key: cord-313379-6sa6oc6u
authors: Bahar, B.; Jacquot, C.; Mo, Y. D.; DeBiasi, R.; Delaney, M.
title: Kinetics of viral clearance and antibody production across age groups in SARS-CoV-2 infected children
date: 2020-08-07
journal: nan
DOI: 10.1101/2020.08.06.20162446
sha: 
doc_id: 313379
cord_uid: 6sa6oc6u

Objectives: To improve understanding of transition from viral infection to viral clearance, and antibody response in pediatric patients with SARS-CoV-2 infection. Study design: This retrospective analysis of children tested for SARS-CoV-2 by RT-PCR and IgG antibody at a quaternary-care, free-standing pediatric hospital between March 13th, 2020 to June 21st, 2020 included 6369 patients who underwent PCR testing and 215 patients who underwent antibody testing. During the initial study period, testing focused primarily on symptomatic children; the later study period included asymptomatic patients who underwent testing as preadmission or preprocedural screening. We report the proportion of positive and negative tests, time to viral clearance, and time to seropositivity. Results: The rate of positivity varied over time due to viral circulation in the community and transition from targeted testing of symptomatic patients to more universal screening of hospitalized patients. Median duration of viral shedding (RT-PCR positivity) was 19.5 days and RT-PCR negativity from positivity was 25 days. Of note, patients aged 6 to 15 years demonstrated a longer period of RT-PCR negativity from positivity, compared to patients aged 16 to 22 years (median=32 versus 18 days, p=0.015). Median time to seropositivity from RT-PCR positivity was 18 days while median time to reach adequate levels of neutralizing antibodies (defined as equivalent to 160 titer) was 36 days. Conclusions: The majority of patients demonstrated a prolonged period of viral shedding after infection with SARS CoV-2. Whether this correlates with persistent infectivity is unknown. Only 17 of 33 patients demonstrated neutralizing antibodies, suggesting that some patients may not mount significant immune responses to infection. It remains unknown if IgG antibody production correlates with immunity and how long measurable antibodies persist and protect against future infection.

cell fusion of SARS-CoV-2 is mediated by the trimeric structure of the two functional subunits of 79 the S protein, namely S1 and S2, after binding to angiotensin-converting enzyme 2 (ACE2). 5 80 Antibodies formed against the receptor binding domain (RBD) on the S1 subunit have the 81 potential to neutralize SARS-CoV-2 by disabling virus-ACE2 binding and endocytosis. 6, 7 In 82 addition, the competitive RBD binding capacity of these antibodies versus ACE2 correlates with 83 neutralizing activity. COVID-19 from China. 8 The authors found that the median age was seven years, greater than 87 90% of the cases had a disease spectrum ranging from asymptomatic to moderate disease, and 88 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint the proportion of severe and critical cases increased with age. 8 Similar findings have 89 subsequently been reported from Europe, the middle east, and the US. 9-12 90 91 Although there is emerging data regarding timing of viral clearance and immunological 92 response in adults with COVID-19 13 , there is a scarcity of data in the pediatric population. 93

Furthermore, a lack of knowledge regarding factors affecting time-to-seropositivity is observed 94 in both pediatric and adult patients. 14 We report viral and antibody testing results from our 95 pediatric patient population in order to contribute to a better understanding of timing of viral 96 clearance and antibody production in children with In addition to the RT-PCR results, qualitative and quantitative serologic testing results, age and 107 sex were also included in the data extracts. Age stratifications were defined as 0 to 5 years, 6 to 108 15 years and 16 to 22 years (see supplemental figure S1 for kernel density estimate of age). 109 110 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint Nasopharyngeal samples were collected from patients. Samples were transferred to the 111 laboratory in viral transport medium as recommended by the manufacturer or in liquid Amies 112 medium as validated by the laboratory. For detection of the virus by RT-PCR, four systems were 113 utilized due to high testing volume, which were (1) GenMark ePlex SARS-CoV-2 Test, (2 CoV-2. The ORF1ab region encodes well-conserved non-structural proteins and therefore is less 131 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 7, 2020. Antibody detection was performed from serum or plasma samples, collected in appropriate 149 separator tubes, using DiaSorin Liaison XL SARS-CoV-2 IgG S1/S2 assay (DiaSorin, Saluggia, Italy). 150

