key: cord-0977342-fckrx3ep
authors: Thiruvengadam, R.; Chattopadhyay, S.; Mehdi, F.; Desiraju, B. K.; Chaudhuri, S.; Singh, S.; Bhartia, V.; Kshetrapal, P.; Natchu, U. C. M.; Wadhwa, N.; Sopory, S.; Gosain, M.; Pandey, A. K.; Das, A.; Anand, N.; Sharma, N.; Sharma, P.; Saxena, S.; Sindhu, D.; Sindhu, B.; Sharma, D.; Dang, N.; Batra, G.; Kang, G.; Bhatnagar, S.; Research, DBT India Consortium for COVID-19
title: Longitudinal serology in SARS-CoV-2 infected individuals in India - a prospective cohort study
date: 2021-02-08
journal: nan
DOI: 10.1101/2021.02.04.21251140
sha: c1af23766ecdd922a3de5ab7a4f9a09386b64e6c
doc_id: 977342
cord_uid: fckrx3ep

Clinical and epidemiological characteristics of SARS-CoV-2 infection are now widely available, but there are little data on longitudinal serology in large cohorts, particularly from low-and middle-income countries. We established an ongoing prospective cohort of 3840 SARS-CoV-2 RT-PCR positive individuals in the Delhi-National Capital Region of India, to document clinical and immunological characteristics during illness and convalescence. The IgG responses to the receptor-binding domain (RBD) and nucleocapsid were assessed at 0-7, 10-28 days, and 6-10 weeks after infection. The clinical predictors of seroconversion were identified by multivariable regression analysis. The seroconversion rates in the post-infection windows of 0-7 days, 10-28 days, and 6-10 weeks were 46%, 84.7%, and 85.3% respectively (n=782). The proportion with a serological response increased with the severity of COVID-19 disease. All participants with severe disease, 89.6% with mild to moderate infection, and 77.3% of asymptomatic participants had IgG antibodies to the RBD antigen. The threshold values in the nasopharyngeal viral RNA RT-PCR in a subset of asymptomatic and symptomatic seroconverters were comparable (p-value: 0.48), with similar results among non-seroconverters (p-value: 0.16) (n=169). This is the first report of longitudinal humoral immune responses to SARS-CoV-2 infection over a period of ten weeks from South Asia. The low seropositivity in asymptomatic participants and differences between assays highlight the importance of contextualizing the understanding of population serosurveys.

Coronavirus disease 2019 , caused by SARS-CoV-2, has emerged as one of the most significant global public health challenges in the twenty-first century. The clinical phenotype of SARS-CoV-2 infection varies across a spectrum of asymptomatic to mild (moderate) illness to severe COVID- 19 We measured IgG antibodies against SARS-CoV-2 RBD and NC protein in participants enrolled into a multi-hospital based prospective cohort from northern India up to at least six weeks post-infection and correlated the clinical and demographic differences between seroconverters and non-seroconverters.

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A trained research team collected clinical data and biospecimens at enrolment and follow-up. The demography, clinical characteristics focussed on symptoms, comorbidities, drug history, and treatment details were collected by electronic data capture based on a priori developed SOP. Venous blood samples were collected, and transported following biosafety protocols recommended by the Government of India and the World Health Organization [4] . Serum was separated and stored in the biorepository at THSTI for subsequent analyses.

Anti-RBD IgG ELISA:

Anti-SARS-CoV-2 RBD IgG antibody was detected using an ELISA method as described earlier [5] . Briefly, samples and controls were diluted to 1:50 in the sample diluent, and 100μl was added per well of stabilized RBD coated plate. After 30 min of incubation at room temperature (RT) (23±2°C), the plates were washed six times with wash buffer. Fifty μ l of ready to use conjugate (HRP-labelled Goat anti-Human IgG Fcγ specific antibody) was added per well and incubated for 30 min at RT, followed by six washes. Hundred μ l of TMB substrate was added to each well, incubated at RT for 10 minutes and the reaction was stopped with 100 μ l per well of stop solution. The absorbance was measured on a microplate reader at 450 nm with 650 nm as a reference wavelength. The pooled negative control and pooled positive control were run on each plate. The cut-off from each plate was calculated by taking the average OD of triplicate of negative control and the addition of 0.2 to this value. Signal to the cut-off . 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 doi: medRxiv preprint ratio (S/Co) was calculated as the ratio of OD value from the test sample to the cut-off value. S/Co ratio of ≥ 1 was considered positive.

