Pediatric humoral immune responses and infection risk after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and two-dose vaccination during SARS-CoV-2 omicron BA.5 and BN.1 variants predominance in South Korea

Background Humoral immune responses and infection risk after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) vaccination during the Omicron BA.5 and BN.1 variants predominant period remains unexplored in pediatric population. Methods We examined anti-spike (anti-S) immunoglobulin G (IgG) responses in a total of 986 children aged 4−18 years who visited outpatient clinics between June 2022 and January 2023, with a history of SARS-CoV-2 infection alone, completed two doses of COVID-19 vaccination alone, vaccine-breakthrough infection (i.e., infection after the single dose of vaccination), and no antigenic exposure. Furthermore, to determine SARS-CoV-2 infection risk, the incidence of newly developed SARS-CoV-2 infection was investigated up to March 2023. Results The anti-S IgG levels in the ‘vaccine-breakthrough infection’ group exceeded those in the ‘infection alone’ and ‘vaccination alone’ groups (both P <0.01). Furthermore, the ‘vaccination alone’ group experienced more rapid anti-S IgG waning than the ‘infection alone’ and ‘vaccine-breakthrough infection’ groups (both P <0.01). We could not identify newly developed SARS-CoV-2 infection in the ‘vaccine-breakthrough infection’ group. Conclusion Our findings suggest that hybrid immunity, acquired from SARS-CoV-2 infection and COVID-19 vaccination, was a potentially higher and longer-lasting humoral immune response and protected against SARS-CoV-2 infection in pediatric population during Omicron BA.5 and BN.1 variants predominant.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus that causes coronavirus disease 2019 (COVID-19) (1).South Korea, located in eastern Asia, has a population of 51.4 million, and it achieved significant success in mitigating the COVID-19 pandemic via public health and social measures alone without lockdown during the 2020-2021 period (2,

Study design
A cross-sectional serosurvey was conducted between 16 June 2022 and 5 January 2023.Serum samples were conveniently collected from individuals aged 4-18 years who visited the outpatient clinics of four national regional pediatric hospitals located in Jeollanam, Jeollabuk, Gyeongsangsnam, and Gyeongsangsbuk provinces.
Children were invited to participate alongside an adult legal guardian.All guardians who had consented to participate were asked to respond to an in-person survey administered by trained staff and allow their children to provide blood specimens, which were collected at the respective pediatric hospitals.The exclusion criteria were as follows: (1) the presence of any respiratory symptom, (2) a history of COVID-19-related home quarantine, and/or (3) any contact history with a COVID-19 case within the preceding 14 days.Participant data included age, sex, comorbidities, history of previous SARS-CoV-2 infection, and COVID-19 vaccination status.Furthermore, with approval from the study participants, the specific, individual dates of previous SARS-CoV-2 infections and COVID-19 vaccinations were extracted from the national infectious disease notification and vaccine registry database of the Korea Disease Control and Prevention Agency to avoid recall bias.By that time, mRNA vaccines (BNT161b2) had been administered to pediatric population in South Korea, and each dose was recorded 14 days after administration.This study was approved by the institutional review boards of Chonnam National University Hospital (IRB No. CNUH-2022-118), Chonbuk National University Hospital (IRB No. CUH-2022-06-031-007), Kyungpook National University Chilgok Hospital (IRB No. KNUCH-2022-05-008-001), and Pusan National University Hospital (IRB No. PNUH-05-2022-103).Informed consent was obtained from all study participants (over 5-year-old) and all study participants' guardians.

Laboratory immunoassay
To detect anti-SARS-CoV-2 antibodies, two commercially available anti-SARS-CoV-2 immunoglobulin immunoassays were employed.The Elecsys ® Anti-SARS-CoV-2 N immunoassay (Roche Diagnostics International Ltd., Rotkreuz, Switzerland), with a sensitivity of 99.8% and specificity of 99.1% (14-16), was used to detect antibodies against the SARS-CoV-2 nucleocapsid protein (N), which are known to develop during a natural infection only (17).Therefore, anti-N IgG was measured to identify the previous SARS-CoV-2 infection in this study.Similarly, to measure antibodies against the SARS-CoV-2 spike protein (S), which are known to develop from both natural SARS-CoV-2 infection and COVID-19 vaccination (18), the Elecsys ® Anti-SARS-CoV-2 S immunoassay (Roche Diagnostics International Ltd., Rotkreuz, Switzerland), which detects the immunoglobulin specific for the S1 domain of the SARS-CoV-2 spike protein with a sensitivity of 99.6% and specificity of 99.8%, was used (15,19).Both assays ran on the Cobas ® e801 analyzer (Roche Diagnostics International Ltd., Rotkreuz, Switzerland).The anti-N IgG immunoassay results were produced as a cut-off index (COI), where results ≥1.00 COI were considered positive.The anti-N and anti-S IgG measurement range were 1.00-125 COI, and 0.40-250 U/mL, respectively.Values lower than the limit of quantification was reported as <0.4 U/mL.Values above the upper limit were diluted up to 50-fold and reported as >12,500 U/mL.All tests were performed according to the manufacturer's instructions.

