Severe Lower Respiratory Tract Disease Research Articles

Risk of respiratory syncytial virus infection in infants with congenital cystic lung disease.

Congenital cystic lung disease (CCLD), which includes congenital cystic adenomatoid malformation, bronchopulmonary sequestration, and congenital lobar emphysema, has been reported to increase the risk of recurrent respiratory infection. In particular, respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in high-risk infants. The objective of this study was to investigate the risk of severe RSV infection in infants with CCLD. Infants antenatally diagnosed as having CCLD and admitted to a neonatal intensive care unit at the National Center for Child Health and Development in Tokyo between September 2002 and October 2011 were included in this study. We investigated retrospectively whether the infants were hospitalized with RSV infection by 24 months of age using their medical records. Forty-eight infants were antenatally diagnosed as having CCLD. Of the 48 infants, four (8.3%) were hospitalized with RSV infection by 24 months of age. Infants with CCLD have increased risk of severe RSV infection.

Head-to-head comparison of the diagnostic accuracies of BD Veritor™ System RSV and Quidel® Sofia® RSV FIA systems for respiratory syncytial virus (RSV) diagnosis

BackgroundRespiratory syncytial virus (RSV) is one of the most common causes of severe lower respiratory tract disease among infants and young children. BD Veritor™ System RSV (BD) and Quidel® Sofia® RSV FIA (QD) are the new generation lateral flow digital immunoassay (DIA) tests with an instrumented read for the qualitative detection of RSV viral antigens. ObjectiveTo compare the diagnostic accuracies of BD and QD for RSV detection using fresh nasopharyngeal aspirates and nasopharyngeal swab specimens collected in universal transport media during 2013–2014 respiratory season. Study designThe two DIA tests were performed simultaneously on randomly selected specimens on a weekly basis during the RSV season until 200 fresh remnant specimens were enrolled. Real-time RT-PCR assay results were used to compare and evaluate the performance of both RSV DIA assays. ResultsAmong 200 specimens tested, RSV real-time RT-PCR assay detected RSV in 104 samples, while QD detected 84 samples and BD detected 74 samples as positive. The overall sensitivity for detection of RSV in comparison to PCR was 71.15% (61.3–79.4) for BD and 80.77% (71.6–87.6) for QD system (P=0.36). The specificity was 100% (95.2–100) for both systems. The work flow analysis revealed that the overall specimen processing time was significantly lower for BD as compared with the QD assay. ConclusionsIn comparison with the real-time PCR, the QD system showed a higher sensitivity than that of the BD system, but the difference did not reach statistical significance (P=0.36). Both BD and QD systems were found comparable in terms of specificity.

The aberrant gene-end transcription signal of the matrix M gene of human parainfluenza virus type 3 downregulates fusion F protein expression and the F-specific antibody response in vivo.

Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is a major viral cause of severe lower respiratory tract disease in infants and children. The gene-end (GE) transcription signal of the HPIV3 matrix (M) protein gene is identical to those of the nucleoprotein and phosphoprotein genes except that it contains an apparent 8-nucleotide insert. This was associated with an increased synthesis of a readthrough transcript of the M gene and the downstream fusion (F) protein gene. We hypothesized that this insert may function to downregulate the expression of F protein by interfering with termination/reinitiation at the M-F gene junction, thus promoting the production of M-F readthrough mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses from which this insert in the M-GE signal was removed were generated. The M-GE mutants exhibited a reduction in M-F readthrough mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wild-type (wt) HPIV3 both in vitro and in vivo. However, the F-protein-specific serum antibody response in hamsters was increased for the mutants compared to wt HPIV3. This study identifies a previously undescribed viral mechanism for attenuating the host adaptive immune response. Repairing the M-GE signal should provide a means to increase the antibody response to a live attenuated HPIV3 vaccine without affecting viral replication and attenuation. The HPIV3 M-GE signal was previously shown to contain an apparent 8-nucleotide insert that was associated with increased synthesis of a readthrough mRNA of the M gene and the downstream F gene. However, whether this had any significant effect on the synthesis of monocistronic F mRNA or F protein, virus replication, virion morphogenesis, and immunogenicity was unknown. Here, we show that the removal of this insert shifts F gene transcription from readthrough M-F mRNA to monocistronic F mRNA. This resulted in a substantial increase in the amount of F protein expressed in the cell and packaged in the virus particle. This did not affect virus replication but increased the F-specific antibody response in hamsters. Thus, in wild-type HPIV3, the aberrant M-GE signal operates a previously undescribed mechanism that reduces the expression of a major neutralization and protective antigen, resulting in reduced immunogenicity. This has implications for the design of live attenuated HPIV3 vaccines; specifically, the antibody response against F can be elevated by "repairing" the M-GE signal to achieve higher-level F antigen expression, with no effect on attenuation.

