Engineering,Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.     

Suppression of pattern-recognition receptor TLR4 sensing does not alter lung responses to pneumovirus infection

Toll-like receptors (TLR) are an important component in the innate immune response to a wide variety of pathogens. Recently, a series of studies has addressed the hypothesis that TLR4 also participates in the host innate response against respiratory syncytial virus (RSV), the leading cause of lower respiratory tract infections in infants and young children. In most of the studies available, RSV, which is not a natural pathogen of mice, has been systematically used in mouse models of human bronchiolitis, with conflicting results. Pneumonia virus of mice (PVM), a member of the pneumovirus genus, shares many similarities with RSV. The serological and structural relationships that exist between them suggest that the immune response to these viruses may be similar in their respective natural hosts. To determine the role of TLR4 in host defense against PVM, TLR4-competent and TLR4-deficient mice were intranasally infected with PVM. Variation of body weight, pulmonary function values, histopathology, and pulmonary viral loads were analyzed. None of the investigated clinical, functional, histological and virological parameters was different between strains, which demonstrates that the sensitivity of the mouse to its natural pneumovirus infection is independent of the presence or absence of TLR4 sensing.     

Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocirculate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HLA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathology in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytokines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.     

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.     

Full-Genome Analysis of a Canine Pneumovirus Causing Acute Respiratory Disease in Dogs,Italy

An outbreak of canine infectious respiratory disease (CIRD) associated to canine pneumovirus (CnPnV) infection is reported. The outbreak occurred in a shelter of the Apulia region and involved 37 out of 350 dogs that displayed cough and/or nasal discharge with no evidence of fever. The full-genomic characterisation showed that the causative agent (strain Bari/100-12) was closely related to CnPnVs that have been recently isolated in the USA, as well as to murine pneumovirus, which is responsible for respiratory disease in mice. The present study represents a useful contribution to the knowledge of the pathogenic potential of CnPnV and its association with CIRD in dogs. Further studies will elucidate the pathogenicity and epidemiology of this novel pneumovirus, thus addressing the eventual need for specific vaccines.     

A fluorescence polarization assay using an engineered human respiratory syncytial virus F protein as a direct screening platform

Human respiratory syncytial virus (hRSV) typically affects newborns and young children. Even though it can cause severe and, in some cases, lifelong respiratory infections, there are currently no Food and Drug Administration (FDA)-approved therapeutics that control this virus. The hRSV F protein facilitates viral fusion, a critical extracellular event that can be targeted for therapeutic intervention by disrupting the assembly of a postfusion 6-helix bundle (6HB) within the hRSV F protein. Here we report the development of a fluorescence polarization (FP) assay using an engineered hRSV F protein 5-helix bundle (5HB). We generated the 5HB and validated its ability to form a 6HB in an FP assay. To test the potential of 5HB as a screening tool, we then investigated a series of truncated peptides derived from the “missing” sixth helix. Using this FP-based 5HB system, we have successfully demonstrated that short peptides can prevent 6HB formation and serve as potential hRSV fusion inhibitors. We anticipate that this new 5HB system will provide an effective tool to identify and study potential antivirals to control hRSV infection.     

Understanding the transmission dynamics of respiratory syncytial virus using multiple time series and nested models

The nature and role of re-infection and partial immunity are likely to be important determinants of the transmission dynamics of human respiratory syncytial virus (hRSV). We propose a single model structure that captures four possible host responses to infection and subsequent reinfection: partial susceptibility, altered infection duration, reduced infectiousness and temporary immunity (which might be partial). The magnitude of these responses is determined by four homotopy parameters, and by setting some of these parameters to extreme values we generate a set of eight nested, deterministic transmission models. In order to investigate hRSV transmission dynamics, we applied these models to incidence data from eight international locations. Seasonality is included as cyclic variation in transmission. Parameters associated with the natural history of the infection were assumed to be independent of geographic location, while others, such as those associated with seasonality, were assumed location specific. Models incorporating either of the two extreme assumptions for immunity (none or solid and lifelong) were unable to reproduce the observed dynamics. Model fits with either waning or partial immunity to disease or both were visually comparable. The best fitting structure was a lifelong partial immunity to both disease and infection. Observed patterns were reproduced by stochastic simulations using the parameter values estimated from the deterministic models.     

