RESULTS:
This study was conducted at CMC Vellore with recruitment between November 2020 and August 2021. The sample size at 80% power and two-sided 5% significance to test a sample correlation coefficient (0.54) of IgG levels and time post-symptom onset against a population correlation coefficient of 0.72 required recruitment of 92 children. Total of 103 children were recruited, but 24 were excluded (loss to follow-up or missing data or insufficient blood for antibody testing). Samples for antibody testing from the cohort (N=79), were collected between December 2020-August 2021. Based on sequencing data from CMC Vellore, predominantly wild type (Wu Hu-1/D614G) or delta (B.1.617.2) were detected from June 2020-March 2021 and April 2021- August 2021 (end of the study period), respectively.18
Out of the total of 79 children recruited, 29% (23/79) were asymptomatic and incidentally detected as PCR positive and 59% (47/79) presented with mild-to-moderate disease not requiring intensive care. The remaining 11% (9/79) manifested severe disease at presentation including 2 children with a diagnosis with MIS-C. There was no mortality in our cohort. The month-wise distribution of date of respiratory and blood sample collection of study subjects is shown in Figure 1. Sixteen (20%) of the 79 children tested PCR positive during the wild-type wave and 59 (75%) during the Delta wave and corresponding sera collected within the period of circulation were designated “wild-type infected” and “delta infected”, respectively. Four samples PCR positive during the wild-type wave, had blood collected during the delta wave, were classified as ‘SARS CoV-2 infected’. We compared between variants among the “wild-type infected” (N=16) and “delta infected” (N=59) sera.
Baseline characteristics of children recruited in the study are presented in Table 1. The median (IQR) age of recruited subjects was 9(11) years. Wild type infection resulted in severe disease in 25% of children while majority of Delta infections (69%) were mild-to-moderate. The mean (SD) Ct value were 33.6 (3.4) and 26.3 (6.7) for wild-type and delta infection, respectively (P<0.0001). Wild-type infected sera showed 100% positivity to both anti-N and anti-S antibodies, while delta-infected sera showed 93 and 95% seropositivity, respectively. Anti-S levels were observed to be higher than anti-N levels (p<0.0001) in both wild-type and delta-infected sera.
We assessed, if difference in antibody levels observed, were due to viral load in the respiratory samples collected 4-6 weeks prior to blood collection. We stratified detected Ct value into quartiles - Q1(range 10.8-24.8), Q2(range 24.9-27.8), Q3 (range 27.9-32.7) and Q4 (range 32.8-42.1) and compared antibody levels across them. We found anti-S levels to be higher within each Ct quartile (p<0.001). When comparing levels between quartiles, we found that for both anti-N and anti-S antibody, Q1 levels were higher than Q3 (p=0.02 and 0.03, respectively) (Figure 2A). When comparing levels by variant, we found delta-infected sera (Figure 2B) to show significant difference in anti-S levels between Q1 and Q3 Ct quartiles . We also noted higher anti-S levels at Q3 loads between wild-type and delta-infected sera (Figure 2B). These results suggest a viral load driver of antibody response and a differential antibody reactivity between wild-type and delta variants.
We further examined if the observed difference in Ct value between wild-type and delta varied by clinical severity at presentation. The mean(SD) Ct of SARS CoV-2 in respiratory samples among asymptomatic and mild-to-moderate disease was 30.7(4.8) and 26.3(6.8) (p=0.03), respectively, while among severe disease was 29.7(8.5). Comparing between variants, we found higher mean (SD) Ctvalues of wild-type [32.5 (4.5)] than Delta [25.0(6.4)] in mild-to-moderate disease (ANOVA; pairwise comparison p=0.0083) (Figure 3). No difference in wild-type Ct was seen across severity strata (Figure 3). These results indicate that for delta infections a higher viral load (lower Ct) was a driver of illness severity in mild-to-moderate disease. Notably, the Ctvalue range for delta in respiratory samples in our cohort was 10.7-37.7 [~log10(9) difference] among mild-to-moderate disease (Figure 3).
We further explored antibody levels across severity strata and found higher anti-S levels compared to anti-N among asymptomatic [median (IQR) 119.4 (200) and 18.6(66.4), p=0.02] and mild-to-moderate disease [176.6(147.8) and 50.2(68.9)], p<0.0001], but similar levels of anti-N and anti-S among severe disease (Figure 4A). When comparing levels by variant, we found only delta-infected sera to show differential levels of anti-N and anti-S only for mild-to-moderate disease [160 (155.1) versus 59.1 (71.0) , p=0.003], but not other strata (Figure 4B). In the severe disease strata, higher levels of anti-S among wild type sera compared to delta sera were seen (P=0.02). These suggest a skewing of antibody response towards S in asymptomatic and mild-to-moderate infections as well as a differential anti-S response between variants.