Dual proinflammatory and antiviral properties of pulmonary eosinophils in respiratory syncytial virus vaccine-enhanced disease.

Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.

Visualizing the replication of respiratory syncytial virus in cells and in living mice.

Respiratory syncytial virus (RSV) is the most important cause of severe lower-respiratory tract disease in calves and young children, yet no human vaccine nor efficient curative treatments are available. Here we describe a recombinant human RSV reverse genetics system in which the red fluorescent protein (mCherry) or the firefly luciferase (Luc) genes are inserted into the RSV genome. Expression of mCherry and Luc are correlated with infection rate, allowing the monitoring of RSV multiplication in cell culture. Replication of the Luc-encoding virus in living mice can be visualized by bioluminescent imaging, bioluminescence being detected in the snout and lungs of infected mice after nasal inoculation. We propose that these recombinant viruses are convenient and valuable tools for screening of compounds active against RSV, and can be used as an extremely sensitive readout for studying effects of antiviral therapeutics in living mice.Supplementary informationThe online version of this article (doi:10.1038/ncomms6104) contains supplementary material, which is available to authorized users.

Regulation of Host Cell Signaling Pathways by Respiratory Syncytial Virus Nonstructural Protein NS1 and NS2

Human Respiratory Syncytial virus (hRSV) is a leading cause of severe lower respiratory tract diseases in the pediatric population.hRSV frequently causes severe morbidity and mortality in high risk groups including infants with congenital heart disease and the immunosuppressed patients. Although hRSV is recognized as a major public health threat and economic burden worldwide, there is no licensed vaccine and effective therapeutic agent. Viral nonstructural (NS) proteins have been known to play multiple functions for efficient viral replication and pathogenesis. Especially, diverse functions of influenza A virus NS1 have been extensively studies. Recent studies demonstrated that NS1 and NS2 of RSV also exert diverse functions to modulate cellular environment and antiviral immune responses. Since NS proteins of RSV are required for efficient replication and pathogenesis, NS mutant viruses have been tested as live-attenuated vaccines. This review will outline the recent progress in understanding the various functions of RSV NS1 and NS2.

Curcumin Prevents Replication of Respiratory Syncytial Virus and the Epithelial Responses to It in Human Nasal Epithelial Cells

The human nasal epithelium is the first line of defense during respiratory virus infection. Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma and severe lower respiratory tract disease in infants and young children. We previously reported in human nasal epithelial cells (HNECs), the replication and budding of RSV and the epithelial responses, including release of proinflammatory cytokines and enhancement of the tight junctions, are in part regulated via an NF-κB pathway. In this study, we investigated the effects of the NF-κB in HNECs infected with RSV. Curcumin prevented the replication and budding of RSV and the epithelial responses to it without cytotoxicity. Furthermore, the upregulation of the epithelial barrier function caused by infection with RSV was enhanced by curcumin. Curcumin also has wide pharmacokinetic effects as an inhibitor of NF-κB, eIF-2α dephosphorylation, proteasome and COX2. RSV-infected HNECs were treated with the eIF-2α dephosphorylation blocker salubrinal and the proteasome inhibitor MG132, and inhibitors of COX1 and COX2. Treatment with salubrinal, MG132 and COX2 inhibitor, like curcumin, prevented the replication of RSV and the epithelial responses, and treatment with salubrinal and MG132 enhanced the upregulation of tight junction molecules induced by infection with RSV. These results suggest that curcumin can prevent the replication of RSV and the epithelial responses to it without cytotoxicity and may act as therapy for severe lower respiratory tract disease in infants and young children caused by RSV infection.

