MIP-1alpha is produced but it does not control pulmonary inflammation in response to respiratory syncytial virus infection in mice

The intent of this study was to compare the cellular and biochemical inflammatory responses of mice infected with the paramyxovirus pathogens respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM). Although RSV is not a natural pathogen of mice, it has been used extensively in mouse models of the human disease, as a limited respiratory infection can be established via intranasal inoculation of virus at high titer. In earlier work, we found that acute infection with the natural rodent pathogen, PVM, elicited a rapid and sustained pulmonary inflammatory response (peak, 1.7 x 10(6) leukocytes/ml BAL fluid) that was dependent on both local production of MIP-1alpha and signaling via its receptor, CCR1. We find here that MIP-1alpha is also produced in response to RSV, although relatively few leukocytes (<200 ml BAL fluid) are recruited to the lungs in response. Further experiments with CCR1-deficient mice confirm the finding that although MIP-1alpha is produced in response to RSV infection, leukocytes do not respond via this pathway. Among the explanations for these findings, we propose that there are other, as yet to be identified proinflammatory mediators elicited in response to PVM (but not in response to RSV) that serve to prime the leukocytes in vivo, thus enabling them to respond to MIP-1alpha signaling via CCR1. Furthermore, the differences in disease pathogenesis seen in response to each of these pneumovirus infections in mice raise questions regarding the extent to which primary RSV infection in mice can be used as a model of primary RSV infection in humans.     

The specific features of the developing T cell compartment of the neonatal lung are a determinant of respiratory syncytial virus immunopathogenesis

The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4⁺ and CD8⁺ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity.     

Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)₂ form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

Viral infection of the lungs through the eye

Respiratory syncytial virus (RSV) is the foremost respiratory pathogen in newborns and claims millions of lives annually. However, there has been no methodical study of the pathway(s) of entry of RSV or its interaction with nonrespiratory tissues. We and others have recently established a significant association between allergic conjunctivitis and the presence of RSV in the eye. Here we adopt a BALB/c mouse model and demonstrate that when instilled in the live murine eye, RSV not only replicated robustly in the eye but also migrated to the lung and produced a respiratory disease that is indistinguishable from the standard, nasally acquired RSV disease. Ocularly applied synthetic anti-RSV small interfering RNA prevented infection of the eye as well as the lung. RSV infection of the eye activated a plethora of ocular cytokines and chemokines with profound relevance to inflammation of the eye. Anticytokine treatments in the eye reduced ocular inflammation but had no effect on viral growth in both eye and lung, demonstrating a role of the cytokine response in ocular pathology. These results establish the eye as a major gateway of respiratory infection and a respiratory virus as a bona fide eye pathogen, thus offering novel intervention and treatment options.     

Reduced clearance of respiratory syncytial virus infection in a preterm lamb model

Respiratory syncytial virus (RSV) causes significant respiratory disease in children worldwide. For the study of severe RSV disease seen in preterm infants, a suitable animal model is lacking. The novel hypothesis of this study was that preterm lambs are susceptible to bovine RSV (bRSV) infection, an analogous pneumovirus with ruminant host specificity, and that there would be age-dependent differences in select RSV disease parameters. During RSV infection, preterm lambs had elevated temperatures and respiration rates with mild anorexia and cough compared to controls. Gross lesions included multifocal consolidation and atelectasis with foci of hyperinflation. Microscopic lesions included multifocal alveolar septal thickening and bronchiolitis. Immunohistochemistry localized the RSV antigen to all layers of bronchiolar epithelium from a few basal cells to numerous sloughing epithelia. A few mononuclear cells were also immunoreactive. To assess for age-dependent differences in RSV infection, neonatal lambs were infected similarly to the preterm lambs or with a high-titer viral inoculum. Using morphometry at day 7 of infection, preterm lambs had significantly more cellular immunoreactivity for RSV antigen (P <0.05) and syncytial cell formation (P <0.05) than either group of neonatal lambs. This work suggests that perinatal RSV clearance is age-dependent, which may explain the severity of RSV infection in preterm infants. The preterm lamb model is useful for assessing age-dependent mechanisms of severe RSV infection.     

Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection

The respiratory syncytial virus (RSV) causes potentially fatal lower respiratory tract infection in infants. The molecular mechanism of RSV infection is unknown. Our data show that RSV colocalizes with intercellular adhesion molecule-1 (ICAM-1) on the HEp-2 epithelial cell surface. Furthermore, a neutralizing anti-ICAM-1 mAb significantly inhibits RSV infection and infection-induced secretion of proinflammatory chemokine RANTES and mediator ET-1 in HEp-2 cells. Similar decrease in RSV infection is also observed in A549, a type-2 alveolar epithelial cell line, and NHBE, the normal human bronchial epithelial cell line when pretreated with anti-ICAM-1 mAb prior to RSV infection. Incubation of virus with soluble ICAM-1 also significantly decreases RSV infection of epithelial cells. Binding studies using ELISA indicate that RSV binds to ICAM-1, which can be inhibited by an antibody to the fusion F protein and also the recombinant F protein can bind to soluble ICAM-1, suggesting that RSV interaction with ICAM-1 involves the F protein. It is thus concluded that ICAM-1 facilitates RSV entry and infection of human epithelial cells by binding to its F protein, which is important to viral replication and infection and may lend itself as a therapeutic target.     

Respiratory syncytial virus (RSV) infects neuronal cells and processes that innervate the lung by a process involving RSV G protein

Respiratory syncytial virus (RSV) is a primary cause of morbidity and life-threatening lower respiratory tract disease in infants and young children. Children with acute RSV bronchiolitis often develop respiratory sequelae, but the disease mechanisms are poorly understood. Mounting evidence suggests that RSV may mediate persistent infection. Using immunohistochemistry to identify RSV and RSV-infected cell types, we show that RSV infects primary neurons and neuronal processes that innervate the lungs through a process that involves RSV G protein and the G protein CX3C motif. These findings suggest a mechanism for disease chronicity and have important implications for RSV disease intervention strategies.     

The immune response to human metapneumovirus is associated with aberrant immunity and impaired virus clearance in BALB/c mice

Human metapneumovirus (HMPV), recently identified in isolates from children hospitalized with acute respiratory tract illness, is associated with clinical diagnosis of pneumonia, asthma exacerbation, and acute bronchiolitis in young children. HMPV has been shown to cocirculate with respiratory syncytial virus (RSV) and mediate clinical disease features similarly to RSV. Little is known regarding the pathophysiology or immune response associated with HMPV infection; thus, animal models are needed to better understand the mechanisms of immunity and disease pathogenesis associated with infection. In this study, we examine features of the innate and adaptive immune response to HMPV infection in a BALB/c mouse model. Primary HMPV infection elicits weak innate and aberrant adaptive immune responses characterized by induction of a Th2-type cytokine response at later stages of infection that coincides with increased interleukin-10 expression and persistent virus replication in the lung. Examination of the cytotoxic T lymphocyte and antibody response to HMPV infection revealed a delayed response, but passive transfer of HMPV-specific antibodies provided considerable protection. These features are consistent with virus persistence and indicate that the immune response to HMPV is unique compared to the immune response to RSV.     

Crawling with Virus: Translational Insights from a Neonatal Mouse Model on the Pathogenesis of Respiratory Syncytial Virus in Infants

The infant immune response to respiratory syncytial virus (RSV) remains incompletely understood. Here we review the use of a neonatal mouse model of RSV infection to mimic severe infection in human infants. We describe numerous age-specific responses, organized by cell type, observed in RSV-infected neonatal mice and draw comparisons (when possible) to human infants.     

Isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelial cells

Respiratory syncytial virus (RSV) infection causes severe lower respiratory diseases in infancy, early childhood and the elderly. RSV infections respond poorly to current therapies. Therefore, we initiated a search for novel drug targets by investigating the characteristics and identity of RSV adhesion receptors on mammalian cells. Soluble human lectins, complex polysaccharides and a low molecular selectin antagonist, TBC1269, were used to characterise and isolate the RSV receptor on a human epithelial cell line (Hep2 cells). The binding characteristics of the RSV receptor on Hep2 cells were similar to those reported for L-selectin. The carbohydrate-based selectin antagonists, fucoidan and TBC 1269, inhibit RSV infection both in vitro and in a mouse model of infection. Furthermore, we have isolated annexin II as a potential RSV receptor on Hep2 cells. The expression of annexin II was increased after RSV infection. Recombinant annexin II binds to RSV G-protein, heparin and plasminogen and the binding is inhibited by a selectin antagonist, TBC1269. These findings indicate that inhibitors of annexin II could have potential in treating RSV infection.     

