Respiratory syncytial virus (RSV) is responsible for a large burden of disease globally and can present as a variety of clinical syndromes in children of all ages. Bronchiolitis in infants under 1 year of age is the most common clinical presentation hospitalizing 24.2 per 1000 infants each year in the United Kingdom. RSV has been shown to account for 22% of all episodes of acute lower respiratory tract infection in children globally. RSV hospitalization, that is, RSV severe disease, has also been associated with subsequent chronic respiratory morbidity. Routine viral testing in all children is not currently recommended by the United Kingdom National Institute for Health and Care Excellence (NICE) or the American Academy of Pediatrics (AAP) guidance and management is largely supportive. There is some evidence for the use of ribavirin in severely immunocompromised children. Emphasis is placed on prevention of RSV infection through infection control measures both in hospital and in the community, and the use of the RSV-specific monoclonal antibody, palivizumab, for certain high-risk groups of infants. New RSV antivirals and vaccines are currently in development. Ongoing work is needed to improve the prevention of RSV infection, not only because of the acute morbidity and mortality, but also to reduce the associated chronic respiratory morbidity after severe infection.

Respiratory syncytial virus (RSV) infection is ubiquitous with almost all infants having been infected by 2 years of age and lifelong repeated infections common. It is the second largest cause of mortality, after malaria, in infants outside the neonatal period and causes up to 200,000 deaths per year worldwide. RSV results in clinical syndromes that include upper respiratory tract infections, otitis media, bronchiolitis (up to 80% of cases) and lower respiratory tract disease including pneumonia and exacerbations of asthma or viral-induced wheeze. For the purposes of this review we will focus on RSV bronchiolitis in infants in whom the greatest disease burden lies. For infants requiring hospital admission, the identification of the causative respiratory virus is used to direct cohorting or isolation and infection control procedures to minimize nosocomial transmission. Nosocomial RSV infections are associated with poorer clinical outcomes, including increased mortality, the need for mechanical ventilation and longer length of hospital stay. Numerous clinical guidelines for the management of infants with bronchiolitis have been published, although none are specific for RSV bronchiolitis. Ribavirin is the only licensed drug for the specific treatment of RSV infection but due to drug toxicity and minimal clinical benefit it has not been recommended for routine clinical use. There is currently no licensed vaccine to prevent RSV infection but passive immunoprophylaxis using a monoclonal antibody, palivizumab, reduces the risk of hospitalization due to RSV infection by 3978% in various high-risk infants predisposed to developing severe RSV disease. The current management of RSV bronchiolitis is purely supportive, with feeding support and oxygen supplementation until the infant immune system mounts a response capable of controlling the disease. The development of a successful treatment or prophylactic agent has the potential to revolutionize the care and outcome for severe RSV infections in the world's most vulnerable infants.

