Experimental trials of predicted CD4+ and CD8+ T-cell epitopes of respiratory syncytial virus.

Background: Respiratory syncytial virus (RSV) is the most common cause of viral lower respiratory tract infections (LRTIs) in young children around the world and an important cause of LRTI in the elderly. The available treatments and FDA-approved vaccines for RSV only lessen the severity of the infection and are recommended for infants and elderly people. Methods: We focused on developing a broad-spectrum vaccine that activates the immune system to directly combat RSV. The objective of this study is to identify CD4+ and CD8+ T-cell epitopes using an immunoinformatics approach to develop RSV vaccines. The efficacy of these peptides was validated through in-vitro and in-vivo studies involving healthy and diseased animal models. Results: For each major histocompatibility complex (MHC) class-I and II, we found three epitopes of RSV proteins including F, G, and SH with an antigenic score of >0.5 and a projected SVM score of <5. Experimental validation of these peptides on female BALB/c mice was conducted before and after infection with the RSV A2 line 19f. We found that the 3RVMHCI (CD8+) epitope of the F protein showed significant results of white blood cells (19.72 × 103 cells/µl), neutrophils (6.01 × 103 cells/µl), lymphocytes (12.98 × 103 cells/µl), IgG antibodies (36.9 µg/ml), IFN-γ (86.96 ng/L), and granzyme B (691.35 pg/ml) compared to control at the second booster dose of 10 µg. Similarly, 4RVMHCII (CD4+) of the F protein substantially induced white blood cells (27.08 × 103 cells/µl), neutrophils (6.58 × 103 cells/µl), lymphocytes (16.64 × 103 cells/µl), IgG antibodies (46.13 µg/ml), IFN-γ (96.45 ng/L), and granzyme B (675.09 pg/ml). In-vitro studies showed that 4RVMHCII produced a significant level of antibodies in sera on day 45 comparable to mice infected with the virus. 4RVMHCII also induced high IFN-γ and IL-2 secretions on the fourth day of the challenge compared to the preinfectional stage. Conclusion: In conclusion, epitopes of the F protein showed considerable immune response and are suitable for further validation.

Immune Prophylaxis Targeting the Respiratory Syncytial Virus (RSV) G Protein.

The respiratory syncytial virus (RSV) causes significant respiratory disease in young infants and the elderly. Immune prophylaxis in infants is currently limited to palivizumab, an anti-RSV fusion (F) protein monoclonal antibody (mAb). While anti-F protein mAbs neutralize RSV, they are unable to prevent aberrant pathogenic responses provoked by the RSV attachment (G) protein. Recently, the co-crystal structures of two high-affinity anti-G protein mAbs that bind the central conserved domain (CCD) at distinct non-overlapping epitopes were solved. mAbs 3D3 and 2D10 are broadly neutralizing and block G protein CX3C-mediated chemotaxis by binding antigenic sites γ1 and γ2, respectively, which is known to reduce RSV disease. Previous studies have established 3D3 as a potential immunoprophylactic and therapeutic; however, there has been no similar evaluation of 2D10 available. Here, we sought to determine the differences in neutralization and immunity to RSV Line19F infection which recapitulates human RSV infection in mouse models making it useful for therapeutic antibody studies. Prophylactic (24 h prior to infection) or therapeutic (72 h post-infection) treatment of mice with 3D3, 2D10, or palivizumab were compared to isotype control antibody treatment. The results show that 2D10 can neutralize RSV Line19F both prophylactically and therapeutically, and can reduce disease-causing immune responses in a prophylactic but not therapeutic context. In contrast, 3D3 was able to significantly (p < 0.05) reduce lung virus titers and IL-13 in a prophylactic and therapeutic regimen suggesting subtle but important differences in immune responses to RSV infection with mAbs that bind distinct epitopes.

