Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: a proof of concept using cyclopamine.

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening.

Longitudinal study of the immune response and memory following natural bovine respiratory syncytial virus infections in cattle of different age.

Human and bovine respiratory syncytial virus (HRSV and BRSV) are closely genetically related and cause respiratory disease in their respective host. Whereas HRSV vaccines are still under development, a multitude of BRSV vaccines are used to reduce clinical signs. To enable the design of vaccination protocols to entirely stop virus circulation, we aimed to investigate the duration, character and efficacy of the immune responses induced by natural infections. The systemic humoral immunity was monitored every two months during two years in 33 dairy cattle in different age cohorts following a natural BRSV outbreak, and again in selected individuals before and after a second outbreak, four years later. Local humoral and systemic cellular responses were also monitored, although less extensively. Based on clinical observations and economic losses linked to decreased milk production, the outbreaks were classified as moderate. Following the first outbreak, most but not all animals developed neutralising antibody responses, BRSV-specific IgG1, IgG2 and HRSV F- and HRSV N-reactive responses that lasted at least two years, and in some cases at least four years. In contrast, no systemic T cell responses were detected and only weak IgA responses were detected in some animals. Seronegative sentinels remained negative, inferring that no new infections occurred between the outbreaks. During the second outbreak, reinfections with clinical signs and virus shedding occurred, but the signs were milder, and the virus shedding was significantly lower than in naïve animals. Whereas the primary infection induced similar antibody titres against the prefusion and the post fusion form of the BRSV F protein, memory responses were significantly stronger against prefusion F. In conclusion, even if natural infections induce a long-lasting immunity, it would probably be necessary to boost memory responses between outbreaks, to stop the circulation of the virus and limit the potential role of previously infected adult cattle in the chain of BRSV transmission.

Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis.

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases. IMPORTANCE: Respiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.

Control of IFN-I responses by the aminopeptidase IRAP in neonatal C57BL/6 alveolar macrophages during RSV infection.

Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract infection in infants, in whom, the sensing of RSV by innate immune receptors and its regulation are still poorly described. However, the severe bronchiolitis following RSV infection in neonates has been associated with a defect in type I interferons (IFN-I) production, a cytokine produced mainly by alveolar macrophages (AMs) upon RSV infection in adults. In the present study, neonatal C57BL/6 AMs mobilized very weakly the IFN-I pathway upon RSV infection in vitro and failed to restrain virus replication. However, IFN-I productions by neonatal AMs were substantially increased by the deletion of Insulin-Responsive AminoPeptidase (IRAP), a protein previously involved in the regulation of IFN-I production by dendritic cells. Moreover, neonatal IRAPKO AMs showed a higher expression of IFN-stimulated genes than their wild-type C57BL/6 counterpart. Interestingly, depletion of IRAP did not affect adult AM responses. Finally, we demonstrated that newborn IRAPKO mice infected with RSV had more IFN-I in their lungs and eliminated the virus more efficiently than WT neonates. Taken together, early-life susceptibility to RSV infection may be related to an original age-dependent suppressive function of IRAP on the IFN-I driven-antiviral responses in neonatal AMs.

Single-Shot Vaccines against Bovine Respiratory Syncytial Virus (BRSV): Comparative Evaluation of Long-Term Protection after Immunization in the Presence of BRSV-Specific Maternal Antibodies.

The induction of long-lasting clinical and virological protection is needed for a successful vaccination program against the bovine respiratory syncytial virus (BRSV). In this study, calves with BRSV-specific maternally derived antibodies were vaccinated once, either with (i) a BRSV pre-fusion protein (PreF) and MontanideTM ISA61 VG (ISA61, n = 6), (ii) BRSV lacking the SH gene (ΔSHrBRSV, n = 6), (iii) a commercial vaccine (CV, n = 6), or were injected with ISA61 alone (n = 6). All calves were challenged with BRSV 92 days later and were euthanized 13 days post-infection. Based on clinical, pathological, and proteomic data, all vaccines appeared safe. Compared to the controls, PreF induced the most significant clinical and virological protection post-challenge, followed by ΔSHrBRSV and CV, whereas the protection of PreF-vaccinated calves was correlated with BRSV-specific serum immunoglobulin (Ig)G antibody responses 84 days post-vaccination, and the IgG antibody titers of ΔSHrBRSV- and CV-vaccinated calves did not differ from the controls on this day. Nevertheless, strong anamnestic BRSV- and PreF-specific IgG responses occurred in calves vaccinated with either of the vaccines, following a BRSV challenge. In conclusion, PreF and ΔSHrBRSV are two efficient one-shot candidate vaccines. By inducing a protection for at least three months, they could potentially improve the control of BRSV in calves.

