Abstract
In 1967, two toddlers immunized with a formalin-inactivated vaccine against respiratory syncytial virus (FIRSV) in the United States died from enhanced RSV disease (ERD), a severe form of illness resulting from aberrant priming of the antiviral immune response during vaccination. Up to 80% of immunized children subsequently exposed to wild-type virus were hospitalized. These events hampered RSV vaccine development for decades. Here, we provide a characterization of the clinical, immunopathological, and transcriptional signature of fatal human ERD, outlining evidence for safety evaluation of RSV vaccines and a framework for understanding disease enhancement for pathogens in general.
Fatal flaws unearthed
Two toddlers vaccinated with a formalin-inactivated vaccine against respiratory syncytial virus (FIRSV) died in 1967 from enhanced RSV disease (ERD), and most FIRSV-immunized children required hospitalization upon RSV infection. Here, Polack et al. characterized the clinical, immunopathological, and transcriptional profiles of ERD from the original autopsy reports and lung sections of the toddlers who died compared with age- and race-matched controls and identified respiratory eosinophils and CCL5 as markers that correlated with ERD. Type I IFN responses were suppressed, and pulmonary immune responses showed a shift toward type 2 polarization, thus validating observations in murine RSV models. These findings provide critical insights into ERD and how it can inform RSV vaccine development.
INTRODUCTION
Respiratory syncytial virus (RSV) is currently a primary cause of hospitalization in infants and young children globally (1). An estimated 118,000 children under 5 years of age died in 2015 because of RSV infection, with about 50% dying at home (1). In recent years, there has been a surge of interest in RSV vaccine development with the advent of new immunization strategies, a variety of vaccine constructs, and better characterization of RSV-neutralizing antigens (2–4). However, RSV vaccine development has proven to be a major challenge for decades. Numerous previous attempts have failed, including one of the most marked vaccine trials in history resulting in the death of two toddlers in the 1960s (5).
In the winter of 1966–1967, a formalin-inactivated vaccine against RSV (FIRSV) was tested in infants and young children in the United States (5, 6). FIRSV primed for enhanced RSV disease (ERD), an atypical and enhanced presentation of RSV illness in the lower respiratory tract. ERD resulted in hospitalization rates up to 80% and two deaths in immunized infants subsequently infected with RSV as toddlers (5, 6). These events nearly halted RSV vaccine development for decades.
Identification of vaccine candidates that prime for ERD is imperative to inform human trials. For this purpose, numerous hypotheses derived from studies in animal models were advanced over the years to define correlates of vaccine safety (7–10). However, information about the two fatal human cases from 1967 has been limited. Here, we report a detailed characterization of the clinical manifestations, immunopathology, and transcriptional footprint of ERD in the respiratory tract of both toddlers who died in 1967.
RESULTS
Clinical presentations
The two male toddlers who died by ERD in January 1967 in Washington, DC (5) were compared with 25 infants and children who died by wild-type RSV (wtRSV) infection in Nashville, TN (n = 1), Santiago, Chile (n = 4) (11), and Buenos Aires, Argentina (n = 20) between 1999 and 2013 (12). The two boys with ERD were significantly older than those with wtRSV disease [median age in months, 15 (interquartile range, 3.25) versus 2 (interquartile range, 3); P = 0.032].
Both toddlers were previously healthy; one of them had experienced an episode of acute bronchiolitis at 3 months of age. Neither toddler with ERD had congenital comorbidities, but congenital risk factors were frequent and included congenital heart disease (3 of 25), prematurity (7 of 24), and Down’s syndrome (1 of 25) in infants with wtRSV. Five of 18 children with wtRSV, for which information was available, had experienced previous episodes of bronchiolitis. Both toddlers with ERD presented to the hospital after experiencing prolonged cough, tachypnea, and rhinorrhea complicated by high fever (Table 1). Forty percent of patients admitted with wtRSV had fever, with 65% presenting with tachypnea.
Table 1.
