Viral load drives disease in humans experimentally infected with respiratory syncytial virus

Abstract

Rationale: Respiratory syncytial virus (RSV) is the leading cause of childhood lower respiratory infection, yet viable therapies are lacking. Two major challenges have stalled antiviral development: ethical difficulties in performing pediatric proof-of-concept studies and the prevailing concept that the disease is immune-mediated rather than being driven by viral load.

Objectives: The development of a human experimental wild-type RSV infection model to address these challenges.

Methods: Healthy volunteers (n = 35), in five cohorts, received increasing quantities (3.0-5.4 log plaque-forming units/person) of wild-type RSV-A intranasally.

Measurements and main results: Overall, 77% of volunteers consistently shed virus. Infection rate, viral loads, disease severity, and safety were similar between cohorts and were unrelated to quantity of RSV received. Symptoms began near the time of initial viral detection, peaked in severity near when viral load peaked, and subsided as viral loads (measured by real-time polymerase chain reaction) slowly declined. Viral loads correlated significantly with intranasal proinflammatory cytokine concentrations (IL-6 and IL-8). Increased viral load correlated consistently with increases in multiple different disease measurements (symptoms, physical examination, and amount of nasal mucus).

Conclusions: Viral load appears to drive disease manifestations in humans with RSV infection. The observed parallel viral and disease kinetics support a potential clinical benefit of RSV antivirals. This reproducible model facilitates the development of future RSV therapeutics.

Figure 1.

Screening immunology and viral outcomes of cohorts. (A) Histogram of serum respiratory syncytial virus (RSV)–neutralizing antibody titers to Memphis 37 strain in screened and entered volunteers. (B) Each successive cohort of volunteers was inoculated with increasing quantities of RSV. The mean quantity of RSV administered to volunteers within individual cohorts was measured as log plaque-forming units (PFU) in an HEp2 cell quantitative culture assay (plaque assay). A volunteer was defined as infected if two successive respiratory secretion collections contained detectable RSV by quantitative reverse transcriptase-polymerase chain reaction (qPCR). The mean amounts of RSV in each cohort are illustrated here in two ways. The top row of bar graphs plots the mean area under the curve (AUC) of the viral loads for each of the volunteers in each cohort. All volunteers were inoculated with RSV but only 71–86% in each cohort produced detectable virus, meeting the definition of infection. The viral AUC (calculated by including only volunteers who were defined as infected) are represented by solid columns and the viral AUC calculated from all volunteers in the cohort are represented by open columns. The bottom row of bar graphs plots the mean peak viral loads of the volunteers in each cohort. These peak viral loads were measured by quantitative culture (qCulture) (log PFU/ml; open columns) or by qPCR (log PFU equivalents/ml [log PFUe/ml]; solid columns). No significant differences in mean viral AUC or peak viral loads existed between cohorts. Error bars represent the SD.

Figure 2.

Viral load and disease over time in human volunteers. (a and b) Viral and disease measurements for selected individual volunteers over time. Volunteers were inoculated with respiratory syncytial virus (RSV) on Day 0 and evaluated in quarantine until Day 12. Twice-daily symptom scores (open columns), once-daily directed physical examination (DPE) scores (solid columns), and daily quantification of nasal mucus weight (striped columns) are measures of RSV disease severity. Viral testing was performed on nasal washes collected twice daily. The nonquantitative twice-daily spin-enhanced culture (SEC) and daily RSV antigen test results are indicated as either + or − in the top rows of graphs. Quantitative real-time reverse transcriptase PCR (qPCR, dashed line) and quantitative culture (qCulture; plaque assay on HEp-2 cells, solid line) were measured twice daily. (c and d) Mean daily viral loads (qPCR and quantitative culture) with normalized incubation periods. The timing of individual volunteer viral load curves was normalized by inserting a common incubation period so that their first positive viral measurement occurred arbitrarily on Infection Day 1. (c) Viral load measured by qPCR; (d) viral load measured by quantitative culture. Error bars represent the SD. (e) Timing of mean viral load and symptomatic disease. Mean data from all infected volunteers from each collection time point starting from Day 1 postinoculation are shown. No adjustment for the variable incubation periods is included in this panel. This makes the breadth of the curves wider and the magnitude of the curves lower than when incubation periods are normalized [as in (c) and (d)]. Initial rises in mean viral loads (qPCR and quantitative culture) correlate with the timing of onset of the rise in mean symptom scores. The timing of peak viral load correlates with the occurrence of peak symptom severity. log PFUe/ml = log plaque-forming unit equivalents per milliliter.

Figure 3.

Disease measures over time in infected and uninfected volunteers. Volunteers were defined as infected if two successive samples of nasal washes, collected between Study Days 2 and 8 inclusive, contained detectable respiratory syncytial virus (RSV). (a–c) Volunteers who became infected; (d–f) volunteers who failed to reach the definition of infected. Infected volunteers showed significantly higher measures of disease than did uninfected volunteers (all days combined). Mean total daily symptom scores, P = 0.021 (a vs. d), mean total daily directed physical examination (DPE) scores, P = 0.181 (b vs. e) and mean daily nasal mucus weights, P = 0.0056 (c vs. f). Error bars represent the SD.

Figure 4.

Relationships between quantitative viral and disease measures. All volunteers were inoculated with respiratory syncytial virus (RSV) and are represented in (a–c). (d–f) Analyses restricted to those volunteers who met the definition of infected. Disease measures (total symptom scores, total directed physical examination [DPE] scores, and mucus weights) for individual volunteers are plotted against their viral area under the curve (AUC). Viral AUC is the area under the viral load curves calculated for individual volunteers. P values represent the probability that the slopes of the regression lines do not include a slope of zero. The dashed curved lines indicate the 95% confidence interval of the slopes of the regression line (solid line). Similar statistically significant direct relationships were observed between viral and disease measures when viral AUC was measured by quantitative real-time reverse transcriptase-polymerase chain reaction. qCulture = quantitative culture.

Figure 5.

Relationships between IL-6 concentration, disease severity, and quantity of respiratory syncytial virus (RSV). Concentrations of IL-6 were measured in respiratory secretions of volunteers and were compared with disease measures and viral quantities. (a) The cumulative sum of the IL-6 concentrations versus disease severity as measured by individual volunteer cumulative symptom scores. (b) The cumulative sum of IL-6 concentrations versus disease severity as measured by individual volunteer cumulative nasal mucus weight. (c) Comparison of the cumulative sum of IL-6 concentrations versus area under the curve (AUC) viral load (quantitative real-time reverse transcriptase-polymerase chain reaction, qPCR). P values represent the probability that the slopes of the regression lines do not include a slope of zero. The dashed curved lines indicate the 95% confidence interval of the slopes of the regression line (solid line). Similar statistically significant direct relationships were observed when viral AUC was measured by quantitative culture.