An RSV Live-Attenuated Vaccine Candidate Lacking G Protein Mucin Domains Is Attenuated, Immunogenic, and Effective in Preventing RSV in BALB/c Mice.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading viral respiratory pathogen in infants. The objective of this study was to generate RSV live-attenuated vaccine (LAV) candidates by removing the G-protein mucin domains to attenuate viral replication while retaining immunogenicity through deshielding of surface epitopes. METHODS: Two LAV candidates were generated from recombinant RSV A2-line19F by deletion of the G-protein mucin domains (A2-line19F-G155) or deletion of the G-protein mucin and transmembrane domains (A2-line19F-G155S). Vaccine attenuation was measured in BALB/c mouse lungs by fluorescent focus unit (FFU) assays and real-time polymerase chain reaction (RT-PCR). Immunogenicity was determined by measuring serum binding and neutralizing antibodies in mice following prime/boost on days 28 and 59. Efficacy was determined by measuring RSV lung viral loads on day 4 postchallenge. RESULTS: Both LAVs were undetectable in mouse lungs by FFU assay and elicited similar neutralizing antibody titers compared to A2-line19F on days 28 and 59. Following RSV challenge, vaccinated mice showed no detectable RSV in the lungs by FFU assay and a significant reduction in RSV RNA in the lungs by RT-PCR of 560-fold for A2-line19F-G155 and 604-fold for A2-line19F-G155S compared to RSV-challenged, unvaccinated mice. CONCLUSIONS: Removal of the G-protein mucin domains produced RSV LAV candidates that were highly attenuated with retained immunogenicity.

Metabolic Reprogramming of Nasal Airway Epithelial Cells Following Infant Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a seasonal mucosal pathogen that infects the ciliated respiratory epithelium and results in the most severe morbidity in the first six months of life. RSV is a common cause of acute respiratory infection during infancy and is an important early-life risk factor strongly associated with asthma development. While this association has been repeatedly demonstrated, limited progress has been made on the mechanistic understanding in humans of the contribution of infant RSV infection to airway epithelial dysfunction. An active infection of epithelial cells with RSV in vitro results in heightened central metabolism and overall hypermetabolic state; however, little is known about whether natural infection with RSV in vivo results in lasting metabolic reprogramming of the airway epithelium in infancy. To address this gap, we performed functional metabolomics, 13C glucose metabolic flux analysis, and RNA-seq gene expression analysis of nasal airway epithelial cells (NAECs) sampled from infants between 2-3 years of age, with RSV infection or not during the first year of life. We found that RSV infection in infancy was associated with lasting epithelial metabolic reprogramming, which was characterized by (1) significant increase in glucose uptake and differential utilization of glucose by epithelium; (2) altered preferences for metabolism of several carbon and energy sources; and (3) significant sexual dimorphism in metabolic parameters, with RSV-induced metabolic changes most pronounced in male epithelium. In summary, our study supports the proposed phenomenon of metabolic reprogramming of epithelial cells associated with RSV infection in infancy and opens exciting new venues for pursuing mechanisms of RSV-induced epithelial barrier dysfunction in early life.

Functional Features of the Respiratory Syncytial Virus G Protein.

Respiratory syncytial virus (RSV) is a major cause of serious lower respiratory tract infections in children <5 years of age worldwide and repeated infections throughout life leading to serious disease in the elderly and persons with compromised immune, cardiac, and pulmonary systems. The disease burden has made it a high priority for vaccine and antiviral drug development but without success except for immune prophylaxis for certain young infants. Two RSV proteins are associated with protection, F and G, and F is most often pursued for vaccine and antiviral drug development. Several features of the G protein suggest it could also be an important to vaccine or antiviral drug target design. We review features of G that effect biology of infection, the host immune response, and disease associated with infection. Though it is not clear how to fit these together into an integrated picture, it is clear that G mediates cell surface binding and facilitates cellular infection, modulates host responses that affect both immunity and disease, and its CX3C aa motif contributes to many of these effects. These features of G and the ability to block the effects with antibody, suggest G has substantial potential in vaccine and antiviral drug design.