The microbiota plays a critical role in the reactivity of lung immune components to innate ligands.

The gut microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the gut and/or lung microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota in general can modulate the reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific-pathogen-free (SPF) and germ-free (GF) mice. Thus, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice after intranasal instillation to lipopolysaccharide (LPS), a component of Gram-negative bacteria. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to the environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs.

The Role of Insulin Regulated Aminopeptidase in Endocytic Trafficking and Receptor Signaling in Immune Cells.

Insulin regulated aminopeptidase (IRAP) is a type II transmembrane protein with broad tissue distribution initially identified as a major component of Glut4 storage vesicles (GSV) in adipocytes. Despite its almost ubiquitous expression, IRAP had been extensively studied mainly in insulin responsive cells, such as adipocytes and muscle cells. In these cells, the enzyme displays a complex intracellular trafficking pattern regulated by insulin. Early studies using fusion proteins joining the IRAP cytosolic domain to various reporter proteins, such as GFP or the transferrin receptor (TfR), showed that the complex and regulated trafficking of the protein depends on its cytosolic domain. This domain contains several motifs involved in IRAP trafficking, as demonstrated by mutagenesis studies. Also, proteomic studies and yeast two-hybrid experiments showed that the IRAP cytosolic domain engages in multiple protein interactions with cytoskeleton components and vesicular trafficking adaptors. These findings led to the hypothesis that IRAP is not only a cargo of GSV but might be a part of the sorting machinery that controls GSV dynamics. Recent work in adipocytes, immune cells, and neurons confirmed this hypothesis and demonstrated that IRAP has a dual function. Its carboxy-terminal domain located inside endosomes is responsible for the aminopeptidase activity of the enzyme, while its amino-terminal domain located in the cytosol functions as an endosomal trafficking adaptor. In this review, we recapitulate the published protein interactions of IRAP and summarize the increasing body of evidence indicating that IRAP plays a role in intracellular trafficking of several proteins. We describe the impact of IRAP deletion or depletion on endocytic trafficking and the consequences on immune cell functions. These include the ability of dendritic cells to cross-present antigens and prime adaptive immune responses, as well as the control of innate and adaptive immune receptor signaling and modulation of inflammatory responses.

Regulatory B Lymphocytes Colonize the Respiratory Tract of Neonatal Mice and Modulate Immune Responses of Alveolar Macrophages to RSV Infection in IL-10-Dependant Manner.

Respiratory syncytial virus (RSV) is the prevalent pathogen of lower respiratory tract infections in children. The presence of neonatal regulatory B lymphocytes (nBreg) has been associated with a poor control of RSV infection in human newborns and with bronchiolitis severity. So far, little is known about how nBreg may contribute to neonatal immunopathology to RSV. We tracked nBreg in neonatal BALB/c mice and we investigated their impact on lung innate immunity, especially their crosstalk with alveolar macrophages (AMs) upon RSV infection. We showed that the colonization by nBreg during the first week of life is a hallmark of neonatal lung whereas this population is almost absent in adult lung. This particular period of age when nBreg are abundant corresponds to the same period when RSV replication in lungs fails to generate a type-I interferons (IFN-I) response and is not contained. When neonatal AMs are exposed to RSV in vitro, they produce IFN-I that in turn enhances IL-10 production by nBreg. IL-10 reciprocally can decrease IFN-I secretion by AMs. Thus, our work identified nBreg as an important component of neonatal lungs and pointed out new immunoregulatory interactions with AMs in the context of RSV infection.

