Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in December 2019 and has caused more than 102 million confirmed cases worldwide as of 31 January 2021.1
Despite intensive study throughout the science and medical communities, many clinical and biological aspects of the disease, especially in the pediatric population, remain to be elucidated. As data emerged from the initial outbreaks in China, the number of COVID-19 cases in children appeared to be low, with reports indicating that less than 1% were patients under the age of 10, 1.2% had between 10 and 19 years old and only 9 patients were infants with mild symptoms.2
In the United States, pediatric infection cases accounted for only 7% of total cases in August 2020.2,3 The US Centers for Disease Control and Prevention (CDC) reported that, as of September 19, In 2020, only 4.1% of confirmed COVID-19 cases nationwide were in school-age pediatric patients (ages 5 to 17).4 Although the causes of these differences remain unclear, the majority of children with SARS-CoV-2 infection are asymptomatic or present mild symptoms5 -7 and have a low risk of developing severe respiratory disease.8,9
The CDC reported that the average weekly incidence of COVID-19 cases among adolescents ages 12 to 17 was approximately twice that of children ages 5 to 11.4 Only a relatively small number of pediatric patients have experienced severe illness during the acute phase of COVID-19.
However, these patients are at risk of serious complications from multisystem inflammatory syndrome in children (MIS-C), an emerging entity believed to occur as a sequelae of acute SARS-CoV-2 infection. 10,11 Therefore, there appear to be differences in the pathophysiological responses to SARS-CoV-2 depending on age.
Although the physiological mechanisms remain unclear, evidence suggests that SARS-CoV-2-specific antibody responses may be different in children and adolescents compared to those in adults, 12 potentially modulating different clinical manifestations.
There is controversy over whether children have an attenuated adaptive immune response, leading to tolerance to SARS-CoV-2 infection, 8 or whether the innate immune response in children plays a more active role against SARS-CoV-2. than in adults.13 Furthermore, the binding avidity of SARS-CoV-2 virus-specific antibodies, which represents the quality of the antibody response, has not been fully characterized in pediatric patients.
As the 2020-2021 school year has resumed, with approximately 56 million school-aged children and adolescents in the United States participating in in-person and/or remote classes, it is imperative to better understand immune responses specific to the SARS-CoV virus. -2 in pediatric patients.
In this study, we evaluated the magnitude of total antibody levels, immunoglobulin (Ig) G levels, and surrogate neutralizing antibody (SNAb) activities, as well as antibody binding avidity in children, adolescents, and young adults. .
Unlike other studies, which primarily focused on hospitalized pediatric patients, this study investigated antibody responses during the convalescent stage of previously asymptomatic or mildly ill non-hospitalized patients, which is more representative of the general pediatric population of COVID patients. -19.
| Methods |
> Patients and data sources
This study was conducted at NewYork–Presbyterian Hospital/Weill Cornell Medical Center with institutional review board approval. The board granted a waiver of informed consent because only leftover serum samples were used. A total of 31,426 SARS-CoV-2 antibody tests were performed between April 9 and August 31, 2020, on serum samples from children (aged 1 to 18 years) and adults (aged > 18 years) , including inpatients and outpatients.
Infants younger than 1 year were not included in this study to exclude the possibility of transfer of maternal antibodies. The results of these tests were used to calculate the SARS-CoV-2 antibody positivity rate for each age group. Among them, the positive results of 85 pediatric cases and 3,648 adults tested with the Pylon COVID-19 IgG assay from April 9 to June 21, 2020 were used to evaluate and compare IgG levels.
Results from samples tested with the Siemens assay from June 22 to August 31, 2020 were not used for semiquantitative IgG studies because approximately 50% of positive results exceeded the upper limit of the reportable range, which prevented an accurate quantitative comparison of antibodies.
The remaining SARS-CoV-2 antibody-positive sera were randomly collected from a biobank between April 22 and August 31, 2020, of which samples from 126 outpatients aged 1 to 24 years were used for serological analysis. additional. These patients did not report any COVID-19-like symptoms at the time of antibody testing.
