the INSPIRE study, including study aims, design, recruitment results, and enrolled population characteristics.
The cohort is a population based longitudinal birth cohort of term healthy infants enrolled during the first months of life over a two year period. Respiratory infection surveillance was conducted from November to March of the first year of life, through surveys administered every two weeks. In-person illness visits were conducted if infants met pre-specified criteria for a respiratory illness visit. Infants will be followed annually to ages 3-4 years for assessment of the primary endpoint: wheezing illness. Nasal, urine, stool and blood samples were collected at various time points throughout the study for measurements of host and viral factors that predict wheezing illness. Nested case-control studies will additionally be used to address other primary and secondary hypotheses.
In the INSPIRE study, 1952 infants (48% female) were enrolled during the two enrollment years and follow-up will continue through 2016. The mean age of enrollment was 60 days. During winter viral season, more than 14,000 surveillance surveys were carried out resulting in 2,103 respiratory illness visits on 1189 infants. First year follow-up has been completed on over 95% percent of participants from the first year of enrollment. With ongoing follow-up for wheezing and childhood asthma outcomes, the INSPIRE study will advance our understanding of the complex causal relationship between RSV infection and early childhood wheezing and asthma.
BMC Pulmonary Medicine 12/2015; 15(1). DOI:10.1186/s12890-015-0040-0 · 2.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children and causes disease throughout life. Understanding biology of infection, including virus binding to the cell surface, should help develop anti-viral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin binding domains. G also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G binding to CX3CR1 is not important to infection of immortalized cell lines but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines, HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was markedly increased by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and approximately 50% of RSV-infected cells in HAECs were CX3CR1 positive. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES, IL-8, and fractalkine production while down-regulated IL-15, IL1-RA and MCP-1. Thus, the RSV G protein CX3C/CX3CR1 interaction is likely important in infection and infection-induced responses of airway epithelium, the primary site for human infection.
Journal of General Virology 06/2015; DOI:10.1099/vir.0.000218 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5bfl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5bfl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5bfl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.
[Show abstract][Hide abstract] ABSTRACT: A first step in primary disease prevention is identifying common, modifiable risk factors that contribute to a significant proportion of disease development. Infant respiratory viral infection and childhood asthma are the two most common acute and chronic diseases of childhood, respectively. Common clinical features and links between these diseases have long been recognized, with early life respiratory syncytial virus (RSV) and rhinovirus (RV) lower respiratory tract infections (LRTI) being strongly associated with increased asthma risk. However, there has long been debate over the role of these respiratory viruses in asthma inception. In this article, we will systematically review the evidence linking early life RSV and RV LRTI with asthma inception and whether they could therefore be targets for primary prevention efforts.
American Journal of Respiratory and Critical Care Medicine 11/2014; 191(1). DOI:10.1164/rccm.201405-0901PP · 11.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice, however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss, but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein.
Journal of Virology 10/2014; 89(1). DOI:10.1128/JVI.02472-14 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in infants and a high priority for vaccine development. We previously reported that RSV virus-like particles (VLPs) expressing either the fusion (F) or attachment (G) glycoprotein could confer protection against RSV challenge in BALB/c mice. Here, we tested the hypothesis that RSV VLP vaccine efficacy can be enhanced by mixing RSV VLP F and RSV VLP G, and we analyzed host responses to these RSV VLPs. Mice were immunized with VLP F, VLP G, or VLP F + VLP G. Lung viral loads in BALB/c mice following RSV strain A2-line19F challenge were lower in mice vaccinated with RSV VLP F + VLP G compared to VLP F- or VLP G-vaccinated mice. Vaccination with VLP F or VLP F + VLP G induced similar levels of neutralizing antibodies. The enhanced protection against RSV challenge induced by vaccination with RSV VLP F + VLP G correlated with CD8 T cells producing T helper type 1 cytokines. VLP G vaccination alone followed by challenge resulted in immunopathology similar to formalin-inactivated RSV vaccination and RSV challenge. Taken together, mixed VLP F + VLP G provided a high level of protection against RSV without vaccine-induced immunopathology, but VLP G vaccination enhanced disease when used alone.
Antiviral Research 09/2014; 111. DOI:10.1016/j.antiviral.2014.09.005 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Some investigators find a deficiency in IFN production from airway epithelial cells infected with human rhinovirus in asthma, but whether this abnormality occurs with other respiratory viruses is uncertain.
To assess the effect of influenza A virus (IAV) and respiratory syncytial virus (RSV) infection on IFN production and viral level in human bronchial epithelial cells (hBECs) from subjects with and without asthma.