Mechanical ventilation enhances lung inflammation and caspase activity in a model of mouse pneumovirus infection

Severe infection with respiratory syncytial virus (RSV) in children can progress to respiratory distress and acute lung injury (ALI). Accumulating evidence suggests that mechanical ventilation (MV) is an important cofactor in the development of ALI by modulating the host immune responses to bacteria. This study investigates whether MV enhances the host response to pneumonia virus of mice (PVM), a mouse pneumovirus that has been used as a model for RSV infection in humans. BALB/c mice were inoculated intranasally with diluted clarified lung homogenates from mice infected with PVM strain J3666 or uninfected controls. Four days after inoculation, the mice were subjected to 4 h of MV (tidal volume, 10 ml/kg) or allowed to breathe spontaneously. When compared with that of mice inoculated with PVM only, the administration of MV to PVM-infected mice resulted in increased bronchoalveolar lavage fluid concentrations of the cytokines macrophage inflammatory protein (MIP)-2, MIP-1alpha (CCL3), and IL-6; increased alveolar-capillary permeability to high molecular weight proteins; and increased caspase-3 activity in lung homogenates. We conclude that MV enhances the activation of inflammatory and caspase cell death pathways in response to pneumovirus infection. We speculate that MV potentially contributes to the development of lung injury in patients with RSV infection.     

Altered pathogenesis of severe pneumovirus infection in response to combined antiviral and specific immunomodulatory agents

We report here the responses of mice with symptomatic pneumovirus infection to combined antiviral and specific immunomodulatory agents. Mice infected with pneumonia virus of mice, a natural mouse pathogen that replicates the signs and symptoms of severe infection with respiratory syncytial virus (RSV), responded to the antiviral agent ribavirin when it was administered in the setting of endogenous (gene deletion) or exogenous (antibody-mediated) blockade of the MIP-1alpha proinflammatory signaling cascade. Although neither treatment is effective alone, together they offer a dramatic reduction in symptoms and pathology, the most impressive of which is a significant reduction in morbidity and mortality. The findings presented are consistent with the notion of unique and independent contributions of virus replication and ongoing inflammation to the pathogenesis of severe respiratory virus infection, and they provide the impetus for the study of this treatment regimen in RSV-infected humans.     

Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS

Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.     

Safety and efficacy of immune-stimulating complex-based antigen delivery systems for neonatal immunisation against respiratory syncytial virus infection

To protect against human respiratory syncytial virus (hRSV)-induced bronchiolitis in early infancy, vaccines need to be designed which are effective in the neonatal period. To test the safety and efficacy of adjuvants in neonatal mice, we injected hRSV surface proteins combined with immune-stimulating complexes (ISCOMs) prepared from fractions A, C or A + C of Quillaja saponins. All were well tolerated in adults, but A + C ISCOMS proved lethal in neonates; A or C fractions alone were well tolerated by neonates up to the adult dose. hRSV-ISCOM A induced antibody responses similar to combined fractions, and potent in vitro cytotoxic T cell responses. Adult-like in vitro cytotoxicity against hRSV-infected targets and precursor cytotoxic T cell frequencies were observed within one week of neonatal priming and hRSV-ISCOM A-primed neonates showed virtually complete protection against subsequent viral challenge. hRSV challenge was associated with some pulmonary eosinophilia in both age groups, with higher IL-4 production by lung CD4+ T cells in mice primed as neonates. This was, however, accompanied by only minor (approximately 10%) and transient illness and weight loss. Thus, the identification of hRSV antigen delivery systems with an age-appropriate adjuvanticity/reactogenicity balance may be feasible even in the vulnerable early-life period.     