Anti-NC IgG Chemiluminescence assay:

The anti-NC IgG chemiluminescence assay (SARS-CoV-2 IgG) by Abbott Diagnostics Division, Ireland, was performed as per manufacturer's instructions, with calibrator and positive and negative controls run before each batch of antibody testing as per manufacturer's protocol. The results are reported by dividing the chemiluminescent signal from sample by the mean of chemiluminescent signals from three calibrator replicates. The default result unit is Index (S/C) which above a cut-off of 1.4 is considered positive.

Seroconverters were defined as those who tested seropositive by either of the assay at least once during the study period. The clinical phenotypes of COVID-19 were stratified into severe, mild to moderate and asymptomatic based on their most severe symptoms and/or the need for treatment at enrolment or at any time in their follow up period.

Participants were considered to have severe COVID-19 if they died or required either of the following, oxygen supplementation or ventilatory support, or treatment in the intensive care unit for cardiopulmonary or multiorgan dysfunction. Mild to moderate disease was defined by the presence of symptoms of COVID-19 but not fulfilling the criteria for severe disease. Those who did not report any symptoms at enrolment or throughout the follow-up period were classified as asymptomatic COVID-19. The clinical and epidemiological characteristics of the participants were described as median and . 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 doi: medRxiv preprint interquartile range (IQR) for continuous and as percentages for categorical variables.

The univariate analyses were performed using chi-squared test for comparison of proportions and Mann Whitney U test for difference in distributions between different clinical groups (seroconverters and non-seroconverters). Multivariable regression analysis was done to identify independent predictors of seroconversion. The difference in cycle threshold values between asymptomatic and symptomatic individuals in both seroconversion and non-seroconversion categories were tested for significance using Mann Whitney U test. All statistical analyses were performed using R.

The cohort has 3790 COVID-19 participants on December 18, 2020, with the majority of enrolled from Lok Nayak Hospital, New Delhi (45%) and ESIC Medical College Hospital, Faridabad (44.6%). We present clinical results of the first 2504 (2139 from the hospital and 365 from testing centres) participants enrolled between April 2020 to October 2020, who were 6-10 weeks post-diagnosis. The median age was 44 years (IQR: 30, 57) with more than two-thirds males. Nearly two-thirds presented with symptoms, most commonly fever (61%), cough (48%), breathlessness (35%), sore throat (27%) and bodyache (22%). Only one in five individuals had a history of primary contact defined as direct contact for 15 minutes without a mask with someone who tested COVID-19 positive. A history of secondary contact, defined as direct contact for 15 minutes without mask with someone who in turn had a primary contact history was noted in 2%. Among those who gave a contact history, the median duration since contact was four days . 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 doi: medRxiv preprint (IQR: 2, 7). The baseline characteristics of the participants included for serological analysis (n=782) are provided in table -1.

The longitudinal serological evaluation included participants who contributed all three samples (n=782). The seroconversion rates in the windows 0-7 days, 10-28 days and 6-10 weeks were 46%, 84.7% and 85.3% respectively ( Table 2 ). The proportion positive increased with severity of COVID-19 disease, 100% with severe disease, 89.6% with mild to moderate disease and 77.3% of asymptomatic positives had IgG antibodies to RBD antigen. As shown in Figure 1 and Table 3 , the response to NC antigen by anti-NC CLIA was lower (severe-98.3%, mild-moderate 85.9%, and asymptomatic-69.9%). The higher seroconversion with disease severity was consistent when we evaluated the signal to cut-off ratio of both anti-RBD and anti-NC IgG antibody In all those who mounted an IgG response, there was no demonstrable fall as seen by the signal-cut off ratio up to 10 weeks of follow up.

