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.

Since the COVID-19 pandemic, functional non-neutralizing antibody responses to SARS-CoV-2, including antibody-dependent cell-mediated cytotoxicity (ADCC), are poorly understood. We developed an ADCC assay utilizing a stably transfected, dual-reporter target cell line with inducible expression of a SARS-CoV-2 spike protein on the cell surface. Using this assay, we analyzed 61 convalescent serum samples from adults with PCR-confirmed COVID-19 and 15 samples from healthy uninfected controls. We found that 56 of 61 convalescent serum samples induced ADCC killing of SARS-CoV-2 S target cells, whereas none of the 15 healthy controls had detectable ADCC. We then found a modest decline in ADCC titer over a median 3-month follow-up in 21 patients who had serial samples available for analysis. We confirmed that the antibody-dependent target cell lysis was mediated primarily via the NK FcγRIIIa receptor (CD16). This ADCC assay had high sensitivity and specificity for detecting serologic immune responses to SARS-CoV-2.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. In the present study, we investigated the effect of prophylactic treatment with the intact and F(ab')2 forms of an anti-G protein monoclonal antibody (MAb), 131-2G, on the humoral and cellular adaptive immune responses to RSV rA2-line19F (r19F) challenge in BALB/c mice. The F(ab')2 form of 131-2G does not decrease virus replication, but intact 131-2G does. The serum specimens for antibodies and spleen cells for memory T cell responses to RSV antigens were analyzed at 30, 45, 75, and 95 days postinfection (p.i.) with or without prior treatment with 131- 2G. The ratios of Th2 to Th1 antibody isotypes at each time p.i indicated that both forms of MAb 131-2G shifted the subclass response from a Th2 (IgG1 and IgG2b) to a Th1 (IgG2A) bias. The ratio of IgG1 to IgG2A antibody titer was 3-fold to 10-fold higher for untreated than MAb-treated mice. There was also some increase in IgG (22%±13% increase) and neutralization (32% increase) in antibodies with MAb 131-2G prophylaxis at 75 days p.i. Treatment with 131-2G significantly (P≤0.001) decreased the percentage of interleukin-4 (IL-4)-positive CD4 and CD8 cells in RSV-stimulated spleen cells at all times p.i., while the percentage of interferon gamma (IFN-γ) T cells significantly (P≤0.001) increased≥75 days p.i. The shift from a Th2- to a Th1-biased T cell response in treated compared to untreated mice likely was directed by the much higher levels of T-box transcription factor (T-bet) (≥45% versus<10%) in CD4 and CD8 T cells and lower levels of Gata-3 (≤2% versus≥6%) in CD4 T cells in peptide-stimulated, day 75 p.i. spleen cells. These data show that the RSV G protein affects both humoral and cellular adaptive immune responses, and induction of 131-2G-like antibodies might improve the safety and long-term efficacy of an RSV vaccine. © 2014, American Society for Microbiology.

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 β-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.

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. Assays that inform on mesenchymal stromal cell (MSC) immune potency need to be defined in advanced clinical trials. Chinnadurai et al. tested an in vitro assay matrix approach combining molecular genetic and secretome analysis, elements of which could be deployed to define MSC immune modulatory potency.

Respiratory syncytial virus (RSV) causes substantial lower respiratory tract disease in children and at-risk adults. Though there are no effective anti-viral drugs for acute disease or licensed vaccines for RSV, palivizumab prophylaxis is available for some high risk infants. To support anti-viral and vaccine development efforts, we developed an RSV virus-like particle (VLP) platform to explore the role RSV F and G protein interactions in disease pathogenesis. Since VLPs are immunogenic and a proven platform for licensed human vaccines, we also considered these VLPs as potential vaccine candidates. We developed two RSV VLP platforms, M+P and M+M2-1 that had F and G, F and a G peptide, or a truncated F and G on their surface. Immunoblots of sucrose gradient purified particles showed co-expression of M, G, and F with both VLP platforms. Electron microscopy imaging and immunogold labeling confirmed VLP-like structures with surface exposed projections consistent with F and G proteins. In mice, the VLPs induced both anti-F and -G protein antibodies and, on challenge, reduced lung viral titer and inflammation. These data show that these RSV VLP platforms provide a tool to study the structure of F and G and their interactions and flexible platforms to develop VLP vaccines in which all components contribute to RSV-specific immune responses.