CD8+ T-Cell Epitope Mapping for Pneumonia Virus of Mice in H-2b Mice

The paramyxovirus pneumonia virus of mice (PVM) is a rodent model of human respiratory syncytial virus (hRSV) pathogenesis. Here we characterized the PVM-specific CD8⁺ T-cell repertoire in susceptible C57BL/6 mice. In total, 15 PVM-specific CD8⁺ T-cell epitopes restricted by H-2D^b and/or H-2K^b were identified. These data open the door for using widely profiled, genetically manipulated C57BL/6 mice to study the contribution of epitope-specific CD8⁺ T cells to PVM pathogenesis.     

Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion

Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins. They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV. Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus. However, in other members, only HR2 gives strong inhibition whereas HR1 does not. Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately. Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known. In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed. It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form.     

A newly discovered human pneumovirus isolated from young children with respiratory tract disease

From 28 young children in the Netherlands, we isolated a paramyxovirus that was identified as a tentative new member of the Metapneumovirus genus based on virological data, sequence homology and gene constellation. Previously, avian pneumovirus was the sole member of this recently assigned genus, hence the provisional name for the newly discovered virus: human metapneumovirus. The clinical symptoms of the children from whom the virus was isolated were similar to those caused by human respiratory syncytial virus infection, ranging from upper respiratory tract disease to severe bronchiolitis and pneumonia. Serological studies showed that by the age of five years, virtually all children in the Netherlands have been exposed to human metapneumovirus and that the virus has been circulating in humans for at least 50 years.     

Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames.

The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.     

Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters

Small animal models are needed in order to evaluate the efficacy of candidate vaccines and antivirals directed against the severe acute respiratory syndrome coronavirus (SARS CoV). We investigated the ability of SARS CoV to infect 5-week-old Golden Syrian hamsters. When administered intranasally, SARS CoV replicates to high titers in the lungs and nasal turbinates. Peak replication in the lower respiratory tract was noted on day 2 postinfection (p.i.) and was cleared by day 7 p.i. Low levels of virus were present in the nasal turbinates of a few hamsters at 14 days p.i. Viral replication in epithelial cells of the respiratory tract was accompanied by cellular necrosis early in infection, followed by an inflammatory response coincident with viral clearance, focal consolidation in pulmonary tissue, and eventual pulmonary tissue repair. Despite high levels of virus replication and associated pathology in the respiratory tract, the hamsters showed no evidence of disease. Neutralizing antibodies were detected in sera at day 7 p.i., and mean titers at day 28 p.i. exceeded 1:400. Hamsters challenged with SARS CoV at day 28 p.i. were completely protected from virus replication and accompanying pathology in the respiratory tract. Comparing these data to the mouse model, SARS CoV replicates to a higher titer and for a longer duration in the respiratory tract of hamsters and is accompanied by significant pathology that is absent in mice. Viremia and extrapulmonary spread of SARS CoV to liver and spleen, which are seen in hamsters, were not detected in mice. The hamster, therefore, is superior to the mouse as a model for the evaluation of antiviral agents and candidate vaccines against SARS CoV replication.     

Measuring lung function in mice: the phenotyping uncertainty principle.

Measuring lung function in mice is essential for establishing the relevance of murine models to human lung disease. However, making such measurements presents particular technical challenges due to the small size of the animal, particularly with regard to the measurement of respiratory flows. In this review, we examine the various methods currently available for assessment of lung function in mice and contrast them in terms of a concept we call the phenotyping uncertainty principle; each method can be considered to lie somewhere along a continuum on which noninvasiveness must be traded off against experimental control and measurement precision. Unrestrained plethysmography in conscious mice represents the extreme of noninvasiveness and is highly convenient but provides respiratory measures that are so tenuously linked to respiratory mechanics that they cannot be considered as meaningful indicators of lung function. At the other extreme, the measurement of input impedance in anesthetized, paralyzed, tracheostomized mice is precise and specific but requires that an animal be studied under conditions far from natural. In between these two extremes lie methods that sacrifice some precision for a reduction in the level of invasiveness, a promising example being the measurement of transfer impedance in conscious, restrained mice. No method is optimal in all regards; therefore, the appropriate technique to use depends on the application.     

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy,Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.