A Single Shot Pre-fusion-Stabilized Bovine RSV F Vaccine is Safe and Effective in Newborn Calves with Maternally Derived Antibodies.

Achieving safe and protective vaccination against respiratory syncytial virus (RSV) in infants and in calves has proven a challenging task. The design of recombinant antigens with a conformation close to their native form in virus particles is a major breakthrough. We compared two subunit vaccines, the bovine RSV (BRSV) pre-fusion F (preF) alone or with nanorings formed by the RSV nucleoprotein (preF+N). PreF and N proteins are potent antigenic targets for neutralizing antibodies and T cell responses, respectively. To tackle the challenges of neonatal immunization, three groups of six one-month-old calves with maternally derived serum antibodies (MDA) to BRSV received a single intramuscular injection of PreF, preF+N with MontanideTM ISA61 VG (ISA61) as adjuvant or only ISA61 (control). One month later, all calves were challenged with BRSV and monitored for virus replication in the upper respiratory tract and for clinical signs of disease over one week, and then post-mortem examinations of their lungs were performed. Both preF and preF+N vaccines afforded safe, clinical, and virological protection against BRSV, with little difference between the two subunit vaccines. Analysis of immune parameters pointed to neutralizing antibodies and antibodies to preF as being significant correlates of protection. Thus, a single shot vaccination with preF appears sufficient to reduce the burden of BRSV disease in calves with MDA.

De novo protein design enables the precise induction of RSV-neutralizing antibodies.

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.

Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen.

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.

Paradigms of Lung Microbiota Functions in Health and Disease, Particularly, in Asthma.

Improvements in our knowledge of the gut microbiota have broadened our vision of the microbes associated with the intestine. These microbes are essential actors and protectors of digestive and extra-digestive health and, by extension, crucial for human physiology. Similar reconsiderations are currently underway concerning the endogenous microbes of the lungs, with a shift in focus away from their involvement in infections toward a role in physiology. The discovery of the lung microbiota was delayed by the long-held view that the lungs of healthy individuals were sterile and by sampling difficulties. The lung microbiota has a low density, and the maintenance of small numbers of bacteria seems to be a critical determinant of good health. This review aims to highlight how knowledge about the lung microbiota can change our conception of lung physiology and respiratory health. We provide support for this point of view with knowledge acquired about the gut microbiota and intestinal physiology. We describe the main characteristics of the lung microbiota and its functional impact on lung physiology, particularly in healthy individuals, after birth, but also in asthma. We describe some of the physiological features of the respiratory tract potentially favoring the installation of a dysbiotic microbiota. The gut microbiota feeds and matures the intestinal epithelium and is involved in immunity, when the principal role of the lung microbiota seems to be the orientation and balance of aspects of immune and epithelial responsiveness. This implies that the local and remote effects of bacterial communities are likely to be determinant in many respiratory diseases caused by viruses, allergens or genetic deficiency. Finally, we discuss the reciprocal connections between the gut and lungs that render these two compartments inseparable.

Pulmonary Susceptibility of Neonates to Respiratory Syncytial Virus Infection: A Problem of Innate Immunity?

Human respiratory syncytial virus (RSV) is a common and highly contagious viral agent responsible for acute lower respiratory infection in infants. This pathology characterized by mucus hypersecretion and a disturbed T cell immune response is one of the major causes of infant hospitalization for severe bronchiolitis. Although different risk factors are associated with acute RSV bronchiolitis, the immunological factors contributing to the susceptibility of RSV infection in infants are not clearly elucidated. Epidemiological studies have established that the age at initial infection plays a central role in the severity of the disease. Thus, neonatal susceptibility is intrinsically linked to the immunological characteristics of the young pulmonary mucosa. Early life is a critical period for the lung development with the first expositions to external environmental stimuli and microbiota colonization. Furthermore, neonates display a lung immune system that profoundly differs to those from adults, with the predominance of type 2 immune cells. In this review, we discuss the latest information about the lung immune environment in the early period of life at a steady state and upon RSV infection and how we can modulate neonatal susceptibility to RSV infection.

Proteome analysis of bronchoalveolar lavage from calves infected with bovine respiratory syncytial virus-Insights in pathogenesis and perspectives for new treatments.