The upper airway – which consists mainly of the naso- and oro-pharynx - is the first point of contact between the respiratory system and microbial organisms that are ubiquitous in the environment. It has evolved highly specialised functions to address these constant threats whilst facilitating seamless respiratory exchange with the lower respiratory tract. Dysregulation of its critical homeostatic and defence functions can lead to ingress of pathogens into the lower respiratory tract, potentially leading to serious illness. Systems-wide proteomic tools may facilitate a better understanding of mechanisms in the upper airways in health and disease. In this study, we aimed to develop a mass spectrometry based proteomics method for characterizing the upper airways proteome. Naso- and oropharyngeal swab samples used in all our experiments had been eluted in the Universal Transport Media (UTM) containing significantly high levels of bovine serum albumin. Our proteomic experiments tested the optimal approach to characterize airway proteome on swab samples eluted in UTM based on the number of proteins identified without BSA depletion (Total proteome: Protocol A) and with its depletion using a commercial kit; Allprep, Qiagen (cellular proteome: Protocol B, Ci, and Cii). Observations and lessons drawn from protocol A, fed into the design and implementation of protocol B, and from B to protocol Ci and finally Cii. Label free proteome quantification was used in Protocol A (n = 6) and B (n = 4) while commercial TMT 10plex reagents were used for protocols Ci and ii (n = 83). Protocols Ci and ii were carried out under similar conditions except for the elution gradient: 3 h and 6 h respectively. Swab samples tested in this study were from infants and children with and without upper respiratory tract infections from Kilifi County Hospital on the Kenyan Coast. Protocol A had the least number of proteins identified (215) while B produced the highest number of protein identifications (2396). When Protocol B was modified through sample multiplexing with TMT to enable higher throughput (Protocol Ci), the number of protein identified reduced to 1432. Modification of protocol Ci by increasing the peptide elution time generated Protocol Cii that substantially increased the number of proteins identified to 1875. The coefficient of variation among the TMT runs in Protocol Cii was <20%. There was substantial overlap in the identity of proteins using the four protocols. Our method was were able to identify marker proteins characteristically expressed in the upper airway. We found high expression levels of signature nasopharyngeal and oral proteins, including BPIFA1/2 and AMY1A, as well as a high abundance of proteins related to innate and adaptive immune function in the upper airway. We have developed a sensitive systems-level proteomic assay for the systematic quantification of naso-oro-pharyngeal proteins. The assay will advance mechanistic studies of respiratory pathology, by providing an untargeted and hypothesis-free approach of examini...

No abstract

Rationale: Pneumonia is a leading cause of mortality in infants and young children. The mechanisms that lead to mortality in these children are poorly understood. Studies of the cellular immunology of the infant airway have traditionally been hindered by the limited sample volumes available from the young, frail children who are admitted to hospital with pneumonia. This is further compounded by the relatively low frequencies of certain immune cell phenotypes that are thought to be critical to the clinical outcome of pneumonia. To address this, we developed a novel in-silico deconvolution method for inferring the frequencies of immune cell phenotypes in the airway of children with different survival outcomes using proteomic data.Methods: Using high-resolution mass spectrometry, we identified > 1,000 proteins expressed in the airways of children who were admitted to hospital with clinical pneumonia. 61 of these children were discharged from hospital and survived for more than 365 days after discharge, while 19 died during admission. We used machine learning by random forest to derive protein features that could be used to deconvolve immune cell phenotypes in paediatric airway samples. We applied these phenotype-specific signatures to identify airway-resident immune cell phenotypes that were differentially enriched by survival status and validated the findings using a large retrospective pneumonia cohort. Main Results:We identified immune-cell phenotype classification features for 33 immune cell types. Eosinophil-associated features were significantly elevated in airway samples obtained from pneumonia survivors and were downregulated in children who subsequently died. To confirm these results, we analyzed clinical parameters from >10,doi: bioRxiv preprint results of this retrospective analysis mirrored airway deconvolution data and showed that survivors had significantly elevated eosinophils at admission compared to fatal pneumonia. Conclusions:Using a proteomics bioinformatics approach, we identify airway eosinophils as a critical factor for pneumonia survival in infants and young children. doi: bioRxiv preprint 1ml of nasopharyngeal and oropharyngeal swab samples obtained from children was centrifuged at 17,000xg for 7 minutes, after which 800 l of the supernatant was removed and discarded. The remaining 200µl were split into two aliquots of 100µl each. The first aliquot was used for neutrophil phenotyping assays and the other was used for neutrophil phagocytosis assays. 20µl of a pre-constituted cocktail of the following antibodies (from ThermoFisher) was used to label both aliquots -CD45, CD16, CD14, CD3, CD19, HLA-DR, CD66b, CD11b and a Live-dead marker. With the exception of the live/dead marker, all other antibodies were diluted 1:100 in FACS buffer. The live/dead marker was prepared at a 1:1000 dilution in FACS buffer. For the phagocytosis assay tube, 20 l of opsonised Escherichia coli (E.coli) was added to the tube (pHrodo Red E. coli BioParticles;ThermoFisher). The bacteria was initially prepared by mixing...