Temporal patterns of anti-SARS-CoV-2 IgG response with infection alone, vaccination alone, and vaccine breakthrough infection groups
This study's primary outcome of interest was the SARS-CoV-2 anti-S IgG level observed over 365 days post-COVID-19 vaccination/SARS-CoV-2 infection (i.e., the date of the last vaccination or that of laboratory-confirmed infection, respectively).Serum anti-S IgG levels were compared among the following three groups: (1) 'infection alone' group (i.e., those who had a previous history of SARS-CoV-2 infection and did not receive COVID-19 vaccination), (2) 'vaccination alone' group (i.e., those who had no history of SARS-CoV-2 infection but received two doses of COVID-19 vaccination), and (3) 'vaccine-breakthrough infection' group (i.e., those who were infected with SARS-CoV-2 after a single dose of mRNA vaccine).
Considering the small number of participants who had received a single vaccine dose after infection (n=16) and a booster dose (i.e., third dose) of the COVID-19 vaccine (n=41), only those who had received the second vaccine dose (n=148) were considered.
To identify the different temporal kinetics among the three groups, we assessed the temporal trend of anti-S IgG values in samples.The smoothing spline was applied to the anti-S IgG values with the function of the last antigenic exposure date to visualize the temporal trends.Furthermore, to compare the anti-S IgG decay kinetics among the three groups, a regression model interaction term of vaccine/ infection history and sampling time was conducted.The coefficient obtained from the model were used to estimate the waning rates (11).

Adjusted comparisons of anti-S IgG response with infection alone, vaccination alone, vaccine-breakthrough infection groups
Adjusted and unadjusted comparisons of anti-S IgG among the three groups were conducted using univariable and multivariable linear regressions.These regressions considered possible predictors of the humoral immune response of anti-S IgG to SARS-CoV-2 infection and/or vaccination in the study population.To normalize data, anti-S IgG levels were converted to log 10 values, and coefficients were subsequently exponentiated to enable result interpretation.The predictors included sex, age, comorbidities, and serum-specimen sampling time (i.e., time from the date of serum-specimen collection to that of last COVID-19 vaccination or laboratory-confirmed date of SARS-CoV-2 infection).

Comparison of newly developed SARS-CoV-2 infection to infection alone, vaccination alone, and no antigenic exposure groups
To assess SARS-CoV-2 infection risk after previous infection and/or vaccination, newly confirmed COVID-19 cases among the study participants were evaluated up to 15 March 2023 using national infectious disease notification registry.Thereafter, logistic regression models were used to assess the association between predictor variables and SARS-CoV-2 infection risk.The covariates included in the models were: age, sex, region, comorbidity, antigenic exposure, and observation period (i.e., the time after sample collection by 15 March 2023).We defined the individuals who had no previous history of SARS-CoV-2 infection and COVID-19 vaccination with negative anti-S and anti-N IgG as a 'no antigenic exposure group'.In the 'antigenic exposure' group, the 'vaccine-breakthrough infection' category did not record any new infections; hence, it was not included in the model.Crude and adjusted coefficients with 95% confidence intervals (CIs) were subsequently estimated.
Descriptive statistics for demographic characteristics were calculated with Fisher exact test.Mann-Whiteney U test and Kruskal Walis variance analysis were used to examine the differences of the data.A P-value < 0.05 was considered to indicate statistical significance in all analyses.All statistical analyses were performed in R (version 4.0.1 for Windows, R Foundation for Statistical, Computing, Vienna, Austria).

