Antibody and Local Cytokine Response to Respiratory Syncytial Virus Infection in Community-Dwelling Older Adults

Abstract

Respiratory syncytial virus (RSV) is increasingly recognized for causing severe morbidity and mortality in older adults, but there are few studies on the RSV-induced immune response in this population. Information on the immunological processes at play during RSV infection in specific risk groups is essential for the rational and targeted design of novel vaccines and therapeutics. Here, we assessed the antibody and local cytokine response to RSV infection in community-dwelling older adults (≥60 years of age). During three winters, serum and nasopharyngeal swab samples were collected from study participants during acute respiratory infection and recovery. RSV IgG enzyme-linked immunosorbent assays (ELISA) and virus neutralization assays were performed on serum samples from RSV-infected individuals (n = 41) and controls (n = 563 and n = 197, respectively). Nasal RSV IgA and cytokine concentrations were determined using multiplex immunoassays in a subset of participants. An in vitro model of differentiated primary bronchial epithelial cells was used to assess RSV-induced cytokine responses over time. A statistically significant increase in serum neutralization titers and IgG concentrations was observed in RSV-infected participants compared to controls. During acute RSV infection, a statistically significant local upregulation of beta interferon (IFN-β), IFN-λ1, IFN-γ, interleukin 1β (IL-1β), tumor necrosis factor alpha (TNF-α), IL-6, IL-10, CXCL8, and CXCL10 was found. IFN-β, IFN-λ1, CXCL8, and CXCL10 were also upregulated in the epithelial model upon RSV infection. In conclusion, this study provides novel insights into the basic immune response to RSV infection in an important and understudied risk population, providing leads for future studies that are essential for the prevention and treatment of severe RSV disease in older adults.IMPORTANCE Respiratory syncytial virus (RSV) can cause severe morbidity and mortality in certain risk groups, especially infants and older adults. Currently no (prophylactic) treatment is available, except for a partially effective yet highly expensive monoclonal antibody. RSV therefore remains a major public health concern. To allow targeted development of novel vaccines and therapeutics, it is of great importance to understand the immunological mechanisms that underlie (protection from) severe disease in specific risk populations. Since most RSV-related studies focus on infants, there are only very limited data available concerning the response to RSV in the elderly population. Therefore, in this study, RSV-induced antibody responses and local cytokine secretion were assessed in community-dwelling older adults. These data provide novel insights that will benefit ongoing efforts to design safe and effective prevention and treatment strategies for RSV in an understudied risk group.

Keywords: IgA; IgG; cytokine; elderly; interferon; mucosa.

FIG 1

Schematic overview of the experimental design of the study. (A) The complete data set includes RSV-infected individuals (n = 41), from whom samples were taken during acute infection (<72 h after onset of fever) and recovery (8 weeks later). In addition, controls without RSV infection (n = 563) were included, some of whom were noninfected and some of whom had respiratory infections other than RSV. Serum and nasopharyngeal swabs were collected and used for various assays. (B) During the 2014-2015 season, symptomatic participants underwent an additional early-recovery sampling at 2 weeks after the acute-phase sampling. This resulted in a subset of RSV-infected individuals (n = 16) from whom samples were taken at three time points: acute phase, early recovery (+2 weeks), and recovery (+8 weeks). (C) To analyze the local IgA and cytokine responses to RSV infection, we selected 10 symptomatic participants with MLPA-confirmed RSV infection and no other detectable viral infections during the acute phase. All of the selected participants were negative for any respiratory viral infection during the recovery phase. As healthy controls, we selected 10 age- and sex-matched participants without symptoms who were negative for any respiratory viral infection during sampling. ELISA, enzyme-linked immunosorbent assay; IgA/IgG, immunoglobulin A/G; M/F, male/female; MIA, multiplex immunoassay; MLPA, multiplex ligation-dependent probe amplification; PRNT, plaque reduction neutralization test; RSV, respiratory syncytial virus.