Autocrine regulation of pulmonary inflammation by effector T-cell derived IL-10 during infection with respiratory syncytial virus

Respiratory syncytial virus (RSV) infection is the leading viral cause of severe lower respiratory tract illness in young infants. Clinical studies have documented that certain polymorphisms in the gene encoding the regulatory cytokine IL-10 are associated with the development of severe bronchiolitis in RSV infected infants. Here, we examined the role of IL-10 in a murine model of primary RSV infection and found that high levels of IL-10 are produced in the respiratory tract by anti-viral effector T cells at the onset of the adaptive immune response. We demonstrated that the function of the effector T cell -derived IL-10 in vivo is to limit the excess pulmonary inflammation and thereby to maintain critical lung function. We further identify a novel mechanism by which effector T cell-derived IL-10 controls excess inflammation by feedback inhibition through engagement of the IL-10 receptor on the antiviral effector T cells. Our findings suggest a potentially critical role of effector T cell-derived IL-10 in controlling disease severity in clinical RSV infection.     

Respiratory Syncytial Virus Human Experimental Infection Model: Provenance,Production, and Sequence of Low-Passaged Memphis-37 Challenge Virus

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children and is responsible for as many as 199,000 childhood deaths annually worldwide. To support the development of viral therapeutics and vaccines for RSV, a human adult experimental infection model has been established. In this report, we describe the provenance and sequence of RSV Memphis-37, the low-passage clinical isolate used for the model''s reproducible, safe, experimental infections of healthy, adult volunteers. The predicted amino acid sequences for major proteins of Memphis-37 are compared to nine other RSV A and B amino acid sequences to examine sites of vaccine, therapeutic, and pathophysiologic interest. Human T- cell epitope sequences previously defined by in vitro studies were observed to be closely matched between Memphis-37 and the laboratory strain RSV A2. Memphis-37 sequences provide baseline data with which to assess: (i) virus heterogeneity that may be evident following virus infection/transmission, (ii) the efficacy of candidate RSV vaccines and therapeutics in the experimental infection model, and (iii) the potential emergence of escape mutants as a consequence of experimental drug treatments. Memphis-37 is a valuable tool for pre-clinical research, and to expedite the clinical development of vaccines, therapeutic immunomodulatory agents, and other antiviral drug strategies for the protection of vulnerable populations against RSV disease.     

Acute and Chronic Airway Disease After Human Respiratory Syncytial Virus Infection in Cotton Rats (Sigmodon hispidus)

