Recently, numerous reports have suggested association of pediatric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and inflammation similar to Kawasaki disease (KD), termed multisystem inflammatory syndrome of children (MIS-C).1 KD is a major cause of childhood acquired heart disease and vasculitis in the pediatric population. Epidemiologic patterns suggest that KD is related to an infectious agent; however, the etiology remains unknown.2 As past reports have considered other coronaviruses to be related to KD,3,4 we postulated that a similar postcoronavirus humoral immune response could account for the similar pathology seen in children with KD and postacute SARS-CoV-2 MIS-C. If there is similar antibody targeting that accounts for this shared pathology, we would see targeting of SARS-CoV-2 proteins from children presenting with KD before the pandemic. We addressed this hypothesis by assessing the antigen targeting of biobanked plasma samples of febrile children, including those with KD, against SARS-CoV-2 proteins.
To establish our assays and identify controls, we enrolled patients who were current inpatients in Kaleida health system with fever and respiratory distress who were tested and were SARS-CoV-2 polymerase chain reaction (PCR) positive under approval of the University at Buffalo STUDY00004340. Our institution utilizes the Abbott SARS-CoV-2 RealTime assay (Abbott Park, IL)., which has a claimed limit of detection of 100 copies/mL, which a preprint study estimates a clinical sensitivity of 90%.5 We also use the Abbott SARS-CoV-2 IgG assay for clinical testing of SARS-CoV-2 which has an estimated sensitivity of 100% and specificity of 99.6%.6 The enzyme-linked immunosorbent assay protocol used followed a recently published SARS-CoV-2-related protocol7 with minor adjustments [use of Goat Anti-Human Ig-horseradish peroxidase (HRP, Southern Biotech, Birmingham, AL)] secondary and 3,3′,5,5′-tetramethylbenzidine Ultra (Thermo Fisher, Grand Island, NY) as developer. This was supported by SUNY Research Seed Grant Program 2019-2020. KD subject samples were collected under approval of the University at Buffalo Institutional Review Board (MODCR00000185) with funding support by the Wildermuth Foundation through the Variety Club of Buffalo as previously described.8 Assignment to having KD depended on hospital admission, infectious disease consultation agreement, independent review agreement and receipt of intravenous immunoglobulin (IVIG). Statistical analysis was performed using GraphPad Prism 8 (v8.4.2, San Diego, CA).
Three enzyme-linked immunosorbent assays were developed using commercially available proteins [Sino Biological (Wayne, PA) SARS-CoV-2 proteins: receptor binding domain (RBD) Cat no. 40592-V08H; nucleocapsid protein (NP) Cat no. 40588-V08B; and spike protein, S1 + S2 extracellular domain, Cat no. 40589-V08B1]. These showed high specificity of binding when tested with rabbit polyclonal antibodies (also obtained from Sino Biological, data not shown). We then tested our 14 SARS-CoV-2 PCR-positive patients’ plasma samples (data not shown). There was variability in positivity of the early samples (3–10 days of illness). Enrollees with blood samples taken after day 10 of illness were positive for all 3 antigens, and we used convalescent samples as controls for further study.
We then tested plasma samples, collected from 2013 to 2019, of 36 febrile controls, 24 pre-IVIG KD samples, 12 post-IVIG KD samples and 3 convalescent KD samples (Fig. 1). This represented a total of 27 children with KD, all who had consistent febrile courses, with 15 of the 27 meeting criteria for complete KD per 2017 American Heart Association guidelines, and the other 12 were incomplete. Consistent with other reports, conjunctivitis was present in 88% (24/27), rash in 85% (23/27), extremity changes in 48% (13/27), cervical lymphadenopathy in 48% (13/27) and mucositis in 81% (22/27). All children with incomplete KD had 2–3 symptoms present except for 1 patient who solely had extremity changes but also had both artery, left coronary artery (Z score max: 3.2) and right coronary artery (Z score max: 6.2), aneurysms without an alternative diagnosis. This child was the only one to have received multiple doses of IVIG and also received methylprednisolone as part of their care. Aneurysms were also present in 3 children with typical KD. Notably, one other child had some signs of coronary artery enlargement (Z score: 2.0–2.5) and 2 other children had gallbladder hydrops seen on imaging. A SARS-CoV-2 PCR-positive patient’s convalescent serum was assayed on each plate as a positive control assayed at 1:50, 1:500 and 1:5000 dilution and is shown in the Figure 1.