Performance evaluation of antibody tests for detecting infant respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is a major cause of respiratory tract disease in young children and throughout life. Infant infection is also associated with later respiratory morbidity including asthma. With a prospective birth cohort study of RSV and asthma, we evaluated the performance of an RSV antibody enzyme-linked immunoassay (EIA) for detecting prior infant RSV infection. Infant RSV infection was determined by biweekly respiratory illness surveillance plus RSV polymerase chain reaction (PCR) testing in their first RSV season and serum RSV antibodies after the season at approximately 1 year of age. RSV antibodies were detected by RSV A and B lysate EIA. Antibody and PCR results on 1707 children included 327 RSV PCR positive (PCR+) and 1380 not RSV+. Of 327 PCR+ children, 314 (96%) were lysate EIA positive and 583 out of 1380 (42%) children not PCR+ were positive. We compared the lysate EIA to RSV F, group A G (Ga), and group B G (Gb) protein antibody EIAs in a subset of 226 sera, 118 PCR+ children (97 group A and 21 group B) and 108 not PCR+. In this subset, 117 out of 118 (99%) RSV PCR+ children were positive by both the F and lysate EIAs and 103 out of 118 (87%) were positive by the Ga and/or Gb EIAs. Comparison of the two G EIAs indicated the infecting group correctly in 100 out of 118 (86%) and incorrectly in 1 out of 118 (1%). The lysate and F EIAs are sensitive for detecting infant infection and the two G EIAs can indicate the group of an earlier primary infection.

Development and optimization of a Zika virus antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Zika virus (ZIKV) has become a global public health issue due to its teratogenicity and ability to cause Guillain-Barré syndrome in adults. Although anti-ZIKV envelope protein neutralizing antibodies correlate with protection, the non-neutralizing function of ZIKV antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) is incompletely understood. To study the role of ADCC antibodies during ZIKV infections, we generated a stably transfected, dual-reporter target cell line with inducible expression of a chimeric ZIKV prM-E protein on the cell surface as the target cell for the assay. By using this assay, nine of ten serum samples from ZIKV-infected patients had >20% ADCC killing of target cells, whereas none of the 12 healthy control sera had >10% ADCC killing. We also observed a time-dependent ADCC response in 2 patients with Zika. This demonstrates that this assay can detect ZIKV ADCC with high sensitivity and specificity, which could be useful for measurement of ADCC responses to ZIKV infection or vaccination.

A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion.

This study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

Type I and Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

Sex-specific association between prenatal life stress exposure and infant pro-inflammatory cytokine levels during acute respiratory infection.

BACKGROUND: Prenatal life stress exposure is linked to dysregulated immune function and chronic inflammatory disease in offspring, but we know little about its effects on infant immune response during viral infection. METHOD: To address this issue, we examined associations between prenatal life stress exposure and infant upper-airway inflammatory markers during acute respiratory infection (ARI) using data from a prospective, population-based birth-cohort study (N = 180). Infant inflammation was measured as a continuous latent factor within a structural equation modeling framework using nasal wash concentrations of interleukin-1ß, interleukin-6, and tumor necrosis factor-α. We hypothesized that infants exposed to prenatal life stress would have greater levels of nasal inflammation during ARI and increased risk for ARI-related morbidity in early childhood. RESULTS: Our findings contradicted these hypotheses and provided evidence of sexually dimorphic effects of prenatal stress exposure on infant immune functioning during ARI. Among boys, but not girls, prenatal stress was negatively associated with nasal inflammation and indirectly associated with both lower ARI severity and reduced likelihood of subsequent ARI-related hospitalization in the 2nd and 3rd years of life. CONCLUSION: These data suggest that prenatal stress exposure may be beneficial for infant boys in the context of respiratory viral infections; however, it will be critical to determine if these benefits are offset by increased risk for chronic inflammatory diseases in later childhood. As the participants in this cohort are being followed longitudinally through age 8, we will be able to evaluate long-term health outcomes in future studies.