To date, there is a limited understanding of the role of the airway microbiome in the early life development of respiratory diseases such as asthma, partly due to a lack of simple and minimally invasive sample collection methods. In order to characterize the baseline microbiome of the upper respiratory tract (URT) in infants, a comparatively non-invasive method for sampling the URT microbiome suitable for use in infants was developed. Microbiome samples were collected by placing filter paper in the nostrils of 33 healthy, term infants enrolled as part of the Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure (INSPIRE) study. After bacterial genomic DNA was extracted from the filters, amplicons were generated with universal primers targeting the V1–V3 region of the 16S rRNA gene. This method was capable of capturing a wide variety of taxa expected to inhabit the nasal cavity. Analyses stratifying subjects by demographic and environmental factors previously observed or predicted to influence microbial communities were performed. Microbial community richness was found to be higher in infants who had been delivered via Cesarean section and in those who had been formula-fed; an association was observed between diet and delivery, which confounds this analysis. We have established a baseline URT microbiome using a non-invasive filter paper nasal sampling for this population, and future studies will be performed in this large observational cohort of infants to investigate the relationship between viral infections, the URT microbiota, and the development of childhood wheezing illnesses.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another preclinical tool to assess the safety of RSV vaccines.

Background: Respiratory syncytial virus (RSV) infection in infants causes significant morbidity and is the strongest risk factor associated with asthma. Metabolites, which reflect the interactions between host cell and virus, provide an opportunity to identify the pathways that underlie severe infections and asthma development. Objective: To study metabolic profile differences between infants with RSV infection, and human rhinovirus (HRV) infection, and healthy infants. To compare infant metabolic differences between children who do and do not wheeze. Methods: In a term birth cohort, urine was collected while healthy and during acute viral respiratory infection with RSV and HRV. We used 1 H-NMR to identify urinary metabolites. Multivariate and univariate statistics were used to discriminate metabolic profiles of infants with either RSV ARI, or HRV ARI, and healthy infants. Multivariable logistic regression was used to assess the association of urine metabolites with 1st-, 2nd-, and 3rd-year recurrent wheezing. Results: Several metabolites in nicotinate and nicotinamide metabolism pathways were down-regulated in infants with RSV infection compared to healthy controls. There were no significant differences in metabolite profiles between infants with RSV infection and infants with HRV Infection. Alanine was strongly associated with reduced risk of 1st-year wheezing (OR 0.18[0.0, 0.46]) and 2nd-year wheezing (OR 0.31[0.13, 0.73]), while 2-hydroxyisobutyric acid was associated with increased 3rd-year wheezing (OR 5.02[1.49, 16.93]) only among the RSV infected subset. Conclusion: The metabolites associated with infant RSV infection and recurrent-wheezing are indicative of viral takeover of the cellular machinery and resources to enhance virulence, replication, and subversion of the host immune-response, highlighting metabolic pathways important in the pathogenesis of RSV infection and wheeze development.

The accumulating evidence suggests that viral-bacterial interactions can affect short- and long-term outcomes of acute respiratory infections (ARIs) due to respiratory syncytial virus (RSV) in infancy. In particular, prior studies have found that in young children with RSV ARIs, a higher relative abundance of Haemophilus in the nasopharynx is associated with an increased viral load,1 delayed viral clearance,2 a different gene expression profile,3 , 4 and more severe disease.3 , 4 However, the mechanisms underlying these associations are largely unknown. To address this gap in knowledge, we examined the association of the nasopharyngeal relative abundance of Haemophilus with viral load and 52 local immune mediators in 105 infants with an RSV-only ARI (ie, no coinfections) who were enrolled in the Infant Susceptibility to Pulmonary Infections and Asthma following RSV Infection in Infancy (INSPIRE) study.