Marked induction of matrix metalloproteinase‐10 by respiratory syncytial virus infection in human nasal epithelial cells

Respiratory syncytial virus (RSV) is an important pathogen of bronchiolitis, asthma, and severe lower respiratory tract disease in infants and young children. Matrix metalloproteinases (MMPs) play key roles in viral infection, inflammation and remodeling of the airway. However, the roles and regulation of MMPs in human nasal epithelial cells (HNECs) after RSV infection remain unclear. To investigate the regulation of MMP induced after RSV infection in HNECs, an RSV-infected model of HNECs in vitro was used. It was found that mRNA of MMP-10 was markedly increased in HNECs after RSV infection, together with induction of mRNAs of MMP-1, -7, -9, and -19. The amount of MMP-10 released from HNECs was also increased in a time-dependent manner after RSV infection as was that of chemokine RANTES. The upregulation of MMP-10 in HNECs after RSV infection was prevented by inhibitors of NF-κB and pan-PKC with inhibition of RSV replication, whereas it was prevented by inhibitors of JAK/STAT, MAPK, and EGF receptors without inhibition of RSV replication. In lung tissue of an infant with severe RSV infection in which a few RSV antibody-positive macrophages were observed, MMP-10 was expressed at the apical side of the bronchial epithelial cells and alveolar epithelial cells. In conclusion, MMP-10 induced by RSV infection in HNECs is regulated via distinct signal transduction pathways with or without relation to RSV replication. MMP-10 may play an important role in the pathogenesis of RSV diseases and it has the potential to be a novel marker and therapeutic target for RSV infection.

Commentary

Commentary

A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus

BackgroundHuman respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial.ResultsWe have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 μM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9.ConclusionsA robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.

Advances in adult asthma diagnosis and treatment in 2012: Potential therapeutics and gene-environment interactions

In the Journal of Allergy and Clinical Immunology in 2012, research reports related to asthma in adults clustered around mechanisms of disease, with a special focus on their potential for informing new therapies. There was also consideration of the effect of the environment on health from pollution, climate change, and epigenetic influences, underlining the importance of understanding gene-environment interactions in the pathogenesis of asthma and response to treatment.

Novel polymeric inhibitors of HCoV-NL63

The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.

Antibodies to the Central Conserved Region of Respiratory Syncytial Virus (RSV) G Protein Block RSV G Protein CX3C-CX3CR1 Binding and Cross-Neutralize RSV A and B Strains

Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein-CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein-CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines.

Dual Role of Respiratory Syncytial Virus Glycoprotein Fragment as a Mucosal Immunogen and Chemotactic Adjuvant

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.

Increased Prevalence of H1N1-Induced Severe Lower Respiratory Tract Diseases in Children With Atopic Sensitization

PurposeViral infection is the most common aggravating factor for childhood asthma. Asthma may be a risk factor for severe respiratory symptoms in children with lower respiratory tract infections of viral etiology. Influenza A infection enhances Th2-polarization to house dust mites during the acute phase and leads to lung dysfunction in a mouse model. However, there are no data on the relationship between atopic sensitization and H1N1 (Influenza A) infection in humans. To investigate whether atopic sensitization is associated with the severity of H1N1 pneumonia, we compared clinical features and the atopic sensitization rate between children with and without H1N1 infection.MethodsUsing reverse transcription-polymerase chain reactions, we investigated H1N1 virus infection in 214 children who were hospitalized with high fever and respiratory symptoms from September 2009 to February 2010. We also performed immunoassays for total and specific IgEs to six common aeroallergens. Atopy was defined as positivity for more than one specific IgE. The clinical severity of pneumonia was evaluated based on intensive care unit admission, oxygen therapy, steroid therapy, and atelectasis.ResultsThere were 70 H1N1-positive children, 42.9% of whom had pneumonia. Children with H1N1 infection were older and had a higher prevalence of atopic sensitization and pneumonia compared with H1N1-negative children. The rate of atelectasis was higher in children with H1N1 pneumonia than in children with non-H1N1 pneumonia. Among children with H1N1 viral infection, those with atopic sensitization had a higher prevalence of intensive care unit admission and oxygen therapy, and a longer duration of hospitalization than non-atopic children. There were no differences between atopic and non-atopic children without H1N1 viral infection.ConclusionsThe prevalence of H1N1-induced severe lower respiratory tract diseases is higher in children with atopic sensitization.