Primary-culture hBECs from subjects with mild to severe asthma (n = 11) and controls without asthma (hBECs; n = 7) were infected with live or ultraviolet-inactivated IAV (WS/33 strain), RSV (Long strain), or RSV (A/2001/2-20 strain) with multiplicity of infection 0.01 to 1. Levels of virus along with IFN-β and IFN-λ and IFN-stimulated gene expression (tracked by 2′-5′-oligoadenylate synthetase 1 and myxovirus (influenza virus) resistance 1 mRNA) were determined up to 72 hours postinoculation.
After IAV infection, viral levels were increased 2-fold in hBECs from asthmatic subjects compared with nonasthmatic control subjects (P < .05) and this increase occurred in concert with increased IFN-λ1 levels and no significant difference in IFNB1, 2′-5′-oligoadenylate synthetase 1, or myxovirus (influenza virus) resistance 1mRNA levels. After RSV infections, viral levels were not significantly increased in hBECs from asthmatic versus nonasthmatic subjects and the only significant difference between groups was a decrease in IFN-λ levels (P < .05) that correlated with a decrease in viral titer. All these differences were found only at isolated time points and were not sustained throughout the 72-hour infection period.
The results indicate that IAV and RSV control and IFN response to these viruses in airway epithelial cells is remarkably similar between subjects with and without asthma.
Journal of Allergy and Clinical Immunology 09/2014; 134(6). DOI:10.1016/j.jaci.2014.07.013 · 11.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The prevalence of allergic diseases has doubled in developed countries in the past several decades. Cyclooxygenase (COX)-inhibiting drugs augmented allergic diseases in mice by increasing allergic sensitization and memory immune responses. However, whether COX inhibition can promote allergic airway diseases by inhibiting immune tolerance is not known.
To determine the role of the COX pathway and prostaglandin I2 (PGI2) signaling through the PGI2 receptor (IP) in aeroallergen-induced immune tolerance.
Wild-type (WT) BALB/c mice and IP knockout mice were aerosolized with ovalbumin (OVA) to induce immune tolerance prior to immune sensitization with an intraperitoneal injection of OVA/alum. The COX inhibitor indomethacin or vehicle was administered in drinking water to inhibit enzyme activity during the sensitization phase. Two weeks after sensitization, the mice were challenged with OVA aerosols. Mouse bronchoalveolar lavage fluid was harvested for cell counts and TH2 cytokine measurements.
WT mice treated with indomethacin had greater numbers of total cells, eosinophils, and lymphocytes, and increased IL-5 and IL-13 protein expression in BAL fluid compared to vehicle-treated mice. Similarly, IP knockout mice had augmented inflammation and TH2 cytokine responses compared to WT mice. In contrast, the PGI2 analog cicaprost attenuated the anti-tolerance effect of COX inhibition.
COX inhibition abrogated immune tolerance by suppressing PGI2 IP signaling, suggesting that PGI2 signaling promotes immune tolerance and that clinical use of COX-inhibiting drugs may increase the risk of developing allergic diseases.
Journal of Allergy and Clinical Immunology 09/2014; 134(3). DOI:10.1016/j.jaci.2014.06.004 · 11.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in children for which there is no licensed vaccine. Live-attenuated RSV vaccines are the most clinically advanced in children, but achieving an optimal balance of attenuation and immunogenicity is challenging. One way to potentially retain or enhance immunogenicity of attenuated virus is to mutate virulence genes that suppress host immune responses. The NS1 and NS2 virulence genes of the RSV A2 strain were codon deoptimized according to either human or virus codon usage bias, and the resulting recombinant viruses (dNSh and dNSv, respectively) were rescued by reverse genetics. RSV dNSh exhibited the desired phenotype of reduced NS1 and NS2 expression. RSV dNSh was attenuated in BEAS-2B and primary differentiated airway epithelial cells but not in HEp-2 or Vero cells. In BALB/c mice, RSV dNSh exhibited a lower viral load than did A2, and yet it induced slightly higher levels of RSV-neutralizing antibodies than did A2. RSV A2 and RSV dNSh induced equivalent protection against challenge strains A/1997/12-35 and A2-line19F. RSV dNSh caused less STAT2 degradation and less NF-κB activation than did A2 in vitro. Serial passage of RSV dNSh in BEAS-2B cells did not result in mutations in the deoptimized sequences. Taken together, RSV dNSh was moderately attenuated, more immunogenic, and equally protective compared to wild-type RSV and genetically stable.