The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography.

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

Progress in Vaccine-Preventable and Respiratory Infectious Diseases-First 10 Years of the CDC National Center for Immunization and Respiratory Diseases, 2006-2015.

The need for closer linkages between scientific and programmatic areas focused on addressing vaccine-preventable and acute respiratory infections led to establishment of the National Center for Immunization and Respiratory Diseases (NCIRD) at the Centers for Disease Control and Prevention. During its first 10 years (2006-2015), NCIRD worked with partners to improve preparedness and response to pandemic influenza and other emergent respiratory infections, provide an evidence base for addition of 7 newly recommended vaccines, and modernize vaccine distribution. Clinical tools were developed for improved conversations with parents, which helped sustain childhood immunization as a social norm. Coverage increased for vaccines to protect adolescents against pertussis, meningococcal meningitis, and human papillomavirus-associated cancers. NCIRD programs supported outbreak response for new respiratory pathogens and oversaw response of the Centers for Disease Control and Prevention to the 2009 influenza A(H1N1) pandemic. Other national public health institutes might also find closer linkages between epidemiology, laboratory, and immunization programs useful.

Potency Analysis of Mesenchymal Stromal Cells Using a Combinatorial Assay Matrix Approach.

Assays that can characterize MSC immune potency need to be identified for use in advanced clinical trials. MSCs possess a number of putative regenerative and immunomodulatory properties, and an assay matrix approach may best capture involved effector pathways. We have tested two assay systems to measure the potency of MSCs derived from human subjects: MSC secretome analysis and a quantitative RNA-based array for genes specific to immunomodulatory and homing properties of MSCs. Secretome analysis identified a unique cytokine signature that is upregulated by MSCs or downregulated in responder PBMCs and correlated with T cell suppression. Use of interferon-γ as a surrogate for the action of activated PBMCs on MSCs served as an alternative for the use of human PBMCs as responder cells in a potency assay. Our approach and results define and simplify the multifunctional or matrix responses of MSCs and may serve as a platform for robust potency analysis.

A Built-In CpG Adjuvant in RSV F Protein DNA Vaccine Drives a Th1 Polarized and Enhanced Protective Immune Response.

Human respiratory syncytial virus (RSV) is the most significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection.

The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection.

Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182-186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.

Immune dysfunctionality of replicative senescent mesenchymal stromal cells is corrected by IFNγ priming.

Industrial-scale expansion of mesenchymal stromal cells (MSCs) is often used in clinical trials, and the effect of replicative senescence on MSC functionality is of mechanistic interest. Senescent MSCs exhibit cell-cycle arrest, cellular hypertrophy, and express the senescent marker ß-galactosidase. Although both fit and senescent MSCs display intact lung-homing properties in vivo, senescent MSCs acquire a significant defect in inhibiting T-cell proliferation and cytokine secretion in vitro. IFNγ does not upregulate HLA-DR on senescent MSCs, whereas its silencing did not reverse fit MSCs' immunosuppressive properties. Secretome analysis of MSC and activated peripheral blood mononuclear cell coculture demonstrate that senescent MSCs are significantly defective in up (vascular endothelial growth factor [VEGF], granulocyte colony-stimulating factor [GCSF], CXCL10, CCL2) or down (IL-1ra, IFNγ, IL-2r, CCL4, tumor necrosis factor-α, IL-5) regulating cytokines/chemokines. Unlike indoleamine 2,3 dioxygenase (IDO), silencing of CXCL9, CXCL10, CXCL11, GCSF, CCL2, and exogenous addition of VEGF, fibroblast growth factor-basic do not modulate MSCs' immunosuppressive properties. Kynurenine levels were downregulated in senescent MSC cocultures compared with fit MSC counterparts, and exogenous addition of kynurenine inhibits T-cell proliferation in the presence of senescent MSCs. IFNγ prelicensing activated several immunomodulatory genes including IDO in fit and senescent MSCs at comparable levels and significantly enhanced senescent MSCs' immunosuppressive effect on T-cell proliferation. Our results define immune functional defects acquired by senescent MSCs, which are reversible by IFNγ prelicensing.

