ABSTRACT
Objective: To determine the potential longer-term effects of maternal antenatal respiratory syncytial virus (RSV) vaccination, we examined the association between cord-blood RSV-neutralizing antibodies (RSV-NA) and RSV infections in the first 2-years of life, RSV-NA at 3-years, and respiratory health to age 5-years.
Methods: Two community-based Australian birth cohorts were combined. For children with at least one atopic parent, paired serum RSV-NA levels were compared in cord-blood and at age 3-years. Weekly nasal swabs were collected in one cohort and during acute respiratory infections (ARI) in the other. Wheeze history up to age 5-years and physician-diagnosed asthma at 5-years was collected by parent report.
Results: In 264 children, each log10increase of cord-blood RSV-NA level was associated with 37% decreased risk (adjusted incidence-rate-ratio (aIRR) 0.63; 95% confidence interval (CI): 0.40–1.01) of RSV-ARI and 49% decreased risk (aIRR 0.51; 95%CI: 0.25–1.02) of RSV acute lower respiratory infections (ALRI) at 12–24 months of age. However, higher cord-blood RSV-NA was associated with increased risk of all-cause ALRI (aIRR 1.29; 95%CI: 0.99–1.69), wheeze-associated ALRI (aIRR 1.75; 95%CI: 1.08–2.82) and severe ALRI (aIRR 2.76; 95%CI: 1.63–4.70) at age 6–<12 months. Cord-blood RSV-NA was not associated with RSV-ARI in the first 6-months, RSV-NA levels at 3-years, or wheeze or asthma at 5-years.
Conclusions: Higher levels of cord-blood RSV-NA did not protect against RSV infections during the first 6-months-of-life, time-to-first RSV-ARI, or wheeze or asthma in the first 5-years of life. Additional strategies to control RSV-related illness in childhood are needed.
INTRODUCTION Respiratory syncytial virus (RSV) is the most common respiratory virus identified in young children with acute lower respiratory infections (ALRI)1. It imposes a substantial global health burden in children aged <5-years with an estimated 33.1 million episodes of RSV-ALRI, 3.2 million hospital admissions, and almost 60,000 inpatient deaths globally in 20151. In Australia, most RSV-associated hospitalizations are in children aged <6-months, with an estimated hospital admission rate for this age group of 2,224 per 100,000 child years2. Severe RSV infections in early life are associated with recurrent wheezing episodes3 and asthma in later childhood4. However, debate exists over whether this relationship is causal5,6. Since the burden of severe RSV-ALRI requiring hospitalization is greatest during the first few months of life, before sufficient time has passed to allow active immunization to induce a protective response, any protection of newborns during this vulnerable period must come from another source.
A potential prevention strategy is maternal antenatal vaccination, which seeks to induce high-levels of RSV-neutralizing antibodies (NA) that cross the placenta and protect young infants7. Maternal vaccination is widely recommended for preventing other infectious diseases in infants, including tetanus, influenza, and pertussis8. In a recent phase III placebo-controlled randomized controlled trial (RCT) of 4,500 pregnant women in 11 countries, there was a 44.4% (95% confidence interval (CI): 19.6%–61.5%) reduction in RSV-related hospitalization due to ALRI in babies of vaccinated mothers9. However, the study did not meet the prespecified primary endpoint of decreasing RSV-associated, medically significant ALRI in the first 90-days of life (vaccine efficacy 39.4%; 97.5%CI: -1.0–63.7). Nevertheless, the concept of maternal vaccination is supported by RSV monoclonal antibody administration to preterm infants reducing severe RSV-ALRI10,11 and observational studies linking high cord-blood NA to a lower risk of severe RSV infections resulting in hospitalization12,13.