Comparison of clinical and laboratory manifestations in fatal ERD and wtRSV in children.
| ERD 1 | ERD 2 | wtRSV (n = 25) | |
|---|---|---|---|
| Clinical presentation | |||
| Age in months, median | 16 | 14 | 2 |
| Days of symptoms at presentation | 14 | 4 | 3 (1–16) |
| Fever | Yes | Yes | 8 of 25 |
| Cough | Yes | Yes | 7 of 20 |
| Days of cough, median (range) | 14 | 4 | NA |
| Tachypnea, % | Yes | Yes | 24 of 25 |
| Bilateral wheezing | Yes | Yes | 6 of 20 |
| Laboratory results | |||
| WBC count admission, mean (range) | 20,800 | 12,100 | 4800 (2300–7300) |
| Neutrophils, mean % (%bands) | 71 (2) | 50 (8) | 65 (30–91) |
| Lymphocytes, mean % | 21 | 26 | 23 (6–70) |
| Eosinophils, mean % | ND | <5 | <5 |
| Hgb on admission, g | 12.8 | 10 | 9.4 (7.3–13.1) |
| Adequate platelets on admission | Yes | Yes | NA |
| Chest x-ray, infiltrates on admission | Bilateral | Bilateral | 9 of 20 |
| Hospital course | |||
| Tmax in °C, median, range | 38.7 | 39.8 | 38.2 (37–38.4) |
| Clinical impression | Bronchopneumonia | Bronchopneumonia | Bronchiolitis (17 of 25) |
| Sepsis | No | No | 17 of 25 |
| Post-mortem cultures | |||
| Viral: Throat/PCR in NP aspirate/swab | RSV | RSV | 25 of 25 |
| Viral: Lungs | RSV | RSV | ND |
| Bacterial: Lungs | Sterile | E. coli | ND |
| Bacterial: Spleen | Sterile | E. coli | ND |
| Bacterial: Trachea | K. pneumoniae | E. coli | ND |
| Bacterial: Lungs | K. pneumoniae | E. coli | ND |
NA, not available; WBC, white blood cell; Hgb, hemoglobin; PCR, polymerase chain reaction; K. pneumoniae, Klebsiella pneumoniae; E. coli., Escherichia coli; NP, nasopharyngeal; ND, not done.
Patients with ERD had moderately elevated white blood cell counts, with elevated neutrophil counts (neutrophilia). In those with wtRSV, neutrophilia was infrequent (27%). Despite the emphasis on blood eosinophilia in previous studies as a potential peripheral biomarker of ERD (5, 8–10, 13–16), no patient in either group had ≥5% eosinophils in their white blood cell count.
After hospital admission, febrile toddlers with ERD progressed to respiratory failure and died within 2 days. Twenty-four of 25 (96%) children with wtRSV were ventilated before death. Median time to death after hospitalization with wtRSV was 3 days. Antemortem blood cultures were sterile in both children with ERD. Twenty-two of 25 (88%) fatal wtRSV cases experienced bacterial sepsis (17 of 25; 68%) (Table 1).
Lung histopathology
The specificity of neutrophils versus eosinophils as biomarkers for ERD has been debated for decades (5, 6, 9, 13). Lung sections from both toddlers with ERD were compared with tissue sections from a 15-month-old toddler with congenital heart disease killed in a traffic accident while experiencing wtRSV bronchiolitis (Fig. 1 and figs. S1 and S2) (17).
Fig. 1. Lung immunopathology of fatal ERD and wtRSV.
Lung sections from the two toddlers who died of ERD (A and B) and a toddler who died in a traffic accident with wtRSV infection in the absence of vaccination (C). H&E (10× fields), neutrophil infiltration [Anti-MP, anti-myeloperoxidase antibody (1:100): 10× fields], and eosinophil infiltration [Anti-EPX, anti-eosinophil peroxidase antibody (1:100): 10× fields]. Scale bars, 200 μm.