Samples were stored at 4 °C for no more than 2 days and then frozen until further testing. These samples were evaluated for levels of SARS-CoV-2 IgG, total receptor binding domain (RBD) binding antibodies (TAb), and SNAb activity, as well as antibody binding avidity to RBD.
Demographic characteristics, medical history, and clinical and laboratory findings related to COVID-19 of these 126 patients were obtained from the electronic medical record (EMR; Epic Systems). None of the patients were reported to have MIS-C.
| SARS-CoV-2 Antibody Assays |
> SARS-CoV-2 IgG Assay and SARS-CoV-2 Total Antibody Assay for Clinical Testing
The COVID-19 IgG Assay on the Pylon 3D Analyzer (ET Healthcare) was used as a laboratory-developed test under New York State Department of Health regulations from April 9 to June 21, 2020. This assay Enhanced cyclic fluorescence targets the S-RBD protein and recombinant nucleocapsid, as described above.
The ADVIA Centaur SARS-CoV-2 Total Antibody Assay (COV2T), used from June 22 to October 25, 2020, is a chemiluminescent immunoassay approved by the US Food and Drug Administration’s Emergency Use Authorization. UU to detect antibodies against SARS-CoV-2. Both assays were used for COVID-19 clinical serological testing. Method validation was performed to demonstrate the equivalence of the 2 assays in reporting positive and negative results.
>SArs-CoV-2 TAb, SNAb and Avidity assays
The TAb assay measures overall binding between SARS-CoV-2 antibodies and the RBD of the virus spike protein (i.e., S). The SNAb assay, designed as a competitive binding assay, is based on SARS-CoV-2 antibody-mediated inhibition of the interaction between the angiotensin-converting enzyme 2 (ACE2) protein and the RBD.
The assay readout is the percentage of RBD-ACE2 binding (%B/B0), which is inversely associated with SNAb activity. The avidity assay measures the dissociation rate of SARS-CoV-2 antibodies from the RBD, which is inversely associated with antibody avidity. TAb, SNAb, and avidity assays are fully automated on the Pylon 3D analyzer.
| Statistic analysis |
Bivariate associations were evaluated using Fisher’s exact test between 2 categorical variables, while the Kruskal-Wallis test or Wilcoxon rank sum test was used between numerical variables and categorical variables.
Dunn’s procedure was used as a posttest for pairwise comparisons of antibody levels between age groups after significant Kruskal-Wallis tests. Two numerical variables were evaluated using Spearman’s correlation coefficient.
Summary statistics are presented as mean with SD or median with interquartile range (IQR) for continuous variables and frequency with proportion for categorical variables. A 2-tailed p < 0.05 was considered statistically significant. Analyzes were performed using SAS version 9.4 (SAS Institute) or Prism version 9.0.0 (GraphPad Software)
| Results |
> Antibody positivity rates against SARS-CoV-2 From April 9 to August 31, 2020
A total of 31,426 SARS-CoV-2 antibody tests were performed (19,797 [63.0%] female patients), including 1,194 tests in pediatric patients (mean [SD] age, 11.0 [5.3] years ) and 30,232 in adult patients (mean [SD] age, 49.2 [17.1] years.
Overall, 197 (16.5%; 95% CI, 14.4%-18.7%) and 5630 (18.6%; 95% CI, 18.2%-19.1%) results were positive in pediatric and adult patients, respectively.
Positivity rates for pediatric and adult patients were not significantly different (p = 0.06). Positivity rates were then evaluated for different age groups. Young adults, ages 19 to 24 (242 of 990 [24.4%; 95% CI, 21.8%-27.3%]) and those ages 25 to 30 (816 of 3,468 [23. 5%, 95% CI, 22.1%-25.0%]) had the highest positivity rates compared to other age groups.
Children 1 to 10 years (76 of 500 [15.2%; 95% CI, 12.2%-18.6%]), patients 61 to 70 years (714 of 4494 [15.9%; CI 95%, 14.8%-17.0%]), patients aged 71 to 80 years (365 of 2824 [12.9%; 95% CI, 11.7%-14.2%]), and patients Those older than 80 years (161 of 1208 [13.3%; 95% CI, 11.5%-15.4%]) had lower positivity rates.