One concern with maternal vaccination is potential interference with developing B-cell mediated protective immunity against RSV infection14. Maternal vaccination may suppress the infant’s immune response, with even low maternal antibody transference inhibiting infant RSV-specific B-cell and antibody responses15,16. However, unlike with monoclonal antibodies17,18, few studies have evaluated the impact of maternal RSV antibodies upon respiratory infection or lung health beyond early infancy13,19,20. The aim of this study was to use data from two prospective community-based Australian birth cohorts to examine the association between maternally-derived RSV-NA levels in cord-blood and (i) RSV infections and ALRI in the first 2-years of life, (ii) RSV-NA levels at age 3-years, and (iii) respiratory health to 5-years of age.METHODS Study populations Data from two prospective community-based Australian birth cohorts, the Childhood Asthma Study (CAS)21 and Observational Research in Childhood Infectious Diseases (ORChID) project22, were combined. Both studies collected cord-blood from participants and are described in detail elsewhere3,21-24. In brief, based in Perth, Western Australia, the CAS was a prospective birth cohort study of 263 infants at high-risk of atopy (at least one parent with a history of physician-diagnosed eczema, hayfever, or asthma)3,21. Participants were born between July 1996 and July 1999. Parents completed daily symptom diaries for the first 5-years of life. Whenever any respiratory illness occurred, the research team was contacted, and nasopharyngeal aspirates were collected in the child’s home within 48-hours of symptom onset and returned immediately to the laboratory for processing and storage at -80°C. A history of wheeze and asthma was collected annually. Blood was collected at 3-years of age.
The ORChID study progressively enrolled 158 unselected healthy, term newborn infants in Brisbane, Queensland, between September 2010 and October 201222. Participants were followed from birth until their second birthday. Parents completed a daily respiratory symptom diary and collected anterior nasal swabs weekly using a single swab for both nostrils. Specimens were mailed to the laboratory, taking a median 3-days (interquartile range 2–4 days) to reach the laboratory where they were stored at -80°C. ORChID participants were subsequently invited to have an annual clinical and respiratory assessment review until 7-years of age as part of the Early Life Lung Function (ELLF) study25. Blood was collected as part of the 3-year assessment. To more closely align with the CAS cohort, a high-risk sub-group of the ORChID cohort was identified. Children were included if there was a history of parental asthma or eczema. At enrollment, participants from both the ORChID and CAS cohorts had socio-demographic and clinical information recorded, while breastfeeding and childcare attendance were collected progressively.
The King Edward Memorial and Princess Margaret Hospital Ethics Committees approved the CAS study (RE95-17.9 and ECO2-53.9). The Royal Brisbane and Women’s Hospital (HREC/10/QRBW125) Human Research Ethics Committee (HREC) approved the ORChID study. The Children’s Health Queensland (HREC/10/QRCH/16 and HREC/12/QRCH/23) and The University of Queensland (2010000820 and 2014000212) HRECs approved both the ORChID and ELLF studies. Informed parental consent was obtained for all participants in each study.
Acute respiratory infections and asthma categorizationAcute respiratory infections (ARI) were categorized hierarchically as either ALRI or upper respiratory infections (URI)26. Similar clinical definitions were used in both cohorts (Supplementary Table 1). An ALRI was any combination of rattly breathing, moist cough, shortness of breath, wheeze, or physician-diagnosed pneumonia. If wheeze was present, an ALRI was sub-categorized as a wheeze-associated ALRI (wARLI), while an ALRI with temperature > 38°C was classified as a severe ALRI (sALRI). A URI included at least one of the following: nasal discharge/congestion, dry cough, or physician-diagnosed acute otitis media. Symptomatic RSV episodes in the ORChID study were identified if symptoms were first detected in the week prior to, or were present in the week after, the first virus detection27. Only symptomatic episodes were identified in the CAS study. Asthma was defined by physician diagnosis of asthma and wheeze present in the previous year.
Laboratory tests (i) Analysis of nasal swabs and nasopharyngeal aspiratesValidated real-time polymerase chain reaction (PCR) assays were used to batch-test stored respiratory specimens for RSV21,22.