We found diffuse alveolar consolidation in ERD sections, as well as intense transmural bronchiolar inflammation and luminal filling with cells and granular debris (Fig. 1 and figs. S1 and S2). In contrast, wtRSV infection was associated with inflammatory responses centered primarily on bronchioles and mostly confined to the submuscular compartment of the bronchiolar wall (Fig. 1 and figs. S1 and S2).
The bronchiolar inflammatory infiltrates in ERD were composed predominantly of neutrophils and eosinophils, with admixed mononuclear cells. Bronchiolar lumens were filled with polymerphonuclear leukocytes, which were a mix of neutrophils and eosinophils based on myeloperoxidase and eosinophil peroxidase (EPX) stains (Fig. 1). Eosinophils were abundant and linked to ERD and nearly absent in wtRSV sections (mean ± SD eosinophil count per 40× field is 40.25 ± 1.75 in ERD versus 0.2 ± 0.01 in wtRSV; P < 0.001; Fig. 1).
In contrast, macrophages and lymphocytes predominated in wtRSV, whereas neutrophils were a minor component of the inflammatory response (mean ± SD neutrophil count per 40× field is 73.4 ± 16.0 in ERD versus 41.0 ± 11.8 in wtRSV; P < 0.001). These findings agree with previously reported assessments of the histopathological signatures of wtRSV (17).
Transcriptional profiles
We compared transcriptional profiles in the lungs of toddlers who experienced ERD with age-, sex-, and race-matched previously healthy toddlers who died from nonpulmonary causes (Fig. 2, fig. S3, and data files S2 to S4).
Fig. 2. Heatmaps of gene expression of two toddlers killed by ERD compared to LungMAP controls of age-, sex-, and race-matched toddlers who died of nonpulmonary causes.
(A) Volcano plot of differentially expressed genes using EdgeR output. (B to F) Heatmaps of statistically significant differences in the two toddlers killed by ERD compared to controls including (B) epithelial integrity genes, (C) cell injury/repair genes, (D) IFN genes, (E) type 2 genes, and (F) eosinophil genes.
At the time of death, both toddlers with ERD had experienced severe symptoms for a minimum of 5 to 6 days. There was a profound defect in the expression of transcripts from type II pneumocytes, including surfactant proteins SFPTA1, SFPTA2, SFPTB, and SFPTC. In contrast, genes associated with ionocytes (CFTR and FOXI1) were mildly decreased, whereas FOXJ1, a marker of ciliated epithelium, was up-regulated (Fig. 2, A and B).
The transcriptional response was substantially reduced in genes responsible for down-regulating the complement response including CR1, CFH, FCGR2, and C5 (18–20). In addition, there was a marked increase in FASLG expression, a mediator of cell death, and suppressed expression of AREG, an important mediator of lung repair during viral infections (Fig. 2C).
We found dramatic evidence for suppression in genes implicated in regulation of type I interferon (IFN) production and the type I IFN–mediated signaling pathway, including expression levels of ZBP1, TLR2, IBKBE, HERC5, and IFNAR2 (Fig. 2D). Transcriptomics confirmed a widely hypothesized phenotype of ERD derived from murine models of illness, which is the emergence of an increased type 2 signature (Fig. 2E) (7–10). In contrast, genes linked to interferon gamma production and type 1 signalling pathways, including STAT-1, HLA, and JAK1, were reduced. No clear differences were observed between groups for genes associated with interleukin-17 (IL-17) and IL-17 + IL-22 (fig. S3). This type 2 bias was associated with increased expression of genes in Gene Ontology terms involved in eosinophil activation (F2RL1, CD300A, HRH1, and ADAM8) and eosinophil chemotaxis (CCL5; Fig. 2F).