> Comparison of SARS-CoV-2 IgG levels in each age group
The SARS-CoV-2 IgG levels of 85 positive samples from pediatric patients and 3648 positive samples from adult patients measured using a single platform (Pylon 3D) were compared, representing 43.1% (95% CI, 36. 1%-50.4%) and 64.8% (95% CI, 63.5%-66.0%) of positive pediatric and adult results, respectively.
The IgG level in the pediatric population exhibited a moderate but significant negative correlation with age (r = −0.45; p < 0.001), and the adult population showed a weak positive correlation with age (r = 0.24; p < 0.001).
Notably, the 32 children aged 1 to 10 years showed significantly higher (IQR) median SARS-CoV-2 IgG levels than the 127 young adults aged 19 to 24 years (443 [188-851] RFU vs. 95 [188-851] RFU 47-180] RFU; P <0.001), the 611 adults aged 25 to 30 years (99 [44-180] RFU; P <0.001), the 956 adults aged 31 to 40 years (104 [48-224] RFU; P < 0.001). 001), the 688 adults aged 41 to 50 years (137 [50-319] RFU; p = 0.001) and the 69 patients over 80 years of age (165 [24-518] RFU; p = 0.01).
Young adults aged 19 to 24 years and 25 to 30 years exhibited the lowest median (IQR) SARS-CoV-2 IgG levels (95 [47-180] RFU and 99 [44-180] RFU, respectively), without any significant difference between these 2 age groups.
Patients aged 19 to 24 years showed significantly lower IgG levels than 612 adults aged 51 to 60 years (95 [47-180] RFU vs 195 [65-585]; P < 0.001), 415 aged 61 to 70 years (225 [65-660] RFU; P <0.001), and the 170 aged 71 to 80 years (233 [62-675] RFU; P <0.001), and patients aged 25 to 30 years showed a significantly lower median (IQR) IgG levels than adults over 41 years of age (e.g., vs. patients 41-50 years of age: 99 [44-180] RFU vs. 137 [50-319] RFU; P < 0.001) but not those aged 81 years or older (165 [24-518] RFU; P > 0.99).
Adults 19 to 24 years (children vs. young adults: 2393 [1362-4346] RFU vs. 370 [125-697] RFU; p < 0.001; adolescents vs. young adults: 961 [290-2074] RFU vs. 370 [125-697]; P = 0.006).
SARS-CoV-2 SNAb activity and binding avidity assays were used to evaluate SARS-CoV-2 antibody quality. %B/B0 was positively correlated with age (r = 0.50; P < 0.001), indicating an inverse correlation between SNAb activity and age.
Similar to IgG and TAb, the median (IQR) of SNAb activities was higher in children (aged 1 to 10 years) than in adolescents (aged 11 to 18 years) (%B/B0: 21.5% [10, 3% to 30.0%] vs 44.0% [25.0% to 65.3%]; P = 0.002) and young adults (19 to 24 years) (%B/B0: 66.0% [37.5% to 79.5%]; P < 0.001).
Adolescents also exhibited higher median (IQR) SNAb activity than young adults (%B/B0: 44.0% [25.0% to 65.3%] vs. 66.0% [37.5% to 79.5%]; p = 0.04).
The relative dissociation rate between SARS-CoV-2 antibodies and RBD showed a weak but significantly positive correlation with age (r = 0.29; P < 0.001), indicating a negative correlation between binding avidity What about the age.
While there were no significant differences between each age group, children aged 1 to 10 years tended to exhibit lower median relative dissociation rates (IQR) and therefore higher antibody binding avidity than young adults. . (5.7 × 10−4 [5.2 × 10−4 to 6.5 × 10−4] vs 6.3 × 10−4 [5.5 × 10−4 to 7.8 × 10−4]; P = .07).
> Evaluation of the quantity and quality of SARS-CoV-2 antibodies in children, adolescents and young adult patients
The authors focused more on pediatric patients (aged 1 to 18 years) and young adults (aged 19 to 24 years) to understand their characteristic profiles of SARS-CoV-2 antibody responses. More extensive SARS CoV-2 serological testing was performed on 126 outpatients aged 1 to 24 years. None of the 126 patients were admitted to the hospital due to COVID-19 before serum sample collection, and they were asymptomatic at the time of antibody testing.