(ii) RSV NA assays Virus neutralization in serum from cord-blood and blood collected from 3-year-old participants was assessed against recombinant green fluorescence protein (GFP)-expressing RSV (rgRSV) derived from D53, strain A2 that was provided by Prof Mark Peeples28. Vero cells were seeded into 96 well, optical bottom/black wall cell culture plates (Thermo Fisher Scientific, Swedesboro, NJ, USA) at a density of 4x104 cells per well in 100µl of Opti-MEM (Gibco, Thermo Fisher Scientific, Swedesboro, NJ, USA) with 3% fetal calf serum and incubated overnight at 37°C in 5% CO2. The following day, human plasma samples were titrated by 4-fold dilutions in Opti-MEM within a round bottom 96 well plate. Plasma dilutions were then mixed with 2 x 103 plaque-forming units/mL of rgRSV and incubated for 1-hour at room temperature before addition to Vero cells. Following 6-days incubation at 37°C in 5% CO2, GFP fluorescence intensity was measured on a CLARIOstar Microplate Reader (BMG LABTECH, Melbourne, VIC, Australia). Fluorescence was graphed against plasma dilution and a three-parameter dose-response curve was fitted by nonlinear regression using GraphPad Prism v7.0.0 (GraphPad Software, San Diego, CA, USA).
OutcomesThe childhood respiratory outcomes available for assessment were RSV-related ARI and ALRI, and all-cause ALRI, wALRI and sALRI in the first 2-years of life; serum RSV-NA levels at age 3-years; wheeze history up to 5-years of age and physician-diagnosed asthma by age 5-years.
AnalysisSummary statistics are presented as frequency (percentage) for categorical variables. Serum RSV-NA half-maximal inhibitory concentration (IC50) values were normalized by log10 transformation. Associations between cord-blood RSV-NA level and rate of RSV infections in the first 2-years of life were presented as incidence rate ratios (IRR) using mixed-effects Poisson regression with study (CAS/ORChID) included as a random-effect. Models were offset by the natural logrithim of each child’s time-at-risk of infection. First, univariable models were constructed, then models were adjusted for the potentially confounding variables season of birth and presence of an older child in the household at birth. Secondly, time-to-event analyses were conducted using Cox proportional hazards regression models. Sensitivity analysis was conducted using all ORChID children who provided cord-blood to evaluate whether there were differences between symptomatic and asymptomatic RSV infections. Thirdly, the association between cord-blood RSV-NA level and serum RSV-NA levels at age 3-years was analyzed using linear regression. Finally, the association between cord-blood RSV-NA level and risk of wheeze at ages 1 to 5-years and asthma at 5-years of age was analyzed using both univariable and multivariable mixed-effects logistic regression models. Study was included as a random effect. Multivariable models were adjusted for season of birth and presence of older child in the household at birth. Data were analyzed using Stata v13 (StataCorp, College Station, TX, USA).
RESULTS Participants Cord-blood RSV-NA data were available for 109/158 (69%) ORChID and 214/236 (91%) CAS children. Fifty children from the ORChID cohort had at least one atopic parent and were combined with the 214 children from the CAS cohort to form the high-risk group (Figure). Characteristics of high-risk children by each study cohort are described in Table 1. All had RSV-NA detected in cord-blood (log10IC50 values ranged from 1.57 to 4.19) and except for paternal atopy, their characteristics were similar across the study cohorts.