DISCUSSION
This report describes a detailed characterization of the clinical presentation, lung immunopathology, and transcriptional signature of fatal human ERD. Toddlers who died by ERD were older, presented with high fever and leukocytosis, experienced bronchopneumonia with no overt evidence of secondary bacterial infections, and lacked the comorbidities typically observed in infants dying from wtRSV (11, 12). ERD lungs were diffusely consolidated by an exuberant inflammatory response, which filled and expanded alveolar spaces and bronchioles in a confluent fashion, obscuring the underlying architecture. In contrast, the wtRSV inflammatory response was moderate, affecting primarily bronchioles without evidence of bronchiolar obstruction or alveolar damage. Consistent with severe alveolar injury seen on histopathology in ERD, we found a substantial reduction in surfactant proteins, associated with injury or dysfunction of type II pneumocytes and a significant down-regulation of antiviral type I IFN genes.
Two cardinal immune signs proposed to characterize ERD pathogenesis are low avidity, nonprotective anamnestic antibodies against wtRSV triggering complement-mediated injury (18–20) and a T helper 2 cell (TH2) polarization of the immune response as observed in murine models of illness (7–10). Kim et al. (5) had postulated a role for immune complexes in ERD pathogenesis in 1969. Murine models suggested that C3a contributes to bronchoconstriction and TH2 polarization, modulated by the anaphylatoxin C5a (20–22). We found profound defects in expression of transcripts for CFH, encoding for the soluble inhibitor of complement activation and amplification factor H; CR1, whose deficit is associated with immune complex-mediated diseases; and FCGR2, which is linked to phagocytosis of immune complexes from circulation and modulation of B cell antibody production. In the end, the entire complement transcriptional response was suppressed.
This study confirms a type 2 pulmonary bias in human ERD. Foundational work described high IL-4 levels in mice, establishing a paradigm for decades of studies (7). Subsequent work demonstrated a role for TH2 cytokines in enhancing pulmonary pathology, mucus secretion, and airways resistance (10). Lung eosinophilia is another manifestation of type 2 polarization and had been linked to carbonyl groups on formaldehyde-treated vaccine antigens and the absence of cytotoxic T lymphocyte priming during vaccination (8, 9). Here, EPX-staining demonstrated that although the original report described only nominal eosinophil involvement (5), the level of eosinophil lung infiltration and degranulation in ERD was considerably higher. Our manuscript supports that eosinophils are likely a better biomarker of disease enhancement than neutrophils (8, 9, 13–15).
The role of eosinophils in ERD pathogenesis remains unclear. Previous murine studies suggested that eosinophils do not modify the ERD clinical phenotype (10) and several animal models of enhanced disease lack pulmonary eosinophilia (14, 15). In addition, IL-5 levels in respiratory secretions were associated with protection against severe wtRSV disease in a population of TLR4+/− infants with increased susceptibility for the virus (16). Whether eosinophils are present to enhance or mitigate ERD is still unknown. Detection of eosinophils in the lungs, along with increased expression of CCL5, which is expressed by eosinophils and T cells and binds to receptors on a wide range of immune cells, may be helpful in distinguishing ERD from typical illness.
Fatal victims had normal eosinophil counts in peripheral blood. Only 20% of children with ERD presented with blood eosinophilia in a FIRSV trial in California in 1967 (6). Blood eosinophilia should raise concerns for disease enhancement, but lack of it should not be misconstrued as an error-proof safety clearance.
Our manuscript has some limitations. The only materials available from the 1967 events, a handful of formalin-fixed, paraffin-embedded (FFPE) lung sections from each child, limited our choice of stains and allowed analysis of a single terminal time point in disease course. In addition, we were unable to compare transcripts to signatures in wtRSV deaths because all our patients undergoing autopsy, as is frequently the case in other studies in the United States and world-wide (11, 12), experienced secondary bacterial infections and/or other secondary conditions, and our investigations were limited by necessity to the only two toddlers who experienced fatal ERD in history (5). Last, a potential effect from differential processing of samples 54 years versus 21 years ago cannot be excluded, although our findings generally fit well with the current understanding of ERD pathogenesis.