Of 118 patients with documentation in the EMR, 56 patients (47.5%) were previously symptomatic, while 62 (52.5%) never had COVID-like symptoms. Nine patients (19.1%) had a positive SARS-CoV-2 reverse transcription polymerase chain reaction test. Most patients (87 [69.0%]) underwent antibody testing due to prior exposure or previous COVID-19-like symptoms.
Other reasons for antibody testing included preprocedure testing (4 [3.2%]), annual checkup (3 [2.4%]), requesting attendance at school or camp (11 [8.7%] ) or to confirm a positive SARS-CoV-2 antibody after testing at another hospital (4 [3.2%]).
Among patients who had self-reported symptom onset dates, there was no correlation between age and days between serologic testing and symptom onset (mean (SD) time, 108 [48] days; r = 0.16 ; p = 0.27). Similar to what was observed in the general patient population, the SARS-CoV-2 IgG level in this subset of patients showed a moderate but significantly negative correlation with age (r = −0.51; P < 0.001).
Children aged 1 to 10 years had significantly higher median (IQR) SARS-CoV-2 IgG levels than adolescents aged 11 to 18 years (473 [233-656] RFU vs. 191 [82-349] RFU; P = 0.01) and young adults aged 19 to 24 years (85 [38-150] RFU; P < 0.001).
Adolescents also exhibited a significantly higher IgG level than young adults (191 [82-349] vs. 85 [35-150]; P = 0.003). Similarly, SARS-CoV-2 TAb levels in this subset of patients showed a negative correlation with age (r = −0.52; P < 0.001). Pediatric patients, both children aged 1 to 10 years and adolescents aged 11 to 18 years, showed higher mean [IQR] SARS-CoV-2 TAb levels than young people.
| Discussion |
There is a compelling need to understand the pathophysiological basis underlying the different disease manifestations of SARS-CoV-2 infection in children compared to adults. However, knowledge of the immune response against SARS-CoV-2 in children and young adults is limited.
Although seroprevalence in the pediatric and adult patient populations was similar, indicating that children were just as likely as adults to be infected with SARS-CoV-2, the production of SARS-CoV IgG antibodies was found to be -2 was clearly different in children, adolescents and different age groups of adults.
Furthermore, comprehensive evaluation focusing on quantitative and qualitative SARS-CoV-2 antibody profiles in pediatric and young adult patients revealed key differences in humoral antibody responses against SARS-CoV-2 by age.
| Therefore, the findings suggest that differences in the clinical manifestations of COVID-19 in pediatric patients compared to adult patients could be partly due to age-related immune responses. |
Similar to what was seen with SARS-CoV and Middle East respiratory syndrome coronavirus infections, SARS-CoV-2 appears to cause fewer symptoms and less severe illness in children than in adults.9, 18 Exact mechanisms underlying different SARS-CoV-2 age-based immune responses remain unclear; however, some possibilities have been suggested. It has been proposed that children may have blunted immune responses, resulting in tolerance to the virus.8
In a 2021 study of 47 pediatric patients (16 with MIS-C and 31 without MIS-C) and 32 adult patients (19 plasma donors and 13 hospitalized patients), Weisberg et al12 demonstrated that pediatric patients generated more antibody responses. close to infection in terms of isotypes, which were largely limited to anti-S IgG antibodies, and an overall lower level of neutralizing antibody response than adults.
In contrast, other studies have suggested that milder manifestations of the disease in children may be due to more active innate immune responses, healthier airways due to less exposure to air pollution or cigarette smoke, and fewer comorbidities. .13
Furthermore, it has also been proposed that trained immunity may play a role19 and that innate immune memory generated by other vaccines, particularly attenuated vaccines such as measles, mumps, and rubella, may confer a nonspecific protective effect against SARS. CoV-2.20
An additional explanation that has been offered as a possibility for higher antibody levels in children is cross-reactivity with other human coronaviruses, given that multiple putative epitopes for B and T cells are conserved between SARS-CoV-2 and human coronavirus 0C43 and HKU1.21
The results indicate that the anti-SARS-CoV-2 immune response in younger children is more robust than that of adolescents and young adults, not only in the magnitude of total antibody and IgG levels, but also with respect to neutralizing activity. functional.