Targeting of the RBD was most specific as a number of positive reactions to NP and spike were observed in the febrile controls. We did not see any significant reactivity to SARS-CoV-2 RBD, NP or spike protein within the KD samples. In those KD samples, IVIG infusion did seem to associate with higher binding on all 3 assays, consistent with recent reports on IVIG containing reactivity to SARS-CoV-2.9 The ratios of the means of post-IVIG to pre-IVIG were 1.47, 1.73 and 1.75, respectively, for RBD, NP and spike proteins.
From this study, we would conclude that there is not an obvious adaptive immune cross-reactivity from previous circulating coronaviruses to SARS-CoV-2 that explains the parallel pathology of KD and MIS-C. From these data, it appears unlikely that MIS-C is due to cross coronavirus immunity or antigen mimicry.
Changes in binding observed post-IVIG infusion imply nonspecific interaction due to higher level of circulating immunoglobulins after IVIG infusion. While this change was statistically significant in all 3 assays, NP and spike were more pronounced. As has been shown elsewhere, IVIG preparations show some SARS-CoV-2 cross-reactivity9 which may also partially explain these minor elevations. As a majority of our post-IVIG samples were collected at day 10, we would have expected more significant elevation if this post-IVIG increase was from seroconversion.
We also confirm other observations about positive serology >10 days from symptom onset when compared with subjects who were tested ≤10 days from symptom onset.10 Additional studies in children are needed to see if this pattern applies to children as well.
As both SARS-CoV-2 and KD appear to incite a large inflammatory response in certain children, continued research is needed to provide the pathophysiologic explanation of these events in SARS-CoV-2 and KD.
1. Centers for Disease Control and Prevention (CDC). Multisystem Inflammatory Syndrome in Children (MIS-C) Associated with Coronavirus Disease 2019 (COVID-19). 2020. Available at: https://emergency.cdc.gov/han/2020/han00432.asp
. Accessed July 14, 2020.
2. McCrindle BW, Rowley AH, Newburger JW, et al.; American Heart Association Rheumatic Fever, Endocarditis, and Kawasaki Disease Committee of the Council on Cardiovascular Disease in the Young; Council on Cardiovascular and Stroke Nursing; Council on Cardiovascular Surgery and Anesthesia; and Council on Epidemiology and Prevention. Diagnosis, treatment, and long-term management of Kawasaki disease: a scientific statement for health professionals from the American Heart Association. Circulation. 2017; 135:e927–e999
3. Shirato K, Imada Y, Kawase M, et al. Possible involvement of infection with human coronavirus 229E, but not NL63, in Kawasaki disease. J Med Virol. 2014; 86:2146–2153
4. Esper F, Shapiro ED, Weibel C, et al. Association between a novel human coronavirus and Kawasaki disease. J Infect Dis. 2005; 191:499–502
5. Arnaout R, Lee RA, Lee GR, et al. SARS-CoV2 testing: the limit of detection matters. bioRxiv. 2020
6. U.S. Food & Drug Administration. EUA Authorized Serology Test Performance. Available at: https://www.fda.gov/medical-devices/emergency-situations-medical-devices/eua-authorized-serology-test-performance
. Accessed July 14, 2020.
7. Stadlbauer D, Amanat F, Chromikova V, et al. SARS-CoV-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup. Curr Protoc Microbiol. 2020; 57:e100
8. Martin M, Wrotniak BH, Hicar M. Suppressed plasmablast responses in febrile infants, including children with Kawasaki disease. PLoS One. 2018; 13:e0193539
9. Díez JM, Romero C, Gajardo R. Currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus 2 antigens. Immunotherapy. 2020; 12:571–576
10. Long QX, Liu BZ, Deng HJ, et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. 2020; 26:845–848