Baculovirus-based Vaccine Displaying Respiratory Syncytial Virus Glycoprotein Induces Protective Immunity against RSV Infection without Vaccine-Enhanced Disease

BackgroundRespiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile.MethodsWe have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G.ResultsBac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection.ConclusionOur data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen.

Atopic Sensitization is Associated With Severe Lower Respiratory Illness in Children With Pandemic H1N1 Influenza Viral Infection

Although most influenza A virus infections result in self-limited uncomplicated disease, it causes significant morbidity and mortality in children.1 Influenza A accounts for the highest rates of influenza-related hospital admissions, especially in young children and elderly people, and pneumonia is one of the most common complications (10%-26%).2,3 Furthermore, it was previously reported that 35% of influenza A (H1N1)-related deaths in children were due to pneumonia.4 In addition to seasonal influenza A infection, there was an outbreak of pandemic H1N1 influenza A viral infection (pandemic H1N1 influenza) that was first reported in patients from Mexico and the United States in early April 2009. An outbreak of pandemic H1N1 influenza during 2009-2010 was responsible for severe respiratory disease and death, especially in children and elderly people with chronic respiratory diseases, even though its overall impact did not appear to cause more severe disease than seasonal influenza A.5,6 A recently published case series of 122 children with pneumonia due to pandemic H1N1 influenza reported up to a 60% prevalence of underlying medical conditions, including asthma (29%), and described the need for intensive care in 20% of patients and mechanical ventilation in 10%.7 The relationship between viral infection and asthma has been investigated during the last few decades and evidence for such a link has been reported; however, the question of whether all respiratory viruses are equipotent remains unclear. As described in a recent review by Holt et al.8, it was assumed that respiratory syncitial virus (RSV) was overwhelmingly the most important trigger of severe lower respiratory illness (LRI) in the first two years of life, and compelling data on the association between early RSV-induced LRI and subsequent development of persistent asthma have been published from independent prospective cohort studies. More recently, it was revealed that the contribution of human rhinovirus to LRI during infancy and the preschool years was considerably higher than previously believed, and that these infections are strongly linked to the risk of subsequent asthma.8 Although the pathogenic nature of the relationship between asthma and H1N1 viral infection is unclear, several studies have reported that asthma is a risk factor for worsening respiratory conditions in pandemic H1N1 influenza patients.6,7 However, there are several other reports that pandemic H1N1 influenza causes high rates of severe LRI (pneumonia, atelectasis), aggravation of asthma, and admission to intensive care units in patients with exisiting asthma or atopy.6,9,10 In a recent animal study, high concentrations of virus-infected cells were found in the central and lower respiratory tracts of ferrets inoculated with H1N1 influenza virus compared to ferrets inoculated with seasonal influenza virus. This result suggests that pandemic H1N1 influenza replicated more extensively in the lower respiratory tract, causing more severe LRI than that caused by seasonal influenza virus.11 In this issue of Allergy, Asthma & Immunology Research, Kim et al.12 report an increased prevalence of H1N1-induced severe lower respiratory tract disease in children with atopic sensitization. In aforementioned study, a high proportion of atopic children with pandemic H1N1 influenza had atelectasis and required ICU care and oxygen and steroid therapy. Based on these findings and the results of several animal studies, the authors suggest that atopic sensitization combined with pandemic H1N1 influenza infection can induce severe LRI. In addition, eosinophil recruitment into the lungs and enhancement of IL-10 production from CD4+ T cells may play a role in the development of severe LRI observed in pandemic H1N1 influenza patients.13,14 Due to the small sample size, Kim et al.12 could not confirm whether atopic sensitization itself, and not asthma, was associated with pandemic H1N1 influenza-induced severe LRI. Although the authors could not directly confirm that the atopic sensitization itself was the cause of severe LRI in pandemic H1N1 influenza, they demonstrated this relationship using proper statistical analyses. There was a recent study that also supports the notion that pandemic H1N1 influenza greatly increases the risk of severe LRI regardless of the severity of previous asthma or the presence of asthma in patients with atopic sensitization.9 In addition, patients with atopy have been shown to exhibit low natural killer cell activity,15 suggesting that both abnormal innate immune responses in atopic individuals and the affinity of the pandemic H1N1 virus for the respiratory tract may lead to increased susceptibility to severe LRI induced by pandemic H1N1 influenza. Based on several recent studies, including the study by Kim et al.12 reported in this issue, it is suggested that atopic sensitization itself can cause severe LRI. This has highlighted the importance of influenza prevention through vaccination, especially in children with atopic sensitization, even if the patients have no obvious history of clinical asthma or any other allergic disease.