Human and bovine respiratory syncytial viruses (HRSV/BRSV) are major causes of severe lower respiratory tract infections in children and calves, respectively. Shared epidemiological, clinical, pathological and genetic characteristics of these viruses make comparative research highly relevant. To characterise the host response against BRSV infection, bronchoalveolar lavage supernatant (BAL) from i) non-vaccinated, BRSV-infected ii) vaccinated, BRSV-infected and iii) non-infected calves was analysed by tandem mass spectrometry. Proteins were semi-quantified and protein expression was validated by immunoblotting. Correlations between selected proteins and pathology, clinical signs and virus shedding were investigated. Calves with BRSV-induced disease had increased total protein concentrations and a decreased number of proteins identified in BAL. The protein profile was characterised by neutrophil activation and a reduction in identified antioxidant enzymes. The presence of neutrophils in alveolar septa, the expression level of neutrophil-related or antioxidant proteins and LZTFL1 correlated significantly with disease. Citrullinated histone 3, an indicator of extracellular traps (ETs), was only detected in non-vaccinated, BRSV-infected animals. By bringing disequilibrium in the release and detoxification of reactive oxygen species, generating ETs and causing elastine degradation, exaggerated neutrophil responses might exacerbate RSV-induced disease. Neutrophil-mitigating or antioxidant treatments should be further explored.

Respiratory Syncytial Virus Infects Regulatory B Cells in Human Neonates via Chemokine Receptor CX3CR1 and Promotes Lung Disease Severity.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.

Bacteria isolated from lung modulate asthma susceptibility in mice.

Asthma is a chronic, non-curable, multifactorial disease with increasing incidence in industrial countries. This study evaluates the direct contribution of lung microbial components in allergic asthma in mice. Germ-Free and Specific-Pathogen-Free mice display similar susceptibilities to House Dust Mice-induced allergic asthma, indicating that the absence of bacteria confers no protection or increased risk to aeroallergens. In early life, allergic asthma changes the pattern of lung microbiota, and lung bacteria reciprocally modulate aeroallergen responsiveness. Primo-colonizing cultivable strains were screened for their immunoregulatory properties following their isolation from neonatal lungs. Intranasal inoculation of lung bacteria influenced the outcome of allergic asthma development: the strain CNCM I 4970 exacerbated some asthma features whereas the pro-Th1 strain CNCM I 4969 had protective effects. Thus, we confirm that appropriate bacterial lung stimuli during early life are critical for susceptibility to allergic asthma in young adults.

RSV N-nanorings fused to palivizumab-targeted neutralizing epitope as a nanoparticle RSV vaccine.

Respiratory syncytial virus (RSV) is the leading cause of acute respiratory infections in children, yet no vaccine is available. The sole licensed preventive treatment against RSV is composed of a monoclonal neutralizing antibody (palivizumab), which targets a conformational epitope located on the fusion protein (F). Palivizumab reduces the burden of bronchiolitis but does not prevent infection. Thus, the development of RSV vaccines remains a priority. We previously evaluated nanorings formed by RSV nucleoprotein (N) as an RSV vaccine, as well as an immunostimulatory carrier for heterologous antigens. Here, we linked the palivizumab-targeted epitope (called FsII) to N, to generate N-FsII-nanorings. Intranasal N-FsII immunization elicited anti-F antibodies in mice that were non-neutralizing in vitro. Nevertheless, RSV-challenged animals were better protected against virus replication than mice immunized with N-nanorings, especially in the upper airways. In conclusion, an N-FsII-focused vaccine is an attractive candidate combining N-specific cellular immunity and F-specific antibodies for protection.

Non-invasive epicutaneous vaccine against Respiratory Syncytial Virus: Preclinical proof of concept.