Association between cord-blood RSV-NA level and risk of RSV-ARIs, including RSV-ALRIs, and all-cause ALRIs in the first 2-years of life The association between cord-blood RSV-NA levels and ARI episodes are displayed in Table 2. Each log10 IC50 increase in cord-blood RSV-NA level was associated with a 37% decreased risk of RSV-ARI (adjusted IRR 0.63; 95%CI: 0.40–1.01) and a 49% decreased risk of RSV-ALRI (aIRR 0.51; 95%CI: 0.25–1.02) in the second-year of life. In contrast, in the first-year of life higher RSV-NA was associated with an increased risk of all-cause ALRI (age 0–<6 months; aIRR 1.41; 95%CI: 0.98–2.04; age 6–<12 months; aIRR 1.29; 95%CI 0.99–1.69), which at age 6–<12 months was significant for wALRI (aIRR 1.75; 95%CI: 1.08–2.82) and sALRI (aIRR 2.76; 95%CI: 1.63–4.70).Time-to-first RSV detectionRSV-NA level was not associated with time-to-first symptomatic RSV detections (Supplmentary Table 2 and Supplementary Figure 1). With each log10 IC50 increase in RSV-NA levels, the hazard ratio (HR) decreased slightly by 11% (adjusted HR (aHR) 0.89; 95%CI: 0.62–1.29) for RSV-ARI, and by 10% (aHR 0.90; 95%CI: 0.54–1.51) for RSV-ALRI. To test the sensitivity of this result, we repeated the analysis using all 109 ORChID children who provided cord-blood (Supplementary Table 3), and RSV-NA level was not associated with time-to-first RSV detection (Supplementary Table 4).Association between serum RSV-NA levels in cord-blood and at age 3-years There was no significant association between paired serum RSV-NA levels in cord-blood and at 3-years of age in the 192 children for whom paired data were available (Supplementary Figure 2).
Cord-blood RSV-NA levels and wheezing episodes during the first 5-years of life and asthma at age 5-years No significant associations between cord-blood RSV-NA levels and risk of wheeze during the first 5-years of life and physician-diagnosed asthma at age 5-years were observed (Table 3).
DISCUSSION In this combined cohort of community-based children at increased risk of asthma, there was weak evidence that each log10increase in maternally-derived RSV-NA levels in cord-blood was associated with a lower risk of both RSV-ARI and RSV-ALRI in the second year of life. Cord-blood RSV-NA levels were not associated with the timing of symptomatic RSV infections in the first 2-years of life. In contrast, each log10 increase in cord-blood RSV-NA level may be associated with increased risk of all-cause ALRI in the first-year of life, with this association statistically significant for wALRI and sALRI episodes in children aged 6–<12 months. There was no association between RSV-NA cord-blood levels and wheezing in the first 5-years of life or an asthma diagnosis at age 5-years.
Both hospital and community-based studies that have shown higher cord-blood RSV antibody levels are associated with older age at infection, decreased hospitalization, and overall less severe disease13,29-31. However, this is not a universal finding and studies in higher-risk populations from Alaska, Kenya, and Nepal have not shown a protective effect for cord-blood antibodies on early RSV infection risk or severity32-34. Our study found that that higher cord-blood RSV-NA levels were associated with a decreased risk of RSV infections at 12–24 months, but not in the first 6-months when a substantial decline in maternally-derived antibody levels occurs. A possible explanation for our failure to identify an association between cord-blood RSV-NA and the timing of RSV infections during infancy is that our cohorts were community-based, and there were relatively few cases in the first 6-months of life. In contrast, hospital-based studies reporting high cord-blood RSV-NA levels protect against RSV disease during infancy are comprised predominantly of infants experiencing severe infections in the first-months of life12,30.
Our finding that children at high risk of atopy who had the highest cord-blood RSV-NA levels were also at increased risk of all-cause ALRI, and especially wALRI and sALRI, episodes in the first-year of life, but at lower risk of RSV-ARI and RSV-ALRI in their second-year was unexpected. It may be that high RSV-NA levels represent maternal exposure to multiple other viruses, rather than RSV alone, and the child develops immunity from prior RSV exposure in their second 6-months of life. While potential confounding factors such as season of birth and presence of children in the household at birth were included as covariables in the regression analyses, additional unmeasured environmental/lifestyle and genetic factors may have influenced the result. This finding was similar to a Danish birth cohort study, where they reported that although elevated cord-blood RSV-NA levels protected infants against hospitalization from RSV infection in the first 6-months of life (aIRR 0.74; 95%CI: 0.62–0.87), they were also associated with increased risk of recurrent wheeze (aIRR 1.28; 95%CI: 1.04–1.57) in the same age group35. While serum antibodies may protect against ALRI, there is emerging evidence that local mucosal immunity, especially for URI, may also be important. Adult human RSV challenge trials found that pre-existing nasal IgA levels correlated with protection, whereas no such relationship was observed for serum NA levels, despite these being moderately high in the healthy volunteers36. Furthermore, although the pivotal studies use of monoclonal RSV antibodies in young infants significantly reduced RSV-ALRI hospitalizations, they did not affect milder RSV infections, including URI episodes10, which comprise the greatest overall community burden of disease27.
