DISCUSSION:
We recruited a cohort of SARS CoV-2 naturally-infected children under 16 years across the infection spectrum (asymptomatic to severe disease) spanning the wild-type (Wu Hu-1/D614G) and Delta (B.1.617.2) infection waves in Vellore and examined the relationship of viral load, anti-nucleoprotein (anti-N) and spike (anti-S) antibodies in them. The sampling design allowed a comparison of dynamics of natural infection between variants (wild-type and delta). We showed that variants (delta) with increased replicative fitness demonstrated increased viral loads in mild-to-moderate disease and contributed to a robust antibody response with a skew towards anti-S. An impaired anti-S immune response was seen in severe disease.
Our study showed evidence of viral load driving antibody response. For both anti-N and anti-S, higher viral load (Q1), resulted in higher antibody levels compared to lower (Q3) loads. In the natural history of SARS CoV-2 infection, virus levels reduce over time due to development of virus-specific immunity14 that enhances virus clearance. Lower Ct values are associated with increased peak of IgG levels. However, children mount a lower antibody responses compared to adults,15,19 possibly due to continual innate immune stimulation and lower viral replication from pre-existing coronavirus immunity.20 Higher viral loads (e.g. Q1), may result in inefficient innate immune clearance , thus stimulating a stronger adaptive response, manifesting as higher antibody levels. Low viral levels (Q3), on the other hand, could result in efficient virus clearance by the innate immunity resulting in lower activation of adaptive responses. We show that delta exhibiting increased replication efficiency as a driver of immune response.2,21,22
We observed a difference in anti-S levels between wild-type and delta among quartile 3. This indicates differing dynamics between variants.22.The viral loads [mean (SD) Ct] in respiratory samples (Table 1) was 33.6 (3.4) and 26.3(6.7) for wild-type and delta, respectively, representing a ~100-fold difference (Ctdifference of 3.3 ~log10(1) viral load difference). Sampling earlier in the illness course leads to lower observed Ct values.23 We excluded sampling date as a cause of low Ct as children in our cohort were sampled median 2 days post-symptom-onset for both wild and delta infections. While Ct values are reported to be similar between asymptomatic and symptomatic infections in children,9 we found viral loads (Ct value) in delta infections (Fig 3) to be ~50X (Ctdifference 4.8) higher in mild-to-moderate disease compared to asymptomatic infections. We found virus loads for delta were ~1000X higher (Ct difference 9.5) than wild-type in mild-to-moderate disease similar to previous reports.24 This asynchrony in Ct seen in delta is noteworthy as clinical severity for wild and delta variants in children were similar, as has been shown previously.25 When Ct value is used as a metric of replication kinetics,24,26 a higher load observed in delta compared to wild-type suggests improved replication fitness in the former.
In adults with severe disease, antibodies (including nAbs) produced are abundant and have increased reactivity to all viral proteins (breadth) compared to mild disease.22 In children however, antibody levels are similar between mild and severe disease27 and importantly skewed towards anti-spike antibodies.15 Higher anti-S antibody compared to anti-N, a characteristic seen among children15 is recapitulated in our cohort. Varying half-life dynamics between anti-S IgG (145 days) versus anti-N IgG (86 days)28 could partly explain differential levels observed in both wild-type and delta sera. The absence of skewing seen among severe disease could be due to a lowered anti-S or elevated anti-N levels. A higher anti-N levels are associated with poorer outcome.29 This was also seen in MIS-C compared to minimal or severe disease.30Elevated anti-N levels have been seen in antibody-dependent enhancement (ADE), which causes robust inflammatory response and increased viremia29 resulting in severe disease . Anti-S is considered protective13 and poor antibody response is associated with severe COVID-19 in children.31 The lowered levels suggest an impairment of response towards the spike, indicating a dysregulated immune response, as is also seen in MIS-C.6 This is in contrast to adults who mount robust responses after severe disease.14,15,32
Through the pandemic, majority of the under-12 children globally have acquired SARS CoV-2 immunity by natural infection. Understanding the dynamics of virus replication, clinical disease and antibody response in natural infection in children has implications for spread of future variants and potentially informing targeted vaccination when widely approved for use in children (under 12 years of age). Infection by future variants are likely to result in a robust immune response but higher replication fitness may lead to increased transmissibility. Our finding of an impaired anti-spike response among children with severe disease is a cause for concern. Hybrid immunity resulting from vaccination would likely boost protection, especially against these future variants.33 Omicron (including subvariants BA.4/5) which are more immune-evasive, have increased ACE2-binding affinity and replication fitness than Delta3. Repeated exposure to spike protein will provide an improved breadth of protection against newer emerging variants.34
We note a few limitations of the study. Inclusion of adults would have allowed direct comparison of immune dynamics with that in children. Immune response to SARS-CoV-2 initiates 7-10 days after symptom onset and reaches a maximum 2-4 weeks after illness. No sampling was conducted in the early stages of infection to assess early immune dynamics. Neutralizing antibodies and cellular immunity were not assessed due to cost constraints.
CONCLUSION: We compared the dynamics of antibody response at 4-6 weeks after PCR confirmation across SARS CoV-2 variants (wild-type, delta), virus load and disease spectrum. Our study reveals the differing dynamics of SARS CoV-2 infection in children with respiratory virus loads driving both anti-N and anti-S antibody levels and skewed, protein-specific antibody response across severity strata, with impaired anti-spike immunity in severe disease .
ACKNOWLEDGEMENTS: AJP acknowledges CMC Research office for intramural research funds. MM acknowledges Department of Biotechnology, India, for financial support through grant, BT/PR40390/COT/142/1/2020.
CONFLICTS OF INTEREST: The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT: Original data can be accessed upon request.
ETHICAL APPROVAL: The Institutional Review Board of CMC, Vellore (IRB No. 13700)