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Demographic and clinical characteristics associated with seroconversion are described in Table 4 . More than 95% (n=105) participants over 60 years of age were IgG antibody positive; this proportion reduced with decreasing age. Interestingly, participants who reported no primary contact or those who were uncertain about the contact history seroconverted more than those who had a history of exposure. Seroconverters reported a longer exposure duration per day than those who did not show IgG response (median exposure: 24 hours vs 8 hours) ( Table 5 ). Presence of pre-existing comorbidities such as chronic hypertension (p-value <0.001) and diabetes mellitus (p-value <0.001) were associated with seroconversion (Table 4 ). The multivariable model after adjusting for confounders showed that age (years) (aOR: 1.03; 95%CI: 1.02,1.05) and presence of symptoms at presentation (aOR: 3.23; 95%CI: 2.01,5.17) were independent predictors of seroconversion.

Age and symptomatic status were independent positive predictors of seroconversion in our Indian cohort. The higher antibody response in severe COVID infection was has reported lower seroconversion rates (45%) among asymptomatic individuals [15] .

. 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. [16] . It has been shown that the nasopharyngeal viral load is significantly higher among individuals with severe COVID-19 as compared to mild illness [17] .

The lower seroconversion rate among asymptomatic RT-PCR positive participants has critical public health significance particularly for interpreting community seroprevalence in highly affected geographical areas. Results from seroprevalence studies from India . 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 doi: medRxiv preprint and other South Asian countries [18] [19] [20] [21] [22] may need to be adjusted in the light of our findings.

The signal-cut off ratio for both the assays reached their maxima between 15-28 days and plateaued suggesting a stable IgG response up to 10 weeks of illness. The longevity of IgG response has been reported to vary between 5 weeks to 4 months. [13, [22] [23] [24] [25] . As shown in another global study [27] , asymptomatic participants in our study did not show any decline of IgG to either antigens over a period of 10 weeks. This is in contrast to initial evidence showing early decline of IgG in asymptomatic infected individuals [3,13]. We will follow up the participants in this cohort for at least a year to evaluate the longevity of IgG response.

The major strengths of the study are the prospective data collection with high follow-up rates, and the use of two validated antibody assays. However, there are some limitations. Due to lack of robust IgM and IgA assays, we were unable to evaluate the immune response with respect to these isotypes. Further, due to the heterogeneity of RT-PCR assays used by the participating hospitals for molecular diagnosis, we do not have a comparable viral load data for all participants in our study. However, analysis in a subset of the individuals who had a similar RT-PCR assay showed no difference in nasopharyngeal viral load between seroconverters and non-seroconverters.

In conclusion, we systematically report clinical and epidemiological characteristics, and longitudinal humoral immune response to SARS-CoV-2 infection in a large Indian cohort. We will continue to study the kinetics of the immune response in a unique platform to evaluate the more complex and emerging questions on cellular immunity, . 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. All authors contributed in revision and approved the final draft of the manuscript.

We deeply thank the Department of Biotechnology, Government of India for supporting the consortium. We are grateful to the leadership and administration of all partner institutions in the consortium for their help and support. We thank all the clinical, laboratory and data management staff for their contributions to this work and the consortium at large. . 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 February 8, 2021. . 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 February 8, 2021. . 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 February 8, 2021. Participants were considered to have severe COVID-19 if they died or required either of the following, oxygen supplementation or ventilatory support, or treatment in the intensive care unit for cardiopulmonary or multiorgan dysfunction. Mild to moderate disease was defined by the presence of symptoms of COVID-19 but not fulfilling the criteria for severe disease. Those who did not report any symptoms at enrolment or throughout the follow-up period were classified as asymptomatic COVID-19.

. 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 doi: medRxiv preprint The seropositivity is represented as absolute numbers and percentages . 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 doi: medRxiv preprint The last column denotes the overall seropositivity rates.

The number in the paranthesis denotes the 95% confidence intervals of the percentage estimates od seroconverters.

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The copyright holder for this preprint this version posted February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 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) . 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) . 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) . 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 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. . 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. Naso-/oropharyn geal swab . 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. 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 February 8, 2021. ; https://doi.org/10.1101/2021.02.04.21251140 doi: medRxiv preprint Anti-NC and anti-RBD IgG denote anti-nucleocapsid and anti-RBD Immunoglobulin-G respectively.

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