We validated the analytical and clinical performance of this assay with similar outcomes as 151 other researchers who previously reported satisfactory results. 16, 17 The test is based on 152 chemiluminescent detection of antibodies against S1 and S2 glycoproteins of the virus using 153 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint magnetic beads. Seropositivity was defined at presence of anti-SARS-CoV-2 IgG antibodies ³ 15 154 absorbance units per milliliters (AU/mL), as recommended by the manufacturer. In addition to 155 the qualitative results, quantitative test results were also included to reflect the amount of 156 circulating IgG antibodies in patient samples at the time of blood draw. As demonstrated by the 157 manufacturer, antibody results ³ 80 AU/mL were comparable to a titer of 160 by plaque 158 reduction neutralization testing (Liaison SARS-CoV-2 S1/S2 IgG (REF 311450)). According to 159 these criteria, results were grouped as 'adequate for neutralization' and 'not adequate for 160 neutralization', based on the 80 AU/mL threshold. In addition to routine testing ordered by 161 clinical providers (n=194), we retrieved available leftover serum or plasma samples from 162 patients who underwent RT-PCR testing but were not tested for antibodies (n=19). Serologic 163 testing was also performed on these samples and their results were included in the present 164 study. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint age groups did not demonstrate a significant difference (χ 2 =1.6, p=0.4) (Figure 4c ). After 219 adjustment for sex, the various age groups also did not demonstrate significant differences in 220 time to seropositivity (χ 2 =0.6, p=0.7). Only 17 of 33 patients demonstrated antibody levels ³ 80 221 AU/mL. The median time to reach adequate levels of neutralizing antibodies as defined by the 222 manufacturer and this study was 36 days (95% CI=18-NA) (Figure 5a) . No significance was found 223 for sex (χ 2 =1.1, p=0.3) (Figure 5b) , age (χ 2 =0.9, p=0.6), or age stratified for sex (χ 2 =1.7, p=0. In this study, we demonstrated that IgG class antibodies directed against S1 and S2 228 glycoproteins could be detected in blood samples of children before viral clearance. Previous 229 studies revealed that antibodies bound to the RBD epitope of SARS-CoV-2's S1 glycoprotein are 230 able to disrupt the virus-ACE2 interaction, thus blocking viral entry into human cells and 231 enabling neutralizing capacity to the antibodies. 6 While the RBD is located on the S1 subunit, 232 the S2 subunit plays a crucial role in membrane fusion of the virus by conformation changes. 18, 233 19 It was previously hypothesized for SARS-CoV that multiple antibodies targeting different 234 epitopes might act synergistically. 20, 21 As noted earlier, the antibody detection assay utilized in 235 this study does not measure only antibodies targeting RBD but includes all antibodies to 236 epitopes on S1 and S2 glycoproteins. We believe that this assay design may be beneficial in 237 assessing antibody response in individuals with a polyclonal immune response to both S1 and 238 S2 antigens with synergistic anti-viral activity. 239 240 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. antibodies with an increase in neutralization activity over time. 23 The authors reported that the 248 variables associated with high neutralizing activity in a multivariate model included time to 249 blood sampling from symptom onset, high BMI and male sex. 23 We demonstrated that females 250 between the ages of 6 to 15 years required a longer period for viral clearance when compared 251 to other groups. This may be due to the age-dependent expression of ACE2 in the nasal 252 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint A strength of our study was the inclusion of patients from multiple pediatric age groups with 262 sequential PCR testing, which allowed comparison between age groups and sex serologic assays for SARS-CoV-2 are still in early phases of development. As of July 26, 2020, no 279 commercial assay was approved by FDA for quantitative reporting of the results. 15 In the 280 present study, we showed that time to reach a quantitative result corresponding to an antibody 281 titer of 160 was associated with time to viral clearance. This titer level has also been 282 recommended by the FDA to identify potential convalescent plasma donors. However, it has 283 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. In summary, given the significant volume of testing performed, we provide a timeline of viral 300 clearance and humoral response to COVID-19 in pediatric patients with additional comparisons 301 amongst age groups and sex. We demonstrated that females between 6 to 15 years of age 302 experienced longer persistence of viral genome in nasopharyngeal samples. It should be noted 303 that presence of viral genome may not correlate with transmissibility. Antibodies were 304 detectable in low titer preceding viral clearance. The timing of antibodies reaching titers that 305 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 7, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted August 7, 2020. . https://doi.org/10.1101/2020.08.06.20162446 doi: medRxiv preprint

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