Ability of device to collect bacteria from cough aerosols generated by adults with cystic fibrosis.

Background: Identifying lung pathogens and acute spikes in lung counts remain a challenge in the treatment of patients with cystic fibrosis (CF). Bacteria from the deep lung may be sampled from aerosols produced during coughing. Methods: A new device was used to collect and measure bacteria levels from cough aerosols of patients with CF. Sputum and oral specimens were also collected and measured for comparison. Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Streptococcus mitis were detected in specimens using Real-Time Polymerase Chain Reaction (RT-PCR) molecular assays. Results: Twenty adult patients with CF and 10 healthy controls participated. CF related bacteria (CFRB) were detected in 13/20 (65%) cough specimens versus 15/15 (100%) sputum specimens. Commensal S. mitis was present in 0/17 (0%, p=0.0002) cough specimens and 13/14 (93%) sputum samples. In normal controls, no bacteria were collected in cough specimens but 4/10 (40%) oral specimens were positive for CFRB. Conclusions: Non-invasive cough aerosol collection may detect lower respiratory pathogens in CF patients, with similar specificity and sensitivity to rates detected by BAL, without contamination by oral CFRB or commensal bacteria.

Seasonal Timing of Infant Bronchiolitis, Apnea and Sudden Unexplained Infant Death.

Rates of Sudden Unexplained Infant Death (SUID), bronchiolitis, and central apnea increase in winter in temperate climates. Though associations between these three conditions are suggested, more work is required to establish if there is a causal pathway linking bronchiolitis to SUID through inducing central apnea. Utilizing a large population-based cohort of infants studied over a 20-year period (n = 834,595, from birth years 1989-2009)), we analyzed ecological associations between timing of SUID cases, bronchiolitis, and apnea healthcare visits. Data were analyzed between 2013 and 2015. We used a Cox Proportional Hazards model to analyze possible interactions between maternal smoking and maternal asthma with infant bronchiolitis on time to SUID. SUID and bronchiolitis both occurred more frequently in winter. An increase in bronchiolitis clinical visits occurred within a few days prior to apnea visits. We found a temporal relationship between infant bronchiolitis and apnea. In contrast, no peak in SUID cases was seen during peaks of bronchiolitis. Among those without any bronchiolitis visits, maternal smoking was associated with an increased risk of SUID: Hazard Ratio (HR) of 2.38 (95% CI: 2.11, 2.67, p-value <0.001). Maternal asthma was associated with an increased risk of SUID among infants with at least one bronchiolitis visit: HR of 2.40 (95% CI: 1.04, 5.54, p-value = 0.04). Consistent trends between bronchiolitis, apnea, and SUID were not established due to small numbers of SUID cases. However, interaction analysis revealed potential differential associations of bronchiolitis and SUID by maternal smoking, maternal asthma status.

Secretory Expression and Purification of Respiratory Syncytial Virus G and F Proteins in Human Cells.

Respiratory syncytial virus (RSV) is one of the leading causes of range of symptoms from mild upper to serious lower respiratory virus infections in infants, immunocompromised individuals, and the elderly. Despite many decades of research and development, a licensed RSV vaccine is not available for use in human. Since the RSV F and G proteins induce neutralizing antibodies and confer protection from infection, they are important for understanding disease and for developing vaccines and access to purified, expressed proteins is important to RSV research and diagnostics. We describe methods to produce recombinant RSV F and G proteins in human cells and purify these proteins using Ni Sepharose affinity chromatography.