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. In the present study, we investigated the effect of prophylactic treatment with the intact and F(ab’)2 forms of an anti-G protein monoclonal antibody (mAb), 131-2G, on the humoral and cellular adaptive immune response to RSV rA2-line19F (r19F) challenge in BALB/c mice. The F(ab’)2 form of 131-2G does not decrease virus replication but intact 131-2G does. The serum specimens for antibodies and spleen cells for memory T cell responses to RSV antigens were analyzed at 30, 45, 75 and 95 p.i. with/without prior treatment with 131-2G. The ratios of Th2/Th1 antibody isotypes at each time p.i indicated that both forms of mAb 131-2G shifted the subclass response from a Th2 (IgG1 and IgG2b) to a Th1 (IgG2A) bias. The ratio of IgG1/IgG2A antibody titer was 3-fold to 10-fold higher for untreated than mAb treated mice. There was also some increase in IgG (22%±13 increase) and neutralization (32% increase) in antibodies with mAb 131-2G prophylaxis at 75 days p.i. Treatment with 131-2G significantly (p≤0.001) decreased the percent of IL-4 positive CD4 and CD8 in RSV stimulated spleen cells at all times p.i. while percent of IFN-γ T cells significantly (p≤0.001) increased ≥75 days p.i. The shift from a Th2 to a Th1 biased T cell response in treated compared to untreated mice likely was directed by the much higher levels of T-box transcription factor (Tbet) (≥45% vs <10%) in CD4 and CD8 T cells and lower levels of Gata-3 (≤2% vs ≥6%) in CD4 T cells in peptide stimulated, day 75 p.i. spleen cells. These data show that the RSV G protein affects both humoral and cellular adaptive immune responses and induction of 131-2G-like antibodies might improve the safety and long term efficacy of an RSV vaccine.
Journal of Virology 08/2014; 88(18). DOI:10.1128/JVI.01503-14 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory syncytial virus (RSV) is a leading pediatric pathogen that is responsible for a majority of infant hospitalizations due to viral disease. Despite its clinical importance, no vaccine prophylaxis against RSV disease or effective antiviral therapeutic is available. In this study, we established a robust high-throughput drug screening protocol by using a recombinant RSV reporter virus to expand the pool of RSV inhibitor candidates. Mechanistic characterization revealed that a potent newly identified inhibitor class blocks viral entry through specific targeting of the RSV fusion (F) protein. Resistance against this class was induced and revealed overlapping hotspots with diverse, previously identified RSV entry blockers at different stages of preclinical and clinical development. A structural and biochemical assessment of the mechanism of unique, broad RSV cross-resistance against structurally distinct entry inhibitors demonstrated that individual escape hotspots are located in immediate physical proximity in the metastable conformation of RSV F and that the resistance mutations lower the barrier for prefusion F triggering, resulting in an accelerated RSV entry kinetics. One resistant RSV recombinant remained fully pathogenic in a mouse model of RSV infection. By identifying molecular determinants governing the RSV entry machinery, this study spotlights a molecular mechanism of broad RSV resistance against entry inhibition that may affect the impact of diverse viral entry inhibitors presently considered for clinical use and outlines a proactive design for future RSV drug discovery campaigns.
Proceedings of the National Academy of Sciences 08/2014; 111(33). DOI:10.1073/pnas.1405198111 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines.
[Show abstract][Hide abstract] ABSTRACT: 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.
IMPORTANCE 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.
Journal of Virology 06/2014; 88(17). DOI:10.1128/JVI.03299-13 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Th17 cytokines interleukin-17A (IL-17A), IL-17F, and IL-22 are critical for the lung immune response to a variety of bacterial
pathogens, including Klebsiella pneumoniae. Th2 cytokine expression in the airways is a characteristic feature of asthma and allergic airway inflammation. The Th2 cytokines
IL-4 and IL-13 diminish ex vivo and in vivo IL-17A protein expression by Th17 cells. To determine the effect of IL-4 and IL-13 on IL-17-dependent lung immune responses
to acute bacterial infection, we developed a combined model in which allergic airway inflammation and lung IL-4 and IL-13
expression were induced by ovalbumin sensitization and challenge prior to acute lung infection with K. pneumoniae. We hypothesized that preexisting allergic airway inflammation decreases lung IL-17A expression and airway neutrophil recruitment
in response to acute K. pneumoniae infection and thereby increases the lung K. pneumoniae burden. As hypothesized, we found that allergic airway inflammation decreased the number of K. pneumoniae-induced airway neutrophils and lung IL-17A, IL-17F, and IL-22 expression. Despite the marked reduction in postinfection airway
neutrophilia and lung expression of Th17 cytokines, allergic airway inflammation significantly decreased the lung K. pneumoniae burden and postinfection mortality. We showed that the decreased lung K. pneumoniae burden was independent of IL-4, IL-5, and IL-17A and partially dependent on IL-13 and STAT6. Additionally, we demonstrated
that the decreased lung K. pneumoniae burden associated with allergic airway inflammation was both neutrophil and CCL8 dependent. These findings suggest a novel
role for CCL8 in lung antibacterial immunity against K. pneumoniae and suggest new mechanisms of orchestrating lung antibacterial immunity.
Infection and immunity 06/2014; 82(9). DOI:10.1128/IAI.00035-14 · 4.16 Impact Factor