To put a Respiratory Syncytial Virus (RSV) vaccine onto the market, new vaccination strategies combining scientific and technical innovations need to be explored. Such a vaccine would also need to be adapted to the vaccination of young children that are the principal victims of acute RSV infection. In the present project, we describe the development and the preclinical evaluation of an original epicutaneous RSV vaccine that combines two technologies: Viaskin® epicutaneous patches as a delivery platform and RSV N-nanorings (N) as a subunit antigen. Such a needle-free vaccine may have a better acceptability for the vaccination of sensible population such as infants since it does not require any skin preparation. Moreover, this self-applicative vaccine would overcome some issues associated to injectable vaccines such as the requirement of sterile medical devices, the need of skilled health-care professionals and the necessity of stringent store conditions. Here, we demonstrate that Viaskin® patches loaded with a formulation containing N-nanorings (Viaskin®-N) are highly immunogenic in mice and promotes a Th1/Th17 oriented immune response. More importantly, Viaskin®-N epicutaneous vaccine confers a high level of protection against viral replication upon RSV challenge in mice, without exacerbating clinical symptoms. In swine, which provides the best experimental model for the transcutaneous passage of drug/antigen in human skin, we have shown that GFP fluorescent N-nanorings, delivered epicutaneously with Viaskin® patches, are taken up by epidermal Langerhans cells. We have also demonstrated that Viaskin®-N induced a significant RSV N-specific T-cell response in pig. In conclusion, Viaskin®-N epicutaneous vaccine seems efficient to protect against RSV infection in animal model.

Flt3 ligand improves the innate response to respiratory syncytial virus and limits lung disease upon RSV reexposure in neonate mice.

Respiratory syncytial virus (RSV) causes severe bronchiolitis in infants worldwide. The immunological factors responsible for RSV susceptibility in infants are poorly understood. Here, we used the BALB/c mouse model of neonatal RSV infection to study the mechanisms leading to severe disease upon reexposure to the virus when adults. Two major deficiencies in neonatal lung innate responses were found: a poor DCs mobilization, and a weak engagement of the IFNI pathway. The administration of Flt3 ligand (Flt3-L), a growth factor that stimulates the proliferation of hematopoietic cells, to neonates before RSV-infection, resulted in increased lung DC number, and reconditioned the IFNI pathway upon RSV neonatal infection. Besides, neonates treated with Flt3-L were protected against exacerbated airway disease upon adult reexposure to RSV. This was associated with a reorientation of RSV-specific responses toward Th1-mediated immunity. Thus, the poor lung DCs and IFNI responses to RSV in neonates may be partly responsible for the deleterious long-term consequences revealed upon adult reexposure to RSV, which could be prevented by Flt3-L treatment. These results open new perspectives for developing neonatal immuno-modulating strategies to reduce the burden of bronchiolitis.

A bovine respiratory syncytial virus model with high clinical expression in calves with specific passive immunity.

BACKGROUND: Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in cattle worldwide. Calves are particularly affected, even with low to moderate levels of BRSV-specific maternally derived antibodies (MDA). Available BRSV vaccines have suboptimal efficacy in calves with MDA, and published infection models in this target group are lacking in clinical expression. Here, we refine and characterize such a model. RESULTS: In a first experiment, 2 groups of 3 calves with low levels of MDA were experimentally inoculated by inhalation of aerosolized BRSV, either: the Snook strain, passaged in gnotobiotic calves (BRSV-Snk), or isolate no. 9402022 Denmark, passaged in cell culture (BRSV-Dk). All calves developed clinical signs of respiratory disease and shed high titers of virus, but BRSV-Snk induced more severe disease, which was then reproduced in a second experiment in 5 calves with moderate levels of MDA. These 5 calves shed high titers of virus and developed severe clinical signs of disease and extensive macroscopic lung lesions (mean+/-SD, 48.3+/-12.0% of lung), with a pulmonary influx of inflammatory cells, characterized by interferon gamma secretion and a marked effect on lung function. CONCLUSIONS: We present a BRSV-infection model, with consistently high clinical expression in young calves with low to moderate levels of BRSV-specific MDA, that may prove useful in studies into disease pathogenesis, or evaluations of vaccines and antivirals. Additionally, refined tools to assess the outcome of BRSV infection are described, including passive measurement of lung function and a refined system to score clinical signs of disease. Using this cognate host calf model might also provide answers to elusive questions about human RSV (HRSV), a major cause of morbidity in children worldwide.