FIG 2

Serologic analyses of RSV-infected participants and controls. (A) Virus neutralization titers were determined by PRNT for controls without RSV (n = 197; white circles) and RSV-infected persons during acute infection and recovery (n = 40; black circles). (B) RSV-A-specific serum IgG concentrations were determined by ELISA for controls without RSV (n = 563; white circles) and RSV-infected persons during acute infection and recovery (n = 41; black circles). PRNT and log-transformed ELISA data were analyzed using an ordinary one-way ANOVA with Tukey’s multiple-comparison test. (C) Participant samples were taken <72 h after fever onset, ranging from 0 to 3 days. The plot shows acute-phase serum neutralization titers, according to the interval between fever onset and sampling. PRNT data were analyzed using an ordinary one-way ANOVA. (D) Correlation between PRNT and ELISA data for the controls without RSV (n = 196). Data were assessed by Pearson correlation. (E) Virus neutralization titers were determined by PRNT for RSV-infected persons (n = 15) during acute infection, early recovery (+2 weeks), and recovery (+8 weeks). (F) RSV-A-specific serum IgG concentrations were determined by ELISA for RSV-infected persons (n = 16) during acute infection, early recovery (+2 weeks), and recovery (+8 weeks). Data in panels E and F were analyzed using the Friedman test with Dunn’s multiple-comparison test. (G and H) RSV prefusion F-specific (G) and nucleoprotein (N)-specific (H) nasal IgA concentrations were determined by multiplex immunoassay in a subset of participants. All data points represent individual participants, and lines indicate geometric means and 95% confidence intervals. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. AU/mL, arbitrary units per milliliter; ELISA, enzyme-linked immune sorbent assay; IgA/IgG, immunoglobulin A/G; PRNT, plaque reduction neutralization test.

FIG 3

Analysis of RSV disease symptoms in relation to acute-phase serum neutralization titers. (A) Self-reported start and end dates of disease symptoms were recorded. All data points represent individual participants, and lines indicate means and 95% confidence intervals. (B and C) Plots showing the correlation between acute-phase serum neutralization titers (PRNT60) and duration of coughing (B; n = 37) and rhinitis (C; n = 36) for RSV-infected participants. For some participants, only the start date of coughing and rhinitis was recorded, and they were excluded from the analysis (n = 3 and n = 4, respectively). Data were assessed by Spearman correlation. PRNT, plaque reduction neutralization test.

FIG 4

Upregulation of nasal cytokines in RSV-infected older adults. A multiplex immunoassay was used to determine the nasal concentration of IFN-β (A), IFN-λ1 (B), IFN-λ2/3 (C), IFN-γ (D), IL-1β (E), TNF-α (F), IL-6 (G), IL-10 (H), CXCL8 (I), and CXCL10 (J). Nasopharyngeal swab samples were collected from study participants within 72 h of presenting with fever (acute RSV; n = 10) and controls without respiratory viral infection (healthy; n = 10). Samples were taken from RSV-infected individuals again during recovery, 8 weeks later (recovery; n = 10). Data points represent individual participants, and lines indicate geometric mean concentrations and 95% confidence intervals. Measurements below the detection limit were set to 0.5 times the lower limit of detection. Unpaired samples from healthy and acutely RSV-infected individuals were compared using a nonparametric Mann-Whitney test. Paired samples from individuals during acute RSV infection and recovery were compared using a nonparametric Wilcoxon matched-pairs signed-rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Dotted lines indicate the lower limit of detection. IFN, interferon; IL, interleukin; RSV, respiratory syncytial virus; TNF, tumor necrosis factor.

FIG 5

RSV infection of primary human airway epithelial (HAE) cultures at the air-liquid interface. (A) Schematic representation of the experimental setup. (B) Titration (TCID₅₀) of apical wash samples collected 12 to 336 h postinfection from RSV-infected HAE cultures derived from three individual donors during two independent experiments. Lines indicate geometric mean titers with standard deviations. (C and D) HAE cultures were infected with RSV-A2 or RSV-X or mock infected, as indicated, and incubated for 12, 24, and 48 h (C) and 336 h (D). Goblet cells, RSV-infected cells, and ciliated cells were subsequently visualized by staining for mucin 5B (MUC5B; purple), RSV G protein (green), and β-tubulin (β-TUB; red), respectively. Nuclei are stained with DAPI (blue). Images are from one representative donor. Bars, 20 μm.

FIG 6

Upregulation of cytokines in basolateral medium and apical wash samples of RSV-infected HAE cultures. A multiplex immunoassay was used to determine the concentration of IFN-β (A and B), IFN-λ1 (C and D), IFN-λ2/3 (E and F), CXCL8 (G and H), and CXCL10 (I and J). Basolateral medium and apical wash samples were collected from 12 to 336 h postinfection from mock- and RSV-infected HAE cultures derived from three individual donors in two independent experiments. Values are geometric mean concentrations and standard deviations. Measurements below the detection limit were set to 0.5 times the lower limit of detection. Dotted lines indicate the lower limit of detection. HAE, human airway epithelial; IFN, interferon; RSV, respiratory syncytial virus.