Discussion
To the best of our knowledge, this is the first real-world study comparing the protective effectiveness in the pediatric population of immunity acquired from the two doses of COVID-19 vaccination and infection after the single dose of vaccination against SARS-CoV-2 infection during Omicron BA.5 and BN.1 variant predominant period.
Our analysis showed that more rapid decay IgG kinetics were observed for those in the vaccination-alone group compared to the infection-alone group.However, anti-S IgG titer in the population who received vaccination was larger than that of the SARS-CoV-2 infection alone group.This finding indicates that hybrid immunity acquired from both COVID-19 vaccination and SARS-CoV-2 infection potentially elicits greater humoral immunogenicity and longer durability than either two-dose COVID-19 vaccination alone or SARS-CoV-2 infection alone in the pediatric population.
Our findings also identified that children with two-dose mRNA vaccination did not exhibit statistically significant protection against SARS-CoV-2 infection during the Omicron BA.5 and BN.1 variant circulating period, compared with those with infection alone and no antigenic exposure group.This finding is in line with a previous study demonstrating that a single dose mRNA vaccination in the pediatric population did not confer significant protection against Omicron BA.4 and BA5 infection (20).
In this study, we could not identify newly developed SARS-CoV-2 infection in the 'vaccine-breakthrough infection' group.This is likely due to the higher humoral immune response after vaccination and infection than the vaccination-only or infectiononly group (21,22).Therefore, our finding provides insights into  hybrid immunity, which affords a more robust protective effect against SARS-CoV-2 during Omicron BA.5 and BN.1 predominant period compared to immunity induced solely by infection and a two-dose vaccination.This interpretation is supported by previous studies that demonstrated the hybrid immune response provided superior protection against the Delta and Omicron (BA.1 and BA.2) variant than infection-or a single-dose vaccine-induced immunity alone (9,21,(23)(24)(25).However, this also should be interpreted cautiously as COVID-19 vaccination reduces Omicron-associated hospitalization and severity of illness in the pediatric population (26).
We identified that 82% of the study population had an anti-S IgG response, which was positively associated with COVID-19 vaccination (27).This finding is in line with 89% of the child population had booster doses immunization in South Korea as of December 2022 (28).We also identified that 74% of this study's population had an anti-N IgG response, which is known to be positively associated with previous SARS-CoV-2 infection (Supplementary Figure 1; Supplementary Figure 3).This indicates  that a vast proportion of children had an immune response against natural SARS-CoV-2 infection.In this study, based on the anti-N seroprevalence and SARS-CoV-2 infection notification registry database in South Korea, 9.5% (95% CI: 7.8%−11.5%) of individuals did not report their SARS-CoV-2 infection to public health authorities.This finding is consistent with that of a previous report wherein 66% of the child population in South Korea was infected by SARS-CoV-2 between January 2020 and June 2022 (29).This finding needs to be interpreted cautiously owing to anti-N antibody waning, which reportedly persists for approximately 1 year after infection (30).However, in this study, we found that the temporal trend of the median anti-N IgG titer after SARS-CoV-2 infection exhibited a decreasing trend after 6 months but persisted for more than 11 months (Figure 3A), and a sizeable proportion of children in South Korea were infected after January 2022 during the Omicron wave.A previous study suggested that the decay rate of anti-N IgG was different in elderly (31).However, we could not identify any statistical difference in anti-N IgG distributions between the different age groups in our study (Figure 3B).Previous studies have revealed differences between vaccinationinduced and SARS-CoV-2-induced seroprevalence across varying demographics (13,32).However, this study could not identify any significant sex-and age-related disparities in anti-S and anti-N IgG possibly because of the considerable number of children who had been infected by SARS-CoV-2 and had undergone COVID-19 vaccination in South Korea.
The key strength of this study is that it is the first pediatric population-based study that included a two-dose COVID-19 vaccination population and estimated infection risk during Omicron BA.5 and BN.1 predominant period.Furthermore, we used the South Korean national SARS-CoV-2 infection notification database, where the extended community COVID-19 screening center operated during the study period (4), and this is likely to  provide improved estimates of the SARS-CoV-2 infection risk.However, it also has several limitations.First, the information of exact timing of SARS-CoV-2 infection is limited (33).Therefore, the reported anti-N and anti-S IgG titers could have been affected by the time of report.Second, the study population might have been biased because individuals who visited the medical facilities for sample collection were likely to be more health conscious.Furthermore, the study population might not represent the healthy pediatric population as the population was mainly recruited in outpatient clinics, and 52% had comorbidities.Therefore, a seroprevalence study using nationally representative samples of the general pediatric population may overcome this limitation.Third, we did  not conduct neutralization assays to support our findings further.However, electrochemiluminescence immunoassay, which we conducted in this study, has strong positive correlation with neuralization assay (34,35) and been widely used in serosurvey of large populations (11,13,19,36).Fourth, cell-mediated immunity, which entails an immune response not involving antibodies, plays a major role in SARS-CoV-2 infection (37).However, this study could not include this aspect during infection risk estimations.Fifth, public health and social measures implemented may affect the infection risk.However, most of measures were relaxed in South Korea during the study period (5).