Nonetheless, our study provides an extensive map of ERD clinical manifestations, immunopathology, and transcriptional footprint [RNA sequencing (RNA-seq) database available in the Supplementary Materials]. Our findings provide insight into pathways linked to enhancement, including suppressed surfactant proteins and type I IFN, and specific candidate disease biomarkers from lung eosinophils including chemokine ligand 5 (CCL5). Last, in the context of the COVID-19 pandemic, these observations serve as a phenotype for human vaccine-enhanced disease to better understand this immune-mediated phenomenon overall.
In summary, we provide a comprehensive characterization of fatal ERD, a tragedy that hampered RSV vaccine development for decades. Disease is characterized by high fever and bronchopneumonia with a substantial reduction in surfactant and type I IFN transcriptional signatures. Type 2 polarization of the immune response in the lungs presented with a profuse pulmonary eosinophilia and increased CCL5 expression. Peripheral blood eosinophil counts could be normal and uninformative. This characterization of the human ERD phenotype should facilitate the evaluation of RSV vaccines advancing in human trials and provide a framework for understanding vaccine-enhanced disease in general.
MATERIALS AND METHODS
Study design
We compared the clinical, immunopathological, and transcriptional responses of the only two individuals who died from ERD to that of infants and toddlers who died from wtRSV infection. Data were collected from the original autopsy reports from 1967 and lung slides from both toddlers affected by ERD in 1967.
Population
Comparisons for age between groups with ERD and wtRSV (n = 23 versus 2) were conducted using a Student’s t test. Lung sections were available from the two children with ERD and five victims of wtRSV. Since four of five infants dying with wtRSV experienced severe lung bacterial coinfections, control lung sections for hematoxylin and eosin (H&E), myeloperoxidase and EPX were obtained from a 15-month-old toddler with congenital heart disease killed in a traffic accident in 1999 while experiencing wtRSV bronchiolitis (Fig. 1) (17). The original autopsy reports of both toddlers with ERD are available in the Supplementary Materials (data file S1).
We compared gene expression profiles in lung sections of the two male African American toddler victims of ERD to control lung sections from two previously healthy age-, sex-, and race-matched toddlers dying from nonpulmonary illness (brain deaths; courtesy of G. S. Pryhuber, University of Rochester Medical Center). The study was approved by the Institutional Review Board from Vanderbilt University (IRB 110485).
Histopathology and immunohistochemistry
Paraffin-embedded lung sections were stained with H&E, anti-myeloperoxidase antibody (clone 2C7; Abcam) to detect activated neutrophils, and the primary monoclonal EPX antibody (clone 144B; homebrew from J. J. Lee, Mayo Clinic, Arizona). Ten 40× fields per slide were counted for statistical comparisons in myeloperoxidase- and EPX-stained slides. A generalized mixed model was used to compare mean scores between subject types, accounted for repeated measures within subjects.
Histomorphometric quantification
H&E-stained lung sections were digitally scanned with an Aperio slide scanner and analyzed using HALO software (Indica Labs, Albuquerque, NM) to determine average bronchiole wall thickness and percentage of lung containing inflammatory fluid in ERD1, ERD2, and wtRSV. To determine the average bronchiole wall thickness, 10 bronchioles were randomly selected from each slide. Semi-circular annotations were drawn along the inner and outer margins of each bronchiole by a pathologist (inner, bronchiole smooth muscle layer and outer, the margin of inflammation). The thickness of each bronchiole was determined by taking the mean of 10 measurements, at regular intervals, between the inner and outer margins. The average bronchiole thickness for each patient was reported as the mean of 10 randomly selected bronchioles from each patient ± SD.
To determine the percentage of lung containing inflammatory fluid, regions of interest were drawn around each section of lung and then a tissue segmentation algorithm (random forest) was trained to identify the following tissue classes: inflammatory fluid, cellular infiltrate, lung, stroma, and glass. Accuracy of the algorithm was confirmed by a pathologist (R.V.B.). The inflammatory fluid was quantified by dividing the area of lung containing inflammatory fluid by the total area analyzed, generating the percentage of lung affected.