Developing Vaccines against Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The F and G glycoprotein of RSV are potentially important targets for protective antiviral immune responses. Here, recombinant replication-deficient adenovirus-based vaccines, rAd/3xG, expressing the soluble core domain of G glycoprotein (amino acids 131 to 230), engineered by codon optimization and tandem repetition for higher level expression, were constructed and evaluated for their potential as RSV vaccine in murine model. Strong serum IgG as well as mucosal IgA responses were induced by intranasal immunization of rAd/3xG. A single intranasal immunization of rAd/3xG vaccines provided potent protection against RSV challenge without vaccine-enhanced immunopathology. We also tested the efficacy of our vaccines against RSV field isolates from Korean pediatric patients and the data showed that our vaccine confers complete protection against RSV-A isolate challenge. We also show that sublingual or intranasal immunization of a procaryotically expressed and purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel selfadjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of our vaccine candidates elicit beneficial protective immunity and represent promising vaccine regimens preventing RSV infection.

A nuclear factor-κB signaling pathway via protein kinase C δ regulates replication of respiratory syncytial virus in polarized normal human nasal epithelial cells

Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma, and severe lower respiratory tract disease in infants and young children. The airway epithelium, which has a well-developed barrier regulated by tight junctions, is the first line of defense during respiratory virus infection. In upper airway human nasal epithelial cells (HNECs), however, the primary site of RSV infection, the mechanisms of replication and budding of RSV, and the epithelial cell responses, including the tight junctional barrier, remain unknown. To investigate the detailed mechanisms of replication and budding of RSV in HNECs and the epithelial cell responses, we established an RSV-infected model using human telomerase reverse transcriptase--transfected HNECs. We first found that the expression and barrier function of tight junction molecules claudin-4 and occludin were markedly induced together with production of proinflammatory cytokines interleukin 8 and tumor necrosis factor-α in HNECs after RSV infection, and the induction of tight junction molecules possibly contributed to budding of RSV. Furthermore, the replication and budding of RSV and the epithelial cell responses in HNECs were regulated via a protein kinase C δ/hypoxia-inducible factor-1α/nuclear factor-κB pathway. The control of this pathway in HNECs may be useful not only for prevention of replication and budding of RSV, but also in therapy for RSV-induced respiratory pathogenesis.

Dynamics of the bacterially expressed conserved immunogenic region of the human respiratory syncytial virus G protein

AbstractDespite all efforts, there is still no effective vaccine available against the human respiratory syncytial virus (HRSV) that is a major cause of severe lower respiratory tract disease in infants and the elderly. In this review, we examined the potential of the conserved immunogenic region (residues 122–226) of the HRSV glycoprotein G alone as the inducer of neutralizing antibodies against this virus. The Escherichia coli produced recombinant conserved region of G (designated as G domain), which was used for rabbit immunization. Although rabbit is a semipermissive host for HRSV, our result showed that the polyclonal antibodies against the G domain protein could strongly neutralize the virus (69.3%), suggesting that the G immunogenic region of HRSV alone has a great potential in vaccine development. To our knowledge, this is the first report in which neutralizing antibodies to respiratory syncytial virus have been evoked using bacterially expressed G immunogenic domain protein without any adjuvant.