Infection with respiratory syncytial virus (RSV) generally presents as a mild, upper airway disease in human patients but may cause severe lower airway disease in the very young and very old. Progress toward understanding the mechanisms of RSV pathogenesis has been hampered by a lack of relevant rodent models. Mice, the species most commonly used in RSV research, are resistant to upper respiratory infection and do not recapitulate the pattern of virus spread in the human host. To address the need for better rodent models of RSV infection, we have characterized the acute and chronic pathology of RSV infection of a relatively permissive host, cotton rats (Sigmodon hispidus). We demonstrate that virus delivered to the upper airway results in widespread RSV replication in the ciliated respiratory epithelial cells of the nasal cavity and, to a lesser extent, of the lung. Although acute inflammation is relatively mild and rapidly eliminated after viral clearance, chronic, eosinophilic lung pathology persists. These data support the use of cotton rats as a robust rodent model of human RSV disease, including the association between RSV pneumonia and subsequent development of allergic asthma.Abbreviations: BAL, bronchoalveolar lavage; RSV, human respiratory syncytial virusHuman respiratory syncytial virus (RSV) is the primary cause of lower airway disease in infants and children worldwide.³⁹ Although generally limited to the upper airway, RSV infection can also manifest as bronchiolitis and pneumonia in young children and the elderly and has been implicated as a major cause of middle ear infections (otitis media).^3,55 In addition to the problems associated with acute illness, children who experience severe RSV disease in infancy are at increased risk for development of asthma and recurrent wheezing later in childhood.^35,53,56RSV is ubiquitous worldwide, with most children infected in infancy, and essentially all children are infected by 3 y of age.²⁰ Although reinfection throughout life has been documented, the true incidence of upper airway infection is difficult to quantify, given that treatment for such infections typically is not sought. In comparison, lower airway infection is more likely to come to medical attention, and it is currently estimated that at least 33.8 million episodes of RSV-induced acute pulmonary infection in children younger than 5 y occur yearly and that as many as 200,000 of those episodes are fatal.⁴⁰ Although supportive treatment for severe RSV disease is highly effective in infants, access to such treatment is generally only available in industrialized countries. More than 90% of fatal RSV cases occur in the developing world.⁴⁰Mice have been used extensively to study RSV infection, yet there are key limitations of the mouse model for the study of human RSV disease and immunity. The most important of these is the poor permissiveness of the mouse for human RSV. RSV replicates to a limited extent in the mouse lung, and large viral loads delivered in a relatively large volume are generally used with this model. Even with intranasal inoculation, primarily lower, and not upper, airway infection is achieved in WT mice. This scenario is unlike the human disease, in which the upper airway is the primary target of RSV replication.^24,25 Mice are essentially resistant to upper airway infection, with little to no virus detected in nasal washes evaluated by plaque assay, and only rare RSV-antigen–positive cells are detected by immunohistochemistry.^13,18,21 In addition to this altered route of entry, the pattern of lung infection is markedly divergent between humans and mice. In humans, RSV primarily infects ciliated bronchiolar epithelial cells and, to a much lesser extent, alveolar cells.³² In contrast, bronchiolar epithelial cells are infected only rarely in the mouse lung; instead RSV targets the pneumocytes.³⁷Unlike mice, cotton rats are susceptible to RSV infection of the upper airway, and the pattern of lower airway infection mirrors that seen in human patients. The cotton rat was established as a model of RSV infection more than 3 decades ago and has since emerged as the preferred rodent model in which to evaluate RSV therapeutics and vaccine candidates.^{19,23,33,41,49,57,60} In addition, as the availability of species-specific reagents has improved, cotton rats have become an increasingly useful model in which to study RSV pathogenesis.⁶ However, whereas the susceptibility of cotton rats to RSV infection has been established firmly, the pathology of RSV infection in this species has not yet been extensively characterized.Asthma clearly is a multifactorial disease, dependent on both genetic and environmental factors (see references 28 and 30 for recent reviews), and many studies have pointed to a role for respiratory virus infection in the induction of asthma. In one study,¹⁷ the combination of atopy and the presence of virus in nasal secretions synergistically increased the odds ratio for wheezing in children 25-fold, and another study²⁹ showed that repeated rhinovirus infections in the first 3 y of life increased by 26-fold the risk of developing asthma by the age of 6 y. This relationship is obviously a complex one, influenced both by the nature and the timing of the viral infection. Nonetheless, as many as 80% of acute asthma exacerbations in children and approximately 50% in adults are associated with viral infection.^33,42 The majority of these infections are attributed to rhinoviruses, but other respiratory viruses, including influenza virus, RSV, and coronaviruses, can provoke these attacks.^33,42 Beyond the exacerbation of established asthma, evidence that severe RSV disease in infancy is correlated with development of asthma and recurrent wheezing in later childhood is mounting. An association between lower airway RSV infection and subsequent development of recurrent wheezing and asthma was demonstrated more than 30 y ago,¹⁶ and several recent prospective studies have strengthened this correlation.^27,53In the current study, we expand upon existing knowledge of the cotton rat model of RSV infection by characterizing the spread of virus after droplet inhalation and the early and late inflammatory response to viral challenge. We confirm the observation that cotton rats are relatively permissive in regard to RSV infection as compared with mice, and we demonstrate that the infection of cotton rats, like that in the human host, is primarily an upper airway phenomenon. In addition, we show that primary RSV infection of the cotton rat lung, even at the low levels in this study, induces chronic changes consistent with those in cases of human allergic asthma. This finding is of interest because of the substantial literature associating early, severe respiratory infection with the development of asthma later in life. The studies of RSV infection in cotton rats that we describe here suggest that this species may be predisposed to atopy and that, long after virus clearance, changes associated with allergic inflammation persist in human hosts. Therefore, in addition to the usefulness of cotton rats for testing RSV treatments and vaccine candidates, we suggest that this species may serve as a model system for determining the involvement of virus infection in the development of allergic asthma.     

Modulation of the host immune response by respiratory syncytial virus proteins

Respiratory syncytial virus (RSV) causes severe respiratory disease in both the very young and the elderly. Nearly all individuals become infected in early childhood, and reinfections with the virus are common throughout life. Despite its clinical impact, there remains no licensed RSV vaccine. RSV infection in the respiratory tract induces an inflammatory response by the host to facilitate efficient clearance of the virus. However, the host immune response also contributes to the respiratory disease observed following an RSV infection. RSV has evolved several mechanisms to evade the host immune response and promote virus replication through interactions between RSV proteins and immune components. In contrast, some RSV proteins also play critical roles in activating, rather than suppressing, host immunity. In this review, we discuss the interactions between individual RSV proteins and host factors that modulate the immune response and the implications of these interactions for the course of an RSV infection.     

Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.