Tissue processing and RNA extraction
H&E-stained slides were soaked overnight in fresh xylene. Coverslips were removed, and sections were scraped into a micro-centrifuge tube using a sterile razor blade. Tissue was rehydrated in ethanol/ribonuclease (RNase)–free water dilutions (100, 95, and 75%) for 15 min per wash. Slides were washed in RNase-free water for 10 min (2×). Tissues were then incubated in proteinase K solution proteinase K digestion buffer (PKD) buffer with the addition of proteinase K (QIAGEN) at 56°C for 3 hours. TRIzol (1 ml; Thermo Fisher Scientific) was added, and RNA was extracted according to the TRIzol protocol. RNA concentration was determined with the Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Quality RNA integrity number scores and fragment sizes (DV200 metrics) were obtained using either the Agilent 2100 Bioanalyzer or the Agilent 4200 TapeStation.
Exome capture RNA-seq
The only tissues available from the victims of ERD were stained slides. Therefore, we optimized a protocol to perform RNA-seq in previously stained FFPE lung sections (23). Sequencing library preparation was performed as previously described using a minimum of 25 ng of RNA according to Illumina’s TruSeq RNA Access Library Preparation protocol. Indexed, pooled libraries were then sequenced on the Illumina NextSeq 550 platform with high-output flow cell–producing stranded, single end reads (1× 75 base pairs). A target count of 50 million reads per sample was used to plan indexing and sequencing runs. Raw reads were quality checked, trimmed, and mapped to the reference genome (GRCh37/hg19) using STAR with National Center for Biotechnology Information RefSeq-annotated genes as transcriptome index data. Raw read counts were normalized across all samples and then used for differential expression analysis using DESeq, EdgeR, and Cuffdiff (Slug Genomics, UC Santa Cruz) (available as data files S2 to S4, respectively). Using log2-normalized fragments per kilobase million (FPKM) values, heatmaps were generated in R (Gene Expression Omnibus accession number GSE18343).
Statistical analysis
For histomorphometric quantifications, a Welch’s t test was used to test the means between patients in a pairwise fashion for statistically significant differences. EdgeR output was used to generate the volcano plot and heatmaps. For the volcano plot, we used a log2 fold change of >5 and an adjusted P < 10−25. The full data output is in the Supplementary Materials. The edgeR program in Bioconductor uses the Benjamini-Hochberg procedure for calculating the adjusted P values (24).
Supplementary Material
Acknowledgments:
We thank the donating families and G. Pryhuber and her team with the IIAM and NDRI Research Organ Recovery Organizations and the LungMAP Consortium supported by NHLBI Molecular Atlas of Lung Development Program Human Tissue Core grants (U01HL122700 and U01HL148861) for the control human tissue samples. We also thank the technical support of A. Lawrence at Vanderbilt University and W. Horne and K. Song for library preparation and RNA sequencing.
Funding:
Supported by the Bill & Melinda Gates Foundation (F.P.P.), the National Institute of Environmental Health Sciences (S.R.K. and F.P.P.), and R35HL139930 (to J.K.K.).
Footnotes
Competing interests: The authors declare that they have no competing interests.
View the article online
Data and materials availability:
All data associated with this study are present in the paper or the Supplementary Materials. Complete autopsy reports from 1967 are available as data file S1. RNA-seq data have been deposited in the Gene Expression Omnibus (GSE accession number GSE18343). In addition, this information has also been provided in the Supplementary Materials (data files S2 to S4). Access to slides stained with H&E and anti-MP from all cases are available at 10.5281/zenodo.5523314.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All data associated with this study are present in the paper or the Supplementary Materials. Complete autopsy reports from 1967 are available as data file S1. RNA-seq data have been deposited in the Gene Expression Omnibus (GSE accession number GSE18343). In addition, this information has also been provided in the Supplementary Materials (data files S2 to S4). Access to slides stained with H&E and anti-MP from all cases are available at 10.5281/zenodo.5523314.