Epidemiological and clinical data from China, the United States and other countries suggest that younger children and adolescents may be affected differently by SARS-CoV-2 infection, with children younger than 10 years having milder symptoms. than adolescents.9,13 This phenomenon is consistent with the differences in antibody profile between younger children and adolescents observed in this study, with children showing significantly higher IgG and SNAb than adolescents.
It is unclear why patients aged 19 to 30 years showed lower levels of IgG antibodies against SARS-CoV-2 than children and older adults. An increase in SARS-CoV-2 antibody levels would have been expected with increasing age, as individuals expand their catalog of memory B and T cells through accumulated immunological memory.
Furthermore, the inflection of the SARS-CoV-2 antibody response would have been expected to occur at an older age, when the aging immune system fails to mount a robust response to new antigenic challenges.
One possibility may be the increase in comorbidities, such as obesity, hypertension or diabetes, commonly associated with increasing age in Western society. A multivariate analysis by Racine-Brzostek and colleagues 22 revealed that when adjusted for age, sex, race, and time from symptom onset to testing, SARS-CoV-2 IgG levels were significantly associated with obesity.
It could be postulated that a higher baseline level of inflammatory cytokines associated with such comorbidities could have a stimulatory effect on the SARS-CoV-2 humoral response. 23 The SARS-CoV-2 antibody avidity in this study showed the same pattern of negative correlation. with age than the levels of IgG, total and functional antibodies.
The present analysis demonstrated, for the first time to our knowledge, a significantly negative correlation between craving and age in the pediatric and young adult cohort. Although it did not reach a significant level which may be due to the limited sample size, children showed higher antibody binding avidity than young adults.
Because antibody avidity in this assay is a measure of the propensity of the antibody to remain bound to RBD, 24 higher avidity could increase the antiviral effects of antibodies in children, which could help mitigate clinical manifestations. in that group.
Measurement of antibody avidity could be of great value in understanding the functional efficiency of antibodies and providing information on antibody-mediated immune protection. It may also be useful in the selection of plasma donors for the treatment of COVID-19 and will be important for understanding the differences in responses elicited by different SARS CoV-2 vaccines.
| Limitations |
This study has limitations. First, there may be selection bias in terms of who is being tested for SARS CoV-2 antibodies. Because the study was retrospective and based on existing data, it relied on the ordering physician to justify SARS-CoV-2 antibody testing.
Children and adults were tested for several reasons, as mentioned above. Second, the time between disease onset and antibody testing was unknown for most patients, especially in asymptomatic patients. However, as serum samples were collected between April and August 2020 (a few months after the COVID-19 outbreak in New York City), patients who had positive SARS-CoV-2 serology were in their early recovery phase.
Furthermore, longitudinal samples were not available for patients and therefore dynamic changes in the magnitude and avidity of SARS-CoV-2 antibodies could not be investigated. Fourth, young children may not be able to describe their symptoms as clearly as adolescents or adults, and the study relied on parent reports.
| Conclusions |
The findings of this study suggest that there are distinct SARS-CoV-2 virus-specific antibody response profiles that vary by age, with younger children exhibiting higher levels of IgG, total, and functional antibody activity than younger children. adolescents and young adults.
The data could partly explain the lower overall rate of symptoms and cases of severe illness in children infected with SARS-CoV-2.
However, the lower incidence of symptoms and decreased disease severity in pediatric patients raises the possibility that this population may represent an important reservoir of viral transmission in the community . Therefore, increased screening of school-aged children, even those without obvious symptoms or exposure, may be an important step in curbing the pandemic.
Furthermore, these age-based differences in disease manifestations suggest the need for age-targeted treatment strategies. Measurements of antibody quantity and quality, as described here, could also help guide rational age-based vaccine choice and deployment.