An alternative prevention strategy to maternal vaccination is newborn monoclonal antibody administration. Palivizumab has been licensed since 1998. However, it has no effect upon RSV-URI and its impact upon RSV hospitalizations on a population basis is limited by its high cost. This has restricted its use to a small number of babies deemed to be at highest risk of severe RSV infections, such as those requiring home oxygen for chronic neonatal lung disease or with complex congenital cyanotic heart disease37. The new monoclonal anti-RSV antibody, Nirsevimab, has more than three-times the half-life of palivizumab11. Thus one dose can potentially provide protection for the typical RSV season of 5–6 months. In a phase III placebo-controlled, RCT involving 1,500 preterm infants from 23 countries, there was a 78.4% (95%CI: 51.9–90.3) reduction in RSV-associated hospitalizations in infants receiving Nirsevimab compared to placebo up to 150-days after the dose was administered11. This strategy is seen as providing protection during the vulnerable period in the first months of life when the lower airways are of small caliber, the immune system is immature, and maternal RSV NA levels are declining. While maternal antenatal RSV vaccines and long-acting monoclonal RSV antibodies may protect against severe ALRIs in young infants, which of itself is important, their overall impact on RSV disease will be limited in older infants and children and will require additional strategies, such as that provided by approved RSV vaccines.
While passive immunoprophylaxis can reduce RSV-associated hospitalization, the preventative effect on subsequent wheeze is still unclear. A recent systematic review and meta-analysis of observational studies and RCTs evaluated the association of RSV-ALRI on subsequent wheezing illness38. Eight immunoprophylaxis studies contributed to the analysis, and although the odds point estimate of subsequent wheezing illnesses was higher in those who had not received RSV monoclonal antibodies (palivizumab or motavizumab), the effect was weak (odds ratio 1.21; 95%CI: 0.73–1.99). A 2020 World Health Organization Report reached the same conclusion that there was insufficient evidence to support a causal relationship between RSV-ALRI and subsequent wheezing illnesses5. The major strength of the present study is that we combined data from two high-quality Australian birth cohorts. Although the two studies were conducted by different research teams, and consequently the study procedures were not identical, we worked with both research teams to combine the different data sets and standardize results. The RSV-NA from all samples were measured in the same laboratory, ensuring consistent results. There are however several limitations. Firstly, parent-reported symptoms were captured, and only otitis media and pneumonia were doctor-diagnosed. To reduce misclassification of symptoms, parents were trained to recognize the respiratory symptoms itemized in the daily diary26. Secondly, the diagnosis of asthma relied upon assessment by a physician and was not supplemented by more objective measures, such as the forced oscillation technique, to determine airway resistance and compliance. Nevertheless, and although controversial, clinical assessment is still the most reliable means of diagnosing asthma in this age group39. However, a history of parent-reported wheeze is subject to information bias, including reporting error with differential misclassification. Thirdly, we were not able to assess the protective effect of maternal RSV-NA on RSV-ALRI occurring in the first 3-months due to the small number (n=6) of cases. Fourthly, CAS captured only symptomatic RSV episodes and thus asymptomatic RSV infections in this cohort went undetected. Finally, while serum RSV-NA represents the main immune correlate of protection, a standardized protective threshold has not been defined40.
RSV remains an important cause of morbidity and mortality in infants worldwide. Although RSV vaccines are a global priority, none are licensed at present. The data from the present study suggest that in contrast to studies focusing upon hospitalization, high levels of transplacental RSV-NA that might be achieved by maternal immunization may not decrease overall RSV infections in the first 6-months of life and may not protect against wheeze and asthma developing in early childhood at a community level. Additional strategies to control RSV disease beyond the first-months of life are needed.