B A
In summary, our results suggested that hybrid immunity acquired from both SARS-CoV-2 infection and COVID-19 vaccination could provide a higher and more persistent effect humoral immune response against SARS-CoV-2 Omicron infection than infection-induced or vaccine-induced immunity alone.Furthermore, during the predominance period of SARS-CoV-2 Omicron BA.5 and BN.1 variants, hybrid immunity conferred better protection against the SARS-CoV-2 infection compared to that of infection-induced or vaccine-induced immunity alone.Further studies are required to assess longerterm protection against infection by novel SARS-CoV-2 variants using additional COVID-19 booster vaccination.

1
FIGURE 1 Temporal kinetics of anti-SARS-CoV-2 spike protein (anti-S) IgG by different types of antigenic exposures by time since SARS-CoV-2 infection or COVID-19 vaccination.Dots represent anti-S IgG values induced by (A) SARS-CoV-2 infection alone, (B) COVID-19 vaccination alone, and (C) vaccine breakthrough infection.The thick curve indicates the trend in anti-Spike IgG using smoothing splines with a 95% confidence interval (shaded area).

FIGURE 2
FIGURE 2Unadjusted comparison of anti-S IgG by category of SARS-CoV-2 infection and/or COVID-19 vaccination.P-values determined using the Kruskal-Wallis test.The comparisons do not adjust for sampling time, which is different for every group.The box plots denote median, the 25th percentile, and the 75th percentile of the anti-S (y-axis) and each group (x-axis) representing SARS-CoV-2 infection alone (light yellow), vaccination alone (light green), and vaccine breakthrough infection (pink).
Temporal kinetics of anti-SARS-CoV-2 nucleocapsid (anti-N) IgG in SARS-CoV-2 infected groups (n=536) and comparison of anti-N IgG by different age groups.Dots represent an individual sample, and horizontal lines in the box indicate the median value with the interquartile range with (A) time in months from infection to collect samples and (B) different age groups.P-values were determined using the Kruskal-Wallis test.Abbreviation: COI, cut-off index.

TABLE 1
Clinical and demographic characteristics of study participants by SARS-CoV-2 and vaccination history.

TABLE 2
Longitudinal linear mixed modelling to estimate decay kinetics of log 10 anti-S IgG response to antigenic exposure .
a Reference: Infection alone group.bSamplingtime refers to the last antigenic exposure (either time since the final vaccine was received or infection, whatever is latest).cThe× indicates interactions among the variables.

TABLE 3
Association between anti-S IgG, and demographic factors, type of antigenic exposure and sampling time in child population, South Korea.
a Adjusted for age group, sex, region, comorbidity, group, and sampling time.b Others: Cardiovascular, endocrine, respiratory, hepatorenal, malignant, neurological psychological diseases.c Sampling time refers to last antigenic exposure (time since final dose of vaccine or infection, whatever is latest).

TABLE 4
Demographics of newly developed COVID-19 in child population during the observation period (time between sample collection and 25 May 2023) in South Korea.

TABLE 4 Continued
b Observation period refers to the time after sampling collection by 15 March 2023.

TABLE 5
Association between newly confirmed SARS-CoV-2 infection risk and covariates including demographic factors, type of antigenic exposure and sampling time in pediatric population, South Korea.

TABLE 5 Continued
CI, confidence interval.a Adjusted for age group, sex, region, comorbidity, antigenic exposure, sampling time, and observation period.b Others: Cardiovascular, endocrine, respiratory, hepatorenal, malignant, neurological, and psychological diseases.c Sampling time refers to last antigenic exposure (time since final dose of vaccine or infection, whatever is latest).d Observation period refers to the time after sampling collection by 15 March 2023.